de la Brousse F C, Shan B, Chen J L
Tularik Inc., South San Francisco, CA 94080, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4096-101. doi: 10.1073/pnas.93.9.4096.
Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis-regulatory elements. A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box. Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts. When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression. When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active. Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression. Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells. These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene.
采用引物延伸法和RACE（cDNA末端快速扩增）分析法来鉴定小鼠ob mRNA的5′末端并进行测序。该序列用于获得一个包含编码基因首个外显子的重组噬菌体。对小鼠ob基因首个外显子上游紧邻区域的DNA序列分析揭示了与假定顺式调控元件相对应的DNA序列。在转录起始位点上游30 - 34个碱基对处观察到一个典型的TATA框，并且在TATA框上游紧邻区域鉴定出一个转录因子C/EBP家族成员的假定结合位点。从原代脂肪细胞制备的核提取物含有一种DNA结合活性，它能够与假定的C/EBP反应元件进行强烈且特异性的相互作用；针对C/EBPα的抗体中和了脂肪细胞核提取物中存在的DNA结合活性。当与萤火虫荧光素酶报告基因连接并转染到原代脂肪细胞中时，小鼠ob基因的假定启动子促进了荧光素酶的表达。当转染到缺乏C/EBPα的HepG2细胞中时，小鼠ob启动子活性微弱。通过与C/EBPα表达载体共转染来补充C/EBPα可显著刺激荧光素酶的表达。最后，在C/EBP反应元件处发生突变的ob启动子变体在原代脂肪细胞和HepG2细胞中均无活性。这些观察结果为鉴定一个能够指导小鼠ob基因表达的功能性启动子提供了证据。
