28
and centrifuged again. The pellet was suspended in 4 ml of 0.50 M HCIO^
and incubated at 85C for 20 min. to hydrolyze DNA and RNA, cooled and
centrifuged. The top 3 ml of the supernatant was used for DNA analysis.
The pellet was suspended in 3 ml of 1.0 N NaOH and heated in a boiling
water bath until the precipitated protein was dissolved. This solution
was used for analysis of soluble proteins.
Protein determination
The protein concentrations were determined by the Lowry method (175) .
Dissolved bovine serum albumin served as the standard.
DNA determination
DNA concentrations were determined by the indole method of Hubbard
(176). Calf thymus DNA served as a standard. A sample of 0.05 ml was
added to 0.05 ml of 10% trichloroacetic acid and 1 ml of 0.04% indole-2
N HC1 solution. The mixture was shaken and incubated at 97C for 15 min.
and then cooled in ice. The mixture was then extracted with chloroform
three times by adding 2 ml of CHCl^ to the mixture, vortexing and centri
fuging 2000 rpm, 5 min. The chloroform phase was removed after each ex
traction. The absorbance of the aqueous phase at 490 mm was then determined.
RNA polymerase assay
Conditions for the assay of RNA polymerase were similar to those
described by Preston ert al. (177). The routine mixture contained in a
total volume of 0.10 ml: 0.05 M Tris-HCl pH 7.9, 0.005 M MgC^, 0.001
M MnCl2, 0.001 M DTT, 0.01 M KC1, 0.001 M of each unlabeled ribonucleoside
triphosphate, 0.032 mM or 0.004 mM or 0.016 mM (2 Ci/mmole of ^H-labeled
ribonucleoside triphosphate, 100 pg/ml of denatured calf thymus DNA and
enzyme. Assay mixtures were incubated for 10 min. (or the time indicated)