Oligo(dT)-primed synthesis of cDNA by reverse transcriptase in mycobacteria.
The possibility that mRNA from Mycobacterium tuberculosis and M. bovis BCG may present polyadenylation at the 3' end was investigated. The total RNA, extracted from the bacterial cells and treated with DNase, was used as substrate for reverse transcriptase (RT)-dependent cDNA synthesis. The RT reaction was primed with oligo(dT) and with downstream specific primers for the genes of the antigens 65 KDa and 85-C. PCR probing of the reaction products for cDNAs of the two mycobacterial genes yielded the expected 225 and 307 bp bands when RT synthesis was primed by oligo(dT) and by downstream specific primers. Reaction products from oligo(dT)-primed RT of RNase-treated RNA and untranscribed RNA, probed by PCR, failed to generate the 225 and 307 bp specific bands. These findings support the existence of polyadenylated tracts in mRNA of mycobacteria that can be targeted by oligo(dT) primers to initiate RT-dependent cDNA synthesis. This may result in an advance in the study of gene expression in these and possibly other bacteria.
Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia Via San Zeno, 35/39 - 56127, Pisa, Italy.
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D000942:Antigens, Bacterial / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D009163:Mycobacterium bovis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*
D000942:Antigens, Bacterial; D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012333:RNA, Messenger; C027903:oligo (dT); D011061:Poly A; D012194:RNA-Directed DNA Polymerase