CLONING, NUCLEOTIDE-SEQUENCE AND EXPRESSION OF THE BIFUNCTIONAL DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE FROM GLYCINE-MAX

cDNAs encoding the bifunctional dihydrofolate reductase-thymidylate sy nthase from Glycine maw were isolated and sequenced. The 1794 base ful l length cDNA contains a single open reading frame of 1593 bases. The predicted size of the encoded protein is 530 amino acids with a molecu lar 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 s ynthase (TS) domain, which is over 60% identical to eukaryotic TS enzy mes. The whole protein sequence is greater than 75% identical to DHFR- TS sequences from two other plants, Daucus carota and Arabidopsis thal iana. The sequence of two tryptic peptides obtained from DHFR preparat ions matched the predicted amino acid sequence, one peptide lying in t he DHFR domain and the other in the TS domain. These results indicate that DHFR and TS exist in a bifunctional polypeptide in Glycine max. T he coding region of the cDNA was inserted downstream of the T-7 promot er and translation initiation signals in the vector pET-3a. This const ruct (pDR-TS) was transformed into Escherichia coli BL21 (DE) [plysS] which produces T-7 RNA polymerase upon induction by isopropyl-beta-D-t hiogalactopyranoside (IPTG). The expression of the bifunctional enzyme was confirmed by detection of both DHFR and TS activities. The purifi ed enzyme has a sllbunit molecular mass of 60 kDa. This is the first r eport of expression of a plant DHFR-TS cDNA.