Identification and characterization of the promoter for the cytotactin gene.
Identification and characterization of the promoter for the cytotactin gene.
复制标题
细胞趋化素基因启动子的鉴定和表征。
DOI：
10.1073/pnas.87.17.6497
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发表时间：
1990
影响因子：
11.1
通讯作者：
Edelman,GM
中科院分区：
文献类型：
--
作者：
Jones,FS;Crossin,KL;Cunningham,BA;Edelman,GM
在鸡胚发育过程中，细胞外糖蛋白cytokerin以一种特征性的、复杂的时空序列表达。为了确定其表达的各种控制元件，已经分离并表征了cytokerin基因的启动子区。通过使用cDNA序列5'端附近的片段从基因组文库中分离克隆。发现该cDNA片段的序列分布在由大的第一内含子分隔的两个外显子上。通过S1核酸酶和引物延伸作图确定转录起始位点。对一个4.3 kb的基因组DNA克隆进行测序，该克隆在RNA起始位点上游含有3986个碱基对（bp），第一个外显子和第一个内含子的一部分，揭示了许多与真核基因的调控和表达有关的序列基序。这些包括CCAAT盒，佛波酯反应元件，增强子元件，和位于主要RNA帽位点上游24 bp的共识TATA序列。侧翼序列也包含了一些二分体对称和直接重复独特的细胞色素蛋白，以及一系列的A + T丰富的序列，类似于engrailed元素的区域。将含有融合到氯霉素乙酰转移酶无启动子基因的cytokrin基因上游区片段的构建体瞬时转染到鸡胚成纤维细胞中以确定功能性启动子序列。虽然从-721到+121的序列显示出最小的启动子活性，但在鸡胚成纤维细胞中产生最大表达需要-3986到+374之间的整个区域。将-3986/+374氯霉素乙酰转移酶质粒转染入人U251 MG星形细胞瘤细胞而非HT 1080纤维肉瘤细胞导致氯霉素乙酰转移酶表达，这与观察到的仅由U251 MG细胞系合成细胞色素蛋白一致。这些数据表明，鸡cytokrin启动子可以控制在另一个物种的细胞内的细胞类型特异性的方式表达。这些研究提供了一个基础的顺式元件和反式因子，支配的细胞色素基因的发育表达的解剖。
The extracellular glycoprotein cytotactin is expressed in a characteristic and complex spatiotemporal sequence during development of the chicken embryo. To identify the various control elements underlying its expression, the promoter region of the cytotactin gene has been isolated and characterized. Clones were isolated from genomic libraries by using a fragment near the 5' end of the cDNA sequence. The sequence of this cDNA fragment was found to be distributed over two exons separated by a large first intron. The site of transcription initiation was determined by S1 nuclease and primer-extension mapping. Sequencing of a 4.3-kilobase (kb) genomic DNA clone that contains 3986 base pairs (bp) upstream of the RNA start site, the first exon, and part of the first intron revealed a number of sequence motifs implicated in the regulation and expression of eukaryotic genes. These included CCAAT boxes, phorbol ester-responsive elements, enhancer elements, and a consensus TATA sequence located 24 bp upstream of the major RNA cap site. The flanking sequence also contained a number of regions of dyad symmetry and direct repeats unique to cytotactin, as well as an array of A + T-rich sequences that resemble engrailed elements. Constructs containing fragments of the upstream region of the cytotactin gene fused to a promoterless gene for chloramphenicol acetyltransferase were transiently transfected into chicken embryo fibroblasts to define functional promoter sequences. Although sequences from -721 to +121 exhibited minimal promoter activity, the entire region between -3986 to +374 was required to yield maximal expression in chicken embryo fibroblasts. Transfection of the -3986/+374 chloramphenicol acetyltransferase plasmid into the human U251MG astrocytoma cells but not HT1080 fibrosarcoma cells resulted in chloramphenicol acetyltransferase expression, consistent with the observed synthesis of cytotactin protein only by the U251MG cell line. These data indicate that the chicken cytotactin promoter can control expression in a cell type-specific fashion within cells of another species. These studies provide a basis for the dissection of cis elements and trans factors that govern the developmental expression of the cytotactin gene.