WO1997035974A9 - Sequences ahead of gene sm22, vectors containing same, and therapeutical uses thereof, particularly for treating 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

 CLASSIFIED SEQUENCES OF SM 22 GENE, VECTORS CONTAINING THEM AND THEIR THERAPEUTIC USES,

IN PARTICULAR IN THE TREATMENT OF VASCULAR DISEASES

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

It further relates to the use of these vectors and sequenced in therapy, for example for the production of active polypeptides locally or systemically, including polypeptides ⁱ mmunogènes, endogenous regulatory polypeptides such as cytok ⁱ nes, or for the production of RNA of therapeutic interest _, for example an antisense RNA In a particular way, the subject of the invention is the use of vectors and nucleotide sequences in the treatment of vascular diseases.

Atherosclerosis is a degenerative disease of the arteries assoc ⁱ ant lesions of arteriosclerosis and atherosclerosis, and combining a ⁱ nsi hardening of the arteries and fatty degeneration of their internal tun that ^i. This is generally remedied coronary heart disease by percutaneous coronary angioplasty of ⁱ ntroduire in the coronary arterial system a catheter provided at its extrem ⁱ ty of a balloon, advancing the catheter _j ntil the stenosis and inflating the balloon to crush the lesion against the wall and thereby restore a coronal caliber allowing sufficient myocardial perfusion

This myocardial revasculansation technique is widely used (50 000 procedures per year in France and 500,000 ntervent ^{i i} ons per year in the US) but is limited by the réappar ⁱ tion, in the months following the operation, d a new lesion s ⁱ te dilated, or restenosis, in about 30% of cases.

The d ⁱ to proposed treatments to treat this type of injury are summarized in the article by Feldman and Steg "Perspective intensive t ⁱ e ⁱ that Gen. restenosis" (Medicine / Science, Synthesis 1996 12, 47-55) Two mechanisms are responsible for restenosis: mimal hyperplasia and arterial remodeling 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 voluminous extracellular matrix This activity would be induced by the local release of growth factors and cytokines. as well as by suppressing the synthesis of certain antiproliferative endothelial peptides. The second mechanism, arterial remodeling, consists of a reduction in the size of the artery without modifying the thickness of the wall, and therefore without proliferation of cells.

These authors describe various attempts to inhibit iπtimal hyperplasia, by gene therapy, but the results have proved unsatisfactory, for various reasons The inefficiency of the transfer is one of the reasons generally invoked to explain these results This problem was partially However, other problems have emerged, related to the immunogenicity of these viruses, the risk of recombination with wild adenoviruses or the risk of dissemination of these vectors into the systemic circulation. These authors also cite the use of specific promoter sequences of vascular cells, such as preproendothelin, or adenovirus-ligand complexes, as constituting very interesting research pathways, without developing these aspects.

The application WO 96/05351 (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 actiπe α smooth muscle is cited in the context of this embodiment. No example of use of such a construction illustrates this request.

The proteins constituting the smooth muscles were the subject of piecemeal studies. Thus, the SM 22 protein, which constitutes one of the first markers to appear during the differentiation of smooth muscle cells, was studied and the coding part of its gene was sequenced in mice (SOLWAY and AL, 1995, J Biol Chem, 270,22, 13460-13469) and in rats (KEMP and AL, 1995, BIOCHEM J 310, 1037-1043). Chicken gene and gene sequences human also been determ ⁱ nes (Nishida et al, 1991, Biochem Int, 23, 663-668 for chicken gene, Thweatt et al 1992 Biochem Biophyse Res Comm, 187 1-7 to the human gene)

The g ^ene encoding the SM protein 22 alpha in the mouse is composed of five exons and includes 6.2 kb II 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 level of transcription of a reporter gene, in primary cell cultures of rat aortae and in the kjr lines. They also showed that the messenger RNAs of this gene were expressed at significant levels in the aorta, uterus, lungs and intestine

KEMP 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 made in the fragment showed that the promoter region 65 between the nucleotides -303 and eta ⁱ ent more active a fragment of 1 kb 5 comprising firstly ⁱ e ⁱ mportant in the region of untranslated gene, which would suggest the presence of regulatory sequences in the end 5 'from the promoter

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

It will be noted that these studies were done in vitro using primary cell cultures of the aorta, and not in vivo, however, these cond ⁱ t ⁱ ons experimentation are not representative of the _{ctivityreport} genes in cells of the body

In addition, they only concern a very limited region of the non-coding part of the gene located at the 5 'end, that is to say the downstream part of the nucleotide -441

More recently r ^e Li et al (1996, J Cell Biol, 132, 849-859) have ^é studies the regulation of the SM22 gene using transgenic mice Nevertheless, this article does not mention any application of vectors carrying fragments of this gene and in any case no therapeutic application.

In addition, a limited number of constructs have been manufactured, and in general only the effect on embryos, and not on adults, has been tested.

The authors deduce from their study that the sequences between nucleotides -2735 and -445 do not contain any essential muscular regulatory elements. The applicant endeavored to identify in vivo, on adult individuals, the activity of the various regions constituting the region upstream of the expressed coding sequence of the gene of the SM 22 II protein has also sought to determine whether modifications in this sequence induced changes in the expression of the gene in the different tissues.

He has surprisingly demonstrated that it is possible, by modifying the 5 'region of the gene of the mouse SM 22 protein, to obtain, in adult individuals, a specific expression of a reporter gene in the smooth muscles of the arteries and especially the aorta

It has furthermore been shown that the sequences identified by Ll et al (1996, previously cited) as containing no essential regulatory sequences, on the contrary contained sequences indispensable for a specific expression of the SM 22 gene in the adult.

Finally, it has surprisingly shown that intronic sequences were necessary for the induction of specific expression in the arteries.

The subject of the present invention is therefore 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 hybridizing sequence under conditions highly consistent with said upstream sequence, said fragment being capable of inducing a specific expression in vivo of this gene in cells of the arteries, and a sequence encoding a protein or RNA of therapeutic interest

The invention also relates to any nucleotide sequence or ohgonucleotide of natural origin or obtained by chemical synthesis having a homology with SEQ ID No. 1 sequence of at least 70%

By nucleotide sequence is meant naturally occurring, a complementary DNA fragment obtained by reverse transcription of ^cellular mRNA or a genomic DNA fragment obtained after cleavage of cellular DNA using enzymes restriction

Nucleotide sequence obtained by chemical synthesis is understood to mean a fragment of DNA of known sequence generated by automatic synthesis of polynucleotides, for example using a suitable automatic apparatus.

By protein or RNA of therapeutic interest is meant a molecule capable of inhibiting the growth of smooth muscle cells, of stimulating 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 stπngeπtes conditions" is used in the sense given by Maniatis et al, 1982 (Molecular cloning A Laboratory Manual, CSH Cold Spπng Harbor Lab, NY USA or one of his recent reissues. preferred, the hybridization conditions are as described by Maniatis (1982 edition). Three washes to remove non-hybrid fragments are required 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 the nucleotides -2126 and +4135, whose sequence SEQ ID No. 1 is given in the appendix, of the sequence comprised between the nucleotides

-2126 and +65 whose sequence SEQ ID No. 2 is given in the appendix or the sequence between nucleotides -2126 and -445 whose sequence SEQ ID No. 3 is given in the appendix It may also comprise a fragment of the sequence SEQ ID No. 4

Such a fragment may in particular comprise at least part of the sequence SEQ ID No. 5 which corresponds to the portion upstream of the 5 'end of the coding portion between the nucleotides -2126 and -1 340 Such a sequence SEQ ID No. 5, 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 included respectively in the plasmids p2126nlz and p2126INTnlz carried by the strains deposited on March 25, 1996 with the National Collection of Culture of Microorganisms of the Pasteur Institute (CNCM) respectively under No. 1-1685 and 1-1686

The invention also relates to hybπdant sequences under conditions of high stringency with the sequences SEQ ID No. 1 SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 5

The sequences including the sequence of the first intron of the SM 22 gene, and in particular the sequence SEQ ID No. 1, constitute particularly advantageous embodiments of the present invention, because they allow an expression of proteins or RNAs of therapeutic interest. , in adults, specific to the arteries

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 an expression of a gene in the cells of the arteries. Thus, those skilled in the art can advantageously refer to the article by KEMP and AL (1995, cited above) which decπt the sequence upstream of the rat gene of the SM 22 protein. The present invention also covers any sequence comprising fragments. 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 may be a protein inducing the formation of a cytotoxic compound, such as the thymidine kiπase of the herpes virus. This protein has the particularity of transforming ganciclovir (Merck Index, reference 4262), an analogue of the guanosine, in a drift which blocks the synthesis of the DNA in the cells in rephcation The introduction of a gene coding for this protein in the sequences according to the present invention thus makes it possible to block the proliferation of the cells in the case of the intimal hyperplasia A sequence coding for thymidine kinase is described by Caruso and Klatzmann (1992, Proc Natl Acad Sci, USA, 89, 182-186).

