Tanaka M, Taniguchi T, Yamamoto I, Sakaguchi K, Yoshizato H, Ohkubo T, Nakashima K
Department of Biochemistry, Faculty of Medicine, Mie University, Tsu, Japan.
DNA Cell Biol. 1998 Oct;17(10):859-68. doi: 10.1089/dna.1998.17.859.
To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.
为了解脊椎动物中胰岛素样生长因子-I（IGF-I）基因表达和加工的综合机制，我们研究了牙鲆（Paralichthys olivaceus）中该基因的基因结构、启动子和转录起始位点、可变剪接和聚腺苷酸化位点以及cDNA结构。牙鲆IGF-I基因由5个外显子和4个内含子组成，跨度为17.5 kb。通过Northern印迹分析，在肝脏中发现了4.7 kb和2.9 kb两种主要的mRNA类别。cDNA克隆和逆转录聚合酶链反应（RT-PCR）分析表明，这两种mRNA类别是由两个聚腺苷酸化信号的交替使用产生的两个不同大小的3'-非编码区导致的。通过RT-PCR和测序进一步分析发现，这些mRNA类别都包含编码两种形式的IGF-I前体肽preproIGF-I-1和preproIGF-I-2的两个mRNA亚类。两种形式的preproIGF-I共享相同的信号肽和成熟IGF-I结构域，但由于外显子3中的可变剪接而包含不同的E结构域。牙鲆IGF-I的成熟形式由68个氨基酸残基组成，分子量较小，为7486。在5'-侧翼区域，通过核糖核酸酶保护试验在翻译起始密码子上游-230至-130之间鉴定出一个主要转录起始位点和四个次要转录起始位点，但在其上游区域直至-724未检测到典型的TATA盒或GC盒。结果表明，一些未知的转录起始因子在促进IGF-I基因表达中发挥作用。
