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tRNA like sequences in the human genome

A human foetal DNA library cloned in bacteriophage lambda Charon 4A was screened for human tRNA gene sequences using a X. laevis tDNA as a probe (3.18 Kb X. laevis DNA fragment containing 8 different tRNA genes, cloned in pBR 322). From the screeing procedure one lambda clone was isolated that showed positive hybridization. This clone, lht3, was further characterized by hybridization of [32P] labelled human 4S RNA probe and the individual subclones of X. laevis tDNA. Of the 5 different subclones of X. laevis tDNA, only the tDNA Tyr subclone showed positive hybridization. In addition the hybridization pattern observed was similar to that observed with a [32p] labelled human 4S RNA probe. A restriction cleavage map of lht3 was constructed, and the Hindlll restriction fragments of lht3 DNA showing positive hybridization were subsequently subcloned in plasmid pAT153 as pNB 1 and pNB4. These subclones were tested in Xenopus oocyte nuclei and HeLa cell extract for their transcriptional activity. A fine structure restriction map of the subclones was constructed. The smallest restriction fragments of pNB1 and pNB4 showing positive hybridization to [32P] labelled human 4S RNA probe were identified as 0.6 Kb Smal-Hindlll restriction fragment of pNB 1 and 0.9Kb PstI restriction fragment of pNB4. The DNA nucleotide sequence of the 0.6Kb Smal - Hindlll fragment was determined. This nucleotide sequence was examined for tRNA gene sequences and found to contain no full length tRNAs. However when the 0.6Kb sequence was compared with the 3.18Kb DNA sequence of X. laevis, one region of homology was identified. This was a stretch of 17 nucleotides long that showed 82% homology to 3' end of X. laevis tRNA Tyr gene. The 0.6Kb DNA sequence also contains an incomplete human Alu sequence and the stretch of 17 nucleotides homologous to Xenopus tRNA Tyr gene is contained within the 31 bp human insert present in the Alu sequence.