Chromatid Fragments Research Articles

Identification of kinetochores and DNA synthesis in micronuclei induced by mitomycin C and colchicine in Chinese hamster ovary cells.

The presence of kinetochore and DNA synthesis in micronuclei (MN) induced in Chinese hamster ovary (CHO) cells by clastogenic and aneuploidogenic substances such as mitomycin C (MMC) and colchicine was determined by immunofluorescence technique using CREST antikinetochore antibodies and anti-bromodeoxyuridine (BrdUrd) antibodies. A cytofluorimetric analysis was also performed. Colchicine significantly increased micronucleated cells at least up to 96 h from the end of treatment. As expected, among colchicine-induced micronucleated cells the majority contained at least one CREST + MN. MMC induced a significant increase in micronucleated cells up to 120 h from the end of treatment and the great majority of MN lacked kinetochore fluorescence, indicating that MMC-induced MN were derived from acentric fragments. However, colchicine and MMC at 48 and 72 h from the end of treatment, induced a significant increase of CREST- and CREST + MN, respectively, suggesting an induction of clastogenicity by colchicine and aneuploidy by MMC. The clastogenic effect of colchicine after 48 h was also confirmed by the presence of chromatid fragments in metaphase cells. A cytofluorimetric analysis indicated that, as expected, colchicine and MMC interfere with the G2/M and S phases, respectively; however, a slight interference of colchicine with the S phase was also observed. DNA synthesis was present in MN and it was in most cases synchronous with synthesis in the main nucleus. The frequency of cells with MN in S phase observed in untreated or MMC-treated cells is in agreement with the proportion of cells without MN showing DNA synthesis. On the contrary, the frequency of cells with MN in S phase observed in colchicine-treated cells was significantly lower than that observed in control and MMC-treated cells.

Genotoxic effects of some systemic pesticides: in vivo chromosomal aberrations in bone marrow cells in rats.

The genotoxic effects of five pesticides (benomyl, 2,4-D, dimecron, monocrotophos, and vitavax) were evaluated in the rat bone marrow cytogenetic assay. The spectrum of aberrations observed included chromatid breaks, chromatid fragments, ring chromosomes, dicentric chromosomes, and chromosome fragments. It was observed that 2,4-D, dimecron, and vitavax were clastogenic, but the results obtained with benomyl and monocrotophos were equivocal.

Potential uses of sea urchin embryos for identifying toxic chemicals: description of a bioassay incorporating cytologic, cytogenetic and embryologic endpoints.

A method for evaluating pollutant genotoxicity, embryotoxicity and teratogenicity using sea urchin embryos has been developed and was tested using benzo(a)pyrene (BP). Initial results suggested that the bioassay may be a sensitive indicator of pollutant toxicity and mutagenicity since several endpoints can be simultaneously assessed. The bioassay is rapid, inexpensive and appears applicable to a variety of toxicants and delivery methods. The test is based upon the standard 48 h sea urchin development assay and incorporates cytologic-cytogenetic analysis of embryos. Following toxic exposure of gametes, fertilization success is assessed. Embryos then develop for 48 h at which time survival and teratogenesis are evaluated. A subsample of embryos is stained and dissociated into monolayers and mitotic configurations are examined using light microscopy. Embryo mitotic rates are used as an indicator of overall embryonic health. Cytotoxic effects are concomitantly evaluated. Genotoxicity is measured using two methods: (1) anaphase aberration analysis, a technique which assesses abnormalities in the chromosome configurations (such as bridges and fragments) as the groups of chromosomes move to opposite poles and (2) micronucleus formation, a procedure examining the incidence of smaller, secondary nuclei composed of whole chromosomes or chromatid fragments. These two measurements preclude the need to examine individual chromosomes for deletions and exchanges, a laborious process in most aquatic organisms which possess numerous relatively small chromosomes. This genotoxicity-teratogenicity test appears promising for laboratory evaluations of individual substances or of complex chemical mixtures as well as for environmental monitoring of nearshore areas. The standard development assay has been used to screen pharmaceuticals and environmental contaminants and some recent investigations have included mitotic aberration analysis. Experiments in our laboratory suggest that the genotoxicity-teratogenicity test may be a feasible approach to field monitoring. Mutagen loads of spawning adult urchins could be assessed by conducting cytologic-cytogenetic analysis of resulting embryos although initial studies suggest that this method is less sensitive than direct embryo exposures.

