Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".
Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".
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通过“屏蔽 RIP”技术使编码线粒体蛋白输入受体 MOM19 的粗糙脉孢菌基因失活。
DOI：
10.1093/genetics/136.1.107
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发表时间：
1994
期刊：
影响因子：
3.3
通讯作者：
Nargang,FE
中科院分区：
文献类型：
--
作者：
Harkness,TA;Metzenberg,RL;Schneider,H;Lill,R;Neupert,W;Nargang,FE
我们使用了一种称为“保护性RIP”（重复诱导点突变）的技术来创建粗神经孢子虫的mom-19基因突变体，该基因编码核编码线粒体前体蛋白的输入受体。保护性RIP允许在一个细胞核中隔离突变基因，即使该基因对生物体的生存至关重要，通过将携带突变基因的细胞核与未受影响的细胞核隔离在异核体中。此外，含有RIPed基因的细胞核含有一个可选择的标记，因此有可能将异核体中的核比率转变为在选择性条件下生长的培养物中含有RIPed基因的细胞核占主导地位的状态。这导致靶基因产物应该存在于非常不理想的水平，并允许突变表型的研究。该方法产生的mom-19的一个等位基因包含44个过渡，导致18个氨基酸取代。当含有该等位基因的异核体在有利于RIPed细胞核的条件下生长时，在菌株的线粒体中检测不到MOM19蛋白。含有RIPed等位基因的同核菌株表现出复杂且极慢的生长表型，这表明mom-19基因的产物在草稻中很重要。
We have used a technique referred to as "sheltered RIP" (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa.