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C J Leaver

Publications and source records attributed to C J Leaver.

At least 55 records · Page 3Linked to original sources
PubMed1992

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.

Amino Acid Sequence↗
PubMed1991

Mitochondrial genome organization and expression associated with cytoplasmic male sterility in sunflower (Helianthus annuus).

A comparative investigation of the organization and expression of the mitochondrial genome in fertile and cytoplasmic male sterile (CMS) sunflower (Helianthus annuus) has been undertaken. A region of mitochondrial genome variation between the two phenotypes has been located in the 3' flanking region of the gene encoding the alpha subunit of the F1 ATPase (atpA). Physical mapping and sequence analysis have been used to show that a rearrangement involving an inversion and an insertion has occurred immediately downstream of the atpA coding region in the mitochondrial DNA from sterile sunflower. This rearrangement has resulted in the creation of a new open reading frame (ORFc) which is co-transcribed with atpA in sterile sunflower. In organello labelling of mitochondrial translation products from the two types of sunflower shows that a 15 kDa protein is synthesized by the mitochondria from sterile sunflower but not by those from fertile plants. The ORFc sequence could encode this 15 kDa protein which may be causally related to the CMS phenotype.

Amino Acid Sequence↗
PubMed1990

Developmental regulation of expression of the malate synthase gene in transgenic plants.

The cucumber malate synthase (MS) gene, including 1856 bp of 5' non-transcribed sequence, has been transferred into Petunia (Mitchell) and Nicotiana plumbaginifolia plants using an Agrobacterium binary vector. The transferred gene is found in variable copy number in different transformants, and is stably transmitted in each case as a single Mendelian character. Transgene mRNA accumulates in the seedling during the first three days of germination, then declines in amount as the cotyledons emerge from the seed. The decline is more pronounced in light-grown seedlings than in dark-grown seedlings. Expression of the MS transgene is also detected at a low level in petals of transformed Petunia plants. In these respects the pattern of MS gene expression is similar in cucumber and in transformed plants, showing that the transferred DNA fragment contains a functional MS gene. A 1076 bp fragment of 5' sequence was linked to the beta-glucuronidase reporter gene and transferred into Nicotiana, where it was shown to direct temporal and spatial patterns of expression similar to that of the complete MS gene. However, histochemical localisation of beta-glucuronidase activity demonstrated that the chimaeric gene is expressed not only in cotyledons of transgenic plants, but also in endosperm and some hypocotyl cells during early germination. The relevance of these findings to the control of malate synthase gene expression is discussed.

Base Sequence↗
PubMed1989

Two genes encode the adenine nucleotide translocator of maize mitochondria. Isolation, characterisation and expression of the structural genes.

Southern blot hybridisation of maize (Zea mays) genomic DNA with a specific cDNA probe and sequence analysis of several cDNAs have revealed that the mitochondrial adenine nucleotide translocator (ADP/ATP carrier) is encoded by two genes, both of which are expressed in dark-grown coleoptile tissue. The structural genes (designated ANT-G1 and ANT-G2), encoding the two translocator polypeptides, have been isolated from a maize genomic library and characterised by DNA sequence analysis, mung bean nuclease mapping and primer extension. The two genes each contain two introns and encode very similar proteins. Transcripts from both genes have long (approximately 200 nucleotides) 5' untranslated leaders which are unusual in that they contain several ATG codons upstream of the initiation codon. Transcript analysis of RNA extracted from serial sections of developing maize leaves reveals that the translocator genes are most actively transcribed in the basal meristem, while transcripts were undetectable in green leaves.

Amino Acid Sequence↗
PubMed1989

Evolution of plant mitochondrial genomes via substoichiometric intermediates.

Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor. Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates. Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution.

Base Sequence↗
PubMed1989

The malate synthase gene of cucumber.

The complete sequences of a full-length cDNA clone and a genomic clone encoding the Cucumis sativus glyoxysomal enzyme malate synthase, have been determined. The sequences have enabled us to identify putative control regions at the 5' end of the gene, three introns, and possible alternative polyadenylation sites at the 3' end. The deduced amino acid sequence predicts a polypeptide of 64,961 molecular weight, which has 48% identity with that of Escherichia coli. Comparison of the sequence of malate synthase from cucumber with that from E. coli and with other glyoxysomal and peroxisomal enzymes, shows that a conserved C-terminal tripeptide is a common feature of those enzymes imported into microbodies.

Amino Acid Sequence↗
PubMed1989

Subunit Composition of Cytochrome c Oxidase in Mitochondria of Zea mays.

