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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• This application relates generally to proteins implicated in coronary artery disease.
• PTC A percutaneous transluminal coronary angioplasty
• the initial response is primarily inflammatory in nature involving T-lymphocytes and macrophages which secrete many different cytokines and growth factors seminal to the local inflammatory response.
• the second phase involves the principle cellular component ofthe restenotic lesion, the vascular smooth muscle cell (VSMC), which in response the these factors migrates into the intima, secrete soluble growth and chemotactic factors, and proliferate [Libby etal, (1992), cited above; Clowes et al, (1983), cited above; R. Ross, Nature. 362:801-809 (1993); and E. R. O'Brien et al, Circ. Res., 73:223-231 (1993)]. These activated VSMC also secrete extracellular matrix.
• the third phase may last several weeks post-injury and is characteristic of a chronic fibroproliferative lesion formation [Ross (1993), cited above].
• Ross (1993) cited above.
• the cellular response to balloon angioplasty is fairly well characterized, there is still a lack of effective anti-restenotic pharmacological adjuncts to prevent neointima formation associated with PTCA.
• ATF allograft inhibitory factor
• the present invention provides novel uses for the polynucleotide and arnino acid sequences of RC-9, which has been isolated from other cellular materials with which it is naturally associated. Also provided are novel uses for vectors containing these sequences, recombinantly produced RC-9 proteins, and anti-RC-9 antibodies.
• the present invention provides methods of diagnosing proliferative arterial disease and vascular restenosis. These methods utilize the nucleic acid and arnino acid sequences of RC-9 and anti-RC-9 antibodies described herein.
• the present invention provides methods of identifying compounds which specifically bind to a selected RC-9 sequence, including agonists and antagonists, using the RC-9 nucleic acid sequences, proteins, and antibodies described herein.
• the present invention provides for compounds or drugs produced by use ofthe above methods.
• the present invention provides methods of treating proliferative arterial disease and vascular restenosis. These methods may utilize the compounds or drugs, nucleic acid and amino acid sequences of RC-9 and, preferably, the anti-RC-9 antibodies described herein.
• Fig. 1 provides the partial DNA sequences [SEQ ID NO:l] of clone RC9 obtained from differential display. This sequence has been assigned GenBank Accession No. U10894.
• Fig. 2 provides genomic DNA sequences [SEQ ID NO:2] of RC-9. This sequence has been assigned GenBank Accession No. U33471.
• Fig. 3 illustrates the cDNA [SEQ ED NO:3] and putative amino acid sequences [SEQ EO NO:4] ofrat RC-9.
• Fig. 4 illustrates the cDNA [SEQ ID NO:5] and amino acid sequences [SEQ ID NO:6] of human RC-9.
• the present invention provides novel uses for a gene, termed RC-9, and the encoded protein, in detecting and treating proliferative arterial disease and vascular restenosis.
• a gene termed RC-9
• the nucleotide and amino acid sequences and antibodies provided herein are useful in diagnosis and monitoring such vascular disease and damage. These sequences and antibodies are further useful in generating compounds useful in treatment of atherosclerosis and other conditions characterized by vascular stenosis.
• Figs. 1-4 provide DNA sequences ofthe novel transcript ofthe invention.
• Fig. 1 provides the nucleotide sequences of clone RC9 [SEQ ID NO:l], obtained as described in Example 1 below. This partial sequence is 424 bases in length and was used as a probe to obtain the longer sequence of Fig. 2.
• Fig. 2 provides genomic DNA sequences of RC-9 [SEQ ID NO:2], including two introns. Intron 1 spans base pairs 121-772 and intron 2 spans base pairs 1000-1276. PolyC tracts are located at base pairs 864-873 and 1254-1276.
• Branch point consensus sequences are located at base pairs 851-857 and 1212-1218 of Fig. 2.
• Fig. 3 provides the cDNA sequences of rat RC-9 [SEQ ID NO:3].
• the transcription initiation codon is located at bp 143-145 and the stop codon is located at bp 581-583 of Fig. 3.
• Fig. 4 provides the 636 bp DNA sequences of human RC-9 [SEQ ID NO:5].
• These sequences include a 444 bp open reading frame (bp 72-516), which encodes a 147 amino acid protein [SEQ ID NO :6], Fragments of these sequences may prove useful for a variety of uses.
• these functional fragments are at least about 15 nucleotides in length and encode a desired amino acid sequence, e.g. an epitope, a therapeutically useful peptide desirably characterized by RC-9-like biological activity, or the like.
• a desired amino acid sequence e.g. an epitope
• a therapeutically useful peptide desirably characterized by RC-9-like biological activity, or the like.
• These RC-9 nucleotide sequences may be isolated by conventional uses of polymerase chain reaction or cloning techniques. Alternatively, these sequences may be constructed using conventional genetic engineering or chemical synthesis techniques.
• the genomic RC-9 DNA sequence (Fig. 1 SEQ ID NO:l and Fig. 2 SEQ ID NO:2) or a cDNA sequence (Fig. 3 SEQ ID NO:3 and Fig. 4 SEQ U> NO:5) ⁇ ding for the encoded RC-9 protein or a functional fragment thereof may be modified.
• sequence data provided herein, it is within the skill ofthe art to obtain other polynucleotide sequences encoding the RC-9 proteins useful in the invention.
• modifications at the nucleic acid level include, for example, modifications to the nucleotide sequences which are silent or which change the amino acids, e.g. to improve expression or secretion.
• polynucleotide sequences useful herein may be modified by adding readily assayable tags to facilitate quantitation, where desirable.
• Nucleotides may be substituted, inserted, or deleted by known techniques, including, for example, in vitro mutagenesis and primer repair. Also included are allehc variations, caused by the natural degeneracy ofthe genetic code.
• this invention also encompasses other nucleic acid sequences, including those complementary to the illustrated DNA sequences, such as antisense sequences.
• Useful DNA sequences also include those sequences which hybridize under high or moderately high stringency conditions [see, T. Maniatis et al., Molecular Cloning (A Laboratory Manual). Cold Spring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences illustrated in Figs. 1-4 [SEQ ED NO:l, 2, 3, 5].
• An example of a highly stringent hybridization condition is hybridization at 4XSSC at 65°C, followed by a washing in 0.1XSSC at 65°C for an hour.
• an exemplary highly stringent hybridization condition is in 50% formamide, 4XSSC at 42°C.
• Other, moderately high stringency conditions may also prove useful, e.g. hybridization in 4XSSC at 55°C, followed by washing in 0.1XSSC at 37°C for an hour.
• an exemplary moderately high stringency hybridization condition is in 50% formamide, 4XSSC at 30°C.
• the RC-9 nucleic acid sequences encoding these proteins are useful for a variety of diagnostic and therapeutic uses.
• nucleic acid sequences are useful as diagnostic probes and antisense probes for use in the detection and diagnosis of proliferative arterial disease and vascular restenosis, among other conditions associated with undesirable RC-9 levels or expression.
• Oligonucleotide probes may be useful in such standard diagnostic techniques as Southern blotting and polymerase chain reaction.
• the RC-9 nucleic acid sequences may be used to produce RC-9 proteins useful in the methods ofthe invention. Once constructed, or isolated, these DNA sequences or suitable fragments are preferably employed to obtain proteins of this invention, in vitro or in vivo.
• RC-9 Amino Acid Sequences The methods ofthe invenuon may utilize RC-9 arnino acid sequences, including the
• RC-9 proteins provided herein and suitable functional fragments thereof.
• the RC-9 rat protein [SEQ DD NO:4] is encoded by the cDNA sequences [SEQ ID NO:3] illustrated in Fig. 3 and the RC-9 human protein [SEQ ED NO: 6] is encoded by the cDNA sequences of Fig 4 [SEQ ID NO:5].
• the rat protein [SEQ ID NO:4] is 147 amino acids in length and has a molecular weight of 16824.9 Daltons.
• the human protein is also 147 amino acids in length [SEQ ID NO: 6].
• Also useful in the methods ofthe invention are biologically active fragments of RC-9. These functional fragments are desirably at least five amino acids in length and may encompass an epitope or other desired amino acid sequence.
• analogs, or modified versions, ofthe RC-9 protein are also useful in invention.
• analogs or modified versions, ofthe RC-9 protein.
• Such analogs differ by only 1, 2, 3 or 4 codon changes and are characterized by RC-9-like biological activity.
• Examples include polypeptides with minor arnino acid variations from the illustrated amino acid sequences of RC-9 (Figs. 3 and 4, SEQ TD NOs: 4 and 6); in particular, conservative amino acid replacements. Conservative replacements are those that take place within a family of amino acids that are related in their side chains and chemical properties.
• the RC-9 proteins useful in the invention may be modified, for example, to improve production thereof, to enhance protein stability or other characteristics, e.g. binding activity or bioavailability, to enhance its use for screening competitive compounds or to confer some other desired property upon the protein.
• the RC-9 protein and RC-9 protein fragments described herein are useful in therapeutic compositions, as described in more detail below.
• the methods ofthe invention also include the use of these proteins in diagnostic applications, as well as for generation of other therapeutic and diagnostic reagents, such as anti-RC-9 antibodies.
• these RC-9 proteins may also serve in screening assays or as research tools. More desirably, the RC-9 proteins are also useful for the screening and development of chemical therapeutic agents useful for preventing the action of RC-9, e.g., in restenosis. m. Recombinant Expression of RC-9
• the DNA sequences described herein may be used to produce recombinant RC-9 proteins.
• the resulting proteins may be used in the methods ofthe invention, or the method ofthe invention may involve in vivo expression ofthe proteins.
• the RC-9 DNA sequences may be inserted into a suitable expression system.
• a recombinant molecule or vector is constructed in which the cDNA encoding RC- 9 is operably linked to a heterologous expression control sequence permitting expression of the RC-9 protein.
• appropriate expression vectors and host cell systems are known in the art for mammalian (including human) expression, insect, e.g., baculovirus expression, yeast, fungal, and bacterial expression, by standard molecular biology techniques. The transformation of these vectors into appropriate host cells can result in expression ofthe selected RC-9 proteins.
• Other appropriate expression vectors of which numerous types are known in the art, can also be used for this purpose.
• Suitable host cells or cell lines for transfection by this method include insect cells, such as Spodoptera frugipedera (Sf9) cells. Methods for the construction and transformation of such host cells are well-known. [See, e.g. Miller et al. , Genetic Engineering. 8:277-298 (Plenum Press 1986) and references cited therein].
• mammalian cells such as Human 293 cells, rat aortic vascular cell lines [E. H. Ohlstein etal, Eur. J. Pharmacol. - Mol. Pharmacol. Section. 225:347-350 (1992)], Chinese hamster ovary cells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived from Swiss, Balb-c or NTH mice may be used.
• Suitable mammalian host cells and methods for transformation, culture, amplification, screening, production and purification are known in the art. [See, e.g., Gething and Sambrook, Nature. 293:620-625 (1981), or alternatively, Kaufman etal., Mol. CeU. Biol.. 5(7): 1750-1759 (1985) or Howley et al, U. S. Patent 4,419,446].
• Another suitable ⁇ iamrnalian cell line is the CV-1 cell line.
• bacterial cells e.g., HB101, MC1061, and strains used in the following examples
• E. coli e.g., HB101, MC1061, and strains used in the following examples
• B. subtilis, Pseudomonas, other bacilli and the like may also be employed in this method.
• yeast cells known to those skilled in the art are also available as host cells for expression ofthe polypeptides useful in the methods ofthe invention.
• Other fungal cells may also be employed as expression systems.
• the present invention provides a method for producing a recombinant RC-9 protein which involves transforming a host cell with at least one expression vector containing a recombinant polynucleotide encoding a RC-9 protein under the control ofa transcriptional regulatory sequence, e.g., by conventional means such as transfection or electroporations.
• the transformed host cell is then cultured under suitable conditions that allow expression of the RC-9 protein.
• host cells e.g., rat aortic vascular smooth muscle cells, may be transfected with sufficient vectors that they are capable of overexpressing the RC-9 protein, making them useful for screening compounds which inhibit RC-9 expression.
• the expressed protein is recovered, isolated, and purified from the culture medium (or from the cell, if expressed intracellularly) by appropriate means known to one of skill in the art.
• the proteins may be isolated following cell lysis in soluble form, or extracted in guanidine chloride.
• the RC-9 proteins ofthe invention may be produced as a fusion protein.
• Suitable fusion partners for the RC-9 proteins described herein are well known to those of skill in the art and include, among others, b-galactosidase and poly-histidine.
• RC-9 proteins as well as modified versions or analogs thereof, or cells expressing same, are useful as antigens for the development of antibodies to RC-9.
• Antibodies useful in the methods of this invention include monoclonal, polyclonal, chimeric, single chain and humanized antibodies, as well as Fab fragments. These antibodies may be produced by conventional methods, including the Kohler and Milstein hybridoma technique, recombinant techniques, such as described by Huse et al. , Science. 246: 1275-1281 (1988), or any other modifications thereof known to the art. Techniques described for the production of single chain antibodies (US Patent No.
• 4,946,778 can be adapted to produce single chain antibodies to the RC-9 proteins descried herein.
• transgenic mice, or other organisms such as mammals may be used to express humanized antibodies to a RC-9 protein or RC-9- derived protein.
• the antibodies of this invention may themselves be used to generate anti- idiotype antibodies. Techniques for generating such antibodies are well-known in the art.
• the antibodies ofthe invention may be utilized in protein form.
• the antibodies ofthe invention may be utilized in the form ofa polynucleotide, which expresses the antibody or a functional fragment thereof (e.g., a single chain or a Fab fragment) in vivo.
• RC-9 proteins, antibodies, and polynucleotide sequences may be used as diagnostic reagents for diagnosing certain vascular disorders, e.g., atherosclerosis, associated with production or excessive production of RC-9.
• a RC-9 protein, antibody, or polynucleotide may be utilized to diagnose vascular damage characteristic of such a condition.
• diagnostic labels such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods.
• the reagents may measure RC-9 levels in selected mammalian tissue in conventional diagnostic assays, e.g., Southern blotting, Northern and Western blotting, polymerase chain reaction and the like.
• diagnostic agents the polynucleotide sequences may be employed to detect or quantitate normal RC-9 mRNA or detect mutations in target gene RNA in a patient sample.
• Such a method may utilize PCR primers complementary to the nucleic acid sequence of Figs. 1-4 [SEQ ID NO:l, 2, 3, 5].
• a specific DNA sequence i.e., RC-9
• metibods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, e.g., restriction fragment length polymo ⁇ hisms (RFLP) and Southern blotting of genomic DNA.
• restriction enzymes e.g., restriction fragment length polymo ⁇ hisms (RFLP) and Southern blotting of genomic DNA.
• Other suitable assays utilize a RC-9 protein, protein fragment, or anti-RC-9 antibody as a reagent. These assays include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. The selection ofthe appropriate assay format and label system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.
• the present invention provides methods for the use of these RC-9 protein- antibody or polynucleotide reagents in the diagnosis of disorders characterized by vascular restenosis, such as atherosclerosis.
• the methods may involve contacting a selected sample, e.g., blood, plasma, serum, or other suitable cells with the selected reagent, protein, antibody or DNA sequence, and measuring or detecting the amount of RC-9 present in the sample in a selected assay format based on the binding or hybridization or the reagent to the sample.
• the invention further provides methods for treatment of vascular restenosis. More particularly, this method involves administration of an anti-RC-9 antibody for blocking RC-9 activity in mammalian tissue. Also useful as therapeutic reagents of this invention are anti- idiotype antibodies, which can be used to block binding of RC-9 to its corresponding receptor.
• the therapeutic reagent may be a RC-9 nucleic acid sequence, a vector containing the nucleic acid sequences or an RC-9 protein. Alternatively, the therapeutic reagent may be a drug obtained using the methods described below.
• the therapeutic reagents may be administered by appropriate routes in a pharmaceutical composition.
• the composition contains between about 10 mg to about 10 mg ofthe active agent (e.g., anti-RC-9 antibody, or a vector or polynucleotide described herein) per kg body weight.
• the active agent e.g., anti-RC-9 antibody, or a vector or polynucleotide described herein
• Suitable pharmaceutical carriers are well known to those of skill in the art and can be readily selected from among saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof, among others.
• compositions ofthe invention may optionally contain other active ingredients, or other desirable components, e.g., pH adjusters, preservatives, and the like.
• Appropriate routes may be readily determined by one of skill in the art and include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, interdermal, oral, vaginal, anal, intranasal, and topical routes.
• the preferred method of administration is intravenous.
• one of skill in the art can readily select another appropriate route of administration. Dosing may be repeated as needed or desired.
• the present invention also provides methods using the RC-9 proteins, antibodies and polynucleotide sequences described herein in the screening and development of chemical compounds or proteins which have utility as therapeutic drugs for the treatment of atherosclerosis and other vascular disorders.
• such a compound is capable of binding to RC-9 and either enhancing (act as an agonist) or blocking (act as an antagonist) its biological activity.
• Such compounds are anticipated to be useful as a drug component for the treatment or prevention of vascular disorders, as described above.
• conventional assays and techniques exist for the screening and development of drugs capable of competitively binding to selected regions of RC-9. These include the use of phage display system for expressing the RC-9 proteins or portions thereof, and using a culture of transfected K coli or another microorganism to produce the proteins for binding studies of potential binding compounds . See, for example, the techniques described in G. Cesarini, FEBS Letters. 307(l):66-70 (July 1992); H.
• a method for identifying compounds which specifically bind to RC-9 DNA sequences can include simply the steps of contacting a selected RC-9 DNA fragment with a test compound to permit binding ofthe test compound to the DNA fragment; and determining die amount of test compound, if any, which is bound to the DNA fragment. Such a method may involve the incubation ofthe test compound and the RC-9 DNA fragment immobilized on a solid support.
• Another method of identifying compounds which specifically bind to RC-9 sequences can include the steps of contacting a RC-9 DNA fragment immobilized on a solid support with both a test compound and the protein sequence which is a receptor for RC-9 to permit binding ofthe receptor to die RC-9 DNA fragment; and determining the amount ofthe receptor which is bound to ⁇ ie DNA fragment. The inhibition of binding ofthe normal protein by the test compound thereby indicates binding ofthe test compound to the RC-9.
• RC-9-like effects of potential agonists, or the effects of potential antagonists may be measured, for instance, by deterriiining activity ofa reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of RC-9 or molecules that are found to elicit the same effects as RC-9.
• Reporter systems that may be useful in this regard include but are not limited to colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in RC-9 activity and binding assays known in the art. Other suitable methods are well known to those of skill in the art.
• the present invention provides compounds capable of interacting with RC-9 or portions thereof, and either enhancing or decreasing its biological activity, as desired.
• Such compounds are encompassed by this invention.
• the following examples which disclose the cloning and expression of RC-9 are for illustrative pu ⁇ oses only, and should not be construed as limiting this invention in any way.
• the catheter was guided a fixed distance down the common carotid artery to the aortic arch, inflated with a fixed volume of fluid, and withdrawn back to the site of insertion. This procedure was performed a total of three times. Once completed, the catheter was removed, and the wound was closed (9-mm Autoclips; Clay Adams, Franklin Lakes, NJ) and swabbed with Povadyne surgical scrub (7.5% Povidone-Iodine- Chaston Dayville, CT). Animals were housed in Plexiglass cages under a 12-hour light ⁇ dark cycle with access to standard laboratory chow and drinking water ad libitum until required for tissue collection.
• RNA isolation To isolate the carotid arteries, rats were exsanguinated via the vena cava under barbiturate anesthesia (100 mg/kg, i.p.). Left common carotid arteries were rapidly cleared of adherent tissue in situ, isolated, and placed directly in guanidine thiocyanate (Promega Company, Madison, WI). These vessels were then immediately flash frozen in liquid N 2 and stored at -80°C until required for RNA isolation. For subsequent Northern analysis, tissues were examined from naive animals (control) and from animals that had undergone angioplasty 6 hours, 3 days, and 14 days before to study die differential expression of transcripts in a more detailed, temporal fashion. Northern analysis was also performed on sham vessels.
• RNA isolation and Northern analysis For each time point studied, four or five left carotid arteries were pooled, or
• RNA from culture isolated and total RNA obtained as described [P. Chomczynski and N. Sacchi, Ann. Biochem.. 162:156-159 (1987)]. Equal amounts of RNA were loaded and separated on a 1.3% agarose/formaldehyde gel, transferred to nitrocellulose, and hybridized (0.25MNaCl, 1% sodium dodecyl sulfite, 50% formamide, 2X Denhardt's solution, 25mg denatured salmon sperm DNA, 5% dextran sulphate 42°C overnight) with the indicated probe.
• Relative intensities of hybridization signals were obtained by densiometric scanning (RFLP-Scan Software, Scanalytics, Inc.) of autoradiograms exposed widiin die linear range ofthe film (Kodak X-OMAT). Human and multiple tissue Northern blots were purchased from Clonetech, Inc. (Palo Alto, CA).
• DNA was removed from total RNA using a sequential procedure of DNA digestion, (1 U DNase; Gibco-BRL, Gaithersberg, MD), phenol/chloroform extraction, and ethanol precipitation according to the manufacturer's protocol.
• Purified total RNA (0.2 mg) was reverse transcribed using modified oligo dT primers (reverse transcriptase from GenHunter Co ⁇ oration, Brookline, MA) [P. Liang et al, Science. 257:967-971 (1992)].
• cDNA was amplified and labeled by including 10% of die reverse transcription reaction widi the appropriate 3' oligo dT primer, dNTPs, [ ⁇ 5 S]dATP, and an arbitrary 10-mer 5' primer (GenHunter). Forty PCR cycles were run widi die following parameters: 94°C for 30 seconds; 40°C for 120 seconds; 72°C for 45 seconds, widi die last cycle followed by a 72°C soak for 7 minutes. The labeled cDNAs obtained from this reaction were separated on a 6% acrylamide sequencing gel, which was dried and exposed to autoradiographic film 12 to 48 hours.
• day 3 vessels represent early molecular events in the pauiogenesis of lesion formation (i.e., those events occurring before die physical formation of a neointimal lesion), whereas day 14 vessels represent later events (i.e., when approximately 80% of die final lesion volume has formed in ie neointima) [S. A. Douglas et al, Eur. J. Pharmacol.. 255:81-89 (1994)].
• RNA from diese samples was reverse transcribed using all four oligo dT anchor primers, and die resulting cDNA was amplified using two different 5' decamers.
• Selected cDNAs were excised from die sequencing gel, eluted, and reamplified using d e same primers as previously described [Liang et al. , cited above] .
• the reamplified PCR product was visualized on 2.0% agarose gel, stained with ethidium bromide, and recovered by glass extraction (Bio 101, La Jolla, CA).
• Reamplified PCR products were labeled as probes as described above and were cloned into die pCRJJ vector using die Invitrogen TA cloning system (San Diego, CA), and DNA was sequenced on an applied biosystems model 373 A automated sequencing apparatus.
• the DNA segments were recovered from die acrylamide sequencing gel by excision and elution and PCR amplified using the same primers and reaction conditions. Reamplification yielded single products, die size of which are consistent widi weir apparent sizes displayed on die acrylamide sequencing gel (data not shown). These reamplification products were tiien glass-purified from die gel and used as probes for Northern analysis.
• RNA showed a strong signal of approximately 1.35 kb, slighdy larger dian die 1.1 kb observed in carotid arteries. A second, much weaker signal of approximately 1.1 kb was detected in spleen.
• this data indicates that die expression of dus transcript is tissue specific, and die differences in size from testes to spleen suggest processing of ti is mRNA transcript, or die presence of similar, messages in different tissues.
• RNA from naive rat carotid arteries and from carotid arteries isolated at tiiree timepoints after balloon angioplasty were probed widi tiiese DNA fragments in Northern analysis to confirm tiieir differential expression.
