Inactivation of the Neurospora crassa gene encoding the mitochondrial protein import receptor MOM19 by the technique of "sheltered RIP".

Abstract

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.

A crucial role of the mitochondrial protein import receptor MOM19 for the biogenesis of mitochondria

“…The novel technique of sheltered RIP for inactivating individual genes in N. crassa provides a useful tool to analyze the roles of important or essential genes. RIP is highly specific, since transformation of a strain carrying the inactivated gene with a wild-type copy leads to restoration of the original phenotype (Harkness et al, 1994). We took advantage of sheltered RIP to investigate the function of MOM19 in vivo.…”

“…LGI, A; LGIV, pyr-1 + mom49 + mtr s trp-4; LGV, am inl inv mei-2; (c) TI28.3, derived from H-IV by integrating an ectopic copy of morn-19 + into an unknown chromosome (linkage group, LG); and (d) 28.17, a heterokaryon containing one nucleus with chromosome IV derived from M-IV and another nucleus containing chromosome IV derived from TI28.3 in which the two copies of morn-19 have been inactivated by RIP (for genetic details see Harkness et al, 1994).…”

“…An outline for generating morn-19 mutants using this method is given in Fig. 1; a description of the genetic details of the procedure will be published elsewhere (Harkness et al, 1994). Briefly, the N. crassa strain H-IV was transformed with genomic mom-19 DNA to generate the TI28.3 isolate containing a single ectopic copy of mom-19 in addition to the endogenous one.…”

“…The method referred to as "sheltered RIP" makes use of RIP for destruction of a target gene, but allows the expression and maintenance of the mutated alleles in a heterokaryon in which the normal copy of the gene, present in another nucleus, shelters the cell against potentially lethal effects. We have described the genetic details underlying the technique elsewhere (Harkness et al, 1994). In brief, the duplicated sequence of a target gene present in one nucleus of the mating pair in the ascogenous hyphae will undergo alterations by RIP with a fairly high frequency as the nucleus passes through a genetic cross.…”

“…For the study of the consequences of inactivating the target gene, growth of cells containing the unaffected nucleus is inhibited by addition of a drug to which the mutant nucleus carries a resistance marker. Growth in the presence of the drug thus selects for cells harboring the mutant allele, and makes it possible to study the mutant phenotype (for details see Harkness et al, 1994). Sheltered RIP provides a generally applicable procedure for the generation of mutants in any cloned gene of interest in N. crassa, since duplicated genes can easily be produced by transformation and integration of that gene into an ectopic site of the genome (Akins and Lambowitz, 1985).…”