Cytochrome c oxidase has been purified from Zea mays mitochondria by a solubilization with dodecyl maltoside followed by a simple and rapid two step fast protein liquid chromatographic method involving anion exchange on Mono Q and size exclusion chromatography on Superose 12. The preparation obtained was resolved by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a subunit composition comprising polypeptides of apparent molecular masses of 48, 31, and 25 kilodaltons at least one at 16 and 11 kilodaltons and three subunits below 10 kilodaltons. Comparison with a purified yeast cytochrome c oxidase revealed that the four largest subunits showed similar electrophoretic mobilities. Subunits I and II cross-reacted with antibodies raised against the yeast homologous polypeptides. Polypeptides of the plant ubiquinone:cytochrome c reductase complex have also been identified by cross-reaction with antibodies raised against yeast cytochrome b and c(1) subunits and by inference from comigration.

Journal Article↗
PubMed1987

Stoichiometric differences in DNA molecules containing the atpA gene suggest mechanisms for the generation of mitochondrial genome diversity in maize.

Four genomic arrangements of the maize mitochondrial atpA gene (encoding the alpha subunit of the F(1) ATPase), have been characterized. Most N (fertile) and S (male-sterile) cytoplasms contain two atpA arrangements of equal abundance. Prolonged exposure of blots of maize mitochondrial DNA probed with atpA-specific sequences show that cytoplasms previously reported to lack one of the atpA arrangements do contain the second arrangement but at low levels. Similarly, restriction fragments containing the atpA gene previously thought unique to male-sterile S and T cytoplasms are present in low abundance in fertile cytoplasms. These observations suggest that fertile and male-sterile cytoplasms of maize may be more closely related than previously thought, and suggest possible mechanisms to explain the observed mitochondrial genome diversity.

Journal Article↗
PubMed1986

Mitochondrial genome rearrangement leads to extension and relocation of the cytochrome c oxidase subunit I gene in sorghum.

The mitochondrial gene (COXI) encoding cytochrome c oxidase subunit I (COI) was isolated from two cytoplasmic genotypes of sorghum that synthesize different COI polypeptides. The Milo COI (Mr approximately 38,000) is encoded by a 530 codon structural gene sharing 98% homology with the corresponding maize gene. A variant COI observed in 9E cytoplasm (Mr approximately 42,000) is encoded by a 631 codon structural gene that diverges completely from the Milo COXI gene both 100 bp 5' to the presumed initiator methionine and within the 3' coding sequence. The 3' divergence results in a 101 C-terminal extension of the 9E COI that is not homologous to any known mitochondrial polypeptide. The novel 9E COXI, apparently arising from at least two rearrangements, affects transcription and gene product.

Base Sequence↗
PubMed1986

Transcript termini of messenger RNAs in higher plant mitochondria.

The 3'-termini of the mRNAs for subunit II of the cytochrome oxidase (COX II) and for the alpha-subunit of the mitochondrial ATPase (ATPA) have been determined in Oenothera mitochondria by two independent methods. Analysis of both transcripts by S1 protection experiments and of cloned cDNAs show an identical terminal 50 nucleotide sequence, to which homology is found 3' to some gene sequences in the maize mitochondrial genome. These regions can be folded into potential secondary structures similar to bacterial terminators.

DNA↗
PubMed1986

A protein specific to mitochondria from S-type male-sterile cytoplasm of maize is encoded by an episomal DNA.

Mitochondria of S-type cytoplasmic male sterile maize contain two linear double-stranded DNA molecules, S1 and S2. Two open reading frames (ORF1 and ORF2) are present in S2 DNA. Fragments from ORF1 were inserted into plasmids to achieve expression in Escherichia coli. Cells transformed with recombinant plasmids produced mRNA which hybridized with ORF1 and corresponding polypeptides were synthesized by in vivo and in vitro systems. Antiserum against a lacZ/S2 fusion protein precipitated the anticipated polypeptides from transformed E. coli cells and was therefore used to detect homologous peptide sequences in protein preparations of mitochondria from different maize cytoplasms. The antiserum detected a protein of 125 000 M(r) present in mitochondria from male sterile B73S but absent from the fertile B73N cytoplasm.

Journal Article↗
PubMed1986

Glyoxysomal Malate Synthase of Cucumber: Molecular Cloning of a cDNA and Regulation of Enzyme Synthesis during Germination.

