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M W Gray

Publications and source records attributed to M W Gray.

At least 37 records · Page 2Linked to original sources
PubMed1999

A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii.

In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity.

Acanthamoeba↗
PubMed1999

Identification and characterization of T3/T7 bacteriophage-like RNA polymerase sequences in wheat.

Using PCR-based methods, we assembled two wheat cDNA sequences, wheat-G and wheat-C, that encode T3/T7 bacteriophage-like RNA polymerases (RNAPs) sharing 45% amino acid identity. In phylogenetic analyses using maximum likelihood, parsimony and distance methods, the predicted protein sequence of wheat-G (1005 amino acids, 113 kDa) clusters with sequences of previously assigned mitochondrial RNAPs from dicotyledonous plants (Arabidopsis thaliana, Chenopodium album); likewise, in such analyses, the wheat-C sequence (949 amino acids, 107 kDa) affiliates specifically with the Arabidopsis sequence that encodes a phage-like RNAP thought to function in chloroplasts. To confirm biochemically the assignment of the gene encoding the putative wheat mitochondrial RNAP, we isolated a ca. 100 kDa wheat mitochondrial protein that is enriched in fractions displaying specific in vitro transcription activity and that reacts with an antibody raised against a recombinant maize phage-type RNAP. Internal peptide sequence information obtained from the 100-kDa polypeptide revealed that it corresponds to the predicted wheat-G cDNA sequence, providing direct evidence that the wheat-G gene (which we propose to call RpoTm) encodes the wheat mitochondrial RNAP.

Amino Acid Sequence↗
PubMed1999

Complete sequence of the mitochondrial DNA of the red alga Porphyra purpurea. Cyanobacterial introns and shared ancestry of red and green algae.

The mitochondrial DNA (mtDNA) of Porphyra purpurea, a circular-mapping genome of 36,753 bp, has been completely sequenced. A total of 57 densely packed genes has been identified, including the basic set typically found in animals and fungi, as well as seven genes characteristic of protist and plant mtDNAs and specifying ribosomal proteins and subunits of succinate:ubiquinone oxidoreductase. The mitochondrial large subunit rRNA gene contains two group II introns that are extraordinarily similar to those found in the cyanobacterium Calothrix sp, suggesting a recent lateral intron transfer between a bacterial and a mitochondrial genome. Notable features of P. purpurea mtDNA include the presence of two 291-bp inverted repeats that likely mediate homologous recombination, resulting in genome rearrangement, and of numerous sequence polymorphisms in the coding and intergenic regions. Comparative analysis of red algal mitochondrial genomes from five different, evolutionarily distant orders reveals that rhodophyte mtDNAs are unusually uniform in size and gene order. Finally, phylogenetic analyses provide strong evidence that red algae share a common ancestry with green algae and plants.

Amino Acid Sequence↗
PubMed1999

The complete mitochondrial DNA sequences of Nephroselmis olivacea and Pedinomonas minor. Two radically different evolutionary patterns within green algae.

Green plants appear to comprise two sister lineages, Chlorophyta (classes Chlorophyceae, Ulvophyceae, Trebouxiophyceae, and Prasinophyceae) and Streptophyta (Charophyceae and Embryophyta, or land plants). To gain insight into the nature of the ancestral green plant mitochondrial genome, we have sequenced the mitochondrial DNAs (mtDNAs) of Nephroselmis olivacea and Pedinomonas minor. These two green algae are presumptive members of the Prasinophyceae. This class is thought to include descendants of the earliest diverging green algae. We find that Nephroselmis and Pedinomonas mtDNAs differ markedly in size, gene content, and gene organization. Of the green algal mtDNAs sequenced so far, that of Nephroselmis (45,223 bp) is the most ancestral (minimally diverged) and occupies the phylogenetically most basal position within the Chlorophyta. Its repertoire of 69 genes closely resembles that in the mtDNA of Prototheca wickerhamii, a later diverging trebouxiophycean green alga. Three of the Nephroselmis genes (nad10, rpl14, and rnpB) have not been identified in previously sequenced mtDNAs of green algae and land plants. In contrast, the 25,137-bp Pedinomonas mtDNA contains only 22 genes and retains few recognizably ancestral features. In several respects, including gene content and rate of sequence divergence, Pedinomonas mtDNA resembles the reduced mtDNAs of chlamydomonad algae, with which it is robustly affiliated in phylogenetic analyses. Our results confirm the existence of two radically different patterns of mitochondrial genome evolution within the green algae.

Animals↗
PubMed1999

Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria.

To investigate the transcriptional apparatus in wheat mitochondria, mitochondrial extracts were subjected to column chromatography and protein fractions were analyzed by in vitro transcription and mobility shift assays. Fractions eluting from DEAE-Sephacel between 0.2 and 0.3 M KCl displayed DNA-binding activity and supported specific transcription initiated from a wheat cox2 promoter. The active DEAE-Sephacel pool was further resolved by chromatography on phosphocellulose. Fractions that exhibited DNA-binding activity and that stimulated both specific and nonspecific transcription in vitro were highly enriched in a 63-kDa protein (p63). From peptide sequence obtained from purified p63, a cDNA encoding the protein was assembled. The predicted amino acid sequence (612 amino acid residues, 69 kDa) contains a basic N-terminal targeting sequence expected to direct transport of the protein into mitochondria. The p63 sequence also features an acidic domain characteristic of transcriptional activation factors, as well as sequence blocks displaying limited similarity to positionally equivalent regions in sigma factors from eubacteria related to mitochondria. Recombinant p63 possesses DNA-binding activity, exhibiting an affinity for the core cox2 promoter element and upstream regions in gel shift assays and having the ability to enhance specific transcription in vitro. Transcripts encoding p63 are expressed at an early stage in the germination of isolated wheat embryos, in a temporal pattern parallelling that of newly synthesized precursors of cox2, a mitochondrial gene. Taken together, these data suggest a role for p63 in transcription in wheat mitochondria.

Amino Acid Sequence↗
PubMed1999

Mitochondrial genome evolution and the origin of eukaryotes.

Recent results from ancestral (minimally derived) protists testify to the tremendous diversity of the mitochondrial genome in various eukaryotic lineages, but also reinforce the view that mitochondria, descendants of an endosymbiotic alpha-Proteobacterium, arose only once in evolution. The serial endosymbiosis theory, currently the most popular hypothesis to explain the origin of mitochondria, postulates the capture of an alpha-proteobacterial endosymbiont by a nucleus-containing eukaryotic host resembling extant amitochondriate protists. New sequence data have challenged this scenario, instead raising the possibility that the origin of the mitochondrion was coincident with, and contributed substantially to, the origin of the nuclear genome of the eukaryotic cell. Defining more precisely the alpha-proteobacterial ancestry of the mitochondrial genome, and the contribution of the endosymbiotic event to the nuclear genome, will be essential for a full understanding of the origin and evolution of the eukaryotic cell as a whole.

Animals↗
PubMed1998

Processing of precursor rRNA in Euglena gracilis: identification of intermediates in the pathway to a highly fragmented large subunit rRNA.

We have identified and characterized the stable steady-state intermediates that appear during formation of the cytoplasmic rRNA in Euglena gracilis. A 10.2 kb RNA is the precursor to both the small subunit (SSU) rRNA and 14 discrete fragments that comprise the large subunit (LSU) rRNA. The SSU rRNA is produced via two intermediates of 4.4 kb and 3.2 kb, whereas the LSU rRNA is generated by way of two RNA species of 5.8 kb and 5.3 kb. A number of unique intermediates are associated with a novel processing pathway by which the 14 mature fragments of the LSU rRNA are produced. Analysis of transcripts mapping within ITS1, the internal transcribed spacer separating the SSU and LSU rRNA coding regions, revealed that the LSU1 (=5.8S) rRNA is heterogeneous at its 5'-end, with a major cluster of primer extension products terminating approx. 4-5 nucleotides upstream from the predominant, mature 5'-end and a second, low-level extension product appearing further upstream within ITS1. The results reported here define the pre-rRNA processing pathway in E. gracilis and provide the basis for further studies of the mechanism of excision of the novel ITSs in this system.

Animals↗
PubMed1998

Genome structure and gene content in protist mitochondrial DNAs.

Although the collection of completely sequenced mitochondrial genomes is expanding rapidly, only recently has a phylogenetically broad representation of mtDNA sequences from protists (mostly unicellular eukaryotes) become available. This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, gene content, ribosomal RNA, introns, transfer RNAs and the genetic code and phylogenetic implications. We also illustrate the utility of a comparative genomics approach to gene identification by providing evidence that orfB in plant and protist mtDNAs is the homolog of atp8 , the gene in animal and fungal mtDNA that encodes subunit 8 of the F0portion of mitochondrial ATP synthase. Although several protist mtDNAs, like those of animals and most fungi, are seen to be highly derived, others appear to be have retained a number of features of the ancestral, proto-mitochondrial genome. Some of these ancestral features are also shared with plant mtDNA, although the latter have evidently expanded considerably in size, if not in gene content, in the course of evolution. Comparative analysis of protist mtDNAs is providing a new perspective on mtDNA evolution: how the original mitochondrial genome was organized, what genes it contained, and in what ways it must have changed in different eukaryotic phyla.