Said protein of therapeutic interest may 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 et al., 1994 , Circulation, 90, 1357-1362)

It may also be a protein exhibiting lipolytic activity, such as 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). -1 1417) It may also be an endothelial cell growth factor Such a protein may be in particular an interleukin

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

It may be a protein inducing an immune response as described in WO 90 1 1092, and in particular a viral antigen such as glycoprotein gp120 or HIV Nef protein (human immunodeficiency virus). protein may be one or more proteins of therapeutic or vaccinal interest, such as proteins of immunotherapeutic interest, for example interleukins, growth factors, for example fibroblast growth factors (FGF) or NGF (Nerve Growth). Factor), proteins that can induce an immune response, be it a humoral, cytotoxic or cellular immunity, or proteins that make it possible to complement the activity of a gene normally expressed in the individual to be treated but which is no longer , either by mutation or deletion of its sequence This sequence may also encode an RNA of therapeutic interest. Such an RNA may be the antisense RNA of the p53 protein or an RNA as described in the application WO 90 1 1092 filed by the company VICAL. The present invention furthermore for object 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 effective origin of rephcation in the cells of the arteries, particularly in smooth muscle cells

Such a vector may comprise sequences allowing homologous recombination in the treated organism, 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 gene undesired, present in the treated organism will be replaced by the gene carried by the vector or the sequence and that one wishes to be expressed in the organism

Such a homologous recombination method may be of the type described by Le Mouellic et al (1990, Proc Nat Acad Sa USA 87, 4712-4716) or in PCT application WO 91/06 667.

Such a vector may be a vector derived from an adenovirus Adenoviruses adapted to the implementation of the present invention are in particular those described by Feldman and STEG (1996, cited above) or OHNO et al (1994, Sciences, 265, 781-784) or in the application FR 94 03 151 (Institut Pasteur, Inserm) These viruses are generally rejected by the individual, because of their excessive immunogenicity Nevertheless, strains of these viruses have been selected to reduce their In any case, these vectors, even having a high 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 rejection times of adenoviruses by the immune system of the host However, it will 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 traced by DNA comprising these sequences. The nucleic acid sequences according to the present invention. invention can be introduced after covalent coupling of the nucleic acid with compounds promoting their penetration into the cells or their transport to the nucleus, the resulting conjugates being optionally encapsulated in polymeric microparticles, as in international application WO 94/27238 of Meidsorb International Technologies

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 of 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 channel-covered balloon catheter, as described by Feldmann and STEG (1996). , previously cited)

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

These sequences or vectors may 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 may be a poly-L-lysine and lactose complex, as described. by MIDOUX (1993, Nucleic Acid Research, 21, No. 4, 871-878) or poloxamer 407 as described by PASTORE (1994, Circulation, 90 I-517) can also be dissolved in a buffered solution or be associated with liposomes

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

Such sequences, vectors or compositions can be advantageously used for the manufacture of medicaments for 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 However, they can also be used for the manufacture of a medicament for the treatment of mutations weakening the vessels. Such a medicament could, for example, comprise a sequence or a vector according to US Pat. invention capable of expressing a gene coding for a normal desmin protein or a CAM-type muscle adhesion protein

The subject of the present invention is also RNAs expressed from the sequences and vectors that 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, in order to test their activity on the regulatory sequences of the gene encoding the SM 22 protein. Such a method will comprise, for example, a first step according to which transfections of 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 encoding 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 transfection are advantageously smooth muscle cells, in particular cells of the aorta, 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 Boehπnger under the reference 1 669 893 ( Boehππger- catalog Mannheim - "Biochemicals" 1996, page 236. beta-Gal Reporter Gene Assay, Chemilummescent)

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 of the diagnostic kit marketed by Boehπnger under the reference 1,758,241 (Boehπnger-Mannheim catalog "Biochemicals", 1,996, 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 of the sequence upstream of the coding part of the SM22 protein gene. Such mutations are in fact capable of modifying the expression of the gene coding for the SM22 protein, in particular reduce or even abolish the expression of this gene in the smooth muscle Such mutations would be likely to cause a change in smooth muscle function, the SM22 protein being related to a family of proteins involved in the regulation of calcium binding, of the calponme type (Ayme-Southgate et al. 1989, J Cell Biol) Such a method for detecting mutations can advantageously be the process that is the subject of the French patent application FR-93 10821

In a complementary manner, 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 gene-encoding sequences encoding the SM 22 protein. Molecules can be administered to mice, and after sacrifice, histological sections are performed to highlight the tissues. colored by the reporter gene For the implementation of the present invention, the skilled person can advantageously refer to the following manual "SAMBROK et al (Molecular Cloning, A Laboratory Manual, Cold Spong Harbor

Laboratory Press, New York, 1989), or one of his recent reissues

The present invention is illustrated without being limited by the following examples

Figure 1 shows the restriction map and the exon / intron structure of the SM 22 site, including the region -2474 to +6030 (relative to the transcription initiation site). The exons are represented by white squares while the arrows indicate the position of the repeated sequences of type B1

FIG. 2 represents the alignment of three B1 type repeat sequences, in which the basic exchanges are indicated. FIG. 3 is an autoradiogram illustrating the identification of the transcription initiation site. The well P corresponds to the primer used.

FIG. 4A represents the sequence of the 5 'region upstream of the transcription initiation site, and the exon of the SM 22 gene. The binding sites of the various factors are indicated, as well as the various sequences of the consensus. The broken lines indicate the position of the repeated sequences of type B1.

Figure 4B is a schematic restriction map of the SM 22 gene.

Figure 4C is a detailed restriction map of the SM 22 gene.

Figure 5 illustrates the manufacture of plasmid p352nlz

Figure 6 illustrates the manufacture of plasmid p2126 nlz.

Figure 7 illustrates the manufacture of plasmid p2126INTn1z.

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

Figures 9A-9D show histological sections of embryos stained with lacZ. Figure 9A shows a sectional view of an embryo at the 12.5 day heart stage, showing intense staining exclusively in the right ventricular myocardium (RV) and in the artery walls (abbreviations AA elbow of the fourth aortic artery, CA carotid artery, esophagus, iv left ventricle PA proximal portion of aorta, PT pulmonary trunk, right atrium RV right ventricle, trachea T, right and left cardinal vein) Figure 9B shows a sectional view of through the abdominal region of a 14.5-day stage embryo, showing umbilical artery (UA) staining and the absence of visceral staining (B bladder, D duodenum, H large intestine L liver, M small bowel, ST stomach) Figure 9D is a section through the 12.5-day-old tail segments showing myotome (my) and tail (a) artery staining.

Figure 9C is an enlargement of the portion of the photograph corresponding to the umbilical artery at the 12.5 day stage, showing exclusive labeling of the muscular layer (m) and absence of endothehum (e) labeling.

FIGS. 10A to 10C illustrate the expression of traπsgene in adult mice

Figure 10A shows a top view of the entire heart showing intense staining of the aorta (a) and pulmonary artery (PA) and absence of vena cava (vc) labeling. 10C are sections through the smooth muscle layer (sm) of the adult colon and an adjacent mesenteric artery (ma). LacZ labeling (Figure 10B) shows that transgene expression is restricted to artery while immunofluorescence with antibodies SM 22 (Figure 10C) demonstrates the expression of the endogenous SM 22 gene in the smooth muscle cells of both artery and colon

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

Figures 12A to 12D illustrate the expression of the SM 445 nlz construct in 15.5 day old PC embryos (Figures 12A to 12C) and in a 2 month old adult (Figure 12D) EXAMPLES: Materials and methods

1. Cloning and characterization of the mouse gene SM 22

Approximately 10 ⁶ recombinant phages of a c57 / b1 mouse genomic library constructed in delta-EMBL phage 3 (provided by Dr. D Plachov Munster Germany) were screened using the 890 bp BAL 1 / Eco RV fragment. DNA complementary to the SM22 mouse protein (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. The 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 re-screenings under identical conditions. one of the isolated clones was found to contain an entire SM 22 locus II was then mapped with restriction enzymes and subcloned. In order to avoid rearrangements during cloning procedures, hybridization analysis of the type Southern genomic DNA from Mice and genomic clones were performed and restriction patterns 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 kit (United States Biochemical, Clevelaπd, Ohio)

2. Northern Hybridization, Primer Extension, 5'RACE, and RNAse Protection Analysis

Total RNA of cell lines and adult mouse tissue was isolated by the method of Chirgwiπ et al., (1979, Biochemistry, 1794-5299) modified for the isolation of messenger RNAs, the Oligotex kit (Quiagen Inc., Chatsworth, CA) was used following the manufacturer's instructions

Northern hybridization analyzes were performed using agarose gels containing formaldehyde and by capillary hybridization on Hyboπd-N membranes (Amersham Life Science UK Little Chalfont) according to methods known to those skilled in the art. For the primer extension assays, a synthetic oligonucleotide of 30 nucleotides (P27N1) complementary to the +36 to +65 sequence of the complementary DNA of the SM 22 gene having the sequence SEQ ID No. 6 (5'-AAGGCTTGGTCGTTTGTGGACTGGAAGGAG-3 ), was labeled with T4 polyanucleotide kinase. 5 μg of mouse uterine mRNA was hybridized at 55 ° C with 1-2 X 10 ⁵ cpm of purified probe and the primer extension reaction was The results were 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 transcription initiation site

The 5'RACE method was carried out using two 18 bp antisense synthetic oligonucleotides corresponding to bases +1 12 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 assays, a 417 bp PCR fragment comprising the region -352 to +65 of the SM 22 gene was made and cloned by the sticky tip method into the HinC II site of the pBluescπpt vector. SK + The sequence of the fragment obtained by PCR was verified In order to generate a radioactively labeled antisense RNA probe, the construct was linearized with Sph I at position -203 of the SM 22 gene and transcribed with T7 RNA polymerase in the presence of 32p_Qjp [ _{α] SO} 3 (c10) was hybridized with 5 μg mouse uterine messenger RNA at 42 ° C. overnight. After hybridization, the samples were incubated with RNases A and T (1 hr). at 37 ° C), precipitates, and assays as described for primer extension analysis

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., USA) at 37 ° C. C, in a Co2 atmosphere at 7% For transfection experiments, confluent cells were plated, subcultured in 6 cm Petri dishes, and allowed to grow to approximately 75% confluence prior to addition of the transfection mixture.

Transfections were performed using Lipofectamine Reagent marketed by Life Technologies Inc.

In summary, 24 μg of Lipofectamine was added to 6 μg of plasmid carrying the supereπrouled reporter gene and 2 μg of gay PCMVβ as an internal control. After addition of 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 again changed after 12 hours. The expression of the product of the reporter gene was analyzed after 24 hours. For experiments st ⁱ emulation by serum, transfected cells were placed in DMEM containing 20% serum 24 hours before being harvested

4. Determination of CAT and β-galactosidase

The cells were taken from petri dishes with a cell scraper, centrifuged, resuspended in 150 μl of buffer Z (100 mM sodium phosphate, pH 7.5, 1 mM MgCl 2, 10 nM KCl, 50 nMβ-mercaptoethanol ) and lysed by three cycles of freezing / thawing. The debris was eliminated by low speed centrifugation and the β-galactosidase activity was determined from 2 to 20 μl of supernatant according to the methods known to those skilled in the art. For the determination of the CAT activity according to GORMAN et al (1982 Mol Cell Biol, 2, 1044-1051), from 10 to 100 μl of lysate were used. To account for variations in transfection efficiency. CAT activity was normalized with βgal activity.

5- Production of the transgenic mice The plasmids pnlz2126 and p2126INTnlz mserts. deposited with the CNCM respectively under No. l-1685 and No. l-1686, were isolated by digestion with Sal I / Pme I, followed by gel electrophoresis, and then purification using the Geneclean kit ( Bio 101. La Jolla, CA) and then resuspended in 10mM Tris Hcl pH 7.5, 0.25mM EDTA The transgenic animals were produced as previously described by Ll et al (1993, Development, 177/3, 947-959).

Mice carrying the transgene were identified by PCR and Southern hybridization using a DNA probe The pnlz2126 construct yielded three positive animals on 17 mice, two of them transmitting and expressing this construct.

For the p2126INTnlz construct two positive animals out of the 28 tested were obtained and again only one expresses and transmits this construct. The transgenic mice were backcrossed with c57 / bl mice and the histochemical stains were performed with hemizygous mice for Transgene 6. Histochemical stains Whole embryos or tissues of adults were fixed for 15-30 minutes at 4 ° C in 1% formaldehyde in buffer A (100 mM sodium phosphate pH 7.3). 2 mM MgCl 2, 0.1% sodium deoxycholate, and 0 2% NP-40). After rinsing, the specimens were stained overnight at 30 ° C in buffer A containing 1 mg / ml X-gal (Sigma, St. Louis, Missouri), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 20 mM Tns-CI pH 7.3 The stained samples were then rinsed, dehydrated with increasing concentrations of ethanol, clarified in xylene, and embedded in paraffin. The histological sections were performed with thickness of 7 to 10 μm and again stained with Ehrhch hematoxylia and eosiπe The embryos were again clarified with a mixture of benzyl alcohol and benzoate of beπzyle

For immunohistocoloration, the mouse tissues were fixed in 3% 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. 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 Cy3 labeled secondary antibody (Biological Detection Systems USA) The sections were examined using a fluorescence microscope

EXAMPLE 1

Characterization of the SM Locus 22 Hybridization screening 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 Figure 1 was established and the exons were localized by hybridization using synthetic ohgonucleotide probes The regions of interest were subcloned and sequenced

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

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

Two potential polyadenylation signals have been located at positions 5905 and 5913 and several TGT regions downstream of the polyadenylation site are likely to be involved in the termination of transcription.

The analysis of the sequence further reveals the presence of three repetitive sequences (FIG. 2) which exhibit significant homologies with the B1-type repeat elements, some of which are transcribed by RNA polymerase III and encode 5S RNAs. all the repeated sequences are in a reverse orientation with respect to the SM locus 22

The starting site of the transcription was mapped by primer extension analysis (FIG. 3). An antisense oligonucleotide complementary to the 3 'end of exoπ 1 leads to obtaining four extension products which differ only in size. by a pair of bases (bp) in length, the longest of them being 65 bp. The transcription product therefore starts with a G, 77bp upstream of the transcription initiation codon.

The shortest extension products were likely caused by premature termination of primer extension due to 5'-end modifications (cappmg) of the messenger RNA

These results were confirmed by the protection analysis by the

RNAse, which leads to a 65bp protected fragment and by cloning and sequencing 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 cited above). Minor sequence differences between these two results are likely due to allelic variations between the different mouse strains.

The TTTAAA sequence at position -28bp (FIG. 4) is closely related to the TATAAA sequence and is likely to have 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 that are known to contribute to the regulation of muscle gene transcription (Figure 4). A total of 1 1 E-type (CANNTG) plates, four Mef-2 / rSRF (YTAWAAATAR) units, four potential SRF linkers (CC (A / T) 6GG), five AP-2 binding sites (CCCMNSSS ) and five SP1 motifs (GGGCGG) were located. Finally, an element similar to the TGT3-3 motif, recently identified as a binding site of a nuclear factor of smooth muscle and as contributing to the regulation of smooth muscle alpha actin transcription, has been located (Figure 4). EXAMPLE 2 Cloning and Construction of Recombinant Plasmids for the Creation of Transgenic Mice 1. Manufacture of pnlz

Plasmid pGEM7 (i.e., plasmid pGEM-72F (+/-), marketed by Promega Corp., Madison, Wi, USA.) Sequences available in Gen Bank, under the following numbers: ^• X65310 and X 6531 1) comprises an ampiciliin resistance gene, a lac Z gene and a replication origin II comprises 3000 base pairs II was digested with Nae 1 / Bsp 1201 The outgoing end Bsp 1201 of the vector was made " blunt ends with the Klenow enzyme, and the plasmid was closed in the presence of a hnker Sal I of 10 bp. The lacZ fragment of pGEM 7 was therefore deleted, a Sal I site introduced, and a Bsp 1201 site restored.

A Pme I site was introduced by inserting a Pme I hnker 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 pDes2.2πlz (Ll 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 III and Sac I sites of the modified pGEM7 vector and verified for their orientation. This procedure gives the promoterless pnlz vector.

2. Manufacture of p352nlz

Figure 5 illustrates the manufacture of this plasmid

A 417 bp PCR fragment covering the region -352 to +65 of the SM 22 gene, whose sequence is found in the sequence SEQ ID No. 1 which comprises the sequence of nucleotides - 2126 to + 4135, was produced and inserted in the Hinc II site made end-free of the vector pBluescrιptSK + The sequence of the PCR fragment was 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 p2126nlz

To create p2126nlz, a 1.9 kb Bsp 1201 / SphI fragment covering the region -2126 to -206 of the SM 22 locus, the sequence of which is found in the sequence SEQ ID No. 1 which comprises the nucleotide sequence - 2126 a + 4135, was inserted into the respective sites of p352nlz This plasmid was deposited on March 25, 1996 with the CNCM under No. 1-1685 Its manufacture is illustrated by Figure 6 4. Manufacturing of p2126INTnlz

The manufacture of this plasmid is illustrated in FIG. 7. A 5.8 kb Bsp 1201 / Hind III fragment covering the -2126 to +3651 region of the SM 22 locus was inserted into the corresponding sites of pnlz giving pINt-nlz. 496 bp PCR fragment of base +3648 to +4143 of locus SM 22 was generated using an upstream primer and a downstream primer designed to introduce a Hind III site at position 41. 35-4140 This PCR product was digested with Hind III and heed in the HindIII site of pINT-nlz to give p2126INT-nlz The p2126INTniz construct therefore contains 2126 base pairs of the 5 'region of the transcription start, the first exon, the first whole intron and base pairs of 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 The portion of the insert derived from the SM promoter 22 content in plasmid p2 126 INTnlz is constituted by the sequence SEQ ID No. 1 This plasmid was deposited on March 25, 1996 with the CNCM under No. l-1686 Its manufacture is illustrated in Figure 7 EXAMPLE 3: Expression of the constructions p2126 nlz and p2126 INTnlz in transgenic animals

Two constructs using the lacZ reporter gene with SV40 virus nuclear localization signal were made, as described in Example 2. The first construct, p2126nlz contains 2126 bp of 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

Embryos obtained from 12.5 to 15.5 days after coitus (dpc) were stained to demonstrate βgai activity. Expressions of both constructs are identical at all stages observed although p2126INTnlz gives a color a little more intense

The expression was first detected at day 8 in the embryo heart region and in the cardiac envelope vessels. At day 9, expression of lacZ in the heart increases and is confined to the right ventricle. to the arteries

Between 12.5 and 15 days segregation of the labeling as a function of the regions becomes evident, the labeling being confined to the right ventricle (FIGS. 8A and 8B). The sections show that the expression has occurred in the myocardium of the right ventricle, with minor markings in left ventricle and right adrium (Figure 9A)

This expression in the heart is only transient and does not appear in the adult It is probably inhibited in the late stages of fetal development Early expression is also observed in segments of the rostrum from 9.5 days extends to caudal segments at 10.5 days, and reaches high levels at 11, 5 days

The expression decreases from 12.5 days in the segments of the rostrum (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. 9 D).

Most of the expression in the vascular system of the developing embyron starts at 9.5 days, the expression is detected in the dorsal aorta at 10.5 days, the coloring of the aorta dorsal and aortic bends increases and at 11, 5 days lacZ expression is clearly visible in the dorsal aorta, the aortic bends, the iliac arteries, the 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 date when the intercostal vessels can be easily seen (Figure 8A)

At 5 days (Figure 8B) lacZ is present in the major vessels, including those of the limbs and the tail, and the staining of the pulmonary trunk becomes visible. Expression in the system vascular persistence in adults and is clearly visible in the aorta, pulmonary trunk and right pulmonary artery (Figure 10A), as well as in the vessels of the intestines (Figure 10B), in the bladder and in the uterus Histological sections better reveal differences in transgene expression in the arteries and veins. The section of the embryo at 12.5 days (FIG. 9A) shows a very high level of expression in the muscle layers of the carotid arteries, the fourth artery of the aortic elbow, the proximal pulmonary trunk and the proximal to the ascending aorta, while the left and right parts of the anterior cardinal veins remain uncoloured The absence of expression of the transgene in the muscle veins is evident from the ductus venosus sections, as well as the portal and portal veins. Umbilical The same situation is observed in the adult, where the expression can never be detected in the cellar veins (figure 10A) nor in the pulmonary veins.

These observations highlight differences that previously had not been found between the smooth muscle layers of the arteries and veins. Other views of the arteries show that the expression is restricted to the muscle layer and is absent from the endothelium (Figure 9C).

It is also clear from all the observations (FIGS. 8A, 8B) that the expression of the transgene is absent from the smooth muscles of the viscera. This is unexpected, since the endogenous SM 22 gene is supposed to be expressed in the Vascular smooth muscle tissue and viscera. In histological sections of the 14-day embryo abdominal region, the developing stomach, intestine, and bladder muscle layers are easily recognizable (Figure 9C). 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 intestine and large intestine, and bladder where none lacZ expression is observed. In addition, no expression is observed in the esophagus and trachea (Figure 9A), as well as in the bronchi of the lung The staining of the muscle tissue of the adult gives essentially the same results. FIG. 10B shows that the expression of the transgene is absent in the muscles of the colon, whereas the mesenteric vessel is very strongly stained by lacZ. On the other hand, staining by immunofluorescence of the section of a similar intestinal tract shows that the endogenous SM 22 protein is present in both tissues (FIG. 10C) The absence of expression of the transgene in the visceral smooth muscles of the adult is further confirmed by stains of the esophagus, trachea, bladder of the vas deferens and uterus for βgal activity In no case can βgal expression be detected in the muscle layers of these tissues

Curiously, an important staining 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

Fabrication of a construct comprising regulatory regions of the SM 22 gene and the gene encoding thymidine kinase of the herpes virus (SM 22 INT-TK1.

The HIV-LTR fragment (-167, +80) is deleted from the plasmid pLTR-TK by Hind III digestion whose ends are made blunt with the Klenow enzyme, followed by Xho I digestion. The plasmid closed by inserting a hnker containing the 5 'XhoI sites, the Hind III and Bsp 1201 sites in the 5' -> 3 'order and a 3' end to give the p-TK vector A Bsp 1201 / Hind III fragment of 5.8 kb covering the region -2126 to +3648 of the SM 22 locus is inserted into p-TK to obtain SM 22 INT-TK This fragment is included in the sequence of nucleotides of SEQ ID No. 1

This construction is schematically represented in FIG.

EXAMPLE 5

Fabrication of plasmid p445 nlz carrying the fragment -445 to +65 of the SM22 gene fused to the lacZ gene, and obtaining transgenes Materials and methods Construction of p445nlz

Plasmid p2126nlz was cut by Xba I which has a unique site in the 5 '' polyhnker 'upstream of the SM 22 sequence. The 5' protuding ends of the fragment were filled with Klenow polymerase, dNTP

After precipitation the fragment was cut with Pst I which has a unique site at position -445 relative to the transcription start nucleotide +1 The protruding 3 'ends of the Pst I cleavage were hydrolysed to Klenow polymerase without dNTP The fragment comprising the plasmid sequences plus the sequence SM 22 from - 445 to +65 fused to the lacZ gene was purified from an agarose gel and closed by the T4 ligase For transgenesis experiments the fragment injected into the mouse eggs was purified from a double digestion Pst 1 / Nsι I plasmid p2126nlz

The results obtained with this construction and those obtained with p2126 INTIacZ and p2126lacZ for comparison are shown in the table below.

The transcreptional regulatory properties of the sequence of the SM 22 gene between nucleotides -445 and +65 were analyzed in vivo in transgenic mice by two methods. The first method called transient analysis consists in analyzing the expression pattern of the lacZ gene directly. in FO founding embryos at a given stage The second method is to establish stable lineages from adult FO founders 1. Transient Analysis

The transient analysis yielded four embryos that integrated the p445πlz transgene into their genome out of a total of 1 3 embryos. Of the four positive embryos analyzed at 12.5 days of development, one embryo did not express the transgene. expression identical to that of the line p2126 INT nlz at the same stage, that is to say specific arterial smooth muscle cells and finally a third embryo presented the same arterial expression territories added with various sites of ectopic expression essentially diffuse in the embryonic mesenchyme

2. Stable lines Two independent stable lines were established from a male (line # 1) and a female (line # 9)

The expression of the transgene was analyzed in the embryo at the stage

15.5 days for line 1 and 17.5 days of development in line 9 Both lines have the same type of expression in the arteries in the heart, the transgene is expressed in the dorsal aorta and the pulmonary trunk arterial

Unlike the lines obtained with the p2126nlz and p2126 INTnlz transgenes, the two p445nlz lines express lacZ very strongly in the walls of the esophagus and respiratory trachea which are composed of smooth muscle at this stage.

The two lineages show differences between them in certain regions of the embryo For line n ^D 1, the transgene is strongly expressed in the bronchioles in continuity of the trachea at 15.5 days, which is not observed in the line no. 9 at 17.5 days where lacZ is found only in the trachea This difference may be due either to a modification of the regulation due to different sites of integration in the two lines, or to the difference of stage which makes the expression of lacZ can be repressed between day 15,5 and 17,5

In both lines we thus find an expression of the transgene in organs which contain a strong component of smooth muscle cells. It is interesting to note that this is at least a part of the smooth muscle cells of the respiratory type, the trachea , and part of the visceral smooth muscle cells, the esophagus

Finally, different ectopic expression sites, ie where the presence of the endogenous SM 22 protein or RNA has not been reported, have been found in both lines. strong expression very localized in the two hind fingers of each limb Line 9 strongly expresses lacZ in the anterior regions of the brain The expression of this construction at two stages of evolution is illustrated by FIGS. 12A to 12D, which represent 15.5-day-old (12A, 12B, 12C) and 2-month-old (12D) SM 445 nlz embryos stained for β-galactosidase activity. The expression in the smooth muscle tissue of the arteries, esophagus and bronchi is shown in Figures 12A, B, C as well as in ectopic sites such as the spinal cord (12B) or the two hind fingers of each limb ( 12A) There is also no expression in the thoracic aorta or pulmonary arterial trunk in adults (12D). Only an expression in the trachea is maintained.

The results obtained show that a sequence of 450 base pairs is sufficient to keep the same expression in the embryo and the fetus but not in the adult.

BOARD

SEQUENCE LIST

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: UNIVERSITY PARIS 7

(B) STREET: 1 PLACE JUSSIEU

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75005

(ii) TITLE OF THE INVENTION: SEQUENCES IN AI-.ONT OF GENE S.-J22; iii) NUMBER OF SEQUENCES: S

(iv) COMPUTER-DEPENDABLE FORM:

(A) SUPPORT TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPO)

INFORMATIONSPOURLASEQIDNO ^; 1:

₍ i ₎ CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 6261 base pairs

(B) TYPE: nuclcoride

(C) NUMBER OF BRINS: double

(D) CONFIGURATION: linear

(ϋ) TYPE OF MOLECULE: DNA (senomics)

(iii) Hypothesis: No

(vi) ORIGIN:

(A) ORGANIZATION: MOUSE GG 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

_A ^A J ^GTGACCCT 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 GGGTTTTTTTTT TGTTTGGGGGGGGGGTGTTTTTTGGTTTTTT

TTTTTTGAGA CAGGGTTTCT CTGTGTGGCC CTGGCCCTCC TGGAACCCAC TCTGTAGACC

AGGCTGGCCT TGAACTCAGA AATCTGCCTG CCTCTGCCTC CCGAGTGCTG GGATTAAAGG

: _Λ ⁽ : ^ ^C ! ^{TGCCCA TCGAGGAGGG} AGATTTTATT TAGATTATAA AAAGGACGGG ATTTGGGGAA

^ ^c _to ^c J ^{TGAA GGA} TTCGG ACGTAATCAG TGGCTGGGAA GCAAGAGCTC TAGAGGAGCT

CCAGCTTATT ATGACCCTTC CTTCAGATGC CACAAGGAGG TGCTGGAGTT CTATGCACCA

^A I ^AGCTTAAA CCAGCCAGGC TGGCTGTAGT GGATTGAGCG TCTGAGGCTG CACCTCTCTG

^ Ji ^CTGCAGCC AGTTCTGGGT GAGACTGACC CTGCCTGAGG GTTCTCTCCT TCCCTCTCTC n _Λ ^ SS £ ?? ^{CC TCC} CTCTCCC TCTCCCTCTC TCTGTTTCCT GAGGTTTCCA

VJAAΓTGGGGA

^ - _Γ ÎSS? _™ ^{CAG AGA} CACCACT AAAGCCTTAC CTTTTAAGAA GTTGCATTCA GTGAGTGTGT

_T i ^ S ^ SiP ^ACAGATAGGG GCAGAGGAGA GCTGGTTCTG TCTCCACTGT GTTTGGTCTT τ ^ ?? ^ J ^{GAA CTCAGA} CCAT CAGGTGTGAT AGCAGTTGTC TTTAACCCTA ACCCTGAGCC n Λ ^ l ^ ^{A C} JR ^{Cτ GTCCCT} TCCC AAGACCACTG AAGCTAGGTG CAAGATAAGT GGGGACCCTT

_A JS? ^" _Λ ^ ^{TGG TAGGA} TCTTT CACGATAAGG ACTATTTTGA AGGGAGGGAG GGTGACACTG

^ _T ^ ^C ^ T ^{GTCCT CTTA} CCCTAG TGTCTCCAGC CTTGCCAGGC CTTAAACATC CGCCCATTGT

_{AA ^} S ^ ¹⁰ ™ ^GAAGGGG CCA CCCTTGACTT GCTGCTAAAC AAGGCACTCC CTAGAGAAGA 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 TGATTTTTTTTTTTGGTTTTTTTTTTTTTTT

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

^GTCCTG CAGT ^TCAGAA CTTT CTGGAGACTG AGAAGTGCAT GTGAGGACAC TCTCCTCCCA

TCTTTTCCTC TAGTGGCTAG TGATGTTTGG TTTTTTGTTT TGAGACAGGG TTTCTCTGTA

_I ^AGCCCTAGC TATCCTGGAA CTCACTTTGT AGATCAGGCT GGCCTCCAAC TCAGAAATCT

GCCTGCCTCT GCCTCCCGAG TGCTGGGACT AAAGGCGTGC GCCACCACTG TCCAGTCAGG

^"A 5î.t ^GAAGGA ^ CTCTAAGG TGCTTGAGAC AGGCTGAGTA GAGGCTAGGA GGAAGGGGCA _{r, Λ,} Ξ ^C 2 ^{CAGTCAC CGG} CTCCATG ACTCTGTGAC TTTTGTGGTT CCTTGTCGCA GCGGTTCCTG

_.DELTA.R ^ ii ^ ΞSS ^TGGTCGGGG <î TTGGGGGGAG GGGGCAGGCC ACACAGTGGG GTGTGGGAGG _{Δ r?} ^ Y ^{G AGC} TC I ^TGACAACT CCAACAGAAA CCAGGCTTTT GAGTCCTCCA GGGTAGCTTG _{n 1 A} ^{AGA GGG ACT} I CAGAAAGCCG TGTCCATGTC CCCTTTCCTT CACCTCAGGG AAGTAAGTTG

- _rr - ^ ïïiϋS? ^{31 TGTCAT} TTCA ATGAGGTCTT CTGGTTATTC TGTTTTTCTC TCAATGTTGG _τ J ^ Y ^ SS ^{10 AGGGAA} TGCT TTGGAGAAGG TGGTGGGAAC TGGAGAAGGG .AAGATCAGTT 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 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 TGC1 1 TTTT TTCCTTTTCT

CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCTCTCTCT CTTTCCTTTT

CTCTCTTTCA CTCTCTCTTT CT.AATTTCTT TTTCTTTTTT TCTTTCTTTT CTTTAGACAG

GGTTTCTCTG TGTAGCCCTG GCTGTTCTGG AACTCACTCT TTAGACCAGG CTGGCCTCGA

ATCTCAGAAA TCTACCTGCC TCTGCCTCCC AAGTGCTGGG ATTAAAGGCG TGTGCCACCA

CTGCCCAGCT AAGGTTTGCT TTTTGATGGC AGCTTGGTCC AGTTTGAAAG TAGGAGGTCA TACTGTGTAA ACTCACTGGT TAAAGTACCC CCTCCCTTCC ACCCTGCAAT ACACACACAC

ACACACACACAC ACACACACAC ACACACACAC ACCCCATCTC GAAGAGCTCT ATTAAGCTCC

AGGTGCACTG TAGTTCACAG ACTGCATCTT 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 CCCAGAGAGC 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

₃ ^ ⁽ ?? _Λ ^K3 2 ^{AGGAA AACGG} AACA.A AAACACAAAC TATAAAACAA AGAGAAGGCC GAGGACAAAG

_™ ^TAGCAATG CATACTTCCC TTTCTATGTG AAGCCCTGGG CTCCACCAGT ACTGCAGAAA

" ^GAAGCAAGCA ATGAGGGACA GGAGGTTGGC TCTAGGCCCA GGGGTTGTCA AAATAGTCCA

^ ^AGGCCAAAG OCAGCCTGAT 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

CTGCCTC

(2) INFORMATION FOR SEQ ID NO: 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 2190 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF BRINS: double

(D) CONFIGURATION: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) Hypothesis: No

(vi) ORIGIN:

(A) ORGANIZATION: MOUSE

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA AGAGGCGGGT CTGGGTCTTTTTTTTTGAGC TTTGTTCTAT GTTCCTCCATGCTACGGTTGT 1 CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGGATTTGGGGTC 1

TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 2-

TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 3 (

GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 3;

TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 4:

CACCACGATAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 4 _Î

GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 5- '

AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 60

TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 6 (

ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 7:

TGGGATTAAA GGCGTGTGCC CATAGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 7E

GGATTTGGGG AATCCTGTCT AGTGAATTCA GGACGTAATC AGTGGCTGGG AAGCAAGAGC 8 ^

TCTAGAGGAG CTCCAGCTTA TTATGACCCT TCCTTCAGAT GCCACAAGGA GGTGCTGGAG 9C

TTCTATGCAC CAATAGCTTA AACCAGCCAG GCTGGCTGTA GTGGATTGAG CGTCTGAGGC 96

TGCACCTCTC TGGCCTGCAG CCAGTTCTGG GTGAGACTGA CCCTGCCTGA GGGTTCTCTC 102

CTTCCCTCTC TCTACTCCTT CCTCCCTCTC CCTCTCCCTC TCTCTGTTTC CTGAGGTTTC 10E

CAGAATTGGG GATGGGACTC AGAGACACCA CTAAAGCCTT ACCTTTTAAG AAGTTGCATT 114

CAGTGAGTGT GTGAGACATA GCACAGATAG GGGCAGAGGA GAGCTGGTTC TGTCTCCACT 12C

GTGTTTGGTC TTGGGTACTG AACTCAGACC ATCAGGTGTG ATAGCAGTTG TCTTTAACCC £ 12

TAACCCTGAG CCTGTCTCAC CTGTCCCTTC CCAAGACCAC TGAAGCTAGG TGCAAGATAA 132

GTGGGGACCC TTTCTGAGGT GGTAGGATCT TTCACGATAA GGACTATTTT GAAGGGAGGG 13B

AGGGTGACAC TGTCCTAGTC CTCTTACCCT AGTGTCTCCA GCCTTGCCAG GCCTTAAACA 144

TCCGCCCATT GTCACCGCTC TAGAAGGGGC CACCCTTGAC TTGCTGCTAA ACAAGGCACT 15C

CCCTAGAGAA GATACCATAC CTGTGGGCAG GATGACCCAT GTTCTGCCAT GCACTTGGTA 156

GCCTTGGAAA GGCCACTTTG AACCTCAATT TTCTCAACTG TTAAATGGAG TGGTAACTGC 162

TATCTCATAA TAAAGGGGAA CGTGAGGAAG GCGTTTGGAT AGTGCCTGGT TGCGGCCAGG 168

CTGCAGTCAA GACTAGTTCC CACCAACTCG ATTTTAAAGC CTTGCAAGAA GGTGGCTTGT 174 TTGTCCCTTG CAGGTTCCTT TGCTCGGGCC AAACTCTAGA ATGCCTCCCC CTTTCTTTCT 18 ι

CATTGAAGAG CAGACCCAAG TCCGGGTAAC AAGGAAGGGT TTCAGGGTCC TGCCCATAAA 18 '

AGGTTTTTCC CGGCCGCCCT CAGCACCGCC CCGCCCCGAC CCCCGCAGCA TCTCCAAAGC 19:

ATGCAGAGAA TGTCTCCGGC TGCCCCCGAC AGACTGCTCC AACTTGGTGT CTTTCCCCAA 19,

ATATGGAGCC TGTGTGGAGT GAGTGGGGCG GCCCGGGGTG GTGAGCCAAG CAGACTTCCA 2 0 -

TGGGCAGGGA GGGGCGCCAC GGGGCGGCAG AGGGGTGACA TCACTGCCTA GGCGGCCTTT 21 (

CCCAGCCGGC CCGCCGGCC GCCCCGGCC GTCTGCCCCA GCCCAGACAC CGAAGCTACT 2 1 -

CTCCTTCCAG TCCACAAACG ACCAAGCCTT 2 1:

(2) INFORMATION FOR SEQ ID NO: -,

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: ιeβ ₁ .Base pairs

(B) TYPE: nuclέotidV

(C) NUMBER OF BRINS: doubling>

(D) CONFIGURATION: linear

(ii) MOLECULE TYPE: DNA (geometric)

(ii) HYPOTHETICS: NO

(Vi) ORIGIN:

(A) ORGANIZATION: MOUSE

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: -5 GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA AGAGGCGGGT CTGGGTCTTTTTTTTTGAGC TTTGTTCTAT GTTCCTCCATGCTACGGTTG CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC

TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 2

TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 3

GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 3

TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 4

CACCACGATAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 4

GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 5

AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 6

TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 6

ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 7

TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 7

GGATTTGGGG AATCCTGTCT AGTGAATTCA GGACGTAATC AGTGGCTGGG AAGCAAGAGC 8

TCTAGAGGAG CTCCAGCTTA TTATGACCCT TCCTTCAGAT GCCACAAGGA GGTGCTGGAG 9

TTCTATGCAC CAATAGCTTA AACCAGCCAG GCTGGCTGTA GTGGATTGAG CGTCTGAGGC 9 '

TGCACCTCTC TGGCCTGCAG CCAGTTCTGG GTGAGACTGA CCCTGCCTGA GGGTTCTCTC 10

CTTCCCTCTC TCTACTCCTT CCTCCCTCTC CCTCTCCCTC TCTCTGTTTC CTGAGGTTTC 10

CAGAATTGGG GATGGGACTC AGAGACACCA CTAAAGCCTT ACCTTTTAAG AAGTTGCATT 11 -

CAGTGAGTGT GTGAGACATA GCACAGATAG GGGCAGAGGA GAGCTGGTTC TGTCTCCACT 12 '

GTGTTTGGTC TTGGGTACTG AACTCAGACC ATCAGGTGTG ATAGCAGTTG TCTTTAACCC 12

TAACCCTGAG CCTGTCTCAC CTGTCCCTTC CCAAGACCAC TGAAGCTAGG TGCAAGATAA 13

GTGGGGACCC TTTCTGAGGT GGTAGGATCT TTCACGATAA GGACTATTTT GAAGGGAGGG 13 '

AGGGTGACAC TGTCCTAGTC CTCTTACCCT AGTGTCTCCA GCCTTGCCAG GCCTTAAACA 14 *

TCCGCCCATT GTCACCGCTC TAGAAGGGGC CACCCTTGAC TTGCTGCTAA ACAAGGCACT 15 ⁽

CCCTAGAGAA GATACCATAC CTGTGGGCAG GATGACCCAT GTTCTGCCAT GCACTTGGTA 15 {

GCCTTGGAAA GGCCACTTTG AACCTCAATT TTCTCAACTG TTAAATGGAG TGGTAACTGC 16:

TATCTCATAA TAAAGGGGAA CGTGAGGAAG GCGTTTGGAT AGTGCCTGGT TGCGGCCAGG 16 i C 40

INFORMATION FOR SEQ ID NO: ₄

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH:. base pairs

(B) TYPE: nuclcotide

(C) NUMBER OF BRINS: double

(D) CONFIGURATION: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) Hypothesis: No

(vi) ORIGIN:

(A) ORGANIZATION: MOUSE

RECTIFIED SHEET (RULE 91) ISA / EP TCTAGAGGCC ACGGAACGGT GCCAAGCACA CAGTCCCTTT TGCCTCTTTC ACGGGAGCAG

GAGTCCCAGT GCCTGTCGTG GAAAGGGAGG AACATGCCAG GTCCCTGTGT GTCCTTGGCC

CTGTCTCACC AAAGGACTCA GGGCTGGTTT CTGAGTTTCC GTCCAGTATT TAGCCAAGTC

CTGTGTTAGT CACGTAGGCC TAAGAGCCTT GGCGTTTACA 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 GGGTTTTTTTTT TGTTTGGGGGGGGGGTGTTTTTTGGTTTTTT

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 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

AAGGGGAACG TGAGGAAGGC GTTTGGATAG TGCCTGGTTG CGGCCAGGCT GCAGTCAAGA

CTAGTTCCCA CCAACTCGAT TTTAAAGCCT TGCAAGAAGG TGGCTTGTTT GTCCCTTGCA

GGTTCCTTTG CTCGGGCCAA ACTCTAGAAT GCCTCCCCCT TTCTTTCTCA TTGAAGAGCA

GACCCAAGTC CGGGTAACAA GGAAGGGTTT CAGGGTCCTG CCCATAAAAG G l I H TCCCG

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 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 CTCTCTCTCTCTCTCTCTCTCTCTCTCTTT CTTTCC I ι i I _^^

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 TGATTTTTTTTTTTGGTTTTTTTTTTTTTTT

TTGTTTTAAG TTAGCATACA AAGTAATACA TTTCATCATG GCATTTGGAC ATACATATAT

ATTTTATTTG CTCTCCTGGC CTCTTCTCAA AGAGACTTCT CTGGACTTTC

TTGTA i I i M

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

TTTCTCTGTA

TAGCCCTAGC TATCCTGGAA CTCACTTTGT AGATCAGGCT GGCCTCCAAC

TCAGAAATCT

GCCTGCCTCT GCCTCCCGAG TGCTGGGACT AAAGGCGTGC GCCACCACTG

TCCAGTCAGG

AGTAGAAGGA AACTGTAAGG TGCTTGAGAC AGGCTGAGTA GAGGCTAGGA

GGAAGGGGCA

CCGCAGTCAC CGGCTCCATG ACTCTGTGAC TTTTGTGGTT 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

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 CCCAGAGAGC 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

CTGCCTCAAC ATG (2) INFORMATION FOR SEQ ID NO: _Ξ

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 785 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF BRINS: double

(D) CONFIGURATION: linear

(ii) MOLECULE TYPE: DNA (oenomic)

(iii) Hypothesis: No

(vi) ORIGIN:

(A) ORGANIZATION: MOUSE

(xi) DESCRIPTION- OF THE SEQUENCE: SEQ ID NO:?

GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAoCTCTTG GGCTGGGAGA t.

AGAGGCGGGT CTGGGTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG II

CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC 1E

TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 24

TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 3C

GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 36

TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 4:

CACCACGATAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 48

GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 5 '

AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 6C

TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC £ 6

ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 7:

TGGGATTAAA GGCGTGTGCC CATGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 7E GGATT- 7E

References

Almendral, J.M., Santaren, J.F., Perera, J., Zerial, M. and Bravo, R. (1989). Expression, cloning and cDNA sequence of a serum-regulated fibroblast gene encoding a putative actin-associated protein (ρ27). Exp. Cell Res., 181, 518-530.

Babij, P., Kelly, C. and Periasamy, M. (1991). Characterization of a mammalian smooth 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.

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

Bour, B.A., O'Brien, M.A., Lockwood, W.L., Goldstein, E.S., Bodmer, R., Taghert, P.H., Abmayr S.M. and Nguyen, H.T. (1995). Drosophila MEF2, a transcription factor that is essential for myogenesis. Genes & Dev., 9, 730-741.

Chamley-Campell, J.H. and Campell, G.R. (1981). Artherosclerosis, 40, 347-357.

Chirgwin, JM, Przybyla, EA, MacDonald, RJ and Rutter, WJ (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18, 5294-5299. Duband, J.-L., Gimona, M-, Scatena, ML, Sartore, S. and Small JV (1993). Calponin and SM 22 as differentiation of smooth muscle markers: spatiotemporal distribution during avian embryonic development. Differentiation, 55, 1-11.

Dynan, W. S. and Tijan, R. (19S3) The promoter-specific transcription factor SP 1 binds to upstream sequences in the SV 40 early promoter. Cell. 35, 79-87.

Frohman, MA, Dush, MK and Martin, GR (1988). Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl. Acad. Sci, _r 85, 8998-9002.

Gimona, M., Sparrow, 2ME. P., Strasser, P., Herzog, M. and Small, J. V. (1992). Calponin and SM 22 isoforms in avian and mammalian smooth muscle. Absence of phosphorylation in vivo. Eur. J. Biochem ... 205. 1067-1075.

Kelly, R., Alonso, S., Tajbakhsh, S, Cossu, G. and Buckingham, M. (1995). Myosin light chain 3F regulatory sequences conferregionalized cardiac and skeletal muscle expression in transgenic mice. J. of Cell Biol, 129/2, 353-396.

Gorman, CM. , Moffat L.F. and B.H.H. (1982). Recombinant genomes which express chloramphenicol acetyl-transferase in mammalian cells. Mol. Cel

Biol. , 2: 1044-1051.

Kemp, P.R., Osbourn, JK, DJ Grainger and Metcalfe, J. C (1995). Cloning and analysis of the promoter region of the rat SM 22α gene. Biochem. J., 310, 1037-1043. Kim, J.-H., Busbel, PR and Kumar, CC (1993). Smooth muscle α-actin promoter activity is induced by serum stimulation of fibroblast cells, Biochem. Biophys. Res. Com., 190/3, 1115-1121.

Kramerov, D.A., Lekakh, I.V., Samarina, O.P. and Ryskov, A.P. (1982). Sequences homologous to major interspersed B1 and B2 repeats of mouse genome are present in mRNA and small cytoplasmic poly (A) + RNA. Nuc. Acid Res., 10/23, 7477-7491.

