Molecular Cloning, Sequence Analysis, Expression, and Tissue Distribution of Suppressin, a Novel Suppressor of Cell Cycle Entry

Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990)J. Biol. Chem. 265, 158–165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5′- and 3′-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at ∼2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator inDrosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J.15, 1961–1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types. Suppressin (SPN) is an inhibitor of cell proliferation that was originally identified and purified to homogeneity from bovine pituitaries (LeBoeuf, R. D., Burns, J. N., Bost, K. L., and Blalock, J. E. (1990)J. Biol. Chem. 265, 158–165). In this report we have cloned the full-length cDNA encoding rat SPN and have identified the tissue distribution of SPN expression. The cDNA of SPN is 1882 nucleotides with a 1488-base coding region and 55 and 339 nucleotides of 5′- and 3′-untranslated sequences, respectively. Northern gel analysis of rat pituitary mRNA showed a single hybridizing species at ∼2 kilobases. Sequence analyses showed that the nucleotide and deduced amino acid sequences of SPN are novel and unrelated to any known vertebrate inhibitors of proliferation. However, the deduced amino acid sequence of SPN contains two domains that have extensive sequence identity with a recently cloned transcription activator inDrosophila, deformed epidermal autoregulatory factor-1 (DEAF-1, see Gross, C. T., and McGinnis, W. (1996) EMBO J.15, 1961–1970) suggesting that SPN represents a vertebrate cognate of deformed epidermal autoregulatory factor-1. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses showed that the SPN mRNA and the SPN protein are expressed in every tissue examined including testis, spleen, skeletal muscle, liver, kidney, heart, and brain suggesting that SPN may be involved in the control of proliferation in a variety of cell types. Suppressin (SPN) 1The abbreviations used are: SPN, suppressin; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; mAb, monoclonal antibody; Ab, antibody; RACE, 5′-rapid amplification of cDNA ends; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; FGF-1, fibroblast growth factor-1; EST, expression sequence-tagged; DEAF-1, deformed epidermal autoregulatory factor-1; PBMC, peripheral blood mononuclear cells; PNGase F, peptide-N 4-(N-acetyl-β-glucosaminyl)-asparagine amidase. 1The abbreviations used are: SPN, suppressin; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; mAb, monoclonal antibody; Ab, antibody; RACE, 5′-rapid amplification of cDNA ends; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; FGF-1, fibroblast growth factor-1; EST, expression sequence-tagged; DEAF-1, deformed epidermal autoregulatory factor-1; PBMC, peripheral blood mononuclear cells; PNGase F, peptide-N 4-(N-acetyl-β-glucosaminyl)-asparagine amidase. is a 63-kDa monomeric protein identified and purified to homogeneity from the bovine pituitary based on its ability to inhibit mitogen-stimulated murine splenocyte proliferation (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar). Initial immunologic and species-specificity studies indicated that SPN was a structurally conserved molecule. Specifically, (i) anti-SPN antibodies prepared against purified bovine SPN cross-reacted with human, mouse, and rat SPN (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar, 2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar, 3Carr D.J. Blalock J.E. Green M.M. LeBoeuf R.D. J. Neuroimmunol. 1990; 30: 179-187Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 4LeBoeuf R.D. Blalock J.E. Ann. N. Y. Acad. Sci. 1990; 594: 393-395Crossref Scopus (4) Google Scholar), and (ii) purified bovine SPN was active on mouse, rat, and human cells. Other cross-species activities could also be shown for SPN (e.g. human SPN was active on rat cells). In the rat pituitary, SPN production is restricted to five hormone-secreting cell phenotypes (somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and mammosomatotrophs) in the anterior pituitary (2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar). The primary biological activity, inhibition of cell proliferation, has been most extensively studied in vitro in murine and human lymphocytes (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar, 3Carr D.J. Blalock J.E. Green M.M. LeBoeuf R.D. J. Neuroimmunol. 1990; 30: 179-187Abstract Full Text PDF PubMed Scopus (11) Google Scholar). The inhibition of cell proliferation by SPN does not occur by either a cytotoxic mechanism or by increasing the rate of apoptosis (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar). The results of cell cycle analyses on SPN-treated lymphocytes have shown that SPN arrests cells in G0 or early G1 stages of the cell cycle (3Carr D.J. Blalock J.E. Green M.M. LeBoeuf R.D. J. Neuroimmunol. 1990; 30: 179-187Abstract Full Text PDF PubMed Scopus (11) Google Scholar). Suppressin also inhibits the proliferation of tumor cells. The addition of exogenous SPN to cultures of leukemia, lymphoma, and thymoma cells and tumor cells from brain, adrenal, breast, and pituitary resulted in markedly reduced proliferation (4LeBoeuf R.D. Blalock J.E. Ann. N. Y. Acad. Sci. 1990; 594: 393-395Crossref Scopus (4) Google Scholar). The results of metabolic labeling have shown that SPN is synthesized and secreted as an active molecule by human and mouse lymphocytes and GH3 cells (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar, 3Carr D.J. Blalock J.E. Green M.M. LeBoeuf R.D. J. Neuroimmunol. 1990; 30: 179-187Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 5LeBoeuf R.D. Carr D.J.J. Green M.M. Blalock J.E. Prog. Neuroendocrinimmunol. 1990; 3: 176-187Google Scholar). Moreover, neutralization of secreted SPN in culture supernatants by anti-SPN antibodies (Ab) increases proliferation in the absence of exogenous growth factors (6Ban E.M. LeBoeuf R.D. Immunol. Res. 1995; 13: 1-9Crossref Scopus (10) Google Scholar) showing that SPN acts as an autocrine/paracrine inhibitor of entry into the cell cycle. Collectively, the results of our studies show that SPN is a fundamental component of a regulatory circuit that functions to maintain cells in a nondividing state. To understand the structure, function, and regulation of SPN, we cloned, sequenced and characterized the full-length SPNcDNA from the rat pituitary. The molecular cloning of SPN was accomplished by immunoscreening a pituitary cDNA library with an anti-SPN Ab, by DNA hybridization screening of cDNA libraries with a partial SPN cDNA, and by polymerase chain reaction (PCR) and 5′-rapid amplification of cDNA ends (RACE) using rat pituitary mRNA. The results of sequence analyses and comparisons showed that SPN is a novel vertebrate regulatory molecule. However, SPN is highly homologous to DEAF-1, a recently cloned molecule fromDrosophila (7Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (126) Google Scholar). In addition, we provide results from studies on the tissue distribution of SPN expression, on structural characteristics of SPN, and on the expression of recombinant SPN. Whole pituitaries were surgically removed from male Sprague-Dawley rats (Harlan) as described previously (2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar), and total RNA was isolated by the guanidinium thiocyanate/cesium chloride method (8Chirgwin J. Przybyla R. MacDonald R. Rutter W. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16608) Google Scholar). Poly(A)+ mRNA was obtained by two rounds of chromatography over oligo(dT)-cellulose type 7 (Pharmacia Biotech Inc.) as described previously (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Total RNA (10–20 μg) or poly(A+) mRNA (1–5 μg) was denatured in 50% (v/v) formamide and 2.2 m formaldehyde and resolved by agarose-formaldehyde gel electrophoresis, and the gel was transferred to Biotrans (ICN, Irvine, CA) nylon membranes by capillary diffusion in 10 × SSC using standard procedures (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Membranes were baked for 2 h at 80 °C in a vacuum oven and prehybridized and hybridized in the following buffer (0.5 m NaHPO4, pH 7.2, 1 mm EDTA, 7% SDS, and 0.1% (w/v) bovine serum albumin).32P-Labeled SPN cDNA probes were prepared from gel-purified cDNAs using a Nick Translation System (Promega, Madison, WI) and [α-32P]dCTP (3000 Ci/mmol, DuPont). For hybridization, radiolabeled SPN cDNA probes were added at 2 × 106 cpm/ml. Pre-hybridization (4–6 h) and hybridization (10–12 h) were done at 42 °C in 50% formamide in 5 × SSC, and membranes were washed in 2 × SSC, 0.1% SDS at 50 °C followed by two washes in 0.2 × SSC, 0.1% SDS at 50 °C. The membrane was then exposed to Kodak XAR film at −70 °C for 10–20 h. Slot blot analyses were performed with total rat RNA, and the membranes were prehybridized, hybridized, and washed as described above except the temperature for these procedures was 45 °C. Two oligodeoxynucleotides were used as probes as follows: one that was complementary to the cDNA sequence containing the largest open reading frame (5′-TGATGGCTTCTCAGTAGAG-3′), and the complement to this oligodeoxynucleotide that is homologous to the cDNA sequence containing the open reading frame. Both oligodeoxynucleotides were labeled with 32P using T4 polynucleotide kinase by standard procedures (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). An oligo(dT) adaptor-primed unidirectional rat pituitary cDNA library was constructed from rat pituitary poly(A)+ mRNA using a Uni-Zap XR/Gigapack Cloning system (Stratagene, La Jolla, CA). This cDNA library was immunoscreened (1 × 106individual plaque-forming units) using a previously characterized (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar) monospecific polyclonal anti-SPN Ab and Ab-positive clonal plaques detected using a BioStain Super ABC alkaline-phosphatase immunodetection kit (Biomeda, Foster City, CA). After tertiary replating, the Ab-positive clonal plaques were analyzed for clonality and cDNA insert size by PCR with vector-specific primers that flank the XhoI/EcoRI cloning site in the Uni-Zap XR vector as described previously (10LeBoeuf R.D. Green M.M. Berecek K.H. Swords B.H. Blalock J.E. Neth. J. Med. 1991; 39: 295-305PubMed Google Scholar). The largest cDNA insert (691 bp) was direct-sequenced and then used as a probe to rescreen the rat pituitary Uni-Zap XR cDNA library. After tertiary replating, one larger clone was obtained by nucleic acid hybridization screening, with an insert of 924 bp corresponding exactly to the SPN 691-bp cDNA sequence but extended at its 5′ end. The SPN 924-bp cDNA was subcloned in M13mp18 and mp19 by standard methods (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and the sequence of this cDNA was determined in both orientations. DNA sequencing was performed using the Sanger dideoxy sequencing method with Sequenase (U. S. Biochemical Corp.). Sequence analyses were performed with the Genepro program (Riverside Scientific, Bainbridge Island, WA). Homology searches and protein structure analyses were performed using the GCG programs (Genetics Computer Group, Madison, WI) against all available public sequence data bases. The partial SPN cDNA sequence (924 bp) was used to designSPN-specific reverse transcriptase (RT) and PCR primers for use in a 5′-RACE system (Life Technologies Inc.) to obtain the full-length cDNA sequence of SPN. Briefly, an antisense SPN-specific primer (SSP-1) that was approximately 100 bp downstream from the 5′ end of the 924-bp SPN cDNA sequence was used in a reverse transcription reaction (RT) with one μg of rat pituitary poly(A)+ mRNA. An oligo(dC) anchor sequence was added to the 3′ end of RT reaction products with terminal deoxynucleotidyltransferase. The oligo(dC)-tailed cDNA was amplified by PCR using a nested antisense SPN-specific primer (SSP-2) that was approximately 50 bp upstream of SSP-1 and the anchor primer provided in the 5′-RACE system. PCR was performed for 50–60 cycles with AmpliTaq DNA polymerase under standard reaction conditions (10LeBoeuf R.D. Green M.M. Berecek K.H. Swords B.H. Blalock J.E. Neth. J. Med. 1991; 39: 295-305PubMed Google Scholar). Reaction products were analyzed by agarose gel electrophoresis and selected products were excised from the gel and purified using a Qiaex II gel extraction kit (Qiagen, Chatsworth, CA). The purified cDNA was cloned in the pGEM-T vector (Promega, Madison, WI), and both strands of the cDNA insert were completely sequenced. Four iterations of this procedure were required to obtain the complete cDNA sequence of the SPN transcript. Once the complete SPN cDNA sequence was obtained, a 5′ sense SPN primer (5′-CGGGATCCCATGGCGGCCGAATCTG-3′) which contained aBamHI site and a 3′ antisense SPN primer (5′-TGATGGCTTCTCAGTAGAG-3′) which contained a Bpu1102I site were synthesized and used in PCR with oligo(dT)-primed cDNA synthesized from rat pituitary poly(A)+ mRNA as template. For these reactions the thermostable proofreading DNA polymerase Pfu (Stratagene, La Jolla, CA) was used in PCR. This reaction yielded a 1583-bp SPN cDNA that contained the complete coding sequence beginning with the first in-frame start codon and 40 bp of 3′-untranslated region. This cDNA was cloned in the pGEM-T vector, and both strands from three independent clones were sequenced. The 120-amino acid peptide