The tRNA(Tyr) multigene family of Nicotiana rustica: genome organization, sequence analyses and expression in vitro

Abstract

Tobacco tRNA(Tyr) genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA(Tyr)-specific probe to Southern 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 lambda gtWES.lambda B 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 tRNA1(Tyr) (G psi A) and seven code for tRNA2(Tyr) (G psi A). The two tRNA species differ in one nucleotide pair at the basis of the T psi C 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.