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. 2004 Oct;78(20):10888–10905. doi: 10.1128/JVI.78.20.10888-10905.2004

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Copyright © 2004, American Society for Microbiology

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FIG.2. — (A) Schematic representation of pBEL and the pBEL-derived plasmids. Boxes indicate the protein coding regions. The CMV promoter is indicated. The probes used in Northern blots are indicated above the diagram. The CMV probe detects all mRNAs produced from the CMV-driven plasmids and has been described previously (9). Numbers refer to nucleotide positions in the HPV-16R sequence. (B) Northern blots of total RNA extracted from HeLa cells transfected with pBEL and pBELM are shown hybridized to the indicated radiolabeled probes. UE, unspliced early mRNAs; L1, the spliced late mRNA; 880/3358, early mRNA spliced from the 5′ ss at position 880 to the 3′ss at position 3358 followed by polyadenylation at pAE. The same samples were hybridized to a human β-actin probe to control for loading. The data variation in each transfection experiment was less than 20%. (C) RT-PCR on the RNA samples shown in Fig. 2B using primers 757S or 880S in combination with either E4A or E5A (Fig. 1). Negative control, RNA sample from cells transfected with unrelated plasmid. (D) Northern blot of total RNA extracted from HeLa cells transfected with pBEL, pBELM, pBEL-pAE, pBELM-pAE, ors pBELMDC hybridized to the L1 probe. Both L2/L1 and L1 mRNAs are spliced from the 5′ ss at position 880 to the 3′ splice site at position 3358. The L2/L1 mRNA then remains unspliced until polyadenylation at pAL, whereas the L1 mRNA is spliced also between the 5′ss at position 3632 and the 3′ ss at position 5639. The truncated L1 mRNA is polyadenylated at a previously identified cryptic poly(A) signal at position 5170 in the HPV-16R genome (33). Spliced mRNA as a percentage of total late RNA in each lane is indicated at the bottom of the gel. The same samples were hybridized to a human β-actin probe to control for loading. The data variation in each transfection experiment was less than 20%. (E) RT-PCR of the RNA samples in Fig. 2D using primers 757S and L1A (left panel) or primers E4S and L1A (right panel). All RT-PCR products were cloned and sequenced. (F) Total, cytoplasmic, and nuclear RNAs were extracted from HeLa cells transfected with pBEL or pBELM. The blotted RNA was probed with the L1 probe.

FIG.2. — (A) Schematic representation of pBEL and the pBEL-derived plasmids. Boxes indicate the protein coding regions. The CMV promoter is indicated. The probes used in Northern blots are indicated above the diagram. The CMV probe detects all mRNAs produced from the CMV-driven plasmids and has been described previously (9). Numbers refer to nucleotide positions in the HPV-16R sequence. (B) Northern blots of total RNA extracted from HeLa cells transfected with pBEL and pBELM are shown hybridized to the indicated radiolabeled probes. UE, unspliced early mRNAs; L1, the spliced late mRNA; 880/3358, early mRNA spliced from the 5′ ss at position 880 to the 3′ss at position 3358 followed by polyadenylation at pAE. The same samples were hybridized to a human β-actin probe to control for loading. The data variation in each transfection experiment was less than 20%. (C) RT-PCR on the RNA samples shown in Fig. 2B using primers 757S or 880S in combination with either E4A or E5A (Fig. 1). Negative control, RNA sample from cells transfected with unrelated plasmid. (D) Northern blot of total RNA extracted from HeLa cells transfected with pBEL, pBELM, pBEL-pAE, pBELM-pAE, ors pBELMDC hybridized to the L1 probe. Both L2/L1 and L1 mRNAs are spliced from the 5′ ss at position 880 to the 3′ splice site at position 3358. The L2/L1 mRNA then remains unspliced until polyadenylation at pAL, whereas the L1 mRNA is spliced also between the 5′ss at position 3632 and the 3′ ss at position 5639. The truncated L1 mRNA is polyadenylated at a previously identified cryptic poly(A) signal at position 5170 in the HPV-16R genome (33). Spliced mRNA as a percentage of total late RNA in each lane is indicated at the bottom of the gel. The same samples were hybridized to a human β-actin probe to control for loading. The data variation in each transfection experiment was less than 20%. (E) RT-PCR of the RNA samples in Fig. 2D using primers 757S and L1A (left panel) or primers E4S and L1A (right panel). All RT-PCR products were cloned and sequenced. (F) Total, cytoplasmic, and nuclear RNAs were extracted from HeLa cells transfected with pBEL or pBELM. The blotted RNA was probed with the L1 probe.

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