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.

ga89: A Temperature-Sensitive, Activating Mutation in let-60 ras.

David M. Eisenmann, Stuart K. Kim

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
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.