WO1994026930A1 - Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities - Google Patents

Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities Download PDF

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WO1994026930A1
WO1994026930A1 PCT/US1994/004496 US9404496W WO9426930A1 WO 1994026930 A1 WO1994026930 A1 WO 1994026930A1 US 9404496 W US9404496 W US 9404496W WO 9426930 A1 WO9426930 A1 WO 9426930A1
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Carlo Croce
Eli Canaani
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Thomas Jefferson University
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Definitions

  • the present invention relates to the field of methods for diagnosis and treatment of human leukemias wherein hematopoietic cells of patients have translocations in a small region of chromosome 11 designated as ALL-l. Diagnostics and therapeutics based on nucleic acid and amino acid sequences are 10 provided.
  • Chromosome translocations have been shown to play an important role in the pathogenesis of human leukemias and lymphomas by either activating cellular protooncogenes or by leading to the formation of chimeric genes capable of transforming hematopoietic cells.
  • Erikson et al .
  • C L chronic myelogenous leukemia
  • Translocations t (1,-19) , t(15;17) , and t(6;9) are other examples of gene fusions, involving in the first two cases transcription factors (Nourse et al . , Cell 1990, 60, 535-545; Kamps et al . , Cell 1990, 60, 547-555; Kakizuka et al .
  • the alternative molecular consequence of translocations is deregulation of protooncogenes by their juxtapositioning to an enhancer or promoter which is active in the type of cell from which the tumor arises.
  • the immunoglobulin (Ig) and T cell receptor (TCR) enhancers participate in at least 15 different translocations associated with Burkitt lymphoma, chronic ly phocytic leukemia, follicular lymphoma, mantle cell lymphoma, and acute T or B cell leukemia.
  • Ig immunoglobulin
  • TCR T cell receptor
  • Chromosomal region llq23 has been shown to be involved in different chromosomal translocations in human acute leukemias of different hematopoietic lineages. Ilq23 chromosome abnormalities have been reported in acute lymphoblastic leukemia and in acute nonlymphoblastic leukemia
  • Chromosome 11 band q23 is frequently rearranged in acute lymphocytic (ALL) , in acute myelomonocytic (AMMOL) , acute monocytic (AMOL) and acute myeloid (AML) leukemias, mostly in reciprocal exchanges with various translocation partners.
  • ALL acute lymphocytic
  • AMMOL acute myelomonocytic
  • AMOL acute monocytic
  • AML acute myeloid leukemias
  • Reciprocal translocation between llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17q25 and 19pl3 are found in 5-6% of AML.
  • Heim and Mitelman, supra are found in 5-6% of AML.
  • interstitial deletions in llq23 have been detected both in ALL and AML.
  • Reciprocal translocations between chromosome region llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17p25 and 19pl3 are found in 5-6% of ANLL.
  • t(4;ll) chromosome translocation In clinical terms, rearrangements of llq23, in particular the t(4;ll) chromosome translocation, have some distinct features.
  • the patients are often quite young; t(4;ll) accounts for the vast majority of cytogenetically abnormal ALLs in infants.
  • the leukemic cells show both B-cell and myeloid marker (Stong et al . Blood 1986, 67, 391-397) and the disease is consequently considered "biphenotypic.
  • the leukemic cells have a CD10-/CD19+ early B cell precursor phenotype and most of them express a myeloid associated antigen (CD15) ; Pui et al . , Blood 1991, 77, 440-447. Myelomonocytic and biphenotypic leukemias carrying the t(4;ll) aberration have also been reported; Nagasaka et al . , Blood 1983, 61, 1174-1181.
  • the cDNA sequence of the ALL-l gene on chromosome 11 is provided.
  • a partial sequence of the AF-4 gene is also provided in the context of the sequences of two reciprocal endproducts of a translocation.
  • Amino acid sequences corresponding to the cDNA sequences of the entire ALL-l gene and the partial sequence of the AF-4 gene, and sequences relating to chimeric genes formed by chromosome translocations with chromosome 4, 9 and 19, respectively, are provided.
  • Probes are provided for detecting chromosome abnormalities involving the ALL-l gene on chromosome 11, including probes for detecting chimeric genes generated by translocations.
  • Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.
  • Figure 1 is a drawing depicting a physical map of YAC B22B, which has been described in Rowley et al. , Proc. Natl . Acad. Sci . USA 1990, 87, 9358-9362.
  • ura and trp correspond to the termini of the vector.
  • a 40 kb segment located towards the ura end and lacking No I and MluI sites is not included in the map.
  • Pulse field analysis indicates two or three Sfil sites located to the left of cosmid 43.
  • Figure 2 is a photograph showing the results of Southern blot analysis of tumor D ⁇ As . Blots were hybridized to the radiolabeled 0.7 kb Ddel fragment derived from the terminus of cosmid 53. Aliquots of 10 ⁇ g were analyzed.
  • Figure 3 is a drawing showing mapping of tumor breakpoints .
  • the internal N ⁇ tl fragment of YAC is shown in the same orientation as in Figure 1.
  • the dotted line represents a region not cloned in the cosmids. Restriction sites within this region are deduced from the size of the relevant germline fragments detected in genomic Southern blots using the indicated probe. Additional EcoRV and Xbal sites are not shown. Some of the samples were not analyzed with BamHI . Lines below the map correspond to the smallest genomic fragments found rearranged.
  • the breakpoint cluster region is believed to span approximately the region encompassed by the two nearest BamHI sites flanking the arrow; more specifically, the breakpoint cluster region is believed to span exons 6-12 illustrated in Figure 10.
  • Figure 4 is a photograph showing the results of Northern blot analysis of RNA from cell lines and a primary leukemia using pooled probes. 10-20 ⁇ g aliquots of total RNA were analyzed on a formaldehyde gel. Following hybridization, blots were washed in a solution containing 0.1% SSC and 0.1% SOS at 700. RNAs were obtained from: a) K562 cells; b) the glioblastoma T98G cell line; c) the SupB pre B ALL cell line; d) the MV4;11 cell line; and e) a patient with t(9;ll) .
  • Figure 5 is a photograph showing the results of Southern blot analysis of DNAs from primary tumors and cell lines with llq23 abnormalities using a modified 0.5 kb Ddel probe.
  • the germline Ba.mHI and Xbal fragments are of 9 and 12 kb, respectively.
  • Figure 6 is a photograph showing the results of Northern blot analysis of RNAs from cell lines using a 1.5 kb EcoRI probe generated from cosmid 20. Lanes included SK DHL (a) ; KC122 (b) ; MV 4;11 (c) ; T98G (d) ; All-1 (e) ; Bl (f) ; K562 (g) ; Jurkat (h) ; GM607 (i) ; 697 (j) ; RS4;11 (k) ; GM1500 (1) ; LNCaPFGC (m) ; PC3 (n) . 28S and 18S indicate migration of ribosomal RNA.
  • Figure 7 shows physical maps of ALL-l cD ⁇ A and gene.
  • Figure 8 shows nucleotide sequence and predicted amino acid sequence of ALL-l cD ⁇ A.
  • Figure 9 depicts homology between ALL-l and Drosophila trithorax (D. Trx) proteins (top and center) , and the structure of ALL-l zinc finger-like domains (bottom) . Bars indicate identical residues. One dot and two dots indicate first and second degree conservative differences, respectively.
  • Figure 10 A-C shows exon-intron structure of ALL-l breakpoint cluster region Figure 10A and partial sequence of the two reciprocal ALL-l/AF-4 fused transcripts (Figure 10B and Figure IOC) .
  • exons containing the zinc finger- like domains (8-12) are represented by cross-hatched boxes.
  • t(4;ll) breakpoints shown (arrowheads in Figure 10A) , included are those of the MV4;11 (MV) , RS4;11 (RS) , and Bl (Bl) cell lines.
  • CL. and I.V. represent leukemic cells with t(4,-ll) from two patients.
  • B, R, G, X, H correspond to sites for the enzymes BamHI, .EcoRI, Bglll, Xbal, and Hindlll, respectively.
  • small and large letters represent introns and exons, respectively. Cytosine in position 4141 of ALL-l sequence ( Figure 2) is replaced by thymidine in clone 25, resulting in alteration of Leucine into Phenylalanine ( Figure IOC) .
  • Figure 11 A-E shows the non ALL-l sequences within the fused RNAs unique to cells with t(4;ll) chromosome translocations (Figure 11A-C) which originate from chromosome 4 ( Figure 11D and HE) .
  • ALL-l cD ⁇ A probe ( Figure HA) , to non ALL-l sequences from cD ⁇ A clone 16 ( Figure 11B) , and to non ALL-l sequences from cD ⁇ A clone 25 ( Figure 11C) .
  • ALL-l is a Philadelphia-chromosome positive cell line (B cell leukemia) lacking Hq23 aberrations (Erikson et al . , Proc Natl . Acad . Sci . USA 1986, 83, 1807- 1811) .
  • K562 originated from chronic myelogenous leukemia (Lozzio, CB and Lozzio, BB, Blood 1975, 45, 321-324) .
  • SKDHL is a B cell lymphoma cell line (Saito et al . , Proc . Natl . Acad . Sci . USA 1983, 80, 7476-7480) .
  • the second and third probes were also used in hybridization to Southern blots ( Figure 11D and HE, respectively) with D ⁇ As from Chinese hamster ovary (CHO cells and CHO cells containing chromosome 4 (CHO/4) .
  • "Fused 1" and "fused 2" correspond to the altered ALL-l RNAs of 14 kb and 12.7 kb, respectively.
  • Figure 12A-C depicts the genomic analysis of the t(6:ll) (q27:q23) chromosome translocation.
  • FIG. 12B Chromosome 6-specific probe XRO .5 detects DNA rearrangement in the bone marrow from a patient, whose karyotype showed Hq23 deletion;
  • Figure 12C Sequence of the t(6;ll) breakpoint region. Cen and Tel denote the direction of the telomeres and centromeres of the two chromosomes . Open vertical boxes represent defined exons. Restriction sites: B, BamHI, H., HindHI, G, Bglll; Rm, EcoRI and X, Xbal.
  • Figure 13A-C shows the cloning and sequencing of AF-6 cDNA and of ALL-l/AG-6 fusion transcript.
  • Figure 13A AF-6 cDNA clones. Dashed lines indicate different sequences possibly representing alternative non-coding exons. Restriction sites: A, Apal; B, BamHI,-, H, HindHI and S, Sad .
  • Figure 13B Predicted amino acid sequence of AF-6 cDNA coding region. Arrow indicates the RNA fusion point.
  • Figure 14 shows a comparison of the GLGF repeat within the AF-6 protein to GLGF repeats of other patients.
  • GLGF repeats are the third GLGF in human ZO-1 (ZO-1 3) ; the second GLGF in rat PSD95 (PSD95 2) , and the third GLGF in Drosophila large disc tumor suppressor gene (dlq3) .
  • Bold amino acids are consensus amino acids conserved among the four proteins.
  • Figure 15 depicts a Northern analysis of AF-6 RNA in human cell lines. 5-10 ⁇ g of polyadenylated RNA were analyzed on agarose gel containing formaldehyde. RNAs were obtained from the lines KC122, K562, B-l, MV4;11, SKDHL, T98G, 293 (a-g, respectively) .
  • Figure 16A and 16B shows genomic analysis of the t(ll,-17) chromosome translocation.
  • Figure 16A Physical map of the genomic junction of patient GUS [der (17)] and a map of the corresponding normal region (chr. Hq23) . Numbered open boxes in the top line represent ALL-l exons. Darkened segment of der (17) correspond to chromosome 17 sequences, and open box therein represents an exon. Fragment Rl .7 was used as a probe for the genomic Southern analysis as well as for cDNA screening. Cen and Tel show directions of the centromeres and telomeres, respectively.
  • R EcoRI; H, HindHI; B, BamHI; G, Bglll, X, Xbal.
  • Figure 16B Southern genomic analysis of a DNA from patient GE with AML and t(ll;17) , and a normal DNA (lanes b and a, respectively) . DNAs were digested with EcoRV and hybridized with the Rl.7 probe. Germline fragment is 18 kb.
  • Figure 17A-C shows cloning and sequencing of AF-17 cDNA and of the junction within ALL-l/AF-17 fusion transcript.
  • Figure 17A Physical map of AF-17 cDNA clones. Restriction sites: S, Sad; H, HindHI; H2, Hindi. Initiation (ATG) and termination (TAA) are shown by arrows.
  • Figure 17B Predicted amino acid sequence of AF-17 protein. Cysteines within the cysteine-rich region at the N-terminus are underlined. Also underlined is the leucine zipper at positions 729-764. Arrow indicates point of fusion with the ALL-l protein.
  • Figure 17C All-l/AF-17 RNA junction cloned from the leukemic cells of patient GUS.
  • Figure 18A and 18B depicts ho ology between the AF-17 protein and the human Brl40 (peregrin) protein.
  • Figure 18A Alignment of AF-17 and Brl40 cysteine-rich domains. Bars indicate identical residues; one dot and two dots indicate first and second degree conservative differences, respectively.
  • Figure 18B Potential zinc fingers within the cysteine-rich domain of AF-17.
  • Figure 19 shows Northern analysis of AF-17 RNA in human cell lines. 5-10 ⁇ g of polyadenylated RNA were analyzed on agarose gel containing formaldehyde . RNAs were obtained from the cell lines KCl-122, MV4;11, ALL-l, GM-607, B 1, 380, PC3, GM 1500, K562, T93G, 679 (a to j, respectively) .
  • Figure 20 depicts landmarks, common motifs and homologous sequences within the partner proteins AF-4, AF-9, ENL, AF-6 and AF-17, and within the ALL-l protein. Arrows indicate fusion points between ALL-l and the partner proteins. Striped regions in AF-9 and ENL indicate domains of highest homology between the two proteins. NTS, nuclear targeting sequence, LZ, leucine zipper, MTase, methyl transferase.
  • Figure 21A and 21B shows use of the B859 probe in detecting ALL-l abnormalities.
  • Figure 21A The B859 probe and the breakpoint cluster region of the ALL-l gene (BCRHq23) . Numbered boxes are the exons of the ALL-l gene. Thin lines display the subclones used for sequencing. Cen. and Tel. denote the centromere and telomere.
  • Figure 21B Southern analysis of the ALL-l gene rearrangements in patients with acute leukemia. Patient's DNA samples were digested with BamHI and probed with the B859 probe. Numbers in each lane correspond to the case numbers in Table 2.
  • Figure 22 shows the nucleotide sequence of the breakpoint cluster region within the ALL-l gene. The predicted amino acid sequences of each exon are shown under the corresponding nucleotide sequences. A consensus sequence for topoisomerase II recognition site is underlined.
  • Figure 23 is a schematic representation of the exons, Alu repeats, and the breakpoints in the breakpoint cluster region in the ALL-l gene. Filled boxes are exons. Alu repeats are shown as open boxes. Arrows point to the positions of the breakpoints with their corresponding case numbers presented in Table 2. Hatched box represents a 130 bp novel repetitive sequence .
  • Figure 24a and 24b shows Southern analysis of ALL-l gene rearrangements in adult AML patients without cytogenetic evidence of Hq23 translocations.
  • the label above each lane corresponds to a unique patient identification number taken from (Caligiuri et al . , Cancer Res . 1994 54, 370-373) .
  • Patients nos . 23 and 24 had trisomy 11 as a sole cytogenetic abnormality whereas patient no. 1 had a normal karyotype. Arrows indicate rearranged bands. N, normal control.
  • Figure 24a Blots examined with the B859 probe.
  • B859 is a cDNA probe (Caligiuri et al . , Cancer Res .
  • Figure 25a-c shows the structure of partial duplication of the ALL-l gene.
  • Figure 25a Restriction enzyme maps of lambda clones ( ⁇ 23 and ⁇ 24) corresponding to rearranged BamHI fragments from two AML patients with trisomy 11. Boxes represent ALL-l exon positions determined by subcloning and partial DNA sequence analysis. The junction point of the duplication is indicated by the juncture of the black and shaded bars. Position of the SASl probe is shown. B, BamHI; R, EcoRI; H, HindHI; X, Xbal.
  • Figure 25b Proposed structure of the partially duplicated ALL-l gene contains a direct tandem duplication spanning exons 2-6.
  • Figure 25c DNA sequence across the junction points of clones ⁇ 23 and ⁇ 24 are aligned with sequences from introns 1 and 6 of the ALL-l gene. ⁇ 24 has a 2 bp N-segment. Heptamer-like signal sequences (Akira et al . , Science 1987 238, 1134-1138) near the junction points in both clones are underlined. Nonamer-like signal sequences are not present.
  • Figure 26a and b shows RNA-PCR analysis of trisomy 11 patient samples.
  • Figure 26a Agarose gel of RNA-PCR products
  • the ALL-l gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 2, 4, 6, 9, 10, 15, 17 or 19.
  • the gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of approximately 4,000 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene including cysteine-rich regions which can be folded into six zinc finger-like domains.
  • the breakpoint cluster region within ALL-l spans approximately 8 kb and encompasses several small exons (including exons 5-12) , most of which begin in the same phase of the open reading frame .
  • the t(4;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 4.
  • This gene on chromosome 4 is termed "AF-4" while the chimeric gene resulting from the t(4;ll) translocation is termed "ALL-l/AF-4. " It is believed that the Hq23 abnormality of translocation with 4q21 gives rise to one or two specific oncogenic fusion proteins.
  • the t(9;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 9.
  • This gene on chromosome 9 is termed "AF-9” while the chimeric gene resulting from the t(9;ll) translocation is termed "ALL-l/AF-9. " It is believed that the Hq23 abnormality of translocation with 9p22 gives rise to one or two specific oncogenic fusion proteins.
  • the t(ll;19) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 19. This gene on chromosome 19 is termed "ENL” while the chimeric gene resulting from the t(ll;19) translocation is termed "ALL-1/ENL.” It is believed that the t(ll;19) translocation gives rise to one or two specific oncogenic fusion proteins.
  • the gene on chromosome 17 is termed AF-17 and the chimeric gene resulting from the t (11:17) translocation is termed ALL-l/AF-17.
  • a DNA fragment which detects DNA rearrangements by Southern analysis in the majority of patients with t(4;ll) , t(9;ll) and t(ll;19) chromosomal aberrations has been cloned from chromosome 11.
  • This locus is referred to as ALL-l for acute lymphocytic leukemia, although the same locus is also involved in acute myelomonocytic, myelogenous and monocytic leukemias carrying translocations involving Hq23.
  • DNAs and RNAs were extracted from cell lines and primary tumors by conventional methods. Southern and Northern analysis were performed as described in Shtivelman et al . , Na ture 1985, 315, 550-554) .
  • cosmids were, digested with a variety of restriction enzymes, and analyzed by Southern blotting for fragments which do not react with radiolabeled total human DNA. End fragments of cosmids were identified by hybridizing cosmids' digests to radiolabeled oligonucleotides corresponding to the recognition sequences for T7 and T3 RNA polymerases . If the end fragments contained human repeats, they were isolated, digested with frequent cutters and analyzed as described above. The 0.7 kb Ddel probe was thus obtained from a terminal 3.5 kb Ec ⁇ RV fragment of cosmid 53.
  • the insert was partially digested by a combined application of dam methylase and the restriction endonuclease Mbol, Hoheisel et al . , Nuc . Acid Res . 1989, 17, 9571-9582. Both enzymes act on the sequence GATC, but Mbol is unable to cut the methylated form. More than a hundred cosmid clones, detected with a probe for human repetitive sequences, were obtained. The cosmids were mapped by screening for those with sites for NotI and Mlul enzymes, and for those hybridizing to CD3, trp and ura probes. Some cosmids were established using unique (repeat free) probes obtained from termini of cosmids.
  • a 0.7 kb Ddel fragment derived from the terminus of cosmid 53 detected rearranged fragments in tumor D ⁇ As digested with EcoRV, Xbal, or Bair-HI . Examples of these analyses are shown in Figure 2.
  • the leukemic cells from patients A.G., E.C, A.L., B.H., I.B., G.F., P.P., and V.S. contained novel EcoRV or Xbal fragments of various sizes. This probe detected rearrangements in 6/7, 4/5, and 3/4 patients with the t(4;ll) , t(9;ll) and t(ll;19) translocations, respectively.
  • pooled unique fragments from cosmid 20 were labeled, together with the terminal fragment of cosmid 53, and were used to probe RNAs from cell lines and patients with or without Hq23 translocations (Figure 4) .
  • the pooled probe detected 5 kb and 10 kb RNA species in the K562, glioblastoma T986 and Sup B cell lines (lanes a, b, c) . It also hybridized with a 5 kb RNA from patients with t(4;ll) , t(9;ll) , and t(ll;19) ( Figure 4, lanes d, e,) . In another patient with t(4;ll) the probe detected the 10 kb RNA species alone.
  • tumor DNAs from the leukemic cells of patients with t (6,-H) (q27;q23) , t (1;11) (p34 ;q23) , t (10;11) (pll-15;q23) and del (11) (q23) were digested with BamHI, Xbal, EcoRV and HindHI enzymes and subjected to Southern analysis using the modified 0.5 kb Ddel fragment as a probe.
  • This probe was obtained from the 0.7 kb Ddel probe by digestion with Alul, which ultimately improved performance by removing a 0.24 kb internal fragment that had caused a higher background in Southern analyses .
  • the internal fragment and the two end fragments were electrophoresed to isolate the two terminal fragments, which were then ligated to form a 0.5 kb fragment which was cloned into a plasmid vector.
  • Results of Southern blotting are shown in Figure 5. Rearranged fragments were found in the DNAs of patients with t(6;ll) , t(l;ll) and t(10,-ll) (lanes a, d, e, respectively) and in two patients
  • segments of cosmid 20 found fully or partially free of repetitive sequences were examined as probes to polyadenylated RNAs obtained from a variety of hematopoietic and nonhematopoietic cell lines.
  • Three ALL cell lines, MV 4;11, RS 4;11 and Bl containing the t(4,-ll) chromosome translocation were included in the analysis. These three cell lines had rearrangements at the breakpoint cluster region, as shown in Figure 5, lanes b and c.
  • a 1.5 kb EcoRI DNA segment generated from cosmid 20 was used as a probe and identified a 12.5 kb RNA in all cell lines ( Figure 6) .
  • the breakpoint in the RC-K8 cell line containing the t(ll;14) (q23;q32) is apparently telomeric to the locus discussed here.
  • other unidentified loci on chromosome 11 could be involved.
  • the ALL-l locus might also be affected in these patients, but this may occur at a different site.
  • RNA species of similar size have recently been reported by others. For example, Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA
  • (#1) which is derived from the same general location, detects predominantly the 12.5 kb species. While the instant probe detects 11 kb transcript solely in leukemic cells with the t(4,-ll) chromosome translocation, the Ziemin-van der Poel et al . study identifies an 11 kb mRNA in the RS4;11 cell line, as well as in small amounts in all cells tested. The results show, however, a clear qualitative alteration in expression of a region adjacent to the breakpoint cluster region on chromosome 11 in cells with the t(4;ll) chromosome translocation.
  • telomeric to the cluster region detected two additional transcripts of 11.5 and 11 kb in the RS 4;11 cell line, as well as in all hematopoietic and nonhematopoietic cells tested (Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA 1991, 88, 10735-10739) .
  • Translocation junction fragments were cloned from leukemic cells with t(4;ll) and showed clustering of the breakpoints in an area of 7-8 kb on chromosome 4.
  • Sequencing analysis indicated heptamer and nonamer-like sequences, associated with rearrangements of immunoglobulin and T cell receptor genes, near the breakpoints. These sequences suggested a direct involvement of the VDJ recombinase in the Hq23 translocations.
  • RNAs transcribed from the der (11) and der (4) chromosomes were analyzed using segments of cloned DNAs as probes. Probes from both sides of the region identified a major transcript of 15-16 kb (previously estimated as 12.5 kb) (Cimino et al . , Cancer Research 1991, 51, 6712- 6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) in cells with or without Hq23 abnormalities. The gene coding for these RNAs was termed ALL-l. Leukemic cells with the t(4;ll) chromosome translocation contained, in addition to the normal species, shorter RNAs transcribed from the der (11) and der (4) chromosomes .
  • ALL-l RNA was extended to clone and sequence ALL-l RNA, to further characterize the ALL-l gene, and to identify chimeric transcripts produced in cells with the t(4,-ll) chromosome translocation. Structure of the ALL-l gene and cDNA
  • a human fibroblast cDNA library and a K562 cDNA library were screened (Chu et al . , EMBO J. 1990, 9, 985-993; Shtivelman et al . , Nature 1985, 315, 550-554) . Positive clones were used as probes for further screening.
  • clones mg 11.1, cl4, and gc3 were obtained from a genomic DNA library made in the EMBL-3 vector (Stratagene) . Restriction enzyme mapping of the cDNA and genomic clones and analysis of the hybridization pattern of cDNA fragments to genomic DNA indicated that the ALL-l gene is composed of a minimum of 21 exons, some of them (6-12) very small (shorter than 150 bp) . The first intron was found to be the largest, spanning approximately 35 kb of DNA.
  • the nucleotide sequence of ALL-l cDNA was determined using an automatic sequencer (ABI) .
  • the sequence revealed a single long open reading frame predicting a protein of approximately 4,000 amino acids with molecular weight of approximately 400,000 Daltons ( Figure 8) .
  • GenBank and SWISS data bases were screened by the FASTA program.
  • Nucleotides 9353-9696 were found to be nearly identical to an anonymous sequence (EST00626) cloned from human fetal brain cDNA library (Adams et al. , Nature 1992, 355, 632- 634) .
  • the third region of homology constitutes the extreme C-terminus of the two proteins; both species end in an identical sequence.
  • the first homology region is cysteine-rich and contains sequence motifs analogous to four zinc finger domains (3-6) within the trithorax gene (Mazo et al . , supra) .
  • the second region of homology is also cysteine-rich and corresponds to zinc fingers 7 and 8 of the Drosophila gene.
  • the human putative zinc finger structures are shown at the bottom of Figure 9.
  • the multiple conserved cysteines and histidines at the 3' end of the motifs allow two or three arrangements of the putative fingers.
  • the structure of these cysteine-rich domains appears to be unique to the trithorax and ALL-l genes.
  • RNAs resulting from the t(4;ll) chromosome translocations Clustering of t(4;ll) breakpoints has previously been found within a small segment of the ALL-l locus (Cimino et al . , Cancer Research 1991, 51, 6712-6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) . This region includes 7 coding exons (6-12) containing 74, 132, 114, 147, 96, 121, and 123 bp respectively. Exons 8-12 contain four zinc finger motifs. Exons 7-11 all begin in the first nucleotide of a codon.
  • t(4;ll) translocations should result in fusion of the ALL-l gene to the gene aforementioned and, consequently, in production of two reciprocal chimeric RNAs .
  • a cD ⁇ A library was constructed from R ⁇ A extracted from the RS4;11 leukemic cell line established from a patient with the t(4;ll) chromosome translocation (Stong, RG, and Kersey, JH, Blood 1985, 66, 439-443) .
  • This RS4;H cD ⁇ A library was constructed by treating polyadenylated RNA with 1 mM methyl mercury for 10 minutes at room temperature, followed by neutralization with 10 mM mercaptoethanol and alcohol precipitation.
  • cD ⁇ A was prepared by using the Time Saver kit (Pharmacia) and was cloned into the lambda ZAP II vector (Stratagene) .
  • the library (2 X 10 6 clones) was screened with a probe composed of exons 3-13. Twenty positive clones were purified and mapped. Two clones varied from normal ALL-l cDNA and were further analyzed by sequencing.
  • Clone 16 contained normal ALL-l sequences 3' to the beginning of exon 9. 5' to this position, ALL-l information was substituted with a new DNA fragment composed of an open reading frame (ORF) that joins in phase the rest of ALL-l ORF ( Figure 10B) .
  • Clone 25 had a reciprocal configuration in which exon 7 of ALL-l is linked to a new DNA segment containing an open reading frame. Here again, the two ORFs are joined in phase ( Figure IOC) .
  • RNAs specific to cells with the t(4;ll) chromosome translocation Two such transcripts were identified: a 14 kb RNA (previously estimated as 11.5 kb) containing 3' ALL-l sequences and a 12.7 kb RNA (previously estimated as 11 kb) hybridizing to 5' ALL-l probe. These RNAs were transcribed from chromosome derivatives 4 and 11, respectively.
  • RNAs from cell lines with or without the t(4;ll) chromosome translocation As a control, the RNAs were first hybridized to 3' ALL-l cDNA probe which detected the major normal transcript of 15-16 kb
  • Clone 16 probe identified a 9.5 kb RNA in all cells examined and a 14 kb transcript in RS4;H, MV4;H and B-l cells ( Figure HB) . It was concluded that clone 16 originated from the 14 kb altered ALL-l transcript and that the non-ALL-1 sequence within this RNA is expressed in human cells as a 9.5 kb transcript, which corresponds to the normal AF-4 transcript on a non-rearranged chromosome 4.
  • clone 25 originated from the second altered 12.7 kb ALL-l
  • ALL-l gene Cloning and sequence analysis of the ALL-l gene indicates that it encodes an unusually large protein of 4,000 amino acids with a mass of approximately 400 kD .
  • the striking feature of the protein is its homology to the Drosophila trithorax gene. The homology is reflected in three ways. First, the transcripts and proteins have a similar size; the Drosophila gene is transcribed into a 15 kb RNA encoding a protein of 3759 amino acids (Mozer, BA, and David, IB, Proc . Na tl . Acad. Sci . USA 1989, 86, 3738-3742; Mazo et al . , Proc . Natl . Acad . Sci . USA 1990, 87, 2112-2116) .
  • the trithorax gene in Drosophila acts to maintain spatially-restricted expression patterns of the Antennapedia and Bithorax complexes during fruit fly development (Ingham, PW, Cold Spring Harbor Symp . Quant . Biol . 1985, 50, 201-208) .
  • Trithorax activates transcription of multiple genes of the two complexes and, as such, counteracts the activity of Polycomb group genes which act as repressors of transcription for the same genes (McKeon, J and Brock, HW, Roux' s Arch . Dev. Biol . 1991, 199, 387-396) .
  • antibodies to the protein react with specific regions of the chromatin in the salivary glands of Drosophila.
  • ALL-l gene is a transcription factor and that it is involved in regulation of genes controlling human development and/or differentiation. While expression of ALL-l during embryonic development has not yet been investigated, the isolation of ALL-l sequences from a human fetal cDNA library indicates transcription of the gene during fetal development. Previous studies (Cimino et al . , Cancer Research 1992, 52, 3811-3813) demonstrated ALL-l RNA in a variety of hematopoietic cell lines, as well as in tumors originating from precursors of epithelial and glial cells.
  • t(4;ll) chromosome translocation cleaves the ALL-l gene within the coding region and results in fusion of the open reading frames of ALL-l and a gene on chromosome 4 (termed AF-4) in phase.
  • the breakpoints on chromosome 11 cluster in a region containing several small exons, 5 of them (exons 7-11) begin in the first letter of a codon. Splicing from the same exon on chromosome 4, adjacent to the breakpoint in RS4;H, to each one of the five exons on chromosome 11 will retain the two open reading frames fused in phase.
  • Two chimeric proteins from the 12.7 and 14 kb R ⁇ As are predicted for cells with the t(4;ll) chromosome translocation.
  • the lack of information about the normal AF-4 protein precludes at this time the determination if it is also a transcription factor that exchanges functional domains with ALL-l to give a chimeric transcription factor. This occurs in the t(l;19) and t(15,-17) chromosome translocations (Kamps et al . , Cell 1990, 60, 547-555; ⁇ ourse et al . , Cell 1990, 60, 535-545; Kakizuka et al., Cell 1991, 66, 663-674; de The et al .
  • the present invention provides methods of diagnosis for human leukemia by providing a tissue sample from a person suspected of having acute lymphocytic, myelomonocytic, monocytic or myelogenous leukemia, and determining if there are breakpoints on chromosome 11 in the ALL-l locus.
  • the sequence of the ALL-l cDNA can be used to generate probes to detect chromosome abnormalities in the ALL-l breakpoint cluster region. These probes may be generated from both the sense and antisense strands of double-stranded DNA.
  • ALL-l probe refers to both genomic and cDNA probes derived from the ALL-l gene.
  • genomic probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences from at least 10 kb centromeric to at least 10 kb telomeric to the breakpoint cluster region, which has been shown to span at least exons 6-9, and may span exons 5-12 of the ALL-l gene. It is believed that cDNA probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences ranging from 2 kb centromeric to 2 kb telomeric to the breakpoint cluster region.
  • preferred embodiments of the present invention for detecting chromosomal abnormalities involving ALL-l provide genomic and cDNA probes spanning the chromosome 11 regions described above. cDNA probes are more preferred, and probes comprising the exons included in the breakpoint cluster region are most preferred.
  • Part or all of the ALL-l cDNA sequence may be used to create a probe capable of detecting aberrant transcripts resulting from chromosome 11 translocations.
  • the EcoRI probe for example, was derived from a genomic clone but its location lies within an exon.
  • preferred embodiments of the present invention for detecting aberrant transcripts provide cDNA probes spanning the ALL-l gene.
  • ALL-l/AF-4 sequences provided in SEQ ID NO: 23 and SEQ ID NO:24 can be used to create probes to detect t(4;ll) chromosome abnormalities and aberrant transcripts corresponding to t(4,-ll) translocations. Additional sequences (see below) include those specific for the ALL-l/AF-6, ALL-l/AF-9 and ALL- l/AF-17 chimeric genes. Also included in the invention and described below are specific ALL-l probes capable of detecting chromosomal abnormalities in the ALL-l gene irrespective of the nature of the fusion partner gene.
  • PCR Polymerase Chain Reaction
  • restriction fragment length analysis oligonucleotide hybridization using, for example, Southern and Northern blotting and in si tu hybridization.
  • PCR technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in PCR Protocols : A Guide to Methods and Applications, Innis, M.A. et al .
  • PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5' and 3' primers that hybridize to sequences present in a DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the DNA sequence between the primers with the free nucleotides to produce a complementary strand of DNA.
  • the enzyme will fill in the complementary sequences between probes only if both the 5' primer and 3' primer hybridize to DNA sequences on the same strand of DNA.
  • one of the two probes can be generated from the ALL-l cDNA and one probe from the AF-4 gene.
  • RNA is isolated from hematopoietic cells of a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia, and cDNA is generated from the mRNA. If the cDNA of the chimeric ALL-l/AF-4 gene is present, both primers will hybridize to the cDNA and the intervening sequence will be amplified.
  • the PCR technology therefore provides a straightforward and reliable method of detecting the chimeric gene.
  • the preferred primers for PCR are selected, one from a portion of SEQ ID NO: 1, corresponding to the ALL-l cDNA, and one from a portion of either SEQ ID NO: 19 or SEQ ID NO: 22, corresponding to AF-4 gene sequences.
  • the sequences chosen from SEQ ID NO: 1 comprise at least a portion of SEQ ID NO: 20, which corresponds to exon 9, or SEQ ID NO: 21, which corresponds to exon 7.
  • diagnostic kits can be assembled which are useful to practice oligonucleotide hybridization methods of distinguishing chromosome 11 abnormalities from non-rearranged chromosomes 11.
  • diagnostic kits comprise a labelled oligonucleotide which hybridizes, for example, to the chimeric transcript that results from t(4;ll) translocations but which does not hybridize to nucleic acid transcripts not associated with aberrations.
  • diagnostic kits of the present invention comprise, for example, a labelled probe that includes ALL-l and AF-4 sequences which make up the chimeric transcript associated with t(4;ll) translocations.
  • probes comprise oligonucleotides having at least a portion of the sequence of the ALL-l/AF-4 gene of SEQ ID NO: 23 or SEQ ID NO: 24.
  • labelled probes of the oligonucleotide diagnostic kits according to the present invention are labelled with a radionucleotide.
  • the oligonucleotide hybridization-based diagnostic kits according to the invention preferably comprise DNA samples that represent positive and negative controls.
  • a positive control DNA sample is one that comprises a nucleic acid molecule which has a nucleotide sequence that is fully complementary to the probes of the kit such that the probes will hybridize to the molecule under assay conditions.
  • a negative control DNA sample is one that comprises at least one nucleic acid molecule, the nucleotide sequence of which is partially complementary to the sequences of the probe of the kit. Under assay conditions, the probe will not hybridize to the negative control DNA sample.
  • Probes useful as diagnostics can be used not only to diagnose the onset of illness in a patient, but may also be used to assess the status of a patient who may or may not be in remission. It is believed that emergence of a patient from remission is characterized by the presence of cells containing chromosome abnormalities. Thus, patients believed to be in remission may be monitored using the probes of the invention to determine their status regarding progression or remission from disease. Use of such probes will thus provide a highly sensitive assay the results of which may be used by physicians in their overall assessment and management of the patient's illness .
  • Antisense oligonucleotides which hybridize to at least a portion of an aberrant transcript resulting from chromosome 11 abnormalities involving the ALL-l gene are also contemplated by the present invention.
  • the oligonucleotide may match the target region exactly or may contain several mismatches.
  • molecules which bind competitively to RNA coded by, for example, the chimeric ALL-l/AF-4 gene, for example are envisioned for therapeutics.
  • Preferred embodiments include antisense oligonucleotides capable of binding to at least a portion of SEQ ID NO: 23 and SEQ ID NO: 24.
  • Preferred embodiments of the present invention include antisense oligonucleotides capable of binding to a region of the ALL-l/AF-4 mRNA corresponding to the ALL-l sequences which encode a peptide having homology with the Drosophila trithorax protein and antisense oligonucleotides capable of binding to a region of the mRNA encoding a zinc finger-like domain in the ALL-l protein.
  • oligonucleotide sequences shorter than 15 bases may be less specific in hybridizing to the target and may be more easily destroyed by enzymatic degradation. Hence, oligonucleotides having at least 15 nucleotides are preferred. Sequences longer than 21 nucleotides may be somewhat less effective in interfering with ALL-l expression because of decreased uptake by the target cell. Therefore, oligonucleotides of 15-21 nucleotides are most preferred.
  • oligonucleotide as used herein includes both ribonucleotides and deoxyribonucleotides, and includes molecules which may be long enough to be termed “polynucleotides . " Oligodeoxyribonucleotides are preferred since oligoribonucleotides are more susceptible to enzymatic attack by ribonucleotides than deoxyribonucleotides. It will also be understood that the bases, sugars or internucleotide linkages may be chemically modified by methods known in the art. Modifications may be made, for example, to improve stability and/or lipid solubility.
  • oligonucleotides of the present invention may be synthesized by any of the known chemical oligonucleotide synthesis methods. See for example, Gait, M.J., ed. (1984) , Oligonucleotide Synthesis (IRL, Oxford) .
  • antisense oligonucleotides hybridizable with any portion of these sequences may be prepared by the synthetic methods known by those skilled in the art . It is generally preferred to apply the therapeutic agent in accordance with this invention internally such as intravenously, transdermally or intramuscularly. Other forms of administration such as topically or interlesionally may also be useful. Inclusion in suppositories is presently believed to be likely to be highly useful. Use of pharmacologically acceptable carriers is also preferred for some embodiments .
  • the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a pharmaceutical carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • the liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells (Arad et al . , Biochem . Biophy. Acta . 1986, 859, 88-94) .
  • the antisense oligonucleotides may be administered in an amount effective to result in extracellular concentrations approximating in vi tro concentrations described below.
  • the actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors.
  • the daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required.
  • the antisense oligonucleotides may be administered in amounts effective to kill leukemic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the nature and extent of the leukemia, the particular oligonucleotide utilized, the relative sensitivity of the leukemia to the oligonucleotide, and other factors. Concentrations from about 10 to 100 ⁇ g/ml per 10 5 cells may be employed, preferably from about 40 to about 60 ⁇ g/ml per 10 5 cells . Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment.
  • dosages from about 2 to about 20 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 12 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
  • the present invention is also directed to monoclonal antibodies capable of binding to chimeric ALL-l/AF proteins including ALL-l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17, and includes monoclonal antibodies capable of binding to a region of the protein having homology with the Drosophila trithorax protein and monoclonal antibodies capable of binding to a zinc finger-like domain.
  • Such monoclonal antibodies are useful as diagnostic and therapeutic agents for leukemias characterized by t(4;ll) , (t(6;ll) , t(9;ll) and t(H;17) translocations.
  • the present invention encompasses immunoassays for detecting at least portions of either the ALL- l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17 proteins.
  • the instant invention contemplates diagnostic kits comprising a monoclonal antibody to at least a portion of the ALL-l fusion proteins listed above in combination with conventional diagnostic kit components.
  • the present invention is also directed to pharmaceutical compositions comprising monoclonal antibodies and a suitable pharmaceutical carrier, which are well known in the pharmaceutical art, and are described, for example, in
  • Polyclonal antibodies to the instant polypeptides are also within the ambit of the invention.
  • Such polyclonal antibodies may be produced using standard techniques, for example, by immunizing a rabbit or a rat with a protein or peptide of the invention, removing serum from the rabbit, and harvesting the resultant polyclonal antibodies from the serum.
  • the polyclonal antibodies may be used as an IgG fraction or may be further purified in varying degrees. Procedures for preparing, harvesting and purifying polyclonal antibodies are well known in the art, and are described, for example, in Methods in Immunology: A Laboratory Text for Instruction and Research, Garvey et al . , Ed., W.A. Benjamin, Reading MA, 1977, 3rd ed. , chapter 22, 24-30.
  • Example 1 provides further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(4;ll) translocations.
  • the information provided in example 1 includes complete cDNA sequences encoding AF-4. These sequences may be used design probes of at least 15 nucleotides which are capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes comprise, an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:25 or SEQ ID NO:27. The procedures for using such probes are described above.
  • Example 2 Provide further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(9;ll) translocations.
  • the information provided in example 2 may be used design probes of at least 15 nucleotides which is capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes may comprise at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. Further, probes capable of identifying chromosome abnormalities within the AF-9 gene of chromosome 9 may be designed.
  • probes comprise an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. The procedures for using such probes are described above.
  • the experiments reported in Examples 3 and 4 describe the cloning and sequencing of ALL-l/AF-6 and ALL-l/AF-17 genes, respectively.
  • the experiments reported in Example 5 describe a probe capable of detecting abnormalities in the ALL-l region irrespective of the nature of the fusion gene, and the experiments reported in Example 6 describe duplications of the ALL-l region in cells of some patients with leukemia.
  • the invention must be construed to include each of these genes, their products and probes derived therefrom as being useful for the diagnosis and treatment of patients with these types of leukemias.
  • each example must be construed to include the other named fusion genes as being useful in the methods and compositins of the invention.
  • a method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations may be performed by first providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; then isolating RNA from the sample followed by generating cDNA from said RNA and amplifying a chimeric gene sequence in said cD ⁇ A which is generated by said translocation using a set of PCR primers if said chimeric gene is present such that detecting the presence of amplified DNA indicates the tissue sample is derived from an individual suffering from lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations.
  • the method may be performed using sets of primers which can be used to amplify a chimeric gene generated by the translocation.
  • primers can be designed, for example, using the sequence information in SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34.
  • primers include SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
  • Monoclonal antibody capable of binding to at least a portion of for example, the chimeric ALL-l/AF-9 protein may be produced by standard techniques.
  • Examples of such a monoclonal antibodies, which can bind specifically to at least a portion of the amino acid sequences encoded by SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13, may be produced using peptides which comprise at least a portion of SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13.
  • tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia is examined to detect the ALL-l/AF-9 chimeric protein or a portion of the chimeric ALL-l/AF-9 protein.
  • a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein is used.
  • the present invention provides antisense oligonucleotides capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA.
  • antisense oligonucleotides include those capable of binding to at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34.
  • Method of treating acute lymphoblastic or nonlymphoblastic leukemia comprise administering an antisense oligonucleotide capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA or, alternatively, administering a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein.
  • the formulation and administration of therapeutics are outlined above.
  • a third cDNA clone, k 1.1 represents another RNA variant probably resulting from alternative splicing; an in frame termination codon is present in this clone immediately 3' to the divergence point.
  • AF-4 encodes 2 or more proteins varying at their termini.
  • AF-4 contains an unusually long 3' untranslated region of 5 3kb. This region includes multiple AATAAA sequences located 20 nucleotides 5' of the poly A, as well as in several upstream positions; it also contains several stretches of T.
  • AF-4 protein sequence was searched for homology to other proteins and for the presence of motifs.
  • the sequence AKKRK at positions 811-815 matched the consensus nuclear targeting sequence - (RKTA) KK (RQNTSG) K- (Gomez-Marquez and Segada, 1988) .
  • AF-4 was relatively rich in serine (16%) and proline (11%) compared to the average frequency of these amino acids (7.1% and 4.6%, respectively) .
  • the protein encloses a nuclear targeting sequence
  • AF-9 protein is serine rich (20%) and includes a remarkable uninterrupted stretch of 42 serines at positions 149-190; it also contains proline at a frequency of 7% which is above the average frequency of 4.1%.
  • a homology search showed, unexpectedly, that the predicted protein shared high similarity with the ENL protein SEQ ID NO:31.
  • the latter is located on chromosome 19 and is fused to the ALL-1/HRX gene in t(ll;19) chromosome translocations.
  • the two proteins show 56% identity and 68% similarly.
  • the homology is highest within the 140 amino acids at the N terminus where the proteins are 82% identical, and 92% similar, and within the 67 amino acids at the C terminus where the corresponding values are 82% and 91%.
  • the der 11 chromosome was due to a break in the other ALL-l DNA strands within the intron flanked by exons 6 and 7, and to a breakage of the second AF-9 DNA strand within an intron located 5' of the AF-9 exon mentioned above.
  • RNA 11 is transcribed into an RNA corresponding to cDNA clone E2.
  • a BamHI-Stul cDNA probe detected some normal genomic fragments, which were also detected by the 0.4 kb Hindlll-Avall probe-derived from the genomic junction cloned from DNA of patient CO.
  • the AF-9 protein is linked at position 375 to the ALL-l moiety, while in patient FI the junction point is at amino acids 444 or 477 of AF-9.
  • the reading frames of the two genes are joined in phase.
  • Hq23 abnormalities are the multitude of chromosome partners participating in translocations with the ALL-l locus.
  • This promiscuity in partners for rearrangement and fusion could suggest that the only critical event in all these different translocations is the separation of a DNA binding domain (either the zinc fingers or the AT hooks in the ALL-l gene) from a positive or negative regulatory element, and that the proteins encoded by the partner genes solely provide initiation or termination codons .
  • High molecular weight DNAs from patients with t(9;ll) chromosome translocation were partially digested with Mbol enzyme and cloned into the EMBL-3 phage vector (Promega) .
  • the libraries were plated into the host bacteria CES200 (Wyman et al . , 1986) .
  • the libraries were screened with an ALL-l specific probe (Cimino et al . , 1992) and positive clones were mapped with restriction enzymes.
  • cytoplasmic RNA was extracted by standard techniques (Berger & Chirgwin, 1989) and polyadenylated RNA purified on an oligo dT column.
  • cDNA was prepared using the Timesaver kit of Pharmacia and cloned into the lambda ZAPII vector (Stratagene) . Construction of cDNA libraries from K562 or fibroblasts RNA was described (Shtivelman et al . , 1985; Chu et al . , 1990) .
  • AF-4 cDNA clones kl.l, kl.2, kll and kl2 originated from the K562 library and the clones kcl 6, kcl 10, and kcl 12 were cloned from the KC122 library.
  • AF-9 cDNA clones v4 and v7 were obtained from the fibroblasts library, and k 16 was cloned from the K562 library.
  • RNA from a patient FI Two micrograms of RNA from a patient FI were reverse transcribed in a reaction utilizing the AF-9 oligonucleotide TCCTCAGGATGTTCCAGATGT (SEQ ID NO:39) or the ALL-l oligonucleotide GGCTCACAACAGACTTGGCAA (SEQ ID NO:40) as primers.
  • the cDNAs were amplified with Taq 1 polymerase
  • the AF-9/ALL-1 RNA function of patient C() was obtained in a similar way using the ALL-l primer CAGCGAACACACTTGGTACAG (SEQ ID NO:43) for synthesis of cDNA and the same primer together with the AF-9 primer CAACGTTACCGCCATTTGAT (SEQ ID NO:44) for PCR amplification.
  • ALL-l primer CAGCGAACACACTTGGTACAG SEQ ID NO:43
  • AF-9 primer CAACGTTACCGCCATTTGAT SEQ ID NO:44
  • AML(M4) Her karyotype was 46XX, t(6;ll) (q27,-q23) in 20/20 of bone marrow cells.
  • Patient Ed was a male diagnosed as AML(M5) with a karyotype of 46 XY del(llq23) .
  • the cell lines used for RNA analysis included K562 and KC122 (erythroid and myeloid acute phase of chronic myeloid leukemia) (Lozzio et al . , Blood 1975 45, 321-324; and Kubonishi et al . , Int . J.
  • the rearranged genomic fragments of ALL-l patients 01 and Ed were cloned into the EMBL-3 phage vector (Promega) after partial digestion of the DNAs with the Mbol enzyme and size selection. Phage libraries were screened using a 0.86 kb Bam HI fragment derived from ALL-l cDNA and spanning exons 5-11. Normal genomic library was constructed in a similar way from normal white blood cell DNA. cDNA library was constructed utilizing a kit from Pharmacia. Cytoplasmic poly A-selected RNA was prepared from KC122 cells.
  • RNA samples were reverse transcribed utilizing the AF-6 oligonucleotide 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO:79] .
  • the cDNA was amplified using the same AF-6 primer together with the All-1 primer 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO: 80] .
  • Both primers contained cloning sites at their 5' termini.
  • the amplified products were cloned into the SK plasmid vector and sequenced.
  • cDNAs and genomic DNAs were excised from the phage vectors and recloned into the SK plasmid vector. Sequencing was performed using the ABI automatic sequencer. Sequence was analyzed using the FASTA, TFASTA and motifs programs.
  • a rearranged ALL-l segment was cloned from the genomic DNA of leukemic cells of patient 01. Mapping of this segment indicated that it originated from the der (6) chromosome (Fig.
  • DNA of the patient Ed with AML and the del(llq23) aberration contained a rearranged BamHI fragment of 12 kb (Fig. 12B) .
  • XRO-5 probe hybridized to human DNA within Chinese hamster cell hybrids containing human chromosome 6. This indicated that the cloned DNA spanned a breakpoint cluster region and that a cytogenetic pattern of del(llq23) could correspond to a t(6;ll) translocation.
  • AF-6 encodes a protein of 1612 amino acids.
  • cDNA clone K10 we find two additional amino acids - glutamic acid at position 101, and a lysine in position 139; both are probably due to alteration in splicing similar to those which we previously detected in ALL-l (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631- 4635; and Ma et al . , Proc . Natl . Acad . Sci . USA 1993 90, 6350- 6354) .
  • RT-PCR reactions on RNAs from patients 01 and Ed using ALL-l and AF-6 primers flanking the expected junction region.
  • AF-6 protein residue 745-925, shows 23.2% identity over 181 amino acids with the C-terminus of yeast myosin-1 isoform (Johnston et al . , J. Cell Biol . 1991 113, 539-551) .
  • AF-6 protein also shows high similarity, though low identity, (66% similarity plus identity) over amino acids 1000-1594 to amino acids 1400-1980 of the myosin heavy chain from Dictyostelium discoideum (Warrick et al., Proc . Natl . Acad. Sci . USA 1986 83, 9433-9437) .
  • this region is part of the tail domain which assumes, due to a high helical potential, a rod structure.
  • a striking homology was detected in the polypeptide spanning amino acids 997-1080.
  • a series of amino acids in this domain are conserved (Fig. 14) in three other proteins - in the human tight junction protein ZO-1 (Willott et al. , Proc .
  • RNAs extracted from several cell lines Fig. 15
  • An 8 kb transcript was detected in cell lines of myeloid (a) , erythroid (b) , lymphoid (c-e) , glia (f) and epithelial (g) origin.
  • myeloid a
  • erythroid b
  • lymphoid c-e
  • glia f
  • epithelial g
  • t(6,-ll) (q27;q23) translocation is one of the most frequent translocations involving Hq23. Cloning of the AF-6 gene involved in this abnormality would enable now the use of
  • AF-6 One of the main reasons for cloning AF-6 was to see if it is related to the partner genes AF-4, AF-9, and ENL. Among these, AF-9 and ENL are highly related. However, AF-6 showed no sequence homology to any of the three partner genes. Short domains rich in prolines, serines and acidic amino acids were the only motifs shared by the four genes. The C-terminus AF-6 showed homology to the tail domain of myosin-1 isoform from yeast and myosin heavy chain from Dictvostelium discoideum; this domain presumably confers the rod structure to the myosin protein.
  • AF-6 displays a remarkable homology to the GLGF repeat found in the ZO-1, PSD- 95 and dig proteins from human, rat, and Drosophila respectively.
  • the first and the third proteins are presumably homologous and are thought to play a role in signal transduction on the cytoplasmic surface of intracellular junctions (Willott et al . , Proc . Natl . Acad. Sci . USA 1993 90, 7834-7838; Woods et al . , Cell 1991 66, 451-464) .
  • the second protein localizes to synaptic junctions and is thought to be involved in synaptic signalling or organization (Willott et al., Proc . Natl . Acad .
  • AF-6 cytoplasmic or associated with membranes.
  • the presence of this domain in AF-6 raises the possibility that AF- 6 is not a nuclear protein. Indeed, unlike AF-4, AF-9 and ENL, AF-6 does not contain a nuclear targeting sequence.
  • AML patients GUS and GE showed the chromosome translocation t(ll;17) (q23;q21) in their leukemic cells.
  • the cell lines used for RNA analysis included K562 and KC1-22 (erythroid and myeloid acute phase of chronic myeloid leukemia) , MV4:H and B-l (ALLs with the 4:11 translocation) , 380, ALL-l, 697, GM607, (ALLs) , GM1500 (EBV transformed lymphoblastoid cell line) , T98G (glioblastoma) , PC3 (prostate carcinoma) , (Prasad et al . , Cancer 1993 53, 5624-5628; Licht et al., Na ture 1990, 346, 76-79)
  • the junction fragment of patient GUS was cloned from a library prepared from a partial digest of genomic DNA clones into the EMBL-3 phage vector.
  • the library was screened with a 0.86 kb BamHI cDNA probe spanning ALL-l exons 5-11.
  • cDNA libraries were prepared from ALL-l and KCl-22 cytoplasmic RNAs utilizing a kit manufactured by Pharmacia, and the lambda ZAPII vector of Stratagene. RT-PCR reaction was performed as described (Nakamura et al . , Proc . Na tl . Acad. Sci .
  • DNA from patient GUS with AML and t(ll;17) was partially digested with Mbol enzyme, and following size selection was cloned into the EMBL-3 phage vector.
  • the library was screened with a cDNA probe spanning the breakpoint cluster region.
  • a clone composed of a rearranged ALL-l segment was identified among positive clones. Comparison between the physical maps of this clone and the corresponding normal ALL-l DNA (Fig. 16A) indicated that ALL-l sequences upstream of exon 6 were substituted with new DNA; the latter was subsequently found to be derived from chromosome 17.
  • a 1.7 kb EcoRI fragment (R1.7) was found to be devoid of repetitive sequences. This fragment was used as a probe to analyze by the Southern technique DNA from a second patient (GE) with AML and the t(ll;17) aberration. In that DNA we detected an 11.6 kb rearranged EcoRV fragment (Fig. 16B, lane b) . This indicated that in both patients the breaks occurred in the same region on chromosome 17. Fragment Rl .7 was next used as a probe on cDNA libraries derived from RNAs of the cell lines KC1-22 and ALL-l.
  • AF-17 cDNA contains an open reading frame spanning 3279 nucleotides. The first ATG shows a good fit to a Kozak consensus sequence and is preceded by an in-frame termination codon. The predicted protein spans 1093 amino acids. It contains relatively high concentrations of serines, glycines, alanines, leucines and prolines (15%, 11%, 10%, 10%, 10%, respectively) often concentrated in short stretches. In addition, it has a glutamine-rich region (41%) between amino acids 935 and 984 (Fig. 17B) .
  • the same region shows high concentration of hydrophobic amino acids, in particular leucines.
  • domains rich in alanines Liicht et al . , Nature 1990, 346, 76-79] , glycines (Shi et al., Cell 1991 67, 377-388) , glutamines and prolines (Madden et al . , Science 1991 253, 1550-1553) were implicated in transcriptional repression.
  • regions with high concentration of serines and prolines (Gill et al . , Proc . Natl . Acad . Sci . USA 1993 91, 192-196) or glutamines intercalated with hydrophobic amino acids (Theill et al . , Nature 1989 342, 945-948) were found to be involved in transcriptional activation.
  • AF-17 showed 48% identity and 67% similarity to a region within the protein Brl40, previously named peregrin (Accession No. M91585) .
  • This domain is cysteine-rich in both proteins and can be arranged into three zinc fingers according to the consensus C - X 2 - C - X 10 _ 13 C- X 2 _ 4 - C (Fig. 18B) .
  • Related consensus sequences are present in the adenovirus E1A protein and in the steroid receptor superfamily.
  • the human Brl40 protein has a second cysteine- rich domain and is located in the nucleus; the function of this protein is unknown.
  • Inspection of AF-17 predicted protein sequence revealed a leucine zipper dimerization motif between amino acids 729 and 764 (Fig. 17B) . Unlike many leucine zippers, the one in AF-17 is not preceded by a basic region.
  • ALL-l/AF-17 fused gene is transcribed into a chimeric RNA
  • cDNA and genomic DNA sequence information to design primers for amplification by RT-PCR of a putative ALL-l/AF-17 RNA junction from the leukemic cells of patient GUS.
  • An amplification product was indeed found to contain the RNA junction (Fig. 17C) .
  • the open reading frames of the two genes were found to be linked in phase.
  • the t(ll;17) abnormality results in production of an RNA encoding a chimeric ALL-l/AF-17 protein.
  • the cloning and sequence analysis of the partner genes which recombine with ALL-l in Hq23 translocations provides information and reagents which can be used in the diagnosis, prognosis and monitoring of human acute leukemias.
  • this cloning enables construction of biologically active molecules, and might provide insights into the mechanism of leukemogenesis .
  • the most notable feature of AF-17 protein is the leucine zipper protein dimerization motif. Following the t(ll;17) chromosome translocation, this motif will be included in the ALL-l/AF-17 chimeric protein which is presumed to be the critical product of the aberration.
  • AF-17 Since the leucine zipper of AF-17 is not preceded by a basic region required for interaction with DNA, and because leucine zippers are found not only in transcription factors but also in other proteins with diverse functions, it is concurrently not clear whether AF-17 is a transcription factor.
  • AF-17 is the fifth partner gene involved in Hq23 abnormalities to be cloned and characterized. Schematic representation of the proteins encoded by these genes and by ALL-l is shown in Figure 20. Inspection of the sequences within the segments of the partner proteins (right side of the arrows) linked to ALL-l sequences (left side of the fusion point within the top scheme) in the chimeric proteins thought to be critical for leukemogenesis, does not reveal a common motif. AF-9 and ENL are the only partner genes which share sequence homology (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635) .
  • AF-9 and ENL proteins contain nuclear targeting sequences and are probably nuclear proteins .
  • the AF-6 polypeptide linked to the N-terminus of ALL-l contains the GLGF motif (Prasad et al . , Cancer 1993 53, 5624-5628) whose function is not known, as well as short regions very rich in acidic amino acids, basic amino acids or prolines.
  • the GLGF motif is found in cytoplasmic or membrane-associated proteins and this suggests that AF-6 is not located in the nucleus.
  • AF-4 polypeptide within the ALL-l/AF-4 protein includes several segments with high concentration of serines or prolines
  • the AF-4 protein includes a nuclear targeting sequence and therefore is probably associated with the nucleus. Finally, each of the normal five partner genes is expressed in all cell lines analyzed, both of hematopoietic and non hematopoietic lineages.
  • AF-4, AF-6, and AF-17 polypeptides linked to the ⁇ - terminus of ALL-l each contain stretches of one or more of those amino acids, the analogous polypeptide of AF-9 as well as its homologous C-terminal region in E ⁇ L are devoid of these amino acids.
  • the AF-6 protein is probably located in the cytoplasm or the membrane of the cell, and therefore does not play a role in transcriptional regulation. Considering the above we find it less likely that the partner polypeptides of AF-6, AF-9 and E ⁇ L contribute domains involved in direct activation or repression of transcription.
  • Inactivation could occur by nonproductive binding to the promoter of the normal target (s) for ALL-l or by dimerization of the chimeric protein to the normal protein and sequestering the latter either to a complex with other proteins or into another cellular compartment.
  • the partner polypeptides could best play a role in the elimination of the normal protein activity through dimerization. They could make the dimer nonfunctional by virtue of their presence within, or by sequestering it through interaction with other cellular proteins.
  • the leucine zipper dimerization motif in AF-17 and the GLGF motif in AF-6 could represent protein-protein interaction domains of partner polypeptides.
  • Sequencing reactions were performed by using an automatic sequencer (ABI) . Sequences were reassembled and analyzed in the Genetic Computer Group system. Alu sequences were analyzed by the Pythia service.
  • ABSI automatic sequencer
  • FIG. 21 shows DNA rearrangements detected by B859 probe in some of the various Hq23 aberrations studied in this report.
  • a phage clone, mgll.l which spans the breakpoint cluster region in the ALL-l gene (Gu et al . , Cell 1992 71, 701- 708) , was subcloned into plasmids for sequencing. The complete sequence of the 8342 bp BamHI fragment is presented in Fig. 22. The exons included in this region are shown.
  • the AF4 probe (Cimino et al . , Cancer Res . 1992 52, 3811-3813 and Gu et al . , Proc . Na tl . Acad. Sci . USA 1992 89, 10464-10468) , a modified Ddel fragment, spans nucleotides 3071 to 3261 and 3502 to 3754 ( Fig . 22 ) .
  • the 8342 bp sequence was first screened for Alu repeats. Eight Alu repeats were identified and their positions are indicated in Table 1. The orientation of these Alu repeats is the same as that of the ALL-l gene. Classification of these Alu repeats was based on recently published diagnostic criteria (Milosavljevic et al . , J " . ⁇ ol . Evol . 1991 32, 105-121) . After the ALL-l exons and Alu repeats were precisely identified, the rest of sequence was searched for other homologous sequence (s) in GenBank.
  • a 130 bp fragment encompassing nucleotides 7429 to 7559 in intron 9, shows around 80 percent sequence identity to genomic sequences in several genes such as TRE17, ApoA4, Factor VIII c subunit, Factor IX, a nuclear gene for mitochondrial ATP synthase c subunit, and G6PD gene (GenBank accessions: X63596, M14642, M88636, K02402, X69907, and Z29527, respectively) . These similar sequences were located in 5' regulatory regions, or in 3' segments, or in introns, suggesting that they may represent a group of repetitive elements with low frequency in the genome.
  • the DNA rearrangements in the ALL-l gene involved in acute leukemia can be detected by a single probe, B859. Digestion with BamHI is normally sufficient for the analysis. However, if only one or no rearranged fragments are detected, the sample DNA should be digested by other restriction enzymes such as HindHI, and probed with B859.
  • the germline sequence of the partner chromosome at the breakpoint should have been Alu. However, this is not the case in any of the five translocations. Nevertheless, the high concentration of the Alu sequences within the region, in particular, within the area spanned by exons 6 and 7, suggested a possible role for the Alu in the translocations. This indirect role might be destabilization of the region so as to make it more prone to breaks.
  • topoisomerase II recognition sites were found in three out of ten cases when three mismatches were allowed in the consensus sequence. Thus, in the majority of the de novo All-1 rearrangements topoisomerase II recognition sites are not present at the breakpoints, and the enzyme is probably not involved. It will be necessary to sequence the breakpoint in secondary leukemias to determine whether in these cases topoisomerase II recognition sites are consistently associated with the breakpoints.
  • i Gu et al., Proc . Natl . Acad. Sci . USA 1992 89, 10464- 10468 ii: Prasad et al . , Cancer Res . 1993 53, 5581-5585 iii: Nakamura, et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635
  • Genomic DNA was extracted from bone marrow aspirates by a standard procedure (Gustincich et al . , BioTechniques 1991, 11, 298-301) . Approximately 8 ⁇ g of genomic DNA was digested to completion with BamHI or HindHI. Restriction enzyme digests were separated by electrophoresis on 0.7% agarose gels and blotted onto positively charged nylon membranes. Southern blotting, probe radiolabeling, and hybridization were performed by standard techniques. A single blot was prepared. After probing with SASl, the blot was stripped, then probed again with B859.
  • Clones corresponding to the rearranged ALL-l BamHI fragments were isolated from bacteriophage XEMBL3 libraries made from size-fractionated BamHI digests of patient DNA. Recombinants were identified in phage libraries by filter hybridization using the B859 probe. Construction of libraries, screening, phage purification, and restriction enzyme mapping were done by standard techniques . Subclones were constructed in the pBluescript II plasmid vector. DNA sequence of selected portions of subclones was determined by cycle sequencing using an Applied Biosystems 373A DNA sequencer. Programs from Genetics Computer Group (GCG) system (Devereux et al . , Nucl . Acids Res . 1984, 12, 387-395) were used for data analysis.
  • GCG Genetics Computer Group
  • RNAzolTM Biotecx Laboratories
  • RT Reverse transcriptase
  • RNA-PCR amplification was performed with rTth DNA polymerase.
  • Nested PCR amplification was performed with Taq DNA polymerase.
  • Oligonucleotide primers were used without further purification.
  • Primers are 3.1c (AGGAGAGAGTTTACCTGCTC) [SEQ ID NO: 83] from exon 3, 5.3 (GGAAGTCAAGCAAGCAGGTC) [SEQ ID NO:84] from exon 5, 6.1 (GTCCAGAGCAGAGCAAACAG) [SEQ ID NO:85] from exon 6, and 3.2c (ACACAGATGGATCTGAGAGG) [SEQ ID NO:86] from exon 3.
  • Primers used in reactions are as follows: 1) RT reaction - 3.1c, 2) RNA-PCR amplication - 5.3/3.1C, 3) nested PCR amplification - 6.1/3.2c. RT reaction was performed for 15 minutes at 57°C using 500 ng RNA.
  • RNA-PCR amplification was performed for 35 cycles (95 C, 1 minutes; 53°C, 1 minutes; 72°C, 1 minute) .
  • Nested PCR amplification was performed using 0.5 ⁇ l of the RNA- PCR product for 30 cycles (95°C, 1 minute; 60°C 1 minute; 72°C, 1 minute) .
  • PCR products were analyzed by 2% agarose gel electrophoresis .
  • Figure 24 shows Southern blot rearrangements in the
  • AML cytogenetic evidence of Hq23 translocations.
  • the rearrangements were detected with a cDNA probe (B859) (Gu et al . , Cell 1992 71, 701-708 and Caligiuri et al . , Cancer Res . 1994 54, 370-373) which spans the ALL-l breakpoint cluster region.
  • Two of these patients (no ⁇ . 23 and 24) had trisomy 11 as a sole cytogenetic abnormality whereas one patient (no. 1) had a normal karyotype (Caligiuri et al . , Cancer Res . 1994 54, 370-373) .
  • a single rearranged ALL-l band is seen for each patient in both BamHI and HindHI restriction enzyme digests.
  • Clones corresponding to the rearranged BamHI fragments from the two trisomy 11 patients were isolated and characterized. Each clone begins and ends with a portion of ALL-l exon 5 delineated by the BamHI cloning site within this exon (Fig. 25A) . The 5'- 3' order of ALL-l exons within each clone is 5-6-2-3-4-5. This novel exon structure indicates that the ALL-l rearrangement in each patient is the result of a direct tandem duplication of a portion of the ALL-l gene (Fig. 25B) . The junction point of this duplication fuses the 5' portion of intron 6 to the 3' portion of intron 1. The precise junction points for the two clones are different. DNA sequence across the junctions (Fig.
  • 25C shows a 1 bp N-segment in one clone ( ⁇ 24) and heptamer- like signal sequences (Akira et al . , Science 1987 238, 1134- 1138) near the junction points in both clones.
  • RNA-PCR was performed using oligonucleotide primers specific for the ALL-l duplication. Discrete bands of the predicted size were detected for the two patients with trisomy 11 (Fig. 26A) . Sequence analysis of nested PCR products (Fig. 26B) shows an in-frame fusion of exon 6 with exon 2.
  • the partial ALL-l duplication creates a novel type of fusion protein in which a truncated polypeptide chain encoded by ALL-l exons 1-6 is fused near the amino-terminus of the native ALL-l protein.
  • the partially duplicated protein may be involved in cellular transformation, as postulated for other ALL-l fusions (Cimino et al. , Cancer Res . 1991 51, 6712-6714; Gu et al., Cell 1992 71, 701-708; Tkachuk et al . , Cell 1992 71, 691-700; Morrissey et al . , Blood 1993 81, 1124-1131; Nakamura et al., Proc. Natl . Acad.
  • ALL-l amino- terminal domains from their normal protein environments is the critical structural alteration leading to ALL-l associated leukemogenesis. Because the ALL-l gene is fused with itself, it follows that partner genes from other chromosomes are not necessary for involvement of ALL-l in leukemia.
  • Trisomy 11 is a rare recurrent finding in AML, estimated to occur at a frequency of about 0.7% (CALGB AML cytogenetic data base) . Trisomy of other chromosomes is reported frequently in hematologic malignancy, sometimes in association with disease progression (Heim et al .
  • TCT TCA AAA AGG ACA GAT GCA ACC ATT GCT AAG CAA CTC TTA CAG 945 Ser Ser Lys Arg Thr Asp Ala Thr lie Ala Lys Gin Leu Leu Gin 305 310 315
  • AGC AGA AGG TAT TCA GTG TCG GAG AGA AGT TTT GGA TCT AGA ACG 1440 Ser Arg Arg Tyr Ser Val Ser Glu Arg Ser Phe Gly Ser Arg Thr 470 475 480
  • AGG TGG ACT TCT TTA AAG CAT TCT AGG TCA GAG CCA CAA TAC TTT 1710 Arg Trp Thr Ser Leu Lys His Ser Arg Ser Glu Pro Gin Tyr Phe 560 565 570
  • AGT AGA GAG AGA GAC CGG GAG AGA GAA AAG GAG AAT AAG CGG GAG 2475 Ser Arg Glu Arg Asp Arg Glu Arg Glu Lys Glu Asn Lys Arg Glu 815 820 825
  • AAA 3060 lie Lys His Val Cys Arg Arg Ala Ala Val Ala Leu Gly Arg Lys 1010 1015 1020
  • Trp Glu Arg Glu Lys lie Leu Ser Ser Met Gly Asn Asp Asp 1040 1045 1050
  • ATC CCT GTA AAA CAA AAA CCA AAA GAA AAG GAA AAA CCA CCT CCG 3915 lie Pro Val Lys Gin Lys Pro Lys Glu Lys Glu Lys Pro Pro Pro 1295 1300 1305
  • ATA ACA CCC AGG GTG GTT TGC TTT CTC TGT GCC
  • AGT GGG CAT 4140 lie Thr Pro Arg Val Val Cys Phe Leu Cys Ala Ser Ser Gly His 1370 1375 1380
  • AAG CGA GGC AAG AGA TCA GCT GAA GGA CAG GTG GAT GGG GCC GAT 7830 Lys Arg Gly Lys Arg Ser Ala Glu Gly Gin Val Asp Gly Ala Asp 2600 2605 2610
  • GGT AAA GAA AAT GGA ACA GAG AAC TTA AAG ATT GAT AGA CCT GAA 8100 Gly Lys Glu Asn Gly Thr Glu Asn Leu Lys He Asp Arg Pro Glu 2690 2695 2700
  • CAG CAT GTC AGT TCT GTA ACC CAA AAC CAA AAA CAA TAT GAT ACA 180 Gin His Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr 50 55 60
  • MOLECULE TYPE DNA (genomic)
  • FEATURE FEATURE
  • AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT GGT CAA GAT CAG GCC CCT 1332 Lys Pro Thr Ala Tyr Val Arg Pro Met Asp Gly Gin Asp Gin Ala Pro 290 295 300
  • GAG CCC CCA CAA AGG CAA ACC GTT GGA ACC AAA CAA CCC AAA AAA CCT 2244 Glu Pro Pro Gin Arg Gin Thr Val Gly Thr Lys Gin Pro Lys Lys Pro 595 600 605
  • GCCACAGCAC ATGGGGTGCC ACCTCGAGGT CTGCACAGGA GGACTTGGCG CTGCCATTTC 4850
  • CAGAGTAAGA CAAATGCAAA GCATCTTAAG GAGTGAAAAT AGAGTCTAAA TCTTGCCTTT 6830

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Abstract

Methods are provided for the diagnosis and treatment of human leukemias involving breakpoints on chromosome 11 in the ALL-1 locus. The ALL-1 breakpoint region, an approximately 8 kb region on chromosome 11, is also disclosed. The ALL-1 region is involved in translocations in acute lymphocytic, myelomonocytic, monocytic and myelogenous leukemias. Probes which identify chromosome aberrations involving the ALL-1 breakpoint region on chromosome 11 are also provided. cDNA sequences of the ALL-1 gene on chromosome 11, the AF-9 gene on chromosome 9, the AF-4 gene on chromosome 4, the AF-6 gene on chromosome 6 and the AF-17 gene on chromosome 17 and corresponding amino acid sequences are also provided. Probes are provided for detecting chromosome abnormalities involving these genes. Chimeric genes involved in translocations are disclosed. Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.

Description

METHODS FOR SCREENING AND TREATING LEUKEMIAS RESULTING FROM ALL-1 REGION CHROMOSOME
ABNORMALITIES
FIELD OF THE INVENTION
5 The present invention relates to the field of methods for diagnosis and treatment of human leukemias wherein hematopoietic cells of patients have translocations in a small region of chromosome 11 designated as ALL-l. Diagnostics and therapeutics based on nucleic acid and amino acid sequences are 10 provided.
BACKGROUND OF THE INVENTION
Specific reciprocal chromosome translocations are very frequently found in human lymphomas and leukemias . These chromosomal abnormalities alter normal cellular genes leading
15 to their deregulation. Chromosome translocations have been shown to play an important role in the pathogenesis of human leukemias and lymphomas by either activating cellular protooncogenes or by leading to the formation of chimeric genes capable of transforming hematopoietic cells. Erikson et al . ,
20 Proc . Na tl . Acad . Sci . USA 1983, 80, 519-523; Tsujimoto et al . , Science 1984, 226, 1097-1099; Tsujimoto et al . , Science 1984, 224, 1403-1406; Shtivelman et al . , Na ture 1985, 315, 35-354; Mellentin et al . , Science 1989, 246, 379-382.
Translocations can lead to gene fusion resulting in
25 a chimeric oncoprotein whose transforming activity is derived from both genes. The prototype of such events is the t(9;22) of chronic myelogenous leukemia (C L) which leads to a BCR-ABL fusion mRΝA and protein (Shtivelman, supra) . Translocations t (1,-19) , t(15;17) , and t(6;9) are other examples of gene fusions, involving in the first two cases transcription factors (Nourse et al . , Cell 1990, 60, 535-545; Kamps et al . , Cell 1990, 60, 547-555; Kakizuka et al . , Cell 1991, 66, 663-674; de The et al . , Cell 1991, 66, 675-684; von Lindern et al . , Mol . Cell . Biol . 1990, 10, 4016-4026) .
The alternative molecular consequence of translocations is deregulation of protooncogenes by their juxtapositioning to an enhancer or promoter which is active in the type of cell from which the tumor arises. The immunoglobulin (Ig) and T cell receptor (TCR) enhancers participate in at least 15 different translocations associated with Burkitt lymphoma, chronic ly phocytic leukemia, follicular lymphoma, mantle cell lymphoma, and acute T or B cell leukemia. (Croce, CM, Cell 1987, 49, 155-156; Rabbitts, TH, Cell 1991, 67, 641-644; Solomon et al . , Science 1991, 254, 1153-1160) .
Chromosomal region llq23 has been shown to be involved in different chromosomal translocations in human acute leukemias of different hematopoietic lineages. Ilq23 chromosome abnormalities have been reported in acute lymphoblastic leukemia and in acute nonlymphoblastic leukemia
(A LL) , most commonly of the M4 and M5A subtypes. Heim and
Mitelman, Cancer Cytogenetics, Alan R. Liss, New York 1987.
Chromosome 11 band q23 is frequently rearranged in acute lymphocytic (ALL) , in acute myelomonocytic (AMMOL) , acute monocytic (AMOL) and acute myeloid (AML) leukemias, mostly in reciprocal exchanges with various translocation partners. The t (4;11) (q21;q23) , t(ll;19) (q23;pl3) , and t (1;11) (p32;q23) are found in 10%, 2% and <1% of ALL, respectively. Reciprocal translocation between llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17q25 and 19pl3 are found in 5-6% of AML. Heim and Mitelman, supra . In addition, interstitial deletions in llq23 have been detected both in ALL and AML.
The same segment on chromosome 11 is apparently involved in the t (11;19) (q23 ;pl3) and t (1;11) (p32 ;q23) translocations in ALL as well as in translocations with the chromosomal regions 9p21, 2p21 6q27, 17q25 and 19pl3 associated with 5-6% of acute myelogenous leukemias (AML) . Heim and Mitelman, Cancer Cytogenetics, Alan R. Liss, New York 1987. Reciprocal translocations between chromosome region llq23 and chromosomal regions 9p22, 6q27, lp21, 2p21, lOpll, 17p25 and 19pl3 are found in 5-6% of ANLL.
In clinical terms, rearrangements of llq23, in particular the t(4;ll) chromosome translocation, have some distinct features. The patients are often quite young; t(4;ll) accounts for the vast majority of cytogenetically abnormal ALLs in infants. In the majority of patients, the leukemic cells show both B-cell and myeloid marker (Stong et al . Blood 1986, 67, 391-397) and the disease is consequently considered "biphenotypic. "
Among children, most patients with the t(4;ll) abnormality are less than one year of age and have a poor prognosis. The leukemic cells have a CD10-/CD19+ early B cell precursor phenotype and most of them express a myeloid associated antigen (CD15) ; Pui et al . , Blood 1991, 77, 440-447. Myelomonocytic and biphenotypic leukemias carrying the t(4;ll) aberration have also been reported; Nagasaka et al . , Blood 1983, 61, 1174-1181.
There remains an unmet need for identification of the breakpoint cluster region and the genes involved in chromosome 11 aberrations associated with acute leukemias in order to provide diagnostics and therapeutics for these diseases.
SUMMARY OF THE INVENTION
The cDNA sequence of the ALL-l gene on chromosome 11 is provided. A partial sequence of the AF-4 gene is also provided in the context of the sequences of two reciprocal endproducts of a translocation. Amino acid sequences corresponding to the cDNA sequences of the entire ALL-l gene and the partial sequence of the AF-4 gene, and sequences relating to chimeric genes formed by chromosome translocations with chromosome 4, 9 and 19, respectively, are provided. Probes are provided for detecting chromosome abnormalities involving the ALL-l gene on chromosome 11, including probes for detecting chimeric genes generated by translocations. Monoclonal antibodies for diagnosis and treatment and antisense oligonucleotides for treatment of acute leukemias are also described.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a drawing depicting a physical map of YAC B22B, which has been described in Rowley et al. , Proc. Natl . Acad. Sci . USA 1990, 87, 9358-9362. ura and trp correspond to the termini of the vector. A 40 kb segment located towards the ura end and lacking No I and MluI sites is not included in the map. Pulse field analysis indicates two or three Sfil sites located to the left of cosmid 43.
Figure 2 is a photograph showing the results of Southern blot analysis of tumor DΝAs . Blots were hybridized to the radiolabeled 0.7 kb Ddel fragment derived from the terminus of cosmid 53. Aliquots of 10 μg were analyzed.
Figure 3 is a drawing showing mapping of tumor breakpoints . The internal Nσtl fragment of YAC is shown in the same orientation as in Figure 1. The dotted line represents a region not cloned in the cosmids. Restriction sites within this region are deduced from the size of the relevant germline fragments detected in genomic Southern blots using the indicated probe. Additional EcoRV and Xbal sites are not shown. Some of the samples were not analyzed with BamHI . Lines below the map correspond to the smallest genomic fragments found rearranged. Ν = NotI ; B = BamKI; RV = EcoRV ; X = Xbal. The breakpoint cluster region is believed to span approximately the region encompassed by the two nearest BamHI sites flanking the arrow; more specifically, the breakpoint cluster region is believed to span exons 6-12 illustrated in Figure 10.
Figure 4 is a photograph showing the results of Northern blot analysis of RNA from cell lines and a primary leukemia using pooled probes. 10-20 μg aliquots of total RNA were analyzed on a formaldehyde gel. Following hybridization, blots were washed in a solution containing 0.1% SSC and 0.1% SOS at 700. RNAs were obtained from: a) K562 cells; b) the glioblastoma T98G cell line; c) the SupB pre B ALL cell line; d) the MV4;11 cell line; and e) a patient with t(9;ll) .
Figure 5 is a photograph showing the results of Southern blot analysis of DNAs from primary tumors and cell lines with llq23 abnormalities using a modified 0.5 kb Ddel probe. a) patient CH. with t(6;ll) ; b) the Bl cell line with t(4;ll) ; c) the RS 4;11 cell line with t(4;ll) ; d) patient J.B. with t(10;ll) ; e) patient M.L. with t(l;ll) ; f) patient S.O. with del (11) (q23) ; g) patient R.E. with del (11) (q23) . Numbers indicate kilobases . The germline Ba.mHI and Xbal fragments are of 9 and 12 kb, respectively.
Figure 6 is a photograph showing the results of Northern blot analysis of RNAs from cell lines using a 1.5 kb EcoRI probe generated from cosmid 20. Lanes included SK DHL (a) ; KC122 (b) ; MV 4;11 (c) ; T98G (d) ; All-1 (e) ; Bl (f) ; K562 (g) ; Jurkat (h) ; GM607 (i) ; 697 (j) ; RS4;11 (k) ; GM1500 (1) ; LNCaPFGC (m) ; PC3 (n) . 28S and 18S indicate migration of ribosomal RNA. Figure 7 shows physical maps of ALL-l cDΝA and gene.
All NotI (Ν) , Hiπdlll (H) , BamHI (B) , and EcoRI (R) sites of the cDΝA are shown; only some EcoRI sites are indicated within the gene and Hiπdlll or Baι*πHI sites within the 5' 25 kb of the first intron are not shown. Exons are depicted as rods or boxes extending above and below the line. Cen and Tel correspond to direction of the centromere and telomere, respectively. cDΝA clones SKV2, SKV3, and SKV18 were obtained from K562 cDΝA library. Clones V1-V26 were obtained from a normal fibroblast cDΝA library. The 9B1 clone originated from a Burkitt lymphoma cDΝA library.
Figure 8 shows nucleotide sequence and predicted amino acid sequence of ALL-l cDΝA.
Figure 9 depicts homology between ALL-l and Drosophila trithorax (D. Trx) proteins (top and center) , and the structure of ALL-l zinc finger-like domains (bottom) . Bars indicate identical residues. One dot and two dots indicate first and second degree conservative differences, respectively. Figure 10 A-C shows exon-intron structure of ALL-l breakpoint cluster region Figure 10A and partial sequence of the two reciprocal ALL-l/AF-4 fused transcripts (Figure 10B and Figure IOC) . In Figure 10A exons containing the zinc finger- like domains (8-12) are represented by cross-hatched boxes. Among the five t(4;ll) breakpoints shown (arrowheads in Figure 10A) , included are those of the MV4;11 (MV) , RS4;11 (RS) , and Bl (Bl) cell lines. CL. and I.V. represent leukemic cells with t(4,-ll) from two patients. B, R, G, X, H correspond to sites for the enzymes BamHI, .EcoRI, Bglll, Xbal, and Hindlll, respectively. In sequences within Figure 10A, small and large letters represent introns and exons, respectively. Cytosine in position 4141 of ALL-l sequence (Figure 2) is replaced by thymidine in clone 25, resulting in alteration of Leucine into Phenylalanine (Figure IOC) .
Figure 11 A-E shows the non ALL-l sequences within the fused RNAs unique to cells with t(4;ll) chromosome translocations (Figure 11A-C) which originate from chromosome 4 (Figure 11D and HE) . Cell lines with t(4;ll) chromosome translocations included: RS4;11 (Stong, RG, and Kersey, JH, Blood 1985, 66, 439-443) , MV4;11 (Lange et al . , Blood 1987, 70, 192-198) and Bl (Cohen et al . , Blood 1991, 78, 94-102) . Northern blots with RNAs from cell lines with translocations t(4;ll)-B-l (a, a') , MV4;11 (b, b' ) and RS4;ll (c, c', c") , and RΝAs from control cell lines without the translocation: ALL-l
(d, d' , d") , K562 (e, e' ) , SKDHL (f, f) , were hybridized to 5'
ALL-l cDΝA probe (Figure HA) , to non ALL-l sequences from cDΝA clone 16 (Figure 11B) , and to non ALL-l sequences from cDΝA clone 25 (Figure 11C) . ALL-l is a Philadelphia-chromosome positive cell line (B cell leukemia) lacking Hq23 aberrations (Erikson et al . , Proc Natl . Acad . Sci . USA 1986, 83, 1807- 1811) . K562 originated from chronic myelogenous leukemia (Lozzio, CB and Lozzio, BB, Blood 1975, 45, 321-324) . SKDHL is a B cell lymphoma cell line (Saito et al . , Proc . Natl . Acad . Sci . USA 1983, 80, 7476-7480) . The second and third probes were also used in hybridization to Southern blots (Figure 11D and HE, respectively) with DΝAs from Chinese hamster ovary (CHO cells and CHO cells containing chromosome 4 (CHO/4) . "Fused 1" and "fused 2" correspond to the altered ALL-l RNAs of 14 kb and 12.7 kb, respectively.
Figure 12A-C depicts the genomic analysis of the t(6:ll) (q27:q23) chromosome translocation. Figure 12A:
Physical map of the t(6;ll) junction, as well as of the corresponding regions from chromosomes 11 and 6. The RVPO .5 probe was used to isolate the corresponding normal DNA of 6q27
(Figure 12B) . Chromosome 6-specific probe XRO .5 detects DNA rearrangement in the bone marrow from a patient, whose karyotype showed Hq23 deletion; (Figure 12C) . Sequence of the t(6;ll) breakpoint region. Cen and Tel denote the direction of the telomeres and centromeres of the two chromosomes . Open vertical boxes represent defined exons. Restriction sites: B, BamHI, H., HindHI, G, Bglll; Rm, EcoRI and X, Xbal.
Figure 13A-C shows the cloning and sequencing of AF-6 cDNA and of ALL-l/AG-6 fusion transcript. Figure 13A: AF-6 cDNA clones. Dashed lines indicate different sequences possibly representing alternative non-coding exons. Restriction sites: A, Apal; B, BamHI,-, H, HindHI and S, Sad . Figure 13B: Predicted amino acid sequence of AF-6 cDNA coding region. Arrow indicates the RNA fusion point. Figure 13C Fusion transcript of ALL-l and AF-6 cloned from the RNAs of patients 01 and Ed. Figure 14 shows a comparison of the GLGF repeat within the AF-6 protein to GLGF repeats of other patients. GLGF repeats are the third GLGF in human ZO-1 (ZO-1 3) ; the second GLGF in rat PSD95 (PSD95 2) , and the third GLGF in Drosophila large disc tumor suppressor gene (dlq3) . Bold amino acids are consensus amino acids conserved among the four proteins.
Figure 15 depicts a Northern analysis of AF-6 RNA in human cell lines. 5-10 μg of polyadenylated RNA were analyzed on agarose gel containing formaldehyde. RNAs were obtained from the lines KC122, K562, B-l, MV4;11, SKDHL, T98G, 293 (a-g, respectively) .
Figure 16A and 16B shows genomic analysis of the t(ll,-17) chromosome translocation. Figure 16A: Physical map of the genomic junction of patient GUS [der (17)] and a map of the corresponding normal region (chr. Hq23) . Numbered open boxes in the top line represent ALL-l exons. Darkened segment of der (17) correspond to chromosome 17 sequences, and open box therein represents an exon. Fragment Rl .7 was used as a probe for the genomic Southern analysis as well as for cDNA screening. Cen and Tel show directions of the centromeres and telomeres, respectively. R, EcoRI; H, HindHI; B, BamHI; G, Bglll, X, Xbal. Figure 16B: Southern genomic analysis of a DNA from patient GE with AML and t(ll;17) , and a normal DNA (lanes b and a, respectively) . DNAs were digested with EcoRV and hybridized with the Rl.7 probe. Germline fragment is 18 kb.
Figure 17A-C shows cloning and sequencing of AF-17 cDNA and of the junction within ALL-l/AF-17 fusion transcript. Figure 17A: Physical map of AF-17 cDNA clones. Restriction sites: S, Sad; H, HindHI; H2, Hindi. Initiation (ATG) and termination (TAA) are shown by arrows. Figure 17B: Predicted amino acid sequence of AF-17 protein. Cysteines within the cysteine-rich region at the N-terminus are underlined. Also underlined is the leucine zipper at positions 729-764. Arrow indicates point of fusion with the ALL-l protein. Figure 17C All-l/AF-17 RNA junction cloned from the leukemic cells of patient GUS. Figure 18A and 18B depicts ho ology between the AF-17 protein and the human Brl40 (peregrin) protein. Figure 18A: Alignment of AF-17 and Brl40 cysteine-rich domains. Bars indicate identical residues; one dot and two dots indicate first and second degree conservative differences, respectively. Figure 18B: Potential zinc fingers within the cysteine-rich domain of AF-17.
Figure 19 shows Northern analysis of AF-17 RNA in human cell lines. 5-10 μg of polyadenylated RNA were analyzed on agarose gel containing formaldehyde . RNAs were obtained from the cell lines KCl-122, MV4;11, ALL-l, GM-607, B 1, 380, PC3, GM 1500, K562, T93G, 679 (a to j, respectively) .
Figure 20 depicts landmarks, common motifs and homologous sequences within the partner proteins AF-4, AF-9, ENL, AF-6 and AF-17, and within the ALL-l protein. Arrows indicate fusion points between ALL-l and the partner proteins. Striped regions in AF-9 and ENL indicate domains of highest homology between the two proteins. NTS, nuclear targeting sequence, LZ, leucine zipper, MTase, methyl transferase.
Figure 21A and 21B shows use of the B859 probe in detecting ALL-l abnormalities. Figure 21A: The B859 probe and the breakpoint cluster region of the ALL-l gene (BCRHq23) . Numbered boxes are the exons of the ALL-l gene. Thin lines display the subclones used for sequencing. Cen. and Tel. denote the centromere and telomere. Figure 21B: Southern analysis of the ALL-l gene rearrangements in patients with acute leukemia. Patient's DNA samples were digested with BamHI and probed with the B859 probe. Numbers in each lane correspond to the case numbers in Table 2.
Figure 22 shows the nucleotide sequence of the breakpoint cluster region within the ALL-l gene. The predicted amino acid sequences of each exon are shown under the corresponding nucleotide sequences. A consensus sequence for topoisomerase II recognition site is underlined.
Figure 23 is a schematic representation of the exons, Alu repeats, and the breakpoints in the breakpoint cluster region in the ALL-l gene. Filled boxes are exons. Alu repeats are shown as open boxes. Arrows point to the positions of the breakpoints with their corresponding case numbers presented in Table 2. Hatched box represents a 130 bp novel repetitive sequence .
Figure 24a and 24b shows Southern analysis of ALL-l gene rearrangements in adult AML patients without cytogenetic evidence of Hq23 translocations. The label above each lane corresponds to a unique patient identification number taken from (Caligiuri et al . , Cancer Res . 1994 54, 370-373) . Patients nos . 23 and 24 had trisomy 11 as a sole cytogenetic abnormality whereas patient no. 1 had a normal karyotype. Arrows indicate rearranged bands. N, normal control. Figure 24a: Blots examined with the B859 probe. B859 is a cDNA probe (Caligiuri et al . , Cancer Res . 1994 54, 370-373) which spans the ALL-l breakpoint cluster region defined by exons 5-11 of the ALL-l gene (Gu et al . , Cell 1992 71, 701-708) . Germline 8.3 kb (BamHI) and 14 kb (HindHI) bands are indicated. Figure 24b: Blots examined with the SASl probe. SASl is a 289 bp DNA probe from intron 1 of the ALL-l gene (see Fig. 25A) . Germline kb (BamHI) and 3.3 kb (HindHI) bands are indicated. The rearranged BamHI band for patient no. 1 is presumably coincident with the germline 20 kb band. Rearranged bands detected with the SASl probe comigrate with the rearranged bands detected by the B859 probe.
Figure 25a-c shows the structure of partial duplication of the ALL-l gene. Figure 25a: Restriction enzyme maps of lambda clones (λ23 and λ24) corresponding to rearranged BamHI fragments from two AML patients with trisomy 11. Boxes represent ALL-l exon positions determined by subcloning and partial DNA sequence analysis. The junction point of the duplication is indicated by the juncture of the black and shaded bars. Position of the SASl probe is shown. B, BamHI; R, EcoRI; H, HindHI; X, Xbal. Figure 25b: Proposed structure of the partially duplicated ALL-l gene contains a direct tandem duplication spanning exons 2-6. Only the BamHI and HindHI sites giving rise to bands detected on Southern blot (Figure 24) are indicated. Figure 25c: DNA sequence across the junction points of clones λ23 and λ24 are aligned with sequences from introns 1 and 6 of the ALL-l gene. λ24 has a 2 bp N-segment. Heptamer-like signal sequences (Akira et al . , Science 1987 238, 1134-1138) near the junction points in both clones are underlined. Nonamer-like signal sequences are not present.
Figure 26a and b shows RNA-PCR analysis of trisomy 11 patient samples. Figure 26a: Agarose gel of RNA-PCR products
(left-hand lanes) using oligonucleotide primers specific for the ALL-l partial duplication. Right-hand lanes show the results of standard PCR amplification of an aliquot of the RNA- PCR product using nested oligonucleotide primers. Discrete bands of the size predicted from the ALL-l cDNA sequence (Gu et al., Cell 1992 71, 701-708) were detected for both RNA-PCR (619 bp) and nested PCR (228 bp) products. Lanes are labeled with patient identification numbers (Caligiuri et al . , Cancer Res . 1994 54, 370-373) . Figure 26b: Sequence analysis of nested PCR products shows an in-frame fusion of ALL-l exon 6 with exon 2. Amino acid translation is shown beneath the DNA sequence.
DETAILED DESCRIPTION OF THE INVENTION
The ALL-l gene located at human chromosome 11 band q23 is rearranged in acute leukemias with interstitial deletions or reciprocal translocations between this region and chromosomes 1, 2, 4, 6, 9, 10, 15, 17 or 19. The gene spans approximately 100 kb of DNA and contains at least 21 exons. It encodes a protein of approximately 4,000 amino acids containing three regions with homology to sequences within the Drosophila trithorax gene including cysteine-rich regions which can be folded into six zinc finger-like domains. The breakpoint cluster region within ALL-l spans approximately 8 kb and encompasses several small exons (including exons 5-12) , most of which begin in the same phase of the open reading frame . It is to be understood from the description given below that each of the examples describing the practice of the invention are applicable to each of the now cloned and sequenced AF-4, AF-9, AF-6 and AF-17 genes and their respective ALL-l fusion genes ALL-l/AF-4, ALL-l/AF-9, ALL-l/AF-6 and ALL- l/AF-17.
The t(4;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 4. This gene on chromosome 4 is termed "AF-4" while the chimeric gene resulting from the t(4;ll) translocation is termed "ALL-l/AF-4. " It is believed that the Hq23 abnormality of translocation with 4q21 gives rise to one or two specific oncogenic fusion proteins.
The t(9;ll) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 9. This gene on chromosome 9 is termed "AF-9" while the chimeric gene resulting from the t(9;ll) translocation is termed "ALL-l/AF-9. " It is believed that the Hq23 abnormality of translocation with 9p22 gives rise to one or two specific oncogenic fusion proteins.
The t(ll;19) chromosome translocation results in two reciprocal fusion products coding for chimeric proteins derived from ALL-l and from a gene on chromosome 19. This gene on chromosome 19 is termed "ENL" while the chimeric gene resulting from the t(ll;19) translocation is termed "ALL-1/ENL." It is believed that the t(ll;19) translocation gives rise to one or two specific oncogenic fusion proteins.
In translocations involving the ALL-l gene and chromosome 6, t(6;ll), the gene on chromosome 6 is termed AF-6 and the chimeric gene resulting from the t(6;ll) translocation is termed ALL-l/AF-6. Similarly, in translocations involving the ALL-l gene and chromosome 17, t(ll;17) , the gene on chromosome 17 is termed AF-17 and the chimeric gene resulting from the t (11:17) translocation is termed ALL-l/AF-17.
A DNA fragment which detects DNA rearrangements by Southern analysis in the majority of patients with t(4;ll) , t(9;ll) and t(ll;19) chromosomal aberrations has been cloned from chromosome 11. This locus is referred to as ALL-l for acute lymphocytic leukemia, although the same locus is also involved in acute myelomonocytic, myelogenous and monocytic leukemias carrying translocations involving Hq23. DNAs and RNAs were extracted from cell lines and primary tumors by conventional methods. Southern and Northern analysis were performed as described in Shtivelman et al . , Na ture 1985, 315, 550-554) . To obtain unique (repeat free) probes, cosmids were, digested with a variety of restriction enzymes, and analyzed by Southern blotting for fragments which do not react with radiolabeled total human DNA. End fragments of cosmids were identified by hybridizing cosmids' digests to radiolabeled oligonucleotides corresponding to the recognition sequences for T7 and T3 RNA polymerases . If the end fragments contained human repeats, they were isolated, digested with frequent cutters and analyzed as described above. The 0.7 kb Ddel probe was thus obtained from a terminal 3.5 kb EcσRV fragment of cosmid 53. A portion of the Washington University's human DNA-containing YAC library (Green et al . , Proc . Na tl . Acad . Sci . USA 1990, 87, 9358-9362) was screened for CD3 DNA sequences (van Den Elsen et al . , Proc . Na tl . Acad . Sci . USA 1986, 83, 2944-2948) by a polymerase chain reaction (PCR) -based screening protocol (Green et al . , supra) . The YAC clone obtained appeared to be identical to the one described by Rowley et al . , Proc . Na tl . Acad. Sci . USA 1990, 87, 9358-9362, and spanned the translocation breakpoint in a t(4;ll) cell line as evidenced by hybridization analysis. By pulse field electrophoretic analysis, the size of the insert was estimated as 350 kb. A 310 kb version of the insert, generated by spontaneous deletion at the left (telomeric) side, predominated in the population of DNA molecules and was mapped (Figure 1) . To obtain specific segments of the insert, the YAC was purified by pulse field electrophoresis and shotgun cloned into the Supercos (Stratagene) cosmid vector. For this purpose the insert was partially digested by a combined application of dam methylase and the restriction endonuclease Mbol, Hoheisel et al . , Nuc . Acid Res . 1989, 17, 9571-9582. Both enzymes act on the sequence GATC, but Mbol is unable to cut the methylated form. More than a hundred cosmid clones, detected with a probe for human repetitive sequences, were obtained. The cosmids were mapped by screening for those with sites for NotI and Mlul enzymes, and for those hybridizing to CD3, trp and ura probes. Some cosmids were established using unique (repeat free) probes obtained from termini of cosmids. The positions of 3 cosmids mapped to the center of the YAC are shown in Figure 1. Unique probes from these cosmids as well as from cosmids mapped to other regions of the YAC were used to screen Southern blots of DΝAs from tumors exhibiting translocations.
A 0.7 kb Ddel fragment derived from the terminus of cosmid 53 detected rearranged fragments in tumor DΝAs digested with EcoRV, Xbal, or Bair-HI . Examples of these analyses are shown in Figure 2. The leukemic cells from patients A.G., E.C, A.L., B.H., I.B., G.F., P.P., and V.S. contained novel EcoRV or Xbal fragments of various sizes. This probe detected rearrangements in 6/7, 4/5, and 3/4 patients with the t(4;ll) , t(9;ll) and t(ll;19) translocations, respectively. Upon determination of the smallest genomic fragment in which rearrangement could be identified, (Figure 3) it became apparent that most or all breakpoints clustered within a small DNA region of approximately 8 kb. In three other patients two rearranged fragments (as well as a germline species) were detected, probably due to the presence of the breakpoint in these patients within the 0.7 kb Ddel segment corresponding to the probe. Finally, normal fibroblast DNAs from 7 additional individuals were used for comparison to show the germline fragments after digestions with EcoRV, Xbal or BamHI.
As a first step toward identification of genes neighboring the breakpoint cluster region, pooled unique fragments from cosmid 20 were labeled, together with the terminal fragment of cosmid 53, and were used to probe RNAs from cell lines and patients with or without Hq23 translocations (Figure 4) . The pooled probe detected 5 kb and 10 kb RNA species in the K562, glioblastoma T986 and Sup B cell lines (lanes a, b, c) . It also hybridized with a 5 kb RNA from patients with t(4;ll) , t(9;ll) , and t(ll;19) (Figure 4, lanes d, e,) . In another patient with t(4;ll) the probe detected the 10 kb RNA species alone.
It has been discovered that in leukemic cells of patients with the t (4;11) , t(9;ll) and t (11,-19) translocations, the breakpoints on chromosome 11 cluster in a small region of approximately 8 kb. Other translocations in acute leukemias affecting Hq23 are believed to map to the same locus. This locus has been designated ALL-l for acute lymphocytic leukemia, although the ALL-l locus is also involved in translocations in acute myelomonocytic, monocytic and myelogenous leukemias. The tight clustering of breaks suggests that the gene involved is close to the breakpoints . The Northern analysis indicates that DNA sequences adjacent to the breakpoints are expressed. However, no new transcript was detected in the leukemic cells. Moreover, only one of the transcripts (usually the 5 kb species) found in cells without the translocation was detected in the patients .
The finding of tight clustering of the breakpoints on chromosome 11 in the three most common Hq23 abnormalities raised the possibility that the same region is rearranged in other chromosomal aberrations involving Hq23. To test this, tumor DNAs from the leukemic cells of patients with t (6,-H) (q27;q23) , t (1;11) (p34 ;q23) , t (10;11) (pll-15;q23) and del (11) (q23) were digested with BamHI, Xbal, EcoRV and HindHI enzymes and subjected to Southern analysis using the modified 0.5 kb Ddel fragment as a probe. This probe was obtained from the 0.7 kb Ddel probe by digestion with Alul, which ultimately improved performance by removing a 0.24 kb internal fragment that had caused a higher background in Southern analyses . Following digestion with Alul, the internal fragment and the two end fragments were electrophoresed to isolate the two terminal fragments, which were then ligated to form a 0.5 kb fragment which was cloned into a plasmid vector. Results of Southern blotting are shown in Figure 5. Rearranged fragments were found in the DNAs of patients with t(6;ll) , t(l;ll) and t(10,-ll) (lanes a, d, e, respectively) and in two patients
(lanes f, g) out of five with interstitial deletion in Hq23
(the 3 negative patients had del 11 (q21;q23) ) . The patients with t(6;ll) and t(10;ll) , as well as one of those with del (11) (q23) showing rearrangement had AML; the rest of the patients tested had ALL.
To further analyze transcription of the genomic DNA adjacent to the breakpoint cluster region, segments of cosmid 20 found fully or partially free of repetitive sequences were examined as probes to polyadenylated RNAs obtained from a variety of hematopoietic and nonhematopoietic cell lines. Three ALL cell lines, MV 4;11, RS 4;11 and Bl containing the t(4,-ll) chromosome translocation were included in the analysis. These three cell lines had rearrangements at the breakpoint cluster region, as shown in Figure 5, lanes b and c. A 1.5 kb EcoRI DNA segment generated from cosmid 20 was used as a probe and identified a 12.5 kb RNA in all cell lines (Figure 6) . A minor species of 11.5 kb was detected in most of the samples without involvement of Hq23, but it was not possible to determine if this RNA was present in the cells with the t(4,-ll) translocation. A transcript of 11 kb was detected in the three cell lines with the t(4,-ll) chromosome translocation (Figure 6; lanes c, f, k) . The width of this band on the autoradiogram suggests that it corresponds to two comigrating RΝA species. The 11 kb RΝA was not detected in any of the cell lines lacking Hq23 aberrations (Figure 6) .
These results show that the same breakpoint cluster region is rearranged in at least seven different Hq23 abnormalities, including six types of translocations, as well as interstitial deletions. Three samples with H(q21;q23) deletions, one sample with t(ll,-15) (q23;q22) , and one sample with t(H;X) (q23;q26) did not show rearrangements within the locus. In addition, in 1 of 12, 1 of 9, and 2 of 9 patients with t(4;ll) , t(9;ll) , and t(ll;19) chromosome translocations respectively, rearrangements were not detected using the Ddel probe. Finally, the breakpoint in the RC-K8 cell line containing the t(ll;14) (q23;q32) is apparently telomeric to the locus discussed here. In all of these cases, other unidentified loci on chromosome 11 could be involved. Alternatively, the ALL-l locus might also be affected in these patients, but this may occur at a different site.
Using a new probe, three polyadenylated transcripts were identified. Two of them, a 12.5 and an 11.5 kb species, are expressed as detected by Northern analysis in most or all cell lines, but the third, an 11 kb RNA, was detected solely in cell lines with the t(4;ll) abnormality. RNA species of similar size have recently been reported by others. For example, Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA
1991, 88, 10735-10739. However, while the instant probe which is located centromeric to the breakpoints, detects all three
RNAs; Ziemin-van der Poel et al . reported that their probe
(#1) , which is derived from the same general location, detects predominantly the 12.5 kb species. While the instant probe detects 11 kb transcript solely in leukemic cells with the t(4,-ll) chromosome translocation, the Ziemin-van der Poel et al . study identifies an 11 kb mRNA in the RS4;11 cell line, as well as in small amounts in all cells tested. The results show, however, a clear qualitative alteration in expression of a region adjacent to the breakpoint cluster region on chromosome 11 in cells with the t(4;ll) chromosome translocation.
Using either somatic cell hybrids (Savage et al . , Cytogenet. Cell Genet . 1988, 49, 289-292; Wei et al . , Cancer Genet . Cytogenet . 1990, 46, 1-8; Yunis et al . , Genomics 1989, 5, 84-90) , or the fluorescent in si tu hybridization (FISH) technique (Rowley et al . , Proc . Natl . Acad. Sci . USA 1990, 87, 9358-9362) , it was possible to position the breakpoints on chromosome 11 to a region between the CD3 and PBGD genes. Rowley et al . , supra , used a CD3-gamma probe to clone a 350 kb human DΝA fragment from a yeast artificial chromosome (YAC) library. This YAC spanned the t(4;ll) , t(9;ll) , t(ll;19) , and t(6;ll) breakpoints as indicated by FISH analysis. Using probes derived from both sides of the breakpoint cluster region, Rowley et al . identified a 12.5 kb RNA in cells with or without Hq23 abnormalities. Further, a probe located telomeric to the cluster region detected two additional transcripts of 11.5 and 11 kb in the RS 4;11 cell line, as well as in all hematopoietic and nonhematopoietic cells tested (Ziemin-van der Poel et al . , Proc . Natl . Acad. Sci . USA 1991, 88, 10735-10739) .
From a YAC clone similar to the one used by Rowley et al . , a DΝA segment was obtained which detected rearrangements in leukemic cells from patients with the t(l;ll) , t(4;ll) , t(6,-ll) , t(9;ll) , t(10;ll) , t(ll;19) or del (Hq23) chromosome abnormalities on Southern blots (Cimino et al . , Cancer Research 1991, 51, 6712-6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) . The breakpoints clustered within a small region of approximately 8 kb termed the ALL-l locus. Translocation junction fragments were cloned from leukemic cells with t(4;ll) and showed clustering of the breakpoints in an area of 7-8 kb on chromosome 4. Sequencing analysis indicated heptamer and nonamer-like sequences, associated with rearrangements of immunoglobulin and T cell receptor genes, near the breakpoints. These sequences suggested a direct involvement of the VDJ recombinase in the Hq23 translocations.
Transcription of the genomic DNA adjacent to the breakpoint cluster region was analyzed using segments of cloned DNAs as probes. Probes from both sides of the region identified a major transcript of 15-16 kb (previously estimated as 12.5 kb) (Cimino et al . , Cancer Research 1991, 51, 6712- 6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) in cells with or without Hq23 abnormalities. The gene coding for these RNAs was termed ALL-l. Leukemic cells with the t(4;ll) chromosome translocation contained, in addition to the normal species, shorter RNAs transcribed from the der (11) and der (4) chromosomes . These studies were extended to clone and sequence ALL-l RNA, to further characterize the ALL-l gene, and to identify chimeric transcripts produced in cells with the t(4,-ll) chromosome translocation. Structure of the ALL-l gene and cDNA
Utilizing a repeat-free genomic DNA segment located 10 kb centromeric to the breakpoint cluster region on chromosome 11 (Cimino et al . , Cancer Research 1992, 52, 3811- 3813) , a human fibroblast cDNA library and a K562 cDNA library were screened (Chu et al . , EMBO J. 1990, 9, 985-993; Shtivelman et al . , Nature 1985, 315, 550-554) . Positive clones were used as probes for further screening. 5-10 μg aliquots of polyadenylated RNAs were electrophoresed on 1.1% agarose gels in formaldehyde, blotted onto nitrocellulose filters and analyzed by hybridization. (Gale, RP and Canaani, Proc . Natl . Acad . Sci . USA 1984, 81, 5648-5652) . 20 μg aliquots of high molecular weight DNA were digested with BamHI and analyzed by the Southern technique. 3' and 5' ALL-l probes were composed of phages VI and SKV2 sequences, respectively (Figure 7) . Non ALL-l probes were generated from clones 16 and 25 by PCR. A series of overlapping clones spanning 14.7 kb
(Figure 7 top) was obtained. These cDNAs presumably originated from the major ALL-l transcript. All cDNA sequences were found to hybridize to genomic DNA within the 95 kb internal Not I fragment of the YAC B22B (Cimino et al . , Cancer Research 1991, 51, 6712-6714) . This region was previously subcloned into cosmids 20, 43, and 53 and into phages gc3 , cl4, and mg 11.1 (Figure 7) . The cloning of cosmids 20, 43, and 53 from YAC B22B has been described (Cimino et al . , Cancer Research 1991, 51, 6712-6714) and clones mg 11.1, cl4, and gc3 were obtained from a genomic DNA library made in the EMBL-3 vector (Stratagene) . Restriction enzyme mapping of the cDNA and genomic clones and analysis of the hybridization pattern of cDNA fragments to genomic DNA indicated that the ALL-l gene is composed of a minimum of 21 exons, some of them (6-12) very small (shorter than 150 bp) . The first intron was found to be the largest, spanning approximately 35 kb of DNA.
The nucleotide sequence of ALL-l cDNA was determined using an automatic sequencer (ABI) . The sequence revealed a single long open reading frame predicting a protein of approximately 4,000 amino acids with molecular weight of approximately 400,000 Daltons (Figure 8) . To search for homologous nucleotide sequences and protein sequences the GenBank and SWISS data bases were screened by the FASTA program. Nucleotides 9353-9696 were found to be nearly identical to an anonymous sequence (EST00626) cloned from human fetal brain cDNA library (Adams et al. , Nature 1992, 355, 632- 634) .
Three regions demonstrated homology to the trithorax gene of Drosophila (Mazo et al., Proc . Natl . Acad . Sci . USA 1990, 87, 2112-2116) . Thus, predicted amino acids 1021-1221, 1462-1570, and 3348-3562 showed 64%, 66%, and 82% similarity, and 43%, 50%, and 61% identity, respectively, to the Drosophila gene (Figure 9) . The third region of homology constitutes the extreme C-terminus of the two proteins; both species end in an identical sequence. The first homology region is cysteine-rich and contains sequence motifs analogous to four zinc finger domains (3-6) within the trithorax gene (Mazo et al . , supra) . The second region of homology is also cysteine-rich and corresponds to zinc fingers 7 and 8 of the Drosophila gene. The human putative zinc finger structures are shown at the bottom of Figure 9. The multiple conserved cysteines and histidines at the 3' end of the motifs allow two or three arrangements of the putative fingers. The structure of these cysteine-rich domains appears to be unique to the trithorax and ALL-l genes.
Chimeric RNAs resulting from the t(4;ll) chromosome translocations Clustering of t(4;ll) breakpoints has previously been found within a small segment of the ALL-l locus (Cimino et al . , Cancer Research 1991, 51, 6712-6714; Cimino et al . , Cancer Research 1992, 52, 3811-3813) . This region includes 7 coding exons (6-12) containing 74, 132, 114, 147, 96, 121, and 123 bp respectively. Exons 8-12 contain four zinc finger motifs. Exons 7-11 all begin in the first nucleotide of a codon. Precise mapping of five t(4;ll) breakpoints localized them to introns between exons 6 and 7, 7 and 8, and 8 and 9 (Figure 10A) . These breaks in chromosome 11 result in removal of the N-terminal 996 amino acids from the ALL-l protein, as well as in disjoining of the 5' noncoding region of the gene.
If the breaks on chromosome 4 occur within a gene positioned with its 5' terminus toward the centromere, t(4;ll) translocations should result in fusion of the ALL-l gene to the gene aforementioned and, consequently, in production of two reciprocal chimeric RNAs . To investigate this possibility, a cDΝA library was constructed from RΝA extracted from the RS4;11 leukemic cell line established from a patient with the t(4;ll) chromosome translocation (Stong, RG, and Kersey, JH, Blood 1985, 66, 439-443) . This RS4;H cDΝA library was constructed by treating polyadenylated RNA with 1 mM methyl mercury for 10 minutes at room temperature, followed by neutralization with 10 mM mercaptoethanol and alcohol precipitation. cDΝA was prepared by using the Time Saver kit (Pharmacia) and was cloned into the lambda ZAP II vector (Stratagene) .
The library (2 X 106 clones) was screened with a probe composed of exons 3-13. Twenty positive clones were purified and mapped. Two clones varied from normal ALL-l cDNA and were further analyzed by sequencing.
Clone 16 contained normal ALL-l sequences 3' to the beginning of exon 9. 5' to this position, ALL-l information was substituted with a new DNA fragment composed of an open reading frame (ORF) that joins in phase the rest of ALL-l ORF (Figure 10B) . Clone 25 had a reciprocal configuration in which exon 7 of ALL-l is linked to a new DNA segment containing an open reading frame. Here again, the two ORFs are joined in phase (Figure IOC) . Since, in the RS4;11 cell line, the breakpoint on chromosome 11 is within an intron located between ALL-l exons 7 and 8 (Figure 10A) , it was expected that in the putative chimeric RNAs sequences of these exons will be directly linked to the new cDNA sequence. This is indeed the case in clone 25 but not in clone 16. In the latter, it was assumed that exon 8 was excluded from the fused transcript by a mechanism involving alternative splicing. Skipping this exon retains the fused ORFs in phase.
The identification of new sequences linked to ALL-l cDNA in RS4;11 leukemic cells suggested that they originated from altered RNAs specific to cells with the t(4;ll) chromosome translocation. Previously, two such transcripts were identified: a 14 kb RNA (previously estimated as 11.5 kb) containing 3' ALL-l sequences and a 12.7 kb RNA (previously estimated as 11 kb) hybridizing to 5' ALL-l probe. These RNAs were transcribed from chromosome derivatives 4 and 11, respectively.
A radiolabelled probe composed of non ALL-l sequences of clone 16 was examined for hybridization to RNAs from cell lines with or without the t(4;ll) chromosome translocation. As a control, the RNAs were first hybridized to 3' ALL-l cDNA probe which detected the major normal transcript of 15-16 kb
(previously estimated as 12.5 kb) in all cell lines and an altered 14 kb RNA (previously estimated as 11.5 kb) in the three cell lines with t(4;ll) (Figure HA) .
Clone 16 probe identified a 9.5 kb RNA in all cells examined and a 14 kb transcript in RS4;H, MV4;H and B-l cells (Figure HB) . It was concluded that clone 16 originated from the 14 kb altered ALL-l transcript and that the non-ALL-1 sequence within this RNA is expressed in human cells as a 9.5 kb transcript, which corresponds to the normal AF-4 transcript on a non-rearranged chromosome 4.
In an analogous experiment, a probe composed of non- ALL-1 sequences in clone 25 hybridized to the 12.7 kb altered RΝA present in the RS4;H cell line and to a 9.5 kb RΝA species present in RS4;11 cells and in control cells (Figure HC) . Thus, clone 25 originated from the second altered 12.7 kb ALL-l
RΝA unique to cells with the t(4;ll) chromosome translocation.
The chromosome from which the new sequences of clones
16 and 25 originated was then identified. High molecular weight DΝAs from lines of Chinese hamster ovary (CHO) cells with or without human chromosome 4 were digested with Ba.mHI enzyme and analyzed by Southern blotting for hybridization to the non ALL-l sequence in clone 16 (Figure HD) and clone 25 (Figure HE) . The cell lines showed an 11 kb or a 6.6 kb band representing CHO cell DΝA cross-reacting with the probes. A fragment of 4.8 kb and fragments of 7.7 and 19.5 kb were detected in the somatic cell hybrid line containing human chromosome 4 (CHO/4) after hybridization with non ALL-l sequences of clones 16 and 25, respectively (Figures HD and E) . The non-ALL-1 sequences in clone 25 hybridized to a specific segment within cloned chromosome 4 DΝA spanning the RS4;H breakpoint. Thus, clones 16 and 25 correspond to the two reciprocal fused transcripts of the ALL-l gene and a gene on chromosome 4. The latter is denominated "AF-4" for ALL-l fused gene from chromosome 4. Cloning and sequence analysis of the ALL-l gene indicates that it encodes an unusually large protein of 4,000 amino acids with a mass of approximately 400 kD . The striking feature of the protein is its homology to the Drosophila trithorax gene. The homology is reflected in three ways. First, the transcripts and proteins have a similar size; the Drosophila gene is transcribed into a 15 kb RNA encoding a protein of 3759 amino acids (Mozer, BA, and David, IB, Proc . Na tl . Acad. Sci . USA 1989, 86, 3738-3742; Mazo et al . , Proc . Natl . Acad . Sci . USA 1990, 87, 2112-2116) .
Second, there is strong sequence homology in three regions, two of which contain zinc finger-like domains unique to the trithorax gene and presumably utilized in interaction with target DΝA. The third region shows 82% similarity and 61% identity across 220 amino acids which end both proteins at their C-terminus.
Finally, there is colinearity of the homologous sequences in the two proteins. Although the sequence homology does not extend to other parts of the protein, the two genes very possibly evolved from a common ancestor and may carry out similar function(s) . In this context, it has been previously noted that structural homology between Drosophila and mammalian genes such as the Antennapedia class homeobox genes, is frequently limited to the functional domains, e.g., the homeodomain (McGinnis, W, and Krumlauf, R. , Cell 1992, 68, 283- 302) .
The trithorax gene in Drosophila acts to maintain spatially-restricted expression patterns of the Antennapedia and Bithorax complexes during fruit fly development (Ingham, PW, Cold Spring Harbor Symp . Quant . Biol . 1985, 50, 201-208) . Trithorax activates transcription of multiple genes of the two complexes and, as such, counteracts the activity of Polycomb group genes which act as repressors of transcription for the same genes (McKeon, J and Brock, HW, Roux' s Arch . Dev. Biol . 1991, 199, 387-396) . Thus, mutations in the trithorax gene frequently result in homeotic transformations (Capdevila, MP and Garcia-Bellido, A., Roux ' s Arch . Dev. Biol . 1981, 190, 339- 350) . The discovery of zinc finger-like domains in the predicted amino acid sequence strongly suggested that the trithorax protein is a transcription factor which binds to DΝA
(Mazo et al . , Proc. Na tl . Acad . Sci . USA 1990, 87, 2112-2116) .
Indeed, antibodies to the protein react with specific regions of the chromatin in the salivary glands of Drosophila.
Based on what is known about the Drosophila gene, it is very likely that the ALL-l gene is a transcription factor and that it is involved in regulation of genes controlling human development and/or differentiation. While expression of ALL-l during embryonic development has not yet been investigated, the isolation of ALL-l sequences from a human fetal cDNA library indicates transcription of the gene during fetal development. Previous studies (Cimino et al . , Cancer Research 1992, 52, 3811-3813) demonstrated ALL-l RNA in a variety of hematopoietic cell lines, as well as in tumors originating from precursors of epithelial and glial cells. It was also found that the t(4;ll) chromosome translocation cleaves the ALL-l gene within the coding region and results in fusion of the open reading frames of ALL-l and a gene on chromosome 4 (termed AF-4) in phase. The breakpoints on chromosome 11 cluster in a region containing several small exons, 5 of them (exons 7-11) begin in the first letter of a codon. Splicing from the same exon on chromosome 4, adjacent to the breakpoint in RS4;H, to each one of the five exons on chromosome 11 will retain the two open reading frames fused in phase. This situation is similar to the situation in the t(9,-22) chromosome translocations where the breakpoints cluster near two BCR exons whose splicing to ABL exon 11 maintain the fused open reading frames in phase (Shtivelman et al . , Nature 1985, 315, 550-554; Heisterkamp et al . , Nature 1985, 315, 758- 761) . The clustering of breakpoints must also reflect the specific biological properties of the fused proteins and probably is also due to the presence of recombination signals in this region.
Two chimeric proteins from the 12.7 and 14 kb RΝAs are predicted for cells with the t(4;ll) chromosome translocation. The lack of information about the normal AF-4 protein precludes at this time the determination if it is also a transcription factor that exchanges functional domains with ALL-l to give a chimeric transcription factor. This occurs in the t(l;19) and t(15,-17) chromosome translocations (Kamps et al . , Cell 1990, 60, 547-555; Νourse et al . , Cell 1990, 60, 535-545; Kakizuka et al., Cell 1991, 66, 663-674; de The et al . , Cell 1991, 66, 675- 684) . Both the 12.7 and the 14 kb fused RNAs are found in the three cell lines with t(4;ll) , therefore it is not possible at this time to establish which of the two products is oncogenic. However, the presence of the three trithorax homologous domains within the 14 kb transcript makes it an attractive candidate. The substitution of the N-terminus 996 amino acids of ALL-l with an AF-4 polypeptide could result in at least two scenarios, both based on the assumption that ALL-l and ALL-l/AF-4 activate transcription of the same gene(s) . First, the substitution could place ALL-l DNA binding domain under the control of a new effector domain activated by either ubiquitous or tissue specific factors. This will result in transcription of the target genes in the wrong cells. Second, the fusion product may function as a dominant negative inhibitor of ALL-l by forming inactive heterodimers or by occupying target DNA sites.
The present invention provides methods of diagnosis for human leukemia by providing a tissue sample from a person suspected of having acute lymphocytic, myelomonocytic, monocytic or myelogenous leukemia, and determining if there are breakpoints on chromosome 11 in the ALL-l locus. The sequence of the ALL-l cDNA can be used to generate probes to detect chromosome abnormalities in the ALL-l breakpoint cluster region. These probes may be generated from both the sense and antisense strands of double-stranded DNA. The term "ALL-l probe" refers to both genomic and cDNA probes derived from the ALL-l gene.
It is believed from the data described above and those data described below that genomic probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences from at least 10 kb centromeric to at least 10 kb telomeric to the breakpoint cluster region, which has been shown to span at least exons 6-9, and may span exons 5-12 of the ALL-l gene. It is believed that cDNA probes capable of detecting chromosomal translocations involving the ALL-l breakpoint cluster region span sequences ranging from 2 kb centromeric to 2 kb telomeric to the breakpoint cluster region. Thus, preferred embodiments of the present invention for detecting chromosomal abnormalities involving ALL-l provide genomic and cDNA probes spanning the chromosome 11 regions described above. cDNA probes are more preferred, and probes comprising the exons included in the breakpoint cluster region are most preferred.
Part or all of the ALL-l cDNA sequence may be used to create a probe capable of detecting aberrant transcripts resulting from chromosome 11 translocations. The EcoRI probe, for example, was derived from a genomic clone but its location lies within an exon. Thus, preferred embodiments of the present invention for detecting aberrant transcripts provide cDNA probes spanning the ALL-l gene.
The ALL-l/AF-4 sequences provided in SEQ ID NO: 23 and SEQ ID NO:24 can be used to create probes to detect t(4;ll) chromosome abnormalities and aberrant transcripts corresponding to t(4,-ll) translocations. Additional sequences (see below) include those specific for the ALL-l/AF-6, ALL-l/AF-9 and ALL- l/AF-17 chimeric genes. Also included in the invention and described below are specific ALL-l probes capable of detecting chromosomal abnormalities in the ALL-l gene irrespective of the nature of the fusion partner gene.
Using the probes of the present invention, several methods are available for detecting chromosome abnormalities in the ALL-l gene on chromosome 11. Such methods include, for example, Polymerase Chain Reaction (PCR) technology, restriction fragment length analysis, and oligonucleotide hybridization using, for example, Southern and Northern blotting and in si tu hybridization. PCR technology is practiced routinely by those having ordinary skill in the art and its uses in diagnostics are well known and accepted. Methods for practicing PCR technology are disclosed in PCR Protocols : A Guide to Methods and Applications, Innis, M.A. et al . , Eds., Academic Press, San Diego, CA 1990, and RT- PCR, Clontech Laboratories (1991) , which are incorporated herein by reference. Applications of PCR technology are disclosed in Polymerase Chain Reaction, Erlich, H.A. et al . , Eds., Cold Spring Harbor Press, Cold Spring Harbor, NY 1989, which is incorporated herein by reference.
PCR technology allows for the rapid generation of multiple copies of DNA sequences by providing 5' and 3' primers that hybridize to sequences present in a DNA molecule, and further providing free nucleotides and an enzyme which fills in the complementary bases to the DNA sequence between the primers with the free nucleotides to produce a complementary strand of DNA. The enzyme will fill in the complementary sequences between probes only if both the 5' primer and 3' primer hybridize to DNA sequences on the same strand of DNA.
To detect rearrangements involving for example, chromosomes 11 and 4, one of the two probes can be generated from the ALL-l cDNA and one probe from the AF-4 gene. RNA is isolated from hematopoietic cells of a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia, and cDNA is generated from the mRNA. If the cDNA of the chimeric ALL-l/AF-4 gene is present, both primers will hybridize to the cDNA and the intervening sequence will be amplified. The PCR technology therefore provides a straightforward and reliable method of detecting the chimeric gene.
The preferred primers for PCR are selected, one from a portion of SEQ ID NO: 1, corresponding to the ALL-l cDNA, and one from a portion of either SEQ ID NO: 19 or SEQ ID NO: 22, corresponding to AF-4 gene sequences. Preferably, the sequences chosen from SEQ ID NO: 1 comprise at least a portion of SEQ ID NO: 20, which corresponds to exon 9, or SEQ ID NO: 21, which corresponds to exon 7.
According to the invention, diagnostic kits can be assembled which are useful to practice oligonucleotide hybridization methods of distinguishing chromosome 11 abnormalities from non-rearranged chromosomes 11. Such diagnostic kits comprise a labelled oligonucleotide which hybridizes, for example, to the chimeric transcript that results from t(4;ll) translocations but which does not hybridize to nucleic acid transcripts not associated with aberrations. Accordingly, diagnostic kits of the present invention comprise, for example, a labelled probe that includes ALL-l and AF-4 sequences which make up the chimeric transcript associated with t(4;ll) translocations. Such probes comprise oligonucleotides having at least a portion of the sequence of the ALL-l/AF-4 gene of SEQ ID NO: 23 or SEQ ID NO: 24.
It is preferred that labelled probes of the oligonucleotide diagnostic kits according to the present invention are labelled with a radionucleotide. The oligonucleotide hybridization-based diagnostic kits according to the invention preferably comprise DNA samples that represent positive and negative controls. A positive control DNA sample is one that comprises a nucleic acid molecule which has a nucleotide sequence that is fully complementary to the probes of the kit such that the probes will hybridize to the molecule under assay conditions. A negative control DNA sample is one that comprises at least one nucleic acid molecule, the nucleotide sequence of which is partially complementary to the sequences of the probe of the kit. Under assay conditions, the probe will not hybridize to the negative control DNA sample. Probes useful as diagnostics can be used not only to diagnose the onset of illness in a patient, but may also be used to assess the status of a patient who may or may not be in remission. It is believed that emergence of a patient from remission is characterized by the presence of cells containing chromosome abnormalities. Thus, patients believed to be in remission may be monitored using the probes of the invention to determine their status regarding progression or remission from disease. Use of such probes will thus provide a highly sensitive assay the results of which may be used by physicians in their overall assessment and management of the patient's illness .
Antisense oligonucleotides which hybridize to at least a portion of an aberrant transcript resulting from chromosome 11 abnormalities involving the ALL-l gene are also contemplated by the present invention. The oligonucleotide may match the target region exactly or may contain several mismatches. Thus, molecules which bind competitively to RNA coded by, for example, the chimeric ALL-l/AF-4 gene, for example, are envisioned for therapeutics. Preferred embodiments include antisense oligonucleotides capable of binding to at least a portion of SEQ ID NO: 23 and SEQ ID NO: 24. Preferred embodiments of the present invention include antisense oligonucleotides capable of binding to a region of the ALL-l/AF-4 mRNA corresponding to the ALL-l sequences which encode a peptide having homology with the Drosophila trithorax protein and antisense oligonucleotides capable of binding to a region of the mRNA encoding a zinc finger-like domain in the ALL-l protein.
While any length oligonucleotide may be utilized, sequences shorter than 15 bases may be less specific in hybridizing to the target and may be more easily destroyed by enzymatic degradation. Hence, oligonucleotides having at least 15 nucleotides are preferred. Sequences longer than 21 nucleotides may be somewhat less effective in interfering with ALL-l expression because of decreased uptake by the target cell. Therefore, oligonucleotides of 15-21 nucleotides are most preferred.
The term "oligonucleotide" as used herein includes both ribonucleotides and deoxyribonucleotides, and includes molecules which may be long enough to be termed "polynucleotides . " Oligodeoxyribonucleotides are preferred since oligoribonucleotides are more susceptible to enzymatic attack by ribonucleotides than deoxyribonucleotides. It will also be understood that the bases, sugars or internucleotide linkages may be chemically modified by methods known in the art. Modifications may be made, for example, to improve stability and/or lipid solubility. For instance, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting a methyl group or sulfur atom for a phosphate oxygen in the internucleotide phosphodiester linkage. The phosphorothioates, in particular, are stable to nuclease cleavage and soluble in lipid. Modified oligonucleotides are termed "derivatives." The oligonucleotides of the present invention may be synthesized by any of the known chemical oligonucleotide synthesis methods. See for example, Gait, M.J., ed. (1984) , Oligonucleotide Synthesis (IRL, Oxford) . Since the entire sequence of the ALL-l gene has been provided along with partial sequences of the AF-4 gene, antisense oligonucleotides hybridizable with any portion of these sequences may be prepared by the synthetic methods known by those skilled in the art . It is generally preferred to apply the therapeutic agent in accordance with this invention internally such as intravenously, transdermally or intramuscularly. Other forms of administration such as topically or interlesionally may also be useful. Inclusion in suppositories is presently believed to be likely to be highly useful. Use of pharmacologically acceptable carriers is also preferred for some embodiments .
For in vivo use, the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solution of dextrose, and the like. In addition to administration with conventional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendai virus envelopes have been successfully used to deliver RNA and DNA to cells (Arad et al . , Biochem . Biophy. Acta . 1986, 859, 88-94) .
For in vivo use, the antisense oligonucleotides may be administered in an amount effective to result in extracellular concentrations approximating in vi tro concentrations described below. The actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required.
It is also possible to administer the antisense oligonucleotides ex vivo by isolating white blood cells from peripheral blood, treating them with the antisense oligonucleotides, then returning the cells to the donor's blood. Ex vivo techniques have been used in the treatment of cancer patients with interleukin-2 activated lymphocytes.
For ex vivo application, for example, in bone marrow purging, the antisense oligonucleotides may be administered in amounts effective to kill leukemic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the nature and extent of the leukemia, the particular oligonucleotide utilized, the relative sensitivity of the leukemia to the oligonucleotide, and other factors. Concentrations from about 10 to 100 μg/ml per 105 cells may be employed, preferably from about 40 to about 60 μg/ml per 105 cells . Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment. Thus, for purging bone marrow containing 2xl07 per ml of marrow volume, dosages from about 2 to about 20 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 12 mg/ml. Greater or lesser amounts of oligonucleotide may be employed.
The present invention is also directed to monoclonal antibodies capable of binding to chimeric ALL-l/AF proteins including ALL-l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17, and includes monoclonal antibodies capable of binding to a region of the protein having homology with the Drosophila trithorax protein and monoclonal antibodies capable of binding to a zinc finger-like domain. Such monoclonal antibodies are useful as diagnostic and therapeutic agents for leukemias characterized by t(4;ll) , (t(6;ll) , t(9;ll) and t(H;17) translocations. Thus, the present invention encompasses immunoassays for detecting at least portions of either the ALL- l/AF-4, ALL-l/AF-6, ALL-l/AF-9 and ALL-l/AF-17 proteins. In addition, the instant invention contemplates diagnostic kits comprising a monoclonal antibody to at least a portion of the ALL-l fusion proteins listed above in combination with conventional diagnostic kit components.
The present invention is also directed to pharmaceutical compositions comprising monoclonal antibodies and a suitable pharmaceutical carrier, which are well known in the pharmaceutical art, and are described, for example, in
Remington's Pharmaceutical Sciences , Gennaro, A.R., ed., Mack
Publishing Co., Easton, PA 1985. The useful dosage will vary depending upon the age, weight, and particular patient treated.
Polyclonal antibodies to the instant polypeptides are also within the ambit of the invention. Such polyclonal antibodies may be produced using standard techniques, for example, by immunizing a rabbit or a rat with a protein or peptide of the invention, removing serum from the rabbit, and harvesting the resultant polyclonal antibodies from the serum. If desired, the polyclonal antibodies may be used as an IgG fraction or may be further purified in varying degrees. Procedures for preparing, harvesting and purifying polyclonal antibodies are well known in the art, and are described, for example, in Methods in Immunology: A Laboratory Text for Instruction and Research, Garvey et al . , Ed., W.A. Benjamin, Reading MA, 1977, 3rd ed. , chapter 22, 24-30.
Experiments reported in Example 1 provide further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(4;ll) translocations. The information provided in example 1 includes complete cDNA sequences encoding AF-4. These sequences may be used design probes of at least 15 nucleotides which are capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes comprise, an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:25 or SEQ ID NO:27. The procedures for using such probes are described above.
Experiments reported in Example 2 provide further data for designing methods of diagnosing and treating acute lymphoblastic or nonlymphoblastic leukemia, particularly those involving a chimeric gene in t(9;ll) translocations. The information provided in example 2 may be used design probes of at least 15 nucleotides which is capable of identifying chromosome abnormalities within the ALL-l gene of chromosome 11. Examples of such probes may comprise at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. Further, probes capable of identifying chromosome abnormalities within the AF-9 gene of chromosome 9 may be designed. Examples of such probes comprise an oligonucleotide sequence or derivatives thereof comprising at least a portion of SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. The procedures for using such probes are described above.
The experiments reported in Examples 3 and 4 describe the cloning and sequencing of ALL-l/AF-6 and ALL-l/AF-17 genes, respectively. The experiments reported in Example 5 describe a probe capable of detecting abnormalities in the ALL-l region irrespective of the nature of the fusion gene, and the experiments reported in Example 6 describe duplications of the ALL-l region in cells of some patients with leukemia. Thus, the invention must be construed to include each of these genes, their products and probes derived therefrom as being useful for the diagnosis and treatment of patients with these types of leukemias. Although specific examples are given, each example must be construed to include the other named fusion genes as being useful in the methods and compositins of the invention. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations may be performed by first providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; then isolating RNA from the sample followed by generating cDNA from said RNA and amplifying a chimeric gene sequence in said cDΝA which is generated by said translocation using a set of PCR primers if said chimeric gene is present such that detecting the presence of amplified DNA indicates the tissue sample is derived from an individual suffering from lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations. The method, which is generally described in detail above, may be performed using sets of primers which can be used to amplify a chimeric gene generated by the translocation. Examples of such primers can be designed, for example, using the sequence information in SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34. Examples of primers include SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO:42; and SEQ ID NO:43 and SEQ ID NO:44.
Monoclonal antibody capable of binding to at least a portion of for example, the chimeric ALL-l/AF-9 protein may be produced by standard techniques. Examples of such a monoclonal antibodies, which can bind specifically to at least a portion of the amino acid sequences encoded by SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13, may be produced using peptides which comprise at least a portion of SEQ ID NO: 9, SEQ ID NO:11 or SEQ ID NO:13.
In one method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia, tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia is examined to detect the ALL-l/AF-9 chimeric protein or a portion of the chimeric ALL-l/AF-9 protein. In one embodiment of such a method, a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein is used.
The present invention provides antisense oligonucleotides capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA. Such antisense oligonucleotides include those capable of binding to at least a portion of SEQ ID NO:32, SEQ ID NO:33 or SEQ ID NO:34.
Method of treating acute lymphoblastic or nonlymphoblastic leukemia are provide which comprise administering an antisense oligonucleotide capable of binding to at least a portion of the chimeric ALL-l/AF-9 mRNA or, alternatively, administering a monoclonal antibody capable of binding to at least a portion of the chimeric ALL-l/AF-9 protein. The formulation and administration of therapeutics are outlined above. Example 1
Experiments were performed to determine the CDNA sequence of AF-4 and study ALL-l/AF-4 chimeric genes. Clonincf and Sequencing AF-4-cDNA. cDNA clones containing the two reciprocal ALL-l/AF-4 RNA junctions were cloned from RNA of the RS4 11 cell line carrying the t(4:ll) chromosome translocation. AF-4 specific probes obtained from these clones were used to screen cDNA libraries prepared from RNAs of the K562 and KC122 hematopoietic cell lines. Positive clones were sequenced and utilized to prepare end probes for further screening. Overlapping clones spanning most or all of the 9.5 kb AF-4 transcript were obtained. Analysis of the longest cDNA composite indicated an open reading frame initiated with a consensus ATG and coding for a protein of 1210 amino acids (SEQ ID NO:25 and SEQ ID NO:27; and SEQ ID NO:26 and SEQ ID NO:28, respectively) . cDNA clone k 12, SEQ ID NO:25, diverged from cDNA clone kcl 6, SEQ ID NO:27, at nucleotide 435 of the latter. 5' of this position the two sequences completely varied. The open reading frames of clones kcl 6 and k 12 started 5 and 12 codons, respectively 5' of the divergence point. This suggests an alternative first exon for AF-4. A third cDNA clone, k 1.1, represents another RNA variant probably resulting from alternative splicing; an in frame termination codon is present in this clone immediately 3' to the divergence point. Thus, AF-4 encodes 2 or more proteins varying at their termini. AF-4 contains an unusually long 3' untranslated region of 5 3kb. This region includes multiple AATAAA sequences located 20 nucleotides 5' of the poly A, as well as in several upstream positions; it also contains several stretches of T.
Using the Swiss, Prosite and Profilescan data bases, the complete AF-4 protein sequence was searched for homology to other proteins and for the presence of motifs. The sequence AKKRK at positions 811-815 matched the consensus nuclear targeting sequence - (RKTA) KK (RQNTSG) K- (Gomez-Marquez and Segada, 1988) . AF-4 was relatively rich in serine (16%) and proline (11%) compared to the average frequency of these amino acids (7.1% and 4.6%, respectively) .
Inspection of AF-4 sequence at the fusion point to ALL-l RNA in the RS4 :11 cell line indicates that three nucleotides (1959-1961) of AF-4 RNA are missing from cDNA clone 25 corresponding to ALL-l/AF-4 fused RNA; these nucleotides might have been excluded through an error in the splicing process where an Ag at positions 1960-1961 was mistaken to the 3' end of an intron.
We have previously shown that in leukemic cells with t(4:ll) abnormalities the breakpoints cluster in a region of approximately 8 kb on chromosome 4. This region corresponds to a single intron flanked by an exon located within a 1 kb BamHI- EcoRI fragment, and an exon positioned >20 kb away towards the telomere. Example 2
Cloning of AF-9/ALL-l Genomic Junctions
The nonavailability of cell lines with the t(9;ll) abnormality made it impossible to obtain intact mRNA in amounts sufficient for preparation of a cDNA library and cloning from it fused ALL-l/AF-9 cDNA. To circumvent this problem, we first cloned (clone C19) to genomic junction fragment from the leukemic cells of patient CO with acute myeloid leukemia (AML) and t(9,-ll) . We also cloned (clone F2) the genomic junction fragment from tumor cells of patient FI with acute lymphocytic leukemia (ALL) and t(9,-ll) . The cloned genomic fragments were derived from the der 9 chromosomes of the patients. Mapping and hybridization analysis of the non-ALL-1 segments within the two phage clones indicated no homology between them.
A 1 kb HindHI fragment from non-ALL-1 region in clone F2 was used to clone the corresponding normal DNA. A 0.4 kb
HindHI fragment from clone 3 and 0.4 kb HindHI-AvalI probe from clone C19 hybridized to human DNA within Chinese hamster cell hybrids containing human chromosome 9. This established that in both patients' DNAs the ALL-l gene is linked to chromosome 9 sequences . Subsequent work showed that both sequences are included in a single gene which we term AF-9, for ALL-l fused gene from chromosome 9.
The same repeat-free fragments were used as probes for detecting rearrangements in DNAs from leukemic cells with t(9;ll) chromosome translocations. Samples from three patients with ALL and from five patients with AML were studied. The 0.4 kb HindHI fragment detected rearrangement in DNA of the ALL patient CU. The HindHI-Avail probe showed rearrangements in patients TA, SU and AG, all with AML. This indicated that at least two regions in the AF-9 gene are involved in recurrent t(9;ll) aberrations. Presently, it is not known whether one region is preferentially rearranged in AML and the second in ALL; it is also not clear whether the AF-9 gene is involved in all t(9;ll) abnormalities. Characterization of Normal and Chimeric cDNAs of AF-9
Repeat-free fragments from AF-9 DNA for hybridization to cDNA libraries were examined. The lkb HindHI fragment reacted with several overlapping cDNAs spanning 3.4 kb. These cDNAs reacted in northern analysis with a major 5 kb transcript expressed in several hematopoietic cell lines.
Nucleotide sequence analysis of AF-9 cDNA revealed an open reading frame beginning in a consensus initiation codon
(SEQ ID NO:29) and coding for a protein of 568 amino acids (SEQ
ID NO:30) . The protein encloses a nuclear targeting sequence
AKKQK at positions 297-301. AF-9 protein is serine rich (20%) and includes a remarkable uninterrupted stretch of 42 serines at positions 149-190; it also contains proline at a frequency of 7% which is above the average frequency of 4.1%.
A homology search showed, unexpectedly, that the predicted protein shared high similarity with the ENL protein SEQ ID NO:31. The latter is located on chromosome 19 and is fused to the ALL-1/HRX gene in t(ll;19) chromosome translocations. The two proteins show 56% identity and 68% similarly. The homology is highest within the 140 amino acids at the N terminus where the proteins are 82% identical, and 92% similar, and within the 67 amino acids at the C terminus where the corresponding values are 82% and 91%.
To demonstrate chimeric ALL-l/AF-9 RNAs, we designed primers supposed to flank the RNA junction points in the two genes and used them in RT-PCR reactions with RNA from patient FI . Two reciprocal cDNA products were amplified SEQ ID NO:32 and SEQ ID NO:34 (encoding protein products SEQ ID NO: 33 and SEQ ID NO:35 respectively) . Close examination of sequences at the RNA junctions showed a stretch of 11 nucleotides of AF-9 (ATTCTTGAAGT; SEQ ID NO:38) at both RNA junctions. In an attempt to understand this, we sequenced the genomic junction in clone F2 and determined exon-intron boundaries of AF-9 exons in this region. This analysis suggested that the two derivative chromosomes of patient FI were formed by staggered breaks in the DNAs of chromosomes 9 and 11 resulting in a small overlapping AF-9 genomic DNA segment and consequently in the overlapping of 11 nucleotides of AF-9 at the RNA junction points . The der 9 chromosome resulted from a break within exon 7 of ALL-l and a break within an exon of AF-9 (11 nucleotides 3' of the intron-exon boundary) . The hybrid exon spans the fusion point in cDNA clone EN (ALL-l exon 8 was skipped during splicing) . The der 11 chromosome was due to a break in the other ALL-l DNA strands within the intron flanked by exons 6 and 7, and to a breakage of the second AF-9 DNA strand within an intron located 5' of the AF-9 exon mentioned above. The der
11 is transcribed into an RNA corresponding to cDNA clone E2.
A BamHI-Stul cDNA probe detected some normal genomic fragments, which were also detected by the 0.4 kb Hindlll-Avall probe-derived from the genomic junction cloned from DNA of patient CO. This enabled designing primers predicted to flank the RNA fusion point of patient CO and use them in a RT-PCR reaction to amplify AF-9/ALL-1 RNA SEQ ID NO:36 (encoding protein SEQ ID NO:37) . In this patient the AF-9 protein is linked at position 375 to the ALL-l moiety, while in patient FI the junction point is at amino acids 444 or 477 of AF-9. In the three junctions examined the reading frames of the two genes are joined in phase.
Perhaps the most unusual feature of Hq23 abnormalities is the multitude of chromosome partners participating in translocations with the ALL-l locus. Using a probe containing sequences of ALL-l exons 5 and 11, which flank the breakpoint cluster region, we have been able to detect rearrangements in 10 types of Hq23 chromosome translocations. This promiscuity in partners for rearrangement and fusion could suggest that the only critical event in all these different translocations is the separation of a DNA binding domain (either the zinc fingers or the AT hooks in the ALL-l gene) from a positive or negative regulatory element, and that the proteins encoded by the partner genes solely provide initiation or termination codons . Our sequence analysis of AF-4 and AF-9 proteins and a comparison to the sequence of the ENL protein is not consistent with such interpretation. The finding that AF-9 and ENL share extensive sequence homology indicates that the two proteins have similar biological function and that presumably they contribute an identical activity to the chimeric proteins. Possibly, other genes participating in Hq23 aberrations have also sequence homology with AF-9 and ENL. Moreover, these two proteins share with AF-4 several common motifs: 1) a nuclear targeting sequence (NTS) (suggesting that the three proteins are nuclear) , 2) serine-rich domains, the most prominent being an uninterrupted stretch of 42 serines in AF-9, 3) stretches rich in proline or in basic amino acids reaching frequency of -30% in some regions. While serine-rich regions have not yet been implicated in function of transcription factors, domains with abundant prolines were shown to act as transcription activators, and domains rich in positively charged amino acids were found to bind DNA. These common structural motifs suggest that AF-4, AF-9, and ENL are involved in transcription regulation, possibly representing a new class of transcription factors. Proteins coded by the other genes involved in Hq23 chromosome translocations might belong to this class.
Inspection of the position of the elements discussed above in relation to the fusion point (s) with the ALL-l protein shows that the NTS of AF-4 is linked to the N-terminus of ALL-l containing the AT hooks, while AF-4 domains rich in serine, proline, or basic amino acids are fused to both reciprocal products of ALL-l cleavage. In patient FI with t(9;ll) , the NTS and most of AF-9 domains rich in specific amino acids are linked to the C-terminus of ALL-l which contains the zinc tingers. In leukemic cells with t(ll;19) all landmarks observed in the ENL protein will be linked to the N-termininus of ALL-l; this may suggest that N-ALL-l/ENL-C is the oncogenic product of the t(ll;19) abnormality. The opposite distribution of the common elements in AF-9 fusion products in patients such as FI raises the possibility that in these cases N-AF-9/ALL-1-C is the oncogenic species. Determination of which one (or both) of the fusion products of Hq23 translocations induce malignancy should be resolved by biological assays in cells in culture and in transgenic mice. Transcription assays utilizing elements of AF-4, AF-9 and ENL should help in understanding the normal function of these elements, as well as their role in the fused proteins.
DNA and Sequencing Analysis
Aliquots (20 micrograms) of high molecular weight DNAs were digested with excess of restriction enzymes and analyzed by the Southern technique using the Probe Tech™2 system (ONCOR) . Sequencing was done with an automatic sequencer
(ABI) . Genomic and cDNA libraries
High molecular weight DNAs from patients with t(9;ll) chromosome translocation were partially digested with Mbol enzyme and cloned into the EMBL-3 phage vector (Promega) . To reduce the frequency of rearrangements during propagation in bacteria, the libraries were plated into the host bacteria CES200 (Wyman et al . , 1986) . The libraries were screened with an ALL-l specific probe (Cimino et al . , 1992) and positive clones were mapped with restriction enzymes. To construct a cDNA library from RNA of the KC122 cell line, cytoplasmic RNA was extracted by standard techniques (Berger & Chirgwin, 1989) and polyadenylated RNA purified on an oligo dT column. cDNA was prepared using the Timesaver kit of Pharmacia and cloned into the lambda ZAPII vector (Stratagene) . Construction of cDNA libraries from K562 or fibroblasts RNA was described (Shtivelman et al . , 1985; Chu et al . , 1990) . AF-4 cDNA clones kl.l, kl.2, kll and kl2 originated from the K562 library and the clones kcl 6, kcl 10, and kcl 12 were cloned from the KC122 library. AF-9 cDNA clones v4 and v7 were obtained from the fibroblasts library, and k 16 was cloned from the K562 library. RT PCR
Two micrograms of RNA from a patient FI were reverse transcribed in a reaction utilizing the AF-9 oligonucleotide TCCTCAGGATGTTCCAGATGT (SEQ ID NO:39) or the ALL-l oligonucleotide GGCTCACAACAGACTTGGCAA (SEQ ID NO:40) as primers. The cDNAs were amplified with Taq 1 polymerase
(Boeringer) using the same primers together with the ALL-l primer ACCTACTACAGGACCGCCAAG (SEQ ID NO:41) , and the AF-9 primer CAGATGAAGTGGAGGATAACG (SEQ ID NO:42), respectively. The reaction products were purified by gel electrophoresis and cloned into the SK plasmid vector (Stratagene) . Recombinants with AF-9/ALL-1 or ALL-l/AF-9 DNA were identified by colony hybridization and were subsequently sequenced. The AF-9/ALL-1 RNA function of patient C() was obtained in a similar way using the ALL-l primer CAGCGAACACACTTGGTACAG (SEQ ID NO:43) for synthesis of cDNA and the same primer together with the AF-9 primer CAACGTTACCGCCATTTGAT (SEQ ID NO:44) for PCR amplification. Ex-ample 3 Cloning and Sequencing of AF-6 cDNA The patient 01 was a 47 year old female, diagnosed as
AML(M4) . Her karyotype was 46XX, t(6;ll) (q27,-q23) in 20/20 of bone marrow cells. Patient Ed was a male diagnosed as AML(M5) with a karyotype of 46 XY del(llq23) . The cell lines used for RNA analysis included K562 and KC122 (erythroid and myeloid acute phase of chronic myeloid leukemia) (Lozzio et al . , Blood 1975 45, 321-324; and Kubonishi et al . , Int . J. Cell Cloning 1 1983 1, 105-117) , B-l and MV4.11 - ALL with the t(4;ll) abnormality (Cohen et al . , Blood 1991 78, 93-102; and Lange et al., Blood 1987, 70, 192-198) , SKDHL (B-cell lymphoma) Saito et al., Proc . Natl . Acad. Sci . USA 1983 80, 7476-7480, T98G (glioblastoma) (Stein, J. Cell Physiol . 1979 99, 43-54) and the 293 cell line derived from kidney (Graham et al . , Virology 1978 86, 10-21) .
The rearranged genomic fragments of ALL-l patients 01 and Ed were cloned into the EMBL-3 phage vector (Promega) after partial digestion of the DNAs with the Mbol enzyme and size selection. Phage libraries were screened using a 0.86 kb Bam HI fragment derived from ALL-l cDNA and spanning exons 5-11. Normal genomic library was constructed in a similar way from normal white blood cell DNA. cDNA library was constructed utilizing a kit from Pharmacia. Cytoplasmic poly A-selected RNA was prepared from KC122 cells. For RT-PCR reactions, aliquots of 2 μg of patients' RNAs were reverse transcribed utilizing the AF-6 oligonucleotide 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO:79] . The cDNA was amplified using the same AF-6 primer together with the All-1 primer 5' ATC TGA ATT CTC CGC TGA CAT GCA CTT CAT AG 3' [SEQ ID NO: 80] .
Both primers contained cloning sites at their 5' termini. The amplified products were cloned into the SK plasmid vector and sequenced. cDNAs and genomic DNAs were excised from the phage vectors and recloned into the SK plasmid vector. Sequencing was performed using the ABI automatic sequencer. Sequence was analyzed using the FASTA, TFASTA and motifs programs.
A rearranged ALL-l segment was cloned from the genomic DNA of leukemic cells of patient 01. Mapping of this segment indicated that it originated from the der (6) chromosome (Fig.
12A) . Sequencing of the junction region (Fig. 12C) showed neither extra neucleotides nor haptamer-like signal at the junction point. Therefore, unlike two t(4;ll) and one (9;11) translocation points that we previously studied (Gu et al . , Proc . Na tl . Acad . Sci . USA 1992 89, 10464-10468) , here the VDJ recombinase was probably not involved in the recombination process . We used now a repeat free EcoRV-PstI 0.5 kb fragment
(RVP 0.5) as a probe to clone the corresponding region from normal DNA (Fig. 12A bottom) . To examine whether this region of chromosome 6 is altered in other patients with Hq23 abnormalities and rearranged ALL-l, we probed genomic blots of patients' DNAs with the 0.5 kb Xbal-EcoRI (XR0.5) radiolabelled fragment. While the DNA of another patient with AML and t(6;ll) showed only germ line configuration of this region, the
DNA of the patient Ed with AML and the del(llq23) aberration contained a rearranged BamHI fragment of 12 kb (Fig. 12B) . The
XRO-5 probe hybridized to human DNA within Chinese hamster cell hybrids containing human chromosome 6. This indicated that the cloned DNA spanned a breakpoint cluster region and that a cytogenetic pattern of del(llq23) could correspond to a t(6;ll) translocation.
The entire area of 30 kb cloned from 6q27 was searched for segments reacting with clones from a normal cDNA library. A 0.6 kb Hinfl DNA reacted with the K12 cDNA clone (Fig. 13A) . The overlapping cDNA clones which spanned the complete coding region of the gene were cloned. We named the latter AF-6 for
ALL-l fused gene from chromosome 6. AF-6 encodes a protein of 1612 amino acids. In cDNA clone K10 we find two additional amino acids - glutamic acid at position 101, and a lysine in position 139; both are probably due to alteration in splicing similar to those which we previously detected in ALL-l (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631- 4635; and Ma et al . , Proc . Natl . Acad . Sci . USA 1993 90, 6350- 6354) . To directly demonstrate a fused transcript we performed RT-PCR reactions on RNAs from patients 01 and Ed using ALL-l and AF-6 primers flanking the expected junction region.
Products of the reactions were cloned, screened for hybridization to ALL-l and AF-6 probes and sequenced. The RT- PCR products of both patients showed identical chimeric ALL- l/AF-6 RNAs transcribed from the der (11) chromosome (Fig. 13C) . The two open reading frames were linked in phase.
The nucleotide and the amino acid sequences of AF-6 were examined for motifs and homology to other genes. Beginning around amino acid 1290 up to the C-terminus of the protein there exist several small domains rich in prolines, serines, acidic amino acids, or glutamines . AF-6 protein, residue 745-925, shows 23.2% identity over 181 amino acids with the C-terminus of yeast myosin-1 isoform (Johnston et al . , J. Cell Biol . 1991 113, 539-551) . AF-6 protein also shows high similarity, though low identity, (66% similarity plus identity) over amino acids 1000-1594 to amino acids 1400-1980 of the myosin heavy chain from Dictyostelium discoideum (Warrick et al., Proc . Natl . Acad. Sci . USA 1986 83, 9433-9437) . In the latter protein this region is part of the tail domain which assumes, due to a high helical potential, a rod structure. A striking homology was detected in the polypeptide spanning amino acids 997-1080. A series of amino acids in this domain are conserved (Fig. 14) in three other proteins - in the human tight junction protein ZO-1 (Willott et al. , Proc . Natl . Acad. Sci . USA 1993 90, 7834-7838) , in the rat PSD-92 protein present in brain synapses Cho et al . , Neuron 1992 9, 929-942) , and in a tumor suppressor gene of Drosophila (dig) located at septate junctions, which are thought to be the invertebrate equivalent of tight junctions (Woods et al . , Cell 1991 66, 451-464) . In this domain, termed the GLGF repeat (Cho et al . , Neuron 1992 9, 929-942) , AF-6 shows identity of 28%, 36% and 42%, and similarity of 57%, 59%, and 67% to the human, rat and Drosophila proteins, respectively.
To examine the expression of AF-6 in different cell types, we performed a Northern analysis on RNAs extracted from several cell lines (Fig. 15) . An 8 kb transcript was detected in cell lines of myeloid (a) , erythroid (b) , lymphoid (c-e) , glia (f) and epithelial (g) origin. Thus, it appears that AF-6 is expressed in a variety of hematopoietic and nonhematopoietic cells.
The t(6,-ll) (q27;q23) translocation is one of the most frequent translocations involving Hq23. Cloning of the AF-6 gene involved in this abnormality would enable now the use of
Southern blotting and the RT-PCR technique to identify relevant patients whose karyotype was different, complex, or not clear. In addition it is possible now to examine residual disease in patients in remission. The analysis reported here of the patient Ed illustrates the first point. This patient showed a typical del(llq23) abnormality. Using the molecular approaches we found here that he had the ALL-l/AF-6 fusion product. Presumably, del(llq23) and t(6;ll) are difficult to distinguish cytogenetically. Using chromosome 6-specific probes and FISH analysis, others have recently concluded that some patients with del(llq23) in fact carry the t(6;ll) chromosome translocation (Shannon et al . , Genes, Chromosomes & Cancer 1993 7, 204-208) .
One of the main reasons for cloning AF-6 was to see if it is related to the partner genes AF-4, AF-9, and ENL. Among these, AF-9 and ENL are highly related. However, AF-6 showed no sequence homology to any of the three partner genes. Short domains rich in prolines, serines and acidic amino acids were the only motifs shared by the four genes. The C-terminus AF-6 showed homology to the tail domain of myosin-1 isoform from yeast and myosin heavy chain from Dictvostelium discoideum; this domain presumably confers the rod structure to the myosin protein. Within this region AF-6 displays a remarkable homology to the GLGF repeat found in the ZO-1, PSD- 95 and dig proteins from human, rat, and Drosophila respectively. The first and the third proteins are presumably homologous and are thought to play a role in signal transduction on the cytoplasmic surface of intracellular junctions (Willott et al . , Proc . Natl . Acad. Sci . USA 1993 90, 7834-7838; Woods et al . , Cell 1991 66, 451-464) . The second protein localizes to synaptic junctions and is thought to be involved in synaptic signalling or organization (Willott et al., Proc . Natl . Acad . Sci . USA 1993 90, 7834-7838) . The three proteins are cytoplasmic or associated with membranes. The presence of this domain in AF-6 raises the possibility that AF- 6 is not a nuclear protein. Indeed, unlike AF-4, AF-9 and ENL, AF-6 does not contain a nuclear targeting sequence. Example 4
Cloning and Sequencing of AF-17 cDNA
AML patients GUS and GE showed the chromosome translocation t(ll;17) (q23;q21) in their leukemic cells. The cell lines used for RNA analysis included K562 and KC1-22 (erythroid and myeloid acute phase of chronic myeloid leukemia) , MV4:H and B-l (ALLs with the 4:11 translocation) , 380, ALL-l, 697, GM607, (ALLs) , GM1500 (EBV transformed lymphoblastoid cell line) , T98G (glioblastoma) , PC3 (prostate carcinoma) , (Prasad et al . , Cancer 1993 53, 5624-5628; Licht et al., Na ture 1990, 346, 76-79)
The junction fragment of patient GUS was cloned from a library prepared from a partial digest of genomic DNA clones into the EMBL-3 phage vector. The library was screened with a 0.86 kb BamHI cDNA probe spanning ALL-l exons 5-11. cDNA libraries were prepared from ALL-l and KCl-22 cytoplasmic RNAs utilizing a kit manufactured by Pharmacia, and the lambda ZAPII vector of Stratagene. RT-PCR reaction was performed as described (Nakamura et al . , Proc . Na tl . Acad. Sci . USA 1993 90, 4631-4635) utilizing as primers an ALL-l oligonucleotide with BamHI site attached at the 5' end CGGGATCCCGACCTACTACAGGACCGCCAAG [SEQ ID NO:81] and AF-17 oligonucleotide with EcoRI site at the 5' end ATCTGAATTCTGGTGGAGATAGAAGCAGAA [SEQ ID NO: 82] . Sequencing was performed in the ABI automatic sequencer with cDNAs and genomic fragments excised from phase vectors and cloned into the SK plasmid vector. The sequence was analyzed using the FASTA, TFASTA and motifs program.
DNA from patient GUS with AML and t(ll;17) was partially digested with Mbol enzyme, and following size selection was cloned into the EMBL-3 phage vector. The library was screened with a cDNA probe spanning the breakpoint cluster region. A clone composed of a rearranged ALL-l segment was identified among positive clones. Comparison between the physical maps of this clone and the corresponding normal ALL-l DNA (Fig. 16A) indicated that ALL-l sequences upstream of exon 6 were substituted with new DNA; the latter was subsequently found to be derived from chromosome 17. Within the non-ALL-1 segment of the junction clone, a 1.7 kb EcoRI fragment (R1.7) was found to be devoid of repetitive sequences. This fragment was used as a probe to analyze by the Southern technique DNA from a second patient (GE) with AML and the t(ll;17) aberration. In that DNA we detected an 11.6 kb rearranged EcoRV fragment (Fig. 16B, lane b) . This indicated that in both patients the breaks occurred in the same region on chromosome 17. Fragment Rl .7 was next used as a probe on cDNA libraries derived from RNAs of the cell lines KC1-22 and ALL-l. Inserts from positive clones were subcloned into the SK plasmid vector and mapped. Clones 1, 3, 13, and a4 (Fig. 17A) were subjected to sequencing analysis. AF-17 cDNA contains an open reading frame spanning 3279 nucleotides. The first ATG shows a good fit to a Kozak consensus sequence and is preceded by an in-frame termination codon. The predicted protein spans 1093 amino acids. It contains relatively high concentrations of serines, glycines, alanines, leucines and prolines (15%, 11%, 10%, 10%, 10%, respectively) often concentrated in short stretches. In addition, it has a glutamine-rich region (41%) between amino acids 935 and 984 (Fig. 17B) . The same region shows high concentration of hydrophobic amino acids, in particular leucines. It should be noted that domains rich in alanines (Licht et al . , Nature 1990, 346, 76-79] , glycines (Shi et al., Cell 1991 67, 377-388) , glutamines and prolines (Madden et al . , Science 1991 253, 1550-1553) were implicated in transcriptional repression. Also, regions with high concentration of serines and prolines (Gill et al . , Proc . Natl . Acad . Sci . USA 1993 91, 192-196) or glutamines intercalated with hydrophobic amino acids (Theill et al . , Nature 1989 342, 945-948) were found to be involved in transcriptional activation.
Homology search in GenBank indicated 90% identity over amino acids 45-139 between AF-17 and an anonymous human cDNA sequence (Accession No. T06113) . Furthermore, over 118 residues (Fig. 18A) AF-17 showed 48% identity and 67% similarity to a region within the protein Brl40, previously named peregrin (Accession No. M91585) . This domain is cysteine-rich in both proteins and can be arranged into three zinc fingers according to the consensus C - X2 - C - X10_13 C- X2_ 4 - C (Fig. 18B) . Related consensus sequences are present in the adenovirus E1A protein and in the steroid receptor superfamily. The human Brl40 protein has a second cysteine- rich domain and is located in the nucleus; the function of this protein is unknown. Inspection of AF-17 predicted protein sequence revealed a leucine zipper dimerization motif between amino acids 729 and 764 (Fig. 17B) . Unlike many leucine zippers, the one in AF-17 is not preceded by a basic region.
To prove that ALL-l/AF-17 fused gene is transcribed into a chimeric RNA, we used cDNA and genomic DNA sequence information to design primers for amplification by RT-PCR of a putative ALL-l/AF-17 RNA junction from the leukemic cells of patient GUS. An amplification product was indeed found to contain the RNA junction (Fig. 17C) . Within the fused RNA the open reading frames of the two genes were found to be linked in phase. Thus, the t(ll;17) abnormality results in production of an RNA encoding a chimeric ALL-l/AF-17 protein.
To examine the expression of the normal AF-17 gene we performed a Northern blot analysis. A major transcript of 7.5 kb and a minor diffuse species of 5 kb were detected in a variety of hematopoietic and non-hematopoietic cell lines (Fig.
19) .
The cloning and sequence analysis of the partner genes which recombine with ALL-l in Hq23 translocations provides information and reagents which can be used in the diagnosis, prognosis and monitoring of human acute leukemias. In addition, this cloning enables construction of biologically active molecules, and might provide insights into the mechanism of leukemogenesis . The most notable feature of AF-17 protein is the leucine zipper protein dimerization motif. Following the t(ll;17) chromosome translocation, this motif will be included in the ALL-l/AF-17 chimeric protein which is presumed to be the critical product of the aberration. Since the leucine zipper of AF-17 is not preceded by a basic region required for interaction with DNA, and because leucine zippers are found not only in transcription factors but also in other proteins with diverse functions, it is concurrently not clear whether AF-17 is a transcription factor. The presence at the N-terminus of AF-17 of a cysteine-rich domain, with high homology to the nuclear protein Brl40 suggests that AF-17 is also located within the nucleus.
AF-17 is the fifth partner gene involved in Hq23 abnormalities to be cloned and characterized. Schematic representation of the proteins encoded by these genes and by ALL-l is shown in Figure 20. Inspection of the sequences within the segments of the partner proteins (right side of the arrows) linked to ALL-l sequences (left side of the fusion point within the top scheme) in the chimeric proteins thought to be critical for leukemogenesis, does not reveal a common motif. AF-9 and ENL are the only partner genes which share sequence homology (Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635) . The highly homologous C-terminal polypeptides contributed by both genes to the chimeric proteins, do not contain obviously recognized motifs and are not particularly rich in serines or prolines (as do other regions of these two proteins) . AF-9 and ENL proteins contain nuclear targeting sequences and are probably nuclear proteins . The AF-6 polypeptide linked to the N-terminus of ALL-l contains the GLGF motif (Prasad et al . , Cancer 1993 53, 5624-5628) whose function is not known, as well as short regions very rich in acidic amino acids, basic amino acids or prolines. The GLGF motif is found in cytoplasmic or membrane-associated proteins and this suggests that AF-6 is not located in the nucleus. The
AF-4 polypeptide within the ALL-l/AF-4 protein includes several segments with high concentration of serines or prolines
(Nakamura et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-
4635) . The AF-4 protein includes a nuclear targeting sequence and therefore is probably associated with the nucleus. Finally, each of the normal five partner genes is expressed in all cell lines analyzed, both of hematopoietic and non hematopoietic lineages.
The high homology between AF-9 and ENL has previously prompted us to speculate (Nakamura et al . , Proc . Na tl . Acad. Sci . USA 1993 90, 4631-4635) that the partner polypeptides are related and possibly contribute a similar function to the chimeric protein. One such possible function would be a transcriptional activation or repression. Domains with these activities were characterized in a number of transcription factors and were found to be rich in particular amino acids such as serines, prolines, glutamines, acidic amino acids, alanines, or glycines (Mitchell et al . , Science 1989 245, 371- 378; Licht et al . , Na ture 1990, 346, 76-79; Shi et al . , Cell 1991 67, 377-388; Madden et al . , Science 1991 253, 1550-1553) While the AF-4, AF-6, and AF-17 polypeptides linked to the Ν- terminus of ALL-l, each contain stretches of one or more of those amino acids, the analogous polypeptide of AF-9 as well as its homologous C-terminal region in EΝL are devoid of these amino acids. In addition, the AF-6 protein is probably located in the cytoplasm or the membrane of the cell, and therefore does not play a role in transcriptional regulation. Considering the above we find it less likely that the partner polypeptides of AF-6, AF-9 and EΝL contribute domains involved in direct activation or repression of transcription.
The multiplicity and variance between the partner polypeptides which is unprecedented in leukemias associated with chromosome translocations suggests that the partner polypeptides play only a secondary role in Hq23 pathogenesis. This idea is consistent with the recent identification of several patients with AML in which ALL-l is rearranged by tandem duplication of exons 2-6 with no involvement of partner genes. It is believed that the critical outcome of Hq23 abnormalities is the loss of function of ALL-l, and that the normal protein is directly involved in the differentiation of lymphoid and myeloid cells. Further, it is suggested that the chimeric protein would act in a dominant negative fashion to inactivate the normal ALL-l protein encoded by the intact ALL-l allele present in the leukemic cells. Inactivation could occur by nonproductive binding to the promoter of the normal target (s) for ALL-l or by dimerization of the chimeric protein to the normal protein and sequestering the latter either to a complex with other proteins or into another cellular compartment. In this scenario the partner polypeptides could best play a role in the elimination of the normal protein activity through dimerization. They could make the dimer nonfunctional by virtue of their presence within, or by sequestering it through interaction with other cellular proteins. The leucine zipper dimerization motif in AF-17 and the GLGF motif in AF-6 could represent protein-protein interaction domains of partner polypeptides.
Postulating that the partner polypeptides play an accessory role in abolishing the activity of the ALL-l protein relaxes the requirements demanded from such proteins and allows a larger variety of them to be involved in Hq23 aberrations. Although chromosome translocations are usually associated with overexpression or activation of oncogenes, there is a recent example for a translocation which apparently involve loss of function and a dominant negative effect. Thus, in the t(15;17) chromosome translocation associated with acute promyelocytic leukemia, the effect of the fusion protein PML/RAR is sequestering of the normal PML protein and inhibiting its organization into nuclear macromolecular organelles (Dyck et al., Cell 1994 76, 333-343 and Weiss et al . , Cell 1994 76, 345- 356) . Example 5
Sequence Analysis of the ALL-l Breakpoint Cluster Region in the ALL-l Gene Frozen bone marrow samples of patients diagnosed with acute leukemia were obtained from the Hospital of University of Pennsylvania, St. Jude Children's Research Hospital, and Roswell Park Cancer Institute. The cytogenetic analyses were performed at the time of diagnosis. Genomic DNA was extracted from either bone marrow of leukemia patients or the cell lines. Aliquots (10 μg) of high molecular weight DNA were digested with BamHI, separated by electrophoreses on 0.7% agarose gels, and blotted onto nylon membrane. The probe was radiolabeled by using the Boehringer Mannheim random-primer kit .
An 859 bp BamHI fragment which spans exons 5-11 of the ALL-l gene was isolated from the V26 cDNA clone (Fig. 21 and Gu et al . , Cell 1992 71, 701-708) and subcloned into the pBluescript SK vector. This probe was named B859. The genomic region corresponding to B859, an 8.3 kb BamHI fragment, was included in the phage clone, mg 11.1 (Gu et al . , Cell 1992 71, 701-708) . For constructing a genomic library, patient or normal DNA was either partially digested with Sau3A or digested to completion with BamHI, and subsequently ligated with a phage vector, XEMBL3 (Stratagene) using standard techniques.
Sequencing reactions were performed by using an automatic sequencer (ABI) . Sequences were reassembled and analyzed in the Genetic Computer Group system. Alu sequences were analyzed by the Pythia service.
In previous studies, we have defined a breakpoint cluster region in the ALL-l locus/gene disrupted in acute leukemia with Hq23 aberrations (Gu et al . , Cell 1992 71, 701- 708; Cimino et al . , Cancer Res . 1992 52, 3811-3813 and Gu et al., Proc . Natl . Acad. Sci . USA 1992 89, 10464-10468) . We have also noticed that exons within this region all started in the same phase within the open reading frame. We have now developed a new probe, a 859 bp cDNA that spans exons 5-11. The probe is supposed to detect two rearranged fragments in all reciprocal translocations. Fig. 21 shows DNA rearrangements detected by B859 probe in some of the various Hq23 aberrations studied in this report. A phage clone, mgll.l, which spans the breakpoint cluster region in the ALL-l gene (Gu et al . , Cell 1992 71, 701- 708) , was subcloned into plasmids for sequencing. The complete sequence of the 8342 bp BamHI fragment is presented in Fig. 22. The exons included in this region are shown. The AF4 probe (Cimino et al . , Cancer Res . 1992 52, 3811-3813 and Gu et al . , Proc . Na tl . Acad. Sci . USA 1992 89, 10464-10468) , a modified Ddel fragment, spans nucleotides 3071 to 3261 and 3502 to 3754 ( Fig . 22 ) .
To search for the repetitive sequences in the breakpoint cluster region, the 8342 bp sequence was first screened for Alu repeats. Eight Alu repeats were identified and their positions are indicated in Table 1. The orientation of these Alu repeats is the same as that of the ALL-l gene. Classification of these Alu repeats was based on recently published diagnostic criteria (Milosavljevic et al . , J". Λol . Evol . 1991 32, 105-121) . After the ALL-l exons and Alu repeats were precisely identified, the rest of sequence was searched for other homologous sequence (s) in GenBank. A 130 bp fragment, encompassing nucleotides 7429 to 7559 in intron 9, shows around 80 percent sequence identity to genomic sequences in several genes such as TRE17, ApoA4, Factor VIII c subunit, Factor IX, a nuclear gene for mitochondrial ATP synthase c subunit, and G6PD gene (GenBank accessions: X63596, M14642, M88636, K02402, X69907, and Z29527, respectively) . These similar sequences were located in 5' regulatory regions, or in 3' segments, or in introns, suggesting that they may represent a group of repetitive elements with low frequency in the genome.
Ten out of twenty patient DNAs studied were analyzed by sequencing at the breakpoint junctions. The relevant sequences of the corresponding normal regions from chromosomes 1, 4, 6, 9, and intron 1 of the ALL-l gene were also analyzed. Table 2 lists the results of cytogenetic and molecular studies from twenty patients, and the positions of the breakpoints from ten patients. Five of these breakpoints were located in three different Alu repeats, but none of the breaks on the partner chromosome is in the Alu sequence. Two breaks were located in exon 7 of the ALL-l gene, and the last three were located in intron sequences (Fig. 23) . All together, several of the breaks occurred in the Alu-rich region delineated by exons 6 and 7 (Fig. 23) . Using the B859 probe it was previously possible to detect rearrangements in DNAs of patients with therapy-related acute myeloid leukemia, or secondary leukemia (all with Hq23 aberrations) (Felix et al . , Cancer Res . 1993 53, 2954-2956; Hunger et al . , Blood 1993, 81, 3197-3203; Negrini et al . , Cancer Res . 1993 53, 4489-4492) . These secondary leukemias were linked to the treatment of the patients with inhibitors of topoisomerase II. One topoisomerase II recognition site which fits with the consensus 5' A/GNT/CNNCNNGT/CNGG/TTNT/CNT/C3 ' (Spitzner, et al . , Nucleic Acids Res . 1988 16, 5533-5556) was found in exon 9 (Fig. 22) . When one or two mismatches were allowed in the consuses, a total of 11 and 129 sites, respectively, were found within the two strands of the breakpoint cluster region. In patients 7 and 12 the breaks were located within the imperfect recognition sites on the minus strand after allowing two mismatches . When three mismatches were allowed, a total of 703 sites were found at the breakpoint in one additional patient, case 1, was located within such consensus sequence on the minus strand.
The DNA rearrangements in the ALL-l gene involved in acute leukemia can be detected by a single probe, B859. Digestion with BamHI is normally sufficient for the analysis. However, if only one or no rearranged fragments are detected, the sample DNA should be digested by other restriction enzymes such as HindHI, and probed with B859.
In order to search for features within the breakpoint cluster region of the ALL-l gene which might predispose it to translocations, we have sequenced and analyzed the 8342 bp genomic BamHI fragment spanned by the B859 cDNA probe. The positions of the ALL-l exons, Alu repeats and the breakpoints have been established as shown in Fig. 23. Breaks/mutations mediated by Alu sequences, particularly homologous recombination events, have been observed in a number of human diseases (Li et al, Am. J. Hum. Genet . 1993 53, 140-149) . Five breakpoints were located within Alu sequences. If the Alu sequence mediate homologous recombination in these translocations, the germline sequence of the partner chromosome at the breakpoint should have been Alu. However, this is not the case in any of the five translocations. Nevertheless, the high concentration of the Alu sequences within the region, in particular, within the area spanned by exons 6 and 7, suggested a possible role for the Alu in the translocations. This indirect role might be destabilization of the region so as to make it more prone to breaks. The previous detection of the ALL-l rearrangements in therapy-related leukemia patients indicated that the consequences of the translocations in both de novo and secondary leukemia, inhibition of topoisomerase II apparently trigger the disease. We searched for topoisomerase II recognition sites in the region. Such sites were found in three out of ten cases when three mismatches were allowed in the consensus sequence. Thus, in the majority of the de novo All-1 rearrangements topoisomerase II recognition sites are not present at the breakpoints, and the enzyme is probably not involved. It will be necessary to sequence the breakpoint in secondary leukemias to determine whether in these cases topoisomerase II recognition sites are consistently associated with the breakpoints.
TABLE 1
POSITIONS OF ALL-l EXONS AND ALU REPEATS WITHIN THE BREAKPOINT CLUSTER REGION AND CLASSIFICATION OF ALU REPEATS
ALL-l/Exon Position Alu Class* Strandy
5 <l-263
6 593-666
799-1108 a J +
1119-1420 b Sx +
1432-1716 c SbO +
1921-2216 d J +
7 2353-2484
8 3032-3145
3973-4268 e SbO +
4764-5094 f J +
6072-6362 g S +
9 6788-6934
7164-7427 h Sx + TABLE 1
POSITIONS OF ALL-l EXONS AND ALU REPEATS WITHIN THE BREAKPOINT CLUSTER REGION AND CLASSIFICATION OF ALU REPEATS
ALL-l/Exon Position Alu Class* Strandy
10 7967-8062
11 8304->8342
x: Based on the diagnostic criteria in Negrine et al . , Cancer Res . 1993 53, 4489-4492. y: "+" Strand corresponds to the coding strand of ALL-l.
TABLE 2
CLINICAL AND MOLECULAR DIAGNOSTIC DATA OF PATIENTS WITH ACUTE LEUKEMIA
Case Age/Sex Karotype B859' Breakpoint*7 Re
1 46, -- t (1;11) (p32- R 3562/3563 34;q23)
2 0.6/F 46,XX,inv(l) (p34; R ND q21) , t (1;11) (p34;q23)
3 10/M 46,XY,t (4;11) (q21; R 1161/1162 i q23)
4 32/F 46,XY,t(4;ll) (q21; R 2530/2531 i q23)
5 14/M 45,XY,der (1) t (1;8) R ND (p36;ql3) , -4, +6, -9,der (10) t (1;10) (qll;pl5) ,der(11) t (4; 11) (q21,q23)
6 47/F 46,XX,t (6;11) (q27; R 720/721 i q23)
7 5/M 46,XY,del (11) (q23) R 1564/1565
8 0.8/F 46,XX,del(H) (q23) R 2415/2416
9 0.5/M 46,XY, t (9;11) (p21; R ND q23) /47,XY,+6,t (9;11) (p21;q23)
10 2/M 46,XY,t (9;11) (p21; R ND q23)
11 5/F 47,XX,X, t (9;11) (p21; R 2437/2438 ii q23) TABLE 2
CLINICAL AND MOLECULAR DIAGNOSTIC DATA OF PATIENTS WITH ACUTE LEUKEMIA
Figure imgf000059_0001
a: R is denoted for DNA rearrangements detected by B859 probe; b: The numbers correspond to nucleotide sequence in Fig. 22. ND=not determined. i: Gu et al., Proc . Natl . Acad. Sci . USA 1992 89, 10464- 10468 ii: Prasad et al . , Cancer Res . 1993 53, 5581-5585 iii: Nakamura, et al . , Proc . Natl . Acad . Sci . USA 1993 90, 4631-4635
Example 6 Partial Duplication of ALL-l in Acute Leukemia
Genomic DNA was extracted from bone marrow aspirates by a standard procedure (Gustincich et al . , BioTechniques 1991, 11, 298-301) . Approximately 8 μg of genomic DNA was digested to completion with BamHI or HindHI. Restriction enzyme digests were separated by electrophoresis on 0.7% agarose gels and blotted onto positively charged nylon membranes. Southern blotting, probe radiolabeling, and hybridization were performed by standard techniques. A single blot was prepared. After probing with SASl, the blot was stripped, then probed again with B859. Clones corresponding to the rearranged ALL-l BamHI fragments were isolated from bacteriophage XEMBL3 libraries made from size-fractionated BamHI digests of patient DNA. Recombinants were identified in phage libraries by filter hybridization using the B859 probe. Construction of libraries, screening, phage purification, and restriction enzyme mapping were done by standard techniques . Subclones were constructed in the pBluescript II plasmid vector. DNA sequence of selected portions of subclones was determined by cycle sequencing using an Applied Biosystems 373A DNA sequencer. Programs from Genetics Computer Group (GCG) system (Devereux et al . , Nucl . Acids Res . 1984, 12, 387-395) were used for data analysis.
Total cellular RNA was isolated using RNAzol™ (Biotecx Laboratories) . Reverse transcriptase (RT) reaction and RNA-PCR amplification were performed with rTth DNA polymerase. Nested PCR amplification was performed with Taq DNA polymerase. Oligonucleotide primers were used without further purification. Primers are 3.1c (AGGAGAGAGTTTACCTGCTC) [SEQ ID NO: 83] from exon 3, 5.3 (GGAAGTCAAGCAAGCAGGTC) [SEQ ID NO:84] from exon 5, 6.1 (GTCCAGAGCAGAGCAAACAG) [SEQ ID NO:85] from exon 6, and 3.2c (ACACAGATGGATCTGAGAGG) [SEQ ID NO:86] from exon 3. Primers used in reactions are as follows: 1) RT reaction - 3.1c, 2) RNA-PCR amplication - 5.3/3.1C, 3) nested PCR amplification - 6.1/3.2c. RT reaction was performed for 15 minutes at 57°C using 500 ng RNA. RNA-PCR amplification was performed for 35 cycles (95 C, 1 minutes; 53°C, 1 minutes; 72°C, 1 minute) . Nested PCR amplification was performed using 0.5 μl of the RNA- PCR product for 30 cycles (95°C, 1 minute; 60°C 1 minute; 72°C, 1 minute) . PCR products were analyzed by 2% agarose gel electrophoresis . Figure 24 shows Southern blot rearrangements in the
ALL-l gene for three adult patients with acute myeloid leukemia
(AML) lacking cytogenetic evidence of Hq23 translocations. The rearrangements were detected with a cDNA probe (B859) (Gu et al . , Cell 1992 71, 701-708 and Caligiuri et al . , Cancer Res . 1994 54, 370-373) which spans the ALL-l breakpoint cluster region. Two of these patients (noε. 23 and 24) had trisomy 11 as a sole cytogenetic abnormality whereas one patient (no. 1) had a normal karyotype (Caligiuri et al . , Cancer Res . 1994 54, 370-373) . A single rearranged ALL-l band is seen for each patient in both BamHI and HindHI restriction enzyme digests. Clones corresponding to the rearranged BamHI fragments from the two trisomy 11 patients were isolated and characterized. Each clone begins and ends with a portion of ALL-l exon 5 delineated by the BamHI cloning site within this exon (Fig. 25A) . The 5'- 3' order of ALL-l exons within each clone is 5-6-2-3-4-5. This novel exon structure indicates that the ALL-l rearrangement in each patient is the result of a direct tandem duplication of a portion of the ALL-l gene (Fig. 25B) . The junction point of this duplication fuses the 5' portion of intron 6 to the 3' portion of intron 1. The precise junction points for the two clones are different. DNA sequence across the junctions (Fig. 25C) shows a 1 bp N-segment in one clone (λ24) and heptamer- like signal sequences (Akira et al . , Science 1987 238, 1134- 1138) near the junction points in both clones.
We next examined the genomic DNA of the three AML patients with a probe from intron 1 (SASl) designed to detect specifically the rearrangement associates with the ALL-l direct tandem duplication. The location of this probe is indicated in Fig. 25A. For all three patients, the SASl probe shows rearranged bands on Southern blot (Fig. 24B) that comigrate with the rearranged bands detected by the ALL-l breakpoint cluster region probe (Fig. 24A) . This result indicates that the ALL-l partial duplication occurs in an AML patient (no. 1) with a normal karyotype, as well as in the two AML patients
(nos. 23 and 24) with trisomy 11. Additional reported cases
(Caligiuri et al . , Cancer Res . 1994 54, 370-373) of ALL-l rearrangements without Hq23 translocations lacked adequate material for study. To determine whether the partially duplicated ALL-l gene is transcribed, RNA-PCR was performed using oligonucleotide primers specific for the ALL-l duplication. Discrete bands of the predicted size were detected for the two patients with trisomy 11 (Fig. 26A) . Sequence analysis of nested PCR products (Fig. 26B) shows an in-frame fusion of exon 6 with exon 2. These results demonstrate that the partially duplicated ALL-l gene is transcribed into mRΝA capable of encoding a partially duplicated protein. The partial ALL-l duplication creates a novel type of fusion protein in which a truncated polypeptide chain encoded by ALL-l exons 1-6 is fused near the amino-terminus of the native ALL-l protein. The partially duplicated protein may be involved in cellular transformation, as postulated for other ALL-l fusions (Cimino et al. , Cancer Res . 1991 51, 6712-6714; Gu et al., Cell 1992 71, 701-708; Tkachuk et al . , Cell 1992 71, 691-700; Morrissey et al . , Blood 1993 81, 1124-1131; Nakamura et al., Proc. Natl . Acad. Sci . USA 1993 90, 4631-4635; Prasad et al., Cancer Res . 1993 53, 5624-5628) . The structure of the partial duplication suggests that dissociation of ALL-l amino- terminal domains from their normal protein environments is the critical structural alteration leading to ALL-l associated leukemogenesis. Because the ALL-l gene is fused with itself, it follows that partner genes from other chromosomes are not necessary for involvement of ALL-l in leukemia.
We have reported previously (Caligiuri et al . , Cancer Res . 1994 54, 370-373) a high incidence (3 of 4 cases) of ALL-l rearrangement associated with trisomy 11 as a sole chromosomal abnormality in AML. The ALL-l partial duplications characterized in this report were cloned from two of these trisomy 11 cases. Trisomy 11 is a rare recurrent finding in AML, estimated to occur at a frequency of about 0.7% (CALGB AML cytogenetic data base) . Trisomy of other chromosomes is reported frequently in hematologic malignancy, sometimes in association with disease progression (Heim et al . , Cancer Cytogenetics 1987 (Liss, New York) ) . Examples include trisomy 8 in AML and transformed chronic granulocytic leukemia (Mitelman et al . , "Report of the Committee on Chromosome Changes in Neoplasia", Chromosome Coordina ting Meeting 1992 pp. 700-726; Cuticchia et al . (eds.) , Genome Priori ty Reports , vol. 1, 1993, Basel, Karger) , trisomy 21 in AML, and trisomy 12 in chronic lymphocytic leukemia (Mitelman et al . , "Report of the Committee on Chromosome Changes in Neoplasia", Chromosome Coordinating Meeting 1992 pp. 700-726; Cuticchia et al . (eds.) , Genome Priori ty Reports, vol. 1, 1993 Basel, Karger) . It has been postulated that trisomy, which occurs in somatic cells by nondisjunction, contributes to the neoplastic phenotype through a gene dosage effect (Mitelman, "Tumor Etiology and Chromosome Pattern: Evidence from Human and Experimental Neoplasms" in Arrighi et al . (eds.) , Genes, Chromosomes and Neoplasia 1981 335-350, Raven Press, New York) . Our findings suggest that, in many cases, the presence of trisomy in malignancy may indicate the partial duplication of a cellular protooncogene.
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCG GCG GCG GCG GCG GGA AGC AGC GGG GCT GGG GTT CCA GGG GGA 45 Ala Ala Ala Ala Ala Gly Ser Ser Gly Ala Gly Val Pro Gly Gly 5 10 15
GCG GCC GCC GCC TCA GCA GCC TCC TCG TCG TCC GCC TCG TCT TCG 90 Ala Ala Ala Ala Ser Ala Ala Ser Ser Ser Ser Ala Ser Ser Ser 20 25 30
TCT TCG TCA TCG TCC TCA GCC TCT TCA GGG CCG GCC CTG CTC CGG 135 Ser Ser Ser Ser Ser Ser Ala Ser Ser Gly Pro Ala Leu Leu Arg 35 40 45
GTG GGC CCG GGC TTC GAC GCG GCG CTG CAG GTC TCG GCC GCC ATC 180 Val Gly Pro Gly Phe Asp Ala Ala Leu Gin Val Ser Ala Ala lie 50 55 60
GGC ACC AAC CTG CGC CGG TTC CGG GCC GTG TTT GGG GAG AGC GGC 225 Gly Thr Asn Leu Arg Arg Phe Arg Ala Val Phe Gly Glu Ser Gly 65 70 75
GGG GGA GGC GGC AGC GGA GAG GAT GAG CAA TTC TTA GGT TTT GGC 270 Gly Gly Gly Gly Ser Gly Glu Asp Glu Gin Phe Leu Gly Phe Gly 80 85 90
TCA GAT GAA GAA GTC AGA GTG CGA AGT CCC ACA AGG TCT CCT TCA 315 Ser Asp Glu Glu Val Arg Val Arg Ser Pro Thr Arg Ser Pro Ser 95 100 105
GTT AAA ACT AGT CCT CGA AAA CCT CGT GGG AGA CCT AGA AGT GGC 360 Val Lys Thr Ser Pro Arg Lys Pro Arg Gly Arg Pro Arg Ser Gly 110 115 120
TCT GAC CGA AAT TCA GCT ATC CTC TCA GAT CCA TCT GTG TTT TCC 405 Ser Asp Arg Asn Ser Ala lie Leu Ser Asp Pro Ser Val Phe Ser 125 130 135
CCT CTA AAT AAA TCA GAG ACC AAA TCT GGA GAT AAG ATC AAG AAG 450 Pro Leu Asn Lys Ser Glu Thr Lys Ser Gly Asp Lys lie Lys Lys 140 145 150
AAA GAT TCT AAA AGT ATA GAA AAG AAG AGA GGA AGA CCT CCC ACC 495 Lys Asp Ser Lys Ser lie Glu Lys Lys Arg Gly Arg Pro Pro Thr 155 160 165
TTC CCT GGA GTA AAA ATC AAA ATA ACA CAT GGA AAG GAC ATT TCA 540 Phe Pro Gly Val Lys lie Lys lie Thr His Gly Lys Asp lie Ser 170 175 180
GAG TTA CCA AAG GGA AAC AAA GAA GAT AGC CTG AAA AAA ATT AAA 585 Glu Leu Pro Lys Gly Asn Lys Glu Asp Ser Leu Lys Lys lie Lys 185 190 195
AGG ACA CCT TCT GCT ACG TTT CAG CAA GCC ACA AAG ATT AAA AAA 630 Arg Thr Pro Ser Ala Thr Phe Gin Gin Ala Thr Lys lie Lys Lys 200 205 210
TTA AGA GCA GGT AAA CTC TCT CCT CTC AAG TCT AAG TTT AAG ACA 675 Leu Arg Ala Gly Lys Leu Ser Pro Leu Lys Ser Lys Phe Lys Thr 215 220 225
GGG AAG CTT CAA ATA GGA AGG AAG GGG GTA CAA ATT GTA CGA CGG 720 Gly Lys Leu Gin lie Gly Arg Lys Gly Val Gin lie Val Arg Arg 230 235 240
AGA GGA AGG CCT CCA TCA ACA GAA AGG ATA AAG ACC CCT TCG GGT 765 Arg Gly Arg Pro Pro Ser Thr Glu Arg lie Lys Thr Pro Ser Gly 245 250 255
CTC CTC ATT AAT TCT GAA CTG GAA AAG CCC CAG AAA GTC CGG AAA 810 Leu Leu lie Asn Ser Glu Leu Glu Lys Pro Gin Lys Val Arg Lys 260 265 270
GAC AAG GAA GGA ACA CCT CCA CTT ACA AAA GAA GAT AAG ACA GTT 855 Asp Lys Glu Gly Thr Pro Pro Leu Thr Lys Glu Asp Lys Thr Val 275 280 285
GTC AGA CAA AGC CCT CGA AGG ATT AAG CCA GTT AGG ATT ATT CCT 900 Val Arg Gin Ser Pro Arg Arg lie Lys Pro Val Arg lie lie Pro 290 295 300
TCT TCA AAA AGG ACA GAT GCA ACC ATT GCT AAG CAA CTC TTA CAG 945 Ser Ser Lys Arg Thr Asp Ala Thr lie Ala Lys Gin Leu Leu Gin 305 310 315
AGG GCA AAA AAG GGG GCT CAA AAG AAA ATT GAA AAA GAA GCA GCT 990 Arg Ala Lys Lys Gly Ala Gin Lys Lys lie Glu Lys Glu Ala Ala 320 325 330
CAG CTG CAG GGA AGA AAG GTG AAG ACA CAG GTC AAA AAT ATT CGA 1035 Gin Leu Gin Gly Arg Lys Val Lys Thr Gin Val Lys Asn lie Arg 335 340 345
CAG TTC ATC ATG CCT GTT GTC AGT GCT ATC TCC TCG CGG ATC ATT 1080 Gin Phe lie Met Pro Val Val Ser Ala lie Ser Ser Arg lie lie 350 355 360
AAG ACC CCT CGG CGG TTT ATA GAG GAT GAG GAT TAT GAC CCT CCA 1125 Lys Thr Pro Arg Arg Phe lie Glu Asp Glu Asp Tyr Asp Pro Pro 365 370 375
ATT AAA ATT GCC CGA TTA GAG TCT ACA CCG AAT AGT AGA TTC AGT 1170 lie Lys lie Ala Arg Leu Glu Ser Thr Pro Asn Ser Arg Phe Ser 380 385 390
GCC CCG TCC TGT GGA TCT TCT GAA AAA TCA AGT GCA GCT TCT CAG 1215 Ala Pro Ser Cys Gly Ser Ser Glu Lys Ser Ser Ala Ala Ser Gin 395 400 405
CAC TCC TCT CAA ATG TCT TCA GAC TCC TCT CGA TCT AGT AGC CCC 1260 His Ser Ser Gin Met Ser Ser Asp Ser Ser Arg Ser Ser Ser Pro 410 415 420
AGT GTT GAT ACC TCC ACA GAC TCT CAG GCT TCT GAG GAG ATT CAG 1305 Ser Val Asp Thr Ser Thr Asp Ser Gin Ala Ser Glu Glu lie Gin 425 430 435
GTA CTT CCT GAG GAG CGG AGC GAT ACC CCT GAA GTT CAT CCT CCA 1350 Val Leu Pro Glu Glu Arg Ser Asp Thr Pro Glu Val His Pro Pro 440 445 450
CTG CCC ATT TCC CAG TCC CCA GAA AAT GAG AGT AAT GAT AGG AGA 1395 Leu Pro lie Ser Gin Ser Pro Glu Asn Glu Ser Asn Asp Arg Arg 455 460 465
AGC AGA AGG TAT TCA GTG TCG GAG AGA AGT TTT GGA TCT AGA ACG 1440 Ser Arg Arg Tyr Ser Val Ser Glu Arg Ser Phe Gly Ser Arg Thr 470 475 480
ACG AAA AAA TTA TCA ACT CTA CAA AGT GCC CCC CAG CAG GAG ACC 1485 Thr Lys Lys Leu Ser Thr Leu Gin Ser Ala Pro Gin Gin Glu Thr 485 490 495
TCC TCG TCT CCA CCT CCA CCT CTG CTG ACT CCA CCG CCA CCA CTG 1530 Ser Ser Ser Pro Pro Pro Pro Leu Leu Thr Pro Pro Pro Pro Leu 500 505 510
CAG CCA GCC TCC AGT ATC TCT GAC CAC ACA CCT TGG CTT ATG CCT 1575 Gin Pro Ala Ser Ser lie Ser Asp His Thr Pro Trp Leu Met Pro 515 520 525
CCA ACA ATC CCC TTA GCA TCA CCA TTT TTG CCT GCT TCC ACT GCT 1620 Pro Thr lie Pro Leu Ala Ser Pro Phe Leu Pro Ala Ser Thr Ala 530 535 540
CCT ATG CAA GGG AAG CGA AAA TCT ATT TTG CGA GAA CCG ACA TTT 1665 Pro Met Gin Gly Lys Arg Lys Ser lie Leu Arg Glu Pro Thr Phe 545 550 555
AGG TGG ACT TCT TTA AAG CAT TCT AGG TCA GAG CCA CAA TAC TTT 1710 Arg Trp Thr Ser Leu Lys His Ser Arg Ser Glu Pro Gin Tyr Phe 560 565 570
TCC TCA GCA AAG TAT GCC AAA GAA GGT CTT ATT CGC AAA CCA ATA 1755 Ser Ser Ala Lys Tyr Ala Lys Glu Gly Leu lie Arg Lys Pro lie 575 580 585 TTT GAT AAT TTC CGA CCC CCT CCA CTA ACT CCC GAG GAC GTT GGC 1800 Phe Asp Asn Phe Arg Pro Pro Pro Leu Thr Pro Glu Asp Val Gly 590 595 600
TTT GCA TCT GGT TTT TCT GCA TCT GGT ACC GCT GCT TCA GCC CGA 1845 Phe Ala Ser Gly Phe Ser Ala Ser Gly Thr Ala Ala Ser Ala Arg 605 610 615
TTG TTT TCG CCA CTC CAT TCT GGA ACA AGG TTT GAT ATG CAC AAA 1890 Leu Phe Ser Pro Leu His Ser Gly Thr Arg Phe Asp Met His Lys 620 625 630
AGG AGC CCT CTT CTG AGA GCT CCA AGA TTT ACT CCA AGT GAG GCT 1935 Arg Ser Pro Leu Leu Arg Ala Pro Arg Phe Thr Pro Ser Glu Ala 635 640 645
CAC TCT AGA ATA TTT GAG TCT GTA ACC TTG CCT AGT AAT CGA ACT 1980 His Ser Arg lie Phe Glu Ser Val Thr Leu Pro Ser Asn Arg Thr 650 655 660
TCT GCT GGA ACA TCT TCT TCA GGA GTA TCC AAT AGA AAA AGG AAA 2025 Ser Ala Gly Thr Ser Ser Ser Gly Val Ser Asn Arg Lys Arg Lys 665 670 675
AGA AAA GTG TTT AGT CCT ATT CGA TCT GAA CCA AGA TCT CCT TCT 2070 Arg Lys Val Phe Ser Pro lie Arg Ser Glu Pro Arg Ser Pro Ser 680 685 690
CAC TCC ATG AGG ACA AGA AGT GGA AGG CTT AGT AGT TCT GAG CTC 2115 His Ser Met Arg Thr Arg Ser Gly Arg Leu Ser Ser Ser Glu Leu 695 700 705
TCA CCT CTC ACC CCC CCG TCT TCT GTC TCT TCC TCG TTA AGC ATT 2160 Ser Pro Leu Thr Pro Pro Ser Ser Val Ser Ser Ser Leu Ser lie 710 715 720
TCT GTT AGT CCT CTT GCC ACT AGT GCC TTA AAC CCA ACT TTT ACT 2205 Ser Val Ser Pro Leu Ala Thr Ser Ala Leu Asn Pro Thr Phe Thr 725 730 735
TTT CCT TCT CAT TCC CTG ACT CAG TCT GGG GAA TCT GCA GAG AAA 2250 Phe Pro Ser His Ser Leu Thr Gin Ser Gly Glu Ser Ala Glu Lys 740 745 750
AAT CAG AGA CCA AGG AAG CAG ACT AGT GCT CCG GCA GAG CCA TTT 2295 Asn Gin Arg Pro Arg Lys Gin Thr Ser Ala Pro Ala Glu Pro Phe 755 760 765
TCA TCA AGT AGT CCT ACT CCT CTC TTC CCT TGG TTT ACC CCA GGC 2340 Ser Ser Ser Ser Pro Thr Pro Leu Phe Pro Trp Phe Thr Pro Gly 770 775 780
TCT CAG ACT GAA AGA GGG AGA AAT AAA GAC AAG GCC CCC GAG GAG 2385 Ser Gin Thr Glu Arg Gly Arg Asn Lys Asp Lys Ala Pro Glu Glu 785 790 795
CTG TCC AAA GAT CGA GAT GCT GAC AAG AGC GTG GAG AAG GAC AAG 2430 Leu Ser Lys Asp Arg Asp Ala Asp Lys Ser Val Glu Lys Asp Lys 800 805 810
AGT AGA GAG AGA GAC CGG GAG AGA GAA AAG GAG AAT AAG CGG GAG 2475 Ser Arg Glu Arg Asp Arg Glu Arg Glu Lys Glu Asn Lys Arg Glu 815 820 825
TCA AGG AAA GAG AAA AGG AAA AAG GGA TCA GAA ATT CAG AGT AGT 2520 Ser Arg Lys Glu Lys Arg Lys Lys Gly Ser Glu lie Gin Ser Ser 830 835 840 TCT GCT TTG TAT CCT GTG GGT AGG GTT TCC AAA GAG AAG GTT GTT 2565 Ser Ala Leu Tyr Pro Val Gly Arg Val Ser Lys Glu Lys Val Val 845 850 855
GGT GAA GAT GTT GCC ACT TCA TCT TCT GCC AAA AAA GCA ACA GGG 2610 Gly Glu Asp Val Ala Thr Ser Ser Ser Ala Lys Lys Ala Thr Gly 860 865 870
CGG AAG AAG TCT TCA TCA CAT GAT TCT GGG ACT GAT ATT ACT TCT 2655 Arg Lys Lys Ser Ser Ser His Asp Ser Gly Thr Asp lie Thr Ser 875 880 885
GTG ACT CTT GGG GAT ACA ACA GCT GTC AAA ACC AAA ATA CTT ATA 2700 Val Thr Leu Gly Asp Thr Thr Ala Val Lys Thr Lys lie Leu lie 890 895 900
AAG AAA GGG AGA GGA AAT CTG GAA AAA ACC AAC TTG GAC CTC GGC 2745 Lys Lys Gly Arg Gly Asn Leu Glu Lys Thr Asn Leu Asp Leu Gly 905 910 915
CCA ACT GCC CCA TCC CTG GAG AAG GAG AAA ACC CTC TGC CTT TCC 2790 Pro Thr Ala Pro Ser Leu Glu Lys Glu Lys Thr Leu Cys Leu Ser 920 925 930
ACT CCT TCA TCT AGC ACT GTT AAA CAT TCC ACT TCC TCC ATA GGC 2835 Thr Pro Ser Ser Ser Thr Val Lys His Ser Thr Ser Ser lie Gly 935 940 945
TCC ATG TTG GCT CAG GCA GAC AAG CTT CCA ATG ACT GAC AAG AGG 2880 Ser Met Leu Ala Gin Ala Asp Lys Leu Pro Met Thr Asp Lys Arg 950 955 960
GTT GCC AGC CTC CTA AAA AAG GCC AAA GCT CAG CTC TGC AAG ATT 2925 Val Ala Ser Leu Leu Lys Lys Ala Lys Ala Gin Leu Cys Lys lie 965 970 975
GAG AAG AGT AAG AGT CTT AAA CAA ACC GAC CAG CCC AAA GCA CAG 2970 Glu Lys Ser Lys Ser Leu Lys Gin Thr Asp Gin Pro Lys Ala Gin 980 985 990
GGT CAA GAA AGT GAC TCA TCA GAG ACC TCT GTG CGA GGA CCC CGG 3015 Gly Gin Glu Ser Asp Ser Ser Glu Thr Ser Val Arg Gly Pro Arg 995 1000 1005
ATT AAA CAT GTC TGC AGA AGA GCA GCT GTT GCC CTT GGC CGA AAA 3060 lie Lys His Val Cys Arg Arg Ala Ala Val Ala Leu Gly Arg Lys 1010 1015 1020
CGA GCT GTG TTT CCT GAT GAC ATG CCC ACC CTG AGT GCC TTA CCA 3105 Arg Ala Val Phe Pro Asp Asp Met Pro Thr Leu Ser Ala Leu Pro 1025 1030 1035
TGG GAA GAA CGA GAA AAG ATT TTG TCT TCC ATG GGG AAT GAT GAC 3150 Trp Glu Glu Arg Glu Lys lie Leu Ser Ser Met Gly Asn Asp Asp 1040 1045 1050
AAG TCA TCA ATT GCT GGC TCA GAA GAT GCT GAA CCT CTT GCT CCA 3195 Lys Ser Ser lie Ala Gly Ser Glu Asp Ala Glu Pro Leu Ala Pro 1055 1060 1065
CCC ATC AAA CCA ATT AAA CCT GTC ACT AGA AAC AAG GCA CCC CAG 3240 Pro lie Lys Pro lie Lys Pro Val Thr Arg Asn Lys Ala Pro Gin 1070 1075 1080
GAA CCT CCA GTA AAG AAA GGA CGT CGA TCG AGG CGG TGT GGG CAG 3285 Glu Pro Pro Val Lys Lys Gly Arg Arg Ser Arg Arg Cys Gly Gin 1085 1090 1095 TGT CCC GGC TGC CAG GTG CCT GAG GAC TGT GGT GTT TGT ACT AAT 3330 Cys Pro Gly Cys Gin Val Pro Glu Asp Cys Gly Val Cys Thr Asn 1100 1105 1110
TGC TTA GAT AAG CCC AAG TTT GGT GGT CGC AAT ATA AAG AAG CAG 3375 Cys Leu Asp Lys Pro Lys Phe Gly Gly Arg Asn lie Lys Lys Gin 1115 1120 1125
TGC TGC AAG ATG AGA AAA TGT CAG AAT CTA CAA TGG ATG CCT TCC 3420 Cys Cys Lys Met Arg Lys Cys Gin Asn Leu Gin Trp Met Pro Ser 1130 1135 1140
AAA GCC TAC CTG CAG AAG CAA GCT AAA GCT GTG AAA AAG AAA GAG 3465 Lys Ala Tyr Leu Gin Lys Gin Ala Lys Ala Val Lys Lys Lys Glu 1145 1150 1155
AAA AAG TCT AAG ACC AGT GAA AAG AAA GAC AGC AAA GAG AGC AGT 3510 Lys Lys Ser Lys Thr Ser Glu Lys Lys Asp Ser Lys Glu Ser Ser 1160 1165 1170
GTT GTG AAG AAC GTG GTG GAC TCT AGT CAG AAA CCT ACC CCA TCA 3555 Val Val Lys Asn Val Val Asp Ser Ser Gin Lys Pro Thr Pro Ser 1175 1180 1185
GCA AGA GAG GAT CCT GCC CCA AAG AAA AGC AGT AGT GAG CCT CCT 3600 Ala Arg Glu Asp Pro Ala Pro Lys Lys Ser Ser Ser Glu Pro Pro 1190 1195 1200
CCA CGA AAG CCC GTC GAG GAA AAG AGT GAA GAA GGG AAT GTC TCG 3645 Pro Arg Lys Pro Val Glu Glu Lys Ser Glu Glu Gly Asn Val Ser 1205 1210 1215
GCC CCT GGG CCT GAA TCC AAA CAG GCC ACC ACT CCA GCT TCC AGG 3690 Ala Pro Gly Pro Glu Ser Lys Gin Ala Thr Thr Pro Ala Ser Arg 1220 1225 1230
AAG TCA AGC AAG CAG GTC TCC CAG CCA GCA CTG GTC ATC CCG CCT 3735 Lys Ser Ser Lys Gin Val Ser Gin Pro Ala Leu Val lie Pro Pro 1235 1240 1245
CAG CCA CCT ACT ACA GGA CCG CCA AGA AAA GAA GTT CCC AAA ACC 3780 Gin Pro Pro Thr Thr Gly Pro Pro Arg Lys Glu Val Pro Lys Thr 1250 1255 1260
ACT CCT AGT GAG CCC AAG AAA AAG CAG CCT CCA CCA CCA GAA TCA 3825 Thr Pro Ser Glu Pro Lys Lys Lys Gin Pro Pro Pro Pro Glu Ser 1265 1270 1275
GGT CCA GAG CAG AGC AAA CAG AAA AAA GTG GCT CCC CGC CCA AGT 3870 Gly Pro Glu Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser 1280 1285 1290
ATC CCT GTA AAA CAA AAA CCA AAA GAA AAG GAA AAA CCA CCT CCG 3915 lie Pro Val Lys Gin Lys Pro Lys Glu Lys Glu Lys Pro Pro Pro 1295 1300 1305
GTC AAT AAG CAG GAG AAT GCA GGC ACT TTG AAC ATC CTC AGC ACT 3960 Val Asn Lys Gin Glu Asn Ala Gly Thr Leu Asn lie Leu Ser Thr 1310 1315 1320
CTC TCC AAT GGC AAT AGT TCT AAG CAA AAA ATT CCA GCA GAT GGA 4005 Leu Ser Asn Gly Asn Ser Ser Lys Gin Lys lie Pro Ala Asp Gly 1325 1330 1335
GTC CAC AGG ATC AGA GTG GAC TTT AAG GAG GAT TGT GAA GCA GAA 4050 Val His Arg lie Arg Val Asp Phe Lys Glu Asp Cys Glu Ala Glu 1340 1345 1350 AAT GTG TGG GAG ATG GGA GGC TTA GGA ATC TTG ACT TCT GTT CCT 4095 Asn Val Trp Glu Met Gly Gly Leu Gly lie Leu Thr Ser Val Pro 1355 1360 1365
ATA ACA CCC AGG GTG GTT TGC TTT CTC TGT GCC AGT AGT GGG CAT 4140 lie Thr Pro Arg Val Val Cys Phe Leu Cys Ala Ser Ser Gly His 1370 1375 1380
GTA GAG TTT GTG TAT TGC CAA GTC TGT TGT GAG CCC TTC CAC AAG 4185 Val Glu Phe Val Tyr Cys Gin Val Cys Cys Glu Pro Phe His Lys 1385 1390 1395
TTT TGT TTA GAG GAG AAC GAG CGC CCT CTG GAG GAC CAG CTG GAA 4230 Phe Cys Leu Glu Glu Asn Glu Arg Pro Leu Glu Asp Gin Leu Glu 1400 1405 1410
AAT TGG TGT TGT CGT CGT TGC AAA TTC TGT CAC GTT TGT GGA AGG 4275 Asn Trp Cys Cys Arg Arg Cys Lys Phe Cys His Val Cys Gly Arg 1415 1420 1425
CAA CAT CAG GCT ACA AAG CAG CTG CTG GAG TGT AAT AAG TGC CGA 4320 Gin His Gin Ala Thr Lys Gin Leu Leu Glu Cys Asn Lys Cys Arg 1430 1435 1440
AAC AGC TAT CAC CCT GAG TGC CTG GGA CCA AAC TAC CCC ACC AAA 4365 Asn Ser Tyr His Pro Glu Cys Leu Gly Pro Asn Tyr Pro Thr Lys 1445 1450 1455
CCC ACA AAG AAG AAG AAA GTC TGG ATC TGT ACC AAG TGT GTT CGC 4410 Pro Thr Lys Lys Lys Lys Val Trp lie Cys Thr Lys Cys Val Arg 1460 1465 1470
TGT AAG AGC TGT GGA TCC ACA ACT CCA GGC AAA GGG TGG GAT GCA 4455 Cys Lys Ser Cys Gly Ser Thr Thr Pro Gly Lys Gly Trp Asp Ala 1475 1480 1485
CAG TGG TCT CAT GAT TTC TCA CTG TGT CAT GAT TGC GCC AAG CTC 4500 Gin Trp Ser His Asp Phe Ser Leu Cys His Asp Cys Ala Lys Leu 1490 1495 1500
TTT GCT AAA GGA AAC TTC TGC CCT CTC TGT GAC AAA TGT TAT GAT 4545 Phe Ala Lys Gly Asn Phe Cys Pro Leu Cys Asp Lys Cys Tyr Asp 1505 1510 1515
GAT GAT GAC TAT GAG AGT AAG ATG ATG CAA TGT GGA AAG TGT GAT 4590 Asp Asp Asp Tyr Glu Ser Lys Met Met Gin Cys Gly Lys Cys Asp 1520 1525 1530
CGC TGG GTC CAT TCC AAA TGT GAG AAT CTT TCA GGT ACA GAA GAT 4635 Arg Trp Val His Ser Lys Cys Glu Asn Leu Ser Gly Thr Glu Asp 1535 1540 1545
GAG ATG TAT GAG ATT CTA TCT AAT CTG CCA GAA AGT GTG GCC TAC 4680 Glu Met Tyr Glu lie Leu Ser Asn Leu Pro Glu Ser Val Ala Tyr 1550 1555 1560
ACT TGT GTG AAC TGT ACT GAG CGG CAC CCT GCA GAG TGG CGA CTG 4725 Thr Cys Val Asn Cys Thr Glu Arg His Pro Ala Glu Trp Arg Leu 1565 1570 1575
GCC CTT GAA AAA GAG CTG CAG ATT TCT CTG AAG CAA GTT CTG ACA 4770 Ala Leu Glu Lys Glu Leu Gin He Ser Leu Lys Gin Val Leu Thr 1580 1585 1590
GCT TTG TTG AAT TCT CGG ACT ACC AGC CAT TTG CTA CGC TAC CGG 4815 Ala Leu Leu Asn Ser Arg Thr Thr Ser His Leu Leu Arg Tyr Arg 1595 1600 1605 CAG GCT GCC AAG CCT CCA GAC TTA AAT CCC GAG ACA GAG GAG AGT 4860 Gin Ala Ala Lys Pro Pro Asp Leu Asn Pro Glu Thr Glu Glu Ser 1610 1615 1620
ATA CCT TCC CGC AGC TCC CCC GAA GGA CCT GAT CCA CCA GTT CTT 4905 He Pro Ser Arg Ser Ser Pro Glu Gly Pro Asp Pro Pro Val Leu 1625 1630 1635
ACT GAG GTC AGC AAA CAG GAT GAT CAG CAG CCT TTA GAT CTA GAA 4950 Thr Glu Val Ser Lys Gin Asp Asp Gin Gin Pro Leu Asp Leu Glu 1640 1645 1650
GGA GTC AAG AGG AAG ATG GAC CAA GGG AAT TAC ACA TCT GTG TTG 4995 Gly Val Lys Arg Lys Met Asp Gin Gly Asn Tyr Thr Ser Val Leu 1655 1660 1665
GAG TTC AGT GAT GAT ATT GTG AAG ATC ATT CAA GCA GCC ATT AAT 5040 Glu Phe Ser Asp Asp He Val Lys He He Gin Ala Ala He Asn 1670 1675 1680
TCA GAT GGA GGA CAG CCA GAA ATT AAA AAA GCC AAC AGC ATG GTC 5085 Ser Asp Gly Gly Gin Pro Glu He Lys Lys Ala Asn Ser Met Val 1685 1690 1695
AAG TCC TTC TTC ATT CGG CAA ATG GAA CGT GTT TTT CCA TGG TTC 5130 Lys Ser Phe Phe He Arg Gin Met Glu Arg Val Phe Pro Trp Phe 1700 1705 1710
AGT GTC AAA AAG TCC AGG TTT TGG GAG CCA AAT AAA GTA TCA AGC 5175 Ser Val Lys Lys Ser Arg Phe Trp Glu Pro Asn Lys Val Ser Ser 1715 1720 1725
AAC AGT GGG ATG TTA CCA AAC GCA GTG CTT CCA CCT TCA CTT GAC 5220 Asn Ser Gly Met Leu Pro Asn Ala Val Leu Pro Pro Ser Leu Asp 1730 1735 1740
CAT AAT TAT GCT CAG TGG CAG GAG CGA GAG GAA AAC AGC CAC ACT 5265 His Asn Tyr Ala Gin Trp Gin Glu Arg Glu Glu Asn Ser His Thr 1745 1750 1755
GAG CAG CCT CCT TTA ATG AAG AAA ATC ATT CCA GCT CCC AAA CCC 5310 Glu Gin Pro Pro Leu Met Lys Lys He He Pro Ala Pro Lys Pro 1760 1765 1770
AAA GGT CCT GGA GAA CCA GAC TCA CCA ACT CCT CTG CAT CCT CCT 5355 Lys Gly Pro Gly Glu Pro Asp Ser Pro Thr Pro Leu His Pro Pro 1775 1780 1785
ACA CCA CCA ATT TTG AGT ACT GAT AGG AGT CGA GAA GAC AGT CCA 5400 Thr Pro Pro He Leu Ser Thr Asp Arg Ser Arg Glu Asp Ser Pro 1790 1795 1800
GAG CTG AAC CCA CCC CCA GGC ATA GAA GAC AAT AGA CAG TGT GCG 5445 Glu Leu Asn Pro Pro Pro Gly He Glu Asp Asn Arg Gin Cys Ala 1805 1810 1815
TTA TGT TTG ACT TAT GGT GAT GAC AGT GCT AAT GAT GCT GGT CGT 5490 Leu Cys Leu Thr Tyr Gly Asp Asp Ser Ala Asn Asp Ala Gly Arg 1820 1825 1830
TTA CTA TAT ATT GGC CAA AAT GAG TGG ACA CAT GTA AAT TGT GCT 5535 Leu Leu Tyr He Gly Gin Asn Glu Trp Thr His Val Asn Cys Ala 1835 1840 184
TTG TGG TCA GCG GAA GTG TTT GAA GAT GAT GAC GGA TCA CTA AAG 5580 Leu Trp Ser Ala Glu Val Phe Glu Asp Asp Asp Gly Ser Leu Lys 1850 1855 1860 AAT GTG CAT ATG GCT GTG ATC AGG GGC AAG CAG CTG AGA TGT GAA 5625 Asn Val His Met Ala Val He Arg Gly Lys Gin Leu Arg Cys Glu 1865 1870 1875
TTC TGC CAA AAG CCA GGA GCC ACC GTG GGT TGC TGT CTC ACA TCC 5670 Phe Cys Gin Lys Pro Gly Ala Thr Val Gly Cys Cys Leu Thr Ser 1880 1885 1890
TGC ACC AGC AAC TAT CAC TTC ATG TGT TCC CGA GCC AAG AAC TGT 5715 Cys Thr Ser Asn Tyr His Phe Met Cys Ser Arg Ala Lys Asn Cys 1895 1900 1905
GTC TTT CTG GAT GAT AAA AAA GTA TAT TGC CAA CGA CAT CGG GAT 5760 Val Phe Leu Asp Asp Lys Lys Val Tyr Cys Gin Arg His Arg Asp 1910 1915 1920
TTG ATC AAA GGC GAA GTG GTT CCT GAG AAT GGA TTT GAA GTT TTC 5805 Leu He Lys Gly Glu Val Val Pro Glu Asn Gly Phe Glu Val Phe 1925 1930 1935
AGA AGA GTG TTT GTG GAC TTT GAA GGA ATC AGC TTG AGA AGG AAG 5850 Arg Arg Val Phe Val Asp Phe Glu Gly He Ser Leu Arg Arg Lys 1940 1945 1950
TTT CTC AAT GGC TTG GAA CCA GAA AAT ATC CAC ATG ATG ATT GGG 5895 Phe Leu Asn Gly Leu Glu Pro Glu Asn He His Met Met He Gly 1955 1960 1965
TCT ATG ACA ATC GAC TGC TTA GGA ATT CTA AAT GAT CTC TCC GAC 5940 Ser Met Thr He Asp Cys Leu Gly He Leu Asn Asp Leu Ser Asp 1970 1975 1980
TGT GAA GAT AAG CTC TTT CCT ATT GGA TAT CAG TGT TCC AGG GTA 5985 Cys Glu Asp Lys Leu Phe Pro He Gly Tyr Gin Cys Ser Arg Val 1985 1990 1995
TAC TGG AGC ACC ACA GAT GCT CGC AAG CGC TGT GTA TAT ACA TGC 6030 Tyr Trp Ser Thr Thr Asp Ala Arg Lys Arg Cys Val Tyr Thr Cys 2000 2005 2010
AAG ATA GTG GAG TGC CGT CCT CCA GTC GTA GAG CCG GAT ATC AAC 6075 Lys He Val Glu Cys Arg Pro Pro Val Val Glu Pro Asp He Asn 2015 2020 2025
AGC ACT GTT GAA CAT GAT GAA AAC AGG ACC ATT GCC CAT AGT CCA 6120 Ser Thr Val Glu His Asp Glu Asn Arg Thr He Ala His Ser Pro 2030 2035 2040
ACA TCT TTT ACA GAA AGT TCA TCA AAA GAG AGT CAA AAC ACA GCT 6165 Thr Ser Phe Thr Glu Ser Ser Ser Lys Glu Ser Gin Asn Thr Ala 2045 2050 2055
GAA ATT ATA AGT CCT CCA TCA CCA GAC CGA CCT CCT CAT TCA CAA 6210 Glu He He Ser Pro Pro Ser Pro Asp Arg Pro Pro His Ser Gin 2060 2065 2070
ACC TCT GGC TCC TGT TAT TAT CAT GTC ATC TCA AAG GTC CCC AGG 6255 Thr Ser Gly Ser Cys Tyr Tyr His Val He Ser Lys Val Pro Arg 2075 2080 2085
ATT CGA ACA CCC AGT TAT TCT CCA ACA CAG AGA TCC CCT GGC TGT 6300 He Arg Thr Pro Ser Tyr Ser Pro Thr Gin Arg Ser Pro Gly Cys 2090 2095 2100
CGA CCG TTG CCT TCT GCA GGA AGT CCT ACC CCA ACC ACT CAT GAA 6345 Arg Pro Leu Pro Ser Ala Gly Ser Pro Thr Pro Thr Thr His Glu 2105 2110 2115 ATA GTC ACA GTA GGT GAT CCT TTA CTC TCC TCT GGA CTT CGA AGC 6390 He Val Thr Val Gly Asp Pro Leu Leu Ser Ser Gly Leu Arg Ser 2120 2125 2130
ATT GGC TCC AGG CGT CAC AGT ACC TCT TCC TTA TCA CCC CAG CGG 6435 He Gly Ser Arg Arg His Ser Thr Ser Ser Leu Ser Pro Gin Arg 2135 2140 2145
TCC AAA CTC CGG ATA ATG TCT CCA ATG AGA ACT GGG AAT ACT TAC 6480 Ser Lys Leu Arg He Met Ser Pro Met Arg Thr Gly Asn Thr Tyr 2150 2155 2160
TCT AGG AAT AAT GTT TCC TCA GTC TCC ACC ACC GGG ACC GCT ACT 6525 Ser Arg Asn Asn Val Ser Ser Val Ser Thr Thr Gly Thr Ala Thr 2165 2170 2175
GAT CTT GAA TCA AGT GCC AAA GTA GTT GAT CAT GTC TTA GGG CCA 6670 Asp Leu Glu Ser Ser Ala Lys Val Val Asp His Val Leu Gly Pro 2180 2185 2190
CTG AAT TCA AGT ACT AGT TTA GGG CAA AAC ACT TCC ACC TCT TCA 6615 Leu Asn Ser Ser Thr Ser Leu Gly Gin Asn Thr Ser Thr Ser Ser 2195 2200 2205
AAT TTG CAA AGG ACA GTG GTT ACT GTA GGC AAT AAA AAC AGT CAC 6660 Asn Leu Gin Arg Thr Val Val Thr Val Gly Asn Lys Asn Ser His 2210 2215 2220
TTG GAT GGA TCT TCA TCT TCA GAA ATG AAG CAG TCC AGT GCT TCA 6705 Leu Asp Gly Ser Ser Ser Ser Glu Met Lys Gin Ser Ser Ala Ser 2225 2230 2235
GAC TTG GTG TCC AAG AGC TCC TCT TTA AAG GGA GAG AAG ACC AAA 6750 Asp Leu Val Ser Lys Ser Ser Ser Leu Lys Gly Glu Lys Thr Lys 2240 2245 2250
GTG CTG AGT TCC AAG AGC TCA GAG GGA TCT GCA CAT AAT GTG GCT 6795 Val Leu Ser Ser Lys Ser Ser Glu Gly Ser Ala His Asn Val Ala 2255 2260 2265
TAC CCT GGA ATT CCT AAA CTG GCC CCA CAG GTT CAT AAC ACA ACA 6840 Tyr Pro Gly He Pro Lys Leu Ala Pro Gin Val His Asn Thr Thr 2270 2275 2280
TCT AGA GAA CTG AAT GTT AGT AAA ATC GGC TCC TTT GCT GAA CCC 6885 Ser Arg Glu Leu Asn Val Ser Lys He Gly Ser Phe Ala Glu Pro 2285 2290 2295
TCT TCA GTG TCG TTT TCT TCT AAA GAG GCC CTC TCC TTC CCA CAC 6930 Ser Ser Val Ser Phe Ser Ser Lys Glu Ala Leu Ser Phe Pro His 2300 2305 2310
CTC CAT TTG AGA GGG CAA AGG AAT GAT CGA GAC CAA CAC ACA GAT 6975 Leu His Leu Arg Gly Gin Arg Asn Asp Arg Asp Gin His Thr Asp 2315 2320 2325
TCT ACC CAA TCA GCA AAC TCC TCT CCA GAT GAA GAT ACT GAA GTC 7020 Ser Thr Gin Ser Ala Asn Ser Ser Pro Asp Glu Asp Thr Glu Val 2330 2335 2340
AAA ACC TTG AAG CTA TCT GGA ATG AGC AAC AGA TCA TCC ATT ATC 7065 Lys Thr Leu Lys Leu Ser Gly Met Ser Asn Arg Ser Ser He He 2345 2350 2355
AAC GAA CAT ATG GGA TCT AGT TCC AGA GAT AGG AGA CAG AAA GGG 7110 Asn Glu His Met Gly Ser Ser Ser Arg Asp Arg Arg Gin Lys Gly 2360 2365 2370 AAA AAA TCC TGT AAA GAA ACT TTC AAA GAA AAG CAT TCC AGT AAA 7155 Lys Lys Ser Cys Lys Glu Thr Phe Lys Glu Lys His Ser Ser Lys 2375 2380 2385
TCT TTT TTG GAA CCT GGT CAG GTG ACA ACT GGT GAG GAA GGA AAC 7200 Ser Phe Leu Glu Pro Gly Gin Val Thr Thr Gly Glu Glu Gly Asn 2390 2395 2400
TTG AAG CCA GAG TTT ATG GAT GAG GTT TTG ACT CCT GAG TAT ATG 7245 Leu Lys Pro Glu Phe Met Asp Glu Val Leu Thr Pro Glu Tyr Met 2405 2410 . 2415
GGC CAA CGA CCA TGT AAC AAT GTT TCT TCT GAT AAG ATT GGT GAT 7290 Gly Gin Arg Pro Cys Asn Asn Val Ser Ser Asp Lys He Gly Asp 2420 2425 2430
AAA GGC CTT TCT ATG CCA GGA GTC CCC AAA GCT CCA CCC ATG CAA 7335 Lys Gly Leu Ser Met Pro Gly Val Pro Lys Ala Pro Pro Met Gin 2435 2440 2445
GTA GAA GGA TCT GCC AAG GAA TTA CAG GCA CCA CGG AAA CGC ACA 7380 Val Glu Gly Ser Ala Lys Glu Leu Gin Ala Pro Arg Lys Arg Thr 2450 2455 2460
GTC AAA GTG ACA CTG ACA CCT CTA AAA ATG GAA AAT GAG AGT CAA 7425 Val Lys Val Thr Leu Thr Pro Leu Lys Met Glu Asn Glu Ser Gin 2465 2470 2475
TCC AAA AAT GCC CTG AAA GAA AGT AGT CCT GCT TCC CCT TTG CAA 7470 Ser Lys Asn Ala Leu Lys Glu Ser Ser Pro Ala Ser Pro Leu Gin 2480 2485 2490
ATA GAG TCA ACA TCT CCC ACA GAA CCA ATT TCA GCC TCT GAA AAT 7515 He Glu Ser Thr Ser Pro Thr Glu Pro He Ser Ala Ser Glu Asn 2495 2500 2505
CCA GGA GAT GGT CCA GTG GCC CAA CCA AGC CCC AAT AAT ACC TCA 7560 Pro Gly Asp Gly Pro Val Ala Gin Pro Ser Pro Asn Asn Thr Ser 2510 2515 2520
TGC CAG GAT TCT CAA AGT AAC AAC TAT CAG AAT CTT CCA GTA CAG 7605 Cys Gin Asp Ser Gin Ser Asn Asn Tyr Gin Asn Leu Pro Val Gin 2525 2530 2535
GAC AGA AAC CTA ATG CTT CCA GAT GGC CCC AAA CCT CAG GAG GAT 7650 Asp Arg Asn Leu Met Leu Pro Asp Gly Pro Lys Pro Gin Glu Asp 2540 2545 2550
GGC TCT TTT AAA AGG AGG TAT CCC CGT CGC AGT GCC CGT GCA CGT 7695 Gly Ser Phe Lys Arg Arg Tyr Pro Arg Arg Ser Ala Arg Ala Arg 2555 2560 2565
TCT AAC ATG TTT TTT GGG CTT ACC CCA CTC TAT GGA GTA AGA TCC 7740 Ser Asn Met Phe Phe Gly Leu Thr Pro Leu Tyr Gly Val Arg Ser 2570 2575 2580
TAT GGT GAA GAA GAC ATT CCA TTC TAC AGC AGC TCA ACT GGG AAG 7785 Tyr Gly Glu Glu Asp He Pro Phe Tyr Ser Ser Ser Thr Gly Lys 2585 2590 2595
AAG CGA GGC AAG AGA TCA GCT GAA GGA CAG GTG GAT GGG GCC GAT 7830 Lys Arg Gly Lys Arg Ser Ala Glu Gly Gin Val Asp Gly Ala Asp 2600 2605 2610
GAC TTA AGC ACT TCA GAT GAA GAC GAC TTA TAC TAT TAC AAC TTC 7875 Asp Leu Ser Thr Ser Asp Glu Asp Asp Leu Tyr Tyr Tyr Asn Phe 2615 2620 2625 ACT AGA ACA GTG ATT TCT TCA GGT GGA GAG GAA CGA CTG GCA TCC 7920 Thr Arg Thr Val He Ser Ser Gly Gly Glu Glu Arg Leu Ala Ser 2630 2635 2640
CAT AAT TTA TTT CGG GAG GAG GAA CAG TGT GAT CTT CCA AAA ATC 7965 His Asn Leu Phe Arg Glu Glu Glu Gin Cys Asp Leu Pro Lys He 2645 2650 2655
TCA CAG TTG GAT GGT GTT GAT GAT GGG ACA GAG AGT GAT ACT AGT 8010 Ser Gin Leu Asp Gly Val Asp Asp Gly Thr Glu Ser Asp Thr Ser 2660 2665 2670
GTC ACA GCC ACA ACA AGG AAA AGC AGC CAG ATT CCA AAA AGA AAT 8055 Val Thr Ala Thr Thr Arg Lys Ser Ser Gin He Pro Lys Arg Asn 2675 2680 2685
GGT AAA GAA AAT GGA ACA GAG AAC TTA AAG ATT GAT AGA CCT GAA 8100 Gly Lys Glu Asn Gly Thr Glu Asn Leu Lys He Asp Arg Pro Glu 2690 2695 2700
GAT GCT GGG GAG AAA GAA CAT GTC ACT AAG AGT TCT GTT GGC CAC 8145 Asp Ala Gly Glu Lys Glu His Val Thr Lys Ser Ser Val Gly His 2705 2710 2715
AAA AAT GAG CCA AAG ATG GAT AAC TGC CAT TCT GTA AGC AGA GTT 8190 Lys Asn Glu Pro Lys Met Asp Asn Cys His Ser Val Ser Arg Val 2720 2725 2730
AAA ACA CAG GGA CAA GAT TCC TTG GAA GCT CAG CTC AGC TCA TTG 8235 Lys Thr Gin Gly Gin Asp Ser Leu Glu Ala Gin Leu Ser Ser Leu 2735 2740 2745
GAG TCA AGC CGC AGA GTC CAC ACA AGT ACC CCC TCC GAC AAA AAT 8280 Glu Ser Ser Arg Arg Val His Thr Ser Thr Pro Ser Asp Lys Asn 2750 2755 2760
TTA CTG GAC ACC TAT AAT ACT GAG CTC CTG AAA TCA GAT TCA GAC 8325 Leu Leu Asp Thr Tyr Asn Thr Glu Leu Leu Lys Ser Asp Ser Asp 2765 2770 2775
AAT AAC AAC AGT GAT GAC TGT GGG AAT ATC CTG CCT TCA GAC ATT 8370 Asn Asn Asn Ser Asp Asp Cys Gly Asn He Leu Pro Ser Asp He 2780 2785 2790
ATG GAC TTT GTA CTA AAG AAT ACT CCA TCC ATG CAG GCT TTG GGT 8415 Met Asp Phe Val Leu Lys Asn Thr Pro Ser Met Gin Ala Leu Gly 2795 2800 2805
GAG AGC CCA GAG TCA TCT TCA TCA GAA CTC CTG AAT CTT GGT GAA 8460 Glu Ser Pro Glu Ser Ser Ser Ser Glu Leu Leu Asn Leu Gly Glu 2810 2815 2820
GGA TTG GGT CTT GAC AGT AAT CGT GAA AAA GAC ATG GGT CTT TTT 8505 Gly Leu Gly Leu Asp Ser Asn Arg Glu Lys Asp Met Gly Leu Phe 2825 2830 2835
GAA GTA TTT TCT CAG CAG CTG CCT ACA ACA GAA CCT GTG GAT AGT 8550 Glu Val Phe Ser Gin Gin Leu Pro Thr Thr Glu Pro Val Asp Ser 2840 2845 2850
AGT GTC TCT TCC TCT ATC TCA GCA GAG GAA CAG TTT GAG TTG CCT 8595 Ser Val Ser Ser Ser He Ser Ala Glu Glu Gin Phe Glu Leu Pro 2855 2860 2865
CTA GAG CTA CCA TCT GAT CTG TCT GTC TTG ACC ACC CGG AGT CCC 8640 Leu Glu Leu Pro Ser Asp Leu Ser Val Leu Thr Thr Arg Ser Pro 2870 2875 2880 ACT GTC CCC AGC CAG AAT CCC AGT AGA CTA GCT GTT ATC TCA GAC 8685 Thr Val Pro Ser Gin Asn Pro Ser Arg Leu Ala Val He Ser Asp 2885 2990 2895
TCA GGG GAG AAG AGA GTA ACC ATC ACA GAA AAA TCT GTA GCC TCC 8730 Ser Gly Glu Lys Arg Val Thr He Thr Glu Lys Ser Val Ala Ser 2900 2905 2910
TCT GAA AGT GAC CCA GCA CTG CTG AGC CCA GGA GTA GAT CCA ACT 8775 Ser Glu Ser Asp Pro Ala Leu Leu Ser Pro Gly Val Asp Pro Thr 2915 2920 2925
CCT GAA GGC CAC ATG ACT CCT GAT CAT TTT ATC CAA GGA CAC ATG 8820 Pro Glu Gly His Met Thr Pro Asp His Phe He Gin Gly His Met 2930 2935 2940
GAT GCA GAC CAC ATC TCT AGC CCT CCT TGT GGT TCA GTA GAG CAA 8865 Asp Ala Asp His He Ser Ser Pro Pro Cys Gly Ser Val Glu Gin 2945 2950 2955
GGT CAT GGC AAC AAT CAG GAT TTA ACT AGG AAC AGT AGC ACC CCT 8910 Gly His Gly Asn Asn Gin Asp Leu Thr Arg Asn Ser Ser Thr Pro 2960 2965 2970
GGC CTT CAG GTA CCT GTT TCC CCA ACT GTT CCC ATC CAG AAC CAG 8955 Gly Leu Gin Val Pro Val Ser Pro Thr Val Pro He Gin Asn Gin 2975 2980 2985
AAG TAT GTG CCC AAT TCT ACT GAT AGT CCT GGC CCG TCT CAG ATT 9000 Lys Tyr Val Pro Asn Ser Thr Asp Ser Pro Gly Pro Ser Gin He 2990 2995 3000
TCC AAT GCA GCT GTC CAG ACC ACT CCA CCC CAC CTG AAG CCA GCC 9045 Ser Asn Ala Ala Val Gin Thr Thr Pro Pro His Leu Lys Pro Ala 3005 3010 3015
ACT GAG AAA CTC ATA GTT GTT AAC CAG AAC ATG CAG CCA CTT TAT 9090 Thr Glu Lys Leu He Val Val Asn Gin Asn Met Gin Pro Leu Tyr 3020 3025 3030
GTT CTC CAA ACT CTT CCA AAT GGA GTG ACC CAA AAA ATC CAA TTG 9135 Val Leu Gin Thr Leu Pro Asn Gly Val Thr Gin Lys He Gin Leu 3035 3040 3045
ACC TCT TCT GTT AGT TCT ACA CCC AGT GTG ATG GAG ACA AAT ACT 9180 Thr Ser Ser Val Ser Ser Thr Pro Ser Val Met Glu Thr Asn Thr 3050 3055 3060
TCA GTA TTG GGA CCC ATG GGA GGT GGT CTC ACC CTT ACC ACA GGA 9225 Ser Val Leu Gly Pro Met Gly Gly Gly Leu Thr Leu Thr Thr Gly 3065 3070 3075
CTA AAT CCA AGC TTG CCA ACT TCT CAA TCT TTG TTC CCT TCT GCT 9270 Leu Asn Pro Ser Leu Pro Thr Ser Gin Ser Leu Phe Pro Ser Ala 3080 3085 3090
AGC AAA GGA TTG CTA CCC ATG TCT CAT CAC CAG CAC TTA CAT TCC 9315 Ser Lys Gly Leu Leu Pro Met Ser His His Gin His Leu His Ser 3095 3100 3105
TTC CCT GCA GCT ACT CAA AGT AGT TTC CCA CCA AAC ATC AGC AAT 9360 Phe Pro Ala Ala Thr Gin Ser Ser Phe Pro Pro Asn He Ser Asn 3110 3115 3120
CCT CCT TCA GGC CTG CTT ATT GGG GTT CAG CCT CCT CCG GAT CCC 9405 Pro Pro Ser Gly Leu Leu He Gly Val Gin Pro Pro Pro Asp Pro 3125 3130 3135 CAA CTT TTG GTT TCA GAA TCC AGC CAG AGG ACA GAC CTC AGT ACC 9450 Gin Leu Leu Val Ser Glu Ser Ser Gin Arg Thr Asp Leu Ser Thr 3140 3145 3150
ACA GTA GCC ACT CCA TCC TCT GGA CTC AAG AAA AGA CCC ATA TCT 9495 Thr Val Ala Thr Pro Ser Ser Gly Leu Lys Lys Arg Pro He Ser 3155 3160 3165
CGT CTA CAG ACC CGA AAG AAT AAA AAA CTT GCT CCC TCT AGT ACC 9540 Arg Leu Gin Thr Arg Lys Asn Lys Lys Leu Ala Pro Ser Ser Thr 3170 3175 3180
CCT TCA AAC ATT GCC CCT TCT GAT GTG GTT TCT AAT ATG ACA TTG 9585 Pro Ser Asn He Ala Pro Ser Asp Val Val Ser Asn Met Thr Leu 3185 3190 3195
ATT AAC TTC ACA CCC TCC CAG CTT CCT AAT CAT CCA AGT CTG TTA 9630 He Asn Phe Thr Pro Ser Gin Leu Pro Asn His Pro Ser Leu Leu 3200 3205 3210
GAT TTG GGG TCA CTT AAT ACT TCA TCT CAC CGA ACT GTC CCC AAC 9675 Asp Leu Gly Ser Leu Asn Thr Ser Ser His Arg Thr Val Pro Asn 3215 3220 3225
ATC ATA AAA AGA TCT AAA TCT AGC ATC ATG TAT TTT GAA CCG GCA 9720 He He Lys Arg Ser Lys Ser Ser He Met Tyr Phe Glu Pro Ala 3230 3235 3240
CCC CTG TTA CCA CAG AGT GTG GGA GGA ACT GCT GCC ACA GCG GCA 9765 Pro Leu Leu Pro Gin Ser Val Gly Gly Thr Ala Ala Thr Ala Ala 3245 3250 3255
GGC ACA TCA ACA ATA AGC CAG GAT ACT AGC CAC CTC ACA TCA GGG 9810 Gly Thr Ser Thr He Ser Gin Asp Thr Ser His Leu Thr Ser Gly 3260 3265 3270
TCT GTG TCT GGC TTG GCA TCC AGT TCC TCT GTC TTG AAT GTT GTA 9855 Ser Val Ser Gly Leu Ala Ser Ser Ser Ser Val Leu Asn Val Val 3275 3280 3285
TCC ATG CAA ACT ACC ACA ACC CCT ACA AGT AGT GCG TCA GTT CCA 9900 Ser Met Gin Thr Thr Thr Thr Pro Thr Ser Ser Ala Ser Val Pro 3290 3295 3300
GGA CAC GTC ACC TTA ACC AAC CCA AGG TTG CTT GGT ACC CCA GAT 9945 Gly His Val Thr Leu Thr Asn Pro Arg Leu Leu Gly Thr Pro Asp 3305 3310 3315
ATT GGC TCA ATA AGC AAT CTT TTA ATC AAA GCT AGC CAG CAG AGC 9990 He Gly Ser He Ser Asn Leu Leu He Lys Ala Ser Gin Gin Ser 3320 3325 3330
CTG GGG ATT CAG GAC CAG CCT GTG GCT TTA CCG CCA AGT TCA GGA 10035 Leu Gly He Gin Asp Gin Pro Val Ala Leu Pro Pro Ser Ser Gly 3335 3340 3345
ATG TTT CCA CAA CTG GGG ACA TCA CAG ACC CCC TCT ACT GCT GCA 10080 Met Phe Pro Gin Leu Gly Thr Ser Gin Thr Pro Ser Thr Ala Ala 3350 3355 3360
ATA ACA GCG GCA TCT AGC ATC TGT GTG CTC CCC TCC ACT CAG ACT 10125 He Thr Ala Ala Ser Ser He Cys Val Leu Pro Ser Thr Gin Thr 3365 3370 3375
ACG GGC ATA ACA GCC GCT TCA CCT TCT GGG GAA GCA GAC GAA CAC 10170 Thr Gly He Thr Ala Ala Ser Pro Ser Gly Glu Ala Asp Glu His 3380 3385 3390 TAT CAG CTT CAG CAT GTG AAC CAG CTC CTT GCC AGC AAA ACT GGG 10215 Tyr Gin Leu Gin His Val Asn Gin Leu Leu Ala Ser Lys Thr Gly 3395 3400 3405
ATT CAT TCT TCC CAG CGT GAT CTT GAT TCT GCT TCA GGG CCC CAG 10260 He His Ser Ser Gin Arg Asp Leu Asp Ser Ala Ser Gly Pro Gin 3410 3415 3420
GTA TCC AAC TTT ACC CAG ACG GTA GAC GCT CCT AAT AGC ATG GGA 10305 Val Ser Asn Phe Thr Gin Thr Val Asp Ala Pro Asn Ser Met Gly 3425 3430 3435
CTG GAG CAG AAC AAG GCT TTA TCC TCA GCT GTG CAA GCC AGC CCC 10350 Leu Glu Gin Asn Lys Ala Leu Ser Ser Ala Val Gin Ala Ser Pro 3440 3445 3450
ACC TCT CCT GGG GGT TCT CCA TCC TCT CCA TCT TCT GGA CAG CGG 10395 Thr Ser Pro Gly Gly Ser Pro Ser Ser Pro Ser Ser Gly Gin Arg 3455 3460 3465
TCA GCA AGC CCT TCA GTG CCG GGT CCC ACT AAA CCC AAA CCA AAA 10440 Ser Ala Ser Pro Ser Val Pro Gly Pro Thr Lys Pro Lys Pro Lys 3470 3475 3480
ACC AAA CGG TTT CAG CTG CCT CTA GAC AAA GGG AAT GGC AAG AAG 10485 Thr Lys Arg Phe Gin Leu Pro Leu Asp Lys Gly Asn Gly Lys Lys 3485 3490 3495
CAC AAT GTT TCC CAT TTG CGG ACC AGT TCT TCT GAA GCA CAC ATT 10530 His Asn Val Ser His Leu Arg Thr Ser Ser Ser Glu Ala His He 3500 3505 3510
CCA GAC CAA GAA ACG ACA TCC CTG ACC TCA GGC ACA GGG ACT CCA 10575 Pro Asp Gin Glu Thr Thr Ser Leu Thr Ser Gly Thr Gly Thr Pro 3515 3520 3525
GGA GCA GAG GCT GAG CAG CAG GAT ACA GCT AGC GTG GAG CAG TCC 10620 Gly Ala Glu Ala Glu Gin Gin Asp Thr Ala Ser Val Glu Gin Ser 3530 3535 3540
TCC CAG AAG GAG TGT GGG CAA CCT GCA GGG CAA GTC GCT GTT CTT 10665 Ser Gin Lys Glu Cys Gly Gin Pro Ala Gly Gin Val Ala Val Leu 3545 3550 3555
CCG GAA GTT CAG GTG ACC CAA AAT CCA GCA AAT GAA CAA GAA AGT 10710 Pro Glu Val Gin Val Thr Gin Asn Pro Ala Asn Glu Gin Glu Ser 3560 3565 • 3570
GCA GAA CCT AAA ACA GTG GAA GAA GAG GAA AGT AAT TTC AGC TCC 10755 Ala Glu Pro Lys Thr Val Glu Glu Glu Glu Ser Asn Phe Ser Ser 3575 3580 3585
CCA CTG ATG CTT TGG CTT CAG CAA GAA CAA AAG CGG AAG GAA AGC 10800 Pro Leu Met Leu Trp Leu Gin Gin Glu Gin Lys Arg Lys Glu Ser 3590 3595 3600
ATT ACT GAG AAA AAA CCC AAG AAA GGA CTT GTT TTT GAA ATT TCC 10845 He Thr Glu Lys Lys Pro Lys Lys Gly Leu Val Phe Glu He Ser 3605 3610 3615
AGT GAT GAT GGC TTT CAG ATC TGT GCA GAA AGT ATT GAA GAT GCC 10890 Ser Asp Asp Gly Phe Gin He Cys Ala Glu Ser He Glu Asp Ala 3620 3625 3530
TGG AAG TCA TTG ACA GAT AAA GTC CAG GAA GCT CGA TCA AAT GCC 10935 Trp Lys Ser Leu Thr Asp Lys Val Gin Glu Ala Arg Ser Asn Ala 3535 3540 3545 CGC CTA AAG CAG CTC TCA TTT GCA GGT GTT AAC GGT TTG AGG ATG 10980 Arg Leu Lys Gin Leu Ser Phe Ala Gly Val Asn Gly Leu Arg Met 3550 3555 3560
CTG GGG ATT CTC CAT GAT GCA GTT GTG TTC CTC ATT GAG CAG CTG 11025 Leu Gly He Leu His Asp Ala Val Val Phe Leu He Glu Gin Leu 3565 3570 3575
TCT GGT GCC AAG CAC TGT CGA AAT TAC AAA TTC CGT TTC CAC AAG 11070 Ser Gly Ala Lys His Cys Arg Asn Tyr Lys Phe Arg Phe His Lys 3580 3585 . 3590
CCA GAG GAG GCC AAT GAA CCC CCC TTG AAC CCT CAC GGC TCA GCC 11115 Pro Glu Glu Ala Asn Glu Pro Pro Leu Asn Pro His Gly Ser Ala 3595 3600 3605
AGG GCT GAA GTC CAC CTC AGG AAG TCA GCA TTT GAC ATG TTT AAC 11160 Arg Ala Glu Val His Leu Arg Lys Ser Ala Phe Asp Met Phe Asn 3610 3615 3620
TTC CTG GCT TCT AAA CAT CGT CAG CCT CCT GAA TAC AAC CCC AAT 11205 Phe Leu Ala Ser Lys His Arg Gin Pro Pro Glu Tyr Asn Pro Asn 3625 3630 3635
GAT GAA GAA GAG GAG GAG GTA CAG CTG AAG TCA GCT CGG AGG GCA 11250 Asp Glu Glu Glu Glu Glu Val Gin Leu Lys Ser Ala Arg Arg Ala 3640 3645 3650
ACT AGC ATG GAT CTG CCA ATG CCC ATG CGC TTC CGG CAC TTA AAA 11295 Thr Ser Met Asp Leu Pro Met Pro Met Arg Phe Arg His Leu Lys 3655 3660 3665
AAG ACT TCT AAG GAG GCA GTT GGT GTC TAC AGG TCT CCC ATC CAT 11340 Lys Thr Ser Lys Glu Ala Val Gly Val Tyr Arg Ser Pro He His 3670 3675 3680
GGC CGG GGT CTT TTC TGT AAG AGA AAC ATT GAT GCA GGT GAG ATG 11385 Gly Arg Gly Leu Phe Cys Lys Arg Asn He Asp Ala Gly Glu Met 3685 3690 3695
GTG ATT GAG TAT GCC GGC AAC GTC ATC CGC TCC ATC CAG ACT GAC 11430 Val He Glu Tyr Ala Gly Asn Val He Arg Ser He Gin Thr Asp 3700 3705 3710
AAG CGG GAA AAG TAT TAC GAC AGC AAG GGC ATT GGT TGC TAT ATG 11 75 Lys Arg Glu Lys Tyr Tyr Asp Ser Lys Gly He Gly Cys Tyr Met 3715 3720 3725
TTC CGA ATT GAT GAC TCA GAG GTA GTG GAT GCC ACC ATG CAT GGA 11520 Phe Arg He Asp Asp Ser Glu Val Val Asp Ala Thr Met His Gly 3730 3735 3740
AAT GCT GCA CGC TTC ATC AAT CAC TCG TGT GAG CCT AAC TGC TAT 11565 Asn Ala Ala Arg Phe He Asn His Ser Cys Glu Pro Asn Cys Tyr 3745 3750 3755
TCT CGG GTC ATC AAT ATT GAT GGG CAG AAG CAC ATT GTC ATC TTT 11610 Ser Arg Val He Asn He Asp Gly Gin Lys His He Val He Phe 3760 3765 3770
GCC ATG CGT AAG ATC TAC CGA GGA GAG GAA CTC ACT TAC GAC TAT 11655 Ala Met Arg Lys He Tyr Arg Gly Glu Glu Leu Thr Tyr Asp Tyr 3775 3780 3785
AAG TTC CCC ATT GAG GAT GCC AGC AAC AAG CTG CCC TGC AAC TGT 11700 Lys Phe Pro He Glu Asp Ala Ser Asn Lys Leu Pro Cys Asn Cys 3790 3795 3800 GGC GCC AAG AAA TGC CGG AAG TTC CTA AAC TAA AGC TGC TCT TCT 11745 Gly Ala Lys Lys Cys Arg Lys Phe Leu Asn
3805 3810
CCCCCAGTGT TGGAGTGCAA GGAGGCGGGG CCATCCAAAG CAACG 11790
CTGAAGGCCT TTTCCAGCAG CTGGGAGCTC CCGGATTGCG TGGCACAGCT 11840
GAGGGGCCTC TGTGATGGCT GAGCTCTCTT ATGTCCTATA CTCACATCAG 11890
ACATGTGATC ATAGTCCCAG AGACAGAGTT GAGGTCTCGA AGAAAAGATC 11940
CATGATCGGC TTTCTCCTGG GGCCCCTCCA ATTGTTTACT GTTAGAAAGT 11990
GGGAATGGGG TCCCTAGCAG ACTTGCCTGG AAGGAGCCTA TTATAGAGGG 12040
TTGGTTATGT TGGGAGATTG GGCCTGAATT TCTCCACAGA AATAAGTTGC 12090
CATCCTCAGG TTGGCCCTTT CCCAAGCACT GTAAGTGAGT GGGTCAGCCA 12140
AAGCCCCAAA TGGAGGGTTG GTTAGATTCC TGACAGTTTG CCAGCCAGCC 12190
GCCACCTACA GCGTCTGTCG AACAAACAGA GGTCTGGTGG TTTTCCCTAC 12240
TGTCCTCCCA CTCGAGAGTT CACTTCTGGT TGGGAGACAG GATTCCTAGC 12290
ACCTCCGGTG TCAAAAGGCT GTCATGGGGT TGTGCCAATT AATTACCAAA 12340
CATTGAGCCT GCAGGCTTTG AGTGGGAGTG TTGCCCCCAG GAGCCTTATC 12390
TCAGCCAATT ACCTTTCTTG ACAGTAGGAG CGGCTTCCCT CTCCCATTCC 12440
CTCTTCACTC CCTTTTCTTC CTTTCCCCTG TCTTCATGCC ACTGCTTTCC 12490
CATGCTTCTT TCGGTTGTAG GGGAGACTGA CTGCCTGCTC AAGGACACTC 12540
CCTGCTGGGC ATAGGATGTG CCTGCAAAAA GTTCCCTGAG CCTGTAAGCA 12590
CTCCAGGTGG GGAAGTGGAC AGGAGCCATT GGTCATAACC AGACAGAATT 12640
TGGAAACATT TTCATAAAGC TCCATGGAGA GTTTTAAAGA AACATATGTA 12690
GCATGATTTT GTAGGAGAGG AAAAAGATTA TTTAAATAGG ATTTAAATCA 12740
TGCAACAACG AGAGTATCAC AGCCAGGATG ACCCTTGGGT CCCATTCCTA 12790
AGACATGGTT ACTTTATTTT CCCCTTGTTA AGACATAGGA AGACTTAATT 12840
TTTAAACGGT CAGTGTCCAG TTGAAGGCAG AACACTAATC AGATTTCAAG 12890
GCCCACAACT TGGGGACTAG ACCACCTTAT GTTGAGGGAA CTCTGCCACC 12940
TGCGTGCAAC CCACAGCTAA AGTAAATTCA ATGACACTAC TGCCCTGATT 12990
ACTCCTTAGG ATGTGGTCAA AACAGCATCA AATGTTTCTT CTCTTCCTTT 13040
CCCCAAGACA GAGTCCTGAA CCTGTTAAAT TAAGTCATTG GATTTTACTC 13090
TGTTCTGTTT ACAGTTTACT ATTTAAGGTT TTATAAATGT AAATATATTT 13140
TGTATATTTT TCTATGAGAA GCACTTCATA GGGAGAAGCA CTTATGACAA 13190
GGCTATTTTT TAAACCGCGG TATTATCCTA ATTTAAAAGA AGATCGGTTT 13240
TTAATAATTT TTTATTTTCA TAGGATGAAG TTAGAGAAAA TATTCAGCTG 13290 TACACACAAA GTCTGGTTTT TCCTGCCCAA CTTCCCCCTG GAAGGTGTAC 13340
TTTTTGTTGT TTAATGTGTA GCTTGTTTGT GCCCTGTTGA CATAAATGTT 13390
TCCTGGGTTT GCTCTTTGAC AATAAATGGA GAAGGAAGGT CACCCAACTC 13440
CATTGGGCCA CTCCCCTCCT TCCCCTATTG AAGCTCCTCA AAAGGCTACA 13490
GTAATATCTT GATACAACAG ATTCTCTTCT TTCCCGCCTC TCTCCTTTCC 13540
GGCGCAACTT CCAGAGTGGT GGGAGACGGC AATCTTTACA TTTCCCTCAT 13590
CTTTCTTACT TCAGAGTTAG CAAACAACAA GTTGAATGGC AACTTGACAT 13640
TTTTGCATCA CCATCTGCCT CATAGGCCAC TCTTTCCTTT CCCTCTGCCC 13690
ACCAAGTCCT CATATCTGCA GAGAACCCAT TGATCACCTT GTGCCCTCTT 13740
TTGGGGCAGC CTGTTGAAAC TGAAGCACAG TCTGACCACT CACGATAAAG 13790
CAGATTTTCT CTGCCTCTGC CACAAGGTTT CAGAGTAGTG TAGTCCAAGT 13840
AGAGGGTGGG GCACCCTTTT CTCGCCGCAA GAAGCCCATT CCTATGGAAG 13890
TCTAGCAAAG CAATACGACT CAGCCCAGCA CTCTCTGCCC CAGGACTCAT 13940
GGCTCTGCTG TGCCTTCCAT CCTGGGCTCC CTTCTCTCCT GTGACCTTAA 13990
GAACTTTGTC TGGTGGCTTT GCTGGAACAT TGTCACTGTT TTCACTGTCA 14040
TGCAGGGAGC CCAGCACTGT GGCCAGGATG GCAGAGACTT CCTTGTCATC 14090
ATGGAGAAGT GCCAGCAGGG GACTGGGAAA AGCACTCTAC CCAGACCTCA 14140
CCTCCCTTCC TCCTTTTGCC CATGAACAAG ATGCAGTGGC CCTAGGGGTT 14190
CCACTAGTGT CTGCTTTCCT TTATTATTGC ACTGTGTGAG GTTTTTTTGT 14240
AAATCCTTGT ATTCC 14255
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Arg Ala Leu Cys Phe Leu Cys Gly Ser Thr Gly Leu Asp Pro Leu 5 10 15
He Phe Cys Ala Cys Cys Cys Glu Pro Tyr His Gin Tyr Cys Val 20 25 30
Gin Asp Glu Tyr Asn Leu Lys His Gly Ser Phe Glu Asp Thr Thr 35 40 45
Leu Met Gly Ser Leu Leu Glu Thr Thr Val Asn Ala Ser Thr Gly 50 55 60
Pro Ser Ser Ser Leu Asn Gin Leu Thr Gin Arg Leu Asn Trp Leu 65 70 75
Cys Pro Arg Cys Thr Val Cys Tyr Thr Cys Asn Met Ser Ser Gly 80 85 90
Ser Lys Val Lys Cys Gin Lys Cys Gin Lys Asn Tyr His Ser Thr 95 100 105 Cys Leu Gly Thr Ser Lys Arg Leu Leu Gly Ala Asp Arg Pro Leu 110 115 120
He Cys Val Asn Cys Leu Lys Cys Lys Ser Cys Ser Thr Thr Lys 125 130 135
Val Ser Lys Phe Val Gly Asn Leu Pro Met Cys Thr Gly Cys Phe 140 145 150
Lys Leu Arg Lys Lys Gly Asn Phe Cys Pro He Cys Gin Arg Cys 155 160 165
Tyr Asp Asp Asn Asp Phe Asp Leu Lys Met Met Glu Cys Gly Asp 170 175 180
Cys Gly Gin Trp Val His Ser Lys Cys Glu Gly Leu Ser Asp Glu 185 190 195
Gin Tyr Asn Leu Leu Ser Thr Leu Pro Glu Ser He Glu Phe He 200 205 210
Cys Lys Lys Cys Ala Arg Arg Asn 215
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 109
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Asp Thr Arg Met Cys Leu Phe Cys Arg Lys Ser Gly Glu Gly Leu 5 10 15
Ser Gly Glu Glu Ala Arg Leu Leu Tyr Cys Gly His Asp Cys Trp 20 25 30
Val His Thr Asn Cys Ala Met Trp Ser Ala Glu Val Phe Glu Glu 35 40 45
He Asp Gly Ser Leu Gin Asn Val His Ser Ala Val Ala Arg Gly 50 55 60
Arg Met He Lys Cys Thr Val Cys Gly Asn Arg Gly Ala Thr Val 65 70 75
Gly Cys Asn Val Arg Ser Cys Gly Glu His Tyr His Tyr Pro Cys 80 85 90
Ala Arg Ser He Asp Cys Ala Phe Leu Thr Asp Lys Ser Met Tyr 95 100 105
Cys Pro Ala His 109
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 210
(B) TYPE: amino acid (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Glu Leu Glu Glu Asn Ala Tyr Asp Cys Ala Arg Cys Glu Pro Tyr 5 10 15
Ser Asn Arg Ser Glu Tyr Asp Met Phe Ser Trp Leu Ala Ser Arg 20 25 30
His Arg Lys Gin Pro He Gin Val Phe Val Gin Pro Ser Asp Asn 35 40 45
Glu Leu Val Pro Arg Arg Gly Thr Gly Ser Asn Leu Pro Met Ala 50 55 60
Met Lys Tyr Arg Thr Leu Lys Glu Thr Tyr Lys Asp Tyr Val Gly 65 70 75
Val Phe Arg Ser His He His Gly Arg Gly Leu Tyr Cys Thr Lys 80 85 90
Asp He Glu Ala Gly Glu Met Val He Glu Tyr Ala Gly Glu Leu 95 100 105
He Arg Ser Thr Leu Thr Asp Lys Arg Glu Arg Tyr Tyr Asp Ser 110 115 120
Arg Gly He Gly Cys Tyr Met Phe Lys He Asp Asp Asn Leu Val 125 130 135
Val Asp Ala Thr Met Arg Gly Asn Ala Ala Arg Phe He Asn His 140 145 150
Cys Cys Glu Pro Asn Cys Tyr Ser Lys Val Val Asp He Leu Gly 155 160 165
His Lys His He He He Phe Ala Val Arg Arg He Val Gin Gly 170 175 180
Glu Glu Leu Thr Tyr Asp Tyr Lys Phe Pro Phe Glu Asp Glu Lys 185 190 195
He Pro Cys Ser Cys Gly Ser Lys Arg Cys Arg Lys Tyr Leu Asn 200 205 210
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TGAATTTTTT AGGTCCA 17
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) AN I-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAAAAGGTGA GGAGAG 16 ( 2 ) INFORMATION FOR SEQ ID NO : 7 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: TTGGCTCCTT CGGAAAAA 18
(2) INFORMATION FOR SEQ ID NO: 8 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCIPTION: SEQ ID NO: 8: TTTAAGGTAA AGGTGT 16
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTCTCTCCAC AGGAGGAT 18
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATAGAGGTAA GGCATC 16
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear ( iv) ANTI - SENSE : No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTCTTACTAT AGTTTGTG 18
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ACAAAGGTAC AAAACT 16
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATTTTCTTAC AGCAGCTG 18
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GTCTGGGTGA GTTATA 16
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CTTCTTTTCT AGATCTGT 18 (2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
AAAGGTACCC AAAA 14
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
CTTTGCTTTC AGGAAAC 17
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: GAAGGTTGGA GTCT 14
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
GTT GCA ATG CAG CAG AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT 45 Val Ala Met Gin Gin Lys Pro Thr Ala Tyr Val Arg Pro Met Asp
5 10 15
GGT CAA GAT CAG GCC CCT AGT GAA TCC CCT GAA CTG AAA CCA CTG 90 Gly Gin Asp Gin Ala Pro Ser Glu Ser Pro Glu Leu Lys Pro Leu 20 25 30 CCG GAG GAC TAT CGA CAG CAG ACC TTT GAA AAA ACA GAC TTG AAA 135 Pro Glu Asp Tyr Arg Gin Gin Thr Phe Glu Lys Thr Asp Leu Lys 35 40 45
GTG CCT GCC AAA GCC AAG CTC ACC AAA CTG AAG ATG CCT TCT CAG 180 Val Pro Ala Lys Ala Lys Leu Thr Lys Leu Lys Met Pro Ser Gin 50 55 60
TCA GTT GAG 189
Ser Val Glu 63
(2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
TTT GTG TAT TGC CAA GTC TGT TGT GAG CCC TTC CAC AAG TTT TGT 45 Phe Val Tyr Cys Gin Val Cys Cys Glu Pro Phe His Lys Phe Cys 5 10 15
TTA GAG GAG AAC GAG CGC CCT CTG GAG GAC CAG CTG GAA AAT TGG 90 Leu Glu Glu Asn Glu Arg Pro Leu Glu Asp Gin Leu Glu Asn Trp 20 25 30
TGT TGT CGT CGT TGC AAA TTC TGT CAC GTT TGT GGA AGG CAA CAT 135 Cys Cys Arg Arg Cys Lys Phe Cys His Val Cys Gly Arg Gin His 35 40 45
CAG GCT ACA AAG 147
Gin Ala Thr Lys 49
(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 132
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
GAA AAA CCA CCT CCG GTC AAT AAG CAG GAG AAT GCA GGC ACT TTG 45 Glu Lys Pro Pro Pro Val Asn Lys Gin Glu Asn Ala Gly Thr Leu 5 10 15
AAC ATC TTC AGC ACT CTC TCC AAT GGC AAT AGT TCT AAG CAA AAA 90 Asn He Phe Ser Thr Leu Ser Asn Gly Asn Ser Ser Lys Gin Lys 20 25 30
ATT CCA GCA GAT GGA GTC CAC AGG ATC AGA GTG GAC TTT AAG 132 He Pro Ala Asp Gly Val His Arg He Arg Val Asp Phe Lys 35 40
(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
ACC TAC TCC AAT GAA GTC CAT TGT GTT GAA GAG ATT CTG AAG GAA 45 Thr Tyr Ser Asn Glu Val His Cys Val Glu Glu He Leu Lys Glu 5 10 15
ATG ACC CAT TCA TGG CCG CCT CCT TTG ACA GCA ATA CAT ACG CCT 90 Met Thr His Ser Trp Pro Pro Pro Leu Thr Ala He His Thr Pro 20 25 30
AGT ACA GCT GAG CCA TCC AAG TTT CCT TTC CCT ACA AAG GAC TCT 135 Ser Thr Ala Glu Pro Ser Lys Phe Pro Phe Pro Thr Lys Asp Ser 35 40 45
CAG CAT GTC AGT TCT GTA ACC CAA AAC CAA AAA CAA TAT GAT ACA 180 Gin His Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr 50 55 60
TCT TCA AAA ACT CAC TCA AAT TCT CAG CAA GGA ACG TCA TCC ATG 225 Ser Ser Lys Thr His Ser Asn Ser Gin Gin Gly Thr Ser Ser Met 65 70 75
CTC GAA GAC GAC CTT CAG CTC AGT GAC AGT GAG GAC AGT GAC AGT 270 Leu Glu Asp Asp Leu Gin Leu Ser Asp Ser Glu Asp Ser Asp Ser 80 85 90
(2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
GTT GCA ATG CAG CAG AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT 45 Val Ala Met Gin Gin Lys Pro Thr Ala Tyr Val Arg Pro Met Asp 5 10 15
GGT CAA GAT CAG GCC CCT AGT GAA TCC CCT GAA CTG AAA CCA CTG 90 Gly Gin Asp Gin Ala Pro Ser Glu Ser Pro Glu Leu Lys Pro Leu 20 25 30
CCG GAG GAC TAT CGA CAG CAG ACC TTT GAA AAA ACA GAC TTG AAA 135 Pro Glu Asp Tyr Arg Gin Gin Thr Phe Glu Lys Thr Asp Leu Lys 35 40 45
GTG CCT GCC AAA GCC AAG CTC ACC AAA CTG AAG ATG CCT TCT CAG 180 Val Pro Ala Lys Ala Lys Leu Thr Lys Leu Lys Met Pro Ser Gin 50 55 60
TCA GTT GAG TTT GTG TAT TGC CAA GTC TGT TGT GAG CCC TTC CAC 225 Ser Val Glu Phe Val Tyr Cys Gin Val Cys Cys Glu Pro Phe His 65 70 75
AAG TTT TGT TTA GAG GAG AAC GAG CGC CCT CTG GAG GAC CAG CTG 270 Lys Phe Cys Leu Glu Glu Asn Glu Arg Pro Leu Glu Asp Gin Leu 80 85 90 GAA AAT TGG TGT TGT CGT CGT TGC AAA TTC TGT CAC GTT TGT GGA 315 Glu Asn Trp Cys Cys Arg Arg Cys Lys Phe Cys His Val Cys Gly 95 100 105
AGG CAA CAT CAG GCT ACA AAG 336
Arg Gin His Gin Ala Thr Lys 110
(2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(iv) ANTI-SENSE: No
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
GAA AAA CCA CCT CCG GTC AAT AAG CAG GAG AAT GCA GGC ACT TTG 45 Glu Lys Pro Pro Pro Val Asn Lys Gin Glu Asn Ala Gly Thr Leu 5 10 15
AAC ATC TTC AGC ACT CTC TCC AAT GGC AAT AGT TCT AAG CAA AAA 90 Asn He Phe Ser Thr Leu Ser Asn Gly Asn Ser Ser Lys Gin Lys 20 25 30
ATT CCA GCA GAT GGA GTC CAC AGG ATC AGA GTG GAC TTT AAG ACC 135 He Pro Ala Asp Gly Val His Arg He Arg Val Asp Phe Lys Thr 35 40 45
TAC TCC AAT GAA GTC CAT TGT GTT GAA GAG ATT CTG AAG GAA ATG 180 Tyr Ser Asn Glu Val His Cys Val Glu Glu He Leu Lys Glu Met 50 55 60
ACC CAT TCA TGG CCG CCT CCT TTG ACA GCA ATA CAT ACG CCT AGT 225 Thr His Ser Trp Pro Pro Pro Leu Thr Ala He His Thr Pro Ser 65 70 75
ACA GCT GAG CCA TCC AAG TTT CCT TTC CCT ACA AAG GAC TCT CAG 279 Thr Ala Glu Pro Ser Lys Phe Pro Phe Pro Thr Lys Asp Ser Gin 80 85 90
CAT GTC AGT TCT GTA ACC CAA AAC CAA AAA CAA TAT GAT ACA TCT 315 His Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr Ser 95 100 105
TCA AAA ACT CAC TCA AAT TCT CAG CAA GGA ACG TCA TCC ATG CTC 360 Ser Lys Thr His Ser Asn Ser Gin Gin Gly Thr Ser Ser Met Leu 110 115 120
GAA GAC GAC CTT CAG CTC AGT GAC AGT GAG GAC AGT GAC AGT 402 Glu Asp Asp Leu Gin Leu Ser Asp Ser Glu Asp Ser Asp Ser 125 130
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9391 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) ( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 421..4053
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GGCAATTTCT TTTCCTTTCT AACTGTGGCC CGCGTTGTGC TGTTGCTGGG CAGGCGTTGG 60
GCGCCGGCGG TCTTCGAGCG TGGGGGCCCG CTGGCTTTCC CTTCTCAGAA ACTGCGCCGG 120
GGGCGCTCGC TTGCCCCGGA TTCGGACGCG GCGCTCCCCG GGCTCGTCTG AAGTGCAGAT 180
CGCCGCAGAG GCCCCAGTGC CCGGATGTCC ATCAGGATTA GCGCGAGCCA ATACGGGCCG 240
AGCCCGGGGC TGCGCCGAGG ACGCCCGGGG CTCGAGAGCA GGTAGTCCCG TAACATCGGG 300
GCGCCGCGCC GGGACGCGTC CCCGCCCGGC TCCGCCAAAT GGTGAGCGCG GCGCTGGCAG 360
CAGGGCCCGC GGGGTGAAGG CGCTCATGGA CGGAAGACCC CTGGCTCTAT AAGCTGAATT 420
ATG GCA GCC CAG TCA AGT TTG TAC AAT GAC GAC AGA AAC CTG CTT CGA 468 Met Ala Ala Gin Ser Ser Leu Tyr Asn Asp Asp Arg Asn Leu Leu Arg 1 5 10 15
ATT AGA GAG AAG GAA AGA CGC AAC CAG GAA GCC CAC CAA GAG AAA GAG 516 He Arg Glu Lys Glu Arg Arg Asn Gin Glu Ala His Gin Glu Lys Glu 20 25 30
GCA TTT CCT GAA AAG ATT CCC CTT TTT GGA GAG CCC TAC AAG ACA GCA 564 Ala Phe Pro Glu Lys He Pro Leu Phe Gly Glu Pro Tyr Lys Thr Ala 35 40 45
AAA GGT GAT GAG CTG TCT AGT CGA ATA CAG AAC ATG TTG GGA AAC TAC 612 Lys Gly Asp Glu Leu Ser Ser Arg He Gin Asn Met Leu Gly Asn Tyr 50 55 60
GAA GAA GTG AAG GAG TTC CTT AGT ACT AAG TCT CAC ACT CAT CGC CTG 660 Glu Glu Val Lys Glu Phe Leu Ser Thr Lys Ser His Thr His Arg Leu 65 70 75 80
GAT GCT TCT GAA AAT AGG TTG GGA AAG CCG AAA TAT CCT TTA ATT CCT 708 Asp Ala Ser Glu Asn Arg Leu Gly Lys Pro Lys Tyr Pro Leu He Pro 85 90 95
GAC AAA GGG AGC AGC ATT CCA TCC AGC TCC TTC CAC ACT AGT GTC CAC 756 Asp Lys Gly Ser Ser He Pro Ser Ser Ser Phe His Thr Ser Val His 100 105 110
CAC CAG TCC ATT CAC ACT CCT GCG TCT GGA CCA CTT TCT GTT GGC AAC 804 His Gin Ser He His Thr Pro Ala Ser Gly Pro Leu Ser Val Gly Asn 115 120 125
ATT AGC CAC AAT CCA AAG ATG GCG CAG CCA AGA ACT GAA CCA ATG CCA 852 He Ser His Asn Pro Lys Met Ala Gin Pro Arg Thr Glu Pro Met Pro 130 135 140
AGT CTC CAT GCC AAA AGC TGC GGC CCA CCG GAC AGC CAG CAC CTG ACC 900 Ser Leu His Ala Lys Ser Cys Gly Pro Pro Asp Ser Gin His Leu Thr 145 150 155 160
CAG GAT CGC CTT GGT CAG GAG GGG TTC GGC TCT AGT CAT CAC AAG AAA 948 Gin Asp Arg Leu Gly Gin Glu Gly Phe Gly Ser Ser His His Lys Lys 165 170 175
GGT GAC CGA AGA GCT GAC GGA GAC CAC TGT GCT TCG GTG ACA GAT TCG 996 Gly Asp Arg Arg Ala Asp Gly Asp His Cys Ala Ser Val Thr Asp Ser 180 185 190
GCT CCA GAG AGG GAG CTT TCT CCC TTA ATC TCT TTG CCT TCC CCA GTT 1044 Ala Pro Glu Arg Glu Leu Ser Pro Leu He Ser Leu Pro Ser Pro Val 195 200 205
CCC CCT TTG TCA CCT ATA CAT TCC AAC CAG CAA ACT CTT CCC CGG ACG 1092 Pro Pro Leu Ser Pro He His Ser Asn Gin Gin Thr Leu Pro Arg Thr 210 215 220
CAA GGA AGC AGC AAG GTT CAT GGC AGC AGC AAT AAC AGT AAA GGC TAT 1140 Gin Gly Ser Ser Lys Val His Gly Ser Ser Asn Asn Ser Lys Gly Tyr 225 230 235 240
TGC CCA GCC AAA TCT CCC AAG GAC CTA GCA GTG AAA GTC CAT GAT AAA 1188 Cys Pro Ala Lys Ser Pro Lys Asp Leu Ala Val Lys Val His Asp Lys 245 250 255
GAG ACC CCT CAA GAC AGT TTG GTG GCC CCT GCC CAG CCG CCT TCT CAG 1236 Glu Thr Pro Gin Asp Ser Leu Val Ala Pro Ala Gin Pro Pro Ser Gin 260 265 270
ACA TTT CCA CCT CCC TCC CTC CCC TCA AAA AGT GTT GCA ATG CAG CAG 1284 Thr Phe Pro Pro Pro Ser Leu Pro Ser Lys Ser Val Ala Met Gin Gin 275 280 285
AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT GGT CAA GAT CAG GCC CCT 1332 Lys Pro Thr Ala Tyr Val Arg Pro Met Asp Gly Gin Asp Gin Ala Pro 290 295 300
AGT GAA TCC CCT GAA CTG AAA CCA CTG CCG GAG GAC TAT CGA CAG CAG 1380 Ser Glu Ser Pro Glu Leu Lys Pro Leu Pro Glu Asp Tyr Arg Gin Gin 305 310 315 320
ACC TTT GAA AAA ACA GAC TTG AAA GTG CCT GCC AAA GCC AAG CTC ACC 1428 Thr Phe Glu Lys Thr Asp Leu Lys Val Pro Ala Lys Ala Lys Leu Thr 325 330 335
AAA CTG AAG ATG CCT TCT CAG TCA GTT GAG CAG ACC TAC TCC AAT GAA 1476 Lys Leu Lys Met Pro Ser Gin Ser Val Glu Gin Thr Tyr Ser Asn Glu 340 345 350
GTC CAT TGT GTT GAA GAG ATT CTG AAG GAA ATG ACC CAT TCA TGG CCG 1524 Val His Cys Val Glu Glu He Leu Lys Glu Met Thr His Ser Trp Pro 355 360 365
CCT CCT TTG ACA GCA ATA CAT ACG CCT AGT ACA GCT GAG CCA TCC AAG 1572 Pro Pro Leu Thr Ala He His Thr Pro Ser Thr Ala Glu Pro Ser Lys 370 375 380
TTT CCT TTC CCT ACA AAG GAC TCT CAG CAT GTC AGT TCT GTA ACC CAA 1620 Phe Pro Phe Pro Thr Lys Asp Ser Gin His Val Ser Ser Val Thr Gin 385 390 395 400
AAC CAA AAA CAA TAT GAT ACA TCT TCA AAA ACT CAC TCA AAT TCT CAG 1668 Asn Gin Lys Gin Tyr Asp Thr Ser Ser Lys Thr His Ser Asn Ser Gin 405 410 415
CAA GGA ACG TCA TCC ATG CTC GAA GAC GAC CTT CAG CTC AGT GAC AGT 1716 Gin Gly Thr Ser Ser Met Leu Glu Asp Asp Leu Gin Leu Ser Asp Ser 420 425 430
GAG GAC AGT GAC AGT GAA CAA ACC CCA GAG AAG CCT CCC TCC TCA TCT 1764 Glu Asp Ser Asp Ser Glu Gin Thr Pro Glu Lys Pro Pro Ser Ser Ser 435 440 445
GCA CCT CCA AGT GCT CCA CAG TCC CTT CCA GAA CCA GTG GCA TCA GCA 1812 Ala Pro Pro Ser Ala Pro Gin Ser Leu Pro Glu Pro Val Ala Ser Ala 450 455 460
CAT TCC AGC AGT GCA GAG TCA GAA AGC ACC AGT GAC TCA GAC AGT TCC 1860 His Ser Ser Ser Ala Glu Ser Glu Ser Thr Ser Asp Ser Asp Ser Ser 465 470 475 480
TCA GAC TCA GAG AGC GAG AGC AGT TCA AGT GAC AGC GAA GAA AAT GAG 1908 Ser Asp Ser Glu Ser Glu Ser Ser Ser Ser Asp Ser Glu Glu Asn Glu 485 490 495
CCC CTA GAA ACC CCA GCT CCG GAG CCT GAG CCT CCA ACA ACA AAC AAA 1956 Pro Leu Glu Thr Pro Ala Pro Glu Pro Glu Pro Pro Thr Thr Asn Lys 500 505 510
TGG CAG CTG GAC AAC TGG CTG ACC AAA GTC AGC CAG CCA GCT GCG CCA 2004 Trp Gin Leu Asp Asn Trp Leu Thr Lys Val Ser Gin Pro Ala Ala Pro 515 520 525
CCA GAG GGC CCC AGG AGC ACA GAG CCC CCA CGG CGG CAC CCA GAG AGT 2052 Pro Glu Gly Pro Arg Ser Thr Glu Pro Pro Arg Arg His Pro Glu Ser 530 535 540
AAG GGC AGC AGC GAC AGT GCC ACG AGT CAG GAG CAT TCT GAA TCC AAA 2100 Lys Gly Ser Ser Asp Ser Ala Thr Ser Gin Glu His Ser Glu Ser Lys 545 550 555 560
GAT CCT CCC CCT AAA AGC TCC AGC AAA GCC CCC CGG GCC CCA CCC GAA 2148 Asp Pro Pro Pro Lys Ser Ser Ser Lys Ala Pro Arg Ala Pro Pro Glu 565 570 575
GCC CCC CAC CCC GGA AAG AGG AGC TGT CAG AAG TCT CCG GCA CAG CAG 2196 Ala Pro His Pro Gly Lys Arg Ser Cys Gin Lys Ser Pro Ala Gin Gin 580 585 590
GAG CCC CCA CAA AGG CAA ACC GTT GGA ACC AAA CAA CCC AAA AAA CCT 2244 Glu Pro Pro Gin Arg Gin Thr Val Gly Thr Lys Gin Pro Lys Lys Pro 595 600 605
GTC AAG GCC TCT GCC CGG GCA GGT TCA CGG ACC AGC CTG CAG GGG GAA 2292 Val Lys Ala Ser Ala Arg Ala Gly Ser Arg Thr Ser Leu Gin Gly Glu 610 615 620
AGG GAG CCA GGG CTT CTT CCC TAT GGC TCC CGA GAC CAG ACT TCC AAA 2340 Arg Glu Pro Gly Leu Leu Pro Tyr Gly Ser Arg Asp Gin Thr Ser Lys 625 630 635 640
GAC AAG CCC AAG GTG AAG ACG AAA GGA CGG CCC CGG GCC GCA GCA AGC 2388 Asp Lys Pro Lys Val Lys Thr Lys Gly Arg Pro Arg Ala Ala Ala Ser 645 650 655
AAC GAA CCC AAG CCA GCA GTG CCC CCC TCC AGT GAG AAG AAG AAG CAC 2436 Asn Glu Pro Lys Pro Ala Val Pro Pro Ser Ser Glu Lys Lys Lys His 660 665 670
AAG AGC TCC CTC CCT GCC CCC TCT AAG GCT CTC TCA GGC CCA GAA CCC 2484 Lys Ser Ser Leu Pro Ala Pro Ser Lys Ala Leu Ser Gly Pro Glu Pro 675 680 685
GCG AAG GAC AAT GTG GAG GAC AGG ACC CCT GAG CAC TTT GCT CTT GTT 2532 Ala Lys Asp Asn Val Glu Asp Arg Thr Pro Glu His Phe Ala Leu Val 690 695 700
CCC CTG ACT GAG AGC CAG GGC CCA CCC CAC AGT GGC AGC GGC AGC AGG 2580 Pro Leu Thr Glu Ser Gin Gly Pro Pro His Ser Gly Ser Gly Ser Arg 705 710 715 720 ACT AGT GGC TGC CGC CAA GCC GTG GTG GTC CAG GAG GAC AGC CGC AAA 2628 Thr Ser Gly Cys Arg Gin Ala Val Val Val Gin Glu Asp Ser Arg Lys 725 730 735
GAC AGA CTC CCA TTG CCT TTG AGA GAC ACC AAG CTG CTC TCA CCG CTC 2676 Asp Arg Leu Pro Leu Pro Leu Arg Asp Thr Lys Leu Leu Ser Pro Leu 740 745 750
AGG GAC ACT CCT CCC CCA CAA AGC TTG ATG GTG AAG ATC ACC CTA GAC 2724 Arg Asp Thr Pro Pro Pro Gin Ser Leu Met Val Lys He Thr Leu Asp 755 760 765
CTG CTC TCT CGG ATA CCC CAG CCT CCC GGG AAG GGG AGC CGC CAG AGG 2772 Leu Leu Ser Arg He Pro Gin Pro Pro Gly Lys Gly Ser Arg Gin Arg 770 775 780
