Suissa M, Suda K, Schatz G
EMBO J. 1984 Aug;3(8):1773-81. doi: 10.1002/j.1460-2075.1984.tb02045.x.
To isolate nuclear genes specifying imported mitochondrial proteins, a yeast genomic clone bank was screened by an RNA hybridization-competition assay. This assay exploited the fact that mRNAs for imported mitochondrial proteins are enriched in polysomes which are bound to the mitochondrial surface in cycloheximide-inhibited yeast cells. Clones selectively hybridizing to these enriched mRNAs were further screened by hybrid-selected translation and immunoprecipitation with monospecific antisera against individual mitochondrial proteins. Thirty-six clones were isolated which contained complete or partial copies of 16 different genes for imported mitochondrial proteins. Several of these clones caused expression of the corresponding precursor polypeptide in Escherichia coli or over-expression of the corresponding mature protein in yeast. The gene for the matrix enzyme citrate synthase was sequenced; the derived amino acid sequence of the precursor polypeptide revealed an amino-terminal extension containing basic but no acidic residues.
为了分离编码导入线粒体蛋白的核基因，通过RNA杂交竞争试验筛选了酵母基因组克隆文库。该试验利用了这样一个事实，即在环己酰亚胺抑制的酵母细胞中，导入线粒体蛋白的mRNA在与线粒体表面结合的多核糖体中富集。通过杂交选择翻译和用针对单个线粒体蛋白的单特异性抗血清进行免疫沉淀，进一步筛选与这些富集mRNA选择性杂交的克隆。分离出36个克隆，其中包含16种不同的导入线粒体蛋白基因的完整或部分拷贝。其中一些克隆导致相应前体多肽在大肠杆菌中表达或在酵母中相应成熟蛋白的过表达。对基质酶柠檬酸合酶的基因进行了测序；前体多肽的推导氨基酸序列显示出一个氨基末端延伸，其中含有碱性但不含酸性残基。
