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 misincor poration in reverse transcription were investigated. The distinctive featur e of the constructive strategy is that the cDNA library was prepared in con nection with the charging activity of the tRNA. The aminoacyl-tRNA was capt ured selectively by using a biotin-avidin system. After hydrolysis of the e ster 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 termina l deoxynucleotidyl transferase, and the cDNA was amplified by PCR. The doub le-stranded cDNA was used for transformation of Escherichia coli JM109, Seq uence 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 trans cription. (C) 2001 Elsevier Science B.V. All rights reserved.