Expression of individual members of a tRNA(Gly)1 multigene family in vivo follows the same pattern as in vitro.
Individual members of a tRNA(Gly)1 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 tRNA(Gly)1 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 tRNA(Gly)1 reached very high levels, whereas the transcripts arising from tRNA(Gly)1-6:7 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.
Sharma
S
S
Department of Microbiology and Cell Biology and Centre for Genetic Engineering, Indian Institute of Science, Bangalore.
Sriram
S
S
Patwardhan
L
L
Gopinathan
K P
KP
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D002460:Cell Line; D018076:DNA, Complementary; D017344:Genes, Insect; D008969:Molecular Sequence Data; D005810:Multigene Family; D009841:Oligonucleotides / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D009841:Oligonucleotides; D012346:RNA, Transfer, Amino Acyl