FR2641792A1 - Nucleotide sequences encoding calmodulin in man and their biological applications - Google Patents

Abstract

Nucleotide sequences comprising all or part of a sequence encoding calmodulin in man. These sequences allow a high production of human calmodulin.

Description

NUCLEOTIDE SEQUENCES ENCODING CALMODULIN
IN MAN AND THEIR BIOLOGICAL APPLICATIONS
The invention relates to nucleotide sequences encoding calmodulin in humans and their biological applications.

 Calmodulin is a ubiquitous protein, a structure highly conserved during evolution, and which binds calcium. It acts as an intracellular mediator for the effects of calcium and is involved in the control of cell proliferation, DNA repair or gene expression.

 We know that calmodulin plays an activating role against adenyl cyclase excreted by B, Dertussis (Gram-negative bacteria) and B. anthracis (Gram-positive bacteria), agents, respectively, of whooping cough. and carbon, which results in a high production of cyclic AMP (cAMP).

 The interaction between adenyl cyclase and - calmodulin - is used in the gene cloning process described in applications FR 87/10614 of 24.7.87 and 88/93952 of 25.10.88 in the name of the applicant.

According to this process, the cloning of genes coding for adenyl cyclase respectively is carried out.
B. Dertussis and B. anthracis in a receptor strain deficient in adenyl cyclase carrying a plasmid containing a synthetic calmodulin gene and expressing high levels of calmodulin. Microbiological detection of the presence of cAMP allows the cloning of genes expressing adenyl cyclase.

 The symmetrical technique has proven to be usable for cloning the complementary DNA (cDNA) of the messenger ar (mRNA) of calmodulin in humans.

 The invention therefore aims to provide sequences carrying the coding sequences for calmodulin in humans and more specifically the AD.Nc of the mRNA of human calmodulin.

 The nucleotide sequence according to the invention is characterized in that it includes all or part of the sequences coding for human calmodulin.

 This sequence of the invention is capable of hybridizing with a DNA strand of the exons of a gene coding for calmodulin in humans or the cDNA of their mRNA.

 According to another arrangement of the invention, the above nucleotide sequence is characterized in that it carries the inFormatior, for the expression of human calmodulin.

 The invention also relates to a nucleotide sequence as defined above, or fragments thereof, coding respectively for human calmodulin or fragments thereof, the calmodulin or its fragments being capable of forming an immunological complex with antibodies directed respectively against human or mammalian calmodulin in general or fragments of human calmodulin.

 According to another aspect, the invention relates to a recombinant nucleotide sequence, characterized in that it comprises a sequence as defined above, if necessary, associated with a promoter capable of controlling the transcription of the sequence and, optionally, a DNA sequence encoding transcription termination signals.

 The recombinant nucleotide sequence of the invention can be associated with a promoter and with an operator making it possible to control transcription, and optionally with a signal sequence allowing the secretion of the protein in the periplasmic space or in the external medium.

