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Mitochondrial DNA differentiation in the critically endangered Berg River redfin (Pseudobarbus burgi).

The Berg River redfin (Pseudobarbus burgi) is a critically endangered endemic cyprinid from South Africa. We investigated mitochondrial DNA control region variation among specimens representative of five populations drawn from two adjacent river systems. Phylogenetic analyses, a minimum spanning network, and an analysis of molecular variance underscore the pronounced genetic separation of redfins originating from the geographically closely allied Verlorevlei and Berg Rivers, two populations that may have remained isolated since the Pleistocene. Despite a lack of geographic structuring within the Berg River, historic female gene flow among the upper and middle/lower parts of the river appears to be limited and the contemporary populations are probably isolated due to deterioration of the mainstream of the river. Our results suggest that the Berg and Verlorevlei populations should be managed as distinct conservation units. We encourage the use of sanctuaries, particularly by private landowners within both river systems, as this approach may contribute effectively to preserving genetic diversity within the species.

Animals↗
PubMed1989

Molecular analysis of the linear 2.3 kb plasmid of maize mitochondria: apparent capture of tRNA genes.

The nucleotide sequence and transcription pattern of the linear 2.3 kb plasmid of maize mitochondria was analyzed in order to elucidate its possible function in the organelle. The plasmid has 170 bp inverted repeats at its termini composed, in turn, of shorter repetitive sequences. An open reading frame within the plasmid is transcribed and can potentially specify a 33 kD product. In addition the plasmid contains two tRNA genes homologous to chloroplast sequences; the tRNApro(CAA) and the tRNAtrp(UGG). Both of the tRNA genes of the plasmid are transcribed, but apparently only the tRNAtrp is processed to the correct size. These tRNA sequences are found in the main mitochondrial genome of all higher plants tested, and in most maize relatives. An exception is the close maize relative Northern teosinte in which the tRNAtrp gene is also carried on a plasmid. These results suggest that the 2.3 kb plasmid has acquired the tRNA sequences from the main mitochondrial DNA. It is possible that the plasmid-encoded tRNAtrp gene is essential for organelle function thereby ensuring the maintenance of the plasmid in the mitochondrion.

Base Sequence↗
PubMed1990

In vitro processing of mitochondrial and plastid derived tRNA precursors in a plant mitochondrial extract.

A lysate of purified mitochondria of the higher plant Oenothera processes in vitro synthesized tRNA precursors to the mature tRNA size. In vitro synthesized transcripts containing genuine plant mitochondrial tRNAs and analogous RNAs from mitochondrial loci with plastid derived tRNA sequences are accurately processed by an RNAase P-like activity to yield the mature 5'-terminus. A four nucleotide deletion in the anticodon stem-loop structure, however, prevents processing. The results show that in vitro transcripts containing tRNAs from sequence fragments of plastid origin integrated in plant mitochondrial genomes can be processed correctly in plant mitochondria, if tRNA sequences and structures are intact.

Base Sequence↗
PubMed1995

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase.

Identity elements of tRNA(Thr) towards Saccharomyces cerevisiae threonyl-tRNA synthetase were examined using in vitro transcripts. By mutation studies, a marked decrease in aminoacylation with threonine showed that the first base pair in the acceptor stem and the second and third positions of the anticodon are major identity elements of tRNA(Thr), which are essentially the same as those of Escherichia coli tRNA(Thr). Base substitution of the discriminator base, A73, by G73 or C73 impaired the threonine accepting activity, but not that by U73, suggesting that this position contributes to discrimination from other tRNAs possessing G73 or C73. No effects on aminoacylation were observed with substitutions at the second base pair in the acceptor stem. These are in contrast to E.coli tRNA(Thr) where the second base pair is required for the specific aminoacylation, with the discriminator base playing no roles. Of several mutations at the third base pair in the acceptor stem, only the G3-U70 mutation impaired the activity, suggesting that the G3-U70 wobble pair, the identity determinant of tRNAAla, acts as a negative element for threonyl-tRNA synthetase. These findings indicate that while the first base pair in the acceptor stem and the anticodon nucleotides have been retained as major recognition sites between S. cerevisiae and E.coli tRNA(Thr), the mechanism by which the synthetase recognizes the vicinity of the top of the acceptor stem seems to have diverged with the species.

Acylation↗
PubMed1997

Two step synthesis of (-) strong-stop DNA by avian and murine reverse transcriptases in vitro.

Retroviral reverses transcriptases (RTs) are RNA- and DNA-dependent DNA polymerases that use a tRNA bound at the so-called primer binding site (PBS) located near the 5'end of the genomic RNA as primer. Thus, RTs must be able to accommodate both RNA and DNA in the primer strand. To test whether the natural primer confers some advantages to the priming process, we compared initiation of reverse transcription of avian and murine retroviral RNAs, using either their natural tRNA primer, tRNATrp and tRNAPro, respectively, or synthetic 18mer oligodeoxyribonucleotides (ODNs) and oligoribonucleotides (ORNs) complementary to their PBS. In both retroviral systems, the initial extension of ODNs was fast and processive. The initial extension of ORNs, tRNATrp and tRNAPro was much slower and distributive, giving rise to the transient accumulation of short pausing products. Synthesis of (-) strong-stop DNA was delayed when using ORNs and tRNAs, compared to ODNs. Even though ORNs and tRNAs were initially extended at the same rate, the short pausing products were more rapidly extended when using the tRNA primers. As a consequence, synthesis of (-) strong-stop DNA was much more efficient with tRNA primers, compared to ORNs. Taken together, these results suggest that the tRNA-primed synthesis of (-) strong-stop DNA is a two-step process, as already observed for HIV-1. The initiation mode corresponds to the initial non-processive nucleotide addition and extension of the short pausing products. It is more efficient with the natural primers than with ORNs. Initiation is followed by a more processive and unspecific elongation mode. Elongation is observed when the primer strand is DNA, i.e. when using the ODNs as primers or when the ORN and tRNA primers have been extended by a sufficient number (depending on the retroviral system) of deoxyribonucleotides.

Animals↗
PubMed1997

Large, rapidly evolving intergenic spacers in the mitochondrial DNA of the salamander family Ambystomatidae (Amphibia: Caudata).

We report the presence, in the mitochondrial DNA (mtDNA) of all of the sexual species of the salamander family Ambystomatidae, of a shared 240-bp intergenic spacer between tRNAThr and tRNAPro. We place the intergenic spacer in context by presenting the sequence of 1,746 bp of mtDNA from Ambystoma tigrinum tigrinum, describe the nucleotide composition of the intergenic spacer in all of the species of Ambystomatidae, and compare it to other coding and noncoding regions of Ambystoma and several other vertebrate mtDNAs. The nucleotide substitution rate of the intergenic spacer is approximately three times faster than the substitution rate of the control region, as shown by comparisons among six Ambystoma macrodactylum sequences and eight members of the Ambystoma tigrinum complex. We also found additional inserts within the intergenic spacers of five species that varied from 87-444 bp in length. The presence of the intergenic spacer in all sexual species of Ambystomatidae suggests that it arose at least 20 MYA and has been a stable component of the ambystomatid mtDNA ever since. As such, it represents one of the few examples of a large and persistent intergenic spacer in the mtDNA of any vertebrate clade.

Ambystoma↗
PubMed2005

tRNA actively shuttles between the nucleus and cytosol in yeast.

Previous evidence suggested that transfer RNAs (tRNAs) cross the nuclear envelope to the cytosol only once after maturing in the nucleus. We now present evidence for nuclear import of tRNAs in yeast. Several export mutants accumulate mature tRNAs in the nucleus even in the absence of transcription. Import requires energy but not the Ran cycle. These results indicate that tRNAs shuttle between the nucleus and cytosol.

Active Transport, Cell Nucleus↗
PubMed2000

Biogeography of the southeastern United States: a comparison of salamander phylogeographic studies.

Most phylogeographic studies of species from the southeastern United States have shown a simple east-west division of mtDNA variation. However, a study of the salamander Ambystoma maculatum resulted in a more complex pattern that includes a close affinity between populations from the Central Highlands of Missouri and Arkansas and the Coastal Plain separated by a genetically distinct central group of populations. We test the generality of this observation by surveying mitochondrial DNA (mtDNA) variation in the closely related species A. talpoideum. An Ambystoma-specific intergenic spacer was amplified and sequenced. The 26 resulting haplotypes varied from 380 to 800 base pairs, and alignments, including the outgroup, required 101 insertions/deletions. Sequence divergence among haplotypes ranged from 0.001 to 0.758. Population subdivision was extensive (theta = 0.64). Phylogenetic analysis of A. talpoideum mtDNA sequence reveals a close relationship between the populations from the Central Highlands and the Coastal Plain. This result is similar to that obtained for A. maculatum, although the A. talpoideum clade is not as well differentiated from its sister clades. We discuss the differences and similarities between the two Ambystoma species and previous studies and call for increased focus on multiple species with similar ecologies as a way to detect subtle biogeographic events.

Ambystoma↗
PubMed2004

Mitochondrial DNA variation in the Japanese marten Martes melampus and Japanese sable, Martes zibellina.

Genetic relationship among Japanese sables, Martes zibellina and the introduced Japanese martens, Martes melampus in northern Japan was revealed by analyzing a 521-524bp DNA sequence from the cytochrome b (112bp)/transfer RNA-threonine (67bp)/tRNA-proline (65bp) and control region (277-280bp) of the mitochondrial genome. Intraspecific differences in sequences of M. zibellina and M. melampus (3.8-15.0% and 1.9-16.4%, respectively) were similar to interspecific differences between these two species (5.8-16.6%). Comparison of sequence data exhibited five haplotypes of M. melampus and four haplotypes of M. zibellina, which clustered into two groups (clusters-A and-B). Cluster-A included two haplotypes of M. melampus and two haplotypes of M. zibellina, whereas cluster-B included three haplotypes of M. melampus and two haplotypes of M. zibellina. Results of this study lead three possible explanations. Firstly, past hybridization between M. zibellina and M. melampus might have occurred. Secondary, these two species might have similar heteroplasmy of mtDNA. Thirdly, these haplotypes might have come from nuclear genome. Although further intensive studies are needed to make a conclusion, detection of hybridization with the Japanese marten are occurred or not is quite important to conserve the Japanese sable.

Animals↗
PubMed2006

[Sequence and structure analysis of mitochondrial tRNApro and tRNAthr genes in domestic goose breeds].

We report here the results of the sequence and structure analysis of mitochondrial tRNApro and tRNAthr genes in domestic goose breeds by sequencing the mitochondrial DNA from a total of 25 samples from 6 breeds of Chinese geese and 2 breeds of domestic Europe geese. Sequences and the cloverleaf structure of tRNApro (69 bp) and tRNAthr (68 bp) in domestic goose breeds were described and analysed They were compared amongst the three domestic goose breeds as well as between Anseriformes (Anser cygnoides) and Galliformes (Gallus gallus domesticus, Genbank accession number NC001323). Both goose tRNApro and tRNAthr genes have normal cloverleaf secondary structures. The amino acid arm and the anticodon loop of the cloverleaf structure of tRNApro and tRNAthr are very conservative among Anser albifrons, Anser anser and Anser cygnoides. The gene sequences in this study were deposited to GenBank under accession numbers AY427800-AY427805 and AY427812-AY427814.

Animals↗
PubMed1992

A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.

Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.

Amino Acid Sequence↗
PubMed1998

Structure and expression of several bean (Phaseolus vulgaris) nuclear transfer RNA genes: relevance to the process of tRNA import into plant mitochondria.

Bean nuclear genes for tRNA(Pro), tRNA(Thr) and tRNA(Leu) were isolated. Expression of the tRNA(Pro) genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNA(Pro) gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNA(Thr) genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNA(Thr). The tRNA(Leu) gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNA(Leu) however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNA(Leu) and tRNA(Thr). From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group.

Base Sequence↗
PubMed1996

Mutations in both the U5 region and the primer-binding site influence the selection of the tRNA used for the initiation of HIV-1 reverse transcription.

The initiation of HIV-1 reverse transcription is primed by a cellular tRNA(Lys),3 molecule which is bound to a complementary sequence near the 5' end of the viral RNA genome designated as the primer-binding site (PBS). Recent studies have suggested that sequences upstream of the PBS within U5 consisting of a stretch of adenine nucleotides (referred to as the A-loop) might be important in the selection and positioning of tRNALys,3 primer used to initiate reverse transcription. To further explore the role that the A-loop plays in reverse transcription, we have constructed proviral genomes in which the PBS was changed so as to be complementary to the 3'-terminal 18 nucleotides of tRNA(Ile), tRNA(Pro), or tRNA(Trp) [pHXB(Ile), pHXB(Pro), or pHXB(Trp), respectively]; a second set of proviral genomes was constructed which contained additional mutations so that the A-loop regions were complementary to the anticodon region of tRNA(Ile) [pHXB(Ile-AC)], tRNA(Pro) [pHXB(Pro-AC)], or tRNA(Trp) [pHXB(Trp-AC)]. Transfection of the proviruses into COS-1 cells followed by coculture with SupT1 cells resulted in production of infectious virus. PCR was used to amplify the PBS regions which were subcloned into M13mp18 followed by DNA sequence analysis. After short-term culture, the PBSs of proviruses derived from pHXB(Ile), pHXB(Pro), and pHXB(Trp) reverted to be complementary to tRNA(Lys),3. The PBSs of the viruses derived from pHXB(Ile-AC) also reverted to be complementary to tRNA(Lys),3; the A-loop region was still complementary to tRNA(Ile). In contrast, viruses derived from transfection of pHXB(Pro-AC) initially maintained a PBS complementary to tRNA(Pro). Upon extended culture, we identified proviruses which contained PBSs complementary to two additional tRNAs: tRNA(Ile) and tRNA(Lys),3. Furthermore, we found proviruses which contain two PBSs within the same genome: one complementary to tRNA(Lys),3 and a second complementary to tRNA(Pro) or tRNA(Ile). Viruses derived from transfection of pHXB(Trp-AC) were the most delayed in appearance following transfection. Analysis of the PBS revealed that early after transfection, the majority of the PBSs were complementary to tRNA(Trp). After further in vitro culture, proviruses were identified with a PBS complementary to a new tRNA, tRNA(Met). Finally, upon extended culture, the viruses derived from the transfection of pHXB(Ile-AC), pHXB(Pro-AC), and pHXB(Trp-AC) contained mutations upstream from the PBS in U5 that created a stretch of 3 adenine nucleotides. The results of these studies then highlight the flexibility that exists with respect to the selection of the tRNA primer used to initiate HIV-1 reverse transcription.

Animals↗