AAA GCA GAA GAT AAA CAG CCG CCC GCA GGG AAG AAG CAC AGC TCT GAG 2820 Lys Ala Glu Asp Lys Gin Pro Pro Ala Gly Lys Lys His Ser Ser Glu 785 790 795 800
AAG AGG AGC TCA GAC AGC TCA AGC AAG TTG GCC AAA AAG AGA AAG GGT 2868 Lys Arg Ser Ser Asp Ser Ser Ser Lys Leu Ala Lys Lys Arg Lys Gly 805 810 815
GAA GCA GAA AGA GAC TGT GAT AAC AAG AAA ATC AGA CTG GAG AAG GAA 2916 Glu Ala Glu Arg Asp Cys Asp Asn Lys Lys He Arg Leu Glu Lys Glu 820 825 830
ATC AAA TCA CAG TCA TCT TCA TCT TCA TCC TCC CAC AAA GAA TCT TCT 2964 He Lys Ser Gin Ser Ser Ser Ser Ser Ser Ser His Lys Glu Ser Ser 835 840 845
AAA ACA AAG CCC TCC AGG CCC TCC TCA CAG TCC TCA AAG AAG GAA ATG 3012 Lys Thr Lys Pro Ser Arg Pro Ser Ser Gin Ser Ser Lys Lys Glu Met 850 855 860
CTC CCC CCG CCA CCC GTG TCC TCG TCC TCC CAG AAG CCA GCC AAG CCT 3060 Leu Pro Pro Pro Pro Val Ser Ser Ser Ser Gin Lys Pro Ala Lys Pro 865 870 875 880
GCA CTT AAG AGG TCA AGG CGG GAA GCA GAC ACC TGT GGC CAG GAC CCT 3108 Ala Leu Lys Arg Ser Arg Arg Glu Ala Asp Thr Cys Gly Gin Asp Pro 885 890 895
CCC AAA AGT GCC AGC AGT ACC AAG AGC AAC CAC AAA GAC TCT TCC ATT 3156 Pro Lys Ser Ala Ser Ser Thr Lys Ser Asn His Lys Asp Ser Ser He 900 905 9.10
CCC AAG CAG AGA AGA GTA GAG GGG AAG GGC TCC AGA AGC TCC TCG GAG 3204 Pro Lys Gin Arg Arg Val Glu Gly Lys Gly Ser Arg Ser Ser Ser Glu 915 920 925
CAC AAG GGT TCT TCC GGA GAT ACT GCA AAT CCT TTT CCA GTG CCT TCT 3252 His Lys Gly Ser Ser Gly Asp Thr Ala Asn Pro Phe Pro Val Pro Ser 930 935 940
TTG CCA AAT GGT AAC TCT AAA CCA GGG AAG CCT CAA GTG AAG TTT GAC 3300 Leu Pro Asn Gly Asn Ser Lys Pro Gly Lys Pro Gin Val Lys Phe Asp 945 950 955 960
AAA CAA CAA GCA GAC CTT CAC ATG AGG GAG GCA AAA AAG ATG AAG CAG 3348 Lys Gin Gin Ala Asp Leu His Met Arg Glu Ala Lys Lys Met Lys Gin 965 970 975
AAA GCA GAG TTA ATG ACG GAC AGG GTT GGA AAG GCT TTT AAG TAC CTG 3396 Lys Ala Glu Leu Met Thr Asp Arg Val Gly Lys Ala Phe Lys Tyr Leu 980 985 990 GAA GCC GTC TTG TCC TTC ATT GAG TGC GGA ATT GCC ACA GAG TCT GAA 3444 Glu Ala Val Leu Ser Phe He Glu Cys Gly He Ala Thr Glu Ser Glu 995 1000 1005
AGC CAG TCA TCC AAG TCA GCT TAC TCT GTC TAC TCA GAA ACT GTA GAT 3492 Ser Gin Ser Ser Lys Ser Ala Tyr Ser Val Tyr Ser Glu Thr Val Asp 1010 1015 1020
CTC ATT AAA TTC ATA ATG TCA TTA AAA TCC TTC TCA GAT GCC ACA GCG 3540 Leu He Lys Phe He Met Ser Leu Lys Ser Phe Ser Asp Ala Thr Ala 1025 1030 1035 1040
CCA ACA CAA GAG AAA ATA TTT GCT GTT TTA TGC ATG CGT TGC CAG TCC 3588 Pro Thr Gin Glu Lys He Phe Ala Val Leu Cys Met Arg Cys Gin Ser 1045 1050 1055
ATT TTG AAC ATG GCG ATG TTT CGT TGT AAA AAA GAC ATA GCA ATA AAG 3636 He Leu Asn Met Ala Met Phe Arg Cys Lys Lys Asp He Ala He Lys 1060 1065 1070
TAT TCT CGT ACT CTT AAT AAA CAC TTC GAG AGT TCT TCC AAA GTC GCC 3684 Tyr Ser Arg Thr Leu Asn Lys His Phe Glu Ser Ser Ser Lys Val Ala 1075 1080 1085
CAG GCA CCT TCT CCA TGC ATT GCA AGC ACA GGC ACA CCA TCC CCT CTT 3732 Gin Ala Pro Ser Pro Cys He Ala Ser Thr Gly Thr Pro Ser Pro Leu 1090 1095 1100
TCC CCA ATG CCT TCT CCT GCC AGC TCC GTA GGG TCC CAG TCA AGT GCT 3780 Ser Pro Met Pro Ser Pro Ala Ser Ser Val Gly Ser Gin Ser Ser Ala 1105 1110 1115 1120
GGC AGT GTG GGG AGC AGT GGG GTG GCT GCC ACT ATC AGC ACC CCA GTC 3828 Gly Ser Val Gly Ser Ser Gly Val Ala Ala Thr He Ser Thr Pro Val 1125 1130 1135
ACC ATC CAG AAT ATG ACA TCT TCC TAT GTC ACC ATC ACA TCC CAT GTT 3876 Thr He Gin Asn Met Thr Ser Ser Tyr Val Thr He Thr Ser His Val 1140 1145 1150
CTT ACC GCC TTT GAC CTT TGG GAA CAG GCC GAG GCC CTC ACG AGG AAG 3924 Leu Thr Ala Phe Asp Leu Trp Glu Gin Ala Glu Ala Leu Thr Arg Lys 1155 1160 1165
AAT AAA GAA TTC TTT GCT CGG CTC AGC ACA AAT GTG TGC ACC TTG GCC 3972 Asn Lys Glu Phe Phe Ala Arg Leu Ser Thr Asn Val Cys Thr Leu Ala 1170 1175 1180
CTC AAC AGC AGT TTG GTG GAC CTG GTG CAC TAT ACA CGA CAG GGT TTT 4020 Leu Asn Ser Ser Leu Val Asp Leu Val His Tyr Thr Arg Gin Gly Phe 1185 1190 1195 1200
CAG CAG CTA CAA GAA TTA ACC AAA ACA CCT TAATGGAGCC CCAGGTTGAT 4070 Gin Gin Leu Gin Glu Leu Thr Lys Thr Pro 1205 1210
TCAATGCCTT GGGAACTATT TTTGCACATT GGAAGCCTCA AAAACAGTCC AGACGTTTGT 4130
TTCATCAGGA CACCAAACTC TAAAAAAGAA GCACCACGAG ATGGCCAGGA CATTTGTCCA 4190
CTTAAACTCT CAACAACAGT GTGATCATTG GTTGGACACT GTGGTTATGC AGAAGCAGAG 4250
ATGAGGAGGC TGGCCCCAGA GATGATCTTG CCCTTCCTAA CTAAAGGACA GAAGTGCAAT 4310
TTAGCTTAAA TGGGTGTATG AATGGTCTAG AAACATTTCT ATTTTTTTTT TAAACCAGCA 4370
GGATACAAGT TGCAAATGAA ATGAGGAGAA ACAGTTTCAA CTCTGAAAGT GAATTTCACG 4430 TCATCTCAGT AGCCACGCTA GTCCATTCCC AGAAGGAAAT TTTTTTTTTT AACAATGACT 4490
TTTGGT.,j-G GGTTTTGTGG ATGATTTTTT TTCTTTTGAG TTTTGGGAGA AATATTTGTT 4550
TAATAACTTC TAATGGCCAT CTGTAAACCA TAAGTAATGA AGGACTCCAC TGTGCCCCAC 4610
TTTCTGCCAA TGAACAGTGG CTTGATAATA CCAAGTATTG TTGTAATTTA TAAAATTGAA 4670
GGCAACCCCC GCTCCTGCCG CCCCCAATCT CCCCATTGCC TAGAGCGCTG CACATTGACC 4730
CCAGCTCTGA CTTCTCATTA CTGTGCTGAA AGTCAGCCCA CGTCGGAGCG GTGAGGAGGA 4790
GCCACAGCAC ATGGGGTGCC ACCTCGAGGT CTGCACAGGA GGACTTGGCG CTGCCATTTC 4850
CTACCCCTGC CATTTCCCAC CCCTGCTTCA GCGAAAGGGA CTCTCTAACA GGGCAGTCAC 4910
TGTTGACTCT ATTCTGAATT TCCTCCCTTG GGGAAGAAGG GAACCAACAT TTATACCTGA 4970
CCAGATGGCT AAAGTGCTTT TAAAGTTTTG TTTAAGTAGA GCTGGAATTT GAGGTGCTGA 5030
TCTGTGGTCT ACAGTTATGT GGTAACTCAT GTTGTCCAGC CAACTCAGAG TTTCGTCAGT 5090
GAACAAGAAA CATGAAATCT GCTTCTTAGA GAGGCTATAT TTTTCTGCTA CAAATATTTT 5150
ATATTTATAG CAAAACTAGA CTTTCAGAGT CCTTGATTGT CTAGGGGAAG TTAACTCCCT 5210
GAGAGGATGT AGAGATTTGG GGTGGTTGAT TAGACTTTTG AAAAACTCAT CACCACATGC 5270
CTTCACTCCA GAGTGTTCTC AGCTAGATTT GATTTGGTTG AGGAGGAACT GTGGCCCTCC 5330
GTAAGTTATT GCCATAGTGT ATGCATTAAA CCAAGTCCAT TTTGAATGAC CTAAAATGAA 5390
GTAACACAAT CAGAAATCCC ATGTGCCCAT AAGCACAGAT TTTTCTTTTT CATTGAAACT 5450
TTAAAGGTTA TTATTGGAAA CATTACTTTG AGTGCAGTGT TTTTAAAAGC CAATTCTTTT 5510
TTATCCCTTT TAGAAGTAGA ATTTGCACAC TTACTACAAT TGAGGAGTGT CATCTCTATA 5570
ACTTTTTCTC CGCCTTTGTC CCATTCTGCC CCTGGACATG TTTCCTACCA AGCATGTTTC 5630
ACATTTTCCT ATTAGTGGAG GAGGGAGAAC CATATTTATT TATAATGAAG ACATCTAAGA 5690
TCCCTATGAT GAATGCAGGA ACTCTCTTGG TAGTTTGTAA ATACACAAAG GGATGTGTCG 5750
AGGGATGGGA GCGATGCTTA TCTCTCACAG TGTGAGTGGT CTGTGTGAGG CTGTTCCTTC 5810
AGTTCTTCTC CAGACTGTTC TTTGGTTGTC ACTTAAGTCA GAGGTCTGGT CCCTCATGTT 5870
TAGGTGAAAG CCAGAGAATG ACAGCTGTAG TCATATCTGA GCATAAGACC TTGATGTGTG 5930
ATTCCTGATG ACCGGTTTCA TTTATTCATG TAATAAAGCA AAGGCCCTGG TCCTTTTTAA 5990
ACTACTAGTT TTAAAAACCT GTGTTAAATG AACAGTAATT GCCTGGTAGG TTTGGTGTGT 6050
GTGTAGCATT GTGTGTCCAT CTGTTATATG TAAAGGACAA GGCACCAGAA TCAGGCTTTA 6110
TTTCGATATT GAAGATGTTA TTTAACATCT TTCTTTTTTC CTTACTCCCT TAGCCATCCC 6170
CTCCCCTTTT GTCCTATCAT TCCCTAGAAC AAGCCACCTG TCAATTGTGA AGGGTTGTGT 6230
TCTTTATGGC AGGTTCTATG CAGATTGTGC CAGAGCATGT GCGTGTTCTG TTGGCAAGCC 6290
ACAGTGCTCC CTTGACTGAA GACATTTCCA GGTAGATTTC TCAGCCAGCT CTAAAACAGA 6350
TTGCTTTTTC AGTGGCCTTA CTCTTTGTGG GTTTTTTTTT TTCTCTGAAC TTGATATAAA 6410
GATTTTATTT GTCCCTTGAA AAAGTAACAA ATGTGCATAG ATCAATTTGT ACTACTTTGG 6470 TCATTGGATA TTTCTGATCC TTATTGCATT GTACCTAAAG GAGAGTAACT AATGGTAACC 6530
TTTTTAATAG AGTATGTGAA AGGTAGTGGC TGATGAATCC TTAACGTTCA TAGGGTCTTT 6590
TTGCTGTTAC GGTTGTATAT AGAGGTCTGA AGGATTTTTA AAATGATTTG CACTTTTTCA 6650
CTGCATGCTT ACAATTCCCA AAGGCAAAAT CTGTACTGAG GTAGATCATT TGAAAGGGCT 6710
AGATTATAAA ATTAAGCCTT AGAGTATGGA AAGTTCTTAT AACAATAATA GTACACACTT 6770
CAGAGTAAGA CAAATGCAAA GCATCTTAAG GAGTGAAAAT AGAGTCTAAA TCTTGCCTTT 6830
GGCACTACAA GGTGTGTGTG TGTGTGTGTG TTGTGTGTCT TTAGTAGGAA ATGGAAGAAC 6890
ACTGTTTTAT TTTTTAAAGT GTTTAATGTT TCTGTCCTTT CTGTGAATTA TTGAATTTAA 6950
GAGCCCTGCT AAATAATGAA AAAACACTTT ACTAAAATTT ATCAAATTAT ACTGGGTTCG 7010
GATTGTGAAA ACATTGGCCA CCTAGTAGCA GTGGTGAGGA GTGGGAGGGC CCAGCAAGCA 7070
TTTATCAGAA ATAGAATCAC AATAGGAGGA GAATTTGGCT GTCTGATATT ATGATTTGAT 7130
TACAATACTG AATGGGAAAA GTATCTAATA TTTTGTAACA AAAAGACCTT CATATTATCT 7190
GTTTTGACCA AAATATGTAG CTATTTCCCT TACACAGATT GGACCGCACT TATCTCCCTT 7250
GTCCTGTATC CTTTAATTTC AGGTCTCAGG ATGTTTAGAA AGCTAAAACC CCCTACCCCT 7310
TTCTGGCTGA AAACTTGCCT TATTTGGTAT CTTACACATT AATGTTACTA GCATCAGGAG 7370
CTTACTGTTT TATTATGATT CATCTTCAGT AATTTTTAGA AGCAAGAAGA AAGCCATTGT 7430
GTCCTCTACA AATTAACAAA ACTTATCTCT GATATACAAA GGGATATAAA TATATACACT 7490
TAAATAGAGA AAAAGAGGTT GATTGAATTG TGCCTTTGAG TGAACCCAGT TTTTAAATAC 7550
CGCTGTGTTT GTTTCGCCAT GGCTTCAGGG ATGCTACATG GCTCTTGCAC CTTTTACTCC 7610
TCTGCTTTAT GAAGTTTGAG TTGTATTTGT GCATCTTAAA GTAGGTTGAG GCTTGAGGCT 7670
GGGCTTTCGG GTTTTTTTGT TTTTTGTTTT GTTTTGTTTT GTTTTGTTTT CTTGTACTTA 7730
AACCTGCTTG CTTCCTACCA CAGATTCTTT ATTTTCCCAA ACACTACAAA AAAACTTTTA 7790
AAACTTTGCC ATTTCATCTG TTTACACTCT TTGCCACTGA TTAGCAGTAT TTAAATCTTG 7850
CAAGAATATT TTGTGCTTTC TTTAGAAACA CAAGAGTAGA GATTTTTCT ACTGAAAAGT 7910
GAGAGTTACG CATTGCAGCC ATGAAGGGAT GCTAGGATCA ATTATGGCAG TACCTTTTTT 7970
CCCCTCCTGT TCTTGAGCCA GTTGTCTCTT TTGTGTTGGG TCCCACTTAG GATTAACGGA 8030
TGTAAGGTAT TTTCCTGTGC CTTTATTTTG TGTCATTCTA TTGGAAGGAG GTGTAACGGC 8090
AGAATAGCAT CGTGTTGGGG GTTTTCCTTC AAACACTGCA AGTGATATTG CCACCATGTG 8150
AACCTCAAAT ATGCAATCCA GTTGTGTTGG TTTCTCGGTG ACTTGGAGTG TTCATCTCTT 8210
CATGAATTGT GAGCACTGAC CATGTTCTTC AGTTCTTAAT TATGGTGAGT TGACAAATAC 8270
CAACTACTGC TTTTCTTTAG GTGGCTATAA ATTTCTTACT GTCAGGAGGA AATGACATTA 8330
TATTCTGTTC CACTGAACGT CAGAGATCAG CAGGCACTGT ACTGGGTAGA GAAGTGCCTA 8390
TACTTCTCTA CCTAAGAGGG CAGGAGGGAA ACCCTACAGC TCCTTGTGAG CCTATATATT 8450
AGTATATCGG CCTGGAGAGG ACAAGGGAAT AAGACCACTC ATAGTGAGGC TGGCCAAGCT 8510 GCACTGGTCG GACCAGGCAG TGGCTGACCT AAGGAAGGCA ACTTGCTTTG CTTAAAAGTA 8570
GATTTTTTAA GCAATGCTTA ACACAGGCAG CATTCACCTT TGTTCAGGCC ATCGACATGT 8630
ATTGTTAAAA TTACTGCATA TCCCCCTCAG ATATCAAGTA TACACTGTTC ATGTTGGGGT 8690
TGTGTGTGTG TATGTGTGTA TGTACGCACG CATGTGTCCC AAATCTTGTT TTAATTTTTT 8750
TTTTCTGAAT GTGATCATGT TTTGGATAAT ACCTGAGCAG GGTTGCCTTT TTTTTATTTA 8810
TTACCATTAT ATATTATATT ATATTATATA TTTTTTGCTT TCTTATAACT TTGGAGGAAA 8870
GTCAAATCTT GGTATTATTA AAATTGTTTT AAAAAGGAGT AAATTTTCCA GTTGATAAAT 8930
GAAAATCACT GGCCTATGTT TAATAAGTTT TTCTTTAATT ACTGTGGAAT AACGTGCCAG 8990
CTATCATCAA CACAATGATT TTGTACATAG GGTAGGGAAG CAGTGATGCT CTCAATGGGA 9050
AGATGTGCAA CACAAATTAA GGGGAACTCC ATGTATTTTA CCTACTTCAG CAATGGAACT 9110
GCAACTTGGG GCTTTGTGAA TAAAATTTAG CTGCCTTGTA TAGTCGTTTG AAAGAATATG 9170
TGATCTGTGA GAGAATTATA GTTTTTTTTT AGAAGAAAAA TCTGCAAAAG ATCTTTCCAA 9230
AGACAATGTG CCACAGATCT TTTGTTCTCT GTAATGAGGA TTAATTGCTG TTTAAACAAA 9290
AATGTAATTG TTCATCTTTA AATTCTTTCC TTTTCATAAG AGGATCAAGC TGTAAAAAAA 9350
CAAAAAAATT AATAAAAATT TCGAGAAATC AAAAAAAAAA A 9391
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1210 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Ala Ala Gin Ser Ser Leu Tyr Asn Asp Asp Arg Asn Leu Leu Arg 1 5 10 15
He Arg Glu Lys Glu Arg Arg Asn Gin Glu Ala His Gin Glu Lys Glu 20 25 30
Ala Phe Pro Glu Lys He Pro Leu Phe Gly Glu Pro Tyr Lys Thr Ala 35 40 45
Lys Gly Asp Glu Leu Ser Ser Arg He Gin Asn Met Leu Gly Asn Tyr 50 55 60
Glu Glu Val Lys Glu Phe Leu Ser Thr Lys Ser His Thr His Arg Leu 65 70 75 80
Asp Ala Ser Glu Asn Arg Leu Gly Lys Pro Lys Tyr Pro Leu He Pro 85 90 95
Asp Lys Gly Ser Ser He Pro Ser Ser Ser Phe His Thr Ser Val His 100 105 110
His Gin Ser He His Thr Pro Ala Ser Gly Pro Leu Ser Val Gly Asn 115 120 125 He Ser His Asn Pro Lys Met Ala Gin Pro Arg Thr Glu Pro Met Pro 130 135 140
Ser Leu His Ala Lys Ser Cys Gly Pro Pro Asp Ser Gin His Leu Thr 145 150 155 160
Gin Asp Arg Leu Gly Gin Glu Gly Phe Gly Ser Ser His His Lys Lys 165 170 175
Gly Asp Arg Arg Ala Asp Gly Asp His Cys Ala Ser Val Thr Asp Ser 180 185 190
Ala Pro Glu Arg Glu Leu Ser Pro Leu He Ser Leu Pro Ser Pro Val 195 200 205
Pro Pro Leu Ser Pro He His Ser Asn Gin Gin Thr Leu Pro Arg Thr 210 215 220
Gin Gly Ser Ser Lys Val His Gly Ser Ser Asn Asn Ser Lys Gly Tyr 225 230 235 240
Cys Pro Ala Lys Ser Pro Lys Asp Leu Ala Val Lys Val His Asp Lys 245 250 255
Glu Thr Pro Gin Asp Ser Leu Val Ala Pro Ala Gin Pro Pro Ser Gin 260 265 270
Thr Phe Pro Pro Pro Ser Leu Pro Ser Lys Ser Val Ala Met Gin Gin 275 280 285
Lys Pro Thr Ala Tyr Val Arg Pro Met Asp Gly Gin Asp Gin Ala Pro 290 295 300
Ser Glu Ser Pro Glu Leu Lys Pro Leu Pro Glu Asp Tyr Arg Gin Gin 305 310 315 320
Thr Phe Glu Lys Thr Asp Leu Lys Val Pro Ala Lys Ala Lys Leu Thr 325 330 335
Lys Leu Lys Met Pro Ser Gin Ser Val Glu Gin Thr Tyr Ser Asn Glu 340 345 350
Val His Cys Val Glu Glu He Leu Lys Glu Met Thr His Ser Trp Pro 355 360 365
Pro Pro Leu Thr Ala He His Thr Pro Ser Thr Ala Glu Pro Ser Lys 370 375 380
Phe Pro Phe Pro Thr Lys Asp Ser Gin His Val Ser Ser Val Thr Gin 385 390 395 400
Asn Gin Lys Gin Tyr Asp Thr Ser Ser Lys Thr His Ser Asn Ser Gin 405 410 415
Gin Gly Thr Ser Ser Met Leu Glu Asp Asp Leu Gin Leu Ser Asp Ser 420 425 430
Glu Asp Ser Asp Ser Glu Gin Thr Pro Glu Lys Pro Pro Ser Ser Ser 435 440 445
Ala Pro Pro Ser Ala Pro Gin Ser Leu Pro Glu Pro Val Ala Ser Ala 450 455 460
His Ser Ser Ser Ala Glu Ser Glu Ser Thr Ser Asp Ser Asp Ser Ser 465 470 475 480
Ser Asp Ser Glu Ser Glu Ser Ser Ser Ser Asp Ser Glu Glu Asn Glu 485 490 495
Pro Leu Glu Thr Pro Ala Pro Glu Pro Glu Pro Pro Thr Thr Asn Lys 500 505 510
Trp Gin Leu Asp Asn Trp Leu Thr Lys Val Ser Gin Pro Ala Ala Pro 515 520 525
Pro Glu Gly Pro Arg Ser Thr Glu Pro Pro Arg Arg His Pro Glu Ser 530 535 540
Lys Gly Ser Ser Asp Ser Ala Thr Ser Gin Glu His Ser Glu Ser Lys 545 550 555 560
Asp Pro Pro Pro Lys Ser Ser Ser Lys Ala Pro Arg Ala Pro Pro Glu 565 570 575
Ala Pro His Pro Gly Lys Arg Ser Cys Gin Lys Ser Pro Ala Gin Gin 580 585 590
Glu Pro Pro Gin Arg Gin Thr Val Gly Thr Lys Gin Pro Lys Lys Pro 595 600 605
Val Lys Ala Ser Ala Arg Ala Gly Ser Arg Thr Ser Leu Gin Gly Glu 610 615 620
Arg Glu Pro Gly Leu Leu Pro Tyr Gly Ser Arg Asp Gin Thr Ser Lys 625 630 635 640
Asp Lys Pro Lys Val Lys Thr Lys Gly Arg Pro Arg Ala Ala Ala Ser 645 650 655
Asn Glu Pro Lys Pro Ala Val Pro Pro Ser Ser Glu Lys Lys Lys His 660 665 670
Lys Ser Ser Leu Pro Ala Pro Ser Lys Ala Leu Ser Gly Pro Glu Pro 675 680 685
Ala Lys Asp Asn Val Glu Asp Arg Thr Pro Glu His Phe Ala Leu Val 690 695 700
Pro Leu Thr Glu Ser Gin Gly Pro Pro His Ser Gly Ser Gly Ser Arg 705 710 715 720
Thr Ser Gly Cys Arg Gin Ala Val Val Val Gin Glu Asp Ser Arg Lys 725 730 735
Asp Arg Leu Pro Leu Pro Leu Arg Asp Thr Lys Leu Leu Ser Pro Leu 740 745 750
Arg Asp Thr Pro Pro Pro Gin Ser Leu Met Val Lys He Thr Leu Asp 755 760 765
Leu Leu Ser Arg He Pro Gin Pro Pro Gly Lys Gly Ser Arg Gin Arg 770 775 780
Lys Ala Glu Asp Lys Gin Pro Pro Ala Gly Lys Lys His Ser Ser Glu 785 790 795 800
Lys Arg Ser Ser Asp Ser Ser Ser Lys Leu Ala Lys Lys Arg Lys Gly 805 810 815
Glu Ala Glu Arg Asp Cys Asp Asn Lys Lys He Arg Leu Glu Lys Glu 820 825 830
He Lys Ser Gin Ser Ser Ser Ser Ser Ser Ser His Lys Glu Ser Ser 835 840 845 Lys Thr Lys Pro Ser Arg Pro Ser Ser Gin Ser Ser Lys Lys Glu Met 850 855 860
Leu Pro Pro Pro Pro Val Ser Ser Ser Ser Gin Lys Pro Ala Lys Pro 865 870 875 880
Ala Leu Lys Arg Ser Arg Arg Glu Ala Asp Thr Cys Gly Gin Asp Pro 885 890 895
Pro Lys Ser Ala Ser Ser Thr Lys Ser Asn His Lys Asp Ser Ser He 900 905 910
Pro Lys Gin Arg Arg Val Glu Gly Lys Gly Ser Arg Ser Ser Ser Glu 915 920 925
His Lys Gly Ser Ser Gly Asp Thr Ala Asn Pro Phe Pro Val Pro Ser 930 935 940
Leu Pro Asn Gly Asn Ser Lys Pro Gly Lys Pro Gin Val Lys Phe Asp 945 950 955 960
Lys Gin Gin Ala Asp Leu His Met Arg Glu Ala Lys Lys Met Lys Gin 965 970 975
Lys Ala Glu Leu Met Thr Asp Arg Val Gly Lys Ala Phe Lys Tyr Leu 980 985 990
Glu Ala Val Leu Ser Phe He Glu Cys Gly He Ala Thr Glu Ser Glu 995 1000 1005
Ser Gin Ser Ser Lys Ser Ala Tyr Ser Val Tyr Ser Glu Thr Val Asp 1010 1015 1020
Leu He Lys Phe He Met Ser Leu Lys Ser Phe Ser Asp Ala Thr Ala 1025 1030 1035 1040
Pro Thr Gin Glu Lys He Phe Ala Val Leu Cys Met Arg Cys Gin Ser 1045 1050 1055
He Leu Asn Met Ala Met Phe Arg Cys Lys Lys Asp He Ala He Lys 1060 1065 1070
Tyr Ser Arg Thr Leu Asn Lys His Phe Glu Ser Ser Ser Lys Val Ala 1075 1080 1085
Gin Ala Pro Ser Pro Cys He Ala Ser Thr Gly Thr Pro Ser Pro Leu 1090 1095 1100
Ser Pro Met Pro Ser Pro Ala Ser Ser Val Gly Ser Gin Ser Ser Ala 1105 1110 1115 1120
Gly Ser Val Gly Ser Ser Gly Val Ala Ala Thr He Ser Thr Pro Val 1125 1130 1135
Thr He Gin Asn Met Thr Ser Ser Tyr Val Thr He Thr Ser His Val 1140 1145 1150
Leu Thr Ala Phe Asp Leu Trp Glu Gin Ala Glu Ala Leu Thr Arg Lys 1155 1160 1165
Asn Lys Glu Phe Phe Ala Arg Leu Ser Thr Asn Val Cys Thr Leu Ala 1170 1175 1180
Leu Asn Ser Ser Leu Val Asp Leu Val His Tyr Thr Arg Gin Gly Phe 1185 1190 1195 1200
Gin Gin Leu Gin Glu Leu Thr Lys Thr Pro 1205 1210
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9370 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 469..4032
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GGCAATTTCT TTTCCTTTCT AACTGTGGCC CGCGTTGTGC TGTTGCTGGG CAGGCGTTGG 60
GCGCCGGCGG TCTTCGAGCG TGGGGGCCCG CTGGCTTTCC CTTCTCAGAA ACTGCGCCGG 120
GGGCGCTCGC TTGCCCCGGA TTCGGACGCG GCGCTCCCCG GGCTCGTCTG AAGTGCAGAT 180
CGCCGCAGAG GCCCCAGTGC CCGGATGTCC ATCAGGATTA GCGCGAGCCA ATACGGGCCG 240
AGCCCGGGGC TGCGCCGAGG ACGCCCGGGG AGTCTGAGAG GCGTGGAGAA TTTTGCTTGT 300
GCAAGATTAT TTCAGAGCAA GGTCGTGCGG TGTGTGTAGA AGATGAACAG ACTAGCCACT 360
TTGCATTGAC TGGAAACAAT GGCATTTACA GAAAGAGTCA ACAGCAGTGG CAACAGTTTG 420
TACAATGACG ACAGAAACCT GCTTCGAATT AGAGAGAAGG AAAGACGC AAC CAG GAA 477
Asn Gin Glu
1
GCC CAC CAA GAG AAA GAG GCA TTT CCT GAA AAG ATT CCC CTT TTT GGA 525 Ala His Gin Glu Lys Glu Ala Phe Pro Glu Lys He Pro Leu Phe Gly 5 10 15
GAG CCC TAC AAG ACA GCA AAA GGT GAT GAG CTG TCT AGT CGA ATA CAG 573 Glu Pro Tyr Lys Thr Ala Lys Gly Asp Glu Leu Ser Ser Arg He Gin 20 25 30 35
AAC ATG TTG GGA AAC TAC GAA GAA GTG AAG GAG TTC CTT AGT ACT AAG 621 Asn Met Leu Gly Asn Tyr Glu Glu Val Lys Glu Phe Leu Ser Thr Lys 40 45 50
TCT CAC ACT CAT CGC CTG GAT GCT TCT GAA AAT AGG TTG GGA AAG CCG 669 Ser His Thr His Arg Leu Asp Ala Ser Glu Asn Arg Leu Gly Lys Pro 55 60 65
AAA TAT CCT TTA ATT CCT GAC AAA GGG AGC AGC ATT CCA TCC AGC TCC 717 Lys Tyr Pro Leu He Pro Asp Lys Gly Ser Ser He Pro Ser Ser Ser 70 75 80
TTC CAC ACT AGT GTC CAC CAC CAG TCC ATT CAC ACT CCT GCG TCT GGA 765 Phe His Thr Ser Val His His Gin Ser He His Thr Pro Ala Ser Gly 85 90 95
CCA CTT TCT GTT GGC AAC ATT AGC CAC AAT CCA AAG ATG GCG CAG CCA 813 Pro Leu Ser Val Gly Asn He Ser His Asn Pro Lys Met Ala Gin Pro 100 105 110 115
AGA ACT GAA CCA ATG CCA AGT CTC CAT GCC AAA AGC TGC GGC CCA CCG 861 Arg Thr Glu Pro Met Pro Ser Leu His Ala Lys Ser Cys Gly Pro Pro 120 125 130
GAC AGC CAG CAC CTG ACC CAG GAT CGC CTT GGT CAG GAG GGG TTC GGC 909 Asp Ser Gin His Leu Thr Gin Asp Arg Leu Gly Gin Glu Gly Phe Gly 135 140 145
TCT AGT CAT CAC AAG AAA GGT GAC CGA AGA GCT GAC GGA GAC CAC TGT 957 Ser Ser His His Lys Lys Gly Asp Arg Arg Ala Asp Gly Asp His Cys 150 155 160
GCT TCG GTG ACA GAT TCG GCT CCA GAG AGG GAG CTT TCT CCC TTA ATC 1005 Ala Ser Val Thr Asp Ser Ala Pro Glu Arg Glu Leu Ser Pro Leu He 165 170 175
TCT TTG CCT TCC CCA GTT CCC CCT TTG TCA CCT ATA CAT TCC AAC CAG 1053 Ser Leu Pro Ser Pro Val Pro Pro Leu Ser Pro He His Ser Asn Gin 180 185 190 195
CAA ACT CTT CCC CGG ACG CAA GGA AGC AGC AAG GTT CAT GGC AGC AGC 1101 Gin Thr Leu Pro Arg Thr Gin Gly Ser Ser Lys Val His Gly Ser Ser 200 205 210
AAT AAC AGT AAA GGC TAT TGC CCA GCC AAA TCT CCC AAG GAC CTA GCA 1149 Asn Asn Ser Lys Gly Tyr Cys Pro Ala Lys Ser Pro Lys Asp Leu Ala 215 220 225
GTG AAA GTC CAT GAT AAA GAG ACC CCT CAA GAC AGT TTG GTG GCC CCT 1197 Val Lys Val His Asp Lys Glu Thr Pro Gin Asp Ser Leu Val Ala Pro 230 235 240
GCC CAG CCG CCT TCT CAG ACA TTT CCA CCT CCC TCC CTC CCC TCA AAA 1245 Ala Gin Pro Pro Ser Gin Thr Phe Pro Pro Pro Ser Leu Pro Ser Lys 245 250 255
AGT GTT GCA ATG CAG CAG AAG CCC ACG GCT TAT GTC CGG CCC ATG GAT 1293 Ser Val Ala Met Gin Gin Lys Pro Thr Ala Tyr Val Arg Pro Met Asp 260 265 270 275
GGT CAA GAT CAG GCC CCT AGT GAA TCC CCT GAA CTG AAA CCA CTG CCG 1341 Gly Gin Asp Gin Ala Pro Ser Glu Ser Pro Glu Leu Lys Pro Leu Pro 280 285 290
GAG GAC TAT CGA CAG CAG ACC TTT GAA AAA ACA GAC TTG AAA GTG CCT 1389 Glu Asp Tyr Arg Gin Gin Thr Phe Glu Lys Thr Asp Leu Lys Val Pro 295 300 305
GCC AAA GCC AAG CTC ACC AAA CTG AAG ATG CCT TCT CAG TCA GTT GAG 1437 Ala Lys Ala Lys Leu Thr Lys Leu Lys Met Pro Ser Gin Ser Val Glu 310 315 320
CAG ACC TAC TCC AAT GAA GTC CAT TGT GTT GAA GAG ATT CTG AAG GAA 1485 Gin Thr Tyr Ser Asn Glu Val His Cys Val Glu Glu He Leu Lys Glu 325 330 335
ATG ACC CAT TCA TGG CCG CCT CCT TTG ACA GCA ATA CAT ACG CCT AGT 1533 Met Thr His Ser Trp Pro Pro Pro Leu Thr Ala He His Thr Pro Ser 340 345 350 355
ACA GCT GAG CCA TCC AAG TTT CCT TTC CCT ACA AAG GAC TCT CAG CAT 1581 Thr Ala Glu Pro Ser Lys Phe Pro Phe Pro Thr Lys Asp Ser Gin His 360 365 370
GTC AGT TCT GTA ACC CAA AAC CAA AAA CAA TAT GAT ACA TCT TCA AAA 1629 Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr Ser Ser Lys 375 380 385
ACT CAC TCA AAT TCT CAG CAA GGA ACG TCA TCC ATG CTC GAA GAC GAC 1677 Thr His Ser Asn Ser Gin Gin Gly Thr Ser Ser Met Leu Glu Asp Asp 390 395 400
CTT CAG CTC AGT GAC AGT GAG GAC AGT GAC AGT GAA CAA ACC CCA GAG 1725 Leu Gin Leu Ser Asp Ser Glu Asp Ser Asp Ser Glu Gin Thr Pro Glu 405 410 415
AAG CCT CCC TCC TCA TCT GCA CCT CCA AGT GCT CCA CAG TCC CTT CCA 1773 Lys Pro Pro Ser Ser Ser Ala Pro Pro Ser Ala Pro Gin Ser Leu Pro 420 425 430 435
GAA CCA GTG GCA TCA GCA CAT TCC AGC AGT GCA GAG TCA GAA AGC ACC 1821 Glu Pro Val Ala Ser Ala His Ser Ser Ser Ala Glu Ser Glu Ser Thr 440 445 450
AGT GAC TCA GAC AGT TCC TCA GAC TCA GAG AGC GAG AGC AGT TCA AGT 1869 Ser Asp Ser Asp Ser Ser Ser Asp Ser Glu Ser Glu Ser Ser Ser Ser 455 460 465
GAC AGC GAA GAA AAT GAG CCC CTA GAA ACC CCA GCT CCG GAG CCT GAG 1917 Asp Ser Glu Glu Asn Glu Pro Leu Glu Thr Pro Ala Pro Glu Pro Glu 470 475 480
CCT CCA ACA ACA AAC AAA TGG CAG CTG GAC AAC TGG CTG ACC AAA GTC 1965 Pro Pro Thr Thr Asn Lys Trp Gin Leu Asp Asn Trp Leu Thr Lys Val 485 490 495
AGC CAG CCA GCT GCG CCA CCA GAG GGC CCC AGG AGC ACA GAG CCC CCA 2013 Ser Gin Pro Ala Ala Pro Pro Glu Gly Pro Arg Ser Thr Glu Pro Pro 500 505 510 515
CGG CGG CAC CCA GAG AGT AAG GGC AGC AGC GAC AGT GCC ACG AGT CAG 2061 Arg Arg His Pro Glu Ser Lys Gly Ser Ser Asp Ser Ala Thr Ser Gin 520 525 530
GAG CAT TCT GAA TCC AAA GAT CCT CCC CCT AAA AGC TCC AGC AAA GCC 2109 Glu His Ser Glu Ser Lys Asp Pro Pro Pro Lys Ser Ser Ser Lys Ala 535 540 545
CCC CGG GCC CCA CCC GAA GCC CCC CAC CCC GGA AAG AGG AGC TGT CAG 2157 Pro Arg Ala Pro Pro Glu Ala Pro His Pro Gly Lys Arg Ser Cys Gin 550 555 560
AAG TCT CCG GCA CAG CAG GAG CCC CCA CAA AGG CAA ACC GTT GGA ACC 2205 Lys Ser Pro Ala Gin Gin Glu Pro Pro Gin Arg Gin Thr Val Gly Thr 565 570 575
AAA CAA CCC AAA AAA CCT GTC AAG GCC TCT GCC CGG GCA GGT TCA CGG 2253 Lys Gin Pro Lys Lys Pro Val Lys Ala Ser Ala Arg Ala Gly Ser Arg 580 585 590 595
ACC AGC CTG CAG GGG GAA AGG GAG CCA GGG CTT CTT CCC TAT GGC TCC 2301 Thr Ser Leu Gin Gly Glu Arg Glu Pro Gly Leu Leu Pro Tyr Gly Ser 600 605 610
CGA GAC CAG ACT TCC AAA GAC AAG CCC AAG GTG AAG ACG AAA GGA CGG 2349 Arg Asp Gin Thr Ser Lys Asp Lys Pro Lys Val Lys Thr Lys Gly Arg 615 620 625
CCC CGG GCC GCA GCA AGC AAC GAA CCC AAG CCA GCA GTG CCC CCC TCC 2397 Pro Arg Ala Ala Ala Ser Asn Glu Pro Lys Pro Ala Val Pro Pro Ser 630 635 640
AGT GAG AAG AAG AAG CAC AAG AGC TCC CTC CCT GCC CCC TCT AAG GCT 2445 Ser Glu Lys Lys Lys His Lys Ser Ser Leu Pro Ala Pro Ser Lys Ala 645 650 655 CTC TCA GGC CCA GAA CCC GCG AAG GAC AAT GTG GAG GAC AGG ACC CCT 2493 Leu Ser Gly Pro Glu Pro Ala Lys Asp Asn Val Glu Asp Arg Thr Pro
660 665 670 675
GAG CAC TTT GCT CTT GTT CCC CTG ACT GAG AGC CAG GGC CCA CCC CAC 2541 Glu His Phe Ala Leu Val Pro Leu Thr Glu Ser Gin Gly Pro Pro His 680 685 690
AGT GGC AGC GGC AGC AGG ACT AGT GGC TGC CGC CAA GCC GTG GTG GTC 2589
Ser Gly Ser Gly Ser Arg Thr Ser Gly Cys Arg Gin Ala Val Val Val 695 700 705
CAG GAG GAC AGC CGC AAA GAC AGA CTC CCA TTG CCT TTG AGA GAC ACC 2637
Gin Glu Asp Ser Arg Lys Asp Arg Leu Pro Leu Pro Leu Arg Asp Thr 710 715 720
AAG CTG CTC TCA CCG CTC AGG GAC ACT CCT CCC CCA CAA AGC TTG ATG 2685
Lys Leu Leu Ser Pro Leu Arg Asp Thr Pro Pro Pro Gin Ser Leu Met 725 730 735
GTG AAG ATC ACC CTA GAC CTG CTC TCT CGG ATA CCC CAG CCT CCC GGG 2733
Val Lys He Thr Leu Asp Leu Leu Ser Arg He Pro Gin Pro Pro Gly
740 745 750 755
AAG GGG AGC CGC CAG AGG AAA GCA GAA GAT AAA CAG CCG CCC GCA GGG 2781
Lys Gly Ser Arg Gin Arg Lys Ala Glu Asp Lys Gin Pro Pro Ala Gly 760 765 770
AAG AAG CAC AGC TCT GAG AAG AGG AGC TCA GAC AGC TCA AGC AAG TTG 2829
Lys Lys His Ser Ser Glu Lys Arg Ser Ser Asp Ser Ser Ser Lys Leu 775 780 785
GCC AAA AAG AGA AAG GGT GAA GCA GAA AGA GAC TGT GAT AAC AAG AAA 2877
Ala Lys Lys Arg Lys Gly Glu Ala Glu Arg Asp Cys Asp Asn Lys Lys 790 795 800
ATC AGA CTG GAG AAG GAA ATC AAA TCA CAG TCA TCT TCA TCT TCA TCC 2925
He Arg Leu Glu Lys Glu He Lys Ser Gin Ser Ser Ser Ser Ser Ser 805 810 815
TCC CAC AAA GAA TCT TCT AAA ACA AAG CCC TCC AGG CCC TCC TCA CAG 2973
Ser His Lys Glu Ser Ser Lys Thr Lys Pro Ser Arg Pro Ser Ser Gin
820 825 830 835
TCC TCA AAG AAG GAA ATG CTC CCC CCG CCA CCC GTG TCC TCG TCC TCC 3021
Ser Ser Lys Lys Glu Met Leu Pro Pro Pro Pro Val Ser Ser Ser Ser 840 845 850
CAG AAG CCA GCC AAG CCT GCA CTT AAG AGG TCA AGG CGG GAA GCA GAC 3069
Gin Lys Pro Ala Lys Pro Ala Leu Lys Arg Ser Arg Arg Glu Ala Asp 855 860 865
ACC TGT GGC CAG GAC CCT CCC AAA AGT GCC AGC AGT ACC AAG AGC AAC 3117
Thr Cys Gly Gin Asp Pro Pro Lys Ser Ala Ser Ser Thr Lys Ser Asn 870 875 880
CAC AAA GAC TCT TCC ATT CCC AAG CAG AGA AGA GTA GAG GGG AAG GGC 3165
His Lys Asp Ser Ser He Pro Lys Gin Arg Arg Val Glu Gly Lys Gly 885 890 895
TCC AGA AGC TCC TCG GAG CAC AAG GGT TCT TCC GGA GAT ACT GCA AAT 3213
Ser Arg Ser Ser Ser Glu His Lys Gly Ser Ser Gly Asp Thr Ala Asn
900 905 910 915
CCT TTT CCA GTG CCT TCT TTG CCA AAT GGT AAC TCT AAA CCA GGG AAG 3261
Pro Phe Pro Val Pro Ser Leu Pro Asn Gly Asn Ser Lys Pro Gly Lys 920 925 930 CCT CAA GTG AAG TTT GAC AAA CAA CAA GCA GAC CTT CAC ATG AGG GAG 3309 Pro Gin Val Lys Phe Asp Lys Gin Gin Ala Asp Leu His Met Arg Glu 935 940 945
GCA AAA AAG ATG AAG CAG AAA GCA GAG TTA ATG ACG GAC AGG GTT GGA 3357 Ala Lys Lys Met Lys Gin Lys Ala Glu Leu Met Thr Asp Arg Val Gly 950 955 960
AAG GCT TTT AAG TAC CTG GAA GCC GTC TTG TCC TTC ATT GAG TGC GGA 3405 Lys Ala Phe Lys Tyr Leu Glu Ala Val Leu Ser Phe He Glu Cys Gly 965 970 975
ATT GCC ACA GAG TCT GAA AGC CAG TCA TCC AAG TCA GCT TAC TCT GTC 3453 He Ala Thr Glu Ser Glu Ser Gin Ser Ser Lys Ser Ala Tyr Ser Val 980 985 990 995
TAC TCA GAA ACT GTA GAT CTC ATT AAA TTC ATA ATG TCA TTA AAA TCC 3501 Tyr Ser Glu Thr Val Asp Leu He Lys Phe He Met Ser Leu Lys Ser 1000 1005 1010
TTC TCA GAT GCC ACA GCG CCA ACA CAA GAG AAA ATA TTT GCT GTT TTA 3549 Phe Ser Asp Ala Thr Ala Pro Thr Gin Glu Lys He Phe Ala Val Leu 1015 1020 1025
TGC ATG CGT TGC CAG TCC ATT TTG AAC ATG GCG ATG TTT CGT TGT AAA 3597 Cys Met Arg Cys Gin Ser He Leu Asn Met Ala Met Phe Arg Cys Lys 1030 1035 1040
AAA GAC ATA GCA ATA AAG TAT TCT CGT ACT CTT AAT AAA CAC TTC GAG 3645 Lys Asp He Ala He Lys Tyr Ser Arg Thr Leu Asn Lys His Phe Glu 1045 1050 1055
AGT TCT TCC AAA GTC GCC CAG GCA CCT TCT CCA TGC ATT GCA AGC ACA 3693 Ser Ser Ser Lys Val Ala Gin Ala Pro Ser Pro Cys He Ala Ser Thr 1060 1065 1070 1075
GGC ACA CCA TCC CCT CTT TCC CCA ATG CCT TCT CCT GCC AGC TCC GTA 3741 Gly Thr Pro Ser Pro Leu Ser Pro Met Pro Ser Pro Ala Ser Ser Val 1080 1085 1090
GGG TCC CAG TCA AGT GCT GGC AGT GTG GGG AGC AGT GGG GTG GCT GCC 3789 Gly Ser Gin Ser Ser Ala Gly Ser Val Gly Ser Ser Gly Val Ala Ala 1095 1100 1105
ACT ATC AGC ACC CCA GTC ACC ATC CAG AAT ATG ACA TCT TCC TAT GTC 3837 Thr He Ser Thr Pro Val Thr He Gin Asn Met Thr Ser Ser Tyr Val 1110 1115 1120
ACC ATC ACA TCC CAT GTT CTT ACC GCC TTT GAC CTT TGG GAA CAG GCC 3885 Thr He Thr Ser His Val Leu Thr Ala Phe Asp Leu Trp Glu Gin Ala 1125 1130 1135
GAG GCC CTC ACG AGG AAG AAT AAA GAA TTC TTT GCT CGG CTC AGC ACA 3933 Glu Ala Leu Thr Arg Lys Asn Lys Glu Phe Phe Ala Arg Leu Ser Thr 1140 1145 1150 1155
AAT GTG TGC ACC TTG GCC CTC AAC AGC AGT TTG GTG GAC CTG GTG CAC 3981 Asn Val Cys Thr Leu Ala Leu Asn Ser Ser Leu Val Asp Leu Val His 1160 1165 1170
TAT ACA CGA CAG GGT TTT CAG CAG CTA CAA GAA TTA ACC AAA ACA CCT 4029 Tyr Thr Arg Gin Gly Phe Gin Gin Leu Gin Glu Leu Thr Lys Thr Pro 1175 1180 1185
TAATGGAGCC CCAGGTTGAT TCAATGCCTT GGGAACTATT TTTGCACATT GGAAGCCTCA 4089
AAAACAGTCC AGACGTTTGT TTCATCAGGA CACCAAACTC TAAAAAAGAA GCACCACGAG 4149 ATGGCCAGGA CATTTGTCCA CTTAAACTCT CAACAACAGT GTGATCATTG GTTGGACACT 4209
GTGGTTATGC AGAAGCAGAG ATGAGGAGGC TGGCCCCAGA GATGATCTTG CCCTTCCTAA 4269
CTAAAGGACA GAAGTGCAAT TTAGCTTAAA TGGGTGTATG AATGGTCTAG AAACATTTCT 4329
ATTTTTTTTT TAAACCAGCA GGATACAAGT TGCAAATGAA ATGAGGAGAA ACAGTTTCAA 4389
CTCTGAAAGT GAATTTCACG TCATCTCAGT AGCCACGCTA GTCCATTCCC AGAAGGAAAT 4449
TTTTTTTTTT AACAATGACT TTTGGTAAAG GGTTTTGTGG ATGATTTTTT TTCTTTTGAG 4509
TTTTGGGAGA AATATTTGTT TAATAACTTC TAATGGCCAT CTGTAAACCA TAAGTAATGA 4569
AGGACTCCAC TGTGCCCCAC TTTCTGCCAA TGAACAGTGG CTTGATAATA CCAAGTATTG 4629
TTGTAATTTA TAAAATTGAA GGCAACCCCC GCTCCTGCCG CCCCCAATCT CCCCATTGCC 4689
TAGAGCGCTG CACATTGACC CCAGCTCTGA CTTCTCATTA CTGTGCTGAA AGTCAGCCCA 4749
CGTCGGAGCG GTGAGGAGGA GCCACAGCAC ATGGGGTGCC ACCTCGAGGT CTGCACAGGA 4809
GGACTTGGCG CTGCCATTTC CTACCCCTGC CATTTCCCAC CCCTGCTTCA GCGAAAGGGA 4869
CTCTCTAACA GGGCAGTCAC TGTTGACTCT ATTCTGAATT TCCTCCCTTG GGGAAGAAGG 4929
GAACCAACAT TTATACCTGA CCAGATGGCT AAAGTGCTTT TAAAGTTTTG TTTAAGTAGA 4989
GCTGGAATTT GAGGTGCTGA TCTGTGGTCT ACAGTTATGT GGTAACTCAT GTTGTCCAGC 5049
CAACTCAGAG TTTCGTCAGT GAACAAGAAA CATGAAATCT GCTTCTTAGA GAGGCTATAT 5109
TTTTCTGCTA CAAATATTTT ATATTTATAG CAAAACTAGA CTTTCAGAGT CCTTGATTGT 5169
CTAGGGGAAG TTAACTCCCT GAGAGGATGT AGAGATTTGG GGTGGTTGAT TAGACTTTTG 5229
AAAAACTCAT CACCACATGC CTTCACTCCA GAGTGTTCTC AGCTAGATTT GATTTGGTTG 5289
AGGAGGAACT GTGGCCCTCC GTAAGTTATT GCCATAGTGT ATGCATTAAA CCAAGTCCAT 5349
TTTGAATGAC CTAAAATGAA GTAACACAAT CAGAAATCCC ATGTGCCCAT AAGCACAGAT 5409
TTTTCTTTTT CATTGAAACT TTAAAGGTTA TTATTGGAAA CATTACTTTG AGTGCAGTGT 5469
TTTTAAAAGC CAATTCTTTT TTATCCCTTT TAGAAGTAGA ATTTGCACAC TTACTACAAT 5529
TGAGGAGTGT CATCTCTATA ACTTTTTCTC CGCCTTTGTC CCATTCTGCC■CCTGGACATG 5589
TTTCCTACCA AGCATGTTTC ACATTTTCCT ATTAGTGGAG GAGGGAGAAC CATATTTATT 5649
TATAATGAAG ACATCTAAGA TCCCTATGAT GAATGCAGGA ACTCTCTTGG TAGTTTGTAA 5709
ATACACAAAG GGATGTGTCG AGGGATGGGA GCGATGCTTA TCTCTCACAG TGTGAGTGGT 5769
CTGTGTGAGG CTGTTCCTTC AGTTCTTCTC CAGACTGTTC TTTGGTTGTC ACTTAAGTCA 5829
GAGGTCTGGT CCCTCATGTT TAGGTGAAAG CCAGAGAATG ACAGCTGTAG TCATATCTGA 5889
GCATAAGACC TTGATGTGTG ATTCCTGATG ACCGGTTTCA TTTATTCATG TAATAAAGCA 5949
AAGGCCCTGG TCCTTTTTAA ACTACTAGTT TTAAAAACCT GTGTTAAATG AACAGTAATT 6009
GCCTGGTAGG TTTGGTGTGT GTGTAGCATT GTGTGTCCAT CTGTTATATG TAAAGGACAA 6069
GGCACCAGAA TCAGGCTTTA TTTCGATATT GAAGATGTTA TTTAACATCT TTCTTTTTTC 6129
CTTACTCCCT TAGCCATCCC CTCCCCTTTT GTCCTATCAT TCCCTAGAAC AAGCCACCTG 6189 TCAATTGTGA AGGGTTGTGT TCTTTATGGC AGGTTCTATG CAGATTGTGC CAGAGCATGT 6249
GCGTGTTCTG TTGGCAAGCC ACAGTGCTCC CTTGACTGAA GACATTTCCA GGTAGATTTC 6309
TCAGCCAGCT CTAAAACAGA TTGCTTTTTC AGTGGCCTTA CTCTTTGTGG GTTTTTTTTT 6369
TTCTCTGAAC TTGATATAAA GATTTTATTT GTCCCTTGAA AAAGTAACAA ATGTGCATAG 6429
ATCAATTTGT ACTACTTTGG TCATTGGATA TTTCTGATCC TTATTGCATT GTACCTAAAG 6489
GAGAGTAACT AATGGTAACC TTTTTAATAG AGTATGTGAA AGGTAGTGGC TGATGAATCC 6549
TTAACGTTCA TAGGGTCTTT TTGCTGTTAC GGTTGTATAT AGAGGTCTGA AGGATTTTTA 6609
AAATGATTTG CACTTTTTCA CTGCATGCTT ACAATTCCCA AAGGCAAAAT CTGTACTGAG 6669
GTAGATCATT TGAAAGGGCT AGATTATAAA ATTAAGCCTT AGAGTATGGA AAGTTCTTAT 6729
AACAATAATA GTACACACTT CAGAGTAAGA CAAATGCAAA GCATCTTAAG GAGTGAAAAT 6789
AGAGTCTAAA TCTTGCCTTT GGCACTACAA GGTGTGTGTG TGTGTGTGTG TTGTGTGTCT 6849
TTAGTAGGAA ATGGAAGAAC ACTGTTTTAT TTTTTAAAGT GTTTAATGTT TCTGTCCTTT 6909
CTGTGAATTA TTGAATTTAA GAGCCCTGCT AAATAATGAA AAAACACTTT ACTAAAATTT 6969
ATCAAATTAT ACTGGGTTCG GATTGTGAAA ACATTGGCCA CCTAGTAGCA GTGGTGAGGA 7029
GTGGGAGGGC CCAGCAAGCA TTTATCAGAA ATAGAATCAC AATAGGAGGA GAATTTGGCT 7089
GTCTGATATT ATGATTTGAT TACAATACTG AATGGGAAAA GTATCTAATA TTTTGTAACA 7149
AAAAGACCTT CATATTATCT GTTTTGACCA AAATATGTAG CTATTTCCCT TACACAGATT 7209
GGACCGCACT TATCTCCCTT GTCCTGTATC CTTTAATTTC AGGTCTCAGG ATGTTTAGAA 7269
AGCTAAAACC CCCTACCCCT TTCTGGCTGA AAACTTGCCT TATTTGGTAT CTTACACATT 7329
AATGTTACTA GCATCAGGAG CTTACTGTTT TATTATGATT CATCTTCAGT AATTTTTAGA 7389
AGCAAGAAGA AAGCCATTGT GTCCTCTACA AATTAACAAA ACTTATCTCT GATATACAAA 7449
GGGATATAAA TATATACACT TAAATAGAGA AAAAGAGGTT GATTGAATTG TGCCTTTGAG 7509
TGAACCCAGT TTTTAAATAC CGCTGTGTTT GTTTCGCCAT GGCTTCAGGG ATGCTACATG 7569
GCTCTTGCAC CTTTTACTCC TCTGCTTTAT GAAGTTTGAG TTGTATTTGT GCATCTTAAA 7629
GTAGGTTGAG GCTTGAGGCT GGGCTTTCGG GTTTTTTTGT TTTTTGTTTT GTTTTGTTTT 7689
GTTTTGTTTT CTTGTACTTA AACCTGCTTG CTTCCTACCA CAGATTCTTT ATTTTCCCAA 7749
ACACTACAAA AAAACTTTTA AAACTTTGCC ATTTCATCTG TTTACACTCT TTGCCACTGA 7809
TTAGCAGTAT TTAAATCTTG CAAGAATATT TTGTGCTTTC TTTAGAAACA CAAGAGTAGA 7869
GATTTTTCTC ACTGAAAAGT GAGAGTTACG CATTGCAGCC ATGAAGGGAT GCTAGGATCA 7929
ATTATGGCAG TACCTTTTTT CCCCTCCTGT TCTTGAGCCA GTTGTCTCTT TTGTGTTGGG 7989
TCCCACTTAG GATTAACGGA TGTAAGGTAT TTTCCTGTGC CTTTATTTTG TGTCATTCTA 8049
TTGGAAGGAG GTGTAACGGC AGAATAGCAT CGTGTTGGGG GTTTTCCTTC AAACACTGCA 8109
AGTGATATTG CCACCATGTG AACCTCAAAT ATGCAATCCA GTTGTGTTGG TTTCTCGGTG 8169
ACTTGGAGTG TTCATCTCTT CATGAATTGT GAGCACTGAC CATGTTCTTC AGTTCTTAAT 8229 TATGGTGAGT TGACAAATAC CAACTACTGC TTTTCTTTAG GTGGCTATAA ATTTCTTACT 8289
GTCAGGAGGA AATGACATTA TATTCTGTTC CACTGAACGT CAGAGATCAG CAGGCACTGT 8349
ACTGGGTAGA GAAGTGCCTA TACTTCTCTA CCTAAGAGGG CAGGAGGGAA ACCCTACAGC 8409
TCCTTGTGAG CCTATATATT AGTATATCGG CCTGGAGAGG ACAAGGGAAT AAGACCACTC 8469
ATAGTGAGGC TGGCCAAGCT GCACTGGTCG GACCAGGCAG TGGCTGACCT AAGGAAGGCA 8529
ACTTGCTTTG CTTAAAAGTA GATTTTTTAA GCAATGCTTA ACACAGGCAG CATTCACCTT 8589
TGTTCAGGCC ATCGACATGT ATTGTTAAAA TTACTGCATA TCCCCCTCAG ATATCAAGTA 8649
TACACTGTTC ATGTTGGGGT TGTGTGTGTG TATGTGTGTA TGTACGCACG CATGTGTCCC 8709
AAATCTTGTT TTAATTTTTT TTTTCTGAAT GTGATCATGT TTTGGATAAT ACCTGAGCAG 8769
GGTTGCCTTT TTTTTATTTA TTACCATTAT ATATTATATT ATATTATATA TTTTTTGCTT 8829
TCTTATAACT TTGGAGGAAA GTCAAATCTT GGTATTATTA AAATTGTTTT AAAAAGGAGT 8889
AAATTTTCCA GTTGATAAAT GAAAATCACT GGCCTATGTT TAATAAGTTT TTCTTTAATT 8949
ACTGTGGAAT AACGTGCCAG CTATCATCAA CACAATGATT TTGTACATAG GGTAGGGAAG 9009
CAGTGATGCT CTCAATGGGA AGATGTGCAA CACAAATTAA GGGGAACTCC ATGTATTTTA 9069
CCTACTTCAG CAATGGAACT GCAACTTGGG GCTTTGTGAA TAAAATTTAG CTGCCTTGTA 9129
TAGTCGTTTG AAAGAATATG TGATCTGTGA GAGAATTATA GTTTTTTTTT AGAAGAAAAA 9189
TCTGCAAAAG ATCTTTCCAA AGACAATGTG CCACAGATCT TTTGTTCTCT GTAATGAGGA 9249
TTAATTGCTG TTTAAACAAA AATGTAATTG TTCATCTTTA AATTCTTTCC TTTTCATAAG 9309
AGGATCAAGC TGTAAAAAAA CAAAAAAATT AATAAAAATT TCGAGAAATC AAAAAAAAAA 9369
A 9370
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1187 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Asn Gin Glu Ala His Gin Glu Lys Glu Ala Phe Pro Glu Lys He Pro 1 5 10 15
Leu Phe Gly Glu Pro Tyr Lys Thr Ala Lys Gly Asp Glu Leu Ser Ser 20 25 30
Arg He Gin Asn Met Leu Gly Asn Tyr Glu Glu Val Lys Glu Phe Leu 35 40 45
Ser Thr Lys Ser His Thr His Arg Leu Asp Ala Ser Glu Asn Arg Leu 50 55 60
Gly Lys Pro Lys Tyr Pro Leu He Pro Asp Lys Gly Ser Ser He Pro 65 70 75 80
Ser Ser Ser Phe His Thr Ser Val His His Gin Ser He His Thr Pro 85 90 95
Ala Ser Gly Pro Leu Ser Val Gly Asn He Ser His Asn Pro Lys Met 100 105 110
Ala Gin Pro Arg Thr Glu Pro Met Pro Ser Leu His Ala Lys Ser Cys 115 120 125
Gly Pro Pro Asp Ser Gin His Leu Thr Gin Asp Arg Leu Gly Gin Glu 130 135 140
Gly Phe Gly Ser Ser His His Lys Lys Gly Asp Arg Arg Ala Asp Gly 145 150 155 160
Asp His Cys Ala Ser Val Thr Asp Ser Ala Pro Glu Arg Glu Leu Ser 165 170 175
Pro Leu He Ser Leu Pro Ser Pro Val Pro Pro Leu Ser Pro He His 180 185 190
Ser Asn Gin Gin Thr Leu Pro Arg Thr Gin Gly Ser Ser Lys Val His 195 200 205
Gly Ser Ser Asn Asn Ser Lys Gly Tyr Cys Pro Ala Lys Ser Pro Lys 210 215 220
Asp Leu Ala Val Lys Val His Asp Lys Glu Thr Pro Gin Asp Ser Leu 225 230 235 240
Val Ala Pro Ala Gin Pro Pro Ser Gin Thr Phe Pro Pro Pro Ser Leu 245 250 255
Pro Ser Lys Ser Val Ala Met Gin Gin Lys Pro Thr Ala Tyr Val Arg 260 265 270
Pro Met Asp Gly Gin Asp Gin Ala Pro Ser Glu Ser Pro Glu Leu Lys 275 280 285
Pro Leu Pro Glu Asp Tyr Arg Gin Gin Thr Phe Glu Lys Thr Asp Leu 290 295 300
Lys Val Pro Ala Lys Ala Lys Leu Thr Lys Leu Lys Met Pro Ser Gin 305 310 315 320
Ser Val Glu Gin Thr Tyr Ser Asn Glu Val His Cys Val Glu Glu He 325 330 335
Leu Lys Glu Met Thr His Ser Trp Pro Pro Pro Leu Thr Ala He His 340 345 350
Thr Pro Ser Thr Ala Glu Pro Ser Lys Phe Pro Phe Pro Thr Lys Asp 355 360 365
Ser Gin His Val Ser Ser Val Thr Gin Asn Gin Lys Gin Tyr Asp Thr 370 375 380
Ser Ser Lys Thr His Ser Asn Ser Gin Gin Gly Thr Ser Ser Met Leu 385 390 395 400
Glu Asp Asp Leu Gin Leu Ser Asp Ser Glu Asp Ser Asp Ser Glu Gin 405 410 415
Thr Pro Glu Lys Pro Pro Ser Ser Ser Ala Pro Pro Ser Ala Pro Gin 420 425 430
Ser Leu Pro Glu Pro Val Ala Ser Ala His Ser Ser Ser Ala Glu Ser 435 440 445
Glu Ser Thr Ser Asp Ser Asp Ser Ser Ser Asp Ser Glu Ser Glu Ser 450 455 460 Ser Ser Ser Asp Ser Glu Glu Asn Glu Pro Leu Glu Thr Pro Ala Pro 465 470 475 480
Glu Pro Glu Pro Pro Thr Thr Asn Lys Trp Gin Leu Asp Asn Trp Leu 485 490 495
Thr Lys Val Ser Gin Pro Ala Ala Pro Pro Glu Gly Pro Arg Ser Thr 500 505 510
Glu Pro Pro Arg Arg His Pro Glu Ser Lys Gly Ser Ser Asp Ser Ala 515 520 525
