Identification of a 38-kDa Heparin-binding Glycoprotein (gp38k) in Differentiating Vascular Smooth Muscle Cells as a Member of a Group of Proteins Associated with Tissue Remodeling
1995; Elsevier BV; Volume: 270; Issue: 22 Linguagem: Inglês
10.1074/jbc.270.22.13076
ISSN1083-351X
AutoresLisa M. Shackelton, David Mann, Albert J.T. Millis,
Tópico(s)Galectins and Cancer Biology
ResumoCultured aortic smooth muscle cells (SMC) exhibit morphological and phenotypic modulation characterized by a change from a substrate attached monolayer culture to a multilayered nodular cell culture in which SMC are imbedded into the extracellular matrix. Associated with nodule formation is a change in the pattern of SMC gene expression including increased expression of a well characterized marker of smooth muscle cell differentiation, SM α-actin, and a 38-kDa glycoprotein (gp38k). gp38k has sequence homology with proteins reported to be correlated with tissue remodeling. To characterize the gp38k mRNA we designed degenerate oligonucleotides based on partial polypeptide sequencing to select a cDNA encoding the full-length gp38k. Southern analysis indicates that porcine gp38k is present as a single copy gene. Northern analysis indicates that the increase in gp38k is correlated with an increase in the steady state level of gp38k mRNA; and is present in cultures that have initiated the formation of multilayered foci and nodules. The correlation between SMC differentiation and gp38k expression is further established by using culture conditions that facilitate SMC differentiation. Cultures seeded onto reconstituted extracellular matrix show rapid formation of nodules and increased expression of gp38k mRNA. Comparison of the gp38k and cDNA sequences with nucleotide and protein sequences available through GenBank and SwissProt data banks revealed that molecules homologous to gp38k were present in human, mouse, bovine, and Drosophila tissues, suggesting that the gp38k may be a member of a gene family. Although a function for gp38k has not been identified, this report represents the first report of its correlation with a specific process important in phenotypic and morphological modulation of vascular SMC. Cultured aortic smooth muscle cells (SMC) exhibit morphological and phenotypic modulation characterized by a change from a substrate attached monolayer culture to a multilayered nodular cell culture in which SMC are imbedded into the extracellular matrix. Associated with nodule formation is a change in the pattern of SMC gene expression including increased expression of a well characterized marker of smooth muscle cell differentiation, SM α-actin, and a 38-kDa glycoprotein (gp38k). gp38k has sequence homology with proteins reported to be correlated with tissue remodeling. To characterize the gp38k mRNA we designed degenerate oligonucleotides based on partial polypeptide sequencing to select a cDNA encoding the full-length gp38k. Southern analysis indicates that porcine gp38k is present as a single copy gene. Northern analysis indicates that the increase in gp38k is correlated with an increase in the steady state level of gp38k mRNA; and is present in cultures that have initiated the formation of multilayered foci and nodules. The correlation between SMC differentiation and gp38k expression is further established by using culture conditions that facilitate SMC differentiation. Cultures seeded onto reconstituted extracellular matrix show rapid formation of nodules and increased expression of gp38k mRNA. Comparison of the gp38k and cDNA sequences with nucleotide and protein sequences available through GenBank