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M W Gray

Publications and source records attributed to M W Gray.

At least 91 records · Page 5Linked to original sources
PubMed1991

Sequence analysis of wheat mitochondrial transcripts capped in vitro: definitive identification of transcription initiation sites.

To identify transcription initiation sites in wheat mitochondria, the nascent 5'-ends of transcripts were specifically labeled by incubation of wheat mitochondrial RNA with [alpha-32P]GTP in the presence of the enzyme guanylyltransferase. After separation of the resulting capped transcripts by electrophoresis in polyacrylamide gels, individual RNAs were recovered and directly sequenced. Four RNA sequences obtained in this way were localized upstream of the protein-coding genes atpA, coxII, coxIII and orf25. Comparison of mRNA and gene sequences allowed precise positioning of transcription initiation sites for these four genes. Sequence similarities immediately upstream of these sites define a conserved motif that we suggest as a candidate regulatory element in wheat mtDNA. The relationship between this motif and putative mitochondrial promoters in other plant species is discussed.

Base Sequence↗
PubMed1991

Short dispersed repeats localized in spacer regions of Chlamydomonas reinhardtii mitochondrial DNA.

In the mtDNA of Chlamydomonas reinhardtii, a unicellular green alga, we have identified a set of short repeated sequences up to 65 nucleotides long, each of which contains the palindromic consensus motif CTCGG(N4-14)CCGAG. Most of these repeated elements are localized in spacer regions that flank the transcribed coding regions of C. reinhardtii mtDNA. These algal mitochondrial repeats have features reminiscent of short repeats in some fungal mtDNAs, such as GC clusters in Saccharomyces cerevisiae and PstI palindromes in Neurospora crassa. The location of these elements suggests that they could play a role in gene expression, e.g., post-transcriptional processing, in C. reinhardtii mitochondria.

Base Sequence↗
PubMed1991

Organisation and expression of small subunit ribosomal RNA genes encoded by a 35-kilobase circular DNA in Plasmodium falciparum.

A restriction map of the 35-kb circular DNA molecule of Plasmodium falciparum showed that a region of about 6 kb, encoding both a large and a small subunit ribosomal RNA gene, has been duplicated in inverted orientation. The complete sequence of one small subunit rRNA gene is presented as well as an analysis of transcripts from erythrocytic stage parasites. Comparative sequence analysis of the rRNA gene and the proposed secondary structure of the rRNA suggest that it is of organellar origin. Intriguingly, while some characteristics of the small subunit rRNA gene are similar to mitochondrial sequences, others are more like those of plastids. The origin of the circular DNA molecule and evolutionary implications of its genetic content are discussed.

Animals↗
PubMed1991

Accurate transcription of a plant mitochondrial gene in vitro.

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

Base Sequence↗
PubMed1990

Sequence heterogeneity in the duplicate large subunit ribosomal RNA genes of Tetrahymena pyriformis mitochondrial DNA.

In the ciliated protozoan, Tetrahymena pyriformis, the mitochondrial large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two discrete RNA species: a 280-nucleotide LSU alpha (constituting the 5'-portion) and a 2315-nucleotide LSU beta (corresponding to the remaining 3'-portion of this rRNA). The T. pyriformis mitochondrial genome contains two copies of the LSU alpha.beta gene complex, and we have previously provided evidence that both copies are transcribed (Heinonen, T. Y. K., Schnare, M. N., Young, P. G., and Gray, M. W. (1987) J. Biol. Chem. 262, 2879-2887). We now report the complete sequences of the two copies of the LSU alpha.beta gene complex. These are not identical, but differ at 5 out of the 2595 positions by single nucleotide substitutions in one sequence relative to the other. In the secondary structure model we propose here, two of these differences are located in base-paired regions of the LSU rRNA; however, they do not interrupt the complementary interactions in these helices. The other three differences occur in single-stranded regions of the secondary structure. The base substitutions documented here are not localized to those regions of LSU rRNA that are the most highly conserved in global phylogenetic comparisons, and therefore it seems unlikely that they are of fundamental functional significance. Whether they might exert more subtle effects on ribosome function remains to be determined.

Animals↗
PubMed1990

Differences in editing at homologous sites in messenger RNAs from angiosperm mitochondria.

