Cloning of the DNA repair gene, uvsF, by transformation of Aspergillus nidulans.
As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector. Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny. Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued. Their inserts had a short internal region in common, giving evidence of rearrangement(s). In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries. Three types of genomic and two overlapping cDNA clones were identified. The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids. Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone. In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.
Oza
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Department of Biology, McGill University, Montreal, Canada.
Käfer
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D001233:Aspergillus nidulans / Q000235:genetics*; D015152:Blotting, Northern; D015139:Blotting, Southern; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal; D008938:Mitosis; D010957:Plasmids; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal