Hostname: page-component-78c5997874-j824f Total loading time: 0 Render date: 2024-11-19T07:28:48.677Z Has data issue: false hasContentIssue false
  • English
  • Français

Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression

Published online by Cambridge University Press:  01 March 2004

M. DĄBROWSKA ,
E. JAGIELSKA ,
J. CIEŚLA ,
A. PŁUCIENNICZAK ,
J. KWIATOWSKI ,
M. WRANICZ ,
P. BOIREAU  and
W. RODE
M. DĄBROWSKA
Affiliation:
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland
E. JAGIELSKA
Affiliation:
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland
J. CIEŚLA
Affiliation:
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland
A. PŁUCIENNICZAK
Affiliation:
Institute of Biotechnology and Antibiotics, 5 Starościńska Street, 02-516 Warszawa, Poland
J. KWIATOWSKI
Affiliation:
Institute of Botany, Warsaw University, 00-478 Warszawa, Poland
M. WRANICZ
Affiliation:
Institute of Parasitology, Polish Academy of Sciences, 51/55 Twarda Street, 00-818 Warszawa, Poland
P. BOIREAU
Affiliation:
Molecular Biology and Immunology of Parasites and Fungi, UMR INRA-AFSSA-ENVA-UPVM, 22 rue Pierre Curie, 94703, Maison-Alfort, France
W. RODE
Affiliation:
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland
Get access Rights & Permissions [Opens in a new window]

Abstract

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequencedNucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the Accession number AF162691., allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35 582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Kmapp values describing interactions with dUMP (28·8 μMvs. 3·9 μM) and (6RS, αS)-N5,10-methylenetetrahydrofolate (383 μMvs. 54·7 μM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Keywords

thymidylate synthasehistone H.3-likeTrichinella spiralismRNAgene expressioncell cycle
Type
Research Article
Information
Parasitology , Volume 128 , Issue 2 , February 2004 , pp. 209 - 221
Copyright
2004 Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

ALTSHUL, S. F., MADDEN, T. L., SCHAFFER, A. A., ZHANG, J., MILLER, W. & LIPMAN, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402.CrossRefGoogle Scholar
AYUSAWA, D., SHIMIZU, K., KOYAMA, H., KANEDA, S., TAKEISHI, K. & SENO, T. (1986). Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. Journal of Molecular Biology 190, 559–567.CrossRefGoogle Scholar
BELFORT, M., MALEY, G. F. & MALEY, F. (1983). Characterization of the Escherichia coli thyA gene and its amplified thymidylate synthetase product. Proceedings of the National Academy of Sciences, USA 80, 1858–1861.CrossRefGoogle Scholar
CARPENTER, N. J. (1973). Thymidylate synthetase in mutants of Drosophila melanogaster. Genetics 75, 113–122.Google Scholar
CARRERAS, C. W. & SANTI, D. V. (1995). The catalytic mechanism and structure of thymidylate synthase. Annual Review of Biochemistry 64, 721–762.CrossRefGoogle Scholar
CHENCHIK, A., DIATCHENKO, L., MOQADAM, F., TARABYKIN, V., LUKYANOV, S. & SIEBERT, P. D. (1996). Full-length cDNA cloning and determination of mRNA 5′ and 3′ ends by amplification of adaptor ligated cDNA. BioTechniques 21, 526–534.Google Scholar
CHUNG, C. T. & MILLER, R. H. (1988). A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Research 16, 3580.CrossRefGoogle Scholar
CIEŚLA, J., WEINER, K. X. B., WEINER, R. S., RESTON, J. T., MALEY, G. F. & MALEY, F. (1995). Isolation and expression of rat thymidylate synthase cDNA: phylogenetic comparison with human and mouse thymidylate synthases. Biochimica et Biophysica Acta 1261, 233–242.CrossRefGoogle Scholar
DALLEY, B. K. & GOLOMB, M. (1992). Gene expression in the Caenorhabditis elegans dauer larva: developmental regulation of Hsp90 and other genes. Developmental Biology 151, 80–90.CrossRefGoogle Scholar
DĄBROWSKA, M., ZIELIŃSKI, Z., WRANICZ, M., MICHALSKI, R., PAWEŁCZAK, K. & RODE, W. (1996). Trichinella spiralis thymidylate synthase: developmental pattern, isolation, molecular properties and inhibition by substrate and cofactor analogues. Biochemical and Biophysical Research Communications 228, 440–445.CrossRefGoogle Scholar
DAVISON, A. J. & SCOTT, J. E. (1986). The complete DNA sequence of varicella-zoster virus. Journal of General Virology 67, 1759–1816.CrossRefGoogle Scholar
DYBCZYŃSKI, I. & PŁUCIENNICZAK, A. (1988). A protocol for DNA fragment extraction from polyacrylamide gels. BioTechniques 6, 924–926.Google Scholar
EDMAN, U., EDMAN, J. C., LUNDGREN, B. & SANTI, D. V. (1989). Isolation and expression of the Pneumocystis carinii thymidylate synthase gene. Proceedings of the National Academy of Sciences, USA 86, 6503–6507.CrossRefGoogle Scholar
FROHMAN, M. A., DUSH, M. K. & MARTIN, G. R. (1988). Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences, USA 85, 8998–9002.CrossRefGoogle Scholar
GAMARRO, F., YU, P. L., ZHAO, J., EDMAN, U., GREENE, P. J. & SANTI, D. (1995). Trypanosoma brucei dihydrofolate reductase-thymidylate synthase: gene isolation and expression and characterization of the enzyme. Molecular and Biochemical Parasitology 72, 11–22.CrossRefGoogle Scholar
GOULD, S. E. (1970). Clinical pathology: diagnostic laboratory procedures. In Trichinosis in Man and Animals (ed. Gould, S. E.), pp. 190–221. Charles C. Thomas, Springfield, USA.
GRIBAUDO, G., RIERA, L., RUDGE, T. L., CAPOSIO, P., JOHNSON, L. F. & LANDOLFO, S. (2002). Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts. Journal of General Virology 83, 2983–2993.CrossRefGoogle Scholar
HONESS, R. W., BODEMER, W., CAMERON, K. R., NILLER, H. H., FLECKENSTEIN, B. & RANDALL, R. E. (1986). The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proceedings of the National Academy of Sciences, USA 83, 3604–3608.CrossRefGoogle Scholar
HORIE, N., NOZAWA, R. & TAKEISHI, K. (1992). Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase. Biochemical and Biophysical Research Communications 185, 127–133.CrossRefGoogle Scholar
HUELSENBECK, J. P. & RONQUIST, F. (2001). MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755.CrossRefGoogle Scholar
HUELSENBECK, J. P., RONQUIST, F., NIELSEN, R. & BOLLBACK, J. P. (2001). Bayesian inference of phylogeny and its impact on evolutionary biology. Science 294, 2310–2314.CrossRefGoogle Scholar
