Figure 4. Transcription in oocyte and egg extracts.

A. pCMV-EYFP plasmid was incubated in oocyte or egg extracts in the presence of ³²P-α-ATP α-amanitin was added or the plasmid pre-incubated with TBP protein, as indicated. The reaction products were stopped and RNA extracted with TRIzol. Precipitated RNA was directly counted in a scintillation counter and resulting transcription radioactivity was scaled to the oocyte plasmid control.

B. pCMV-EYFP plasmid was incubated with TBP protein and then in egg extract in the presence of ³²P-α-ATP and the reactions stopped and RNA extracted at 0, 30, 60, 90, and 120 minutes after addition. Resulting radioactive incorporation is plotted (cpm) demonstrating transcription over time.

C. Topoisomerase-I relaxed pCMV-EYFP plasmid or plasmid pre-bound with TBP was incubated in egg extracts and stopped at indicated time. The reactions were deproteinized and the DNA was run on an agarose gel and stained with EtBr. Supercoiled and relaxed positions are indicated.