The Vacuolar Atpase of Neurospora Crassa Is Indispensable: Inactivation of the Vma-1 Gene by Repeat-Induced Point Mutation |
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| Authors: | T. L. Ferea and B. J. Bowman |
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| Abstract: | 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. |
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