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Porcine follistatin gene structure supports two forms of mature follistatin produced by alternative splicing
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 7] 7-723
April 29, 1988
Porcine Follistatin Gene Structure Supports Two F o r m s of M a t u r e Follistatin Produced by Alternative Splicing
Shunichi Shimasaki, Makoto Koga, Fred Esch*, Maluz Mercado, Karen Cooksey, Ann Koba, and Nicholas Ling Laboratories for Neuroendocrinology, The Salk Institute, 10010 N. TorreyPines Road, La Jolla, CA, and *Athena Neurosciences, Inc. 887-D Industrial Road, San Carlos, CA Received March ii, 1988
Follistatin (FS), a novel gonadal protein which inhibits specifically the secretion of pituitary follicle stimulating hormone(FSH), has recently been isolated from porcine follicular fluid, cDNA cloning of the porcine ovarian FS precursor revealed two populations of cDNAs which differed at the Y-region of the open reading frames; one population encodes a precursor of 317 amino acids while the other encodes another precursor having the same 317 amino acids, but with an additional 27 amino acids at the carboxy-terminal. Herein, we report the cloning of the porcine FS gene whose DNA structure reveals that the two populations of mRNA are generated by alternative splicing. In addition, restriction endonuclease mapping and DNA sequencing show that the FS gene is approximately 6 Kb long and consists of six exons separated by five introns. The first exon encodes the putative signal sequence, followed by four exons which encode the four domains of FS, three of which are highly homologous to each other. The last exon encodes the extra 27-amino acid carboxy-terminal domain of the 344-residued precursor, o 1988 Academic P ..... Inc.
The name inhibin was coined by McCullagh in 1932 (1), to denote a gonadal polypeptide responsible for negative feedback inhibition of pituitary follicle stimulating hormone (FSH) secretion. In 1985, two proteins of relative molecular size (Mr) 32,000 that fit the definition of inhibin were isolated from porcine ovarian follicular fluid (2-4), and from bovine follicular fluid a homologous Mr 32,000 protein was also purified (5,6). The two porcine inhibins are heterodimers composed of a common a-subunit of Mr 18,000, and one of two distinct, but highly homologous Mr 14,000 [3-subunits. Using the amino acid sequence information, cDNA clones encoding the subunits of the porcine and bovine inhibins were identified and sequenced to reveal their complete primary structures (7,8). Moreover, the structures of human (9), rat (10), and ovine (11) inhibins have also been determined by molecular cloning using the porcine or bovine inhibin cDNAs as hybridization probes. Comparison of the predicted amino acid sequences showed that each of the inhibin subunits is highly conserved between species. Throughout the isolation of the inhibins, we consistently observed one side fraction which could also inhibit pituitary FSH secretion. The active material in this side fraction has now
Abbreviations: follistatin, FS; follicle stimulating hormone, FSH
717
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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been purified to yield two glycosylated single-chain proteins of Mr 35,000 and 32,000, respectively (12). The amino acid composition of the two proteins are similar and their amino-terminal amino acid sequences are identical, suggesting that they may differ in the state of glycosylation or carboxy-terminal truncation. The structure of the precursor for these two proteins, named "follistatins" (FSs), was subsequently deduced by molecular cloning from a porcine ovarian cDNA library and two mature proteins were predicted, one composed of 315 amino acids, while the other was a carboxy-terminal truncated counterpart containing 288 amino acids (13). Independently, Robertson et al. (14) reported the isolation of three singlechain proteins with FSH release-inhibitory activity from bovine follicular fluid, all having the same amino-terminal amino acid sequence as porcine FS. To elucidate the origin of the two porcine FS precursors as well as to determine the genomic structure, we have cloned the porcine FS gene and sequenced its DNA. The sequencing results revealed that the origin of the two precursors was derived from alternative splicing of the primary transcript.
M A T E R I A L S AND METHODS Southern Blot Hvbridization. DNA was prepared from porcine liver as described (15) and 4-ug aliquots were digested with restriction enzymes for Southern blotting. The digests were electrophoresed on a 0.6% agarose gel and transferred onto a nylon membrane filter. Hybridization was performed with a 32p-labelled porcine eDNA probe encoding the FS precursor at 37°C for 15 hrs in 1M NaC1, 1% SDS, 10% dextran sulfate, 50% formamide, and 200 ~g/ml denatured salmon sperm DNA. The membrane filters were washed in 0.1X SSC and 0.1% SDS at 65°C for 30 minutes. Autoradiography was carried out with an intensifying screen at -80°C overnight. Qvarian RNA Preoaration and Northern Blot Hybridization. Porcine ovaries were obtained from adult females and the tissues were snap frozen in liquid nitrogen immediately after excision, and stored at -8&C until ready for RNA preparation. Total RNA was prepared by the conventional guanidine isothiocyanate method (16) and poly(A) + RNA was purified by oligo(dT)-cellulose column chromatography. Five micrograms of poly(A) + RNA was electrophoresed on a formaldehyde-agarose gel and then transferred onto a nylon membrane filter. Hybridization and washing of the filter were performed under the same conditions as those that were used for Southern blot hybridization. Cloning and DNA Sequencing. Fifty micrograms of porcine liver DNA were digested with EcoRI and then electrophoresed on a 0.6% agarose gel. Since EcoRI digestion had yielded a single positive band at - 10Kb in Southern analysis, DNA fragments from 9 to llKb containing the FS gene were recovered from the gel using NA45 DEAE anionic exchange membrane (Schleicher & Schuell, Inc.). An aliquot of the recovered DNA fragments was ligated to the EcoRI/BamHI double-digested EMBL 4-vector arms and then packaged in vitro with packaging extract purchased from Stratagene, Inc.(San Diego, CA). The recombinant phages were used to infect the bacterial strain P2392 (P2 lysogen of LE392). Thirteen thousand plaques were screened with a 32p-labelled porcine eDNA probe. Hybridization and washing conditions were the same as those used for the genomic Southern blot hybridization. One positive phage clone was identified and the inserted DNA fragment excised with EcoRI and subcloned into pUC18. The DNA was mapped for restriction sites and sequenced by the dideoxynudeotide chain-termination method (17) using synthetic primers based on the porcine FS eDNA sequences. 718
Vol. 152, No. 2, 1988
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RESULTS AND DISCUSSION When we cloned the porcine FS cDNAs from an ovarian library, two populations of eDNA clones were identified, although the Northern blot analysis showed a single band for the FS mRNA in the porcine ovary (13). One population of cDNAs encodes a 317 amino acid precursor while the other has an additional 27 amino acids immediately following the carboxy-terminal of the same 317-amino acid precursor. No adequate explanation for the origin of the two different termination signals could be derived from the limited data provided in the previous paper (13). To elucidate the origin of the two precursors, we have now cloned and sequenced the porcine FS gene. Prior to the cloning experiment, Southern analysis of porcine genomic DNAwas performed to find a suitable restriction enzyme for construction of the gene library. EcoRI digestion showed a single - 10 Kb positive band that hybridized with the porcine eDNA probe (see Fig. 3). As a result, DNA fragments ranging from 9 to 11 Kb recovered from EcoRI digestion of porcine liver DNA were ligated to EMBL 4-vector arms, packaged in vitro and then used to infect bacterial strain P2392. Out of the 1.3 x 104 independent phage plaques, one positive clone was identified. The restriction map and the exon-intron organization of this clone is shown in Fig. 1. The FS gene contains five introns, two of which, introns 3 and 4 in Fig. 1, correspond to introns 1 and 2, respectively, which was identified in eDNA clone FS18 in the previous paper (13). The first exon (I) encodes the signal sequence, the following four exons (II-V) encode four contiguous domains three of which are highly homologous to each other (HI,IV,V) as was pointed out in the previous paper (13). The last exon (VI) encodes the extra 27 amino acid domain which is present only in the FS344 precursor. Fig. 2 shows the DNA sequence of the porcine FS gene. The putative initiation methionine occurs at nucleotides 798-800 which is located within a sequence favored for eukaryotic initiation sites, A/G-X-X-A-U-G-G (18). There is no other methionine codon between this codon and the upstream in-frame codon at nucleotides 414-416. The typical promoter consensus sequences (TATA and CcAAT) are not found in the 797 nucleotides upstream of the initiation methionine. The common DNA sequences in both populations of the previously characterized FS eDNA clones are located in the coding regions I-V of the gene. However, the two eDNA populations differ at the location immediately following the 3'- ter-
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Fig.1. Schematic representation of the porcine FS gene and its restriction enzyme map.
The cloned fragment is represented by a box bound by the solid and broken lines and the wavy lines correspond to the vector. The region of the gene that was sequenced is represented by the solid lines of the box. The shaded areas correspond to the coding regions. The arrows denote the alternative splicing pattern to generate the two forms of eDNA. 719
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rninal nucleotide of the coding region V; in one population exon V is joined to nucleotide #5453 and in the other, exon V is linked to #5741 in the DNA sequence. The two nucleotides 5' to nucleotides #5453 and #5741 in the gene are "AG", which is the consensus acceptor site for splicing. Southern blotting analysis of the porcine FS genomic DNA and the cloned FS gene showed an identical restriction pattern for both DNAs (Fig. 3) implicating a single FS gene in the porcine genome. These findings strongly suggest that the two kinds of FS mRNAs are generated from one gene by alternative splicing during the RNA processing. Since two molecular forms of FS with Mr 35K and 32K were isolated from porcine follicular fluid, it is tempting to assign the structure of the two proteins to the product predicted from the two forms of the FS mRNA, respectively. Re-analysis of the porcine FS mRNA by Northern blotting showed its size to be - 2.5 Kb (Fig. 4) instead of 1.9 Kb, as reported previously (13). The discrepancy was caused by misalignment of the marker standards on the electrophoresis gel in the earlier paper. Although a single broad band was observed in Fig. 4, this does not rule out the possibility that the band may contain two forms of FS mRNA which differ by 288 nucleotides. Such a small difference in message size could not be separated by the electrophoretic analysis. Although there are many examples of two different proteins generated from one gene by alternative splicing of a precursor mRNA, the pair of proteins produced usually contains different amino acid sequences besides the common residues. The two mature FSs produced by alternative splicing are a unique example because one of the two splicing patterns at intron 5 generates a stop codon immediately following the common amino acid sequence. As a result, one of the two FS mRNAs encodes a carboxy-terminal truncated form of the other. Aside from the possibility that they encode the respective Mr 35K and 32K FS proteins isolated from porcine follicular fluid, the physiological significance of the two forms of FS mRNA is not clear. Since the two proteins have practically identical potency on the inhibition of FSH release from rat pituitary cells (19), the extra 27-amino acid domain at the carboxy-terminal of the 35K FS might have no physiological function and thus, under evolution pressure, be deleted by alternative splicing. On the other hand, the extremely acidic amino acid sequence (Glu-Asp-Thr-Glu-Glu-Glu-Glu-Glu-Asp-Glu-Asp-Gln-Asp) contained in that domain might have an as yet unknown function which is associated with the 35K protein. Thorough physiological studies with these two forms of mature FS proteins will be required to answer these questions. Fig.2 Nucleotide and deduced protein sequences of porcine FS gene.
The nucleotides are numbered at both ends, and amino acids in one-letter code, are numbered throughout. Two potential N-linked glycosylation sites are marked by the stars. The first upstream in-frame stop codon in the 5' untranslated sequence is boxed. Two types of splicing involving introns 5 and 5' create a termination codon28~TGA) and the extra 27 amino acid sequence, respectively, immediately following the Asn . Sequence comparison of the FS gene with the FS eDNA clone # 18 previously reported (13) revealed 10 nucleotide differences. Three of them exist in exon II, III and V at positions 2869 ( C [gene] / T [eDNA] ), 3563 (G/A), 4856 (C/T), respectively. However, none of these nucleotide substitutions change the corresponding amino acids. The remaining 7 nucleotide differences exist in intron 3 and 4 at positions 3771 (additional T in eDNA), 4201 (A/T), 4475 (additional T in eDNA), 4531 (G/C), 4555 (A/C), 4669 (C/T) and 4749 (A/G). 721
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ACKNOWLEDGEMENTS We thank Dr.R.Guillemin for his advice and encouragement and Ms.D.Higgins for secretarial assistance. This research was supported by NICHD Contract N01HD-6-2944, Program Project Grants HD-09690 and DK-18811 from the NIH, and a grant from the Robert. and Helen C. Kleberg Foundation.
REFERENCES 1. McCullagh, D.R. (1932) Science 76, 19-20. 2. Ling, N., Ying, S-Y., Ueno, N., Esch, F., Denoroy, L., and Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82, 7217-7221. 722
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3. Miyamoto, K., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K, and Matsuo, H. (1985) Biochem. Biophys. Res. Commun. 129, 396-403. 4. Rivier, J., Spiess, J., McClintock, R., Vaughan, J., and Vale, W. (1985) Biochem. Biophys. Res. Commun. 133, 120-127. 5. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K., and Matsuo, H. (1986) Mol. Cell. Endocrinol. 44, 55-60. 6. Robertson, D.M., deVos, F.L., Foulds, L.M., McLachlan, R. I., Burger, H.G., Morgan, F.J., Hearn, M.T.W., and deKretser, D.M. (1986) Mol. Cell. Endocrinol. 44, 271-277. 7. Mason, A.J., Hayflick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S-Y., Guillemin, R., Niall, H., and Seeburg, P. (1985) Nature 318, 659-663. 8. Forage, R.G., Ring, J.M., Brown, R.W., Mclnerney, B.V., Cobon, G.S., Gregson, R.P., Robertson, D.M., Morgan, F.J., Hearn, M.T.W., Findlay, J.K., Wettenhall, R.E.H., Burger, H.G., and deKretser, D.M. (1986) Proc. Natl. Acad. Sci. USA 83, 3091-3095. 9. Mason, A., Niall, H., and Seeburg, P. (1986) Biochem. Biophys. Res. Commun. 135, 957964. 10. Esch, F., Shimasaki, S., Cooksey, IC, Mercado, M., Mason, A., Ying, S-Y., Ueno, N., and Ling, N. (1987) Molec. Endocrinol. 1,388-396. 11. Forage, R.G., Brown, R.W., Ring, J.M., Stewart, A.G., Milborrow, H.M., Oliver, K.J., Atrache, B.T., Devine, P.L., Hudson, G.C., Gross, N.H., Tolstoshev, P., Robertson, D.M., Doughton, B., deKretser, D.M., Burger, H.G., and Findlay, J.K. (1986) In Serono Symposia Publications 42. "Inhibin-non-steroidal regulation of follicle stimulating hormone secretion", (Burger, H.G., deKretser, D.M., Findlay, J.K., and Igarashi, M., Eds.),, pp. 89-103. Raven Press, New York. 12. Ueno, N., Ling, N., Ying, S-Y., Esch, F., Shimasaki, S., and Guillemin, R. (1987) Proc. Natl. Acad. SCl.USA 84, 8282-8286. 13. Esch, F.S., Shimasaki, S., Mercado, M., Cooksey, K., Ling, N., Ying, S-Y., Ueno, N., and Guillemin, R. (1987) Mol. Endoo-inol. 1, 849-855. 14. Robertson, D.M., Klein, R., deVos, F.L., McLachlan, R.I., Wettenhall, R.E.H., Hearn, M.T.W., Burger, H.G., and deKretser, D.M. (1987) Biochem. Biophys. Res. Commun. 149, 744-749. 15. Blin, N. and Stafford, D.W. (1976) Nucleic Adds Res. 3, 2303-2308. 16. Chirgwin, J., Przybyla, A., MacDonald, J., and Rutter, W. (1979) Biochemistry 18, 52945299. 17. Sanger, F., Nicklen, S., and Coulseon, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 54635467. 18. Kozak, M. (1981) Nucleic Acid Res. 9, 5233-5252. 19. Ying, S-Y., Becker, A., Swanson, G., Tan, P., Ling, N., Esch, F., Ueno, N., Shimasaki, S., and Guillemin, R. (1987) Biochem. Biophys. Res. Commun. 149, 133-139.
723
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS Pages 7] 7-723
April 29, 1988
Porcine Follistatin Gene Structure Supports Two F o r m s of M a t u r e Follistatin Produced by Alternative Splicing
Shunichi Shimasaki, Makoto Koga, Fred Esch*, Maluz Mercado, Karen Cooksey, Ann Koba, and Nicholas Ling Laboratories for Neuroendocrinology, The Salk Institute, 10010 N. TorreyPines Road, La Jolla, CA, and *Athena Neurosciences, Inc. 887-D Industrial Road, San Carlos, CA Received March ii, 1988
Follistatin (FS), a novel gonadal protein which inhibits specifically the secretion of pituitary follicle stimulating hormone(FSH), has recently been isolated from porcine follicular fluid, cDNA cloning of the porcine ovarian FS precursor revealed two populations of cDNAs which differed at the Y-region of the open reading frames; one population encodes a precursor of 317 amino acids while the other encodes another precursor having the same 317 amino acids, but with an additional 27 amino acids at the carboxy-terminal. Herein, we report the cloning of the porcine FS gene whose DNA structure reveals that the two populations of mRNA are generated by alternative splicing. In addition, restriction endonuclease mapping and DNA sequencing show that the FS gene is approximately 6 Kb long and consists of six exons separated by five introns. The first exon encodes the putative signal sequence, followed by four exons which encode the four domains of FS, three of which are highly homologous to each other. The last exon encodes the extra 27-amino acid carboxy-terminal domain of the 344-residued precursor, o 1988 Academic P ..... Inc.
The name inhibin was coined by McCullagh in 1932 (1), to denote a gonadal polypeptide responsible for negative feedback inhibition of pituitary follicle stimulating hormone (FSH) secretion. In 1985, two proteins of relative molecular size (Mr) 32,000 that fit the definition of inhibin were isolated from porcine ovarian follicular fluid (2-4), and from bovine follicular fluid a homologous Mr 32,000 protein was also purified (5,6). The two porcine inhibins are heterodimers composed of a common a-subunit of Mr 18,000, and one of two distinct, but highly homologous Mr 14,000 [3-subunits. Using the amino acid sequence information, cDNA clones encoding the subunits of the porcine and bovine inhibins were identified and sequenced to reveal their complete primary structures (7,8). Moreover, the structures of human (9), rat (10), and ovine (11) inhibins have also been determined by molecular cloning using the porcine or bovine inhibin cDNAs as hybridization probes. Comparison of the predicted amino acid sequences showed that each of the inhibin subunits is highly conserved between species. Throughout the isolation of the inhibins, we consistently observed one side fraction which could also inhibit pituitary FSH secretion. The active material in this side fraction has now
Abbreviations: follistatin, FS; follicle stimulating hormone, FSH
717
0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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been purified to yield two glycosylated single-chain proteins of Mr 35,000 and 32,000, respectively (12). The amino acid composition of the two proteins are similar and their amino-terminal amino acid sequences are identical, suggesting that they may differ in the state of glycosylation or carboxy-terminal truncation. The structure of the precursor for these two proteins, named "follistatins" (FSs), was subsequently deduced by molecular cloning from a porcine ovarian cDNA library and two mature proteins were predicted, one composed of 315 amino acids, while the other was a carboxy-terminal truncated counterpart containing 288 amino acids (13). Independently, Robertson et al. (14) reported the isolation of three singlechain proteins with FSH release-inhibitory activity from bovine follicular fluid, all having the same amino-terminal amino acid sequence as porcine FS. To elucidate the origin of the two porcine FS precursors as well as to determine the genomic structure, we have cloned the porcine FS gene and sequenced its DNA. The sequencing results revealed that the origin of the two precursors was derived from alternative splicing of the primary transcript.
M A T E R I A L S AND METHODS Southern Blot Hvbridization. DNA was prepared from porcine liver as described (15) and 4-ug aliquots were digested with restriction enzymes for Southern blotting. The digests were electrophoresed on a 0.6% agarose gel and transferred onto a nylon membrane filter. Hybridization was performed with a 32p-labelled porcine eDNA probe encoding the FS precursor at 37°C for 15 hrs in 1M NaC1, 1% SDS, 10% dextran sulfate, 50% formamide, and 200 ~g/ml denatured salmon sperm DNA. The membrane filters were washed in 0.1X SSC and 0.1% SDS at 65°C for 30 minutes. Autoradiography was carried out with an intensifying screen at -80°C overnight. Qvarian RNA Preoaration and Northern Blot Hybridization. Porcine ovaries were obtained from adult females and the tissues were snap frozen in liquid nitrogen immediately after excision, and stored at -8&C until ready for RNA preparation. Total RNA was prepared by the conventional guanidine isothiocyanate method (16) and poly(A) + RNA was purified by oligo(dT)-cellulose column chromatography. Five micrograms of poly(A) + RNA was electrophoresed on a formaldehyde-agarose gel and then transferred onto a nylon membrane filter. Hybridization and washing of the filter were performed under the same conditions as those that were used for Southern blot hybridization. Cloning and DNA Sequencing. Fifty micrograms of porcine liver DNA were digested with EcoRI and then electrophoresed on a 0.6% agarose gel. Since EcoRI digestion had yielded a single positive band at - 10Kb in Southern analysis, DNA fragments from 9 to llKb containing the FS gene were recovered from the gel using NA45 DEAE anionic exchange membrane (Schleicher & Schuell, Inc.). An aliquot of the recovered DNA fragments was ligated to the EcoRI/BamHI double-digested EMBL 4-vector arms and then packaged in vitro with packaging extract purchased from Stratagene, Inc.(San Diego, CA). The recombinant phages were used to infect the bacterial strain P2392 (P2 lysogen of LE392). Thirteen thousand plaques were screened with a 32p-labelled porcine eDNA probe. Hybridization and washing conditions were the same as those used for the genomic Southern blot hybridization. One positive phage clone was identified and the inserted DNA fragment excised with EcoRI and subcloned into pUC18. The DNA was mapped for restriction sites and sequenced by the dideoxynudeotide chain-termination method (17) using synthetic primers based on the porcine FS eDNA sequences. 718
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RESULTS AND DISCUSSION When we cloned the porcine FS cDNAs from an ovarian library, two populations of eDNA clones were identified, although the Northern blot analysis showed a single band for the FS mRNA in the porcine ovary (13). One population of cDNAs encodes a 317 amino acid precursor while the other has an additional 27 amino acids immediately following the carboxy-terminal of the same 317-amino acid precursor. No adequate explanation for the origin of the two different termination signals could be derived from the limited data provided in the previous paper (13). To elucidate the origin of the two precursors, we have now cloned and sequenced the porcine FS gene. Prior to the cloning experiment, Southern analysis of porcine genomic DNAwas performed to find a suitable restriction enzyme for construction of the gene library. EcoRI digestion showed a single - 10 Kb positive band that hybridized with the porcine eDNA probe (see Fig. 3). As a result, DNA fragments ranging from 9 to 11 Kb recovered from EcoRI digestion of porcine liver DNA were ligated to EMBL 4-vector arms, packaged in vitro and then used to infect bacterial strain P2392. Out of the 1.3 x 104 independent phage plaques, one positive clone was identified. The restriction map and the exon-intron organization of this clone is shown in Fig. 1. The FS gene contains five introns, two of which, introns 3 and 4 in Fig. 1, correspond to introns 1 and 2, respectively, which was identified in eDNA clone FS18 in the previous paper (13). The first exon (I) encodes the signal sequence, the following four exons (II-V) encode four contiguous domains three of which are highly homologous to each other (HI,IV,V) as was pointed out in the previous paper (13). The last exon (VI) encodes the extra 27 amino acid domain which is present only in the FS344 precursor. Fig. 2 shows the DNA sequence of the porcine FS gene. The putative initiation methionine occurs at nucleotides 798-800 which is located within a sequence favored for eukaryotic initiation sites, A/G-X-X-A-U-G-G (18). There is no other methionine codon between this codon and the upstream in-frame codon at nucleotides 414-416. The typical promoter consensus sequences (TATA and CcAAT) are not found in the 797 nucleotides upstream of the initiation methionine. The common DNA sequences in both populations of the previously characterized FS eDNA clones are located in the coding regions I-V of the gene. However, the two eDNA populations differ at the location immediately following the 3'- ter-
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20 10 811 A G CA C C A G C C C G G C G G G C T A T GC C T C C T G C T G C T ~ CTG C T C T G C C A G T T C A T G G A G G A C C G C A G C G C C C A G G ~ T A A G A G A A G G G G G A T G C K H Q P G G L C L L L L L L C Q F M E D R S A Q ~ mtron1 9@I G C T G G G A G G C T CGG C A G G C G A C A G G G G T T T G TAC"I'TTTC A G T C G T C T C C A C T G GT C T G T T C T G A G C C C T G C T C T T A G A T G T T G G A C C A A C 9~I T T A G G A A G T C C G G A T G G A G A G G A G T G G G A A ~ T T C A A G G C A G T G A C A C T T T A G G A C T G G G A G C C G A G T T T G G A G C G G G G C G A T C C T T T A A I~I A A G G A A A G G G A G T G A G C T G G A G T C T G T C T C A G T A G T T A G G G T T A G A T G C T C C A A G T T C T G G A C A T A A T G A G T T T A G A T G T T A T C T C C C C T 1171 G T T T G G C ` P C T G A A G A A G G G G A A A A G C A T C A G T A G C C T G G A G A T T T C T G T C G A C C C A C T T A C A G T C T A C C T C T C T A C C T C C T A C A T C T ~ 1261 G G A T A C T T T A A A A A A T G A C T G C A T T G G G G A G G G G G A T A C T G A A A T T T C T T C G G T G G • T G A G G G A C G T G G T T T c A G A A A C T T T G C C C A A A C 1351 T T C G A A A T G T G G G T G C C G A A ~ T T T T A G A G G G A A G T A G C T T T ~ T G C C T A G T G G A T G C T T C T T C C A A C A C C C T C C C C C A A A C C ~ G C A A A A A 1441 ~ T A C C C C T G A C T G T T T T C A G T A G T T T A C A A T T T T T T T T C T T T G C C T C A T T C C T C A T G T T A A C T T T C T G T A G I$31 A A C A G T G A A A A G C C C A C A A T T G A T T T A ~ T T C T G A G A T G C A G T A A G A G C A A A G T A T T T T T T T A A T T A G A T T T A T T T A T T T C T G C T A G T C T 1621 T T G G G C A G A C T T T T T T A A A A T C A T A A A A G C A G C T T G C T G T T C A A T A T A T T G T T A A G A T T A T A G G G A T G A T T C C C G A G A G G T C T T T C T G A C 1711 C A A A T C C G A A C A T T A ~ G G A G G A G G G G G G G G G A A T T A C T T G C A A A G A T ~ / ~ T C T T G G T A G G T A T G A G A T A T G T A T T A C C A T G A G A C G G T G 1801 G A A G C C ~ C T C C C C C C T T T A T T A T T A T T A T T T A C T T G G T T G G T T G G G T T ~ T C C ~ C C T A C A C T c T T C T T G A G A C C ~ A C T T G T A A C T T T ~ C 1891 T G G C T T T A G C A T T A A C A T T T T A T G T G T G T T G T A A A C T G G C C A C C G G G C A C T G C C C C T C G C G G T C T G C A A C G C A C C A C T C T G A G T T G ~ C C G I~I CCTTTCGGGGCGAGGCGCTGCCTAGGTGC•GGGGACGTGGGCGcCGGGGAAAGCAGCCCTCGGAGATTTCGAAAGATCGCGGTGGCCC•CG /2,71 G G c G T C C T C T G G A T • T G G • G G A T T G C A A A A C G G G A C C G T • G G G A G G G C T G • G C T T G A G G C C G C A C G G G G C A G C T C C G G G • A A G G C T C A G T 2161 T G C T T T T G C T T T T T T G C T T T C A T T T T T C T C A A G T G C C T T T T G C T T G A T C T G T T C C T G T A G G T G T C T G T C T C A G C C T C T C C A • • C T C C • C • 2251 C C T T T T T T C T T T C T C T T A A G T T C T C C T A C T T T C T T A A C C C C T G T C A C A A A T A A T A C T T T T A C C G C C C A C A T G A A A C C A A CATG T A A A A A C