Recent work has shown that amino acid sequence comparisons can be used to infer sites of C-to-U RNA editing in plant mitochondrial mRNAs (1). In order to test such predictions further and to search for conserved mRNA structural motifs that might provide insight into the mechanism of recognition of editing sites, the complete sequences of the cytochrome c oxidase subunit II (COXII) mRNAs of wheat, maize and pea were determined by reverse transcriptase sequencing. The results affirm the high reliability of editing predictions based on amino acid sequence alignments, and prompt us to make the further inference that COXI (cytochrome oxidase subunit I) mRNA is extensively edited in dicotyledonous plants but not in monocotyledons. In plant COXII mRNAs, additional non-predicted editing occurs such that the resulting derived amino acid sequences are more similar to those of non-plants than is indicated by the respective plant COXII DNA sequences. A number of homologous sites show differences in editing among species, and certain positions show partial editing within a species. Despite some deviation from expected nucleotide frequencies in the vicinity of editing sites, no extensive conserved primary or secondary structural motifs are apparent. The relevance of these data to the mechanism of RNA editing in plant mitochondria is discussed.

Amino Acid Sequence↗
PubMed1990

Sixteen discrete RNA components in the cytoplasmic ribosome of Euglena gracilis.

We have isolated cytoplasmic ribosomes from Euglena gracilis and characterized the RNA components of these particles. We show here that instead of the four rRNAs (17-19 S, 25-28 S, 5.8 S and 5 S) found in typical eukaryotic ribosomes, Euglena cytoplasmic ribosomes contain 16 RNA components. Three of these Euglena rRNAs are the structural equivalents of the 17-19 S, 5.8 S and 5 S rRNAs of other eukaryotes. However, the equivalent of 25-28 S rRNA is found in Euglena as 13 separate RNA species. We demonstrate that together with 5 S and 5.8 S rRNA, these 13 RNAs are all components of the large ribosomal subunit, while a 19 S RNA is the sole RNA component of the small ribosomal subunit. Two of the 13 pieces of 25-28 S rRNA are not tightly bound to the large ribosomal subunit and are released at low (0 to 0.1 mM) magnesium ion concentrations. We present here the complete primary sequences of each of the 14 RNA components (including 5.8 S rRNA) of Euglena large subunit rRNA. Sequence comparisons and secondary structure modeling indicate that these 14 RNAs exist as a non-covalent network that together must perform the functions attributed to the covalently continuous, high molecular weight, large subunit rRNA from other systems.

Animals↗
PubMed1990

Fourteen internal transcribed spacers in the circular ribosomal DNA of Euglena gracilis.

Cytoplasmic ribosomes from Euglena gracilis contain 16 rRNA components. These include the typical 5 S, 5.8 S and 19 S rRNAs that are found in other eukaryotes as well as 13 discrete small RNAs that interact to form the equivalent of eukaryotic 25-28 S rRNA (accompanying paper). We have utilized DNA sequencing techniques to establish that genes for all of these RNAs, with the exception of 5 S rRNA, are encoded by the 11,500 base-pair circular rDNA of E. gracilis. We have determined the relative positions of the coding regions for the 19 S rRNA and the 14 components (including 5.8 S rRNA) of the large subunit rRNA, thereby establishing that the genes for each of these rRNAs are separated by internal transcribed spacers. We conclude that sequences corresponding to these spacers are removed post-transcriptionally from a high molecular weight pre-rRNA, resulting in a multiply fragmented large subunit rRNA. Internal transcribed spacers, in positions analogous to some of these additional Euglena rDNA spacers, have been found in the rDNA of other organisms and organelles. This finding supports the view that at least some internal transcribed spacers may have been present at an early stage in the evolution of rRNA genes.

Animals↗
PubMed1990

Processing of transfer RNA precursors in a wheat mitochondrial extract.

To investigate mechanisms for processing of plant mitochondrial RNAs, we studied the fate of wheat mitochondrial tRNA precursors in a homologous soluble extract. Artificial precursor transcripts were synthesized in vitro using T3 or T7 RNA polymerase and DNA templates containing wheat mitochondrial tRNA genes and flanking sequences. We found that the mitochondrial extract supports processing of precursors containing both native and chloroplast-like (Joyce, P. B. M., and Gray, M. W. (1989) Nucleic Acids Res. 77, 5461-5476) wheat mitochondrial tRNA sequences. Incubation of precursor transcripts with the extract results in processing of tRNAs via precise 5'- and 3'-endonucleolytic cleavages. However, these cleavages are not ordered in vitro because intermediates composed of 5'-leader + tRNA and tRNA + 3'-trailer are present simultaneously throughout the course of the reaction. Sequence analysis of processed products confirmed that endonucleolytic cleavages occur at the expected positions, generating tRNAs with 5'-phosphoryl and 3'-hydroxyl termini. The mitochondrial extract also contains a tRNA nucleotidyltransferase activity that adds -CCAOH termini to the 3'-ends of processed tRNAs. This cell-free RNA processing system provides the basis for biochemical characterization of the various enzymes involved in the production and maturation of plant mitochondrial tRNAs.

Base Sequence↗
PubMed1990

RNA sequence and the nature of the CuA-binding site in cytochrome c oxidase.

