Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
1991-2-28
pubmed:databankReference
pubmed:abstractText
The coding sequence of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from a moderately pyrimethamine-resistant strain (HB3) of Plasmodium falciparum was assembled in a pUC expression vector. The coding sequence possesses unique Nco1 and Xba1 sites which flank 243 bp of the DHFR gene that include all point mutations thus far linked to pyrimethamine resistance. Wild-type (3D7) and highly pyrimethamine-resistant (7G8) TS-DHFRs were made from this vector by cassette mutagenesis using Nco1-Xba1 fragments from the corresponding cloned TS-DHFR genes. Catalytically active recombinant TS-DHFRs were expressed in Escherichia coli, albeit at low levels. Both TS and DHFR coeluted upon gel filtration and copurified upon affinity and anion exchange chromatography. Gel filtration and SDS-PAGE indicated that the enzyme was a dimer with identical 67-kDa subunits, characteristic of protozoan TS-DHFRs. Amino-terminal sequencing gave 10 amino acids which perfectly matched the sequence predicted from the nucleotide sequence. The recombinant TS-DHFR was purified to homogeneity by 10-formylfolate affinity chromatography followed by Mono Q FPLC. The inhibition properties of pyrimethamine toward the purified recombinant enzymes show that the point mutations are the molecular basis of pyrimethamine resistance in P. falciparum.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
4
pubmed:volume
29
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
10779-85
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:2176883-Amino Acid Sequence, pubmed-meshheading:2176883-Animals, pubmed-meshheading:2176883-Base Sequence, pubmed-meshheading:2176883-Chromatography, pubmed-meshheading:2176883-Cloning, Molecular, pubmed-meshheading:2176883-DNA Restriction Enzymes, pubmed-meshheading:2176883-Drug Resistance, pubmed-meshheading:2176883-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:2176883-Escherichia coli, pubmed-meshheading:2176883-Gene Expression, pubmed-meshheading:2176883-Genetic Vectors, pubmed-meshheading:2176883-Kinetics, pubmed-meshheading:2176883-Molecular Sequence Data, pubmed-meshheading:2176883-Multienzyme Complexes, pubmed-meshheading:2176883-Mutagenesis, Insertional, pubmed-meshheading:2176883-Plasmids, pubmed-meshheading:2176883-Plasmodium falciparum, pubmed-meshheading:2176883-Pyrimethamine, pubmed-meshheading:2176883-Recombinant Proteins, pubmed-meshheading:2176883-Tetrahydrofolate Dehydrogenase, pubmed-meshheading:2176883-Thymidylate Synthase
pubmed:year
1990
pubmed:articleTitle
Heterologous expression of active thymidylate synthase-dihydrofolate reductase from Plasmodium falciparum.
pubmed:affiliation
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't