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Science Technology & Engineering |
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Location:
Mitochondrial Genetics Laboratory |
Contact Information:
Office Phone: +61 3 9479 2322 |
Research Interests:
Mitochondria are found in almost all eukaryotic cells. They are the powerhouse of the cell, generating energy in form of ATP, and have other important functions in haem and lipid biosynthesis, intracellular signal transduction and programmed cell death. According to the endosymbiotic hypothesis, the organelles evolved from once free-living proteobacteria, which have been engulfed by primitive anaerobic eukaryotes. Today, many characteristics of the mitochondria still point to their bacterial origin. Surrounded by two membranes, the outer and the inner membrane, the organelles divide in much the same way as bacteria do, and they generate energy using the same series of electron-transfer reactions. And as a remnant of their bacterial ancestors, mitochondria contain their own genome. The contemporary organelle genomes, however, have been greatly reduced in size as well as in the number of proteins they encode. During evolution, many of the encoded proteins became obsolete in the internal environment of the host cell, and their genes have been eliminated from the mitochondrial DNA (mtDNA). The majority of essential organellar genes has been transferred to the nuclear genome of the cell, their products are now synthesised on ribosomes in the cytoplasm and are subsequently targeted to and imported into the organelle. .
Although the number of proteins still encoded in the mitochondrial genome is relatively small, the organellar genome has to be maintained, replicated and transcribed, and the encoded gene products have to be synthesised on ribosomes in the mitochondrial matrix. The genetic processes involved are quite different from those in the nucleus and in the cytoplasm of the eukaryotic host - in fact they are strikingly similar to the genetic processes found in bacteria. However, the proteins that mediate mitochondrial replication, transcription and protein synthesis are now all nuclear-encoded.
In order to investigate mitochondrial biogenesis and function, we are currently studying the genetic processes and the protein components mediating mitochondrial DNA replication and transcription using the cellular slime mould Dictyostelium discoideum as a model organism. Dictyostelium has a circular mitochondrial genome (56,564 bp in size), which codes for 33 polypeptides, 17 transfer RNAs (tRNAs) and two ribosomal RNAs (rRNAs). In Northern hybridisation studies and primer extension experiments the mtDNA was found to be transcribed into eight major polycistronic transcripts (see Figure). Most of the polycistronic transcripts are subsequently processed into smaller mono-, di- or tricistronic RNA molecules. The processing mechanisms appear to be similar to those reported in mammalian mitochondria, where maturation of the polycistronic transcripts involves cleavage using tRNAs as excision signals. By mapping the 5' ends of the polycistronic transcripts, potential transcription start sites and promoter regions have been identified. Their role as binding sites for various regulatory elements is of specific interest.
Relevant Publications:
Lab Members and Their Research Projects:
Mitochondrial Transcription: RNA polymerase, transcription factors and
promoter sequences in Dictyostelium mitochondria
The 5' ends of the eight polycistronic RNA transcripts in the mitochondria of Dictyostelium discoideum have been mapped in primer extension experiments. These ends could represent genuine transcription start sites or they could have been generated by p rocessing of even larger, primary transcripts that were not detected in the Northern Hybridisation studies. In a series of Reverse Transcription Polymerase Chain Reactions (RT-PCR) we were able to ampify some of the regions overlapping the eight major transcripts, indicating that larger transcripts do exist. Furthermore, results obtained from Southern hybridisation analyses of DNA fragments representing the regions between the eight transcripts with in vitro capped mitochondrial RNA suggested the presence of only one single promoter. This promoter site is now being characterised by employing DNA mobility shift assays and foot printing experiments.
For the above experiments, the nuclear gene of the Dictyostelium mitochondrial RNA polymerase has been cloned and sequenced (AY040092) allowing us to express the gene in E. coli cells and to purify the protein. Most mitochondrial and chloroplast genomes are transcribed by a homologous family of small nuclear-encoded RNA polymerases that share high sequence similarity with the single-subunit RNA polymerase transcribing the T3/T7 bacteriophage genome. The Dictyostelium mitochondrial RNA polymerase is a T3/T7 bacteriophage-like enzyme that shows highest homology to plant mitochondrial RNA polymerases. Pairwise alignments revealed the Dictyostelium protein to be closest related to mitochondrial RNA polymerases of Chenopodium album and Arabidopsis thaliana.
