Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. IV. Genetic transformation using amplified genes from Djungarian hamster cells highly resistant to colchicine

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.     

Amplification of portions of the genome in mammalian somatic cells resistant to colchicine. II. Marker chromosomes with long homogeneously staining regions in colchicine-resistant cells of the Djungarian hamster

The series of sublines 170-750 times more resistant to colchicine were obtained from 10 independent clones of Djungarian hamster cells possessing 16-22-fold resistance to the drug. From each clone, several sublines with different levels of colchicine-resistance were developed. The drug resistance was unstable. 2,7-4,0% of cells per population doubling lost resistance to selective dosages of colchicine. The loss of resistance was stepwise. The chromosomes stained by trypsin G-banding technique were studied in 17 sublines. 15 sublines derived from 9 independent clones contained chromosomes with long homogeneously staining regions (HSRs). These were, as a rule, primarily localized in the long arm of chromosome 4. During cultivation, HSRs were transferred from chromosome 4 into other chromosomes. Evidently, transposition of HSRs was due to translocations of different chromosomes of HSRs in the chromosome 4 and to subsequent breakages of the resulting dicentrics within HSRs. A great number of different chromosomal rearrangements was also found in the cells containing HSRs. Possibly, formation of HSR leads to destabilization of the karyotype and to the variability of the genome. The length of HSRs varied in different cells of each subline. The levels of colchicine-resistance in different sublines did not correlate with the average length of HSRs in their cells. The lack of connection between the lengths of HSRs and the levels of drug resistance as well as the existence of highly resistant sublines with gene amplification, but without HSRs, suggest that amplified genes are localized in Djungarian hamster colchicine-resistant cells both in chromosomes and extrachromosomally.     

Amplification of genome regions in the somatic cells of mammals resistant to colchicine. III. Localization of the amplified genes in minute chromatin bodies

Small chromatin bodies (SCB) were revealed in Djungarian hamster cells resistant to colchicine. They looked like single bodies or like clusters of small particles. SCB were localized both in nucleus and cytoplasm. Similar formations were earlier observed in oocytes of insects with amplified extrachromosomal rDNA genes. DNA in the SCB was able to replicate not only during the S phase but also during other phases of the cell cycle. The restriction analysis showed that in cells with SCB DNA amplified sequences were replicated autonomously too. These data indicate that SCB in colchicine-resistant cells contain amplified genes. Besides, SCB double-minute chromosomes (DMs) were observed in some resistant sublines. In one of them, DMs were the only karyotypic alteration. The relationship between SCB, chromosomal homogeneously staining regions (HSRs) and DMs was studied. Single SCB and DMs appeared at the early stage of the development of colchicine-resistance (the level of drug resistance is 16-22). Selection of variants 170-220-fold resistant to colchicine was usually accompanied by the decrease in the cell number with SCB and DMs and by the increase in the amount of cells containing the chromosomes with HSRs. During the further enhancement of drug resistance (700-750), some decrease in the number of cells with HSRs and the appearance of the great number of cells containing large groups of SCB were found. The loss of colchicine-resistance observed during cultivation in colchicine free medium was accompanied by the disappearance of HSRs, emergence of SCB and DMs and further elimination of SCB and DMs from cells. The quantity of autonomously replicating amplified DNA fragments after digestive by HindIII was increased with the enhancement of SCB number in cultures.     

Amplification of portions of the genome in the somatic cells of mammals resistant to colchicine. V. The induction of gene amplification in the cells of the Djungarian hamster and the mouse

The influence of some agents on gene amplification in Djungarian hamster and mouse cells was studied. The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), the epidermal growth factor (EGF), insulin, and 5-bromodeoxyuridine (BUdR) increase the incidence of colchicine-resistance, connected with amplification of the genes, which probably encode the polypeptide p22. The highest frequency of gene amplification was observed after the pretreatment of cells with TPA, which enhanced the number of colchicine-resistant colonies 44-200-fold. Mitostatic agents colchicine and colcemid increased the number of methotrexate-resistant cells, 2.0-6.5 times. These cells usually arise as the result of amplification of dihydrofolate reductase genes. Dexamethasone and ethidium bromide did not change the portion of cells resistant to colchicine. Ethylmethane sulfonate (EMS) decreased the number of colchicine-resistant cells. The cells of two Djungarian hamster colchicine-resistant clones obtained after treatment with TPA did not differ from those of spontaneously derived colchicine-resistant clones. Both have similar survival patterns in the medium with different colchicine concentrations, unstable inheritance of the drug resistance, the additional chromosome 4 and small chromatin bodies-the structures containing the amplified genes. Possible mechanisms of the induction of gene amplification by the agents used are discussed.     

Amplification of regions of the genome in the somatic cells of mammals resistant to colchicine. VII. Localization of the initial and amplified copies of gene mdr in one and the same segment of chromosome 4 of the Djungarian hamster

By in situ hybridization technique, the mdr gene which is amplified during the development of multiple drug resistance was mapped in the 4q15--21 segment of normal Djungarian hamster chromosome 4. As was shown earlier, this chromosomal region is specific for the location of amplified mdr gene copies. These results, as well as some data obtained by other authors, suggest that recombinations of amplified DNAs occur preferentially in or near the sites bearing homologous sequences.     

DNA sequences amplified in cancer cells: an interface between tumor biology and human genome analysis.

There is growing evidence that amplification of specific genes is associated with tumor progression. While several proto-oncogenes are known to be activated by amplification, it is clear that not all the genes involved in DNA amplification in human tumors have been discovered. Our approach to the identification of such genes is based on the 'reverse genetics' methodology. Anonymous amplified DNA fragments are cloned by virtue of their amplification in a given tumor. These sequences are mapped in the normal genome and hence define a new genetic locus. The amplified domain is isolated by long-range cloning and analyzed along three lines of investigation: new genes are sought that can explain the biological significance of the amplification; the structure of the domain is studied in normal cells and in the amplification unit in the cancer cell; attempts are made to identify molecular probes of diagnostic value within the amplified domain. This application of genome technology to cancer biology is demonstrated in our study of a new genomic domain at chromosome 10q26 which is amplified specifically in human gastric carcinomas.     

Gene amplification in methotrexate-resistant mouse cells. IV. Different DNA sequences are amplified in different resistant lines

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.     

Cre-stimulated recombination at loxP-containing DNA sequences placed into the mammalian genome.

The cre gene of coliphage P1 encodes a 38 kDa protein which efficiently promotes both intra- and intermolecular recombination at specific 34 bp sites called loxP. To demonstrate that the Cre protein can promote DNA recombination at loxP sites resident on a mammalian chromosome, a mouse cell line was constructed containing two directly repeated loxP sites flanking a 2.5 kb yeast DNA fragment and inserted between the SV40 promoter and the neo structural gene to disrupt expression of the neo gene. Expression of the cre gene in this cell line results in excision of the intervening yeast DNA and thus permits sufficient expression of the neo gene to allow cell growth in high concentrations of G418. Southern analysis indicated that Cre-mediated excision occurred at the loxP sites. In the absence of the cre gene such excisive events are quite rare. Cre-mediated recombination should thus be quite useful in effecting a variety of genomic rearrangements in eukaryotic cells.     

Amplification of polyomavirus DNA sequences stably integrated in rat cells.

To investigate the mechanism by which the polyomavirus large T antigen (T-Ag) promotes amplification of integrated viral sequences, we constructed a rat cell line, Hy2-ts5, carrying two different inserts of polyomavirus DNA. The first insert, designated the middle T (pmt) locus, was devised to analyze homologous recombination between two defective copies of pmt lying 3.3 kb apart on the same chromosome. Reconstitution of a functional pmt by spontaneous recombination occurred at a rate of about 2 x 10(-7) per cell generation. The second locus contained the polyomavirus large T (plt) gene carrying a temperature-sensitive mutation and producing a nonfunctional large T-Ag at 39 degrees C. A shift to the permissive temperature for as little as 24 h induced the production of a functional large T-Ag which, in turn, promoted homologous recombination in the pmt locus at a rate close to 1.0 per cell generation. The particularity of this system is that it allowed recombination products to be analyzed as early as a single cell doubling following the initial recombinational event. Amplification occurred by successive duplications of a discrete sequence in the viral insert. Unequal sister chromatid exchange was ruled out as the recombination mechanism promoted by large T-Ag. Instead, we proposed a model of nonconservative recombination involving mispairing between homologous sequences.     

Instability of plasmid DNA integrated into mammalian somatic cell genome

The phenomenon of loosing exogenic DNA from the mammalian somatic cell genome is under investigation. It is found that foreign DNA incorporated into cell genome as a result of transfection by electrophoretion may be lost with the frequency from 1/100 up to 1/100 000 per cell division during cultivation. This effect is not dependent of the nature of cell line and vector DNA. It is actual for different cell lines: A23, human fibroblasts AG 11395, murine embryonic line F9, and for different plasmid vectors: p16, p.39, pATR4 and pcDNA3.1-Higr (WRN). Integration of pDNA into genome and the following loosing of this DNA is registered by selection markers G418 and hygromycin B resistance and gancyclovir sensibility. The presence of foreign DNA in the genome was controlled by PCR. It is found that true foreign DNA deletion from the genome takes place rather than gene expression changes. For closely linked plasmid genes deletion of both genes at once as well as loosing any one gene separately is shown. Thus, the phenomenon of selective deletion of exogenic DNA from genome has been demonstrated for different mammalian cells.     

Random amplified polymorphic DNA analysis of eel genome

Eel family is a huge one, in which many kinds of eels especially some migratory eels, bear strong resemblance to each other, and are therefore difficult to be identified. In this study 29 random primers were used to make RAPD analysis for Japaneses eel (Anguilla japonica), European eel (Anguilla anguilla) and Pike eel (Muraenesox cinereus).And totally 299 fragments were counted.Shared or specific fragments were counted and genetic similarity or genetic distance were calculated.The genetic similarity between Japanese eel and Pike eel is 0.68 and the genetic distance between them is 0.32;those between European eel and Pike eel are 0.72 and 0.28 respectively,and between Japanese eel and European eel are 0.74 and 0.25 respectively.The method has been shown to be suitable to molecular identification of eels.It provides an alternative approach to determine the relationship between species.     

The existence of DNA sequences homologous to adenovirus 5 DNA in the genome of normal rat cells.

Three discrete bands specifically hybridizing to adenovirus 5 DNA were found in the rat liver DNA restricted BY Bam HI endonuclease and fractionated electrophoretically. The hybridization with different regions of the viral genome takes place. Similar bands are present in the DNA from different lines of adenovirus 5 transformed cells, but in these cases high molecular weight DNA fragments containing the integrated viral genomes can also be found.     

Repair of DNA in replicated and unreplicated portions of the human genome.

Portions of the human genome that have replicated after ultraviolet light irradiation and those that remain unreplicated have both been examined for the distribution of pyrimidine dimers and the extent of repair replication following their removal. The data indicate that the number of unrepaired dimers and the extent of repair replication seen after their excision are equal in the replicated and unreplicated DNA. Furthermore, the daughter strand of replicated DNA is larger than the average interdimer distance found in the parental strand. Hence, DNA replication in normal human fibroblasts is clearly capable of getting past pyrimidine dimers, and a preferential repair of such lesions in DNA that is about to be or has been replicated does not operate to any visible extent in these cells.     

Restriction analysis of the Frankia spp. genome

Abstract Total cellular DNAs of 10 Frankia isolates from Alnus, Elaeagnus and Colletia spp. root nodules were cleaved with ten site-specific restriction endonucleases and analysed by agarose gel electrophoresis. Among the endonucleases tested, Bam HI, Bgl II, Sal I and Sma I proved to be the most suitable for the genome analysis in Frankia spp. DNA restriction banding patterns were reproducible and characteristic of each Frankia strain. The patterns of different strains differed marked indicating considerable genotypic heterogeneity among the isolates. The approach can be used for strain identification in Frankia spp. as well as for differentiation between phenotypically similar strains.     

Isolation of DNA probes for the detection of sequences amplified in colchicine-resistant cells

Earlier we have found that the development of resistance to colchicine in mammalian cells in vitro is due to gene amplification leading to decreased plasma membrane permeability to the selective agent and some other unrelated drugs. By a stepwise self-renaturation procedure followed by chromatography on hydroxyapatite we isolated the fraction of middle-repeated sequences (DNAc0t = 10-250) enriched in amplified DNA from the DNA of colchicine-resistant Djungarian hamster cell line. Blotting-hybridization with [32P]DNAc0t = 10-250 performed in the presence of the excess of unlabelled DNA from wild type cells reveals amplified sequences in resistant cell lines. The comparison of DNAs from cell lines resistant to colchicine, adriablastin and actinomycin D showed that common but not identical DNA sequences are amplified in these cases. In situ hybridization with [3H]DNAc0t = 10-250 indicates that amplified sequences are located in the long homogeneously staining regions (HSRs) of the marker chromosomes. These results suggest that DNAc0t = 10-250 may be used for screening of recombinant molecules containing amplified sequences.     

Restriction enzyme analysis of satellite DNA components from the bovine genome

A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.     

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated HR-15 and HR-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. HR-15 and HR-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that HR-15 and HR-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.