and SwissProt data banks revealed that molecules homologous to gp38k were present in human, mouse, bovine, and Drosophila tissues, suggesting that the gp38k may be a member of a gene family. Although a function for gp38k has not been identified, this report represents the first report of its correlation with a specific process important in phenotypic and morphological modulation of vascular SMC. INTRODUCTIONThe principal function of aortic medial smooth muscle cells (SMC)1( 1The abbreviations used are: SMCsmooth muscle cellsSMsmooth musclegp38k38-kDa glycoproteinPCRpolymerase chain reactionbpbase pair(s)Pipes1,4-piperazinediethanesulfonic acid.) is to maintain the vessel wall tension and contractility (1Campbell G.R. Campbell J.H. Exp. Mol. Pathol. 1985; 42: 139-162Crossref PubMed Scopus (317) Google Scholar, 2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9929) Google Scholar). Medial SMC are embedded in an interstitial extracellular matrix and in response to endothelial injury migrate into the intima, proliferate, and synthesize components of the extracellular matrix (3Mosse P.R. Campbell G.R. Wang Z.L. Campbell J.H. Lab. Invest. 1985; 53: 556-562PubMed Google Scholar, 4Fingerle J. Johnson R. Clowes A.W. Majesky M.W. Reidy M.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8412-8416Crossref PubMed Scopus (347) Google Scholar, 5Dilley R.J. McGeachie J.K. Prendergast F.J. Atherosclerosis. 1987; 63: 99-107Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 6Schwartz S.M. Campbell G.R. Campbell J.H. Circ. Res. 1986; 58: 427-444Crossref PubMed Scopus (633) Google Scholar, 7Ross R. Klebanoff S.J. J. Cell Biol. 1971; 50: 159-171Crossref PubMed Scopus (235) Google Scholar). The phenotypic modulation of medial SMC from a quiescent differentiated phenotype to a migrating synthetic phenotype is considered to be a key event in the development of atherosclerotic plaques in response to injury (8Clowes A.W. Reidy M.A. Clowes M.M. Lab. Invest. 1983; 49: 327-333PubMed Google Scholar, 9Nobuyoshi M. Kimura T. Ohishi H. Horiuchi H. Nosaka H. Hamasaki N. Yokoi H. Kim K. J. Am. Coll. 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Chem. 1986; 261: 14740-14745Abstract Full Text PDF PubMed Google Scholar, 15Frid M.G. Shekhonin B.V. Koteliansky V.E. Glukhova M.A. Dev. Biol. 1992; 153: 185-193Crossref PubMed Scopus (247) Google Scholar, 16Birukov K.G. Frid M.G. Rogers J.D. Shirinsky V.P. Koteliansky V.E. Campbell J.H. Campbell G.R. Exp. Cell Res. 1993; 204: 46-53Crossref PubMed Scopus (52) Google Scholar, 17Glukhova M.A. Koteliansky V. Fondacci C. Marotte F. Rappaport L. Dev. Biol. 1993; 157: 437-447Crossref PubMed Scopus (99) Google Scholar, 18Shanahan C.M. Weissberg P.L. Metcalfe J.C. Circ. Res. 1993; 73: 193-204Crossref PubMed Scopus (320) Google Scholar, 19Holycross B.J. Blank R.S. Thompson M.M. Peach M.J. Owens G.K. Circ. Res. 1992; 71: 1525-1532Crossref PubMed Scopus (133) Google Scholar). In the appropriate environment, cultured SMC can express proteins that appear to be related to cell differentiation, including clusterin (20Diemer V.D. Hoyle M. Baglioni C. Millis A.J.T. J. Biol. Chem. 1992; 267: 5257-5264Abstract Full Text PDF PubMed Google Scholar, 21Thomas-Salgar S. Millis A.J.T. J. Biol. Chem. 1994; 269: 17879-17885Abstract Full Text PDF PubMed Google Scholar) and bone morphogenetic protein (22Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (896) Google Scholar).After subcultivation in serum containing medium, medial SMC initially form a confluent cell monolayer. Within the monolayer multicellular foci form which subsequently develop into morphologically and molecularly distinct nodules that contain differentiated non-proliferating SMC embedded in a carbohydrate-rich extracellular matrix (22Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (896) Google Scholar, 23Brennan M.J. Millis A.J.T. Fritz K.E. J. Cell. Physiol. 