Evidence that sister chromatid exchanges and chromatid breaks are two independent events

The relative frequencies of sister chromatid exchanges (SCE) and chromatid breaks in BrdU (5-bromodeoxyuridine) — sensitive site (lq22 lq23) in Chinese hamster cells after BrdU incorporation were studied. The results show that chromatid breaks do not follow the exchange hypothesis and provide evidence that chromatid breaks and SCEs are two independent events despite some common features.     

Deoxycytidine reverses the suppression of nucleolar organizer regions' activity caused by BrdU

Summary In this article we report that 5-bromodeoxyuridine (BrdU) significantly suppresses the nucleolar organizer regions (NORs) activity of Chinese hamster cells (both dipliod cells and cell line Wg3-h) (P<0.001). One of the most obvious characteristics of the suppression is a significant decrease in the total number of the Ag-NORs per cell rather than in a frequency variation of the associated Ag-NORs. The decrease in the Ag-NORs number is mainly because of the decrease in number of chromosomes bearing 2 Ag-NORs. The degree of the suppression increases with increase in BrdU concentration in the culture medium. There is a close relationship between the suppression and the BrdU-treatment time, i.e. for a given concentration of the BrdU, the longer the BrdU-treatment time, the stronger the suppression. When the BrdU-treated cells are transferred into BrdU-free medium and allowed to grow in it for another 30 h, NORs activity can be restored. Therefore, the suppression of NORs activity may be due to BrdU toxicity. When deoxycytidine (dC) is added into medium containing 30 g/ml of BrdU, the total number of both the Ag-NORs and the chromosomes bearing Ag-NORs per cell increases to the level of untreated cells. Our results thus indicate that the addition of dC reverses the suppression of the NORs activity caused by BrdU.     

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

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 g/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.     

Sister chromatid differential staining and NOR activity of BrdU-resistant sublines

Summary Two 30 g/ml BrdU-resistant sublines and two 60 g/ml BrdU-resistant sublines are induced from a Chinese hamster cell line Wg3h (HGPRT^–) by one-step and two-step selections, respectively. By inoculating the cells into BrdU-free medium or by adding more BrdU into the culture medium for 26–27 h, it was found that the two BrdU-resistant sublines analysed have very clear sister chromatid differential (SCD) staining patterns. This indicates that some of the nuclear DNA of the BrdU-resistant cells incorporate with BrdU to reach a kinetic balance. Frequencies of sister chromatid exchange (SCE) of the resistant cells are twice to four times as high as those of the Wg3h cells, depending on which BrdU-resistant subline is analysed. The SCE frequencies of the resistant cells also increase with the BrdU concentration in the medium. Analysis of silver-stained nucleolar organizer regions (NORs) indicates that the NOR activity of three out of the four BrdU-resistant sublines is significantly suppressed, i.e., averages of the Ag-NOR number and number of the chromosomes bearing Ag-NORs per cell decrease significantly. The degree of suppression for different BrdU-resistant sublines may be quite different. The suppressed NOR activity of the resistant cells can gradually be restored when the cells are inoculated into BrdU-free medium, but the recovery speed is far lower than that of the Wg3h cells. The suppression of the NOR activity of the BrdU-resistant sublines should be due to BrdU toxicity.     

Radiation induced isostaining: fact or fiction?

Two different experiments were set up to induce differential staining in M-1¹ cells or to induce isostaining in M-2 cells after -irradiation of human lymphocytes. Neither of these two unusual staining patterns previously described by Luchnik and Porjadkova (1977) were observed. Possible explanations for this disagreement are discussed.Abbreviations SCE Sister chromatid exchange - IS Isostained regions - S-1, S-2, S-3 and S-4 First, second, third and fourth DNA synthetic periods after addition of 5-bromodeoxyuridine - M-1, M-2, M-3, M-4 and M-5 First, second, third, fourth and fifth mitosis after addition of 5-bromodeoxyuridine - BrdU 5-bromodeoxyuridine - EMEM Eagle's Minimum Essential Medium     

