AU751632B2 - Laminins and uses thereof - Google Patents

Description

WO 99/19348 PCT/US98/21391 1 LAMININS AND USES THEREOF BACKGROUND OF THE INVENTION The invention relates to the laminin 12, laminin subunit y3, and laminin subunit pi1, and methods of making and using these molecules.

SUMMARY OF THE INVENTION The present invention is based, in part, on the discovery of a novel member of the laminin family, laminin 12. Accordingly, the present invention features a purified or isolated preparation or a recombinant preparation of laminin 12 which includes an a2 subunit, a P1 subunit and a y3 subunit.

In a preferred embodiment, the a2 subunit has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human a2 subunit, the human a2 subunit of SEQ ID NO:7. The a2 subunit can be identical to a human a2 sequence, e.g., that of SEQ ID NO:7. In another embodiment, the a2 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:8. In addition, the a2 subunit can have substantially the same electrophoretic mobility as human a2 subunit, it appears as a 205 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an o2 subunit which is reactive with an a2-specific antibody, an antibody which binds to the epitope recognized by mAb 5H2. a2 specific antibodies can be made by methods known in the art.

Another preferred embodiment of the invention features a 131 subunit having at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about to about 95%, and most preferably at least about 99% sequence identity with human pi subunit, the human p1 subunit of SEQ ID NO:9. Preferably, the pi subunit has the identical amino acid sequence of human pi subunit, that of SEQ ID NO:9. In another embodiment, the pi subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID In addition, the pi subunit can have substantially the same electrophoretic mobility as human p1 subunit, it appears as a 185 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an 1p subunit which is reactive with an p1-specific antibody, an antibody which binds to the epitope recognized by mAb 545.

11-specific antibodies can be made by methods known in the art.

In yet another preferred embodiment, the y3 subunit of laminin 12 has at least 60% to about 70%, more preferably at least about 80%, even more preferably at least about 90% to about 95%, and most preferably at least about 99% sequence identity with human y3 subunit, WO 99/19348 PCT/US98/21391 2 the y3 subunit of SEQ ID NO:3. The y3 subunit can be identical to a naturally occuring human y3 subunit, that of SEQ ID NO:3. In another embodiment, the y3 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:4. In addition, the y3 subunit can have substantially the same electrophoretic mobility as human y3 subunit, it appears as a 170 kDa electrophoretic band on reducing gels. Yet another preferred embodiment of the invention features an y3 subunit which is reactive with an y3-specific antibody. y3-specific antibodies can be made by methods known in the art and taught herein.

In a preferred embodiment, the laminin 12 is a trimer which can be found in, or can be isolated from human placental chorionic villi. In another embodiment, the laminin 12 is expressed by a recombinant cell, a bacterial cell, a cultured cell a cultured eukaryotic cell) or a cell of a non-human transgenic animal. Cultured cells can include CHO cells or SF8 cells. Expression of laminin 12 in a transgenic animal can be general or can be under the control of a tissue specific promoter. Preferably, one or more sequences which encode subunits of the laminin 12 trimer are expressed in a preferred cell-type by. a tissue specific promoter, a milk specific promoter.

The present invention is also based, in part, on the discovery of a novel laminin subunit, y3. Accordingly, the invention features a recombinant or substantially pure or isolated preparation of a y3 polypeptide.

In a preferred embodiment, the y3 polypeptide has the following biological acitivities: 25 1) it promotes adhesion between tissue elements; 2) provides a site for insertion of nerves into the basement membrane. In other preferred embodiments: the y3 polypeptide includes an amino acid sequence with at least 60%, 80%, 90%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO:3; the y3 polypeptide includes an amino acid sequence essentially the same as the amino acid sequence in SEQ ID NO:3; the y3 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the y3 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least oo* 50, 100, or 150 contiguous amino acids from SEQ ID NO:3; the y3 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring y3 subunit; the y3 polypeptide is a vertebrate, a mammalian, e.g. a primate, a human, y3 polypeptide.

In a preferred embodiment, the invention includes ay3 polypeptide encoded by a DNA insert of a plasmid deposited with ATCC, Rockville Maryland on 10 October 1997, as Accession No: 209357. In another embodiment, the y3 polypeptide is a polypeptide encoded by nucleotide sequences of the overlapping DNA inserts of more than one, preferably all seven of the plasmids deposited with ATCC as Accession No: 209357.

In preferred embodiments: the y3 polypeptide is encoded by the nucleic acid in SEQ ID NO:4, or by a nucleic acid having at least about 85%, more preferably at least about to about 95%, and most preferably at least about 99% sequence identity with the nucleic acid S from SEQ ID NO: 4.

WO 99/19348 PCT/US98/21391 3 In preferred embodiments, the y3 polypeptide includes a nidogen-binding domain.

Generally, the nidogen-binding domain is at least 5 residues in length and preferably, has about 70, 80, 90, or 95% sequence identity with the nidogen-binding domain of the protein shown in SEQ ID NO: 3 (amino acid residues 750-755). In another embodiment, the y3 polypeptide includes at least 5, preferably 6 to 7, and most preferably 8 of the cysteins found in native y3 protein. In yet another embodiment of the invention features a y3 polypeptide that does not include or has an inactivated nidogen-binding domain which serves as an antagonist to y3 biological activities. Furthermore, a y3 polypeptide which has antagonist activity can have inactivated or excluded regions which comprise at least one cystein found in native y 3 protein.

In a preferred embodiment, the y3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 3. In other preferred embodiments, the y3 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 of the residues from a sequence in SEQ ID NO: 3. Preferably, the differences are such that: the y3 polypeptide exhibits a y3 biological activity, the y3 polypeptide retains a biological activity of a naturally occurring y3 subunit.

In preferred embodiments the y3 polypeptide includes a y3 subunit sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.

In preferred embodiments, the y3 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO: 3, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO: 3.

In yet other preferred embodiments, the y3 polypeptide is a recombinant fusion protein having a first y 3 portion and a second polypeptide portion, a second polypeptide portion having an amino acid sequence unrelated to y3. The second polypeptide portion can be, any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.

In a preferred embodiment the y3 polypeptide includes amino acid residues 750-755 of SEQ ID NO:3. In another embodiment, the y3 polypeptide encodes domains IV-VI of the y3 subunit.

In preferred embodiments the y3 polypeptide has antagonistic activity, and is capable of: inhibiting adhesion between connective tissues.

In a preferred embodiment, the y3 polypeptide is a fragment of a naturally occurring y 3 which inhibits connective tissue adhesion.

Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and alternative translational and postranslational events. The y3 polypeptide can be expressed in systems, cultured cells, which result in substantially the same postranslationai modifications present when expressed y3 is expressed in a native cell, or in systems which WO 99/19348 PCT/US98/21391 4 result in the omission of postranslational modifications present when expressed in a native cell.

The invention includes an immunogen which includes a y3 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the y3 polypeptide, a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, a unique determinant, from a protein represented by SEQ ID NO: 3.

The present invention also includes an antibody preparation specifically reactive with an epitope of the y3 immunogen or generally of a y3 polypeptide, preferably an epitope which consists all or in part of residues from the the amino acid sequence of SEQ ID NO:3, or an epitope, which when bound to an antibody, results in themodulation of a biological activity.

In preferred embodiments the y3-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has a molecular weight of 170 kDa as determined by SDS-PAGE.

In another embodiment, the y3 polypeptide comprises amino acid residues 100-1761 of SEQ ID NO: 3.

In a preferred embodiment, the y3 polypeptide has one or more of the following characteristics: it has the ability to promote adhesion between connective tissues; (ii) it has a molecular weight, amino acid composition or other physical characteristic of y3 subunit of SEQ ID NO:3; (iii) it has an overall sequence similarity of at least 50%, preferably at least more preferably at least 70, 80, 90, or 95%, with a y3 polypeptide of SEQ ID NO:3; (iv) it can be isolated from human placenta chorionic villi; it has a nidogen-binding domain which is preferably about 70%, 90% or 95% with amino acid residues 750-755 of SEQ ID NO:3; (vi) it can colocalize with protein ubiquitin carboxy terminal hydroxylase I; (vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found amino acid sequence of native y3.

Also included in the invention is a composition which includes a y3 polypeptide (or a nucleic acid which encodes it) and one or more additional components, a carrier, diluent, or solvent. The additional component can be one which renders the composition useful for in vitro and in vivo pharmaceutical or veterinary use.

In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a y3 polypeptide, a y3 polypeptide described herein.

A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 85% sequence identity to a nucleotide sequence of SEQ ID NO:4. In other preferred embodiments, the y 3 polypeptide is encoded by a nucleic acid WO 99/19348 PCT/US98/21391 molecule having a nucleotide sequence with at least about 90% to about 95%, and more preferably about 98% to about 99% sequence identity to the nucleotide sequence from SEQ ID NO:4. In another preferred embodiment, the y3 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:4.

In prefered embodiments, the isolated nucleic acid molecule includes the nucleotide sequence of at least one and preferably all of the DNA inserts of the plasmids deposited with ATCC as Accession No: 209357.

In preferred embodiments, the subject y3 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the y3 gene sequence (also referred to as LAMG3), to render the y3 gene sequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes a y3 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides of SEQ ID NO:4. More preferably, the nucleic acid probe corresponds to at least 20 consecutive nucleotides from SEQ ID NO: 4.

The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide sequence-which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 4, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includes a label group attached thereto.

The label group can be, a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.

The invention involves nucleic acids, RNA or DNA, encoding a y3 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.

In another aspect, the invention features a cell or purified preparation of cells which include a y3 subunit transgene, or which otherwise misexpress a y3 gene. The cell preparation can consist of human or non human cells, rodent cells, mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a y3 transgene, a heterologous form of a y3 gene, a gene derived from humans (in the case of a non-human cell). The y3. transgene can be misexpressed, overexpressed or underexpressed. In-other preferred embodiments, the cell or cells include a gene which misexpress an endogenous y3 gene, a gene the expression of which is disrupted, a knockout. Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed y3 alleles or for use in drug screening.

In another aspect, the invention features a transgenic y3 animal, a rodent, a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a y3 gene, a gene WO 99/19348 PCT/US98/21391 6 derived from humans. In a further embodiment, the y3 transgene includes a tissue specific promoter, a milk-specific promoter. In other preferred embodiments, the animal has an endogenous y3 gene which is misexpressed, a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed y3 alleles or for use in drug screening.

The invention is also based, in part, on the discovery of a novel laminin subunit, p4.

Accordingly, the invention features a recombinant or substantially pure preparation of a 34 polypeptide.

In preferred embodiment, the p4 polypeptide has the following biological activities: 1) it promotes adhesion between tissue elements; 2) it aids in wound healing. In other preferred embodiments: the p4 polypeptide includes an amino acid sequence with at least 65%, 95%, 98%, or 99% sequence identity to an amino acid sequence from SEQ ID NO: 1; the p4 polypeptide includes an amino acid sequence essentially the same as an amino acid sequence in SEQ ID NO: 1; the 04 polypeptide is at least 5, 10, 20, 50, 100, or 150 amino acids in length; the p4 polypeptide includes at least 5, preferably at least 10, more preferably at least 20, most preferably at least 50, 100, or 150 contiguous amino acids from SEQ ID NO:1; the p4 polypeptide is either, an agonist or an antagonist, of a biological activity of a naturally occurring p4 subunit; the p4 polypeptide is a vertebrate, a mammalian, e.g. a primate, a human, p4 polypeptide.

In preferred embodiments: the p4 polypeptide is encoded by the nucleic acid in SEQ ID NO:2, or by a nucleic acid having at least about 65% to about 70%, more preferably at least 80%, even more preferably at least about 90% to about 95%, and most preferably about 99% sequence identity with the nucleic acid from SEQ ID NO: 2.

In preferred embodiments, the 04 polypeptide includes domains VI and V found in native p4 subunits. Amino acid residues from about 221-262 and 263-535 of SEQ ID NO: 1 are exemplary of domains VI and V, respectively, of p4. Generally, domain VI is at least 33 residues in length and has preferably at least about 60%, more preferably about 70% to about and most preferably about 90% to about 95% sequence identity with the amino acid residues 221-262 of the 04 protein shown in SEQ ID NO: 1. Domain V is at least 272 residues in length and has preferably at least about 60%, more preferably about 70% to about 80%, and most preferably about 90% to about 95% sequence identity with the amino acid residues 263-535 of the p4 protein shown in SEQ ID NO: 1. In another embodiment, the 34 polypeptide has at least 5, preferably 6 or 7, and most preferably 8 cysteins as found in native P4. In yet another embodiment, a p4 polypeptide which has antagonist activity has inactivated or excluded regions which comprise at least one of the cysteins found in native 34 protein.

In a preferred embodiment, the p4 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 residues, from a sequence in SEQ ID NO: 1. In other preferred embodiments, the p4 polypeptide differs in amino acid sequence at up to 1, 2, 3, 5, or 10 of the residues WO 99/19348 PCT/US98/21391 from a sequence in SEQ ID NO: 1. Preferably, the differences are such that: the 34 polypeptide exhibits a p4 biological activity, the p4 polypeptide retains a biological activity of a naturally occurring p4 subunit.

In preferred embodiments the p4 polypeptide includes a p4 sequence described herein as well as other N-terminal and/or C-terminal amino acid sequence.

In preferred embodiments, the p4 polypeptide includes all or a fragment of an amino acid sequence from SEQ ID NO:1, fused, in reading frame, to additional amino acid residues, preferably to residues encoded by genomic DNA 5' to the genomic DNA which encodes a sequence from SEQ ID NO:1.

In yet other preferred embodiments, the p4 polypeptide is a recombinant fusion protein having a first P4 portion and a second polypeptide portion, a second polypeptide portion having an amino acid sequence unrelated to 04. The second polypeptide portion can be, any of glutathione-S-transferase, a DNA binding domain, or a polymerase activating domain. In preferred embodiment the fusion protein can be used in a two-hybrid assay.

In preferred embodiments the p4 polypeptide has antagonistic activity, and is capable of: inhibiting the adhesion of connective tissues.

Preferably, the p4 polypeptide is a fragment of a naturally occurring p4 which inhibits connective tissue adhesion.

Polypeptides of the invention include those which arise as a result of the existence of multiple genes, alternative transcription events, alternative RNA splicing events, and 25 alternative translational and postranslational events. In one aspect of the invention, the 34 polypeptide is a splice variant of the p4 subunit. In another preferred embodiment, the 34 splice variant is encoded by a nucleic acid molecule identical to the nucleotide sequence of SEQ ID NO:6. The polypeptide can be expressed in systems, cultured cells, which result in substantially the same postranslational modifications present when expressed p4 is expressed in a native cell, or in systems which result in the omission of postranslational modifications present when expressed in a native cell.

The invention includes an immunogen which includes a p4 polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for the p4 polypeptide, a humoral response, an antibody response, or a cellular response. In preferred embodiments, the immunogen comprising an antigenic determinant, a unique determinant, from a protein represented by SEQ ID NO: 1.

The present invention also includes an antibody preparation specifically reactive with an epitope of the p4 immunogen or generally of a p4 polypeptide, preferably an epitope which consists all or in part of residues from the amino acid sequence of SEQ ID NO:1, or an epitope, which when bound to an antibody, results in the modulation of a biological activity.

In preferred embodiments the p4-like polypeptide, as expressed in the cells in which it is normally expressed or in other eukaryotic cells, has an estimated molecular weight of 200 kDa as determined by SDS-PAGE.

WO 99/19348 PCT/US98/21391 8 In a preferred embodiment, the p4 polypeptide has one or more of the following characteristics: it has the ability to promote adhesion between connective tissues; (ii) it has a molecular weight, amino acid composition or other physical characteristic of 14 subunit of SEQ ID NO:1; (iii) it has an overall sequence similarity of at least 50%, preferably at least more preferably at least 70, 80, 90, or 95%, with a p4 polypeptide of SEQ ID NO: 1; (iv) it can be isolated from human placenta chorionic villi; it can associate with a3 or y2 subunits; (vi) it has coiled coils in domains I and II.

(vii) it has at least 5, preferably 6 or 7, and most preferably 8 of the cysteins found in native p4 sequence.

Also included in the invention is a composition which includes a p4 polypeptide (or a nucleic acid which encodes it) and one or more additional components, a carrier, diluent, or solvent. The additional component can be one which renders the composition for in vitro and in vivo pharmaceutical or veterinary use. Such uses can include aiding in wound healing or promotion of the adhesion of dermal and epidermal cells.

In another aspect, the invention provides an isolated or substantially pure nucleic acid having or comprising a nucleotide sequence which encodes a 34 polypeptide, a 34 polypeptide described herein.

A preferred embodiment of the invention features a nucleic acid molecule having a nucleotide sequence at least about 65% sequence identity to a nucleotide sequence of SEQ ID NO:2. In other preferred embodiments, the p4 polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence with at least 70%, preferably 80%, more preferably about 90% to about 95%, and even more preferably about 99% sequence identity to the nucleotide sequence from SEQ ID NO:2. In another preferred embodiment, the 34 polypeptide is encoded by the nulceic acid molecule of SEQ ID NO:2.

In preferred embodiments, the subject p4 nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the 04 gene sequence (also referred to as LAMB4), to render the 04 gene sequence suitable for use.as an expression vector.

In yet a further preferred embodiment, the nucleic acid which encodes a 34 polypeptide of the invention, hybridizes under stringent conditions to a nucleic acid probe corresponding to at least 12 consecutive nucleotides from SEQ ID NO:2, more preferably to at least 20 consecutive nucleotides from SEQ ID NO:2.

In a preferred embodiment, the nucleic acid differs by at least one nucleotide from a nucleotide sequence of SEQ ID NO:2, nucleotides 4686-5870, The invention also provides a probe or primer which includes or comprises a substantially purified oligonucleotide. The oligonucleotide includes a region of nucleotide WO 99/19348 PCT/US98/21391 9 sequence which hybridizes under stringent conditions to at least 10 consecutive nucleotides of sense or antisense sequence from SEQ ID NO: 2, or naturally occurring mutants thereof. In preferred embodiments, the probe or primer further includesa label group attached thereto.

The label group can be, a radioisotope, a fluorescent compound, an enzyme, and/or an enzyme co-factor. Preferably the oligonucleotide is at least 10 and less than 20, 30, 50, 100, or 150 nucleotides in length.

The invention involves nucleic acids, RNA or DNA, encoding a P4 polypeptide of the invention. This includes double stranded nucleic acids as well as coding and antisense single strands.

In another aspect, the invention features a cell or purified preparation of cells which include a P4 transgene, or which otherwise misexpress a p4 gene. The cell preparation can consist of human or non human cells, rodent cells, mouse or rat cells, rabbit cells, or pig cells. In preferred embodiments, the cell or cells include a p4 transgene, a heterologous form of a p4 gene, a gene derived from humans (in the case of a nonhuman cell). The 34 transgene can be misexpressed, overexpressed or underexpressed.

In other preferred embodiments, the cell or cells include a gene which misexpress an endogenous P4 gene, a gene the expression of which is disrupted, a knockout.-Such cells can serve as a model for studying disorders which are related to mutated or misexpressed p4 alleles or for use in drug screening.

In another aspect, the invention features a transgenic 14 animal, a rodent, a mouse or a rat, a rabbit, a pig, a goat, or a cow. In preferred embodiments, the transgenic animal includes (and preferably express) a heterologous form of a p4 gene, a gene derived from humans. In a further embodiment, the 34 transgene includes a tissue specific promoter, a milk-specific promoter. In other preferred embodiments, the animal has an endogenous p4 gene which is misexpressed, a knockout. Such a transgenic animal can serve as a model for studying disorders which are related to mutated or mis-expressed 34 alleles or for use in drug screening.

In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting-one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, laminin 12, or y3 (or-a laminin trimer which inlcudes y3), sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, by topical appication or by injection, or administered in a systemic fashion.

A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements include, skin cells, epidermal or dermal cells, neuronal-cells, nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, non- WO 99/19348 PCT/US98/21391 traumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.

In preferred embodiments the molecule is exogenous administered to a subject) or is recombinant.

In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.

In preferred embodiments the first tissue is healthy tissue, skin tissue, and the second tissue is wounded, burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, burned or abraided tissue.

In preferred embodiments the first tissue and second tissue element are normally adhered but have become detached from one another due to trauma, bur or other physical injury, disease, or age.

In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve; the first tissue element is a retinal cell or retina tissue and the second tissue element is a cell or structure which in normal, nontraumatized, or non-diseased tissue is adjascent or adhered to the a retinal cell or retina tissue, the first tissue is a nerve and the second tissue is basement membrane.

The administration of laminin can be repeated.

In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, laminin 12, y3 (or a laminin trimer which inlcudes y3), sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, by topical appication or by injection, or administered in a systemic fashion.

The wound can be in any tissue, but preferably ina tissue in which the laminin normally occurs. Examples skin, central or peripheral nervous tissue, tissues of the eye, e.g., the retinal, the basement membrane, or any tissue which in normal, non-traumatized, or nondiseased tissue is adjascent or adhered thereto.

In preferred embodiments the molecule is exogenous administered to a subject) or is recombinant.

In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraided.

WO 99/19348 M PCTl5WL 1 11 The administration of laminin can be repeated.

In another aspect, the invention features a method of promoting nerve growth or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, laminin 12, or y3 (or a laminin trimer which inlcudes y3), sufficient to promote nerve growth or regeneration. The administration can be directed to the site where nerve growth or regeneration is desired, by topical appication or by injection, or administered in a systemic fashion.

In preferred embodiments the molecule is exogenous administered to a subject) or is recombinant.

In preferred embodiments the nerve growth or regeneration is promoted at a wound site.

The administration of laminin can be repeated.

In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a gene which encodes a laminin described herein, y 3 or laminin 12.

Such disorders include, a disorder associated with the misexpression of a laminin, e.g., laminin 12, or misexpression of the y3 subunit; a disorder of the central or peripheral nervous system; a disorder associated with a genetic lesion at chromosome 9, region q31-34; Fukuyama-type muscular dystrophy; muscle-eye-brain disease; Walker-Warburg Syndrome (hydrocephalus, ageria, and retinal displasia); a retinal disorder; a disorder associated with abnormal levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder; a disorder associated with the testis, spleen, placenta, thymus, ovary, small intestine, lung, or liver.

The method includes one or more of the following: S 30 detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the y3 gene, or other gene which encodes a subunit of laminin 12, detecting the presence or absence of a mutation in a region which controls the expression of the gene, a mutation in the 5' control region; detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of the y3 gene, or other gene which encodes a subunit of laminin 12; detecting, in a tissue of the subject, the misexpression of they3 gene, or other gene which encodes a subunit of laminin 12 at the mRNA level, detecting a non-wild type So level of a y3, or an other laminin 12 subunit mRNA; detecting, in a tissue of the subject, the misexpression of the y3 gene, or other gene which encodes a subunit of laminin 12, at the protein level, detecting a non-wild type level of a y3, or an other laminin 12 subunit polypeptide.

In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the y3 gene, or other gene which encodes a WO 99/19348 PCT/US98/21391 12 subunit of laminin 12; an insertion of one or more nucleotides into the gene, a point mutation, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:4, or naturally occurring mutants thereof or or 3' flanking sequences naturally associated with the LAMG3 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the y3 gene, or other gene which encodes a subunit of laminin 12; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the y3 gene, or other gene which encodes a subunit of laminin 12; or a non-wild type level of y3, or other subunit of laminin 12.

Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structure of a y3 gene, or other gene which encodes a subunit of laminin 12, an abnormal structure being indicative of risk for the disorder.

In preferred embodiments the method includes contacting a sample form the subject with an antibody to the laminin protein or a nucleic acid which hybridizes specifically with the y3 gene, or other gene which encodes a subunit of laminin 12.

In another aspect, the invention features, a method of promoting adhesion of a first tissue element to a second tissue element. The method includes contacting one or both of the first tissue element and the second tissue element with an amount of a laminin molecule described herein, P4, sufficient to promote adhesion. The method can be performed in vivo, or in vitro. In in vivo methods-the laminin is administered to the subject. The administration can be directed to the site where adhesion is desired, by topical application or by injection, or administered in a systemic fashion.

A tissue element can be a cell or a multi-cellular on acellular structure. Examples of tissue elements incude, skin cells, epidermal or dermal cells, neuronal cells, nerve cells, retinal cells, central or pereipheral nervous system components, basement membrane or components of the basement membrane, or any cell or structure which in normal, nontraumatized, or non-diseased tissue is adjascent or adhered to a specific tissue element recited herein.

In preferred embodiments the molecule is exogenous administered to.a subject) or is recombinant.

WO 99/19348 PCT/US98/21391 13 In preferred embodiments the method is an vivo method. In vivo methods can be autologous, allogeneic, or xenogeneic. In autologous methods, adhesion between two tissue elements from the subject is promoted. In allogeneic methods, adhesion between a recipient tissue element and a donor tissue element from an allogeneic donor is promoted. In xenogeneic methods, adhesion between a recipient tissue element and a donor tissue element from a xenogeneic donor is promoted. Thus, one element can be a donor tissue element which is implanted into a recipient subject.

In preferred embodiments the first tissue is healthy tissue, skin tissue, and the second tissue is wounded, burned, diseased, traumatized, cut, and the tissue, or is a wound bed. For example, the first tissue is skin tissue, from the subject or from a donor, and the second tissue is wounded, burned or abraided tissue.

In preferred embodiments: the first tissue element is a dermal cell and the second tissue element is an epidermal cell; the first tissue element is a nerve cell or nerve and the second tissue element is a cell or structure which in normal, non-traumatized, or non-diseased tissue is adjascent or adhered to the nerve cell or nerve; the first tissue is a nerve and the second tissue is basement membrane.

The administration of laminin can be repeated.

In another aspect, the invention features a method of promoting wound healing in a subject. The method includes administering an amount of a laminin molecule described herein, sufficient to promote healing to the wound. The administration can be directed to the site where healing is desired, by topical appication or by injection, or administered in a systemic fashion.

The wound can be in any tissue, but preferably in a tissue in which the laminin normally occurs in fetal or adult life. Examples examples include skin the basement membrane.

In preferred embodiments the molecule is exogenous administered to a subject) or is recombinant.

In preferred embodiments the wound tissue is burned, diseased, traumatized, cut, the subject of immune attack, e.g, autoimmune attack, or abraded.

The administration of laminin can be repeated.

In another aspect, the invention features a method of promoting tissue growth, development, or regeneration in a subject. The method includes administering an amount of a laminin molecule described herein, P4, sufficient to promote tissue growth, development, or regeneration in a subject. The administration can be directed to the site where nerve growth or regeneration is desired, by topical appication or by injection, or administered in a systemic fashion.

in preferred embodiments the molecule is exogenous administered to a subject) or is recombinant.

WO 99/19348 PCT/US98/21391 14 In preferred embodiments the nerve growth or regeneration is promoted at a wound site.

The administration of laminin can be repeated.

In another aspect, the invention provides, a method of determining if a subject is at risk for a disorder related to a lesion in or the misexpression of a laminin molecule described herein, p4.

Such disorders include, a disorder associated with the misexpression of a laminin, 1 4; a disorder associated with a genetic lesion at chromosome region 7 q22-q31.2; a developmetnal disorder; a disorder associated with abnormal levels, abnormally low levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder, an epidermal or dermal, disorder.

The method includes one or more of the following: detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of the p4 gene, e.g, detecting the presence or absence of a mutation in a region which controls the expression of the gene, a mutation in the 5' control region; detecting, in a tissue of the subject, the presence-or absence of a mutation which alters the structure of the p4 gene; detecting, in a tissue of the subject, the misexpression of the p4 gene, detecting a non-wild type level of a p4 mRNA; detecting, in a tissue of the subject, the misexpression of the p4, at the protein level, detecting a non-wild type level of a p4 polypeptide.

In preferred embodiments the method includes: ascertaining the existence of at least one of: a deletion of one or more nucleotides from the p4; an insertion of one or more nucleotides into the gene, a point mutation, a substitution of one or more nucleotides of the p4 gene, a gross chromosomal rearrangement of the 04 gene, a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: providing a probe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence from SEQ ID NO:2, or naturally occurring mutants thereof or or 3' flanking sequences naturally associated with the LAMB4 gene; (ii) exposing the probe/primer to nucleic acid of the tissue; and detecting, by hybridization, in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

In preferred embodiments: detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of the P 4; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the p4; or a non-wild type level of 04.

Methods of the invention can be used prenatally or to determine if a subject's offspring will be at risk for a disorder.

WO 99/19348 PCT/US98/21391 In preferred embodiments the method includes determining the structure of the a p4, an abnormal structure being indicative of risk for the disorder.

In preferred embodiments the method includes contacting a sample form the subject with an antibody to the p4 protein or a nucleic acid which hybridizes specifically with the p4.