1982; 112: 284-290Crossref PubMed Scopus (17) Google Scholar, 24May J.F. Paule W.J. Rounds D.E. Blankenhorn D.H. Zemplenyi T. Virchows Arch. B. Cell Pathol. 1975; 18: 205-211PubMed Google Scholar). In contrast to representative cells in monolayer culture, cells in the nodule contain myofilaments and dense bodies. A comparison of the proteins synthesized and secreted by nodular SMC cultures with those secreted by monolayer SMC cultures revealed increased expression of two glycoproteins: clusterin (20Diemer V.D. Hoyle M. Baglioni C. Millis A.J.T. J. Biol. Chem. 1992; 267: 5257-5264Abstract Full Text PDF PubMed Google Scholar, 21Thomas-Salgar S. Millis A.J.T. J. Biol. Chem. 1994; 269: 17879-17885Abstract Full Text PDF PubMed Google Scholar, 25Rosenberg M.E. Dvergsten J. Correa-Rotter R. J. Lab. Clin. Med. 1993; 121: 205-214PubMed Google Scholar) and a heparin-binding glycoprotein of 38-kDa (gp38k) (26Millis A.J.T. Hoyle M. Reich E. Mann D.M. J. Biol. Chem. 1985; 260: 3754-3761Abstract Full Text PDF PubMed Google Scholar, 27Millis A.J.T. Hoyle M. Kent L. J. Cell. Physiol. 1986; 127: 366-372Crossref PubMed Scopus (32) Google Scholar). The factors regulating differential expression of those genes, and SMC modulation, in general, are not presently known. However, it is known that cultured SMC respond to soluble factors including cytokines, growth factors, and extracellular matrix macromolecules (28D'Amore P.A. Smith S.R. Growth Factors. 1993; 8: 61-75Crossref PubMed Scopus (146) Google Scholar, 29Brennan M.J. Millis A.J.T. Mann D. Fritz K.E. Dev. Biol. 1983; 97: 391-397Crossref PubMed Scopus (14) Google Scholar, 30Hedin U. Thyberg J. Differentiation. 1987; 33: 239-246Crossref PubMed Scopus (120) Google Scholar, 31Thyberg J. Palmberg L. Nilsson J. Ksiazek T. Sjoelund M. Differentiation. 1983; 25: 156-167Crossref PubMed Scopus (140) Google Scholar, 32Kunzelmann U. Dartsch P.C. Cell. Physiol. Biochem. 1992; 2: 49-56Crossref Google Scholar, 33Majack R.A. J. Cell Biol. 1987; 105: 465-471Crossref PubMed Scopus (177) Google Scholar, 34Owens G.K. Geisterfer A.A.T. Yang Y.W-H. Komlriya A. J. Cell Biol. 1988; 107: 771-780Crossref PubMed Scopus (234) Google Scholar, 35Pauly R.R. Passaniti A. Crow M. Kinsella J.L. Papadopoulous N. Monticone R. Lakatta E.G. Martin G.R. Circulation. 1992; 86: 68-73Google Scholar, 36Davidson J.M. Zoia O. Liu J-M. J. Cell. Physiol. 1993; 155: 149-156Crossref PubMed Scopus (99) Google Scholar). It is possible that SMC modulation is mediated by proteins secreted by SMC in response to unidentified signals (23Brennan M.J. Millis A.J.T. Fritz K.E. J. Cell. Physiol. 1982; 112: 284-290Crossref PubMed Scopus (17) Google Scholar, 29Brennan M.J. Millis A.J.T. Mann D. Fritz K.E. Dev. Biol. 1983; 97: 391-397Crossref PubMed Scopus (14) Google Scholar).In order to characterize the porcine SMC gp38k and begin to develop an understanding of its role in SMC modulation, we used degenerate oligonucleotides, based on polypeptide sequence information to isolate a cDNA encoding full-length gp38k from a nodular SMC cDNA library. The deduced amino acid sequence of the gp38k cDNA indicates that it encodes a protein composed of 383 amino acid residues, has a putative signal sequence, contains a single consensus sequence for N-linked glycosylation, and a heparin-binding consensus sequence. The deduced sequence reveals it to be a member of a family of related proteins whose expression is not limited to SMC. Homologues have been sequenced from human, bovine, murine, and Drosophila cDNAs and all of the family members show partial sequence homology to a group of proteins which cleave the invertebrate matrix polysaccharide chitin (37Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar, 38Chang, N. C. A., Liu, C. H., Chang, A. C., (1993) Accession #S27879, submitted to data bank June, 1993.Google Scholar, 39Sendai Y. Abe H. Kikuchi M. Satoh T. Hoshi H. Biol. Reprod. 