Skip to main content
. 1999 Sep;19(9):6260–6268. doi: 10.1128/mcb.19.9.6260

FIG. 6.

FIG. 6

Ku is associated with the Ty1 cDNA in vivo. (A) In vivo formaldehyde cross-linking was performed on the wild-type and yku70 mutant strains, both carrying the pGTy1 plasmid. Whole-cell extracts were prepared and used in immunoprecipitation reactions with an antibody directed against Yku70p. DNA from the I or immunoprecipitated P fraction was isolated and analyzed by PCR and Southern blotting using primers that give a specific product for the Ty1 cDNA. Lanes: 1 and 2, I and P fractions from yku70 mutant cells grown in glucose; 3 and 4, I and P fractions from yku70 mutant cells grown in galactose; 5 and 6, I and P fractions from wild-type cells grown in glucose; 7 and 8, I and P fractions from wild-type cells grown in galactose. PCRs using DNA isolated from a partially purified VLP fraction (lane 9), plasmid DNA (pGTy1, lane 10), and the P fraction from wild-type yeast not containing the pGTy1 plasmid (W303, lane 11) as the substrate are shown, demonstrating the specificity of the PCR product. (B) Whole-cell extracts prepared from formaldehyde-treated wild-type cells not carrying the pGTy1 plasmid (W303) were mixed with whole-cell extracts from formaldehyde-treated yku70 mutant cells carrying the pGTy1 plasmid (yku70/Ty1). Immunoprecipitations and Ty1 DNA analysis using the mixed extracts were performed as described above, and I and P fractions are shown in lanes 1 and 2. In lanes 3 and 4, as a positive control, the immunoprecipitation and Ty1 DNA analysis were performed in parallel by using wild-type cells carrying the pGTy1 plasmid (W303/Ty1). (C) In vivo formaldehyde cross-linking and immunoprecipitations were performed as described above on strain JDY1 (rad52::TRP1) containing integration-deficient pGTy1-in2600 (lanes 1 and 2), JC297 (containing the genomic HIS3AI-marked Ty1 element under the control of its endogenous promoter [11]; lanes 3 and 4), or JDY15 (yku80::URA3 in JC297; lanes 5 and 6). (D) Transposition levels were measured for the genetically marked endogenous Ty1 element in wild-type (JC297) and yku80 mutant (JDY15) yeast strains. Measurements were done five times for each strain. (E) Northern blot analysis of 10 μg of total RNA using probes directed against either the Ty1 element or actin.