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1.
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   

2.
Mouse L-cell lines (B-82, tk-) were obtained using the stepwise selection method, their aminopterin (AP) resistance being 10(3)-5 X 10(4) times higher than that of parental cells. This resistance increase results from dihydrofolate reductase (DHFR) gene amplification which was determined from the 15-120-fold rise of the enzyme activity and with the cytogenetical techniques. The development and loss of AP resistance have been studied and karyological analysis of the lines obtained carried out. Two types of karyological changes were found in stable DM and HSR cells which correspond to extrachromosomal and intrachromosomal forms of the amplified material organization. Localization of the DHFR gene in HSR was proved using the in situ hybridization technique. Extrachromosomal localization of the amplified genes in DM providing unstable AP resistance is dominant at the early stages of the development of resistance and for a long time. It was demonstrated that DM and HSR can exist in one cell during the prolonged period. DHFR gene copy number in such cells is regulated by a change in the DM number, whereas the HSR size and localization are highly stable. HSR covers 1.7-1.9% of the genome length and 38-40% of the marker chromosome length. The genes localized in HSR provide stable AP resistance. Evidence on some intermediate, relative stabilization of the resistance has been obtained. This stabilization is mediated by temporary integration of DHFR copies into other chromosomal sites, in addition to HSR.  相似文献   

3.
A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.  相似文献   

4.
Summary Conventional and molecular cytogenetic analyses of three murine cancer cell lines that had been induced in male athymic mice by the injection of three different human prostate cancer cell lines revealed selective amplification of the Y chromosome. In particular, analysis of metaphase and interphase nuclei by fluorescence in situ hybridization (FISH) with the mouse Y chromosome-specific DNA painting probe revealed the presence of various numbers of Y chromosomes, ranging from one to eight, with a large majority of nuclei showing two copies (46.5–60.1%). In Interphase nuclei, the Y chromosomes showed distinct morphology, allowing identification irrespective of whether the preparations were treated for 15 min or for 5 h with Colcemid, a chemical known to cause chromosome condensation. However, FISH performed on human lymphocyte cultures with chromosome-specific DNA painting probes other than the Y chromosome did not reveal condensed chromosome morphology in interphase nuclei even after 12 h of Colcemid treatment. Our FISH results indicate that (1) the Y chromosome is selectively amplified in all three cell lines; (2) the mouse Y chromosome number is comparable in both interphase and metaphase cells; (3) the Y chromosome number varies between one and eight, with a large majority of cells showing two or three copies in most interphase nuclei; (4) the condensation of the Y chromosome is not affected by the duration of Colcemid treatment but by its inherent DNA constitution; and (5) the number of copies of the Y chromosome is increased and retained not only in human prostate tumor cell lines but also in murine tumors induced by these prostate tumor cell lines.  相似文献   

5.
HSRs (homogeneously staining regions) are the cytological correlates of DNA amplification. In the house mouse, Mus musculus, many populations are polymorphic for the presence or absence of HSRs on chromosome 1. In the semispecies M. m. domesticus the amplified DNA is present within one HSR, whereas in M. m. musculus chromosomes 1 with two HSRs are found. Hybridization of HSR-specific probes to Southern blots of HSR-carrying genomic DNAs from different localities and semispecies revealed similar complex band patterns. the remaining variation is restricted to sequences with a low degree of amplification. Variation is higher between semispecies than within one semispecies. It is assumed that HSRs are derived from one original amplification event and that unequal recombination is the mechanism underlying the length variation of HSRs present today in both semispecies. Evidence from G-banding and in situ hybridization shows that the two HSRs of M. m. musculus originated from a single HSR by means of a paracentric inversion, where one break-point was located within the single HSR and the second outside the HSR. As a consequence of the paracentric inversion the two HSRs of M. m. musculus are permanently linked together. Since exchange of genes between the two semispecies is restricted to a narrow hybrid zone the amplification that gave rise to the HSR most probably occurred prior to the divergence into the semispecies M. m. domesticus and M. m. musculus about 1 million years ago.by D. Schweizer  相似文献   

6.
A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library ( NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB ). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere- NF1-CRYB1-CHRNB1-TP53-GH1-P4HB , was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53 , previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14–22.  相似文献   

7.
Molecular cloning of genomic sequences altered in cancer cells is believed to lead to the identification of new genes involved in the initiation and progression of the malignant phenotype. DNA amplification is a frequent molecular alteration in tumor cells, and is a mode of proto-oncogene activation. The cytologic manifestation of this phenomenon is the appearance of chromosomal homogeneously staining regions (HSRs) or double minute bodies (DMs). The gastric carcinoma cell line KATO III is characterized by a large HSR on chromosome 11. In-gel renaturation analysis confirmed the amplification of DNA sequences in this cell line, yet none of 42 proto-oncogenes that we tested is amplified in KATO III DNA. We employed the phenol-enhanced reassociation technique (PERT) to isolate 21 random DNA fragments from the amplified domain, and used 6 of them to further clone some 150 kb from that genomic region. While in situ hybridization performed with some of these sequences indicated that in KATO III they are indeed amplified within the HSR on chromosome 11, somatic cell hybrid analysis and in situ hybridization to normal lymphocyte chromosomes showed that they are derived from chromosome 10, band q26. The same sequences were found to be amplified in another gastric carcinoma cell line, SNU-16, which contains DMs, but were not amplified in other 70 cell lines representing a wide variety of human neoplasms. One of these sequences was highly expressed in both KATO III and SNU-16. Thus, the cloned sequences supply a starting point for identification of novel genes which might be involved in the pathogenesis of gastric cancers, and are located in a relatively unexplored domain of the human genome.  相似文献   

8.
This study centers on marker chromosomes carrying expanded chromosomal regions which were observed in two independent derivatives of the AA12 murine fibrosarcoma line, the 10–3 M MTX-res H2 and the 5×10–7 M MTX-res E. Previous characterization of the marker chromosomes of MTX-res variants showed their common derivation from a marker chromosome (m) of the parental line, endowed with two interstitial C-bands. Cytogenetic evidence pointed to one C-band ofm as the site involved in the chromosomal rearrangements leading to the HSR/ASR chromosomes. ISH of a3H-labeled satellite DNA probe allowed satellite sequences flanking the HSR/ASR in the marker chromosomes, where the C-band was no longer visible, to be detected. FISH experiments using biotinylated DHFR and satellite DNA probes showed that the respective target sequences are contiguous in new marker chromosomes. They also allowed inter- and intrachromosomal rearrangements to be seen at DHFR amplicons and satellite sequences. Double-color FISH using digoxygenated satellite DNA and biotinylated pDHFR7 showed that in a marker chromosome from the H2 cell line the two target sequences are not only adjacent, but closer than 3 Mb, as indicated by overlapping of the different fluorescence signals given by the two probes. Another marker chromosome in the E variant was shown to display a mixed ladder structure consisting of a head-to-head tandem of irregularly-sized satellite DNA blocks, with two symmetrical interspersed DHFR clusters.Abbreviations DHFR dihydrofolate reductase - MTX Methotrexate - HSR Homogeneously Staining Region - ASR Abnormally Staining Region - DM Double Minute - ISH In Situ Hybridization - FISH FluorescenceIn Situ Hybridization  相似文献   

9.
10.
A yeast artificial chromosome (YAC) library has been constructed from a somatic cell hybrid containing a t(1p;19q) chromosome and chromosome 17. After amplification, part of this library was analyzed by high-density colony filter screening with a repetitive human DNA probe (Alu). The human YACs distinguished by the screening were further analyzed by Alu fingerprinting and Alu PCR. Fluorescent in situ hybridization (FISH) was performed to localize the YACs to subchromosomal regions of chromosome 1p, 17, or 19q. We have obtained a panel of 123 individual YACs with a mean size of 160 kb, and 77 of these were regionally localized by FISH: 33 to 1p, 10 to 17p, 25 to 17q, and 9 to 19q. The YACs cover a total of 19.7 Mb or 9% of the 220 Mb of human DNA contained in the hybrid. No overlapping YACs have yet been detected. These YACs are available upon request and should be helpful in mapping studies of disease loci, e.g., Charcot-Marie-Tooth disease, Miller-Dieker syndrome, hereditary breast tumor, myotonic dystrophy, and malignant hyperthermia.  相似文献   

11.
A cloned DNA sequence that is specific for a germ line homogeneously staining region (HSR) on chromosome 1 of the mouse was found to be homologous to a single copy sequence in non-HSR mice. By in situ hybridization, the sequence in non-HSR mice was localized to approximately the same site as the insertion site of the HSR on chromosome 1 of HSR mice, indicating in situ amplification of the HSR.  相似文献   

12.
Transfection of a mouse dihydrofolate reductase (DHFR) cDNA contained in a plasmid "expression vector" into DHFR deficient Chinese hamster cells, followed by progressive selection of cells in increasing concentrations of methotrexate (MTX), leads to marked amplification of the exogenous DHFR sequences in the recipient hamster cells. This gene amplification is evident at the cytological level, in the form of homogeneously staining chromosomal regions (HSRs), at a gene expression level, in the form of fluorescein-methotrexate binding, and at the DNA level. Flow sorting, based on variable fluorescein-MTX binding, or direct cellular cloning, followed by chromosome analysis, revealed intercellular heterogeneity of HSRs in size and distribution. This suggested that there was a rapid evolution of HSRs in MTX-resistant transfectants. Chromosomal analysis of HSR evolution in situ, by examining individual colonies presumably derived from one or a few cells, underscored this impression of chromosome structural fluidity. Rates of HSR change in excess of 0.01 per cell division, increased by low doses of the recombinogen, mitomycin C, were detected. The Chinese hamster DHFR transfectants described should be amenable to detailed, coordinate cytological and molecular characterization. Such an analysis should contribute to an understanding of processes such as homologous recombination in mediating HSR evolution in mammalian chromosomes.  相似文献   

13.
Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.  相似文献   

14.
Breakage-fusion-bridge cycles contribute to chromosome aberrations and generate large DNA palindromes that facilitate oncogene amplification in cancer cells. At the molecular level, large DNA palindrome formation is initiated by chromosome breaks, and genomic architecture such as short inverted repeat sequences facilitates this process in mammalian cells. However, the prevalence of DNA palindromes in cancer cells is currently unknown. To determine the prevalence of DNA palindromes in human cancer cells, we have developed a new microarray-based approach called Genome-wide Analysis of Palindrome Formation (GAPF, Tanaka et al., Nat Genet 2005; 37: 320-7). This approach is based on a relatively simple and efficient method to purify "snap-back DNA" from large DNA palindromes by intramolecular base-pairing, followed by elimination of single-stranded DNA by nuclease S1. Comparison of Genome-wide Analysis of Palindrome Formation profiles between cancer and normal cells using microarray can identify genome-wide distributions of somatic palindromes. Using a human cDNA microarray, we have shown that DNA palindromes occur frequently in human cancer cell lines and primary medulloblastomas. Significant overlap of the loci containing DNA palindromes between Colo320DM and MCF7 cancer cell lines suggests regions in the genome susceptible to chromosome breaks and palindrome formation. A subset of loci containing palindromes is associated with gene amplification in Colo320DM, indicating that the location of palindromes in the cancer genome serves as a structural platform that supports subsequent gene amplification.  相似文献   

15.
The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.  相似文献   

16.
B P Kopnin  A V Gudkov 《Genetika》1983,19(6):864-871
DNA-mediated transfer of colchicine-resistance from Djungarian hamster DM5/7 cell line, 750-fold resistant to the drug, was studied. The resistance to colchicine of DM5/7 cells is due to amplification of the genes, possibly coding for the polypeptide p22. Both high-molecular weight DNA (presumably, chromosomal DNA) and low-molecular weight DNA (presumably, extrachromosomal DNA) effectively transferred the colchicine-resistance to Djungarian hamster and mouse cells. DNA of sensitive to colchicine but resistant to ouabain mouse cells CAK-143OuaR was not capable to transfer colchicine-resistance, but effectively transferred ouabain-resistance connected with a mutation in Na+/K+-dependent ATP-ase locus. The differences in genetic transformation with amplified p22 genes and mutant Na+/K+-dependent ATP-ase genes were revealed. All cells of 3 colchicine-resistant transformants of DM-15 cells and all 10 spontaneously derived resistant clones contain the additional chromosome 4. The role of trisomy 4 in the development of colchicine-resistance in DM-15 cells is discussed.  相似文献   

17.
Tyramide signal amplification (TSA)-FISH was used to map one mouse and two human DNA probes of less than 1 kb in size. The two human probes were 319 and 608 bp, and the mouse probe was 855 bp. Probes, made from PCR products, were labeled by incorporating biotin-11-dUTP (human) and biotin-16-dUTP (mouse) during PCR amplification. Signals were readily observed in both interphase and metaphase cells following TSA-FISH for all three genes, whereas conventional FISH experiments produced no signals. The two human ATP-binding cassette (ABC) genes, EST883227 (GenBank Accession No. AA243820) and EST990006 (GenBank Accession No. AA348546), mapped to human chromosomes 7p21 and 17q25. The mouse gene, cmyc (exon 2) mapped to band D2 of mouse chromosome 15. These findings demonstrate the ability of this technique to map small probes (PCR products and expressed sequence tags) of less than 1 kb through highly increased signal amplification.  相似文献   

18.
A family of endogenous retroviruses (enJSRV) closely related to Jaagsiekte sheep retrovirus (JSRV) is ubiquitous in domestic and wild sheep and goats. Southern blot hybridization studies indicate that there is little active replication or movement of the enJSRV proviruses in these species. Two approaches were used to investigate the distribution of proviral loci in the sheep genome. Fluorescence in situ hybridization (FISH) to metaphase chromosome spreads using viral DNA probes was used to detect loci on chromosomes. Hybridization signals were reproducibly detected on seven sheep chromosomes and eight goat chromosomes in seven cell lines. In addition, a panel of 30 sheep-hamster hybrid cell lines, each of which carries one or more sheep chromosomes and which collectively contain the whole sheep genome, was examined for enJSRV sequences. DNA from each of the lines was used as a template for PCR with JSRV gag-specific primers. A PCR product was amplified from 27 of the hybrid lines, indicating that JSRV gag sequences are found on at least 15 of the 28 sheep chromosomes, including those identified by FISH. Thus, enJSRV proviruses are essentially randomly distributed among the chromosomes of sheep and goats. FISH and/or Southern blot hybridization on DNA from several of the sheep-hamster hybrid cell lines suggests that loci containing multiple copies of enJSRV are present on chromosomes 6 and 9. The origin and functional significance of these arrays is not known.  相似文献   

19.
Using a mouse cDNA probe for ornithine decarboxylase (ODC), we have identified and isolated an ODC cDNA clone from a lambda gt11 recombinant library prepared from human liver cell mRNA. The 2.0-kb insert of this clone hybridizes with several mouse genomic ODC DNA restriction fragments under conditions of low stringency, but reacts with only few human DNA fragments and a polyA+ RNA species of 2.2 kb under both nonstringent and stringent hybridization conditions. This suggests that, unlike the mouse genome, there are only few ODC genes in the human genome. The human ODC DNA fragments segregate with chromosome regions 2pter----p23 and 7cen----qter in mouse X human somatic cell hybrid clones containing normal, translocated, and deleted human chromosomes. Sequences of the short arm of chromosome 2 containing the NMYC oncogene at 2p23----p24 are often involved in DNA amplification in neuroblastomas and small-cell lung cancers. However, in at least three cases--one neuroblastoma cell line, one neuroblastoma tumor, and one lung carcinoma--the ODC sequences are not coamplified with the NMYC oncogene.  相似文献   

20.
A M Saunders  M F Seldin 《Genomics》1990,6(2):324-332
The syntenic relationship of the myotonic dystrophy (DM) gene region on human chromosome 19q and proximal mouse chromosome 7 was examined using an interspecific backcross between C3H/HeJ-gld/gld mice and Mus spretus. Segregation analyses were used to order homologs of nine human loci linked with the DM gene. Their order from the centromere was Prkcg, [Apoe, Atpa-2, Ckmm, D19S19h, Ercc-2], Cyp2b, Mag, Lhb. Two other murine loci, D7Rp2 and Ngfg, were also positioned within this interval. Homologs for five human chromosome 11 and 15 loci (Calc, Fes, Hras-1, Igflr, Tyr) were localized within an 18-cM span telomeric to Lhb. Comparison of the gene orders indicates an inversion extending from Prkcg through the interval between Mag and Lhb. This study establishes a detailed map of proximal mouse chromosome 7 that will be useful in identifying and determining whether new human chromosome 19 probes are linked to the DM region.  相似文献   

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