ISOLATION AND CHARACTERIZATION OF THE PROMOTER FOR THE GENE CODING FOR THE 68 KDA CARNITINE PALMITOYLTRANSFERASE FROM THE RAT
ISOLATION AND CHARACTERIZATION OF THE PROMOTER FOR THE GENE CODING FOR THE 68 KDA CARNITINE PALMITOYLTRANSFERASE FROM THE RAT
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DOI：
10.1042/bj2860779
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发表时间：
1992-09-15
影响因子：
4.1
通讯作者：
BRADY, LJ
中科院分区：
文献类型：
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作者：
BRADY, PS;PARK, EA;BRADY, LJ
肉毒碱棕榈酰转移酶（CPT）调节长链脂肪酸进入线粒体的流量，用于随后的β-氧化。采用聚合酶链反应从大鼠DASH基因组文库中分离出编码68 kDa CPT基因的启动子的485 bp片段。该启动子含有CREB（环AMP反应元件结合蛋白）在-153至-166处和C/EBP-α（CCAAT/增强子结合蛋白）在-115至-128处的共有结合序列。使用从大鼠肝细胞核中分离的蛋白质进行的DNA酶I足迹分析表明，核蛋白结合的几个区域的存在，最显著的是在-95至-130、-273至-295和包括-395至-465的宽区域。使用纯化的CREB和C/EBP-α进行的DNA酶I足迹研究证实，蛋白质与DNA的结合发生在共有序列预测的位点。将含有481 bp 5'侧翼序列和181 bp非翻译mRNA的片段连接到氯霉素乙酰转移酶（CAT）的结构基因上。当将该质粒转染到Hep G2细胞中时，通过加入1 mM-8-溴-环AMP（8-Br-cAMP）或共转染编码蛋白激酶A（PKA）催化亚基的表达载体，CAT活性被刺激7倍。在共转染实验中测试了几种已知的第二信使和转录因子刺激68 kDa CPT启动子-CAT报告子转录的能力。68 kDa CPT启动子-CAT报告基因转录活性被8-Br-cAMP刺激7倍，而这种诱导被佛波醇酯抑制50%。当68 kDa CPT启动子-CAT报告基因与CREB或C/EBP-α的表达载体共转染时，转录分别增加3倍和10倍。在每种因子存在的情况下，8-Br-cAMP均引起额外的8倍诱导，分别产生25倍和80倍诱导。c-jun表达载体的共转染也增加了由68 kDa CPT启动子驱动的CAT活性，而与c-fos表达载体的共转染没有影响。当C-jun和c-fos的表达载体与68 kDa CPT启动子共转染时，c-fos抑制单独c-jun的诱导。
Carnitine palmitoyltransferase (CPT) regulates the flux of long-chain fatty acids into the mitochondria for subsequent beta-oxidation. A 485 bp segment of the promoter for the gene encoding the 68 kDa CPT was isolated from a rat lambda-DASH genomic library using the polymerase chain reaction. The promoter contained a consensus binding sequence for CREB (cyclic AMP response element binding protein) at -153 to -166, and for C/EBP-alpha (CCAAT/enhancer binding protein) at -115 to -128. DNAase I footprinting using proteins isolated from rat liver nuclei indicated the presence of several regions of nuclear protein binding most notably at -95 to -130, at -273 to -295, and at a wide region encompassing -395 to -465. DNAase I footprinting studies with purified CREB and C/EBP-alpha confirmed that protein binding to DNA occurred at the sites predicted by the consensus sequences. The segment containing 481 bp of 5' flanking sequence plus 181 bp of untranslated mRNA was ligated to the structural gene for chloramphenicol acetyltransferase (CAT). When this plasmid was transfected into Hep G2 cells, CAT activity was stimulated 7-fold by addition of 1 mM-8-bromo-cyclic AMP (8-Br-cAMP) or co-transfection of the expression vector coding for the catalytic subunit of protein kinase A (PKA). The ability of several known second messengers and transcription factors to stimulate transcription of 68 kDa CPT promoter-CAT reporter was tested in co-transfection experiments. 68 kDa CPT promoter-CAT reporter transcription activity was stimulated 7-fold by addition of 8-Br-cAMP, and this induction was depressed 50% by the addition of phorbol esters. When the 68 kDa CPT promoter-CAT reporter was co-transfected with an expression vector for CREB or C/EBP-alpha, transcription was increased 3- and 10-fold respectively. 8-Br-cAMP caused an additional 8-fold induction in the presence of each factor to yield 25- and 80-fold induction respectively. Co-transfection of the expression vector for c-jun also increased the CAT activity driven by the 68 kDa CPT promoter, while co-transfection with the expression vector for c-fos had no effect. When expression vectors for both C-jun and c-fos were co-transfected with the 68 kDa CPT promoter, c-fos depressed the induction seen with c-jun alone.