Reverse transcription of yeast double-stranded RNA and ribosomal RNA using synthetic oligonucleotide primers

Abstract

The ability of the four oligodeoxyribonucleotide primers oligo(dT)12-18, oligo(dA)12-18, oligo(dG)12-18 and oligo(dC)12-18 to act as primers for avian myeloblastosis virus reverse transcriptase on denatured yeast double-stranded (ds) RNA templates was investigated. Oligo(dT) and oligo(dA) were found to prime the synthesis of 1.1 and 1.0 kb reverse transcripts, respectively, using denatured M dsRNA as a template. The oligo(dT)- and oligo(dA)-primed cDNAs of M dsRNA hybridized to the region of the M dsRNA that encoded the killer toxin and to each other. Addition of oligo(dT) to reverse transcription reactions of denatured L dsRNA produced a 4.3 kb cDNA. During the course of this investigation oligo(dC) was observed to be a highly efficient primer for reverse transcription of yeast 18 S ribosomal RNA. Oligo(dC) primed the synthesis of a 1.0 kb transcript of 18 S rRNA which hybridized to the large Eco RI fragment of the 18 S rRNA gene. Reverse transcription of double-stranded RNA and 25 S ribosomal RNA was found to occur to some extent in the absence of added oligonucleotide primer.