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.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Glycine and asparagme tRNA sequences from the archaebacteriuin,Methanobacterium thermoautotrophicum

Two tRNA sequences from Methanobacterium thermoautotrophium are reported. Both tRNA^Gly_GCC and tRNA_NUU^Asn, the first tRNA sequences from methanogens, were determined by partial hydrolyses (both chemical and enzymatic) and analyzed by gel electrophoresis. The two tRNAs contain the unusual T-loop modifications, Cm and m¹I, which are present in other archaebacterial tRNAs. Finally the presence of an unknown modification in the D-loop has been inferred by a large jump in the sequence ladder. These tRNAs are approximately equidistant from eubacterial or eukaryotic tRNAs.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

Multiple sequence rearrangements accompanying the duplication of a tRNAPro gene in wheat mitochondrial DNA

In the course of isolating tRNA genes from wheat mtDNA, we have found the same tRNA^Pro gene in two different Hind III restriction fragments, H-P1 (0.7 kbp) and H-P2 (1.7 kbp). Sequences immediately flanking these duplicate genes are closely related, although not identical; sequence comparisons suggest that multiple rearrangements have occurred in the vicinity of the H-P2 tRNA^Pro gene, relative to the H-P1 version. The chimeric nature of H-P2 is emphasized by the presence of sequences that are also found upstream of the wheat mitochondrial 26S rRNA gene, as well as sequences derived from chloroplast DNA. Comparison of H-P2 with H-P1 plus upstream sequences provides some insight into possible molecular events that might have generated H-P2. In particular, such comparisons suggest a model in which the homologous sequences in H-P2 are seen to be derived from H-P1 plus upstream sequences as a result of an intragenomic, site-specific rearrangement event, followed by amplification of the product, its fixation in the mitochondrial genome, and subsequent sequence divergence (single base changes as well as insertions/deletions of up to 50 nucleotides). The results reported here implicate particular primary sequence motifs in certain of the rearrangements that characterize H-P2.     

Localization of tRNA genes on the Petunia hybrida 3704 mitochondrial genome

22 tRNA genes corresponding to 17 tRNA species were localized on the master circle of Petunia hybrida mitochondrial (mt) DNA. Genes for trnN, trnM, trnS-GGA, trnW and trnH are of the chloroplast-like type and presumably originate from promiscuous chloroplast (cp) DNA sequences inserted into the petunia mitochondrial genome. A comparison of the mt tRNAs or tRNA genes population present in two monocotyledonous plants (wheat and maize) and two dicotyledonous plants (petunia and potato) show slight differences in the genetic origin of individual tRNAs. The organization of the petunia mt tRNA genes as well as the number of tRNA gene copies, compared to other plant species, is discussed.     

Queuine in plants and plant tRNAs: Differences between embryonic tissue and mature leaves

In eubacterial and eukaryotic tRNAs specific for Asn, Asp, His and Tyr the modified deazaguanosinederivative queuosine occurs in position 34, the first position of the anticodon. Analysis of unfractionated tRNAs from wheat and from tobacco leaves shows that these tRNAs contain high amounts of guanosine (G) in place of queuosine (Q). This was measured by the exchange of G34 for [³H]guanine catalysed by the specific tRNA guanine transglycosylase from E. coli. Upon gel electrophoretic separation of the labeled tRNAs, seven Q-deficient tRNA species including isoacceptors are detectable. Two are identified as cytoplasmic tRNAs^Tyr and tRNA^Asp and two represent chloroplast tRNA^Tyr isoacceptors. In contrast to leaf cytoplasm and chloroplasts, wheat germ has low amounts of tRNAs with G34 in place of Q.A new enzymatic assay is described for quantitation of free queuine in cells and tissues. Analysis of queuine in plant tissues shows that wheat germ contains about 200 ng queuine per g wet weight. In wheat and tobacco leaves queuine is present, if at all, in amounts lower than 10 ng/g wet weight. The absence of Q in tRNAs from plant leaves is therefore caused by a deficiency of queuine. Tobacco cells cultivated in a synthetic medium without added queuine do not contain Q in tRNA, indicating that these rapidly growing cells do not synthesize queuine de novo.     

Structure and Evolution of the Atypical Mitochondrial Genome of <Emphasis Type="Italic">Armadillidium vulgare</Emphasis> (Isopoda,Crustacea)

The crustacean isopod Armadillidium vulgare is characterized by an unusual ∼42-kb-long mitochondrial genome consisting of two molecules co-occurring in mitochondria: a circular ∼28-kb dimer formed by two ∼14-kb monomers fused in opposite polarities and a linear ∼14-kb monomer. Here we determined the nucleotide sequence of the fundamental monomeric unit of A. vulgare mitochondrial genome, to gain new insight into its structure and evolution. Our results suggest that the junction zone between monomers of the dimer structure is located in or near the control region. Direct sequencing indicated that the nucleotide sequences of the different monomer units are virtually identical. This suggests that gene conversion and/or replication processes play an important role in shaping nucleotide sequence variation in this mitochondrial genome. The only heteroplasmic site we identified predicts an alloacceptor tRNA change from tRNA^Ala to tRNA^Val. Therefore, in A. vulgare, tRNA^Ala and tRNA^Val are found at the same locus in different monomers, ensuring that both tRNAs are present in mitochondria. The presence of this heteroplasmic site in all sequenced individuals suggests that the polymorphism is selectively maintained, probably because of the necessity of both tRNAs for maintaining proper mitochondrial functions. Thus, our results provide empirical evidence for the tRNA gene recruitment model of tRNA evolution. Moreover, interspecific comparisons showed that the A. vulgare mitochondrial gene order is highly derived compared to the putative ancestral arthropod type. By contrast, an overall high conservation of mitochondrial gene order is observed within crustacean isopods.     

A tRNA Val(GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed

A tRNA^Val (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA^Val gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA^Val is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA^Val species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.     

Gene rearrangements in gekkonid mitochondrial genomes with shuffling,loss, and reassignment of tRNA genes

Background

Vertebrate mitochondrial genomes (mitogenomes) are 16–18 kbp double-stranded circular DNAs that encode a set of 37 genes. The arrangement of these genes and the major noncoding region is relatively conserved through evolution although gene rearrangements have been described for diverse lineages. The tandem duplication-random loss model has been invoked to explain the mechanisms of most mitochondrial gene rearrangements. Previously reported mitogenomic sequences for geckos rarely included gene rearrangements, which we explore in the present study.

Results

We determined seven new mitogenomic sequences from Gekkonidae using a high-throughput sequencing method. The Tropiocolotes tripolitanus mitogenome involves a tandem duplication of the gene block: tRNA^Arg, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 4. One of the duplicate copies for each protein-coding gene may be pseudogenized. A duplicate copy of the tRNA^Arg gene appears to have been converted to a tRNA^Gln gene by a C to T base substitution at the second anticodon position, although this gene may not be fully functional in protein synthesis. The Stenodactylus petrii mitogenome includes several tandem duplications of tRNA^Leu genes, as well as a translocation of the tRNA^Ala gene and a putative origin of light-strand replication within a tRNA gene cluster. Finally, the Uroplatus fimbriatus and U. ebenaui mitogenomes feature the apparent loss of the tRNA^Glu gene from its original position. Uroplatus fimbriatus appears to retain a translocated tRNA^Glu gene adjacent to the 5’ end of the major noncoding region.

Conclusions

The present study describes several new mitochondrial gene rearrangements from Gekkonidae. The loss and reassignment of tRNA genes is not very common in vertebrate mitogenomes and our findings raise new questions as to how missing tRNAs are supplied and if the reassigned tRNA gene is fully functional. These new examples of mitochondrial gene rearrangements in geckos should broaden our understanding of the evolution of mitochondrial gene arrangements.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-930) contains supplementary material, which is available to authorized users.     

Codon recognition mechanisms in plant chloroplasts

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA^Ala(U^*GC), cp tRNA^Pro(U^*GG) and cp tRNA^Arg(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that U:U and U:C wobble mechanisms can operate to allow the reading of these codons by cp tRNA^Leu (UAm⁷G).     

Sequences of three transfer RNAs from mosquito mitochondria

The sequences of three transfer RNAs from mosquito cell mitochondria, tRNA_UCG^Arg, tRNA_GUC^Asp, and tRNA_GAU^Ile, determined using a combination of rapid ladder and fingerprinting procedures are reported. These were compared with hamster mitochondrial tRNA_UCG^Arg and tRNA_GUC^Asp determined similarly, and a bovine mitochondrial tRNA_GAU^Ile determined using a somewhat different approach. The primary sequences of the mosquito tRNAs were 35 to 65% homologous to the corresponding mammalian mitochondrial species, and bore little homology to “conventional” (bacterial or eucaryotic cytoplasmic) tRNA. The modification status of the mosquito mitochondrial tRNAs resembled that of mammalian mitochondrial tRNA. The results contribute to the generalization that metazoan mitochondrial tRNA constitutes a distinctive, albeit loosely structured, phylogenetic group.     

Platyhelminth mitochondrial DNA: Evidence for early evolutionary origin of a tRNAserAGN that contains a dihydrouridine arm replacement loop,and of serine-specifying AGA and AGG codons

Summary The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the liver flukeFasciola hepatica (phylum Platyhelminthes, class Trematoda) has been determined, within which have been identified the genes for tRNA^ala, tRNA^asp, respiratory chain NADH dehydrogenase subunit I (ND1), tRNA^asn, tRNA^pro, tRNA^ile, tRNA^lys, ND3, tRNA^serAGN, tRNA^trp, and cytochromec oxidase subunit I (COI). The 11 genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of theF. hepatica mitochondrial genes differs from what is found in other metazoan mtDNAs. All of the sequenced tRNA genes except the one for tRNA^serAGN can be folded into a secondary structure with four arms resembling most other metazoan mitochondrial tRNAs, rather than the tRNAs that contain a TψC arm replacement loop, found in nematode mtDNAs. TheF. hepatica mitochondrial tRNA^serAGN gene contains a dihydrouridine arm replacement loop, as is the case in all other metazoan mtDNAs examined to date. AGA and AGG are found in theF. hepatica mitochondrial protein genes and both codons appear to specify serine. These findings concerningF. hepatica mtDNA indicate that both a dihydrouridine arm replacement loop-containing tRNA^serAGN gene and the use of AGA and AGG codons to specify serine must first have occurred very early in, or before, the evolution of metazoa.     

Transfer RNA genes associated with the 16S and 23S rRNA genes of Euglena chloroplast DNA

Hybridization studies of Euglena chloroplast ¹²⁵I-labeled tRNAs to restriction fragments of Euglena chloroplast DNA have shown that the spacer between the 16S and 23S rRNA genes, in two and possibly all three of the ribosomal DNA units, contains genes for tRNA^Ile and tRNA^Ala, whereas a tRNA gene (for either tRNA^Trp or tRNA^Glu) is located before probably all four 16S rRNA genes present on the chloroplast DNA molecule.     

Nucleotide sequence of Rattus norvegiens mitochondrial DNA that includes the genes for tRNAile, tRNAgln and tRNAf-met

The nucleotide sequence of a segment of mtDNA from Rattus norvegiens (rat) which contains the genes for tRNA^ile, tRNA^gl and tRNA^f-met has been determined. A detailed comparison has been made between this sequence and the corresponding sequences of mouse, human and bovine mtDNAs with regard to the primary and secondary structure of the tRNA genes, the regions connecting the tRNA genes, and the regions flanking the tRNA genes which code for the carboxyl terminus of URF-1 and the amino terminus of URF-2. No differences were found in the nucleotide sequences of the genes for tRNA^ile, tRNA^gln and tRNA^f-met in mtDNAs from three different female lines of rats (SASCO-1, SASCO-2 and Wild-UT) that differ by substitutions of 0.8% to 1.8% of their total nucleotides.     

Insertional Editing of Mitochondrial tRNAs of Physarum polycephalum and Didymium nigripes

tRNAs encoded on the mitochondrial DNA of Physarum polycephalum and Didymium nigripes require insertional editing for their maturation. Editing consists of the specific insertion of a single cytidine or uridine relative to the mitochondrial DNA sequence encoding the tRNA. Editing sites are at 14 different locations in nine tRNAs. Cytidine insertion sites can be located in any of the four stems of the tRNA cloverleaf and usually create a G·C base pair. Uridine insertions have been identified in the T loop of tRNA^Lys from Didymium and tRNA^Glu from Physarum. In both tRNAs, the insertion creates the GUUC sequence, which is converted to GTΨC (Ψ = pseudouridine) in most tRNAs. This type of tRNA editing is different from other, previously described types of tRNA editing and resembles the mRNA and rRNA editing in Physarum and Didymium. Analogous tRNAs in Physarum and Didymium have editing sites at different locations, indicating that editing sites have been lost, gained, or both since the divergence of Physarum and Didymium. Although cDNAs derived from single tRNAs are generally fully edited, cDNAs derived from unprocessed polycistronic tRNA precursors often lack some of the editing site insertions. This enrichment of partially edited sequences in unprocessed tRNAs may indicate that editing is required for tRNA processing or at least that RNA editing occurs as an early event in tRNA synthesis.