HUGHES, D. E., SHONEKAN, O. A. & SIMPSON, L. (1989). Structure, genomic organization and transcription of the bifunctional dihydrofolate reductase – thymidylate synthase gene from Crithidia fasciculata. Molecular and Biochemical Parasitology 34, 155–166.CrossRefGoogle Scholar
JASMER, D. P. (1995). Trichinella spiralis: subversion of differentiated mammalian skeletal muscle cells. Parasitology Today 11, 185–188.CrossRefGoogle Scholar
JENH, C.-H., GEYER, P. K. & JOHNSON, L. F. (1985). Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse line. Molecular and Cellular Biology 5, 2527–2532.CrossRefGoogle Scholar
KURATLI, S., LINDH, J. G., GOTTSTEIN, B., SMITH, D. F. & CONNOLLY, B. (1999). Trichinella spp: differential expression of two genes in the muscle larva of encapsulating and nonencapsulating species. Experimental Parasitology 93, 153–159.CrossRefGoogle Scholar
LAZAR, G., ZHANG, H. & GOODMAN, H. M. (1993). The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana. Plant Journal 3, 657–668.CrossRefGoogle Scholar
LEE, Y., SHEN, G. & JOHNSON, L. F. (1999). Complex transcriptional initiation patteren of the thymidylate synthase promoter in mouse tissues. Archives of Biochemistry and Biophysics 372, 389–392.CrossRefGoogle Scholar
LIVI, L. L., EDMAN, U., SCHNEIDER, G. P., GREENE, P. J. & SANTI, D. V. (1994). Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans. Gene 150, 221–226.Google Scholar
LUKYANOV, K., DIATCHENKO, L., CHENCHLIK, A., NANISETTI, A., SIEBERT, P., USMAN, N., MATZ, M. & LUKYANOV, S. (1997). Construction of cDNA libraries from small amounts of total RNA using the suppression PCR effect. Biochemical and Biophysical Research Communications 230, 285–288.CrossRefGoogle Scholar
LUO, M., PIFFANELLI, P., RASTELLI, L. & CELLA, R. (1993). Molecular cloning and analysis of a cDNA for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota. Plant Molecular Biology 22, 427–435.CrossRefGoogle Scholar
MACLEA, K. S., KRIESER, R. J. & EASTMAN, A. (2003). A family history of deoxyribonuclease II: surprises from Trichinella spiralis and Burkholderia pseudomallei. Gene 305, 1–12.CrossRefGoogle Scholar
MAK, C. H., SU, K. W. & KO, R. C. (2001). Identification of some heat-induced genes of Trichinella spiralis. Parasitology 123, 293–300.CrossRefGoogle Scholar
MIKIEWICZ, D., WROBEL, B., WĘGRZYN, G. & PŁUCIENNICZAK, A. (1997). Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene. Plasmid 38, 210–219.CrossRefGoogle Scholar
NELSON, K., ALONSO, G., LANGER, P. J. & BEVERLEY, S. M. (1990). Sequence of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of Leishmania amazonensis. Nucleic Acids Research 18, 2819.Google Scholar
PERRYMAN, S. M., ROSSANA, C., DENG, T., VANIN, E. F. & JOHNSON, L. F. (1986). Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme. Molecular Biology of Evolution 3, 313–321.Google Scholar
PESTALOZZI, B. C., McGINN, C. J., KINSELLA, T. J., DRAKE, J. C., GLENNON, M. C., ALLEGRA, C. J. & JOHNSTON, P. G. (1995). Increased thymidylate synthase protein levels are principally associated with proliferation but not cell cycle phase in asynchronous human cancer cells. British Journal of Cancer 7, 1151–1157.CrossRefGoogle Scholar
PINTER, K., DAVISSON, V. J. & SANTI, D. V. (1988). Cloning, sequencing and expression of the Lactobacillus casei thymidylate synthase gene. DNA 7, 235–241.CrossRefGoogle Scholar
PŁUCIENNICZAK, G., JAGIEŁŁO, A., PŁUCIENNICZAK, A., HOLODY, D. & STRZEŻEK, J. (1999). Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma. Molecular Reproduction and Development 52, 303–309.3.0.CO;2-7>CrossRefGoogle Scholar
RICHTER, J., PUCHTLER, I. & FLECKENSTEIN, B. (1988). Thymidylate synthase gene of herpesvirus ateles. Journal of Virology 62, 3530–3535.Google Scholar
RODE, W., DĄBROWSKA, M., ZIELIŃSKI, Z., GOŁOS, B., WRANICZ, M., FELCZAK, K. & KULIKOWSKI, T. (2000). Trichinella spiralis and Trichinella pseudospiralis: developmental patterns of enzymes involved in thymidylate biosynthesis and thymidylate synthase as a potential target in chemotherapy. Parasitology 120, 593–600.CrossRefGoogle Scholar
RODE, W. & SZYMANOWSKA, H. (1976). Developmental pattern of thymidylate synthetase activity in the silkworm Bombyx mori. Insect Biochemistry 6, 333–337.CrossRefGoogle Scholar
ROOS, D. S. (1993). Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. Journal of Biological Chemistry 268, 6269–6280.Google Scholar
RUSSO, J. J., BOHENZKY, R. A., CHIEN, M. C., CHEN, J., YAN, M., MADDALENA, D., PARRY, J. P., PERUZZI, D., EDELMAN, I. S., CHANG, Y. & MOORE, P. S. (1996). Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proceedings of the National Academy of Sciences, USA 93, 14862–14867.CrossRefGoogle Scholar
RUSTUM, Y. M., HARSTRICK, A., CAO, S., VANHOEFER, U., YIN, M.-B., WILKE, H. & SEEBER, S. (1997). Thymidylate synthase inhibitors in cancer therapy: direct and indirect inhibitors. Journal of Clinical Oncology 15, 398–400.CrossRefGoogle Scholar
SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
SCHLICHTHERLE, I. M., ROOS, D. S. & VAN HOUTEN, J. L. (1996). Cloning and molecular analysis of the bifunctional dihydrofolate reductase-thymidylate synthase gene in the ciliated protozoan Paramecium tetraurelia. Molecular and General Genetics 250, 665–673.Google Scholar
SAGATA, N. (1996). Meiotic metaphase arrest in animal oocytes: its mechanisms and biological significance. Trends in Cell Biology 6, 22–28.CrossRefGoogle Scholar
SINGER, S. C., RICHARDS, C. A., FERONE, R., BENEDICT, D. & RAY, P. (1989). Cloning, purification, and properties of Candida albicans thymidylate synthase. Journal of Bacteriology 171, 1372–1378.CrossRefGoogle Scholar
SNEWIN, V. A., ENGLAND, S. M., SIMS, P. F. & HYDE, J. E. (1989). Characterisation of the dihydrofolate reductase-thymidylate synthetase gene from human malaria parasites highly resistant to pyrimethamine. Gene 76, 41–52.CrossRefGoogle Scholar
STEPHENS, R. S., KALMAN, S., LAMMEL, C., FAN, J., MARATHE, R., ARAVIND, L., MITCHELL, W., OLINGER, L., TATUSOV, R. L., ZHAO, Q., KOONIN, E. V. & DAVIS, R. W. (1998). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754–759.CrossRefGoogle Scholar
TAKEISHI, K., KANEDA, S., AYUSAWA, D., SHIMIZU, K., GOTOH, O. & SENO, T. (1985). Nucleotide sequence of a functional cDNA for human thymidylate synthase. Nucleic Acids Research 13, 2035–2043.CrossRefGoogle Scholar
TAYLOR, G. R., LAGOSKY, P. A., STORMS, R. K. & HAYNES, R. H. (1987). Molecular characterization of the cell cycle-regulated thymidylate synthase gene of Saccharomyces cerevisiae. Journal of Biological Chemistry 262, 5298–5307.Google Scholar
TELFORD, E. A., WATSON, M. S., AIRD, H. C., PERRY, J. & DAVISON, A. J. (1995). The DNA sequence of equine herpesvirus 2. Journal of Molecular Biology 249, 520–528.CrossRefGoogle Scholar
THOMPSON, J. D., GIBSON, T. J., PLEWNIAK, F., JEANMOUGIN, F. & HIGGINS, D. G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882.CrossRefGoogle Scholar
THOMPSON, J. D., HIGGINS, D. G. & GIBSON, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.CrossRefGoogle Scholar
VASQUEZ, J. R., GOOZE, L., KIM, K., GUT, J., PETERSEN, C. & NELSON, R. G. (1996). Potential antifolate resistance determinants and genotypic variation in the bifunctional dihydrofolate reductase – thymidylate synthase gene from human and bovine isolates of Cryptosporidium parvum. Molecular and Biochemical Parasitology 79, 153–165.CrossRefGoogle Scholar
WAHBA, A. J. & FRIEDKIN, M. (1961). Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate. Journal of Biological Chemistry 236, PC11–12.Google Scholar
WANG, M., RATNAM, S. & FREISHEIM, J. H. (1995). Cloning, nucleotide sequence and expression of the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max. Biochimica et Biophysica Acta 1261, 325–336.CrossRefGoogle Scholar
WRANICZ, M. J., GUSTOWSKA, L., GABRYEL, P., KUCHARSKA, E. & CABAJ, W. (1998). Trichinella spiralis: induction of the basophilic transformation of muscle cells by synchronous newborn larvae. Parasitology Research 84, 403–407.CrossRefGoogle Scholar
YASUMASU, I., SAITOH, M., FUJIMOTO, N. & KUSUNOKI, S. (1979). Changes in activities of thymidylate synthase and dihydrofolate reductase in sea urchin eggs after fertilization. Development Growth and Differentiation 21, 237–243.CrossRefGoogle Scholar