More specifically, the invention relates to a nucleotide sequence capable of hybridizing with a probe formed from the sequence having the following sequence of nucleotides (I), or the complementary sequence of this sequence
10 20 30 40 50 60
ATTAGTCCGA GTGGAGAGAG CGAGCTGAGT GGTTGTGTGG TCGCGTCTCG GAAACCGGTA
70 80 90 100 110 120
GCGCTTGCAG CATGGCTGAC CAAC TGAC TG AAG AGCAGAT TGCAGAATTC AAAGAAGCTT
130 140 150 160 170 180
TTTCACTATT TGACAAAGAT GGTGATGGAA CTATAACAAC AAAGGAATTG GGAACTGTAA
190 200 210 220 230 240
TGAGATCTCT TGGGCAGAAT CCCACAGAAG CAGAGTTACA GGACATGATT AATGAAGTAG
250 260 270 280 290 300
ATGCTGATGG TAATGGCACA ATTGACTTCC CTGAATTTCT GACAATGATG GCAAGAAAAA
310 320 330 340 350 360
TGAAAGACAC AGACAGTGAA GAAGAAATTA GAGAAGCATT CCGTGTGTTT GATAAGGATG
370 380 390 400 410 420
GCAATGGCTA TATTAGTGCT GCAGAACTTC GCCATGTGAT GACAAACCTT GGAGAGAAGT
430 440 450 460 470 480
TAACAGATGA AGAAGTTGAT GAAATGATCA GGGAAGCAGA TATTGATGGT GATGGTCAAG
490 500 510 520 530 540
TAAACTATGA AGAGTTTGTA CAAATGATGA CAGCAAAGTG AAGACCTTGT ACAGAATGTG
550 560 570 580 590 600
TTAAATTTCT TGTACAAAAT TGTTTATTTG CCTTTTCTTT GTTTGTAACT TATCTGTAAA
610 620 630 640 650 660
AGGTTTCTCC CTACTGTCAA AAAAATATGC ATGTATAGTA ATTAGGACTT CATTCCTCCA
670 680 690 700 710 720
TGTTTTCTTC CCTTATCTTA CTGTCATTGT CCTAAAACCT TATTTTAGAA AATTGATCAA
730 740 750 760 770 780 GTAACATGTT GCATGTGGCT TACTCTGGAT ATATCTAAGC CCTTCTGCAC ATCTAAACTT
790 800 810 820 830 840 AGATGGAGTT GG TCAAATGA GGGAACATCT GGGTTATGCC TTTTTTAAAG TAGTTTTCTT
850 860 870 880 890 900
TAGGAACTGT CAGCATGTTG TTGTTGAAGT GTGGAGTTGT AACTCTGCGT GGACTATGGA
910 920 930 940 950 960
CAGTCAACAA TATGTACTTA AAAGTTGCAC TATTGCAAAA CGGGTGTATT ATCCAGGTAC
970 980 990 1000 1010 1020
TCGTACACTA TTTTTTTGTA CTGCTGGTCC TGTACCAGAA ACATTTTCTT TTATTGTTAC
1030 1040 1050 1060 1070 1080
TTGCTTTTTA AACTTTGTTT AGCCACTTAA AATCTGCTTA TGGCACAATT TGCCTCAAAA
1090 1100 1110 1120 1130 1140 TCCATTCCAA GTTGTATATT TGTTTTCCAA TAAAAAAATT ACAATTTACC C. (poly A termination)
Sequences according to the invention are characterized in that they comprise the sequence (I) defined above or consist of all or part of this sequence, the total sequence corresponding to the cDNA of the mRNA discomfort from human calmodulin. In this sequence, the beginning of the coding sentence is in position 72 and the end in position 519.

 It goes without saying that the bases of the nucleotide sequence considered can be in a different order from that found in the sequence above and / or that these bases can be, if necessary, substituted, as soon as a probe developed from such a sequence gives a characteristic and unequivocal response as to the ability to recognize the presence of coding sequences vis-à-vis human calmodulin.

 The nucleotide sequences according to the invention are advantageously obtained according to the process for cloning applicants mentioned above.

According to one embodiment of the method of the invention, a defective cva derivative of the bacteria
E. coli, which is less restricted in order to accept foreign DNA, is first transformed by a plasmid carrying the gene for adenyl cyclase from B. pertussis.

The resulting bacteria remain phenotypically cyclase-, and are white on Mc Conkey medium supplemented with 1% maltose. Any activation of adenyl cyclase is reflected by the formation of a red colony on the same medium. A library of human complementary DNA is transferred into this strain and several red clones are obtained. The corresponding plasmid DNA is isolated, purified, and the cDNA sequenced (sequence I). The use of the universal genetic code shows the presence of a polypeptide sequence identical to that of mammalian calmodulins (preserved without change during evolution in most)
Note that this is a cloning of eukaryotic genes carried out after complementation of a deficient function of a prokaryote, which has been done only very rarely. Thus according to the invention, it is demonstrated that It is possible to clone the human brain cDNA encoding the synthesis of calmodulin although the host strain does not normally have a calmodulin function.

 Cloning with several partners is preferably carried out with plasmids having different incompatibility groups, so that they remain stable, in the presence of each other in the same cell.

 Recombinant expression and cloning vectors capable of transforming an appropriate host cell also fall within the scope of the invention. These vectors comprise at least part of a nucleotide sequence of the invention under the control of regulatory elements allowing its expression.

 The transformed microorganism strains also form part of the invention. These strains comprise one of the nucleotide sequences defined above or also a recombinant vector as defined above.

 The invention also relates to the biological applications of the nucleotide sequences, of the corresponding proteins and of the monoclonal or polyclonal antibodies.