Amino Acid Sequence↗
PubMed1998

The Organelle Genome Database Project (GOBASE).

The taxonomically broad organelle genome database (GOBASE) organizes and integrates diverse data related to organelles (mitochondria and chloroplasts). The current version of GOBASE focuses on the mitochondrial subset of data and contains molecular sequences, RNA secondary structures and genetic maps, as well as taxonomic information for all eukaryotic species represented. The database has been designed so that complex biological queries, especially ones posed in a comparative genomics context, are supported. GOBASE has been implemented as a relational database with a web-based user interface (http://megasun.bch.umontreal.ca/gobase/gobas e.html ). Custom software tools have been written in house to assist in the population of the database, data validation, nomenclature standardization and front-end design. The database is fully operational and publicly accessible via the World Wide Web, allowing interactive browsing, sophisticated searching and easy downloading of data.

Amino Acid Sequence↗
PubMed1997

The cytochrome oxidase subunit 1 gene (cox1) from the dinoflagellate, Crypthecodinium cohnii.

To date, no genes have been characterized from dinoflagellate mitochondrial DNA. Here we present the complete sequence of the gene (cox1) encoding subunit 1 of cytochrome c oxidase in the dinoflagellate, Crypthecodinium cohnii. Analysis of nucleotide and deduced amino acid sequences predicts a protein of 523 amino acids that is translated using universal initiation, stop and tryptophan codons. COX1 amino acid identity and phylogenetic tree analyses strongly support a close evolutionary relationship between dinoflagellates and apicomplexans; however, inclusion of the ciliates in this clade is less well supported, a result likely due to the highly derived nature of ciliate COX1 sequences.

Amino Acid Sequence↗
PubMed1997

An ancestral mitochondrial DNA resembling a eubacterial genome in miniature.

Mitochondria, organelles specialized in energy conservation reactions in eukaryotic cells, have evolved from eubacteria-like endosymbionts whose closest known relatives are the rickettsial group of alpha-proteobacteria. Because characterized mitochondrial genomes vary markedly in structure, it has been impossible to infer from them the initial form of the proto-mitochondrial genome. This would require the identification of minimally derived mitochondrial DNAs that better reflect the ancestral state. Here we describe such a primitive mitochondrial genome, in the freshwater protozoon Reclinomonas americana. This protist displays ultrastructural characteristics that ally it with the retortamonads, a protozoan group that lacks mitochondria. R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase. Features of gene content together with eubacterial characteristics of genome organization and expression not found before in mitochondrial genomes indicate that R. americana mtDNA more closely resembles the ancestral proto-mitochondrial genome than any other mtDNA investigated to date.

Animals↗
PubMed1997

On the evolution of the single-subunit RNA polymerases.

Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

Amino Acid Sequence↗
PubMed1996

A 5 S rRNA gene is present in the mitochondrial genome of the protist Reclinomonas americana but is absent from red algal mitochondrial DNA.

Except in the case of land plants, mitochondrial ribosomes apparently lack a 5 S rRNA species, even though this small RNA is a component of all prokaryotic, chloroplast and eukaryotic cytosol ribosomes. In plants, the mitochondrial 5 S rRNA is encoded by mtDNA and differs in sequence from the 5 S rRNA specified by plant nuclear and chloroplast genomes. A distinctive 5 S rRNA component has not been found in the mitochondrial ribosomes of non-plant eukaryotes and, with the notable exception of the chlorophycean alga, Prototheca wickerhamii, a 5 S rRNA gene has not been identified in those non-plant mtDNAs characterized to date. Here, we report the presence of a 5 S rRNA gene in the mtDNA of the heterotrophic flagellate Reclinomonas americana. This unicellular eukaryote is a member of the jakobid flagellates, an early-diverging group of protists that share ultrastructural characteristics with the retortamonads, primitive protists that lack mitochondria. We report sequence data from the mtDNAs of the red algae Porphyra purpurea and Gracilariopsis lemaneiformis, which we use to evaluate a recent claim that a 5 S rRNA gene exists in the mtDNA of a third rhodophyte alga, Chondrus crispus. Our results lead us to the opposite conclusion: that a 5 S rRNA gene is not encoded by red algal mtDNA. In view of the accumulating evidence favoring a monophyletic origin of the mitochondrial genome, it is likely that a 5 S rRNA gene was present in an ancestral proto-mitochondrial genome, and that contemporary mtDNA-encoded 5 S rRNA genes have all descended from this ancestral gene. Considering the highly restricted phylogenetic distribution of identified mtDNA-encoded 5 S rRNA genes, it follows that the mitochondrial 5 S rRNA gene must have been lost multiple times during evolutionary diversification of the eukaryotic lineage.

Animals↗