deduced from the 691-bp cDNA was analyzed for highly antigenic regions using the Hoop and Woods hydropathy index to predict antigenic regions within this peptide. One such region, a nonapeptide NH2-QRKVWKDHQ-COOH, was synthesized, purified by high pressure liquid chromatography, covalently coupled to keyhole limpet hemocyanin as a carrier immunogen (2.5 mg of peptide + 2.5 mg of keyhole limpet hemocyanin) using a standard glutaraldehyde coupling protocol (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), and then used to immunize a New Zealand White rabbit (500 μg/injection). The rabbit received booster injections at 10-day intervals. The presence of anti-peptide antibodies was assayed by enzyme-linked immunosorbent assay using the peptide covalently linked to a 96-well COBIND microtiter plate and an alkaline phosphatase-conjugated anti-rabbit IgG as the secondary Ab. Thirty days after immunization, serum was obtained from the rabbit, and the Ig fraction of the sera was purified by protein G-Sepharose affinity chromatography (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To determine if the Abs in the postimmune Ig fraction would specifically recognize SPN, homogeneous pituitary SPN was covalently attached to COBIND microtiter wells and assayed for specific binding by the Abs in the Ig fraction using an enzyme-linked immunosorbent assay. BamHI and Bpu1102I endonuclease restriction sites flanking the coding region of the SPN cDNA were constructed by PCR using the 1583-bp SPN cDNA in pGEM-T as template and Pfu polymerase. The reaction product was sequentially digested with BamHI and Bpu1102I, gel purified, and ligated in-frame in the BamHI/Bpu1102I site of expression vector pET-15b (Novagen, Madison, WI). The resulting fusion protein contained six consecutive histidine residues (His-Tag) on its amino terminus. The orientation and sequence of the SPN cDNA in the pET plasmid were confirmed by DNA sequencing. The SPN cDNA was expressed in the E. coli strain BL21 (DE53, pLysS) by growing the bacterial cells at 30 °C and following an induction protocol previously described (11Popov K.M. Kedishvili N.Y. Zhao Y. Shimomura Y. Crabb D.W. Harris R.A. J. Biol. Chem. 1992; 268: 26602-26606Abstract Full Text PDF Google Scholar); the recombinant protein was purified by metal chelation chromatography (12Hoffman A. Roeder R.G. Nucleic Acids Res. 1991; 19: 6337-6338Crossref PubMed Scopus (249) Google Scholar). Poly(A)+mRNA from several rat tissues were obtained commercially (CLONTECH, Palo Alto, CA). mRNA (250 ng) from each tissue was used as template in an oligo(dT)-primed first-strand cDNA synthesis with Superscript RT (Life Technologies, Inc.) using the protocol provided by the manufacturer. Each reaction was treated with DNase I before the addition of RT and cDNA synthesis using a standard protocol (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A replicate first-strand cDNA reaction in which RT was not added was performed for each tissue as a control for the presence of genomic DNA. One-tenth of the first-strand cDNA synthesis reaction was used as template with a sense SPN primer (5′-TGGAGATGTCAGAGCATCG-3′) and an antisense SPN primer (5′-TGATGGCTTCTCAGTAGAG-3′) that will amplify a 402-bp target sequence of the SPN cDNA. The products from each set (with and without RT) of PCR reactions were analyzed by agarose gel electrophoresis. Restriction analysis was performed on part of each reaction and yielded the expected restriction fragments for the SPN target sequence (data not shown). Rat tissues were collected and fixed in 5% glacial acetic acid in 95% ethanol at −20 °C for 24 h before embedding in paraffin. Serial sections 4 μm thick were cut and stained on glass slides. Staining for SPN expression was performed using an anti-SPN monoclonal antibody (mAb) (3F10; Ref. 2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar). The specificity of this anti-SPN mAb has been previously demonstrated on intracellular SPN in rat pituitary cells (2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar). 3F10 binding to intracellular SPN from rat pituitary cells is specifically blocked by preincubation of the mAb with pure native SPN (2Ban E.M. Propst S.M. Blalock J.E. LeBoeuf R.D. Endocrinology. 