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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
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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

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