68
blocked terminus and/or methylated nucleoside moieties can not be ruled
out.
The Solubilization and Partial Purification of
Euglena Mitochondrial RNA Polymerase
The final objectives were to solubilize and purify the mitochondrial
DNA-directed RNA polymerase, to characterize its activity, to determine
its requirements for product synthesis, and to compare it to nuclear RNA
polymerase activities with respect to these requirements. In order to
accomplish this it was necessary to prepare mitochondria which were devoid
of contaminating nuclear DNA. This was achieved by obtaining a crude 5P
mitochondrial pellet, as described in Figure 1, and washing it twice in
STM Buffer before incubating the mitochondria in the presence of deoxyri-
bionuclease I (DNase I) for thirty minutes, zero degrees centigrade.
These mitochondria were shown to be devoid of nuclear DNA by work substan
tiated in this laboratory and no further purification was necessary. The
buoyant densities of the DNA in the mitochondrial preparations in CsCl are
demonstrated in Figure 10. The crude washed 5P mitochondria yield mostly
Euglena nuclear DNA (Tube a) which has a density of 1.707 g/cm but after
five minutes of incubation in DNase I at 4 C, most of the nuclear DNA was
removed (Tube b). Thirty minutes of incubation in DNase removes all of
the contaminating nuclear DNA and only mitochondrial DNA remains, which
3
has a density of 1.691 g/cm (Tube c). Further treatment with DNase for
one hour shows that the mitochondrial DNA is still present and is there
fore protected from digestion by DNase I by the mitochondrial membranes.
The mitochondria purified in this manner demonstrated the same ribonucle
otide incorporating activities as the mitochondria purified on renografin
gradients. Table 9 shows that the DNased treated mitochondria incorpora-