The analysis of exchanges in tritium-labelled meiotic chromosomes

The autoradiographic analysis of exchanges in tritium-labelled meiotic chromosomes is potentially a useful approach to the study of meiotic exchange events since this method differentially labels meiotic chromatids along their entire length. The main problem encountered in earlier autoradiographic studies is that of distinguishing label exchanges generated at chiasmata from label exchanges generated by sister chromatid exchange. This problem was overcome in the present study by the choice of a meiotic system (male meiosis of Stethophyma grossum) where chiasmata are limited to just one proximally localised chiasma in each bivalent. This system allows the positive identification of chiasma-generated label exchanges and demonstrates convincingly the origin of chiasmata through breakage and rejoining of homologous non-sister chromatids. Sister chromatid exchanges are also readily detected in labelled meiotic chromosomes of this species, where they occur with a mean frequency of 0.35 per chromosome. This frequency is similar to that found in mitotic spermatogonial cells and the exchanges are randomly distributed both within and between chromosomes. These features of meiotic sister chromatid exchanges suggest that they are unrelated to non-sister chiasmatic exchanges and they probably have no special meiotic significance.     

Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes

Summary Lymphocytes from 20 normal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU—Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G groups had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.     

Colchicine promotes a change in chromosome structure without loss of sister chromatid cohesion in prometaphase I-arrested bivalents

In somatic cells colchicine promotes the arrest of cell division at prometaphase, and chromosomes show a sequential loss of sister chromatid arm and centromere cohesion. In this study we used colchicine to analyse possible changes in chromosome structure and sister chromatid cohesion in prometaphase I-arrested bivalents of the katydid Pycnogaster cucullata. After silver staining we observed that in colchicine-arrested prometaphase I bivalents, and in contrast to what was found in control bivalents, sister kinetochores appeared individualised and sister chromatid axes were completely separated all along their length. However, this change in chromosome structure occurred without loss of sister chromatid arm cohesion. We also employed the MPM-2 monoclonal antibody against mitotic phosphoproteins on control and colchicine-treated spermatocytes. In control metaphase I bivalents this antibody labelled the tightly associated sister kinetochores and the interchromatid domain. By contrast, in colchicine-treated prometaphase I bivalents individualised sister kinetochores appeared labelled, but the interchromatid domain did not show labelling. These results support the notion that MPM-2 phosphoproteins, probably DNA topoisomerase IIalpha, located in the interchromatid domain act as "chromosomal staples" associating sister chromatid axes in metaphase I bivalents. The disappearance of these chromosomal staples would induce a change in chromosome structure, as reflected by the separation of sister kinetochores and sister axes, but without a concomitant loss of sister chromatid cohesion.     

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.     

Detection of sister chromatid exchanges by 4′-6-diamidino-2-phenylindole fluorescence

This paper describes a 4-6-diamidino-2-phenylindole (DAPI) fluorescent technique for differentiation of sister chromatids and for the study of sister chromatid exchanges (SCE) in mouse chromosomes. The advantages of the DAPI fluorescent technique are also described. Differences in the occurrence of SCE between the centromeric heterochromatin (C-banded) and the chromosomal arm chromatin were studied in mouse cells (RAG) with or without mitomycin C treatment. Single strand exchanges between the DNA double helices in the sister chromatids were not detected. SCE and chromosome breakage appeared to occur more frequently in the centromeric region than in the chromosomal arm. This might play an important role in chromosome evolution in mice.     

Sister chromatid exchanges in barley

Summary A mean frequency of 20.6 sister chromatid exchanges (SCEs) per cell has been observed in a reconstructed karyotype of Hordeum vulgare by application of the FPG technique after unifilar incorporation of BrdU into chromosomes. The involvement in SCEs of the 48 segments into which the chromosome set had been subdivided was, with a single deviation, length proportional and independent of the segment's heterochromatin content. Asymmetric bands, indicative of an uneven distribution of adenine and thymidine between the DNA strands in adenine (A)-thymidine (T) rich chromosome regions, could not be detected after incubation of the cells in BrdU for one cycle of DNA replication.     

