FIG. 3.

VLP formation is normal in yku70 mutant cells. (A) Transcription levels in wild-type (squares) and yku70 mutant (diamonds) cells assayed by using a reporter plasmid containing a lacZ construct under the control of the same galactose-inducible promoter that is on the Ty1 plasmid construct. Cells were grown in the presence of galactose and assayed at various time points after induction for β-galactosidase activity by measuring ONPG cleavage. (B) RT activity profile of sucrose gradient fractions. Whole-cell extracts of the wild-type and yku70 mutant strains containing pGTy1 and grown under inducing conditions were run over sucrose gradients and analyzed for RT activity. (C) Southern analysis of Ty1 cDNA prepared from VLPs from the wild-type and yku70 mutant strains. The DNA was hybridized to a radiolabeled fragment of the pGTy1 plasmid. One and 10 ng, respectively, of the BamHI-XhoI fragment of pGTy1 are shown in the last two lanes. (D) Ty1 cDNAs isolated from VLPs from the wild-type and yku70 mutant strains were digested with SpeI, radiolabeled, and analyzed by nondenaturing PAGE. (E) SpeI cleavage pattern of the Ty1 element. (F) Western blot analysis of VLPs prepared from the wild-type and yku70 mutant strains using monoclonal antibody 8B11 directed at a portion of the integrase (IN) coding sequence (20). It recognizes both the TYA/TYB precursor protein and the mature form of integrase (p90-TYB; IN).