In another aspect, the invention features, a method of evaluating a compound for the ability to interact with, bind, a subject laminin polypeptide, laminin 12, y3, a laminin trimer which inlcudes y3, P4, or a laminin trimer which includes p4. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating ability of the compound to interact with, to bind or form a complex with the subject laminin polypeptide. This method can be performed in vitro, in a cell free system, or in vivo, in a two-hybrid interaction trap assay. This method can be used to identify naturally occurring molecules which interact with subject laminin polypeptide. It can also be used to find natural or synthetic inhibitors of subject laminin polypeptide.

In another aspect, the invention features, a method of evaluating a compound, a polypeptide, a naturally occurring ligand of or a naturally occuring substrate to which binds a subject laminin polypeptide, of laminin 12, y3, a laminin trimer which inlcudes y 3, 14, or a laminin trimer which includes p4, for the ability to bind a subject laminin polypeptide. The method includes: contacting the compound with the subject laminin polypeptide; and evaluating the ability of the compound to interact with, to bind or form a complex with the subject laminin polypeptide, the ability of the compound to inhibit a subject laminin polypeptide/ligand interaction. This method can be performed in vitro, e.g., in a cell free system, or in vivo, in a two-hybrid interaction trap assay. This method can be used to identify compounds, fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.

In another aspect, the invention features, a method of evaluating a first compound, a subject laminin polypeptide, laminin 12, y3, a laminin trimer which inlcudes y3, P 4, or a laminin trimer which includes p4, for the ability to bind a second compound, a second polypeptide, a naturally occurring ligand of or substrate to which binds a subject laminin polypeptide. The method includes: contacting the first compound with the second compound; and evaluating the ability of the first compound to form a complex with the second compound. This method can be performed in vitro, in a cell free system, or in vivo, in a two-hybrid interaction trap assay. This method can be used to identify compounds, fragments or analogs of a subject laminin polypeptide, which are agonists or antagonists of a subject laminin polypeptide.

In yet another aspect, the invention features a method for evaluating a compound, e.g., for the ability to modulate an interaction, the ability to inhibit an interaction of a subject laminin polypeptide, of laminin 12, y-3, a laminin trimer which inlcudes y3, P4, or a laminin trimer which includes 04, with a second polypeptide, a polypeptide, a natural ligand of the of or a substrate wo which binds a subject laminin polypeptide, or a WO 99/19348 PCT/US98/21391 16 fragment thereof. The method includes the steps of combining the second polypeptide (or preferably a purified preparation thereof), a subjectlaminin polypeptide, (or preferably a purified preparation thereof), and a compound, under conditions wherein in the absence of the compound, the second polypeptide, and the subject laminin polypeptide, are able to interact, to bind or form a complex; and (ii) detecting the interaction, detecting the formation (or dissolution) of a complex which includes the second polypeptide, and the subject laminin polypeptide. A change, a decrease or increase, in the formation of the complex in the presence of a compound (relative to what is seen in the absence of the compound)-is indicative of a modulation, an inhibition or promotion, of the interaction between the second polypeptide, and the subject laminin polypeptide. In preferred embodiments: the second polypeptide, and the subject laminin polypeptide, are combined in a cell-free system and contacted with the compound; the cell-free system is selected from a group consisting of a cell lysate and a reconstituted protein mixture; the subject laminin polypeptide, and the second polypeptide are simultaneously expressed in a cell, and the cell is contacted with the compound, e.g. in an interaction trap assay a two-hybrid assay).

In yet another aspect, the invention features a two-phase method a method having an in vitro, in a cell free system, and an in vivo phase) for evaluating a compound, for the ability to modulate, to inhibit or promote, an interaction of a subject laminin polypeptide subject laminin polypeptide, of laminin 12, y3, a laminin trimer which inlcudes y3, p4, or a laminin trimer which includes P4, with a second compound, a second polypeptide, a naturally occurring ligand of or a substrate to which binds a subject laminin polypeptide, or a fragment thereof. The method includes steps and (ii) of the method described immediately above performed in vitro, and further includes: (iii) determining if the compound modulates the interaction in vitro, in a cell free system, and if so; (iv) administering the compound to a cell or animal; and (v) evaluating the in vivo effect of the compound on an interaction, inhibition, of a subject laminin polypeptide, with a second polypeptide.

In another aspect, the invention features, a method of evaluating a compound for the ability to bind a nucleic acid encoding a subject laminin polypeptide, a laminin 12, y3, a laminin trimer.which inlcudes y3, 34, or a laminin trimer which includes p4 polypeptide regulatory sequence. The method includes: contacting the compound with the nucleic acid; and evaluating ability of the compound to form a complex with the nucleic acid.

In another aspect, the invention features a method of making a y3 or p4 polypeptide, a peptide having a non-wild type activity, an antagonist, agonist, or super agonist of a naturally occurring y3 or p4 polypeptide, a naturally occurring y3 or p4 polypeptide.

The method includes: altering the sequence of a y3 or p4 polypeptide, altering the sequence by substitution or deletion of one or more residues of a non-conserved region, a domain or residue disclosed herein, and testing the altered polypeptide for the desired activity.

WO 99/19348 PCT/US98/21391 17 In another aspect, the invention features a method of making a fragment or analog of a y3 or p4 polypeptide having a biological activity of a naturally occurring y 3 or 134 polypeptide. The method includes: altering the sequence, by substitution or deletion of one or more residues, of a y3 or p4 polypeptide, altering the sequence of a non-_ conserved region, or a domain or residue described herein, and testing the altered polypeptide for the desired activity.

In another aspect, the invention features, a human cell, a hematopoietic stem cell, transformed with nucleic acid which encodes a subject laminin polypeptide, a laminin 12, y3, a laminin trimer which inlcudes y3, p4, or a laminin trimer which includes 04.

In another aspect, the invention includes: a y3, p4 nucleic acid, a y3, p4 nucleic acid inserted into a vector; a cell transformed with a y3, p4 nucleic acid; a y3, p4 made by culturing a cell transformed with a y3, p4 nucleic acid; and a method of making a y3, P4 polypeptide including culturing a a cell transformed with a y3, 14 nucleic acid.

The inventors have shown that y3 forms laminin 12 in association with a2 and p1.

However, we are unsure of the chain associations of y3 within other tissues. It is very likely that y3 can also associate with y3, a3, a4, and a5; with 02, 13, 14 and p5. Therefore, our results predict 25 new laminins: laminins 12-37. y3 and p4 polypetides of the invention can be expressed with, assembled with, or administered with other laminin subunits in any of the methods described herein. y3 can be assembled with an a and a 1 subunit to form a laminin trimer. p4 can be assembled with an a and a P subunit to form a laminin trimer.

In any treatment or therapeutic application which administers y3, a p2 subunit can also be administered.

A "heterologous promoter", as used herein is a promoter which is not naturally associated with a gene or a purified nucleic acid.

A "purified"or "substantially pure" or isolated "preparation" of a polypeptide, as used herein, means a polypeptide that has been separated from other proteins, lipids, and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances, antibodies or gel matrix, polyacrylamide, which are used to purify it.

Preferably, the polypeptide constitutes at least 10, 20, 50 70, 80 or 95% dry weight of the purified preparation. Preferably, the preparation contains: sufficient polypeptide to allow protein sequencing; at least 1, 10, or 100 pLg of the polypeptide; at least 1, 10, or 100 mg of the polypeptide.

A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

A "treatment", as used herein, includes any therapeutic treatment, e.g.,.the administration of a therapeutic agent or substance, a drug.

WO 99/19348 PCT/US98/21391 18 An "isolated" or" pure nucleic acid", a substantially pure DNA, is a nucleic acid which is one or both of: not immediately contiguous with either one or both of the sequences, coding sequences, with which it is immediately contiguous one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the nucleic acid is derived; or which is substantially free of a nucleic acid sequence with which it occurs in the organism from which the nucleic acid is derived. The term includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. Substantially pure DNA can also includes a recombinant DNA which is part of a hybrid gene encoding sequence.

"Sequence identity or homology", as used herein, refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous or sequence identical at that position. The percent of homology or sequence identity between two sequences is a function of the number of matching or homologous identical positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10, of the positions in two sequences are the same then the two sequences are 60% homologous or have 60% sequence identity. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology or sequence identity. Generally, a comparison is made when two sequences are aligned to give maximum homology.

The terms "peptides", "proteins", and "polypeptides" are used interchangeably herein.

As used herein, the term "transgene" means a nucleic acid sequence (encoding, e.g., one or more subject laminin polypeptides), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of the selected nucleic acid, all operably linked to the selected nucleic acid, and may include an enhancer sequence.

As used herein, the term "transgenic cell" refers to a cell containing a transgene.

As used herein, a "transgenic animal" is any animal in which one or more, and preferably essentially all, of the cells of the.animal includes a transgene. The transgene can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a W-0O 99/19348 PCTIUS98/21391 19 recombinant virus. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

As used herein, the term "tissue-specific promoter" means a DNA sequence that serves as a promoter, regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in specific cells of a tissue, such as mammary tissue. The term also covers so-called "leaky" promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.

"Unrelated to a y3 or P4 amino acid or nucleic acid sequence" means having less than sequence identity, less than 20% sequence identity, or, preferably, less than homology with a naturally occuring y3 or p4 sequence disclosed herein.

A polypeptide has y3 biological activity if it has one or more of the properties of y3 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or superagonist of a polypeptide having one of the properties of y3 disclosed herein.

A polypeptide has p4 biological activity if it has one or more of the properties of P4 disclosed herein. A polypeptide has biological activity if it is an antagonist, agonist, or superagonist of a polypeptide having one of the properties of P4 disclosed herein.

"Misexpression", as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

Subject, as used herein, can refer to a mammal, a human, or to an experimental or animal or disease model. The subject can also be a non-human animal, a horse, cow, goat, or other domestic animal.

As described herein, one aspect of the invention features a substantially pure (or recombinant) nucleic acid which includes a nucleotide sequence encoding a y3 or P4 polypeptide and/or equivalents of such nucleic acids. The term nucleic acid as used herein can include fragments and equivalents. The term equivalent refers to nucleotide sequences encoding functionally equivalent polypeptides. Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as 20 allelic variants, and include sequences that differ from the nucleotide sequences disclosed herein by degeneracy of the genetic code.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature.

See, for example, Molecular Cloning A Laboratory Manual, 2 nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II Glover ed., 1985); Oligonucleotide Synthesis Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization Hames S.J.

Higgins eds. 1984); Transcription And Translation (B.D.

Hames S.J. Higgins eds. 1984); Culture Of Animal Cells I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A 20 Practical Guide To Molecular Cloning (1984); the treatise, i Methods In Enzymology (Academic Press, Inc., Gene Transfer Vectors For Mammalian Cells Miller and M.P.

Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), 25 Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); 'i Handbook Of Experimental Immunology, Volumes I-IV (D.M.

Weir and C.C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold 30 Spring Harbor, 1986).

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the H:\janel\Keep\Speci\10765-99.doc 28/06/01 P 20a references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

DETAILED DESCRIPTION- The drawings are briefly described.

Figure 1 depicts the cDNA sequence for human a2 subunit.

20 Figure 2 depicts the predicted amino acid sequence for human a2 subunit.

i' Figure 3 depicts the cDNA sequence for human 34 subunit.

Figure 4 depicts the predicted amino acid sequence for human 34 subunit Figure 5 depicts an alignment of the amino acid 30 sequence of human 34 of SEQ ID NO: 1 and p4 splice varient of SEQ ID NO:5 AND LAMININ 31, 32, and 33 subunits.

Figure 6 provides a comparison of the similarities of laminin 34 domains with the domains of other known laminin 3 subunits.

\O H:\janel\Keep\Speci\10765-99.doc 28/06/01 11 F WO 99/19348 PCT/US98/21391 21 Isolation of laminin 12 Laminin 12 was isolated from human placental choronic villi. Briefly, human chorionic placental villi were frozen in liquid nitrogen, ground in a Waring blender and washed in 1 M NaCI. The final tissue pellet (200g, wet weight) was suspended in 1 L of extraction buffer (50 mM Tris-HCl 50 mM, pH=7.8; NaCl 0.5M, EDTA 10mM, 625 mg/l of N-ethylmaleimide, 150 mg/l ofphenylmethylsulphonyl fluoride. The suspension was incubated at 4 0 C with stirring for 48 h. Unless otherwise noted, all subsequent steps were performed at 40C. The soluble fraction was collected following centrifugation (30000 x g, min) and precipitated by 300g/l of Ammonium Sulfate. The precipitated proteins were collected by centrifugation (30000 x g, 60 min) and redissolved into chromatography buffer (2M Urea, 25 mM NaCl, 5 mM EDTA, and 50 mM Tris-HC1, pH=7.8). The sample was then dialyzed against the same buffer. Following dialysis, 0.5 volumes of buffer equilibrated DEAE-cellulose (DE-52, Whatman) was added and the mixture shaken overnight. Material not bound to DEAE-cellulose was collected by filtration on a Buchner funnel (Whatman filter 4) and precipitated by addition of 300g/l of ammonium sulfate. The proteins were collected by centrifugation (30000 x g, 60 min), redissolved in the Concanavalin-A buffer (0.5 M NaC1, 5 mM CaCl2, 5 mM MgC12, and Tris-HCI 50 mM, pH=7.8) and dialyzed against the same buffer overnight. The fraction was applied to a 2.5 x 5 cm Concanavalin-A sepharose column (Pharmacia), and unbound material was removed by extensive washing. Bound proteins were first eluted with 10 mM a-D- Mannopyrannoside (Sigma, St. Louis, MO) and secondly with 1 M a-D-Glucopyrannoside (Sigma, St. Louis, MO). A third elution with 1M a-D-Manno-pyrannoside (Sigma, St. Louis, MO) allowed the recovery of the proteins of interest. Each fraction was independently concentrated to 10 ml on a AmiconTM concentrator kDa membrane) and applied to a-2.5 x 100 cm Sephacryl S-500 column in a 0.5 M NaC1, 50 mM Tris-HCl, pH=7.8 buffer. The fractions of interest were pooled, dialyzed against Mono-Q buffer (0.1 M NaCl, 25 mM Tris-HC1, pH=7.8) and applied to the 1 x 5 cm Mono-Q column (Pharmacia). Elution was achieved with a 60 ml 0.1-0.5 M NaCl gradient.

The final fraction of interest resulting from the above protocol contains multiple laminins. The laminin 12 was resolved from this mixture by SDS-PAGE polyacrylamide) under non-reducing conditions. Six band were resolved. Only the bands at approximately 560 kDa and at the top of the gel were shown to be reactive with polyclonal anti-laminin antiserum (Sigma, St. Louis, MO).

Isolation of a2. 01.y3 subunits from laminin 12 Laminin 12 was excised, equilibrated and reduced in 10% 2-me SDS-PAGE sample buffer, and resolved by 5% SDS-PAGE. Three bands were resolved, which were approximately 205 kDa, 185 kDa, and 170 kDa. The band at 185 kDa reacted with monoclonal antibody 545, specific to the laminin 1 subunit. Each of the three bands were WO 99/19348 PCT/US98/21391 22 digested with trypsin and the peptides were resolved by HPLC. The selected resolves were subject to peptide sequencing.

Sequencing of the a2. 11 subunits of laminin2 Protein sequencing was done according to Aebersold et al. (1987). The complex laminin 5-laminin 7 was run on a polyacrylamide gel in the presence of 2 -mercaptoethanol and blotted onto a nitrocellulose membrane (Biorad). The 190 kDa band of p2 and the 165 kDa a3 band were separately excised and digested by protease trypsin. The digested product was separated by HPLC and one fragment was sequenced on an Applied Biosystems sequenator (Applied Biosystems, Foster City, CA). The 205 kDa chain contained a sequence identical to human laminin a2, and was thus identified as human laminin a2 subunit. The 185 kDa produced two peptides identical to human 11, and was thus identified as human laminin pi subunit. The band at 170 kDa contained three sequences not contained in any known laminin chain. A N-terminal sequence of the 170 kDa chain was also determined. In addition, the N-terminal sequence was not identical to any known laminin sequence.

Identification of the y3 subunit The cDNA sequences of human yl and y2 were used to probe the National Center for Biomedical Information (NCBI) dBest T M data base by BLAST search and a clone was isolated that was homologous, but not identical to yl and y2. This clone was extended by PCR at the 5' end using Marathon cDNA from human placenta from Clonetech (Palo Alto, CA). The resulting sequence was determined to be 100% identical to all three of the 170 kDa band peptide sequences.

Comparison of the nucleotide sequence of the isolated y3 subunit to yl, demonstrated about 80% sequence identity.

Structural Analysis of y3 encoding DNA The human cDNA encoding y3, which is approximately 4710 nucleotides in length, encodes a protein having an estimated molecular weight of approximately 146 kDa (including post-translational modifications) and which is approximately 1570 amino acid residues in length. The human y3 protein contains a nidogen-binding domain, which can be found, for example, from about amino acids 750-755 of SEQ ID NO:3. The y3 amino acid sequence and the nucleotide sequence encoding human laminin y3 is shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.

By Northern analysis the size of the. y3 mRNA is approximately 5 kb, which is consistent with other laminin y subunits. The y3 mRNA transcript is expressed in human tissues including spleen, testis, brain, placenta, lung, and possibly liver. Chromosomal mapping using the y3 cDNA sequence indicates that the human y3 gene is located on WO 99/19348 PCT/US98/21391 23 chromosome 9q31-34. The location of y3 on chromosome 9 was confirmed by FISH analysis using a 1.3 kb y3 cDNA probe within the predicted domains I and II, which are the regions of the least sequence identity among y subunits. Four human genes associated with Walker- Walburg syndrome, Fukuyama muscular dystrophy, retinitis pigmentosa-deafness syndrome and Eye, Muscle, Brain disease have also been mapped to chromosome 9q31-34.

Production of a y3 specific antibody and tissue localization of y3 The 170 kDa (y3) chain was excised from the reducing SDS-PAGE gel described above and injected into a rabbit for antibody production. The resulting serum (rabbit 16) was evaluated by Western analysis and shown to react with the 170 kDa y3 chain, and showed minor crossreactivity with other laminin chains.

Using immunofluorescence, this antiserum shows localization of y3 to the following tissue areas: 1) sites of insertions of nerves into the dermal-epidermal junction basement membrane of human skin; 2) the inner nuclear layers, outer nuclear layers, and outer limiting membranes of human, mouse and rat neural retina; 3) the Purkinje cells, and molecular layers, and (perhaps) the glial cells of the mouse and rat cerebellum; 4) the neuromuscular junctions of skeletal muscle; and, 5) the taste buds of the cow tongue.

The y3 was also shown to colocalize with protein ubiquitin carboxy terminal hydrolase I using antibody pGp 9.5. The y3 subunit also appears to colocalize with the a2 subunit in the same tissue sections.

Isolation and Sequencing of cDNA encoding 04 The initial 350 bp fragment of human laminin p4 cDNA was amplified by touchdown RT-PCR from cultured human keratinocyte total RNA using nested primers made from the published chicken laminin p x 503 bp cDNA sequence (as described in Ybot- Gonzalez et al. (1995)). Subsequent cDNA clones were isolated by nested PCR directly from a human placenta cDNA library packaged in lambda-gtl 1 (Clontech, Palo Alto, CA) or by nested PCR directly from human placenta Marathon-Ready cDNA (Clontech, Palo Alto, CA).

The 5' end of the cDNA was cloned using the 5'-RACE technique from human placenta total RNA. The Expanded Long Template PCR System (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used for all PCR reactions. The PCR products were ligated into the pCR2.1 vector (Invitrogen, San Diego, CA) and recombinant plasmids purified for sequencing using the QIAprepTM kit (Qiagen). The DNA sequence was determined using either the Sequenase version 2.0 DNA Sequencing Kit (Amersham) and 3 5 S-dATP or the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (Amersham) and 3 3

P-

ddNTPs. At least two independent cDNA subclones were sequenced to rule out Taq polymerase-generated nucleotide substitutions. In some cases, PCR product bands were sequenced directly by cycle sequencing after excision from a TAE-EtBr agarose gel and purification using QIAquick Gel Extraction kit (Qiagen).

WO 99/19348 PCTIUS98/21391 24 Structural Analysis of DNA encoding 14 The human cDNA encoding a long form P4, which is approximately 5.87 kb, encodes a protein having an estimated molecular weight of approximately 200 kDa and which is approximately 1761 amino acid residues in length. The human p4 protein retains the highest amino acid sequence identity with domains VI and V, which can be found, for example, from about amino acids 221-262 and about 263-535 of SEQ ID NO:1. In addition, a short form, splice variant of 14, which is approximately 3.84 kb and an estimated molecular weight of 120 kDa, was also isolated. The splice variant has 132 nucleotide sequence identical to the long form of p4, with the sequence diverging at nucleotide 3375 and spliced into a unique 3' untranslated region. The short form cDNA encodes a truncated 14 subunit which contains only the short arm of the 34 subunit and is missing the domains necessary for heterodimerization. The p4 amino acid sequence and the nucleotide sequence encoding human laminin p4 is shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

Northern analysis was performed using total RNA prepared from JAR cell, cultured human keratinocytes and human placenta using either Trizol (Gibco BRL, Bethesda, MD) or RNeasyTM (Qiagen) which was denatured, separated on a formaldehyde agarose gel and blotted onto nitrocellulose according to standard protocols (Sambrook, et al., 1989). In addition, A human multiple tissue northern blot (Clontech, Palo Alto, CA) and Human Northern Territory normal tissue blots and custom fetal skin northern blot (Invitrogen, San Diego, CA) were used. Hybridization and washing were performed using NorthernMAX

T

buffer system (Ambion) by manufacturer's recommended protocols. 32P-dCTP-labelled probes were generated from gel-purified restriction fragments using Rediprime T M random primer labeling kit (Amersham). 32P-UTP-labelled antisense RNA probes were generated using the RNA transcription kit (Stratagene, La Jolla, CA) from cDNAs subcloned into Bluescript II KS+ (Stratagene, La Jolla, CA).

Northern blotting showed that human laminin p4 is expressed in JAR cells, derived from undeveloped chronic villi and in placenta. By RT-PCR, it is also expressed in cultured keratinocytes. Using a northern blot of human fetal skin developmental progression, 34 subunit (long form) demonstrates strong expression at week twelve of fetal development and persists until birth, but expression is barely detectable in adult skin. The 04 splice variant, however, is expressed in various tissues including adult heart, brain, lung, liver, skeletal muscle, kidney, spleen, stomach, esophagus, intestine, colon, uterus, bladder, adipose tissue and pancreas. Chromosomal mapping with a p4 cDNA probe indicates that the human 34 subunit is located at locus 7q22-q31.2. The gene encoding 31 is located near, but not on, this position of chromosome 7. Statistical analysis of the mapping data using markers for 11 and P4 suggest that the gene encoding pi is linked to both ends of the gene encoding 04. In addition,-neonatal cutis laxa with manifold phenotype has been mapped near, but not in the same position, as the gene encoding 04.

WD 99/19348 PCT/US98/21391 In situ hybridization to wounded human skin grafted into nude mice suggests that laminin 0 x is expressed in the dermis underneath the migrating epidermal tongues during wound closure.

A GenBankTM search using the human nucleotide sequence encoding 34 as shown in SEQ ID NO:3 revealed an EST, which corresponds to nucleotides 4686-5870 of the human nucleotide sequence encoding 04 depicted in SEQ ID NO:3. Alignment of cDNA encoding 34 with the genes encoding human laminin 1p and laminin p2 shows 61% and 59% sequence identity, respectively, as shown in Figure Production of a 14 specific antibody and tissue localization of p4 Antibodies were raised in rabbits against a 26 kDa bacterial fusion protein which corresponds to the 175 amino acid residues of domain VI from about amino acid residues 221-262) of SEQ ID NO:1. Briefly the fusion protein was made by PCR amplification of nucleotides 302-785 of the cDNA encoding p4 using adapter primers and cloned in-frame into the NdeI and SacII sites of pET-15b (Novagen). The fusion protein construct was confirmed by restriction mapping and DNA sequencing. Expression of the fusion protein was induced and separated from E. coli proteins using reducing SDS-PAGE.

Bands corresponding to the fusion protein were excised from the gel, equilibrated and homogenized using Freud's adjuvant. The same fusion protein was also western blotted on nitrocellulose, dissolved in DMSO and used to immunize mice for monoclonal antibody production.

The polyclonal antisera raised in mice against the fusion protein reacted well with 04, as well as, p1 and p2 polypeptides.

Structural Analysis of the 04 subunit and the 14 splice variant The p4 subunit contains six domains, and a interruption and a signal peptide. The signal peptide and domain VI can be found, for example, at about amino acid residues 1-262 of SEQ ID NO:1. Domain V can be found, for example, at about amino acid residues 263- 535 of SEQ ID NO:1. Domains IV and III can be found, for example, at about amino acid residues 536-767 and 768-1178 of SEQ ID NO:1, respectively. Domain I can be found, for example, at about amino acid residues 1409-1761 of SEQ ID NO:1.

The p4 subunit (long form) is most similar in size and domain structure to laminin pi with an amino acid sequence identity of 42.5%. p4 retains the highest levels of amino acid identity with the other laminin P subunits in domains VI and V, and the lowest levels in domains I and II, as shown in Figure 6. Using the MulticoiTM program, it was determined that only domains I and II of 34 have a high probability of forming coiled coil structures.

Domains I and II of 14 look most similar to human p3. Both p4 and p3 are epithelial and the coiled coil structures in domains I and II dictate the a and y subunits with which the p WO 99/19348 PCT/US98/21391 26 subunits are associated. Thus, it is likely that 34 associates with aC3 and y2, as does the laminin p3 subunit.

The cDNA encoding the splice variant of p4 contains only the short arm of the 34 subunit, and is missing the EGF repeat of domain III, as shown in Figure 5. Thus, the 34 polypeptide encoded by the p4 c DNA splice variant is missing the coiled coil structures in domains I and II, rendering the short subunit unable to associate into a laminin heterotrimer.

PCR amplification of human genomic DNA suggest that the exon which encodes the alternative short form 3' untranslated region is located downstream from the carboxyl-most common exon, exon 23, and is splices out of the p4 subunit, long form, by exon skipping.

Analogs of y3 and 34 Analogs can differ from naturally occurring y3 or p4 in amino acid sequence or in ways that do not involve sequence, or both. Non-sequence modifications include in vivo or in vitro chemical derivatization ofy3 or p4. Non-sequence modifications include changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.

Preferred analogs include y3 or 04 (or biologically active fragments thereof) whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the y3 or p4 biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative substitutions can be taken from the table below.

WO 99/19348 WO 9919348PCTIUS98/2 1391 27 TABLE I CONSERVATIVE AMINO ACID REPLACEMENTS For Amino Acid Code Replace with any of Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homno-Arg, D-homo-Arg, Met, le, D- Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, f3-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Mrg, D-Arg, homo-Arg, D- Met, D-Met, le, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, le, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, or 5-phenyiproline, cis-3,4, or Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or L- 1 ________oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D- Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D- Met(O), Val, D-Val- Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine 1V D-Val, Leu, D-Leu, le, D-Ile, Met, D-Met Other analogs within the invention are those with modifications which increase peptide stability; such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: analogs that include residues other than naturally occurring L-amino acids, D-amnino acids or nonnaturally occurring or synthetic amino acids, 03 or 'y amino acids; and cyclic analogs.

Gen Therapy WO 99/19348 PCT/US98/21391 28 The gene constructs of the invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of a y3 or P 4 polypeptide. The invention features expression vectors for in vivo transfection and expression of a y3 or p4 polypeptide in particular cell types so as to reconstitute the function of, or alternatively, antagonize the function of y3 or p4 polypeptide in a cell in which that polypeptide is misexpressed. Expression constructs of y3 or p4 polypeptides, may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the 73 or p4 gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo.

A preferred approach for in vivo introduction of nucleic acid into a cell is.by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding a y3 or p4 polypeptide. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans.

These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.

(1990) Blood 76:271). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques.

Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include vCrip, pCre, V2 and 4 Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:.1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460- WO 99/19348 PCT/US98/21391 29 6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc.

Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993)J. Immunol. 150:4104-4115; U.S.

Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present invention utilizes adenovirusderived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155.

Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 d1324 or other strains of adenovirus Ad2, Ad3, Ad7 etc.) are known to those skilled in the art.

Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992) cited supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).

Yet another viral vector system useful for delivery of the subject gene is the adenoassociated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing as little as 300.base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.

A variety of nucleic acids have been introduced into different cell types using AAV vectors WO 99/19348 PCT/US98/21391 (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32- 39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.

268:3781-3790).

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of a y3 or 34 polypeptide in the tissue of an animal. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject y3 or p4 gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding a y3 or p4 polypeptide can be entrapped in liposomes bearing positive charges on their surface lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication W091/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic y3 or p4 gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.

For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by Stereotactic injection Chen et al. (1994) PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced in tact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

Transgenic Animals The invention includes transgenic animals which include cells (of that animal) which contain a y3 or p4 transgene and which preferably (though optionally) express (or misexpress) an endogenous or exogenous y3 or p4 gene in one or more cells in the animal.

