Mitochondrial Gene Regions Research Articles

Amphi-Panamic Geminates of Snook (Percoidei: Centropomidae) Provide a Calibration of the Divergence Rate in the Mitochondrial DNA Control Region of Fishes

The mitochondrial DNA control region is one of the most frequently utilized sequences for both intra- and interspecific genetic studies of fishes, yet a tenable divergence rate specifically for fish control regions has not been established. We attempted to establish a rate through a comparative study of control region sequences and those of a protein-coding mitochondrial gene region from geminate species of snook (Centropomus) assumed to have been separated by the emergence of the Isthmus of Panama 3.5 million years ago. The divergence rate suggested from the control region alignments between the geminates was markedly higher than rates currently applied in many studies, as was the rate suggested from the ND 5/6 protein coding region alignments. However, the suggested ND 5/6 rate when applied to alignments with the outgroup species was not concordant with the scant centropomid fossil record and therefore the assumed separation time of 3.5 million years seemed implausible. An average control region divergence rate was then estimated based on separation times of snook species derived assuming a divergence rate of 1% per million years for transversion substitutions at third codon positions in the ND 5/6 region. Using these separation times, a tenable average divergence rate for fish control regions of approximately 3.6% per million years ± 0.46% SE was calculated.

Post-transcriptional and developmental regulation of a CMS-associated mitochondrial gene region by a nuclear restorer gene.

Transcripts of the mitochondrial gene region orf224/atp6, which is associated with the Polima or pol cytoplasmic male sterility (CMS) of Brassica napus, differ among fertile, sterile and nuclear-restored plants. We show here that the effects of the restorer gene Rfp on orf224/atp6 transcripts varies among different floral organs. Relative to monocistronic atp6 transcripts, levels of the dicistronic transcripts spanning orf224 and atp6 are dramatically reduced in petals, stamens and carpels, but not sepals, of restored flowers. In pol CMS plants, the relative levels of different orf224/atp6 transcripts are similar among the floral organs. Analysis of guanylyltransferase-labeled mtRNA indicates that only the dicistronic 2.2 and 1.9 kb orf224/atp6 transcripts carry an initiator 5' terminus; hence the 1.4 and 1.3 kb transcripts of restored plants, as well as the 1.1 kb atp6 transcript common to all genotypes, are generated by RNA processing and not de novo initiation. Although steady-state levels of dicistronic transcripts in flower buds are lower in restored than in sterile plants, run-on transcription experiments show that these transcripts are synthesized at the same rate in both types of flowers. These findings imply that the restorer gene acts by conditioning the removal of sequences from the 5' end of dicistronic transcripts in a developmentally regulated manner. Run-on transcription experiments indicate that the single 1.1 kb atp6 transcript of nap cytoplasm is also generated by removal of sequences from the 5' end of a precursor. We suggest that specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may represent a common mechanism for tailoring transcripts in plant mitochondria.

Genetic Relationship between Masu and Amago Salmon Examined through Sequence Analysis of Nuclear and Mitochondrial DNA

Historically, the taxonomy and nomenclature of Japanese salmon have been in a state of confusion. Masu, amago and biwa salmon have been variously classified as distinct species, subspecies, or often conflicting or overlapping combinations of the two. In particular, the taxonomy of masu and amago salmon is obscured by their similarity in ecological and morphological traits. Here, DNA sequence analyses of nuclear and mitochondrial DNA were applied to clarify the genetic relationship between masu and amago salmon. No fixed differences were detected in the mitochondrial ND3 gene and control (D-loop) region, or in the nuclear growth hormone type-2 gene (GH2). However, the frequency of single nucleotide substitution alleles within GHZ intron C and size variants at a microsatellite locus nested within intron D differed markedly, providing genetic evidence to support a taxonomic distinction between the two types. The genetic data were related to previous mitochondrial DNA sequence analyses and alternative classification schemes for masu and amago salmon. The best-supported scheme arranges the two types as subspecies; masu as Oncorhynchus masou masou Brevoort and amago as Oncorhynchus masou ishikawae Jordan and McGregor.

Molecular evolution of a portion of the mitochondrial 16S ribosomal gene region in scleractinian corals.

Relationships among families and suborders of scleractinian corals are poorly understood because of difficulties 1) in making inferences about the evolution of the morphological characters used in coral taxonomy and 2) in interpreting their 240-million-year fossil record. Here we describe patterns of molecular evolution in a segment of the mitochondrial (mt) 16S ribosomal gene from taxa of 14 families of corals and the use of this gene segment in a phylogenetic analysis of relationships within the order. We show that sequences obtained from scleractinians are homologous to other metazoan 16S ribosomal sequences and fall into-two distinct clades defined by size of the amplified gene product. Comparisons of sequences from the two clades demonstrate that both sets of sequences are evolving under similar evolutionary constraints: they do not differ in nucleotide composition, numbers of transition and transversion substitutions, spatial patterns of substitutions, or in rates of divergence. The characteristics and patterns observed in these sequences as well as the secondary structures, are similar to those observed in mt 16S ribosomal DNA sequences from other taxa. Phylogenetic analysis of these sequences shows that they are useful for evaluating relationships within the order. The hypothesis generated from this analysis differs from traditional hypotheses for evolutionary relationships among the Scleractinia and suggests that a reevaluation of evolutionary affinities in the order is needed.

Brassica nap cytoplasmic male sterility is associated with expression of a mtDNA region containing a chimeric gene similar to the pol CMS-associated orf224 gene.

Two different cytoplasmic male-sterility (CMS) systems, nap and pol, are found in the oilseed rape (canola) species Brassica napus. Physical mapping studies have previously shown that organizational differences between the sterile pol and fertile cam mitochondrial genomes are restricted to a relatively small region immediately upstream of the atp6 gene. An approximately 4.5-kb pol mtDNA segment containing a chimeric open reading frame (orf224) co-transcribed with atp6 is missing from cam mtDNA and located at a different site on nap mtDNA; expression of the orf224/atp6 gene region is highly correlated with the pol CMS trait. Sequence analysis now shows that the transposed nap segment contains an open reading frame (ORF) related to, but distinct from, pol orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino acids possessing 79% sequence similarity to the predicted product of the pol orf224 gene. nap orf222 is co-transcribed with the third exon of the trans-spliced nad5 gene and another ORF. orf222 transcripts are several times more abundant in nap CMS than in fertility restored nap-cytoplasm plants and qualitative transcript differences for the region between CMS and restored plants are found as well. Expression of the orf222/nad5c/orf139 region is specifically correlated with nap CMS: of 21 mitochondrial gene regions examined, including all the sites of rearrangement between the nap and fertile cam mitochondrial genomes and 22 known genes, only the orf222/nad5c/orf139 region detected transcript differences between maintainer cam cytoplasm, nap CMS- and fertility restored nap cytoplasm-plants. Our results suggest that expression of the orf222/nad5c/orf139 region may be associated with nap CMS, and, more generally, that different forms of CMS may be associated with genes encoding structurally similar proteins.

Phylogenetic relationships among Japanese species of the family Sciuridae (Mammalia, Rodentia), inferred from nucleotide sequences of mitochondrial 12S ribosomal RNA genes.

In order to investigate phylogenetic relationships of the family Sciuridae living in Japan, we sequenced partial regions (379 bases) of mitochondrial 12S rRNA genes in six species of Japanese and other Asian squirrels. Phylogenetic trees constructed by sequence data indicated that two genera of flying squirrels (Petaurista and Pteromys) were clustered in a group distinct from non-flying squirrels, suggesting a possible monophyletic relationships of these flying squirrels. The evolutionary distance between the Japanese squirrel (Sciurus lis) from Honshu island and the Eurasian red squirrel (Sciurus vulgaris) from Hokkaido island was comparable to intraspecific distances of the remaining species examined.

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.

Nuclear Genes Associated With a Single Brassica CMS Restorer Locws Influence Transcripts of Three Different Mitochondrial Gene Regions

Previous studies have shown that the mitochondrial orf224/atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus. We now extend this correlation by showing that the effects of nuclear fertility restoration on orf224/atp6 transcripts cosegregate with the pol restorer gene Rfp1 in genetic crosses. We also show, however, that the recessive rfp1 allele, or a very tightly linked gene, acts as a dominant gene, designated Mmt (modifier of mitochondrial transcripts), in controlling the presence of additional smaller transcripts of the nad4 gene and a gene possibly involved in cytochrome c biogenesis. A common sequence, TTGTGG, maps immediately downstream of the 5' termini of both of the transcripts specific to plants with the Mmt gene and may serve as a recognition motif in generation of these transcripts. A similar sequence, TTGTTG, that may be recognized by the product of the alternate allele (or haplotype), Rfp1, is found within orf224 just downstream of the major 5' transcript terminus specific to fertility restored plants. Our results suggest that Rfp1/ Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes.

PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp.

A pair of conserved PCR primers, designated NMS1 and NMS2, that amplify a region in the mitochondrial small rRNA gene region were designed for fungi belonging to the class Ascomycetes. These primers were tested with members of eight fungal genera (Aspergillus, Fusarium, Magnaporthe, Mycospharella, Neurospora, Saccharomyces, Sclerotinia, Verticillium) and 10 Verticillium species (Verticillium albo-atrum, Verticillium chlamydosporium, Verticillium cinnebarium, Verticillium dahliae, Verticillium fungicola, Verticillium lecanii, Verticillium lateritium, Verticillium nigrescens, Verticillium psaliotae, and Verticillium tricorpus). The primers were also tested with 35 isolates of V. dahliae obtained from diverse geographic areas and diverse hosts. The results of a restriction fragment length polymorphism analysis of the region amplified by the primers differentiated the genera examined and the results of a DNA sequence analysis of the amplified region differentiated the Verticillium species. Two Fusarium species were also differentiated by the results of the restriction fragment length polymorphism analysis. On the basis of the nucleotide sequences of the amplified regions, we obtained a pair of PCR primers that could be used to differentiate V. dahliae from the other fungal isolates tested, including V. albo-atrum, a closely related plant-pathogenic species. The V. dahliae-specific PCR primer may aid in more rapid and specific detection of the pathogen directly in plant and/or soil samples. PCR primers NMS1 and NMS2 may be used as potential mitochondrial markers for studying fungal cytoplasmic inheritance of ascomycetes and for identifying DNA probes that are informative at or below the genus level.

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.

Sequence evolution of mitochondrial tRNA genes and deep-branch animal phylogenetics

Mitochondrial DNA sequences are often used to construct molecular phylogenetic trees among closely related animals. In order to examine the usefulness of mtDNA sequences for deep-branch phylogenetics, genes in previously reported mtDNA sequences were analyzed among several animals that diverged 20-600 million years ago. Unambiguous alignment was achieved for stem-forming regions of mitochondrial tRNA genes by virtue of their conservative secondary structures. Sequences derived from stem parts of the mitochondrial tRNA genes appeared to accumulate much variation linearly for a long period of time: nearly 100 Myr for transition differences and more than 350 Myr for transversion differences. This characteristic could be attributed, in part, to the structural variability of mitochondrial tRNAs, which have fewer restrictions on their tertiary structure than do nonmitochondrial tRNAs. The tRNA sequence data served to reconstruct a well-established phylogeny of the animals with 100% bootstrap probabilities by both maximum parsimony and neighbor-joining methods. By contrast, mitochondrial protein genes coding for cytochrome b and cytochrome oxidase subunit I did not reconstruct the established phylogeny or did so only weakly, although a variety of fractions of the protein gene sequences were subjected to tree-building. This discouraging phylogenetic performance of mitochondrial protein genes, especially with respect to branches originating over 300 Myr ago, was not simply due to high randomness in the data. It may have been due to the relative susceptibility of the protein genes to natural selection as compared with the stem parts of mitochondrial tRNA genes. On the basis of these results, it is proposed that mitochondrial tRNA genes may be useful in resolving deep branches in animal phylogenies with divergences that occurred some hundreds of Myr ago. For this purpose, we designed a set of primers with which mtDNA fragments encompassing clustered tRNA genes were successfully amplified from various vertebrates by the polymerase chain reaction.

Characterization of expression of a mitochondrial gene region associated with the Brassica ?Polima? CMS: developmental influences

The mitochondrial genome of the Polima (pol) male-sterile cytoplasm of Brassica napus contains a chimeric 224-codon open reading frame (orf224) that is located upstream of, and co-transcribed with, the atp6 gene. The N-terminal coding region of orf224 is derived from a conventional mitochondrial gene, orfB, while the origin of the remainder of the sequence is unknown. We show that an apparently functional copy of orfB is present in the pol mitochondrial genome, indicating that the pol CMS is not caused by the absence of an intact, expressed orfB gene. The 5' termini of orf224/atp6 transcripts present in both sterile and fertility-restored (Rf) pol cytoplasm plants are shown to map to sequences resembling mitochondrial transcription-initiation sites, whereas the 5' termini of two transcripts specific to restored lines map to sequences which resemble neither one another nor mitochondrial promoter motifs. It is suggested that the complex orf224/atp6 transcript pattern of Rf plants is generated by a combination of multiple transcription initiation and processing events and that the nuclear restorer gene acts to specifically alter orf224/atp6 transcripts by affecting RNA processing. Northern analyses demonstrate that the effect of the restorer gene on orf224/atp6 transcripts is not tissue or developmental-stage specific. However, the expression of the atp6 region is developmentally regulated in pol plants, resulting in decreased levels of monocistronic atp6 transcripts in floral tissue relative to seedlings. It is suggested that this developmental regulation may be related to the absence of overt phenotypic effects of the CMS mutation in vegetative tissues.

Sequence Similarity in Nuclear and Mitochondrial Gene Regions in Plants

A 114-basepair fragment at 5′-upstream of the gene oee2-A from Nicotiana tabacum encoding the 23kD polypeptide of oxygen-evolving complex of photosystem II was found, through database search, to have high sequence similarity with segments in the mitochondrial genome of Oenothera, watermelon and wheat. This observation is supported by the Southern hybridization data. Repeated sequences were also found in the nuclear-mitochondrial homologous region, However, the function of these repeats is yet to be determined.

Suppression of cytoplasmic male sterility by nuclear genes alters expression of a novel mitochondrial gene region.

To identify regions of the mitochondrial genome that potentially could specify the "Polima" (pol) cytoplasmic male sterility (CMS) of Brassica napus, transcripts of 14 mitochondrial genes from nap (male fertile), pol (male sterile), and nuclear fertility-restored pol cytoplasm plants were analyzed. Transcriptional differences among these plants were detected only with the ATPase subunit 6 (atp6) gene. Structural analysis of the atp6 gene regions of pol and nap mitochondrial DNAs showed that rearrangements in the pol mitochondrial genome occurring upstream of atp6 have generated a chimeric 224-codon open reading frame, designated orf224, that is cotranscribed with atp6. In CMS plants, most transcripts of this region are dicistronic, comprising both orf224 and atp6 sequences. Nuclear restorer genes at either of two distinct loci appear to specifically alter this transcript pattern such that monocistronic atp6 transcripts predominate. The differences in expression of this region appear to result, in part, from differential processing of a tRNA-like element comprising a tRNA pseudogene present immediately upstream of atp6 in both the sterile and fertile mitochondrial DNAs. Possible mechanisms by which expression of the orf224/atp6 locus and the Polima CMS trait may be specifically related are considered.

Suppression of Cytoplasmic Male Sterility by Nuclear Genes Alters Expression of a Novel Mitochondrial Gene Region

Suppression of Cytoplasmic Male Sterility by Nuclear Genes Alters Expression of a Novel Mitochondrial Gene Region

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.

Structure and transcription of the gene coding for subunit 3 of cytochrome oxidase in wheat mitochondria

The wheat mitochondrial (mt) cox3 has been localized and sequenced. The gene exists as a single copy in the wheat mt master chromosome and is transcribed into a single 1.2 kb RNA, whose extremities have been mapped. Comparison of the wheat and Oenothera cox3 sequences gives ambiguous indications concerning the amino acid coded by the codon CGG. Upstream and downstream of the wheat cox3 gene, two short sequences of 43 bp and 69 bp respectively are present, which are almost identical to sequences present in the flanking regions of other plant mitochondrial genes. These common sequences seem to have played a role in the rearrangements which caused sequence divergence of the plant mt genomes during evolution. Furthermore, mapping of wheat and maize cox3 and cob transcripts suggests that some of these common sequences can play a role in the regulation of transcription or processing.

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants.

The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3' end of the gene. The 5' and 3' ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5' region of the 26S rRNA gene or in the 18S rRNA gene.

The ATPase subunit 6 gene of tobacco mitochondria contains an unusual sequence.

We have isolated and characterized the F0-ATPase subunit 6 gene (atp6) from tobacco mitochondria. The tobacco sequence exists as a single copy, is transcribed and contains an open reading frame (ORF) capable of encoding a peptide of 395 amino acids. The first 130 amino acids of the tobacco putative polypeptide show limited homology with the N terminus predicted for the maize ATPase subunit 6. Although poorly conserved at the sequence level, the tobacco and maize amino termini are hydrophilic and have a high percentage of charged amino acids. This portion of the predicted peptide may represent a presequence that is common to the ATPase subunit 6 of plants. Significant homology between tobacco and maize begins with amino acid 131, in a region that is highly conserved among fungal ATPase 6 subunits. In the remainder of the predicted protein, tobacco and maize share approximately 81% homology. A 41 bp sequence and a 175 bp conserved region found upstream from the tobacco atp6 coding region are homologous with sequence elements found in the 5' flanking regions of other plant mitochondrial genes and may be important for regulation and expression of the atp6 gene.