68 Cook and SlaterRNA polymerase II. RNA polymerase II from wheat germ does not
function in this system, indicating that it is a combined effect of RNA
polymerase II and specific factors that mediate accurate and selective
transcription. Fractionated S 100 does contain multiple factors required
for accurate transcription by purified RNA polymerase II (16,22).
5.2. HeLa Whole Cell Lysate (39)
The extract is prepared by gentle lysis of cells with a Dounce homog-
enizer. Ammonium sulfate is added to approx 20% saturation, and the
nuclei lysed. Cell debris and broken nuclei are removed by centrifu-
gation at 175,000g for 3 h. Proteins in the supernatant are resuspended
and dialyzed for 12 h. The dialysate is centrifuged to remove insoluble
material, and supernatant removed to form the extract. This can be
stored in small aliquots with repeated thawing and refreezing for up to
1 yr at -70°C. The extract normally contains 6-30 mg/mL of protein.
The rate of RNA polymerase II elongation is approx 300 nucleotides/
min approx 1/10th that in vivo (53). The in vitro temperature of 30°C
should not be increased to 37°C, otherwise RNase degradation may
become a problem (39). The reaction mixture is detailed in Section 4.2.
Each whole cell extract must be characterized by DNA and extract
titration, since there is a threshold DNA concentration below which no
accurate transcription occurs and an inhibitory effect at high DNA
concentrations. Specific transcription can be obtained when the final
concentration of protein in the extract is in the range 6-18 mg/mL. The
extract contains high levels of 18S and 28S rRNA. Therefore, following
extraction, it is possible that only 50-75% of the sample may be loaded
onto the gel to avoid overloading. Since the extract is made from whole
cells, weak promoters may not be as easily detected, although this does
depend on the level of background nonspecific transcription occurring.
5.3. Nuclear Extracts from Human Cells (40)
The extract is prepared by isolating nuclei in hypotonic buffer, lysis
of cells with a Dounce homogenizer, extraction of the nuclei at 0.42M
NaC1, high-speed centrifugation to clear the supernatant, and concen-
tration of the extracted nucleic acids and various transcription factors
by ammonium sulfate precipitation. Since the nuclei are isolated at
very low ionic strength (0.01M KCI), retention of nuclear components
is facilitated and contaminating cytoplasmic proteins are discarded in
the supernatant following pelleting of the nuclei. Extraction at 0.42M