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Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid ( ovary / gonadotropin / oligonucleotide / precursor )
| Content Provider | Semantic Scholar |
|---|---|
| Author | Forage, R. G. Ring, Jennifer M. Brown, Richard W. McInerney, B. V. Cobon, Gary S. Findlay, John K. Wettenhall, Richard E. H. Burger, H. G. |
| Copyright Year | 2003 |
| Abstract | The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA. The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and'has a pre-pro region of 60 amino acids. The mature B sublinit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa forn of inhibin also isolated from bovine follicular fluid, with subunits qf 20 kJa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit. Considerable evidence has now accumulated to support the concept that the gonads produce a protein termed inhibin (1) that selectively suppresses the pituitary secretion of folliclestimulating hormone (2), a key hormone in controlling folliculogenesis and spermatogenesis. Controversy still exists concerning the nature of inhibin (3) partly because of the fact that some of the bioassays used in its characterization do not monitor follicle-stimulating hormone directly (4). For instance, inhibin-like activity has been detected in seminal plasma; the proteins associated with this activity have been purified and characterized but subsequently they were shown not to be of gonadal origin (5-9). We have isolated inhibin from a gonadal source, bovine follicular fluid, with an apparent molecular mass of 58 kDa, composed of two subunits now designated A and B, of 43 kDa and 15 kDa, respectively, linked together by disulfide bonds (10). Subsequently, 32-kDa inhibin was isolated by others from porcine follicular fluid, with two subunits of 18/20 kDa and 13/14 kDa (11, 12). We have also shown that a 31-kDa form of bFF inhibin, composed of 20-kDa and 15-kDa subunits, is generated during a pH precipitation step in the purification procedure (13) but the precise relationship between the different forms has been unknown. However, the biological activity of the 31-kDa form is neutralized in vitro by an antiserum raised against the 58-kDa form (13), suggesting that the 31-kDa inhibin is a processed form of the 58-kDa inhibin. This article describes the amino acid sequences of the two subunits of the 58-kDa bovine inhibin as determined fromn cDNA sequencing and reports that the 20-kDa subunit of the 31-kDa inhibin is derived from the 43-kDa subunit of the 58-kDa inhibin, thus clarifying the relationship between these different forms. MATERIALS AND MIETIODS NH2-Terminal Amino Acid Sequencing of the 58-kDa Inhibin. The 58-kDa inhibin was isolated from bFF (10). Briefly, this involved a four-step' purjfication procedure: (i) gel permeation chromatography on Sephacryl 3200 in 0.05 M ammonium acetate, (it) gel permeation chromatography on Sephadex G100 in 4M acetic acid, (iii) reversed-phase HPLC on an Ultrapore RPSC column(Beckman) using a 0.1% trifluoroacetic acid-acetonitrile-H2O gradient, and (iv) preparative NaDodSO4/PAGE with electrophoretic elution of the bioactive band. The inhibin was reduced and alkylated, and the separated A and B subunits were isolated by electroelution from NaDodSO4/PAGE gels (14). The NaDodSO4 was removed by precipitation of inhibin in methanol overnight at -20°C. The proteins were dissolved in dilute triethylamine for application to the sequenator. Bovine 58-kDa inhibin (80 pmol), the A subunit (17 pmol), and the B subunit (6 pmol) were analyzed by E4man degradation in a gas-phase sequenator (Applied Biosystems, Foster City, CA); the initial yields of the phenylthiohydantoin-derivatized amino acids (10) given here were-40-50% of the estimated applied sample. cDNA Synthesis. RNA was extracted (15) from bovine granulosa cells obtained from fresh follicular fluid, and the nrRNA fraction was purified by chromatography on oligo(dT)-cellulose (16). Synthesis ofthe first strand ofcDNA was at 42°C using (dT)12_18 primers and reverse transcriptase from avian myeloblastoma virus (Life Sciences, St. Petersburg, FL). Second strand synthesis was by the RNase H (Bethesda Research Laboratories) method but omitting Escherichia coliDNA ligase from the mixture (17). The cDNA was inserted into the Pst I site of pBR322 by the dG-dC homopolymer tailing method (18) and the resultant molecules were transformed (19) into E. coli ED8654 (20). Typically, 5 x 103 to 5 x 104 transformants were obtained per ,ug ofvector and a library of approximately 10,000-14,000 independent colonies was established. Clone Isolation. All possible oligonucleotide sequences that coded for selected regions of the subunit aminc acid sequences were deduced and their complements were synthesized by the phosphoramidite method (21) using an Applied Bioystems model 380A synthesizer. Probe 1 (5' CKCATMAANCCNCC 3')11 was pQoled from four independent synIlFor recent recommendations on nomenclature of incompletely specified bases, see ref. 22. 3091 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) theses having, at positions 2 and 6 from the 5' end, guanosine and adenosine or thymidine and adenosine or guanosine and guanosine or thymidine and guanosine and was used to isolate inhibin A subunit cDNA. Probe 2 (5' CCDATRTCYTTRAA 3') was synthesized in a single batch and was used to isolate inhibin B subunit cDNA. From each synthesis, oligonucleotides of the correct length were purified by preparative clectrophoresis, then labeled with 3 P at the 5' end using T4 polynucleotide kinase (Bethesda Research Laboratories). Colonies on nitrocellulose filters were prepared for hybridization (23) after amplification of their plasmid content (24). The filters were probed with the labeled oligonucleotides at 1 pmol/ml in 10x Denhardt's solution (1 x = 0.02% polyvinylpyrollidone/0.02% Ficoll/0.02% bovine serum albumin) containing 5x NaCl/citrate (lx = 0.15 M NaCl/15 mM sodium citrate) at room temperature overnight and unbound probe was removed by washing at 37°C in 1 x NaCl/citrate/0.1% NaDodSO4. Autoradiography was overnight at -70°C using Fuji RX x-ray film and a Dupont Cronex Hi Plus intensifying screen. DNA Sequencing. Appropriate restriction enzyme fragments were subcloned into bacteriophages M13 mp8 and M13 mp9 for sequencing by the dideoxy chain termination method (25) using a universal primer (17-mer, no. 1211; New England Biolabs) or oligonucleotide primers complementary to the cDNA itself. |
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| Alternate Webpage(s) | http://www.pnas.org/content/83/10/3091.full.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