The size of micronuclei in human lymphocytes varies according to inducing agent used

Micronuclei were induced in vitro in human lymphocytes by mitomycin C, X-rays, vincristine, and colcemid and analyzed in cells with preserved cytoplasm. The micronucleus/cell nucleus ratio was measured. It was found that micronuclei induced by mitomycin C and X-rays were significantly smaller than those formed by vincristine and colcemid. Thus, in spite of the wide size span of human chromosomes, it could be shown that it is possible to differentiate between micronuclei formed by spindle-damaging agents (vincristine and colcemid) and those induced by agents directly damaging the chromosomes (mitomycin C and X-rays). Mitomycin C-induced micronuclei were smaller than those induced by X-rays, probably because the former agent preferentially produces chromatid fragments and the latter chromosome fragments.

Spontaneous chromosomal aberration levels in human peripheral lymphocytes

Spontaneous levels of structural chromosomal aberrations in human peripheral lymphocytes were studied cytogenetically in 49 female and 56 male subjects. With a total of 16 267 metaphase spreads examined, 191 cells were found to contain chromosomal aberrations, giving a rate of 1.17%. The rates of individual aberration types were as follows: chromatid fragments, 0.39%; chromosome fragments, 0.71%; dicentrics, 0.06%; symmtrical and asymmetrical chromatid exchanges, 0.01%; and rings, 0.006%. There were significant differences in aberration yields between females aged 20–29 yr. and 60–70 yr., whereas males aged 20–50 yr. showed no difference. The two sexes differed significantly in chromatid fragments, chromosome fragments and aberrant cells.

Somatic variations on a yellow mutant in Nicotianatabacum L. ( a1+/ a1a2+/ a2) II. Reciprocal genetic events occurring in leaf cells

Twin spots observed on leaves are frequently assumed to be somatic crossovers from heterozygous loci. However, no genetic evidence has been obtained from progeny tests on the two reciprocal products. In this study, plants were regenerated from such reciprocally variant areas by in vitro bud induction, and genetically tested. Reciprocal variations were observed on four different segregants from an initial J 1 mutant ( a 1 +/ a 1 a 2 +/ a 2) 11 and on a fifth genotype obtained from a somatic variation on a 1 +/ a 1 + a 2/ a 2. In eight cases, both variants from one reciprocal variation were obtained; they were therefore considered as two products of one single genetic event. The variants were mono-, di- or trihybrids. Three types of twinned variations were distinguished on the basis of the locus (loci) involved in the modifications, viz., (1) the a 1 +− a 1 locus involved in both products; (2) the a 1 +− a 1 locus involved in one product and a 2 +− a 2 involved in the other (3) either one of the loci a 1 +− a 1 and a 2 +− a 2 involved in one product and a third unknown locus in the other; this third locus segregated independently from the other two known loci and acted additively with the a j + genes. The events proposed to explain these variations are respectively: (1) symmetrical exchange between homologous chromatids, with of without loss of the eschanged fragment(s). It was not possible to show whether or not the exchange was equal; (2) reciprocal or non-reciprocal translocations between chromatids carrying homeologous genes; (3) single translocation of a chromatid fragment to a locus other than a 1 +− a 1 or a 2 +− a 2. A monosomic analysis showed that the a 1 +− a 1 and a 2 +− a 2 loci are located on chromosomes R and S, respectively. There are two arguments that sustain the homeology hypothesis between these loci: (1) the two monosomics, a 1 a 2 +/ a 2 + and a 1 +/ a 1 a 2 +, both carrying a 1 and two wild genes, have the same greenish-yellow J 1 phenotype, and (2) when in a 1 +/ a 1 a 2 +/ a 2, a 2 + is substituted for a 1 by chromatid translocation, the new genotype behaves as a 1 +/ a 1 + a 2 +/ a 2.

