Biomedical subjects

Patrick J Keeling

Publications and source records attributed to Patrick J Keeling.

63 records · Page 4Linked to original sources

PubMedDate not supplied

Re-examining alveolate evolution using multiple protein molecular phylogenies.

Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non-photosynthetic dinoflagellate, Crypthecodinium cohnii: actin, beta-tubulin, hsp70, BiP, hsp90, and mitochondrial hsp10. Beta-tubulin, hsp70, BiP, and hsp90 were found to be useful for intra-alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa-dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.

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PubMedDate not supplied

Molecular phylogeny and surface morphology of Colpodella edax (Alveolata): insights into the phagotrophic ancestry of apicomplexans.

The molecular phylogeny of colpodellids provides a framework for inferences about the earliest stages in apicomplexan evolution and the characteristics of the last common ancestor of apicomplexans and dinoflagellates. We extended this research by presenting phylogenetic analyses of small subunit rRNA gene sequences from Colpodella edax and three unidentified eukaryotes published from molecular phylogenetic surveys of anoxic environments. Phylogenetic analyses consistently showed C. edax and the environmental sequences nested within a colpodellid clade, which formed the sister group to (eu)apicomplexans. We also presented surface details of C. edax using scanning electron microscopy in order to supplement previous ultrastructural investigations of this species using transmission electron microscopy and to provide morphological context for interpreting environmental sequences. The microscopical data confirmed a sparse distribution of micropores, an amphiesma consisting of small polygonal alveoli, flagellar hairs on the anterior flagellum, and a rostrum molded by the underlying (open-sided) conoid. Three flagella were present in some individuals, a peculiar feature also found in the microgametes of some apicomplexans.

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PubMedDate not supplied

Actin and ubiquitin protein sequences support a cercozoan/foraminiferan ancestry for the plasmodiophorid plant pathogens.

The plasmodiophorids are a group of eukaryotic intracellular parasites that cause disease in a variety of economically significant crops. Plasmodiophorids have traditionally been considered fungi but have more recently been suggested to be members of the Cercozoa, a morphologically diverse group of amoeboid, flagellate, and amoeboflagellate protists. The recognition that Cercozoa constitute a monophyletic lineage has come from phylogenetic analyses of small subunit ribosomal RNA genes. Protein sequence data have suggested that the closest relatives of Cercozoa are the Foraminifera. To further test a cercozoan origin for the plasmodiophorids, we isolated actin genes from Plasmodiophora brassicae, Sorosphaera veronicae, and Spongospora subterranea, and polyubiquitin gene fragments from P. brassicae and S. subterranea. We also isolated actin genes from the chlorarachniophyte Lotharella globosa. In protein phylogenies of actin, the plasmodiophorid sequences consistently branch with Cercozoa and Foraminifera, and weakly branch as the sister group to the foraminiferans. The plasmodiophorid polyubiquitin sequences contain a single amino acid residue insertion at the functionally important processing point between ubiquitin monomers, the same place in which an otherwise unique insertion exists in the cercozoan and foraminiferan proteins. Taken together, these results indicate that plasmodiophorids are indeed related to Cercozoa and Foraminifera, although the relationships amongst these groups remain unresolved.

Actins↗

PubMedDate not supplied

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans.

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.

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PubMedDate not supplied

Transfer of Nosema locustae (Microsporidia) to Antonospora locustae n. comb. based on molecular and ultrastructural data.

Nosema locustae is a microsporidian parasite of grasshopper pests that is used as a biological control agent, and is one of the emerging model systems for microsporidia. Due largely to its diplokaryotic nuclei, N. locustae has been classified in the genus Nosema, a large genus with members that infect a wide variety of insects. However, some molecular studies have cast doubt on the validity of certain Nosema species, and on the taxonomic position of N. locustae. To clarify the affinities of this important insect parasite we sequenced part of the rRNA operon of N. locustae and conducted a phylogenetic analysis using the complete small subunit rRNA gene. Nosema locustae is only distantly related to the nominotypic N. bombycis, and is instead closely related to Antonospora scoticae, a recently described parasite of bees. We examined the ultrastructure of mature N. locustae spores, and found the spore wall to differ from true Nosema species in having a multi-layered exospore resembling that of Antonospora (one of the distinguishing features of that genus). Based on both molecular and morphological evidence, therefore, we propose transferring N. locustae to the genus Antonospora, as Antonospora locustae n. comb.

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PubMedDate not supplied

Symbiotic innovation in the oxymonad Streblomastix strix.

Streblomastix strix is an enigmatic oxymonad found exclusively in the hindgut of the damp-wood termite Zootermopsis. Streblomastix has a number of unusual morphological characters and forms a complex but poorly understood symbiosis with epibiotic bacteria. Here we described the ultrastructure of S. strix, with emphasis on the axial cytoskeleton and cell-cell associations, in its normal state and when treated with antibiotics. In untreated cells, epibiotic bacteria were orderly arranged end-to-end on six or seven longitudinal vanes, giving S. strix a stellate appearance in transverse section. The epibiotic bacteria were unusually long bacilli of at least three different morphotypes. Bacteria adhered to the oxymonad host by distinct cell-cell junctions that protruded between the poles of adjacent epibiotic bacteria. Treating termites with the antibiotic carbenicillin led to the loss of most (but not all) of the bacteria and the transformation of S. strix from a long slender cell to a teardrop-shaped cell, where the axostyle was compacted and became bifurcated near the posterior end.

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PubMedDate not supplied

HSP90, tubulin and actin are retained in the tertiary endosymbiont genome of Kryptoperidinium foliaceum.

The dinoflagellate Kryptoperidinium foliaceum has replaced its ancestral peridinin-containing plastid with a fucoxanthin-containing diatom plastid via tertiary endosymbiosis. The diatom endosymbiont of K. foliaceum is much less reduced than well-studied endosymbiotic intermediates, such as cryptophytes and chlorarachniophytes, where relict nuclear genomes are retained in secondary endosymbionts. The K. foliaceum endosymbiont retains a prominent nucleus, multiple four-membrane plastids, and mitochondria, all within a relatively large volume of cytoplasm that is separated from the host cytoplasm by a single membrane. Here we report the first protein-coding gene sequences from the K. foliaceum endosymbiont and host nuclear genomes. We have characterised genes for nucleus-encoded cytosolic proteins, actin (from endosymbiont), alpha-tubulin (from both), beta-tubulin (from host), and HSP90 (from both), in addition to homologues from pennate diatoms Nitzschia thermalis and Phaeodactylum tricornutum. Phylogenetic reconstruction shows that the actin is diatom-derived, the beta-tubulin dinoflagellate-derived, while both diatom- and dinoflagellate-derived alpha-tubulin and HSP90 genes were found. The base composition biases of these genes co-varied with their phylogenetic position, suggesting that the genes still reside in their respective genomes. The presence of these genes implies they are still functional and more generally indicates that the endosymbiont is less genetically reduced than those of cryptophytes or chlorarachniophytes, raising the interesting question of whether any genes have transferred between the two nuclear genomes.

Actins↗

PubMedDate not supplied

A transcriptional fusion of genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase in dinoflagellates.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase are enzymes essential for glycolysis and gluconeogenesis. Dinoflagellates possess several types of both GAPDH and enolase genes. Here, we identify a novel cytosolic GAPDH-enolase fusion protein in several dinoflagellate species. Phylogenetic analyses revealed that the GAPDH moiety of this fusion is weakly related to a cytosolic GAPDH previously reported in dinoflagellates, ciliates, and an apicomplexan. The enolase moiety has phylogenetic affinity with sequences from ciliates and apicomplexans, as expected for dinoflagellate genes. Furthermore, the enolase moiety has two insertions in a highly conserved region of the gene that are shared with ciliate and apicomplexan homologues, as well as with land plants, stramenopiles, haptophytes, and a chlorarachniophyte. Another glycolytic gene fusion in eukaryotes is the mitochondrion-targeted triose-phosphate isomerase (TPI) and GAPDH fusion in stramenopiles (i.e. diatoms and oomycetes). However, unlike the mitochondrial TPI-GAPDH fusion, the GAPDH-enolase fusion protein appears to exist in the same compartment as stand-alone homologues of each protein, and the metabolic reactions they catalyze in glycolysis and gluconeogenesis are not directly sequential. It is possible that the fusion is post-translationally processed to give separate GAPDH and enolase products, or that the fusion protein may function as a single bifunctional polypeptide in glycolysis, gluconeogenesis, or perhaps more likely in some previously unrecognized metabolic capacity.

Amino Acid Sequence↗

PubMedDate not supplied

Microsporidian mitochondrial proteins: expression in Antonospora locustae spores and identification of genes coding for two further proteins.

Microsporidia are obligate intracellular parasites, phylogenetically allied to the fungi. Once considered amitochondriate, now a number of mitochondrion-derived genes have been described from various species, and the relict organelle was recently identified in Trachipleistophora hominis. We have investigated the expression of potential mitochondrial targeted proteins in the spore stage to determine whether the organelle is likely to have a role in the spore or early infection stage. To investigate whether the Antonospora locustae genome codes for a different complement of mitochondrial proteins than Encephalitozoon cuniculi an EST library was searched for putative mitochondrial genes that have not been identified in the E. cuniculi genome project. The spore is the infectious stage of microsporidia, but is generally considered to be metabolically dormant. Fourteen genes for putatively mitochondrion-targeted proteins were shown to be present in purified spore mRNA by 3'-rapid amplification of cDNA ends and EST sequencing. Pyruvate dehydrogenase E1alpha and mitochondrial glycerol-3-phosphate dehydrogenase proteins were also shown to be present in A. locustae and E. cuniculi spores, respectively, suggesting a role for these proteins in the early stages of infection, or within the spore itself. EST sequencing also revealed two mitochondrial protein-encoding genes in A. locustae that are not found in the genome of E. cuniculi. One encodes a possible pyruvate transporter, the other a subunit of the mitochondrial inner membrane peptidase. In yeast mitochondria, this protein is part of a trimeric complex that processes proteins targeted to the inner membrane and the intermembrane space, and its substrate in A. locustae is presently unknown.

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