1993; 133: 241-247Crossref PubMed Scopus (5) Google Scholar). Sections were stained in PBS containing 10% goat serum/Tween 0.05% and 2 μg/ml of anti-SPN mAb. The levels of background staining (negative control) were obtained by incubating the sections with an irrelevant isotype-matched (IgM) mAb (UAB mAb Core Facility). After incubation with the mAb (1.5 h at room temperature), the slides were washed three times in PBS and incubated with PBS/Tween 0.05% containing a biotinylated rabbit anti-mouse IgM (μ-chain specific) antibody (5 μg/ml) (Pharmingen, San Diego, CA). Slides were washed three times in PBS and incubated with streptavidin coupled to fluorescein isothiocyanate at the concentration of 1 μg/ml in PBS/Tween 0.05% for 30 min at room temperature. After three washes in PBS, the slides were mounted in ethanol and photographed using a Leitz Diaplan microscope (Leitz, Wetzlar, Germany). GH3 cells (ATCC, Rockville, MD) were cultured in RPMI, 10% horse serum. PBMC were cultured in RPMI, 5% fetal calf serum for 24 h. Supernatants were collected and concentrated 40 times using CENTRIPREP 30 (Amicon, Beverly, MA). Cell extracts were prepared from 108 cells with standard procedures (7Gross C.T. McGinnis W. EMBO J. 1996; 15: 1961-1970Crossref PubMed Scopus (126) Google Scholar), and proteins were separated by electrophoresis using a 4–20% continuous gradient Tris-HCl acryl/bisacryl gel (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Bio-Rad) by electroblotting under 30 mV for 16 h at 4 °C. Membranes were blocked in Tris borate saline (TBS), 3% casein, 10% goat serum, washed, and then incubated with the 3F10 mAb (2.5 μg/ml) for 1.5 h at room temperature. After three washes, membranes were incubated with a biotinylated goat antibody against mouse immunoglobulin (H + L) (0.2 μg/ml), washed again 3 times and then incubated with avidin conjugated to horse peroxidase (1 μg/ml). Membranes were washed before a 60-s incubation with the enhanced chemiluminescent substrate (Amersham Corp.) and exposed for 5 min to autoradiographic film. Two hundred ng of affinity purified native rat SPN was analyzed for the presence of Asn-linked oligosaccharides by digestion with the glycosidase, PNGase F, according to the protocol provided by the manufacturer (Glyko Inc., Novato, CA). A parallel reaction in which fetuin was digested with PNGase F served as a positive control for enzyme activity. PNGase F and control reactions (without PNGase F) were performed for SPN and the control glycoprotein, fetuin, and the reaction products were analyzed under reducing conditions on 10% SDS-PAGE (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205511) Google Scholar), and protein bands were stained with silver (14Merril C.R. Goldman D. Sedman S.A. Ebert M.A. Science. 1981; 211: 1437-1439Crossref PubMed Scopus (2085) Google Scholar). A polyclonal anti-SPN antibody (Ab) prepared against purified bovine SPN (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar) was used to screen a rat pituitary cDNA library. This Ab cross-reacts with bovine, human, mouse, and rat SPN (1LeBoeuf R.D. Burns J.N. Bost K.L. Blalock J.E. J. Biol. Chem. 1990; 265: 158-165Abstract Full Text PDF PubMed Google Scholar, 3Carr D.J. Blalock J.E. Green M.M. LeBoeuf R.D. J. Neuroimmunol. 1990; 30: 179-187Abstract Full Text PDF PubMed Scopus (11) Google Scholar, 4LeBoeuf R.D. Blalock J.E. Ann. N. Y. Acad. Sci. 1990; 594: 393-395Crossref Scopus (4) Google Scholar). The initial screening (1 × 106 clones) yielded 10 independent clones after tertiary replating, and Southern analysis indicated that all clones were related. Both strands of the largest clone (clone 12) were sequenced, and it had an insert size of 691 bp. One strand of the clone 12 cDNA contained a poly(A) tail, two polyadenylation signal sequences within 50 nucleotides of the poly(A) tail, and an open reading frame (ORF) encoding a 120-amino acid peptide. The results from Northern gel analyses using rat pituitary mRNA and the clone 12 cDNA as a probe showed that this cDNA hybridized to a single mRNA species that was ∼2 kilobases (Fig.1). To identify the coding strand of clone 12, two 32P-labeled oligodeoxynucleotides complementary to each strand were synthesized and used as probes in slot analysis with rat pituitary total RNA. Only the oligodeoxynucleotide that was complementary to the sequence with the poly(A) tail and the ORF hybridized to pituitary total RNA showing that this was the coding strand of the SPN cDNA (Fig.2). At this point we also showed that the clone 12 cDNA sequence was an authentic partial clone of theSPN cDNA. Antibodies prepared to a synthetic peptide derived from the deduced 120-amino acid ORF of clone 