Brown A L, Rechler M M
Growth and Development Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Mol Endocrinol. 1990 Dec;4(12):2039-51. doi: 10.1210/mend-4-12-2039.
We have isolated clones encoding the rat insulin-like growth factor-binding protein-2 (IGFBP-2) gene and determined its organization and nucleotide sequence. The rat IGFBP-2 gene spans at least 8 kilobases and consists of four exons, each of which contains protein-coding sequences. The amino acid sequences of exons 1, 3, and 4 are 32-50% identical to the corresponding exons of human IGFBP-1 and IGFBP-3, and 87-91% identical to those of human IGFBP-2. The 18 cysteines in the mature binding proteins are conserved. Exon 2 shows negligible homology. Primer-extended reverse transcription indicated that the 5' end of IGFBP-2 mRNA is 151 nucleotides up-stream from the translation start site [designated nucleotide (nt) -151]. Consistent with this result, IGFBP-2 mRNA protected a genomic fragment terminating at approximately nt -148, as well as smaller fragments. A 1260 nt fragment containing 1144 nt of 5' flanking region had promoter activity when inserted in the correct orientation into a plasmid containing a promoterless luciferase reporter gene and transiently transfected into BRL-3A rat liver cells, which express IGFBP-2, but not when transfected into H4-II-E cells, which do not express IGFBP-2. The IGFBP-2 gene lacks a TATA box immediately up-stream from the transcription initiation site. It is GC rich (66% between nt -270 and +385) and contains GC boxes that might be recognized by transcription factors Sp1 or ETF. The promoter region contains multiple direct and indirect repeats. One direct repeat contains a variant Sp1 site (-158 to -150) near the consensus Sp1 site at nt -138 to -130. The 5' flanking region also contains motifs that might be recognized by transcription factors AP-1 (Jun/Fos), AP-2, and liver factor B1. The role of these sites in basal and regulated expression of the IGFBP-2 gene remains to be determined.
我们已分离出编码大鼠胰岛素样生长因子结合蛋白2（IGFBP - 2）基因的克隆，并确定了其结构和核苷酸序列。大鼠IGFBP - 2基因跨度至少8千碱基，由四个外显子组成，每个外显子都包含蛋白质编码序列。外显子1、3和4的氨基酸序列与人类IGFBP - 1和IGFBP - 3的相应外显子有32% - 50%的同源性，与人类IGFBP - 2的相应外显子有87% - 91%的同源性。成熟结合蛋白中的18个半胱氨酸是保守的。外显子2显示出可忽略不计的同源性。引物延伸逆转录表明，IGFBP - 2 mRNA的5'端在翻译起始位点上游151个核苷酸处[指定为核苷酸（nt）- 151]。与该结果一致，IGFBP - 2 mRNA保护了一个在大约nt - 148处终止的基因组片段以及较小的片段。当一个包含1144 nt 5'侧翼区域的1260 nt片段以正确方向插入到一个含有无启动子荧光素酶报告基因的质粒中，并瞬时转染到表达IGFBP - 2的BRL - 3A大鼠肝细胞中时，该片段具有启动子活性，但转染到不表达IGFBP - 2的H4 - II - E细胞中时则没有活性。IGFBP - 2基因在转录起始位点上游紧邻处缺乏TATA框。它富含GC（在nt - 270至 + 385之间为66%），并含有可能被转录因子Sp1或ETF识别的GC框。启动子区域包含多个直接和间接重复序列。一个直接重复序列在nt - 138至 - 130处的共有Sp1位点附近含有一个变体Sp1位点（- 158至 - 150）。5'侧翼区域还包含可能被转录因子AP - 1（Jun/Fos）、AP - 2和肝因子B1识别的基序。这些位点在IGFBP - 2基因基础表达和调控表达中的作用尚待确定。
