Gene amplification in methotrexate-resistant mouse cells. II. Rearrangement and amplification of non-dihydrofolate reductase gene sequences accompany chromosomal changes.
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Biomedical subjects
Publications and source records attributed to C J Bostock.
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The additional segments of five large marker chromosomes of methotrexate-resistant mouse melanoma PG19T3 cells are shown to consist of C-banding material. In situ hybridization indicates that these additional segments also contain a high proportion of sequences that will cross hybridize with mouse satellite DNA. Analytical density gradient centrifugation suggests that up to 60% of the DNA in the additional segments may be in the form of satellite DNA. Incorporation of bromodeoxyuridine for one complete S phase and staining with the Hoechst 33258 fluorescence plus Giemsa technique reveals complex asymmetries within the additional segments. These asymmetries are interpreted as showing a large repeating unit, which is likely to be involved in the amplification of the dihydrofolate reductase gene.
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DNA was extracted from various rodent-human somatic cell hybrids that contained single or a few human chromosomes. These DNAs were examined by a combination of restriction endonuclease digestion, gel electrophoresis, and filter hybridisation to radioactive satellite DNA probes following transfer of the denatured restriction fragments from a gel to a nitrocellulose filter. In this way the arrangement of sequences homologous to human satellite III were examined on human chromosomes 1, 7, 11, 15, 22 and X. It was found that the distribution of restriction endonuclease sites within satellite III DNA is different on different chromosomes.
PG19T3 mouse melanoma cells were selected for resistance to methotrexate. Nine sub-lines that are resistant to concentrations of methotrexate ranging from 1.27 x 10(-7) M, to 1 x 10(-4) M methotrexate were selected and characterised in terms of their content of dihydrofolate reductase activity and their chromosomes. The intracellular level of dihydrofolate reductase activity increases with increasing resistance such that at the highest level of resistance PG19T3:MTXR10(-4)M cells contain approximately 1,000 fold more enzyme activity than the parental PG19T3 cells. It is shown that the enhanced activity is due to an increase in the amount of the enzyme rather than any structural change to the enzyme in resistant cells. Comparisons of pH activity profiles, profiles under different activating conditions and titrations with methotrexate suggest that the sensitive and resistant cells contain identical dihydrofolate reductases. Analysis of the chromosomes of resistant cells shows the presence of up to 5 large marker chromosomes which contain homogeneously staining regions after G-banding. These same regions stain intensely after C-banding and fluoresce brightly after staining with Hoechst 33258. The size of homogeneously staining regions increases throughout the process of selection. For one marker chromosome this increase may have been mediated via a ring chromosome.
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The frequency of sister chromatid exchanges (SCEs) has been determined for C band and non-C band regions of chromosomes of the kangaroo rat after staining with the fluorescence plus giemsa (FPG) technique. After one complete round of DNA synthesis in the presence of bromodeoxyuridine (BrdU) staining of the C band regions revealed simple or complex asymmetries between chromatids. After two complete rounds of DNA synthesis in the presence of BrdU "harlequin" chromosomes were observed. Analysis of the distribution of SCE in chromosomes at their 1st and 2nd mitosis showed that relatively few exchanges occur within C band regions, although the frequency of SCEs is high at the junction between C band and non-C band chromosome regions.
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The chromosomes of an established cell line of Dipodomys panamintinus have been characterised in terms of their C, G and Q banding patterns, and the distributions of silver grains in autoradiographs of chromosomes labelled in early or late S phase. No relationship could be established between C, G or Q banding regions of chromosomes and a particular S phase time of replication of the DNA in these banded regions. The implication of this result to the concept of heterochromatin is discussed.
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The course of events by which a macronucleus is formed from a micronucleus after conjugation in the ciliated protozoan, Stylonychia, suggests that genetic diminution may occur. This idea is supported by determinations of the density profiles and melting curves for micro- and macronuclear DNAs. Macronuclear DNA consists of a single density component and melts as if it were a single component. Micronuclear DNA consists of four or more density components and melts as if it were a mixture of several DNAs of different base compositions. These data indicate that at least 60% of the micronuclear DNA components are absent from the macronuclear DNA.