FR2746815A1 - SEQUENCES UPSTREAM OF THE SM 22 GENE, VECTORS CONTAINING THEM AND THEIR THERAPEUTIC USES, ESPECIALLY IN THE TREATMENT OF VASCULAR DISEASES - Google Patents

Abstract

DNA sequences including a fragment of the sequence ahead of the coding portion of the SM22 protein gene, or a sequence hybridisable therewith under highly stringent conditions, said fragment being capable of inducing specific gene expression in eukaryotic cells, as well as a sequence coding for a protein or RNA of therapeutical interest, are disclosed. Said sequence or a vector containing same may be used for treating coronary diseases, particularly restenosis.

Description

 The present invention relates to sequences upstream of a gene expressed in smooth muscle cells, such as SM 22, as well as to vectors containing them.

 It further relates to the use of these vectors and sequences in therapy, for example for the production of polypeptides active locally or systemically, in particular immunogenic polypeptides, endogenous regulatory polypeptides such as cytokines, or also for the production RNA of therapeutic interest, for example an antisense RNA. In particular, the invention relates to the use of vectors and nucleic sequences in the treatment of vascular diseases.

 Atherosclerosis is a degenerative disease of the arteries associating lesions of arteriosclerosis and atheroma, and thus combining a hardening of the arteries and a fatty degeneration of their internal coat.

This coronary insufficiency is generally remedied by percutaneous coronary angioplasty consisting in introducing into the coronary artery network a catheter provided at its end with a balloon, advancing the catheter to the level of the stenosis and inflating the balloon in order to crush the lesion against the wall and thus restore a coronary caliber allowing sufficient myocardial perfusion.

This myocardial revascularization technique is very widely used (50,000 procedures per year in
France and 500,000 operations per year in the United States) but is however limited by the reappearance, in the months following the operation, of a new lesion at the dilated site, or restenosis, in approximately 30% of cases.

 The various treatments proposed to treat this type of lesion are summarized in the article by FELDMAN and STEG: "Perspective of gene therapy for restenosis" (Medicine / Sciences, synthesis 1996, 12, 47-55).

 Two mechanisms are responsible for restenosis of intimal hyperplasia and arterial reshaping. The first of these mechanisms, intimal hyperplasia is due to the proliferation of smooth muscle cells in the intima, which results in the synthesis of a bulky extra cellular matrix. This activation would be induced by the local release of growth factors, and cytokines, as well as by the suppression of the synthesis of certain antiproliferative endothelial peptides. The second mechanism, arterial remodeling, consists in reducing the caliber of the artery without modifying the thickness of the wall, and therefore without proliferation of cells.

 These authors describe various attempts to inhibit intimal hyperplasia, by gene therapy, but the results of which have proved unsatisfactory, for various reasons. The ineffectiveness of the transfer is one of the reasons generally cited to explain these results.

This problem was partially solved by the use of adenoviral vectors, but other problems appeared, related to the immunogenicity of these viruses, to the risk of recombination with wild adenoviruses or to the risk of dissemination of these vectors in the circulation. systemic.

 These authors cite the use of specific promoter sequences of vascular cells, such as preproendothelin, or adenovirus-ligand complexes, as constituting very interesting research avenues, without however developing these aspects.

 Application WO 96/05/05 (Rhone Poulenc Rorer S.A.) also describes defective recombinant adenoviruses comprising a suicide gene for the treatment of restenosis. The suicide gene can be placed under the control of an active promoter in vascular smooth muscle cells. Only the promoter of smooth muscle actinia is cited in the context of this embodiment.

No example of the use of such a construction illustrates this request.

 The proteins constituting smooth muscles have been the subject of fragmented studies. Thus, the protein SM 22, which constitutes one of the first markers to appear during the differentiation of smooth muscle cells, has been studied and the coding part of its gene has been sequenced in mice (SOLWAY et AL, 1995, J. Biol. Chem, 270.22, 13460-13469) and in the rat (KEMP et AL, 1995, BIOCHEM. J.

310.1037-1043).

The sequences of the chicken gene and the human gene have also been determined (Nishida et al, 1991, Biochem. Int., 23,663-668 for the chicken gene; Thweatt et al, 1992,
Biochem. Biophysis. Res. Comm., 187.1-7 for the human gene).

 The gene coding for the protein SM 22 alpha in mice is composed of five exons and comprises 6.2 kb. It is present in a single copy in the genome. SOLWAY et al.

showed that the 441 base pairs upstream of the coding region of the gene were necessary and sufficient to induce a high rate of transcription of a reporter gene, in primary cell cultures of rat aortas and in the A7r5 lines. They also demonstrated that the messenger RNAs of this gene were expressed at high levels in the aorta, in the uterus, in the lungs and in the intestine.

 XEMP et al (1995, BIOCHEM. J. 310, 1037-1043.) Cloned and sequenced the 1.9 kb fragment upstream of the coding sequence of the rat SM 22 gene. Deletions in the fragment have shown that the promoter region between nucleotides +65 and -303 were more active than a 1.5 kb fragment comprising a significant part of the region of the untranslated gene, which would suggest the presence of regulatory sequences at the 5 'end of the promoter.

 OSBOURN and AL (1995, Gene, 154, 249-253) also studied the 5 'region upstream of the rat SM 22 gene. They highlighted the presence of two introns in this region.

 Note that these studies were done in vitro using primary aorta cell cultures, not in vivo. However, these experimental conditions are not representative of the activity of genes in the body's cells.

 In addition, studies carried out on the mouse gene concern only a very limited region of the non-coding part of the gene located at the 5 ′ end, that is to say the part downstream of the nucleotide -441. .

 The applicant set out to identify in vivo the activity of the various regions constituting the region upstream of the expressed coding sequence of the gene for the SM 22 protein. It also set out to determine whether any modifications in this sequence induced changes in gene expression in different tissues.

 He surprisingly demonstrated that it was possible, by modifying the 5 ′ region of the mouse SM 22 protein gene, to obtain a specific expression of a reporter gene in the smooth muscles of the arteries and in particular of the aorta.

The present invention therefore relates to a DNA sequence characterized in that it comprises
a fragment of the sequence upstream of the coding part of the gene for the SM 22 protein, or of a sequence hybridizing under highly stringent conditions to said upstream sequence, said fragment being capable of inducing a specific expression in vivo of this gene in artery cells, and
- a sequence coding for a protein, or an RNA, of therapeutic interest
The invention also relates to any nucleotide sequence of natural origin or obtained by chemical synthesis having homology with the sequence SEQID n'l of at least 70 *.

 The term “nucleotide sequence of natural origin” is intended to mean a fragment of complementary DNA obtained by reverse transcription of cellular messenger RNA or alternatively a fragment of genomic DNA obtained after cleavage of cellular DNA using enzymes. restriction.

 By nucleotide sequence obtained by chemical synthesis is meant a DNA fragment of known sequence generated by automatic synthesis of polynulceotides, for example using a suitable automatic device.

 A protein of therapeutic interest, or RNA, is capable of inhibiting the growth of smooth muscle cells, of activating the growth of endothelial cells, of consolidating the arteries and / or capable of inducing a cellular or humoral immune response.

For the present invention, the term "strongly stringent conditions" is used in the sense given by
Maniatis et al., 1982 (Molecular cloning, A Laboratory
Manual, Cold Spring Harbor Lab. CSH, NY USA or one of its recent reissues. Preferably, the hybridization conditions are as described by Maniatis (1982 edition). Three washes intended to remove the non-hybridized fragments are necessary and are carried out at 65 ° C. in the presence of 0.2 SSC and 0.1% SDS, in the absence of formamide.

 Advantageously, said sequence comprises a fragment of the sequence between nucleotides -2126 and +4135, whose sequence SEQ ID n'l is given in the appendix, or of the sequence between nucleotides -2126 and +65 whose SEQID sequence n 2 is given in the appendix.

Such a fragment can in particular comprise at least part of the sequence SEQ ID N 3 which corresponds to the part upstream of the 5 ′ end of the coding part comprised between the nucleotides -2126 and -1340. Such a sequence SEQ ID N 3, the sequence of which is given in the appendix, constitutes an object of the present invention. The sequences
SEQ ID No. 2 and SEQ ID No. 1 are respectively included in the plasmids p2126nlz and p2126INTnlz carried by the strains deposited on March 25, 1996 with the Collection.
National Culture of Microorganisms of the institute
Pasteur (CNCM) respectively under n I-1685 and I-1686.

The subject of the invention is also the sequences hybridizing under conditions of high stringency with the sequences
SEQ ID no 1, SEQ ID no 2 and SEQ ID no 3.

 The present invention is nevertheless not restricted to sequences containing fragments of the mouse gene, but relates to any other sequence of any other species having the same properties, namely to specifically induce expression of a gene in the cells of arteries. Thus, a person skilled in the art can advantageously refer to the article by KEMP et AL (1995, previously cited) which describes the sequence upstream of the rat gene of the SM 22 protein. The present invention also covers any sequence comprising fragments of the upstream sequences of SM 22 protein genes, modified for example by deletion of certain structures which retain functions identical or similar to those of the complete sequence.

 The protein of therapeutic interest can be a protein inducing the formation of a cytotoxic compound, such as the herpes virus thymidine kinase. This protein has the particularity of transforming ganciclovir (Merck Index, reference 4262), an analogue of guanosine, into a derivative which blocks the synthesis of DNA in replicating cells. The introduction of a gene coding for this protein in the sequences according to the present invention therefore makes it possible to block the proliferation of cells in the case of intimal hyperplasia. A sequence encoding thymidine kinase is described in particular by Caruso and Klatzmann (1992, Proc. Natl. Acad. Sci., USA, 89, 182-186).

Said protein of therapeutic interest can also be a protein exhibiting a cytostatic effect, such as the protein encoded by the Rb gene (Chang et al., 1995, Science, 267, 518-522), or by the eNOS gene (Hamon and al., 1994,
Traffic, 90, 1357-1362).

It can also be a protein exhibiting lipolytic activity, such as a lipoprotein lipase. Such proteins are encoded by the sequences described by
Reyner (1995, Nature Genetics, 10, 28) and Ming Sun Liu (1994, J. Biol. Chem, 269-11417).

 It can also be a growth factor for endothelial cells. Such a protein can in particular be an interleukin.

 It can be a muscle protein, such as myosin, or actin, or a tissue-structure protein such as collagen or elastin.

 It can be a protein inducing an immune response as described in application WO 90 11092, and in particular a viral antigen such as the glycoprotein gpl20 or the Nef protein of HIV (human immunodeficiency virus).

In general, said protein can be one or more proteins of therapeutic or vaccine interest, such as proteins of immunotherapeutic interest, for example interleukins, growth factors, for example fibroblast growth factors (FGF) or NGF (Nerve Growth
Factor), proteins which can induce an immune response, whether humoral, cytotoxic or cellular immunity, or proteins which make it possible to complement the activity of a gene normally expressed in the individual to be treated but which is no longer so , either by mutation or deletion of its sequence.

 This sequence can also code for an RNA of therapeutic interest. Such an RNA can be the antisense RNA of the protein p 53 or an RNA as described in application WO 90 11092, filed by the company VICAL.

 The present invention further relates to vectors characterized in that they contain a sequence according to the invention, as described above. Such a vector may contain any other DNA sequence necessary for the expression of the protein, or RNA, of therapeutic interest in the target tissues, and in particular may contain an origin of efficient replication in the artery cells. , especially in smooth muscle cells.

 Such a vector may comprise sequences allowing homologous recombination in the organism treated, specific for the gene to be replaced, said sequences being placed upstream and downstream of the sequence according to the invention. Due to the presence of such sequences, the unwanted gene present in the treated organism will be replaced by the gene carried by the vector or the sequence which is desired to be expressed in the organism.

Such a homologous recombination method can be of the type described by Le Mouellic et al., (1990,
Proc. Nat. Acad. Sci. USA, 87, 4712-4716) or in PCT application WO 91 / 06.667.

Such a vector can be a vector derived from an adenovirus. Adenoviruses suitable for the implementation of the present invention are in particular those described by
FELDMAN and STEG (1996, previously cited) or OHNO et al (1994, Sciences, 265, 781-784) or in application FR 94.03.151 (Institut Pasteur, Inserm). These viruses are generally rejected by the individual, due to their too strong immunogenicity. Nevertheless, strains of these viruses have been selected in order to reduce their immunogenic character. In any event, these vectors, even having a strong immunogenicity, can be used in the context of the present invention, because the arteries are treated for relatively short periods of time, of the order of a few weeks, compatible with the Adenovirus rejection time by the host immune system.

 It should nevertheless be noted that the introduction of the sequences according to the present invention into the vectors described above is not essential, and that the cells of the arteries can be directly transfected with DNA comprising these sequences.

The nucleic acid sequences according to the present invention can be introduced after covalent coupling of the nucleic acid with compounds promoting their penetration into cells or their transport to the nucleus, the resulting conjugates being optionally encapsulated in polymeric microparticles, as in international application WO 94/27238 from Meidsorb Technologies
International.

 According to another embodiment, the nucleic sequences can be included in a transfection system comprising polypeptides promoting their penetration into cells, as in international application WO 95/10534 from Seikagaku Corporation.

 These vectors or sequences can be administered in situ by any means known to those skilled in the art. Thus, they can be delivered in situ using a balloon catheter covered with channels, as described by FELDMAN and STEG (1996, previously cited).

They can also be administered during an operation, or by stripping the aorta. Another mode of administration consists in introducing a mesh of small mesh impregnated with DNA comprising these sequences, along the aorta; as described by Feldman et al. (1995,
J. Clin. Invest., 95, 2662-2671).

These sequences or vectors can advantageously be administered in the form of a composition containing them, for example in a gel facilitating their transfection into the cells of the arteries. Such a gel can be a poly-L lysine and lactose complex, as described by MIDOUX (1993, Nucleic Acid Research, 21, n 4, 871-878) or poloxamer 407 as described by PASTORE (1994,
Traffic, 90, I-517). They can also be dissolved in a buffered solution or be combined with liposomes.

 The present invention further relates to medicaments containing such sequences, vectors or compositions, as well as to pharmaceutical compositions containing them in pharmaceutically effective amounts as well as pharmaceutically compatible excipients.

Such sequences, vectors or compositions can be advantageously used for the manufacture of medicaments for the delivery to the arteries of a
DNA (cDNA or genomic DNA) which can express in particular a gene coding for a protein of interest, for the treatment of coronary heart diseases, and in particular for the treatment of restenosis. They can nevertheless also be used for the manufacture of a medicament for the treatment of mutations which weaken the vessels. Such a medicament could, for example, comprise a sequence or a vector according to the invention capable of expressing a gene coding for a normal protein of desmin or a muscle adhesion protein of CAM type.

 The present invention also relates to RNAs expressed from the sequences and vectors which are the subject of the present invention, and in particular messenger RNAs.

 The present invention also relates to a method for screening molecules in vitro, with a view to testing their activity on the regulatory sequences of the gene coding for the SM protein 22. Such a method will comprise, for example, a first step according to which one transfects cells with a sequence or a vector according to the invention, comprising a reporter gene placed under the control of all or part of the promoter sequence of the gene coding for the SM 22 protein. In a second step, the cells thus transfected are incubated in the presence of the molecule to be tested, then the expression of the reporter gene is quantified.

 The cells used for the transfection are advantageously smooth muscle cells, in particular aortic cells, either in the form of a primary culture, or in the form of a stable cell line.

In the case where the cells are transfected with constructs such as p2126nlz or p2126INTnlz, the quantification of the beta-galactosidase produced will advantageously be carried out by optical reading, for example using the detection kit marketed by
Boehringer under reference 1 669 893 (catalog
Boehringer-Mannheim - Biochemicals, 1996, page 236, beta-Gal Reporter Gene Assay, Chemiluminescent).

In another embodiment of the in vitro screening method according to the invention, the reporter gene is the gene coding for luciferase. In this case, the quantification of the expression of luciferase is advantageously carried out by chemiluminescence, for example by means of the diagnostic kit marketed by Boehringer under the reference 1 758 241 (catalog
Boehringer-Mannheim Biochemicals, 1996, Luciferase
Reporter Gene Assay).

 The present invention also relates to transgenic animals and in particular to mice carrying a sequence or a vector as defined above in which the gene coding for the protein of therapeutic interest is replaced by a reporter gene.

 The present invention finally relates to a method for detecting mutations in the sequence upstream of the coding part of the gene for the protein SM22. Such mutations are indeed capable of modifying the expression of the gene coding for the protein SM22, in particular reducing or even abolishing the expression of this gene in smooth muscles. Such mutations would be likely to cause a modification of the smooth muscle functioning, the SM22 protein being related to a family of proteins involved in the regulation of calcium fixation, of the calponin type (Ayme-Southgate et al, 1989, J. Cell. Biol.). Such a mutation detection method can advantageously be the method which is the subject of French patent application FR-93 10821.

 In addition, sequences according to the invention modified so that they allow an increase in the expression of the SM22 gene are also part of the invention.

 Such transgenic mice can be used to screen molecules for their activity on the regulatory sequences gene encoding the protein SM 22.

Molecules can be administered to mice, then after sacrifice, histological sections are made in order to highlight the tissues stained by the reporter gene.

For the implementation of the present invention, a person skilled in the art may advantageously refer to the following manual: "SAMBROK et al (Molecular cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New
York, 1989), or one of its recent reissues.

The present invention is illustrated without however being limited by the following examples
FIG. 1 represents the restriction map and the exon / intron structure of the SM 22 site, comprising the region -2474 to +6030 (relative to the site of initiation of transcription). The exons are represented by white squares while the arrows indicate the position of the B1 type repeat sequences.

 FIG. 2 represents the alignment of three repetitive sequences of type B1, in which the basic exchanges are indicated.

 Figure 3 is an autoradiogram illustrating the identification of the site of initiation of transcription.

Well P corresponds to the primer used.

 FIG. 4 represents the sequence of the 5 ′ region upstream of the transcription initiation site, and of the exon of the SM 22 gene. The sites for attachment of the different factors are indicated, as well as the different concensus sequences. The broken lines indicate the position of the repeated sequences of type B1.

 Figure 5 illustrates the manufacture of the plasmid p352nlz.

 FIG. 6 illustrates the manufacture of the plasmid p2126 nlz.

 Figure 7 illustrates the manufacture of the plasmid p2126INTnlz.

 Figures 8A and 8B illustrate the expression of the lacZ gene in transgenic mouse embryos. Figures 8A, 8B show embryos in the 12.5 and 15.5 day stages, respectively.

 Figures 9A to 9D show histological sections of embryos stained with lacZ. FIG. 9A represents a section of an embryo at the 12.5 day stage at the level of the heart, showing an intense staining exclusively in the myocardium of the right ventricle (RV) and in the walls of the arteries. (abbreviations: AA: elbow of the fourth aortic artery; CA: carotid artery; E: esophagus; li: left ventricle; PA: proximal part of the aorta; PT: pulmonary trunk; RA: right atrium; RV: right ventricle; T: trachea; V: right and left cardinal vein).

 FIG. 9B represents a section through the abdominal region of an embryo at the 14.5 day stage, showing a coloration of the umbilical arteries (AU) and the absence of labeling of the viscera (B: bladder; D: duodenum; H: large intestine; L: liver; M: small intestine; ST: stomach).

 Figure 9D is a section through the tail segments at the 12.5 day stage showing the labeling in the myotome (my) and the tail artery (a).

 FIG. 9C is an enlargement of the part of the photograph corresponding to the umbilical artery at the 12.5 day stage, showing an exclusive marking of the muscular layer (m) and an absence of marking of the endothelium (e).

 Figures 10A to 10C illustrate the expression of the transgene in adult mice.

 FIG. 10A represents a top view of the whole of the heart showing an intense marking of the aorta (a) and of the pulmonary artery (pa) and an absence of marking of the vena cava (vc). Figures 10B and 10C are sections through the smooth muscle layer (sm) of the adult colon and an adjacent mesentery artery (ma).

The lacZ labeling (FIG. 10B) shows that the expression of the transgene is restricted to the artery, while the immunofluorescence with SM 22 antibodies (FIG. 10C) highlights the expression of the endogenous SM 22 gene in the cells. smooth muscle from both the artery and the colon.

 FIG. 11 represents a vector comprising the regulatory regions of the SM 22 gene and the gene encoding the thymidine kinase of the herpes virus.

EXAMPLES Materials and methods
1 / Clonaae and characterization of the mouse gene SM 22
About 106 recombinant phages from a c57 / bl mouse genomic library constructed in phage delta-EMBL 3 (supplied by Dr D. Plachov, Münster, Germany) were screened using the BAL I / Eco RV fragment of 890 bp from The complementary DNA of the mouse protein SM22
Almendral et al, 1989; Exp. Cell Res. 181, 518-530), as a probe. The probe was radioactively labeled by random initiation and the DNA was hybridized on filters left overnight at 65 ° C in a buffer.
Church. Filters were then washed in 0.2 X
SSC / 0.1% SDS at 65 c and positive plates were purified and homogenized by three successive screenings under identical conditions. One of the isolated clones was found to contain an entire SM 22 locus. It was then mapped using restriction enzymes and subcloned. In order to avoid rearrangements during the cloning procedures, a type hybridization analysis
Southern mouse genomic DNA and genomic clones were performed and restriction figures of the fragments were compared.

The sequence of the SM 22 gene was determined on both strands by the chain termination method (
Sanger et al, 1977, Proc. Natl. Acad Sci. 74, 5463) using the Sequenase V2.0 kit (United States Biochemical,
Cleveland, Ohio).

2. Hybridization of tvpe Northern extension by primer, 5'RACE, and protection analysis by RNAse
Total RNA from cell lines and adult mouse tissue was isolated by the method of Chirgwin et al, (1979, Biochemistry, 18, 5294-5299) modified. For the isolation of messenger RNAs, the Oligotex kit (Quiagen Inc,
Chatsworth, CA) was used following the manufacturer's instructions.

The analyzes by Northern hybridization were carried out using agarose gels containing formaldehyde and by capillary hybridization on Hybond-N membranes (Amersham Life Science, Little Chalfont,
United Kingdom) according to methods known to those skilled in the art.

For the primer extension analyzes, a synthetic oligonucleotide of 30 nucleotides (P27N1) complementary to the sequence +36 to +65 of the DNA complementary to the SM 22 gene (5'-
AAGGCTTGGTCGTTTGTGGACTGGAAGGAG-3), was labeled with T4 polynucleotide kinase. 5 µg of mouse uterine messenger RNA was hybridized at 55 ° C with 1-2 X 105 cpm of purified probe and the primer extension reaction was carried out using laboratory procedures known to man of career. The results are analyzed by electrophoresis on 6% denaturing polyacrylamide gels. A reaction using the same primer was loaded into an adjacent well to allow direct determination of the transcription initiation site.

 The 5'RACE method was carried out using two synthetic 18 bp antisense oligonucleotides corresponding to the bases +112 to +129 (primer 2) and +162 to +179 (primer 1) of the complementary DNA SM 22, as described in substance by FROHMAN et al, (1988, Proc. Natl.

Acad. Sci, 85, 8998-9002).

For RNase protection analyzes, a fragment obtained by PCR of 417 bp comprising the region -352 to +65 of the SM 22 gene was manufactured and cloned by the sticky ends method in the HinC II site of the vector pBluescript. SK +. The sequence of the fragment obtained by PCR was verified. In order to generate a radioactive labeled antisense RNA probe, the construction was linearized with Sph I at position -203 of the SM 22 gene and transcribed with T7 RNA polymerase in the presence of 32p-CTp, The probe (3 X 105 cpm) was hybridized with 5 pg of mouse uterine messenger RNA at 42-C overnight. After hybridization, the samples were incubated with
RNases A and T (1 hour at 37 ° C.), precipitated, and analyzed as described for the analysis by primer extension.

3. Cell culture and transfection
The NIH 3T3, 10T1 / 2 and 8/47 cell lines were cultured in DMEM (Life Technologies, Inc, Vienna,
Austria) supplemented with 10% fetal calf serum (Hyclone Inc., United States) at 37 C, in an atmosphere of Co2 at 7%.

For the transfection experiments, confluent cells were spread out, subcultured in boxes of
6 cm Petri dish, and allowed to grow until approximately 75% confluence is obtained before adding the transfection mixture.

The transfections were carried out using the reagent Lipofectamine marketed by Life Technologies
Inc ..

In summary, 24 μg of Lipofectamine were added to 6 μg of plasmid carrying the superwound reporter gene and 2 g of PCMVss gal as an internal control. After adding 3 ml of
DMEM, the transfection cocktail was added to the cells. After 6 hours, this mixture was replaced with growth medium containing 10% serum and changed again after 12 hours. The expression of the reporter gene product was analyzed after 24 hours.

For the serum stimulation experiments, transfected cells were placed in DMEM containing 20% serum, 24 hours before being harvested.

4. Determination of CAT and B-aalactosidase
The cells were removed from petri dishes with a cell scraper, centrifuged, resuspended in 150 l of buffer Z (100 mM sodium phosphate, pH 7.5, lmM MgCl2, 10 nM KC1, 50 nMss - mercaptoethanol) and lysed by three freeze / thaw cycles. The debris was removed by low speed centrifugation and the activity of ss-galactosidase was determined from 2 to 20 µl of supernatant according to methods known to those skilled in the art. For the determination of the CAT activity according to GORMAN et al (1982), 10 to 100 μl of lysate were used. In order to account for variations in transfection efficiency, CAT activity was normalized with equal activity.

5. Production of transgenic mice
Inserts of the plasmids pnlz2126 and p2126INTnlz, deposited with the CNCM respectively under nI-1685 and nI-1686, were isolated by digestion with Sal I / Pme I, followed by gel electrophoresis, then purification using the Geneclean kit (Bio 101, la Jolla,
CA) then resuspended in 10 mM Tris Hcl pH 7.5, 0.25 mM
EDTA. Transgenic animals were produced as previously described by LI et al (1993, Development, 177/3, 947-959).

Mice carrying the transgene have been identified by
PCR and Southern hybridization using a probe
DNA. Construction pnlz2126 made it possible to obtain three positive animals on 17 mice, two of them transmitting and expressing this construction.

 For the construction p2126INTnlz two positive animals out of the 28 tested were obtained and again only one expresses and transmits this construction.

 The transgenic mice were backcrossed with c57 / bl mice and the histochemical stains were carried out with hemizygous mice for the transgene.

6. Histochemical stains
Whole embryos or adult tissues were fixed for 15 to 30 minutes at 4 ° C in 1% formaldehyde in buffer A (100 mM sodium phosphate, pH 7.3.2 mM Mgcl2, 0.18 deoxycholate sodium, and 0.2 8
NP-40). After rinsing, the specimens were stained overnight at 30 ° C in buffer A containing 1 mg / ml of X gal (Sigma, St Louis, Missouri), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 20 mM of
Tris-Cl pH 7.3. The colored samples were then rinsed, dried with increasing concentrations of ethanol, clarified in xylene, and coated in paraffin. The histological sections were made with a thickness of 7 to 10 μm and again stained with Ehrlich hematoxline and eosin. The embryos could also be clarified again using a mixture of benzyl alcohol and benzyl benzoate.

 For immunohistocoloration, the tissues of mice were fixed in 3 * of paraformaldehyde in PBS buffer at 4 ° C. for 3 hours, then coated in Tissue-Tek medium (Reichert-Jung, Vienna) and sectioned on a cryo-microtome at thicknesses of 5 to 10 µm. The selected sections were mounted and stained using a monoclonal antibody directed against SM 22 (Duband et al, 1993, Differentiations, 55, 1-11), at a 1/30 dilution as well as using a secondary antibody labeled Cy3 (Biological Detection Systems, USA). The sections were examined using a fluorescence microscope.

Example 1 Characterization of the SM 22 locus
Screening by hybridization of the mouse genomic library led to the isolation of three overlapping clones containing the complete SM 22 gene. A restriction map, which is partially reproduced in FIG. 1, has been established and the exons were localized by hybridization using synthetic oligonucleotide probes.

 The regions of interest have been subcloned and sequenced.

 The sequence was determined between positions -2474 and +110 downstream of the polyadenylation site and was compared in the EMBL database. The gene covers 5923 bp from the start of transcription site to the polyadenylation site and the coding sequence is divided into 5 exons (Figure 1). All exonintron boundaries correspond to consensus sequences.

 The first exon contains only a 5 'leader sequence and the transcription start codon is therefore located in the second exon separated from the transcription start site by more than 4 kbp of intron sequence.

 Two potential polyadenylation signals have been located at positions 5905 and 5913 and several TGT regions placed downstream of the polyadenylation site, presumably take part in the termination of transcription.

The analysis of the sequence also reveals the presence of three repeated sequences (FIG. 2) which have significant homologies with the repeated elements of the type
B1, some of which are transcribed by RNA polymerase III and code for 5S RNA. It will be noted that all the repeated sequences are in an opposite orientation relative to the locus SM 22.

 The transcription start site was mapped by primer extension analysis (Figure 3). An antisense oligonucleotide complementary to the 3 'end of exon 1 leads to the production of four extension products which differ only in base pair (bp) in length, the longest being 65 bp. The transcription product therefore starts with a G, 77bp upstream of the transcription initiation codon.

 The shortest extension products were thought to be caused by premature termination of primer extension due to capping at the 5 'end of the messenger RNA.

 These results were confirmed by the RNAse protection analysis, which leads to a protected fragment of 65bp and by the cloning and sequencing of the extension product derived from an independent reaction with an antisense primer located in the second exon. of the gene.

 These results are in agreement with those of Solway et al (1995 previously cited). Differences in minor sequences between these two results are probably due to allelic variations between the different strains of mice.

 The TTTAAA sequence in position -28bp (Figure 4) is closely related to the TATAAA sequence and probably has the function of a TATA box. Computer analysis of the 5 'sequence reveals the presence of several potential binding sites for factors as well as for elements which are known to contribute to the regulation of muscle gene transcription (Figure 4). A total of 11 E-type boxes (CANNTG), four Mef-2 / rSRF-type patterns (YTAWAAATAR), four potential SRF linkers (CC (A / T) 6GG), five AP-2 link sites (CCCMNSSS) and five SP1 motifs (GGGCGG) have been located. Finally, an element similar to the motif TGT3-3, recently identified as a binding site for a nuclear factor of smooth muscle and as contributing to the regulation of transcription of the alpha actin of smooth muscle, has been located (FIG. 4). .

Example 2
Clonaae and construction of recombinant plasmids for the creation of transgenic mice
1 / Dnlz manufacturing
Plasmid pGEM7 (Promega Inc., Madison, Wi) was digested with Nae I / Bsp 120I. The outgoing end Bsp 1201 of the vector was filled with the Klenow enzyme, and the plasmid was closed in the presence of a 10 bp Sal I linker. The lacZ fragment of pGEM 7 has therefore been deleted, a site
Sal I introduced, and a restored Bsp 120I site.

An SME site was introduced by insertion of a linker
Sme I with Nsi I outgoing single-stranded ends, in the Nsi I construction site.

 Finally, the lacZ gene coupled to the nuclear localization signal was isolated from pDes2.2nlz (LI et al, 1993, previously cited) with Hind III and Sac I giving two fragments of the reporter gene.

The two fragments were inserted between the Hind sites
III and Sac I of the modified pGEM7 vector and verify for their orientation. This procedure gives the promoter-free pnlz vector.

2 / Manufacture of D352nlz
Figure 5 illustrates the manufacture of this plasmid.

 A PCR fragment of 417 bp covering the region -352 to +65 of the SM 22 gene was produced and inserted into the Hinc II site blunted from the vector pBluescriptSK +. The sequence of the PCR fragment has been verified. The fragment is recovered by the Xba I and Xho I sites and inserted into the plasmid pBLCAT 3 in the corresponding sites, generating the recombinant p352CAT. To create p352nlz, the Xba I / XhoI fragment of p352CAT was inserted into the corresponding sites of pnlz.

3 / Manufacture of D2126nlz
To create p2126nlz, a 1.9 kb Bsp 120I / Sph I fragment covering the region -2126 to -206 of the SM 22 locus was inserted into the respective sites of p352nlz. This plasmid was deposited on March 25, 1996 with the CNCM under the number I1685. Its manufacture is illustrated in Figure 6.

4 / Manufacture of D2126INTnlz
The manufacture of this plasmid is illustrated in FIG. 7.

A 5.8 kb Bsp 120I / Hind III fragment covering the region -2126 to +3651 of the SM 22 locus was inserted into the corresponding sites of pnlz, giving pINt-nlz. Then a 496 bp PCR fragment from the base +3648 to +4143 of the SM 22 locus was generated using a primer upstream and a primer downstream designed to introduce a Hind III site at position 4135-4140. This PCR product was digested with
Hind III and linked into the Hind III site of pINT-nlz to give p2126INT-nlz. Construction p2126INT-nlz therefore contains 2126 base pairs from the region 5 'from the start of transcription, the first exon, the first whole intron and 15 base pairs from the second exon with the last four base pairs mutated from CATG to GCTT to remove the translation initiation codon and introduce a Hind III site as mentioned above. This plasmid was deposited on March 25, 1996 with the CNCM under nI-1686. Its manufacture is illustrated in FIG. 7.

EXAMPLE 3 Expression of Constructions in Transcenia Animals
Two constructs using the lacZ reporter gene with nuclear localization signal of the SV 40 virus were manufactured, as described in example 2.

 The first construction, p2126nlz contains 2126 bp from the upstream region and the first exon (65 bp). The second construction p2126INTnlz is identical to the first except for the addition of the first intron and the first 12bp of the second exon.

 As indicated in the Materials and Methods section, each of the constructs gave rise to at least one transgenic mouse expressing the reporter gene.

 The embryos obtained from 12.5 to 15.5 days after coitus (dpc) were stained so as to demonstrate equal activity. The expressions of the two constructions are identical, at all the stages observed, although p2126INTnlz gives a slightly more intense coloration.

 Expression was first detected on day 8.5 in the heart region of the embryo and in the vessels of the cardiac envelope. On day 9.5, the expression of lacZ in the heart increases and is confined to the right ventricle, to the arteries.

 Between 12.5 and 15.5 days a segregation of the marking according to the regions becomes obvious, the marking being confined to the right ventricle (Figures 8A and 8B). The sections show that expression takes place in the myocardium of the right ventricle, with minor markings in the left ventricle and in the right adrium (FIG. 9A).

 This expression in the heart is only transient, and does not appear in adults. It is likely to be inhibited in the late stages of fetal development. Early expression is also observed in the segments of the rostrum from 9.5 days which extends to the caudal segments at 10.5 days, and reaches significant levels & 11.5 days.

 The expression decreases from 12.5 days in the rostrum segments (Figure 8B) and finally is no longer detectable at 14.5 days (Figure 10E).

 The sections show that the expression of the transgene is restricted to the myotomal region of the segments (FIG. 9D).

 Most of the expression takes place in the vascular system of the developing embryo. From 9.5 days, the expression is detected in the dorsal aorta. At 10.5 days, the coloring of the dorsal aorta and aortic elbows increases and at 11.5 days, the lacZ expression is clearly visible in the dorsal aorta, aortic elbows, iliac arteries, umbilical arteries, the carotid arteries and in the major vessels of the head.

 The expression of the transgene in the vessels increases from 12.5 days, when the intercostal vessels can be easily seen (Figure 8A).

 At 15.5 days (Figure 8B), lacZ is present in the major vessels, including those of the limbs and tail, and the coloration of the pulmonary trunk becomes visible. Expression in the vascular system persists in adults and is clearly visible in the aorta, in the pulmonary trunk and in the right pulmonary artery (Figure 10A), as well as in the vessels of the intestines (Figure 10B), the bladder and into the uterus.

 Histological sections better reveal the differences in the expression of the transgene in the arteries and in the veins. The section of the embryo at 12.5 days (FIG. 9A) shows a very high level of expression in the muscular layers of the arteries of the carotid artery, of the fourth artery of the elbow of the aorta, of the proximal pulmonary trunk and of the proximal part of the ascending aorta, while the left and right parts of the anterior cardinal veins remain uncolored. The absence of expression of the transgene in the muscular veins is evident from the sections of the ductus venosus, as well as from the portal and umbilical veins. The same situation is observed in adults, where expression can never be detected in the cava veins (Figure 10A) or in the pulmonary veins.

 These observations highlight differences that had not previously been demonstrated between the smooth muscle layers of the arteries and veins.

Other views of the arteries show that expression is restricted to the muscle layer and is absent from the endothelium (Figure 9C).

 It is moreover evident, in the light of all the observations (FIGS. 8A, 8B), that the expression of the transgene is absent from the smooth muscles of the viscera. This is unexpected because the endogenous SM 22 gene is thought to be expressed in vascular smooth muscle tissue and viscera. In histological sections of the abdominal region of the embryo at 14.5 days, the layers of muscles of the developing stomach, intestine and bladder are easily recognizable (Figure 9C).

The expression of the transgene in this region is clearly detected in the umbilical arteries, but not in the muscle layers of the viscera of the stomach, duodenum, small and large intestine, and bladder, where none lacZ expression is not observed. Furthermore, no expression is observed in the esophagus and in the trachea (FIG. 9A), as well as in the bronchi of the lung.

 The coloring of adult muscle tissue gives essentially the same results. FIG. 10B shows that the expression of the transgene is absent in the muscles of the colon, while the mesentery vessel is very strongly stained by lacZ. On the other hand, immunofluorescence staining of the section of a similar intestinal region shows that the endogenous SM 22 protein is present in both tissues (FIG. 10C). The absence of expression of the transgene in the smooth muscles of the viscera of the adult is further confirmed by staining of the esophagus, trachea, bladder, vas deferens and the uterus for activity. equal. In no case can a Jugal expression be detected in the muscle layers of these tissues.

 Curiously, a significant coloration is observed in the cytoplasm of the epithelial cells of the lumen of the vas deferens and in the epithelium of the renal ducts.

This is probably due to an endogenous galactosidase activity, the coloration being also visible in the non-transgenic control animals.

 Example 4 - Manufacture of a construct comprising regulatory regions of the SM 22 gene and the gene encoding the herpesvirus thvmidine kinase (SM 22 INT-TK).

The HIV-LTR fragment (-167, + 80) is deleted from the plasmid pLTR-TK by Hind III digestion, the ends of which are made blunt with Klenow enzyme, followed by Xho I digestion. The plasmid closed by inserting a linker containing the XhoI sites in 5 ', the Hind III and Bsp 120I sites in the order 5'->3' and a 3 'blunt end to give the vector p-TK. A 5.8 kb Bsp 120I / Hind III fragment covering the region -2126 to + 3648 of the SM 22 locus is inserted into p-TK to obtain SM 22 INT
TK.

 This construction is schematically represented in FIG. 11.

LIST OF SEQUENCES (1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: INSTITUT PASTEUR
(B) STREET: 28 RUE DU DOCTEUR ROUX
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75015
(A) NAME: PARIS 7 UNIVERSITY
(B) STREET: 1 PLACE JUSSIEU
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 75005
(ii) TITLE OF THE INVENTION: SEQUENCES UPSTREAM OF THE SM22 GENE
(iii) NUMBER OF SEQUENCES: 3
(iv) COMPUTER-DETACHABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release +1.0, Version # 1.30 (EPO)
INFORMATION FOR SEQ ID NO; I:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 6261 pairs of bases
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linear
(ti) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIC: NO
(vi) ORIGIN
(A) ORGANIZATION: MOUSE
TCTAGAGGCC ACGGAACGGT GCCAAGCACA CAGTCCCTTT TGCCTCTTTC
ACGGGAGCAG
GAGTCCCAGT GCCTGTCGTG GAAAGGGAGG AACATGCCAG GTCCCTGTGT
GTCCTTGGCC
CTGTCTCACC AAAGGACTCA GGGCTGGTTT CTGAGTTTCC GTCCAGTATT
TAGCCAAGTC
CTGTGTTAGT CACGTAGGCC TAAGAGCCTT GGCGTTTACG GAGTCACCCA
GCTCTGGCCC
CTGGCATTCT GGTCCTTGGC GTTTACAGAG TCACCCAGCT CCAGGCCCCT
GGCACTTTGG
TACTTGGTTG CCCTTCACTC CACCAGGTCC ATTCCAGATG CCAAGAGTGG
GCCCCAGGAA
TGTGTTTCCT TCTCTCCACC ATGTTTTTAT AGCTCTTGGG CTGGGAGAAG
AGGCGGGTCT
GGGTCTTTGT TTCTGAGCTT TGTTCTATGT TCCTCCATGC TACGGTTGCA
ATTGTTTTCT
ATGAACGAGT ACATTCAATA AAGACAACCA GACCTGGGAT TTGGGGTCTT
ACTGATGTGT
TGGGAGGTGC AGGAGCCTCC GTGTCCCATT TATTTTGGCC TTCCCGTCTC
GTTTCTGTGC
GTGGCTACAT TGGGAATGAC CTTCCTTGAT CCCACCAAGC CACCCATTGA
TTCTGTAAAC
AtGTGACCCT TGCTCCAAGC ATTGCTTACA GGAGCAGGAT ACTGAAAGTG
TGTCTGTGCC
CTCTCCTGAT AACCCCTCCC TTCAGCAGGC ACACAGCACC TGACTACCCA
CCACGTATGT
AAACGTCAGT ATCCTTTCCA GCCAGCTCTG CAGATGGGTG TCCAGGCTGT
GCATGATGCA
CCTCAAGTGG GCAGAGCTTG CAGGCCAAGG TTTTAAAGGC TGTTCAGGAA
TGGATGGCAA
GCAGGATCTA AGAGGAGGGG GGGTTGTTGT TGTTTGGGGG GGGGGTGGTT
TTGGTTTGTT
TTTTTTGAGA CAGGGTTTCT CTGTGTGGCC CTGGCCCTCC TGGAACCCAC TCTGTAGACC
AGGCTGGCCT TGAACTCAGA AATCTGCCTG CCTCTGCCTC CCGAGTGCTG
GGATTAAAGG
CGTGTGCCCA TCGAGGAGGG AGATTTTATT TAGATTATAA AAAGGACGGG
ATTTGGGGAA
TCCTGTCTAG TGAATTCAGG ACGTAATCAG TGGCTGGGAA GCAAGAGCTC
TAGAGGAGCT
CCAGCTTATT ATGACCCTTC CTTCAGATGC CACAAGGAGG TGCTGGAGTT
CTATGCACCA
ATAGCTTAAA CCAGCCAGGC TGGCTGTAGT GGATTGAGCG TCTGAGGCTG
CACCTCTCTG
GCCTGCAGCC AGTTCTGGGT GAGACTGACC CTGCCTGAGG GTTCTCTCCT
TCCCTCTCTC
TACTCCTTCC TCCCTCTCCC TCTCCCTCTC TCTGTTTCCT GAGGTTTCCA
GAATTGGGGA
TGGGACTCAG AGACACCACT AAAGCCTTAC CTTTTAAGAA GTTGCATTCA
GTGAGTGTGT
GAGACATAGC ACAGATAGGG GCAGAGGAGA GCTGGTTCTG TCTCCACTGT GlliGGTCTT
GGGTACTGAA CTCAGACCAT CAGGTGTGAT AGCAGTTGTC TTTAACCCTA
ACCCTGAGCC
TGTCTCACCT GTCCCTTCCC AAGACCACTG AAGCTAGGTG CAAGATAAGT
GGGGACCCTT
TCTGAGGTGG TAGGATCTTT CACGATAAGG ACTATTTTGA AGGGAGGGAG
GGTGACACTG
TCCTAGTCCT CTTACCCTAG TGTCTCCAGC CTTGCCAGGC CTTAAACATC
CGCCCATTGT
CACCGCTCTA GAAGGGGCCA CCCTTGACTT GCTGCTAAAC AAGGCACTCC
CTAGAGAAGA
TACCATACCT GTGGGCAGGA TGACCCATGT TCTGCCATGC ACTTGGTAGC
CTTGGAAAGG
CCACTTTGAA CCTCAATTTT CTCAACTGTT AAATGGAGTG GTAACTGCTA
TCTCATAATA
AAGGGGAAGG TGAGGAAGGC GTTTGGATAG TGCCTGGTTG CGGCCAGGCT
GCAGTCAAGA
CTAGTTCCCA CCAACTCGAT TTTAAAGCCT TGCAAGAAGG TGGCTTGTTT
GTCCCTTGCA
GGTTCCTTTG CTCGGGCCAA ACTCTAGAAT GCCTCCCCCT TTCTTTCTCA
TTGAAGAGCA
GACCCAAGTC CGGGTAACAA GGAAGGGTTT CAGGGTCCTG CCCATAAAAG
GTTTTTCCCG
GCCGCCCTCA GCACCGCCCC GCCCCGACCC CCGCAGCATC TCCAAAGCAT
GCAGAGAATG
TCTCCGGCTG CCCCCGACAG ACTGCTCCAA CTTGGTGTCT TTCCCCAAAT
ATGGAGCCTG
TGTGGAGTGA GTGGGGCGGC CCGGGGTGGT GAGCCAAGCA GACTTCCATG
GGCAGGGAGG
GGCGCCACGG GGCGGCAGAG GGGTGACATC ACTGCCTAGG CGGCCTTTAA
ACCCCTCACC
CAGCCGGCGC CCCGGCCCGT CTGCCCCAGC CCAGACACCG AAGCTACTCT
CCTTCCAGTC
CACAAACGAC CAAGCCTTGT AAGTGCAAGT CATGGGAGCA GAAGGGCTGT
GGGCTCAATT
AGATCCCCTA GTCTCTTCTA GTTTGCTGGG TGGAATTGGG TCCCTAGAGA
CCATTCTCTG
TGTTAGACAA AAAGTCTGGG TTAAAATGCC TAGGATGATT TGACTGGGGC
AAAAGAATAA
ATGQGGTGAG AGGGAGGCTC AAATTCAGTC ACTGTCCCAC CCATAGGTGT
ATGGGCTATG
TGTTAGGCCC AAAGAGGTGA CAAATGAGGC CAAGGGAACA ACTCCATCTT
TGGATCTCCA
AGAAGGTGAG GGGCTAAGTT CTGGAAAGCA GTGACCCACT GATGGTCCCC
AGGGCTAATG
CAACTCGGGG GAGCCAGGAG GTAGCCCCCT CAGGCAGTGG AGGACTAAAG
ATCTTATTTT
TTGTAGCGCT AGGGATCAAA CCCCAGGGCG CTATGTGTGG CAGGCATGTG
CTCCATCTAC
CACAGAAGTT TAATCCTTCA GACTAGCCTG GGATAGGGCC TGCTTTTTCT
TTCCTTTTCT
CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTTTTT
CTTTCCTTTT
CTCTCTTTCA CTCTCTCTTT CTAATTTCTT TTTCTTTTTT TCTTTCTTTT CTTTAGACAG
GGTTTCTCTG TGTAGCCCTG GCTGTTCTGG AACTCACTCT TTAGACCAGG
CTGGCCTCGA
ATCTCAGAAA TCTACCTGCC TCTGCCTCCC AAGTGCTGGG ATTAAAGGCG
TGTGCCACCA
CTGCCCAGCT AAGGTTTGCT TTTTGATGGC AGCTTGGTCC AGTTTGAAAG
TAGGAGGTCA
GTCCACTGTA GGCAGATAGG TGACAGGTGG CAGATAGGTG ACAGATAGGT
GACAGGTGGA
GGAGCTTTGG AACTGGGACT GGACAGCCCT GGGACCCTGT TCCTCCCAAA
GGGTCTTGGT GGTTCCCCTT GGGGCTCTCT MAGGATGTC AGTGGGCTGT TGCCACATCT
ATATAAGAGG
ACTAGTCTrC TGGAATTTAG GTGTGATCTC TCAGGGATGC AGAAATGCTC
ACCCTTACTG
TCATTTTATG GGCTGAGGTA CCACAGGCAG ATATACCCTG GTCTGCTTGT
TGTCCAGGGT
CTCTGCTACA TGGAGGCCCC TTTCCACAGC CTAACCTCTC TACCTGCTGA
CAGGAGGGCT
GGATGGCCAC AGGCATCCAA CGTGCGCATC ATGCAGGTGT TTTGCGTTGG
AGCTTTTGTC
TAGAAATACC CTGGTGGGCT GCCAAACCAC CACCCATATC CCTCTCTCCT
CTCTGCTGCC
TCTAAGATGA CAGCTTGATT TTTCTTATAG TGATTTTTTT TTTTGGTTTT GTTTTTTTGT
TTGTTTTAAG TTAGCATACA AAGTAATACA TTTCATCATG GCATTTGGAC
ATACATATAT
ATTTTATTTG CTCTCCTGGC CTCTTCTCAA AGAGACTTCT CTGGAcTTTC
TTGTATTTTT
GGTTGTGAGC CTAGCCTTTA ACGGCTGAGC CATCTCTCCA GCCCTTCTTT
GGACTTTCTA
CTTCATACTT CCCACCAGTC TGGGAAGAAG GGCACATGGA ATCTTGAGAG
CATGACCTGA
CCCAGACCTG ACAGATGTCA AGGCTGCAGT GTATGCTCTT GTTCGTACGG
CTTGTTCTTA
GTCCTGCAGT TCAGAACTTT CTGGAGACTG AGAAGTGCAT GTGAGGACAC
TCTCCTCCCA
TCTTTTCCTC TAGTGGCTAG TGATGTTTGG TTTTTTGTTT TGAGACAGGG TTICTCTGTA
TAGCCCTAGC TATCCTGGAA CTCACTTTGT AGATCAGGCT GGCCTCCAAC
TCAGAAATCT
GCCTGCCTCT GCCTCCCGAG TGCTGGGACT AAAGGCGTGC GCCACCACTG
TCCAGTCAGG
AGTAGAAGGA AACTGTAAGG TGCTTGAGAC AGGCTGAGTA GAGGCTAGGA GGAAGGGGCA
CCGCAGTCAC CGGCTCCATG ACTCTGTGAC WTGTGGTT CCTTGTCGCA
GCGGTTCCTG
GTGGTGGTGG TGGTCGGGGG TTGGGGGGAG GGGGCAGGCC ACACAGTGGG
GTGTGGGAGG
GAATAGCTGT TGACAACTTC CCAACAGAAA CCAGGCTTTT GAGTCCTCCA
GGGTAGCTTG
AGAGGGTACT CAGAAAGCCG TGTCCATGTC CCCTTTCCTT CACCTCAGGG
AAGTAAGTTG
CCTATAGGGT TGTCATTTCA ATGAGGTCTT CTGGTTATTC TGTTTTTCTC TCAATGTTGG
TGTTGGGCTC AGGGAATGCT TTGGAGAAGG TGGTGGGAAC TGGAGAAGGG
AAGATCAGTT
TACTGTGTAA ACTCACTGGT TAAAGTACCC CCTCCCTTCC ACCCTGCAAT
ACACACACAC
ACACACACAC ACACACACAC ACACACACAC ACCCCATCTC GAAGAGCTCT ATTAAGCTCC
AGGTGCACTG TAGTTCACAG ACTGCATCTT CCAGGTTTGC TCCCACTTCA <RTI ID = 32.
CTCCATGCTC ACACAATTTT TCTCTCAACC AATGACCTCT CAGAAGCAGG
GGTTGGTTTG
CAAAATTCTT CAGATACCTC AACAGATGGC ATCCCACTCA GGCTATCCCT GCTGACTAGG
TCTGGCTCCA GCCCTGACTG TATCTACCCA GGGACCTACC TGCCTGCTTT
GCTCCTATAG
CCTTCCTCCG TGTCTGGGTC CCCAGAGAGAGC TGCCGGCATA GGCCTTTGAG
GCAACAGCTG
GCATACAGGC CAGGCTTCCC ATGCTCTGGC TAGCAGATTC TCTGCCCTGG
AGGACTTTGA
CTGCATGGTT TCTCTCACTG CTGCAACAGT CAGAGCTGGC CCACACGGGC ACAACAGCGC
ACTTCCATCT GGGTCTCCCT GAGAATGCCG CTGTTTTCTG AGAACCCTTG
GACTCTGGTG
GCTTTATCAG GTCTTTTTGT CAGCTGCGCT TTGGGGGATG AACTTTGCTC
TTCTGGCTTC
TGGGTCAGAG GGTAAAGATT TGGTGGCAAC CGGTAGCTAG AGAAAGATAG
CTACTGGCTG
AATTTGGAGG ACATGGCTTC TGGAAAACCT CTCTAGTGCT TTTCTGGCTA
GTCTTGGCAA
AGTAAAAATG CTCTGATAGC CAGCCCGGGT GATGCAGGGC TTCCTGTTCG AGGCCTTTCT
GTACAAAATT AGTGAGACAT TGCCTCAAAA CTATGAAACA AGCCAGACTC
TGTTGAAGCA
CGCCTTTAAT CCCAGCACTC AGGAGGCTGA AGCAGGCAAG ATCTCTGTTA
GTTGGAGGCC
AGTCTACAGG AAAGTTCTAC AACAGCAGAG GCCAGACAGT GCAACCCTTT
CTGGGGGTGT
GGGGGAGGAA AACCCAACAA AAACACAAAC TATAAAACAA AGAGAAGGCC
GAGGACAAAG
CTTAGCAATG CATACTTCCC TTTCTATGTG AAGCCCTGGG CTCCACCAGT
ACTGCAGAAA
GAAGCAAGCA ATGAGGGACA GGAGGTTGGC TCTAGGCCCA GGGGTTGTCA AAATAGTCCA
CAGGCCAAAG GCAGCCTGAT GTCTGTTTTT ATAAACAAAA TTTTATTGGC ACACATTGGT
TATGTATCAG CTAGGCTATT TTCATTACAA TAGAGGCCAT ATGGTCTGTA
AAGTCTAAAA
TATTTACTCT GCTGTTTTAC ATAAAAAGTT GACAGACTCT TGCTCTAGAC
TGACAAATAT
CTAAGACCTT GTTTTCTGAG GTTCAAGTTT CAGAGGGGTC TCTGCAGCAA
GTGGGTAAAG
CTGGTCTAGG TCATGCTATG ATGTCTAGGG TCCCCTCAGA GTGGAAGGCC
TGCTTAGCAC
AAATGAAGTA AAGTAACTTG CTGGCTCTTT GTTCTTTTCT CCACACTCTA
TACTTTAGCT
CTGCCTCAACATG (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 2190 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIC: NO
(vi) ORIGIN:
(A) ORGANIZATION: MOUSE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2
GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA 60
AGAGGCGGGT CTGGGTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG 120
CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC 180
TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 240
TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 300
GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 360
TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 420
CACCACGTAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 480
GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 540
AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 600 TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 660
ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 720
TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 780
GGATTTGGGG AATCCTGTCT AGTGAATTCA GGACGTAATC AGTGGCTGGG AAGCAAGAGC 840
TCTAGAGGAG CTCCAGCTTA TTATGACCCT TCCTTCAGAT GCCACAAGGA GGTGCTGGAG 900
TTCTATGCAC CAATAGCTTA AACCAGCCAG GCTGGCTGTA GTGGATTGAG CGTCTGAGGC 960
TGCACCTCTC TGGCCTGCAG CCAGTTCTGG GTGAGACTGA CCCTGCCTGA GGGTTCTCTC 1020
CTTCCCTCTC TCTACTCCTT CCTCCCTCTC CCTCTCCCTC TCTCTGTTTC CTGAGGTTTC 1080 CAGAATTGGG GATGGGACTC AGAGACACCA CTAAAGCCTT ACCTTTTAAG AAGTTGCATT 1140
CAGTGAGTGT GTGAGACATA GCACAGATAG GGGCAGAGGA GAGCTGGTTC TGTCTCCACT 1200
GTGTTTGGTC TTGGGTACTG AACTCAGACC ATCAGGTGTG ATAGCAGTTG TCTTTAACCC 1260
TAACCCTGAG CCTGTCTCAC CTGTCCCTTC CCAAGACCAC TGAAGCTAGG TGCAAGATAA 1320
GTGGGGACCC TTTCTGAGGT GGTAGGATCT TTCACGATAA GGACTATTTT GAAGGGAGGG 1380
AGGGTGACAC TGTCCTAGTC CTCTTACCCT AGTGTCTCCA GCCTTGCCAG GCCTTAAACA 1440
TCCGCCCATT GTCACCGCTC TAGAAGGGGC CACCCTTGAC TTGCTGCTAA ACAAGGCACT 1500
CCCTAGAGAA GATACCATAC CTGTGGGCAG GATGACCCAT GTTCTGCCAT GCACTTGGTA 1560
GCCTTGGAAA GGCCACTTTG AACCTCAATT TTCTCAACTG TTAAATGGAG TGGTAACTGC 1620
TATCTCATAA TAAAGGGGAA CGTGAGGAAG GCGTTTGGAT AGTGCCTGGT TGCGGCCAGG 1680
CTGCAGTCAA GACTAGTTCC CACCAACTCG ATTTTAAAGC CTTGCAAGAA GGTGGCTTGT 1740
TTGTCCCTTG CAGGTTCCTT TGCTCGGGCC AAACTCTAGA ATGCCTCCCC CTTTCTTTCT 1800
CATTGAAGAG CAGACCCAAG TCCGGGTAAC AAGGAAGGGT TTCAGGGTCC TGCCCATAAA 1860
AGGTTTTTCC CGGCCGCCCT CAGCACCGCC CCGCCCCGAC CCCCGCAGCA TCTCCAAAGC 1920
ATGCAGAGAA TGTCTCCGGC TGCCCCCGAC AGACTGCTCC AACTTGGTGT CTTTCCCCAA 1980
ATATGGAGCC TGTGTGGAGT GAGTGGGGCG GCCCGGGGTG GTGAGCCAAG CAGACTTCCA 2040
TGGGCAGGGA GGGGCGCCAC GGGGCGGCAG AGGGGTGACA TCACTGCCTA GGCGGCCTTT 2100
AAACCCCTCA CCCAGCCGGC GCCCCGGCCC GTCTGCCCCA GCCCAGACAC CGAAGCTACT 2160
CTCCTTCCAG TCCACAAACG ACCAAGCCTT 2190 (2) INFORMATION FOR SEQ ID NO: 3
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 785 base pairs
(B) TYPE: nucleotide
(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomic)
(iii) HYPOTHETIC: NO
(vi) ORIGIN:
(A) ORGANIZATION: MOUSE
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID No: 3:
GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA 60
AGAGGCGGGT CTGGGTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG 120
CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC 180
TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 240
TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 300
GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 360
TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 420
CACCACGTAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 480
GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 540
AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 600 TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 660
ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 720
TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 780 WATT: 785
References
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Babij, P., Kelly, C. and Periasarny, M. (1991). Characterization of a mammalian sruooth muscle myosin heavy-chain gene: Complete nucleotide and protein coding sequence and analysis of the 5 'end of the gene. Proc. Natl. Acad Sci, 88, 10676-10680.

Birnsticl, M.L., Busslinger, M. and Strub, K. (1985). Transcription termination and 3 'processing: the end is in site. Cell, 41, 349-359.

Bour, BA, O'Brien, MA, Lockwood, WL, Goldstein, ES, Bodmer, R., Taghert, P.
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LI LI, JM MIANO, B. MERCER and E. OLSON
J. OF CELL BIOLOGY 1996 - VOL 132 N 5, p. 849-859

Claims (33)

 1. DNA sequence characterized in that it comprises  a fragment of the sequence upstream of the coding part of the gene for the SM22 protein, or of a sequence hybridizing under conditions of high stringency to said sequence upstream, said fragment being capable of inducing a specific expression of a gene in eukaryotic cells, and  - a sequence coding for a protein, or an RNA, of therapeutic interest.  2. DNA sequence according to claim 1, characterized in that it comprises  a fragment of the sequence upstream of the coding part of the gene for the protein SM 22, or of a sequence hybridizing under conditions of high stringency to said sequence upstream, said fragment being capable of inducing a specific expression in vivo a gene in artery cells, and  - a sequence coding for a protein, or an RNA, of therapeutic interest.  3. Sequence according to one of claims 1 or 2, characterized in that said protein, or said RNA, is capable of inhibiting the growth of smooth muscle cells, of activating the growth of endothelial cells, of consolidating the walls arteries and / or induce an immune response.  4. Sequence according to one of claims 1 to 3, characterized in that said fragment is included in the sequence located between nucleotides -2126 and +4135 of the mouse gene of the protein SM22 (SEQ ID n 1.)  5. Sequence according to one of claims 1 to 4, characterized in that said fragment is included in the sequence located between the nucleotides - 2126 to +65 of the mouse gene of the protein SM 22 (SEQ ID N-2).  6. Sequence according to one of claims 1 to 5, characterized in that said protein of therapeutic interest is a protein inducing the formation of a cytotoxic compound.  7. Sequence according to claim 6, characterized in that said protein is the thymidine kinase of the herpes virus.  8. Sequence according to one of claims 1 to 5, characterized in that said protein of therapeutic interest has a cytostatic effect.  9. Sequence according to one of claims 1 to 5 characterized in that said protein of therapeutic interest has lipolytic activity.  10. Sequence according to claim 9, characterized in that said protein is lipoprotein lipase.  11. Sequence according to one of claims 1 to 5, characterized in that said protein of therapeutic interest is a growth factor for endothelial cells.  12. Sequence according to claim 10, characterized in that said factor is an interleukin.  13. Sequence according to one of claims 1 to 5, characterized in that said protein of therapeutic interest is a muscle protein or tissue structure.  14. Sequence according to one of claims 1 to 5, characterized in that the RNA of therapeutic interest is the antisense RNA of the protein p53.  15. DNA sequence located upstream of the coding part of the mouse gene of the protein SM 22, characterized in that it comprises at least a part of the following sequence SEQ ID N-3, or a sequence hybridizing with this under stringent conditions GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA AGAGGCGGGT CTGGGTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC CACCACGTAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG GGATT  16. DNA sequence located upstream of the coding part of the mouse gene of the protein SM22, characterized in that it comprises at least a part of the sequence SEQ Following ID # 1, or a sequence hybridizing therewith under stringent conditions TCTAGAGGCC ACGGAACGGT GCCAAGCACA CAGTCCCTTT TGCCTCTTTC ACGGGAGCAG  GAGTCCCAGT GCCTGTCGTG GAAAGGGAGG AACATGCCAG GTCCCTGTGT GTCCTTGGCC  CTGTCTCACC AAAGGACTCA GGGCTGGTTT CTGAGTTTCC GTCCAGTATT TAGCCAAGTC  CTGTGTTAGT CACGTAGGCC TAAGAGCCTT GGCGmACA GAGTCACCCA GCTCTGGCCC  CTGGCATTCT GGTCCTTGGC GTTTACAGAG TCACCCAGCT CCAGGCCCCT GGCACTTTGG  TACTTGGTTG CCCTTCACTC CACCAGGTCC ATTCCAGATG CCAAGAGTGG GCCCCAGGAA  TGTGTTTCCT TCTCTCCACC ATGTTTTTAT AGCTCTTGGG CTGGGAGAAG AGGCGGGTCT  GGGTCTTTGT TTCTGAGCTT TGTTCTATGT TCCTCCATGC TACGGTTGCA ATTGTrTTCT  ATGAACGAGT ACATTCAATA AAGACAACCA GACCTGGGAT TTGGGGTCTT ACTGATGTGT  TGGGAGGTGC AGGAGCCTCC GTGTCCCATT TATTTTGGCC TTCCCGTCTC GTTTCTGTGC  GTGGCTACAT TGGGAATGAC CTTCCTTGAT CCCACCAAGC CACCCATTGA TTCTGTAAAC  ATGTGACCCT TG CTC CAAGC ATTGCTTACA GGAGCAGGAT ACTGAAAGTG TGTCTSTGCC  CTCTCCTGAT AACCCCTCCC TTCAGCAGGC ACACAGCACC TGACTACCCA CCACGTATGT  AAACGTCAGT ATCCTTTCCA GCCAGCTCTG CAGATGGGTG TCCAGGCTGT GCATGATGCA  CCTCAAGTGG GCAGAGCTTG CAGGCCAAGG TTTTAAAGGC TGTTCAGGAA TGGATGGCAA  GCAGGATCTA AGAGGAGGGG GGGTTGTTGT TGTTTGGGGG GGGGGTGGTT TTGGTTTGTT  TTTTTTGAGA CAGGGTTTCT CTGTGTGGCC CTGGCCCTCC TGGAACCCAC TCTGTAGACC  AGGCTGGCCT TGAACTCAGA AATCTGCCTG CCTCTGCCTC CCGAGTGCTG GGATTAAAGG  CGTGTGCCCA TCGAGGAGGG AGATTTTATT TAGATTATAA AAAGGACGGG ATTTGGGGAA  TCCTGTCTAG TGAATTCAGG ACGTAATCAG TGGCTGGGAA GCAAGAGCTC TAGAGGAGCT  CCAGCTTATT ATGACCCTTC CTTCAGATGC CACAAGGAGG TGCTGGAGTT CTATGCACCA  ATAGCTTAAA CCAGCCAGGC TGGCTGTAGT GGATTGAGCG TCTGAGGCTG CACCTCTCTG  GCCTGCAGCC AGTTCTGGGT GAGACTGACC CTGCCTGAGG GTTCTCTCCT TCCCTCTCTC  TACTCCTTCC TCCCTCTCCC TCTCCCTCTC TCTGTTTCCT GAGGTTTCCA GAATTGGGGA  TGGGACTCAG AGACACCACT AAAGCCTTAC CTTTTAAGAA GTTGCATTCA GTGAGTGTGT  GAGACATAGC ACAGATAGGG GCAGAGGAGA GCTGGTTCTG TCTCCACTGT GTTTGGTCTT  GGGTACTGAA CTCAGACCAT CAGGTGTGAT AGCAGTTGTC TTTAACCCTA ACCCTGAGCC  TGTCTCACCT GTC CCTTC CC AAGACCACTG AAGCTAGGTG CAAGATAAGT GGGGACCCTT  TCTGAGGTGG TAGGATCTTT CACGATAAGG ACTATTTTGA AGGGAGGGAG GGTGACACTG TCCTAGTCCTCTTACCCTAGTGTCTCCAGCCTTGCCAGGCCTTAAACATC CGCCCATTGT  CACCGCTCTA GAAGGGGCCA CCCTTGACTT GCTGCTAAAC AAGGCACTCC CTAGAGAAGA  TACCATACCT GTGGGCAGGA TGACCCATGT TCTGCCATGC ACTTGGTAGC CTTGGAAAGG  CCACTTTGAA CCTCAATTTT CTCAACTGTT AAATGGAGTG GTAACTGCTA TCTCATAATA  AAGGGGAACG TGAGGAAGGC GTTTGGATAG TGCCTGGTTG CGGCCAGGCT GCAGTCAAGA  CTAGTTCCCA CCAACTCGAT TTTAAAGCCT TGCAAGAAGG TGGCTTGTTT GTCCCTTGCA  GGTTCCTTTG CTCGGGCCAA ACTCTAGAAT GCCTCCCCCT TTCTTTCTCA TTGAAGAGCA  GACCCAAGTC CGGGTAACAA GGAAGGGTTT CAGGGTCCTG CCCATAAAAG GTTTTTCCCG  GCCGCCCTCA GCACCGCCCC GCCCCGACCC CCGCAGCATC TCCAAAGCAT GCAGAGAATG  TCTCCGGCTG CCCCCGACAG ACTGCTCCAA CTTGGTGTCT TTCCCCAAAT ATGGAGCCTG  TGTGGAGTGA GTGGGGCGGC CCGGGGTGGT GAGCCAAGCA GACTTCCATG GGCAGGGAGG  GGCGCCACGG GGCGGCAGAG GGGTGACATC ACTGCCTAGG CGGCCTTTAA ACCCCTCACC  CAGCCGGCGC CCCGGCCCGT CTGCCCCAGC CCAGACACCG AAGCTACTCT CCTTCCAGTC  CACAAACGAC CAAGCCTTGT AAGTGCAAGT CATGGGAGCA GAAGGGCTGT GGGCTCAATT  AGATCCCCTA GTCTCTTCTA GTTTGCTGGG TGGAATTGGG TCCCTAGAGA CCATTCTCTG  TGTTAGACAA AAAGTCTGGG TTAAAATGCC TAGGATGATT TGACTGGGGC AAAAGAATAA  ATGGGGTGAG AGGGAGGCTCAAATTCAGTCACTGTCCCACCCATAGGTGT ATGGGCTATG  TGTTAGGCCC AAAGAGGTGA CAAATGAGGC CAAGGGAACA ACTCCATCTT TGGATCTCCA  AGAAGGTGAG GGGCTAAGTT CTGGAAAGCA GTGACCCACT GATGGTCCCC AGGGCTAATG  CAACTCGGGG GAGCCAGGAG GTAGCCCCCT CAGGCAGTGG AGGACTAAAG ATCTTATTTT  77GTAGCGCT AGGGATCAAA CCCCAGGGCG CTATGTGTGG CAGGCATGTG CTCCATCTAC  CACAGAAGTT TAATCCTTCA GACTAGCCTG GGATAGGGCC TGCTTTTTCT TTCCTTTTCT  CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTTTTT CTTTCCTTTT  CTCTCTTTCA CTCTCTCTTT CTAATTTCTT TTTCTTTTTT TCTTTCTTTT CTTTAGACAG  GGTTTCTCTG TGTAGCCCTG GCTGTTCTGG AACTCACTCT TTAGACCAGG CTGGCCTCGA  ATCTCAGAAA TCTACCTGCC TCTGCCTCCC AAGTGCTGGG ATTAAAGGCG TGTGCCACCA  CTGCCCAGCT AAGGTTTGCT TTTTGATGGC AGCTTGGTCC AGTTTGAAAG TAGGAGGTCA  GTCCACTGTA GGCAGATAGG TGACAGGTGG CAGATAGGTG ACAGATAGGT GACAGGTGGA GGAGCTTTGG AACTGGGACT GGACAGCCCT GGGACCCTGT TCCTCCCAAA GGGTCTTGGT  GGTTCCCCTT GGGGCTCTCT AAAGGATGTC AGTGGGCTGT TGCCACATCT ATATAAGAGG  ACTAGTCTTC TGGAATTTAG GTGTGATCTC TCAGGGATGC AGAAATGCTC ACCCTTACTG  TCATTTTATG GGCTGAGGTA CCACAGGCAG ATATACCCTG GTCTGCTTGT TGTCCAGGGT  CTCTGCTACA TGGAGGCCCC TTTCCACAGC CTAACCTCTC TACCTGCTGA CAGGAGGGCT  GGATGGCCAC AGGCATCCAA CGTGCGCATC ATGCAGGTGT TTTGCGTTGG AGCTTTTGTC  TAGAAATACC CTGGTGGGCT GCCAAACCAC CACCCATATC CCTCTCTCCT CTCTGCTGCC  TCTAAGATGA CAGCTTGATT TTTCTTATAG TGATTTTTTT TTTTGGTTTT GTTTTTTTGT  TTGTTTTAAG TTAGCATACA AAGTAATACA TTTCATCATG GCATTTGGAC ATACATATAT  ATTTTATTTG CTCTCCTGGC CTCTTCTCAA AGAGACTTCT CTGGAGTTTC TTGTATTTTT  GGTTGTGAGC CTAGCCTTTA ACGGCTGAGC CATGTCTCCA GCCCTTCTTT GGACTTTCTA  CTTCATACTT CCCACCAGTC TGGGAAGAAG GGCACATGGA ATCTTGAGAG CATGACCTGA  CCCAGACCTG ACAGATGTCA AGGCTGCAGT GTATGCTCTT GTTCGTACGG CTTGTTCTTA  GTCCTGCAGT TCAGAACTTT CTGGAGACTG AGAAGTGCAT GTGAGGACAC TCTCCTCCCA  TCTTTTCCTC TAGTGGCTAG TGATGTTTGG TTTTTTGTTT TGAGACAGGG TTTCTCTGTA  TAGCCCTAGC TATCCTGGAA CTCACTTTGT AGATCAGGCT GGCCTCCAAC TCAGAAATCT  GCCTGCCTCT GCCTCCCGAG TGCTGGGACT AAAGGCGTGC GCCACCACTG TCCAGTCAGG  AGTAGAAGGA AACTGTAAGG TGCTTGAGAC AGGCTGAGTA GAGGCTAGGA GGAAGGGGCA  CCGCAGTCAC CGGCTCCATGACTCTGTGACTTTTGTGGTTCCTTGTCGCA GCGGTTCCTG  GTGGTGGTGG TGGTCGGGGG TTGGGGGGAG GGGGCAGGCC ACACAGTGGG GTGTGGGAGG  GAATAGCTGT TGACAACTTC CCAACAGAAA CCAGGCTTTT GAGTCCTCCA GGGTAGCTTG  AGAGGGTACT CAGAAAGCCG TGTCCATGTC CCCTTTCCTT CACCTCAGGG AAGTAAGTTG  CCTATAGGGT TGTCATTTCA ATGAGGTCTT CTGGTTATTC TGTTTTTCTC TCAATGTTGG TGTTGGGCTC AGGGAATGCT TTGGAGAAGG TGGTGGGAAC TGGAGAAGGG AAGATCAGTT  TACTGTGTAA ACTCACTGGT TAAAGTACCC CCTCCCTTCC ACCCTGCAAT ACACACACAC  ACACACACAC ACACACACAC ACACACACAC ACCCCATCTC GAAGAGCTCT ATTAAGCTCC  AGGTGCACTG TAGTTCACAG AGTGCATCTT CCAGGTTTGC TCCCACTTCA CAAGCAGAGA  ACTCATAACT GAAGGGGGTG ACAGCACAGG GGAAGGGAAA GCAAGATGTT TAGAGTCTGA  CAGCTGGCCC GGGACCAGAG CCATGTGGTA ATGTTTGCTC CACTCCCATC CACCTCCACG  GCTGTGATGT GGAGAAGGTC CCCGCTTTCA TGGGAAGGAG GTGGGGGAGC CTGTCATCTG  CTCCATGCTC ACACAATTTT TCTCTCAACC AATGACCTCT CAGAAGCAGG GGTTGGTTTG  CAAAATTCTT CAGATACCTC AACAGATGGC ATCCCACTCA GGCTATCCCT GCTGACTAGG  TCTGGCTCCA GCCCTGACTG TATCTACCCA GGGACCTACC TGCCTGCTTT GCTCCTATAG CCTTCCTCCG TGTCTGGGTC CCCAGAGAGAGC TGCCGGCATA GGCCTTTGAG GCAACAGCTG  GCATACAGGC CAGGCTTCCC ATGCTCTGGC TAGCAGATTC TCTGCCCTGG AGGACTTTGA  CTGCATGGTT TCTCTCACTG CTGCAACAGT CAGAGCTGGC CCACACGGGC ACAACAGCGC  ACTTCCATCT GGGTCTCCCT GAGAATGCCG CTGTTTTCTG AGAACCCTTG GACTCTGGTG  GCTTTATCAG GTCTTTTTGT CAGCTGCGCT TTGGGGGATG AACTTTGCTC TTCTGGCTTC  TGGGTCAGAG GGTAAAGATT TGGTGGCAAC CGGTAGCTAG AGAAAGATAG CTACTGGCTG  AATTTGGAGG ACATGGCTTC TGGAAAACCT CTCTAGTGCT TTTCTGGCTA GTCTTGGCAA  AGTAAAAATG CTCTGATAGC CAGCCCGGGT GATGCAGGGC TTCCTGTTCG AGGCCTTTCT  GTACAAAATT AGTGAGACAT TGCCTCAAAA CTATGAAACA AGCCAGACTC TGTTGAAGCA  CGCCTTTAAT CCCAGCACTC AGGAGGCTGA AGCAGGCAAG ATCTCTGTTA GTTGGAGGCC  AGTCTACAGG AAAGTTCTAC AACAGCAGAG GCCAGACAGT GCAACCCTTT CTGGGGGTGT  GGGGGAGGAA AACCCAACAA AAACACAAAC TATAAAACAA AGAGAAGGCC GAGGACAAAG  CTTAGCAATG CATACTTCCC TTTCTATGTG AAGCCCTGGG CTCCACCAGT ACTGCAGAAA  GAAGCAAGCA ATGAGGGACA GGAGGTTGGC TCTAGGCCCA GGGGTTGTCA AAATAGTCCA  CAGGCCAAAG GCAGCCTGAT GTCTGTTTTT ATAAACAAAA TTTTATTGGC ACACATTGGT  TATGTATCAG CTAGGCTATT TTCATTACAA TAGAGGCCAT ATGOTCTGTA AAGTCTAAAA  TATTTACTCT GCTGTTTTAC ATAAAAAGTT GACAGACTCT TGCTCTAGAC TGACAAATA  CTAAGACCTT GTTTTCTGAG GTTCAAGTTT CAGAGGGGTC TCTGCAGCAA GTGGGTAAAG  CTGGTCTAGG TCATGCTATG ATGTCTAGGG TCCCCTCAGA GTGGAAGGCC TGCTTAGCAC  AAATGAAGTA AAGTAACTTG CTGGCTCTTT GTTCTTTTCT CCACACTCTA TACTTTAGCT CTGCCTCAAC ATG  47. DNA sequence located upstream of the coding part  of the mouse gene of the protein SM22 characterized in  that it comprises at least part of the sequence SEQ ID  next n 2, or a sequence hybridizing with it  5 under stringent conditions GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA 6 AGAGGCGGGT CTGGGTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG 12 CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC 18 TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 240 TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 300 GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 360 TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 420 CACCACGTAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 480 GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 540 AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 600 TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 660 ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 720 TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 780 GGATTTGGGG AATCCTGTCT AGTGAATTCA GGACGTAATC AGTGGCTGGG AAGCAAGAGC 840 TCTAGAGGAG CTCCAGCTTA TTATGACCCT TCCTTCAGAT GCCACAAGGA GGTGCTGGAG 900 TTCTATGCAC CAATAGCTTA AACCAGCCAG GCTGGCTGTA GTGGATTGAG CGTCTGAGGC 960 TGCACCTCTC TGGCCTGCAG CCAGTTCTGG GTGAGACTGA CCCTGCCTGA GGGTTCTCTC 1020 CTTCCCTCTC TCTACTCCTT CCTCCCTCTC CCTCTCCCTC TCTCTGTTTC CTGAGGTTTC 1080 CAGAATTGGG GATGGGACTC AGAGACACCA CTAAAGCCTT ACCTTTTAAG AAGTTGCATT 1140 CAGTGAGTGT GTGAGACATA GCACAGATAG GGGCAGAGGA GAGCTGGTTC TGTCTCCACT 1200 GTGTTTGGTC TTGGGTACTG AACTCAGACC ATCAGGTGTG ATAGCAGTTG TCTTTAACCC 1260 TAACCCTGAG CCTGTCTCAC CTGTCCCTTC CCAAGACCAC TGAAGCTAGG TGCAAGATAA 1320 GTGGGGACCC TTTCTGAGGT GGTAGGATCT TTCACGATAA GGACTATTTT GAAGGGAGGG 1380 AGGGTGACAC TGTCCTAGTC CTCTTACCCT AGTGTCTCCA GCCTTGCCAG GCCTTAAACA 1440 TCCGCCCATT GTCACCGCTC TAGAAGGGGC CACCCTTGAC TTGCTGCTAA ACAAGGCACT 1500 CCCTAGAGAA GATACCATAC CTGTGGGCAG GATGACCCAT GTTCTGCCAT GCACTTGGTA 1560 GCCTTGGAAA GGCCACTTTG AACCTCAATT TTCTCAACTG TTAAATGGAG TGGTAACTGC 1620 TATCTCATAA TAAAGGGGAA CGTGAGGAAG GCGTTTGGAT AGTGCCTGGT TGCGGCCAGG 1680 CTGCAGTCAA GACTAGTTCC CACCAACTCG ATTTTAAAGC CTTGCAAGAA GGTGGCTTGT 1740 TTGTCCCTTG CAGGTTCCTT TGCTCGGGCC AAACTCTAGA ATGCCTCCCC CTTTCTTTCT 1800 CATTGAAGAG CAGACCCAAG TCCGGGTAAC AAGGAAGGGT TTCAGGGTCC TGCCCATAAA l860 AGGTTTTTCC CGGCCGCCCT CAGCACCGCC CCGCCCCGAC CCCCGCAGCA TCTCCAAAGC 1920 ATGCAGAGAA TGTCTCCGGC TGCCCCCGAC AGACTGCTCC AACTTGGTGT CTTTCCCCAA 1980 ATATGGAGCC TGTGTGGAGT GAGTGGGGCG GCCCGGGGTG GTGAGCCAAG CAGACTTCCA 2040 TGGGCAGGGA GGGGCGCCAC GGGGCGGCAG AGGGGTGACA TCACTGCCTA GGCGGCCTTT 2100 AAACCCCTCA CCCAGCCGGC GCCCCGGCCC GTCTGCCCCA GCCCAGACAC CGAAGCTACT 2160 CTCCTTCCAG TCCACAAACG ACCAAGCCTT 2190  18. Strain deposited on March 25, 1996 with the CNCM under the number I-1685 carrying the plasmid p2126nlz comprising the sequence according to claim 17.  19. Strain deposited on March 25, 1996 with the CNCM under the nO I-1686 carrying the plasmid p2126INTnlz comprising the sequence according to claim 16.  20. Vector characterized in that it contains a sequence according to one of claims 1 to 15.  21. Vector according to claim 19, characterized in that it contains an origin of effective replication in artery cells.  22. Vector according to one of claims 20 and 21 characterized in that it is a derivative of an adenovirus.  23. RNA characterized in that it is capable of being expressed by a sequence or a vector according to one of claims 1 to 22.  24. Composition characterized in that it contains a sequence or a vector according to one of claims 1 to 15 and 20 to 22.  25. Composition according to claim 24, characterized in that the sequence or the vector are included in a composition facilitating their transfection in the cells.  26. Composition according to claim 25, characterized in that it comprises a gel which is a poly complex L-lysine and lactose.  27. Medicament, characterized in that it contains a sequence or a vector according to one of claims 1 to 15 and 20 to 22.  28. Pharmaceutical composition characterized in that it contains a pharmaceutically effective amount of a nucleic sequence or of a vector according to one of claims 1 to 15 and 20 to 22, and pharmaceutically compatible excipients.  29. Use of a vector or a sequence according to one of claims 1 to 15 and 20 to 22 for the manufacture of a medicament for the treatment of coronary heart diseases.  30. Use of a vector or a sequence according to one of claims 1 to 15 and 20 to 22 for the manufacture of a medicament for the treatment of restenosis.  31. Use of a vector or a sequence according to one of claims 1 to 15 and 20 to 22 for the manufacture of a medicament for the treatment of mutations which weaken the vessels.  32. Transgenic animals characterized in that they carry a sequence or a vector according to one of claims 1 to 15 and 20 to 22, in which the gene coding for the protein of therapeutic interest is replaced by a reporter gene.  33. A method of screening molecules in vitro for their activity on the regulatory sequences of the gene coding for the protein SM22, comprising the following steps:  - transfection of cells with a sequence or a vector according to one of claims 1 to 15 and 20 to 22, in which the gene coding for the protein of therapeutic interest is replaced by a reporter gene,  - incubation of cells transfected with the test molecule, and  - quantification of the expression of the transporter gene.  34. Method for detecting mutations in the region comprising the sequence according to one of claims 1 to 15 or the vector according to one of claims 20 to 22, characterized by an alteration of the expression of the gene placed downstream of said sequence.  35. Process for the expression of proteins of therapeutic interest, characterized in that it uses the products according to one of claims 1 to 23.