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at 37C. The reaction was stopped by adding stop bath (0.05 M sodium
pyrophosphate, 2 mg/ml bovine serum albumin, 2 mg/ml Torula RNA and 5 mM
of the unlabeled ribonucleotide which was identical to the labeled NTP)
and chilling in ice (1). Macromolecules were precipitated by adding 2
ml of cold 10% trichloroacetic acid containing 20 mM sodium pyrophosphate
(TCAPP) to each reaction tube. After standing in ice for 15 min. the
precipitate was collected by filtration through a glass fiber disc (What
man GF/C) which had been prewashed with 5 ml of cold TCAPP. Each disc
was washed three times with 5-10 ml TCAPP, then three times with 5-10 ml
ether-ethanol (1:3) 37C, followed by two washes 5-10 ml ether.
Filtration was carried out in perforated porcelain crucibles (Gooch)
adapted to a vaccum manifold with ruber crucible holders (Walter).
Filtration was facilitated by maintaining a partial vaccum (ca. 28 in.
Hg). The GF/C filters were dried and placed in scintillation vials
containing 5 ml of toluene (scintillation grade, Mallinkrodt), 0.4% PPO
(2,5-diphenyloxazole), 0.01% POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene),
and counted for 10 min. in a liquid scintillation counter (Beckman LS-133).
A unit of enzyme activity is defined as that which will catalyze the
incorporation of 1 pmole of labeled ribonucleoside monophosphate into acid
precipitable material in 10 min. at 37.
Isolation of CTP labeled product
A sample of 0.30 ml of purified mitochondria (0.18 mg/ml protein)
was incubated in a 3.0 ml RNA polymerase reaction mixture (0.05 mM H-CTP,
2 Ci/mmole) for 15 min. The reaction was stopped by adding 3.0 ml of stop
bath (0.05 M sodium pyrophosphate, 1.09 mg/ml _E. coli RNA and 5 mM of
unlabeled CTP) and brought to 0.20 M NaCl and 0.10 M Tris pH9. Triton X