1994; 50: 927-934Crossref PubMed Scopus (77) Google Scholar, 40Kirkpatrick R.B. Matico R.E. McNulty D.E. Strickler J.E. Rosenberg M. Gene (Amst.). 1995; 153: 147-154Crossref PubMed Scopus (71) Google Scholar).Expression of gp38k mRNA is correlated with multilayered SMC growth and nodule formation and the steady state level of the mRNA is increased during conditions that facilitate nodule formation. Addition of nodular cell culture conditioned medium or seeding SMC onto a preformed reconstituted extracellular matrix promote expression of gp38k and mRNA. Although the role of this protein in phenotypic modulation of SMC remains to be elucidated, this report provides strong support for the conclusion that gp38k is a marker of SMC differentiation by demonstrating that its expression parallels that of SM α-actin (18Shanahan C.M. Weissberg P.L. Metcalfe J.C. Circ. Res. 1993; 73: 193-204Crossref PubMed Scopus (320) Google Scholar, 19Holycross B.J. Blank R.S. Thompson M.M. Peach M.J. Owens G.K. Circ. Res. 1992; 71: 1525-1532Crossref PubMed Scopus (133) Google Scholar) and demonstrates for the first time that gp38k expression can be regulated by components of the extracellular matrix.EXPERIMENTAL PROCEDURESMaterialsMedium 199 was obtained from Life Technologies, Inc. (Grand Island, NY); fetal bovine sera from HyClone Laboratories, Inc. (Logan, UT); [32P]dCTP(3000 Ci/mmol) from Amersham, Inc.; Klenow fragment of DNA polymerase from U. S. Biochemical Corp. (Cleveland, OH); GeneScreen plus from Biotech System (Boston, MA); electrophoresis reagents from Bio-Rad; and TRI reagent from Molecular Research Center, Inc. (Cincinnati, OH).Cell CulturePorcine aortic smooth muscle cell cultures were initiated using explants of the luminal face of the thoracic aorta and cultivated in medium 199 supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and 3% CO2, 97% air as described previously (21Thomas-Salgar S. Millis A.J.T. J. Biol. Chem. 1994; 269: 17879-17885Abstract Full Text PDF PubMed Google Scholar, 23Brennan M.J. Millis A.J.T. Fritz K.E. J. Cell. Physiol. 1982; 112: 284-290Crossref PubMed Scopus (17) Google Scholar). Cells were routinely subcultivated by treatment of confluent monolayer cultures with EDTA and trypsin in isotonic saline. At each passage the cell number was determined using a Coulter counter (Coulter Electronics, Hialeah, FL) and the cultures replated at 2 × 104 cells/cm2 in fresh serum containing medium. Nodular cell cultures were generated by replacing the medium of confluent cultures with fresh medium 199 supplemented with 5% fetal bovine serum. Under these conditions, nodules begin to form after 5-9 days and well nodulated cultures (>70 nodules/cm2 of surface) are present within 2-3 weeks.Substrate PreparationFreshly harvested cells were mixed with equal volumes of soybean trypsin inhibitor, centrifuged for 5 min at 1000 rpm, and immediately suspended in M199 containing 10% serum. These cells were seeded at a density of 7 × 104cells/cm2 on various substrata. Reconstituted basement membrane gel, Matrigel (Collaborative Biomedical Products, Bedford, MA), was