Suen T C, Hung M C
Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Mol Cell Biol. 1990 Dec;10(12):6306-15. doi: 10.1128/mcb.10.12.6306-6315.1990.
A 2.4-kb rat neu genomic DNA fragment that hybridized to the 5'-most coding sequence of the rat neu cDNA was cloned. S1 nuclease mapping identified multiple transcriptional initiation sites. DNA sequence analysis revealed that this fragment contained 64 bp of the first intron, 81 bp of the first exon, and the upstream noncoding sequence of the neu gene. The sequence immediately upstream of the translation start site was G + C rich (greater than 75%) and contained a consensus CCAAT sequence despite the absence of a TATA box. An Sp1-binding site was found, in addition to various sequence motifs common to the promoters of the human neu gene (erbB2), the epidermal growth factor receptor gene, and the simian virus 40 enhancer. A 2.2-kb EcoRI-Narl fragment containing sequences upstream from the 3'-most transcriptional start site was fused to the bacterial chloramphenicol acetyltransferase reporter gene and shown to promote transcription efficiently. A series of promoter deletion constructs was made, and results from transfection and subsequent chloramphenicol acetyltransferase assays suggested the presence of multiple cis-acting elements that contributed either positively or negatively to the transcription activity. Cotransfection competition experiments using subcloned cis-acting elements confirmed the existence of trans-acting factors interacting with these DNA fragments. In addition, a gel retardation assay was performed to demonstrate the physical binding of nuclear factors to certain fragments. The results complemented those of the deletion studies and led us to conclude that transcriptional regulation of the neu proto-oncogene involves at least one negative and three positive trans-acting factors interacting with different cis-acting elements along the neu gene promoter.
克隆了一个与大鼠neu cDNA 5'端最编码序列杂交的2.4kb大鼠neu基因组DNA片段。S1核酸酶图谱分析确定了多个转录起始位点。DNA序列分析表明，该片段包含第一个内含子的64bp、第一个外显子的81bp以及neu基因的上游非编码序列。翻译起始位点上游紧邻的序列富含G + C（大于75%），尽管没有TATA框，但包含一个共有CCAAT序列。除了人neu基因（erbB2）、表皮生长因子受体基因和猿猴病毒40增强子启动子共有的各种序列基序外，还发现了一个Sp1结合位点。一个包含3'端最转录起始位点上游序列的2.2kb EcoRI - Narl片段与细菌氯霉素乙酰转移酶报告基因融合，并显示能有效促进转录。构建了一系列启动子缺失构建体，转染及随后的氯霉素乙酰转移酶检测结果表明存在多个顺式作用元件，它们对转录活性有正向或负向作用。使用亚克隆的顺式作用元件进行的共转染竞争实验证实了与这些DNA片段相互作用的反式作用因子的存在。此外，进行了凝胶阻滞试验以证明核因子与某些片段的物理结合。这些结果补充了缺失研究的结果，使我们得出结论，neu原癌基因的转录调控涉及至少一个负向和三个正向反式作用因子，它们与neu基因启动子上不同的顺式作用元件相互作用。
