Ewulonu U K, Ravi L, Medof M E
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106.
Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4675-9. doi: 10.1073/pnas.88.11.4675.
Decay-accelerating factor (DAF) expression modulates susceptibility of cells to autologous complement attack. To characterize the regulatory region controlling DAF gene transcription, genomic DNA extending from 815 base pairs (bp) upstream to approximately 4 kilobases downstream of DAF's AUG codon (designated +1) was cloned and sequenced. The 5' flanking sequence showed 59-76% G + C content (-355 to +1), at least one GC box(es) (-135 to -131), and variable length sequences (from -629 to -285) conforming to the motifs TCCTCC and TCn. Nuclease S1 digestions and primer extensions localized a major transcriptional start site to -82/-81, 38 bp downstream of a possible TATA variant, (A)TTTAA. In COS cell transfections, the sequence encompassing -815 to -67 functioned 2.5% as efficiently as the Rous sarcoma virus 3' long terminal repeat, but following deletion upstream of -355 its activity increased approximately 4-fold. Two octanucleotides exhibiting partial homology to phorbol 12-myristate 13-acetate (PMA) and cAMP responsive elements (PREs and CREs, respectively) were detected, and the respective modulators enhanced transcriptional efficiency 2- and approximately 10-fold, respectively. Thus, the DAF gene promoter (i) exhibits sequences resembling both conventional and unconventional transcriptional control elements, (ii) possesses a region with negative regulatory activity, and (iii) responds to PMA and cAMP induction presumably via PRE- and CRE-like enhancer elements.
衰变加速因子（DAF）的表达调节细胞对自身补体攻击的敏感性。为了表征控制DAF基因转录的调控区域，克隆并测序了从DAF的AUG密码子（指定为+1）上游815个碱基对（bp）延伸至下游约4千碱基的基因组DNA。5'侧翼序列显示G + C含量为59 - 76%（-355至+1），至少有一个GC框（-135至-131），以及符合TCCTCC和TCn基序的可变长度序列（从-629至-285）。核酸酶S1消化和引物延伸将主要转录起始位点定位到-82 / -81，位于可能的TATA变体（A）TTTAA下游38 bp处。在COS细胞转染中，包含-815至-67的序列的功能效率为劳氏肉瘤病毒3'长末端重复序列的2.5%，但在删除-355上游序列后，其活性增加了约4倍。检测到两个分别与佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）和cAMP反应元件（分别为PRE和CRE）具有部分同源性的八核苷酸，相应的调节剂分别将转录效率提高了2倍和约10倍。因此，DAF基因启动子（i）表现出类似于传统和非传统转录控制元件的序列，（ii）具有一个具有负调控活性的区域，并且（iii）可能通过类似PRE和CRE的增强子元件对PMA和cAMP诱导作出反应。