One-Step and Stepwise Magnification of a BOBBED LETHAL Chromosome in DROSOPHILA MELANOGASTER

Bobbed lethal (bbl) chromosomes carry too few ribosomal genes for homozygous flies to be viable. Reversion of bbl chromosomes to bb or nearly bb+ occurs under magnifying conditions at a low frequency in a single generation. These reversions occur too rapidly to be accounted for by single unequal sister chromatid exchanges and seem unlikely to be due to multiple sister strand exchanges within a given cell lineage. Analysis of several one-step revertants indicates that they are X-Y recombinant chromosomes which probably arise from X-Y recombination at bb. The addition of ribosomal genes from the Y chromosome to the bbl chromosome explains the more rapid reversion of the bbl chromosome than is permitted by single events of unequal sister chromatid exchange. Analysis of stepwise bbl magnified chromosomes, which were selected over a period of 4-9 magnifying generations, shows ribosomal gene patterns that are closely similar to each other. Similarity in rDNA pattern among stepwise magnified products of the same parental chromosome is consistent with reversion by a mechanism of unequal sister strand exchange.     

Comparison of sister chromatid exchanges from three successive cell cycles in Wallabia bicolor chromosomes

An autoradiographic analysis of tritiated thymidine labeled chromosomes of Wallabia bicolor at the second and third metaphases after label incorporations has shown that sister chromatid exchanges (SCE's) from the first and second cell cycles are less than as frequent as SCE's from the third cell cycle after label. Exchange levels per cell cycle estimated at the seconf division are under-estimated due to coincident exchanges. In both methaphases exchanges were largely distributed at random along Wallabia chromosomes with frequencies proportional to chromosome length. The ratio of twin: single SCE's in spontaneoulsy occuring tetraploid cells indicated the first cycle exchanges were marginally more frequent than second cycle exchanges.These data are compatible with exchange probabilities being equal and independent over divisions, but a component of exchanges reducing as tritium content in chromosomes decreases cannot be excluded. This findings that SCE's are primarily independent of tritium cannot be attributed to a saturation of sites for exchange and it is therefore probable that sister exchanges are, in part at least, spontaneous events in Wallabia chromosomes.     

Ring Chromosomes and rDNA Magnification in Drosophila

Tartof showed that ribosomal gene magnification in Drosophila was inhibited in a ring X chromosome. The present studies extend this observation by showing that ring X chromosomes are lost meiotically in male Drosophila undergoing ribosomal gene magnification as evidenced by the recovery of a lower number of ring-bearing progeny under magnifying conditions compared with nonmagnifying conditions. Associated with ring chromosome loss is a highly significant increase in the number of double-sized dicentric ring chromosomes in meiotic cells from magnifying males. These observations explain the failure of ring X chromosomes to magnify and imply that magnification in rod chromosomes occurs via a mechanism of unequal sister chromatid exchange. Our results support the hypothesis that the primary event of magnification is a sister chromatid exchange in the rDNA, that the frequency of sister strand exchanges is increased in magnifying flies, that a significant number of exchanges in magnifying flies occurs meiotically and that some of the exchanges are nonreciprocal. We have also found that autosomal mutations can affect both the frequency of abnormal ring structures and the ability of ring X chromosomes to magnify.     

Mitotic chiasmata in human diplochromosomes

Summary Three placental tissue cultures of spontaneous human abortions showed an unusually high frequency of metaphases with diplochromosomes. In 62 such cells, nine configurations were interpreted as mitotic chiasmata between the two sister chromosomes of a diplochromosome. One U-type exchange between two sister chromosomes was also found. This differs significantly from the 1:1 ratio of adjacent and alternate exchanges in translocations, thus supporting the idea that mitotic chiasmata are in principle different from chromatid translocations. The hypothesis is put forward that the frequency of homologous exchanges is determined by the intimacy of pairing which ranges from meiotic pairing through sister chromatid association, through sister chromosome association in diplochromosomes to accidental pairing of homologous regions in diploid cells.     

Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes]

The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C-negative regions, as well as between G-bands and R-bands, has been produced.     

Sister chromatid exchange in the centromere and centromeric area

Central and peripheral sister chromatid exchanges (SCE) were evaluated separately in human phytohemagglutinin (PHA)-stimulated lymphocytes after culture for 72 h in 5-bromodeoxyuridine (BrdU) containing medium. At the same time, the length of chromosome No. 1 was measured in 10 metaphases per case and the mean value taken as a representative parameter for the contraction of chromosomes. The statistical analysis of regression revealed a close relationship between the percentage of SCE observed in the centromere and the contraction state of chromosomes (P less than or equal to 0.01). A statistically significant increase of central exchanges was seen in more condensed chromosomes, due to the difficulty in differentiating clearly between centric and pericentric exchanges. Consequently, if exchanges in the centromere are omitted from evaluation, this would lead to spuriously low SCE rates in more contracted chromosomes. In order to exclude the variable factor of chromosome contraction in SCE studies, we highly recommend inclusion of counts of central exchanges. Results obtained on chromosomes with twisted chromatids, a situation which tends to stimulate SCE, should be omitted.     

Spo13 maintains centromeric cohesion and kinetochore coorientation during meiosis I

BACKGROUND: The meiotic cell cycle, the cell division cycle that leads to the generation of gametes, is unique in that a single DNA replication phase is followed by two chromosome segregation phases. During meiosis I, homologous chromosomes are segregated, and during meiosis II, as in mitosis, sister chromatids are partitioned. For homolog segregation to occur during meiosis I, physical linkages called chiasmata need to form between homologs, sister chromatid cohesion has to be lost in a stepwise manner, and sister kinetochores must attach to microtubules emanating from the same spindle pole (coorientation). RESULTS: Here we show that the meiosis-specific factor Spo13 functions in two key aspects of meiotic chromosome segregation. In cells lacking SPO13, cohesin, which is the protein complex that holds sister chromatids together, is not protected from removal around kinetochores during meiosis I but is instead lost along the entire length of the chromosomes. We furthermore find that Spo13 promotes sister kinetochore coorientation by maintaining the monopolin complex at kinetochores. In the absence of SPO13, Mam1 and Lrs4 disassociate from kinetochores prematurely during pro-metaphase I and metaphase I, resulting in a partial defect in sister kinetochore coorientation in spo13 Delta cells. CONCLUSIONS: Our results indicate that Spo13 has the ability to regulate both the stepwise loss of sister chromatid cohesion and kinetochore coorientation, two essential features of meiotic chromosome segregation.     

Influence of ataxia telangiectasia gene dosage on bleomycin-induced chromosome breakage and inhibition of replication in human lymphoblastoid cell lines

Long-term lymphoblastoid cell lines, obtained by E-B virus transformation of peripheral blood lymphocytes, retain many of the features of hypersensitivity to environmental agents found in primary cultures and fibroblast strains from patients with genetic diseases. Primary lymphocyte cultures from patients with ataxia telangiectasia, a cancer-prone genetic disease, have increased sensitivity to chromosomal damage induced by the radio-mimetic drug, bleomycin. In order to study the expression of ataxia telangiectasia gene dosage in lymphoblastoid cell lines, we examined chromosomal aberrations in lines containing two, one, or no alleles for ataxia telangiectasia. These were derived from ataxia telangiectasia homozygotes, from ataxia telangiectasia obligate heterozygotes, and from presumably normal donors, respectively. Chromosome preparations were made 46 h after a 2 h exposure to bleomycin and scored for chromosome breakage, for the relative rate of cell replication as assessed by sister chromatid differentiation patterns, and for the frequency of sister chromatid exchanges. Baseline frequencies of chromosome breakage and sister chromatid exchanges, and baseline rates of cell replication were similar in all nine lymphoblastoid cell lines. Following treatment with 25 or 250 mU/ml bleomycin, all the lymphoblastoid cell lines showed increased chromosome breakage and decreased cell replication. The lymphoblastoid cell lines from the ataxia telangiectasia homozygotes had significantly increased chromosome breakage and decreased rate of cell replication after either bleomycin dose in comparison with the normal or with the ataxia telangiectasia heterozygous lines. Sister chromatid exchange frequencies were not altered by bleomycin exposure.     

