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. Author manuscript; available in PMC: 2014 Jan 1.
Published in final edited form as: Traffic. 2012 Oct 17;14(1):57–69. doi: 10.1111/tra.12013

Fig. 5.

Fig. 5

Gag provided in trans restores Ty1fs mRNA level, nuclear export, and formation of cytoplasmic mRNA/Gag foci. (A) Northern analysis of Ty1fs RNA in the presence or absence of Gag expressed from pMPOLΔ. Total RNA was extracted from strains expressing pGfs (expresses Ty1fs RNA) and Gag (pMPOLΔ: expresses a transcript encoding Gag) or pGfs and an empty vector (−) induced with galactose for 48 hr. RNA was hybridized with 32P-labeled riboprobes corresponding to Ty1 GAG and ACT1. Note that the Ty1 GAG probe will detect both the Ty1fs RNA (5.7 kb) and GAG mRNA (2 kb) from pMPOLΔ. Ty1/ACT1 ratios were estimated as described in Fig 4. (B) Ty1fs RNA dynamics when Gag is provided in trans. Cells containing pMPOLΔ and pGfs were grown in media containing galactose (+Gal) and methionine (+Met) to induce pGfs only, or lacking methionine (+Gal − Met) to induce both plasmids, for indicated time lengths prior to FISH-IF analysis. Ty1 mRNA was detected with either GAG-DIG or RT-DIG probes as indicated. Note that the GAG-DIG probe will detect both Ty1fs mRNA and truncated POLΔ mRNA, while the RT-DIG probe will only detect Ty1fs RNA (Fig. 1B). Gag was detected with an anti-Ty1 VLP antiserum. Representative images shown illustrate the percentages of cells containing Ty1 mRNA or Gag foci, propensities for mRNA/Gag (m/g) overlap, and percentages of cytoplasmic (c) or nuclear (n) localization of Ty1 mRNA.