Choi S J, Oh D H, Song C S, Roy A K, Chatterjee B
Bioproducts Research Center, Yonsei University, Seoul, South Korea.
Biochim Biophys Acta. 1995 Nov 7;1264(2):215-22. doi: 10.1016/0167-4781(95)00146-8.
The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70,301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5' untranslated region (UTR), 791 bp of 3' UTR, two putative polyadenylation sites, and a poly(A19-23) tail. Screening of a rat genomic DNA library in the lambda phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position -1140 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing -1140 to +84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at -284 and -93), two GC boxes for Sp1 binding (at -160 and -68), two AP2 sites (at -359 and -25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at -737 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified.
已分离并测序了大鼠过氧化物酶体肉碱辛酰基转移酶（COT）的全长cDNA和天然基因。这个2681bp长的cDNA包含一个编码613个氨基酸的开放阅读框，推导的蛋白质分子量为70301，并且有一个C末端过氧化物酶体靶向序列（丙氨酸 - 组氨酸 - 亮氨酸）。分离出的COT cDNA有51bp的5'非翻译区（UTR），791bp的3'UTR，两个推定的聚腺苷酸化位点，以及一个聚（A19 - 23）尾。用COT cDNA探针筛选λ噬菌体中的大鼠基因组DNA文库，得到了七个重叠克隆，它们共同包含了带有17个外显子的完整COT基因。所有外显子 - 内含子边界序列均符合GT - AG规则。COT基因似乎分布在大鼠基因组的40至60kbp区域。通过引物延伸确定了COT基因的转录起始位点，并确定了至 - 1140位置的启动子序列。该启动子缺乏典型的TATA框，一个包含 - 1140至 + 84碱基位置序列和萤火虫荧光素酶报告基因cDNA的启动子 - 报告基因构建体，在转染的肝癌细胞中使启动子活性增加了约100倍。启动子序列中存在的推定顺式元件的一些共有序列为：两个用于CTF / NF1 / CBP结合的CCAAT基序（在 - 284和 - 93处），两个用于Sp1结合的GC框（在 - 160和 - 68处），两个AP2位点（在 - 359和 - 25处），一个过氧化物酶体增殖物激活受体（PPAR）结合的半位点（TGACCT）在 - 737处的部分回文序列区域内。还鉴定了潜在的调控元件，例如五个不同序列片段的几个回文和重复基序。