Krayev, A.S., Kramerig, D.A., Skryabin, K.G., Ryskov A.P., Bayev, A.A. and Georgiev, G.P. (1980). The nucleotide sequence of the ubiquitous repetitive DNA sequence B1 is complementary to the most abundant class of mouse fold-back RNA. Nuc Acid Res ... 8/6, 1201-1215.

Lees-Miller, J.P., Heeley, B.H., Smillie, L.B. and Kay, C.M. (1987a). Isolation and charakterization of an abundant and novel 22-kDa protein (MS 22) from chicken gizzard smooth muscle, y. Biol Chem., 262/7, 2988-2993.

Lees-Miller, J.P., Heeley, D.H. and Smillie, L.B. (1987b). An abundant and novel protein of 22 kDa (SM 22) is widely distributed in smooth muscles. Biochem. J., 244, 705-709.

Li, Z., Marchand, P., Humbert, J., Babinet C. and Paulin D. (1993). Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice. Development, 177/3, 947-959. Lilly, B., Zhao, B. Ranganayakulu, G., Paterson, BM, Schulz, RA and Olson, EN

(1995). Requirement fo MADS Domain D-MEF2 facter transcription. for muscle formation in drosophila. Science, 267, 688-693.

Luckow ,. B. and Schötz, G. (1987). CAT constructs with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nuc. Acid Res., 15/13, 5490.

Min, B., Foster, D. N. and Strauch, A. R. (1990). The 5'-flanking region of the smooth muscle smooth muscle α-actin gene contains evolutionarily conserved sequence motifs within a functional promoter. J. Biol. Chem., 265/27, 16667-16675.

Miwa, T., Manabe, Y, Kurokawa, K., Kamada, S., Kanda, N., Bruns, G., Ueyama, H. and Kakunaga, T. (1991). Structure, chromosome location and expression of the human smooth muscle (enteric type) γ-actin gene: Evolution of six human actin genes. Mol. Cell. Biol, 11/6, 3296-3306.

Mössler H. (1995). Sm 22 of Mus Musculus. PhD thesis at the University of Salzburg, Austria.

Nishida, W., Kitami, Y., Abe, M. and Hiwada, K. (1991). Gene cloning and nucleotide sequence SM 22α from the smooth muscle gizzard. Biochem. Insem., 23/4, 663-668. Olson, EN (1990). Myo D family: a paradigm for development ?. Genesis and Dev., 4, 1454-

1461.

Osbourn, J.K., Weissberg, P.L. and Shanahan, C.M. (1995). A regulatory element downstream of the rat SM 22a gene transcription start point enhances reporter gene expression in vascular smooth muscle cells. Gene, 154, 249-253.

Pearlstone, J.R., Weber, M., Lees-Miller, J.P., Carpenter, M.R. and Smillie, L.B.

(1987). Amino acid sequence of chicken SM smooth muscle gizard 22α. J. Biol Chem., 262/13, 5985-5991.

Prinjha, R.K., Shapland, C.E., Hsuan, J.J., Totty, N.F., Mason, L.J. and Lawson, D.

(1994). Cloning and sequencing of cDNAs encoding the actin cross-linking protein transgelin dermes to a new faeily of actin-associatcd proteins. Cell Motil and Cytoskel, 28, 243-255.

Reddy, S., Ozgur, K., Lu, M., Chang, W., Mohan, S., Kumar, C. and Ruley, H. E. (1990). Structure of the human smooth muscle α-actin gene. J. Biol Chem., 265/3, 1683-1687.

Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain termination inhibitors. Proc. Natl Acad Sci, 74, 5463ff.

Santaren, JF, Blüthmann, H., MacDonald-Bravo, H. and Bravo, R. (1987). Specific antibody against a protein (p27) present in nonestablished fibroblasts. A putative Microfilament associated protein. Exp. Cell. Res., 173, 341-348. Shanahan ,. CM, Weissberg, PL and Metcalf; J .. C. (1993). Isolation of gene markers of differentiated and proliferating vascular smooth muscle cells. Circ. Res., 73, 193 - 204.

Shanahan, C., Cary, N.R.B., Metcalf, J.C. and Weissberg, P.L. (1994). High Expression of genes for calcification-regulating proteins in human artherosclerotic plaques. J. Clin, Invest, 93, 2393-2402.

Shapland, C., Lowings, P. and Lawson, D. (1988). Identification of new actin associated polypeptides that are modified by viral transformation and changes in cell shape. J. Cell Biol., 107, 153-161.

Shapland, C., Hsuan, J.J. Totty, N.F. and Lawson, D. (1993). Purification and properties of Transgelin: A transformation and shape change sensitive actin-gelling protein. J. Cell Biol 121/5, 1065-1073.

Solway, J, Seltzer, X, Samaha, F., Kim, S. Alger, L.E., Nia, Q., Morrisey, E.E., Ip, H.S. and Parmacek, M.S. (1995). Structure and expression of a smooth muscle cell-specific gene, SM 22α. J. of Biol Chem., 270/22, 13460-13469.

Thweatt ,. R. Lumpkin, CK and Goldstein, S. (1992). A novel gene encoding a smooth muscle protein is overexpressed in senescent human fibroblasts. Biochem. Biophys. Res. Com., 187/1, 1-7. Tzeng, Y.-J, Guhl E. Graessmann, M. and Graessmann, A. (1993). Breast cancer formation in transgenic animals induced by the acidic protein whey SV 40 T antigen (WAP-SV-T) hybrid gene. Oncogene, 8, 1965-1971.

LI LI, J.M. MIANO, B. MERCER and E. OLSON

J. OF CELL BIOLOGY 1996 - VOL 132 No. 5, p. 349-859

Claims

DNA sequence characterized in that it comprises

a fragment of the sequence upstream of the coding part of the gene of the SM22 protein, or of a hybndaπt sequence under conditions of high stringency with said upstream sequence, said fragment being capable of inducing a specific expression of a gene in eukaryotic cells, and

a sequence encoding a protein or 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 of the SM 22 protein, or of a hybridizing sequence under conditions of high stringency with said upstream sequence, said fragment being capable of inducing a specific expression in vivo of a gene in the cells of the arteries, and

a sequence encoding a protein or 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, to activate the growth of endothelial cells to consolidate the walls of the arteries and / or to induce an immune response. 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 SM22 protein ( SEQ ID π ° 1)

Sequence according to one of Claims 1 to 4, characterized in that the said fragment is included in the sequence located between nucleotides - 2126 to +65 of the mouse gene of the SM 22 protein (SEQ ID No. 2)

6 sequence according to one of claims 1 to 5 characterized in that it contains at least a portion of the sequence between the nucleotides -2126 and -445 of the mouse gene of the SM 22 protein (SEQ ID No. 3)

7 Sequence according to one of claims 1 to 6, characterized in that said protein of therapeutic interest is a protein inducing the formation of a cytotoxic compound

8 Sequence according to claim 7, characterized in that said protein is the thymidine kmase of the herpes virus

9 Sequence according to one of claims 1 to 6, characterized in that said protein of therapeutic interest has a cytostatic effect

Sequence according to one of Claims 1 to 6, characterized in that the said protein of therapeutic interest has lipolytic activity.

1 1 sequence according to claim 10, characterized in that said protein is lipoprotein lipase

Sequence according to one of claims 1 to 6, characterized in that said protein of therapeutic interest is an endothelial cell growth factor

Sequence according to claim 11, characterized in that said factor is an interleukin

14 Sequence according to one of claims 1 to 6, characterized in that said protein of therapeutic interest is a muscle protein or tissue structure

Sequence according to one of Claims 1 to 6, characterized in that the RNA of therapeutic interest is the aπtisens RNA of the p53 protein

A DNA sequence located upstream of the coding portion of the mouse gene of the SM 22 protein, characterized in that it comprises at least a part of the following sequence SEQ ID N ^c 5, or a sequence which hybridizes with that under severe 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 G GATT

17. A DNA sequence located upstream of the coding part of the gene of the mouse SM22 protein, characterized in that it comprises at least a portion of SEQ ID NO: 1 next sequence, or a sequence ^hybridizing with it in stringent conditions

GG 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 GGGTTTTTTTTT TGTTTGGGGGGGGGGTGTTTTTTGGTTTTTT

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 GTCCCTTCCC AAGACCACTG AAGCTAGGTG CAAGATAAGT GGGGACCCTT

TCTGAGGTGG TAGGATCTTT CACGATAAGG ACTATTTTGA AGGGAGGGAG GGTGACACTG

TCCTAGTCCT CTTACCCTAG TGTCTCCAGC CTTGCCAGGC CTTAAACATC CGCCCATTGT

CACCGCTCTA GAAGGGGCCA CCCTTGACTT GCTGCTAAAC AAGGCACTCC CTAGAGAAGA 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 TGATTTTTTTTTTTGGTTTTTTTTTTTTTTT

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 TTTCTCTGTA

TAGCCCTAGC TATCCTGGAA CTCACTTTGT AGATCAGGCT GGCCTCCAAC TCAGAAATCT

GCCTGCCTCT GCCTCCCGAG TGCTGGGACT AAAGGCGTGC GCCACCACTG TCCAGTCAGG

AGTAGAAGGA AACTGTAAGG TGCTTGAGAC AGGCTGAGTA GAGGCTAGGA GGAAGGGGCA

CCGCAGTCAC CGGCTCCATG ACTCTGTGAC TTTTGTGGTT 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 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 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 CTCTCTCTCTCTCTCT 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 TACTGTGTAA ACTCACTGGT TAAAGTACCC CCTCCCTTCC ACCCTGCAAT ACACACACAC

