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Plasmids containing a mouse cDNA sequence encoding the enzyme dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been used to study the efficiency of initiation of protein synthesis at an ATG (AUG) translational start codon indigenous to the eukaryotic CDNA. differences in DHFR production assayed phenotypically, enzymatically, and immunologically were correlated with the primary structure of the DNA segment that precedes the translational start codon. Our results indicate that initiation of a structurally discrete and biologically functional eukaryotic protein can occur in bacteria on a fused mRNA molecule, and that the efficiency of expression is strongly affected by: (i) the extent of homology of the translational control region with the 3'-OH end of 16S ribosomal RNA, and (ii) the distance between the protein start codon and the ribosome-binding sequence on the mRNA.

We have examined the pattern of dihydrofolate reductase (DHFR) enzyme and mRNA levels in cell cycle stage-specific populations obtained by centrifugal elutriation in Chinese hamster ovary cells and in a derivative line in which the dihydrofolate reductase gene is amplified approximately 50-fold. On a per cell basis, we observed a 2-fold increase in DHFR activity as cells progressed from G1 to G2/M with a concomitant 2-fold increase in the rate of protein synthesis and steady state level of mRNA. Analysis of DHFR mRNA levels in cell cycle stage-specific mouse 3T6 and human 143 tk- cells gave a similar pattern. We also demonstrate that simple alterations in growth conditions prior to elutriations can dramatically increase the levels of DHFR mRNA in all cell cycle states, thereby indicating that growth response associated with the DHFR gene functions independent of the cell cycle. We conclude that during periods of exponential growth the increases in dihydrofolate reductase activity, rate of protein synthesis, and steady state levels of mRNA parallel the general increases in cell volume and protein content associated with normal progression through the cell cycle, and therefore DHFR cannot be considered a cell cycle-regulated enzyme.

The overproduction of heterologous gene products in mammalian cells is often a prerequisite for studies of protein structure, function, and therapeutic efficacy. We report that recombinant fusion of a nonselectable reporter template into the intronic sequences of an amplifiable minigene gives dramatically enhanced co-expression efficiency in stable, primary transformants. Further incremental selective pressure for marker gene amplification results in the rapid acquisition of very high expression levels from the intronically positioned reporter. Nested within a constitutively expressing gene, these templates can be independently regulated at moderate gene dosage levels. Intronic positioning may therefore be of general utility for a variety of recombinant studies.

Cellulose-bound DNA complementary to ovalbumin mRNA was used in a continous hybridization system to isolate single-stranded DNA molecules containing the ovalbumin gene. Fragmented DNA segments containing the ovalbumin gene were enriched 300-350 fold in one cycle of purification. Two cycles of purification resulted in a DNA fraction which was enriched 2300 fold in the ovalbumin sequence. The method is suitable for purification of the ovalbumin sequence from both sheared DNA fragments, as well as larger molecular weight DNA containing more than twice the number of nucleotides necessary to code for ovalbumin mRNA. The chromatographic procedures were specific and reproducible. In addition, the recovery of ovalbumin DNA was essentially quantitative (80-100%), even when large amounts of starting DNA (70-75 mg) were used. This purification scheme should also be useful for the enrichment of other unique sequence gened form eucaryotic DNA.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.

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.

We have employed the technique of chromosome "walking" to determine the structure of 240 kilobases of amplified DNA surrounding the dihydrofolate reductase gene in methotrexate-resistant mouse cell lines. Within this region, we have found numerous DNA rearrangements which occurred during the amplification process. DNA subclones from regions flanking the dihydrofolate reductase gene were also utilized as hybridization probes in other cell lines. Our results show that: 1) amplification-specific DNA rearrangements or junctions are unique to each cell line; 2) within a given cell line, multiple amplification-specific DNA sequence rearrangements are found; 3) the degree of amplification of sequences flanking the dihydrofolate reductase gene shows quantitative variation among and within cell lines; and 4) both the arrangement of amplified sequences as well as the magnitude of gene amplification may vary with prolonged culture even under maintenance selection conditions. These studies indicate that there is no static repetitive unit amplified in these cells. Rather, a dynamic and complex arrangement of the amplified sequences exists which is continually changing.

We have previously reported the presence of four dihydrofolate reductase messenger RNAs differing in the length of 3' untranslated regions in murine cells (Setzer, D. R., McGrogan, M., Nunberg, J. H., and Schimke, R. T. (1980) Cell 22, 361-370). We have now mapped the 3' ends of these RNAs more precisely and have demonstrated colinearity between their shared sequences. Analysis of three larger dihydrofolate reductase RNAs has shown that these RNA species contain very long 3' noncoding regions, bringing the total number of dihydrofolate reductase RNAs to seven, ranging in length from 750 to 5600 nucleotides. We have determined the nucleotide sequence at and surrounding the polyadenylation sites of the four smaller RNAs. We find no striking structures in this sequence that might constitute multiple polyadenylation signals, but conclude that the putative polyadenylation signal AAUAAA is not required for polyadenylation of at least three of the four dihydrofolate reductase messengers.

