Wheat Mitochondrial DNA Research Articles

A Real-Time Quantitative PCR Detection Method for Pork, Chicken, Beef, Mutton, and Horseflesh in Foods

A rapid real-time quantitative PCR method to detect trace amounts of pork, chicken, beef, mutton, and horseflesh in foods was developed. The primers and TaqMan MGB (minor groove binder) probes were designed on the gene encoding cytochrome b for the specific detection of each species. The limit of quantification of this method was found to be 100 fg/microl of each mitochondrial DNA in 10 ng/microl of the wheat mitochondrial DNA matrix. The calculated R(2) values of the standard curves for the five species ranged between 0.994 and 0.999. This method would be particularly useful in the detection of hidden meat mince in processed foods, which would verify food labeling and gain consumers' trust.

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Transfer of RPS14 and RPL5 from the mitochondrion to the nucleus in grasses.

Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an on-going phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. To better understand the strategies developed by higher plants to transfer organellar genes during evolution, we investigated the fate of the mitochondrial RPL5-RPS14 locus in grasses. While maize mitochondrial genome does not contain RPS14 and RPL5 genes, wheat mitochondrial DNA contains an intact RPL5 gene and a nonfunctional RPS14 pseudogene. RPL5 and PsiRPS14 are co-transcribed and their transcripts are edited. In wheat, the functional RPS14 gene is located in the nucleus, within the intron of the respiratory complex II iron-sulfur subunit gene (SDH2). Its organization and expression mechanisms are similar to those previously described in maize and rice, allowing us to conclude that RPS14 transfer and nuclear activation occurred before divergence of these grasses. Unexpectedly, we found evidence for a more recent RPL5 transfer to the nucleus in wheat. This nuclear wheat RPL5 acquired its targeting information by duplication of an existing targeting presequence for another mitochondrial protein, ribosomal protein L4. Thus, mitochondrial and nuclear functional RPL5 genes appear to be maintained in wheat, supporting the hypothesis that in an intermediate stage of the transfer process, both nuclear and mitochondrial functional genes coexist. Finally, we show that RPL5 has been independently transferred to the nucleus in the maize lineage and has acquired regulatory elements for its expression and a mitochondrial targeting peptide from an unknown source.

Complex II subunit 4 (sdh4) homologous sequences in plant mitochondrial genomes.

Through cDNA analysis a 95-codons-long novel open reading frame (orf) is identified in the Arabidopsis thaliana mitochondrial genome, overlapping the 3'-end region of the cox3 gene. This sequence is conserved in other dicot plants such as Oenothera, pea and sunflower, but is not detected in wheat mitochondrial DNA. The Arabidopsis, sunflower and Oenothera sequences may be pseudogenes, with the first two being shortened by stop codons and transcription of the latter terminating within the orf. However, RNA editing increases the similarity to homologous Marchantia, algal and bacterial polypeptides, suggesting that this orf could code for the complex-II membrane-anchor subunit (SDH4) in at least some higher-plant species.

Inheritance of mitochondrial DNA in the conifer Larix

Restriction fragment length polymorphisms between Larix leptolepis and Larix decidua were identified in heterologous hybridization experiments, using wheat mitochondrial DNA probes specific for atp9, coxI, nad3/rps12, and orf25. Analysis of eight individuals of each reciprocal hybrid of these two species revealed that mitochondrial DNA was maternally inherited. Furthermore, sequences homologous to wheat orf25 were also identified in Larix gmelini, Larix siberica, Larix olgensis, and Larix laricina, as well as Ginkgo biloba, Picea mariana, Picea glauca and Pinus contorta.

Sequence and organization of a 7.2 kb region of wheat mitochondrial DNA containing the large subunit (26S) rRNA gene.

We report the sequence of a 7.2 kilobase pair DNA fragment containing a copy of the wheat mitochondrial gene (rrn26) that encodes the mitochondrial large-subunit ribosomal RNA (26S rRNA). The mature 26S rRNA was determined by direct RNA sequencing to be 3467 nucleotides long, and to share a 5'-terminal pentanucleotide (5'-AUCAU), thought to be important in post-transcriptional processing, with the wheat mitochondrial small-subunit (18S) rRNA. Two other prominent features of the sequence were noted. First, upstream of rrn26 are located two tandem copies of a 70 base pair element containing a putative mitochondrial promoter motif (TCGTATAAAAA). Second, downstream of rrn26 is a sequence element that, if transcribed, would produce an RNA with a secondary structure resembling that of tRNAs but differing sufficiently from the latter structure to preclude any transcript from functioning normally in translation. These upstream and downstream sequence elements may play a role in the expression of rrn26 in wheat mitochondria.

Maize S1 and S2 sequences in wheat mitochondrial DNA

The mitochondrial DNA (mtDNA) of fertile ( Triticum aestivum) and cytoplasmic male sterile (cms) wheat ( T. aestivum nucleus; T. timopheevi mitochondria) was isolated and subjected to restriction enzyme analysis. Restriction fragments of the mtDNAs were probed with DNA specific to the S-episomes of maize mitochondria. Small, independent episomal DNA was not found in the mtDNA of wheat. Sequences related to maize S1 DNA are present in fertile and cms wheat mtDNA but do not appear to be contiguous. Restriction enzyme analyses do not show detectable differences between fertile and cms mtDNA around the maize-like S1 sequences. Sequences related to maize S2 mtDNA may be located at two different positions in both fertile and cms mitochondrial genomes. The fertile and cms mtDNA show restriction fragment length polymorphism at one of two sites.

In vitro processing of transcripts containing novel tRNA-like sequences (?t-elements?) encoded by wheat mitochondrial DNA

We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.

Nucleotide sequence of a tRNA pseudogene from wheat mitochondrial DNA

BamH1 fragments of wheat mtDNA were cloned into a pBR322 plasmid. Screening of the bank was done by colony hybridization using 32 P-labeled wheat mitochondrial tRNA as a probe. For sequence analysis was selected one of the subclones that strongly hybridized with tRNA. The determined sequence is presented here.

Maize and wheat mitochondrial tRNA Pro coding regions have similar sequences but different organizations

The nucleotide sequences of two maize mitochondrial DNA regions containing a tRNA Pro gene and an imcomplete tRNA Pro gene have been compared with the corresponding regions of wheat mitochondrial DNA. These regions have similar sequences but their organization is modified due to different recombination events involving the tRNA Pro immediate environment.

Identification of new mitochondrial genome organizations in wheat plants regenerated from somatic tissue cultures

Plants have been regenerated from short-and long-term in vitro somatic tissue cultures made from immature embryos of the hexaploid wheat cultivar "Chinese Spring". The mitochondrial genome organization of each regenerated plantlet was studied, after one selfing, by probing Sal I-restricted total DNA with cloned Sal I fragments of wheat mitochondrial DNA derived from a segment of the genome, which displays marked structural changes in response to in vitro culture. Short-term in vitro cultures give rise to regenerated plants whose mitochondrial genome organization is either close to that of the parental cultivar or to that of embryogenic callus cultures, except for a single plant which has an organization resembling that of short-term non-embryogenic cultures. In contrast, all but one of the plants regenerated from long-term cultures exhibited a mitochondrial genome organization similar to that of long-term nonembryogenic cultures. In addition, extra labelled bands were detected in some of the regenerated plants with two of the probes used. These results emphasize the importance of the duration of the in vitro step preceding the regeneration process: the longer it is, the higher the probability is of obtaining mitochondrial DNA variability in regenerated plants. Furthermore, since increasing the duration of the in vitro stetp results in the production of regenerated plants with a mitochondrial genome organization resembling that of non-embryogenic tissue cultures, the question is thus raised as to whether regeneration from long-term cultures is suitable for use in plant breeding.

Aspartate and asparagine tRNA genes in wheat mitochondrial DNA: a cautionary note on the isolation of tRNA genes from plants.

We have identified genes encoding a "native" tRNA(Asp) (trnD-GTC) and a "chloroplast-like" tRNA(Asn) (trnN-GTT) on opposite strands and 633 bp apart within a sequenced 1640 bp RsaI restriction fragment of wheat mtDNA. The trnD gene has been found previously at a different location in wheat mtDNA (P.B.M. Joyce et al. (1988) Piant Mol. Biol. 11, 833-843); the duplicate copies of this gene are identical within the coding and immediate flanking regions (9 bp downstream and at least 68 bp upstream), after which obvious sequence similarity abruptly disappears. The trnN gene is identical to its homolog in maize ctDNA; continuation of sequence similarity beyond the coding region suggests that this gene originated as promiscuous ctDNA that is now part of the wheat mitochondrial genome. In the course of this work, we have encountered some unexpected similarities between tRNA gene regions from wheat mitochondria and other sources. Detailed analysis of these similarities leads us to suggest that trnN genes reportedly from petunia nuclear DNA (N. Bawnik et al. (1983) Nucleic Acids Res. 11, 1117-1122) and lupine mtDNA (B. Karpińska and H. Augustyniak (1988) Nucleic Acids Res. 16, 6239) are, in fact, from petunia mtDNA and lupine ctDNA, respectively, whereas a putative wheat nuclear tRNA(Ser) (trnS-TGA) gene (Z. Szwekowska-Kulińska et al. (1989) Gene 77, 163-167) is actually from wheat mtDNA. In these instances, it seems probable that the DNA samples used for cloning contained trace amounts of DNA from another sub-cellular compartment, leading to the inadvertent selection of spurious clones.

Nucleotide sequence of a second glutamine tRNA gene in wheat mitochondrial DNA

We have developed a novel in vitro mutagenesis technique that allows us to introduce mutations at the level of double-stranded DNA and then transcribe the mutant DNA directly. The technique is useful for those wishing to produce recombinant RNA, particularly if the desired recombinant is the result of an insertion or deletion. It is also useful for the preparation of 3'-truncated RNAs with a defined end. The technique is not dependent on the presence of a convenient restriction site within the target gene, and does not involve construction of a clone or amplification of the mutant DNA within a bacterial host. It is intended as a simple and rapid method for the preparation of roughly 100-200 pmol of mutant RNA, which would be sufficient for obtaining sequence information and assessing the functional consequences of the mutation.

Wheat mitochondrial DNA: organization and sequences of the atpA and atp9 genes

Journal Article Wheat mitochondrial DNA: organization and sequences of the atpA and atp9 genes Get access Erika Schulte, Erika Schulte Lehrstuhl für Allgemeine Botanik, Ruhr-Universitāt BochumPostfach 102148, D-4630 Bochum 1, FRG Search for other works by this author on: Oxford Academic PubMed Google Scholar Sabine Staubach, Sabine Staubach Lehrstuhl für Allgemeine Botanik, Ruhr-Universitāt BochumPostfach 102148, D-4630 Bochum 1, FRG Search for other works by this author on: Oxford Academic PubMed Google Scholar Beate Laser, Beate Laser Lehrstuhl für Allgemeine Botanik, Ruhr-Universitāt BochumPostfach 102148, D-4630 Bochum 1, FRG Search for other works by this author on: Oxford Academic PubMed Google Scholar Ulrich Kūck Ulrich Kūck Lehrstuhl für Allgemeine Botanik, Ruhr-Universitāt BochumPostfach 102148, D-4630 Bochum 1, FRG Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 17, Issue 18, 25 September 1989, Page 7531, https://doi.org/10.1093/nar/17.18.7531 Published: 25 September 1989 Article history Received: 02 August 1989 Published: 25 September 1989

Changes in the molecular organization of the mitochondrial genome in albino tissue cultures derived from wheat pollen embryos and in plants regenerated from these cultures

Total DNA was isolated from albino short- and long-term tissue cultures prepared from pollen embryos of wheat cultivars Chinese Spring and Aquila respectively and from the corresponding albino plants regenerated from these cultures. The DNA was digested with Sall, fractionated and probed with cloned restriction fragments of wheat mitochondrial DNA. These probes hybridized to regions of the mitochondrial DNA known to vary in somatic tissue cultures initiated from immature embryos of Aquila and Chinese Spring and in green plants regenerated from Chinese Spring embryogenic tissue cultures. The hybridization patterns obtained showed that the variability detected in albino androgenic cultures was similar to that previously observed in a comparable somatic system. However, all the regenerated albino plantlets within each group had the same mitochondrial genome organization, contrary to plants regenerated from somatic tissue cultures.

Evidence for a direct relationship between mitochondrial genome organization and regeneration ability in hexaploid wheat somatic tissue cultures

Embryogenic and non-embryogenic long-term callus cultures of hexaploid wheat exhibit differences in the organization of their mitochondrial genome. Embryogenic and non-embryogenic fractions of callus cultures initiated from immature embryos of the wheat cultivar “Chinese Spring” have been isolated and subsequently subcultured. DNA-DNA hybridization experiments using labelled cloned wheat mitochondrial DNA fragments have shown that the mitochondrial DNA organization of embryogenic subcultures derived from embryogenic parts of “Chinese Spring” calli is closely related to that of the initial “Chinese Spring” calli, while non-embryogenic subcultures derived from non-embryogenic fragments of “Chinese Spring” calli exhibit a mitochondrial DNA organization similar to that found in non-embryogenic calli derived from cultivar “Aquila”. In addition, somatic tissue cultures initiated from three other non-embryogenic wheat cultivars (“Talent”, “Thesee” and “Capitole”) display mitochondrial DNA arrangements similar to those found in cultivar “Aquila”. These results strongly suggest that, in wheat callus cultures, a particular mitochondrial genome organization is correlated with the ability of cultured cells to regenerate whole plants.

Genes for tRNAAsp, tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNA(Asp) (anticodon GUC), tRNA(Pro) (UGG), tRNA(Tyr) (GUA), and two tRNAs(Ser) (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions ∼ 150 nucleotides upstream and ∼ 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with (32)P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.

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, sequence and expression of the gene coding for tRNAPro (UGG) in plant mitochondria

The four Sal I fragments of wheat mitochondrial DNA containing the 18S and 5S ribosomal RNA genes were screened for the presence of tRNA genes. Upon sequencing, a tRNA(Pro) (UGG) gene was found in two of these four fragments. The localization of the corresponding gene on the maize mitochondrial genome was established. Transcriptional studies have shown that this gene is transcribed in wheat and maize mitochondria. The sequence of the corresponding tRNA(Pro) (UGG) of bean mitochondria was determined using in vitro post-labeling techniques.

Studies on wheat mitochondrial DNA organization. Comparison of mitochondrial DNA from normal and cytoplasmic male sterile varieties of wheat

The mitochondrial genome of fertile, male-sterile and restored cytoplasm lines of wheat has been studied by means of recombinant DNA and hybridization techniques. Using cloned fragments of mitochondrial DNA (mtDNA) from fertile wheat cytoplasms as probes, about 40% of the genome is shown to have a differential hybridization pattern. The use of wheat rRNA and corn cytochrome oxidase subunit II probes indicates that duplication and rearrangement of genes or parts of genes may account for the differences observed. DNA synthesis in isolated mitochondria showed neither preferential labeling of part of the mtDNA nor the presence of extrachromosomal elements.