Mitochondrial Large rRNA Research Articles

Phylogenetic Relationships among Some Catfishes Assessed by Small and Large Mitochondrial rRNA Sequences

Phylogenetic Relationships among Some Catfishes Assessed by Small and Large Mitochondrial rRNA Sequences

Matching symbiotic associations of an endangered orchid to habitat to improve conservation outcomes.

An understanding of mycorrhizal variation, orchid seed germination temperature and the effect of co-occurring plant species could be critical for optimizing conservation translocations of endangered plants with specialized mycorrhizal associations. Focusing on the orchid Thelymitra epipactoides, we isolated mycorrhizal fungi from ten plants within each of three sites; Shallow Sands Woodland (SSW), Damp Heathland (DH) and Coastal Heathland Scrub (CHS). Twenty-seven fungal isolates were tested for symbiotic germination under three 24 h temperature cycles: 12 °C for 16 h-16 °C for 8 h, 16 °C for 16 h-24 °C for 8 h or 27 °C constant. Fungi were sequenced using the internal transcribed spacer (ITS), nuclear large subunit 1 (nLSU1), nLSU2 and mitochondrial large rRNA gene (mtLSU). Orchids were grown to maturity and co-planted with each of ten associated plant species in a glasshouse experiment with tuber width measured at 12 months after co-planting. Two Tulasnella fungal lineages were isolated and identified by phylogenetic analyses, operational taxonomic unit 1 (OTU1) and 'T. asymmetrica'. Fungal lineages were specific to sites and did not co-occur. OTU1 (from the SSW site) germinated seed predominantly at 12-16 °C (typical of autumn-winter temperature) whereas 'T. asymmetrica' (from the DH and CHS sites) germinated seed across all three temperature ranges. There was no difference in the growth of adult orchids germinated with different OTUs. There was a significant reduction in tuber size of T. epipactoides when co-planted with six of the commonly co-occurring plant species. We found that orchid fungal lineages and their germination temperature can change with habitat, and established that translocation sites can be optimized with knowledge of co-occurring plant interactions. For conservation translocations, particularly under a changing climate, we recommend that plants should be grown with mycorrhizal fungi tailored to the recipient site.

A small sequence in domain v of the mitochondrial large ribosomal RNA restores Drosophila melanogaster pole cell determination in uv-irradiated embryos.

The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.

Use of mitochondrial RNA genes for the differentiation of fourTrichinellaspecies by multiplex PCR amplification

Until now, four species of the Trichinella genus have been identified in Europe: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. The aim of this work was to establish a sound polymerase chain reaction (PCR)-based method to differentiate these four species using mitochondrial rDNA as a reliable genetic marker and to evaluate the sensitivity of this method. Full-length DNA sequences coding for the small and large mitochondrial rRNA (mt-rrnS and mt-rrnL) of the four species are described. A multiplex PCR was designed and successfully tested on 24 European isolates. As few as one larva, or 100 pg of genomic DNA was detected, providing equivalent sensitivity to previously described PCR methods. The PCR-based method of mitochondrial rDNA amplification was thereby established as a sensitive and reproductive diagnostic method for the four European Trichinella species.

The Mechanisms Underlying Germline Development in Drosophila Embryos

The separation of germ and somatic cells is one of the oldest problems in developmental biology. In many animal species, a specialized portion of the egg cytoplasm, or germ plasm, is inherited by the cell lineage which gives rise to the germ cells, or germline. Germ plasm contains the maternal factors that are sufficient for germline formation. In Drosophila , germ plasm is referred to as polar plasm and is histologically distinguished by the presence of polar granules, which act as a repository for the maternal factors required for germline formation. We have been focusing on the function of two such factors, mitochondrial large rRNA (mtlrRNA) and nanos mRNA (for review, see Kobayashi, Sato and Hayashi, 2005).

Molecular identification of Trichinella spiralis and Trichinella britovi by diagnostic multiprimer large mitochondrial rRNA amplification.

Trichinella parasites with different epidemiological features still occur in Europe and four species of genus Trichinella have been identified: T. spiralis, T. britovi, T. nativa and T. pseudospiralis. Until now, two of them, T. spiralis and T. britovi, have been identified in Poland. In our studies we selected sequence coding for large mitochondrial rRNA (mt LrDNA) as a genetic marker and developed a sensitive LrDNA multiprimer PCR assay allowing for rapid identification of T. spiralis and T. britovi, parasites present in wild and domestic animals in Poland.

Genetic structure of Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota)

The genetic structure of five Fennoscandian populations of the threatened wood-decay fungus Fomitopsis rosea (Basidiomycota) was investigated using codominant PCR-RFLP, allele specific amplification (ASA) markers, inter simple sequence repeat (ISSR) markers and mating studies. Sequence analyses of a subset of single spore isolates revealed sequence variation in four target sequences; internal transcribed spacer (ITS) and intergenic spacer (IGS1) of the nuclear ribosomal DNA, the translation elongation factor 1 alpha (efa) gene and the super oxide dismutase (sod) gene. No sequence variation was found in amplified portions of the mitochondrial large and small rRNA genes. Genotype distributions were mostly (90%) in accordance with Hardy-Weinberg expectations, and the nrDNA markers (ITS/IGS1), efa and sod were in most cases (87%) in linkage equilibrium, indicating an outcrossing reproductive mode, panmictic conditions and large population sizes of the fungus. Mating tests confirmed that F. rosea exhibits an outcrossing bipolar heterothallic mating system. Mating allele richness was high in two investigated populations. Phylogenetic analyses of ITS and IGS1 sequences from the five geographic populations revealed some geographic sub-structuring of the ITS sequences, but no sub-structuring of IGS1. The nrDNA (ITS/IGS1), efa and sod markers gave a low overall FST (0.013). The ISSR markers gave no clustering of the populations in UPGMA, and the between-population variance component was very low in AMOVA (0.4%), indicating a high level of gene flow.

Three-Dimensional Ultrastructural Analysis of RNA Distribution within Germinal Granules of Xenopus

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.

Mitochondrial small ribosomal RNA is a component of germinal granules in Xenopus embryos.

Mitochondrial large rRNA (mtlrRNA) and small rRNA (mtsrRNA) have been identified as components of germinal granules in Drosophila. We have previously reported that mtlrRNA is present on the germinal granules in Xenopus embryos. Here we report that mtsrRNA is also a common component of the germinal granules. Extra-mitochondrial mtsrRNA is localized on the surface of germinal granules in germ plasm from four-cell to blastula stage, then disappears until the completion of gastrulation. This temporal and spatial distribution pattern is identical to that of mtlrRNA. During the stages when both mitochondrial rRNAs are present around the germinal granules, mitochondrial-type ribosomes, typified by their smaller size, were also present on the surface of the germinal granules.

Delocalization of polar plasm components caused by grandchildless mutations, gs(1)N26 and gs(1)N441, in Drosophila melanogaster.

Two maternal-effect grandchildless (gs) mutations of Drosophila melanogaster, gs(1)N26 and gs(1)N441, cause delay in nuclear arrival at the polar plasm. In mutant embryos, polar plasm loses its ability to induce pole cells during retarded nuclear migration to the posterior pole of embryos. In the present study, it was shown that in N26 and N441 embryos, mitochondrial large rRNA (mtlrRNA), an essential factor for pole cell formation, is delocalized during the delay in nuclear arrival. This suggests that the loss of mtlrRNA causes failure of the mutants to form pole cells. Furthermore, it was shown that all of the other polar plasm components examined, namely Vasa protein, Germ cell-less protein, nanos mRNA and Polar granule component RNA start to be delocalized during the delay in nuclear arrival. This suggests that polar plasm integrity is not maintained in mutant embryos. It was finally shown that Vas is also delocalized in embryos that are inhibited to form pole cells by reducing the amount of mtlrRNA. This indicates that the segregation of polar plasm into pole cells is required to maintain polar plasm integrity. The mechanism regulating polar plasm integrity in embryos is discussed.

Localization of mitochondrial large rRNA in germinal granules and the consequent segregation of germ line.

This article reviews recent studies on cytoplasmic factors for germ-line establishment in Drosophila and Xenopus. In a variety of animal groups, the cytoplasmic factors for germ-line differentiation have been postulated to be localized in the germ plasm. We have found that mitochondrial large rRNA (mtlrRNA) is present in germinal granules, the distinctive morphological markers of germ plasm in the fruit-fly and the frog. MtlrRNA has been identified as a cytoplasmic factor that induce pole cells, the progenitor of the germ line, in uv-sterilized Drosophila embryos. The developmental stages of Xenopus at which mtlrRNA is present in germinal granules correspond to the stages of germ line segregation. Based on our findings and available data on posterior class gene products as well as on classical developmental biology of germ cells, we discuss the probable role of mtlrRNA in germ-line segregation from the somatic line in these animals.

Biosystematic Studies onPhlebia Acerina, P. Rufa, andP. Radiatain North America

Three closely related species, Phlebia rufa, P. acerina, and P. radiata, were analyzed with molecular, morphological, genetic, and physiological methods. Emphasis was placed on the phenotypically similar taxa P. rufa and P. acerina. Phlebia acerina is common and widely distributed in North America, but P. rufa occurs only in Washington, Oregon, and British Columbia. Although often considered conspecific, P. acerina and P. rufa are intersterile. In addition, differences in basidioma and cultural traits support their separation. At the molecular level, these species differ slightly in sequence of an internal transcribed spacer of the nuclear ribosomal DNA repeat and a part of the large mitochondrial rRNA gene. Restriction maps of the small mitochondrial rRNA gene and flanking regions provide further evidence that the species are distinct but closely related. This report includes basidioma and cultural descriptions of both species. Phlebia radiata is genetically distinct but shares many morphological and molecular traits with P. acerina and P. rufa. Cladistic analyses of molecular data showed that the three taxa are closely related relative to the outgroup taxa P. ludoviciana and P. subochracea.

Biosystematic Studies on Phlebia acerina, P. rufa, and P. radiata in North America

Three closely related species, Phlebia rufa, P. acerina, and P. radiata, were analyzed with molecular, morphological, genetic, and physiological methods. Emphasis was placed on the phenotypically similar taxa P. rufa and P. acerina. Phlebia acerina is common and widely distributed in North America, but P. rufa occurs only in Washington, Oregon, and British Columbia. Although often considered conspecific, P. acerina and P. rufa are intersterile. In addition, differences in basidioma and cultural traits support their separation. At the molecular level, these species differ slightly in sequence of an internal transcribed spacer of the nuclear ribosomal DNA repeat and a part of the large mitochondrial rRNA gene. Restriction maps of the small mitochondrial rRNA gene and flanking regions provide further evidence that the species are distinct but closely related. This report includes basidioma and cultural descriptions of both species. Phlebia radiata is genetically distinct but shares many morphological and molecular traits with P. acerina and P. rufa. Cladistic analyses of molecular data showed that the three taxa are closely related relative to the outgroup taxa P. ludoviciana and P. subochracea.

Nucleotide sequence of the large mitochondrial rRNA gene of Penicillium chrysogenum

The nucleotide sequence of a large rRNA (1-rRNA) gene and its flanking regions in the cloned fragments of mitochondrial (mt) DNA from Penicillium chrysogenum NRRL1951 (Sekiguchi J., Ohsaki, T., Yamamoto, H., Koichi, K. and Shida, T. (1990) J. Gen. Microbiol. 136, 535–543) was determined and compared with those in Aspergillus nidulans and Neurospora crassa mitochondrial DNAs. The P. chrysogenum mt 1-rRNA gene has a 1678 bp intron which intervenes between a 2835 bp 5′ exon and a 581 bp 3′ exon, and extensive homology exists between overall sequences of mt 1-rRNA genes of P. chrysogenum and A. nidulans. The P. chrysogenum intron contains a large open reading frame which encodes a polypeptide comprising of 399 amino acids. The intron sequence also suggests that the intron belongs to a self-splicing group IA.

Increased mitochondrial activities in pigmented (melanized) fish cells and nucleotide sequence of mitochondrial large rRNA

Using the substracted probe approach, we had previously isolated eight cDNAs whose corresponding RNAs are more abundant in pigmented (melanized) than in closely related unpigmented goldfish cell lines. We report here that two of these are of mitochondrial origin, suggesting that pigmentation is accompanied by higher content (activity) of mitochondria. We also present the complete nucleotide sequence of the full length cDNA for the large mitochondrial rRNA, showing the presence of a polyA tail, two polyadenylation signals and a long open reading frame potentially encoding for a polypeptide of 166 amino acids and with no known protein homologue. It is however unknown whether such a polypeptide is actually produced.

Integration of a group I intron into a ribosomal RNA sequence promoted by a tyrosyl-tRNA synthetase.

Group I and II introns are mobile elements that propagate by insertion into different genes. Some introns of both types self-splice in vitro by transesterification reactions catalysed by the intron RNA. These transesterifications are reversible, and it has been suggested that reverse splicing followed by reverse transcription and recombination with genomic DNA may be a mechanism for intron transposition. In vivo the splicing of many, if not all, group I and II introns requires protein factors, which may facilitate correct folding of the intron RNAs. Here we show that the Neurospora mitochondrial large rRNA intron, a group I intron that is not self-splicing in vitro, undergoes reverse splicing in a reaction promoted by the CYT-18 protein, the Neurospora mitochondrial tyrosyl-tRNA synthetase, which is required for splicing the intron in vivo. In contrast to known RNA-catalysed reverse splicing reactions, this protein-assisted reverse splicing is sufficiently rapid to compete with forward splicing at low RNA concentrations under physiologically relevant conditions, including high GTP and low Mg2+ concentrations. Our results indicate that proteins that promote splicing could contribute to intron mobility by promoting reverse splicing in vivo.

The Neurospora mitochondrial tyrosyl-tRNA synthetase is sufficient for group I intron splicing in vitro and uses the carboxy-terminal tRNA-binding domain along with other regions.

Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria. Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity. The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro. Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis. We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity. Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity. Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities. Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure. Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity. The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr).

Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing

The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5' splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5' intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.

Restoration of pole-cell-forming ability to u.v.-irradiated Drosophila embryos by injection of mitochondrial IrRNA

Screening a cDNA library generated from poly(A) +RNA of Drosophila cleavage embryos, we selected a cDNA clone (pDE20.6). The cDNA hybridized specifically with a poly(A) +RNA that is capable of restoring embryos from u.v.-caused inability of pole cell formation. The RNA hybrid-selected by pDE20.6 was also able to induce pole cells in the anterior region of embryos, if it was coinjected with u.v.-irradiated polar plasm, although the RNA or irradiated polar plasm alone was not effective. Pole cells thus formed in the anterior or in the u.v.-irradiated posterior region were identified by polar granules and nuclear bodies, morphological markers for normal pole cells. Furthermore, the RNA-induced pole cells were able to migrate into gonadal rudiments. The nucleotide sequence of pDE20.6 cDNA insert was highly homologous with the mitochondrial large rRNA (lrRNA) gene, but not with any nuclear DNA sequences. Using pDE20.6 as a primer, a full-length cDNA of mitochondrial lrRNA was generated and cloned. The RNA transcribed in vitro from the cDNA was able to restore pole cell formation. The cDNA hybridized only with a 1.5 kb poly(A) +RNA on a Northern blot. The 1.5 kb RNA sedimented more with the post-mitochondrial (P3) fraction than with the mitochondrial (P2) fraction, while the majority of transcripts from another mitochondrial gene was detected in the P2 fraction.

Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.