Worm Breeder's Gazette 13(5): 21 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 9430*.
While performing a genetic screen for mutations that cause a p*truding-vulva (Pvl) phenotype (WBG 13.1 pg 69; West Coast WM94 pg 67), we identified an interes*ng mutation in the let-60 gene, which encodes a Ras homolog that functions during vulval development and other processes. This mutation, le*-60(ga89), causes a temperalure sensitive multivulva phenotype, as well as temperature-sensitive defects in brood size and male mating (Table 1). Table l: let-60(ga89) phenotypes In ga89 animals at 25¡, the multivulva phenotype arises when P3.p and/or P4.p (but almost never P8.p) express induced vulval cell fates. ga89 hermaphrodites produce fewer oocytes than wild type, and these oocytes look normal. The embryos in the uterus of ga89 animals do appear abnormal, and often resemble unfertilized oocytes when laid. ga89 hermaphrodites appear bloated or fluid-filled at 25¡ as well. The multivulva, male mating and fertility phenotypes described above are nearly recessive (ga89/ + is 0.6percent Muv at 25¡) The multivulva phenotype caused by ga89 indicates that this mutation activates Ras; this is the first Ras activating mutation to be described which is also temperature-sensitive. In addition to a temperature-sensitive gain-of-function phenotype, ga89 causes a cold sensitive loss-of-function phenotype in certain genetic backgrounds. For example, ga89 results in an Egl phenotype in trans to a let-60(1f) allele or a deficiency, or when other Ras signaling pathway components, such as MAP kinase, are compromised (Table 2). These and other interesting genetic properties led us to characterize ga89 at the molecular level. Sequencing of let-60 from ga89 animals identified a single base change in the entire Ras coding region, predicted to cause a leucine to phenylalanine change at position 19. A leucine at position 19 is found in every eukaryotic Ras protein and some Ras-related GTPases, showing that it is a strongly conserved residue. This residue lies at the start of an alpha helix located carboxy terrninal to the L1 loop which plays an important role in guanine nucleotide binding. Mutations in nearby residues cause a gain-of-function phenotype (residues 12 and 13) or a dominant negative phenotype (residues 15, 16 and 17). No mutations at leucine 19 have been characterized previously in vivo or in vitro in any system. To understand how this mutation affects the function of Ras, we have introduced the L19F change into human H-ras, and are purifying wild-type and mutant proleins from E. coli in order to assay the guanine nucleotide binding, exchange and hvdrolysis properties of the proteins. In addition, we are introducing the H-ras L19F gene into NIH3T3 cells, to see if the mutant Ras protein can cause a transformed phenotype in mammalian cells.