Skip to main content
Figure 1 | Microbial Cell Factories

Figure 1

From: Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae

Figure 1

No transcription or translation from HIV-1 LTR in yeast. (A) Construction of yeast expression plasmids for HIV-1. In HIV-TGAP, the 3′ LTR of the HIV-1 cDNA (with deletion the env gene) was replaced with the terminator for the yeast GAP gene and was cloned into yeast 2μ plasmid pKT10. In PGAP-HIV-TGAP, the 5′ LTR U3 and 3′ LTR of HIV-1 cDNA (with deletion the env gene) were replaced by the promoter and terminator for the yeast GAP gene, respectively and were cloned into pKT10. (B) HIV-1 RNA synthesis in yeast transformed with HIV-1 expression plasmids. Yeast was transformed with HIV-TGAP, HIV-TGAP plus HIV-1 Tat expression plasmids, PGAP-HIV-TGAP, or PGAP-HIV-TGAP plus HIV-1 Rev expression plasmids. Northern blotting of the yeast transformants was performed using minus-strand biotinylated RNA probe for the HIV-1 pol gene. Representative blots were shown. HeLa cells transfected with HIV-1 molecular clone pNL43 were used as control. A 0.24-9.5 kb RNA ladder (GIBCO BRL) were used as molecular weight markers. (C) HIV-1 protein expression in yeast transformed HIV-1 expression plasmids. Yeast was transformed with HIV-1 expression plasmids as described in (B) and whole cell lysate was subjected to Western blotting using anti-HIV-1 p24 antibody. Yeast transformed with HIV-1 Gag expression plasmid was used as control. Representative blots were shown. Prestained 21–113 kDa protein markers (Bio-Rad) were used as molecular weight markers. (D) Expression of HIV-1 Tat and Rev in yeast. Yeast transformed with HIV-1 Tat and Rev expression plasmids were subjected to spheroplast formation and immunostaining using anti-HIV-1 Tat and Rev antibodies, respectively. Nuclei were stained with DAPI. Representative images were shown at the same magnification.

Back to article page