The vacuolar ATPase of Neurospora crassa is indispensable: inactivation of the vma-1 gene by repeat-induced point mutation.
To analyze the phenotype of cells lacking the vacuolar ATPase, we inactivated the vma-1 gene, which encodes the catalytic subunit of the enzyme. Because preliminary experiments suggested the vma-1 gene was essential, we developed a method of simultaneously inactivating the gene and complementing it with a functional copy. We call this method repeat-induced point mutation (RIP) & Rescue. Two strains, both of which contained an extra copy of the vma-1 gene, were mated. Progeny that had inherited a functional copy of the gene at an ectopic site in the genome were selected. In some of these progeny the endogenous vma-1 gene had been altered by the RIP process. Sequencing showed the endogenous vma-1 gene had been inactivated by multiple point mutations. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional copy of the gene cosegregated, indicating that the vacuolar ATPase is essential in Neurospora crassa.
Ferea
T L
TL
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA. ferea@biology.ucsc.edu
Bowman
B J
BJ
D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000235:genetics*; D005800:Genes, Fungal; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D029701:Saccharomyces cerevisiae Proteins; D014170:Transformation, Genetic; D025262:Vacuolar Proton-Translocating ATPases
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae