The induction of aneuploidy by colcemid fed to Drosophila melanogaster females

Colcemid, a chemical closely related to colchicine, was fed to Drosophila melanogaster females (0.0001 and 0.0005%, respectively). In the F₁ the frequency of aneuploid males (XO karyotype) and aneuploid females (either of the XXY or of the XXXY karyotype, with 2 and 3 sets of autosomes, respectively) was significantly higher than in the controls as shown by genetical methods supplemented by cytological tests. A consistent brood pattern effect was observed, possibly but not necessarily reflecting differential stage sensitivity to the action of colcemid. It seems plausible to assume that most of the aneuploid exceptions were produced via colcemid-induced spindle defects leading to lagging of the X-chromosome. Exclusion of the lagging X from the pronucleus (or its precursors) would yield XO males, its inclusion would yield XXY females. Definitely more XO than XXY exceptions were observed.     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

The replication of human heterochromatin in serial culture

The human C group chromosomes late to start replication in asynchronous and in FUdR synchronized cell lines are X chromosomes. These same chromosomes are also heterochromatic during interphase. During metaphase these allocyclic Xs cannot be identified simply by metaphase position or morphology and show a wide range of measurements for arm ratio, centromere index and total length. Replication starts in the short arm and extends over the entire chromosome during the 2nd and 3rd hr of S until by the 4th hr distinction from other C group chromosomes cannot be made by means of the labeling pattern. When the allocyclic X chromosomes start replication the pattern of H³TdR label over interphase sex chromatin and non-specific heterochromatin shifts from unlabeled to labeled in FUdR synchronized human cell lines. The overall time required for replication of the allocyclic X is less than that for the other chromosomes in both asynchronous and FUdR treated cells. A hypothesis is presented for a direct relation between the delay of onset of replication in heterochromatin and its degree of interphase condensation.The present study was supported by research grants: No. HD-00777 from the National Institutes of Health and No. E-487 from the American Cancer Society, Inc.     

X-chromosomal nondisjunction induced by aging oocytes of Drosophila melanogaster: the special susceptibility of mature eggs

In Drosophila melanogaster suppression of oviposition results in an aging of both mature and immature oocytes. When oviposition was suppressed for four days, the incidence of X-chromosomal nondisjunction (XXY exceptions) in mature oocytes was more than doubled, whereas in immature oocytes the nondisjunction frequency was not increased. It is shown that this special susceptibility of mature oocytes to aging-induced nondisjunction has to be considered in experiments on the induction of nondisjunction by chemical agents.     

DNA synthesis in growth and encystment of Acanthamoeba castellanii.

Differentiation of Acanthamoeba castellanii into dormant cysts occurs spontaneously in stationary phase cultures, or can be induced experimentally by starvation. Although no further increase in cell density occurred after induction in either case, incorporation of [H]thymidine into DNA continued at a reduced rate through the period when differentiated products (cyst wall components) were formed. No net accumulation of DNA occurred during differentiation, indicating that the DNA synthesis occurring at this time was balanced by breakdown. When either 5-fluorodeoxyuridine (FUdR) or hydroxyurea was added to exponentially growing cultures, growth was terminated and the subsequent spontaneous encystment was delayed in comparison with untreated stationary phase cultures. A similar delay was observed for experimentally induced encystment of FUdR-pretreated cells. In all cases, delay of encystment was correlated with inhibition of 32PO4 incorporation into DNA, and unexpectedly also into RNA. Addition of FUdR at zero-time of experimental induction of cells not previously exposed to FUdR, on the other hand, had no effect on encystment or on 32PO4 incorporation. The delay of encystment produced by FUdR and hydroxyurea, therefore, appeared to reflect a requirement for normal synthesis of DNA and/or RNA not only during encystment, but also during the period of exponential growth just before encystment induction.     

Sex-chromosome aberrations in wood lemmings (Myopus schisticolor)

The wood lemming displays certain peculiar features: (1) The sex ratio shows a prevalence of females (FRANK, 1966; KALELA and OKSALA, 1966), and some females produce only female offspring (KALELA and OKSALA, 1966). (2) In a considerable proportion (in the present material, slightly less than half) of the females, an XY chromosome complement is found in the somatic tissues, but the Y is absent in the germ line of those studied (Fredga et al., 1976). Therefore, (3) a mechanism of double nondisjunction in early fetal life of XY females has to be postulated, which replaces the Y in the germ line by duplication of the X. It is assumed (4) that the X of XY females bears a sex-reversal factor that affects the male determining action of the Y (Fredga et al., 1977). There is (5) a strong presumption that in most cases the XY females are those that produce daughters only, but (6) a few exceptions may occur (FRANK, unpublished observations), suggesting that the regulation according to assumption 3 (perhaps also to 4) is incomplete in XY females. In the present report, four females are described with a 31,XO karyotype, two females with 33,XYY or 32,XY/33,XYY, respectively, two males with a 33,XXY, and one male with a 32,XX/33,XXY karyotype, as observed in a consecutive series of 502 wood lemmings. The incidence of sex-chromosome anomalies in liveborn and adult animals was 2.3%; the overall incidence, including embryos, was 1.79%. Neither the somatic XO constitution nor the existence of an extra Y in females precludes fertility. However, the XXY condition in the male results in sterility. There is certain evidence that an instability of the proposed mechanism for double mitotic nondisjunction of the sex chromosomes in oogonia accounts for the high rate of sex-chromosome aberrations in wood lemmings, at least when the mother is XY.     

Sex chromosome aneuploidy in wild small mammals

We describe four examples of the XO condition in wild mammals. One XO house mouse (Mus musculus domesticus) was caught in nature and subsequently gave birth to three litters in captivity, confirming for wild mice the fertility observed for XO laboratory mice. Two other XO house mice were produced from laboratory crosses of wild-caught mice. An immature XO common shrew (Sorex araneus) was caught in nature; this appears to be the first XO recorded in the order Insectivora. We collected data from researchers studying chromosome variation in house mice and common shrews and found an overall incidence of 0.22% sex chromosome aneuploidy in 4608 mice and 0.05% in 6625 shrews. The discrepancy related to a much higher frequency of XO's in mice than shrews. Single XXY and XYY shrews and an XXX mouse have been recorded in nature.     

Cytogenetic investigations in mentally retarded and normal males from 14 families with the fragile site at Xq28

Summary Lymphocyte cultures from 27 mentally retarded males aged 1 year to 77 years, and from 11 normal brothers from a total of 14 families with the fragile X segregating have been examined cytogenetically employing three different culture methods including methods for induction of fra(X) by FUdR (fluorodeoxyuridine) or MTX (methotrexate). All mentally retarded males showed unequivocal fra(X) expression. No statistically significant correlation between fra(X) expression and age could be demonstrated. No enhancement with FUdR was observed. Fibroblast cultures from 10 retarded males expressed fra(X) in a dose-response relationship to increasing concentrations of FUdR. None of the normal males showed fra(X). In vivo folic acid treatment of seven mentally retarded males resulted in marked reduction in fra(X) expression in lymphocyte cultures grown in medium 199. However, reinduction was achieved by FUdR or MTX, except in one case who temporarily received very high doses of folic acid.     

A Reappraisal of Xanthine Dehydrogenase and Oxidase in Hypoxic Reperfusion Injury: the Role of NADH as an Electron Donor

Xanthine oxidase (XO) is conventionally known as a generator of reactive oxygen species (ROS) which contribute to hypoxic-reperfusion injury in tissues. However, this role for human XO is disputed due to its distinctive lack of activity towards xanthine, and the failure of allopurinol to suppress reperfusion injury. In this paper, we have employed native gel electrophore-sis together with activity staining to investigate the role human xanthine dehydrogenase (XD) and XO in hypoxic reperfusion injury. This approach has provided information which cannot be obtained by conventional spectrophotometric assays. We found that both XD and XO of human umbilical vein endothelial cells (HUVECs) and lymphoblastic leukaemic cells (CEMs) catalysed ROS generation by oxidising NADH, but not hypoxanthine. The conversion of XD to XO was observed in both HUVECs and CEMs in response to hypoxia, although the level of conversion varied. Purified human milk XD generated ROS more efficiently in the presence of NADH than in the presence of hypoxanthine. This NADH oxidising activity was blocked by the FAD site inhibitor, diphenyleneiodo-nium (DPI), but was not suppressible by the molybdenum site inhibitor, allopurinol. However, in the presence of both DPI and allopwinol the activities of XD/XO were completely blocked with either NADH or hypoxanthine as substrates. We conclude that both human XD and XO can oxidise NADH to generate ROS. Therefore, the conversion of XD to XO is not necessary for post-ischaemic ROS generation. The hypoxic-reperfusion injury hypothesis should be reappraised to take into account the important role played by XD and XO in oxidising NADH to yield ROS.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

DNA replication in mammalian cells after the action of factors of a physical, chemical or biological nature. V. The structural reorganization of replicating DNA in HeLa cells after incubation with 5-fluorodeoxyuridine

A study was made of sedimentation properties of the nucleoid (chromatin) of HeLa cells with radio- and thermostable mode of DNA synthesis induced by 5-fluorodeoxyuridine (FUdR). After the incubation of HeLa cells with FUdR (10(-6) M, 6 h or 24 h) the rate of nucleoid sedimentation was shown to rise by 40 and 25%, respectively. Maximum relaxation of the nucleoid was observed under 5 mg/ml ethidium bromide concentration in sucrose gradients. After the incubation with FUdR the nucleoid relaxes to a lesser extent, and after irradiation its response to ethidium bromide in various concentrations was similar to that of intact nucleoid, and by this property the "FUdR nucleoid" differs essentially from the irradiated "normal nucleoid". A model of chromatin structure of cells exposed to FUdR is proposed, based on the transformation of large domains in small ones, for the explanation of radioresistant DNA synthesis.     

DNA replication in mammalian cells exposed to the action of physical, chemical or biological factors. IV. 5-fluorodeoxyuridine modifies the effect of hyperthermia on DNA synthesis and the survival of HeLa cells

Preliminary incubation of logarithmically growing HeLa cells with FUdR decreases an inhibitory effect of hyperthermia (43 degrees C, 1 hour) on DNA synthesis. The hyperthermia alone inhibits DNA synthesis considerably: the label in acid-precipitable material accounts for 30% of control level. Preliminary incubation of the cells with FUdR (10(-6)) for 24 or 6 hours (plus 18 hours in fresh medium) decreases the effect: the label yields account for 50 or 90% of the respective control levels. A molecular weight of nascent DNA synthetized in the cells after hyperthermia or incubation with FUdR is lower than the control one but it increases rapidly during postincubation. Nucleoid of cells treated with FUdR has a sedimentation velocity which exceeds that of the control cells by more than 25%. Preliminary incubation with FUdR sensitizes the cells to hyperthermia. The effect is not believed to be associated with cells synchronization since the treatment of the cells with FUdR for 2 or 6 hours, when FUdR itself does not exert its toxic effect, brings about sensibilization of cells to hyperthermia. It is suggested that modification of the cell viability and DNA replication are related to some changes of chromatine structure induced by FUdR.     

Microtubule rearrangements during mitosis in multinucleate cells

The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.     

Enhanced sensitivity of lymphoblastoid cells from individuals carrying the mutation for the fragile X syndrome to the clastogenic effects of FUdR

Individuals known to carry the mutation for the fragile X syndrome can sometimes be identified cytogenetically by the presence of a fragile site on the X chromosome at q27.3. The frequency of cells bearing this fragile site is known to be enhanced by culturing the cells in folic acid deficient medium and/or by introducing folic acid metabolism inhibitors such as FUdR. In this study FUdR induction of chromosomal aberrations other than the fragile X was investigated. Lymphoblastoid cells from an obligate carrier, a mentally retarded male and a control were cultured in folic acid deficient medium in the presence of FUdR and harvested at various times after culture initiation. The frequency of chromosome and chromatid breaks was found to be higher in cells from the individuals carrying the mutation for the fragile X syndrome. The frequency of micronuclei, an indirect index of chromosome breakage, was also more elevated in cells from these individuals than in cells from the control. These findings are of potential importance to carrier detection of this common genetic disorder.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

De novo X-chromosome inactivation in somatic hybrid cells between the XO mouse embryonal carcinoma cell and XY rat lymphocyte

Polyethylene glycol-mediated cell fusion between the hypoxanthine phosphoribosyl transferase (HPRT) deficient XO mouse embryonal carcinoma cell line, PSA-6TG1, and thymus or spleen lymphocytes from the normal male WKA/Hok rat gave rise to 35 somatic hybrid cultures. Hybrid cells being products of either a 1 : 1 or a 2 : 1 fusion invariably had morphological characteristics of endodermal cells from early embryos. BrdU-acridine orange (AO) fluorescence microscopy revealed do novo appearance of a late or early replicating, presumably genetically inactivated, mouse X chromosome in a substantial proportion of virtually tetraploid (XXY) or hexaploid (XXXY) hybrid cells.     

Induction of chromosomal aberrations in active oxygen-generating systems. II. A study with hydrogen peroxide-resistant cells in culture

Cells hyper-resistant to hydrogen peroxide (H2O2) were obtained from a Chinese hamster cell line (CHL) by repeated treatments with H2O2 at stepwise increasing concentrations. A clonal line (R-8) was approximately 10 times more resistant to H2O2 than the parental cells, and retained its resistance for about 2 months in normal medium. However, with further passages after the completion of the present study, the elevated resistance gradually decreased. Although the concentration of H2O2 required to induce chromosomal aberrations in 50% of treated cells was about 10 times higher in R-8 than in the parental cells, there were no distinct differences between the cells in the induction of chromosomal aberrations by 3 alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and mitomycin C). The catalase activity of R-8 was 10-fold in comparison with the parental cells, but no obvious differences were seen in the activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase. Therefore, the elevated H2O2-resistance seemed to be associated with the enhanced catalase activity. The induction of chromosomal aberrations in two O2- generating systems--xanthine oxidase plus hypoxanthine (XO + HX), and paraquat--was compared between R-8 cells and the ordinary CHL cells. XO + HX produced chromosomal aberrations in the parental cells but not in the R-8, while paraquat induced almost the same level of aberrations in both cell lines. This finding suggests that different active oxygens are responsible for the induction of aberrations in these two O2- generating systems, i.e., H2O2 in XO + HX and O2- in paraquat.     

Role of xanthine oxidase activation and reduced glutathione depletion in rhinovirus induction of inflammation in respiratory epithelial cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-kappaB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.     

Chromosomal Abnormalities and Their Relation to Disease

When human chromosome anomalies were first described in 1959, it appeared that specific abnormalities might be correlated with specific syndromes. Mongolism and the D and E syndromes are examples of specific syndromes associated with the presence of an extra autosome. Klinefelter''s syndrome may be associated with a variety of different sex chromosome anomalies including XXY, XXYY, XXXY and XXXXY. The lastnamed variant is the only one that frequently presents features distinguishing it from the others. An XO sex chromosome complex is found in many women with gonadal dysgenesis. However, a variety of mosaicisms have been described in association with this condition, including XO/XX, XO/XXX, XO/XX/XXX, XO/XY and XO/XYY. Extra X chromosomes in phenotypical females do not seem to impair fertility or be consistently associated with congenital anomalies. Two families are described in which chromosome anomalies were found, but the association with defects was irregular. In one family the abnormality involved one of the number 16 chromosomes and in the other it involved one of the small acrocentric chromosomes.