Telakowski-Hopkins C A, Rodkey J A, Bennett C D, Lu A Y, Pickett C B
J Biol Chem. 1985 May 10;260(9):5820-5.
Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.
利用多核糖体免疫选择的大鼠肝脏谷胱甘肽S-转移酶mRNA，我们按照古布勒和霍夫曼所述的方法（古布勒，U.，和霍夫曼，B.S.（1983年）《基因》25卷，263 - 269页），使用DNA聚合酶I、核糖核酸酶H和大肠杆菌连接酶（NAD⁺）介导的第二链cDNA合成构建了cDNA克隆。重组克隆pGTB42含有一个900个碱基对的cDNA插入片段，其3'端在杂交选择翻译实验中对Yc mRNA显示出特异性。已确定pGTB42的核苷酸序列，并推导了Yc亚基的完整氨基酸序列。该cDNA克隆包含一个663个核苷酸的开放阅读框，编码一个由221个氨基酸组成的多肽，分子量为25,322。从pGTB42推导的NH₂-末端序列与通过传统蛋白质测序技术确定的Ya - Yc异二聚体的前39个氨基酸一致。将pGTB42的核苷酸序列与我们实验室先前描述的Ya克隆pGTB38的序列（皮克特，C.B.，特拉科夫斯基 - 霍普金斯，C.A.，丁，G.J.-F.，阿根布赖特，L.，和卢，A.Y.H.（1984年）《生物化学杂志》259卷，5182 - 5188页）进行比较，发现在两个克隆的相同区域序列同源性为66%；然而，Ya和Yc mRNA的5'和3'非翻译区在序列上完全不同。Ya和Yc亚基之间的总体氨基酸序列同源性为68%，然而，NH₂-末端结构域比中间或羧基末端结构域更高度保守。我们的数据表明，大鼠肝脏谷胱甘肽S-转移酶的Ya和Yc亚基是两种不同mRNA的产物，这两种mRNA来自两个相关但不同的基因。