 These applications include the high-level production of human calmodulin, which constitutes an ultrastructure for the use of synthetic genes.

 They also include the development, from fragments of the chain (I), of probes for the detection of complementary sequences in the genes producing calmodulin. This elaboration includes, in particular, the denaturation of the double stranded sequences to obtain a single stranded sequence usable as a probe.

 The invention therefore relates to detection probes characterized in that they comprise at least part of a nucleotide sequence defined above, more especially an intragenic fragment of the sequence coding for calmodulin in humans.

 The DNA fragment used as probe contains a sufficient number of nucleotides to obtain the required specificity and the formation of a stable hybrid.

 Probes suitable for this type of detection are advantageously marked with a radioactive element (hot probes) or any other non-radioactive group (cold probes) allowing the recognition of the probe in the hybridized state with the preparation containing 1 ′ DNA to study.

 According to conventional techniques, these probes are brought into contact with a biological sample containing bacteria, or directly with these bacteria or their nucleic acids, under conditions allowing the possible hybridization of the nucleotide sequence of the probe with a complementary sequence, possibly contained in the product tested.

 One can, for example, implement the hybridization method on spots. This method comprises, after denaturation of the DNA previously obtained from cells expressing adenyl cyclase, the deposition of an aliquot of this DNA on nitrocellulose membranes, the hybridization of each membrane under the usual conditions with probe and detection, in a conventional manner, of the hybrid formed.

One can also use a hybridization method on replica, according to the technique of
Southern. This method comprises the electrophoretic separation in agarose gel of the DNA fragments generated after treatment of the ADSs with restriction enzymes, the transfer after alkaline denaturation on appropriate membranes and their hybridization with the probe under the usual conditions. It is not always necessary to carry out the prior expression of DNA. It suffices that the DNA is made accessible to the probe.

 For the implementation of the detection methods considered above based on the use of nucleotide probes, use is advantageously made of kits or kits comprising - a determined quantity of a nucleotide probe according to the invention, - advantageously, a medium suitable respectively for the formation of a hybridization reaction between the sequence to be detected and the probe, advantageously reagents allowing the detection of the hybridization complexes formed between the nucleotide sequence and the probe during the hybridization reaction.

The nucleotide sequences of the invention also allow, among other things
- the evaluation of the level of messenger RNA of the
calmodulin of various cell types,
particular tumor cells, and so the
identification of cells of a particular class,
for example tumor
- labeling of chromosomes,
- the manufacture of kits linked to these methods, and
- cloning of the complete calmodulin gene
human.

- the manufacture of calmodulin anti-messengers,
whether in the form of RNA, DNA or
modified polynucleotides.

 Another application of the nucleotide sequences in accordance with the invention relates to the identification of genetic fingerprints of the calmodulin gene and the search for human genetic polymorphism. The search for human genetic polymorphism means both the genetic identification of an individual and the detection of certain pathologies linked to genetic modifications relating to a calmodulin gene or a genetically linked gene.

The identification of an individual may include the analysis of restriction fragments containing all or part of a gene for human calmodulin.

 In the context of this application, the nucleotide sequences defined above are used as probes. These probes are used on a biological sample, for example a tissue or a fluid prepared to be accessible to these probes. One operates advantageously according to European patent application 0 186 271, the content of which is incorporated in the present description.

 The specific detection of the presence of sequences coding for calmodulin in humans can also be carried out according to methods using DNA amplification techniques (PCR). These techniques are described in particular in US patents 4,683,202 and 4,683,195.

 In these techniques, two primers are used, advantageously about twenty nucleotides, but distant from each other and included in one of the nucleotide sequences defined above. One of the sequences is capable of binding to a nucleotide sequence of one of the strands of the DNA fragment to be amplified, this sequence being located at one end of this fragment (for example the 5 'end) the other sequence is capable of binding to a nucleotide sequence of the second strand of the DNA fragment to be amplified, this latter sequence being located at the end of this fragment opposite to that mentioned above (this latter end being also designated by 3 'end in the example proposed).

 The following indicates, by way of non-limiting example, the materials and methods used to clone and sequence the cDNA of the mRNA of a gene for human calmodulin.

Bacterial strains and culture media
A cva derivative TP610 (1) of the strain of E. coli restriction minus C600SF8 (2) is used and as growth media the rich medium LB or the minimal medium (3). The bacteria are sorted with regard to their capacity to produce the fermentation of sugars on Nc Conkey agar plates containing 1% of the sugar tested.

 The chloramphenicol and ampicillin concentrations are 30 pg / ml and 100 ug / ml respectively.

 The strain TP610 was transformed according to (4) using the plasmid pDI5 (5), a plasmid derived from pACYC184. This transformed strain is unable to synthesize cAMP and is used as a receptor to clone human brain cDNA fragments.

Constructions of cDNA Libraries
The cDNA library is constructed in the plasmid pUC9, which is a derivative of pBR322 (compatible with pACYC184) and the resistance used is resistance to ampicillin.

The construction of the cDNA library was done according to the method of Cathala et al (6) for the extraction of messenger RNA. Then, the library was built on a primer of oligo dT from messenger RNAs terminated with poly A according to the method of Caput (7): briefly, the plasmid pUC9 was linearized by cleavage by PstI and an extension of dG was added by the transferase terminal. The molecules thus elongated were digested with BamHI and the small fragment generated by the BamHI cleavage was eliminated by elution through small columns of
Sepharose CL6B2.

CDNA is produced from 1 g of mRNA template using reverse transcriptase and a synthetic primer to hybridize to poly ends
To messengers (5 'GATCCGGGCCCTl23').

 The residual mRNA is removed by alkaline hydrolysis and the dC ends of the cDNA are added using terminal transferase.

 The 5 'end of the cDNA is modified into a BamHI cohesive end by hybridization with an adapter constituted by a synthetic oligonucleotide (5'Ai2GGGCCCG3') complementary to the primer. Single strand cDNA is ligated to the vector to produce circular molecules.

 Repair of the single strand region is accomplished using the dG end of the vector as a primer and ADS T4 polymerase.

DNA sequence analysis
The DNA sequence was analyzed using subclones in phage M13tgl31 (8). Unidirectional deletions are generated using the Cyclone system (IBI).

 The DNA sequence is determined by the dideoxynucleotide chain termination method of Sanger et al (9) using alpha S3 dATP (Amersham) and 7-deaza-dGTP (Boehringer) in place of dGTP.

BIBLIOGRAPHIC REFERENCES
1. STRUHL, R. et al: Functional genetic expression of
eukaryotic DNA in Escherichia coli, Proc. Natl.

 Acad. Sci. USA 73 (1976), 1471-1475.

2. HEDEGAARD, L. DANCHIN A., The cya gene region of
Erwinia chrvsanthemi B374: organization and gene
products, Mol. Gen. Broom. (1985) 201: 38-42.

3. MILLER, J.H: Experiments in molecular genetics.

Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY, 1972.

4. MANIATIS, T. et al: Molecular cloning: a
laboratorymanual. VCold Spring Harbor Laboratory,
Cold Spring Harbor, NY, 1982.

5. GLASER, P. et al: Mol. Microbiol. 2 (1988) 19-30.

6. CATHALA, G. et al: DNA 2 (1983) 329-335.

7. McKEON, F.D. et al: Nature 319 (1986) 463-468.

8. KIENY, MP et al: New versatile cloning and
vector sequencing based on bacteriophage M13.

 Gene 26 (1983), 91-99.

9. SANGER, F et al: DNA sequencing with chain
terminating inhibitors. Proc. Native. Acad. Sci. USA
74 (19770 5463-5467.

Claims (10)

 1. Nucleotide sequence, characterized in that it includes all or part of the sequences coding for calmodulin in humans.  2. Nucleotide sequence, characterized in that it is capable of hybridizing with a strand of DNA from the exons of a gene coding for calmodulin in humans or the cDNA of their mRNA.  3. Sequence according to claim 1 or 2, characterized in that it carries the information for the expression of calmodulin in humans.  4. Nucleotide sequence, or fragments of this sequence, coding respectively for human calmodulin or fragments thereof, the calmodulin or its fragments being capable of forming an immunological complex with antibodies respectively against human calmodulin or fragments thereof. the latter, calmodulin or its fragments being capable of forming an immunological complex with antibodies directed respectively against human calmodulin, mammals in general, or fragments of human calmodulin.  5. Recombinant nucleotide sequence, characterized in that it comprises a sequence according to any one of claims 1 to 4, if necessary, associated with a promoter capable of controlling the transcription of the sequence and optionally a DNA sequence coding for transcription termination signals.  6. Recombinant nucleotide sequence, characterized in that it comprises a nucleotide sequence according to any one of claims 1 to 4, associated with a promoter and with an operator making it possible to control transcription, and optionally with a signal sequence allowing the secretion of the protein in the periplasmic space or in the external nile.  7. Nucleotide sequence, characterized in that it is capable of hybridizing with a probe formed from the sequence having the following sequence of nucleotides (I)  10 20 30 40 50 60 ATTAGTCCGA GTGGAGAGAG CGAGCTGAGT GGTTGTGTGG TCGCGTCTCG GAAACCGGTA  70 80 90 100 110 120 GCGCTTGCAG CATGGCTGAC CAAC TGACTG AAGAGCAGAT TGCAGAATTC AAAGAAGCTT  130 140 150 160 170 180 TTTCACTATT TGACAAAGAT GGTGATGGAA CTATAACAAC AAAGGAATTG GGAACTGTAA  190 200 210 220 230 240 TGAGATCTCT TGGGCAGAAT CCCACAGAAG CAGAGTTACA GGACATGATT AATGAAGTAG  250 260 270 280 290 300 ATGCTGATGG TAATGGCACA ATTGACTTCC CTGAATTTCT GACAATGATG GCAAGAAAAA  310 320 330 340 350 360 TGAAAGACAC AGACAGTG AA GAAGAAATTA GAGAAGCATT CCGTGTGTTT GATAAGGATG  370 380 390 400 410 420 GCAATGGCTA TATTAGTGCT GCAGAACTTC GCCATGTGAT GACAAACCTT GGAGAGAAGT  430 440 450 460 470 480 TAACAGATGA AGAAGTTGAT GAAATGATCA GGGAAGCAGA TATTGATGGT GATGGTCAAG  490 500 S10 520 530 540 TAAACTATGA AGAGTTTGTA CAAATGATGA CAGCAAAGTG AAGACCTTGT ACAGAATGTG  550 560 570 580 590 600 TTAAATTTCT TGTACAAAAT TGTTTATTTG CCTTTTCTTT GTTTGTAACT TATCTGTAAA  610 620 630 640 650 660 AGGTTTCTCC CTAC TGTCAA AAAAATATGC ATGTATAGTA ATTAGGACTT CATTCCTCCA  670 680 690 700 710 720 TGTTTTCTTC CCTTATCTTA CTGTCATTGT CCTAAAACCT TATTTTAGAA AATTGATCAA  730 740 750 760 770 780 GTAACATGTT GCATGTGGCT TACTCTGGAT ATATCTAAGC CCTTCTGCAC ATCTAAACTT  790 800 810 820 830 840 AGATGGAGTT GGTCAAATGA GGGAACATCT GGGTTATGCC TTTTTTAAAG TAGTTTTCTT  850 860 870 880 890 900 TAGGAACTGT CAGCATGTTG TTGTTGAAGT GTGGAGTTGT AACTCTGCGT GGACTATGGA  910 920 930 940 950 960 CAGTCAACAA TATGTACTTA AAAGTTGCAC TATTGCAAA CGGGTGTATT ATCCAGGTAC  970 980 990 1000 1010 1020 TCGTACACTA TTTTTTTGTA CTGCTGGTCC TGTACCAGAA ACATTTTCTT TTATTGTTAC  1030 1040 1050 1060 1070 1080 TTGCTTTTTA AACTTTGTTT AGCCACTTAA AATCTGCTTA TGGCACAATT TGCCTCAAAA  1090 1100 1110 1120 1130 1140 TCCATTCCAA GTTGTATATT TGTTTTCCAA TAAAAAAATT ACAATTTACC C (poly A termination)  8. Detection probe characterized in that it comprises all or part of the nucleotide sequences according to any one of claims 1 to 7.  9. Use as primers in ADS amplification techniques, of the PCR type, of two nucleotide sequences of approximately at least 20 nucleotides, included in one of the sequences according to any one of claims 1 at 7, one of the sequences being capable of binding at the 5 'end of one strand of the sequence to be amplified and the second sequence at the 3' end of the other strand.  10. Probe for the detection of the polymorphism of restriction fragments of the human genome, characterized in that it comprises all or part of a sequence according to any one of claims 1 to 7.