92
present in the cell. This procedure provided the cleanest mitochondria
feasible without treating them with an exogenous nuclease.
3
The incorporation of label from H-ribonucleoside triphosphates into
acid insoluble products showed no dependence upon the presence of the
other ribonucleotides or that of added DNA. The cpm incorporated with
3 3
H-ATP or H-GTP as substrates were so low that it made it difficult to
determine the dependence of their incorporation upon the presence of the
other ribonucleotides. The presence of mitochondrial DNA and mitochondrial
pools of endogenous ribonucleotides could account for these results. How-
3
ever, H-CTP was the preferred substrate compared to the other tritium
labeled ribonucleoside triphosphates. The kinetics of the incorporation
(Figure 3) allow the presence of nuclease activity in the mitochondria as
3
demonstrated by departure from linearity after 5 to 10 min. With H-UTP
as substrate these mitochondria are capable of carrying out the synthesis
of a product which demonstrates some sensitivity to RNase. The failure
3
of the labeled product, obtained upon incubation with H-CTP, to be de
graded by pancreatic RNase A may be due to either the failure of the nuclease
to get into the mitochondria or the alteration of the RNA 3' terminus thus
preventing the RNase A from binding to the products. It is also possible
that the product is complexed with DNA such that it is RNase insensitive.
The results in Figure 3 also suggest that RNA synthesis based upon at
least partial sensitivity of product to RNase could be better studied by
following the incorporation of UTP by the isolated mitochondria rather
than CTP.
3
The H-CTP incorporating activity appears to be associated with
mitochondrial DNA in that the activity is enriched 125 fold per yg of