Functional genomics of nuclear tRNAs in plants

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Functional analysis of entire tRNA genes in Arabidopsis whose genome had been determined recently. First, expression patterns of 23 different kinds of isoacceptor tRNA gene families were investigated with corresponding gene probes. Northern blot analysis indicated that tRNA^<Leu> (CAA) was tissue-specifically expressed in root and flower-bud. During seed-maturation after fertilization, several tRNA species such as tRNA^<Lys>(CUU),tRNA^<Pro>(UGG),tRNA^<Glu>(CUC) and tRNA^<Gln>(UUG) were abundant in mature seeds, while the level of remaining tRNAs examined were decreased in the same stage. Analysis of tRNAs that were sampled from various stages in the seed maturation with denatured polyacrylamide gel electrophoresis showed degradation of tRNAs in the late stage of the maturation. This result suggests that most of tRNA species were target of ribonuclease, but some of tRNAs were escaped from this degradation via an unknown mechanism.Second, in order to explore the functional expression of … More tRNAs in plant cells, reporter genes such as a GUS and luciferase has been widely used so far. In this study, we have evaluated a GFP marker gene : GFP/amber, in which a premature amber codon was artificially introduced in the fourth codon (Lys) from an initiation codon, was constructed by an in vitro site-directed mutagenesis. When the GFP/amber was introduced in onion epidermal cells with a particle gun, no fluorescence was observed as expected, but when introduced and co-expressed with a gene coding for suppressor tRNA^<Ser> (NtS2-am), weak fluorescence was detected. Therefore, GFP/amber would be used alternatively to explore functional expression of the specific tRNA in plant cells. In parallel, on the basis of the NtS2-am, which is known as the most active suppressor tRNA known so far, a novel in vivo system for analysis of effect of 5'-flanking region of tRNA on its transcription, i.e., substitution of the 5'-flanking sequence of the NtS2 for that of a target gene. We examined five RNA polymerase III genes in this system. Interestingly, relative activity of in vivo transcription of these genes was well correlated to that of transcription of the same genes in the cell-free system of tobacco BY-2. Less