Showing papers on "Sister chromatid exchange published in 1976"

Sister chromatid exchange as an assay for genetic damage induced by mutagen-carcinogens. II. In vitro test for compounds requiring metabolic activation.

Abstract: Sister chromatid exchanges (SCE's) which are easily seen by “harlequin chromosome” techniques can be readily induced in cultured Chinese hamster ovary (CHO) cells by low concentrations of mutagen-carcinogens that do not require metabolic activation. If the cells are simultaneously treated with cyclophosphamide which does require metabolic activation before it becomes mutagenic, and an activating system consisting of an extract of rat liver containing microsomes (S-9 Mix) then numerous SCE's are induced by the compound. This indicates that the induction of sister chromatid exchanges in such cells can be used as an in vitro assay for mutagens that require activation as well as those that do not. The method, which is very simple and quick, is more sensitive than is the usual cytogenetic assay in which chromosome aberrations are assayed.

Differential chromatid staining by in vivo treatment as a mutagenicity test system

Abstract: DIFFERENTIAL staining of sister chromatids can be demonstrated with Hoechst 33258 fluorochrome1 and with Giemsa after special pretreatment2, even if 5-bromodeoxyuridine (BUdR) is present during the first S phase only3. These techniques permit the use of the phenomenon of the sister chromatid exchange (SCE). The average spontaneous rate of 10–14 SCEs per metaphase seems to be fairly constant in all cultures from several species4,5. A variety of mutagenic factors (that is, ultraviolet light and alkylating agents) produce very high frequencies of SCE even at concentrations far below the level necessary for the induction of chromosomal breakage, whereas others, like X rays or certain chemicals, hardly influence the rate of SCE6. Because of the high turnover of BrdU in the liver and the physiologically available thymidine, differential chromatid staining has so far only been produced in in vitro systems (cell cultures) and in the chicken embryo.

Analysis of sister chromatid exchange formation in vivo in mouse spermatogonia as a new test system for environmental mutagens

Abstract: SPERMATOGONIA, as stem cells of male gametes1,2, constitute a highly relevant system for studying the genetic hazards of potential mutagens. Gross chromosome aberrations in spermatogonia have been used as a gauge of the genetic damage induced in animals exposed to agents such as mitomycin C (refs 3–5). Sister chromatid exchanges (SCEs) formed in response to DNA damage constitute even more sensitive indices of the impact of alkylating agents and other clastogens on chromosomes6–8. Analysis of SCE induction in cultured cells has been greatly facilitated by techniques in which substitution of DNA with the base analogue 5-bromodeoxyuridine (BUdR) is detected either with fluorescent dyes9,10 or by modified Giemsa procedures11–13. This approach has so far been limited to in vitro trials, with the exception of one system14 in which SCEs were visualised in chromosomes of chick embryos after exposure to BUdR in ovo. We report here a BUdR technique which enables the detection of SCEs formed in spermatogonial cells of intact mice. As a prototype of a general in vivo mutagenesis test, this procedure has been utilised to demonstrate a several-fold increase in the sister chromatid exchange frequency in mouse spermatogonia after injection of the animals with small amounts of mitomycin C.

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells.

Abstract: BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

Abstract: An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 mug/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.

Analysis of the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodomys ordii)

Abstract: 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.

Study of Mitomycin C-induced chromosomal exchange

Abstract: Mitomycin C-induced sister chromatic exchange and quadriradial formation were studied in chromosomes from Muntiacus muntjac fibroblasts and A9, a transformed mouse cell line. We present the first direct cytological evidence for the induction of true somatic recombination involving homologous chromosomes. Analysis of the quadriradials from Muntjac cells indicates a non-random distribution with respect to the chromosomes involved and with respect to the points of conjunction. Sister chromatid exchange and quadriradial formation may reflect the outcome of repair processes involving a high frequency of homologous exchanges in the interphase nucleus.

Differential rates of sister chromatid exchanges between euchromatin and heterochromatin

Abstract: In two rodent species, the Chinese hamster and the montane vole (Microtus montanus), the rate of sister chromatid exchange was lower in constitutive heterochromatin than in euchromatin.

