Study on construction of a cDNA library corresponding to an amino acid-specific tRNA and influence of the modified nucleotide upon nucleotide misincorporations in reverse transcription.
The construction of a cDNA library corresponding to an amino acid-specific tRNA and the influence of the modified nucleotide in the tRNA upon misincorporation in reverse transcription were investigated. The distinctive feature of the constructive strategy is that the cDNA library was prepared in connection with the charging activity of the tRNA. The aminoacyl-tRNA was captured selectively by using a biotin-avidin system. After hydrolysis of the ester bond, the tRNA was collected as an amino acid-specific tRNA pool, and a poly(A) tail was attached to the CCA terminus for reverse transcription. To the 3'-terminus of the transcribed cDNA, poly (dC) was added by terminal deoxynucleotidyl transferase, and the cDNA was amplified by PCR. The double-stranded cDNA was used for transformation of Escherichia coli JM109. Sequence analyses of the obtained clones bearing the tRNA genes revealed that a few nucleotide substitutions occurred at the location where the modified nucleotides exist. Among them, it was noteworthy that 1-methyladenosine (m(1)A22) in the D-loop of Bacillus subtilis tRNA(Ser) was recognized as G in the reverse transcription and the result revealed different tendency of the misincorporation, which has been shown in the study of HIV-1 reverse transcription.
Matsugi
J
J
Division of Structural Biochemistry, Department of Biochemistry, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, 329-0498, Tochigi, Japan. jmatsugi@jichi.ac.jp
Murao
K
K
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D018076:DNA, Complementary / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D015723:Gene Library; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D012345:RNA, Transfer, Amino Acid-Specific / Q000737:chemistry / Q000235:genetics*; D012694:Serine / Q000235:genetics; D014158:Transcription, Genetic
D018076:DNA, Complementary; D009711:Nucleotides; D012345:RNA, Transfer, Amino Acid-Specific; D012694:Serine