ACACACACACAC ACACACACAC ACACACACAC ACCCCATCTC GAAGAGCTCT ATTAAGCTCC

AGGTGCACTG TAGTTCACAG ACTGCATCTT 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 CCCAGAGAGC 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 AAGTCTAAAΛ TATTTACTCT GCTGTTTTAC ATAAAAAGTT GACAGACTCT TGCTCTAGAC TGACAAATAT

CTAAGACCTT GTTTTCTGAG GTTCAAGTTT CAGAGGGGTC TCTGCAGCAA GTGGGTAAAG

CTGGTCTAGG TCATGCTATG ATGTCTAGGG TCCCCTCAGA GTGGAAGGCC TGCTTAGCAC

AAATGAAGTA AAGTAACTTG CTGGCTCTTT GTTCTTTTCT CCACACTCTA TACTTTAGCT

CTGCCTC

18 A ^DNA sequence located upstream of the coding part of the gene of the mouse SM22 protein characterized in that it comprises at least a portion of the sequence SEQ ID N ° 2 or a sequence ^s' hybπdant with celle- ci in stπngentes conditions

SEQ ID NO: 2 _: GGGCCCCAGG AATGTGTTTC CTTCTCTCCA CCATGTTTTT ATAGCTCTTG GGCTGGGAGA

AGAGGCGGGT CTGGCTCTTT GTTTCTGAGC TTTGTTCTAT GTTCCTCCAT GCTACGGTTG 1

CAATTGTTTT CTATGAACGA GTACATTCAA TAAAGACAAC CAGACCTGGG ATTTGGGGTC 1

TTACTGATGT GTTGGGAGGT GCAGGAGCCT CCGTGTCCCA TTTATTTTGG CCTTCCCGTC 2

TCGTTTCTGT GCGTGGCTAC ATTGGGAATG ACCTTCCTTG ATCCCACCAA GCCACCCATT 3

GATTCTGTAA ACATGTGACC CTTGCTCCAA GCATTGCTTA CAGGAGCAGG ATACTGAAAG 3

TGTGTCTGTG CCCTCTCCTG ATAACCCCTC CCTTCAGCAG GCACACAGCA CCTGACTACC 4

CACCACGATAT GTAAACGTCA GTATCCTTTC CAGCCAGCTC TGCAGATGGG TGTCCAGGCT 4

GTGCATGATG CACCTCAAGT GGGCAGAGCT TGCAGGCCAA GGTTTTAAAG GCTGTTCAGG 5

AATGGATGGC AAGCAGGATC TAAGAGGAGG GGGGGTTGTT GTTGTTTGGG GGGGGGGTGG 6

TTTTGGTTTG TTTTTTTTGA GACAGGGTTT CTCTGTGTGG CCCTGGCCCT CCTGGAACCC 6

ACTCTGTAGA CCAGGCTGGC CTTGAACTCA GAAATCTGCC TGCCTCTGCC TCCCGAGTGC 7

TGGGATTAAA GGCGTGTGCC CATCGAGGAG GGAGATTTTA TTTAGATTAT AAAAAGGACG 7

GGATTTGGGG AATCCTGTCT AGTGAATTCA GGACGTAATC AGTGGCTGGG AAGCAAGAGC 8

TCTAGAGGAG CTCCAGCTTA TTATGACCCT TCCTTCAGAT GCCACAAGGA GGTGCTGGAG 9

TTCTATGCAC CAATAGCTTA AACCAGCCAG GCTGGCTGTA GTGGATTGAG CGTCTGAGGC 9

TGCACCTCTC TGGCCTGCAG CCAGTTCTGG GTGAGACTGA CCCTGCCTGA GGGTTCTCTC 10:

CTTCCCTCTC TCTACTCCTT CCTCCCTCTC CCTCTCCCTC TCTCTGTTTC CTGAGGTTTC 10, CAGAATTGGG GATGGGACTC AGAGACACCA CTAAAGCCTT ACCTTTTAAG AAGTTGCATT 1 1 - ^'

CAGTGAGTGT GTGAGACATA GCACAGATAG GGGCAGAGGA GAGCTGGTTC TGTCTCCACT 12 C

GTGTTTGGTC TTGGGTACTG AACTCAGACC ATCAGGTGTG ATAGCAGTTG TCTTTAACCC 12 t

TAACCCTGAG C CTGTCTCAC CTGTCCCTTC CCAAGACCAC TGAAGCTAGG TGCAAGATAA 1 31

GTGGGGACCC TTTCTGAGGT GGTAGGATCT TTCACGATAA GGACTATTTT GAAGGGAGGG 13 c

AGGGTGACAC TGTCCTAGTC CTCTTACCCT AGTGTCTCCA GCCTTGCCAG GCCTTAAACA 14 -

TCCGCCCATT GTCACCGCTC TAGAAGGGGC CACCCTTGAC TTGCTGCTAA ACAAGGCACT 15 ⁽

CCCTAGAGAA CATAC GATAC CTGTGGGCAG GATGACCCAT GTTCTGCCAT GCACTTGGTA 15 t

GCCTTGGAAA GGCC ACTTTG AACCTCAATT TTCTCAACTG TTAAATGGAG TGGTAACTGC 1 61

TATCTCATAA TAAAGGGGAA CGTGAGGAAG GCGTTTGGAT AGTGCCTGGT TGCGGCCAGG 16 E

CTGCAGTCAA GACTAGTTC C CACCAACTCG ATTTTAAAGC CTTGCAAGAA GGTGGCTTGT 17 -

TTGTCCCTTG CAGGTTCCTT TGCTCGGGCC AAACTCTAGA ATGCCTCCCC CTTTCTTTCT 18 C

CATTGAAGAG CAGACCCAAG TCCGGGTAAC AAGGAAGGGT TTCAGGGTCC TGCCCATAAA 18 £

AGGTTTTTCC CGGCCGCCCT CAGCACCGCC CCGCCCCGAC CCCCGCAGCA TCTCCAAAGC 192

ATGCAGAGAA TGTCTCCGGC TGCCCCCGAC AGACTGCTCC AACTTGGTGT CTTTCCCCAA 19 E

ATATGGAGCC TGTGTGGAGT GAGTGGGGCG GCCCGGGGTG GTGAGCCAAG CAGACTTCCA 204

TGGGCAGGGA GGGGCGCCAC GGGGCGGCAG AGGGGTGACA TCACTGCCTA GGCGGCCTTT 2 1 C

AAACCCCTCA CCCAGCCGGC GCCCCGGCCC GTCTGCCCCA GCCCAGACAC CGAAGCTACT 2 1 Ê

CTCCTTCCAG TCCACAAACG ACCAAGCCTT 2 1 S

19 strain filed March 25, 1996 with the CNCM under No. l-1685 bearing the plasmid p2126πlz comprising the sequence seion claim 18

Strain deposited on March 25, 1996 with the CNCM under the number 1-1686 carrying the plasmid p2126INTnlz comprising the sequence according to claim 17

Vector characterized in that it contains a sequence according to one of claims 1 to 16

Vector according to claim 20, characterized in that it contains an efficient origin of rephcation in the cells of the arteries

Vector according to one of claims 21 and 22, characterized in that it is a derivative of an adenovirus

24 RNA characterized in that it is capable of being expressed by a sequence or a vector according to one of claims 1 to 23

Composition characterized in that it contains a sequence or a vector according to one of Claims 1 to 16 and 21 to 23

Composition according to Claim 25, characterized in that the sequence or the vector are included in a composition facilitating their transfection into the cells

27 Composition according to claim 25, characterized in that it comprises a gel which is a complex of poly-L-lysiπe and lactose

28 characterized in that it contains a sequence or a vector according to one of claims 1 to 16 and 21 to 23

A pharmaceutical composition characterized in that it contains a pharmaceutically effective amount of a nucleic acid sequence or a vector according to any one of claims 1 to 16 and 21 to 23, and pharmaceutically compatible excipients.

Use of a vector or sequence according to one of claims 1 to 16 and 21 to 23 for the manufacture of a medicament for the treatment of coronary heart disease

Use of a vector or sequence according to one of Claims 1 to 16 and 21 to 23 for the manufacture of a medicament for the treatment of restenosis Use of a vector or sequence according to one of claims 1 to 16 and 21 to 23 for the manufacture of a medicament for the treatment of mutations weakening vessels

Transgenic animals characterized in that they carry a sequence or a vector according to one of claims 1 to 16 and 21 to

23, in which the gene coding for the protein of therapeutic interest is replaced by a reporter gene

34 molecules of in vitro screening method for their activity on the regulatory sequences of the gene encoding the protein SM22 comprising the ^steps'

- transfection of cells with a sequence or a vector according to one of claims 1 to 16 and 21 to 23, wherein the gene encoding the protein of therapeutic interest is replaced by a reporter gene, - incubation of the transfected cells with the molecule to be tested, and

- Quantification of the Expression of the Transporter Gene 35. A method for detecting mutations on the region comprising the sequence according to one of Claims 1 to 16 or the vector according to one of Claims 21 to 23, characterized by an alteration of the expression of the gene placed downstream of said sequence.

Process for the expression of proteins of therapeutic interest, characterized in that it uses the products according to one of claims 1 to 24