Ohsako S, Ohtomi M, Sakamoto Y, Uyemura K, Deguchi T
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neurosciences, Japan.
J Biol Chem. 1988 Jun 5;263(16):7534-8.
A cDNA clone encoding the full coding region of chicken liver arylamine acetyltransferase (EC 2.3.1.5) was isolated from lambda gt10 cDNA library by screening with the 32P-labeled oligonucleotide deduced from the amino acid sequence reported in the preceding paper. The complete nucleotide sequence of the cDNA was determined, from which the amino acid sequence of N-acetyltransferase was deduced. It consisted of 1,302 nucleotides including a 861-nucleotide region coding for 287 amino acids with a molecular weight of 32,914. Two methods were applied to confirm that the cDNA encoded arylamine N-acetyltransferase. First, mRNA was synthesized in vitro by Bluescript and subsequently translated in vitro. The molecular mass of the translation product was 34 kDa consistent with that of the enzyme purified from the chicken liver. The translated protein was immunoprecipitated by a monoclonal antibody to N-acetyltransferase. Second, the cDNA was inserted into an expression vector pcDL1 and introduced into Chinese hamster ovary cells. The supernatant of the homogenate of transfected cells showed a high level of N-acetyltransferase activity with a substrate specificity comparable to that of the liver enzyme. Northern blot analysis revealed three mRNAs corresponding to 4.7, 2.0, and 1.4 kilobases. The levels of N-acetyltransferase mRNAs were relatively high in the liver and distributed in various tissues. Genomic Southern blot analysis indicated the presence of only one gene in the chicken haploid genome.
通过用前一篇论文报道的氨基酸序列推导的32P标记寡核苷酸筛选λgt10 cDNA文库，分离出了编码鸡肝芳胺乙酰转移酶（EC 2.3.1.5）完整编码区的cDNA克隆。测定了该cDNA的完整核苷酸序列，并由此推导了N - 乙酰转移酶的氨基酸序列。它由1302个核苷酸组成，包括一个861个核苷酸的区域，编码287个氨基酸，分子量为32914。应用两种方法来证实该cDNA编码芳胺N - 乙酰转移酶。首先，通过Bluescript在体外合成mRNA，随后在体外进行翻译。翻译产物的分子量为34 kDa，与从鸡肝中纯化的酶一致。翻译后的蛋白质被抗N - 乙酰转移酶单克隆抗体免疫沉淀。其次，将该cDNA插入表达载体pcDL1并导入中国仓鼠卵巢细胞。转染细胞匀浆的上清液显示出高水平的N - 乙酰转移酶活性，其底物特异性与肝酶相当。Northern印迹分析揭示了对应于4.7、2.0和1.4千碱基的三种mRNA。N - 乙酰转移酶mRNA的水平在肝脏中相对较高，并分布于各种组织中。基因组Southern印迹分析表明鸡单倍体基因组中仅存在一个基因。
