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1.
P Borst A M Van der Bliek T Van der Velde-Koerts E Hes 《Journal of cellular biochemistry》1987,34(4):247-258
Pulsed field gradient electrophoresis allows the separation of large DNA molecules up to 2,000 kilobases (kb) in length and has the potential to close the resolution gap between standard electrophoresis of DNA molecules (smaller than 50 kb) and standard cytogenetics (larger than 2,000 kb). We have analysed the amplified DNA in four cell lines containing double minute chromosomes (DMs) and two lines containing homogeneously staining regions. The cells were immobilized in agarose blocks, lysed, deproteinized, and the liberated DNA was digested in situ with various restriction endonucleases. Following electrophoretic separation by pulsed field gel electrophoresis, the DNA in the gel was analysed by Southern blotting with appropriate probes for the amplified DNA. We find that the DNA in intact DMs is larger than 1,500 kb. Our results are also compatible with the notion that the DNA in DMs is circular, but this remains to be proven. The amplified segment of wild-type DNA covers more than 550 kb in all lines and possibly up to 2,500 kb in some. We confirm that the repeat unit is heterogeneous in some of the amplicons. In two cell lines, however, with low degrees of gene amplification, we find no evidence for heterogeneity of the repeats up to 750 (Y1-DM) and 800 kb (3T6-R50), respectively. We propose that amplicons start out long and homogeneous and that the heterogeneity in the repeat arises through truncation during further amplification events in which cells with shorter repeats have a selective advantage. Even if the repeats are heterogeneous, however, pulsed field gradient gels can be useful to establish linkage of genes over relatively short chromosomal distances (up to 1,000 kb). We discuss some of the promises and pitfalls of pulsed field gel electrophoresis in the analysis of amplified DNA. 相似文献
2.
D Cohen S M Higman S I Hsu S B Horwitz 《The Journal of biological chemistry》1992,267(28):20248-20254
Two closely related but functionally distinct P-glycoprotein isoforms are encoded by the murine multidrug-resistance genes mdr1a and mdr1b. In a series of independently selected multidrug-resistant (MDR) J774.2 cell lines, mdr gene amplification and/or overexpression and overproduction of either the mdr1a or mdr1b products, or both gene products, correlates with the MDR phenotype. To investigate the possibility that mutations in the promoter regions of the mdr1a or mdr1b genes could influence their differential expression, mdr promoter-specific probes were used to detect and map potential structural alterations. An unusual structural rearrangement was found in the 5'-region of the amplified mdr1a allele in J7.T1, a cell line selected with taxol. To characterize this rearrangement, the regulatory regions of the mdr1a and mdr1b genes were analyzed. Whereas no gross structural alterations were detected by Southern blot hybridization using the mdr1b promoter probe, a novel amplified EcoRI fragment was detected by the mdr1a promoter probe. To determine the precise nature of this mutation, an mdr1a 5'-genomic clone was isolated from J7.T1 cells. Sequence analysis revealed an unusual DNA rearrangement consisting of the mdr1b gene, from its fourth intron toward its 3'-end, upstream of an intact mdr1a promoter on the amplified allele. We propose that this event occurred by an unequal sister chromatid exchange that was mediated by LINE-1 repetitive elements. 相似文献
3.
Application of natural partial digests to pulsed-field gel analysis of the amplified MDR locus 总被引:1,自引:0,他引:1
The analysis by pulsed-field gel electrophoresis of partial digestion products visualized by probes for the human multidrug resistance (MDR) locus has been used to further establish the restriction map of this region. Results place the MDR1 and MDR2 genes on a single SfiI fragment, with partial digestion products establishing the distance between these genes to be 230-250 kb. The feasibility and potential advantages of using "natural" partials to generate detailed restriction maps within an amplified DNA domain are discussed. 相似文献
4.
R C Hightower M L Wong L Ruiz-Perez D V Santi 《The Journal of biological chemistry》1987,262(30):14618-14624
Three independently derived antifolate-resistant Leishmania major cell lines overproduce the bifunctional protein thymidylate synthase-dihydrofolate reductase (TS-DHFR) by amplification of a region of DNA (R-region DNA) that contains the gene for TS-DHFR. On orthogonal-field-alteration gel electrophoresis (OFAGE), the extrachromosomal R-region DNAs are circular molecules, and different forms of R-region DNA within these cell lines are resolved. The R-region DNAs migrate aberrantly on OFAGE with respect to linear DNA and supercoiled plasmid standards. We describe a method for the isolation of these R-region DNA forms from OFAGE. By electron microscopy, we show that the extrachromosomal elements are single supercoiled circular DNA molecules, and are predominantly circular monomers and dimers of the original R-region DNA amplification unit. Using OFAGE, an analysis of cloned isolates shows that individual cells may contain multiple forms of R-region DNA. Furthermore, within a given cell line, certain distinguishable forms appear to have the same size and restriction map, suggesting they may be topoisomers. The multiple forms of R-region DNA are in a dynamic state in the antifolate-resistant populations, and the relative amount of DNA in each form as well as the number of forms within each cell line change through time. As currently understood, the generation of amplified R-region DNA in L. major is summarized. 相似文献
5.
O B Chernova V I Shifrin O I Sokova B P Kopnin A V Gudkov 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1987,(4):14-19
A number of DNA clones containing the amplified DNA sequences were isolated from the genomic library of multidrug-resistant (MDR) Djungarian hamster cells using the DNAC0t 10-250 hybridization probe. Five independent nonoverlapping clones were obtained that covered more than 100 kb of the amplified genomic region. These clones were used as hybridization probes in blot-hybridization with DNA from 7 independently derived MDR Djungarian hamster cell lines selected for the resistance to colchicine or actinomycin D. Some clones contained the DNA sequences amplified in all of the cell lines tested while the others contained the cell line specific amplified sequences. Hybridization in situ was used to localize the amplified DNA in metaphase chromosomes of a MDR cell line that contained about 140 copies of these sequences. The approximate size of an amplicon calculated on the basis of the obtained data is about 1-2 X 10(3) kb. 相似文献
6.
Considerable work with DNA amplification has been carried out in the murine SEWA ascites tumor cell system. In SEWA cells there is 'spontaneous' amplification of the c-myc oncogene, and transitions between different cytogenetic expressions of gene amplification such as DM (double minutes), CM (C-bandless chromosomes) and HSR (homogeneously staining regions) of the amplified DNA have been recorded during serial in vivo transplantations. In SEWA cells it has also been shown that the c-myc-containing DM will he lost under in vitro conditions, but are rapidly recovered if the cells are reinjected into animals. Additional gene amplification has been superimposed on the c-myc amplification in SEWA cells by stepwise selection in vitro, leading to resistance to different drugs, such as methotrexate, actinomycin D, colcemid and vincristine. Cytogenetically, DNA amplification is multifaceted and, in addition to the structures mentioned, it may also take the form of CB (chromatin bodies), which have been shown to be the carriers of resistance genes in hybrids between multidrug-resistant SEWA cells and Chinese hamster CHO cells. In most instances, DM are noncentromeric and distributed by a 'hitch-hiking' mechanism at mitosis; in one colcemid-resistant SEWA line, however, we have shown that the DM carry active centromeres. The molecular mechanism behind DNA amplification appears to be complex. We have shown that in four independently derived multidrug-resistant SEWA sublines the amplicons resided on circular molecules which were about 2500 kb long and carried at least five genes, including the three mouse mdr genes. Within the circles the DNA was unrearranged compared to the organization of the DNA in sensitive cells. 相似文献
7.
Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line. 总被引:17,自引:7,他引:10
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J C Ruiz K H Choi D D von Hoff I B Roninson G M Wahl 《Molecular and cellular biology》1989,9(1):109-115
Gene amplification in human tumor cells is frequently mediated by extrachromosomal elements (e.g., double minute chromosomes [DMs]). Recent experiments have shown that DMs can be formed from smaller, submicroscopic circular precursors referred to as episomes (S. M. Carroll, M. L. DeRose, P. Gaudray, C. M. Moore, D. R. Needham-Vandevanter, D. D. Von Hoff and G. M. Wahl, Mol. Biol. 8:1525-1533, 1988). To investigate whether episomes are generally involved as intermediates in gene amplification, we determined whether they mediate the amplification of the mdr1 gene, which when overexpressed engenders cross resistance to multiple lipophilic drugs. A variety of methods including electrophoresis of undigested DNAs in high-voltage gradients, NotI digestion, and production of double-strand breaks by gamma irradiation were used to distinguish between mdr1 sequences amplified on submicroscopic circular molecules and those amplified within DMs or chromosomal DNA. The gamma-irradiation procedure provides a new method for detecting and determining the size of circular molecules from 50 kilobases (kb) to greater than 1,000 kb. These methods revealed that some of the amplified mdr1 genes in vinblastine-resistant KB-V1 cells are contained in supercoiled circular molecules of approximately 600 and approximately 750 kb. Analysis of the replication of these molecules by a Meselson-Stahl density shift experiment demonstrated that they replicate approximately once in a cell cycle. The data lend further support to a model for gene amplification in which DMs are generally formed from smaller, autonomously replicating precursors. 相似文献
8.
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10.
The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes. 相似文献
11.
Robin A. Lockington Graham G. Taylor Michael Winther Claudio Scazzocchio R.Wayne Davies 《Gene》1982,20(2):135-137
The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit. 相似文献
12.
Gene amplification in methotrexate-resistant mouse cells. IV. Different DNA sequences are amplified in different resistant lines 总被引:10,自引:1,他引:9
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DNA was purified from double minutes isolated from MTX-resistant EL4/8 mouse lymphoma cells, digested to completion with Bam H1 restriction endonuclease and cloned in lambda-1059. The properties of the library suggest that the DNA from which it was made was not detectably contaminated with non-dm chromosome material, and that the library is essentially complete for sequences contained in Bam H1 restriction fragments between 9 and 19 kb. The inserts of some selected lambda-recombinants were subcloned in pBR328 or pAT153 to separate sequences of differing repetition frequency. Clones representative of different classes of sequences were used as probes to Southern transfers of Bam H1 digested total nuclear DNAs of various MTX-resistant cell lines. The results clearly show that the amplified unit of each cell line has a unique structure, and that different amplified units differ widely in their sequence composition. 相似文献
13.
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. 相似文献
14.
The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules 总被引:4,自引:1,他引:3
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A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors. 相似文献
15.
Herpesvirus saimiri DNA in tumor cells--deleted sequences and sequence rearrangements. 总被引:16,自引:14,他引:2
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R C Desrosiers 《Journal of virology》1981,39(2):497-509
Herpesvirus saimiri DNA in continuous lymphoblastoid cell lines obtained from viral induced tumors in marmosets has been analyzed by gel electrophoresis of restricted DNA. Southern transfer to nitrocellulose filters, and hybridization to 32P-labeled viral DNA or DNA fragments. The viral DNA fragments EcoRI-G, -H, -D, and -I, KpnI-A, and BamHI-D and -E were not detected in Southern transfers of DNA from the nonproducing 1670 cell line. For each restriction endonuclease, a new fragment appeared, consistent with a 13.0-megadalton deletion of viral DNA sequences. This deletion encompassed 35 to 48 +/- 0.6 megadaltons from the left end of the unique DNA region. A sequence arrangement map is presented for the major population of H. saimiri DNA sequences in the 1670 cell line. Although H. saimiri DNA in the nonproducing 70N2 cell line can be distinguished from viral DNA in the 1670 cell line by several criteria, the same sequences were found to be deleted in the major population of viral DNA molecules. Unlike 1670 and 70N2 cells, restricted DNA from the virus-producing cell lines 77/5 and 1926 contained all of the DNA fragments present in the parental virion DNA. DNA from 1670, 70N2, and 77/5 cells contained additional viral DNA fragments that did not comigrate with any virion DNA fragments. Most of these unexplained fragments were confined to or highly enriched in partially purified circular or linear DNA fractions. DNA from tumor cells taken directly from a tumor-bearing animal contained viral DNA indistinguishable from the parental virion DNA by the assay conditions used. These results indicate that viral DNA sequence rearrangements can occur upon cultivation of tumor cells in vitro and that excision of DNA sequences from the viral genome may play a role in establishing the nonproducing state of some tumor cell lines. 相似文献
16.
A. Hirai T. Ishibashi A. Morikami N. Iwatsuki K. Shinozaki M. Sugiura 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1985,70(2):117-122
Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants. 相似文献
17.
Double minute chromosomes carrying the human multidrug resistance 1 and 2 genes are generated from the dimerization of submicroscopic circular DNAs in colchicine-selected KB carcinoma cells. 总被引:2,自引:0,他引:2
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P V Schoenlein D W Shen J T Barrett I Pastan M M Gottesman 《Molecular biology of the cell》1992,3(5):507-520
This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads. 相似文献
18.
Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells 总被引:3,自引:0,他引:3
The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells. 相似文献
19.
Analysis of chromosomal alterations during stepwise development of mdr1, dhfr, or CAD gene amplifications in a large number of independently selected Djungarian hamster DM-15 and murine P388 sublines revealed typical patterns of karyotypic evolution, specific for multiplication of each of these genes in each cell type. Some principal similarities of karyotypic evolution were noted in at least two different systems. They include: (i) appearance at the first selection step of a new chromosomal arm bearing the resident gene copy followed at the next selection steps by the formation in these specific chromosomal arms of amplified DNA tandem arrays; (ii) translocations of amplified DNA from its initial site to other, also non-random, chromosomal sites; and (iii) emergence in the cell variants with high degrees of gene amplification of multiple extra-chromosomal elements. The most prominent distinctions among the systems were as follows: (i) different structures, evidently containing amplified DNAs, appeared at the initial steps of amplification of different genes--additional heterogeneously staining regions in specific chromosomal segments in the case of amplification of dhfr or CAD genes in DM-15 cells, and mini-chromosomes in the case of mdr1 gene amplification in both DM-15 and P380 cells; (ii) distinct patterns of location of the amplified mdr1 gene copies are characteristic of Djungarian hamster DM-15 and murine P388 cell derivatives after subsequent steps of selection--at the site of resident gene localization or in some other, also non-random, chromosomal sites in DM-15 sublines, and predominantly extra-chromosomal in P388 sublines. We propose that different mechanisms are responsible for the initial steps of amplification of dhfr and CAD genes on the one hand and the mdr1 gene on the other: non-equal sister-chromatid exchanges and autonomous replication of the extra-chromosomal elements. It seems, however, that both mechanisms may be involved in further rounds of amplification of each of these three genes. 相似文献
20.
Abstract. Nuclear DNA content was assessed in multidrug-resistant (MDR) cells by image and flow cytometry. Two human MDR cell lines (K562-Dox and CEM-VLB) obtained by in vitro drug selection and overexpressing mdr1 gene were compared to their respective sensitive counterparts (K562 and CCRF-CEM) and to the MDR hamster LR73-R cell line obtained by transfection of mouse mdr1 cDNA. Both cell lines obtained by selection displayed a decreased DNA content, as measured by image cytometry after Feulgen staining, or by flow cytometry after staining with propidium iodide, ethidium bromide, or Hoechst 33342. This decrease was not accompanied by changes in cell cycle phase distribution of cells. Moreover, image cytometry of cells stained after various hydrolysis times in 5 M HCl indicated that MDR cells displayed the same hydrolysis kinetics and sensitivity as drug-sensitive cells with a well-preserved stoichiometry of the Feulgen reaction. LR73-R cells transfected with mdr1 cDNA exhibited only a very limited change in propidium iodide staining as compared with sensitive LR73 cells, suggesting that mdr1 gene overexpression alone could not account for the alterations in DNA content observed in the selected MDR cells. 相似文献