We have isolated 4 independent mutations affecting alpha-mannosidase-1, a developmentally regulated activity in Dictyrostelium discoideum. Three of these result in a thermolabile alpha-mannosidase-1 activity. One mutation also affects the substrate affinity (Km) of the activity. In diploids these mutations show a gene dosage effect and are all alleles. The structural gene for alpha-mannosidase-1, as defined by these mutations, defines a new linkage group, linkage group VI. alpha-mammosidase 1 is probably a homopolymer with subunits of 54,000 daltons. We have also mapped two temperature-sensitive-for-growth mutations onto two previously defined linkage groups.

mRNA that encodes the common peptide precursor for the hormones corticotropin and beta-lipotropin was purified from the neurointermediate lobe of bovine pituitaries, and double-stranded cDNA species synthesized from this template were cloned in Escherichia coli X1776 by inserting them into the Pst I endonuclease cleavage site of the pBR322 plasmid using poly(dG)poly(dC) homopolymeric extensions. Certain of the cloned cDNA inserts contain nucleotides corresponding to the complete amino acid sequence of bovine corticotropin and a coding sequence that corresponds to at least the first portion of bovine beta-lipotropin. The nucleotide sequences coding for corticotropin and beta-lipotropin are separated on the cDNA by a 6-base-pair sequence encoding lysine and arginine, indicating that the carboxyl terminus of corticotropin is connected on the precursor peptide with the amino terminus of beta-lipotropin by these two amino acids. In addition, the cloned cDNA insert is characterized by an unusually high C+G nucleotide base content as well as by a number of DNA sequence duplications.

The construction and analysis of bacterial plasmids that contain and phenotypically express a mammalian genetic sequence are described. Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim.

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5' untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.

We have determined whether the gene encoding dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) varies spontaneously in gene copy number in cells in vitro. Cells were stained under nonselective conditions with fluoresceinated methotrexate, which binds quantitatively to dihydrofolate reductase. Cells with the highest fluorescence were collected by a fluorescence-activated cell sorter and subsequently grown in the absence of methotrexate. At no time during the experiment were the cells placed under metabolic stress. After 10 successive rounds of growth and sorting, the derived population showed a 50-fold increase in fluorescence intensity, was highly resistant to methotrexate, and was amplified 40-fold in content of dihydrofolate reductase gene. We also found that cells already having amplified genes can undergo increases or decreases in their fluorescence and in gene copy number even more rapidly (at rates as high as 3 X 10(-2) amplification events per cell division) than do parental cells (ca. 10(-3) events per division). We therefore conclude that gene amplification can occur spontaneously in cells and that the rate of its occurrence varies with gene copy number.

Resistance of mouse cells to the folate analog, methotrexate, results from selection of increasingly resistant cells on progressive increases of methotrexate in the culture medium. High-level resistance is associated with high rates of synthesis of dihydrofolate reductase and correspondingly high numbers of reductase genes. In some variants high resistance and gene copy number are stable in the absence of selection pressure, whereas in others they are unstable. Analogies are made to antibiotic and insecticide resistance wherein selection of organisms with increased capacity to counteract the drug effect results in emergence of resistance. Gene amplification may underlie many such resistance phenomena.

We have used the technique of DNA-mediated gene transfer to introduce dihydrofolate reductase genes into dihydrofolate reductase-deficient Chinese hamster ovary cells. The transferred sequences include: dihydrofolate reductase minigenes, dihydrofolate reductase cDNA clones, and genomic DNA from mouse cells with highly amplified dihydrofolate reductase genes. The hamster cells were capable of utilizing the murine transcription initiation sites, splice junctions, and polyadenylation sites in complete murine dihydrofolate reductase genes. Only a short region of 5'-flanking sequence (160 base pairs (bp] was sufficient for proper initiation of dihydrofolate reductase transcription. In contrast only those clones with all of the dihydrofolate reductase introns and extensive (greater than 6.5 kilobase pairs) 3'-flanking regions utilized the dihydrofolate reductase polyadenylylation sites. Four of nine clones tested regulate the transfected genes normally, synthesizing dihydrofolate reductase preferentially at the onset of S phase. Regulated genes include one which entirely lacks intervening sequences, two with fewer than 420 bp of 5'-flanking sequence, and two in which polyadenylylation occurs predominantly in the adjacent hamster sequences. Two of the five cell lines which do not exhibit normal regulation contain genes which appear to be transcribed primarily from promoters present in the flanking hamster DNA. The other genes which fail to regulate do not appear to differ significantly in the structure of their transcripts or protein products from genes which regulate normally. We conclude that only the coding sequences, a region of less than 340 bp of sequence 5' of the translation initiation codon, and a short region of 3'-flanking sequence are required for regulated production of dihydrofolate reductase. The regulation can be abolished, however, by unknown properties of the site of insertion.

