Isolation and characterization of the krev-1 gene, a novel member of ras superfamily in Neurospora crassa: involvement in sexual cycle progression.

Abstract

Genes belonging to the ras superfamily encode low-molecular-weight GTP/GDP-binding proteins that are highly conserved in wide variety of organisms. We used the polymerase chain reaction (PCR) to isolate a novel member of the ras superfamily from the filamentous fungus Neurospora crassa and obtained a mammalian Krev-1 homolog. We named the gene krev-1 and analyzed its structure and function. The krev-1 gene encodes a polypeptide of 225 amino acids, which is nearly 60% homologous to the mammalian Krev-1 p21. The krev-1 gene product (KREV1) is functionally analogous to mammalian Krev-1 p21 and Rsr1p/Bud1p, a Krev-1 homolog from the yeast Saccharomyces cerevisiae. GAL1-driven expression of KREV1 in a wild-type yeast strain resulted in a random budding pattern, as did its mammalian counterpart Krev-1 p21. We disrupted the krev-1 gene by RIP (repeat-induced point mutation), but the krev-1 disruptants showed no abnormalities. By in vitro mutagenesis, we constructed several mutant krev-1 genes (G21V, A68T, and D128A) which mimic constitutively active mutants of Ha-ras, and the krev-1 (K25N) mutant which is analogous to a dominant-negative mutant of Ha-ras. Each mutant gene was introduced into the wild-type strain and the phenotypes were analyzed. We could not observe any difference in vegetative growth between these transformants. When each strain was used as the female in mating tests, the development of perithecia from protoperithecia was inhibited in all cases. The results indicate that the krev-1 gene may be involved in sexual cycle progression.