NUCLEAR TRANSFER-RNA TYR GENES ARE HIGHLY AMPLIFIED AT A SINGLE CHROMOSOMAL SITE IN THE GENOME OF ARABIDOPSIS-THALIANA

We have examined the organization of tRNA(Tyr) genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 X 10(7) bp. Three tRNA(Tyr) gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA adn sequenced. All Eco RI fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA(Tyr) genes flanked by a tRNA(Ser) gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA(Tyr) genes encode the known cytoplasmic tRNA(G-PSI-A)(Tyr). Both genes contain a 12 bp long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRI-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O)also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggest that the tRNA(Ser)/tRNA(Tyr) cluster evolved 1-5 million year ago, after the Brassicaceae diverged from the other flowering plants about 5-10 million years ago.