Expression of individual members of a tRNA1Gly multigene family in vivo follows the same pattern as in vitro

Sharma, Sujata ; Sriram, Satyanarayana ; Patwardhan, Lara ; Gopinathan, Karumathil P. (1997) Expression of individual members of a tRNA1Gly multigene family in vivo follows the same pattern as in vitro Gene, 194 (2). pp. 257-266. ISSN 0378-1119

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Individual members of a tRNA1Gly multigene family from Bombyx mori are transcribed to different levels in vitro in homologous nuclear extracts but their transcription status in vivo is not known. Two sets of tRNA1Gly belonging to the extreme groups of highly transcribed and barely transcribed copies have been examined for their expression patterns in vivo in B. mori-derived cell lines following transfection. We have developed a sensitive and reliable method for directly quantifying the transcription levels of transfecting tRNA genes without relying on the biological activity of the transcript. The strategy involved the insertion of synthetic oligodeoxyribonucleotide sequences into the coding region of the transfecting gene and monitoring the transcripts in an RNase protection assay using an antisense probe that clearly distinguished them from the endogenous tRNAs. The oligonucleotide insertion did not significantly affect the transcriptional status of the genes, even though the distance between the A and B boxes was enhanced by 10-15 nt. In vivo also the transcription of tRNA1Gly-1 reached very high levels, whereas the transcripts arising from tRNA1Gly-6:7 accounted for only 2-5% of the former, closely resembling the transcription patterns in vitro. These individual gene copies having identical coding sequences and consequently the same internal conserved regions, differed only in their flanking sequences which modulate their transcription levels.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Bombyx mori; Differential Gene Expression; In Vivo Transcription; RNA Polymerase III; Transcriptional Regulation; tRNA Genes
ID Code:23675
Deposited On:26 Nov 2010 09:00
Last Modified:02 Jun 2011 06:03

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