Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.     

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.     

Construction of a dominant selectable marker using a novel dihydrofolate reductase.

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.     

Stable transfer of a mouse dihydrofolate reductase gene into a deficient cell line using human adenovirus vector

A plasmid containing the mouse dihydrofolate reductase (dhfr) gene was rescued in a human adenovirus in early region 3. Analysis of the insert in the recombinant virus revealed that the dhfr sequences were intact in the viral genome, whereas a part of the ampicillin gene in the plasmid sequences was deleted. The recombinant virus could successfully express this gene in a deficient cell line. A permanent dhfr+ cell line was established by stable transfer of the gene using the recombinant virus.     

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.     

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.     

Purification and properties of dihydrofolate reductase from cultured mammalian cells

Dihydrofolate reductase was purified quickly and simply from small quantities of cultured mammalian cells by affinity chromatography. On gel electrophoresis of the purified enzyme, multiple bands of activity resulted from enzyme-buffer interaction at low but not high buffer concentration. A Ferguson plot (Ferguson, 1964) showed that this heterogeneity was due to a charge difference with no alteration in the size of the enzyme. Stimulation of enzyme activity by KCl, urea and p-hydroxymercuribenzoate, and inhibition by methotrexate and trimethoprim, showed only minor differences between the various enzymes.     

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.     

Selective overproduction of human dihydrofolate reductase in a methotrexate-resistant human-mouse somatic cell hybrid

The development of methotrexate (MTX) resistance in cultured cells results in increased levels of the drug's target enzyme dihydrofolate reductase (DHFR). Stepwise-selected MTX-resistant sublines originating from an MTX-sensitive human-mouse hybrid expressed elevated DHFR levels and human-DHFR specific gene sequence amplification. By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance. These findings and those in a parallel study indicate that concurrent with overproduction of human DHFR and amplification DHFR sequences in VB2a-100, a loss of mouse-specific DHFR gene sequences occurred.     

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.     

Steroid-induced nuclear binding of glucocorticoid receptors in intact hepatoma cells

Some of the early steps of steroid hormone action have been studied in cultured hepatoma cells, in which glucocorticoids induce tyrosine aminotransferase. The hypothesis that inducer steroids promote the binding of specific cytoplasmic receptors to the cell nucleus has been examined in intact cells.Binding of steroids such as dexamethasone and cortisol results in a loss of most of the receptor sites from the cytoplasm. This coincides with the binding of an equivalent number of steroid molecules in the nucleus. Both processes occur concomitantly, even when their kinetics are altered by reducing the temperature. When the inducer is removed from the culture, steroid dissociates from the nucleus while the level of cytoplasmic receptor returns to normal, even if protein or RNA synthesis is inhibited. These results suggest that nuclear binding of glucocorticoids is due to the association with the nucleus of the cytoplasmic receptor-steroid complex itself and make it unlikely that the receptor acts as a mere carrier for the intracellular transfer of the steroid.Steroids that differ in their effects on tyrosine aminotransferase induction were also studied. In contrast to those bound with inducer steroids, receptors complexed with the anti-inducer progesterone did not leave the cytosol. Further, a suboptimal inducer (deoxycorticosterone) produced an intermediate level of depletion. Thus, the biological effect of different classes of steroids can be related to their capacity to promote nuclear binding of the receptor. These data support a model proposed earlier, according to which the receptor is an allosteric regulatory protein directly involved in the hormone action, under the control of specific steroid ligands. They further suggest that the conformational state influenced by the inducer is such that a nuclear binding site on the receptor is exposed.Evidence is also presented that a distinct reaction takes place between the binding of the steroid to the receptor and the association of the complex with the nucleus. At 0 °C, this change is rate-limiting. It could correspond to the “activation” of receptor-steroid complexes known to be required for binding of the complexes by isolated nuclei, and thus represent an additional step in hormone action.     

Different states of glucocorticoid receptors in intact cells and cytosol preparations

Binding of the glucocorticoid dexamethasone was studied in intact cells of the mouse lymphoma lines S49. 1, WEHI-7, WEHI-22, and WEHI-112. The number of binding sites per cell varied from 13 000 to 130 000 depending on the cell line. The equilibrium dissociation constant at 37° was in the range of 10 nM. When dexamethasone binding was investigated at 0° in cytosol preparations of the same cell lines significantly lower receptor levels were found and the dissociation constants were about one order of magnitude lower than those determined in whole cells. The data suggest that glucocorticoid receptors exist in different states in intact cells and cell extracts.     

Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.     

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.     

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.