12 specifically bound pure native SPN in a solid phase assay (TableI).Figure 2Slot blot RNA analysis with SPN oligodeoxynucleotides. Rat total RNA was probed with two complementary oligodeoxynucleotides from the same region of the cDNA. One oligodeoxynucleotide was complementary to the sequence of the cDNA strand that contained the largest open reading frame (A) and one was complementary to the opposite strand of the cDNA. Numbers in the middle of the figure indicate the amount of total RNA (μg) added to each slot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEnzyme-linked immusorbent assay analysis of deduced SPN peptide antibody binding to native SPNIg fractionA 405Pre-immune IgG − peptide0.186 ± 0.007Pre-immune IgG + peptide0.181 ± 0.004Post-immune IgG − peptide0.605 ± 0.005Post-immune IgG + peptide0.295 ± 0.009 Open table in a new tab The clone 12 cDNA was used to rescreen a rat pituitary library. One larger clone was isolated in the second round of screening with an insert size of 924 bp. This cDNA was sequenced in both directions, and it was identical to the 691-bp sequence of clone 12 and provided an additional 233 bp of new nucleotide sequence that extended the size of the ORF to 195 amino acids. Preparation of new rat pituitary cDNA libraries and nucleic acid hybridization screening of these libraries failed to produce any larger SPN cDNA clones. The inability to clone larger SPN cDNAs from these libraries using the clone 12 cDNA as a probe was unexpected, because the quality (recombinant titer and average size) of all libraries used were normal, and these libraries have been used to clone cDNAs of similar size to the size of SPN cDNA. It may be that the SPN transcript contains structural features that lead to the generation of truncated transcripts or that SPN recombinants are unstable, and their proportion in the library is reduced. To overcome this problem, PCR and 5′-RACE were used to obtain the remaining sequence of the SPN cDNA. The sequences obtained were used to generate the full-length SPN cDNA sequence and to design primers that were then used to PCR amplify from the 5′ end of the sequence through the complete ORF. This PCR product was cloned in pGEM-T, and three independent clones were sequenced in both directions to obtain the complete cDNA sequence of SPN. The rat full-length SPN cDNA isolated is 1882 nucleotides (Fig.3), and the size of this cDNA agrees with estimates of the size of the SPN mRNA obtained by Northern gel analysis. The complete SPN cDNA contains an open reading frame of 1488 nucleotides that extends from the first translation consensus sequence (15Kozak M. Nucleic Acid Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4148) Google Scholar) of an initiation codon at position 56 to the first termination codon at position 1545. The entire cDNA contains 55 and 339 nucleotides of 5′- and 3′-untranslated sequences, respectively. Translation of the cDNA results in a predicted protein of 496 amino acids with a relative M r of 53,113 (Fig.4), which is less than the molecular mass previously observed for native SPN (∼63 kDa) by SDS-PAGE analysis. Differences in the actual molecular mass of a protein and its electrophoretic mobility in SDS-PAGE analysis could be due to either post-translational modifications (e.g. glycosylation and/or phosphorylation) and/or to specific regions that bind SDS anomalously and affect electrophoretic mobility (16Mattaj I.W. Cell. 1989; 57: 1-3Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 17Query C.C. Bentley R.C. Keene J.D. Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (439) Google Scholar). The results of bacterial expression studies using the SPN coding sequence (Fig.5) suggest that the SPN protein contains structural regions that apparently bind SDS anomalously and affect its electrophoretic mobility (Fig. 5). The expression of recombinant proteins in E. coli typically leads to the production of proteins that are not modified post-translationally. Therefore, analyses of the mass of recombinant forms by SDS-PAGE reveals proteins of lower molecular weight compared with native proteins that are post-translationally modified. However, SDS-PAGE analysis of SPN produced by E. coli (Fig. 5) showed that it migrated at molecular mass higher (∼63 kDa) than predicted by the deduced amino acid sequence (∼53 kDa). Consistent with the idea of structural features of SPN as a major cause of anomalous electrophoretic mobility are the results fr