Bornstein P, Alfi D, Devarayalu S, Framson P, Li P
Department of Biochemistry, University of Washington, Seattle 98195.
J Biol Chem. 1990 Sep 25;265(27):16691-8.
We have isolated the mouse thrombospondin (TS) gene and determined the DNA sequence of the first nine exons and eight introns. Comparison with the human cDNA sequence reveals a high degree of conservation in coding sequences. Exon 3 of the mouse gene, which encodes the heparin-binding domain of TS, has a higher degree of nucleotide substitution than the other exons, but the distribution of charged and hydrophobic amino acids found in the human protein is generally conserved. DNA and protein sequences in exons 6-9, which encode a procollagen homology and motifs very similar to those found in at least two malarial parasite proteins, are highly conserved. The first two of the three malarial homologies in TS, which are also found in properdin and in components C6-9 of the lytic complement complex, are each encoded by a separate exon (8 and 9) in the mouse gene. Since the sequence data did not reveal substantial similarity in sequence between intron I in the human and mouse genes, we have reexamined the role of the first intron in the transcriptional regulation of the human TS gene. In accord with published studies (Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222-11227), we find that deletion of some intronic segments from TS-chloramphenicol acetyltransferase (CAT) constructs reduces CAT activity in NIH 3T3 cells. However, deletion of the same sequences from TS-bovine growth hormone constructs does not affect the expression of bovine growth hormone in these cells. We conclude that differences in the activity of TS-CAT constructs reflect post-transcriptional differences that are peculiar to the resulting chimeric transcripts and that there is currently no evidence for a transcriptional enhancer in the first intron of the human TS gene.
我们已经分离出小鼠血小板反应蛋白（TS）基因，并确定了前九个外显子和八个内含子的DNA序列。与人类cDNA序列比较发现编码序列具有高度保守性。小鼠基因的外显子3编码TS的肝素结合结构域，其核苷酸取代程度高于其他外显子，但人类蛋白质中带电荷和疏水氨基酸的分布总体上是保守的。外显子6 - 9中的DNA和蛋白质序列高度保守，它们编码的原胶原同源性和基序与至少两种疟原虫蛋白质中的非常相似。TS中的三个疟疾同源性中的前两个，也存在于备解素和溶细胞补体复合物的C6 - 9成分中，在小鼠基因中分别由一个单独的外显子（8和9）编码。由于序列数据未揭示人类和小鼠基因中内含子I之间在序列上有实质性相似性，我们重新研究了第一个内含子在人类TS基因转录调控中的作用。与已发表的研究一致（Laherty, C.D., Gierman, T.M., and Dixit, V.M. (1989) J. Biol. Chem. 264, 11222 - 11227），我们发现从TS - 氯霉素乙酰转移酶（CAT）构建体中缺失一些内含子片段会降低NIH 3T3细胞中的CAT活性。然而，从TS - 牛生长激素构建体中缺失相同序列并不影响这些细胞中牛生长激素的表达。我们得出结论，TS - CAT构建体活性的差异反映了转录后差异，这些差异是所产生的嵌合转录本特有的，并且目前没有证据表明人类TS基因的第一个内含子中有转录增强子。
