Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.
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 sequenced, 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 Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). 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.
Dabrowska
M
M
Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, 02-093 Warszawa, Poland.
Jagielska
E
E
Cieśla
J
J
Płucienniczak
A
A
Kwiatowski
J
J
Wranicz
M
M
Boireau
P
P
Rode
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000276:immunology / Q000469:parasitology; D010802:Phylogeny; D018510:RNA, Helminth / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013940:Thymidylate Synthase / Q000235:genetics* / Q000378:metabolism; D017160:Trichinella spiralis / Q000201:enzymology* / Q000235:genetics
D018510:RNA, Helminth; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013940:Thymidylate Synthase