Cloning, nucleotide sequence and expression of the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max.
cDNAs encoding the bifunctional dihydrofolate reductase-thymidylate synthase from Glycine max were isolated and sequenced. The 1794 base full length cDNA contains a single open reading frame of 1593 bases. The predicted size of the encoded protein is 530 amino acids with a molecular weight of 59,707. The protein has two domains: a 226 residue DHFR domain in the N-terminus, which is over 30% identical to human DHFR or the DHFR domain of protozoal DHFR-TS, and a 304 residue thymidylate synthase (TS) domain, which is over 60% identical to eukaryotic TS enzymes. The whole protein sequence is greater than 75% identical to DHFR-TS sequences from two other plants, Daucus carota and Arabidopsis thaliana. The sequence of two tryptic peptides obtained from DHFR preparations matched the predicted amino acid sequence, one peptide lying in the DHFR domain and the other in the TS domain. These results indicate that DHFR and TS exist in a bifunctional polypeptide in Glycine max. The coding region of the cDNA was inserted downstream of the T7 promoter and translation initiation signals in the vector pET-3a. This construct (pDR-TS) was transformed into Escherichia coli BL21 (DE) [plysS] which produces T7 RNA polymerase upon induction by isopropyl-beta-D-thiogalactopyranoside (IPTG). The expression of the bifunctional enzyme was confirmed by detection of both DHFR and TS activities. The purified enzyme has a subunit molecular mass of 60 kDa. This is the first report of expression of a plant DHFR-TS cDNA.
Wang
M
M
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Ratnam
S
S
Freisheim
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013025:Glycine max / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics
D018076:DNA, Complementary; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase