Figure 3

Transcription of HPV58 genes in S. cerevisiae. A. RT-PCR analysis of transcriptional expression of HPV58 early and late genes in the HPV58-Ura transformed yeast. E1 (lanes 1, 2), E2 (lanes 3, 4), E6 (lanes 5, 6), E7 (lanes 7, 8), L1 (lanes 9, 10) and L2 (lanes 11, 12) from the cDNA samples. Total RNA was isolated from HPV58-Ura transformed yeast and digested with DNase I. The DNase I treated RNA was amplified to ensure the complete digestion of contaminating viral DNA (lanes 2, 4, 6, 8, 10 and 12). DNase I completely digested RNA were then analysized by RT-PCR (lanes 1, 3, 5, 7, 9 and 11). B. Relative replication levels of HPV58 genomes in HPV58-Ura, HPV58-Ura-E2mt and HPV58-Ura-E2mt/pDBLeu-E2 transformed yeast. 18S rDNA was used as internal control. The DNA relative replication levels of HPV58-Ura-E2mt (0.03) and HPV58-Ura-E2mt/pDBLeu-E2 (0.54) are relative to that of HPV58-Ura, which was set to 1.0. Standard deviations are indicated by error bars. C. Relative transcription levels of E6 and E7 genes for HPV58-Ura, HPV58-Ura-E2mt and HPV58-Ura-E2mt/pDBLeu-E2. DNase I treated RNA was analysized by qRT-PCR with 18S rRNA as internal control. As for HPV58 genomes have different replication efficiency in the presence or absence of E2 protein (Figure 3B), the transcription levels of E6 and E7 genes were then standardized to the relative DNA replication assay. The E6 and E7 genes transcriptional levels of HPV58-Ura were set to 1.0. The transcription levels of E6 are 8.62 for HPV58-Ura-E2mt and 1.02 for HPV58-Ura-E2mt/pDBLeu-E2. The E7 transcription levels are 1.96 for HPV58-Ura-E2mt and 0.74 for HPV58-Ura-E2mt/pDB Leu-E2 relative to that of HPV58-Ura, which was set to 1.0. Standard deviations are indicated by error bars.