Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi.
We have cloned, sequenced and expressed the Trypanosoma cruzi gene encoding the bifunctional protein dihydrofolate reductase-thymidylate synthase (DHFR-TS). The strategy followed for the isolation of positive clones from a genomic library was based on the construction of a probe by the amplification of highly conserved sequences of the TS domain by the polymerase chain reaction. Translation of the open reading frame of 1563 bp yields a polypeptide of 521 amino acids with a molecular mass of 58829 Da. For heterologous expression of T. cruzi DHFR-TS in Escherichia coli, the entire coding sequence was amplified by polymerase chain reaction and cloned into the plasmid vector pKK223.3. The presence of catalytically active DHFR-TS was demonstrated by complementation of the Thy- E. coli strain chi 2913 and the DHFR- Thy- E. coli strain PA414. The gene is expressed as an active protein which constitutes approximately 2% of the total cell soluble protein. Recombinant bifunctional enzyme and the DHFR domain have been purified by methotrexate-Sepharose chromatography to yield 1-2 mg of active DHFR-TS per litre of culture. Southern and electrophoretic analyses using the coding sequence as probe indicated that the T. cruzi enzyme is encoded by a single copy gene which maps to two bands of approximately 990 kb and 1047 kb. It appears that T. cruzi is diploid for the DHFR-TS gene which is located on two different-sized homologous chromosomes.
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Instituto de Parasitología y Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Arrebola
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R
Olmo
A
A
Santi
D V
DV
Gonzalez-Pacanowska
D
D
Ruiz-Perez
L M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
D016054:DNA, Protozoan; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase