Horowitz B, Farley R A
University of Southern California School of Medicine, Los Angeles 90033.
Prog Clin Biol Res. 1988;268B:85-90.
cDNA fragments coding for the alpha and beta subunits of the Na,K-ATPase were separately ligated into the yeast expression vector YEp1PT in both the sense (YEpNKA(+)) and anti-sense (YEpNKA(-)) orientations with respect to the promoter. The recombinant plasmids were introduced into Saccharomyces cerevisiae strain UT4 by transformation. Total RNA from the transformed strains was isolated and analyzed by Northern hybridization. The resulting autoradiogram revealed strong signals indicative of a high level of transcriptional expression of both subunits in both orientations of the cDNA. 35S-Methionine labeled extracts were immunoprecipitated with antibodies specific for the beta subunit. A beta subunit translation product was produced from YEp beta NKA(+) but not from YEp beta NKA(-). Experiments to detect an alpha specific translation product are in progress.
编码钠钾ATP酶α和β亚基的cDNA片段分别以相对于启动子的正义（YEpNKA(+)）和反义（YEpNKA(-)）方向连接到酵母表达载体YEp1PT中。通过转化将重组质粒导入酿酒酵母菌株UT4。从转化菌株中分离出总RNA，并通过Northern杂交进行分析。所得放射自显影片显示出强信号，表明cDNA两个方向上两个亚基的转录表达水平都很高。用针对β亚基的特异性抗体对35S-甲硫氨酸标记的提取物进行免疫沉淀。YEpβNKA(+)产生了β亚基翻译产物，而YEpβNKA(-)则未产生。检测α特异性翻译产物的实验正在进行中。