WO 99/19348 PCT/US98/21391 31 The y3 or p4 transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs. In preferred embodiments, the expression of the transgene is restricted to specific subsets of cells, or tissues utilizing, for example, cis-acting sequences that control expression in the desired pattern. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns, to restrict production to the milk or other secreted product of the animal.

Production of Fragments and Analogs Generation of Fragments Fragments of a protein can be produced in several ways, recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments.

Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.

Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, peptides of 25 the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.

Generation of Analogs: Production of Altered DNA and Peptide Sequences by Random Methods Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein.) PCR Mutagenesis In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al., 1989, Technique 1:11-15). This is a very powerful and relatively rapid method of introducing random mutations. The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, by using a dGTP/dATP ratio of five and adding Mn 2 to the PCR reaction. The pool of amplified WO 99/19348 PCT/US98/21391 32 DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.

Saturation Mutagenesis Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., 1985, Science 229:242). This technique includes generation of mutations, by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.

Degenerate Oligonucleotides A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector.

The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 2 73-289; Itakura et al.

(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al.

(1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).

Generation of Analogs: Production of Altered DNA and Peptide Sequences by Directed Mutagenesis Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, by substituting first with conserved amino acids and then with more radical choices depending upon results achieved, deleting the target residue, or inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

Alanine Scanning Mutagenesis WO 99/19348 PCT/US98/21391 33 Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989). In aliiine scanning, a residue or group of target residues are identified charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.

Oligonucleotide-Mediated Mutagenesis Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, Adelman et al., (DNA 2:183, 1983).

Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least nucleotides in length are.used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the singlestranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl.Acad. Sci. USA, 5765[1978]).- Cassette Mutagenesis Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al. (Gene, 34:315[1985]). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist,they-may be generated using the above-described oligonucleotide-mediated mutagenesis method to introduce them at appropriate locations in the desired protein subunit WO 99/19348 PCT/US98/21391 34 DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette.

This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.

Combinatorial Mutagenesis Combinatorial mutagenesis can also be used to generate mutants. the amino acid sequences for a group of homologs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.

25 Primary High-Through-Put Methods for Screening Libraries of Peptide Fragments or Homologs Various techniques are known in the art for screening generated mutant gene products.

Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, in this case, binding to other laminin subunits, assembly into a trimeric laminin molecules, binding to natural ligands or substrates, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, by random mutagenesis techniques.

Two Hybrid Systems Two hybrid assays such as the system described above (as with the other screening methods described herein), can be used to identify fragments or analogs. These may include agonists, superagonists, and antagonists. (The subject protein and a protein it interacts with are used as the bait protein and fish proteins.) Display Libraries WO 99/19348 PCT/US98/21391 In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140). In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, receptors, can be used to detect homolog which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of a viral particle.

For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these phage can be applied to affinity matrices at concentrations well over 1013 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH 2 -terminal end of pII and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol.

Chem. 267:16007-16010; Griffiths et al. (1993) EMBOJ 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).

A common approach uses the maltose receptor ofE. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al. (1986) EMBO 5, 3029-3037).

Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, OmpA (Schorr et al. (1991) Vaccines 91, pp. 387-392), PhoE (Agterberg, et al. (1990) Gene 88, 37-45), and PAL (Fuchs et al. (1991) Bio/Tech 9, 1369-1372), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al.

(1989) Appl. Environ. Microbiol. 55, 984-993). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular WO 99/19348 PCT/US98/21391 36 environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion ofpeptides to the subunit protein flagellin offers a dense array of may peptides copies on the host cells (Kuwajima et al. (1988) Bio/Tech. 6, 1080-1083).

Surface proteins of other bacterial species have also served as peptide fusion partners.

Examples include the Staphylococcus protein A and the outer membrane protease IgA of Neisseria (Hansson et al. (1992) J. Bacteriol. 174, 4239-4245 and Klauser et al. (1990) EMBOJ. 9, 1991-1999).

In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein LacI to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89:1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3'end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced.

This fusion retains the natural ability of LacI to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the LacI-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids ineach cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide- DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort ofpeptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B. (Cull et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89- 1869) This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pII and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outwardextending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc. Natl. Acad. Sci.

U.S.A. 87, 6378-6382) A second difference is the set of biological biases affecting the population ofpeptides actually present in the libraries. The LacI fusion molecules are confined-to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the WO 99/19348 PCT/US98/21391 37 periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the LacI and phage libraries may differ significantly as a result of their exposure to different proteolytic activities.

The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al. (1994) J.

Med. Chem. 37(9):1233-1251). These particular biases are not a factor in the LacI display system.

The number of small peptides available in recombinant random libraries is enormous.

Libraries of 107-109 independent clones are routinely prepared. Libraries as large as 1011 recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.

In one application of this method (Gallop et al. (1994) J. Med. Chem. 37(9):1233- 1251), a molecular DNA library encoding 1012 decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ELISA, or for binding specificity in a completion phage ELISA (Barret, et al. (1992) Anal...Biochem 204,357-364). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.

Secondary Screens WO 99/19348 PCT/US98/21391 38 The high through-put assays described above can be followed by secondary screens in order to identify further biological activities which will, allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an assay can be developed in which the ability to inhibit an interaction between a protein of interest and its respective ligand can be used to identify antagonists from a group of peptide fragments isolated though one of the primary screens described above.

Therefore, methods for generating fragments and analogs and testing them for activity are known in the art. Once the core sequence of interest is identified, it is routine to perform for one skilled in the art to obtain analogs and fragments.

Peptide Mimetics The invention also provides for reduction of the protein binding domains of the subject y3 or p4 polypeptides to generate mimetics, e.g. peptide or non-peptide agents. See, for example, "Peptide inhibitors of human papillomavirus protein binding to retinoblastoma gene protein" European patent applications EP-412,762A and EP-B31,080A.

Non-hydrolyzable peptide analogs of critical residues can be generated using benzodiazepine see Freidinger et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine see Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al. (1986) JMed Chem 29:295; and Ewenson et al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, IL, 1985), p-tur dipeptide cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and p-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Communl26:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).

Antibodies The invention also includes antibodies specifically reactive with a subject y3 or P4 polypeptides. Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).

Antibodies which specifically bind y3 or 04 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of y 3 or p4.. Anti y3 or p4 antibodies can be used diagnostically in WO 99/19348 PCT/US98/21391 39 immuno-precipitation and immuno-blotting to detect and evaluate y3 or 34 levels in tissue or bodily fluid as part of a clinical testing procedure.

Another application of antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as Xgtl 1, gtl 8-23, XZAP, and XORF8. Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instance, Xgt 11 will produce fusion proteins whose amino termini consist of B-galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide. Antigenic epitopes of a subject polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with antibodies of the invention. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence ofhomologs can be detected and cloned from other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.

Other Embodiments Included in the invention are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high or low stringency conditions to a nucleic acid which encodes a polypeptide of SEQ ID NO:1 or SEQ ID NO:3 (for definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley Sons, New York, 1989, 6.3.1 6.3.6, hereby incorporated by reference); and, polypeptides specifically bound by antisera to y3 or p4.

Nucleic acids and polypeptides of the invention includes those that differ from the sequences discolosed herein by virtue of sequencing errors in the disclosed sequences.

The invention also includes fragments, preferably biologically active fragments, or analogs of y3 or p4. A biologically active fragment or analog is one having any in vivo or in vitro activity which is characteristic of they3 or p4 shown in SEQ ID NO:3 and SEQ ID NO:1, respectively, or of other naturally occurring y 3 or p4, one or more of the biological activities described above. Especially preferred are fragments which exist in vivo, fragments which arise from post transcriptional processing or which arise from translation of alternatively spliced RNA's. Fragments include those expressed in native or endogenous cells, as a result of post-translational processing, as the result of the removal of an amino-terminal signal sequence, as well as those made in expression systems, in CHO cells. Particularly preferred fragments are fragments, active fragments, which are generated by proteolytic cleavage or alternative splicing events.

WO 99/19348 PCT/US98/21391 Other embodiments are within the following claims.

What is claimed is: EDITORIAL NOTE-NO.10765/99 This specification contains a sequence description and is numbered listing following the as follows: Sequence listing pages 1 to 61 Claim pages 41 to 46 1/61 SEQUENCE LISTING <110> Burgeson, Robert Champliaud, Marie-France Olson, Pamela Koch, Manuel Brunken, William <120> LAMININS AND USES THEREOF <130> 10287/060001 <150> US 09/168,948 <151> 1998-10-09 <150> US 60/061,609 <151> 1997-10-10 <160> <170> FastSEQ for Windows Version <210> 1 <211> 1761 <212> PRT <213> Homo sapiens

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90 Gln Ala Pro Lys Ile 170 Lys Val1 Gln His Asp 250 Leu Ala Leu I le Arg 75 Asn Ser Leu Ala Val 155 Thr Tyr Leu Asp Thr 235 Gly Cys Met Leu Phe Val Glu Phe Ala 140 Phe Ser Ser Asp Leu 220 Leu Trp His Ala Ser Pro Thr Asn Arg 125 Met Lys Gly Asp Pro 205 Val Gly Leu Pro Ser Tyr Tyr Val Gly 110 Phe Leu Tyr Gln Ile 190 Ser Thr Asp Ser Thr Ser Leu Asp Ser Leu Ser Val Phe Ala 175 Glu Phe Leu Ala Tyr Thr Thr Glu Pro Phe Asp His Glu Ala 160 Gln Pro Glu Thr Leu 240 Leu Gly Arg Arg Gln.Asn Asp Ser Leu 245 Lys Tyr Tyr Tyr Ala Leu 255

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Asn Val Cys Ser ccc Pro 520 cag tgt Gin Cys 525 gaa tgc cgc cca Giu Cys Arg Pro cat His 530 gtc act ggc cgt Val Thr Gly Arg agc Ser 535 tgc tct gaa cca Cys Ser Giu Pro 1361 1409 1457 1505 1553 1601 1649 1697 1745 1793 1841 1889 1937 1985 2033 9 9 9 9 9.

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I L G 17/61 agc cgc gcc gac Ser Arg Ala Asp ggc Gly 155 cca tgg gag ccc Pro Trp Glu Pro cag ttc tac agc Gin Phe Tyr Ser gcc tcc Ala Ser 165 595 tgc cag aag Cys Gin Lys gag gac gag Glu Asp Glu 185 tac ggc cgg ccc Tyr Gly Arg Pro gag Glu 175 ggc cag tac ctg Gly Gin Tyr Leu cgc ccc ggc Arg Pro Gly 180 gac atc tcc Asp Ile Ser cgc gtg gcc ttc Arg Val Ala Phe acc tct gag ttc Thr Ser Glu Phe agc Ser 195 ccg ctg Pro Leu 200 agt ggc ggc aac Ser Gly Gly Asn gtg Val1 205 gcc ttc tcc acc Ala Phe Ser Thr c tg Leu 210 gag ggc cgg ccc Giu Gly Arg Pro agc Ser 215 gcc tac aac ttc Ala Tyr Asn Phe gag agc cct ggg Giu Ser Pro Gly cag gag tgg gtc Gin Giu Trp Val acc Thr 230 agc acc gaa ctc Ser Thr Glu Leu ctc Leu 235 atc tct cta gac Ile Ser Leu Asp cgg Arg 240 ctc aac acg ttt Leu Asn Thr Phe ggg gac Gly Asp 245 gac atc ttc Asp Ile Phe tcc gac ttc Ser Asp Phe 265 aag Lys 250 gac ccc aag gtg Asp Pro Lys Val cag tcc tac tat Gin Ser Tyr Tyr tat gcc gtg Tyr Ala Val 260 cat gcc agc His Ala Ser tct gtg ggc ggc Ser Val Gly Gly agg Arg 270 tgc aag tgc aac Cys Lys Cys Asn ggg Gly 275 gag tgc Giu Cys 280 ggc ccc gac gtg Gly Pro Asp Val gca Ala 285 ggc cag ttg gcc Gly Gin Leu Ala cgg tgc cag cac Arg Cys Gin His a.

C.

aac Asn 295 acc acc ggc aca Thr Thr Gly Thr tgt gag cgc tgc Cys Giu Arg Cys ctg Leu 305 ccc ttc ttc cag Pro Phe Phe Gin cgc ccg tgg gcc Arg Pro Trp Ala cgg Arg 315 ggc acc gcc gag Gly Thr Ala Glu gcc cac gag tgt Ala His Giu Cys ctg ccc Leu Pro 325 tgc aac tgc Cys Asn Cys ttc cgc agc Phe Arg Ser 345 ggc cgc tcc gag Gly Arg Ser Glu gaa Glu 335 tgc acg ttt gat Cys Thr Phe Asp cgg gag ctc Arg Giu Leu 340 cgt gac cac Arg Asp His 979 1027 1075 1123 1171 1219 1267 aca ggc cac ggc Thr Gly His Gly ggg Gly 350 cgc tgt cac cac Arg Cys His His tgc Cys 355 aca gct Thr Ala 360 ggg cca cac tgt Gly Pro His Cys gag Glu 365 cgc tgt cag gag Arg Cys Gin Glu ttc tat cac tgg Phe Tyr His Trp gac Asp 375 ccg cgg atg cca Pro Arg Met Pro tgc Cys 380 cag ccc tgt gac Gin Pro Cys Asp tgc Cys 385 cag tcg gca ggc Gin Ser Ala Gly tcc Ser 390 (3'V LC 18/61 cta cac ctc cag Leu His Leu Gin tgc gat gac aca ggc acc tgc gcc tgc aag ccc aca Cys Asp Asp Thr Gly Thr Cys Ala Cys Lys Pro Thr 395 400 405 gtg act ggc Val Thr Gly agt gag gga Ser Giu Gly 425 aag tgt gac cgc Lys Cys Asp Arg tgt Cys 415 ctg coo ggg ttc Leu Pro Gly Phe cac tcg ctc His Ser Leu 420 ggc agc ctg Gly Ser Leu ggc tgo aga ccc Gly Cys Arg Pro act tgo aat ccc Thr Cys Asn Pro got Ala 435 gac acc Asp Thr 440 tgt gac ccc cgo Cys Asp Pro Arg agt Ser 445 ggg cgc tgc ccc Gly Arg Cys Pro aaa gag aat gtg Lys Giu Asn Val gaa Giu 455 ggc aac ota tgt Gly Asn Leu Cys gao Asp 460 aga tgt ogo cog Arg Cys Arg Pro ggg Gly 465 aco ttt aac ctg Thr Phe Asn Leu coo cac aat oca Pro His Asn Pro ggc tgo ago ago Gly Cys Ser Ser ttc tgo tat ggc cac too Phe Cys Tyr Gly ffis Ser 485 aag gtg tgo Lys Val Cys gat ttc cac Asp Phe His 505 gog Ala 490 too act goc cag Ser Thr Ala Gin ttc Phe 495 cag gtg cat cac Gin Val His His ato oto ago Ile Leu Ser 500 cag gga goc gaa Gin Gly Ala Giu ggc tgg tgg goc aga agt gtg ggg ggc Gly Trp Trp Ala Arg Ser Val Gly Gly 510 515 1315 1363 1411 1459 1507 1555 1603 1651 1699 1747 1795 1843 1891 1939 1987 tot gag Ser Giu 520 cac too oca oaa His Ser Pro Gin tgg Trp 525 ago oca aat ggg Ser Pro Asn Gly gto Val1 530 oto ctg ago oca Leu Leu Ser Pro 9* 9* a *a.

a gao gag gag gag Asp Giu Giu Giu aca gca oca. ggg Thr Ala Pro Gly aag Lys 54 5 tto ctg gga gao Phe Leu Gly Asp cag Gin 550 cgg tto ago tat Arg Phe Ser Tyr ggg Gly 555 cag coo oto ata Gin Pro Leu Ile otg Leu 560 aco tto cgg gtg Thr Phe Arg Val coo co Pro Pro 565 ggg gao too Gly Asp Ser goc otg too Ala Leu Ser 585 oca Pro 570 oto cot gta cag Leu Pro Val Gin agg otg gaa ggg Arg Leu Giu Gly aca ggo ttg Thr Gly Leu 580 gat goc agg Asp Ala Arg 00a otg agg cac tot Leu Arg His Ser ago Ser 590 ctg tot ggc ccc Leu Ser Gly Pro gca too Ala Ser 600 cag gga ggt aga Gin Gly Gly Arg cag gtt oca. otg Gin Val Pro Leu cag Gin 610 gag aco too gag Giu Thr Ser Giu gao Asp 615 gtg goc cot oca Val Ala Pro Pro ccc ccc tto cac Pro Pro Phe His cag cgg oto oto Gin Arg Leu Leu

N

(9 ID Li; Li 0, /~1TE 19/61 aac ctg acc agc Asn Leu Thr Ser cgc ctc cgc gtc Arg Leu Arg Val agt Ser 640 ccc ggc ccc agc Pro Gly Pro Ser cct gcc Pro Ala 645 ggt cca gtg Gly Pro Val ctt tcc ccg Leu Ser Pro 665 ttc Phe 650 ctg act gag gtc Leu Thr Giu Val ctc aca tcc gcc Leu Thr Ser Ala cgg cca ggg Arg Pro Gly 660 ccc act ggc Pro Thr Gly cca gcc tcc tgg Pro Ala Ser Trp gtg Va1 670 gag att tgt tca Glu Ile Cys Ser tgt Cys 675 tac acg Tyr Thr 680 ggc cag ttc tgt Gly Gin Phe Cys tcc tgt gct ccg Ser Cys Ala Pro gga Gly 690 tac aag agg gag Tyr Lys Arg Glu atg Met 695 cca cag ggg ggt Pro Gin Gly Gly tat gcc agc tgt Tyr Ala Ser Cys gtc Val 705 ccc tgc acc tgt Pro Cys Thr Cys cag cat ggc acc Gin His Gly Thr tgt Cys 715 gac ccc aac aca Asp Pro Asn Thr atc tgt gtc tgc Ile Cys Val Cys agc cac Ser His 725 cat acc gag His Thr Glu aac cct ttc Asn Pro Phe 745 ggc Gly 730 cca tcc tgt gaa Pro Ser Cys Glu cgc Arg 735 tgt ttg cca ggt Cys Leu Pro Gly ttc tat ggc Phe Tyr Gly 740 ccc tgc cct Pro Cys Pro gcg ggc caa gcc Ala Gly Gin Ala gac Asp 750 gac tgc cag ccc Asp Cys Gin Pro tgt Cys 755 2035 2083 2131 2179 2227 2275 2323 2371 2419 2467 2515 2563 2611 2659 2707 ggc cag Gly Gin 760 tcg gcc tgt acg Ser Ala Cys Thr atc cca gag agc Ile Pro Glu Ser ggg Gly 770 gag gtg gtg tgt Glu Val Val Cys a acc Thr 775 cac tgc ccc ccg His Cys Pro Pro ggc Gly 780 cag aga ggg cgg Gin Arg Gly Arg cgc Arg 785 tgt gag gtc tgt Cys Giu Val Cys gat Asp 790 gat ggc ttt ttt Asp Gly Phe Phe ggg Gly 795 gac ccg ctg ggg Asp Pro Leu Gly ctc Leu 800 ttt ggg cac ccc Phe Gly His Pro cag ccc Gin Pro 805 tgc cac cag tgc cag tgt agc ggg aac gtg gac ccc aat gcc gtg ggc Cys His Gin Cys Gin Cys Ser Gly Asn Val Asp Pro Asn Ala Val Gly 810 815 820 a a a a a sa a.

a. a a a a.

aac tgt gac Asn Cys Asp 825 ccc ctg tct ggc Pro Leu Ser Gly tgc ctg cgc tgc Cys Leu Arg Cys cac aac acc His Asn Thr acg ggt Thr Gly 840 gac cac tgt gag Asp His Cys Glu cac His 845 tgt cag gaa ggc Cys Gin Glu Gly ttc Phe 850 tac ggg agc gcc Tyr Gly Ser Ala ctg Leu 855 gcc cct cga ccc Ala Pro Arg Pro gca Ala 860 gac aaa tgc atg Asp Lys Cys Met tgc agc tgt cac Cys Ser Cys His cca Pro 870

AV

Z__ Lj :iI 20/61 cag ggc tcg gtc Gin Gly Ser Val gag cag atg ccc Glu Gin Met Pro tgc Cys 880 gac cca gtg aca Asp Pro Val Thr ggc caa Gly Gin 885 tgc tcc tgc Cys Ser Cys cct ggc ttc Pro Gly Phe 905 c tg Leu 890 cct cat gtg act Pro His Val Thr gca Ala 895 cgg gac tgc agc Arg Asp Cys Ser cgc tgc tac Arg Cys Tyr 900 agc tgc aag Ser Cys Lys ttc gac ctc cag Phe Asp Leu Gin ggg agg ggc tgc Gly Arg Gly Cys c gg Arg 915 tgt cac Cys His 920 cca ctg ggc tcc Pro Leu Gly Ser gag gac cag tgc Giu Asp Gin Cys cat His 930 ccc aag act gga Pro Lys Thr Gly cag Gin 935 tgc acc tgc cgc Cys Thr Cys Arg cca Pro 940 ggt gtc aca ggc Gly Val Thr Gly cag Gin 945 gcc tgt gac agg Ala Cys Asp Arg cag ctg ggt ttc Gin Leu Gly Phe ggc tcc tca atc Gly Ser Ser Ile aag Lys 960 ggc tgc cgg gcc Gly Cys Arg Ala tgc agg -Gtys Arg 965 tgc tcc cca Cys Ser Pro ctg Leu 970 ggc gct gcc tcg Gly Ala Ala Ser gcc Ala 975 cag tgc cac tat Gin Cys His Tyr aac ggc aca Asn Gly Thr 980 cgc tgc cac Arg Cys His tgc gtg tgc agg Cys Val Cys Arg 985 cct ggc ttc Pro Gly Phe gag ggc Giu Gly 990 tac aaa tgt Tyr Lys Cys gac Asp 99E 2755 2803 2851 2899 2947 2995 3043 3091 3139 3187 3235 3283 3331 3379 3427 tac aac ttc Tyr Asn Phe 1000 ttc ctc acg Phe Leu Thr gca gac Ala Asp 1005 ggc aca cac Gly Thr His tgc cag caa tgt ccg Cys Gin Gin Cys Pro 1010 te a a a a.

a. a a a a a a a a tcc tgc Ser Cys 1015 tac gcc ctg Tyr Ala Leu gtg aag Val Lys 1020 gag gag aca Giu Giu Thr gcc aag Ala Lys 1025 ctg aag gcc Leu Lys Ala aga Arg 1030 ctg act ttg acg Leu Thr Leu Thr gag ggg Giu Gly 1035 tgg ctc caa Trp Leu Gin ggg tcc gac tgt ggc Gly Ser Asp Cys Gly 1040 agt ccc Ser Pro 1045 tgg gga cca cta gac att ctg Trp Gly Pro Leu Asp Ile Leu 1050 tac cag ggc cat cac ctg ctt Tyr Gin Gly His His Leu Leu 1065 ctg gga gag Leu Gly Giu 1055 gcc cca agg ggg gac gtc Ala Pro Arg Gly Asp Val 1060 cca ggg Pro Gly 1070 gct cgg gaa Ala Arg Glu gcc ttc ctg gag Ala Phe Leu Giu 1075 cag atg atg ggc ctc Gin Met Met Gly Leu 1080 cag agg ctg aac aag Gin Arg Leu Asn Lys 1095 gag ggt gct gtc aag Giu Gly Ala Val Lys 1085 ggt gcc cgc tgt gcc Gly Ala Arg Cys Ala 1100 gcc gcc cgg gag cag ctg Ala Ala Arg Giu Gin Leu 1090 cag gcc gga tcc cag Gin Ala Gly Ser Gin 1105 aag Lys 1110 7

C-

1'

C

21/61 acc tgc acc Thr Cys Thr gag gag att Glu Glu Ile cag ctg gca Gin Leu Ala 1115 gac ctg gag gca gtg Asp Leu Glu Ala Val 1120 ctg gag tcc tcg gaa Leu Giu Ser Ser Glu 1125 ctg cat Leu His 1130 gca gct gcc att ctc Ala Ala Ala Ile Leu 1135 gcg tct ctg gag att cct Ala Ser Leu Giu Ile Pro 1140 agc cac ctg gcc ata gag Ser His Leu Ala Ile Glu 1155 cag gaa ggt ccc Gin Giu Gly Pro 1145 agt cag ccg Ser Gin Pro acc aaa Thr Lys 1150 gcc cgt gcc Ala Arg Ala 1160 ctc gcc agg Leu Ala Arg agc cac Ser His 1165 aga gac acc Arg Asp Thr gcc acc Ala Thr 1170 aag atc gca Lys Ile Ala gcc act Ala Thr 1175 gct tgg agg Ala Trp Arg gcc ctg Ala Leu 1180 ctc gcc tcc Leu Ala Ser aac acc Asn Thr 1185 agc tac gcg Ser Tyr Ala ctt Leu 1190 ctc tgg aat ctg Leu Trp Asn Leu ctg gag Leu Glu 1195 gga agg gtg Gly Arg Val gcc cta Ala Leu 1200 gag acc cag Glu Thr Gln cgg gac Arg Asp 1205 ctg gag gac Leu Glu Asp agg tac Arg Tyr 1210 cag gag gtc Gin Glu Val cag gcg Gin Ala 1215 gcc cag aaa Ala Gin Lys gca ctg agg Ala Leu Arg 1220 9 9 acg gct gtg gca Thr Ala Val Ala 1225 gtg cag caa gtt Val Gin Gin Val 1240 tcc ccg gga gct Ser Pro Gly Ala 1255 aag gcg aag gcc Lys Ala Lys Ala gag gtg ctg Glu Val Leu cct gaa Pro Glu 1230 gcg gaa agc Ala Giu Ser gtg ttg gcc acc Val Leu Ala Thr 1235 ggc gca Gly Ala gat aca Asp Thr 1245 gcc ccg tac Ala Pro Tyr ctg gcc Leu Ala 1250 ttg ctg gct Leu Leu Ala ctg ggc ctg Leu Gly Leu 1270 3475 3523 3571 3619 3667 3715 3763 3811 3859 3907 3955 4003 4051 4099 4147 ctg cct cag aag Leu Pro Gin Lys 1260 tcc cgg gct gaa gac Ser Arg Ala Glu Asp 1265 ctg gag Leu Glu 1275 aag aca gtt Lys Thr Val gca tca Ala Ser 1280 tgg cag cac Trp Gin His atg gcc Met Ala 1285 act gag gct Thr Glu Ala gcc cga Ala Arg 1290 acc ctc cag Thr Leu Gin act gct Thr Ala 1295 gcc cag gcg Ala Gin Ala acg cta cgg Thr Leu Arg 1300 99*9 9 9. **99 9 .9 99 9 9.

