Disease relevance of Dhfr

• Expression and purification of the wild-type and mutant DHFR from E. coli cells showed that the S3Mtx enzyme had a reduced binding affinity for the antifolates, MTX and trimethoprim, with 15-fold higher K[d] and K[i] values than those from the wild-type enzyme [1].
• Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus [2].

High impact information on Dhfr

• The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts [2].
• Transformation abrogates an early G1-phase arrest point required for specification of the Chinese hamster DHFR replication origin [2].
• Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance [3].
• Transformed Drosophila Kc cell lines, resistant to methotrexate, an inhibitor of de novo purine and pyrimidine synthesis, have been obtained by calcium phosphate transfection of plasmids containing a sequence coding for a methotrexate-resistant dihydrofolate reductase enzyme (DHFR) [3].
• The Drosophila genome contains a single Dhfr copy at 89E and a selected cell line has not amplified the gene [4].

Biological context of Dhfr

• Using the PCR-amplified partial cDNA as a probe, Dhfr was cloned by screening a Drosophila genomic library [4].
• To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR [5].
• We have shown that two inhibitors of thymidylate (dTMP) synthesis - aminopterin inhibiting dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR) inhibiting thymidylate synthetase - result in a significant increase in meiotic recombination in the yellow/white region on the X chromosome of Drosophila melanogaster [6].
• Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells [5].
• A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line [5].

Anatomical context of Dhfr

• ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen [5].

Associations of Dhfr with chemical compounds

• The most striking effects are obtained with the vgBUR27 strain, which is highly sensitive to aminopterin and to fluorodeoxyuridine and didplas the highest thymidine kinase (TK) and DHFR activities within the strains tested [7].
• Isolation and characterization of a dihydrofolate reductase gene mutation in methotrexate-resistant Drosophila cells [1].
• Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant [8].
• Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM) [9].
• Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor [9].

Other interactions of Dhfr

• Our results suggest that the vg+ gene could be a regulatory gene acting on the DHFR gene [7].
• Candidate target mechanisms of the growth inhibitor cyromazine: studies of phenylalanine hydroxylase, puparial amino acids, and dihydrofolate reductase in dipteran insects [10].

Analytical, diagnostic and therapeutic context of Dhfr

• A Dhfr sequence has now been obtained by nested polymerase chain reaction (PCR) from Drosophila bearing a single gene copy [4].
• Southern blot analysis demonstrated that the gene for the MTX target, dihydrofolate reductase (DHFR), was not significantly amplified in the resistant line [1].
• To determine if these phenotypes could be attributed solely to DHFR inhibition, microarray analysis was undertaken and included MTX-treated females, ovaries, and cell line samples [11].
• Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30 [5].

References