THE TRANSFER RNATYR MULTIGENE FAMILY OF NICOTIANA-RUSTICA - GENOME ORGANIZATION, SEQUENCE ANALYSES AND EXPRESSION INVITRO

Tobacco tRNA(Tyr) genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA(Tyr)-specific probe to Southem blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector lambdagtWES.lambdaB and in vitro packaging. The phage library was screened with a 5'-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA(Tyr). Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA(Tyr) genes code for the known tobacco tRNA1Tyr (GpsiA) and seven code for tRNA2Tyr (GpsiA). The two tRNA species differ in one nucleotide pair at the basis of the TpsiC stem. Only one tRNA(Tyr) gene (pNtY5) contains a point mutation (T54 --> A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs(Tyr) - with one exception - are processed and spliced in both extracts. The tRNA(Tyr) gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.