Kinetics of lymphocyte division in blood cultures studied with the BrdU-giemsa technique

Abstract: Normal human lymphocytes were cultured for 72 h with different doses of BrdU. The analysis of metaphases processed with the BrdU-Giemsa method shows that in leukocyte cultures 3 different lymphocyte populations coexist which are able to perform 1, 2 or 3 rounds of replication in vitro. Moreover, it was concluded that 5 mug/ml is the minimal dose of BrdU inducing good differentiation in the areas of sister chromatid exchanges.

On the continuity of chromosomal subunits: an analysis of induced ring chromosomes in Vicia faba

Abstract: The proposition that subunits of a chromatid are continuous in a directional sense has been tested by observing the behaviour of induced ring chromosomes in Vicia faba. On the simplest hypothesis, that the subunits are the uninterrupted complementary strands of the DNA molecule, the polarity of rejoining should result in free separation of rings following replication in successive cell cycles. Centric and acentric ring chromosomes were separately assessed in both diploid and colchicine-accumulated tetraploid metaphase cells of primary root tips. Contrary to expectation large numbers of single and interlocked rings were observed in both cell cycles. Spontaneous sister chromatid exchanges and other breakage-reunion events can produce the configurations seen; with the postulated level of sister chromatid exchange equating that determined autoradiographically in rod chromosomes of V. faba. Unless the replication of ring chromosomes produces conditions unusual in rod chromosome replication, spontaneous breakage is probably common in replicating or post replication Vicia chromosomes. — A fundamental difference exists between the behaviour of centric and acentric ring chromosomes. Acentric ring chromosomes behave as if the chromatid arm were one DNA molecule, or a number of DNA molecules with identical directional sense. However, centric ring chromosomes behave as if there were a difference at the centromere in at least one (probably the metacentric) chromosome of the Vicia complement. That is, the two “duplication-segregation” subunits which extend the length of the chromosome, may contain a change in polarity at the centromere.

Dependency of sister chromatid exchange in T- and B-cells on the incorporation of deoxyribonucleosides into chromosomal DNA.

Abstract: Human T-cells (CCRF-HSB2) did not incorporate tritiated thymidine ([3H]TDR)--1.0-5.0 muCi/ml--into the nuclei, where.as they readily incorporated tritiated deoxycytidine (E13H]CDR). When contamination with pleuropneumonia-like organisms was ruled out, these findings strongly suggested a deficiency of the enzyme thymidine kinase in the cells. Human B-cells (CCRF-SB) and normal T-lymphocytes (NTL) readily incorporated [3H]CDR, [3H]TDR, and tritiated 5-bromodeoxyuridine, and they clearly exhibited differential staining of the sister chromatids (SCD). When nonisotopic bromodeoxyuridine (BUDR), 10(-6)-10(-4) M, was used with the B-cells and NTL, SCD were clearly evident and sister chromatid exchange (SCE) was relatively infrequent; when the concentration was 10(-7) M, SCD staining was poor but the frequency of SCE was high. SCE frequencies in NTL, measured by autoradiography after incorporation of [3H]CDR, were the same as SCE frequencies measured by staining with BUDR at 10(-4) M. In the case of CCRF-HSB2, 10(-4) M BUDR produced relatively high frequencies of SCE as did 10(-7) M BUDR with the former two cells. However, [3H]CDR with CCRF-HSB2 gave relatively low frequencies of SCE, of the magnitude observed after 10(-4) M BUDR was used with NTL and the B-cells. Thus the high frequency of SCE in CCRF-HSB2 cells may have been due to the staining property of chromosomes that had incorporated low levels of BUDR.

Detection of Hybrid DNA Formed during Mitomycin C-Induced Sister Chromatid Exchange in Chinese Hamster Cells

Abstract: Most cells appear to possess several repair mechanisms capable of removing the damage produced in their DNA by mutagens such as γ-rays, ultraviolet (UV) light, and mitomycin C The most widely studied of these is that involving excision of the damage and patching of the DNA by repair synthesis (Howard-Flanders, 1973) However, it is now known that other systems of repair involve a process of recombination In particular, this second type of mechanism is necessary for the repair of damage involving both strands of the DNA molecule Such damage occurs, for example, when UV-irradiated cells replicate their DNA and gaps are left in the newly synthesized strand opposite pyrimidine dimers in the old strand, or when cells are treated with mitomycin C, an agent that causes cross links to be formed between the two strands (Iyer and Szybalski, 1963) Genetic recombination is greatly stimulated by agents causing this type of damage