52
reported by Duda and Cherry (184) for a poly C polymerase activity in
sugar beet nuclei.
The RNA polymerase assay reaction mixture was scaled up so that the
3
labeled products of the incorporation of H-CMP could be studied. For
this purpose the reaction mixture contained in a total volume of 3.0 ml,
5.0 mM of each unlabeled ribonucleoside triphosphate and 0.05 mM of
3
H-CTP with a specific radioactivity of 2 Ci/mmold. After incubationof
15 min the products were isolated as described in the methods and
material section by phenol extraction followed by ethanol precipitation
of labeled products from the aqueous phase in the presence of carrier
_E. coli RNA and CTP. Zonal centrifugation (Figure 4) on sucrose gradient
was performed in order to determine the size of the tritium labeled pro
duct. The radioactivity remained at the top of the gradient indicating
the material was smaller than that of _E. coli 5S RNA. Therefore, the
sizes of the isolated products were estimated by Biogel P-4 column
filtration (Figure 5). The _E. coli carrier RNA eluted in the void volume
while two adsorbancy peaks were eluted which contained the labeled cyto
sine. Using the elution position of a standard CpC the molecular weight
of the first peak (BGP-1) was estimated to be approximately 800 which
corresponds to the molecular weight of a trinucleotide of poly C. The
molecular weight of the second peak (BGP-2) was estimated to be about
680 which corresponds to the molecular weight of a dinucleotide. It
should be noted that this smaller entity elutes before CpC indicating
that it is larger than a dinucleotide of this structure.
The components in the two peaks eluted from the P-4 column have
ultraviolet absorbancy spectrums very similar if not identical to that
of CpC, showing the characteristic absorption maximum and minimum at