Exogenous primer-independent cDNA synthesis with commercial reverse transcriptase preparations on plant virus RNA templates

Abstract

Upon reverse transcription and cloning manipulations with virion RNAs of several plant viruses, namely beet yellows virus, brome mosaic virus, and potato virus X, we came across a significant background synthesis of cDNA on the virion RNA template in vitro independent of exogenous primers added. When tested with beet yellow virus RNA template, several commercial preparations of avian myeloblastosis virus (AMV) reverse transcriptase showed the background activity monitored by the [alpha-32P]dNTP incorporation in vitro, while the enzyme from murine moloney leukemia virus (MMLV) was found strictly exogenous-primer-dependent. To detect possible nucleic acid contaminations in reverse transcriptase, the enzyme preparations from several commercial sources were incubated with [gamma-32P]ATP and polynucleotide kinase. The labeled material from AMV reverse transcriptase preparations comigrated with a tRNA marker in polyacrylamide gels and was found to be RNase-sensitive. The MMLV reverse transcriptase preparations were free from such a contamination. These results indicate that the exogenous-primer-independent cDNA synthesis by some AMV reverse transcriptases could be due to a contaminating tRNA (or its low-molecular-weight degradation products) serving as an endogenous primer.