Cloning and expression of cDNAs for polymorphic and monomorphic arylamine N-acetyltransferases from human liver.
To elucidate the molecular basis of human N-acetylation polymorphism, cDNA clones encoding human liver N-acetyltransferases (EC 2.3.1.5) were isolated from lambda gt10 cDNA libraries using the 32P-labeled cDNA of rabbit liver N-acetyltransferase recently cloned in this laboratory. Three types of cDNAs (D-14, O-7, and D-24) were isolated and their nucleotide sequences were determined, from which the amino acid sequences of human N-acetyltransferases were deduced. All the cDNAs coded for 290 amino acids. Between D-14 and O-7 cDNAs, there was only a single-base substitution in the coding region, which replaced glutamic acid in D-14 cDNA for glycine in O-7 cDNA. There were considerable differences between O-7/D-14 and D-24 cDNAs, with 80% homology in amino acid sequences. When the cDNAs were inserted into pEF321 expression vector and introduced into Chinese hamster ovary cells, N-acetyltransferase activity was expressed in three groups of the transfected cells. The activity in cells transfected with D-14 cDNA was only 9-17% of the activity in O-7 cells. Immunoblot analysis of the transfected cells indicated that the difference in the enzyme activity between O-7 and D-14 cells was possibly due to a difference in the amount of enzyme proteins. The substrate specificity of the expressed enzymes indicated that O-7 and D-14 cDNAs code for polymorphic N-acetyltransferase whereas D-24 cDNA codes for monomorphic enzyme. Southern blot analysis indicated that the polymorphic and monomorphic N-acetyltransferases were encoded in separate genes and that there was restriction fragment length polymorphism with KpnI digestion in the polymorphic N-acetyltransferase gene.
Ohsako
S
S
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
Deguchi
T
T
D000123:Acetyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D014162:Transfection
D004247:DNA; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase