Design and utility of temperature-sensitive Gal4 mutants for conditional gene expression in Drosophila.
Temperature-sensitive (ts) mutants are valuable tools to study the function of essential genes in vivo. Despite their widespread use, little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Since ts mutants are typically generated by random mutagenesis it is difficult to isolate such mutants without efficient screening procedures. We have recently shown that it is possible to obtain ts mutants at high frequency by targeted mutations at either predicted, buried residues important for protein stability or at functional, ligand binding residues. The former class of residues can be identified solely from amino acid sequence and the latter from Ala scanning mutagenesis or from a structure of the protein:ligand complex. Several ts mutants of Gal4 in yeast were generated by mutating both categories of residues. Two of these ts mutants were also shown to result in tight and rapid ts reporter gene-expression in Drosophila when driven by either the elav or GMR promoters. We suggest possible mechanisms that might be responsible for such transferable ts phenotypes and also discuss some of the limitations and difficulties involved in rational design of ts mutants.
Mondal
Kajari
K
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
VijayRaghavan
K
K
Varadarajan
Raghavan
R
D000818:Animals; D030801:Animals, Genetically Modified; D004268:DNA-Binding Proteins; D004330:Drosophila / Q000235:genetics* / Q000254:growth & development; D029721:Drosophila Proteins / Q000235:genetics; D051959:ELAV Proteins / Q000235:genetics; D005123:Eye / Q000254:growth & development; D015870:Gene Expression; D005800:Genes, Fungal; D017344:Genes, Insect; D017930:Genes, Reporter; D007763:Lac Operon; D009154:Mutation; D010641:Phenotype; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D051959:ELAV Proteins; C498009:ELAV protein, Drosophila; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors