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Construction of gender-enriched cDNA archives for adult Oesophagostomum dentatum by suppressive-subtractive hybridization and a microarray analysis of expressed sequence tags

Published online by Cambridge University Press:  23 January 2006

P. A. COTTEE ,
A. J. NISBET ,
Y. G. ABS EL-OSTA ,
T. L. WEBSTER  and
R. B. GASSER
P. A. COTTEE
Affiliation:
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia
A. J. NISBET
Affiliation:
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland EH26 OPZ, UK
Y. G. ABS EL-OSTA
Affiliation:
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia
T. L. WEBSTER
Affiliation:
Department of Primary Industries, Primary Industries Research Victoria, Bundoora Centre, La Trobe University, Bundoora, Victoria 3086, Australia
R. B. GASSER
Affiliation:
Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia Biotechnology Research Institute, Macquarie University, Sydney, New South Wales 2109, Australia
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Abstract

In the present study, we constructed gender-enriched cDNA libraries for the adult stage of the parasitic nematode Oesophagostomum dentatum (order Strongylida) using suppressive-subtractive hybridization (SSH), sequenced clones from the female-library and male-library (480 from each) and conducted bioinformatic and microarray analyses of the expressed sequence tags (ESTs). In total, 873 ESTs (440 male and 433 female) were obtained, achieving a sequencing success of 91%The nucleotide sequences reported in this article (Tables 1–5) have been deposited in the EMBL, GenBank and DDJB databases under the Accession nos. AM157797–AM158083. Microarray analyses of 516 unique ESTs representing both gender-enriched libraries revealed differential hybridization for 391 of them (75·8%). Of these, 220 (56·3%) had significantly greater signal intensities in the female than in the male, and 154 (70%) of these were predicted to have homologues in C. elegans. These homologues were predicted to be involved in key biological processes, including embryonic nutrition, gametogenesis, molecular binding/transport or metabolism, nucleic acid synthesis and function, and signal transduction. Of the 171 ESTs with statistically higher signal intensities in male O. dentatum, 43·8% had homologues in C. elegans. These homologues included major sperm proteins (MSPs) or MSP-like molecules, keratin-like molecules, molecules involved in metabolism, PDZ domain-containing proteins, sugar binding proteins, protein kinases, serine proteases or protease inhibitors, molecules involved in proteolysis and other proteins, such as enzymes and various putative proteins. Of the 287 ESTs (from both gender-enriched cDNA libraries) with no known homologues in C. elegans, 50 (17·4%) had homologues in other nematodes, 8 had homologues in various other organisms and 104 (36·2%) had no homology to any sequence in current gene databases. The present study lays a foundation for the isolation and molecular, biochemical and functional characterization of selected genes from the gender-enriched cDNA archives established for O. dentatum.

Keywords

Oesophagostomum dentatumNematoda (Strongylida)differential gene expressionmicroarray analysisgender-specific
Type
Research Article
Information
Parasitology , Volume 132 , Issue 5 , May 2006 , pp. 691 - 708
Copyright
2006 Cambridge University Press

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References

REFERENCES

Aboobaker, A. A. and Blaxter, M. L. ( 2004). Functional genomics for parasitic nematodes and platyhelminths. Trends in Parasitology 20, 178–184.CrossRefGoogle Scholar
Arrigo, A.-P. and Landry, J. ( 1994). Expression and function of the low molecular-weight heat shock proteins. In The Biology of Heat Shock Proteins and Molecular Chaperones ( ed. Georgopoulos, G.), pp. 335–373. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ash, L. R. and Orihel, T. C. ( 1987). Parasites: A Guide to Laboratory Procedures and Identification. American Society of Clinical Pathologists, Chicago, Illinois, USA.
Blaxter, M. L., Raghavan, N., Ghosh, I., Guiliano, D., Lu, W., Williams, S. A., Slatko, B. and Scott, A. L. ( 1996). Genes expressed in Brugia malayi infective third stage larvae. Molecular and Biochemical Parasitology 77, 77–93.CrossRefGoogle Scholar
Blaxter, M. L., De Lay, P., Garey, J. R., Liu, L. X., Scheldeman, P., Vierstraete, A., Vanfleteren, J. R., Mackey, L. Y., Dorris, M., Frisse, L. M., Vida, J. T. and Thomas, K. W. ( 1998). A molecular evolutionary framework for the phylum Nematoda. Nature, London 392, 71–75.CrossRefGoogle Scholar
Boag, P. R., Newton, S. E., Hansen, N., Christensen, C. M., Nansen, P. and Gasser, R. B. ( 2000). Isolation and characterization of sex-specific transcripts from Oesophagostomum dentatum by RNA arbitrarily-primed PCR. Molecular and Biochemical Parasitology 108, 217–224.CrossRefGoogle Scholar
Boag, P. R., Ren, P., Newton, S. E. and Gasser, R. B. ( 2003). Molecular characterisation of a male-specific serine/threonine phosphatase from Oesophagostomum dentatum (Nematoda: Strongylida), and functional characterization of homologues in Caenorhabditis elegans. International Journal for Parasitology 33, 313–325.CrossRefGoogle Scholar
Buttery, S. M., Ekman, G. C., Seavy, M., Stewart, M. and Roberts, T. M. ( 2003). Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion. Molecular Biology of the Cell 14, 5082–5088.CrossRefGoogle Scholar
Chen, J. S., Sappington, T. W. and Raikhel, A. S. ( 1997). Extensive sequence conservation among insect, nematode, and vertebrate vitellogenins reveals ancient common ancestry. Journal of Molecular Evolution 44, 440–451.CrossRefGoogle Scholar
Christensen, C. M., Grondahl-Nielsen, C. and Nansen, P. ( 1996). Non-surgical transplantation of Oesophagostomum dentatum to recipient pigs via rectal intubation. Veterinary Parasitology 65, 139–145.CrossRefGoogle Scholar
Cottee, P. A., Nisbet, A. J., Boag, P. R., Larsen, M. and Gasser, R. B. ( 2004). Characterization of major sperm protein genes and their expression in Oesophagostomum dentatum (Nematoda: Strongylida). Parasitology 129, 479–490.CrossRefGoogle Scholar
Dhadialla, T. S. and Raikhel, A. S. ( 1990). Biosynthesis of mosquito vitellogenin. The Journal of Biological Chemistry 265, 9924–9933.Google Scholar
Daugschies, A. and Watzel, C. ( 1999). In vitro development of histotropic larvae of Oesophagostomum dentatum under various conditions of cultivation. Parasitology Research 85, 158–161.CrossRefGoogle Scholar
Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D. and Siebert, P. D. ( 1996). Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proceedings of the National Academy of Sciences, USA 93, 6025–6030.CrossRefGoogle Scholar
Ding, L. and Candido, E. P. ( 2000 a). HSP43, a small heat-shock protein localized to specific cells of the vulva and spermatheca in the nematode Caenorhabditis elegans. The Biochemical Journal 349, 409–412.Google Scholar
Ding, L. and Candido, E. P. ( 2000 b). Association of several small heat-shock proteins with reproductive tissues in the nematode Caenorhabditis elegans. The Biochemical Journal 351, 13–17.Google Scholar
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. ( 1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, London 391, 806–811.CrossRefGoogle Scholar
Foster, J. M., Zhang, Y., Kumar, S. and Carlow, C. K. S. ( 2005). Mining nematode genome data for novel drug targets. Trends in Parasitology 21, 101–104.CrossRefGoogle Scholar
Gasser, R. B. and Newton, S. E. ( 2000). Genomic and genetic research on bursate nematodes: significance, implications and prospects. International Journal for Parasitology 30, 509–534.CrossRefGoogle Scholar
Grant, B. and Hirsh, D. ( 1999). Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Molecular Biology of the Cell 10, 4311–4326.CrossRefGoogle Scholar
Hanazawa, H., Mochii, M., Ueno, N., Kohara, Y. and Iino, Y. ( 2001). Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in Caenorhabditis elegans. Proceedings of the National Academy of Sciences, USA 98, 8686–8691.CrossRefGoogle Scholar
Hartman, D., Donald, D., Nikolaou, S., Savin, K. W., Hasse, D., Presidente, P. J. and Newton, S. E. ( 2001). Analysis of developmentally regulated genes of the parasite Haemonchus contortus. International Journal for Parasitology 31, 1236–1245.CrossRefGoogle Scholar
Hartman, D., Cottee, P. A., Savin, K. W., Bhave, M., Presidente, P. J., Fulton, L., Walkiewicz, M. and Newton, S. E. ( 2003). Haemonchus contortus: molecular characterisation of a small heat shock protein. Experimental Parasitology 104, 96–103.CrossRefGoogle Scholar
Haslbeck, M., Franzmann, T., Weinfurtner, D. and Buchner, J. ( 2005). Some like it hot: the structure and function of small heat-shock proteins. Nature Structural and Molecular Biology 12, 842–846.CrossRefGoogle Scholar
Haupt, W. ( 1966). Ein Beitrag zur Morphologie der Knötchenwuermer des Hausschweines, ihrer Eier sowie der dritten invasionstüchtigen Larvenstadien. Archiv für Experimentelle Veterinärmedizin 20, 701–711.Google Scholar
Hoekstra, R., Visser, A., Otsen, M., Tibben, J., Lenstra, J. A. and Roos, M. H. ( 2000). EST sequencing of the parasitic nematode Haemonchus contortus suggests a shift in gene expression during transition of the parasitic stages. Molecular and Biochemical Parasitology 110, 53–68.CrossRefGoogle Scholar
Issa, Z., Grant, W. N., Stasiuk, S. and Shoemaker, C. B. ( 2005). Development of methods for RNA interference in the sheep gastrointestinal parasite, Trichostrongylus colubriformis. International Journal for Parasitology 35, 935–940.CrossRefGoogle Scholar
Jiang, M., Ryu, J., Kiraly, M., Duke, K., Reinke, V. and Kim, S. K. ( 2001). Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans. Proceedings of the National Academy of Sciences, USA 98, 218–223.CrossRefGoogle Scholar
Joshua, G. W. and Hsieh, C. Y. ( 1995). Stage-specifically expressed genes of Angiostrongylus cantonensis: identification by differential display. Molecular and Biochemical Parasitology 71, 285–289.CrossRefGoogle Scholar
Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., Sohrmann, M., Welchman, D. P., Zipperlen, P. and Ahringer, J. ( 2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature, London 421, 231–237.CrossRefGoogle Scholar
Knox, D. P. ( 2004). Technological advances and genomics in metazoan parasites. International Journal for Parasitology 34, 139–152.CrossRefGoogle Scholar
Ladomery, M., Wade, E. and Sommerville, J. ( 1997). Xp54, the Xenopus homologue of human RNA helicase p54, is an integral component of stored mRNP particles in oocytes. Nucleic Acids Research 25, 965–973.CrossRefGoogle Scholar
Liang, P. and Pardee, A. B. ( 1992). Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257, 967–971.CrossRefGoogle Scholar
Liao, V. H. C. and Freedman, J. H. ( 2002). Differential display analysis of gene expression in invertebrates. Cellular and Molecular Life Sciences 59, 1256–1263.CrossRefGoogle Scholar
Lillibridge, C. D., Rudin, W. and Philipp, M. T. ( 1996). Dirofilaria immitis: ultrastructural localization, molecular characterization and analysis of the expression of p27, a small heat shock protein homolog of nematodes. Experimental Parasitology 83, 30–45.CrossRefGoogle Scholar
MacLennan, K., McLean, K. and Knox, D. P. ( 2005). Serpin expression in the parasitic stages of Trichostrongylus vitrinus, an ovine intestinal nematode. Parasitology 130, 349–357.CrossRefGoogle Scholar
Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. ( 2001). Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi. Current Biology 11, 171–176.CrossRefGoogle Scholar
Maekawa, H., Nakagawa, T., Uno, Y., Kitamura, K. and Shimoda, C. ( 1994). The ste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeast Schizosaccharomyces pombe. Molecular and General Genetics 244, 456–464.CrossRefGoogle Scholar
McCarter, J. P. ( 2004). Genomic filtering: an approach to discovering novel antiparasitics. Trends in Parasitology 20, 462–468.CrossRefGoogle Scholar
Mello, C. C. and Conte, D. Jr. ( 2004). Revealing the world of RNA interference. Nature, London 431, 338–342.CrossRefGoogle Scholar
Miller, M. A., Nguyen, V. Q., Lee, M., Kosinski, M., Schedl, T., Caprioli, R. M. and Greenstein, D. ( 2001). A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation. Science 291, 2144–2147.CrossRefGoogle Scholar
Miller, M. A., Ruest, P. J., Kosinski, M., Hanks, S. K. and Greenstein, D. ( 2003). An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans. Genes and Development 17, 187–200.CrossRefGoogle Scholar
Moriya, H. and Isono, K. ( 1999). Analysis of genetic interactions between DHH1, SSD1 and ELM1 indicates their involvement in cellular morphology determination in Saccharomyces cerevisiae. Yeast 15, 481–496.3.0.CO;2-M>CrossRefGoogle Scholar
Muhlrad, P. J. and Ward, S. ( 2002). Spermiogenesis inititation in Caenorhabditis elegans involves a casein kinase 1 encoded by the spe-6 gene. Genetics 161, 143–155.Google Scholar
Nakamura, N., Komiyama, M., Fujioka, M. and Mori, C. ( 2003). Sorting specificity of spermatogenic cell specific region of mouse hexokinase-s (mHk1-s). Molecular Reproduction and Development 64, 113–119.CrossRefGoogle Scholar
Navarro, R. E., Yong Shim, E., Kohara, Y., Singson, A. and Blackwell, T. K. ( 2001). cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans. Development 128, 3221–3232.Google Scholar
Newton, S. E., Boag, P. R. and Gasser, R. B. ( 2002). Opportunities and prospects for investigating developmentally regulated and sex-specific genes and their expression in intestinal nematodes of humans. In World Class Parasites: Vol. 2. The Geohelminths: Ascaris, Trichuris and Hookworms ( ed. Holland, C. V. and Kennedy, M. W.), pp. 235–268. Kluver, Boston, USA.CrossRef
Nisbet, A. J. and Gasser, R. B. ( 2004). Profiling of gender-specific gene expression for Trichostrongylus vitrinus (Nematoda: Strongylida) by microarray analysis of expressed sequence tag libraries constructed by suppressive-subtractive hybridisation. International Journal for Parasitology 34, 633–643.CrossRefGoogle Scholar
Nisbet, A. J., Cottee, P. and Gasser, R. B. ( 2004). Molecular biology of reproduction and development in parasitic nematodes: progress and opportunities. International Journal for Parasitology 34, 125–138.CrossRefGoogle Scholar
Raghavan, N., Ghosh, I., Eisinger, W. S., Pastrana, D. and Scott, A. L. ( 1999). Developmentally regulated expression of a unique small heat shock protein in Brugia malayi. Molecular and Biochemical Parasitology 104, 233–246.CrossRefGoogle Scholar
Reinke, V., Smith, H. E., Nance, J., Wang, J., Van Doren, C., Begley, R., Jones, S. J. M., Davis, E. B., Scherer, S., Ward, S. and Kim, S. K. ( 2000). A global profile of germline gene expression in C. elegans. Molecular Cell 6, 605–616.CrossRefGoogle Scholar
Roberts, T. M. and Stewart, M. ( 2000). Acting like actin: the dynamics of the nematode Major Sperm Protein (MSP) cytoskeleton indicate a push-pull mechanism for amoeboid cell motility. The Journal of Cell Biology 149, 7–12.CrossRefGoogle Scholar
van Roessel, P. and Brand, A. H. ( 2004). Spreading silence with Sid. Genome Biology 5, 208.Google Scholar
Schmitt-Wrede, H. P., Waldraff, A., Krucken, J., Harder, A. and Wunderlich, F. ( 1999). Characterization of a hexokinase encoding cDNA of the parasitic nematode Haemonchus contortus. Biochimica et Biophysica Acta 1444, 439–444.CrossRefGoogle Scholar
Slotved, H. C., Barnes, E. H., Bjørn, H., Christensen, C. M., Eriksen, L., Roepstorff, A. and Nansen, P. ( 1996). Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel. Veterinary Parasitology 63, 237–245.CrossRefGoogle Scholar
Smyth, G. K., Yang, Y. H. and Speed, T. ( 2003). Statistical issues in cDNA microarray data analysis. Methods in Molecular Biology 224, 111–136.CrossRefGoogle Scholar
Stein, J. and Liang, P. ( 2002). Differential display technology: a general guide. Cellular and Molecular Life Sciences 59, 1235–1240.CrossRefGoogle Scholar
Sugimoto, A. ( 2004). High-throughput RNAi in Caenorhabditis elegans: genome-wide screens and functional genomics. Differentiation 72, 81–91.CrossRefGoogle Scholar
Talvik, H., Christensen, C. M., Joachim, A., Roepstorff, A., Bjørn, H. and Nansen, P. ( 1997). Prepatent periods of different Oesophagostomum spp. isolates in experimentally infected pigs. Parasitology Research 83, 563–568.Google Scholar
Tarr, D. E. K. and Scott, A. L. ( 2005). MSP domain proteins. Trends in Parasitology 21, 224–231.CrossRefGoogle Scholar
THE C. ELEGANS SEQUENCING CONSORTIUM ( 1998). Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282, 2012–2018.
Thompson, F. J., Martin, S. A. and Devaney, E. ( 1996). Brugia pahangi: characterisation of a small heat shock protein cDNA clone. Experimental Parasitology 83, 259–266.CrossRefGoogle Scholar
de Valoir, T., Tucker, M. A., Belikoff, E. J., Camp, L. A., Bolduc, C. and Beckingham, K. ( 1991). A second maternally expressed Drosophila gene encodes a putative RNA helicase of the “DEAD box” family. Proceedings of the National Academy of Sciences, USA 88, 2113–2117.CrossRefGoogle Scholar