Dr. Christian Barth
and the Dictyostelium Mitochondrial Genetics Laboratory
Contact details
Department of Microbiology
Faculty of Science, Technology and Engineering
La Trobe University Victoria 3086
AUSTRALIA
Office Phone: +61 3 9479 2322
Departmental Enquiries: + 61 3 9479 1114
Mitochondrial Genetics Lab: +61 3 9479 2657
Fax: +61 3 9479 1222
Email: c.barth@latrobe.edu.au
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 in a variety of organisms, including the cellular slime mould Dictyostelium discoideum, its close relative Acanthamoeba castellanii, and other protists such as Naegleria gruberi.
The cellular slime mould Dictyostelium discoideum has long been regarded as a valuable and attractive tool for the study of eukaryotic cell biology. The organism combines typical eukaryotic cellular and molecular biology with the experimental tractability of a microorganism in which biochemical, classical and molecular genetics are readily adopted. Its developmental life cycle is unique amongst protists and at the different stages of development Dictyostelium features both plant- and animal-like characteristics. 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). Despite its relatively large size, the Dictyostelium mitochondrial genome is transcribed in a similar way as the human mitochondrial genome: transcription is initiated at a single initiation site, giving rise to a large polycistronic transcript that is co-transcriptionally processed into smaller, mature RNA molecules (see the map below for details; Barth et al., 1999; 2001, 2007; Le et al., 2009). The processing mechanisms also appear to be similar to those reported in mammalian mitochondria, where maturation of the polycistronic transcripts involves cleavage using tRNAs as excision signals. The similarities in transcription and transcript processing between Dictyostelium amd mammals make Dictyostelium an attractive model for the study of the processes and the components involved in the expression of mitochondrial genes.
Click to view high resolution image (3.57 MB)
Relevant Publications:
- Lang, B.F., Gray, M.W., and Burger, G. (1999). Mitochondrial genome evolution and the origin of eukaryotes. Annual Review in Genetics 33, 351-397.
- Tracy, R.L., and Stern, D.B. (1995). Mitochondrial transcription initiation: promoter structures and RNA polymerases. Current Genetics 28, 205-16.
- Clayton, D.A. (2000). Transcription and replication of mitochondrial DNA. Human Reproduction 15, 11-7.
- Ogawa, S., Yoshino, R., Angata, K., Iwamoto, M., Pi, M., Kuroe, K., Matsuo, K., Morio, T., Urushihara, H., Yanagisawa, K., and Tanaka, Y. (2000) The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content and genome organization. Molecular And General Genetics 263, 514-519.
- Barth, C., Greferath, U., Kotsifas, M., and Fisher, P.R. (1999). Polycistronic transcription and editing of the mitochondrial small subunit (SSU) ribosomal RNA in Dictyostelium discoideum. Current Genetics 36, 55-61.
- Barth, C., Greferath, U., Kotsifas, M., Tanaka, Y., Alexander, S., Alexander, H., and Fisher, P.R. (2001). Transcript mapping and processing of mitochondrial RNA in Dictyostelium discoideum. Current Genetics 39, 355-364.
- Barth, C., Le, P. and Fisher, P.R. (2007). Mitochondrial biology and disease in Dictyostelium. International Review of Cytology 263, 207-252.
- Le, P., Fisher, P.R. and Barth, C. (2009). Transcription of the Dictyostelium discoideum mitochondrial genome occurs from a single initiation site. RNA 15, 1-10.
Lab Members and Their Research Projects:
Maggie Mokbel, PhD student:
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.
Luke Kennedy, PhD student:
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.
Leanne Bekhet, PhD student:
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 homologues have no reported role in nuclear DNA repair processes and are therefore likely to be involved in mitochondrial DNA repair. The aim of this PhD project is to verify the mitochondrial localisation of these proteins and to determine their role in mitochondrial DNA repair.
Jessica Accari, PhD student:
Mitochondrial transcription: Transcription and transcript processing in the mitochondria of Acanthamoeba castellanii
Acanthamoeba castellanii is one of the most common protozoa in the soil and fresh water. The interest in the organism is based on its pathogenicity: it can invade the cornea, causing a painful and sight-threatening disease of the eye (amebic keratitis), but it can also spread to the central nervous system in which case it leads to the fatal disease amebic encephalitis. Acanthamoebae have also been associated with various secondary infections associated with immuno-compromised individuals such as AIDS patients and with several diseases in a variety of animals. Moreover, Acanthamoebae can serve as hosts for bacterial pathogens such as Staphylococcus aureus (an important and well known pathogen in hospitals due to its resistance to many antibiotics) or Legionella pneumophilia, the major cause of Legionnaire’s disease, a potentially fatal form of pneumonia.
The mitochondrial genomes of Dictyostelium and Acanthamoeba castellanii are strikingly similar in size, gene content and gene organization. This implies that both organisms share the same mode of transcription and transcript processing. During this project, the knowledge gained in the study of mitochondrial transcription in Dictyostelium will be used to investigate these processes in Acanthamoeba.
Alkouni Saad Ali Makhlouf, PhD student:
Mitochondrial transcription: Transcription in the marine choanoflagellate Monosiga brevicollis
Choanoflagellates are a group of free-living unicellular and colonial flagellate eukaryotes, which are of interest because they are considered to be the closest living relatives to animals. The cells are almost identical in shape and function with the choanocytes, or collar cells of sponges, the most primitive metazoans. Since choanoflagellates and metazoans are closely related, comparisons between the two groups promise to provide insight into the biology of their last common ancestor and the earliest events in metazoan evolution.
To investigate the transcription process in the mitochondria of Monosiga and to identify transcription initiation sites, Northern hybridisation studies, RT-PCRs and 5'-capping experiments will be conducted.
Sam Manna, PhD student
Ashley Harman, student
Previous Lab members:
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.
Elin Berger, PhD student: Characterization of mitochondrial DNA repair enzymes in Dictyostelium
Thuan Phuong, Honours, 2006: Investigation of mitochondrial RNA transcript extremities and identification of polyadenylated RNAs in Dictyostelium discoideum.
Chi Lun Tony Tam, Honours, 2006: Identification and characterisation of two proteins with unknown functions in Dictyostelium discoideum.
Daniela Basa, Honours, 2007: Identification and characterisation of mitochondrial repair proteins in Dictyostelium discoideum.
Sara Wong, Honours, 2008: DNA repair in the mitochondria of Dictyostelium discoideum.
Eunice Lee, Honours, 2009:
Dr Phuong Le, PhD student: RNA polymerase, transcription factors and promoter sequences in Dictyostelium mitochondrial transcription.