Abstract
A cDNA clone encoding the full coding region of polymorphic arylamine N-acetyltransferase was isolated from rabbit liver and expressed in Chinese hamster ovary cells. The expressed enzyme acetylated 2-aminofluorene, procainamide, sulfamethazine, and p-aminobenzoic acid at equivalent rates. N-Acetyltransferase activity was measured in 17 rabbits from an inbred colony which were classified into rapid, intermediate, and slow acetylators. The livers of the rapid and intermediate acetylators efficiently acetylated all four substrates, while the liver from the slow acetylator showed a low but significant activity with p-aminobenzoic acid. Immunoblot and Northern blot analyses of rabbit livers indicated that the differences in N-acetyltransferase activity were due to differences in N-acetyltransferase protein and mRNA content. Genomic clones of N-acetyltransferase were isolated from the rapid and slow acetylator rabbits. The nucleotide sequence of the gene from rapid acetylator rabbit was identical to that of the cDNA, while the sequence of the gene from slow acetylator rabbit was homologous, but not identical, to the cDNA sequence. Genomic Southern blot and polymerase chain reaction analyses of the genomic DNAs and cDNAs from the three types of acetylator indicated that the gene for polymorphic arylamine N-acetyltransferase is totally deleted in the slow acetylator rabbit, while the gene from slow acetylator rabbit is expressed in all rabbits and might encode another N-acetyltransferase. Thus the genetic mechanism of N-acetyltransferase polymorphism in rabbit liver is essentially different from that of human liver as demonstrated in this laboratory (Ohsako, S., and Deguchi, T. (1990) J. Biol. Chem. 265, 4630-4634; Deguchi, T., Mashimo, M., and Suzuki, T. (1990) J. Biol. Chem. 265, 12757-12760).
Highlights
Acetylator indicated that the gene for polymorpahri-c Parts of this study have been reported in abstract form (19, ylamine N-acetyltransferase is totally deleted in the 20). slow acetylator rabbit, while the gene from sloawcetylator rabbit is expressed in all rabbits and might encode another N-acetyltransferase
Nucleotide and Amino Acid Sequences of cDNA for Polymorphic N-Acetyltranferase of Rabbit Liver-AXgtlO cDNA library was constructed from the liver poly(A)+ RNA of an intermediate acetylator rabbit ansdcreened with a 32P-labeled 20-mer oligonucleotide that was deduced from the amino acid sequence [16] anddesigned according to codon usagefrequencies [28]
The cDNAs were analyzed by restriction enzymes
Summary
Nucleotide and Amino Acid Sequences of cDNA for Polymorphic N-Acetyltranferase of Rabbit Liver-AXgtlO cDNA library was constructed from the liver poly(A)+ RNA of an intermediate acetylator rabbit ansdcreened with a 32P-labeled 20-mer oligonucleotide that was deduced from the amino acid sequence [16] anddesigned according to codon usagefrequencies [28]. Out of IO6independent phages, six positive cDNA clones were isolated. The cDNAs were analyzed by restriction enzymes. All clones contained a cDNA insert of similar size and showed an identical fragment pattern upon digestion with various restriction enzymes. Analysis of the nucleotide sequence of a cDNA clone revealed an open reading frame, with the putativeinitiation codon ATG at position 1,containing 870 nucleotides coding for 290 amino acids (Fig. l),with a calculated molecular weightof 33,648. There was a 66-bp 5"noncoding region, a 608-bp 3'noncoding region, and two polyadenylation signals (AATAAA) located at positions 968-973 and 1394-1399. All clones expressed a high enzyme activity with the four substrates, whereas the cells transfected with pSV2neo plasmid alone showed no activity. The expressed enzyme efficiently acetylated 2-aminofluorene, sulfamethazine, procainamide, and p-aminobenzoic acid.
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