Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.
Splicing-defective mutants of the yeast mitochondrial COXI gene can be corrected by transformation with a hybrid maturase gene.
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酵母线粒体 COXI 基因的剪接缺陷突变体可以通过用杂合成熟酶基因转化来纠正。
DOI：
10.1073/pnas.88.13.5592
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发表时间：
1991
影响因子：
11.1
通讯作者：
Butow,RA
中科院分区：
文献类型：
--
作者：
Anziano,PQ;Butow,RA
我们开发了一种重组载体，称为pMIT，用于通过生物转化将基因传递到酵母线粒体的瞬时表达。利用该载体，我们将一个杂交RNA成熟酶（剪接）基因引入rho 0小细胞的线粒体，并证明该基因在杂交中具有功能。杂交成熟酶是由COXI（细胞色素氧化酶）基因的内含子1编码的成熟酶的n端一半与COXI内含子2编码的类似成熟酶的c端一半之间的框架内融合。在COXI内含子1编码的成熟酶合成缺陷的呼吸缺陷菌株中，pMIT转化子可以在杂交中提供功能性成熟酶，以恢复呼吸和COXI多肽的合成；这种转化还将恢复COXI内含子1和3的两个剪接缺陷顺式突变体的呼吸作用。我们检测到一个68-kDa的多肽，其丰度与其他主要线粒体翻译产物相当，可能是杂交成熟酶基因的产物。在杂交成熟酶基因中含有218个氨基酸缺失突变的转化子在杂交中不再表达功能性成熟酶，并产生预期的约40 kDa的缩短多肽；然而，这些转化体仍然恢复COXI顺式突变体的呼吸。这些研究表明pMIT转化系统在酵母线粒体基因的表达和反向遗传分析中的实用性。
We have developed a recombinant vector, termed pMIT, for transient expression of genes delivered to yeast mitochondria by biolistic transformation. Using that vector, we introduced a hybrid RNA maturase (splicing) gene into mitochondria of rho 0 petite cells and showed the gene to be functional in crosses. The hybrid maturase is an in-frame fusion between the N-terminal half of the maturase encoded by intron 1 of the COXI (cytochrome oxidase) gene and the C-terminal half of a similar maturase encoded by COXI intron 2. pMIT transformants can provide a functional maturase in crosses to restore respiration and COXI polypeptide synthesis to a respiratory-deficient strain defective in the synthesis of a maturase encoded by COXI intron 1; the transformant will also restore respiration to two splicing-defective cis mutants of COXI introns 1 and 3. We detect a 68-kDa polypeptide comparable in abundance to other major mitochondrial translation products as a likely product of the hybrid maturase gene. Transformants containing an internal 218-amino acid deletion mutation of the hybrid maturase gene no longer express a functional maturase in crosses and produce the expected shortened polypeptide of approximately 40 kDa; however, those transformants still restore respiration to the COXI cis mutants. These studies show the utility of the pMIT transformation system for the expression and reverse genetic analysis of yeast mitochondrial genes.