In addition, the genes of six potential mitochondrial transcription factors have been identified and cloned. Of the six genes, two that showed the highest potential to encode mitochondrial transcription factors were selected for characterization in DNA mobility shift assays to determine if these proteins interact with the potential promoter region. Further investigations will be conducted to reveal the role of the transcription factors in the regulation of transcription.
Mitochondrial Replication: Cloning and characterisation of
mitochondrial DNA polymerases in Dictyostelium
Mitochondrial DNA polymerase g (mtDNAg) is the sole enzyme found so far to replicate mitochondrial DNA in a variety of organisms, including mammals and fungi. To date, no known enzyme has been found to replicate the mitochondrial genome of Dictyostelium discoideum.
Database searches did not reveal any homologous sequences to the known mitochondrial DNA polymerase gamma, however, we found a DNA segment within the Dictyostelium nuclear genome that shares high sequence homology with the E. coli DNA polymerase I. My project involves the use of this sequence information to identify and clone the complete sequence of the potential Dictyostelium mt DNA polymerase, and to express its gene in E. coli cells in order to purify the protein for further characterization.
Mitochondrial gene expression: Processing of mitochondrial RNA transcripts in Dictyostelium
The primary, polycistronic transcript transcribed from the Dictyostelium discoideum mitochondrial genome is processed to yield multiple mono-, di- and tricistronic RNA molecules. In the maturation if these transcripts, the tRNA sequences located on the transcripts play a crucial role. The tRNA sequences are almost regularly interspersed between the rRNA and protein-coding sequences and they seem to "punctuate" the transcripts, dictating where the processing by endonucleolytic cleavage occurs.
It is of interest that Dictyostelium mitochondria share this unique genetic arrangement and transcript maturation with mammalian mitochondria. My project involves the identification of key components involved in the processing of mitochondrial RNA transcripts and the development of a mitochondrial in vitro processing system.
Mitochondrial DNA maintenance: Characterization of mitochondrial DNA repair enzymes in Dictyostelium
In contrast to the well studied DNA repair processes found in the nucleus of the cell, it has long been held that similar processes do not occur in mitochondria. Early observations even suggested that this was in fact the reason for the accumulation of damaged DNA in the mitochondria, which has been demonstrated to cause aging of the cell and a variety of degenerative diseases in humans and plants. However, more recent reports provide evidence that mitochondria do have a limited DNA repair capacity and several proteins thought to be involved in mitochondrial DNA repair have been identified and characterised.
We have identified putative Dictyostelium proteins with high homologies to proteins that form part of known E. coli DNA repair complexes. The identified Dictyostelium omologues have no reported role in nuclear DNA repair processes and are therefore likely to be involved in mitochondrial DNA repair. The aim of my PhD project is to verify the mitochondrial localisation of these proteins and to determine their role in mitochondrial DNA repair.
Patrizia Novello, Honours, 2000: Identification of Nuclear-Encoded Mitochondrial Proteins by Antisense RNA Inhibition in Dictyostelium discoideum.
Anna Figueiredo, Honours, 2000: Identification and Characterization of the Nuclear-Encoded Mitochondrial RNA Polymerase in Dictyostelium discoideum.
Megan O'Brien, Honours, 2001: Does the Effectiveness of Antisense RNA Inhibition vary depending on which Portion of the RNA Transcript is bound by an Antisense Strand?
Misal Maroun, Honours, 2002: The Identification and Cloning of the Mitochondrial Heat Shock Protein 70 gene in Dictyostelium discoideum.
Beatriz Jayawardena, Masters, 2002: Characterization of the Nuclear Gene coding for the Mitochondrial RNA Polymerase in Dictyostelium discoideum.
Esther Bauer, Honours, 2003: Identification and Cloning of Mitochondrial RNA Helicase genes in Dictyostelium discoideum.
Fabian Carter, Honours, 2003: Impairment of Mitochondrial Function in Dictyostelium discoideum by Manipulation of the Mitochondrial RNA Polymerase Gene.
Michael Smith, Honours, 2004: Mitochondrial Transcript Processing in Dictyostelium discoideum.
Elizabeth Hume, Honours, 2005: Characterisation of potential promoter sequences in the mitochondria of Dictyostelium discoideum.
Matt Donegan, Honours, 2005: Polyadenylation of RNA transcripts in Dictyostelium mitochondria.
Jonathon Brodie, Honours, 2005: Cloning and characterisation of the gene encoding mitochondrial prohibitin in Dictyostelium discoideum.
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Content approved by: Christian Barth
Page maintained by: Craig Lighton
Last updated: 6 March, 2006
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Dr. Christian Barth