Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter.
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.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002333:Carnitine Acyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D005091:Exons; D015723:Gene Library; D006801:Humans; D007438:Introns; D008099:Liver / Q000201:enzymology*; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008297:Male; D008830:Microbodies / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008156:Luciferases; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase