Analysis of T4 bacteriophage deletion mutants that lack td and frd genes.

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.     

Coregulation of dihydrofolate reductase and thymidylate synthase B in bacillus subtilis

A 2.0-kb fragment of Bacillus subtilis 168 chromosomal DNA has been shown to contain both the dihydrofolate reductase (dfrA) and thymidylate synthase B (thyB) genes. In addition to the close proximity of dfrA and thyB, the expression of these genes seems to be regulated coordinately. Mutations that map near or within the dfrA gene resulted in coordinate increases in both dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase and thymidylate synthase B activities. Also, when trimethoprim, a specific inhibitor of dihydrofolate reductase, was added to growing cells, both dihydrofolate reductase and thymidylate synthase B activities increased coordinately.     

Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition.

An important unresolved issue in antifolate pharmacology is the basis for the observation that the major portion of cellular tetrahydrofolate cofactors is preserved after dihydrofolate reductase activity is abolished by antifolates despite the fact that tetrahydrofolate cofactor-dependent purine and pyrimidine biosynthesis ceases. This has been attributed to feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates that accumulate in the presence of antifolates. This report combines network thermodynamic modeling and experimental observations to evaluate the effects of direct inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site with a potent lipophilic quinazoline antifolate PD130883 on folate oxidation in cells. Computer simulations predict and the data indicate that marked PD130883 suppression of thymidylate synthase only slows the rate but not the extent of tetrahydrofolate cofactor interconversion to dihydrofolate upon complete suppression of dihydrofolate reductase with trimetrexate. These observations are consistent with earlier studies from this laboratory with fluorodeoxyuridine inhibition at the deoxyuridylate binding site. Hence, the much weaker inhibition by dihydrofolate polyglutamates at the level of thymidylate synthase cannot account for the apparent preservation of tetrahydrofolate cofactor pools in cells and has virtually no pharmacologic significance under conditions in which antifolates completely suppress dihydrofolate reductase. The extent of interconversion of tetrahydrofolate cofactors to dihydrofolate is strongly influenced by residual dihydrofolate reductase catalytic activity. Exposure of cells to 0.1 microM trimetrexate results in only approximately 60% of maximum dihydrofolate levels achieved when dihydrofolate reductase activity is abolished. Network thermodynamic simulations predict, and experiments verify, that inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate site by PD130883, when dihydrofolate reductase is only partially suppressed (approximately 85%) with 0.1 microM trimetrexate, substantially decreases (31-47%) the net level of interconversion of tetrahydrofolate cofactors to dihydrofolate. Further computer simulations predict that under conditions in which residual dihydrofolate reductase activity persists within the cells (more than about 5%), feedback inhibitory effects of dihydrofolate polyglutamates as well as other weak inhibitors of thymidylate synthase can significantly limit the extent of net interconversion of tetrahydrofolate cofactors to dihydrofolate and produce an apparent "compartmentation phenomenon" in which tetrahydrofolate cofactor pools are preserved within the cell in the presence of antifolates. Residual dihydrofolate reductase activity cannot, however, account for the partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure to high trimetrexate or methotrexate levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis

The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2α (SHMT1 and SHMT2α), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2α is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.     

Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates. Experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamylates in antifolate action in cells

Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase

We have determined the nucleotide sequence of a 1075-base-pair HindIII fragment of the T4 phage genome. This fragment contains the structural gene (frd) for dihydrofolate reductase and part of the gene (td) encoding thymidylate synthase. The fragment contains a 579-base-pair open reading frame, encoding a 193-residue polypeptide with a calculated mass of 21,603 Da, in agreement with our reported subunit molecular mass of 23,000. The deduced amino acid sequence shows partial homology with other dihydrofolate reductases, with most of the identities lying in regions known to be involved in substrate binding and catalysis. The 3' end of the coding strand overlaps the coding region for thymidylate synthase; the sequence - ATGA -includes an opal terminator for the frd gene and an initiating triplet for the td gene. The deduced amino acid sequence from this initiating ATG is identical, for the first 20 residues, with the NH2-terminal 20 residues reported for the td protein (M. Belfort , A. Moelleken , G. F. Maley , and F. Maley (1983) J. Biol. Chem. 258, 2045-2051). The sequenced HindIII fragment was transferred into a high expression plasmid vector for large scale production of homogeneous T4 dihydrofolate reductase. The experimentally determined sequence of 20 residues at the NH2-terminus of this protein is identical with that deduced from the nucleotide sequence for T4 dihydrofolate reductase.     

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.     

Kinetic characterization of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis: a paradigm shift for ts activity and channeling behavior

This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s(-) (1), C. hominis TS activity is 10-40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.     

Electrostatic Channeling in P.?falciparum DHFR-TS: Brownian Dynamics and Smoluchowski Modeling

We perform Brownian dynamics simulations and Smoluchowski continuum modeling of the bifunctional Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (P. falciparum DHFR-TS) with the objective of understanding the electrostatic channeling of dihydrofolate generated at the TS active site to the DHFR active site. The results of Brownian dynamics simulations and Smoluchowski continuum modeling suggest that compared to Leishmania major DHFR-TS, P. falciparum DHFR-TS has a lower but significant electrostatic-mediated channeling efficiency (∼15–25%) at physiological pH (7.0) and ionic strength (150 mM). We also find that removing the electric charges from key basic residues located between the DHFR and TS active sites significantly reduces the channeling efficiency of P. falciparum DHFR-TS. Although several protozoan DHFR-TS enzymes are known to have similar tertiary and quaternary structure, subtle differences in structure, active-site geometry, and charge distribution appear to influence both electrostatic-mediated and proximity-based substrate channeling.     

Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin:NADP+ reductase: site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials.

Transient absorbance measurements following laser flash photolysis have been used to measure the rate constants for electron transfer (et) from reduced Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reversal mutants of Anabaena ferredoxin:NADP+ reductase (FNR). These mutations have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed edge of the FAD cofactor in the protein-protein interaction during et with Fd. The mutant proteins fall into two groups: overall, the K75E, R16E, and K72E mutants are most severely impaired in et, and the K138E, R264E, K290E, and K294E mutants are impaired to a lesser extent, although the degree of impairment varies with ionic strength. Binding constants for complex formation between the oxidized proteins and for the transient et complexes show that the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the protein in which positive charge predominates, and charge reversals have large effects on the calculated local surface electrostatic potential. In contrast, K138, R264, K290, and K294 are located within or close to regions of intense negative potential, and therefore the introduction of additional negative charges have considerably smaller effects on the calculated surface potential. We attribute the relative changes in et kinetics and complex binding constants for these mutants to these characteristics of the surface charge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.     

Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase

Here we report on a Chlamydia trachomatis gene that complements the growth defect of a thymidylate synthase-deficient strain of Escherichia coli. The complementing gene encodes a 60.9-kDa protein that shows low level primary sequence homology to a new class of thymidylate-synthesizing enzymes, termed flavin-dependent thymidylate synthases (FDTS). Purified recombinant chlamydial FDTS (CTThyX) contains bound flavin. Results with site-directed mutants indicate that highly conserved arginine residues are required for flavin binding. Kinetic characterization indicates that CTThyX is active as a tetramer with NADPH, methylenetetrahydrofolate, and dUMP required as substrates, serving as source of reducing equivalents, methyl donor, and methyl acceptor, respectively. dTMP and H(4)folate are products of the reaction. Production of H(4)folate rather than H(2)folate, as in the classical thymidylate synthase reaction, eliminates the need for dihydrofolate reductase, explaining the trimethoprim-resistant phenotype displayed by thyA(-) E. coli-expressing CTThyX. In contrast to the extensively characterized thyA-encoded thymidylate synthases, which form a ternary complex with substrates dUMP and CH(2)H(4)folate and follow an ordered sequential mechanism, CTThyX follows a ping-pong kinetic mechanism involving a methyl enzyme intermediate. Mass spectrometry was used to localize the methyl group to a highly conserved arginine, and site-directed mutagenesis showed this arginine to be critical for thymidylate synthesizing activity. These differentiating characteristics clearly distinguish FDTS from ThyA, making this class of enzymes attractive targets for rational drug design.     

Analysis of thymidylate synthase gene amplification and of mRNA levels in the cell cycle

We report that the gene for thymidylate synthase (TS) is amplified in the mouse cell line L1210:C15 that was selectively grown in increasing concentrations of the competitive inhibitor of thymidylate synthase, CB3717. The gene is amplified 50-fold compared to the parental cell line. Amplification has not been accompanied by any major rearrangements, and the increase in gene copy number is reflected in elevation of thymidylate synthase mRNA levels. The amplification is relatively stable as there was only a 2- to 3-fold decrease in the number of amplified TS genes when cells were grown in the absence of selection for 375 generations. We also observe a 30- to 40-fold increase in number of copies of the dihydrofolate reductase gene with 7-fold elevation of the RNA product, and we suggest that this may be due to cross-inhibition of dihydrofolate reductase by CB3717. Thymidylate synthase mRNA levels in L1210 and L1210:C15 show no variation within the different phases of the cell cycle but are significantly reduced during quiescence.     

Structural analysis of interactions for complex formation between Ferredoxin-NADP+ reductase and its protein partners

The three-dimensional structures of K72E, K75R, K75S, K75Q, and K75E Anabaena Ferredoxin-NADP+ reductase (FNR) mutants have been solved, and particular structural details of these mutants have been used to assess the role played by residues 72 and 75 in optimal complex formation and electron transfer (ET) between FNR and its protein redox partners Ferredoxin (Fd) and Flavodoxin (Fld). Additionally, because there is no structural information available on the interaction between FNR and Fld, a model for the FNR:Fld complex has also been produced based on the previously reported crystal structures and on that of the rat Cytochrome P450 reductase (CPR), onto which FNR and Fld have been structurally aligned, and those reported for the Anabaena and maize FNR:Fd complexes. The model suggests putative electrostatic and hydrophobic interactions between residues on the FNR and Fld surfaces at the complex interface and provides an adequate orientation and distance between the FAD and FMN redox centers for efficient ET without the presence of any other molecule as electron carrier. Thus, the models now available for the FNR:Fd and FNR:Fld interactions and the structures presented here for the mutants at K72 and K75 in Anabaena FNR have been evaluated in light of previous biochemical data. These structures confirm the key participation of residue K75 and K72 in complex formation with both Fd and Fld. The drastic effect in FNR activity produced by replacement of K75 by Glu in the K75E FNR variant is explained not only by the observed changes in the charge distribution on the surface of the K75E FNR mutant, but also by the formation of a salt bridge interaction between E75 and K72 that simultaneously "neutralizes" two essential positive charged side chains for Fld/Fd recognition.     

Subunit complementation of thymidylate synthase.

Each of the two active sites of thymidylate synthase contains amino acid residues contributed by the other subunit. For example, Arg-178 of one monomer binds the phosphate group of the substrate dUMP in the active site of the other monomer [Hardy et al. (1987) Science 235, 448-455]. Inactive mutants of such residues should combine with subunits of other inactive mutants to form heterodimeric hybrids with one functional active site. In vivo and in vitro approaches were used to test this hypothesis. In vivo complementation was accomplished by cotransforming plasmid mixtures encoding pools of inactive Arg-178 mutants and pools of inactive Cys-198 mutants into a host strain deficient in thymidylate synthase. Individual inactive mutants of Arg-178 were also cotransformed with the C198A mutant. Subunit complementation was detected by selection or screening for transformants which grew in the absence of thymidine, and hence produced active enzyme. Many mutants at each position representing a wide variety of size and charge supported subunit complementation. In vitro complementation was accomplished by reversible dissociation and unfolding of mixtures of purified individual inactive Arg-178 and Cys-198 mutant proteins. With the R178F + C198A heterodimer, the Km values for dUMP and CH2H4folate were similar to those of the wild-type enzyme. By titrating C198A with R178F under unfolding-refolding conditions, we were able to calculate the kcat value for the active heterodimer. The catalytic efficiency of the single wild-type active site of the C198A + R178F heterodimer approaches that of the wild-type enzyme.     

Plasmodium falciparum: induction, selection, and characterization of pyrimethamine-resistant mutants

We have selected eight pyrimethamine resistant mutants of a cloned, drug sensitive, Plasmodium falciparum malaria parasite, strain FCR3. The mutants exhibited resistance to between 10 and 200 times higher concentrations of drug than the wild type parasite. The mutants were selected from cultured parasites that were either unmutagenized or N-methyl-N'-nitro-N-nitrosoguanidine mutagenized. One mutant was shown to contain a mutant dihydrofolate reductase enzyme in parasite extracts that exhibited (1) a five- to ninefold reduction in its binding of methotrexate, (2) an undetectable enzyme activity based on the spectrophotometric conversion of dihydrofolate to tetrahydrofolate, and (3) essentially normal amounts of the parasite's bifunctional thymidylate synthetase-dihydrofolate reductase enzyme. Other mutants exhibited both normal dihydrofolate reductase specific activity and normal enzyme sensitivity to the inhibitory activity of the drug.     

Protein-DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single-stranded DNA-binding protein

Our laboratory has reported data suggesting a role for T4 phage gene 32 single-stranded DNA-binding protein in organizing a complex of deoxyribonucleotide-synthesizing enzymes at the replication fork. In this article we examined the effects of gene 32 ablation on the association of these enzymes with DNA-protein complexes. These experiments showed several deoxyribonucleotide-synthesizing enzymes to be present in DNA-protein complexes, with some of these associations being dependent on gene 32 protein. To further understand the role of gp32, we created amber mutations at codons 24 and 204 of gene 32, which encodes a 301-residue protein. We used the newly created mutants along with several experimental approaches--DNA-cellulose chromatography, immunoprecipitation, optical biosensor analysis and glutathione-S-transferase pulldowns--to identify relevant protein-protein and protein-DNA interactions. These experiments identified several proteins whose interactions with DNA depend on the presence of intact gp32, notably thymidylate synthase, dihydrofolate (DHF) reductase, ribonucleotide reductase (RNR) and Escherichia coli nucleoside diphosphate (NDP) kinase, and they also demonstrated direct associations between gp32 and RNR and NDP kinase, but not dCMP hydroxymethylase, deoxyribonucleoside monophosphate kinase, or DHF reductase. Taken together, the results support the hypothesis that the gene 32 protein helps to recruit enzymes of deoxyribonucleoside triphosphates synthesis to DNA replication sites.     

The extremely halophilic archaeon Haloferax volcanii has two very different dihydrofolate reductases

The gene encoding dihydrofolate reductase, hdrA, from the extremely halophilic archaeon Haloferax volcanii was previously isolated from a spontaneous trimethoprim-resistant mutant in a DNA sequence that had undergone amplification. Here, we show that deletion of hdrA did not affect growth in minimal medium and that the strain carrying the deletion remained sensitive to trimethoprim. A spontaneous trimethoprim-resistant colony was isolated in the hdrA deletion strain and found to possess a new DNA amplification. Sequencing of the amplification revealed a second, substantially different, dihydrofolate reductase gene, hdrB, which was found to be located immediately downstream of the thymidylate synthase gene, hts. The physiological role of hDHFR-1 and hDHFR-2 was determined by generating Haloferax volcanii strains in which each gene, hdrA or hdrB, or both genes were deleted. It was found that hdrB alone can support growth of Haloferax volcanii in minimal medium, whereas hdrA alone can support growth of Haloferax volcanii in minimal medium only when the medium is supplemented with thymidine. It was also shown that, in contrast to Escherichia coli, the DeltahdrA, DeltahdrB double deletion mutant is viable in the presence of a functional thymidylate synthase gene. The hdrB gene was overexpressed in Escherichia coli and the enzyme purified to homogeneity. The biochemical properties of the new enzyme (hDHFR-2) are markedly different from those of hDHFR-1. The use of the dihydrofolate reductase and thymidylate synthase genes as stable selectable markers is described.