Park S, Moon Y, Kim K, Ahn Y, Kim Y
Department of Biochemistry and Molecular Biology, the Institute of Genetic Science, Yonsei University College of Medicine, Seoul, South Korea.
Biochim Biophys Acta. 1997 Sep 12;1353(3):236-40. doi: 10.1016/s0167-4781(97)00094-8.
Two phage clones, lambda hgACL21 and lambda hgACL28, harboring the 5' flanking region of human ATP-citrate lyase (ACL) gene were identified by screening about 1.5 X 10(6) recombinant plaques from the lambdaEMBL3-human placental genomic DNA library. The 5' flanking region of ACL had the CAAT box on -92 bp from the transcription initiation site (+1), however, the TATA box was not found. The primer extension and rapid amplification of cDNA end showed that mRNA is transcribed at a thymine extending 12 bp upstream of the reported cDNA end. The sequences of 5' flanking region in 1.5 kb size of human ACL showed 60% homology with those of rat; however, no homology was found in the exon 1 and intron 1 region. Several consensus sequences, including four Sp1 binding sites, were found in the 5' flanking region of this gene. The promoter activity was assayed by transfecting the 3' or 5' deletion clones of ACL-chloramphenicol acetyl transferase (CAT) plasmid into PLC/PRF5 cells. The clone that contains the part of the first intron sequences from -659 to +440 bp showed the highest CAT activity in the transient transfection assay. High promoter activities were maintained until the transcription initiation site was removed. It is suggested that the sequences from -213 to +12 which contain three Sp1-binding sequences, CAAT box, and the transcription initiation site were necessary as a mean of for exerting the basal promoter activity of ACL gene.
通过从λEMBL3-人胎盘基因组DNA文库中筛选约1.5×10⁶个重组噬菌斑，鉴定出两个携带人ATP-柠檬酸裂解酶（ACL）基因5'侧翼区的噬菌体克隆，即λhgACL21和λhgACL28。ACL的5'侧翼区在转录起始位点（+1）上游-92 bp处有CAAT盒，但未发现TATA盒。引物延伸和cDNA末端快速扩增表明，mRNA在报道的cDNA末端上游12 bp处的胸腺嘧啶处转录。人ACL 1.5 kb大小的5'侧翼区序列与大鼠的序列有60%的同源性；然而，在第1外显子和第1内含子区域未发现同源性。在该基因的5'侧翼区发现了几个共有序列，包括四个Sp1结合位点。通过将ACL-氯霉素乙酰转移酶（CAT）质粒的3'或5'缺失克隆转染到PLC/PRF5细胞中来检测启动子活性。在瞬时转染实验中，包含从-659到+440 bp的第一个内含子序列部分的克隆显示出最高的CAT活性。直到转录起始位点被去除，仍保持高启动子活性。提示包含三个Sp1结合序列、CAAT盒和转录起始位点的-213至+12序列作为发挥ACL基因基础启动子活性的一种方式是必要的。
