Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes.
Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)
Yeowell
H N
HN
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
McClellan-Green
P D
PD
Negishi
M
M
Goldstein
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005260:Female; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis; D010641:Phenotype; D011110:Polymorphism, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D009841:Oligonucleotides; D012333:RNA, Messenger; D004247:DNA; D003577:Cytochrome P-450 Enzyme System