Sertoli cells of adult rats “in vitro”

Summary A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied in vitro over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n. 74.00155.04 and n. 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, ItalyPart of this work was presented at the 10th Italian Congress of Electron Microscopy. Ostuni 1–4 October 1975The excellent technical assistance of Miss Laura Vassallo, Daniela Venturini and Mr. Massimo Rosati and Mario Termine is deeply appreciated     

Isolation and subregional mapping of a human cDNA clone detecting a common RELP on chromosome 12

Summary Peripheral blood lymphocytes from three patients with Down syndrome (DS; trisomy 21; aged 5–6 years) and three age-matched control children were studied for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs).Cells in G₀ were exposed to bleomycin (20–100 g/ml) for 3 h, and then cultured in medium containing 5-bromodeoxyuridine and phytohemagglutinin for 66 h. By the sister chromatid differential staining method, chromosome analyses were performed on metaphase cells that had divided one, two, or three or more times after treatment. The results indicate that DS cells exposed to bleomycin are hypersensitive to the production of dicentric and ring chromosomes compared to normal cells. Bleomycin also led to a dose-related increase in the frequency of SCEs, but no difference was found between the SCE frequencies in DS or normal lymphocytes exposed to bleomycin.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff     

Sister chromatid exchange frequency,cellular replication and relative cloning efficiency in human teratocarcinoma-derived cells

To determine the relationships between the induction of specific biological responses and exposure to DNA-damaging agents, human teratocarcinoma-derived cells were exposed to either ethyl methanesulfonate or to methyl methanesulfonate, and sister chromatid exchange, cellular proliferation and relative cloning ability measured. SCE increased while cellular proliferation and relative cloning ability each decreased in a concentration-dependent manner. Methyl methanesulfonate was consistently more efficient in inducing biological responses than was ethyl methanesulfonate. When the individual responses were compared, the decrease in cellular proliferation paralleled the reduction in cloning efficiency. A strong correlation was also observed between the reduction in relative cloning ability and sister chromatid exchange frequency. Because these relationships are similar to those previously described in other mammalian cell lines, the observations in our study suggest that the P3 cell line is an appropriate choice for modeling effects of toxicant exposure in human cells.Abbreviations AGT average generation time - BUdR 5-bromodeoxyuridine - CHO Chinese hamster ovary - EMS ethyl methanesulfonate - ENU N-ethyl-N-nitrosourea - MMS methyl methanesulfonate - MNU N-methyl-Nnitrosourea - SCE sister chromatid exchange     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Cloned β1,4N-acetylgalactosaminyltransferase: subcellular localization and formation of disulfide bonded species

Cloned human 1,4N-acetylgalactosaminyltransferase (GalNAcT) catalyses the synthesis of the glycosphingolipids GM2, GD2, and gangliotriosylceramide. To determine the subcellular location of this enzyme and whether it exists in intermolecular disulfide bonded species, we stably transfected Chinese hamster ovary (CHO) cells with three myc epitope-tagged forms of the GalNAcT gene: the native enzyme; the lumenal domain of GalNAcT fused to the cytoplasmic and transmembrane domains ofN-acetylglucosaminyltransferase I (GNT); and the transmembrane and lumenal domains of GalNAcT fused to the cytoplasmic domain of the Iip33 form of human invariant chain in order to retain the enzyme in the endoplasmic reticulum (ER). Immunoelectron microscopic analysis with anti-myc revealed that GalNAcT/myc was present throughout the Golgi stack, the GNT/GalNAcT/myc form was restricted primarily to the medial Golgi cisternae, and the Iip33/GalNAcT/myc form was restricted to the ER. Cells transfected with each of the three constructs contained high levels of GM2 synthase activityin vitro, but only the GalNAcT/myc form and the GNT/GalNAcT/myc forms were able to synthesize the GM2 productin vivo. The enzyme produced by all three constructs was present in the transfected cells in a disulfide bonded form having a molecular size consistent with that of a homodimer or higher aggregate.Abbreviations GSL glycosphingolipid(s) - CHO Chinese hamster ovary - GSL structures: GM2 GalNAc1,4(NeuAc2,3)Gal1,4GlcCer - GD2 GalNac1,4(NeuAc2,8NeuAc2,3)Gal1,4GlcCer - GM3 NeuAc2,3Gal1,4GlcCer - Gg₃ GalNAc1,4Gal1,4GlcCer - LacCer Gal1,4GlcCer - GlcCer glucosylceramide - PBS-BSA phosphate buffered saline pH 7.4 containing 1% bovine serum albumin - GalNAcT N-acetylgalactosaminyltransferase - GNT N-acetylglucosaminyltransferase I - Iip33 p33 form of human invariant chain - HPTLC high performance thin layer chromatography - PCR polymerase chain reaction - BFA Brefeldin A This paper is dedicated to Professor Sen-itiroh Hakomori on the occasion of his 65th birthday.     

Isolabelling is a radiation-induced phenomenon

Human lymphocytes were incubated during two mitotic cycles in the presence of 5-bromodeoxyuridine and differentiation between chromatids was obtained with combined Hoechst 33258 and azur-eosine staining. Analysis of non-irradiated cells revealed numerous sister chromatid exchanges (SCE) and no abnormalities of harlequine appearance of chromosomes. When, however, the cells were irradiated, an identical staining (IS, isostaining) of some chromosomes or chromosome segments were observed. Production of IS was accompanied by decrease of the frequency of SCE, the total frequency of SCE+IS remained, however, the same as in control. An antagonism between SCE and IS was established: the frequency of SCE decreased in the cells with multiple IS, and chromosomes with both SCE and IS were only rarely observed. Thus, IS is neither an artifact nor a physiologic event but a phenomenon induced by radiation. The reliable existence of IS is considered as an evidence for binemic structure of chromatid. It is suggested that some mechanism of lateral spread of genetic information is involved in the production of SCE. If delayed by radiation, the spread could be restricted only to a fraction of chromosome cross-section resulting in IS.     

Characterization of mitoses with sister chromatid differentiation (SCD) and consequences for the analysis of proliferation kinetics and sister chromatid exchanges in asynchronously growing cells

Summary Labeling cells with bromodeoxyuridine (BrdU) permits the differentiation of mitoses of the first, second, and third generation after the addition of BrdU. The term second mitoses is used for those cells which have incorporated BrdU for two-S-phases and which exhibit sister chromatid differentiation (SCD). However, SCD can also be obtained if the cell was in S-phase at the time of BrdU-addition and had already replicated part of its DNA. Such cells with incomplete BrdU-substitution in the first S-phase can only be differentiated from completely substituted ones by the quality of the SCD and are usually also grouped as second mitoses in the evaluation of experiments. Due to the heterogeneity of the evaluated second mitoses, the determination of proliferation delay and the incidence of sister chromatid exchange-induction can depend on the time of chromosome preparation.     

Sister chromatid exchanges are preferentially induced at expressed and nonexpressed common fragile sites

Summary To investigate the relationship between common fragile sites and sister chromatid exchange (SCE), lymphocyte cultures were treated with aphidicolin and bromodeoxyuridine (BrdU) and analyzed using a sequential GSCE staining protocol. A total of 1163 SCEs were mapped to their corresponding G-band sites, which were assigned to one of the following four categories: fragile sites expressed; fragile sites nonexpressed; nonfragile sites with breaks; or nonfragile sites with no breaks. The designated common fragile sites were found to be preferred locations for SCE formation, not only when these sites were expressed as visible gaps or breaks, but even when they were nonexpressed in the cell. SCEs were also more likely to occur at nonfragile sites with breaks than at nonfragile with no break sites. Further, SCEs were found to be distributed nonrandomly across fragile sites and nonfragile sites, and among the fragile sites, the high frequency SCE sites were highly correlated with the high frequency breakage sites. These data support the hypothesis of common steps in the mechanism of aphidicolin-induced SCE formation and common fragile site expression.     

Reduced N-methyl-N′-nitro-N-nitrosoguanidine sister chromatid exchange induction in chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine

The sequence in which N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 5-bromodeoxyuridine (BrdU) are added to cell cultures affects the number of sister chromatid exchanges (SCE) induced by MNNG. When V79 Chinese hamster cell monolayer cultures were treated with MNNG for 2 h prior to addition of BrdUrd, approximately a 4–5-fold increase in SCE was observed at the second division metaphases compared to controls exposed to BrdU alone. This effect was independent of whether one or three DNA strands had been substituted as a result of incubating the cells through one or two DNA synthesis periods in the presence of BrdU. This increase in SCE also occurred after MNNG exposure and BrdU incubation was extended for three division cycles. In contrast, when BrdU incorporation preceded MNNG treatment, the average number of SCE/metaphase was reduced 70–80% at the second division cycle and 60% relative to the total number found in three division cycles. SCE induction by MNNG does not involve a caffeine sensitive step since caffeine had no effect on the SCE frequency regardless of the treatment protocol. The conditions in which BrdU preceded MNNG exposure may be responsible for either reducing the number of DNA sites available for interaction with MNNG or preventing the expression of SCE.     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

Methyl methane-sulphonate (MMS) induced SCEs are reduced by the BrdU used to visualise them

SCE induction in synchronised CHO cells treated with methyl methane sulphonate (MMS) in G₁ was studied over successive pairs of cell cycles by introducing bromodeoxyuridine (BrdU) at consecutive G₁ stages. When individual cell cycle SCE values were calculated from the data, anomalous results were obtained with ratios of 1.01.82.1 for the first three cycles but a negative value for the fourth cycle. Further studies using different BrdU concentrations showed that MMS induced SCEs were reduced by values exceeding 50% in DNA containing high levels of incorporated BrdU. This reduction was dose dependent and accounted for the anomalous results obtained over successive cycles. Lesions leading to chromatid exchanges were also reduced by the same mechanism. SCEs induced by UV irradiation were also decreased but those induced by the cross-linking agent nitrogen mustard (HN₂) remained unaffected. The results indicate that not only are SCE lesions induced by MMS, UV or HN₂ expressed independently of the spontaneous SCEs induced by BrdU but that SCE lesions are multiple in nature. Mechanisms by which SCE lesions could be repaired in BrdU containing DNA are discussed. SCE lesions in MMS treated cells arrested in G₁ with arginine deprived medium (ADM) are repaired without the presence of BrdU in the DNA. An opposite effect is seen however in the control cells, where SCEs are increased with time spent in ADM arrest. These interactions between the effects of MMS, BrdU and ADM arrest are discussed.     

Genetic variation detected by use of the M13 “DNA fingerprint” probe in Malus,Prunus, and Rubus (Rosaceae)

Summary Recently, DNA fingerprints have been reported in a wide array of organisms. We used the M13 repeat probe on several genera and species in the angiosperm family Rosaceae. Four apple cultivars could be differentiated when any one of five restriction enzymes was used to analyze minisatellite DNA. Similarly, four individual trees of Prunus serotina (black cherry) exhibited different fingerprints with each of four enyzmes. A total of 14 Rubus (blackberries and raspberries) plants representing four species were investigated with two enzymes. Extensive inter-and intraspecific variation was found. However, some closely growing plants had identical fingerprints, probably due to their being derived through vegetative propagation.     

Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis.     

In vivo sister chromatid exchange in cells of various organs of the mouse

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 g/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.