Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants

被引:18
作者
Bhat, A [1 ]
Tamuli, R [1 ]
Kasbekar, DP [1 ]
机构
[1] Ctr Cellular & Mol Biol, Hyderabad 500007, Andhra Pradesh, India
关键词
D O I
10.1534/genetics.103.025171
中图分类号
Q3 [遗传学];
学科分类号
071007 ; 090102 ;
摘要
The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg 3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3(+) and bacterial hph genes. Cotransformation of hph with PCR-amplified DNA of other genes enabled us to construct strains duplicated for the amplified DNA. Using transformation we constructed self-fertile strains that were homoallelic for an ectopic erg-3(+) transgene and a mutant erg-3 allele at the endogenous locus. Self-crosses of these strains yielded erg 3 mutant ascospores that produced colonies with the characteristic morphology on Vogel's sorbose agar described previously for erg-3 mutants of N. crassa. The mutants were generated by repeat-induced point mutation (RIP), a genome defense process that causes numerous G:C to A:T mutations in duplicated DNA sequences. Homozygosity for novel recessive RIP-deficient mutations was signaled by self-crosses of erg-(3)-duplication strains that fail to produce erg-3 mutant progeny. Using this assay we isolated a UV-induced mutant with a putative partial RIP defect. RIP-induced mutants were isolated in rid-1 and sad-1, which are essential genes, respectively, for RIP and another genome defense mechanism called meiotic silencing by unpaired DNA.
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页码:1155 / 1164
页数:10
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