Overexpression of the RAD2 gene of S. cerevisiae: identification and preliminary characterization of Rad2 protein.
The cloned RAD2 gene of S. cerevisiae was tailored into regulatable expression vectors for overexpression of Rad2 protein in E. coli and in yeast. In E. coli both Rad2/beta-galactosidase fusion protein and native Rad2 protein are insoluble, but are extractable with 1% Sarkosyl. In yeast some of the overexpressed native Rad2 protein is also insoluble; however, soluble protein is readily detected by immunoblotting with Rad2-specific antibodies. All forms of the protein detected in transformed or untransformed yeast cells and the insoluble species in E. coli migrate in denaturing polyacrylamide gels with an apparent molecular weight considerably larger than the size predicted from the sequence of the RAD2 coding region. This property is not the result of post-translational glycosylation detectable by binding of concanavalin A, or of phosphorylation of the protein. Overexpression of the RAD2 gene is toxic to yeast. Transformed yeast cells grow much more slowly than untransformed controls and when yeast transformants are serially propagated cultures show considerably colony heterogeneity and concomitant selection for rapidly growing variants which express less Rad2 protein. Antisera raised against Rad2/beta-galactosidase fusion protein expressed in E. coli do not cross-react with Rad1, Rad3 or Rad10 protein in crude extracts of yeast, nor with purified E. coli UvrA, UvrB or UvrC proteins.
Nicolet
C M
CM
Department of Physiological Chemistry, University of Wisconsin, Madison 53706.
Friedberg
E C
EC
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005800:Genes, Fungal; D008970:Molecular Weight; D010957:Plasmids; D011233:Precipitin Tests; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic
D005656:Fungal Proteins