Presence of a Base Line Level of Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER IN VIVO and IN VITRO

Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2'-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 micrograms/ml and 0.25-2.5 micrograms/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).     

小麦愈伤组织细胞的姐妹染色单体交换

用BrdU标记,改良的FPG法染色,建立了一种植物愈伤组织细胞姐妹染色单体分染的方法。并对培养基的不同附加成分对SCE的影响进行了研究。所有培养基上愈伤组织细胞的SCE率都显著高于正常根尖分生组织细胞。6-BA,AgNO_3,高浓度的2,4-D,蔗糖均可诱发SCE。     

Sister Chromatid Exchanges in Tradescantia Paludosa

A modified fluorescence-plus-Giemsa technique is described that allows differential staining of sister chromatids in root tip cells from cuttings of Tradescantia patudesa. With this staining technique, chromatids with both DNA strands unsubstituted are differentiated from chromatids containing 5-bromouracil in place of thymine in one of the strands of the DNA duplex. The baseline level of sister chromatid exchanges was shown to be dependent on the concentration of 5-bromodeoxyuridine in the treatment solution, the mean frequency being 43.5 sister chromatid exchanges per cell for the experimental protocol suggested.     

Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells

Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70(-/-) cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.     

Viability of Homozygous Deficiencies in Somatic Cells of DROSOPHILA MELANOGASTER

The viability of cells made homozygous for different deficiencies by induced mitotic recombination was examined. The deficiencies varied in length from two to 30 polytene chromosome bands and were distributed over the five major chromosome arms. Among a sample of 30, ten deficiencies were cell viable. Our results show that 12% of the genome is necessary for cell survival, supporting previous estimates of about 5,000 genes in the genome of Drosophila.     

限制性内切酶诱发的姊妹染色单体互换

用限制性内切酶PstⅠ,SalⅠ,PvuⅡ和BamHⅠ处理CHO细胞后,发现其SCE率升高,与对照相比,前三种酶具有显著性差异。但这些酶诱导SCE的效应与其致染色体畸变效应相比则较弱,提示引起DNA双链断裂的限制性内切酶不是SCE的强刺激物。实验结果表明,BrdU取代胸苷不能消除限制酶对底物DNA的识别及裂解。     

Aphidicolin-Resistant Mutants of Mouse Lymphoma L5178y Cells with a High Incidence of Spontaneous Sister Chromatid Exchanges

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.     

Behavior of Somatic Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

The behavior in genetic mosaics of 86 EMS-induced sex-linked lethals has been studied. Seventy-five percent of them are autonomous in gynandromorphs. Forty-three lethals nonviable in sex mosaics have been analyzed in X-ray-induced spots in the abdominal tergites and the imaginal wing derivatives. Of the lethals, 90.7% are homozygous viable in mosaic spots, and only 9.3% have been classified as epidermal cell lethal. Thus, the fraction of the Drosophila genome essential for cell viability has been estimated to be about 420 genes. The phenotypes at the cellular level of some cell-viable mutations altering cell parameters (mitotic orientation, differentiation, etc.) are described.     

Aberrations Induced in Chromosomes of Somatic Cells of DROSOPHILA MELANOGASTER Irradiated in C-Metaphase

Experiments conducted on the X irradiation of neural ganglia of Drosophila melanogaster are described. The ganglia were placed in saline containing colchicine. After two hours, they were irradiated and then samples were fixed at 5,15,25,35 minutes from the beginning of irradiation. The results obtained show that the aberration level increases with time subsequent to fixing. This increase takes place first for chromatid deletions and then for isochromatid deletions and chromatid exchanges. Gaps and subchromatid exchanges do not, on the contrary, show any increase with time. We did not observe a difference in radiosensitivity between the sexes. Some hypotheses are put forth in an attempt to explain these results.     

Sister Chromatid Exchanges Induced by Heavy Metals in Vicia Faba

The induction of sister chromatid exchanges (SCE) by chloride and nitrate salts of nickel, cobalt, cadmium and zinc were studied in meristematic root cells of Vicia faba. Salts of nickel, cobalt and cadmium significantly increased the frequency of SCE, whereas chloride and nitrate salts of zinc did not increase the frequency of SCE significantly above the spontaneous level. The reported data demonstrate that the induction of SCE in Vicia faba may represent a valuable bioindicator for detecting the cytogenetic damage of heavy metals.     

Effects of Carvacrol on Sister Chromatid Exchanges in Human Lymphocyte Cultures

Carvacrol is a predominant aromatic compound in oil of oregano. It has naturally remarkable antibacterial, antiviral, antifungal and antiparasital effects. In this study, genotoxic and antigenotoxic activities of carvacrol were investigated by the in vitro sister chromatid exchange (SCE) assay on human peripheral blood lymphocytes. The genotoxicity test was performed with carvacrol in two donors. On the other hand, inhibitory effect of carvacrol was tested in the presence of mitomycin C (MMC) in the same assay. According to data, all doses of carvacrol did not increase the formation of SCE, whereas it inhibited the rate of SCE induced by MMC. In conclusion, carvacrol exhibited a significant antigenotoxic activity in mammalian cells, indicating its potential for use as an antigenotoxic agent.     

Sequential and Combined G-Banding for Identifying Breakpoints in Sister Chromatid Exchanges

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Sequential and Combined G-Banding for Identifying Breakpoints in Sister Chromatid Exchanges

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.