13th International Congress of Protistology

Lauwaet, T., C.C.L. Wong, D. Reiner, B.J. Davids, S.G. Svard, A.G. McArthur, J.R. Yates, & FD. Gillin. 2009. Biological surprises from the Giardia lamblia basal body proteome. Presentation at the 13th International Congress of Protistology, Rio de Janeiro, Brazil.

Encystation and excystation, which are crucial to the pathogenesis of Giardia lamblia, are marked by dramatic cytoskeletal remodeling. However, the underlying signaling pathways are not known. Although basal bodies have been studied for >100 years, their composition and functions remain incompletely understood. As an ancient protist that is motile, differentiates, and causes disease, Giardia makes a valuable model. Since calmodulin, which is needed for excystation, localizes exclusively to the basal bodies in giardial cytoskeletons, we used it as bait to affinity purify and identify interacting proteins that may help regulate differentiation. Among the proteins that bound to calmodulin-Sepharose in a Ca-dependent manner, we found known basal body proteins, validating the approach. We also found several unexpected proteins. Exploring their functions has produced complex and interesting findings. Ribosomal proteins found in basal body proteomes are assumed to be contaminants. However, when we epitope tagged one highly conserved ribosomal protein (RP) that affinity purified with calmodulin, we found that it localized to the nucleoli. Confocal microscopy using co-staining with centrin, showed that the nucleolar RP staining overlapped with a single basal body that had been shown by Benchimol to associate with a nucleus. This suggests distinct function of a single basal body and a novel association between a basal body and the nucleolus, another incompletely understood organelle.

We also identified a unique unknown Giardia protein that affinity-purified and co-localized with calmodulin in vegetative cells. Surprisingly it is targeted to variable numbers of encystation secretory vesicles in encysting cells. This may be the first hint that ESVs may be heterogeneous and is the first direct connection between calmodulin, centrosomes and cyst wall biosynthesis. “Biological surprises” such as these may offer unexpected insights into giardial differentiation and basic cell biology.

15th International Symposium on Pollutant Responses in Marine Organisms

Goldstone, J.V., A.G. McArthur, J. Zanette, M. Jonsson, T. Parente, & J.J. Stegeman. 2009. The total cytochrome P450 complement of zebrafish and the expression of CYP genes during development. Oral presentation at the 15th International Symposium on Pollutant Responses in Marine Organisms, Bordeaux, France.

Kirischian, N., A.G. McArthur, C. Jesuthasan, B. Krattenmacher, & J.Y. Wilson. 2009. Phylogenetic and functional analysis of the vertebrate cytochrome P450 2 family. Poster presentation at the 15th International Symposium on Pollutant Responses in Marine Organisms, Bordeaux, France.

SOT 2009 - Cytochrome P450 genes in zebrafish development

Goldstone, J., M. Jonsson, T. Parente, J. Zanette, A.G. McArthur, B.R. Woodin, D.R. Nelson, & J.J. Stegeman. 2009. Cytochrome P450 genes in zebrafish development. Presentation at the Society of Toxicology 48th Annual Meeting, Baltimore, Maryland.


This presentation summarizes observations on the full complement of CYP genes in zebrafish, a non-mammalian model for pharmacological, toxicological and carcinogenesis research. A total of 88 CYP genes were identified in zebrafish. Homologies between zebrafish and human CYP were inferred from amino acid sequence identity and molecular phylogeny. Functions and regulation of most zebrafish CYP are not known. For CYP that are likely involved principally in endogenous functions there is clear orthology between human genes and their zebrafish counterparts, although there are differences in numbers of genes in some families. Relative to the human CYP complement there is expansion or enhanced diversity in zebrafish CYP families 1, 2 and 3 thought to be most involved in xenobiotic metabolism. There are five CYP1 genes, five CYP3s, and 42 CYP2s in 11 subfamilies. Only two of the zebrafish CYP2s (CYP2R and CYP2U) warrant the same designation as in humans based on sequence identity. However, syntenic analysis reveals that there are some 11 genes in the zebrafish CYP2N, 2P, 2V, and 2AD subfamilies, all of which occur in tandem in a cluster that has shared synteny with the single human CYP2J2, indicating orthology. Studies of CYP2Ns and CYP2Ps in other species indicate functional similarity with human CYP2J2. The multiple CYP2Ks share synteny with CYP2W, but the CYP2Xs and CYP2AAs do not obviously share synteny with any human CYP2 genes. The five zebrafish CYP1s occur in four subfamilies, divided into 2 clades. Zebrafish CYP1s show organ, cell and developmental stage differences in transcript expression and in inducibility by aryl hydrocarbon receptor agonists. CYP1D1, which is most closely related to CYP1A, is distinctly not induced by AH receptor agonists. Oligonucleotide microarrays targeted to all zebrafish CYPs reveal that many CYPs, including 17 CYP2s, are expressed during early zebrafish development.

Transcriptome analysis of Schistosoma mansoni larval development using serial analysis of gene expression (SAGE)

Parasitology 2009, 136(5): 469-485.

Authors: Taft AS, Vermeire JJ, Bernier J, Birkeland SR, Cipriano MJ, Papa AR, McArthur AG, Yoshino TP.

Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21440 SAGE tags and mapped 13381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3' UTR lengths or using existing 3' EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

Plasmodium possesses dynein light chain classes that are unique and conserved across species

Infection, Genetics, & Evolution 2009, 9(3): 337-343.

Authors: Githui EK, De Villiers EP, McArthur AG.

Plasmodium belongs to the phylum Apicomplexa. Within the Apicomplexa, Plasmodium, Toxoplasma and Cryptosporidium are parasites of considerable medical importance while Theileria and Eimeria are animal pathogens. P. falciparum is particularly important as it causes malaria, resulting in more than 1 million deaths each year. The malaria parasite actively invades the host cell in which it propagates and several proteins associated with the apical organelles have been implicated to be crucial in the invasion process. The biogenesis of the apical organelles is not well understood, but several studies indicate that microtubule-based vesicular transport is involved. Vesicular transport proteins are also present in Plasmodium and are presumed to be involved in transcellular transport in infected erythrocytes. Dynein is a multi-subunit motor protein involved in microtubule-based vesicular transport. In this study, we analyzed the cytoplasmic dynein light chains (Dlcs) of P. falciparum since they provide adaptor surface to the cargoes and are likely to be involved in differential transport. Dlcs consist of three different families: TcTex1/2, LC8 and LC7/roadblock. The data presented demonstrate that P. falciparum Dlcs sequences and functional domains show high sequence similarity within the species, but that only the Dlc group 1 (LC8) has a high similarity to human orthologues. TcTex1 and LC7/roadblock have low similarity to human orthologues. This sequence variation could be targeted for vaccine or drug development.

Differential gene expression between Fall- and Spring-run Chinook salmon assessed by Long Serial Analysis of Gene Expression

Transactions of the American Fisheries Society 2008, 137(5): 1378-1388.

Authors: Bernier JC, Birkeland SR, Cipriano MJ, McArthur AG, Banks MA.

Of all Pacific salmonids, Chinook salmon Oncorhynchus tshawytscha display the greatest variability in return times to freshwater. The molecular mechanisms of these differential return times have not been well described. Current methods, such as long serial analysis of gene expression (LongSAGE) and microarrays, allow gene expression to be analyzed for thousands of genes simultaneously. To investigate whether differential gene expression is observed between fall- and spring-run Chinook salmon from California's Central Valley, LongSAGE libraries were constructed. Three libraries containing between 25,512 and 29,372 sequenced tags (21 base pairs/tag) were generated using messenger RNA from the brains of adult Chinook salmon returning in fall and spring and from one ocean-caught Chinook salmon. Tags were annotated to genes using complementary DNA libraries from Atlantic salmon Salmo salar and rainbow trout O. mykiss. Differentially expressed genes, as estimated by differences in the number of sequence tags, were found in all pairwise comparisons of libraries (freshwater versus saltwater = 40 genes; fall versus spring = 11 genes; and spawning versus nonspawning = 51 genes). The gene for ependymin, an extracellular glycoprotein involved in behavioral plasticity in fish, exhibited the most differential expression among the three groupings. Reverse transcription polymerase chain reaction analysis verified the differential expression of ependymin between the fall- and spring-run samples. These LongSAGE libraries, the first reported for Chinook salmon, provide a window of the transcriptional changes during Chinook salmon return migration to freshwater and spawning and increase the amount of expressed sequence data.

NUTMEG 2008

Goldstone, J.V., M. Jonsson, D.R. Nelson, A.G. McArthur, B.R. Woodin, & J.J. Stegeman. 2008. Cytochrome P450 in zebrafish: genomic complement and developmental expression. Poster presentation at the New England Membrane Biochemistry Meeting.

Molecular Parasitology Meeting 2008

Davids, B.J., M.A. Gilbert, J. Liu, D.S. Reiner, C. Lee, A.G. McArthur, & F.D. Gillin. 2008. Novel glutamyl endopeptidase activity needed for Giardia encystation. Presentation at the 19th Annual Molecular Parasitology Meeting, Woods Hole, Massachusetts.

Lauwaet, T., M. Baitaluk, D.S. Reiner, E.P. Romijn, C.C.L. Wong, H. Skarin, B.J. Davids, S.R. Birkeland, M.J. Cipriano, D. Palm, S.P. Preheim, A. Gupta, S.G. Svard, A.G. McArthur, J.R. Yates, A. Ray, & F.D. Gillin. 2008. The Giardia lamblia basal bodies: proteome, transcriptome, and interactome analyses. Presentation at the 19th Annual Molecular Parasitology Meeting, Woods Hole, Massachusetts.

ICAP 2008 - Proteome, transcriptome and interactome analyses reveal key roles of basal bodies in Giardia differentiation

Lauwaet, T., M. Baitaluk, D.S. Reiner, E.P. Romijn, C.C.L. Wong, H. Skarin, B.J. Davids, S.R. Birkeland, M.J. Cipriano, D. Palm, S.P. Preheim, A. Gupta, S.G. Svard, A.G. McArthur, J.R. Yates, A. Ray, & F.D. Gillin. 2008. Proteome, transcriptome and interactome analyses reveal key roles of basal bodies in Giardia differentiation. Presentation at the 4th International Conference on Anaerobic Protists, Taoyuan, Taiwan.

The success of Giardia as a parasite depends on its ability to accurately interpret physiologic signals from its external environment and to respond by differentiating into a dormant infectious cyst that can awaken into a pathogenic trophozoite. Giardia is a unique model for other intestinal protozoa because its life cycle has been completed in vitro. Since excystation is rapid, we proposed that signal transduction pathways are important. We found earlier that calmodulin, PKA, and PP2A, which are crucial to and upregulated in excystation, localize to the basal bodies/centrosomes. We hypothesized that the eight flagellar basal bodies regulate and co-ordinate the cellular reorganization of excystation. We isolated basal bodies from giardial cytoskeletons, analyzed their proteome by Multidimensional Protein Identification Technology (MudPIT), and matched individual peptides to genes in the Giardia genome database. We identified a total of 369 basal body proteins of which 98 are unique to Giardia.

To evaluate their functional significance, we identified human orthologs of giardial basal body proteins and constructed an interaction network (interolog) model of Giardia basal body proteins based on known protein-protein interactions in the human interactome. To determine whether the expression of genes encoding basal body proteins changes during differentiation, we measured their mRNA expression levels during growth, encystation and excystation by SAGE (serial analysis of gene expression). Of 175 genes encoding basal body proteins with SAGE tags, 72 were upregulated in encystation and 46 in cysts and excystation. When we overlaid the interolog network with the mRNA expression data, we found that expression of most protein biosynthesis genes is upregulated during encystation, while that of signaling and cytoskeletal genes is upregulated during excystation. This is consistent with the biological roles of these pathways. We verified the authenticity of the interolog model by using calmodulin, which localizes only to the basal bodies, as bait and affinity-purified proteins from the five major functional groups in the network.

Our proteome, transcriptome, and interactome analyses reveal complex composition and dynamic expression of Giardia basal body components. We propose that these organelles are cellular control centers regulating differentiation and entry into and awakening from dormancy.

Mollusc DNA Extraction - Formalin Perserved Samples

When extracting from fresh or frozen invertebrate specimens, we usually use the CTAB protocol (see my previous post). In the past, we also used this protocol for working with specimens that had been stored in formalin for some period before being transfered to ethanol but the results were hit and miss. The Etter/Rex group at UMass Boston developed a much superior protocol (Chase et al. 1998). It works incredibly well for formalin preserved invertebrates and we highly recommend it. The resulting DNA is very workable for PCR, although formalin still places limits on product lengths. It is basically the France and Kocher (1996) protocol but with the use of a silica-based column instead of ethanol precipitations. Here is our version of the protocol. Please cite Chase et al. (1998) when using it.

The protocol was centered on the QIAamp Tissue Kit. I don't know if this still exists in the same form, but take a look at the QIAamp DNA FFPE Tissue Kit. Be sure to order extra Buffer ATL - it can be ordered separately. Also be sure to prepare the solutions as described in the manual.

1. Mince 250 mg tissue, add 200 ul Buffer ATL, place at 55C for 24 hours.
2. Add 5 ul of 50 mg/ml Proteinase K and an additional 95 ul Buffer ATL. Place at 55C for 72 hours, in a mild shaker or with occasional vortexing.
3. Add 200 ul Buffer AL, mix by vortexing and incubate at 70C for 10 minutes.
4. Add 210 ul of 95% Ethanol, mix by vortexing.
5. Add mixture to column, centrifuge at 8000 rpm for 1 minute.
6. Place spin column in fresh tube, discard used collection tube.
7. Add 500 ul Buffer AW to column, centrifuge at 8000 rpm for 1 minute. Place spin column in fresh tube, discard used collection tube.
8. Add 500 ul Buffer AW to column, centrifuge at full speed for 3 minutes.
9. Place spin column in fresh tube, discard used collection tube.

Read the manual carefully to decide which elution strategy is best for your sample. Here are some options:

• Good Tissue or Lots of Tissue: Elute twice with 200 ul Buffer AE preheated to 70C. Incubate at room temperature for 1 minute before spinning for 1 minute at 8000 rpm. This is the manual's suggested approach. If you think DNA is rare, use less Buffer AE.
• Minute Specimens or Heavily Formalized Tissue: Add 50 ul of 70C preheated Buffer AE. Incubate at 70C for 5 minutes. Spin for 1 minute at 8000 rpm. Do not perform a second elution.