For cytochrome c oxidase subunit II (COXII), DNA and protein sequences suggest that Met-207 (bovine numbering) is conserved in all species except plants. Sequencing of plant mitochondrial COXII mRNAs now indicates that Met-207 is also conserved among plants as a result of a C-to-U type of RNA editing. Considering the strict evolutionary conservation of Met-207 and the homology of COXII to type I (blue) copper proteins and nitrous oxide reductase, we propose a model in which Met-207 is associated with the CuA-binding site (along with Cys-196, Cys-200 and His-204) and plays a role in determining its reduction potential and stability.

Base Sequence↗
PubMed1990

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.

Base Sequence↗
PubMed1990

Physical organization of the 18S and 5S ribosomal RNA genes in the mitochondrial genome of rye (Secale cereale L.).

The mitochondrial 18S and 5S ribosomal RNA (rRNA) genes of rye, plus a total of about 90 kilobase pairs of flanking DNA, have been cloned and maps of restriction enzyme cleavage sites have been constructed. Like their homologs from hexaploid wheat, the rye genes are closely linked and are part of a three-copy family of recombining repeats (the "18S/5S repeat"). The rye repeat probably also contains a mitochondrial tRNA(fMet) gene, which the wheat repeat is known to carry. However, despite the overall organizational similarity between the wheat and rye 18S/5S repeats in the immediate vicinity of their coding regions, extensive rearrangement of flanking sequences has taken place during evolutionary divergence of the two species. Our data provide additional support for an emerging picture of plant mitochondrial genomes as evolving much more rapidly in structure than in sequence.

Base Sequence↗
PubMed1989

RNA editing in plant mitochondria.

A basic principle of molecular biology is that the primary sequence of RNA faithfully reflects the primary sequence of the DNA from which it is transcribed. This concept has been challenged recently by the discovery of RNA editing, broadly defined as any process that changes the nucleotide sequence of an RNA molecule from that of the DNA template encoding it. Examples of RNA editing (see ref. 2 for review) include the insertion and deletion of uridine residues in mitochondrial messenger RNAs in kinetoplastid protozoa, the conversion of a cytidine to uridine in mammalian apolipoprotein-B mRNA, and the appearance of two non-templated guanosine residues in a paramyxovirus transcript. In these cases, RNA editing either re-tailors a non-functional transcript, producing a translatable mRNA, or modifies an already functional mRNA so that it generates a protein of altered amino-acid sequence. Here we report an editing phenomenon that involves the conversion of cytidine to uridine at multiple positions in the mRNA for subunit II of cytochrome c oxidase in wheat mitochondria. Such RNA editing provides an explanation for apparent coding anomalies in plant mitochondria.

Amino Acid Sequence↗
PubMed1989

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.

Base Sequence↗
PubMed1989

Nucleotide sequence of a wheat mitochondrial lysine tRNA gene.

We present the sequence of a wheat mitochondrial (mt) lysine tRNA gene (trnK-UUU). This gene more closely resembles its E. coli counterpart than it does the corresponding gene in fungal or mammalian mtDNA. Hybridization experiments with a trnK-specific probe suggest that at least two copies of this tRNALys gene are present in the wheat mitochondrial genome.

Base Sequence↗
PubMed1989

Chloroplast-like transfer RNA genes expressed in wheat mitochondria.

In the course of a systematic survey of wheat mitochondrial tRNA genes, we have sequenced chloroplast-like serine (trnS-GGA), phenylalanine (trnF-GAA) and cysteine (trnC-GCA) tRNA genes and their flanking regions. These genes are remnants of 'promiscuous' chloroplast DNA that has been incorporated into wheat mtDNA in the course of its evolution. Each gene differs by one or a few nucleotides from the authentic chloroplast homolog previously characterized in wheat or other plants, and each could potentially encode a functional tRNA whose secondary structure shows no deviations from the generalized model. To determine whether these chloroplast-like tRNA genes are actually expressed, wheat mitochondrial tRNAs were resolved by a series of polyacrylamide gel electrophoreses, after being specifically end-labeled in vitro by 3'-CCA addition mediated by wheat tRNA nucleotidyltransferase. Subsequent direct RNA sequence analysis identified prominent tRNA species corresponding to the mitochondrial and not the chloroplast trnS, trnF and trnC genes. This analysis also revealed chloroplast-like elongator methionine, asparagine and tryptophan tRNAs. Our results suggest that at least some chloroplast-like tRNA genes in wheat mtDNA are transcribed, with transcripts undergoing processing, post-transcriptional modification and 3'-CCA addition, to produce mature tRNAs that may participate in mitochondrial protein synthesis.

Base Sequence↗