• Each of diese DNAs was found to represent a discrete gene transcript.
• Northern analysis verified dieir expression pattern.
• RC9 expression is undetectable in naive vessels, is detected at low levels 6 hours postsurgery, reaches rriaximal levels at day 3, and declines in day 14 vessels, cxmfirrning die pattern observed in die differential display gel. No expression of RC9 mRNA is detected in sham control vessels.
• cDNAs were subcloned into die pCRJJ vector and dideoxy sequenced in both directions.
• the RC9 clone is 424 bases (Fig. 1, SEQ JJ) NO: 1) and displays no significant sequence homology to any previously characterized genes deposited in Genbank.
• RC9 is a 424 bp partial genomic DNA fragment [SEQ ID NO:l] isolated from differential display analysis of rat carotid arteries post-balloon angioplasty, and identifies an mRNA of approximately 1.1 kb, as described in Example 1 above. To fully identify and characterize diis transcript, it was essential to obtain die full lengtii gene.
• a rat testes cDNA library (Stratagene, Inc.) was screened at high stringency (0.1X SSPE at 65°C) with the 424 bp RC9 [SEQ ID NO:l]. This probe was used to screen approximately 72,000 plaques, of which diree were positive after diree rounds of screening. These diree clones, each containing an insert of approximately 1.68 kb flanked by EcoRI sites as part ofthe vector polylinker were obtained and shown to be identical by restriction analysis.
• One clone was chosen and shown to be 98% homologous to RC9 by dideoxy nucleotide sequencing using an RC9 specific primer and also used to probe rat carotid arteries subject to balloon angioplasty to confirm the identity of this clone by mRNA expression.
• RNA from naive rat carotid arteries and from carotid arteries isolated at diree timepoints (1, 3 and 7 days) following balloon angioplasty were size fractionated and hybridized with a radiolabeled PCR product representing die insert of RC-9. More specifically, total RNA (10 mg) was separated on a 1.2% agarose/formaldehyde gel, transferred to nitrocellulose, hybridized, and washed as described above. A glyceraldehyde-3 -phosphate dehydrogenase (G3PDH) probe was used as a loading control.
• G3PDH glyceraldehyde-3 -phosphate dehydrogenase
• the clone was sequenced on both strands in its entirety (Sequenase, United States Biochemical Co ⁇ oration) as previously described. DNA and protein sequences were analyzed using die Mac Vector software package (International Biotechnologies, Inc.).
• the 3' region of die 1.68 kb insert from this clone was identified as 98% homologous with die 424 bp fragment obtained from differential display, further corifirming die identity of tiiis clone as the sequence of RC9.
• the nucleic acid clone RC-9-1 is shown in Figure 2, SEQ ID NO:2.
• Subsequent analysis of die sequences of Fig. 2 [SEQ ID NO:2] revealed die presence of polyC tracts at base pairs 864-873 and 1254-1276 and branch point consensus sequences at base pairs 851-857 and 1212-1218. This indicated tiiat the sequence encompassed intronic sequences. Further analysis identified two introns. Intron 1 spans base paris 121-772 of Fig. 2 and intron 2 spans based pairs 1000-1276.
• the sequences of Fig. 2 [SEQ ID NO:2] were used to obtain a RC-9 cDNA clone, as described in Example 3 below.
• SEQ ID NO:2 hybridized to number of restriction fragments from various species under low stringency washing conditions (2X SSC, 0.1% SDS, 50°C), die most prominent being a 24 kb fragment in botii rat and cow, and a 4.4 kb fragment in mouse.
• Otiier bands were detectable in dog, mouse, rat, and cow. No bands were visible in rabbit, chicken, or yeast.
• a number of less prominent bands were also observable at tiiis stringency, including a band approximately 2.3 and 1.0 kb in length visible in human, rat, and cow. A strong signal at 2.5 kb in mouse is also prominent.
