Genetic Map of Bacteriophage T3

About 200 amber mutants of phage T3 were found to lie in 14 different genes. These genes are homologous to known T7 genes. The genetic map of T3 is very similar to that of T7.     

Nucleotide sequences and functional characterization of two tobacco UAG suppressor tRNAGln isoacceptors and their genes

We isolated and sequenced the two major tRNAGln isoacceptors with CUG and UmUG anticodons from the cytoplasm of Nicotiana rustica. These are the first tRNAsGln of nuclear origin characterized in plants. The tRNAGln sequences were used to design probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. The two cloned Nicotiana tRNAGln genes, coding for either of the two isoacceptors, are efficiently transcribed in HeLa cell nuclear extract. In vitro translation in the presence of purified Nicotiana tRNAsGln was carried out in a wheat germ extract partially depleted of endogenous tRNAs. Cytoplasmic (cyt) tRNAGlnCUG and to a lesser extent cyt tRNAGlnUmUG stimulated readthrough over the UAG stop codon present in the tobacco mosaic virus-specific context. The two tRNAGln isoacceptors are the second class of natural UAG suppressors identified in plants, in addition to cyt tRNATyrG A which has previously been characterized as the first natural UAG suppressor.     

Plant tRNA ligases are multifunctional enzymes that have diverged in sequence and substrate specificity from RNA ligases of other phylogenetic origins

Pre-tRNA splicing is an essential process in all eukaryotes. It requires the concerted action of an endonuclease to remove the intron and a ligase for joining the resulting tRNA halves as studied best in the yeast Saccharomyces cerevisiae. Here, we report the first characterization of an RNA ligase protein and its gene from a higher eukaryotic organism that is an essential component of the pre-tRNA splicing process. Purification of tRNA ligase from wheat germ by successive column chromatographic steps has identified a protein of 125 kDa by its potentiality to covalently bind AMP, and by its ability to catalyse the ligation of tRNA halves and the circularization of linear introns. Peptide sequences obtained from the purified protein led to the elucidation of the corresponding proteins and their genes in Arabidopsis and Oryza databases. The plant tRNA ligases exhibit no overall sequence homologies to any known RNA ligases, however, they harbour a number of conserved motifs that indicate the presence of three intrinsic enzyme activities: an adenylyltransferase/ligase domain in the N-terminal region, a polynucleotide kinase in the centre and a cyclic phosphodiesterase domain at the C-terminal end. In vitro expression of the recombinant Arabidopsis tRNA ligase and functional analyses revealed all expected individual activities. Plant RNA ligases are active on a variety of substrates in vitro and are capable of inter- and intramolecular RNA joining. Hence, we conclude that their role in vivo might comprise yet unknown essential functions besides their involvement in pre-tRNA splicing.     

Nucleotide sequences of nuclear tRNACys genes from Nicotiana and Arabidopsis and expression in HeLa cell extract

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA_GCA ^Cys as novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA^Cys genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs^Cys. Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA^Cys genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA^Cys genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs^Cys of plant origin.