Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana).
Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana).
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DOI：
10.1002/1097-010x(20010215)289:3
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发表时间：
2001-02
期刊：
影响因子：
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通讯作者：
Li Wang;Myung Sik Yoo;Hae Mook Kang;W. Im;H. Choi;Jan Bogerd;Hyuk Bang Kwon
中科院分区：
文献类型：
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作者：
Li Wang;Myung Sik Yoo;Hae Mook Kang;W. Im;H. Choi;Jan Bogerd;Hyuk Bang Kwon
我们从牛蛙 (Rana catesbeiana) 大脑中分别分离出了编码 GnRH1 和 GnRH2 前体的 cDNA。第一个 cDNA 由 648 bp 组成，包含 270 个核苷酸的开放阅读框，编码牛蛙 GnRH1 前体。第二个 cDNA 由 1053 bp 组成，包含 255 个核苷酸的开放阅读框，编码牛蛙 GnRH2 前体。两种类型的牛蛙 GnRH 前体具有与其他 GnRH 前体相似的分子结构，由信号肽、随后的 GnRH 十肽、保守的羧基末端酰胺化和蛋白水解加工位点以及 GnRH 相关肽 (GAP) 组成。此外，我们还鉴定了第三种 cDNA，其 GAP 编码区含有 24 个额外的核苷酸。基因组 PCR 和序列分析证实该 cDNA 代表牛蛙 GnRH2 前体 mRNA 的替代剪接变体。牛蛙 GnRH1 前体与其非洲爪蟾和哺乳动物对应物分别表现出 60% 和不到 40% 的氨基酸同一性，而牛蛙 GnRH2 前体与其非哺乳动物对应物表现出 50% 至 60% 的氨基酸同一性，但与其哺乳动物对应物仅具有 25% 的氨基酸同一性。 Northern印迹分析显示，在牛蛙前脑中表达了约0.75kb的单一GnRH1前体mRNA种类，在牛蛙中脑/后脑中表达了约1.1kb的单一GnRH2前体mRNA种类。此外，两种牛蛙 GnRH 前体 mRNA 均表现出差异的时空表达模式。基因组 Southern 印迹分析表明，牛蛙 GnRH 基因均以单拷贝基因形式存在。这是关于从两栖动物物种中分子克隆 GnRH2 前体 cDNA 的第一份报告。此外，我们提供的数据显示，选择性剪接可用于生成不同的 GnRH2 前体 mRNA。 J.Exp。动物园。 289：190-201，2001 年。
We have isolated the cDNAs encoding the GnRH1 and GnRH2 precursors, respectively, from bullfrog (Rana catesbeiana) brain. The first cDNA consists of 648 bp and contains an open-reading frame of 270 nucleotides, encoding the bullfrog GnRH1 precursor. The second cDNA consists of 1053 bp and contains an open-reading frame of 255 nucleotides, encoding the bullfrog GnRH2 precursor. Both types of bullfrog GnRH precursor have a similar molecular architecture as observed in other GnRH precursors, consisting of a signal peptide, followed by the GnRH decapeptide, a conserved carboxy-terminal amidation and proteolytical processing site, and a GnRH-associated peptide (GAP). In addition, we have identified a third cDNA, containing 24 additional nucleotides in its GAP-coding region. Genomic PCR and sequence analysis confirmed that this cDNA represents an alternative splice variant of the bullfrog GnRH2-precursor pre-mRNA. The bullfrog GnRH1 precursor exhibits 60% and less than 40% amino acid identity to its Xenopus and mammalian counterparts, respectively, whereas the bullfrog GnRH2 precursor displays 50% to 60% amino acid identity to that of its nonmammalian counterparts, but shares only 25% amino acid identity with its mammalian counterparts. Northern blot analysis revealed a single GnRH1-precursor mRNA species of approximately 0.75 kilobases, expressed in bullfrog forebrain, and a single GnRH2-precursor mRNA species of approximately 1.1 kilobases, expressed in bullfrog midbrain/hindbrain. Furthermore, both bullfrog GnRH-precursor mRNAs exhibited a differential spatiotemporal expression pattern. Genomic Southern blot analysis indicated that both bullfrog GnRH genes are present as single copy genes. This is the first report on the molecular cloning of a GnRH2-precursor cDNA from an amphibian species. In addition, we present data showing that alternative splicing is utilized to generate different GnRH2-precursor mRNAs. J. Exp. Zool. 289:190-201, 2001.