Miran S G, Lawson J E, Reed L J
Biochemical Institute, University of Texas, Austin 78712.
Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1252-6. doi: 10.1073/pnas.90.4.1252.
The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.
来自酿酒酵母的丙酮酸脱氢酶（PDH）复合体β亚基（E1β）的编码基因已被克隆、测序、破坏并表达。基于酵母和人类E1β亚基的氨基酸序列，用合成寡核苷酸引物通过聚合酶链反应从酵母基因组DNA文库中产生了两个重叠的DNA片段。将这些DNA片段进行亚克隆并测序。复合序列有一个1098个核苷酸的开放阅读框，编码一个33个氨基酸残基的推定前导序列和一个333个残基的成熟蛋白，计算的M（r）= 36,486。酵母和人类E1β表现出62%的序列同一性。mRNA的大小约为1.5千碱基。杂交分析表明，E1β基因（PDHβ1）定位于II号染色体。在营养生长条件下，PDHβ1的破坏并不致命。用单位拷贝质粒上的PDHβ1转化的缺失突变体产生了成熟的E1β和功能性的PDH复合体。
