Transformation of mice by a prokaryotic gene for dihydrofolate reductase

In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.     

Insertion of the bacterial gene for dihydrofolate reductase into colony-forming cells of mouse bone marrow

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.     

Dismutation of dihydrofolate by dihydrofolate reductase

Degradation of 7,8-dihydrofolate (H2folate) in the presence of dihydrofolate reductase (DHFR) has been shown due not to an oxygenase activity of the reductase as previously reported but to dismutation of H2folate to folate and 5,6,7,8-tetrahydrofolate (H4folate). The reaction can be followed spectrophotometrically or by analysis of the reaction mixture by high-performance liquid chromatography (HPLC). The products have also been isolated and characterized. Oxygen uptake during the reaction is much less than stoichiometric with H2folate disappearance and is attributed to autoxidation of the H4folate formed. The dismutation activity is a property of highly purified Streptococcus faecium DHFR isoenzyme 2 (but not isoenzyme 1) and of Lactobacillus casei DHFR, but not of bovine liver DHFR. The activity is dependent on tightly bound NADP+ and/or NADPH. Removal of the nucleotide results in loss of dismutation activity, which is restored by adding NADP+ or NADPH. Maximum activity is obtained when approximately 1 mol equiv of nucleotide is added per mol of DHFR. It is proposed that in the dismutation reaction bound NADP(H) is alternately reduced and oxidized by incoming molecules of H2folate with release of folate and H4folate, respectively. The relatively slow rate of folate formation presumably limits the rate of the overall reaction. The equilibrium constant for the dismutation reaction is 19.4 +/- 7.4 at 22 degrees C and pH 7.0. Calculation of standard oxidation-reduction potentials at pH 7 gave values of -0.230 V for the H2folate/H4 folate pair and -0.268 V for the folate/H2folate pair. The mechanism by which NADP+ is retained by the enzyme from some sources during purification procedures is unclear.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.     

Vectors containing a prokaryotic dihydrofolate reductase gene transform Drosophila cells to methotrexate-resistance.

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). The introduced DNA is stably maintained in the cells as head-to-tail multimeric structures of the initial DNA sequence even after several months of culture in the presence of the selective agent. The introduced sequences are present at a high copy number in the transformed cells and express cytoplasmic RNAs transcribed from the DHFR gene.     

Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.

We investigated various parameters associated with the initial selection of mouse 3T6 cells for resistance to single concentrations of methotrexate and characterized resistant colonies for the presence of additional (amplified) copies of the dihydrofolate reductase gene. Our results indicate that the frequency of occurrence of dihydrofolate reductase gene amplification varies with the selecting concentration of methotrexate and is highly variable between clonally derived sublines of mouse 3T6 cells. Second, we increased the frequency of occurrence of cells with amplified dihydrofolate reductase genes by transiently inhibiting DNA synthesis with hydroxyurea before the selection of cells in single concentrations of methotrexate. This effect was dependent on the concentration of hydroxyurea, the time of exposure to the drug, and the time interval between the removal of hydroxyurea and the selection of cells in methotrexate.     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.     

Effects of 5-fluorouracil on dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells

Growth of methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 5-fluorouracil results in an increase in dihydrofolate reductase mRNA. This increase can be solely attributed to a species of RNA of approximately 3.5 kilobase pairs in size. Although dihydrofolate reductase enzyme activity increases per cell with increasing 5-fluorouracil, there is a decrease of enzyme activity per mg of protein (Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299-13306). The rate of in vivo enzyme synthesis, as assayed by immunoprecipitation and supported by gel electrophoresis, does not decrease and may in fact increase with increasing 5-fluorouracil. Translation of purified dihydrofolate reductase mRNA in vitro shows that the rate of translation is unaffected by 5-fluorouracil incorporation into mRNA. The inhibition of dihydrofolate reductase by a monospecific polyclonal antiserum is reduced with extracts from 5-fluorouracil-treated cells. Inhibition of dihydrofolate reductase by methotrexate is significantly reduced in extracts from 5-fluorouracil-treated cells compared to control extracts. Tight binding of [3H]methotrexate is also different in extracts from 5-fluorouracil-treated cells. This data supports the hypothesis of translational miscoding during protein synthesis as a major mechanism of 5-fluorouracil-mediated cytotoxicity and suggests a new mechanism of 5-fluorouracil-methotrexate antagonism.     

An amplified insect dihydrofolate reductase gene contains a single intron

We have used methotrexate-resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5-kb EcoRI fragment containing an amplified dihydrofolate reductase (DHRF) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx-5011-256 cells, which were 3000-fold more resistant to methotrexate than wild-type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8-kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56-nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186-amino-acid protein with 43-48% similarity to vertebrate DHFR.     

Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells

We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.     

UV radiation facilitates methotrexate resistance and amplification of the dihydrofolate reductase gene in cultured 3T6 mouse cells.

Pretreatment of 3T6 murine cells with the carcinogen UV radiation or N-acetoxy-N-acetylaminofluorene increased the number of methotrexate-resistant colonies. This carcinogen-induced enhancement was seen only at low toxicities. The enhancement was transient and was observed at its maximum when cells were subjected to methotrexate selection 12 to 24 h after treatment. The addition of a tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate, during or after carcinogen treatment further enhanced this effect. A large proportion of the resistant colonies had an increase in the dihydrofolate reductase gene copy number and the relative proportions of colonies with amplified genes were similar, regardless of whether selected cells were untreated, treated with carcinogen, or treated with carcinogen plus promoter. We discuss some of the variables which both enhance the generation and improve the detection of methotrexate-resistant colonies, as well as certain implications of our results for the generation and mechanism of gene amplification.     

The adenovirus Ela gene induces differentiation of F9 teratocarcinoma cells.

Undifferentiated F9 cells transfected with plasmids encoding adenovirus E1a gene products underwent radical morphological changes. They ceased to express the SSEA-1 stem cell marker antigen and started to express a number of the characteristics of the differentiated state that is induced in F9 cells by treatment with retinoic acid. In particular, they expressed keratin intermediate filaments and acquired the ability to synthesise simian virus 40 tumor antigens after virus infection. The transfected cells expressed the E1a proteins, and this expression was necessary to induce the phenotypic changes, since a coisogenic plasmid encoding only a truncated 70-amino-acid E1a polypeptide and the transfection procedure itself did not detectably after the morphology or marker expression of the F9 stem cells. The phenotypic change was induced by both 13S and 12S cDNA plasmids. We discuss these results in the context of known E1a functions and with reference to the other oncogenes and external factors that can cause F9 cell differentiation.     

Organization of a Chinese hamster ovary dihydrofolate reductase gene identified by phenotypic rescue.

We have constructed a genomic DNA library from a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) in the cosmid vector pHC79. By utilizing a murine dihydrofolate reductase (DHFR) cDNA clone, we have identified 66 DHFR+ clones among the 11,000 colonies screened by colony hybridization. To isolate a recombinant cosmid containing the entire DHFR gene, we have tested these colonies for their ability to rescue a DHFR- Chinese hamster ovary cell line, using the spheroplast fusion method of gene transfer developed by W. Schaffner (Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980). One clone (cH1) was able to transform DHFR- cells to the DHFR+ phenotype and was shown in hybridization studies to contain all of the gene except a small portion of the 3' untranslated region. We have mapped cosmid cH1 and several overlapping cosmids with a variety of restriction enzymes and have determined the approximate positions of the five (and possibly six) exons within the DHFR gene. Differences between the sizes of homologous genes in hamster cells (24.5 kilobases [kb]) and in mouse cells (31.5 kb) are shown to reside primarily in the length of the 3' intron, which is 8 kb in the hamster gene and 16 kb in length in the mouse gene. Our studies confirm the utility of cosmid libraries for the isolation of large genes, as previously shown by R. de Saint Vincent et al. (Cell 27:267-277, 1981). In addition, a cosmid that contains a functional DHFR gene will be a useful vector for the co-amplification and subsequent overexpression of other cloned genes.