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Figure 3 | BMC Microbiology

Figure 3

From: Evidence that the RNAseH activity of the duck hepatitis B virus is unable to act on exogenous substrates

Figure 3

DHBV core particles contain enzymatically active P that can synthesize DNA employing the RNAseH substrates as templates. (A) Endogenous polymerase assay in permeabilized DHBV core particles. Viral cores were permeablized by brief treatment at pH 2.5. Following neutralization, the cores were incubated with [α-32P]dNTPs and then the viral DNAs were isolated by proteinase K digestion, phenol and chloroform extraction, and ethanol precipitation. DNA synthesized by P within the cores was separated on a 1.2% agarose gel and detected by autoradiography. Lane 1: wild-type DHBV cores; lane 2: polymerase mutant YMHA cores. RC: relaxed circular DNA; DL, duplex linear DNA; SS, single strand DNA. (B) Reverse transcriptase activity using the exogenous RNAseH substrate as a template (the trans-reaction). Viral cores were permeabilized and the endogenous nucleic acids were removed with micrococcal nuclease. The trans reaction was performed with the indicated exogenous nucleic acids. The products were purified by phenol and chloroform extraction and were resolved by denaturing polyacrylamide electrophoresis. Lane 1 contains internally 32P -labeled DRF+ RNA as a marker. Complementary Oligo, oligonulceotide D2507-; Noncomplementary Oligo, oligonucleotide D2526+. Lane 4 contains the RNAseH substrate (DRF+ RNA annealed to oligonucleotide D2507-). Sizes of single-stranded marker RNAs are indicated.

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