A cDNA clone for the glyoxysomal enzyme malate synthase was isolated from a cDNA library made with polyadenylated RNA from the cotyledons of germinating Cucumis sativus L. This cloned DNA sequence was used as a probe to characterize changes in the amounts of malate synthase gene transcripts in cotyledons of cucumber seeds grown both in the light and in the dark. Malate synthase gene transcripts increase in amount to a peak at day 3 or day 4, and thereafter decline. In the light, this rate of decline is significantly greater than in the dark. Measurement of the changes in the amounts of malate synthase by assaying enzyme activity directly, and by immunological reaction with a specific antiserum indicate that the developmentally regulated synthesis of malate synthase in germinating cucumber is brought about primarily by changes in the amount of malate synthase gene transcripts, rather than through a control of translation. Similarly, the effect of light on the amount of malate synthase correlates precisely with its effect on the abundance of malate synthase gene transcripts.

Journal Article↗
PubMed1985

Isolation and sequence analysis of a cDNA encoding the ATP/ADP translocator of Zea mays L.

A cDNA complementary to the mRNA for the ATP/ADP translocator of maize (Zea mays L.) has been identified by virtue of hybridisation with the homologous gene from yeast. The cloned cDNA has been shown by DNA sequence analysis to contain an open reading frame of 954bp., which encodes a polypeptide of molecular weight 40,519. This polypeptide exhibits a high degree of homology to the translocator polypeptides of beef heart and Neurospora crassa mitochondria.

Amino Acid Sequence↗
PubMed1985

The maize cytochrome c oxidase subunit I gene: sequence, expression and rearrangement in cytoplasmic male sterile plants.

The single copy of the gene for cytochrome c oxidase subunit I (COX I) present in the mitochondrial genome of fertile maize (Zea mays L.) is encoded by a continuous open reading frame of 1584 nucleotides. The predicted polypeptide encoded by the gene has a mol. wt. of 58 219 daltons and shows >60% amino acid sequence homology with the corresponding fungal and animal polypeptides. Two major transcripts of 2400 and 2300 nucleotides can be detected and the 5' end of the larger transcript maps to a sequence from -161 to -153 (relative to the initiator codon) which shows high homology to the yeast mitochondrial promoter. In mitochondrial DNA from the S male-sterile cytoplasm of maize, which also characteristically contain two low mol. wt. linear DNAs (S1 and S2), rearrangements just 5' (at -175) to the COX I gene, generate additional DNA restriction fragments containing entire copies of the gene. These rearrangements involve a sequence identical to the terminal 186 bp of the 208-bp inverted repeat sequence found at either end of the S1 and S2 DNAs.

Journal Article↗
PubMed1985

The mitochondrial genome of fertile maize (Zea mays L.) contains two copies of the gene encoding the alpha-subunit of the F1-ATPase.

In contrast to the situation in animals and fungi the alpha-subunit of the mitochondrial F1-ATPase is encoded by two identical mitochondrial genes (ATP A) in male fertile maize (Zea mays L.). Cytoplasmic male sterile (T, C and S) maize mitochondrial genomes only contain a single copy of the gene. Sequence analysis reveals that the uninterrupted coding region of both copies of the gene is 1,524 bp long and encodes a polypeptide of 508 amino acids with a molecular weight of 55,117. The predicted amino acid sequence shares over 60% homology with the nuclear encoded alpha-subunit from yeast and bovine ATPase and approx. 50% with the corresponding chloroplast and bacterial polypeptides.

Amino Acid Sequence↗
PubMed1984

The apocytochrome b gene in maize mitochondria does not contain introns and is preceded by a potential ribosome binding site.

The apocytochrome b (COB) gene has been isolated from maize (Zea mays L.) mitochondrial DNA. Sequence analysis reveals that the coding region of the gene is 1164 bp long and, in contrast with the homologous gene from yeast, does not contain introns or TGA (Trp) codons. The predicted polypeptide encoded by the gene has a mol. wt. of 42 868 daltons, and shows 48% amino acid sequence homology with the corresponding yeast and mammalian polypeptides. Hydropathic profiles of the polypeptide indicate the presence of nine, membrane spanning hydrophobic domains suggesting that it is organised in the inner mitochondrial membrane in a similar fashion to that proposed for apocytochrome b in other organisms. The COB gene is preceded by a sequence 5' -AGTTGTCA-3' which may act as a ribosome binding site in the mRNA since: (i) it shows 67.5% complementarity with an octanucleotide at the 3' end of the maize mitochondrial 18S rRNA, located in a position homologous to that of the Escherichia coli Shine and Dalgarno sequence, and (ii) a similar sequence precedes several other plant mitochondrial genes at a distance of 15-20 nucleotides from the ATG initiation codon. RNA transcript analysis shows that the gene is transcribed in a complex manner with the presumed mature mRNA ( 2.25 kb) probably being derived by sequential processing from a larger primary transcript.

Journal Article↗