Rosen G D, Birkenmeier T M, Dean D C
Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 1991 May 15;88(10):4094-8. doi: 10.1073/pnas.88.10.4094.
A cDNA for the alpha 4 chain of the alpha 4 beta 1 integrin was described previously [Takada, Y., Elices, M. J., Crouse, C. & Hemler, M. E. (1989) EMBO J. 8, 1361-1368]. Primer extension analysis indicated that alpha 4 mRNA extended well beyond the 5' end of this cDNA. To clone this 5' sequence, a primer extension cDNA library was constructed, and a cDNA extending an additional 660 base pairs was isolated. This cDNA hybridized to multiple mRNAs in both T and B lymphocytes, but no alpha 4 mRNA was found in different tissues or in adherent cell lines. A single alpha 4 gene was detected in a genomic Southern blot when hybridization was done at high stringency; however, additional bands were observed at lower stringency, indicating the presence of alpha 4-related genes. Some of the different mRNAs that hybridize to the alpha 4 cDNA may then be the products of these related genes. Analysis of the alpha 4 genomic sequence revealed a large first exon of 958 base pairs. Interestingly, translation of alpha 4 initiates from the second ATG in this exon (nucleotide + 744). The first ATG (nucleotide +21) is followed by a termination codon 21 amino acids downstream. Such upstream ATG codons have been implicated in translational control of protooncogenes. One major transcriptional start site was identified by using S1 nuclease and primer extension mapping. Consensus sequences for DNA regulatory elements were found upstream of the gene and in exon 1 and intron 1. The alpha 4 gene 5' flanking region acted as a promoter in transfection assays. Detailed characterization of the promoter should provide insight into molecular events regulating expression and tissue specificity of alpha 4.
先前已报道了α4β1整合素α4链的cDNA[高田洋、埃利塞斯·M·J、克劳斯、C和赫姆勒·M·E（1989年）《欧洲分子生物学组织杂志》8卷，1361 - 1368页]。引物延伸分析表明，α4 mRNA延伸至该cDNA 5'端之外。为克隆该5'序列，构建了引物延伸cDNA文库，并分离出一个额外延伸660个碱基对的cDNA。该cDNA与T淋巴细胞和B淋巴细胞中的多种mRNA杂交，但在不同组织或贴壁细胞系中未发现α4 mRNA。当在高严谨度下进行杂交时，在基因组Southern印迹中检测到单个α4基因；然而，在较低严谨度下观察到额外条带，表明存在α4相关基因。与α4 cDNA杂交的一些不同mRNA可能是这些相关基因的产物。对α4基因组序列的分析揭示了一个958个碱基对的大的第一外显子。有趣的是，α4的翻译从该外显子中的第二个ATG（核苷酸+744）起始。第一个ATG（核苷酸+21）之后21个氨基酸处是一个终止密码子。这种上游ATG密码子与原癌基因的翻译控制有关。通过使用S1核酸酶和引物延伸图谱鉴定出一个主要转录起始位点。在基因上游以及外显子1和内含子1中发现了DNA调控元件的共有序列。在转染实验中，α4基因5'侧翼区域起启动子作用。对启动子的详细表征应能深入了解调节α4表达和组织特异性的分子事件。
