Sequence and transcription of tRNAVal gene from Xenopus laevis

A DNA fraction enriched in tRNA genes has been prepared by CsCl density gradient centrifugation of Xenopus laevis DNA in the presence of actinomycin D. This DNA fraction was cut with the restriction endonuclease EcoRI and the fragments 800-900 base pairs in size were cloned into the plasmid pBR325. Recombinant DNAs were screened by hybridization to labeled tRNA and for the ability to support transcription in vitro. The entire sequence of one fragment was determined by sequencing the ends of an overlapping set of deletion fragments. A sequence homologous to tRNAVal from mammalian sources was found in this fragment and it was shown that this sequence corresponds to the region of the fragment that is transcribed. The cloned fragment was also transcribed in vivo after injection into X. laevis oocytes. The RNA that was synthesized in the oocytes was digested with ribonuclease T1 and the oligonucleotides were separated to produce a two-dimensional fingerprint. The results of the analysis of the oligonucleotides are consistent with the sequence determined for the tRNAVal gene. The X. laevis genome has 200-250 copies of the 892 base pair EcoRI fragment and additional copies of a 4100 base pair EcoRI fragment that each contain a tRNAVal gene. Digestion of X. laevis DNA with several other restriction endonucleases reveals that the cloned fragment that contains the tRNAVal gene is part of a longer sequence element that is tandemly repeated in the genome.