used after 1:1 dilution, on ice, in cold serum-free medium M199. Collagen gel-coated cluster dishes were also prepared on ice using a collagen I solution consisting of 8 parts rat tail collagen I (Collaborative Biomedical Products), 1 part 0.01 M NaOH, 1 part 10 × phosphate-buffered saline and the pH brought to 7.4 with 0.1 M NaOH. Each well of a 6-well cluster dish (Falcon, Becton Dickinson, Bedford, MA) was coated with 400 μl of either gel solution and allowed to gel for 30 min at 37°C before addition of SMC (21Thomas-Salgar S. Millis A.J.T. J. Biol. Chem. 1994; 269: 17879-17885Abstract Full Text PDF PubMed Google Scholar).Culture morphology was monitored using a Videoscope imaging camera attached to an Olympus CK2 inverted microscope fitted with a 2 × objective and the image processed with Metamorph imaging software (Universal Imaging Corp., West Chester, PA). The numbers of nodules are calculated by the software directly from the recorded image.Preparation of Conditioned MediaSerum-free conditioned media from monolayer and nodular smooth muscle cell cultures were prepared by washing cultures 3 × with Hank's balanced salt solution before incubation for 24 h in serum-free medium 199. The medium was collected into 1 mM phenylmethylsulfonyl fluoride and 1% aprotinin (Trasylol; Meloy Labs, NY), centrifuged for 15 min at 37,000 × g, and either used immediately or stored frozen (23Brennan M.J. Millis A.J.T. Fritz K.E. J. Cell. Physiol. 1982; 112: 284-290Crossref PubMed Scopus (17) Google Scholar, 26Millis A.J.T. Hoyle M. Reich E. Mann D.M. J. Biol. Chem. 1985; 260: 3754-3761Abstract Full Text PDF PubMed Google Scholar).Gel ElectrophoresisFor polyacrylamide gel electrophoresis the conditioned medium was precipitated with 15% trichloroacetic acid at 4°C for 1 h. Greater than 95% of the total protein was precipitated by this procedure (as estimated from Bio-Rad Protein assays) and all of the immunodetectable gp38k was precipitated (trichloroacetic acid-soluble fractions were concentrated in Amicon ultrafiltrations units and examined by immunoassay). Electrophoresis grade reagents were obtained from Bio-Rad.Polyacrylamide gel electrophoresis was performed using a 10% separating gel and a 5% stacking gel containing 1% SDS. Gels were prepared using the system of Laemmli (41Laemmli U. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206016) Google Scholar) and run at a constant current of 16 mA for 1.5-2 h. Molecular weight standards were myosin (200,000), phosphorylase b(97, 400), bovine serum albumin (69,000), carbonic anhydrase (30Hedin U. Thyberg J. Differentiation. 1987; 33: 239-246Crossref PubMed Scopus (120) Google Scholar, 0), trypsin inhibitor (21Thomas-Salgar S. Millis A.J.T. J. Biol. Chem. 1994; 269: 17879-17885Abstract Full Text PDF PubMed Google Scholar, 500), and lysozyme (14Rovner A.S. Murphy R.A. Owens G.K. J. Biol. Chem. 1986; 261: 14740-14745Abstract Full Text PDF PubMed Google Scholar, 300). Gel lanes were loaded with either equal volumes of conditioned medium or equal amounts of protein (Bio-Rad protein assay).Antibody Preparations and ImmunoblottingPolyclonal antibodies to rat clusterin (Sertoli cell clusterin) were generously provided by Drs. Michael Griswold and Steven Sylvester, Washington State University (42Collard M.W. Griswold M.D. Biochemistry. 1987; 26: 3297-3303Crossref PubMed Scopus (271) Google Scholar). Polyclonal antibodies to gp38k were prepared in this laboratory as described previously (27Millis A.J.T. Hoyle M. Kent L. J. Cell. Physiol. 1986; 127: 366-372Crossref PubMed Scopus (32) Google Scholar). Antibodies to gp38k were affinity purified by adsorption to purified gp38k fixed to nitrocellulose membrane (43Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, San Diego, CA1989: 1-129Google Scholar, 44Hunkapillar M.W. Lujan E. Ostrander F. Hood L.E. Methods Enzymol. 1983; 91: 227-236Crossref PubMed Scopus (683) Google Scholar). Proteins were separated on minigels and transferred from the gel onto nitrocellulose paper (Schleicher & Schuell) in 192 mM glycine, 20% methanol, 25 mM Tris-HCl, pH 8.3, using a Genie electroblotter (Idea Scientific). Following transfer at 25 V for 1 h the blot was incubated in a blocking agent consisting of 5% nonfat dried milk (Carnation, Los Angeles, CA). After incubation with primary antibody (1:2000 dilution of anti-SGP2 or anti-38K protein in rabbit sera) for 2 h at room temperature on a rocker, the membrane was washed and incubated with horseradish peroxidase-conjugated donkey anti-rabbit whole antibody (1:10,000 dilution; Amersham) for 60 min at room temperature. After washing, immobilized antigens were detected using an enhanced chemiluminescence assay (ECL: Amersham). Following immunotransfer, each gel was stained with Coomassie Blue to evaluate the efficiency of transfer.Purification and Amino-terminal Sequencing of gp38kgp38k was purified from nodular SMC conditioned medium by heparin affinity chromatography (Pharmacia, Inc., Pitscataway, NJ), followed by high performance liquid chromatography. Samples were purified using conditions to minimize amino-terminal blocking and further purified via 5-20% linear gradients of polyacrylamide (44Hunkapillar M.W. Lujan E. Ostrander F. Hood L.E. Methods Enzymol. 1983; 91: 227-236Crossref PubMed Scopus (683) Google Scholar). The gel bands were visualized by brief staining with Ponceau S. Amino-terminal sequencing was performed on protein eluted from gel band slices by soaking overnight at 37°C in 0.5 ml of 100 mM Tris, 0.1% SDS, pH 8.0, or by electroeluting in 0.1% SDS, 0.05 M NH4HCO3. This material was loaded onto the hydrophobic portion of a biphasic Hewlett-Packard sequencer column and washed with 0.2% trifluoroacetic acid, and 10% acetonitrile to remove the Tris and glycine. Sequencing was performed using automated Edman degradation chemistry on a Hewlett-Packard G1000A Protein Sequencing system.To obtain internal sequences, samples were concentrated in 50 mM NH4HCO3, using an ISCO electroelution protein concentrator. Each sample was diluted with 5 volumes of 50 mM NH4HCO3 and subsequently precipitated with 8 volumes of acetone at −20°C. The pellet was then washed 3 times with 95% ethanol at −20°C. The pellet was resuspended in 30 μl of Lys-C digestion buffer composed of 25 mM Tris-HCl, pH 8.5, 0.01% SDS, 1 mM EDTA and allowed to dissolve overnight at 4°C. This preparation was digested for 24 h with 5 μg of Lys-C (Boehringer Mannheim, Indianapolis, IN) and fractionated on a C18 reverse phase column (Brownlee RP300) prior to sequencing.From this analysis we obtained a single amino-terminal sequence and 5 internal sequences. Degenerate oligonucleotides were synthesized based on conserved regions in gp38k and mammalian homologues and porcine codon usage (47Wada K-N. Aota S-i. Tsuchiya R. Ishibashi F. Gojobori T. Ikemura T. Nucleic Acids Res. 1990; 18: 2367-2411Crossref PubMed Scopus (138) Google Scholar). Degeneracy was reduced by substituting inosine at the third position of the triplet coding for leucine in the sense primer. To allow directional cloning, the sense primer was designed with a BamHI adaptor at the 5′ end followed by TTYGAYGGNYTIGAYYT-3 and the antisense primer with an EcoRI adaptor at the 5′ end followed by TCRTCRTANCCNACCCA-3′. The primers were synthesized and trityl-specific purified by Ransom Hill Bioscience (Ramona, CA). Amplification of the region between these two primers included nucleotide sequences encoding three of the polypeptide sequences that we obtained by direct amino acid sequencing of purified gp38k.Selection of Clone Expressing gp38k cDNA (pBS38K)To determine the cDNA sequence of gp38k, a strategy employing reverse transcription-PCR and cDNA library screening was used.Reverse TranscriptionRNA isolated from cultured porcine aorta nodular smooth muscle cells was reverse transcribed as follows: 2-4 μg of RNA was brought up to a final volume of 16.5 μl and incubated at 65°C for 5-7 min. After heating, the RNA was quenched on ice for 2 min and centrifuged briefly. A reverse transcription reaction mixture (final volume of 13.5 μl) containing 250 mM Tris-Cl, pH 8.3, 50 mM MgCl2, 300 mM KCl, 50 mM dithiothreitol, 50 mg/ml oligo(dT) (Pharmacia, Upsala, Sweden), 0.5 mM dNTP mixture (U. S. Biochemical Corp.), 1 mg/ml bovine serum albumin, 667 units/ml RNasin (Promega, Madison, WI), and 900 units/ml AMV Reverse Transcriptase (Molecular Genetic Resources, Tampa, FL) was added to the tube containing RNA and the reaction was incubated at 42°C for 1 h. After incubation, 20 μl of double distilled H2O was added to the resulting cDNA before using in subsequent PCR reactions.Polymerase Chain ReactionPolymerase chain reaction was performed in 50-μl samples containing 500 mM KCl, 100 mM Tris-Cl, pH 8.3, 25 mM MgCl2, 200 μM dNTP mixture, 4 mM each of the sense and antisense degenerate primers, 80 units/ml Taq DNA polymerase (Perkin Elmer), and 10 μl of cDNA reverse transcribed under the conditions listed above. Each 50-μl reaction was overlaid with 1 drop of DNase-free, RNase-free mineral oil (Sigma), denatured for 5 min at 94°C, and cycled 30 times using the following parameters; 60 s at 94°C, 90 s at 48°C, and 60 s at 72°C in an Eppendorf Microcycler E (Eppendorf, Fremont, CA)Subcloning and Sequencing of PCR ProductEcoRI and BamHI (40 units) were added to the reaction mixture containing the PCR product and incubated at 37°C for 2.5 h. After electrophoresis in 1% agarose and treatment with ethidium bromide, an amplification product of approximately 600 bp was observed and excised from the gel. The excised DNA was purified and then ligated between the EcoRI and BamHI sites of the polylinker region of pBluescript SK- (Stratagene, La Jolla, CA). Xl-1 Blue bacteria (Stratagene) were transformed with pBluescript containing the insert and incubated overnight at 37°C on LB plates containing ampicillin.White colonies were picked and grown overnight at 37°C in LB medium containing 50 μg/ml ampicillin. Minipreps were performed to isolate plasmid DNA, and the DNA was displayed on a 1% agarose gel next to 2 μg of pBluescript SK- without an insert. The insert was partially sequenced using the chain termination method (45Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Prototcols in Molecular Biology. Vols. 1 and 2. John Wiley and Sons, New York1987-1994Google Scholar) with [35S]dATP (Amersham) and a Sequenase 2.0 DNA Sequencing Kit (U. S. Biochemical Corp.) and double stranded plasmid DNA that was prepared by the alkaline lysis procedure (45Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Prototcols in Molecular Biology. Vols. 1 and 2. John Wiley and Sons, New York1987-1994Google Scholar).cDNA Library ScreeningA Uni-Zap XR cDNA library was prepared using poly(A)+ RNA isolated from nodular SMC (20Diemer V.D. Hoyle M. Baglioni C. Millis A.J.T. J. Biol. Chem. 1992; 267: 5257-5264Abstract Full Text PDF