Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants.
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.
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.
Ashwin
A
Bhat
Ranjan
R
Tamuli
Durgadas P
DP
Kasbekar
D001483:Base Sequence; D002934:Cinnamates; D003433:Crosses, Genetic; D003470:Culture Media; D017931:DNA Primers; D005800:Genes, Fungal / Q000235:genetics; D006921:Hygromycin B / Q000031:analogs & derivatives*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009491:Neurospora / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
D002934:Cinnamates; D003470:Culture Media; D017931:DNA Primers; D006921:Hygromycin B; C026273:hygromycin A; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase