GENETIC ANALYSIS OF PROTEIN TRANSPORT INTO MITOCHONDRIA

酵母线粒体酒精脱氢酶(ADHIII) 啤酒是在细胞质中合成的，并被运输到 线粒体。编码这种同工酶的核基因ADH3已经被 利用其与先前克隆的ADH1的同源性进行克隆 以及编码细胞质同工酶的ADH2基因。完整的 该基因的核苷酸序列将通过化学和化学方法确定 酶测序技术。功能基因的极限将是 通过S1核酸酶分析确定5英尺和3英尺末端的位置 其信使核糖核酸。 线粒体ADH在呼吸和发酵中的作用将是 通过基因破坏来研究。正常基因将被灭活 重组DNA技术和酵母转化及其表型的研究 缺乏功能性ADH3基因的突变体将被研究。 ADHIII进入线粒体的运输及其在线粒体内的位置 通过对新合成的ADHIII多肽进行表征进行研究。这个 将对假定的前体进行鉴定和表征 使用免疫学和蛋白质测序技术。这些研究将 利用一株缺乏ADH1和ADH2基因的酵母菌株，使两种基因都不能 也不存在代表ADHI和ADHIII的多肽。蛋白 推测的前体和成熟蛋白的序列分析将是 以确定它们的氨基末端。 ADHIII不同区域对线粒体运输的作用， 处理和瞄准正确的线粒体内位置将是 学习。ADH2和ADH3之间将进行初步融合，以研究 氨基末端用于运输的必要性和充分性。后续 如果氨基末端不是，ADH3和lac Z之间将发生融合 足够了。 将分离ADHIII线粒体转运改变的酵母突变株 或者通过选择抗烯丙醇或通过选择 对抗菌素的抗药性。ADHIII信号序列突变 并对运输蛋白进行鉴定和表征。一个 还将开发选择或筛选系统来分离有条件的 可能具有多效性运输缺陷的致命性突变体。

The mitochondrial alcohol dehydrogenase (ADHIII) of the yeast Saccharomyces cerevisiae is synthesized in the cytoplasm and transported into the mitochondria. The nuclear gene encoding this isozyme, ADH3, has been cloned by taking advantage of its homology to the previously cloned ADH1 and ADH2 genes, which code for the cytoplasmic isozymes. The complete nucleotide sequence of the gene will be determined by chemical and enzymatic sequencing techniques. The limits of the functional gene will be determined by S1 nuclease analysis to locate the 5 feet and 3 feet ends of its mRNA. The function of mitochondrial ADH in respiration and fermentation will be studied by gene disruption. The normal gene will be inactivated by recombinant DNA techniques and yeast transformation and the phenotype of the mutant lacking a functional ADH3 gene will be studied. The transport of ADHIII into and its location within the mitochondria will be studied by characterizing newly-synthesized ADHIII polypeptides. The identification and characterization of a putative precursor will be done using immunological and protein sequencing techniques. These studies will utilize a yeast strain which lacks ADH1 and ADH2 genes so that neither mRNA nor polypeptides representing ADHI and ADHIII are present. Protein sequence analysis of the putative precursor and the mature protein will be performed in order to identify their amino termini. The function of different regions of ADHIII for mitochondrial transport, processing and targetting to the correct intramitochondrial site will be studied. Initial fusions will be made between ADH2 and ADH3 to study the necessity and sufficiency of the amino terminus for transport. Subsequent fusions will be made between ADH3 and lac Z if the amino terminus is not sufficient. Yeast mutants altered in mitochondrial transport of ADHIII will be isolated either by selection for resistance to allyl alcohol or by selection for resistance to antimycin. Mutants altered in the signal sequence of ADHIII and in transport proteins will be identified and characterized. A selection or screening system will also be developed to isolate conditional lethal mutants which might have pleiotropic transport defects.