RNA-dependent DNA polymerase from rat tissues : distribution, partial purification, and characterization

Moranelli, Francesco (1973) RNA-dependent DNA polymerase from rat tissues : distribution, partial purification, and characterization. Masters thesis, Memorial University of Newfoundland.

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Abstract

RNA-dependent DNA polymerase (RD-DP) has been detected in all rat tissues examined, and a partial purification and characterization carried out. The relative distribution per unit weight of tissue in order of decreasing amount of activity is as follows: thymus, spleen, brain, liver, kidney, testis, heart, plasma, and red blood cells. The activity in crude extracts was found complexed to an endogenous template sensitive to RNase. After partial or complete elimination of the endogenous template, this enzyme was found to respond to externally added templates such as yeast RNA, 16S+23S rRNA from E. coli and QβRNA. The rRNA and QβRNA were more efficient templates than yeast RNA. RNase treatment prior to fractionation was found to abolish a portion of the endogenous template without freeing the enzyme from the high molecular weight (MW) complex. Prolonged RNase treatment was found to shift 30-80% of the RD-DP activity to a lower MW region on a gel filtration column, resulting in a separation of the activity from the bulk of the DNA-dependent and endogenous DNA polymerase activities. The characteristics of the low MW RD-DP differ substantially from those of the DNA-dependent DNA polymerase . Mn⁺⁺ proved to be twice as efficient as Mg⁺⁺ for the RNA-dependent activity with an optimun concentration one-fifth that of Mg⁺⁺, whereas the DNA-dependent polymerase preferred Mg⁺⁺ to Mn⁺⁺. A MW of approximately 120,000 has been estimated for the RD-DP eluting in the lower MW region. The activity versus enzyme concentration curve for the RD-DP was sigmoidal, and that for the DNA-dependent DNA polymerase linear, in the concentration range — examined. The effect of the rifamycin derivatives on the RD-DP paralleled their effect on the RD-DP from RNA oncogenic viruses reported in the literature. An interesting observation was the effect of N-ethylmaleimide and o-chloromercuribenzoate; the RD-DP was relatively unaffected compared to the inhibition effect on the DNA-dependent activity. Some of the later experiments revealed a DNA-dependent enzyme eluting in the same region as the low MW RD-BP. As to whether these two activities originate from the same or different enzymes remains to be determined.

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