MOLECULAR CLONING AND EXPRESSION OF ALPHA INHIBIN

Abstract

Inhibins are gonadal glycoproteins that preferentially inhibit the secretion and synthesis of follicle stimulating hormone (FSH) but not luetinizing hormone (LH). There are two forms of inhibin, designated as inhibin A (??A) and inhibin B (??B), which are composed of a common ? subunit linked by two disulphide bonds to either a ?A or a ?B subunit. In the male, inhibin inhibits spermatogonia DNA synthesis and reduces spermatogonia number. In females, inhibin has been suggested to inhibit oocyte meiosis, increase the number of ovarian follicles and stimulate thecal steriodogenesis in vitro. A targeted deletion of the a-inhibin gene from mouse embryonic stem cells resulted in the development of gonadal stromal tumours. Thus, inhibin has been the first secreted protein shown to have tumour suppressing activity. Ovaries of a New Zealand sheep were obtained from a local abattoir and the total RNA was isolated. The mRNAs were isolated and used to construct a cDNA library. The cDNA library was screened using an internal Inhibins are gonadal glycoproteins that preferentially inhibit the secretion and synthesis of follicle stimulating hormone (FSH) but not luetinizing hormone (LH). There are two forms of inhibin, designated as inhibin A (??A) and inhibin B (??B), which are composed of a common ? subunit linked by two disulphide bonds to either a ?A or a ?B subunit. In the male, inhibin inhibits spermatogonia DNA synthesis and reduces spermatogonia number. In females, inhibin has been suggested to inhibit oocyte meiosis, increase the number of ovarian follicles and stimulate thecal steriodogenesis in vitro. A targeted deletion of the a-inhibin gene from mouse embryonic stem cells resulted in the development of gonadal stromal tumours. Thus, inhibin has been the first secreted protein shown to have tumour suppressing activity. Ovaries of a New Zealand sheep were obtained from a local abattoir and the total RNA was isolated. The mRNAs were isolated and used to construct a cDNA library. The cDNA library was screened using an internal a-inhibin sequence from bovine (probe S5) and five positive plaques were obtained. The DNA from these plaques were then amplified by PCR using ?gt10 specific primers. The largest fragment obtained from the PCR was 880 bp and it was restricted using restriction enzyme, EcoR1 and subcloned into pUC19 vector for sequencing. The sequence indicated the presence of a 863 bp (?JS4) fragment which constituted a partial cDNA. It however had the full-length structural gene of a-inhibin required for expression studies. This sequence was deposited at the Genbank under the accession number L28815. The amplification of the full-length structural gene of a-inhibin was performed using primers designed from the sequence of the ?JS4. The PCR fragment was subcloned directly without any modification into pT7 Blue vector. The recombinant plasmid harbouring the a-inhibin gene in the corect orientation was identified by restriction analysis and subsequently sequenced. The presence of a full-length structural gene of ?-inhibin was confirmed. Inhibins are gonadal glycoproteins that preferentially inhibit the secretion and synthesis of follicle stimulating hormone (FSH) but not luetinizing hormone (LH). There are two forms of inhibin, designated as inhibin A (??A) and inhibin B (??B), which are composed of a common ? subunit linked by two disulphide bonds to either a ?A or a ?B subunit. In the male, inhibin inhibits spermatogonia DNA synthesis and reduces spermatogonia number. In females, inhibin has been suggested to inhibit oocyte meiosis, increase the number of ovarian follicles and stimulate thecal steriodogenesis in vitro. A targeted deletion of the a-inhibin gene from mouse embryonic stem cells resulted in the development of gonadal stromal tumours. Thus, inhibin has been the first secreted protein shown to have tumour suppressing activity. Ovaries of a New Zealand sheep were obtained from a local abattoir and the total RNA was isolated. The mRNAs were isolated and used to construct a cDNA library. The cDNA library was screened using an internal a-inhibin sequence from bovine (probe S5) and five positive plaques were obtained. The DNA from these plaques were then amplified by PCR using ?gt10 specific primers. The largest fragment obtained from the PCR was 880 bp and it was restricted using restriction enzyme, EcoR1 and subcloned into pUC19 vector for sequencing. The sequence indicated the presence of a 863 bp (?JS4) fragment which constituted a partial cDNA. It however had the full-length structural gene of a-inhibin required for expression studies. This sequence was deposited at the Genbank under the accession number L28815. The amplification of the full-length structural gene of a-inhibin was performed using primers designed from the sequence of the ?JS4. The PCR fragment was subcloned directly without any modification into pT7 Blue vector. The recombinant plasmid harbouring the ?-inhibin gene in the corect orientation was identified by restriction analysis and subsequently sequenced. The presence of a full-length structural gene of ?-inhibin was confirmed. ?-inhibin sequence from bovine (probe S5) and five positive plaques were obtained. The DNA from these plaques were then amplified by PCR using ?gt10 specific primers. The largest fragment obtained from the PCR was 880 bp and it was restricted using restriction enzyme, EcoR1 and subcloned into pUC19 vector for sequencing. The sequence indicated the presence of a 863 bp (?JS4) fragment which constituted a partial cDNA. It however had the full-length structural gene of a-inhibin required for expression studies. This sequence was deposited at the Genbank under the accession number L28815. The amplification of the full-length structural gene of a-inhibin was performed using primers designed from the sequence of the ?JS4. The PCR fragment was subcloned directly without any modification into pT7 Blue vector. The recombinant plasmid harbouring the a-inhibin gene in the corect orientation was identified by restriction analysis and subsequently sequenced. The presence of a full-length structural gene of ?-inhibin was confirmed.