Homologous recombination is critical for generating genetic variation in living organisms by exchange and rearrangement of DNA. Most of our knowledge about homologous recombination is limited to processes in bacteria or in eukaryotic nuclei. In E. coli, homologous recombination is dependent on the RecA protein. Higher plant chloroplasts have RecA-like strand exchange activity. However, little is known about these mechanisms in higher plant mitochondria. I have detected a RecA-like strand exchange activity in soybean mitochondria. This activity forms joint molecules in the presence of ATP, Mg2+, and homologous DNA substrates. In addition, the E. coli single-stranded DNA binding (SSB) protein is a non-sequence-specific DNA binding protein that functions as an accessory factor for RecA protein-promoted strand exchange reactions. Our lab has identified an Arabidopsis homologue of E. coli SSB that is targeted to mitochondria (mtSSB). The results of my research shows the mtSSB protein has the same properties as the E. coli SSB protein and it can stimulate the E. coli RecA protein-promoted strand exchange reactions. DNA helicases utilize the energy of ATP to separate the two parental DNA strands at the replicating fork or during recombinational strand exchange. Although higher plant chloroplast helicase activity has been reported, no such activity has heretofore been identified in higher plant mitochondria. We report the characterization of a plant mitochondrial DNA helicase isolated from soybean leaves. ATP is required for this enzyme and this enzyme poorly utilizes any other NTPs or dNTPs. The enzyme requires Mg2+ for activity. This enzyme only has 3' to 5'unwinding activity. The optimal conditions for mitochondrial DNA helicase are 2 mM ATP, 8 to 10 mM Mg2+,100 to 200 mM NaCl and 37-42 oC incubation for one hour or longer time.

Previous studies have shown that transcriptional differences in the orf224-atp6 mitochondrial gene region are correlated with fertility restoration of the pol CMS trait by the dominant nuclear Rfp gene in Brassica napus. Recently, the recessive rfp allele, or a tightly linked gene, was found to act as a dominant gene, designated Mmt, in controlling the production of additional, smaller transcripts of two other mitochondrial loci. The results presented in this thesis reveal that Mmt-specific transcripts lack sequences found at the $5 sp prime$ end of the full-length transcripts of these loci and contain a common sequence, UUGUGG, which maps immediately downstream of their $5 sp prime$ termini. A similar sequence, UUGUUG, is found within orf224 downstream of the major Rfp-specific $5 sp prime$ transcript terminus; these hexanucleotide sequences may serve as recognition motifs in the generation of Mmt- and Rfp-specific transcripts. These results suggest that Rfp/Mmt is a novel nuclear locus affecting the expression of multiple mitochondrial gene regions, with different alleles or haplotypes affecting different mitochondrial genes.

Cytoplasmic male sterility (CMS) in plants is associated with mitochondrial dysfunction. We have proposed in this study that the mitochondrial-encoded chimeric peptide thought to be responsible for cytoplasmic male sterility in Polima system could function as a dominant male sterility inducer when expressed in the nucleus and targeted to the mitochondria. Transgenic plants expressing such mitochondrial targeting constructs exhibited reduction of pollen production that was characterized in fertile Westar (nap ) and restored fertile Westar (pol) plants by homeotic transformation of floral organs and in male-sterile Westar (pol) plants by a reduction in pollen production with shortening of the stamens. Genetic and molecular analysis has shown that the phenotypic changes were correlated with the effective genetic transmission of the inserted transgene through female gametes. Most significantly, we have found that differences in floral morphology induced by transgene expression between pol CMS and fertile Westar plants might be related to differences in transcriptional activity of the APETALA3 MADS box gene. We suggest that the alterations in floral morphology that accompany CMS in several plant species might be due to effects of mitochondria on transcriptional activity of floral organ identity genes.

In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai

Typescript (Photocopy) / Bibliography: leaves i-xii. (3rd paging sequence) / x, 157, xii, 13 leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Botany, 1990

[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.

Thesis (Ph.D.)--University of Western Australia, 2006.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 161-180).

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