Mikawa S, Yoshikawa G, Aoki H, Yamano Y, Sakai H, Komano T
Department of Agricultural Chemistry, Kyoto University, Japan.
Biosci Biotechnol Biochem. 1995 Jan;59(1):87-92. doi: 10.1271/bbb.59.87.
Goat IGF-I (gIGF-I) cDNA was cloned using the reverse transcriptase-polymerase chain reaction (RT-PCR). The cDNA, which was homologous to rat class 1 IGF-I cDNA, was 969 bp long. From the open reading frame found in it, we predicted a 154 amino acid protein consisting of a 49 amino acid signal peptide, a 70 amino acid mature IGF-I peptide, and a 35 amino acid E domain (the COOH-terminal peptide). From the gIGF-I gene, we isolated and sequenced some segments containing four exons that encompassed the entire gIGF-I cDNA sequence. Goat liver RNA was analyzed by RT-PCR, and the nucleotides of the RT-PCR products were sequenced and checked with the nucleotide sequences of the segments from the gIGF-I gene. The gene had three leader exons (1W, 1, and 2, from upstream to downstream) and was transcribed into three kinds of mRNAs (classes 1W, 1, and 2). Another RNA species was detected by RT-PCR analysis of exon 1W. We sequenced it and found that in this transcript, the 3'-portion of exon 1 was inserted between exons 1W and 3, resulting in class 1W-1 del. mRNA. That is to say, the gIGF-I gene had three leader exons and four kinds of mature mRNA.
利用逆转录聚合酶链反应(RT-PCR)克隆了山羊胰岛素样生长因子I(gIGF-I)的互补DNA(cDNA)。该cDNA与大鼠1类IGF-I cDNA同源,长度为969 bp。根据其中发现的开放阅读框,我们预测了一种由154个氨基酸组成的蛋白质,它包括一个49个氨基酸的信号肽、一个70个氨基酸的成熟IGF-I肽和一个35个氨基酸的E结构域(COOH末端肽)。从gIGF-I基因中,我们分离并测序了一些包含四个外显子的片段,这些外显子涵盖了整个gIGF-I cDNA序列。通过RT-PCR分析山羊肝脏RNA,并对RT-PCR产物的核苷酸进行测序,并与来自gIGF-I基因片段的核苷酸序列进行比对。该基因有三个前导外显子(从上游到下游依次为1W、1和2),并转录成三种mRNA(1W类、1类和2类)。通过对1W外显子的RT-PCR分析检测到了另一种RNA。我们对其进行了测序,发现在该转录本中,外显子1的3'部分插入到外显子1W和3之间,产生了1W-1缺失型mRNA。也就是说,gIGF-I基因有三个前导外显子和四种成熟mRNA。