Genetics, Vol. 167, 1155-1164, July 2004, Copyright © 2004
doi:10.1534/genetics.103.025171

Genetic Transformation of Neurospora tetrasperma, Demonstration of Repeat-Induced Point Mutation (RIP) in Self-Crosses and a Screen for Recessive RIP-Defective Mutants

Ashwin Bhat, Ranjan Tamuli and Durgadas P. Kasbekar1

Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

1 Corresponding author: Centre for Cellular and Molecular Biology, Uppal Rd., Hyderabad 500 007, India.
E-mail: kas{at}ccmb.res.in

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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