Eukaryotic RNA Polymerases 67
mixed together, resuspended in hybridization buffer (40 mM PIPES
[pH 6.4], 1 mM EDTA [pH 8.0], 0.4MNaC1 and 50% formamide), and
incubated at 85°C for 10 min to denature the nucleic acids. Hybridiza-
tion follows for 12-16 h, but again the temperature is dependent on the
GC content of the DNA. Nuclease S 1 at 100-1000 U/mL and single-
stranded carrier DNA at 20 Bg/mL are then added for single-strand
digestion, but the temperature depends on the degree of digestion
required. Loops of DNA will be degraded at 20°C, and 37-45°C will
digest single-stranded regions of DNA:RNA hybrids. The reactions
are cooled to 0°C and nuclease stop mixture (containing carrier RNA
at 50 Bg/cm 3) added. The products can then be phenol:chloroform
extracted, and the nuclease S 1-resistant hybrids analyzed by alkaline
or neutral agarose gel electrophoresis. In the case where a radiolabeled
DNA probe has been chosen, this can be analyzed by electrophoresis
through a polyacrylamide/urea gel and an autoradiogram established.
A detailed description of nuclease-S 1 mapping was presented in a
review by Favalaro et al. in 1980 (52).
- Cell and Nuclear Extracts
- I. Soluble Cell Extract Transcription System
for RNA Polymerase II
A soluble transcription system is a cell extract that will faithfully
transcribe an exogenous DNA as template. For this, the extract should
contain all the necessary factors needed for the correct initiation and
elongation of the RNA chain, and be low in nuclease activity to pre-
vent degradation of the exogenous DNA template or transcription
products during the incubation. Establishing soluble systems provides
a starting point for the separation, isolation, and analysis of those
factors required (in addition to RNA polymerase) for accurate tran-
scription in vitro (45).
To date, many eukaryotic and viral genes have been sequenced, and
putative controlling sequences have been deduced. These observa-
tions can be verified by in vitro manipulation of the DNA template and
then using the DNA in an in vitro transcription system to test the effect.
Weil et al. in 1979 (38) prepared a high-speed supernatant (S100)
cellular extract from cultured human cells. This S 100 extract can be
used for transcription experiments once supplemented with purified