• RC-9 primers were chosen from the extreme ends of die RC-9 genomic DNA (Fig. 2, SEQ ID NO:2). More particularly, the sense primer (25 mer ohgonucleotide) corresponds to nt 31- nt 55 of Fig. 2 [SEQ ED NO:2] and has a sequence of 5' - ACT TCA GAC TCT CTC TTC CCT ACG G - 3'; the anti-sense primer (28 mer ohgonucleotide) corresponds to nt 1651-1678 of Fig. 2 [SEQ ED NO:2] and has a sequence of
• PCR Polymerase chain reaction
• a partial cDNA sequence is provided in Fig. 3 [SEQ ID NO:3], togetiier w h die arnino acid sequence encoded tiiereby.
• the sequence of Fig. 3 is 696 basepairs in length [SEQ ID NO: 3].
• Example 4 Function of RC-9
• the RC-9 clone of Example 3 was cut out ofthe pCRII plasmid using die restriction endonuclease BstXl.
• the RC-9 DNA was purified and ligated into the expression vector pRc/CMV (Invitrogen Inc.) which had been pre-cut with BstXl and grown in E. coli. A single bacterial colony was chosen containing RC-9 in the correct orientation for propagation. Purified DNA was obtained with RC-9 in pRc/CMV.
• Rat aortic vascular smootii muscle (RAVSM) cells were transfected widi either pRc/CMV plasmid alone or with plasmid pRc/CMV containing RC-9. Transfection was done using LipofectAMJ-NE Reagent (Life Technologies Inc.) mixed widi die DNA to be transfected (pRc/CMV or pRc/CMV RC-9). The LipofectAMTNE Reagent allows the DNA to pass through die cell membrane, eventually permitting die DNA to be inco ⁇ orated into die host's chromosomes. Two days following transfection, the compound G418 (Geneticin) was added to the cells.
• G418 Geneticin
• G418 kills cells tiiat do not contain the pRc/CMV plasmid, leaving only cells containing pRc/CMV or pRc/CMV RC-9.
• Transfected RAVSM were grown in die presence of G418 for functional studies on RC-9.
• RAVSM containing eitiier pRc/CMV or pRc/CMV RC-9 were seeded onto T-150 flasks at equal densities and left to grow over a period of 5 or 7 days in DMEM + 10% FBS + G418. The cells were then trypsinized and counted using a hemocytometer to look for differences in proliferation. At 5 days, cell counts for pRc/CMV were 4.55 x 10 4 + 8.5 x 10 3 cells/cm 2 (in a T-150 flask @ 150 cm 2 ) and for pRc/CMV RC-9 1.22 x 10 5 ⁇ 5 x IO 2 cells/cm 2 ; a change of 168%.
• cell counts for pRc/CMV were 5.93 x 10 4 ⁇ 5.5 x IO 3 cells/cm 2 (in a T-150 flask @ 150 cm 2 ) and for pRc/CMV RC-9 1.84 x IO 5 ⁇ 3.7 x 10 4 cells cm 2 ; a change of 210%.
• the cell count data clearly shows tiiat RAVSM cells containing RC-9 proliferate at a higher rate tiian control cells.
• MOLECULE TYPE DNA (genomic)
• GGTGGATATA ACACGGTGGG ACCGAGGACC TTCGTGTCAG CAGCATGGGA AGAGAAGAAT 360
• AAAA 424 INFORMATION FOR SEQ ID NO:2:
• MOLECULE TYPE DNA (genomic)
• GTCTCACCCC ATTCTTGGAG CAGCCTGCAG ACTTCAGACT CTCTCTTCCC TACGGAGAAA 60
• GTAACCCTCC CAGCCCCCAC CCCAGGGTCT GGGGAAGAAG TCATTTTTGT GTTTCCTTGG 180
• CTCTCTTTCT ACCCAGCGAA GTACATGGAG TTTGATCTGA ATGGCAATGG AGATATCGAT 1320 ATTATGTCCT TGAAGCGAAT GCTGGAGAAA CTTGGGGTTC CCAAGACCCA TCTAGAGCTG 1380
• Lys Arg Ser Ala lie Leu Lys Met lie Leu Met Tyr Glu Glu Lys Ala 115 120 125

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