Search

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.

Chaperonin-60 and chaperonin-10 form stable binary complexes in the presence of ATP. This phenomenon has been used as the basis for the identification of a chaperonin-10 homologue in potato (Solanum tuberosum L.) mitochondria. ATP-dependent binary complexes formed between chaperonin-60 and chaperonin-10 in potato mitochondrial extracts were isolated by affinity chromatography on a column of immobilised chaperonin-60 antibodies. Partial amino acid sequence analysis of the chaperonin-10 protein indicates that it is related in primary structure to rat mitochondrial chaperonin-10.

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.

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves.

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.

Published methods of ribosome isolation from plant tissues result in a partial and selective recovery of the total ribosome population. The enrichment of polyribosomes obtained may give a false impression of the change in translational capacity of the tissue under study.

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.

Ribosomes from higher-plant mitochondria contain 5S rRNA, in contrast with the mitochondrial ribosomes of animals and fungi, in which such a component has not been detected. In common with the ribosomes of prokaryotes and chloroplasts, higher-plant mitochondrial ribosomes do not appear to contain an RNA equivalent to the 5.8 S rRNA that is found in eukaryoytes hydrogen-bonded to the largest of the cytoplasmic rRNA species.

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.

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.

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.