PLMItRNA, a database for mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

The PLMItRNA database for mitochondrial tRNA molecules and genes in VIRIDIPLANTAE: (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159-162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (RHODOPHYTAE:) and two STRAMENOPILES: The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA.     

PLMItRNA, a database for higher plant mitochondrial tRNAs and tRNA genes.

The PLMItRNA database contains information and multialignments of tRNA genes and molecules detected in higher plant mitochondria. It has been developed from a previous compilation of higher plant mitochondrial tRNA genes [Sagliano,A., Volpicella,M., Gallerani,R. and Ceci,L.R. (1998) Nucleic Acids Res., 26, 154-155] and implemented with data and sequences of tRNA molecules retrieved from the literature. The current version of the database reports information on 171 genes and 16 tRNA molecules from 24 plants. PLMItRNA is accessible via WWW at http://bio-www.ba.cnr.it:8000/srs/     

PLMItRNA,a database on the heterogeneous genetic origin of mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes

The updated version of PLMItRNA reports information and multialignments on 609 genes and 34 tRNA molecules active in the mitochondria of Viridiplantae (27 Embryophyta and 10 Chlorophyta), and photosynthetic algae (one Cryptophyta, four Rhodophyta and two Stramenopiles). Colour-code based tables reporting the different genetic origin of identified genes allow hyper-textual link to single entries. Promoter sequences identified for tRNA genes in the mitochondrial genomes of Angiospermae are also reported. The PLMItRNA database is accessible at http://bighost.area.ba.cnr.it/PLMItRNA/.     

The complete set of tRNA species in Nanoarchaeum equitans

The archaeal parasite Nanoarchaeum equitans was found to generate five tRNA species via a unique process requiring the assembly of seperate 5' and 3' tRNA halves [Randau, L., Munch, R., Hohn, M.J., Jahn, D. and Soll, D. (2005) Nanoarchaeum equitans creates functional tRNAs from separate genes for their 5'- and 3'-halves. Nature 433, 537-541]. Biochemical evidence was missing for one of the computationally-predicted, joined tRNAs designated as tRNA(Trp). Our RT-PCR and sequencing results identify this tRNA as tRNA(Lys) (CUU) joined at the alternative position between bases 30 and 31. We show that the intron-containing tRNA(Trp) was misidentified in the initial Nanoarchaeum equitans genome annotation [E. Waters et al. (2003) The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc. Natl. Acad. Sci. USA 100, 12984-12988]. Along with a previously unidentified joined tRNA(Gln) (UUG), Nanoarchaeum equitans exhibits 44 tRNAs and is enabled to read all 61 sense codons. Features unique to this set of tRNA molecules are discussed.     

tRNA nucleotide 47: an evolutionary enigma.

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.     

Structural analysis of Arabidopsis thaliana chromosome 3. I. Sequence features of the regions of 4,504,864 bp covered by sixty P1 and TAC clones.

Based on the physical map of Arabidopsis thaliana chromosome 3 previously constructed with CIC YAC, TAC, P1 and BAC clones (Sato, S. et al., DNA Res., 5, 163-168, 1998), a total of 60 P1 and TAC clones were sequenced, and the sequence features of the resulting 4,504,864 bp regions were analyzed by applying various computer programs for similarity search and gene modeling. As a result, a total of 1054 potential protein-coding genes were identified. The average density of the genes identified was 1 gene per 4066 bp. Introns were observed in 77% of the genes, and the average number per gene and the average length of the introns were 3.9 and 156 bp, respectively. These sequence features are essentially identical to those of chromosome 5 in our previous reports, but the gene density was slightly higher than that observed for chromosomes 2 and 4. The regions also contained 10 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available through the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/kaos/.     

tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria.

Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis.     

Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures

Mamit-tRNA (http://mamit-tRNA.u-strasbg.fr), a database for mammalian mitochondrial genomes, has been developed for deciphering structural features of mammalian mitochondrial tRNAs and as a helpful tool in the frame of human diseases linked to point mutations in mitochondrial tRNA genes. To accommodate the rapid growing availability of fully sequenced mammalian mitochondrial genomes, Mamit-tRNA has implemented a relational database, and all annotated tRNA genes have been curated and aligned manually. System administrative tools have been integrated to improve efficiency and to allow real-time update (from GenBank Database at NCBI) of available mammalian mitochondrial genomes. More than 3000 tRNA gene sequences from 150 organisms are classified into 22 families according to the amino acid specificity as defined by the anticodon triplets and organized according to phylogeny. Each sequence is displayed linearly with color codes indicating secondary structural domains and can be converted into a printable two-dimensional (2D) cloverleaf structure. Consensus and typical 2D structures can be extracted for any combination of primary sequences within a given tRNA specificity on the basis of phylogenetic relationships or on the basis of structural peculiarities. Mamit-tRNA further displays static individual 2D structures of human mitochondrial tRNA genes with location of polymorphisms and pathology-related point mutations. The site offers also a table allowing for an easy conversion of human mitochondrial genome nucleotide numbering into conventional tRNA numbering. The database is expected to facilitate exploration of structure/function relationships of mitochondrial tRNAs and to assist clinicians in the frame of pathology-related mutation assignments.     

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.     

Complete structure of the chloroplast genome of a legume, Lotus japonicus.

The nucleotide sequence of the entire chloroplast genome (150,519 bp) of a legume, Lotus japonicus, has been determined. The circular double-stranded DNA contains a pair of inverted repeats of 25,156 bp which are separated by a small and a large single copy region of 18,271 bp and 81,936 bp, respectively. A total of 84 predicted protein-coding genes including 7 genes duplicated in the inverted repeat regions, 4 ribosomal RNA genes and 37 tRNA genes (30 gene species) representing 20 amino acids species were assigned on the genome based on similarity to genes previously identified in other chloroplasts. All the predicted genes were conserved among dicot plants except that rpl22, a gene encoding chloroplast ribosomal protein CL22, was missing in L. japonicus. Inversion of a 51-kb segment spanning rbcL to rpsl6 (positions 5161-56,176) in the large single copy region was observed in the chloroplast genome of L. japonicus. The sequence data and gene information are available on our World Wide Web database at http://www.kazusa.or.jp/en/plant/database.html.     

Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z

Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA-snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNA(Gly) genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNA(Gly)-snoR43 precursors. Using transgenic lines expressing a tagged tRNA-snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNA(Gly). In addition, we show that a recombinant RNase Z, the plant tRNA 3' processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNA(Gly). Finally, we describe a similar case in rice implicating a tRNA(Met-e) expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora)

Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA.     

ZYGOTE-ARREST 3 that encodes the tRNA ligase is essential for zygote division in Arabidopsis

In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive.Here, we showed that a mutation in ZYGOTE-ARREST 3(ZYG3) in Arabidopsis led to a tight zygote-lethal phenotype.Map-based cloning revealed that ZYG3 encodes the transfer RNA(tRNA) ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL::cAtRNL-sYFP-complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA.Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.     

tRNAs of Trypanosoma brucei. Unusual gene organization and mitochondrial importation.

In Trypanosoma brucei the U snRNA B gene (J. C. Mottram, K. L. Perry, P. M. Lizardi, R. Lührmann, N. Agabian, and R. G. Nelson (1989) Mol. Cell. Biol. 9, 1212-1223) is very tightly linked with other small RNA genes coding for tRNA(ACGArg), tRNA(CUULys), and a approximately 275-nucleotide RNA (RNA X) of unknown function. A similar genomic organization is found at the U6 snRNA locus, where the U6 gene is linked to tRNA(CGUThr) and tRNA(GUATyr) genes. The tRNA(Lys) and tRNA(Arg) genes are members of a multigene family, whereas the tRNA(Thr) and tRNA(Tyr) genes are single copy. Two additional tRNA(CUULys) genes and one tRNA(UUULys) gene were also isolated and sequenced and, together with a sequence previously published (D. A. Campbell (1989) Nucleic Acids Res. 17, 9479), appear to represent the entire gene family. Probes for tRNA(Lys), tRNA(Arg), tRNA(Thr), and tRNA(Tyr) were found to hybridize with mitochondrial and cytoplasmic tRNAs but not with mitochondrial DNA. This supports the hypothesis that mitochondrial tRNAs may be nuclear-encoded and imported from the cytosol into the mitochondrion.     

The nucleotide sequence of blue-green algae phenylalanine-tRNA and the evolutionary origin of chloroplasts.

Phenylalanine tRNA from the blue-green alga, Agmenellum quadruplicatum, has been purified to homogeneity. The nucleotide sequence of this tRNA was determined to be: (see tests) Comparisons of the sequence and the modified nucleosides of this tRNA with those of other tRNAPhes thus far sequenced, indicate that this blue green algal tRNAPhe is typically prokaryotic and closely resembles the chloroplast tRNAPhes of higher plants and Euglena. The significance of this observation to the evolutionary origin of chloroplasts is discussed.