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Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
rninal nucleotide of the coding region V; in one population exon V is joined to nucleotide #5453 and in the other, exon V is linked to #5741 in the DNA sequence. The two nucleotides 5' to nucleotides #5453 and #5741 in the gene are "AG", which is the consensus acceptor site for splicing. Southern blotting analysis of the porcine FS genomic DNA and the cloned FS gene showed an identical restriction pattern for both DNAs (Fig. 3) implicating a single FS gene in the porcine genome. These findings strongly suggest that the two kinds of FS mRNAs are generated from one gene by alternative splicing during the RNA processing. Since two molecular forms of FS with Mr 35K and 32K were isolated from porcine follicular fluid, it is tempting to assign the structure of the two proteins to the product predicted from the two forms of the FS mRNA, respectively. Re-analysis of the porcine FS mRNA by Northern blotting showed its size to be - 2.5 Kb (Fig. 4) instead of 1.9 Kb, as reported previously (13). The discrepancy was caused by misalignment of the marker standards on the electrophoresis gel in the earlier paper. Although a single broad band was observed in Fig. 4, this does not rule out the possibility that the band may contain two forms of FS mRNA which differ by 288 nucleotides. Such a small difference in message size could not be separated by the electrophoretic analysis. Although there are many examples of two different proteins generated from one gene by alternative splicing of a precursor mRNA, the pair of proteins produced usually contains different amino acid sequences besides the common residues. The two mature FSs produced by alternative splicing are a unique example because one of the two splicing patterns at intron 5 generates a stop codon immediately following the common amino acid sequence. As a result, one of the two FS mRNAs encodes a carboxy-terminal truncated form of the other. Aside from the possibility that they encode the respective Mr 35K and 32K FS proteins isolated from porcine follicular fluid, the physiological significance of the two forms of FS mRNA is not clear. Since the two proteins have practically identical potency on the inhibition of FSH release from rat pituitary cells (19), the extra 27-amino acid domain at the carboxy-terminal of the 35K FS might have no physiological function and thus, under evolution pressure, be deleted by alternative splicing. On the other hand, the extremely acidic amino acid sequence (Glu-Asp-Thr-Glu-Glu-Glu-Glu-Glu-Asp-Glu-Asp-Gln-Asp) contained in that domain might have an as yet unknown function which is associated with the 35K protein. Thorough physiological studies with these two forms of mature FS proteins will be required to answer these questions. Fig.2 Nucleotide and deduced protein sequences of porcine FS gene.
The nucleotides are numbered at both ends, and amino acids in one-letter code, are numbered throughout. Two potential N-linked glycosylation sites are marked by the stars. The first upstream in-frame stop codon in the 5' untranslated sequence is boxed. Two types of splicing involving introns 5 and 5' create a termination codon28~TGA) and the extra 27 amino acid sequence, respectively, immediately following the Asn . Sequence comparison of the FS gene with the FS eDNA clone # 18 previously reported (13) revealed 10 nucleotide differences. Three of them exist in exon II, III and V at positions 2869 ( C [gene] / T [eDNA] ), 3563 (G/A), 4856 (C/T), respectively. However, none of these nucleotide substitutions change the corresponding amino acids. The remaining 7 nucleotide differences exist in intron 3 and 4 at positions 3771 (additional T in eDNA), 4201 (A/T), 4475 (additional T in eDNA), 4531 (G/C), 4555 (A/C), 4669 (C/T) and 4749 (A/G). 721
VoI. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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Fig. 3 Southern blot analyses of porcine FS genomie DNA and cloned FS gene. Genomic DNA (G) and cloned FS gene (C) were digested with the respective restriction enzymes indicated on the top of each pair of lanes. Size markers are x-DNA digested with Hind III. Fig. 4 Northern blot analyses of porcine ovarian poly(A) + RNA. The numbers in Kb correspond to the markers of a RNA ladder (BRL, Gaithersburg, MD).
ACKNOWLEDGEMENTS We thank Dr.R.Guillemin for his advice and encouragement and Ms.D.Higgins for secretarial assistance. This research was supported by NICHD Contract N01HD-6-2944, Program Project Grants HD-09690 and DK-18811 from the NIH, and a grant from the Robert. and Helen C. Kleberg Foundation.
REFERENCES 1. McCullagh, D.R. (1932) Science 76, 19-20. 2. Ling, N., Ying, S-Y., Ueno, N., Esch, F., Denoroy, L., and Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82, 7217-7221. 722
Vol. 152, No. 2, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Miyamoto, K., Hasegawa, Y., Fukuda, M., Nomura, M., Igarashi, M., Kangawa, K, and Matsuo, H. (1985) Biochem. Biophys. Res. Commun. 129, 396-403. 4. Rivier, J., Spiess, J., McClintock, R., Vaughan, J., and Vale, W. (1985) Biochem. Biophys. Res. Commun. 133, 120-127. 5. Fukuda, M., Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K., and Matsuo, H. (1986) Mol. Cell. Endocrinol. 44, 55-60. 6. Robertson, D.M., deVos, F.L., Foulds, L.M., McLachlan, R. I., Burger, H.G., Morgan, F.J., Hearn, M.T.W., and deKretser, D.M. (1986) Mol. Cell. Endocrinol. 44, 271-277. 7. Mason, A.J., Hayflick, J.S., Ling, N., Esch, F., Ueno, N., Ying, S-Y., Guillemin, R., Niall, H., and Seeburg, P. (1985) Nature 318, 659-663. 8. Forage, R.G., Ring, J.M., Brown, R.W., Mclnerney, B.V., Cobon, G.S., Gregson, R.P., Robertson, D.M., Morgan, F.J., Hearn, M.T.W., Findlay, J.K., Wettenhall, R.E.H., Burger, H.G., and deKretser, D.M. (1986) Proc. Natl. Acad. Sci. USA 83, 3091-3095. 9. Mason, A., Niall, H., and Seeburg, P. (1986) Biochem. Biophys. Res. Commun. 135, 957964. 10. Esch, F., Shimasaki, S., Cooksey, IC, Mercado, M., Mason, A., Ying, S-Y., Ueno, N., and Ling, N. (1987) Molec. Endocrinol. 1,388-396. 11. Forage, R.G., Brown, R.W., Ring, J.M., Stewart, A.G., Milborrow, H.M., Oliver, K.J., Atrache, B.T., Devine, P.L., Hudson, G.C., Gross, N.H., Tolstoshev, P., Robertson, D.M., Doughton, B., deKretser, D.M., Burger, H.G., and Findlay, J.K. (1986) In Serono Symposia Publications 42. "Inhibin-non-steroidal regulation of follicle stimulating hormone secretion", (Burger, H.G., deKretser, D.M., Findlay, J.K., and Igarashi, M., Eds.),, pp. 89-103. Raven Press, New York. 12. Ueno, N., Ling, N., Ying, S-Y., Esch, F., Shimasaki, S., and Guillemin, R. (1987) Proc. Natl. Acad. SCl.USA 84, 8282-8286. 13. Esch, F.S., Shimasaki, S., Mercado, M., Cooksey, K., Ling, N., Ying, S-Y., Ueno, N., and Guillemin, R. (1987) Mol. Endoo-inol. 1, 849-855. 14. Robertson, D.M., Klein, R., deVos, F.L., McLachlan, R.I., Wettenhall, R.E.H., Hearn, M.T.W., Burger, H.G., and deKretser, D.M. (1987) Biochem. Biophys. Res. Commun. 149, 744-749. 15. Blin, N. and Stafford, D.W. (1976) Nucleic Adds Res. 3, 2303-2308. 16. Chirgwin, J., Przybyla, A., MacDonald, J., and Rutter, W. (1979) Biochemistry 18, 52945299. 17. Sanger, F., Nicklen, S., and Coulseon, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 54635467. 18. Kozak, M. (1981) Nucleic Acid Res. 9, 5233-5252. 19. Ying, S-Y., Becker, A., Swanson, G., Tan, P., Ling, N., Esch, F., Ueno, N., Shimasaki, S., and Guillemin, R. (1987) Biochem. Biophys. Res. Commun. 149, 133-139.
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