Purification of a virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells that accurately initiates late and very late transcription in vitro.
The virus-induced RNA polymerase from Autographa californica nuclear polyhedrosis virus-infected Spodoptera frugiperda cells was separated from the three host nuclear RNA polymerases by DEAE-Sephadex chromatography and then purified through two more steps: heparin-agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayed in vitro for the ability to perform accurate initiation of transcription on a late (p6.9) and a very late (polyhedrin) template using primer extension analysis. In each case, the ability to accurately initiate transcription of these genes coincided with the virus-induced polymerase activity. Only after the glycerol gradient ultracentrifugation step did significant amounts of nonspecific late initiation occur, but specific late initiation was still readily detectable, suggesting that there is a limited number of late transcription factors, or that the factors are stably bound in a complex. After the glycerol gradient ultracentrifugation step, SDS-PAGE showed fewer than 10 prominent polypeptides remaining in the active fractions, which suggests a high degree of purity of the transcription machinery.
Beniya
H
H
Department of Biochemistry, University of Kansas, Lawrence 66045-2106, USA.
Funk
C J
CJ
Rohrmann
G F
GF
Weaver
R F
RF
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D015967:Gene Expression Regulation, Viral; D006493:Heparin; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000201:enzymology* / Q000235:genetics; D018411:Spodoptera / Q000166:cytology; D014158:Transcription, Genetic
D004279:DNA, Viral; D006493:Heparin; D012321:DNA-Directed RNA Polymerases