Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Radiation-reduced chromosomes provide valuable reagents for cloning and mapping genes, but they require multiple rounds of x-ray deletion mutagenesis to excise unwanted chromosomal DNA while maintaining physical attachment of the desired DNA to functional host centromere and telomere sequences. This requirement for chromosomal rearrangements can result in undesirable x-ray induced chromosome chimeras where multiple non-contiguous chromosomal fragments are fused. We have developed a cloning system for maintaining large donor subchromosomal fragments of mammalian DNA in the megabase size range as acentric chromosome fragments (double-minutes) in cultured mouse cells. This strategy relies on randomly inserted selectable markers for donor fragment maintenance. As a test case, we have cloned random segments of Chinese hamster ovary (CHO) chromosomal DNA in mouse EMT-6 cells. This was done by cotransfecting plasmids pZIPNeo and pSV2dhfr into DHFR-CHO cells followed by isolation of a Neo + DHFR + CHO donor colony and radiation-fusion-hybridization (RFH) to EMT-6 cells. We then selected for initial resistance to G418 and then to increasing levels of methotrexate (MTX). Southern analysis of pulsed-field gel electrophoresis of rare-cutting restriction endonuclease digestions of DNA from five RFH isolates indicated that all five contain at least 600 kb of unrearranged CHO DNA. In situ hybridization with the plasmids pZIPNeo and pSV2dhfr to metaphase chromosomes of MTX-resistant hybrid EMT-6 lines indicated that these markers reside on double-minute chromosomes.
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PMID:Double-minute chromosomes as megabase cloning vehicles. 162 62

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population. The proportion of MTX-resistant cells among the survivors increased logarithmically with dose, up to a 1000-fold increase over unirradiated cells at 1000 cGy, the highest dose tested. The induced frequency of MTX resistance after X-irradiation was greater than the induced frequency of 8-azaguanine resistance, which indicates deletion of the hypoxanthine phosphoribosyltransferase gene. Inhibition of poly(ADP-ribose) polymerase by the addition of 3-aminobenzamide before irradiation increased both cell killing and MTX resistance. Metaphase spreads of chromosomes from EMT-6 cells that had been irradiated and subjected to stepwise increases in MTX concentration showed numerous double minutes. Pulsed-field gel electrophoresis of the DNA from cells containing radiation-induced double minutes showed that many copies of the dhfr gene were present on circular DNA molecules of 10(6), 2 x 10(6), and 3 x 10(6) base pairs. These results suggest a relationship between the induction of chromosome aberrations and the induction of gene amplification.
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PMID:X-ray induction of methotrexate resistance due to dhfr gene amplification. 212 27

The development of double-minute chromosomes (DMs) and subsequent gene amplification are important genomic alterations resulting in increased oncogene expression in a variety of tumors. The molecular mechanisms mediating the development of these acentric extrachromosomal elements have not been completely defined. To elucidate the mechanisms involved in DM formation, we have developed strategies to map amplified circular DM DNA. In this study, we present a long-range restriction map of a 980-kb DM. A cell line cloned from mouse EMT-6 cells was developed by stepwise selection for resistance to methotrexate. This cloned cell line contains multiple copies of the 980-kb DM carrying the dihydrofolate reductase (DHFR) gene. A long-range restriction map was developed in which a hypomethylated CpG-rich region near the DHFR gene served as a landmark. This strategy was combined with plasmid-like analysis of ethidium bromide-stained pulsed-field gels and indicated that a single copy of the DHFR gene was located near a hypomethylated region containing SsII and NotI sites. At least 490 kb of this DM appears to be composed of unrearranged chromosomal DNA.
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PMID:CpG island mapping of a mouse double-minute chromosome. 833 94

Double minute chromosomes (DMs) are the principal genetic vehicles for amplifying oncogenes in human tumors and drug resistance genes in cultured mouse cells. Mouse EMT-6 cells resistant to methotrexate (MTX) generally contain circular DMs, approximately 1 megabase (Mb) in size, that amplify the dihydrofolate reductase (DHFR) gene. The 1 Mb DMs generally have CpG islands located 500 kb upstream of the DHFR gene. The purpose of this study was to determine the relationship between CpG islands and chromosomal breakpoints giving rise to the DM. We show that EMT-6 cells growing in very low levels of MTX that do not yet contain the 1 Mb DHFR-amplifying DM, develop a NotI/EagI site 500 kb upstream of the DHFR gene. This NotI site is close to, if not identical with, one of the chromosomal breakpoints giving rise to the DM. We show that 500 kb of DM DNA from upstream of the DHFR gene is derived from 500 kb of chromosomal DNA upstream of the chromosomal DHFR gene. The downstream breakpoint maps to a region approximately 200 kb downstream of the DHFR gene near a chromosomal SstII/EagI site. Therefore, approximately 700 kb of DM DNA was derived from the genomic region surrounding the DHFR gene. To confirm the organization of the DM DNA, we isolated DNA probes from the 1 Mb DM. Using pulsed field gel electrophoresis and Southern hybridization, we determined the approximate location of each probe with respect to the CpG island in both the DM and the chromosome. Approximately 300 kb of chimeric DNA from a region unrelated to the DHFR gene was incorporated during DM formation. Implications for the mechanism of DM formation are discussed.
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PMID:Chromosome breakpoints near CpG islands in double minutes. 975 10