Fig 3. Mediator subunits influence Ty1 cDNA levels without altering levels of Ty1 RNA or Gag protein.

(A) Quantitative northern blot analysis of sense-strand (Ty1) and antisense-strand (Ty1 AS) RNA. Total RNA was fractioned by gel electrophoresis, blotted, and the membranes probed for Ty1 AS RNA, followed by stripping and probing for Ty1 RNA using strand-specific riboprobes as schematized at the top, and for 18S rRNA as a loading control. The graph on the right shows the quantitation of Ty1 RNA and Ty1 AS RNA levels from two biological replicates, each relative to the 18S subunit rRNA level, normalized to WT levels for Ty1 and to spt3Δ yeast for Ty1 AS. (B) Western blot of total cell lysates probed for Gag using a polyclonal antibody against p18 [24]. Data for med5Δ and med16Δ is derived from two biological replicates; all other values represent data from three biological replicates, and values are normalized to WT. (C) Quantitative Southern blot analysis to determine the level of unintegrated Ty1 cDNA (cDNA) relative to the amount of DNA in bands representing two genomic Ty1 elements (G1 and G2). Image quantification is representative of three biological replicates, only one of which is shown. All experiments were performed using congenic WT, spt3Δ and Mediator subunit deletion strains harboring Ty1his3AI-3114. Bars in (B) and (C) are color coded as in Fig 2, and all error bars represent s.d. except for measurements with n = 2, in which case the range is indicated.