Chicken metaphase chromosomes were partially purified by rate zonal centrifugation, and DNA was prepared from each of the fractions of the sucrose gradient. The DNA was digested with various restriction enzymes and subjected to electrophoresis in agarose gels. The DNA was transferred to nitrocellulose filters (as described by Southern), and the filters were hybridized with cDNA probes. Four globin genes alpha A, alpha D, beta, and rho or epsilon are located on at least two chromosomes, and three of the estrogen-inducible genes of the hen oviduct--ovalbumin, ovomucoid, and transferrin--are on three different chromosomes. These experiments also confirm our earlier assignment of the endogenous viral sequence related to Rous-associated virus-0 to a separate (and larger) chromosome than the cellular sequence related to the transforming gene of avian sarcoma virus (cellular sarc), although it now appears that cellular sarc is on a small macrochromosome, rather than on a microchromosome.

We have examined the karyological consequences of dihydrofolate reductase gene amplification in a series of six rat hepatoma cell lines, all derived from the same clone. Cells of three of these lines express a series of liver-specific functions whereas those of three others fail to express these functions. Cells of each line have been subjected to stepwise selection for methotrexate resistance and, in most cases, resistance is associated with a 40-50-fold amplification of sequences hybridizing to a dihydrofolate reductase cDNA probe. In one line no modified chromosome is observed, whereas in two others the amplified genes are associated with an expanded chromosomal region. R-banding analysis of these karyotypes showed that few changes have occurred. These observations apply to two of the well-differentiated lines, and to a variant able to revert to the differentiated state. In contrast, in the two stably dedifferentiated hepatoma cell lines, amplified dihydrofolate reductase genes are found on large chromosomes of variable size, on ring chromosomes, and on chromosomes containing terminal, median, or multiple centromeres. We conclude that the nature of the chromosomal changes associated with dihydrofolate reductase gene amplification are the result of differences in cell lines rather than in the protocols employed for selection.

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.

The murine dihydrofolate reductase gene codes for mRNAs that differ in the length of their 3' untranslated region as well as in the length of their 5' leader sequence. In addition, the dihydrofolate reductase promoter functions bidirectionally, producing a series of RNAs from the opposite strand than the dihydrofolate reductase mRNAs. We have examined the production of these RNAs and their heterogeneous 5' and 3' termini as mouse 3T6 cells progress through a physiologically continuous cell cycle. We found that all of the transcripts traverse the cell cycle in a similar manner, increasing at the G1/S boundary without significantly changing their ratios relative to one another. We conclude that cell-cycle regulation of dihydrofolate reductase is achieved without recruiting new transcription initiation sites and without a change in polyadenylation sites. It appears that the mechanism responsible for the transcriptional cell-cycle regulation of the dihydrofolate reductase gene is manifested only by transiently increasing the efficiency of transcription at the dihydrofolate reductase promoter.

Methotrexate-resistant Chinese hamster ovary cells selected for high resistance by progressive increments of methotrexate in the culture medium have levels of dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) 200 times that of sensitive cells and a corresponding increase in the number of copies of the dihydrofolate reductase gene. The resistant cells contain an expanded region on the second chromosome (homogeneously staining region) that is not present in sensitive cells. In situ hybridization of DNA complementary to dihydrofolate reductase mRNA shows that the dihydrofolate reductase genes are specifically localized to the homogeneously staining region of this chromosome in the resistant cells.

We constructed a gene library from a murine cell line with amplified dihydrofolate reductase (dhfr) genes by inserting random segments of DNA into lambda Ch4A. From this library, the dhfr gene and 30 kilobase pairs of surrounding DNA were cloned, and the restriction map was determined. All of the coding regions were sequenced and show that the gene spans a total of 31 kilobase pairs and has five intervening sequences in the coding portion of the gene. In addition, two classes of variant dhfr genes were found in the amplified line, which were amplified and present at levels of 10 to 30% of the normal dhfr genes. Numerous repeated sequences were located throughout the gene region, some of which share homology with previously defied families of repeats.

Exposure of Chinese hamster cells to reduced oxygen partial pressure results in a marked enhancement in the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. The frequency of enhanced resistance is a function of the length of exposure to hypoxic conditions and the time after recovery from hypoxia when cells are plated into methotrexate-containing medium. Hypoxia results in an inhibition of DNA synthesis; upon return to normal oxygen atmosphere, greater than 60% of cells in S phase at the time hypoxia was started subsequently undergo overreplication of DNA within a single cell cycle. The cells with the increased frequency of gene amplification are derived from this subset of overreplicated cells. These results are discussed within the context of the hypoxic state of many solid tumors and the high frequency of aneuploidy, chromosomal aberrations, and spontaneously occurring resistances to a number of cancer chemotherapeutic agents.