Abstract
In vitro transcription of herpes simplex virus genes. Partial purification and properties of RNA polymerase II from uninfected and infected Hep-2 cells.
Highlights
These results demonstrate that both Pol11 and PoZII-H synthesize high molecular weight RNA ranging in size fromabout 600 to greater than 5000 nucleotides
Values shownare the percentage of totalradioactivityhybridized andare given as the mean f S.D. for twoautoradiograms of two separate hybridizations.The SUArestriction mapof HSV-1 DNA is shown in Fi”e. 6D
We have described an in vitro transcription system, consisting of partially purified RNA polymerase I1 from HSV-1-infected (PolII-H) and uninfected (PolII) HEp2 cells
Summary
PARTIAL PURIFICATION AND PROPERTIES OF RNA POLYMERASE I1 FROM UNINFECTED AND INFECTED HEp-2 CELLS*. Transcription was initiated by pg of HSV DNA with 150 enzyme units of Sal in 9 mM Tris-HCI, pH the additionof 5-10 p1 of RNA polymerase I1 preparation andallowed 7.6, 6 mM Mg(OAc)p,0.2 mM EDTA, 150 mM NaCl, 100pg/ml bovine to proceed for 30 min a t 37 "C. Was performed essentially as described above (RNA polymerase as- a t 2.2 X loJcpm/pg composed of 27% form I DNA in place of HSV say) but in 25-50-pl reaction volumes containing 50 mM Tris-HCI, pH DNA in the standard transcription mixture, and units of PolII or. EDTA and SDSwere added to 14 mM and 0.2%,respectively, HSV DNA at 50 pg/ml, and p1 of RNA polymerase This gave heated to65 "C for 3 min, and analyzed by electrophoresis througha afinal glycerol concentration o2f 0% and 14-140 units of RNA 1%agarose gel. Incubated 5 minat 37 "C.The sample was extracted as described above and precipitatedwith 2 volumesof ethanol without additionof
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