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Research Interests

    Research in the lab is generally related to the molecular evolution and cell biology of eukaryotes, in particular the protists (i.e. eukaryotes that are not animals, fungi, or plants). Protists are mostly single celled organisms, but are many are extremely complex and sophisticated despite their small size. Protists also represent the greatest part of eukaryotic diversity, although most protists groups are very poorly studied, especially at the molecular level. We use molecular biology, microscopy, and increasingly use genome wide analyses such as EST sequencing and genome sequence surveys to study a number of questions in different lineages of protists. Much of our research focuses on cellular organelles, in particular mitochondria and plastids. These organelles originated by endosymbiosis, or the uptake and retention of a bacterium (the endosymbiont) by a eukaryote (the host). In the case of mitochondria this involved an alpha-proteobacterium and took place around the origin of eukaryotes. Plastids originated more recently from a cyanobacterium. Plastids have also spread between eukaryotic lineages by a process called secondary endosymbiosis. In this case, a plastid-bearing alga is itself taken up by another eukaryote, and its photosynthetic apparatus is retained by this new, secondary, host. Another major focus of the research is the transition of free living organisms to parasitism and how this affects their organelles and metabolism. All this relies on an accurate phylogeny of eukaryotes, so we are also interested in piecing together this huge puzzle:

Below are short descriptions of several projects currently underway.

Protist EST Project. - ESTs from algae with secondary plastids.
Gymnochlora     The lab is beginning EST projects on a number of other algae with secondary plastids though the Genome Canada Protist EST Project (PEP). The intent is to sequence relatively a small number of ESTs from a variety of algae with secondary plastids, including haptophytes, heterokonts, cryptomonads, and chlorarachniophytes, as well as some of their non-photosynthetic relatives. Sequencing is underway for the haptophyte Isochrysis galbana, the dinoflagellate Heterocapsa triquetra, and library construction is underway for several other species. The goal of this work is similar to that for the Bigalowiella EST project, but with a much broader aim of understanding the history of all plastid types in eukaryotes.

PEP homepage.

Origin and evolution of the apicomplexa.
    This is a major and long-term focus of the lab. Apicomplexa are a group of intracellular parasites that include several major disease causing agents, such as Toxoplasma, Cryptosporidium, and the malaria parasite, Plasmodium. In the mid-1990s, apicomplexa were surprisingly shown to contain a plastid, although they are obviously not photosynthetic. The plastid function is now fairly well characterised in Plasmodium and Toxoplasma, where genes for nuclear-encoded plastid targeted proteins involved in the biosynthesis of heme, fatty acids and isoprenoids have been found. We are interested in the origin of this plastid and its metabolic enzymes, and also the distribution of the plastid in extant apicomplexa. To first end, we are studying the phylogeny of each enzyme involved in the major biochemical pathways presently recognised in the plastids of Toxoplasma and Plasmodium. To examine the distribution of the plastid, we are presently looking for evidence of a plastid or plastid genome in the earliest branching apicomplexa, the gregarines. We and other labs have also generated evidence that the apicomplexan plastid is of red algal origin and likely traces back to the same endosymbiotic event that gave rise to plastids in several algal groups, namely cryptomonads, heterokonts, and haptophytes. We are pursuing this question further by developing multigene data sets to test the relationship of these organisms to alveolates. In addition, we are also working to resolve the early branches of the apicomplexan tree to reconstruct the evolution of certain other characters involved in parasitism.

Left: Scanning Electron Micrograph (SEM) of the archigregarine, Selenidium vivax. Gregarines are extremely abundant and diverse apicomplexa, but not very well studied. They are thought to be the earliest branch of the apicopmpelxa, a conclusion now seeing support from molecular data. In addition, the vertebrate parasite Cryptosporidium now appears to be related to gregarines, although exactly how remains unknown. SEM by B. Leander.

Plastid function in Helicosporidium.
    Helicosporidia are little studied intracellular parasites of invertebrates with a complex infectious cyst. The origin of helicosporidia has not been clear historically, but recently they were shown by phylogenetic analyses to be highly derived green algae related to the vertebrate parasite Prototheca. There is no morphological evidence for a plastid in helicosporidian parasites, but plastids have been found in Prototheca and a number of other algal parasites, and there is now evidence for a plastid rRNA in Helicosporidium. To characterise the plastid and its functions in Helicosporidium, we have begun to sequence ESTs (again) in collaboration with the lab of Drion Boucias at the University of Florida. Important functional proteins in the plastid are likely encoded in the nuclear genome for the most part, and targeted to the plastid post-translationally. The aims of this work are to characterise as many genes for plastid targeted proteins as possible so as to determine the functional metabolic pathways that are housed in the plastid, and also to use sequences from plastid-encoded genes to identify the organelle by in situ hybridisation. This will offer a unique chance to compare plastids in highly adapted intracellular parasites that evolved from algae, but with very different ancestors. In the case of apicomplexa, the ancestor contained a red algal secondary plastid, while the Helicosporidium plastid is a primary green plastid.

Molecular Phylogeny of Doughnuts Revealed by Environmental PCR.
    Doughnut biodiversity, and the origin of doughnuts are questions of great biomedical importance that have vexed scientists for many years. We have addressed these problems using a novel environmental sampling technique that simultaneously examines doughnodiversity and the relative abundance of different species in the doughnosphere. Based on the hypothesis that doughnut expression will leave environmental traces, doughnodiversity was sampled from the sub-rimal regions of used double-doubles. Double-doubles were procured at Spring Garden Road, Halifax, and rims were rolled-up manually, exposing traces of doughnofauna in the rim core. In total, 10,000 rim cores were examined between the months of April and May (we are indebted to the members of the Doolittle Lab for field work).

DNA was prepared from isolated rim-doughnofauna, and used as templates in environmental PCR reactions for the Surface Coat Sugar gene, SGS. Sequences were found to fall into three major families, which were identified by immunogold labeling and electron microscopy as representing the classical ring-form doughnut, or Ringozoa, the jelly- and cream-filled forms, or Jellyzoa, and the more recently discovered and putatively primitive Timbitozoa. Within each family the relative abundance of species varied dramatically, with Honi krulleri being the most abundant Ringozoa, and Bostonia creami being the most abundant Jellyzoa. Interestingly, an unexpected abundance of Timbitozoa was discovered in the relatively benign environment of Spring Garden Road, despite the widespread belief that Timbitozoa are restricted to extreme environments such as toddlers' birthday parties, school staff meetings, and of course, campus security stations. Unexpectedly, we also found that the Surface Coat Sugar gene is the product of an ancient gene duplication, resulting in "glazed" and "dusted" phenotypes. Since Ringozoa, Jellyzoa, and Timbitozoa all contain both sugar-glazed and sugar-dusted varieties, we were able to root the universal doughnut tree using this ancient sugar paralogy. Astonishingly, we found Timbitozoa to be sisters to the Jellyzoa, indicating that the Ringozoa are the most primitive family of doughnuts. Clearly, the Timbitozoa are not primitively simple doughnuts, but instead are secondarily derived, perhaps the result of extreme marketing pressure for something different. This finding focuses new importance on the study of the Ringozoa, and also on the characters shared among the three families. We suggest a number of shared characters exist between Jellyzoa and Ringozoa would have been present in the last common ancestor of doughnuts, which we call the "Proughgenut".

Genomics and function of mitochondria in microsporidian spores.
Antonospora     Microsporidia are a large and diverse group of intracellular parasites with a very complex and highly adapted mechanism of infection other cells. Microsporidia were thought for some time to be primitive eukaryotes that lacked a number of otherwise common features, in particular the mitochondrion. Now, however, molecular phylogenies have demonstrated that microsporidia are in fact highly derived fungi, and not especially ancient. In addition, several genes derived from the mitochondrial endosymbiont have now been found in microsporidian nuclear genomes. The first of these were HSP70 and pyruvate dehydrogenase, but the recent completion of the Encephalitozoon genome has provided several more. We are interested in how this organelle has adapted in microsporidia, and what it present functions are. We are using Antonospora locustae as our model system, and have completed a genome sequence survey on this organism. We are now in the process of comparing this genome with that of Encephalitozoon, and characterising the gene sequence, expression pattern, and localisation of each mitochondrial protein. We are also carrying out several genome-wide sampling surveys of microsporidia.

Origin of non-canonical genetic codes and how they affect translation machinery.
    The genetic code is one of the most highly conserved characteristics of biological systems, but it does change very rarely. In cases where the code has changed, it differs only slightly from the "Universal Genetic Code". It is clear that the last ancestor of all extant life used this standard code, and that all these variants arose by making small changes to this ancestral code. This non-canonical codes are very rare in eubacteria, unknown in archaebacteria, and relatively abundant in mitochondrial genomes. In eukaryotic nuclear genomes five lineages have been discovered to use non-canonical codes, and in one (ciliates) the code has changed several times independently. We recently discovered one of these five changes in the oxymond Streblomstix strix. In the standard code TAA and TAG are stop signals, but Streblomastix uses TAA and TAG to encode the amino acid glutamine. Oxymonads are arguably the worst-studied group of protists. They are relatively diverse but are generally uncultavatable and only live in association with animals, predominantly as symbionts or parasites in the gut of wood eating insects such as termites and roaches. Accordingly, very few molecular data are known from oxymonads, so the first goal of this project is to characterise the distribution of this code in oxymonads. Secondly, this non-canonical code is also found in some ciliates, hexamitid diplomonads, and certain green algae, and we are examining two translational proteins in these groups that are likely affected by the altered code, to determine if systematic changes take place in regions of these proteins that are important in maintaining the fidelity of translation.
SEM of Streblomastix strix. Oxymonad symbiont of the termite Zootermopsis angusticolis. Visable are surface "strips" that are actually symbiotic bacteria, and the four flagella in two pairs, characteristic of oxymonads. Streblomastix was found to use a non-canonical genetic code where TAA and TAG encode glutamine rather than "stop". See publication, Keeling & Leander 2003. J. Mol. Biol. 326, 1337-1349 (PDF). SEM by B. Leander.

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