PubMed Google Scholar). XL-1 Blue Escherichia coli (Stratagene) cells were infected with 104 plaque forming units of the Uni-Zap cDNA library and plated onto 100 × 15-mm LB Agar plates. The plates were incubated at 37°C for 8 h.Each plaque lift was transferred onto nitrocellulose (Schleicher & Schuell, Inc.) and denatured by submerging in 1.5 M NaCl, 0.5 M NaOH for 2 min. After neutralization, the DNA was cross-linked to the filters for 30 s using a Stratalinker UV cross-linker (Stratagene).Filters were prehybridized at 42°C for 2 h in a solution containing 50% deionized formamide, 0.5% SDS, 2 × Pipes buffer, pH 6.5, and 100 mg/ml denatured sonicated salmon sperm DNA. A cDNA probe was prepared using the 600-bp EcoRI/BamHI restriction fragment of pBS600 excised from a low melting point agarose gel. The probe was labeled with [32P]dCTP (Amersham) using a MultiPrime DNA Labeling System (Amersham). Hybridization was accomplished using 1 × 107 counts per filter at 42°C for 16 h. To identify positive clones, filters were washed, and then exposed to Kodak X-Omat film overnight at −70°C in a cassette including an intensifying screen. Positive clones were isolated, replated, and prescreened until single isolates were obtained.In Vivo Excision of Potential ClonesClonal isolates were excised in vivo, from the Uni-Zap vector following the procedures described by the manufacturer and used to transform XL-1 Blue bacteria and plated onto LB plates containing 50 μg/ml ampicillin coated with isopropyl-1-thio-β-D-galactopyranoside and 5-bromo-4-chloro-3-indoyl β-D-galactoside. Plates were grown overnight at 37°C. Single white colonies were picked and grown overnight at 37°C in LB broth containing 50 μg/ml ampicillin.DNA sequencing utilized new primers after approximately every 200 nucleotides. During the course of sequencing, the BLAST e-mail server at the National Center for Biotechnology Information was used to search for homologous sequences in various data bases (48Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69072) Google Scholar). In addition, the PC GENE package (Intelligenetics, Mountain View, CA) and FASTA were used to search for homologies against various data bases.Southern BlottingGenomic DNA was isolated from nodular porcine SMC using TRI Reagent, according to the manufacturer's instructions. Genomic DNA (12 μg) was digested with 40 units of BamHI, EcoRI, or HindIII for 20 h at 37°C. Each sample was loaded onto a 0.7% agarose gel containing 1 mg/ml EtBr and run at 60 V until the markers (1 kilobase pair molecular weight ladder; Life Technologies, Inc., Grand Island, NY) were well separated (approximately 5 h).After denaturation in 0.4 M NaOH containing 0.6 M NaCl the gel was incubated twice for 15 min in a neutralization solution (0.5 M Tris-HCl, pH 7.8, 1.5 M NaCl). Size fractionated DNA fragments were transferred onto nylon membrane (GeneScreen, DuPont) using downward capillary transfer for 16 h (49Chomczynski P. Anal. Biochem. 1992; 201: 134-139Crossref PubMed Scopus (493) Google Scholar). The membrane was then washed in neutralization solution and the DNA cross-linked to the membrane using a Stratalinker (Strategene, La Jolla, CA).The membrane was prehybridized in buffer containing 5 × SSC, 5× Denhardt's solution, 1% SDS, and 100 μg/ml denatured salmon sperm DNA at 65°C for 4 h. A 600-bp cDNA (excised from pBS600) was random primer labeled with [32P]dCTP and added to the prehybridization solution at an activity of 1.2 × 106 counts/ml. Hybridization occurred at 65°C for 20 h. Subsequently the membrane was washed 4 tim
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