Further studies with Vicia faba on the ends of experimentally produced chromatid fragments

In a recent study, when X-irradiated chromosomes of Vicia faba were treated with trypsin, we observed two types of ends of chromatid fragments, namely “open” and “closed” ends. To put this qualitative finding on a quantitative basis, the fraction of “open” ends among the total number of ends lassified was determined. It amounted to 58.1% (18/31). When the X-irradiation was replaced by treatment with an effective chromosome-breaking agent, namely FUdR (5-fluorodeoxyuridine), again both “open” and “closed” chromatid fragment ends were observed and a similar fraction of “open” ends was found, namely 43.2% (16/37).

Further studies with Vicia faba on the ends of experimentally produced chromatid fragments

Further studies with Vicia faba on the ends of experimentally produced chromatid fragments

The structure of chromatid ends and of the ends of experimentally procuced chromatid fragments as revealed by trypsin treatment of Vicia faba chromosomes

After digestion with trypsin the metaphase chromatids of Vicia faba reveal two length structures (half-chromatids). This confirms the results of other authors. According to our observations the half-chromatids are connected at their ends in such a way that a U-shaped configuration is formed. This finding can be explained in different ways. The ends of chromatid fragments obtained after X-irradiation are either U-shaped (“closed”) like the “natural” chromatid ends or “open”, i.e. the two half-chromatids do not seem to be connected at their ends. As a by-product of our experiments the structure of X-ray-induced achromatic lesions (gaps) was studied in trypsin-treated chromosomes.

Cytogenetical study of bone-marrow in man after a single local gamma-irradiation: dose- and time-relationships.

SummaryDirect chromosome studies of bone-marrow cells were made in a control group of four tumour patients and in a group of 39 patients at different intervals of time (1 to 96 hours) after a single local irradiation with 60Co gamma-rays at doses of 50, 100, 150, 200 and 250 rads. The data obtained demonstrated that the frequency and type of chromosome aberrations are dependent on the time after irradiation. The most frequent aberrations induced in bone-marrow cells by gamma-rays are chromatid and chromosome fragments. It was shown that the yield of aberrant cells increases linearly with increase in dose.

Chromatin bridges and origin of multinucleate cells in a bovine testicular cell line.

In the course of cytological observations of a cell line of bovine testicular origin since its initiation, many chromatin bridges and binucleate cells were noticed following the apparently spontaneous occurrence of chromatid fragmentation and formation of dicentric chromosomes. Subsequently, the frequency of binucleate cells and their respective nuclear sizes were recorded from permanent slides of material of passages made prior to, during, and after the incidence of chromatid fragmentation. Since the frequency of binucleate cells rose simultaneously with that of the chromatin bridges, and since the same nuclear size was involved in most cases, the frequency of chromatin bridges was taken as an index of their contribution to the production of binucleate cells following the period of chromatid fragmentation.

The relative biological efficiency of single doses of fast neutrons and gamma-rays on Vicia faba roots and the effect of oxygen. Part II. Chromosone damage: the production of micronuclei.

SummaryThe frequency of micronuclei resulting from acentric chromosome and chromatid fragments induced by cobalt-60 gamma radiation and by ∼3 mev fast neutrons has been studied in the root-tip meristematic cells of Vicia faba. Roots irradiated with gamma-ray doses of 107 and 188 rads whilst in aerated water showed a greater micronuclear frequency than roots irradiated with similar doses in the absence of oxygen. The ratio of doses given in nitrogen and in air in order to produce the same micronuclear yield was found to be about 2·4. With roots irradiated with fast-neutron doses of 2·02, 4·95 and 9·94 rads the effect of oxygen is less marked, the nitrogen/air dose-ratio being about 1·4.For a given level of chromosome damage produced under conditions of aeration, neutron radiation was found to be about 10·5 times as effective as gamma-rays. Because of the quantitative difference in the response of the two types of radiation to the presence of oxygen, the relative biological efficiency of neutrons to gamma-r...

Cytological Observations Concerning Inversion and Translocation in the House Mouse

1. Balanced lethal lines of tailless mice containing a crossover suppressor have been examined cytogenetically. A low frequency of chromatid bridges and fragments in anaphase indicates that if inversion is present it is very small. 2. Pseudo-bridges and lagging chromosomes were found rather frequently in spermatogenesis of both tailless and control animals. 3. Chromosome rings indicating segmental interchanges have been found in spermatogenesis of semi-sterile males, belonging to three different semi-sterile lines.

Chromosomal Aberrations in Onion Roots from Plants Grown in an Atmosphere Containing C14O21

1. Sets of the Ebenezer variety of onion grown in the special nutriculture chamber at the Argonne National Laboratory under controlled temperature and humidity conditions had their natural atmospheric carbon dioxide supply supplemented by the addition of C14O2 so that the total concentration varied from 0.03% to 0.1%. The photoperiod was extended to 16 hours by means of incandescent lamps. 2. One leaf from a plant of approximately a dozen grown showed a chlorophyll-deficient streak along its entire length. There were no chlorotic streaks in control plants. 3. Roots from the carbon 14 plants showed both chromosome and chromatid breaks and fragments in dividing cells, bridging, and micronuclei. No aberrancies were observed in dividing cells of control roots.

Effects of Fast Neutrons upon Plants, II

Three different effects (the primary, mitosis-free period and the secondary effects) of neutron bombardment on the root-tips of Vicia faba were described cytologically. The primary effect consists of advancement of the mitotic process from metaphase to telophase, retardation of the same mitotic process owing to the chromosome aberration and relative advancement of chromosome formation from prophase chromonemata. The former may chiefly be attributed to the effect on the cytosome and chromosome (dehydration) which resulted in an increase in the number of mitotic figures in the telophase, while the latter two are attributed to the effect an the chromosomes, namely fragmentation, clumping and hydration. These, moreover, result in an accumulation of mitotic figures in the metaphase and later stages.The mitosis-free period results from the delay in chromosome formation from the resting nuclei, induced after irradiation.The secondary effect, which reveals itself after the mitosis-free period, shows abnormalities appearing in the process of recovery of cells from the effect of the neutron bombardment. Fragmentation and fusion of the chromosomes and irregular mitoses were observed 96 hours after irradiation.The fragments formed were chiefly chromosome fragments and rarely chromatid fragments. The abnormal changes in somatic mitoses produced by the action of neutron rays may be comparable with those seen in the case of the desiccation treatment (cf. Wada 1936).These investigations were carried out according to the program of the Atomic Nucleus Sub-Committee of the Japan Society for the Promotion of Scientific Research to which we wish to express our gratitude. We are indebted to the Japan Wireless Telegraph Company for the electromagnet and other pieces of equipment used for the cyclotron, and to the Mitsui Hoonkwai Foundation, the Tokyo Electric Light Company, and Mr. G. Hattori, Director of K. Hattori Company, for financial aid. We acknowledge the kind assistances given by our colleges of the Nuclear Research Laboratory in connexion with the irradiation of samples.

A Reciprocal Translocation in <i>Lillium Hansonii</i> LEICHT (A Preliminary Note)

Normal plants of Lillium Hansonii (2n=24) form always 12II at IM (in 200 metaphasic plates observed) (Fig. 1). A plant heterozygous for a reciprocal translocation showed invariably 1IV+10II at that stage (in 342 cases observed) (Fig. 2). The metaphasic configuration of the tetravalent was a ring modified by a number of interstitial chiasmata. This type of metaphasic configuration is a conclusive evidence that the translocation is reciprocal.It is an important point revealed by the present study that in this plant the kinetochores of the homologous chromosomes retained paired until the IA in bivalents as well as in tetravalents (Figs. 1-2).In tetravalents the chromosome part between the paired kinetochore and the chiasma nearest to it frequently appears very short (Figures of such cases not shown). Considering the above fact interchanged chromosomes Ab and Ba are obviously the result of a mutual trans-location of the major part of both long arms of the normal chromosomes Aa and Bb (cf. IIA in Fig. 3).2.1 (in 240 cases observed) and 2.2 per cent. (in 831 cases observed) of PMCs at IA was irregular in normal and reciprocal translocation plants respectively. A majority of irregularities are bridges not accompanied by fragments and their configurations indicate that they result from a half-twist between the chromatids of a dyad (Fig. 4, a-i). The others are bridges accompanied by fragments which would have probably originated from chromatid fragmentation and fusion (Fig. 4, j-k).

Chromosome fragmentation produced by crossing-over inTrillium erectum L.

A chromatid fragment lacking an attachment has been found to occur in about 12 per cent. of the pollen mother cells in two plants ofTrillium erectum L.