Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 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.     

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.     

Unbalanced growth in mouse cells with amplified dhfr genes

When grown in the absence of methotrexate, cells carrying unstably amplified dihydrofolate reductase ( dhfr ) genes have a growth disadvantage that is a function of their level of gene amplification. Although this growth disadvantage is thought to drive the loss of unstably amplified dhfr genes in the absence of methotrexate, its mechanism is not understood. The present studies of murine cell lines with different levels of dhfr gene amplification demonstrate that such cells experience increased unbalanced growth (excess RNA and protein content relative to DNA content) with increased levels of dhfr gene amplification. Stathmokinetic analysis of a cell line with unstably amplified dhfr genes showed that the unbalanced growth was associated with a very low rate of G₁/S transit, which suggests that amplified DNA sequences may activate a cell cycle checkpoint at the G₁/S boundary. Hydroxyurea, which is known to induce rapid elimination of amplified genes at sub-cytotoxic concentrations, also inhibits the cell cycle at the G₁/S transition and causes unbalanced growth. Earlier work has shown that hydroxyurea selectively targets those cells within the heterogeneous drug resistant cell populations which have the highest amplified gene dosage. The finding that unstable gene amplification and hydroxyurea have similar effects on the cell suggests that hydroxyurea may achieve this selective targeting by pushing those cells with the highest levels of gene amplification over a critical stress threshold to cause growth arrest or cell death.     

Anchorage of the Chinese hamster dihydrofolate reductase gene to the nuclear scaffold occurs in an intragenic region

We have identified a region near the center of the dihydrofolate reductase gene dhfr in Chinese hamster ovary cells that is attached to nuclear scaffolds isolated by extraction with lithium diiodosalicylate. Detailed analysis presented here reveals the presence of only two closely linked sites in 35,000 base-pairs scanned that mediate attachment of the dhfr gene to the nuclear scaffold. Sequence analysis of one of the sites reveals a high A + T content, the presence of cleavage consensus sequences for topoisomerase II, and direct and inverted repeated sequence motifs that are localized to a small region of the attachment site. Attachment of these two regions to the nuclear scaffold is observed in wild-type, hemizygous, and amplified cell lines. Attachment is also retained in dhfr mutants isolated in our laboratory, in which chromosomal lesions have occurred directly adjacent to the scaffold-associated regions. These two regions are not bound to scaffolds prepared from isolated metaphase chromosomes, suggesting that attachment of the dhfr gene is lost during mitosis.     

Carcinogen-mediated methotrexate resistance and dihydrofolate reductase amplification in Chinese hamster cells.

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.     

Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.     

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

Increased rate of base substitution in a hamster mutator strain obtained during serial selection for gene amplification.

The pattern of mutations produced by a mutator gene (obtained during serial selection for amplification of the dihydrofolate reductase [dhfr] locus) shows a pronounced shift from that found in wild-type cells. The rate of certain types of base substitutions (particularly transitions) is dramatically increased, while gene rearrangements constitute a lower proportion of mutations. These data suggest a lower fidelity of the replication process in the mutator strain.     

Expression of cell-associated and secreted forms of herpes simplex virus type 1 glycoprotein gB in mammalian cells.

The gene for glycoprotein gB1 of herpes simplex virus type 1 strain Patton was expressed in stable Chinese hamster ovary cell lines. Expression vectors containing the dihydrofolate reductase (dhfr) cDNA plus the complete gB1 gene or a truncated gene lacking the 194 carboxyl-terminal amino acids of gB1 were transfected into CHO DHFR-deficient cells. Radioimmunoprecipitation demonstrated that the complete gB1 protein expressed in CHO cell lines was cell associated, whereas the truncated protein was secreted from the cells due to deletion of the transmembrane and C-terminal domains of gB1. Cells expressing the truncated gB1 protein were subjected to stepwise methotrexate selection, and a cell line was isolated in which the gB1 gene copy number had been amplified 10-fold and the level of expression of gB1 had increased over 60-fold. The truncated gB1 protein was purified from medium conditioned by the amplified cell line. N-terminal amino acid sequence analysis of this purified protein identified the signal peptide cleavage site and predicted the cleavage of a 30-amino-acid signal sequence from the primary protein. The immunogenicity of the truncated gB1 protein was also tested in mice, and high levels of antibody and protection from virus challenge were observed.     

Genetic demonstration of mitotic recombination in cultured Chinese hamster cell hybrids

Chinese hamster ovary cell hybrids were constructed that are heterozygous for two markers, leuS and emtB, linked to the long arm of chromosome 2. In addition, the chromosome 2 carrying the wild-type leuS and emtB alleles contains, on its short arm, a homogeneously staining region (hsr) in which the gene encoding dihydrofolate reductase (dhfr) is amplified approximately 50-fold. This provides a convenient cytogenetic and biochemical means to distinguish the chromosome 2s from the different parents. Analysis of emetine-resistant segregants isolated from such hybrids identified three distinct classes of segregants. One rare class of segregants loses the wild-type leuS and emtB gene functions on the long arm of the hsr chromosome 2 (H-2) but retains the amplified dhfr genes on the opposite arm. Detailed genetic analysis of two such segregants that did not arise by chromosome loss or deletion revealed that new gene linkage relationships had been established on the H-2 chromosome in each, demonstrating that the segregation events in these cell lines involved mitotic recombination.