Thr Ser Gin Glu His Ser Glu Ser Lys Asp Pro Pro Pro Lys Ser Ser 530 535 540
Ser Lys Ala Pro Arg Ala Pro Pro Glu Ala Pro His Pro Gly Lys Arg 545 550 555 560
Ser Cys Gin Lys Ser Pro Ala Gin Gin Glu Pro Pro Gin Arg Gin Thr 565 570 575
Val Gly Thr Lys Gin Pro Lys Lys Pro Val Lys Ala Ser Ala Arg Ala 580 585 590
Gly Ser Arg Thr Ser Leu Gin Gly Glu Arg Glu Pro Gly Leu Leu Pro 595 600 605
Tyr Gly Ser Arg Asp Gin Thr Ser Lys Asp Lys Pro Lys Val Lys Thr 610 615 620
Lys Gly Arg Pro Arg Ala Ala Ala Ser Asn Glu Pro Lys Pro Ala Val 625 630 635 640
Pro Pro Ser Ser Glu Lys Lys Lys His Lys Ser Ser Leu Pro Ala Pro 645 650 655
Ser Lys Ala Leu Ser Gly Pro Glu Pro Ala Lys Asp Asn Val Glu Asp 660 665 670
Arg Thr Pro Glu His Phe Ala Leu Val Pro Leu Thr Glu Ser Gin Gly 675 680 685
Pro Pro His Ser Gly Ser Gly Ser Arg Thr Ser Gly Cys Arg Gin Ala 690 695 700
Val Val Val Gin Glu Asp Ser Arg Lys Asp Arg Leu Pro Leu Pro Leu 705 710 715 720
Arg Asp Thr Lys Leu Leu Ser Pro Leu Arg Asp Thr Pro Pro Pro Gin 725 730 735
Ser Leu Met Val Lys He Thr Leu Asp Leu Leu Ser Arg He Pro Gin 740 745 750
Pro Pro Gly Lys Gly Ser Arg Gin Arg Lys Ala Glu Asp Lys Gin Pro 755 760 765
Pro Ala Gly Lys Lys His Ser Ser Glu Lys Arg Ser Ser Asp Ser Ser 770 775 780
Ser Lys Leu Ala Lys Lys Arg Lys Gly Glu Ala Glu Arg Asp Cys Asp 785 790 795 800
Asn Lys Lys He Arg Leu Glu Lys Glu He Lys Ser Gin Ser Ser Ser 805 810 815
Ser Ser Ser Ser His Lys Glu Ser Ser Lys Thr Lys Pro Ser Arg Pro 820 825 830
Ser Ser Gin Ser Ser Lys Lys Glu Met Leu Pro Pro Pro Pro Val Ser 835 840 845
Ser Ser Ser Gin Lys Pro Ala Lys Pro Ala Leu Lys Arg Ser Arg Arg 850 855 860
Glu Ala Asp Thr Cys Gly Gin Asp Pro Pro Lys Ser Ala Ser Ser Thr 865 870 875 880
Lys Ser Asn His Lys Asp Ser Ser He Pro Lys Gin Arg Arg Val Glu 885 890 895
Gly Lys Gly Ser Arg Ser Ser Ser Glu His Lys Gly Ser Ser Gly Asp 900 905 910
Thr Ala Asn Pro Phe Pro Val Pro Ser Leu Pro Asn Gly Asn Ser Lys 915 920 925
Pro Gly Lys Pro Gin Val Lys Phe Asp Lys Gin Gin Ala Asp Leu His 930 935 940
Met Arg Glu Ala Lys Lys Met Lys Gin Lys Ala Glu Leu Met Thr Asp 945 950 955 960
Arg Val Gly Lys Ala Phe Lys Tyr Leu Glu Ala Val Leu Ser Phe He 965 970 975
Glu Cys Gly He Ala Thr Glu Ser Glu Ser Gin Ser Ser Lys Ser Ala 980 985 990
Tyr Ser Val Tyr Ser Glu Thr Val Asp Leu He Lys Phe He Met Ser 995 1000 1005
Leu Lys Ser Phe Ser Asp Ala Thr Ala Pro Thr Gin Glu Lys He Phe 1010 1015 1020
Ala Val Leu Cys Met Arg Cys Gin Ser He Leu Asn Met Ala Met Phe 1025 1030 1035 1040
Arg Cys Lys Lys Asp He Ala He Lys Tyr Ser Arg Thr Leu Asn Lys 1045 1050 1055
His Phe Glu Ser Ser Ser Lys Val Ala Gin Ala Pro Ser Pro Cys He 1060 1065 1070
Ala Ser Thr Gly Thr Pro Ser Pro Leu Ser Pro Met Pro Ser Pro Ala 1075 1080 1085
Ser Ser Val Gly Ser Gin Ser Ser Ala Gly Ser Val Gly Ser Ser Gly 1090 1095 1100
Val Ala Ala Thr He Ser Thr Pro Val Thr He Gin Asn Met Thr Ser 1105 1110 1115 1120
Ser Tyr Val Thr He Thr Ser His Val Leu Thr Ala Phe Asp Leu Trp 1125 1130 1135
Glu Gin Ala Glu Ala Leu Thr Arg Lys Asn Lys Glu Phe Phe Ala Arg 1140 1145 1150
Leu Ser Thr Asn Val Cys Thr Leu Ala Leu Asn Ser Ser Leu Val Asp 1155 1160 1165
Leu Val His Tyr Thr Arg Gin Gly Phe Gin Gin Leu Gin Glu Leu Thr 1170 1175 1180 Lys Thr Pro 1185
(2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3376 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 196..1902
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
TTTGGGGCTG AGTTTAATAA GCGAGCGAGC GAGCAAGCGA GCGCGGGGGG AAAAAGGCAG 60
AGAATGTCCG CCATCTACCC TCCGCTCCTG GGCGCGCTCT CATTCATAGC AGCCTCTTCA 120
TGAATTACAG CTGAGGGGGG GCGGAGGAGG GGGGGGTACC ACACAACACC CCAGCAAACC 180
TCCGGGCCCC CAGGC ATG GCT AGC TCG TGT TCC GTG CAG GTG AAG CTG GAG 231 Met Ala Ser Ser Cys Ser Val Gin Val Lys Leu Glu 1 5 10
CTG GGG CAC CGC GCC CAG GTG AGG AAA AAA CCC ACC GTG GAG GGC TTC 279 Leu Gly His Arg Ala Gin Val Arg Lys Lys Pro Thr Val Glu Gly Phe 15 20 25
ACC CAC GAC TGG ATG GTG TTC GTA CGC GGT CCG GAG CAC AGT AAC ATA 327 Thr His Asp Trp Met Val Phe Val Arg Gly Pro Glu His Ser Asn He 30 35 40
CAG CAC TTT GTG GAG AAA GTC GTC TTC CAC TTG CAC GAA AGC TTT CCT 375 Gin His Phe Val Glu Lys Val Val Phe His Leu His Glu Ser Phe Pro 45 50 55 60
AGG CCA AAA AGA GTG TGC AAA GAT CCA CCT TAC AAA GTA GAA GAA TCT 423 Arg Pro Lys Arg Val Cys Lys Asp Pro Pro Tyr Lys Val Glu Glu Ser 65 70 75
GGG TAT GCT GGT TTC ATT TTG CCA ATT GAA GTT TAT TTT AAA AAC AAG 471 Gly Tyr Ala Gly Phe He Leu Pro He Glu Val Tyr Phe Lys Asn Lys 80 85 90
GAA GAA CCT AGG AAA GTC CGC TTT GAT TAT GAC TTA TTC CTG CAT CTT 519 Glu Glu Pro Arg Lys Val Arg Phe Asp Tyr Asp Leu Phe Leu His Leu 95 100 105
GAA GGC CAT CCA CCA GTG AAT CAC CTC CGC TGT GAA AAG CTA ACT TTC 567 Glu Gly His Pro Pro Val Asn His Leu Arg Cys Glu Lys Leu Thr Phe 110 115 120
AAC AAC CCC ACA GAG GAC TTT AGG AGA AAG TTG CTG AAG GCA GGA GGG 615 Asn Asn Pro Thr Glu Asp Phe Arg Arg Lys Leu Leu Lys Ala Gly Gly 125 130 135 140
GAC CCT AAT AGG AGT ATT CAT ACC AGC AGC AGC AGC AGC AGC AGC AGT 663 Asp Pro Asn Arg Ser He His Thr Ser Ser Ser Ser Ser Ser Ser Ser 145 150 155
AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGT AGC AGC AGC AGC AGC 711 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 160 165 170
AGC AGC AGC AGC AGC AGT AGC AGC AGC AGT AGC AGC AGC AGC AGC AGC 759 Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser - Ill -
175 180 185
AGT AGT ACC AGT TTT TCA AAG CCT CAC AAA TTA ATG AAG GAG CAC AAG 807 Ser Ser Thr Ser Phe Ser Lys Pro His Lys Leu Met Lys Glu His Lys 190 195 200
GAA AAA CCT TCT AAA GAC TCC AGA GAA CAT AAA AGT GCC TTC AAA GAA 855 Glu Lys Pro Ser Lys Asp Ser Arg Glu His Lys Ser Ala Phe Lys Glu 205 210 215 220
CCT TCC AGG GAT CAC AAC AAA TCT TCC AAA GAA TCC TCT AAG AAA CCC 903 Pro Ser Arg Asp His Asn Lys Ser Ser Lys Glu Ser Ser Lys Lys Pro 225 230 235
AAA GAA AAT AAA CCA CTG AAA GAA GAG AAA ATA GTT CCT AAG ATG GCC 951 Lys Glu Asn Lys Pro Leu Lys Glu Glu Lys He Val Pro Lys Met Ala 240 245 250
TTC AAG GAA CCT AAA CCC ATG TCA AAA GAG CCA AAA CCA GAT AGT AAC 999 Phe Lys Glu Pro Lys Pro Met Ser Lys Glu Pro Lys Pro Asp Ser Asn 255 260 265
TTA CTC ACC ATC ACC AGT GGA CAA GAT AAG AAG GCT CCT AGT AAA AGG 1047 Leu Leu Thr He Thr Ser Gly Gin Asp Lys Lys Ala Pro Ser Lys Arg 270 275 280
CCG CCC ATT TCA GAT TCT GAA GAA CTC TCA GCC AAA AAA AGG AAA AAG 1095 Pro Pro He Ser Asp Ser Glu Glu Leu Ser Ala Lys Lys Arg Lys Lys 285 290 295 300
AGT AGC TCA GAG GCT TTA TTT AAA AGT TTT TCT AGC GCA CCA CCA CTG 1143 Ser Ser Ser Glu Ala Leu Phe Lys Ser Phe Ser Ser Ala Pro Pro Leu 305 310 315
ATA CTC ACT TGT TCT GCT GAC AAA AAA CAG ATA AAA GAT AAA TCT CAT 1191 He Leu Thr Cys Ser Ala Asp Lys Lys Gin He Lys Asp Lys Ser His 320 325 330
GTC AAG ATG GGA AAG GTC AAA ATT GAA AGT GAG ACA TCA GAG AAG AAG 1239 Val Lys Met Gly Lys Val Lys He Glu Ser Glu Thr Ser Glu Lys Lys 335 340 345
AAA TCA ACG TTA CCG CCA TTT GAT GAT ATT GTG GAT CCC AAT GAT TCA 1287 Lys Ser Thr Leu Pro Pro Phe Asp Asp He Val Asp Pro Asn Asp Ser 350 355 360
GAT GTG GAG GAG AAT ATA TCC TCT AAA TCT GAT TCT GAA CAA CCC AGT 1335 Asp Val Glu Glu Asn He Ser Ser Lys Ser Asp Ser Glu Gin Pro Ser 365 370 375 380
CCT GCC AGC TCC AGC TCC AGC TCC AGC TCC AGC TTC ACA CCA TCC CAG 1383 Pro Ala Ser Ser Ser Ser Ser Ser Ser Ser Ser Phe Thr Pro Ser Gin 385 390 395
ACC AGG CAA CAA GGT CCT TTG AGG TCT ATA ATG AAA GAT CTG CAT TCT 1431 Thr Arg Gin Gin Gly Pro Leu Arg Ser He Met Lys Asp Leu His Ser 400 405 410
GAT GAC AAT GAG GAG GAA TCA GAT GAA GTG GAG GAT AAC GAC AAT GAC 1479 Asp Asp Asn Glu Glu Glu Ser Asp Glu Val Glu Asp Asn Asp Asn Asp 415 420 425
TCT GAA ATG GAG AGG CCT GTA AAT AGA GGA GGC AGC CGA AGT CGC AGA 1527 Ser Glu Met Glu Arg Pro Val Asn Arg Gly Gly Ser Arg Ser Arg Arg 430 435 440
GTT AGC TTA AGT GAT GGC AGC GAT AGT GAA AGC AGT TCT GCT TCT TCA 1575 Val Ser Leu Ser Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala Ser Ser 445 450 455 460
CCC CTA CAT CAC GAA CCT CCA CCA CCC TTA CTA AAA ACC AAC AAC AAC 1623 Pro Leu His His Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn Asn Asn 465 470 475
CAG ATT CTT GAA GTG AAA AGT CCA ATA AAG CAA AGC AAA TCA GAT AAG 1671 Gin He Leu Glu Val Lys Ser Pro He Lys Gin Ser Lys Ser Asp Lys 480 485 490
CAA ATA AAG AAT GGT GAA TGT GAC AAG GCA TAC CTA GAT GAA CTG GTA 1719 Gin He Lys Asn Gly Glu Cys Asp Lys Ala Tyr Leu Asp Glu Leu Val 495 500 505
GAG CTT CAC AGA AGG TTA ATG ACA TTG AGA GAA AGA CAC ATT CTG CAG 1767 Glu Leu His Arg Arg Leu Met Thr Leu Arg Glu Arg His He Leu Gin 510 515 520
CAG ATC GTG AAC CTT ATA GAA GAA ACT GGA CAC TTT CAT ATC ACA AAC 1815 Gin He Val Asn Leu He Glu Glu Thr Gly His Phe His He Thr Asn 525 530 535 540
ACA ACA TTT GAT TTT GAT CTT TGC TCG CTG GAC AAA ACC ACA GTC CGT 1863 Thr Thr Phe Asp Phe Asp Leu Cys Ser Leu Asp Lys Thr Thr Val Arg 545 550 555
AAA CTA CAG AGT TAC CTG GAA ACA TCT GGA ACA TCC TGAGGATATA 1909 Lys Leu Gin Ser Tyr Leu Glu Thr Ser Gly Thr Ser 560 565
ACAACTGGAT GCATCAAGAA CTATTGTGTT TTTTTTTTTT GGTTTTTTTT TTTTTTGGTT 1969
GTGATTTTTT GTTCTTGTTG TTTATATGAA AACACTCAAA ATGATGCAAC CAAAAGGGAA 2029
AAAATAAAAA TCAAACAACC TTCAGCTTTA TTTTTCTTTA AAGCCAGTCA TCATCTCTTG 2089
ATAAAGGAGA GGTTAAAGCA AACCAGCCTC AGCGGACCAC TCTTCTCTCC AAGGAAATCC 2149
CCGGGAAGAG TTAGCCTGGA TAGCCTTGAA AACAAACAAA TCAAACACAA CACAAGAAAA 2209
CTCAAAGAAT GTGTATGGTA TCATGTATCT CTCTGTGGTG GTTCATTCCA CAGGACGAAT 2269
GCATATTCAA CACACTGCCT TATTACATAA CTGATCTATT TATTATCGCA TACAGATATT 2329
CTAAGTCGTT GAGGGAATGA CACCATCAGA CATTATAAGT ACTTGGTCCC GTGGATGCTC 2389
TTTCAATGCA GCACCCTTGC CATCCCAAGC CCAGTGACCT TACTCGTATA CCGTGCCACT 2449
TTCCACCAAC TTTTTCCAAG TCCTTTAACT CGTTGCAGTC TGTATTTTCC ACCTTTTGTT 2509
TTTCCAGTTC CAGGACACAG ATTATCAACT GGGGGGACCA AATAGCCACC TTGATTTTCT 2569
TCTTTGTGGT CTTTTTCCTG AAAGTTGGGG CCCAGTCCTT GGCTGTATCC ATGTAATGAT 2629
CTTGGACCAT GGTAGAAAAT GCACCAAATA GGATCATATG AATTGCTGTC TAGCCTTAGT 2689
CAATAAACTT GTAGGACTTT TAAACAAAAG TGTACCTGTA AATGTCCTGA ATCCAGCATT 2749
GTTGAGCTGT CATCAACATT CTTGTGTCTG TTTTACTGTT ACAATATTAG GTGAATATGG 2809
AAGTAAAGGC ATTCCACAGG ATCATCATTT AAAAAAAAAG AATTCTGGTC CTGTTTTCTA 2869
AAAAAAAAAA ACTGTTGTAG AAATTCTTAA TTTGGATCTA TTTATTAGTC AGAGTTTCAG 2929
CTTTCTTCAG CTGCCAGTGT GTTACTCATC TTTATCCTAA AAATCTGGAA TCAGAGATTT 2989 TTGTTTGTTC ACATATGATT CTCTTAGACA CTTTTATATT TGAAAAAATT AAAATCTTTC 3049
TTTGGGGAAA AATTCTTGGT TATTCTGCCA TAACAGATTA TGTATTAACT TGTAGATTCA 3109
GTGGTTCAAT ACCTGTTTAG TTGCTTGCTA ATATTTCCAG AAGGATTTCT TGTATTGGTG 3169
AAAGACGGTT GGGGATGGGG GGATTTTTTT GTTCTTGTTG TACCCTTGTT TTGAAACTAG 3229
AAATCTGTCC TGTGGCATGC AAAAGAAAGC AAATTATTTT TAAAAGAAAA AAACCAAAGT 3289
ACTTTTGGTG TCATTATTCC ATCTTCTCCA TAAGTGGAGA AATGAAAAGT AAGAACAGCT 3349
CATCTTCAAA GTTTTTACTA GAAATTC 3376
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 568 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Met Ala Ser Ser Cys Ser Val Gin Val Lys Leu Glu Leu Gly His Arg 1 5 10 15
Ala Gin Val Arg Lys Lys Pro Thr Val Glu Gly Phe Thr His Asp Trp 20 25 30
Met Val Phe Val Arg Gly Pro Glu His Ser Asn He Gin His Phe Val 35 40 45
Glu Lys Val Val Phe His Leu His Glu Ser Phe Pro Arg Pro Lys Arg 50 55 60
Val Cys Lys Asp Pro Pro Tyr Lys Val Glu Glu Ser Gly Tyr Ala Gly 65 70 75 80
Phe He Leu Pro He Glu Val Tyr Phe Lys Asn Lys Glu Glu Pro Arg 85 90 95
Lys Val Arg Phe Asp Tyr Asp Leu Phe Leu His Leu Glu Gly His Pro 100 105 110
Pro Val Asn His Leu Arg Cys Glu Lys Leu Thr Phe Asn Asn Pro Thr 115 120 125
Glu Asp Phe Arg Arg Lys Leu Leu Lys Ala Gly Gly Asp Pro Asn Arg 130 135 140
Ser He His Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 145 150 155 160
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 165 170 175
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser 180 185 190
Phe Ser Lys Pro His Lys Leu Met Lys Glu His Lys Glu Lys Pro Ser 195 200 205
Lys Asp Ser Arg Glu His Lys Ser Ala Phe Lys Glu Pro Ser Arg Asp 210 215 220
His Asn Lys Ser Ser Lys Glu Ser Ser Lys Lys Pro Lys Glu Asn Lys 225 230 235 240 Pro Leu Lys Glu Glu Lys He Val Pro Lys Met Ala Phe Lys Glu Pro 245 250 255
Lys Pro Met Ser Lys Glu Pro Lys Pro Asp Ser Asn Leu Leu Thr He 260 265 270
Thr Ser Gly Gin Asp Lys Lys Ala Pro Ser Lys Arg Pro Pro He Ser 275 280 285
Asp Ser Glu Glu Leu Ser Ala Lys Lys Arg Lys Lys Ser Ser Ser Glu 290 295 300
Ala Leu Phe Lys Ser Phe Ser Ser Ala Pro Pro Leu He Leu Thr Cys 305 310 315 320
Ser Ala Asp Lys Lys Gin He Lys Asp Lys Ser His Val Lys Met Gly 325 330 335
Lys Val Lys He Glu Ser Glu Thr Ser Glu Lys Lys Lys Ser Thr Leu 340 345 350
Pro Pro Phe Asp Asp He Val Asp Pro Asn Asp Ser Asp Val Glu Glu 355 360 365
Asn He Ser Ser Lys Ser Asp Ser Glu Gin Pro Ser Pro Ala Ser Ser 370 375 380
Ser Ser Ser Ser Ser Ser Ser Phe Thr Pro Ser Gin Thr Arg Gin Gin 385 390 395 400
Gly Pro Leu Arg Ser He Met Lys Asp Leu His Ser Asp Asp Asn Glu 405 410 415
Glu Glu Ser Asp Glu Val Glu Asp Asn Asp Asn Asp Ser Glu Met Glu 420 425 430
Arg Pro Val Asn Arg Gly Gly Ser Arg Ser Arg Arg Val Ser Leu Ser 435 440 445
Asp Gly Ser Asp Ser Glu Ser Ser Ser Ala Ser Ser Pro Leu His His 450 455 460
Glu Pro Pro Pro Pro Leu Leu Lys Thr Asn Asn Asn Gin He Leu Glu 465 470 475 480
Val Lys Ser Pro He Lys Gin Ser Lys Ser Asp Lys Gin He Lys Asn 485 490 495
Gly Glu Cys Asp Lys Ala Tyr Leu Asp Glu Leu Val Glu Leu His Arg 500 505 510
Arg Leu Met Thr Leu Arg Glu Arg His He Leu Gin Gin He Val Asn 515 520 525
Leu He Glu Glu Thr Gly His Phe His He Thr Asn Thr Thr Phe Asp 530 535 540
Phe Asp Leu Cys Ser Leu Asp Lys Thr Thr Val Arg Lys Leu Gin Ser 545 550 555 560
Tyr Leu Glu Thr Ser Gly Thr Ser 565
(2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 559 amino acids (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Met Asp Asn Gin Cys Thr Val Gin Val Arg Leu Glu Leu Gly His Arg 1 5 10 15
Ala Gin Leu Arg Lys Lys Pro Thr Thr Glu Gly Phe Thr His Asp Trp 20 25 • 30
Met Val Phe Val Arg Gly Pro Glu Gin Cys Asp He Gin His Phe Val 35 40 45
Glu Lys Val Val Phe Trp Leu His Asp Ser Phe Pro Lys Pro Arg Arg 50 55 60
Val Cys Lys Glu Pro Pro Tyr Lys Val Glu Glu Ser Gly Tyr Ala Gly 65 70 75 80
Phe He Met Pro He Glu Val His Phe Lys Asn Lys Glu Glu Pro Arg 85 90 95
Lys Val Cys Phe Thr Tyr Asp Leu Phe Leu Asn Leu Glu Gly Asn Pro 100 105 110
Pro Val Asn His Leu Arg Cys Glu Lys Leu Thr Phe Asn Asn Pro Thr 115 120 125
Thr Glu Phe Arg Tyr Lys Leu Leu Arg Ala Gly Gly Val Met Val Met 130 135 140
Pro Glu Gly Ala Asp Thr Val Ser Arg Pro Ser Pro Asp Tyr Pro Met 145 150 155 160
Leu Pro Thr He Pro Leu Ser Ala Phe Ser Asp Pro Lys Lys Thr Lys 165 170 175
Pro Ser His Gly Ser Lys Asp Ala Asn Lys Glu Ser Ser Lys Thr Ser 180 185 190
Lys Pro His Lys Val Thr Lys Glu His Arg Glu Arg Pro Arg Lys Asp 195 200 205
Ser Glu Ser Lys Ser Ser Ser Lys Glu Leu Glu Arg Glu Gin Ala Lys 210 215 220
Ser Ser Lys Asp Thr Ser Arg Lys Leu Gly Glu Gly Arg Leu Pro Lys 225 230 235 240
Glu Glu Lys Ala Pro Pro Pro Lys Ala Ala Phe Lys Glu Pro Lys Met 245 250 255
Ala Leu Lys Glu Thr Lys Leu Glu Ser Thr Ser Pro Asn Pro Gly Pro 260 265 270
Pro Pro Pro Pro Pro Pro Pro Pro Arg Ala Ser Ser Lys Arg Pro Ala 275 280 285
Thr Ala Asp Ser Pro Lys Pro Ser Ala Lys Lys Gin Lys Lys Ser Ser 290 295 300
Ser Lys Gly Ser Arg Ser Ala Pro Gly Thr Ser Pro Arg Thr Ser Ser 305 310 315 320
Ser Ser Ser Phe Ser Asp Lys Lys Pro Ala Lys Asp Lys Ser Ser Thr 325 330 335
Arg Gly Glu Lys Val Lys Ala Glu Ser Glu Pro Arg Glu Ala Lys Lys 340 345 350
Ala Leu Glu Val Glu Glu Ser Asn Ser Glu Asp Glu Ala Ser Phe Lys 355 360 365
Ser Glu Ser Ala Gin Ser Ser Pro Ser Asn Ser Ser Ser Ser Ser Asp 370 375 380
Ser Ser Ser Asp Ser Asp Phe Glu Pro Ser Gin Asn His Ser Gin Gly 385 390 395 400
Pro Leu Arg Ser Met Val Glu Asp Leu Gin Ser Glu Glu Ser Asp Glu 405 410 415
Asp Asp Ser Ser Ser Gly Glu Glu Ala Ala Gly Lys Thr Asn Pro Gly 420 425 430
Arg Asp Ser Arg Leu Ser Phe Ser Asp Ser Glu Ser Asp Asn Ser Ala 435 440 445
Asp Ser Ser Leu Pro Ser Arg Glu Pro Pro Pro Pro Gin Lys Pro Pro 450 455 460
Pro Pro Asn Ser Lys Val Ser Gly Arg Arg Ser Pro Glu Ser Cys Ser 465 470 475 480
Lys Pro Glu Lys He Leu Lys Lys Gly Thr Tyr Asp Lys Ala Tyr Thr 485 490 495
Asp Glu Leu Val Glu Leu His Arg Arg Leu Met Ala Leu Arg Glu Arg 500 505 510
Asn Val Leu Gin Gin He Val Asn Leu He Glu Glu Thr Gly His Phe 515 520 525
Asn Val Thr Asn Thr Thr Phe Asp Phe Asp Leu Phe Ser Leu Asp Glu 530 535 540
Thr Thr Val Arg Lys Leu Gin Ser Cys Leu Glu Ala Val Ala Thr 545 550 555
(2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..260
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
CA GAT GAA GTG GAG GAT AAC GAC AAT GAC TCT GAA ATG GAG AGG CCT 47
Asp Glu Val Glu Asp Asn Asp Asn Asp Ser Glu Met Glu Arg Pro 1 5 10 15
GTA AAT AGA GGA GGC AGC CGA AGT CGC AGA GTT AGC TTA AGT GAT GGC 95 Val Asn Arg Gly Gly Ser Arg Ser Arg Arg Val Ser Leu Ser Asp Gly 20 25 30
AGC GAT AGT GAA AGC AGT TCT GCT TCT TCA CCC CTA CAT CAC GAA CCT 143 Ser Asp Ser Glu Ser Ser Ser Ala Ser Ser Pro Leu His His Glu Pro 35 40 45
CCA CCA CCC TTA CTA AAA ACC AAC AAC AAC CAG ATT CTT GAA GTA AAA 191 Pro Pro Pro Leu Leu Lys Thr Asn Asn Asn Gin He Leu Glu Val Lys 50 55 60
ATT CCA GCA GAT GGA GTC CAC AGG ATC AGA GTG GAC TTT AAG TTT GTG 239 He Pro Ala Asp Gly Val His Arg He Arg Val Asp Phe Lys Phe Val 65 70 75
TAT TGC CAA GTC TGT TGT GAG CC 262
Tyr Cys Gin Val Cys Cys Glu 80 85
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Asp Glu Val Glu Asp Asn Asp Asn Asp Ser Glu Met Glu Arg Pro Val 1 5 10 15
Asn Arg Gly Gly Ser Arg Ser Arg Arg Val Ser Leu Ser Asp Gly Ser 20 25 30
Asp Ser Glu Ser Ser Ser Ala Ser Ser Pro Leu His His Glu Pro Pro 35 40 45
Pro Pro Leu Leu Lys Thr Asn Asn Asn Gin He Leu Glu Val Lys He 50 55 60
Pro Ala Asp Gly Val His Arg He Arg Val Asp Phe Lys Phe Val Tyr 65 70 75 80
Cys Gin Val Cys Cys Glu 85
(2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 439 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..436
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
A CCT ACT ACA GGA CCG CCA AGA AAA GAA GTT CCC AAA ACC ACT CCT 46
Pro Thr Thr Gly Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro 1 5 10 15
AGT GAG CCC AAG AAA AAG CAG CCT CCA CCA CCA GAA TCA GGT CCA GAG 94 Ser Glu Pro Lys Lys Lys Gin Pro Pro Pro Pro Glu Ser Gly Pro Glu 20 25 30
CAG AGC AAA CAG AAA AAA GTG GCT CCC CGC CCA AGT ATC CCT GTA AAA 142 Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser He Pro Val Lys 35 40 45
CAA AAA CCA AAA GAA AAG ATT CTT GAA GTG AAA AGT CCA ATA AAG CAA 190 Gin Lys Pro Lys Glu Lys He Leu Glu Val Lys Ser Pro He Lys Gin 50 55 60
AGC AAA TCA GAT AAG CAA ATA AAG AAT GGT GAA TGT GAC AAG GCA TAC 238 Ser Lys Ser Asp Lys Gin He Lys Asn Gly Glu Cys Asp Lys Ala Tyr 65 70 75
CTA GAT GAA CTG GTA GAG CTT CAC AGA AGG TTA ATG ACA TTG AGA GAA 286 Leu Asp Glu Leu Val Glu Leu His Arg Arg Leu Met Thr Leu Arg Glu 80 85 90 95
AGA CAC ATT CTG CAG CAG ATC GTG AAC CTT ATA GAA GAA ACT GGA CAC 334 Arg His He Leu Gin Gin He Val Asn Leu He Glu Glu Thr Gly His 100 105 110
TTT CAT ATC ACA AAC ACA ACA CTT GAT TTT GAT CTT TGC TCG CTG GAC 382 Phe His He Thr Asn Thr Thr Leu Asp Phe Asp Leu Cys Ser Leu Asp 115 120 125
AAA ACC ACA GTC CGT AAA CTA CAG AGT TAC CTG GAA ACA TCT GGA ACA 430 Lys Thr Thr Val Arg Lys Leu Gin Ser Tyr Leu Glu Thr Ser Gly Thr 130 135 140
TCC TGAGGA 439
Ser
145
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 144 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Pro Thr Thr Gly Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro Ser 1 5 10 15
Glu Pro Lys Lys Lys Gin Pro Pro Pro Pro Glu Ser Gly Pro Glu Gin 20 25 30
Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser He Pro Val Lys Gin 35 40 45
Lys Pro Lys Glu Lys He Leu Glu Val Lys Ser Pro He Lys Gin Ser 50 55 60
Lys Ser Asp Lys Gin He Lys Asn Gly Glu Cys Asp Lys Ala Tyr Leu 65 70 75 80
Asp Glu Leu Val Glu Leu His Arg Arg Leu Met Thr Leu Arg Glu Arg 85 90 95
His He Leu Gin Gin He Val Asn Leu He Glu Glu Thr Gly His Phe 100 105 110
His He Thr Asn Thr Thr Leu Asp Phe Asp Leu Cys Ser Leu Asp Lys 115 120 125
Thr Thr Val Arg Lys Leu Gin Ser Tyr Leu Glu Thr Ser Gly Thr Ser 130 135 140
(2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 343 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..341
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
CA ACG TTA CCG CCA TTT GAT GAT ATT GTG GAT CCC AAT GAT TCA GAT 47
Thr Leu Pro Pro Phe Asp Asp He Val Asp Pro Asn Asp Ser Asp 1 5 10 15
GTG GAG GAG AAT ATA TCC TCT AAA TCT GAT TTT GTG TAT TGC CAA GTC 95 Val Glu Glu Asn He Ser Ser Lys Ser Asp Phe Val Tyr Cys Gin Val 20 25 30
TGT TGT GAG CCC TTC CAC AAG TTT TGT TTA GAG GAG AAC GAG CGC CCT 143 Cys Cys Glu Pro Phe His Lys Phe Cys Leu Glu Glu Asn Glu Arg Pro 35 40 45
CTG GAG GAC CAG CTG GAA AAT TGG TGT TGT CGT CGT TGC AAA TTC TGT 191 Leu Glu Asp Gin Leu Glu Asn Trp Cys Cys Arg Arg Cys Lys Phe Cys 50 55 60
CAC GTT TGT GGA AGG CAA CAT CAG GCT ACA AAG CAG CTG CTG GAG TGT 239 His Val Cys Gly Arg Gin His Gin Ala Thr Lys Gin Leu Leu Glu Cys 65 70 75
AAT AAG TGC CGA AAC AGC TAT CAC CCT GAG TGC CTG GGA CCA AAC TAC 287 Asn Lys Cys Arg Asn Ser Tyr His Pro Glu Cys Leu Gly Pro Asn Tyr 80 85 90 95
CCC ACC AAA CCC ACA AAG AAG AAG AAA GTC TGG ATC TGT ACC AAG TGT 335 Pro Thr Lys Pro Thr Lys Lys Lys Lys Val Trp He Cys Thr Lys Cys 100 105 110
GTT CGC TG 343
Val Arg
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Thr Leu Pro Pro Phe Asp Asp He Val Asp Pro Asn Asp Ser Asp Val 1 5 10 15
Glu Glu Asn He Ser Ser Lys Ser Asp Phe Val Tyr Cys Gin Val Cys 20 25 30
Cys Glu Pro Phe His Lys Phe Cys Leu Glu Glu Asn Glu Arg Pro Leu 35 40 45
Glu Asp Gin Leu Glu Asn Trp Cys Cys Arg Arg Cys Lys Phe Cys His 50 55 60
Val Cys Gly Arg Gin His Gin Ala Thr Lys Gin Leu Leu Glu Cys Asn 65 70 75 80
Lys Cys Arg Asn Ser Tyr His Pro Glu Cys Leu Gly Pro Asn Tyr Pro 85 90 95
Thr Lys Pro Thr Lys Lys Lys Lys Val Trp He Cys Thr Lys Cys Val 100 105 110
Arg
( 2 ) INFORMATION FOR SEQ ID NO : 38 :
( i ) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 11
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
ATTCTTGAAG T 11
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
TCCTCAGGAT GTTCCAGATG T 21
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GGCTCACAAC AGACTTGGCA A 21
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
ACCTACTACA GGACCGCCAA G 21
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
CAGATGAAGT GGAGGATAAC G 21
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
CAGCGAACAC ACTTGGTACA G 21 (2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
CAACGTTACC GCCATTTGAT 20
(2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
TGAGGAGAGA TTTGTTTCTC TGCCATTTCT CAGGGATGTA TTCTATTTTG TAGGGAAAAG 60
CCTTATCCTT GACTTCTATG TAGATGGCAG TGGAATTTCT TAAAATTAAG AAA 113
(2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
TTCCTCATAG GAAATAAAAT CTTTTAAATT AGCTTGTTTA GTTCCAGGAA AAAGGAAAAG 60
CCTTATCCTT GACTTCTATG TAGATGGCAG TGGAATTTCT TAAAATTAAG AAA 113
(2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
TTCCTCATAG GAAATAAAAT CTTTTAAATT AGCTTGTTTA GTTCCAGGAA AAAAAGAAAA 60
CCCAACAAAA CCATTGTATT TTTAGTTACT GTTTTCTTAA ATTTATAAAT TAA 113
(2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1612 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Met Ser Ala Gly Gly Arg Asp Glu Glu Arg Arg Lys Leu Ala Asp He 1 5 10 15
He His His Trp Asn Ala Asn Arg Leu Asp Leu Phe Glu He Ser Gin 20 25 30
Pro Thr Glu Asp Leu Glu Phe His Gly Val Met Arg Phe Tyr Phe Gin 35 40 45
Asp Lys Ala Ala Gly Asn Phe Ala Thr Lys Cys He Arg Val Ser Ser 50 55 60
Thr Ala Thr Thr Gin Asp Val He Glu Thr Leu Ala Glu Lys Phe Arg 65 70 75 80
Pro Asp Met Arg Met Leu Ser Ser Pro Lys Tyr Ser Leu Tyr Glu Val 85 90 95
His Val Ser Gly Glu Arg Arg Leu Asp He Asp Glu Lys Pro Leu Val 100 105 110
Val Gin Leu Asn Trp Asn Lys Asp Asp Arg Glu Gly Arg Phe Val Leu 115 120 125
Lys Asn Glu Asn Asp Ala He Pro Pro Lys Ala Gin Ser Asn Gly Pro 130 135 140
Glu Lys Gin Glu Lys Glu Gly Val He Gin Asn Phe Lys Arg Thr Leu 145 150 155
Ser Lys Lys Glu Lys Lys Glu Lys Lys Lys Arg Glu Lys Glu Ala Leu 165 170 175
Arg Gin Ala Ser Asp Lys Asp Asp Arg Pro Phe Gin Gly Glu Asp Val 180 185 190
Glu Asn Ser Arg Leu Ala Ala Glu Val Tyr Lys Asp Met Pro Glu Thr 195 200 205
Ser Phe Thr Arg Thr He Ser Asn Pro Glu Val Val Met Lys Arg Arg 210 215 220
Arg Gin Gin Lys Leu Glu Lys Arg Met Gin Glu Phe Arg Ser Ser Asp 225 230 235
Gly Arg Pro Asp Ser Gly Gly Thr Leu Arg He Tyr Ala Asp Ser Leu 245 250 255
Lys Pro Asn He Pro Tyr Lys Thr He Leu Leu Ser Thr Thr Asp Pro 260 265 270 Ala Asp Phe Ala Val Ala Glu Ala Leu Glu Lys Tyr Gly Leu Glu Lys 275 280 285
Glu Asn Pro Lys Asp Tyr Cys He Ala Arg Val Met Leu Pro Pro Gly 290 295 300
Ala Gin His Ser Asp Glu Lys Gly Ala Lys Glu He He Leu Asp Asp 305 310 315
320
Asp Glu Cys Pro Leu Gin He Phe Arg Glu Trp Pro Ser Asp Lys Gly 325 330 335
He Leu Val Phe Gin Leu Lys Arg Arg Pro Pro Asp His He Pro Lys 340 345 350
Lys Thr Lys Lys His Leu Glu Gly Lys Thr Pro Lys Gly Lys Glu Arg 355 360 365
Ala Asp Gly Ser Val Tyr Gly Ser Thr Leu Pro Pro Glu Lys Leu Pro 370 375 380
Tyr Leu Val Glu Leu Ser Pro Asp Gly Ser Asp Ser Arg Asp Lys Pro 385 390 395 400
Lys Leu Tyr Arg Leu Gin Leu Ser Val Thr Glu Val Gly Thr Glu Lys 405 410 415
Leu Asp Asp Asn Ser He Gin Leu Phe Gly Pro Gly He Gin Pro His 420 425 430
His Cys Asp Leu Thr Asn Met Asp Gly Val Val Thr Val Thr Pro Arg 435 440 445
Ser Met Asp Ala Glu Thr Tyr Val Glu Gly Gin Arg He Ser Glu Thr 450 455 460
Thr Met Leu Gin Ser Gly Met Lys Val Gin Phe Gly Ala Ser His Val 465 470 475 480
Phe Lys Phe Val Asp Pro Ser Gin Asp His Ala Leu Ala Lys Arg Ser 485 490 495
Val Asp Gly Gly Leu Met Val Lys Gly Pro Arg His Lys Pro Gly He 500 505 510
Val Gin Glu Thr Thr Phe Asp Leu Gly Gly Asp He His Ser Gly Thr 515 520 525
Ala Leu Pro Thr Ser Lys Ser Thr Thr Arg Leu Asp Ser Asp Arg Val 530 535 540
Ser Ser Ala Ser Ser Thr Ala Glu Arg Gly Met Val Lys Pro Met He 545 550 555 560
Arg Val Glu Gin Gin Pro Asp Tyr Arg Arg Gin Glu Ser Arg Thr Gin 565 570 575
Asp Ala Ser Gly Pro Glu Leu He Leu Pro Ala Ser He Glu Phe Arg 580 585 590
Glu Ser Ser Glu Asp Ser Phe Leu Ser Ala He He Asn Tyr Thr Asn 595 600 605
Ser Ser Thr Val His Phe Lys Leu Ser Pro Thr Tyr Val Leu Tyr Met 610 615 620
Ala Cys Arg Tyr Val Leu Ser Asn Gin Tyr Arg Pro Asp He Ser Pro 625 630 635 640
Thr Glu Arg Thr His Lys Val He Ala Val Val Asn Lys Met Val Ser 645 650 655
Met Met Glu Gly Val He Gin Lys Gin Lys Asn He Ala Gly Ala Leu 660 665 670
Ala Phe Trp Met Ala Asn Ala Ser Glu Leu Leu Asn Phe He Lys Gin 675 680 685
Asp Arg Asp Leu Ser Arg He Thr Leu Asp Ala Gin Asp Val Leu Ala 690 695 700
His Leu Val Gin Met Ala Phe Lys Tyr Leu Val His Cys Leu Gin Ser 705 710 715 720
Glu Leu Asn Asn Tyr Met Pro Ala Phe Leu Asp Asp Pro Glu Glu Asn 725 730 735
Ser Leu Gin Arg Pro Lys He Asp Asp Val Leu His Thr Leu Thr Gly 740 745 750
Ala Met Ser Leu Leu Arg Arg Cys Arg Val Asn Ala Ala Leu Thr He 755 760 765
Gin Leu Phe Ser Gin Leu Phe His Phe He Asn Met Trp Leu Phe Asn 770 775 780
Arg Leu Val Thr Asp Pro Asp Ser Gly Leu Cys Ser His Tyr Trp Gly 785 790 795
800
Ala He He Arg Gin Gin Leu Gly His He Glu Ala Trp Ala Glu Lys 805 810 815
Gin Gly Leu Glu Leu Ala Ala Asp Cys His Leu Ser Arg He Val Gin 820 825 830 Ala Thr Thr Leu Leu Thr Met Asp Lys Tyr Ala Pro Asp Asp He Pro 835 840 845
Asn He Asn Ser Thr Cys Phe Lys Leu Asn Ser Leu Gin Leu Gin Ala 850 855 860
Leu Leu Gin Asn Tyr His Cys Ala Pro Asp Glu Pro Phe He Pro Thr 865 870 875 880
Asp Leu He Glu Asn Val Val Thr Val Ala Glu Asn Thr Ala Asp Glu 885 890 895
Leu Ala Arg Ser Asp Gly Arg Glu Val Gin Leu Glu Glu Asp Pro Asp 900 905 910
Leu Gin Leu Pro Phe Leu Leu Pro Glu Asp Gly Tyr Ser Cys Asp Val 915 920 925
Val Arg Asn He Pro Asn Gly Leu Gin Glu Phe Leu Asp Pro Leu Cys 930 935 940
Gin Arg Gly Phe Cys Arg Leu He Pro His Thr Arg Ser Pro Gly Thr 945 950 955 960
Trp Thr He Tyr Phe Glu Gly Ala Asp Tyr Glu Ser His Leu Leu Arg 965 970 975
Glu Asn Thr Glu Leu Ala Gin Pro Leu Arg Lys Glu Pro Glu He He 980 985 990
Thr Val Thr Leu Lys Lys Gin Asn Gly Met Gly Leu Ser He Val Ala 995 1000 1005
Ala Lys Gly Ala Gly Gin Asp Lys Leu Gly He Tyr Val Lys Ser Val 1010 1015 1020
Val Lys Gly Gly Ala Ala Asp Val Asp Gly Arg Leu Ala Ala Gly Asp 1025 1030 1035 1040
Gin Leu Leu Ser Val Asp Gly Arg Ser Leu Val Gly Leu Ser Gin Glu
1045 1050 1055
Arg Ala Ala Glu Leu Met Thr Arg Thr Ser Ser Val Val Thr Leu Glu 1060 1065 1070
Val Ala Lys Gin Gly Ala He Tyr His Gly Leu Ala Thr Leu Leu Asn 1075 1080 1085
Gin Pro Ser Pro Met Met Gin Arg He Ser Asp Arg Arg Gly Ser Gly 1090 1095 1100
Lys Pro Arg Pro Lys Ser Glu Gly Phe Glu Leu Tyr Asn Asn Ser Thr 1105 1110 1115 1120 Gin Asn Gly Ser Pro Glu Ser Pro Gin Leu Pro Trp Ala Glu Tyr Ser 1125 1130 1135
Glu Pro Lys Lys Leu Pro Gly Asp Asp Arg Leu Met Lys Asn Arg Ala 1140 1145 1150
Asp His Arg Ser Ser Pro Asn Val Ala Asn Gin Pro Pro Ser Pro Gly 1155 1160 1165
Gly Lys Ser Ala Tyr Ala Ser Gly Thr Thr Ala Lys He Thr Ser Val 1170 1175 1180
Ser Thr Gly Asn Leu Cys Thr Glu Glu Gin Thr Pro Pro Pro Arg Pro 1185 1190 1195 1200
Glu Ala Tyr Pro He Pro Thr Gin Thr Tyr Thr Arg Glu Tyr Phe Thr 1205 1210 1215
Phe Pro Ala Ser Lys Ser Gin Asp Arg Met Ala Pro Pro Gin Asn Gin 1220 1225 1230
Trp Pro Asn Tyr Glu Glu Lys Pro His Met His Thr Asp Ser Asn His 1235 1240 1245
Ser Ser He Ala He Gin Arg Val Thr Arg Ser Gin Glu Glu Leu Arg 1250 1255 1260
Glu Asp Lys Ala Tyr Gin Leu Glu Arg His Arg He Glu Ala Ala Met
1265 1270 1275 1280
Asp Arg Lys Ser Asp Ser Asp Met Trp He Asn Gin Ser Ser Ser Leu
1285 1290 1295
Asp Ser Ser Thr Ser Ser Gin Glu His Leu Asn His Ser Ser Lys Ser 1300 1305 1310
Val Thr Pro Ala Ser Thr Leu Thr Lys Ser Gly Pro Gly Arg Trp Lys 1315 1320 1325
Thr Pro Ala Ala He Pro Ala Thr Pro Val Ala Val Ser Gin Pro He 1330 1335 1340
Arg Thr Asp Leu Pro Pro Pro Pro Pro Pro Pro Pro Val His Tyr Ala 1345 1350 1355 1360
Gly Asp Phe Asp Gly Met Ser Met Asp Leu Pro Leu Pro Pro Pro Pro 1365 1370 1375
Ser Ala Asn Gin He Gly Leu Pro Ser Ala Gin Val Ala Ala Ala Glu 1380 1385 1390
Arg Arg Lys Arg Glu Glu His Gin Arg Trp Tyr Glu Lys Glu Lys Ala 1395 1400 1405 Pro Leu Glu Glu Glu Arg Glu Arg Lys Arg Arg Glu Gin Glu Arg Lys 1410 1415 1420
Leu Gly Gin Met Arg Thr Gin Ser Leu Asn Pro Ala Pro Phe Ser Pro 1425 1430 1435 1440
Leu Thr Ala Gin Gin Met Lys Pro Glu Lys Pro Ser Thr Leu Gin Arg 1445 1450 1455
Pro Gin Glu Thr Val He Arg Glu Leu Gin Pro Gin Gin Gin Pro Arg 1460 1465 1470
Thr He Glu Arg Arg Asp Leu Gin Tyr He Thr Val Ser Lys Glu Glu 1475 1480 1485
Leu Ser Ser Gly Asp Ser Leu Ser Pro Asp Pro Trp Lys Arg Asp Ala 1490 1495 1500
Lys Glu Lys Leu Glu Lys Gin Gin Gin Met His He Val Asp Met Leu 1505 1510 1515 1520
Ser Lys Glu He Gin Glu Leu Gin Ser Lys Pro Asp Arg Ser Ala Glu 1525 1530 1535
Glu Ser Asp Arg Leu Arg Lys Leu Met Leu Glu Trp Gin Phe Gin Lys 1540 1545 1550
Arg Leu Gin Glu Ser Lys Gin Lys Asp Glu Asp Asp Glu Glu Glu Glu 1555 1560 1565
Asp Asp Asp Val Asp Thr Met Leu He. Met Gin Arg Leu Glu Ala Glu 1570 1575 1580
Arg Arg Ala Arg Val Lys Gly Gly Val Leu Trp Leu Cys Pro Ser Val 1585 1590 1595 1600
Val Pro He Leu Ala Ser Ala Cys Phe Pro Trp Gly 1605 1610
(2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..269
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
GT CCA GAG CAG AGC AAA CAG AAA AAA GTG GCT CCC CGC CCA AGT ATC 47
Pro Glu Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser He 1 5 10 15 CCT GTA AAA CAA AAA CCA AAA GAA AAG GAT TTG GAG TTC CAT GGA GTG 95 Pro Val Lys Gin Lys Pro Lys Glu Lys Asp Leu Glu Phe His Gly Val 20 25 30
ATG AGA TTT TAT TTT CAA GAT AAA GCT GCT GGA AAC TTT GCA ACA AAA 143 Met Arg Phe Tyr Phe Gin Asp Lys Ala Ala Gly Asn Phe Ala Thr Lys 35 40 45
TGT ATT CGG GTC TCT AGT ACT GCC ACC ACT CAA GAT GTA ATC GAA ACG 191 Cys He Arg Val Ser Ser Thr Ala Thr Thr Gin Asp Val He Glu Thr 50 55 60
CTC GCG GAG AAA TTT CGA CCT GAT ATG CGA ATG CTG TCC TCT CCC AAG 239 Leu Ala Glu Lys Phe Arg Pro Asp Met Arg Met Leu Ser Ser Pro Lys 65 70 75
TAT TCA CTC TAT GAA GTG CAT GTC AGC GGA G 270
Tyr Ser Leu Tyr Glu Val His Val Ser Gly 80 85
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 89 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Pro Glu Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser He Pro 1 5 10 15
Val Lys Gin Lys Pro Lys Glu Lys Asp Leu Glu Phe His Gly Val Met 20 25 30
Arg Phe Tyr Phe Gin Asp Lys Ala Ala Gly Asn Phe Ala Thr Lys Cys 35 40 45
He Arg Val Ser Ser Thr Ala Thr Thr Gin Asp Val He Glu Thr Leu 50 55 60
Ala Glu Lys Phe Arg Pro Asp Met Arg Met Leu Ser Ser Pro Lys Tyr 65 70 75 80
Ser Leu Tyr Glu Val His Val Ser Gly 85
(2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 85 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Lys Lys Gin Asn Gly Met Gly Leu Ser He Val Ala Ala Lys Gly Ala 1 5 10 15
Gly Gin Asp Lys Leu Gly He Tyr Val Lys Ser Val Val Lys Gly Gly 20 ' 25 30
Ala Ala Asp Val Asp Gly Arg Leu Ala Ala Gly Asp Gin Leu Leu Ser 35 40 45
Val Asp Gly Arg Ser Leu Val Gly Leu Ser Gin Glu Arg Ala Ala Glu 50 55 60
Leu Met Thr Arg Thr Ser Ser Val Val Thr Leu Glu Val Ala Lys Gin 65 70 75 80
Gly Ala He Tyr Pro 85
(2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Arg Lys Gly Asp Ser Val Gly Leu Arg Leu Ala Gly Gly Asn Asp Val 1 5 10 15
Gly He Phe Val Ala Gly Val Leu Glu Asp Ser Pro Ala Ala Lys Glu 20 25 30
Gly Leu Glu Glu Gly Asp Gin He Leu Arg Val Asn Asn Val Asp Phe 35 40 45
Thr Asn He He Arg Glu Glu Ala Val Leu Phe Leu Leu Asp Leu Pro 50 55 60
Lys Gly Glu Glu Val Thr He Leu Ala Gin Lys Lys Lys Asp Val Tyr 65 70 75 80
(2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 86 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Lys Gly Pro Lys Gly Leu Gly Phe Ser He Ala Gly Gly Val Gly Asn 1 5 10 15
Gin His He Pro Gly Asp Asn Ser He Tyr Val Thr Lys He He Glu 20 25 30
Gly Gly Ala Ala His Lys Asp Gly Arg Leu Gin He Gly Asp Lys He 35 40 45
Leu Ala Val Asn Ser Val Gly Leu Glu Asp Val Met His Glu Asp Ala 50 55 60
Val Ala Ala Leu Lys Asn Thr Tyr Asp Val Val Tyr Leu Lys Val Ala 65 70 75 80
Lys Pro Ser Asn Ala Tyr 85
(2) INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
Lys Gly Pro Gin Gly Leu Gly Phe Asn He Val Gly Gly Glu Asp Gly 1 5 10 15
Gin Gly He Tyr Val Ser Phe He Leu Ala Gly Gly Pro Ala Asp Leu 20 25 30
Gly Ser Glu Leu Lys Arg Gly Asp Gin Leu Leu Ser Val Asn Asn Val 35 40 45
Asn Leu Thr His Ala Thr His Glu Glu Ala Ala Gin Ala Leu Lys Thr 50 55 60
Ser Gly Gly Val Val Thr Leu Leu Ala Gin Tyr Arg Pro Glu Glu Tyr 65 70 75 80
(2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1093 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Met Lys Glu Met Val Gly Gly Cys Cys Val Cys Ser Asp Glu Arg Gly 1 5 10 15
Trp Ala Glu Asn Pro Leu Val Tyr Cys Asp Gly His Ala Cys Ser Val 20 25 30
Ala Val His Gin Ala Cys Tyr Gly He Val Gin Val Pro Thr Gly Pro 35 40 45
Trp Phe Cys Arg Lys Cys Glu Ser Gin Glu Arg Ala Ala Arg Val Arg 50 55 60
Cys Glu Leu Cys Pro His Lys Asp Gly Ala Leu Lys Arg Thr Asp Asn 65 70 75 80
Gly Gly Trp Ala His Val Val Cys Ala Leu Tyr He Pro Glu Val Gin 85 90 95
Phe Ala Asn Val Leu Thr Met Glu Pro He Val Leu Gin Tyr Val Pro 100 105 110
His Asp Arg Phe Asn Lys Thr Cys Tyr He Cys Glu Glu Thr Gly Arg 115 120 125
Glu Ser Lys Ala Ala Ser Gly Ala Cys Met Thr Cys Asn Arg His Gly 130 135 140
Cys Arg Gin Ala Phe His Val Thr Cys Ala Gin Met Ala Gly Leu Leu 145 150 155 160
Cys Glu Glu Glu Val Leu Glu Val Asp Asn Val Lys Tyr Cys Gly Tyr 165 170 175
Cys Lys Tyr His Phe Ser Lys Met Lys Thr Ser Arg His Ser Ser Gly 180 185 190
Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Ser Met Gly Gly Gly 195 200 205
Gly Ser Gly Phe He Ser Gly Arg Arg Ser Arg Ser Ala Ser Pro Ser 210 215 220
Thr Gin Gin Glu Lys His Pro Thr His His Glu Arg Gly Gin Lys Lys 225 230 235 240
Ser Arg Lys Asp Lys Glu Arg Leu Lys Gin Lys His Lys Lys Arg Pro 245 250 255
Glu Ser Pro Pro Ser He Leu Thr Pro Pro Val Val Pro Thr Ala Asp 260 265 270
Lys Val Ser Ser Ser Ala Ser Ser Ser Ser His His Glu Ala Ser Thr 275 280 285
Gin Glu Thr Ser Glu Ser Ser Arg Glu Ser Lys Gly Lys Lys Ser Ser 290 295 300
Ser His Ser Leu Ser His Lys Gly Lys Lys Leu Ser Ser Gly Lys Gly 305 310 315 320
Val Ser Ser Phe Thr Ser Ala Ser Ser Ser Ser Ser Ser Ser Ser Ser 325 330 ' 335
Ser Ser Gly Gly Pro Phe Gin Pro Ala Val Ser Ser Leu Gin Ser Ser 340 345 350
Pro Asp Phe Ser Ala Phe Pro Lys Leu Glu Gin Pro Glu Glu Asp Lys 355 360 365
Tyr Ser Lys Pro Thr Ala Pro Ala Pro Ser Ala Pro Pro Ser Pro Ser 370 375 380
Ala Pro Glu Pro Pro Lys Ala Asp Leu Phe Glu Gin Lys Val Val Phe 385 390 395 400
Ser Gly Phe Gly Pro He Met Arg Phe Ser Thr Thr Thr Ser Ser Ser 405 410 415
Gly Arg Ala Arg Ala Pro Ser Pro Gly Asp Tyr Lys Ser Pro His Val 420 425 430
Thr Gly Ser Gly Ala Ser Ala Gly Thr His Lys Arg Met Pro Ala Leu 435 440 445
Ser Ala Thr Pro Val Pro Ala Asp Glu Thr Pro Glu Thr Gly Leu Lys 450 455 460
Glu Lys Lys His Lys Ala Ser Lys Arg Ser Arg His Gly Pro Gly Arg 465 470 475 480
Pro Lys Gly Ser Arg Asn Lys Glu Gly Thr Gly Gly Pro Ala Ala Pro 485 490 495
Ser Leu Pro Ser Ala Gin Leu Ala Gly Phe Thr Ala Thr Ala Ala Ser 500 505 510
Pro Phe Ser Gly Gly Ser Leu Val Ser Ser Gly Leu Gly Gly Leu Ser 515 520 525
Ser Arg Thr Phe Gly Pro Ser Gly Ser Leu Pro Ser Leu Ser Leu Glu 530 535 540 Ser Pro Leu Leu Gly Ala Gly He Tyr Thr Ser Asn Lys Asp Pro He 545 550 555 560
Ser His Ser Gly Gly Met Leu Arg Ala Val Cys Ser Thr Pro Leu Ser 565 570 575
Ser Ser Leu Leu Gly Pro Pro Gly Thr Ser Ala Leu Pro Arg Leu Ser 580 585 590
Arg Ser Pro Phe Thr Ser Thr Leu Pro Ser Ser Ser Ala Ser He Ser 595 600 605
Thr Thr Gin Val Phe Ser Leu Ala Gly Ser Thr Phe Ser Leu Pro Ser 610 615 620
Thr His He Phe Gly Thr Pro Met Gly Ala Val Asn Pro Leu Leu Ser 625 630 635 640
Gin Ala Glu Ser Ser His Thr Glu Pro Asp Leu Glu Asp Cys Ser Phe 645 650 655
Arg Cys Arg Gly Thr Ser Pro Gin Glu Ser Leu Ser Ser Met Ser Pro 660 665 670
He Ser Ser Leu Pro Ala Leu Phe Asp Gin Thr Ala Ser Ala Pro Cys 675 680 685
Gly Gly Gly Gin Leu Asp Pro Ala Ala Pro Gly Thr Thr Asn Met Glu 690 695 700
Gin Leu Leu Glu Lys Gin Gly Asp Gly Glu Ala Gly Val Asn He Val 705 710 715 720
Glu Met Leu Lys Ala Leu His Ala Leu Gin Lys Glu Asn Gin Arg Leu 725 730 735
Gin Glu Gin He Leu Ser Leu Thr Ala Lys Lys Glu Arg Leu Gin He 740 745 750
Leu Asn Val Gin Leu Ser Val Pro Phe Pro Ala Leu Pro Ala Ala Leu 755 760 765
Pro Ala Ala Asn Gly Pro Val Pro Gly Pro Tyr Gly Leu Pro Pro Gin 770 775 780
Ala Gly Ser Ser Asp Ser Leu Ser Thr Ser Lys Ser Pro Pro Gly Lys 785 790 795 800
Ser Ser Leu Gly Leu Asp Asn Ser Leu Ser Thr Ser Ser Glu Asp Pro 805 810 815
His Ser Gly Cys Pro Ser Arg Ser Ser Ser Ser Leu Ser Phe His Ser 820 825 830
Thr Pro Pro Pro Leu Pro Leu Leu Gin Gin Ser Pro Ala Thr Leu Pro 835 840 845
Leu Ala Leu Pro Gly Ala Pro Ala Pro Leu Pro Pro Gin Pro Gin Asn 850 855 860
Gly Leu Gly Arg Ala Pro Gly Ala Ala Gly Leu Gly Ala Met Pro Met 865 870 875 880
Ala Glu Gly Leu Leu Gly Gly Leu Ala Gly Ser Gly Gly Leu Pro Leu 885 890 895 Asn Gly Leu Leu Gly Gly Leu Asn Gly Ala Ala Ala Pro Asn Pro Ala 900 905 910
Ser Leu Ser Gin Ala Gly Gly Ala Pro Thr Leu Gin Leu Pro Gly Cys 915 920 925
Leu Asn Ser Leu Thr Glu Gin Gin Arg His Leu Leu Gin Gin Gin Glu 930 935 940
Gin Gin Leu Gin Gin Leu Gin Gin Leu Leu Ala Ser Pro Gin Leu Thr 945 950 955. 960
Pro Glu His Gin Thr Val Val Tyr Gin Met He Gin Gin He Gin Gin 965 970 975
Lys Arg Glu Leu Gin Arg Leu Gin Met Ala Gly Gly Ser Gin Leu Pro 980 985 . 990
Met Ala Ser Leu Leu Ala Gly Ser Ser Thr Pro Leu Leu Ser Ala Gly 995 1000 1005
Thr Pro Gly Leu Leu Pro Thr Ala Ser Ala Pro Pro Leu Leu Pro Ala 1010 1015 1020
Gly Ala Leu Val Ala Pro Ser Leu Gly Asn Asn Thr Ser Leu Met Ala 1025 1030 1035 1040
Ala Ala Ala Ala Ala Ala Ala Val Ala Ala Ala Gly Gly Pro Pro Val 1045 1050 1055
Leu Thr Ala Gin Thr Asn Pro Phe Leu Ser Leu Ser Gly Ala Glu Gly 1060 1065 1070
Ser Gly Gly Gly Pro Lys Gly Gly Thr Ala Asp Lys Gly Ala Ser Ala 1075 1080 1085
Asn Gin Glu Lys Gly 1090
(2) INFORMATION FOR SEQ ID NO: 56: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 228 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..228
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
CCA CCT ACT ACA GGA CCG CCA AGA AAA GAA GTT CCC AAA ACC ACT CCT 48 Pro Pro Thr Thr Gly Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro 1 5 10 15
AGT GAG CCC AAG AAA AAG CAG CCT CCA CCA CCA GAA TCA GGC ATC TAC 96 Ser Glu Pro Lys Lys Lys Gin Pro Pro Pro Pro Glu Ser Gly He Tyr 20 25 30
ACC AGT AAT AAG GAC CCC ATC TCC CAC AGT GGC GGG ATG CTG CGG GCT 144 Thr Ser Asn Lys Asp Pro He Ser His Ser Gly Gly Met Leu Arg Ala 35 40 45 GTC TGC AGC ACC CCT CTC TCC TCC AGC CTC CTG GGG CCC CCA GGG ACC 192 Val Cys Ser Thr Pro Leu Ser Ser Ser Leu Leu Gly Pro Pro Gly Thr 50 55 60
TCG GCC CTG CCC CGC CTC AGC CGC TCC CCG TTC ACC 228
Ser Ala Leu Pro Arg Leu Ser Arg Ser Pro Phe Thr 65 70 75
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
Pro Pro Thr Thr Gly Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro 1 5 10 15
Ser Glu Pro Lys Lys Lys Gin Pro Pro Pro Pro Glu Ser Gly He Tyr 20 25 30
Thr Ser Asn Lys Asp Pro He Ser His Ser Gly Gly Met Leu Arg Ala 35 40 45
Val Cys Ser Thr Pro Leu Ser Ser Ser Leu Leu Gly Pro Pro Gly Thr 50 55 60
Ser Ala Leu Pro Arg Leu Ser Arg Ser Pro Phe Thr 65 70 75
(2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 188 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Met Lys Glu Met Val Gly Gly Cys Cys Val Cys Ser Asp Glu Arg Gly
1 5 10 15
Trp Ala Glu Asn Pro Leu Val Tyr Cys Asp Gly His Ala Cys Ser Val 20 25 30
Ala Val His Gin Ala Cys Tyr Gly He Val Gin Val Pro Thr Gly Pro 35 40 45
Trp Phe Cys Arg Lys Cys Glu Ser Gin Glu Arg Ala Ala Arg Val Arg 50 55 60
Cys Glu Leu Cys Pro His Lys Asp Gly Ala Leu Lys Arg Thr Asp Asn 65 70 75 80
Gly Gly Trp Ala His Val Val Cys Ala Leu Tyr He Pro Glu Val Gin 85 90 95 Phe Ala Asn Val Leu Thr Met Glu Pro He Val Leu Gin Tyr Val Pro 100 105 110
His Asp Arg Phe Asn Lys Thr Cys Tyr He Cys Glu Glu Thr Gly Arg 115 120 125
Glu Ser Lys Ala Ala Ser Gly Ala Cys Met Thr Cys Asn Arg His Gly 130 135 140
Cys Arg Gin Ala Phe His Val Thr Cys Ala Gin Met Ala Gly Leu Leu 145 150 155 160
Cys Glu Glu Glu Val Leu Glu Val Asp Asn Val Lys Tyr Cys Gly Tyr 165 170 175
Cys Lys Tyr His Phe Ser Lys Met Lys Thr Ser Arg 180 185
(2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Leu Val Asp Glu Asp Ala Val Cys Cys He Cys Asn Asp Gly Glu Cys 1 5 10 15
Gin Asn Ser Asn Val He Leu Phe Cys Asp Met Cys Asn Leu Glu Val 20 25 30
His Gin Glu Cys Tyr Gly Val Pro Tyr He Pro Glu Gly Gin Trp Leu 35 40 45
Cys Arg Arg Cys Leu Gin Ser Pro Ser Arg Ala Val Asp Cys Ala Leu 50 55 60
Cys Pro Asn Lys Gly Gly Ala Phe Lys Gin Thr Asp Asp Gly Arg Trp 65 70 75 80
Ala His Val Val Cys Ala Leu Trp He Pro Glu Val Cys Phe Ala Asn 85 90 95
Thr Val Phe Leu Glu Pro He Asp Ser He Glu His He Pro Pro Ala 100 105 110
Arg Trp Lys Leu Thr Cys Tyr He Cys Lys Gin Arg Gly Ser Gly Ala 115 120 125 Cys He Gin Cys His Lys Ala Asn Cys Tyr Thr Ala Phe His Val Thr 130 135 140
Cys Ala Gin Gin Ala Gly Leu Tyr Met Lys Met Glu Pro Val Arg Glu 145 150 155 160
Thr Gly Ala Asn Gly Thr Ser Phe Ser Val Arg Lys Thr Ala Tyr Cys 165 170 175
Asp He His Thr Pro Pro Gly Ser Ala Arg Arg 180 185
(2) INFORMATION FOR SEQ ID NO:60: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Cys Val Asp Glu Arg Gly Trp Ala Glu Asn Pro Leu Val Tyr Asp Gly 1 5 10 15
His Ala
(2) INFORMATION FOR SEQ ID NO:61: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Arg Lys Glu Ser Gin Glu Arg Ala Ala Arg Val Arg Glu Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS :
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Tyr He Glu Glu Thr Gly Arg Glu Ser Lys Ala Ala Ser Gly Ala Met 1 5 10 15
Thr
(2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2..265 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 595..666 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 2353..2484 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3032..3145 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 6788..6934 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 7967..8062 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 8304..8342
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
G GAT CCT GCC CCA AAG AAA AGC AGT AGT GAG CCT CCT CCA CGA AAG 46
Asp Pro Ala Pro Lys Lys Ser Ser Ser Glu Pro Pro Pro Arg Lys 1 5 10 15
CCC GTC GAG GAA AAG AGT GAA GAA GGG AAT GTC TCG GCC CCT GGG CCT 94 Pro Val Glu Glu Lys Ser Glu Glu Gly Asn Val Ser Ala Pro Gly Pro 20 25 30
GAA TCC AAA CAG GCC ACC ACT CCA GCT TCC AGG AAG TCA AGC AAG CAG 142 Glu Ser Lys Gin Ala Thr Thr Pro Ala Ser Arg Lys Ser Ser Lys Gin 35 40 45
GTC TCC CAG CCA GCA CTG GTC ATC CCG CCT CAG CCA CCT ACT ACA GGA 190 Val Ser Gin Pro Ala Leu Val He Pro Pro Gin Pro Pro Thr Thr Gly 50 55 60
CCG CCA AGA AAA GAA GTT CCC AAA ACC ACT CCT AGT GAG CCC AAG AAA 238 Pro Pro Arg Lys Glu Val Pro Lys Thr Thr Pro Ser Glu Pro Lys Lys 65 70 75
AAG CAG CCT CCA CCA CCA GAA TCA GGT GAGTGAGGAG GGCAAGAAGG 285
Lys Gin Pro Pro Pro Pro Glu Ser Gly 80 85
AATTGCTGAC CCACAAGTAC TAACAAAAAA GCACTGATGT CTCAAACAGC ATTTGAAAGC 345
AGGAAATGTA TGATTTGAAG TCTTCAGTTC AAGAAAATCA GCTCTCTTTC TAACTATTAT 405
GTTTAATAAT AAAGAAACAG AAACAAAAAA AACAGTTAAA TTGGAGGTAT TGTTTTAATT 465
TCCTGTTCGA AGCCTAGAGT TTAAATAGTT ττττττττττ TTTTCTAATG GCCCTTTCTT 525
CACAGGTCAG TCAGTACTAA AGTAGTCGTT GCCAGCATCT GACTGCAATT TATTCTGAAT 585
TTTTTAGGT CCA GAG CAG AGC AAA CAG AAA AAA GTG GCT CCC CGC CCA 633 Pro Glu Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro 1 5 10
AGT ATC CCT GTA AAA CAA AAA CCA AAA GAA AAG GTGAGGAGAG ATTTGTTTCT 686 Ser He Pro Val Lys Gin Lys Pro Lys Glu Lys 15 20 CTGCCATTTC TCAGGGATGT ATTCTATTTT GTAGGGAAAA GCCTTATCCT TGACTTCTAT 746
GTAGATGGCA GTGGAATTTC TTAAAATTAA GAAACTTCAA GTTTAGGCTT TTAGCTGGGC 806
ACGGTGGCTC ACGCTGGTAA TCCCAACACT TAGTGAGGCT GAGGTGGGAG GATTGCTTGA 866
GGCCAGCAGT TCAAGACCAG CCTGGGCAAC ATAGCAAGAC CCTGTCTTTA TTTAAACCAA 926
AAAAAAAAAA AGAAGAAGAA GAAGTTAGCC AGGCATGGTG GCAGTTGCGT GTAGTCCCAG 986
GTACTCAGGA GGCTGAGATA GAAGGATTGT CTTGAGCCCA GGAATTCAAG GCTGTAGTGA 1046
GCTATGATTG TACCACTGCA GTCCAGCCTG GGTGACAAAG CAAAACACTG TCTCCAAAAA 1106
AAATTTAGGC TTGGCAAGGC GCAGCGGCTC ACGCCTGTGA TCCCAGCACT TTGGGAAGCC 1166
GAAGCAGGCA GATCACTTGA GGTCAGGAGT TGGAGACCAG CCTGGCCAAC ATGGTGAAAC 1226
CCTGTCTCTA CTGAAAATAC AAAAATTAGC CGGTTGTGGT AGTGGGTGCT TGGTAATCCT 1286
AGCTACTTGG GAGGCTGAGG CAGGGGGAAT TGCCTGAAAC CTGCGAGGCG GAGGCTGCAG 1346
TGAGCCGAGA TTGCATCATT GCACTCTAGC CTGGACAACA GAGCTAGACT CCATCCCAAA 1406
AAAAAAAAAA AAAAGTAGCC GGGCACGGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA 1466
GGCCGAGGCG GGCGGATCAT GAGGGCAGGA GATCGAGACC ATCCTGGCTA ACACGGTGAA 1526
ACCCTGTCTC TACTAAAAAT ACAAAAAATT AGCCCGGCGA GGTGGCGGGC GCCTGTAGTC 1586
CCAGCTACTC AGGAGAGTGA GCCAGGAGAA TGGCGTGAAC CCGGGGGGCG GAGCCTGCAG 1646
TGAGCCGAGA TCGCGCCACT GCACTCCAGC TTGGGTGACA CCGAGACTCC GTCTCAAAAA 1706
AAAATAAAAA GTTTAGGCTT TAGCCTGTTT CTTTTTTGGT TTCTTCCTTG TTGCTTTTCC 1766
CTTCTTTGTG GCCCCACATG TTCTAGCCTA GGAATCTGCT TATTCTAAAG GCCATTTGGC 1826
GTAATTATTT TTTGACCCCA ACATCCTTTA GCAATTATTT GTCTGTAAAA ATCACCCTTC 1886
CCTGTATTCA CTATTTTTAT TTATTATGGA TAAAGAGATA GTGTGGTGGC TCACATCTAT 1946
AATCCCAGCA CTTTGGGGGC CCAAGGCGGG AGGATCACTT GAGGGCAGGA GCTGGAGACC 2006
AGCCTGGGCA GCACAGTGAC ACACAGTTGC TATAAAAAAT TTAAAACCCA ACTAGGCATG 2066
GTGGCATGCA CCTGTAGTCC CAGCTACTCT TGAGAAGCTG AGGCAGGAGG ATCACGAGCC 2126
CACAAGGTCT AGGCTGCAGT GAGCTGTGAC TGTGCCACTG TATTGCAGCC TAGGCAACAA 2186
AGCAAGACCC AGTCTCTTTT AAAAAAAAAT TCAAAGATTA TTGTTTATGT TGGAAACATG 2246
TTTTTTAGAT CTATTAATAA AATTTGTCAT TTGCATTATT ATCTGTTGCA AATGTGAAGG 2306
CAAATAGGGT GTGATTTTGT TCTATATTCA TCTTTTGTCT CCTTAG GAA AAA CCA 2361
Glu Lys Pro
1
CCT CCG GTC AAT AAG CAG GAG AAT GCA GGC ACT TTG AAC ATC CTC AGC 2409 Pro Pro Val Asn Lys Gin Glu Asn Ala Gly Thr Leu Asn He Leu Ser 5 10 15
ACT CTC TCC AAT GGC AAT AGT TCT AAG CAA AAA ATT CCA GCA GAT GGA 2457 Thr Leu Ser Asn Gly Asn Ser Ser Lys Gin Lys He Pro Ala Asp Gly 20 25 30 35 GTC CAC AGG ATC AGA GTG GAC TTT AAG GTAAAGGTGT TCAGTGATCA 2504 Val His Arg He Arg Val Asp Phe Lys 40
TAAAGTATAT TGAGTGTCAA AGACTTTAAA TAAAGAAAAT GCTACTACCA AAGGTGTTGA 2564
AAGAGGAAAT CAGCACCAAC TGGGGGAATG AATAAGAACT CCCATTAGCA GGTGGGTTTA 2624
GCGCTGGGAG AGCTTTGGAC AGTGTTGTTA GGTCACTGTT TGTGAACTGA CTGCAGAACA 2684
TACATAATGA AACATTCCTA TCCATCCTGA GGAGTATCAG AGGAAGTAAT TCCTTCACAT 2744
GGAAAGTATC AAACCATGAT GATTCCTTGA GTCAGCAAAA CTGTAAGAGA AATTCAATCC 2804
CAGTGTATTT TCGCAATATC TTCACTATGA ATTGAACAAC TAGGTGAGCC TTTTAATAGT 2864
CCGTGTCTGA GATTAAAACT TTTTAAAGCA GCAGTTATTT TTGGACTCAT TGAAATGAAA 2924
TACTCTGACA TTGTGATGTC ACACTAATTT TATGCTTTTC ATCCTTATTT TCCATCCAAA 2984
GTTGTGTAAT TGTAAAACTT TCCTAAGTGA CCTTTCTCTC TCCACAG GAG GAT TGT 3040
Glu Asp Cys
1
GAA GCA GAA AAT GTG TGG GAG ATG GGA GGC TTA GGA ATC TTG ACT TCT 3088 Glu Ala Glu Asn Val Trp Glu Met Gly Gly Leu Gly He Leu Thr Ser 5 10 15
GTT CCT ATA ACA CCC AGG GTG GTT TGC TTT CTC TGT GCC AGT AGT GGG 3136 Val Pro He Thr Pro Arg Val Val Cys Phe Leu Cys Ala Ser Ser Gly 20 25 30 35
CAT GTA GAG GTAAGGCATC CTGCTTCTTT GTACCCCAGG AAGTACATAA 3185 His Val Glu
ATGATTGATC TGGGGATGAG ATTACTATAG TCTGTTTTGT TGGTATTTAG CAGGTACTAT 3245
TCCCTGTTTA AACCAGCTAA AGAAATGTTT TGAAGTATTT TAGAGATTTT AGGAAGGAAT 3305
CTGCTATTAG AGTAGCAAAG TTATTGAGAG TGAAAAGATC AATAATCCCA TCTCTCTTAA 3365
ATTCAGTCTT TATTAGAGTT CTGATCTTTC TGTTAGATGT CTAAATAAGA GAAAAAATTA 3425
TACAGTGGTC TATTAAAAGG GATGCTATTG ATGGTTATTT TATATTGTAT ATCAAAGCCT 3485
CTTCATCTAT AAGGAGCTCT TACCAATTAA TAAGAAAAAG GAATGACATC CAGAAAAAAA 3545
AATAGGCAAA AGACAGAAAT AGATAATTCA CAAAATTAGA AATAAATACA TGTTGGGTGG 3605
CAGGGGGAGG TGAAGGGAGG GTGTCTGTTT TTTAGCCCTC TAGTGACCAA AAACTGGAAA 3665
TTAAAGCATG ATAAAAAAAG AATCCTGAAT AAATGGGGAC TTTCTGTTGG TGGAAAGAAA 3725
TATAGATTAG TTACAATCTT TCTTTCTGAG GGAATTATTT GGAAATATAT ATATCTATCT 3785
TTAAAATAGG TATATCCTCT AACATAGCAA TTGCACTTCA AACACTTATG GATATAATTA 3845
GATAAATTGG CAAATCTGTA GATATAAAGA AGTGTTCATT TCAATATTGC TCATAATAAT 3905
AAAAAACTGG AAACAACCCG AAAGTCCATC TATAGGGAGC ATGGGTTAAA ATAAGCATAG 3965
GGCATATAGC TGGGCACGGT GGCTCACGCC TGTAATCCCA GCACTTTGGG AGGCCAAGGC 4025
AGGCGGATCA CAAGGTCAGG AGATCCAGAC CATCCTGGCT AACACAGTGA AACCCCGTCT 4085
CTATTAAAAA TACAAAAAAA TTAGCCGGGT GTGGTGGCGG GCGCCTGTAG TCCCAGCTAC 4145 TCGAGAGGCT GAGGCAGGAG AACGGCATGA ACCCGGGAGG TGGAGCTTGC AGTGAGCCGA 4205
GATCGCCCCA CTGCACTCCC GCCTGGGCGA CAGAGCAAGA CTCCGTCTCA AAAAAAAATA 4265
AAAGTGTAGG GCATATATAA TGCCAAATAT GAAGTCCTAA AGATAATATA TATTAATATT 4325
ATTAGGTTGG TCCAAAAGTA ATTGCAGTAA TAACATGGAA AGATGTCCAT GACATATCAC 4385
TGAGTGAAAA GAGCAGGTTA CAAGATAATA TATAAAGCAC AATCCCATCT TAGTTTGGAA 4445
AAGTGTTTTT AAAGTATATA TCTAGAAAAC AATCTGGAAG GATTCACACC AAAATATTAA 4505
GAGTGTGGTT GGATTATGGG TGACCTTTAT TTGTTTCTCT GGTTTTTTTT TTTTTAATCT 4565
TTCTGAGTTT TTTGGAGTAT GTACCACCTT TACAATGAGG AAGGAAAAAG TAGCACAATT 4625
TTAAATAGGA AGCAGTAGTT TGTCATTTAT AAGGGACATA TCCTACATCC TTTACAGTTC 4685
TTAAATTCCT GGCAGATACC TCTTTGGCTT ATTACTTACC ACATAAGATA TGTATTCAAA 4745
GGTGGTAAAG AAAATCCACG TCGGGTGCAG TGGCTCACGC CTGTAATCCC AGTACTTTGG 4805
GAGGCTGACG CAGGAGGACC GCTTGAGCTC AGGAGTTCAA GACCAGCCTG AGCACCATAG 4865
TGAGACCTCA TCTCTACTAA AAAAAAAATA AAATACCAGG CATGGTAGCA TGTGCCTGTA 4925
GTCCCAGCTA CTCTAGTCCC AGCTACTTGG GAGGCTGAGG TGAGAGGATC ACTTGAGCCC 4985
AGGAGATCGA GGCTGCAGTG AGCCATTATC ACGCCACTGC ACTCCAGCCT GGGCAACTAA 5045
GCAAGACCCT GTCTCAAAAA AATTTTAAAA AATTTAAAAA ATAAGAAAAT CCAAGCTAGG 5105
TTGAAATCTG AATGTTGAGC AGTCAGTGAG ACACAAACTA GCTAAGAAAG TCAACCCTGC 5165
CCACTTGCCA TTTGAAGTTA TTACTAGCAA AATTACAAAT TATTGCCTAC TATTCATTTA 5225
CTAAGCAAAT ATTCTCTTAG TCCCTATTAC GAACAACTTA TTGTTCTAAG TGCAGAAGTT 5285
CAGATATCAT TGAGACTGAG AATATTCAGT CTACAAGTGC CAGGGGTCTA CTGTATCCTC 5345
TTTTCCGTCT TAATACAGTG CTTTGCACCC ATATATATGC CACCCACAGG AATAACTTTT 5405
TTTATAGCAC CAGTCCTTCA ACTTCTGGGA TTAAACAGAT TTTTTTTCAG GGTATAATTG 5465
TTCTGATCTA AATTCTTTAT AGTTGTACAT AGCAATCTCA CAGGGTTCCT AAAATATAAA 5525
ATAGAGAATA GCATGCTGCC TGCACTGCAC TCCTAAAGCA TGACCAGTGC TTGATAAACT 5585
CTCCTCCATG CGAATTTTTT AAACTTTTTA TGTTGACATG ATTTCAGACT TACAAAAAAA 5645
CTATGAGTTG TACAGAGAAT TCTAAGTACC CCTCACCCAA ATTCCCTAAG TGTTAATATG 5705
TTTCTCTGTG TGTATATATT TTACAAAATA ACAAATAAAA TACATATACA CATTTTACCT 5765
GTAGATACAC ATGTATCTAA AAATTTGAGA ACAAGTTGGA GACATAAACC ATTTTACCTC 5825
TAAATATTTT AGTGTATATT TTTAAAAATC AAGGACGTTC TCGTATTTAA CCATGGTATA 5885
ATTACCAAAT CAGGAAATTA ACACACTGGG ACATTACTAT TATCTGATCT ATAGGCCTTA 5945
TTTAGGTTTG ACCAATTGTC CCAATAATTC CTTTATGGCA AAAGAAAATT CTGGATTATC 6005
CTAGTTAGTA TTTTTGAAAA TCCTATATCA ATATGAAAAT AACTTATTTC TAAAATTAGA 6065
AATGGAGGCT GGGCGTGGTG GCTCACGCCT ATAATCCCAG CACTTTGGGA GGCCGAGGCA 6125
GGCAGATCAC AAGGTCAGGA GATTGAGACC ATCCTCGCTA ACACAGTGAA ACCCCATCTC 6185 TACTAAAAAT ACAAAAAATT AGCCAGGTGT GGTGGGACGC GCCTGTGATC CCAGCTACTC 6245
AGGAGACTGA GGCTGGAGAA TCGCTTGAAC CCAACAGGCG GAGGGTTGCA GTGAGTCGAG 6305
ATCGCACCAC TGCACCCCAG CCTGGGCGAC AGCGAGACTC CGTCTCAAAA AAATAAATAA 6365
ATAAAAATTA AAACAATTAA AAAAATAAAA TTACAAATGG AAAGGACAAA CCAGACCTTA 6425
CAACTGTTTC GTATATTACA GAAAACGTTT AAACCCTCCC TATTTCCCCC ACCCCACTCC 6485
TTTATATTCC CATAGCTCTT TGTTTATACC ACTCTTAGGT CACTTAGCAT GTTCTGTTAA 6545
ATCTTGTATT ATATTTATTT TGTTACTTTC TATTTCCACT GGTATTACCA CTTTAGTACT 6605
CTGAATCTCC CGCAATGTCC AATACTGTAC TTTTTTACAT AGTCATTGCT TAATGAATAT 6665
GTATTGAATT AAATATATGC CAGTGGACTA CTAAAACCCA AAGTATATAA GAAGGGTATG 6725
GTTGATTATG TTTTTCTACA TATTATTTGA CATACTTCTA TCTTCCCATG TTCTTACTAT 6785
AG TTT GTG TAT TGC CAA GTC TGT TGT GAG CCC TTC CAC AAG TTT TGT 6832 Phe Val Tyr Cys Gin Val Cys Cys Glu Pro Phe His Lys Phe Cys 1 5 10 15
TTA GAG GAG AAC GAG CGC CCT CTG GAG GAC CAG CTG GAA AAT TGG TGT 6880 Leu Glu Glu Asn Glu Arg Pro Leu Glu Asp Gin Leu Glu Asn Trp Cys 20 25 30
TGT CGT CGC TGC AAA TTC TGT CAC GTT TGT GGA GGG CAA CAT CAG GCT 6928 Cys Arg Arg Cys Lys Phe Cys His Val Cys Gly Gly Gin His Gin Ala 35 40 45
ACA AAG GTACAAAACT TGGTAATAGA ACTACAGCTG GGCCTCTGTA TCAGTGGGTT 6984 Thr Lys
CTGTATCCCT GGACTCAACC AACCTTGGAT TGAATGTATC TGGGAAAAAA TGAGTAGTTG 7044
CCTCTGTACT CTATGTGAAC AGACTTTTTC TTGTCATTAT TTCCTAAACA ATACAGTATA 7104
ACAACTATTT ACATTGTATT AGGTATGATA AGTAATCTAG AGATAATTTA AAGTATATGG 7164
TGGGCGGATC ACTTGAAGCC AGGAGTTCGA GACCAGCCTG AGCCAACATG GTGAAACCCC 7224
ATCTCTACTA AAAATACAAA AAATTAGCCA GGTGTGGTGG TGGGCACCTG TAGTCCCAGC 7284
TACTTGGGAG GCTGAGGGAG GAAAATCGCT TGAACTTTGG AGGCAGAGGT TGCAGTGAGC 7344
CACTCCAGCC TGTGGTGCAG TCTGTCACTC CAGCCTGGGT GACACAGTGA GACTCCATCT 7404
CAAAAAAAAA AAAAAAAAAA AAACTATATG GGAGGATGTG CATTTTGTTA TATGCAAATG 7464
CTGCACCATT TTGTCTAGGG ACTTGGGCAT CCATGGACTT TGGTATCCTC TGGGGGTCCT 7524
GGAACCAATC CCCCATGGAA ACCAAGGATG ACTGTGCTTA GAGTATTGCT TTCTTTCTTG 7584
ATTTGTATTT CTGTCTTCCA GTTAAGATTT TGTATCTATA TTATTTCTCT TTTTACTTAG 7644
TCTGTCTTTA GCATTTAATT GGGTGTAATC AGTTGCCTAT TTTGTGTTTT AATTTTGGGA 7704
CTATAGCAGA AAACATGATG TTGAATAAAA TTCCAAAAAT AAGTCAAATC TACCTAATAT 7764
GAATACTCAT CACTGAGTGC CTTTGGCAGG AAATAAATCT ATCTCAATGC GTTAATTGGG 7824
AGTAAATAAT GCATGAGGAA ATTTAAACTC ATAATTGTGT GCTGTACTTA CTTGCCAGTA 7884
AATGTGAAAT GGGGTACTAA GTAATAGGTG TTGGGTGAAG GTAATATGAT GCTTATCTTT 7944 TTGCCATTAT ATTTTCTTAC AG CAG CTG CTG GAG TGT AAT AAG TGC CGA AAC 7996
Gin Leu Leu Glu Cys Asn Lys Cys Arg Asn 1 5 10
AGC TAT CAC CCT GAG TGC CTG GGA CCA AAC TAC CCC ACC AAA CCC ACA 8044 Ser Tyr His Pro Glu Cys Leu Gly Pro Asn Tyr Pro Thr Lys Pro Thr 15 20 25
AAG AAG AAG AAA GTC TGG GTGAGTTATA CACATGATGC TCTTTTATAG 8092
Lys Lys Lys Lys Val Trp 30
AGAACCACCA TGTGACTATT GGACTTATGT AACTTGTATT ACAAATATCT ATGCATGAGG 8152
ATGTCAGTAT GACAATCTTT TTCCCTCATT ACTAGGAAAT CATCTCAGGA GAGAAATTAA 8212
ATCTATAAAT GGATGCATTT AAGATCTTTT TAGTTAAGTA AAGATATTAA AAACAAGAAA 8272
TTCCTATTGA ATTTCTTTTC TTCTTTTCTA G ATC TGT ACC AAG TGT GTT CGC 8324
He Cys Thr Lys Cys Val Arg 1 5
TGT AAG AGC TGT GGA TCC 8342
Cys Lys Ser Cys Gly Ser 10
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Asp Pro Ala Pro Lys Lys Ser Ser Ser Glu Pro Pro Pro Arg Lys Pro 1 5 10 15
Val Glu Glu Lys Ser Glu Glu Gly Asn Val Ser Ala Pro Gly Pro Glu 20 25 30
Ser Lys Gin Ala Thr Thr Pro Ala Ser Arg Lys Ser Ser Lys Gin Val 35 40 45
Ser Gin Pro Ala Leu Val He Pro Pro Gin Pro Pro Thr Thr Gly Pro 50 55 60
Pro Arg Lys Glu Val Pro Lys Thr Thr Pro Ser Glu Pro Lys Lys Lys 65 70 75 80
Gin Pro Pro Pro Pro Glu Ser Gly 85
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
Pro Glu Gin Ser Lys Gin Lys Lys Val Ala Pro Arg Pro Ser He Pro 1 5 10 15
Val Lys Gin Lys Pro Lys Glu Lys 20 (2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
Glu Lys Pro Pro Pro Val Asn Lys Gin Glu Asn Ala Gly Thr Leu Asn 1 5 10 - 15
He Leu Ser Thr Leu Ser Asn Gly Asn Ser Ser Lys Gin Lys He Pro 20 25 30
Ala Asp Gly Val His Arg He Arg Val Asp Phe Lys 35 40
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Glu Asp Cys Glu Ala Glu Asn Val Trp Glu Met Gly Gly Leu Gly He 1 5 10 15
Leu Thr Ser Val Pro He Thr Pro Arg Val Val Cys Phe Leu Cys Ala 20 25 30
Ser Ser Gly His Val Glu 35
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Phe Val Tyr Cys Gin Val Cys Cys Glu Pro Phe His Lys Phe Cys Leu 1 5 10 15
Glu Glu Asn Glu Arg Pro Leu Glu Asp Gin Leu Glu Asn Trp Cys Cys 20 25 30
Arg Arg Cys Lys Phe Cys His Val Cys Gly Gly Gin His Gin Ala Thr 35 40 45
Lys
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
Gin Leu Leu Glu Cys Asn Lys Cys Arg Asn Ser Tyr His Pro Glu Cys 1 5 10 15 Leu Gly Pro Asn Tyr Pro Thr Lys Pro Thr Lys Lys Lys Lys Val Trp 20 25 30
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
He Cys Thr Lys Cys Val Arg Cys Lys Ser Cys Gly Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO: 71: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
GCCTGTAGTC CCAGCTACTC AGGAGAGTGA GCCAGGAGAA TGGCGTGAAC CCGGGGGGCG 60
(2) INFORMATION FOR SEQ ID NO: 72: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
GCCTGTAGTC CCAGCTACTC AGGAGAGTGA GTCCTAAAAG TTATATATGT CTTTTAATAT 60
(2) INFORMATION FOR SEQ ID NO: 73: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
TTTAAATTTA AGAGATGAAC CTGCTAATTT GTCCTAAAAG TTATATATGT CTTTTAATAT 60
(2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO.-74:
TTGTACCACT GCAGTCCAGC CTGGGTGACA AAGCAAAACA CTGTCTCCAA AAAAAATTTA 60 (2) INFORMATION FOR SEQ ID NO:75: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
TTGTACCACT GCAGTCCAGC CTGGGTGACT GCATCCAGCA CTCTCCTCAC TGGCATCACG 60
(2) INFORMATION FOR SEQ ID NO: 76: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
CTGAGACCCT AAACCAACCC TTCTCTCCCC ACATCCAGCA CTCTCCTCAC TGGCATCACG 60
(2) INFORMATION FOR SEQ ID NO: 77: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..30
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
AAA CCA AAA GAA AAG GAT GAG CAA TTC TTA 30
Lys Pro Lys Glu Lys Asp Glu Gin Phe Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
Lys Pro Lys Glu Lys Asp Glu Gin Phe Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO:79: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
ATCTGAATTC TCCGCTGACA TGCACTTCAT AG 32
(2) INFORMATION FOR SEQ ID NO:80: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
ATCTGAATTC TCCGCTGACA TGCACTTCAT AG 32
(2) INFORMATION FOR SEQ ID NO: 81: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
CGGGATCCCG ACCTACTACA GGACCGCCAA G 31
(2) INFORMATION FOR SEQ ID NO: 82: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
ATCTGAATTC TGGTGGAGAT AGAAGCAGAA 30
(2) INFORMATION FOR SEQ ID NO: 83: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
AGGAGAGAGT TTACCTGCTC 20
(2) INFORMATION FOR SEQ ID NO: 84: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: GGAAGTCAAG CAAGCAGGTC 20
(2) INFORMATION FOR SEQ ID NO:85: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
GTCCAGAGCA GAGCAAACAG 20
(2) INFORMATION FOR SEQ ID NO:86: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
ACACAGATGG ATCTGAGAGG 20

Claims

1. A prΩbe CΩmprising an ΩligΩnucleΩtide sequence or derivative thereof of at least 15 nucleΩtides which identifies chromosome abnormalities within the AF-4 gene of chromosome 4.
2. The probe of claim 1 comprising an oligonucleΩtide sequence Ωr derivative thereof having at least a portion of SEQ ID NO:25 or SEQ ID NO:27.
3. A method of diagnosing acute lymphoblastic or nΩnlymphΩblastic leukemia CΩmprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; and detecting chromosome abnormalities within the AF-4 gene of chromosome 4 in genetic material from the cells.
4. The method of claim 3 further comprising: obtaining nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Northern analysis using an AF-4 probe; and detecting aberrant transcripts from the Northern analysis.
5. The method of claim 3 wherein said probe identifies t(4;ll) abnormalities.
6. The method of claim 3 further comprising: digesting nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Southern analysis using an ALL-l prΩbe; and detecting chromosome abnormalities in the AF-4 gene.
7. A probe comprising an oligonucleotide sequence or derivative thereof of at least 15 nucleotides which identifies chromosome abnormalities within the AF-9 gene of chromosome 9.
8. The probe of claim 7 comprising an oligonucleotide sequence or derivative thereof having at least a portion of SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
9. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; and detecting chromosome abnormalities within the AF-9 gene of chromosome 9 in genetic material from the cells.
10. The method of claim 9 further comprising: obtaining nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Northern analysis using an AF-9 probe; and detecting aberrant transcripts from the Northern analysis .
11. The method of claim 9 wherein said probe identifies t(9;ll) abnormalities.
12. The method of claim 9 further comprising: digesting nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Southern analysis using an AF-9 probe; and detecting chromosome abnormalities in the AF-9 gene.
13. A monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-9 protein.
14. The monoclcnal antibody of claim 13 which binds tc at least a portion of the amino acid sequences contained within SEQ ID NO:33, SEQ ID NO:35 or SEQ ID NO:37.
15. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-9 protein.
16. The method of claim 15 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-9 protein.
17. The method of claim 15 wherein said protein is detected using a monoclonal antibody selected frΩm the grΩup CΩnsisting of: a monoclonal antibody which binds tc at least a portion of the amino acid sequences contained within SEQ ID NO:33; a monΩclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:35 and a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:37.
18. An antisense oligonucleotide which binds to at least a portiΩn of the chimeric ALL-l/AF-9 mRNA.
19. An antisense oligonucleotide which binds to at least a portion of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
20. A method of treating acute lymphoblastic or nonlymphoblastic leukemia comprising administering an antisense oligonuclectide which binds to at least a portion of the chimeric ALL-l/AF-9 mRNA.
21. The method of claim 20 comprising administering an antisense oligΩnucleΩtide which binds to at least' a portion of SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36.
22. A method of treating acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-9 protein.
23. The method of claim 22 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-9 protein.
24. The method of claim 22 wherein said protein is detected using a monoclonal antibody selected from the group consisting of: a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:33; a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:35 and a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:37.
25. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(9;ll) translocations comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; isΩlating RNA from the sample; generating cDNA from said RNA; amplifying a chimeric gene sequence in said cDNA which is generated by said translocation using a set of PCR primers if said chimeric gene is present; and detecting the presence of amplified DNA.
26. The method of claim 25 wherein said set of PCR primers comprises a set selected from the group consisting Ωf : SEQ ID NO:39 and SEQ ID NO:40; SEQ ID NO:41 and SEQ ID NO: 2; and SEQ ID NO:43 and SEQ ID NO:44.
27. A probe comprising an oligonucleotide sequence or derivative thereof of at least 15 nucleotides which identifies chromosome abnormalities within the AF-6 gene of chromosome 6.
28. The probe of claim 27 comprising an oligonucleotide sequence or derivative thereof having at least a portion of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:49.
29. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; and detecting chromosome abnormalities within the AF-6 gene of chromosome 6 in genetic material from the cells.
30. The method of claim 29 further comprising: obtaining nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Northern analysis using an AF-6 probe; and detecting aberrant transcripts from the Northern analysis .
31. The methΩd of claim 29 wherein said probe identifies t(6;ll) abnΩrmalities .
32. The methΩd of claim 29 further comprising: digesting nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Southern analysis using an ALL-l probe; and detecting chromosome abnormalities in the AF-6 gene.
33. A monΩclΩnal antibody which binds to at least a portion of the chimeric ALL-l/AF-6 protein.
34. The monoclonal antibody of claim 33 which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:48, SEQ ID NO:50 or SEQ ID NO:51.
35. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-6 protein.
36. The method of claim 35 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-6 protein.
37. The method of claim 35 wherein said protein is detected using a monoclonal antibody selected from the group consisting of : a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:48; a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:50 and a monoclonal antibody which binds tΩ at least a pΩrtion of the amino acid sequences contained within SEQ ID NO:51.
38. An antisense oligonucleotide which binds to at least a portion of the chimeric ALL-l/AF-6 mRNA.
39. An antisense ΩligΩnucleΩtide which binds to at least a portion of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 Ωr SEQ ID NO:49.
40. A method of treating acute lymphoblastic or nonlymphoblastic leukemia comprising administering an antisense oligonucleotide which binds to at least a portion of the chimeric ALL-l/AF-6 mRNA.
41. The method of claim 40 comprising administering an antisense oligonucleΩtide which binds to at least a portion of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:49.
42. A method of treating acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-6 protein.
43. The method of claim 42 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-6 protein.
44. The method of claim 42 wherein said protein is detected using a monoclonal antibody selected from the group consisting of: a monoclonal antibody which binds to at least a pΩrtiΩn of the amino acid sequences contained within SEQ ID NO:48; a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:50 and a monΩclΩnal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:51.
45. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(6;ll) translocatiΩns comprising: providing a tissue sample containing hematopΩietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; isolating RNA from the sample; generating cDΝA from said RNA; amplifying a chimeric gene sequence in said cDΝA which is generated by said translocation using a set of PCR primers if said chimeric gene is present; and detecting the presence of amplified DΝA.
46. A probe comprising an oligonucleotide sequence or derivative thereof of at least 15 nucleotides which identifies chromosome abnormalities within the AF-17 gene of chromosome 17.
47. The probe of claim 46 comprising an oligonucleotide sequence or derivative thereof having at least a portion of SEQ ID NO:56.
48. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; and detecting chromosome abnormalities within the AF-17 gene of chromosome 17 in genetic material from the cells.
49. The method of claim 48 further comprising: obtaining nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Northern analysis using an AF-17 probe; and detecting aberrant transcripts from the Northern analysis.
50. The method of claim 48 wherein said probe identifies t(ll;17) abnormalities.
51. The method of claim 48 further comprising: digesting nucleic acid from the hematopoietic cells; subjecting the digested nucleic acid to Southern analysis using an ALL-l probe; and detecting chromosome abnormalities in the AF-17 gene .
52. A monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-17 protein.
53. The monoclonal antibody of claim 52 which binds to at least a portion of the amino acid sequences encoded by SEQ ID NO:55, SEQ ID NO:57 or SEQ ID NO:58.
54. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-17 protein.
55. The method Ωf claim 54 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-17 protein.
56. The method of claim 55 wherein said protein is detected using a monΩclonal antibody selected from the group consisting of: a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO: 55; a monoclΩnal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO: 57 and a monoclonal antibody which binds to at least a portion of the amino acid sequences cotained within SEQ ID NO: 58.
57. An antisense oligonucleotide which binds to at least a portion of the chimeric ALL-l/AF-17 mRNA.
58. An antisense oligonucleotide which binds to at least a portion of SEQ ID NO:56.
59. A method of treating acute lymphoblastic or nonlymphΩblastic leukemia CΩmprising administering an antisense oligonucleotide which binds to at least a portion of the chimeric ALL-l/AF-17 mRNA.
60. The method of claim 59 CΩmprising administering an antisense oligonucleotide which binds to at least a portion of SEQ ID NO:56.
61. A method of treating acute lymphoblastic or nonlymphoblastic leukemia comprising: providing a tissue sample containing hematopoietic cells from a person suspected of having acute lymphocytic or nonlymphoblastic leukemia; and detecting at least a portion of the chimeric ALL-l/AF-17 protein.
62. The method of claim 61 wherein said protein is detected using a monoclonal antibody which binds to at least a portion of the chimeric ALL-l/AF-17 protein.
63. The method of claim 61 wherein said protein is detected using a monoclonal antibody selected from the group consisting of: a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:55; a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:57 and a monoclonal antibody which binds to at least a portion of the amino acid sequences contained within SEQ ID NO:58.
64. A method of diagnosing acute lymphoblastic or nonlymphoblastic leukemia involving a chimeric gene in t(ll;17) translocatiΩns comprising: providing a tissue sample containing hematopΩietic cells frΩm a person suspected of having acute lymphoblastic or nonlymphoblastic leukemia; isolating RNA from the sample; generating cDNA from said RNA; amplifying a chimeric gene sequence in said cDNA which is generated by said translΩcation using a set of PCR primers if said chimeric gene is present; and detecting the presence of amplified DNA.
65. A probe which identifies chromosΩmal abnormalities in the ALL-l gene, said probe comprising B859.
PCT/US1994/004496 1993-05-14 1994-04-22 Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities WO1994026930A1 (en)

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US6040140A (en) * 1991-12-11 2000-03-21 Thomas Jefferson University Methods for screening and treating leukemias resulting from all-1 region chromosome abnormalities
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WO2003057240A2 (en) * 2002-01-09 2003-07-17 Smithkline Beecham P.L.C. Treatment and diagnosis of af4-dependant diseases
WO2003057240A3 (en) * 2002-01-09 2003-09-25 Smithkline Beecham Plc Treatment and diagnosis of af4-dependant diseases
US8124351B2 (en) 2005-11-18 2012-02-28 Board Of Regents, The University Of Texas System Quantification of fusion proteins and their activity from chromosomal translocation
US8349560B2 (en) 2007-06-15 2013-01-08 The Ohio State University Research Method for diagnosing acute lymphomic leukemia (ALL) using miR-222
US9085804B2 (en) 2007-08-03 2015-07-21 The Ohio State University Research Foundation Ultraconserved regions encoding ncRNAs
US8911998B2 (en) 2007-10-26 2014-12-16 The Ohio State University Methods for identifying fragile histidine triad (FHIT) interaction and uses thereof
US9125923B2 (en) 2008-06-11 2015-09-08 The Ohio State University Use of MiR-26 family as a predictive marker for hepatocellular carcinoma and responsiveness to therapy
US8916533B2 (en) 2009-11-23 2014-12-23 The Ohio State University Materials and methods useful for affecting tumor cell growth, migration and invasion
US8946187B2 (en) 2010-11-12 2015-02-03 The Ohio State University Materials and methods related to microRNA-21, mismatch repair, and colorectal cancer
US10758619B2 (en) 2010-11-15 2020-09-01 The Ohio State University Controlled release mucoadhesive systems
US11679157B2 (en) 2010-11-15 2023-06-20 The Ohio State University Controlled release mucoadhesive systems
US8664192B2 (en) 2011-03-07 2014-03-04 The Ohio State University Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer
US9249468B2 (en) 2011-10-14 2016-02-02 The Ohio State University Methods and materials related to ovarian cancer
US9481885B2 (en) 2011-12-13 2016-11-01 Ohio State Innovation Foundation Methods and compositions related to miR-21 and miR-29a, exosome inhibition, and cancer metastasis
US8859202B2 (en) 2012-01-20 2014-10-14 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
US9434995B2 (en) 2012-01-20 2016-09-06 The Ohio State University Breast cancer biomarker signatures for invasiveness and prognosis
WO2020165570A1 (en) * 2019-02-11 2020-08-20 Phoremost Limited Methods relating to disrupting the binding of af9 partner proteins to af9 and/or enl

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