99 caa aca gaa ccc ctc Gin Thr Giu Pro Leu 1305 gcc tcc cag ctg cac Ala Ser Gin Leu His 1320 tca tct gtc cag gct Ser Ser Val Gin Ala 1335 aca atg gcg cga tct Thr Met Ala Arg Ser 1310 cag ggg gcc aga gcc Gin Gly Ala Arg Ala 1325 gcg aca gtg act gtc Ala Thr Val Thr Val 1340 cgg ctc act gca acc ttt Arg Leu Thr Ala Thr Phe 1315 gcc ctg acc cag gct tcc Ala Leu Thr Gin Ala Ser 1330 atg gga gcc agg act Met Gly Ala Arg Thr 1345 ctg Leu 1350 1' id C 2a~ 22/61 ctg got gat ctg gaa gga Leu Ala Asp Leu Giu Gly 1355 atg aag ctg cag ttt ccc cgg ccc aag gac Met Lys Leu Gin Phe Pro Arg Pro Lys Asp 1360 1365 cag gog gca ttg cag Gin Ala Ala Leu Gin 1370 agg aag goa gac tco Arg Lys Ala Asp Ser 1375 gto agt gac aga ctc ctt Val Ser Asp Arg Leu Leu 1380 gca gao aog aga Ala Asp Thr Arg 1385 aag aag acc Lys Lys Thr aag cag Lys Gin 1390 gcg gag agg Ala Glu Arg atg otg gga aac Met Leu Gly Asn 1395 gcg gcc cct Ala Ala Pro 1400 ott tc tc Leu Ser Ser agt gcc Ser Ala 1405 aag aag aag Lys Lys Lys ggo aga Gly Arg 1410 gaa gca gag Glu Ala Glu gtg ttg Val Leu 1415 goc aag gao Ala Lys Asp agt gc Ser Ala 1420 aag ctt gc Lys Leu Ala aag gcc Lys Ala 1425 ttg otg agg Leu Leu Arg gag Glu 1430 cgg aaa cag gcg Arg Lys Gin Ala cac cgc His Arg 1435 cgt gc ago agg otc acc ago cag acg caa Arg Ala Ser Arg Leu Thr Ser Gin iThr Gin 1440 1445 gc aog otc Ala Thr Leu caa cag gcg Gin Gin Ala 1450 too cag cag gtg otg Ser Gin Gin Val Leu 1455 gcg tot gaa goa cgc Ala Ser Giu Ala Arg 1460 aga oag gag otg Arg Gin Glu Leu 1465 gag gaa got Glu Glu Ala gag ogg Glu Arg 1470 gtg ggt got Val Gly Ala ggg otg ago gag Gly Leu Ser Glu 1475 4195 4243 4291 4339 4387 4435 4483 4531 4579 4627 4675 4723 4771 4819 4868 4928 4988 atg gag oag Met Glu Gin 1480 oag ato ogg Gin Ile Arg gaa tcg Glu Ser 1485 ogt ato toa Arg Ile Ser otg gag Leu Glu 1490 aag gao ato Lys Asp Ile 9 9 9 9 gag aoo Glu Thr 1495 ttg toa gag Leu Ser Glu otg ott Leu Leu 1500 goo agg otg Ala Arg Leu ggg tcg Gly Ser 1505 otg gao aoo Leu Asp Thr oat His 1510 oaa goo cca goo Gin Ala Pro Ala oag goo Gin Ala 1515 otg aao gag Leu Asn Glu act oag Thr Gin 1520 tgg goa ota Trp Ala Leu gaa cgc Glu Arg 1525 otg agg otg Leu Arg Leu oag otg Gin Leu 1530 ggo too ccg Gly Ser Pro ggg too Gly Ser 1535 ttg oag agg Leu Gin Arg aaa otc agt Lys Leu *Ser 1540 9 9 0 9*99 9. 9 .9 9 9 otg otg gag oag gaa Leu Leu Giu Gin Glu 1545 too cag oag oag gag Ser Gin Gin Gin Glu 1550 otg oag ato cag ggo tto Leu Gin Ile Gin Gly Phe 1555 gag agt gao Glu Ser Asp 1560 att otg cac Ile Leu His 1575 otc goo gag Leu Ala Glu ato cgo Ile Arg 1565 goo gao aaa Ala Asp Lys oag aao Gin Asn 1570 ctg gag gcc Leu Giu Ala tgagggctgc ago ctg cc gag aao Ser Leu Pro Giu Asn 1580 tgt goo ago tgg oag Cys Ala Ser Trp Gin 1585 ccagatccc ggcacacact cccccacctg otgtttaoat gacccagggg gtgcacacta ccccacaggt gtgcccatao agacattccc cggagccggo tgotgtgaao togaccccgt 23/61 gtggatagtc acactccctg ccgattctgt ctgtggcttc ttccctgcca gcaggactga gtgtgcgtac ccagttcacc tggacatgag tgcacactct cacccctgca catgcataaa cgggcacacc ccagtgtcaa taacatacac acgtgagggt gcatgtctgt gtgtatgacc caaataaaaa aaaaaa <210> <211> 1101 <212> PRT <213> Homo sapiens 5048 5108 5168 5184 <400> Met Ser Gly Cys Gly Tyr Giu His Leu Arg 145 Lys Gly Ser Ile Asn 225 Leu Tyr Giu Gly Asn 305 Ala His Gly Gin 9 S

S

S S

S.

5

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S

a r *54*

S

S

a..

S.

a S 0 4 a

S.

5 S S S 0* *5 S 55 a Gin Lys Asp Gly Glu Asp Pro Vai Ilie 130 Ser Asp Val1 Thr Giu 210 Leu Gly Giu Cys Met 290 Cys Ala Ser Leu His 370 Phe Ala Leu Leu Gin Gin Asp Ser 115 Leu Thr Cys Gly Gly 195 Asn Arg Arg Met Arg 275 Val1 Giu Asp Ser Ser 355 Cys Gin Gin Leu Ser Lys Pro Arg 100 Ile Thr Asp Ala Asp 180 Gly Pro Ile Arg Ile 260 Pro His Arg Leu Arg 340 Gly Asp Leu Asp Val1 Arg Cys Asn Giu Arg Phe Tyr Thr 165 Ile Giu Tyr Asn Gin 245 Val Met Gly Cys Gin 325 Cys Giy Arg Thr Asp Gly Ala Ser 70 Ser Lys Leu Lys Gly 150 Ser Val1 Val1 Ser Phe 230 Asn Arg Gin Gin Lys 310 Asp His Val1 Cys Leu Phe Cys Asn Arg Asn 40 Gin Lys 55 Ile Cys His Thr Lys Trp Asp Leu 120 Thr Phe 135 His Asn Phe Pro Cys Asp Val Leu 200 Pro Tyr 215 Thr Lys Asp Ser Giy Ser Lys Met 280 Cys Vai 295 Asp Phe Asn Aia Phe Asp Cys Giu 360 Arg Pro 375 Ala Cys Leu His 10 Arg Giy 25 Thr Gin Tyr Cys Asp Ser Ile Glu 90 Trp Gin 105 Giu Ala Arg Pro Trp Lys Asn Ile 170 Ser Lys 185 Lys Vai Ile Gin Leu His Leu Asp 250 Cys Phe 265 Arg Gly Cys Gin Phe Gin Cys Arg 330 Met Thr 345 Asp Cys Leu Phe Ile Pro Leu Ala Leu Ile Arg 75 Asn Ser Leu Aia Val1 i55 Thr Tyr Leu Asp Thr 235 Lys Cys Asp His Asp 315 Ser Thr Gin Tyr Cys 395 Gly Trp Cys His Met Ala Leu Ser Phe Pro Val Thr Giu Asn Phe Arg 125 Ala Met 140 Phe Lys Ser Gly Ser Asp Asp Pro 205 Leu Vai 220 Leu Giy Tyr Tyr Asn Giy Vai Phe 285 Asn Thr 300 Aia Pro Cys Ser Tyr Leu His Asn 365 Arg Asp 380 Giu Cys Leu Pro Ser Tyr Tyr Val Giy 110 Phe Leu Tyr Gin Ile Ser Thr Asp Tyr His 270 Ser Asp Trp Cys Aia 350 Thr Pro Asp Ser Tyr is Thr Thr Ser Thr Leu Giu Asp Pro Ser Phe Leu Asp Ser His Val Giu Phe Ala 160 Ala Gin 175 Giu Pro Phe Giu Leu Thr Ala Leu 240 Ala Leu 255 Aia Ser Pro Pro Gly Pro Arg Pro 320 Asn Ser 335 Ser Gly Giu Gly Leu Lys Pro Asp 400 Thr Ile Ser Asp Pro Tyr

A

24/61 Gly Gly Lys Pro Phe 465 Val1 Giy Ala Pro Phe 545 Leu Ala Trp Phe His 625 Asn Lys Pro Tyr Leu 705 Phe Giu Arg Cys Giy 785 Arg Cys Thr Arg Cys 865 Asn Thr Ser Cys Leu 450 Leu Thr Asn Tyr His 530 Ala Gin Val Thr Ala 610 Tyr Pro Pro Thr Phe 690 Val1 Cys Ile Leu Lys 770 Gly Cys His Giy Cys 850 Asn Ile Val1 Asp 435 Gly Thr Giy His Ser 515 Val1 Pro Gly His Giy 595 Val1 Giu Pro Gin Pro 675 Ser Asp Ser Aia Ile 755 Cys Gin Ser Cys Gin 835 Leu Arg Ser Aia 420 Gin Cys Cys Ala Leu 500 Asn Thr Leu Leu Vali 580 Pro Asn Thr Giy Her 660 Ile Gin Her Lys Her 740 Ile His Cys Thr His 820 Cys Ala Phe Gly 405 Gly Cys Gin Asp His 485 His Val Giy Asn Ala 565 Val1 Giy Asn Gin Giy 645 Phe Cys Pro Leu Gin 725 Ala Ser Pro Gin Giy 805 Pro Pro Gly Ala Phe 885 Gly Ile Cys Val Ser Gin Cys Lys Pro Pro Cys 455 Vai Asp 470 Cys Glu Gly Cys Cys Ser Arg Ser 535 Phe Tyr 550 Pro Leu Leu Gly Phe Ala Ile Pro 615 Ser Aia 630 Her Glu Ala Leu Leu Giu Leu Gin 695 Giy Leu 710 Asp Leu Met Gly Met Her Gin Gly 775 Cys Lys 790 Her Tyr Gin Giy Cys His Tyr Phe 855 Giu Leu 870 Leu Asn 440 Asp Thr Giu Her Pro 520 Cys Leu Gly Giu Arg 600 Phe Aia His Pro Pro 680 Gly Ile Asp Pro Ala 760 Her Pro Asp Her Gly 840 Gly Cys Cys 425 His Cys Gly Cys Pro 505 Lys Her Tyr Her Pro 585 Val Pro Asp Cys Aia 665 Asp Giu Pro Giu Gin 745 Lys Val1 Leu Leu Lys 825 Giu Phe Asp Lys Tyr Asn Gin Thr 490 Cys Asn Giu Glu Giu 570 Vali Leu Val1 Trp Ile 650 Ala Val Ser Gin Tyr 730 Vai Leu Giy Vai Gly 810 Asp Val1 Pro Pro His Giu Gly Pro Cys 475 Val Asp Giy Pro Aia 555 Thr Pro Pro Asp Thr 635 Pro Thr Gin His Ile 715 Gin Leu His Her Val1 795 His Thr Her Ser Giu 875 Her Asn Leu Leu 460 Leu Giy Cys Gin Ala 540 Giu Phe Giy Giy Phe 620 Vai Lys Arg Tyr Ala 700 Asn Leu Pro Asp Her 780 Gly His Val1 Gly Cys 860 Thr Asp Val1 Her 445 Gly Cys Tyr Asp Cys 525 Pro Giu Gly Asn Aia 605 Thr Gin Thr Ile Her 685 His Her His Giy Giy 765 Cys Arg Giy Cys Arg 845 His Gly Pro Giu 430 Aia Her Leu Trp Ile 510 Giu Gly Ala Gin Pro 590 Gly Ile Ile Leu Met 670 Ile Her Leu Asn Ala 750 Ala Her Cys Cys Asp 830 Arg Pro Her Ala Leu 415 Gly Ala Thr Asp Leu Pro Her Tyr 480 Gly Leu 495 Gly Gly Cys Arg Tyr Phe Thr Thr 560 Her Pro -5-75 Vai Thr Leu Arg Aia Ile Vai Vai 640 Gin Her 655 Leu Leu Asp Vai His Val Giu Asn 720 Cys Val 735 Cys Giu Val Ala Arg Leu Cys Asp 800 His Pro 815 Gin Val Cys Asp Cys Pro Cys Phe 880 Ile Asp 895 .00 0 ego a 00 S0i 0 0 Cys Gly Giy Thr Thr Gly Arg Asn Cys Giu Arg Cys 890 ~Aii 0;

,C)

1 Gly Tyr Tyr Cys Pro Asp 915 Gin Asn Leu 930 Thr Gly Thr 945 Arg Ile Ser Asp Val Thr Arg Cys Leu 995 Gly His Tyr 1010 His Ala Ser 1025 Cys Leu Cys Thr Gly Leu Pro Gly Arg 1075 25/61 Gly Asn Pro Ser Ser Gly Gin Pro Cys Arg Pro Cys Leu 900 905 910 Asp Pro Ser Ser Asn Gin Tyr Phe Ala His Ser Cys Tyr 920 925 Trp Ser Ser Asp Val Ile Cys Asn Cys Leu Gin Gly Tyr 935 940 Gin Cys Gly Glu Cys Ser Thr Gly Phe Tyr Gly Asn Pro 950 955 960 Gly Ala Pro Cys Gin Pro Cys Ala Cys Asn Asn Asn Ile 965 970 975 Asp Pro Glu Ser Cys Ser Arg Val Thr Gly Glu Cys Leu 980 985 990 His Asn Thr Gin Gly Ala Asn Cys Gin Leu Cys Lys Pro 1000 1005 Gly Ser Ala Leu Asn Gin Thr Cys Arg Arg Cys Ser Cys 1015 1020 Gly Val Ser Pro Met Glu Cys Pro Pro Gly Gly Gly Ala 1030 1035 1040 Asp Pro Val Thr Gly Ala Cys Pro Cys Leu Pro Asn Val 1045 1050 1055 Ala Cys Asp Arg Cys Ala Asp Gly Tyr Trp Asn Leu Val 1060 1065 1070 Gly Cys Gin Ser Cys Asp Cys Asp Pro Arg Thr Ser Gin 1080 1085 a.

Ser Ser His Cys Asp Gin Ala Arg Tyr Phe Lys Ala Tyr 1090 1095 1100 <210> 6 <211> 3754 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (81)...(3383) <400> 6 acatgccccg tttgctgcct gaacctctcc acaaagactc ccagatcctg aattgaattt aatcatctcc tgacaaaaga atg caa ttt caa ctg acc ctt ttt ttg cac ctt Met Gin Phe Gin Leu Thr Leu Phe Leu His Leu 1 5 ggg tgg ctc agt tac tea aaa get caa gat gac tgc aac agg ggt gcc Gly Trp Leu Ser Tyr Ser Lys Ala Gin Asp Asp Cys Asn Arg Gly Ala 15 20 tgt cat ccc acc act ggt gat ctc ctg gtg ggc agg aac acg cag ctt Cys His Pro Thr Thr Gly Asp Leu Leu Val Gly Arg Asn Thr Gin Leu 35 atg get tct tct acc tgt ggg ctg age aga gcc cag aaa tac tgc atc Met Ala Ser Ser Thr Cys Gly Leu Ser Arg Ala Gin Lys Tyr Cys Ile 50 ctc agt tac ctg gag ggg gaa caa aaa tgc tcc ate tgt gac tct aga Leu Ser Tyr Leu Glu Gly Glu Gin Lys Cys Ser Ile Cys Asp Ser Arg 65 70 ttt cca tat gat ccg tat gac caa ccc aac age cac acc att gag aat Phe Pro Tyr Asp Pro Tyr Asp Gin Pro Asn Ser His Thr Ile Glu Asn 85 7Ah 0 26/61 gtc act gta Val Thr Val gaa aat ggt Glu Asn Gly 110 ttt gaa cca gac Phe Giu Pro Asp aga Arg 100 gaa aag aaa tgg Glu Lys Lys Trp tgg caa tct Trp Gin Ser 105 gag gca tta Glu Ala Leu ctt gat oat gto Leu Asp His Val atc aga otg gac Ile Arg Leu Asp tta Leu 120 401 449 497 545 ttt cgg Phe Arg 125 tto agc cac ctt Phe Ser His Leu ctg aco ttt aag Leu Thr Phe Lys ttt cgg cot got Phe Arg Pro Ala gca Ala 140 atg tta gtt gaa Met Leu Val Glu ogt Arg 145 too aca gao tat Ser Thr Asp Tyr gga Gly 150 cac aac tgg aaa His Asn Trp Lys tto aaa tat ttt Phe Lys Tyr Phe aaa gao tgt gc Lys Asp Cys Ala act Thr 165 too ttt cot aac Ser Phe Pro Asn ato aca Ile Thr 170 tct ggo cag Ser Gly Gin tcg gat att Ser Asp Ile 190 go Ala 175 cag gga gtg gga Gin Gly Val Gly gao Asp 180 att gtt tgt gao Ile Val Cys Asp too aaa tao Ser -bys Tyr 185 aaa gtt ttg Lys Val Leu gaa ccc toa aca Glu Pro Ser Thr gga gag gtt gtt Gly Giu Val Val tta Leu 200 gat ccc Asp Pro 205 agt ttt gaa att Ser Phe Giu Ile gaa Glu 210 aao cot tat ago Asn Pro Tyr Ser cc Pro 215 tao ato caa gao Tyr Ile Gin Asp ott Leu 220 gtg aca ttg aca Val Thr Leu Thr otg agg ata aac Leu Arg Ile Asn ttt Phe 230 aco aag otc cac Thr Lys Leu His aco Thr 235 ott ggg gat got Leu Gly Asp Ala ttg Leu 240 ott gga agg agg Leu Gly Arg Arg oaa Gin 245 aat gat too ctt Asn Asp Ser Leu gat aaa Asp Lys 250 9* 99 9 9* 9 tao tao tat Tyr Tyr Tyr aat ggo cat Asn Gly His 270 otg tao gag atg Leu Tyr Glu Met att Ile 260 gtt cgg gga ago Val Arg Gly Ser tgo ttt tgo Cys Phe Cys 265 cgg gga gat Arg Gly Asp got ago gaa tgt Ala Ser Giu Cys cgo Arg 275 cot atg cag aag Pro Met Gin Lys atg Met 280 gtt tto Val Phe 285 ago cot cot gga Ser Pro Pro Gly gtt cac ggt cag Val His Gly Gin gtg tgt cag cac Val Cys Gin His aat Asn 300 aca gat ggt ccg Thr Asp Gly Pro aac Asn 305 tgt gag aga tgc Cys Giu Arg Cys aag Lys 310 gao tto tto cag Asp Phe Phe Gin 1025 1073 got cot tgg agg Ala Pro Trp Arg got gca gac otc Ala Ala Asp Leu oag Gin 325 gao aao got tgo Asp Asn Ala Cys aga tcg Arg Ser 330 7\j/V N

CA

I.

27/61 tgc agc tgt Cys Ser Cys tac ctg gca Tyr Leu Ala 350 aat Asn 335 agc cac tcc agc Ser His Ser Ser cgc Arg 340 tgt cac ttt gac Cys His Phe Asp atg act a g Met Thr Thr 345 gac tgc cag Asp Cys Gin agc ggt ggc ctc Ser Gly Gly Leu agc Ser 355 ggg ggc gtg tgt Gly Gly Val Cys gaa Glu 360 cac aac His Asn 365 act gag ggg cag Thr Glu Gly Gin tgc gac cgc tgc Cys Asp Arg Cys aga Arg 375 ccc ctc ttc tac Pro Leu Phe Tyr agg Arg 380 gac ccg ctc aag Asp Pro Leu Lys acc Thr 385 atc tca gat ccc Ile Ser Asp Pro tac Tyr 390 gcg tgc att cct Ala Cys Ile Pro gaa tgt gac ccc Glu Cys Asp Pro gat Asp 400 ggg acc ata tct Gly Thr Ile Ser ggt Gly 405 ggc att tgt gtg Gly Ile Cys Vai agc cac Ser His 410 tct gat cct Ser Asp Pro aac gtg gaa Asn Val Glu 430 gcc Ala 415 tta ggg tct gtg Leu Gly Ser Val gcc Ala 420 ggc cag tgc ctt Gly Gin Cys Leu tgt aaa gag Cys Lys Glu 425 cac tac gga His Tyr Gly gga gcc aaa tgc Gly Ala Lys Cys gac Asp 435 cag tgc aaa ccc Gin Cys Lys Pro cta agc Leu Ser 445 gcc acc gac ccc Ala Thr Asp Pro ctg Leu 450 ggc tgc cag ccc Gly Cys Gin Pro tgc Cys 455 gac tgt aac ccc Asp Cys Asn Pro 1121 1169 1217 1265 1313 1361 1409 1457 1505 1553 1601 1649 1697 1745 1793 ggg agt ctg cca Gly Ser Leu Pro ttc Phe 465 ttg acc tgt gat Leu Thr Cys Asp gat aca ggc caa Asp Thr GlyGln ttg tgc ctg tca Leu Cys Leu Ser tat Tyr 480 gtc acc gga gca Val Thr Gly Ala tgc gaa gaa tgc Cys Glu Giu Cys act gtt Thr Val 490 a. gga tac tgg Gly Tyr Trp tgt gat att Cys Asp Ile 510 ggc Gly 495 ctg gga aat cat Leu Gly Asn His ctc Leu 500 cat ggg tgt tct His Gly Cys Ser ccc tgt gac Pro Cys Asp 505 aag aat ggg Lys Asn Gly gga ggt gct tat Gly Gly Ala Tyr tct Ser 515 aac gtg tgc tca Asn Val Cys Ser ccc Pro 520 cag tgt Gin Cys 525 gaa tgc cgc cca Glu Cys Arg Pro gtc act ggc cgt Val Thr Gly Arg tgc tct gaa cca Cys Ser Giu Pro gcc Ala 540 cct ggc tac ttc Pro Gly Tyr Phe ttt Phe 545 gct cct ttg aat Ala Pro Leu Asn ttc tat ctc tac gag gca Phe Tyr Leu Tyr Giu Ala 550 555 gag gaa gcc aca Glu Giu Ala Thr aca Thr 560 ctc caa gga ctg Leu Gin Gly Leu cct ttg ggc tcg Pro Leu Gly Ser gag acg Glu Thr 570 i i

C

28/61 ttt ggc cag Phe Gly Gin ggg aac cct Gly Asn Pro 590 agt Ser 575 Oct got gtt cac Pro Ala Val His gtt tta gga gag Vai Leu Gly Glu cca gtt cct Pro Val Pro 585 gtt ctc cct Val Leu Pro gtt aca tgg act Val Thr Trp Thr gga Gly 595 oct gga ttt gcc Pro Gly Phe Ala agg Arg 600 ggg got Gly Ala 605 ggc ttg aga ttt Gly Leu Arg Phe got Ala 610 gto aac aao att Vai Asn Asn Ile ttt cot gtg gao Phe Pro Val Asp ac att gc att Thr Ile Ala Ile cac tat gaa aco cag tot gca got gao tgg act His Tyr Glu Thr Gin Ser Ala Ala Asp Trp Thr 625 630 635 gto cag att gtg Vai Gin Ile Val gtg Va1 640 aac ccc cct gga Asn Pro Pro Gly agt gag oac tgo Ser Giu His Cys ata ccc Ile Pro 650 aag act ota Lys Thr Leu aga ato atg Arg Ile Met 670 oag Gin 655 toa aag cct cag Ser Lys Pro Gin tct Ser 660 ttt gc tta cca Phe Ala Leu Pro gcg got acg Ala MHa Thr 665 gat gta oaa Asp Val Gin otg ott ccc aca Leu Leu Pro Thr ato tgt tta gaa Ile Cys Leu Glu cca Pro 680 4 0 4.

tat too Tyr Ser 685 ata gat gto tat Ile Asp Val Tyr ttt Phe 690 tct cag oct ttg Ser Gin Pro Leu oaa Gin 695 gga gag too cac Gly Giu Ser His 1841 1889 1937 1985 2033 2081 2129 2177 2225 2273 2321 2369 2417 2465 2513 got Ala 700 oat toa oat gto His Ser His Val gtg gao tot ott Val Asp Ser Leu ggc Gly 710 ott att ccc caa Leu Ile Pro Gin ato Ile 715 aat tca ttg gag Asn Ser Leu Glu aat Asn 720 tto tgo ago aag Phe Cys Ser Lys oag Gin 725 gao tta gat gag Asp Leu Asp Glu tat cag Tyr Gin 730 r r ott cac aao Leu His Asn ccg ggt gc Pro Gly Ala 750 gtt gaa att gc Val Giu Ile Ala gca atg gga oct Ala Met Gly Pro oaa gtg otc Gin Val Leu 745 aag otg oat Lys Leu His tgt gaa agg otg Cys Giu Arg Leu ato Ile 755 ato ago atg tct Ile Ser Met Ser goc Ala 760 gat ggg Asp Gly 765 got gtg gco tgo Ala Val Ala Cys tgt cac ccc cag Cys His Pro Gin toa gtc gga too Ser Val Gly Ser ago Ser 780 tgo ago oga ott Cys Ser Arg Leu gga Gly 785 ggc cag tgc oag Gly Gin Cys Gin tgt Cys 790 aaa oct ott gtg Lys Pro Leu Val ggg cgo tgc tgt Gly Arg Cys Cys gao Asp 800 agg tgo tca act Arg Cys Ser Thr gga Gly 805 ago tat gat ttg Ser Tyr Asp Leu ggg cat Gly His 810 A yI 1': 29/61 cac ggc tgt His Gly Cys gta tgt gac Val Cys Asp 830 cca tgt cac tgc Pro Cys His Cys cct caa gga tca Pro Gin Gly Ser aag gac act Lys Asp Thr 825 gag gtg tct Glu Val Ser caa gta aca gga Gin Val Thr Gly cag Gin 835 tgc ccc tgc cat Cys Pro Cys His gga Gly 840 ggc cgc Gly Arg 845 cgc tgt gat cgc Arg Cys Asp Arg ctg gca ggc tac Leu Ala Gly Tyr gga ttt ccc agc Gly Phe Pro Ser tgc Cys 860 cac cct tgc cct His Pro Cys Pro tgt Cys 865 aat agg ttt gct Asn Arg Phe Ala gaa Glu 870 ctt tgt gat cct Leu Cys Asp Pro aca ggg tca tgc Thr Gly Ser Cys aat tgt gga ggc Asn Cys Gly Gly aca act ggc aga Thr Thr Gly Arg aac tgt Asn Cys 890 gaa agg tgt Glu Arg Cys tgt cgt cct Cys Arg Pro 910 att Ile 895 gat ggt tac tat Asp Gly Tyr Tyr gga Gly 900 aat cct tct tca Asn Pro Ser Ser gga cag ccc Gly Gin Pro 905 cag tat ttt Gin Tyr Phe tgc ctg tgt cca Cys Leu Cys Pro gat ccc tca agc Asp Pro Ser Ser aat Asn 920 r r r r r gcc cat Ala His 925 tcc tgt tat cag Ser Cys Tyr Gin aat Asn 930 ctg tgg agc tca Leu Trp Ser Ser gat Asp 935 gta atc tgc aat Val Ile Cys Asn 2561 2609 2657 2705 2753 2801 2849 2897 2945 2993 3041 3089 3137 3185 3233 tgt Cys 940 ctt caa ggt tat Leu Gin Gly Tyr ggt act cag tgt Gly Thr Gin Cys gga Gly 950 gaa tgc tct act Glu Cys Ser Thr ttc tat gga aat Phe Tyr Gly Asn cca Pro 960 aga att tca gga Arg Ile Ser Gly gca Ala 965 cct tgc caa cca Pro Cys Gin Pro tgt gcc Cys Ala 970 tgc aac aac aac ata gat gta acc gat cca gag tcc tgc agc cgg gta Cys Asn Asn Asn Ile Asp Vai Thr Asp Pro Glu Ser Cys Ser Arg Val 975 980 985 aca ggg gag tgc Thr Gly Glu Cys 990 ctt cga tgt Leu Arg Cys ttg cac Leu His 995 aac act cag Asn Thr Gin ggc gca aac tgc Gly Ala Asn Cys 1000 cag ctc tgc aaa cca ggt Gin Leu Cys Lys Pro Gly 1005 cac tat gga tca gcc His Tyr Gly Ser Ala 1010 ctc aat Leu Asn 1015 cag acc tgc Gin Thr Cys aga aga tgc tcc tgc Arg Arg Cys Ser Cys 1020 cat gct tcc ggc gtg His Ala Ser Gly Val 1025 agt ccc atg gag tgt Ser Pro Met Glu Cys 1030 ccc Pro 1035 cct ggt ggg gga Pro Gly Gly Gly gct tgc ctc tgt gac Ala Cys Leu Cys Asp 1040 cct gtc act ggt gca Pro Val Thr Gly Ala 1045 tgt cct Cys Pro 1050 3 i I

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L~ j~ 30/61 tgt ctg ccg aat gtc aca ggc ctg gcc tgt gac cgt tgt gct gat gga Cys Leu Pro Asn Val Thr Gly Leu Ala Cys Asp Arg Cys Ala Asp Gly 1055 1060 1065 tac tgg aat ctg gtc cct ggc aga gga tgt cag tca. tgt gac tgt gac Tyr Trp Asn Leu Val Pro Gly Arg Gly Cys Gin Ser Cys Asp Cys Asp 1070 1075 1080 cct agg acc tct caa agt agc cac tgt gac cag gca aga tac ttt aaa Pro Arg Thr Ser Gin Ser Ser His Cys Asp Gin Ala Arg Tyr Phe Lys 1085 1090 1095 gct tac tagtgcatca aagtgagcat gatagtgaga catggtttct aatgtgtaaa Ala Tyr 1100 gaaagtttct tttatgtact gttgttaatt agtgcattga aacaggatgc cttacaggga tggagtcagc ctctatcaag gaatgaaacc aaaaaagaga atgagcatct caagttcagc ttcgcctact tcagtttccc ctctgtgact gaggaagtca gaattcatac acagtgaaac acagacatca gcctcacctt tcactatttc atacatgtaa ccatagggaa gacctaagaa atagttaatc agaagagatt atgaatcaga atgaaaataa acagatacct tcaaaaccta aaaaaaaaaa aaaaaaaaaa a <210> 7 <211> 3109 <212> PRT <213> Homo sapiens 3281 3329 3377 3493 3553 3613 3673 3733 3754 S. *5 S.

S.

<400> 7 Met Leu Ala Asn Met Pro His Pro Asp Asn 145 Val Leu Lys Leu Ala 225 Ile Pro Gly His Ala Tyr Gin Pro Ser Leu 130 Ser Giu Thr Asp Giu 210 Asp Gly Gly Gin Leu Cys Cys Ile Ile 115 Gin Pro Tyr Leu Asp 195 Asn Asp Ala Val 20 Gin Ile Lys Arg Thr 100 Lys Gin Arg Lys Tyr 180 Giu Gly Pro Ala Gin Arg Thr Leu Ile Asn Asn Val1 Pro Pro 165 Asn Val1 Giu Ser Gly Ala Gly Thr Val1 70 Cys Ala Gly Phe Gly 150 Trp Ile Ile Ile Pro 230 Val1 Gin Leu Asn 55 Glu Asn Ile Ile Gin 135 Asn Gin Tyr Cys His 215 Glu Leu Arg Phe 40 Ala His Gin Asp Glu 120 Ile Trp Tyr Pro Thr 200 Ile Leu Leu Pro Pro Thr Val1 Asn Gly 105 Tyr Ala Ile His Arg 185 Ser Ser Leu Leu 10 Gin Ala Cys Pro Ser 90 Lys His Tyr Leu Ala 170 Thr Phe Leu Giu Leu Gin Val1 Gly Gly 75 Ser Asn Tyr Val Glu 155 Val1 Gly Tyr Ile Phe 235 Leu Gin Leu Giu Gin Asn Thr Val1 Ile 140 Arg Thr Pro Ser Asn 220 Thr Leu Arg Asn Lys Pro Pro Trp Thr 125 Val1 Ser Asp Pro Lys 205 Gly Ser Gly Ser Ala Pro Arg Gin Gin Thr Ala Asp Glu 175 Tyr His Pro Arg Gly Gin Ser Giu Asn Arg Ser Leu Ala Asp 160 Cys Ala Pro Ser Tyr 240 Arg Leu Arg Phe Gin Arg Ile Arg Thr Leu Asn Ala Asp Leu Met 245 250 *255 '7 \IA/y /~3 D ti~

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Met Arg Cys Ser 305 Cys Thr Cys Arg Thr 385 Lys Asp Ala Gly Asp 465 Pro Cys Lys Ser Leu 545 Leu Ala Asn Asp Ile 625 Val Giu Glu Thr Leu 705 Val1 Ser Phe Tyr Tyr 290 Arg Cys Lys Tyr Gly 370 Ala Gly Pro Arg Gly 450 Cys Cys Ser Gly Ser 530 Thr Asp Leu Lys Leu 610 Ile Tyr Giu Phe Tyr 690 Glu Glu Ala His 260 Tyr Tyr 275 Gly His Cys Giu Pro Gly Thr Glu 340 Tyr Asp 355 Lys Tyr Gly Ile Val Ser Ile Gly 420 Arg Gly 435 Val Ser Lys Ala Phe Gly Arg Cys 500 Cys Asp 515 Tyr Trp Asp Leu Ser Pro Pro His 580 Leu Pro 595 Giu Giu Leu Giu Leu His Ser Phe 660 Met Thr 675 Ser Phe Ser Ala Val Cys Lys Asp Ser Val Ala Arg Cys Giu 310 Phe His 325 Cys Giu Giu Asn Ile Gly Asn Cys 390 Pro Asn 405 Ser Leu Leu Ala Cys Asp Cys Asn 470 Pro Cys 485 Lys Ser Giu Cys Thr Tyr Pro Gly 550 Gin Gin 565 Ser Tyr Ala Val Giu Giu Giy Asn 630 Pro Ser 645 Thr Ile Val Leu Gly Met Val Ser 710 Gin Cys 725 Lys Ala 295 His Gin Ala Val1 Gly 375 Giu Tyr Asn Pro Arg 455 Cys Ile Gly Phe Gly 535 Arg Ile Tyr Gly Giu 615 Asp Giu His Ala Asp 695 Tyr Pro Asp 280 Cys Asn Lys Cys Ala 360 Gly Thr Pro Giu Gly 440 Cys Ser Cys Phe Cys 520 Lys Ile Ser Trp Gly 600 Asp Leu Giu Giy Asn 680 Ala Pro Pro Ile Pro Thr Pro Asn 345 Arg Val1 Cys Arg Val1 425 Ser Aia Gly Lys Phe 505 Ser Ile Arg Ile Ser 585 Gin Thr Ser His Thr 665 Leu Ile Thr Giy Val1 745 Ser Leu Cys Trp 330 Cys Arg Cys Thr Pro 410 Cys Cys Arg Leu Giu 490 Asn Giy Gin Val Ser 570 Ala Leu Giu Ile Thr 650 His Lys Phe Asp Tyr 730 Val Asp Gly 315 Arg His Asn Ile Asp 395 Cys Val His Gly Gly 475 Asn Leu Val1 Asp Aia 555 Asn Pro Thr Arg Ser 635 Asn Phe Arg Arg Gly 715 Thr Gly Pro 300 Asp Ala Gly Leu Asn 380 Gly Gin Lys Cys Tyr 460 Ser Val1 Gin Ser Met 540 Pro Aia Ala Phe Val 620 Thr Val Pro Val1 Leu 700 Ser Gly Giy 285 Ala Ser Gly Lys Ser 365 Cys Phe Pro Asp Lys 445 Thr Lys Giu Giu Asn 525 Ser Gin Giu Pro Thr 605 Leu Ala Leu Val Leu 685 Ser Ile Ser Met Thr Cys Thr Ala 350 Leu Thr Phe Cys Giu 430 Thr Gly Asn Gly Asp 510 Arg Gly Gin Ala Tyr 590 Ile Gin Gin Leu Arg 670 Leu Ser Ala Ser Cys Asn Asp Phe 335 Giu Asn Gin Arg His 415 Lys Gly Tyr Giu Giy 495 Asn Cys Trp Asp Arg 575 Leu Ser Leu Asp Leu 655 Arg Gin Val1 Ala Cys 735 Pro Arg Giu Ile Asp Pro Ile Val Thr Arg Ile Lys Gin 320 Leu Giu Ile Asn Pro 400 Cys His Phe Pro Asp 480 Asp Trp Gin Tyr Asp 560 Gin Gly Tyr Met Giu 640 Lys Lys Ile Asn Ala 720 Giu Cys Trp Pro Arg His Arg Arg 740 Asn Gly Thr Ile Phe Gly Gly 750 /1

C

32/61 Ile Cys Glu Pro Cys Gin Cys Phe Gly His Ala Glu Ser Cys Asp Asp 755 760 765 Val Thr Gly Glu Cys Leu Asn Cys Lys Asp His Thr Gly Gly Pro Tyr 770 775 780 Cys Asp Lys Cys Leu Pro Gly Phe Tyr Gly Glu Pro Thr Lys Gly Thr 785 790 795 800 Ser Glu Asp Cys Gin Pro Cys Ala Cys Pro Leu Asn Ile Pro Ser Asn 805 810 815 Asn Phe Ser Pro Thr Cys His Leu Asp Arg Ser Leu Gly Leu Ile Cys 820 825 830 Asp Gly Cys Pro Val Gly Tyr Thr Gly Pro Arg Cys Glu Arg Cys Ala 835 840 845 Glu Gly Tyr Phe Gly Gin Pro Ser Val Pro Gly Gly Ser Cys Gin Pro 850 855 860 Cys Gin Cys Asn Asp Asn Leu Asp Phe Ser Ile Pro Gly Ser Cys Asp 865 870 875 880 Ser Leu Ser Gly Ser Cys Leu Ile Cys Lys Pro Gly Thr Thr Gly Arg 885 890 895 Tyr Cys Glu Leu Cys Ala Asp Gly Tyr Phe Gly Asp Ala Val Asp Ala 900 905 910 Lys Asn Cys Gin Pro Cys Arg Cys Asn Ala Gly Gly Ser Phe Ser Glu 915 920 925 Val Cys His Ser Gin Thr Gly Gin Cys Glu Cys Arg Ala Asn Val Gin 930 935 940 Gly Gin Arg Cys Asp Lys Cys Lys Ala Gly Thr Phe Gly Leu Gin Ser 945 950 955 960 Ala Arg Gly Cys Val Pro Cys Asn Cys Asn Ser Phe Gly Ser Lys Ser 965 970 975 Phe Asp Cys Glu Glu Ser Gly Gin Cys Trp Cys Gin Pro Gly Val Thr 980 985 990 Gly Lys Lys Cys Asp Arg Cys Ala His Gly Tyr Phe Asn Phe Gin Glu 995 1000 1005 Gly Gly Cys Thr Ala Cys Glu Cys Ser His Leu Gly Asn Asn Cys Asp 1010 1015 1020 Pro Lys Thr Gly Arg Cys Ile Cys Pro Pro Asn Thr Ile Gly Glu Lys S 1025 1030 1035 1040 Cys Ser Lys Cys Ala Pro Asn Thr Trp Gly His Ser Ile Thr Thr Gly 1045 1050 1055 Cys Lys Ala Cys Asn Cys Ser Thr Val Gly Ser Leu Asp Phe Gin Cys 1060 1065 1070 Asn Val Asn Thr Gly Gin Cys Asn Cys His Pro Lys Phe Ser Gly Ala S1075 1080 1085 Lys Cys Thr Glu Cys Ser Arg Gly His Trp Asn Tyr Pro Arg Cys Asn 1090 1095 1100 Leu Cys Asp Cys Phe Leu Pro Gly Thr Asp Ala Thr Thr Cys Asp Ser 1105 1110 1115 1120 Glu Thr Lys Lys Cys Ser Cys Ser Asp Gin Thr Gly Gin Cys Thr Cys 1125 1130 1135 Lys Val Asn Val Glu Gly Ile His Cys Asp Arg Cys Arg Pro Gly Lys 1140 1145 1150 Phe Gly Leu Asp Ala Lys Asn Pro Leu Gly Cys Ser Ser Cys Tyr Cys 1155 1160 1165 Phe Gly Thr Thr Thr Gin Cys Ser Glu Ala Lys Gly Leu Ile Arg Thr 1170 1175 1180 Trp Val Thr Leu Lys Ala Glu Gin Thr Ile Leu Pro Leu Val Asp Glu 1185 1190 1195 1200 Ala Leu Gin His Thr Thr Thr Lys Gly Ile Val Phe Gin His Pro Glu 1205 1210 1215 Ile Val Ala His Met Asp Leu Met Arg Glu Asp Leu His Leu Glu Pro 1220 1225 1230 Phe Tyr Trp Lys Leu Pro Glu Gin Phe Glu Gly Lys Lys Leu Met Ala 1235 1240 1245

I

h 33/61 Tyr Gly Gly Lys Leu Lys Tyr Ala Ile Tyr Phe Glu Ala Arg Glu Glu 1250 1255 1260 Thr Gly Phe Ser Thr Tyr Asn Pro Gin Val Ile Ile Arg Gly Gly Thr 1265 1270 1275 1280 Pro Thr His Ala Arg Ile Ile Val Arg His Met Ala Ala Pro Leu Ile 1285 1290 1295 Gly Gin Leu Thr Arg His Glu Ile Glu Met Thr Glu Lys Glu Trp Lys 1300 1305 1310 Tyr Tyr Gly Asp Asp Pro Arg Val His Arg Thr Val Thr Arg Glu Asp 1315 1320 1325 Phe Leu Asp Ile Leu Tyr Asp Ile His Tyr Ile Leu Ile Lys Ala Thr 1330 1335 1340 Tyr Gly Asn Phe Met Arg Gin Ser Arg Ile Ser Glu Ile Ser Met Glu 1345 1350 1355 1360 Val Ala Glu Gin Gly Arg Gly Thr Thr Met Thr Pro Pro Ala Asp Leu 1365 1370 1375 Ile Glu Lys Cys Asp Cys Pro Leu Gly Tyr Ser Gly Leu Ser Cys Glu 1380 1385 1390 Ala Cys Leu Pro Gly Phe Tyr Arg Leu Arg Ser Gin Pro Gly Gly Arg 1395 1400 1405 Thr Pro Gly Pro Thr Leu Gly Thr Cys Val Pro Cys Gin Cys Asn Gly 1410 1415 1420 His Ser Ser Leu Cys Asp Pro Glu Thr Ser Ile Cys Gin Asn Cys Gin 1425 1430 1435 1440 His His Thr Ala Gly Asp Phe Cys Glu Arg Cys Ala Leu Gly Tyr Tyr 1445 1450 1455 Gly Ile Val Lys Gly Leu Pro Asn Asp Cys Gin Gin Cys Ala Cys Pro 1460 1465 1470 Leu Ile Ser Ser Ser Asn Asn Phe Ser Pro Ser Cys Val Ala Glu Gly 1475 1480 1485 Leu Asp Asp Tyr Arg Cys Thr Ala Cys Pro Arg Gly Tyr Glu Gly Gin 1490 1495 1500 S Tyr Cys Glu Arg Cys Ala Pro Gly Tyr Thr Gly Ser Pro Gly Asn Pro 1505 1510 1515 1520 Gly Gly Ser Cys Gin Glu Cys Glu Cys Asp Pro Tyr Gly Ser Leu Pro 1525 1530 1535 Val Pro Cys Asp Pro Val Thr Gly Phe Cys Thr Cys Arg Pro Gly Ala 1540 1545 1550 Thr Gly Arg Lys Cys Asp Gly Cys Lys His Trp His Ala Arg Glu Gly 1555 1560 1565 Trp Glu Cys Val Phe Cys Gly Asp Glu Cys Thr Gly Leu Leu Leu Gly 1570 1575 1580 Asp Leu Ala Arg Leu Glu Gin Met Val Met Ser Ile Asn Leu Thr Gly 1585 1590 1595 1600 Pro Leu Pro Ala Pro Tyr Lys Met Leu Tyr Gly Leu Glu Asn Met Thr 1605 1610 1615 Gin Glu Leu Lys His Leu Leu Ser Pro Gin Arg Ala Pro Glu Arg Leu 1620 1625 1630 Ile Gin Leu Ala Glu Gly Asn Leu Asn Thr Leu Val Thr Glu Met Asn 1635 1640 1645 Glu Leu Leu Thr Arg Ala Thr Lys Val Thr Ala Asp Gly Glu Gin Thr 1650 1655 1660 Gly Gin Asp Ala Glu Arg Thr Asn Thr Arg Ala Lys Ser Leu Gly Glu 1665 1670 1675 1680 Phe Ile Lys Glu Leu Ala Arg Asp Ala Glu Ala Val Asn Glu Lys Ala 1685 1690 1695 Ile Lys Leu Asn Glu Thr Leu Gly Thr Arg Asp Glu Ala Phe Glu Arg 1700 1705 1710 Asn Leu Glu Gly Leu Gin Lys Glu Ile Asp Gin Met Ile Lys Glu Leu 1715 1720 1725 Arg Arg Lys Asn Leu Glu Thr Gin Lys Glu Ile Ala Glu Asp Glu Leu 1730 1735 1740

LC

34/61 Val Ala Ala Glu Ala Leu Leu Lys Lys Val Lys Lys Leu Phe Gly Glu 1745 1750 1755 1760 Ser Arg Gly Glu Asn Glu Glu Met Glu Lys Asp Leu Arg Glu Lys Leu 1765 1770 1775 Ala Asp Tyr Lys Asn Lys Val Asp Asp Ala Trp Asp Leu Leu Arg Glu 1780 1785 1790 Ala Thr Asp Lys Ile Arg Glu Ala Asn Arg Leu Phe Ala Val Asn Gin 1795 1800 1805 Lys Asn Met Thr Ala Leu Glu Lys Lys Lys Glu Ala Val Glu Ser Gly 1810 1815 1820 Lys Arg Gln Ile Glu Asn Thr Leu Lys Glu Gly Asn Asp Ile Leu Asp 1825 1830 1835 1840 Glu Ala Asn Arg Leu Ala Asp Glu Ile Asn Ser Ile Ile Asp Tyr Val 1845 1850 1855 Glu Asp Ile Gin Thr Lys Leu Pro Pro Met Ser Glu Glu Leu Asn Asp 1860 1865 1870 Lys Ile Asp Asp Leu Ser Gin Glu Ile Lys Asp Arg Lys Leu Ala Glu 1875 1880 1885 Lys Val Ser Gin Ala Glu Ser His Ala Ala Gin Leu Asn Asp Ser Ser 1890 1895 1900 Ala Val Leu Asp Gly Ile Leu Asp Glu Ala Lys Asn Ile Ser Phe Asn 1905 1910 1915 1920 Ala Thr Ala Ala Phe Lys Ala Tyr Ser Asn Ile Lys Asp Tyr Ile Asp 1925 1930 1935 Glu Ala Glu Lys Val Ala Lys Glu Ala Lys Asp Leu Ala His Glu Ala 1940 1945 1950 Thr Lys Leu Ala Thr Gly Pro Arg Gly Leu Leu Lys Glu Asp Ala Lys 1955 1960 1965 Gly Cys Leu Gin Lys Ser Phe Arg Ile Leu Asn Glu Ala Lys Lys Leu .o 1970 1975 1980 Ala Asn Asp Val Lys Glu Asn Glu Asp His Leu Asn Gly Leu Lys Thr 1985 1990 1995 2000 Arg Ile Glu Asn Ala Asp Ala Arg Asn Gly Asp Leu Leu Arg Thr Leu 2005 2010 2015 Asn Asp Thr Leu Gly Lys Leu Ser Ala Ile Pro Asn Asp Thr Ala Ala 2020 2025 2030 Lys Leu Gln Ala Val Lys Asp Lys Ala Arg Gin Ala Asn Asp Thr Ala S* 2035 2040 2045 Lys Asp Val Leu Ala Gin Ile Thr Glu Leu His Gin Asn Leu Asp Gly 2050 2055 2060 Leu Lys Lys Asn Tyr Asn Lys Leu Ala Asp Ser Val Ala Lys Thr Asn 2065 2070 2075 2080 Ala Val Val Lys Asp Pro Ser Lys Asn Lys Ile Ile Ala Asp Ala Asp 2085 2090 2095 Ala Thr Val Lys Asn Leu Glu Gin Glu Ala Asp Arg Leu Ile Asp Lys 2100 2105 2110 Leu Lys Pro Ile Lys Glu Leu Glu Asp Asn Leu Lys Lys Asn Ile Ser 2115 2120 2125 Glu Ile Lys Glu Leu Ile Asn Gin Ala Arg Lys Gin Ala Asn Ser Ile 2130 2135 2140 Lys Val Ser Val Ser Ser Gly Gly Asp Cys Ile Arg Thr Tyr Lys Pro 2145 2150 2155 2160 Glu Ile Lys Lys Gly Ser Tyr Asn Asn Ile Val Val Asn Val Lys Thr 2165 2170 2175 Ala Val Ala Asp Asn Leu Leu Phe Tyr Leu Gly Ser Ala Lys Phe Ile 2180 2185 2190 Asp Phe Leu Ala Ile Glu Met Arg Lys Gly Lys Val Ser Phe Leu Trp 2195 2200 2205 Asp Val Gly Ser Gly Val Gly Arg Val Glu Tyr Pro Asp Leu Thr Ile 2210 2215 2220 Asp Asp Ser Tyr Trp Tyr Arg Ile Val Ala Ser Arg Thr Gly Arg Asn 2225 2230 2235 2240 35/61 Gly Thr Ile Ser Val Arg Ala Leu Asp Gly Pro Lys Ala Ser Ile Val 2245 2250 2255 Pro Ser Thr His His Ser Thr Ser Pro Pro Gly Tyr Thr Ile Leu Asp 2260 2265 2270 Val Asp Ala Asn Ala Met Leu Phe Val Gly Gly Leu Thr Gly Lys Leu 2275 2280 2285 Lys Lys Ala Asp Ala Val Arg Val Ile Thr Phe Thr Gly Cys Met Gly 2290 2295 2300 Glu Thr Tyr Phe Asp Asn Lys Pro Ile Gly Leu Trp Asn Phe Arg Glu 2305 2310 2315 2320 Lys Glu Gly Asp Cys Lys Gly Cys Thr Val Ser Pro Gin Val Glu Asp 2325 2330 2335 Ser Glu Gly Thr Ile Gin Phe Asp Gly Glu Gly Tyr Ala Leu Val Ser 2340 2345 2350 Arg Pro Ile Arg Trp Tyr Pro Asn Ile Ser Thr Val Met Phe Lys Phe 2355 2360 2365 Arg Thr Phe Ser Ser Ser Ala Leu Leu Met Tyr Leu Ala Thr Arg Asp 2370 2375 2380 Leu Arg Asp Phe Met Ser Val Glu Leu Thr Asp Gly His Ile Lys Val 2385 2390 2395 2400 Ser Tyr Asp Leu Gly Ser Gly Met Ala Ser Val Val Ser Asn Gin Asn 2405 2410 2415 His Asn Asp Gly Lys Trp Lys Ser Phe Thr Leu Ser Arg Ile Gin Lys 2420 2425 2430 Gin Ala Asn Ile Ser Ile Val Asp Ile Asp Thr Asn Gin Glu Glu Asn 2435 2440 2445 Ile Ala Thr Ser Ser Ser Gly Asn Asn Phe Gly Leu Asp Leu Lys Ala 2450 2455 2460 Asp Asp Lys Ile Tyr Phe Gly Gly Leu Pro Thr Leu Arg Asn Leu Ser 2465 2470 2475 2480 Met Lys Ala Arg Pro Glu Val Asn Leu Lys Lys Tyr Ser Gly Cys Leu 2485 2490 2495 Lys Asp Ile Glu Ile Ser Arg Thr Pro Tyr Asn Ile Leu Ser Ser Pro 2500 2505 2510 Asp Tyr Val Gly Val Thr Lys Gly Cys Ser Leu Glu Asn Val Tyr Thr 2515 2520 2525 Val Ser Phe Pro Lys Pro Gly Phe Val Glu Leu Ser Pro Val Pro Ile 2530 2535 2540 Asp Val Gly Thr Glu Ile Asn Leu Ser Phe Ser Thr Lys Asn Glu Ser 2545 2550 2555 2560 Gly Ile Ile Leu Leu Gly Ser Gly Gly Thr Pro Ala Pro Pro Arg Arg 2565 2570 2575 Lys Arg Arg Gin Thr Gly Gin Ala Tyr Tyr Val Ile Leu Leu Asn Arg 2580 2585 2590 S Gly Arg Leu Glu Val His Leu Ser Thr Gly Ala Arg Thr Met Arg Lys 2595 2600 2605 o Ile Val Ile Arg Pro Glu Pro Asn Leu Phe His Asp Gly Arg Glu His 2610 2615 2620 Ser Val His Val Glu Arg Thr Arg Gly Ile Phe Thr Val Gin Val Asp 2625 2630 2635 2640 Glu Asn Arg Arg Tyr Met Gin Asn Leu Thr Val Glu Gin Pro Ile Glu 2645 2650 2655 Val Lys Lys Leu Phe Val Gly Gly Ala Pro Pro Glu Phe Gin Pro Ser 2660 2665 2670 Pro Leu Arg Asn Ile Pro Pro Phe Glu Gly Cys Ile Trp Asn Leu Val 2675 2680 2685 Ile Asn Ser Val Pro Met Asp Phe Ala Arg Pro Val Ser Phe Lys Asn 2690 2695 2700 Ala Asp Ile Gly Arg Cys Ala His Gin Lys Leu Arg Glu Asp Glu Asp 2705 2710 2715 2720 Gly Ala Ala Pro Ala Glu Ile Val Ile Gin Pro Glu Pro Val Pro Thr 2725 2730 2735 j I .1 36/61 Pro Ala Phe Pro Thr Pro Thr Pro Val Leu Thr Hi-s Gly Pro Cys Ala 2740 2745 2750 Ala Glu Ser Glu Pro Ala Leu Leu Ile Gly Ser Lys Gln Phe Gly Leu 2755 2760 2765 Ser Arg Asn Ser His Ile Ala Ile Ala Phe Asp Asp Thr Lys Val Lys 2770 2775 2780 Asn Arg Leu Thr Ile Glu Leu Glu Val Arg Thr Glu Ala Glu Ser Gly 2785 2790 2795 2800 Leu Leu Phe Tyr Met Ala Ala Ile Asn His Ala Asp Phe Ala Thr Val 2805 2810 2815 Gln Leu Arg Asn Gly Leu Pro Tyr Phe Ser Tyr Asp Leu Gly Ser Gly 2820 2825 2830 Asp Thr His Thr Met Ile Pro Thr Lys Ile Asn Asp Gly Gln Trp His 2835 2840 2845 Lys Ile Lys Ile Met Arg Ser Lys Gln Glu Gly Ile Leu Tyr Val Asp 2850 2855 2860 Gly Ala Ser Asn Arg Thr Ile Ser Pro Lys Lys Ala Asp Ile Leu Asp 2865 2870 2875 2880 Val Val Gly Met Leu Tyr Val Gly Gly Leu Pro Ile Asn Tyr Thr Thr 2885 2890 2895 Arg Arg Ile Gly Pro Val Thr Tyr Ser Ile Asp Gly Cys Val Arg Asn 2900 2905 2910- Leu His Met Ala Glu Ala Pro Ala Asp Leu Glu Gln Pro Thr Ser Ser 2915 2920 2925 Phe His Val Gly Thr Cys Phe Ala Asn Ala Gln Arg Gly Thr Tyr Phe 2930 2935 2940 Asp Gly Thr Gly Phe Ala Lys Ala Val Gly Gly Phe Lys Val Gly Leu 2945 2950 2955 2960 Asp Leu Leu Val Glu Phe Glu Phe Ala Thr Thr Thr Thr Thr Gly Val *O2965 2970 2975 Leu Leu Gly Ile Ser Ser Gln Lys Met Asp Gly Met Gly Ile Glu Met 2980 2985 2990 Ile Asp Glu Lys Leu Met Phe His Val Asp Asn Gly Ala Gly Arg Phe 2995 3000 3005 S Thr Ala Val Tyr Asp Ala Gly Val Pro Gly His Leu Cys Asp Gly Gln 3010 3015 3020 Trp His Lys Val Thr Ala Asn Lys Ile Lys His Arg Ile Glu Leu Thr 3025 3030 3035 3040 Val Asp Gly Asn Gln Val Glu Ala Gln Ser Pro Asn Pro Ala Ser Thr 3045 3050 3055 S Ser Ala Asp Thr Asn Asp Pro Val Phe Val Gly Gly Phe Pro Asp Asp Le ysGn3060 3065 3070 LeuLysGlnPhe Gly Leu Thr Thr Ser Ile Pro Phe Arg Gly Cys Ile Ag er3075 3080 3085 ArgSerLeu Lys Leu Thr Lys Gly Thr Ala Ser His Trp Arg Leu Ile 3090 3095 3100 Leu Pro Arg Pro Trp 3105 :.The <210> 8 <211> 9534 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (50) (9379) <400> 8 cagcgactcc tctggctccc gagaagtgga tccggtcgcg gccactacg atg ccg gga 58 Met Pro Gly ox1 37/61 gcc gcc Ala Ala ggg gtc ctc CtC Gly Val Leu Leu ctg ctg ctc tcc Leu Leu Leu Ser ggc ctc ggg ggc Gly Leu Gly Gly gta Val1 cag gcg cag cgg Gin Ala Gin Arg cag cag cag cgg Gin Gin Gin Arg tca cag gca cat Ser Gin Ala His 106 154 202 caa aga ggt tta Gin Arg Gly Leu cct gct gtc ctg Pro Ala Val Leu aat Asn ctt gct tct aat Leu Ala Ser Asn gct ctt Ala Leu atc acg acc Ile Thr Thr aaa ttg gta Lys Leu Val aat Asn gca aca tgt gga Ala Thr Cys Gly gaa Giu 60 aaa gga cct gaa Lys Gly Pro Giu atg tac tgc Met Tlyr Cys ccg cag tgt Pro Gin Cys gaa cat gtc cct Giu His Val Pro ggg Gly 75 cag cct gtg agg Gin Pro Val Arg aac Asn cga atc Arg Ile tgc aat caa aac Cys Asn Gin Asn agc Ser 90 agc aat cca aac Ser Asn Pro Asn cag 'Gin aga cac ccg att Arg His Pro Ile aca Thr 100 aat gct att gat Asn Ala Ile Asp gga Gly 105 aag aac act tgg Lys Asn Thr Trp tgg Trp 110 cag agt ccc agt Gin Ser Pro Ser 4.

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-Q

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4/ Pt, 38/61 aga ttt Arg Phe 245 oag agg ato cgo Gin Arg Ilie Arg aca Thr 250 otg aat gct gao Leu Asn Ala Asp atg atg ttt gct Met Met Phe Ala cac His 260 aaa gac oca aga Lys Asp Pro Arg gaa Giu 265 att gao ccc att Ile Asp Pro Ile gtc Val 270 aco aga aga tat Thr Arg Arg Tyr tac Tyr 275 826 874 922 tac tog gto aag gat att toa gtt gga ggg atg tgo ato tgo tat ggt Tyr Ser Vai Lys Asp Ile Ser Vai Giy Giy Met Cys Ile Cys Tyr Giy 280 285 290 oat goo agg His Aia Arg gag tgt gag Giu Cys Giu 310 got Ala 295 tgt ooa Ott gat Cys Pro Leu Asp ooa Pro 300 gog aoa aat aaa Ala Thr Asn Lys tot ogo tgt Ser Arg Cys 305 tgo tgt ooa Cys Cys Pro oat aao aoa tgt His Asn Thr Cys ggo Giy 315 gat ago tgt gat Asp Ser Cys Asp gga tto Giy Phe 325 oat oag aaa 000 His Gin Lys Pro aga got gga act Arg Ala Giy Thr ttt Phe 335 ota act aaa act Leu Thr-bys Thr gaa Giu 340 tgt gaa goa tgc Cys Giu Ala Cys aat Asn 345 tgt oat gga aaa Cys His Gly Lys gaa gaa tgc tat Giu Giu Cys Tyr tat Tyr 355 gat gaa aat gtt Asp Giu Asn Val go o Ala 360 aga aga aat ctg Arg Arg Asn Leu agt Ser 365 ttg aat ata ogt Leu Asn Ile Arg gga aag Giy Lys 370

S

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5 4, 5* tao att gga ggg ggt gto tgo att Tyr Ile Giy Gly Gly Vai Cys Ile 375 aat tgt aoo oaa aao act got ggt Asn Cys Thr Gin Asn Thr Ala Gly 380 385 970 1018 1066 1114 1162 1210 1258 1306 1354 1402 1450 1498 ata aao tgo Ile Asn Cys 390 gag aoa tgt aoa Giu Thr Cys Thr gat Asp 395 ggo tto tto aga Giy Phe Phe Arg 000 Pro 400 aaa ggg gta Lys Giy Vai tot ooa Ser Pro 405 aat tat oca agg Asn Tyr Pro Arg ooa Pro 410 tgc oag ooa tgt Cys Gin Pro Cys tgc gat ooa att Cys Asp Pro Ile ggt Giy 420 too tta aat gaa Ser Leu Asn Giu gto Val 425 tgt gtc aag gat Cys Val Lys Asp gag Giu 430 aaa oat got oga Lys His Ala Arg ggt ttg goa cot Gly Leu Ala Pro gga Gly 440 too tgt oat tgo Ser Cys His Cys act ggt ttt gga Thr Gly Phe Gly ggt gtg Gly Val 450 ago tgt gat Ser Cys Asp goo tgt aao Ala Cys Asn 470 tgt goo agg ggo Cys Aia Arg Giy tao Tyr 460 act ggo tao oog Thr Giy Tyr Pro gao tgo aaa Asp Cys Lys 465 cot tgt ttt Pro Cys Phe tgc agt ggg tta Cys Ser Gly Leu ggg Giy 475 ago aaa aat gag Ser Lys Asn Giu

-J

39/61 ggc ccc Gly Pro 485 tgt atc tgc aag Cys Ilie Cys Lys aat gtt gaa gga Asri Val Giu Gly gga Gly 495 gac tgt agt cgt Asp Cys Ser Arg tgc Cys 500 aaa tcc ggc ttc Lys Ser Giy Phe aat ttg caa gag Asn Leu Gin Giu gat Asp 510 aat tgg aaa ggc Asn Trp Lys Giy tgc Cys 515 gat gag tgt ttc Asp Giu Cys Phe tgt Cys 520 tca ggg gtt tca Ser Giy Vai Ser aac Asn 525 aga tgt cag agt Arg Cys Gin Ser tcc tac Ser Tyr 530 tgg acc tat Trp Thr Tyr ctt cct ggc Leu Pro Giy 550 aaa ata caa gat Lys Ile Gin Asp agt ggc tgg tat Ser Giy Trp Tyr ctg act gac Leu Thr Asp 545 ttg gac tca Leu Asp Ser cgc att cga gtg Arg Ile Arg Vai gc t Aia 555 ccc cag cag gac Pro Gin Gin Asp gac Asp 560 cct cag Pro Gin 565 cag atc agc atc Gin Ile Ser Ile aac gcg gag gcc Asn Ala Giu Aia cgg Arg 575 caa gcc ctg ccg Gin Ala Leu Pro c ac His 580 agc tac tac tgg Ser Tyr Tyr Trp gcg ccg gct ccc Ala Pro Ala Pro tat Tyr 590 ctg gga aac aaa Leu Giy Asn Lys ctc Leu 595 cca gca gta gga Pro Aia Vai Giy gga Giy 600 cag ttg aca ttt Gin Leu Thr Phe acc Thr 605 ata tca tat gac Ile Ser Tyr Asp ctt gaa Leu Giu 610 1546 1594 1642 1690 1738 1786 1834 1882 1930 1978 2026 2074 2122 2170 2218 .4 0 4 44~ 4 *4* 4 44*4 S 0 4 4* 44 gaa gag gaa Giu Giu Giu gag ggt aat Giu Giy Asn 630 gaa Giu 615 gat aca gaa cgt Asp Thr Giu Arg ctc cag ctt atg Leu Gin Leu Met att atc tta Ilie Ile Leu 625 gtg tac ctg Val Tyr Leu gac ttg agc atc Asp Leu Ser Ile agc Ser 635 aca gcc caa gat Thr Aia Gin Asp gag Giu 640 cac cca His Pro 645 tct gaa gaa cat Ser Giu Giu His act Thr 650 aat gta ttg tta Asn Val Leu Leu aaa gaa gaa tca Lys Giu Giu Ser acc ata cat ggc Thr Ile His Giy cat ttt cca gtc His Phe Pro Val aga aag gaa ttt Arg Lys Giu Phe atg Met 675 aca gtg ctt gcg Thr Val Leu Aia ttg aag aga gtc Leu Lys Arg Vai ctc Leu 685 cta caa atc aca Leu Gin Ile Thr tac agc Tyr Ser 690 ttt ggg atg Phe Giy Met gct gtc tcc Ala Vai Ser 710 gat Asp 695 gcc atc ttc agg Aia Ile Phe Arg ttg Leu 700 agc tct gtt Ser Ser Val aac ctt gaa tcc Asn Leu Giu Ser 705 gct gta gaa gtg Aia Val Giu Vai 720 tat cct act gat Tyr Pro Thr Asp gga agc att gca gca Giy Ser Ile Aia Aia 715

K

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40/61 tgt cag tgc cca cca ggg tat act ggc tcc tct tgt Cys Gin Cys Pro Pro Gly Tyr Thr Gly Ser Ser Cys 725 730 735 gaa tct tgt tgg Glu Ser Cys Trp cct Pro 740 agg cac agg cga Arg His Arg Arg gtt Val1 745 aac ggc act att Asn Gly Thr Ile ttt Phe 750 ggt ggc atc tgt Gly Gly Ile Cys cca tgt cag tgc Pro Cys Gin Cys ggt cat gcg gag Giy His Aia Giu tcc Ser 765 tgt gat gac gtc Cys Asp Asp Vai act gga Thr Gly 770 gaa tgc ctg Giu Cys Leu aac tgt aag gat cac aca ggt ggc cca tat tgt gat aaa Asn Cys Lys Asp His Thr Gly Gly Pro Tyr Cys Asp Lys 775 780 785 tgt ctt cct ggt ttc tat ggc gag cct act aaa gga acc Cys Leu Pro Giy Phe Tyr Gly Giu Pro Thr Lys Giy Thr 790 795 800 tct gaa gac Ser Giu Asp tgt caa Cys Gin 805 ccc tgt gcc tgt Pro Cys Aia Cys cca Pro 810 ctc aat atc cca Leu Asn Ile Pro tcc Ser 815 aat aac ttt agc Asn Asn 'Phe Ser acg tgc cat tta Thr Cys His Leu cgg agt ctt gga Arg Ser Leu Giy atc tgt gat gga Ile Cys Asp Giy tgc Cys 835 cct gtc ggg tac Pro Vai Giy Tyr aca Thr 840 gga cca cgc tgt Gly Pro Arg Cys gag Giu 845 agg tgt gca gaa Arg Cys Ala Giu ggc tat Gly Tyr 850 2266 2314 2362 2410 2458 2506 2554 2602 2650 2698 2746 2794 2842 2890 2938 ttt gga caa Phe Giy Gin aat gac aac Asn Asp Asn 870 tct gta cct gga.

Ser Val Pro Gly tca tgt cag cca Ser Cys Gin Pro tgc caa tgc Cys Gin Cys 865 agc ttg tct Ser Leu Ser ctt gac ttc tcc Leu Asp Phe Ser atc Ile 875 cct ggc agc tgt Pro Giy Ser Cys gap Asp 880 ggc tcc Gly Ser 885 tgt ctg ata tgt Cys Leu Ile Cys cca ggt aca aca.

Pro Giy Thr Thr ggc Gly 895 cgg tac tgt gag Arg Tyr Cys Glu a a a a.

a a a a a.

a ctc Leu 900 tgt gct gat gga Cys Aia Asp Giy tat Tyr 905 ttt gga gat gca Phe Gly Asp Aia gtt Val 910 gat gcg aag aac Asp Ala Lys Asn cag ccc tgt cgc Gin Pro Cys Arg aat gcc ggt ggc Asn Ala Giy Gly tc t Ser 925 ttc tct gag gtt Phe Ser Giu Val tgc cac Cys His 930 agt caa act Ser Gin Thr gga Gly 935 cag tgt gag tgc Gin Cys Giu Cys aga Arg 940 gcc aac gtt cag Ala Asn Val Gin ggt cag aga Giy Gin Arg 945 tgt gac aaa tgc aag gct ggg acc ttt ggc cta caa tca gca agg ggc Cys Asp Lys Cys Lys Aia Giy Thr Phe Giy Leu Gin Ser Ala Arg Gly 950 955 960 A/ L"

Q

41/61 tgt gtt Cys Val 965 gaa gag Glu Glu 980 ccc tgc aac Pro Cys Asn agt gga caa Ser Gly Gin tgc aat Cys Asn 970 tgt tgg Cys Trp 985 tct ttt ggg Ser Phe Gly tgc caa cct Cys Gin Pro tct aag Ser Lys 975 gga gtc Gly Val 990 tca ttc gac tgt Ser Phe Asp Cys aca ggg aag Thr Gly Lys aaa Lys 995 tgt gac cgc tgt Cys Asp Arg Cys gcc cac Ala His 1000 ggc tat ttc Gly Tyr Phe aac ttc Asn Phe 1005 caa gaa gga Gin Glu Gly ggc tgc Gly Cys 1010 aca gct tgt Thr Ala Cys gaa tgt Glu Cys 1015 tct cat ctg Ser His Leu ggt aat Gly Asn 1020 aat tgt gac Asn Cys Asp cca aag act Pro Lys Thr 1025 ggg cga tgc att Gly Arg Cys Ile 1030 tgt gca ccc aat Cys Ala Pro Asn 1045 tgc cca ccc Cys Pro Pro aat acc Asn Thr 1035 att gga gag Ile Gly Glu aaa tgt tct aaa Lys Cys Ser Lys 1040 acc tgg ggc cac agc att acc act ggt tgt aag gct Thr Trp Gly His Ser Ile Thr Thr Gly Cys Lys Ala 1050 1055 tgt aac tgc agc aca gtg gga tcc ttg gat ttc caa tgc aat gta aat Cys Asn Cys Ser Thr Val Gly Ser Leu Asp Phe Gin Cys Asn Val Asn 1060 1065 1070 1075 .99.

9999*9 aca ggc caa Thr Gly Gin gag tgc agt Glu Cys Ser tgc aac tgt Cys Asn Cys 1080 cat cca aaa ttc tct His Pro Lys Phe Ser 1085 ggt gca aaa tgt aca Gly Ala Lys Cys Thr 1090 2986 3034 3082 3130 3178 3226 3274 3322 3370 3418 3466 3514 3562 3610 3658 cga ggt Arg Gly 1095 cac tgg aac His Trp Asn tac cct Tyr Pro 1100 cgc tgc aat Arg Cys Asn ctc tgt gac Leu Cys Asp 1105 tgc ttc ctc cct ggg Cys Phe Leu Pro Gly 1110 aca gat gcc aca acc Thr Asp Ala Thr Thr 1115 tgt gat tca gag act aaa Cys Asp Ser Glu Thr Lys 1120 aaa tgc tcc tgt agt gat caa act ggg cag tgc act tgt aag gtg aat Lys Cys Ser Cys Ser Asp Gin Thr Gly Gin Cys Thr Cys Lys Val Asn 1125 1130 1135 too 0 gtg gaa Val Glu 1140 ggc atc cac tgt gac Gly Ile His Cys Asp 1145 aga tgc cgg cct ggc Arg Cys Arg Pro Gly 1150 aaa ttc gga ctc Lys Phe Gly Leu 1155 gat gcc aag aat cca ctt ggc tgc agc Asp Ala Lys Asn Pro Leu Gly Cys Ser 1160 agc tgc Ser Cys 1165 tat tgc ttc Tyr Cys Phe ggc act Gly Thr 1170 act acc cag Thr Thr Gin tgc tct gaa gca Cys Ser Glu Ala 1175 aaa gga ctg atc Lys Gly Leu Ile 1180 cgg acg tgg gtg act Arg Thr Trp Val Thr 1185 ctg aag gct gag cag acc att Leu Lys Ala Glu Gin Thr Ilie 1190 cta ccc ctg gta gat Leu Pro Leu Val Asp 1195 gag gct ctg cag Glu Ala Leu Gin 1200 42/61 cac acg acc His Thr Thr 1205 acc aag ggc att gtt Thr Lys Gly Ile Val 1210 ttt caa cat cca gag att gtt gcc Phe Gin His Pro Glu Ile Val Ala 1215 cac atg His Met 1220 gac ctg atg Asp Leu Met aga gaa Arg Glu 1225 gat ctc cat ttg gaa Asp Leu His Leu Glu 1230 cct ttt tat tgg Pro Phe Tyr Trp 1235 aaa ctt cca gaa caa ttt gaa gga aag aag ttg atg gcc tat Lys Leu Pro Glu Gin Phe Glu Gly Lys Lys Leu Met Ala Tyr 1240 1245 ggg ggc Gly Gly 1250 aaa ctc aag Lys Leu Lys tat gca Tyr Ala 1255 ate tat ttc Ile Tyr Phe gag get Glu Ala 1260 cgg gaa gaa Arg Glu Glu aca ggt ttc Thr Gly Phe 1265 tct aca tat aat Ser Thr Tyr Asn 1270 get aga att atc Ala Arg Ile Ile 1285 cct caa gtg Pro Gin Val ate att Ile Ile 1275 cga ggt ggg Arg Gly Gly aca cct act cat Thr Pro Thr His 1280 gtc agg cat atg gct Val Arg His Met Ala 1290 get cct ctg att ggc caa ttg Ala Pro Leu Ile Gly -in Leu 1295 aca agg Thr Arg 1300 cat gaa att His Glu Ile gaa atg Glu Met 1305 aca gag aaa Thr Glu Lys gaa tgg Glu Trp 1310 aaa tat tat ggg Lys Tyr Tyr Gly 1315 gat gat cct cga Asp Asp Pro Arg gtc cat Val His 1320 aga act gtg Arg Thr Val acc cga Thr Arg 1325 gaa gac ttc Glu Asp Phe ttg gat Leu Asp 1330 3706 3754 3802 3850 3898 3946 3994 4042 4090 4138 4186 4234 4282 4330 4378

S

ata cta tat Ile Leu Tyr gat att cat Asp Ile His 1335 tac att ctt ate aaa Tyr Ile Leu Ile Lys 1340 gct act tat gga aat Ala Thr Tyr Gly Asn 1345 ttc atg cga caa Phe Met Arg Gin 1350 age agg att Ser Arg Ile tct gaa Ser Glu 1355 atc tca atg Ile Ser Met gag gta get gaa Glu Val Ala Glu 1360 caa gga cgt Gin Gly Arg 1365 gga aca aca Gly Thr Thr atg act Met Thr 1370 cct cca get Pro Pro Ala gac ttg Asp Leu 1375 att gaa aaa Ile Glu Lys

S

S S *9 tgt gat Cys Asp 1380 tgt ccc ctg Cys Pro Leu ggc tat Gly Tyr 1385 tct ggc ctg Ser Gly Leu tec tgt Ser Cys 1390 gag gca tgc Glu Ala Cys ttg Leu 1395 ccg gga ttt tat Pro Gly Phe Tyr cga ctg Arg Leu 1400 cgt tct caa cca ggt ggc cgc acc cct gga Arg Ser Gin Pro Gly Gly Arg Thr Pro Gly 1405 1410 cca ace ctg Pro Thr Leu ggc acc tgt Gly Thr Cys 1415 gtt cca tgt caa tgt Val Pro Cys Gin Cys 1420 aat gga cac age age Asn Gly His Ser Ser 1425 ctg tgt gac cct gaa aca tcg Leu Cys Asp Pro Glu Thr Ser 1430 ata tgc cag aat tgt Ile Cys Gin Asn Cys 1435 caa cat cac act Gin His His Thr 1440 i IL

I,

43/61 got ggt gac Ala Gly Asp 1445 ttc tgt gaa cga tgt got ott gga tao tat gga att gto Phe Cys Giu Arg Cys Ala Leu Gly Tyr Tyr Gly Ile Val 1450 1455 aag gga Lys Gly 1460 ttg oca aat gao Leu Pro Asn Asp 1465 tgt oag oaa tgt goo tgc cct Cys Gin Gin Cys Ala Cys Pro 1470 ccc tot tgt gto goa gaa gga Pro Ser Cys Val Ala Glu Gly 1485 too agt aao aat Ser Ser Asn Asn tto ago Phe Ser 1480 otg att tct Leu Ile Ser 1475 ott gao gao Leu Asp Asp 1490 tao tgt gaa Tyr Cys Glu 1505 tac cgc tgo Tyr Arg Cys acg got tgt cca ogg Thr Ala Cys Pro Arg 1495 gga tat Gly Tyr 1500 gaa ggc cag Glu Gly Gin agg tgt goo cct Arg Cys Ala Pro 1510 ggo tat act Gly Tyr Thr ggo agt Gly Ser 1515 cca ggo aao Pro Gly Asn cct gga ggo too Pro Gly Gly Ser 1520 tgc caa gaa Cys Gin Glu 1525 tgt gag tgt Cys Giu Cys gat cc Asp Pro 1530 tat ggo toa Tyr Gly Ser otg cct Leu Pro 1535 gtg ccc tgt Val Pro Cys gac cct gto aca Asp Pro Val Thr 1540 aag tgt gao ggo Lys Cys Asp Gly gga tto tgo acg Gly Phe Cys Thr 1545 tgc oga cct gga gco aog gga agg Cys Arg Pro Gly Ala Thr Gly Arg 1550 1555 tgo aag Cys Lys 1560 cac tgg oat His Trp His gca cgc Ala Arg 1565 gag ggo tgg Glu Gly Trp gag tgt Glu Cys 1570 4426 4474 4522 4570 4618 4666 4714 4762 4810 4858 4906 4954 5002 5050 5098 aI 'i d .4 gtt ttt tgt Val Phe Cys gga gat Gly Asp 1575 gag tgo act Glu Cys Thr ggc ctt Gly Leu 1580 ott otc ggt Leu Leu Gly gao ttg got Asp Leu Ala 1585 cgc ctg gag oag atg Arg Leu Glu Gin Met 1590 gto atg ago ato aao Val Met Ser Ile Asn 1595 otc act ggt ccg otg cct Leu Thr Gly Pro Leu Pro 1600 gcg cca tat Ala Pro Tyr 1605 aaa atg otg Lys Met Leu tat ggt Tyr Gly 1610 ott gaa aat Leu Glu Asn atg act Met Thr 1615 oag gar cta Gin Glu Leu aag cac Lys His 1620 ttg otg toa Leu Leu Ser cct oag Pro Gin 1625 ogg goo cca Arg Ala Pro gag agg Glu Arg 1630 ott att oag Leu Ile Gin otg Leu 1635 gca gag ggo aat Ala Glu Gly Asn otg aat aca Leu Asn Thr 1640 otc gtg aoo gaa Leu Val Thr Glu 1645 ac agg got Thr Arg Ala ac aaa gtg Thr Lys Val 1655 aca goa gat ggo gag Thr Ala Asp Gly Glu 1660 aga goa aag too ctg Arg Ala Lys Ser Leu 1675 atg aao gag otg otg Met Asn Glu Leu Leu 1650 oag aoo gga oag gat Gin Thr Gly Gin Asp 1665 gga gaa tto att aag Gly Giu Phe Ile Lys 1680 got gag agg aoo aao aca Ala Glu Arg Thr Asn Thr 1670 9,J C) I, I'B (I~c 45/61 gcc ttc aaa Ala Phe Lys 1925 gct tac agc aat att Ala Tyr Ser Asn Ile 1930 aag gac tat att gat Lys Asp Tyr Ile Asp 1935 gaa get gag Glu Ala Glu aaa gtt Lys Val 1940 gcc aaa gaa Ala Lys Glu gcc aaa Ala Lys 1945 gat ctt gca cat gaa Asp Leu Ala His Glu 1950 get aca aaa Ala Thr Lys ctg Leu 1955 gca aca ggt cct Ala Thr Gly Pro cgg ggt Arg Gly 1960 tta tta aag Leu Leu Lys gaa gat Glu Asp 1965 gcc aaa ggc Ala Lys Gly tgt ctt Cys Leu 1970 cag aaa agc Gin Lys Ser ttc agg Phe Arg 1975 att ctt aac Ile Leu Asn gaa gcc Glu Ala 1980 aag aag tta Lys Lys Leu gca aat gat Ala Asn Asp 1985 gta aaa gaa aat Val Lys Glu Asn 1990 aat get gat get Asn Ala Asp Ala 2005 gaa gac cat Glu Asp His cta aat Leu Asn 1995 ggc tta aaa Gly Leu Lys acc agg ata gaa Thr Arg Ile Glu 2000 aga aat ggg gat ctc Arg Asn Gly Asp Leu 2010 ttg aga act ttg aat gac act Leu Arg Thr Leu Asn *sp Thr 2015 ttg gga Leu Gly 2020 aag tta tea Lys Leu Ser gct att Ala Ile 2025 cca aat gat Pro Asn Asp aca get Thr Ala 2030 get aaa ctg Ala Lys Leu caa Gin 2035 gct gtt aag gac Ala Val Lys Asp aaa gcc Lys Ala 2040 aga caa gcc Arg Gin Ala aac gac Asn Asp 2045 aca gct aaa Thr Ala Lys gat gta Asp Val 2050 5866 5914 5962 6010 6058 6106 6154 6202 6250 6298 6346 6394 6442 6490 6538 a.

a a..

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ctg gca cag Leu Ala Gin att aca gag Ile Thr Glu 2055 ctc cac cag aac ctc Leu His Gin Asn Leu 2060 gat ggc ctg aag aag Asp Gly Leu Lys Lys 2065 aat tac aat aaa Asn Tyr Asn Lys 2070 cta gca gac Leu Ala Asp age gtc Ser Val 2075 gcc aaa acg Ala Lys Thr aat gct gtg gtt Asn Ala Val Val 2080 aaa gat cct Lys Asp Pro 2085 tec aag aac Ser Lys Asn aaa ate Lys Ile 2090 att gcc gat Ile Ala Asp gca gat Ala Asp 2095 gcc act gtc Ala Thr Val ctc aaa ccc Leu Lys Pro 2115 r r r r r c aaa aat Lys Asn 2100 tta gaa cag Leu Glu Gin gaa get gac Glu Ala Asp 2105 cgg cta ata gat aaa Arg Leu Ile Asp Lys 2110 atc aag gaa ctt Ile Lys Glu Leu gag gat Glu Asp 2120 aac cta aag Asn Leu Lys aaa aac atc tct gag Lys Asn Ile Ser Glu 2125 ata aag Ile Lys 2130 gaa ttg ata Glu Leu Ile aac caa get Asn Gin Ala 2135 cgg aaa caa gcc aat Arg Lys Gin Ala Asn 2140 tct ate aaa gta tct Ser Ile Lys Val Ser 2145 gtg tct tea gga ggt gac tgc Val Ser Ser Gly Gly Asp Cys 2150 att cga aca tac aaa Ile Arg Thr Tyr Lys 2155 cca gaa atc aag Pro Glu Ile Lys 2160

X

7 44/61 gag ctt gcc cgg gat gca gaa got gta aat gaa aaa gct ata aaa cta Glu Leu Ala Arg Asp Ala Glu Ala Val Asn Glu Lys Ala Ile Lys Leu 1685 1690 1695 aat gaa Asn Glu 1700 act cta gga Thr Leu Gly act cga Thr Arg 1705 gac gag gcc Asp Glu Ala ttt gag Phe Glu 1710 aga aat ttg Arg Asn Leu gaa Glu 1715 ggg ott cag aaa Gly Leu Gln Lys gag att Glu Ile 1720 gac cag atg Asp Gln Met att aaa Ile Lys 1725 gaa ctg agg Glu Leu Arg agg aaa Arg Lys 1730 aat cta gag Asn Leu Glu aca caa Thr Gln 1735 aag gaa att Lys Glu Ile got gaa Ala Glu 1740 gat gag ttg Asp Glu Leu gta got gca Val Ala Ala 1745 gaa goc ott ctg Glu Ala Leu Leu 1750 aaa aaa gtg Lys Lys Val aag aag Lys Lys 1755 otg ttt gga Leu Phe Gly gag tcc cgg ggg Glu Ser Arg Gly 1760 gaa aat gaa Glu Asn Glu 1765 gaa atg gag Glu Met Glu aag gat Lys Asp 1770 oto cgg gaa Leu Arg Glu aaa ctg Lys Leu 1775 got gao tao Ala Asp Tyr aaa aac Lys Asn 1780 aaa gtt gat Lys Val Asp gat got Asp Ala 1785 tgg gao ott Trp Asp Leu ttg aga Leu Arg 1790 gaa goo aoa Glu Ala Thr gat Asp 1795 aaa ato aga gaa Lys Ile Arg Glu got aat Ala Asn 1800 cgc ota ttt Arg Leu Phe goa gta Ala Val 1805 aat. oag aaa Asn Gln Lys aac atg Asn Met 1810 5146 5194 5242 5290 5338 5386 5434 5482 5530 5578 5626 5674 5722 5770 5818 9 9 act gca ttg Thr Ala Leu gag aaa Glu Lys 1815 aag aag gag Lys Lys Glu got gtt Ala Val 1820 gag ago ggc Glu Ser Gly aaa oga oaa Lys Arg Gln 1825 att gag aao act Ile Glu Asn Thr 1830 tta aaa gaa Leu Lys Glu ggo aat Gly Asn 1835 gao ata oto gat gaa goc aao Asp Ile Leu Asp Glu Ala Asn 1840 ogt ott Arg Leu 1845 oaa act Gln Thr 1860 gca gat gaa ato Ala Asp Glu Ile aao too Asn Ser 1850 ato ata gao Ile Ile Asp tatfgtt Tyr Val 1855 gaa gao ato Glu Asp Ile .9 a 9 4.

9a aaa ttg oca Lys Leu Pro cot atg Pro Met 1865 tot gag gag Ser Glu Glu ott aat Leu Asn 1870 gat aaa ata Asp Lys Ile gat Asp 1875 gao oto too oaa Asp Leu Ser Gln gaa ata Glu Ile 1880 aag gao agg Lys Asp Arg aag ott Lys Leu 1885 gct gag aag Ala Glu Lys gtg too Val Ser 1890 cag got gag Gln Ala Glu ago cac goa Ser His Ala 1895 got cag ttg aat gao Ala Gln Leu Asn Asp 1900 toa tot got gto ott Ser Ser Ala Val Leu 1905 gat gga ato ott gat gag got Asp Gly Ile Leu Asp Glu Ala 1910 aaa aao ato too tto Lys Asn Ile Ser Phe 1915 aat gcc act goa Asn Ala Thr Ala 1920 'A<.7

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46/61 aaa gga agt Lys Gly Ser 2165 tac aat aat att gtt Tyr Asn Asn Ile Val 2170 gtc aac gta aag aca Val Asn Val Lys Thr 2175 get gtt get Ala Val Ala gat aac Asp Asn 2180 ctc ctc ttt Leu Leu Phe tat ctt Tyr Leu 2185 gga agt gcc Gly Ser Ala aaa ttt Lys Phe 2190 att gac ttt Ile Asp Phe ctg Leu 2195 gct ata gaa atg Ala Ile Glu Met cgt aaa ggc Arg Lys Gly 2200 aaa gtc age ttc ctc Lys Val Ser Phe Leu 2205 tgg gat gtt gga Trp Asp Val Gly 2210 tct gga gtt Ser Gly Val gga cgt Gly Arg 2215 gta gag tac Val Glu Tyr cca gat Pro Asp 2220 ttg act att Leu Thr Ile gat gac tca Asp Asp Ser 2225 tat tgg tac cgt Tyr Trp Tyr Arg 2230 tct gtg aga gcc Ser Val Arg Ala 2245 ate gta gca Ile Val Ala tca aga Ser Arg 2235 act ggg aga Thr Gly Arg aat gga act att Asn Gly Thr Ile 2240 ctg gat gga ccc aaa Leu Asp Gly Pro Lys 2250 gcc age att gtg ccc age aca Ala Ser Ile Val Pro -Ser Thr 2255 cac cat His His 2260 tcg acg tct Ser Thr Ser cct cca Pro Pro 2265 ggg tac acg Gly Tyr Thr att cta Ile Leu 2270 gat gtg gat Asp Val Asp gca Ala 2275 aat gca atg ctg Asn Ala Met Leu ttt gtt Phe Val 2280 ggt ggc ctg Gly Gly Leu act ggg Thr Gly 2285 aaa tta aag Lys Leu Lys aag get Lys Ala 2290 6586 6634 6682 6730 6778 6826 6874 6922 6970 7018 7066 7114 7162 7210 7258 4 *r gat gct gta Asp Ala Val cgt gtg att Arg Val Ile 2295 aca ttc act ggc tgc Thr Phe Thr Gly Cys 2300 atg gga gaa aca tac Met Gly Glu Thr Tyr 2305 ttt gac aac aaa Phe Asp Asn Lys 2310 cct ata ggt Pro Ile Gly ttg tgg Leu Trp 2315 aat ttc cga Asn Phe Arg gaa aaa gaa ggt Glu Lys Glu Gly 2320 gac tgc aaa Asp Cys Lys 2325 gga tgc act Gly Cys Thr gtc agt Val Ser 2330 cct cag gtg gaa gat agt gag ggg Pro Gin Val Glu Asp Ser Glu Gly 2335 .4 act att Thr Ile 2340 caa ttt gat Gln Phe Asp gga gaa Gly Glu 2345 ggt tat gca Gly Tyr Ala ttg gtc Leu Val 2350 age cgt ccc Ser Arg Pro att Ile 2355 cgc tgg tac ccc Arg Trp Tyr Pro aac atc Asn Ile 2360 tec act gtc Ser Thr Val atg ttc Met Phe 2365 aag ttc aga Lys Phe Arg aca ttt Thr Phe 2370 tct tcg agt Ser Ser Ser gct ctt ctg Ala Leu Leu 2375 atg tat ctt gcc aca Met Tyr Leu Ala Thr 2380 cga gac ctg aga gat Arg Asp Leu Arg Asp 2385 ttc atg agt gtg gag ctc act Phe Met Ser Val Glu Leu Thr 2390 gat ggg cac ata aaa Asp Gly His Ile Lys 2395 gtc agt tac gat Val Ser Tyr Asp 2400 -J i

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47/61 ctg ggc tca Leu Gly Ser 2405 gga atg gct tcc gtt Gly Met Ala Ser Val 2410 gtc agc aat caa aac Val Ser Asn Gin Asn 2415 cat aat gat His Asn Asp ggg aaa Gly Lys 2420 tgg aaa tca Trp Lys Ser ttc act Phe Thr 2425 ctg tca aga Leu Ser Arg att caa Ile Gin 2430 aaa caa gcc aat Lys Gin Ala Asn 2435 ata tca att gta Ile Ser Ile Val gat ata Asp Ile 2440 gat act aat Asp Thr Asn cag gag Gin Glu 2445 gag aat ata Glu Asn Ile gca act Ala Thr 2450 tcg tct tct Ser Ser Ser gga aac Gly Asn 2455 aac ttt ggt Asn Phe Gly ctt gac Leu Asp 2460 ttg aaa gca Leu Lys Ala gat gac aaa Asp Asp Lys 2465 ata tat ttt ggt ggc Ile Tyr Phe Gly Gly 2470 ctg cca acg ctg aga Leu Pro Thr Leu Arg 2475 aac ttg agt atg aaa gca Asn Leu Ser Met Lys Ala 2480 agg cca gaa Arg Pro Glu 2485 gta aat ctg Val Asn Leu aag aaa Lys Lys 2490 tat tcc ggc Tyr Ser Gly tgc ctc Cys Leu 2495 aaa gat att Lys Asp Ile gaa att Glu Ile 2500 tca aga act Ser Arg Thr ccg tac Pro Tyr 2505 aat ata ctc Asn Ile Leu agt agt Ser Ser 2510 ccc gat tat Pro Asp Tyr gtt Va1 2515 ggt gtt acc aaa gga tgt tcc ctg gag aat gtt tac aca gtt Gly Val Thr Lys Gly Cys Ser Leu Giu Asn Val Tyr Thr Val 2520 2525 agc ttt Ser Phe 2530 7306 7354 7402 7450 7498 7546 7594 7642 7690 7738 7786 7834 7882 7930 7978

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a cct aag cct Pro Lys Pro ggt ttt Gly Phe 2535 gtg gag ctc Val Giu Leu tcc cct Ser Pro 2540 gtg cca att Val Pro Ile gat gta gga Asp Val Gly 2545 aca gaa atc aac Thr Glu Ile Asn 2550 ctt ttg gga agt Leu Leu Gly Ser 2565 ctg tca ttc Leu Ser Phe agc acc Ser Thr 2555 aag aat gag Lys Asn Glu tcc ggc atc att Ser Gly Ile Ile 2560 gga ggg aca cca gca Gly Gly Thr Pro Ala 2570 cca cct agg aga aaa cga agg Pro Pro Arg Arg Lys Arg Arg 2575

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cag act Gin Thr 2580 gga cag gcc Gly Gin Ala tat tat Tyr Tyr 2585 gta ata ctc Val Ile Leu ctc aac Leu Asn 2590 agg ggc cgt Arg Gly Arg ctg Leu 2595 gaa gtg cat ctc Glu Val His Leu tcc aca Ser Thr 2600 ggg gca cga Gly Ala Arg aca atg Thr Met 2605 agg aaa att Arg Lys Ile gtc atc Val Ile 2610 aga cca gag Arg Pro Glu ccg aat ctg ttt cat Pro Asn Leu Phe His 2615 gat gga aga Asp Gly Arg 2620 gaa cat tcc gtt cat Glu His Ser Val His 2625 gta gag cga act aga ggc atc Val Glu Arg Thr Arg Gly Ile 2630 ttt aca gtt caa gtg Phe Thr Val Gin Val 2635 gat gaa aac aga Asp Glu Asn Arg 2640 'v :9 I

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i i> _C 48/61 aga tao atg Arg Tyr Met 2645 caa aao otg Gin Asn Leu aca gtt Thr Val 2650 gaa cag Oct ato gaa Giu Gin Pro Ile Giu 2655 gtt aaa aag Val Lys Lys ott ttc Leu Phe 2660 gtt ggg ggt Val Gly Gly got cca Ala Pro 2665 cot gaa ttt Pro Giu Phe caa cct Gin Pro 2670 too coa oto Ser Pro Leu aga Arg 2675 aat att cot cot Asn Ile Pro Pro ttt gaa Phe Giu 2680 ggo tgC ata Gly Cys Ile tgg aat Trp Asn 2685 ctt gtt att Leu Val Ile aac tot Asn Ser 2690 gtc 000 atg Val Pro Met gac ttt Asp Phe 2695 gca agg cot Ala Arg Pro gtg tcc Val Ser 2700 ttc aaa aat Phe Lys Asn got gao at Ala Asp Ile 2705 ggt cgc tgt gcc Gly Arg Cys Ala 2710 oat cag aaa His Gin Lys oto ogt Leu Arg 2715 gaa gat gaa Giu Asp Giu gat gga goa got Asp Gly Ala Ala 2720 ooa got gaa Pro Ala Giu 2725 ata gtt ato Ile Val Ilie oag oot Gin Pro 2730 gag ooa gtt Glu Pro Val 000 aoo Pro Thr 2735 ooa goo ttt Pro-A*la Phe oot aog Pro Thr 2740 000 aoo ooa Pro Thr Pro gtt otg Val Leu 2745 aoa oat ggt Thr His Gly oot Lgt Pro Cys 2750 got goa gaa Ala Ala Giu toa Ser 2755 gaa ooa got ott Giu Pro Ala Leu ttg ata Leu Ile 2760 ggg ago aag Gly Ser Lys oag tto Gin Phe 2765 ggg ott toa Gly Leu Ser aga aao Arg Asn 2770 8026 8074 8122 8170 8218 8266 8314 8362 8410 8458 8506 8554 8602 8650 8698 4

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agt oao at Ser His Ile goa at Ala Ile 2775 goa ttt gat Ala Phe Asp gao aoo Asp Thr 2780 aaa gtt aaa Lys Val Lys aao ogt oto Asn Arg Leu 2785 aoa att gag Ltg Thr Ile Giu Leu 2790 gaa gta aga Giu Val Arg aoo gaa Thr Giu 2795 got gaa too Ala Giu Ser ggo ttg ott ttt Gly Leu Leu Phe 2800 tao atg got Tyr Met Ala 2805 gog ato aat Ala Ile Asn oat got His Ala 2810 gaL ttt goa Asp Phe Ala aoa gtt Thr Val 2815 oag otg aga Gin Leu Arg

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4*9 4 C C 4.

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aat gga Asn Gly 2820 ttg coo tao Leu Pro Tyr tto ago Phe Ser 2825 tat gao ttg Tyr Asp LeuC aat gat ggo Asn Asp Gly 2845 ggg agt Giy Ser ~830 ggg gao aoo Gly Asp Thr oao His 2835 aoo atg ato coo Thr Met Ilie Pro aco aaa ato Thr Lys Ile 2840 cag tgg oao aag Gln Trp His Lys att aag Ile Lys 2850 ata atg aga Ile Met Arg agt aag Ser Lys 2855 oaa gaa gga Gin Giu Gly att ott Ile Leu 2860 tat gta gaL Tyr Val Asp ggg got too Gly Ala Ser 2865 aao aga aoo ato agt Asn Arg Thr Ile Ser 2870 000 aaa aaa goo gao Pro Lys Lys Ala Asp 2875 ato otg gat gto gtg gga Ile Leu Asp Val Val Gly 2880

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7 49/61 atg ctg tat Met Leu Tyr 2885 gtt ggt ggg Val Gly Gly tta ccc Leu Pro 2890 atc aac tac act ace Ile Asn Tyr Thr Thr 2895 cga aga att Arg Arg Ile ggt cca Gly Pro 2900 gtg acc tat Val Thr Tyr agc att Ser Ile 2905 gat ggc tgc gtc agg Asp Gly Cys Val Arg 2910 gaa caa ccc acc tcc Glu Gin Pro Thr Ser 2925 aat etc cac atg Asn Leu His Met 2915 agc ttc cat gtt Ser Phe His Val 2930 gca gag gcc cct Ala Giu Aia Pro gcc gat ctg Ala Asp Leu 2920 ggg aca tgt Gly Thr Cys ttt gca Phe Ala 2935 aat gct cag Asn Ala Gin agg gga Arg Gly 2940 aca tat ttt Thr Tyr Phe gac gga acc Asp Gly Thr 2945 ggt ttt gcc aaa Gly Phe Ala Lys 2950 gta gaa ttt gaa Val Giu Phe Glu 2965 gca gtt ggt Ala Val Gly gga ttc Gly Phe 2955 aaa gtg gga Lys Val Gly ttg gac ctt ctt Leu Asp Leu Leu 2960 ttc gcg aca act aca Phe Ala Thr Thr Thr 2970 acg act gga gtt ctt ctg ggg Thr Thr Gly Val Leu Leu Gly 2975 atc agt Ile Ser 2980 agt caa aaa Ser Gin Lys atg gat Met Asp 2985 gga atg ggt Gly Met Gly att gaa Ile Glu 2990 atg att gat Met Ile Asp gaa Glu 2995 aag ttg atg ttt Lys Leu Met Phe cat gtg His Val 3000 gac aat ggt Asp Asn Gly gcg ggc Ala Gly 3005 aga ttc act Arg Phe Thr get gtc Ala Val 3010 a a 8746 8794 8842 8890 8938 8986 9034 9082 9130 9178 9226 9274 9322 9370 9429 9489 9534 tat gat gct Tyr Asp Ala ggg gtt Gly Val 3015 cca ggg cat Pro Gly His ttg tgt Leu Cys 3020 gat gga caa tgg cat aaa Asp Gly Gin Trp His Lys 3025 gtc act gcc aac Val Thr Ala Asn 3030 aag atc aaa Lys Ile Lys cac cgc His Arg 3035 att gag ctc Ile Glu Leu aca gtc gat ggg Thr Val Asp Gly 3040 aac cag gtg Asn Gin Val 3045 gaa gcc caa Glu Ala Gin age cca Ser Pro 3050 aac cca gca Asn Pro Ala tct aca Ser Thr 3055 tca gct gac Ser Ala Asp etc aag cag Leu Lys Gin 3075 a a a a.

a.

a aca aat Thr Asn 3060 gac cct gtg Asp Pro Val ttt gtt gga Phe Val Gly 3065 ggc ttc cca gat gac Gly Phe Pro Asp Asp 3070 ttt ggc cta aca Phe Gly Leu Thr acc agt Thr Ser 3080 att ccg ttc Ile Pro Phe cga ggt Arg Gly 3085 tgc atc aga tcc ctg Cys Ile Arg Ser Leu 3090 tta att ttg cca agg Leu Ile Leu Pro Arg 3105 aag ctc acc Lys Leu Thr aaa ggc aca Lys Gly Thr 3095 gca agc cac tgg agg Ala Ser His Trp Arg 3100 ccc tgg aac tgaggggcgt tcaacctgta tcatgcccag ccaactaata aaaataagtg Pro Trp taaceccagg aagagtctgt caaaacaagt atatcaagta aaacaaacaa atatatttta cctatatatg ttaattaaac taatttgtgc atgtacatag aattc '9 C

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Thr 465 Cys His Asp Cys Pro 545 Glu Gin Giu Giu Giu 625 Ser Leu Giu Ser Val1 705 Ser Tyr Asp Thr Cys 785 Arg Gly Cys Arg Gin 865 Gly Arg His Phe Ile Lys Trp Leu Ser 530 Gly Ala Asp Gly Tyr 610 Lys Arg Ser Lys Ser 690 Leu Gly Arg Val Gly 770 Asp Thr Cys Asn Gin 850 Pro Giu Cys Cys Ala Pro Arg Gly Gly 515 Cys Tyr Asn Arg Ala 595 Asp Ala Cys Pro Gly 675 Asp Met Asp Cys Cys 755 Leu Pro Cys Lys Pro 835 Cys Cys Cys Leu Arg 915 Gly Leu Leu 500 Gly Arg Tyr Leu Ile 580 Tyr Ile Val Gly Gly 660 Thr Ser Pro Gly Leu 740 Arg Ala Asn Asn Pro 820 Val Asp Gin Leu Ala 900 Pro Gly Val1 485 Ser Ala Pro Phe Gly 565 Pro Leu Leu Ile Asn 645 Ser Asn Asp Tyr Val 725 Giu Asn Cys Gly Arg 805 Cys Thr Arg Cys Asn 885 Gly Cys Asn 470 Thr Asn Leu His Ala 550 Pro Ser Giu Ile Thr 630 Thr Arg Tyr Val1 Cys 710 Val1 Asn Ile Giu Gly 790 Cys Giu Gly Cys Asn 870 Cys Tyr Pro Cys 455 Pro Gly Asp Asn Met 535 Thr Gly Trp Phe Arg 615 Val Ile Tyr Thr Giu 695 Lys Thr Ser Ile Cys 775 Gin Ala Cys Gin Leu 855 Gly Gin Tyr Cys Cys Gin Leu Asn 520 Ile Leu Val1 Thr Phe 600 Tyr Gin Pro Val Val 680 Ser Ser Asn Arg Phe 760 Asp Cys Pro His Cys 840 Pro His Asp Gly Pro 920 Asp His Asp 505 Ser Gly Asp Ser Gly 585 Ile Giu Arg Asp Val1 665 Arg Pro Leu Ser Ser 745 Ser Pro Gin Gly Leu 825 His Gly Ala Tyr Asp 905 Asp Ser Cys 490 Gly Cys Arg His Ile 570 Ala Asp Pro Pro Asp 650 Leu Leu Tyr Asp Ala 730 Val1 Ile Gin Cys Thr 810 Gin Cys His Asp Thr 890 Pro Gly Giu 475 Asp Cys Phe Gin Tyr 555 Val1 Gly Asn Gin Gly 635 Asp Pro Giu Thr Ile 715 Trp Val1 Ser Gly Arg 795 Phe Gly Phe Trp Asp 875 Met Ile Pro Lys Ser Cys Ala Cys 460 Thr Gin Arg Ala Cys 540 Leu Giu Phe Ile Leu 620 Arg Asn Arg Leu Leu 700 Phe Giu Lys Al a Ser 780 Pro Gly Ser Gin Gly 860 Cys Gly Ile Asp Gly Cys Pro Giu 525 Asn Tyr Arg Val1 Pro 605 Pro Ile Gin Pro Pro 685 Ile Thr Thr Thr Leu 765 Leu Asn Phe Val1 Gly 845 Phe Asp His Gly Ser 925 His Leu Cys 510 Ser Giu Giu Gin Arg 590 Tyr Asp Pro Val Val1 670 Gin Asp Val1 Phe Pro 750 Leu Ser Val1 Gly Asn 830 Val Pro Pro Asn Ser 910 Gly Cys Pro 495 Asp Gly Val1 Ala Tyr 575 Val1 Ser His Thr Val 655 Cys Tyr Ser Gly Gin 735 Met His Ser Val Pro 815 Ala Tyr Ser Val1 Cys 895 Gly Arg 445 Asn Pro Leu Gly Tyr 480 Giu Cys Gin Giu Giu 560 Ile Pro Met Trp Ser 640 Ser Phe Thr Leu Gly 720 Arg Thr Gin Val Gly 800 Ser Phe Ala Cys Thr 880 Giu Asp Gin Arg Ser Cys Tyr Gin Asp Pro Val Thr Leu Gin Leu Ala Cys 7L t 52/61 930 935 940 Val Cys Asp Pro Gly Tyr Ile Gly Ser Arg Cys Asp Asp Cys Ala Ser 945 950 955 960 Gly Tyr Phe Gly Asn Pro Ser Glu Val Gly Gly Ser Cys Gin Pro Cys 965 970 975 Gin Cys His Asn Asn Ile Asp Thr Thr Asp Pro Glu Ala Cys Asp Lys 980 985 990 Glu Thr Gly Arg Cys Leu Lys Cys Leu Tyr His Thr Glu Gly Glu His 995 1000 1005 Cys Gin Phe Cys Arg Phe Gly Tyr Tyr Gly Asp Ala Leu Arg Gin Asp 1010 1015 1020 Cys Arg Lys Cys Val Cys Asn Tyr Leu Gly Thr Val Gin Glu His Cys 1025 1030 1035 1040 Asn Gly Ser Asp Cys Gin Cys Asp Lys Ala Thr Gly Gin Cys Leu Cys 1045 1050 1055 Leu Pro Asn Val Ile Gly Gin Asn Cys Asp Arg Cys Ala Pro Asn Thr 1060 1065 1070 Trp Gin Leu Ala Ser Gly Thr Gly Cys Asp Pro Cys Asn Cys Asn Ala 1075 1080 1085 Ala His Ser Phe Gly Pro Ser Cys Asn Glu Phe Thr Gly Gin Cys Gin 1090 1095 1100 Cys Met Pro Gly Phe Gly Gly Arg Thr Cys Ser Glu Cys Gin G4u Leu 1105 1110 1115 1120 Phe Trp Gly Asp Pro Asp Val Glu Cys Arg Ala Cys Asp Cys Asp Pro 1125 1130 1135 Arg Gly Ile Glu Thr Pro Gin Cys Asp Gin Ser Thr Gly Gin Cys Val 1140 1145 1150 Cys Val Glu Gly Val Glu Gly Pro Arg Cys Asp Lys Cys Thr Arg Gly 1155 1160 1165 Tyr Ser Gly Val Phe Pro Asp Cys Thr Pro Cys His Gin Cys Phe Ala 1170 1175 1180 Leu Trp Asp Val Ile Ile Ala Glu Leu Thr Asn Arg Thr His Arg Phe 1185 1190 1195 1200 Leu Glu Lys Ala Lys Ala Leu Lys Ile Ser Gly Val Ile Gly Pro Tyr 1205 1210 1215 S Arg Glu Thr Val Asp Ser Val Glu Arg Lys Val Ser Glu Ile Lys Asp 1220 1225 1230 Ile Leu Ala Gin Ser Pro Ala Ala Glu Pro Leu Lys Asn Ile Gly Asn 1235 1240 1245 Leu Phe Glu Glu Ala Glu Lys Leu Ile Lys Asp Val Thr Glu Met Met 1250 1255 1260 Ala Gin Val Glu Val Lys Leu Ser Asp Thr Thr Ser Gin Ser Asn Ser 1265 1270 1275 1280 Thr Ala Lys Glu Leu Asp Ser Leu Gin Thr Glu Ala Glu Ser Leu Asp 1285 1290 1295 Asn Thr Val Lys Glu Leu Ala Glu Gin Leu Glu Phe Ile Lys Asn Ser 1300 1305 1310 Asp Ile Arg Gly Ala Leu Asp Ser Ile Thr Lys Tyr Phe Gin Met Ser 1315 1320 1325 Leu Glu Ala Glu Glu Arg Val Asn Ala Ser Thr Thr Glu Pro Asn Ser T 1330 1335 1340 Thr Val Glu Gin Ser Ala Leu Met Arg Asp Arg Val Glu Asp Val Met 1345 1350 1355 1360 S Met Glu Arg Glu Ser Gin Phe Lys Glu Lys Gin Glu Glu Gin Ala Arg 1365 1370 1375 Leu Leu Asp Glu Leu Ala Gly Lys Leu Gin Ser Leu Asp Leu Ser Ala 1380 1385 1390 Ala Ala Glu Met Thr Cys Gly Thr Pro Pro Gly Ala Ser Cys Ser Glu 1395 1400 1405 Thr Glu Cys Gly Gly Pro Asn Cys Arg Thr Asp Glu Gly Glu Arg Lys 1410 1415 1420 Cys Gly Gly Pro Gly Cys Gly Gly Leu Val Thr Val Ala His Asn Ala 53/61 1425 1430 1435 1440 Trp Gin Lys Ala Met Asp Leu Asp Gin Asp Val Leu Ser Ala Leu Ala 1445 1450 1455 Glu Val Giu Gin Leu Ser Lys Met Val Ser Giu Ala Lys Leu Arg Ala 1460 1465 1470 Asp Giu Ala Lys Gin Ser Ala Giu Asp Ile Leu Leu Lys Thr Asn Ala 1475 1480 1485 Thr Lys Giu Lys Met Asp Lys Ser Asn Giu Giu Leu Arg Asn Leu Ile 1490 1495 1500 Lys Gin Ile Arg Asn Phe Leu Thr Gin Asp Ser Ala Asp Leu Asp Ser 1505 1510 1515 1520 Ile Giu Ala Val Ala Asn Giu Val Leu Lys Met Giu Met Pro Ser Thr 1525 1530 1535 Pro Gin Gin Leu Gin Asn Leu Thr Giu Asp Ile Arg Giu Arg Val Giu 1540 1545 1550 Ser Leu Ser Gin Val Giu Val Ile Leu Gin His Ser Ala Ala Asp Ile 1555 1560 1565 Ala Arg Ala Giu Met Leu Leu Giu Giu Ala Lys Arg Ala Ser Lys Ser 1570 1575 1580 Ala Thr Asp Val Lys Val Thr Ala Asp Met Val Lys Giu Ala Leu Giu 1585 1590 1595 1600 Giu Ala Giu Lys Ala Gin Val Ala Ala Giu Lys Ala Ile Lys Gin Ala 1605 1610 1615 Asp Giu Asp Ile Gin Gly Thr Gin Asn Leu Leu Thr Ser Ile Giu Ser 1620 1625 1630 Giu Thr Ala Ala Ser Giu Giu Thr Leu Phe Asn Ala Ser Gin Arg Ile 1635 1640 1645 Ser Giu Leu Giu Arg Asn Val Giu Giu Leu Lys Arg Lys Ala Ala Gin 1650 1655 1660 Asn Ser Gly Giu Ala Giu Tyr Ile Giu Lys Val Val Tyr Thr Val Lys Gl e5 Ala Giu Asp ValLys Lys Thr Leu Asp 5 Gly Giu Leu Asp 1680 *1685 1690 1695 Lys Tyr Lys Lys Val Giu Asn Leu Ile Ala Lys Lys Thr Giu Giu Ser :Aa1700 1705 1710 AaAsp Ala Arg Arg Lys Ala Giu Met Leu Gin Asn Giu Ala Lys Thr Lu 1715 1720 1725 LuLeu Ala Gin Ala Asn Ser Lys Leu Gin Leu Leu Lys Asp Leu Giu 1730 1735 1740 Arg Lys Tyr Giu Asp Asn Gin Arg Tyr Leu Giu Asp Lys Ala Gin Giu 1745 1750 1755 1760 Leu Ala Arg Leu Giu Gly Giu Val Arg Ser Leu Leu Lys Asp Ile Ser 1765 1770 1775 Gin Lys Val Ala Val Tyr Ser Thr Cys Leu <20>1780 1785 210 <211> 5613 <212> DNA <213> Homno sapiens <220> <221> CDS <222> (118) (5475) <400> cccggagcag ggcgagagct cgcgtcgccg gaaaggaaga cgggaagaaa gggcaggcgg ctcggcgggc gtcttctcca ctcctctgcc gcgtccccgt ggctgcaggg agccggc atg 120 Met 54/61 ggg ctt ctc cag ttg cta gct ttc Gly Leu Leu Gin Leu Leu Ala Phe ttc tta gcc ctg Phe Leu Ala Leu tgc aga gcc Cys Arg Ala gca gaa ggc Ala Giu Gly cga gtg cgc Arg Val Arg gct cag gaa ccc Ala Gin Glu Pro ttc agc tac ggc Phe Ser Tyr Gly tgc Cys agc tgc tat ccc gcc acg ggc gac ctt ctc atc ggc Ser Cys Tyr Pro Ala Thr Gly Asp Leu Leu Ile Gly 40 cga gca cag aag Arg Ala Gin Lys ctt Leu tcg gtg acc tcg Ser Val Thr Ser tgc ggg ctg cac Cys Gly Leu His ccc gaa ccc tac Pro Glu Pro Tyr atc gtc agc cac Ile Val Ser His t tg Leu cag gag gac aaa Gin Giu Asp Lys aaa Lys 75 tgc ttc ata tgc Cys Phe Ile Cys aat tcc Asn Ser caa gat cct Gin Asp Pro aat gtg gtc Asn Vai Val 100 cat gag acc ctg His Giu Thr Leu cct gac agc cat Pro Asp Ser His ctc gaa Leu Ile Glu tgg tgg caa Trp Trp Gin act aca ttt gct Thr Thr Phe Ala cca Pro 105 aac cgc ctt aag Asn Arg Leu Lys C V

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tct gaa Ser Giu 115 aat ggt gtg gaa Asn Gly Val Giu gta act atc caa Val Thr Ilie Gin c tg Leu 125 gat ttg gaa gca Asp Leu Giu Ala 408 456 504 552 600 gaa Glu 130 ttc cat ttt act Phe His Phe Thr cat His 135 ctc ata atg act Leu Ile Met Thr t tc Phe 140 aag aca ttc cgt Lys Thr Phe Arg cca Pro 145 gct gct atg ctg Ala Ala Met Leu gaa cga tcg tcc Giu Arg Ser Ser gac Asp 155 ttt ggg aaa acc Phe Gly Lys Thr tgg ggt Trp Gly 160 gtg tat aga Val Tyr Arg tca act ggc Ser Thr Gly 180 ttc gcc tat gac Phe Ala Tyr Asp tgt Cys 170 gag gcc tcg ttt Giu Ala Ser Phe cca ggc att Pro Gly Ile 175 gat tct cga Asp Ser Arg ccc atg aaa aaa Pro Met Lys Lys gat gac ata att Asp Asp Ilie Ile tgt Cys 190 tat tct Tyr Ser 195 gac att gaa ccc Asp Ile Glu Pro tca Ser 200 act gaa gga gag Thr Giu Gly Giu gtg Vai 205 ata ttt cgt gct Ile Phe Arg Ala tta Leu 210 gat cct gct ttc Asp Pro Ala Phe aaa Lys 215 ata gaa gat cct Ile Glu Asp Pro agc cca agg ata Ser Pro Arg Ile aat tta tta aaa Asn Leu Leu Lys acc aac ttg aga Thr Asn Leu Arg atc Ile 235 aag ttt gtg aaa ctg cat Lys Phe Vai Lys Leu His 840

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55/61 act ttg gga Thr Leu Giy tat tat tat Tyr Tyr Tyr 260 gat Asp 245 aac ctt ctg gat Asn Leu Leu Asp tcc Ser 250 agg atg gaa atc Arg Met Giu Ile aga gaa aag Arg Giu Lys 255 tgc ttc tgc Cys Phe Cys 888 936 gca gtt tat gat Ala Val Tyr Asp gtg gtt cga gga Val Val Arg Gly aat Asn 270 tat ggt Tyr Gly 275 cat gcc agc gaa His Ala Ser Glu tgt Cys 280 gcc cct gtg gat Ala Pro Val Asp gga Gly 285 ttc aat gaa gaa Phe Asn Giu Glu gtg Val1 290 gaa gga atg gtt Giu Gly Met Val gga cac tgc atg Gly His Cys Met agg cat aac acc Arg His Asn Thr aag Lys 305 ggc tta aac tgt Gly Leu Asn Cys gaa Glu 310 ctc tgc atg gat Leu Cys Met Asp ttc Phe 315 tac cat gat tta Tyr His Asp Leu cct tgg Pro Trp 320 aga cct gct Arg Pro Ala aat gaa cat Asn Giu His 340 gaa Glu 325 ggc cga aac agc Gly Arg Asn Ser aac Asn 330 gcc tgt aaa aaa Ala Cys Lys Lys tgt aac tgc Cys Asn Cys 335 tac ctg gcc Tyr Leu Ala tcc atc tct tgt Ser Ile Ser Cys c ac His 345 ttt gac atg gct Phe Asp Met Ala gtt Val 350 *9 9 a a a 9 4 a a..

