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Polyadenylylation in mycobacteria: evidence for oligo(dT)-primed cDNA synthesis

Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK¹

Author for correspondence: Colin Ratledge. Tel: +44 1482 465243. Fax: +44 1482 465458. e-mail: c.ratledge{at}biosci.hull.ac.uk

The ability of mRNA to direct synthesis of cDNA in the presence of oligo(dT) was analysed using a novel application of fluorescein-11-dUTP incorporation into cDNA by reverse transcriptase. Evidence is provided for the first time that a majority of the mycobacterial mRNA pool is polyadenylylated. mRNA transcripts of hsp65 were also amplified with specific primers from the oligo(dT)-primed cDNA preparation in Mycobacterium bovis BCG, M. smegmatis and M. vaccae. Furthermore, PCR amplication of cDNAs for genes entD, entC and trpE2 from M. bovis BCG yielded the expected products when reverse transcription was primed with oligo(dT), suggesting that polyadenylylation is a general phenomenon in mycobacteria.

Keywords: mycobacteria, polyadenylylation, mRNA, oligo d(T), fluorescein-11-dUTP

Abbreviations: DEPC, diethylpyrocarbonate

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