Analysis of dominant-negative mutations of the Caenorhabditis elegans let-60 ras gene.
The let-60 gene of Caenorhabditis elegans controls the choice between vulval and hypodermal differentiation in response to an inductive signal from the gonad. let-60 encodes a ras protein that acts downstream of the let-23 receptor tyrosine kinase in a signal transduction pathway. Dominant-negative mutations of let-60 [let-60(dn)] cause a reduction of the gene activity in let-60(dn)/+ heterozygotes and a vulva-less mutant phenotype. We have found that nine let-60(dn) mutations cause replacements of conserved residues. Four are in two novel positions; others are in positions known previously to cause dominant-negative mutations in mammalian cells. The locations of these lesions suggest that they disrupt the ability of the ras protein to bind guanine nucleotides. Four let-60(dn) mutant genes were introduced into wild-type animals in the form of extrachromosomal arrays and were found to generate three dominant phenotypes--lethality, vulva-less, or multivulva--depending on gene dose and alleles. The dominant lethality caused by high-dose transgenic let-60(dn) genes suggests a toxic effect of these mutant genes in early development. The dominant-negative effects of these mutations in heterozygotes are likely to be caused by competition between let-60(dn) and let-60(+) protein for a positive regulator. All let-60(dn) mutations interfere with let-60(+) activity, but some alleles have partial constitutive activity, suggesting that the ability to interact with the activator is separable from the ability to exert a physiological effect (stimulation of vulval differentiation). These dn mutations might be useful for interfering with ras-mediated signal transduction pathways in other multicellular organisms.
Han
M
M
Howard Hughes Medical Institute, Division of Biology, California Institute of Technology, Pasadena 91125.
Sternberg
P W
PW
D000818:Animals; D030801:Animals, Genetically Modified; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D002107:Caenorhabditis / Q000235:genetics*; D029742:Caenorhabditis elegans Proteins; D002454:Cell Differentiation / Q000235:genetics; D003062:Codon; D005260:Female; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D011905:Genes, ras; D005838:Genotype; D006150:Guanine Nucleotides / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics*; D009154:Mutation; D010641:Phenotype; D014844:Vulva / Q000166:cytology; D018631:ras Proteins
D029742:Caenorhabditis elegans Proteins; D003062:Codon; D006150:Guanine Nucleotides; D015801:Helminth Proteins; C066646:let-60 protein, C elegans; D001216:Asparagine; D001120:Arginine; D018631:ras Proteins