4 *4.a a A a acg ggg Thr Gly 355 aac gtc agc gga Asn Val Ser Gly ggc Gly 360 gtg tgt gat gac Val Cys Asp Asp cag cac aac acc Gin His Asn Thr atg Met 370 ggg cgc aac tgt Gly Arg Asn Cys gag Giu 375 cag tgc aag ccg Gin Cys Lys Pro ttt Phe 380 tac tac cag cac Tyr Tyr Gin His 984 1032 1080 1128 1176 1224 1272 1320 1368 1416 1464 1512 1560 gag agg gac atc Glu Arg Asp Ile cga Arg 390 gat cct aat ttc Asp Pro Asn Phe gaa cga tgt acg Glu Arg Cys Thr tgt gac Cys Asp 400 a *944 9 4 act.

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9 a, *9 *9 9 9 50 *4* p cca gct ggc Pro Ala Gly tct act ggt Ser Thr Gly 420 caa aat gag gga Gin Asn Glu Gly att Ile 410 tgt gac agc tat Cys Asp Ser Tyr act gat ttt Thr Asp Phe 415 aat gtg gaa Asn Val Giu ctc att gct ggc Leu Ile Ala Gly cag Gin 425 tgt cgg tgt aaa Cys Arg Cys Lys gga gaa Gly Glu 435 cat tgt gat gtt His Cys Asp Val aaa gaa ggc ttc Lys Giu Giy Phe tat Tyr 445 gat tta agc agt Asp Leu Ser Ser gaa Glu 450 gat cca ttt ggt Asp Pro Phe Gly tgt Cys 455 aaa tct tgt gct Lys Ser Cys Ala aat cct ctg gga aca Asn Pro Leu Gly Thr 465 att cct gga ggg Ile Pro Gly Gly cct tgt gat tcc Pro Cys Asp Ser gag Giu 475 aca ggt cac tgc Thr Gly His Cys tac tgc Tyr Cys 480 3

C.

56/61 aag cgt ot~g Lys Arg Leu tgg ggc tta Trp Gly Leu 500 aca gga cag cat Thr Gly Gin His tgt Cys 490 gac cag tgc ct~g Asp Gin Cys Leu cca gag cac Pro Giu His 495 gao tgt gac Asp Cys Asp agc aat gat ttg Ser Asn Asp Leu gga tgt cga cca Gly Cys Arg Pro tgt Cys 510 ott ggg Leu Giy 515 gga goo tt~a aac Gly Ala Leu Asn aac Asn 520 agt tgo ttt gog Ser Cys Phe Ala toa. ggo oag tgc Ser Gly Gin Cys tcoa Ser 530 tgo ogg cot oac Cys Arg Pro His atg Met 535 at~t gga ogt cag Ile Gly Arg Gin tgo Cys 540 aao gaa gt~g gaa Asn Glu Val Giu ct Pro 545 ggt tac tao ttt Giy Tyr Tyr Phe aco ot~g gat cac Thr Leu Asp His tao Tyr 555 cto tat gaa gog Leu Tyr Giu Ala gag gaa Giu Giu 560 gco aao tt~g Ala Asn Leu gao ogg att Asp Arg Ile 580 ggg Gly 565 cot ggg gtt ago Pro Giy Vai Ser at~a Ile 570 gt~g gag cgg oaa Val Giu Arg Gin tat-&tc cag Tyr Ile Gin 575 gt~g cot gaa Val Pro Giu coo too tgg act Pro Ser Trp Thr gga Giy 585 goc ggo tto gto Ala Giy Phe Vai oga Arg 590 0@ 0 0 0 S *00 0 00 q 0 00 0 @099 0@

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O 0 ggg got Gly Ala 595 tat ttg gag ttt Tyr Leu Giu Phe ttc Phe 600 at~t gao aao at~a Ile Asp Asn Ile oca Pro 605 tat tcc atg gag Tyr Ser Met Giu 1608 1656 1704 1752 1800 1848 1896 1944 1992 2040 2088 2136 2184 2232 2280 tao Tyr 610 gao ato ota att Asp Ile Leu Ilie ogo tao gag oca cag ot~a coo gao cac tgg gaa Arg Tyr Glu Pro Gin Leu Pro Asp His Trp Giu 615 620 625 aaa got gtco ato Lys Ala Val Ile aca Thr 630 gt~g cag oga cot Val Gin Arg Pro gga Gly 635 agg att oca aco Arg Ile Pro Thr ago ago Ser Ser 640 oga tgt ggt Arg Cys Gly tcoa oca ggo Ser Pro Gly 660 aat Asn 645 aco atco coo gat Thr Ile Pro Asp gat Asp 650 gao aac cag gtg Asp Asn Gin Val gt~g toa tta Val Ser Leu 655 tgc ttt gag Cys Phe Giu toa aga tat gtc Ser Arg Tyr Val gtco Val 665 ot~t cot cgg cog Leu Pro Arg Pro gtg Val1 670 aag gga Lys Gly 675 aca aac tao acg Thr Asn Tyr Thr agg ttg gag ot~g Arg Leu Giu Leu ct Pro 685 cag tao aco too Gin Tyr Thr Ser gat ago gao gt~g Asp Ser Asp Val ago coo tao acg Ser Pro Tyr Thr ato gat tot ott Ile Asp Ser Leu gtt Val1 705 otco atg oca tac Leu Met Pro Tyr tgt Cys 710 aaa toa otg gao Lys Ser Leu Asp ato ttco aco gtg gga ggt toa Ile Phe Thr Val Gly Gly Ser 715 720

II

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57/61 gga gat ggg Gly Asp Gly oga tgt cta Arg Cys Leu 740 gtc aco aac agt Val Thr Asn Ser gocc Ala 730 tgg gaa acc ttt Trp Giu Thr Phe cag aga tac Gin Arg Tyr 735 atg aca gat Met Thr Asp gag aac ago aga Giu Asn Ser Arg gtt gtg aaa aca Val Vai Lys Thr cog Pro 750 gtt tgo Val Cys 755 aga aao ato ato Arg Asn Ile Ile ago att tot goo Ser Ile Ser Ala otg Leu 765 tta cao oag aoa Leu His Gin Thr ggc Giy 770 ctg got tgt gaa Leu Aia Cys Giu tgc Cys 775 gao oct oag ggt Asp Pro Gin Gly tog Ser 780 tta agt too gtg Leu Ser Ser Val gat ccc aao gga Asp Pro Asn Giy ggc Gly 790 cag tgo cag tgo Gin Cys Gin Cys ogg Arg 795 ccc aac gtg gtt Pro Asn Val Val gga aga Giy Arg 800 aco tgc aac Thr Cys Asn tgc aaa cot Cys Lys Pro 820 aga tgt gca cot gga act ttt ggo ttt ggo ccc agt gga Arg Cys Aia Pro Gly Thr Phe Gly Phe Gly Pro Ser Giy 805 810 815 tgt gag tgc cat Cys Giu Cys His ctg Leu 825 oaa gga tot gtc Gin Giy Ser Val aa t Asn 830 goc tto tgo Aia Phe Cys 0@ OS S S 0 0 0 0 0 0 o aat ccc Asn Pro 835 gto act ggc Val Thr Giy cag tgc Gin Cys 840 cac tgt ttc oag His Cys Phe Gin gga Gly 845 gtg tat got cgg Val Tyr Ala Arg 2328 2376 2424 2472 2520 2568 2616 2664 2712 2760 2808 2856 2904 2952 3000 cag Gin 850 tgt gat cgg tgo Cys Asp Arg Cys tta Leu 855 cot ggg cac tgg Pro Gly His Trp ttt oca agt tgc Phe Pro Ser Cys ccc tgc cag tgo Pro Cys Gin Cys aat Asn 870 ggc cac goc gat Gly His Ala Asp gao Asp 875.

tgo gao oca gtg Cys Asp Pro Val act ggg Thr Gly 880 gag tgo ttg Giu Cys Leu aac Asn 885 tgo oag gao tao Cys Gln Asp Tyr ac 0 Thr 890 atg ggt oat aao Met Gly His Asn tgt gaa agg Cys Giu Arg 895 tgo ttg got ggt tao tat ggc gac ccc ato att ggg toa ggt gat cac Cys Leu Ala Gly Tyr Tyr Gly Asp Pro Ile Ile Gly Ser Giy Asp His 900 905 910 tgc ogo Cys Arg 915 cot tgo cot tgc Pro Cys Pro Cys gat ggt coo gac AspD Gly Pro Asp gga cgc cag ttt Gly Arg Gin Phe gc Ala 930 agg ago tgc tao Arg Ser Cys Tyr gat cot gtt act Asp Pro Val Thr tta Leu 940 cag ott goc tgt Gin Leu Ala Cys tgt gat cot gga Cys Asp Pro Giy tao Tyr 950 att ggt too aga Ile Gly Ser Arg gao gao tgt gc Asp Asp Cys Ala toa gga Ser Gly 960

'N

:7) i Y, 58/61 tac ttt ggc Tyr Phe Gly tgt cac aac Cys His Asn 980 act ggg agg Thr Gly Arg 995 cca tca gaa gtt Pro Ser Giu Val ggg Gly 970 ggg tcg tgt cag Gly Ser Cys Gin aac att gac acg Asn Ile Asp Thr aca Thr 985 gac cca gaa gcc Asp Pro Glu Ala tgt Cys 990 cct tgc cag Pro Cys Gin 975 gac aag gag Asp Lys Giu gaa cac tgt Giu His Cys tgt ctc aag Cys Leu Lys tgc ctg Cys Leu 1000 tac cac acg Tyr His Thr gaa ggg Giu Gly 1005 cag ttc Gin Phe 1010 tgc cgg ttt Cys Arg Phe gga tac Gly Tyr 1015 tat ggt gat Tyr Gly Asp gcc ctc Ala Leu 1020 cgg cag gac Arg Gin Asp tgt Cys 1025 cga aag tgt gtc Arg Lys Cys Val tgt aat Cys Asn 1030 tac ctg ggc acc gtg caa gag cac Tyr Leu Giy Thr Val Gin Giu His 1035 tgt aac Cys Asn 1040 ggc tct gac Gly Ser Asp tgc cag Cys Gin 1045 tgc gac aaa Cys Asp Lys gcc act Ala Thr 1050 ggt cag tgc Gly Gin Cys ttg -bgt ctt Leu Cys Leu 1055 cct aat gtg atc ggg cag aac Pro Asn Vai Ile Gly Gin Asn 1060 tgt gac cgc tgt gcg ccc aat acc tgg Cys Asp Arg Cys Ala Pro Asn Thr Trp 1065 1070 00:000 cag ctg gcc Gin Leu Ala 1075 agt ggc act Ser Gly Thr ggc tgt Gly Cys 1080 gac cca tgc Asp Pro Cys aac tgc Asn Cys 1085 aat gct gct Asn Ala Ala 3048 3096 3144 3192 3240 3288 3336 3384 3432 3480 3528 3576 3624 3672 3720 cat tcc His Ser 1090 ttc ggg cca Phe Gly Pro tct tgc Ser Cys 1095 aat gag ttc Asn Giu Phe acg ggg Thr Gly 1100 cag tgc cag Gin Cys Gin tgc Cys 1105 atg cct ggg ttt Met Pro Gly Phe gga ggc Gly Gly 1110 cgc acc tgc Arg Thr Cys agc gag Ser Glu 1115 tgc cag gaa ctc ttc Cys Gin Glu Leu Phe 1120 tgg gga gac ccc gac gtg gag tgc Trp Gly Asp Pro Asp Val Giu Cys 1125 cga gcc Arg Ala 1130 tgt gac tgt gac ccc agg Cys Asp Cys Asp Pro Arg 1135 acg ggc cag tgt gtc tgc Thr Gly Gin Cys Val Cys 1150 ggc att gag acg cca Gly Ile Glu Thr Pro 1140 cag tgt gac cag tcc Gin Cys Asp Gin Ser 1145 gtt gag ggt gtt gag ggt cca cgc tgt gac aag tgc acg cga ggg tac Val Giu Gly Val Giu Gly Pro Arg Cys Asp Lys Cys Thr Arg Gly Tyr 1155 1160 1165 tcg ggg gtc ttc Ser Gly Val Phe 1170 tgg gat gtg atc Trp Asp Val Ilie cct gac tgc aca ccc Pro Asp Cys Thr Pro 1175 att gcc gag ctg acc Ile Ala Glu Leu Thr 1190 tgc cac cag tgc ttt Cys His Gin Cys Phe 1180 aac agg aca cac aga Asn Arg Thr His Arg 1195 gct ctc Ala Leu 1185 ttc ctg Phe Leu 1200 V) 59/61 gag aaa gcc aag gcc Glu Lys Ala Lys Ala 1205 ttg aag ate agt ggt Leu Lys Ile Ser Gly 1210 gtg ate ggg cct tac cgt Val Ile Gly Pro Tyr Arg 1215 gag act gtg gac Glu Thr Val Asp 1220 tcg gtg gag Ser Val Glu agg aaa Arg Lys 1225 gtc age gag Val Ser Glu ata aaa gac atc Ile Lys Asp Ile 1230 ctg gcg cag Leu Ala Gin 1235 age ccc gca Ser Pro Ala gca gag Ala Glu 1240 cca ctg aaa Pro Leu Lys aac att Asn Ile 1245 ggg aat ctc Gly Asn Leu ttt gag Phe Glu 1250 gaa gca gag Glu Ala Glu aaa ctg Lys Leu 1255 att aaa gat Ile Lys Asp gtt aca Val Thr 1260 gaa atg atg Glu Met Met get Ala 1265 caa gta gaa gtg Gin Val Glu Val aaa tta Lys Leu 1270 tct gac aca Ser Asp Thr act tcc Thr Ser 1275 caa age aac Gin Ser Asn age aca Ser Thr 1280 gcc aaa gaa Ala Lys Glu ctg gat Leu Asp 1285 tct cta cag Ser Leu Gin aca gaa Thr Glu 1290 gcc gaa age Ala Glu Ser cta gac aac Leu Asp Asn 1295 act gtg aaa gaa ctt Thr Val Lys Glu Leu 1300 get gaa caa ctg gaa Ala Glu Gin Leu Glu 1305 ttt ate aaa aac tea gat Phe Ile Lys Asn Ser Asp 1310 r att cgg ggt Ile Arg Gly 1315 gcc ttg gat Ala Leu Asp age att Ser Ile 1320 acc aag tat Thr Lys Tyr ttc cag Phe Gin 1325 atg tct ctt Met Ser Leu 3768 3816 3864 3912 3960 4008 4056 4104 4152 4200 4248 4296 4344 4392 4440 gag gca Glu Ala 1330 gag gag agg Glu Glu Arg gtg aat Val Asn 1335 gcc tcc acc Ala Ser Thr aca gaa Thr Glu 1340 ccc aac age Pro Asn Ser act Thr 1345 gtg gag cag tca Val Glu Gin Ser gcc ctc atg Ala Leu Met 1350 aga gac aga gta gaa Arg Asp Arg Val Glu 1355 gac gtg atg atg Asp Val Met Met 1360 gag cga gaa Glu Arg Glu tec cag Ser Gin 1365 ttc aag gaa Phe Lys Glu aaa caa Lys Gin 1370 gag gag cag Glu Glu Gin get cgc ctc Ala Arg Leu 1375 o ctt gat gaa ctg gca Leu Asp Glu Leu Ala 1380 gcc gaa atg acc tgt Ala Glu Met Thr Cys 1395 ggc aag cta caa age Gly Lys Leu Gin Ser 1385 gga aca ccc cca ggg Gly Thr Pro Pro Gly 1400 cta gac ctt tca gcc get Leu Asp Leu Ser Ala Ala 1390 gcc tcc tgt tec gag act Ala Ser Cys Ser Glu Thr 1405 gaa tgt ggc Glu Cys Gly 1410 ggg Gly cca aac tgc aga act Pro Asn Cys Arg Thr 1415 tgt ggt ggt ctg gtt Cys Gly Gly Leu Val 1430 gac gaa gga Asp Glu Gly 1420 act gtt gca Thr Val Ala 1435 gag agg aag Glu Arg Lys tgt Cys 1425 ggg ggg cct ggc Gly Gly Pro Gly f) i cac aac gcc tgg His Asn Ala Trp 1440 4 U 60/61 cag aaa gcc atg gac Gin Lys Ala Met Asp 1445 gtg gaa cag ctc tcc Val Glu Gin Leu Ser 1460 ttg gac caa gat gtc Leu Asp Gin Asp Val 1450 aag atg gtc tct gaa Lys Met Val Ser Glu 1465 ctg agt gcc ctg get gaa Leu Ser Ala Leu Ala Glu 1455 gca aaa ctg agg gca gat Ala Lys Leu Arg Ala Asp 1470 gag gca Glu Ala I 1475 aaa gaa Lys Glu 1490 aaa caa agt get Lys Gin Ser Ala gaa gac Glu Asp 1480 att ctg ttg Ile Leu Leu aag aca Lys Thr 1485 aat get ace Asn Ala Thr aaa atg gac Lys Met Asp aag age Lys Ser 1495 aat gag gag Asn Glu Glu ctg aga Leu Arg 1500 aat cta ate Asn Leu Ile aag Lys 1505 caa ate aga aac Gin Ile Arg Asn ttt ttg Phe Leu 1510 ace cag gat Thr Gin Asp agt get Ser Ala 1515 gat ttg gac age att Asp Leu Asp Ser Ile 1520 atg cct age acc cca Met Pro Ser Thr Pro 1535 gaa gca gtt Glu Ala Val get aat gaa Ala Asn Glu 1525 gta ttg aaa atg gag Val Leu Lys Met Glu 1530 cag cag tta cag Gin Gin Leu Gin 1540 aac ttg aca Asn Leu Thr gaa gat Glu Asp 1545 ata cgt gaa Ile Arg Glu cga gtt gaa age Arg Val Glu Ser 1550 r ctt tct caa Leu Ser Gin 1555 gta gag gtt Val Glu Val att ctt Ile Leu 1560 cag cat agt Gin His Ser get get Ala Ala 1565 gac att gcc Asp Ile Ala 4488 4536 4584 4632 4680 4728 4776 4824 4872 4920 4968 5016 5064 5112 5160 aga get Arg Ala 1570 gag atg ttg Glu Met Leu tta gaa Leu Glu 1575 gaa get aaa Glu Ala Lys aga gca Arg Ala 1580 age aaa agt Ser Lys Ser gca Ala 1585 aca gat gtt aaa Thr Asp Val Lys gtc act Val Thr 1590 gca gat atg Ala Asp Met gta aag Val Lys 1595 gaa get ctg Glu Ala Leu gaa gaa Glu Glu 1600 gca gaa aag Ala Glu Lys gcc cag Ala Gin 1605 gtc gca gca Val Ala Ala gag aag Glu Lys 1610 gca att aaa Ala Ile Lys caa gca gat Gin Ala Asp 1615 gaa gac att caa gga Glu Asp Ile Gin Gly 1620 ace cag aac ctg tta Thr Gin Asn Leu Leu 1625 act tcg att gag tct gaa Thr Ser Ile Glu Ser Glu 1630 aca gca get Thr Ala Ala 1635 gag tta gag Glu Leu Glu 1650 tct gag gaa Ser Glu Glu ace ttg ttc Thr Leu Phe 1640 aac gcg tec Asn Ala Ser 1645 aag egg aaa Lys Arg Lys 1660 cag cgc ate age Gin Arg Ile Ser agg aat gtg gaa gaa ctt Arg Asn Val Glu Glu Leu 1655 get gcc caa Ala Ala Gin aac Asn 1665 tec ggg gag gca Ser Gly Glu Ala gaa tat att gaa aaa Glu Tyr Ile Glu Lys 1670 gta gta tat act gtg Val Val Tyr Thr Val 1675 aag caa Lys Gin 1680

~K"

ID

I")

61/61 agt gca gaa gat gtt Ser Ala Giu Asp Val 1685 aag aag act tta gat Lys Lys Thr Leu Asp 1690 ggt gaa. ctt gat gaa aag Gly Giu Leu Asp Giu Lys 1695 aaa act gaa gag tca gct Lys Thr Glu Glu Ser Ala 1710 tat aaa aaa gta Tyr Lys Lys Val 1700 gaa aat tta Giu Asn Leu att gcc Ile Ala 1705 gat gec aga Asp Ala Arg 1715 agg aaa gcc Arg Lys Ala gaa atg Giu Met 1720 cta caa aat Leu Gin Asn gaa gca Glu Ala 1725 aaa act ctt Lys Thr Leu tta gct Leu Ala 1730 caa gca aat Gin Ala Asn agc aag Ser Lys 1735 ctg caa ctg Leu Gin Leu ctc aaa.

Leu Lys 1740 gat tta gaa Asp Leu Giu aga Arg 1745 5208 5256 5304 5352 5400 5448 5495 5555 5613 aaa tat gaa gac Lys Tyr Giu Asp aat caa Asn Gin 1750 aga tac tta Arg Tyr Leu gaa gat Giu Asp 1755 aaa gct caa Lys Ala Gin gaa tta Glu Leu 1760 gca aga ctg Ala Arg Leu gaa gga Giu Gly 1765 gaa gtc cgt Giu Val Arg tca ctc cta Ser Leu Leu 1770 aag gat ata agc cag Lys Asp Ile Ser Gin 1775 aaa gtt gct gtg tat Lys Val Ala Val Tyr 1780 agc aca tgc ttg taacagagga gaataaaaaa Ser Thr Cys Leu 1785

S

tggctgaggt gaacaaggta aaacaactac attttaaaaa ctgacttaat gctcttcaaa ataaaacatc acctatttaa tgtttttaat cacattttgt atgagttaaa taaagccc

Claims (34)

1. An isolated laminin 12 y3 subunit.

2. The isolated y3 subunit of claim 1, having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:3.

3. The isolated y3 subunit of claim 1 or claim 2, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:3.

4. The isolated y3 subunit of any one of claims 1 to 3, having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:3. The isolated y3 subunit of any one of claims 1 to 4, encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of 20 the nucleic acid sequence of SEQ ID NO:4.

6. An isolated y3 subunit comprising the amino acid sequence of SEQ ID NO:3. 25 7. An isolated y3 subunit obtainable by reducing laminin 12 in 10% 2-mereaptoethanol, SDS-PAGE sample buffer, resolving three bands by 5% SDS-PAGE, obtaining the 170 kDa band, digesting the band with trypsin, and .resolving the band by HPLC.

8. An isolated nucleic acid molecule having at least sequence identity to the nucleic acid sequence of SEQ ID NO:4 or the nucleic acid sequence of ATCC Accession No:209357.

9. The nucleic acid molecule of claim 8, having at least 90% sequence identity to the nucleic acid sequence of L, U \\melb_files\homeS\WendyS\Keep\species\10765-99 GenHosp.doc 21/11/00 Y) 0 PA 42 SED ID NO:4 or the nucleic acid sequence of ATCC Accession No:209357. The nucleic acid molecule of claim 8 or claim 9, having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4 or the nucleic acid sequence of ATCC Accession No:209357.

11. An isolated nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid sequence SEQ ID NO:4 or the nucleic acid sequence of ATCC Accession No:209357.

12. A vector comprising the nucleic acid of any one of claims 8 to 11.

13. A cell comprising the purified nucleic acid of any one of claims 8 to 11. 20 14. An isolated laminin 12 molecule comprising an a2 subunit, a l subunit and a y3 subunit. The laminin 12 of claim 14, wherein the y3 subunit is a y3 subunit of any of claims 1 to 7.

16. The laminin 12 of claim 14, wherein the a2 subunit has at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:7. 30 17. The laminin 12 of claim 14 or claim 15, wherein the a2 subunit has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:7.

18. The laminin 12 of any one of claims 14 to 17, wherein the a2 subunit has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7. L \\melb_files\home$\WendyS\Keep\species\10765-99 GenHosp.doc 21/11/00 43

19. The laminin 12 of claim 14, wherein the a2 subunit comprises the amino acid sequence of SEQ ID NO:7. The laminin 12 of claim 14, wherein the a2 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID NO:8.

21. The laminin 12 of claim 14 or claim 20, wherein the pi subunit has at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:9.

22. The laminin 12 of claim 14 or claim 20 or claim 21, wherein the pi subunit has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:9.

23. The laminin 12 of any one of claims 14 or 20 to 22, wherein the P1 subunit has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:9. S

24. The laminin 12 of claim 14, wherein the pi subunit comprises the amino acid sequence of SEQ ID NO:9.

25. The laminin 12 of claim 14, wherein the pi 25 subunit is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence shown in SEQ ID 30 26. An isolated laminin 12 P4 subunit.

27. The isolated P4 subunit of claim 26, having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1.

28. The isolated p4 subunit of claim 26 or claim 27 ~having at least 85% sequence identity with the amino acid U-I i \\melb_files\home$\WendyS\Keep\species\10765-99 GenHosp.doc 21/11/00 U- 44 sequence of SEQ ID NO:1.

29. The isolated P4 subunit of any one of claims 26 to 28, having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1. The isolated 34 subunit of any one of claims 26 to 29, having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1.

31. The isolated P4 subunit of claim 26, encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the nucleic acid sequence of SEQ ID NO:2.

32. An isolated P4 subunit comprising the amino acid sequence of SEQ ID NO:1.

33. An isolated nucleic acid molecule having at least 20 80% sequence identity to the nucleic acid sequence of SEQ woo. 0:00: ID NO:2. oe *e

34. The nucleic acid sequence of claim 33, having at least 85% sequence identity to the nucleic acid sequence of 25 SEQ ID NO:2.

35. The nucleic acid sequence of claim 33 or claim 34, having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:2. o

36. The nucleic acid sequence of any one of claims 33 to 35, having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:2.

37. An isolated nucleic acid molecule which hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:2. Sri \\melb_files\home$\WendyS\Keep\species\10765-99 GenHosp.doc 21/11/00 13-LL 1 Y- 0 45

38. A vector comprising the nucleic acid of any one of claims 33 to 37.

39. A cell containing the purified nucleic acid of any one of claims 33 to 37. An isolated laminin 12 y3 subunit according to claim 1, substantially as herein described with reference to the examples and drawings.

41. An isolated laminin 12 P4 subunit according to claim 26, substantially as herein described with reference to the examples and drawings.

42. An isolated laminin 12 molecule according to claim 14, substantially as herein described with reference to the examples and drawings.

43. Use of a laminin 12 subunit, according to any one of claims 1, 14 or 26, for the preparation of a medicament for the inhibition of connective tissue adhesion; the promotion of wound healing; adhesion of dormant dermal and epidermal cells, nerve growth or regeneration; treatment of a disorder associated with the misexpression of a 25 laminin; a disorder of the central or peripheral nervous system; a disorder associated with a genetic lesion at chromosome 9, region q31-34; Fukuyama-type muscular dystrophy; muscle-eye-brain disease; Walker-Warburg Syndrome (hyudrocephalus, ageria, and retinal displasia); a retinal disorder, a disorder associated with abnormal levels, of adhesion between tissues; a disorder associated with the basement membrane; a skin disorder; a disorder associated with the testis, spleen, placenta, thymus, ovary, small intestine, lung, or liver.

44. An isolated nucleic acid molecule according to -AM ~any one of claims 8, 10, 33 and 37, substantially as H: \ane Keep\Speci\1076599do 28/06/01 CO H:\janel\Keep\Speci\10765-99.doc 28/06/01 46 herein described with reference to the examples and drawings. Dated this 28th day of June 2001 THE GENERAL HOSPITAL CORPORATION By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade'Mark Attorneys of Australia 9. C P H:\janel\Keep\Speci\10765-99.doc 28/06/01