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

Incomplete sister chromatid separation of long chromosome arms

Chromosome segregation ensures the equal partitioning of chromosomes at mitosis. However, long chromosome arms may pose a problem for complete sister chromatid separation. In this paper we report on the analysis of cell division in primary cells from field vole Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome painting with probes specific for the X and the Y chromosomes showed that these long chromosomes are prone to mis-segregate, producing DNA bridges between daughter nuclei and micronuclei. Analysis of mitotic cells with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner CENtromere Protein (INCENP)/Aurora B to the spindle midzone and furrow formation occur while the two groups of daughter chromosomes are still connected by sex chromosome arms. Late cytokinetic processes are not efficiently inhibited by the incomplete segregation as in a significant number of cell divisions cytoplasmic abscission proceeds while Aurora B is at the midbody. Live-cell imaging during late mitotic stages also revealed abnormal cell division with persistent sister chromatid connections. We conclude that late mitotic regulatory events do not monitor incomplete sister chromatid separation of the large X and Y chromosomes of Microtus agrestis, leading to defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient chromosome transmission through mitosis.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Polyamine deprivation causes major chromosome aberrations in a polyamine-dependent Chinese hamster ovary cell line

This paper describes the effects of polyamine deprivation on chromosomes of a polyamine-dependent Chinese hamster ovary cell line which grows continuously in serum-free medium. After 6 days' polyamine deprivation the number of mitoses decreased drastically and most of them showed major chromosome aberrations. There were several gaps and breaks in many chromosomes, some cells exhibiting extensive chromosome fragmentation. Elongation of chromosomes, indicative for unpacking of the chromosome fibres, were also seen. The time course of Harlequin staining of the chromosomes revealed prolongation of the cell cycle in the polyamine-starved cells. The mean number of sister chromatid exchanges per cell was a little higher in the polyamine-starved than in the control cells. The difference is statistically significant, but it has to be taken with some caution, because the experiments could not be carried out under strictly comparable conditions.     

THE FREQUENCY OF SISTER CHROMATID EXCHANGES FOLLOWING EXPOSURE TO VARYING DOSES OF H3-THYMIDINE OR X-RAYS

In the Chinese hamster cell line CHEF-125, sister chromatid exchanges occurred at a rate of a little higher than one per three chromosomes for each cell cycle. The exchanges were detectable by labeling with H³-thymidine and autoradiographic analyses of chromosomes at the second and subsequent metaphases after labeling had occurred. To test the hypothesis that sister chromatid exchanges are caused by radiation, cells were incubated in media with different amounts of H³-thymidine. No statistically significant change in the exchange rate was detected over 100-fold range of variation in the amount of incorporated H³-thymidine (determined by grain counts of autoradiographs). We have concluded that sister chromatid exchanges are not caused by tritium radiation and therefore are spontaneous events. Cultures were also irradiated with acute doses of x-rays up to 200 r and scored for sister chromatid exchanges. Between zero and 50 r there was a statistically significant increase in the rate of exchanges. This is interpreted as evidence that x-rays can induce some exchanges, although the majority of these events are probably spontaneous.     

Human sister chromatid exchange caused by methylazoxymethanol acetate

The incidence of sister chromatid exchange was determined in human leucocyte cultures treated with methylazoxymethanol acetate. In all individuals examined, treated cultures manifested a significantly higher frequency of sister chromatid exchanges than controls. Two concentrations of MAM AC were tested in blood cultures from nine individuals. The concentrations varied from individual to individual since they were determined by means of individual dose-response curves, which involved [3H]-thymidine incorporation in PHA-stimulated short-term lymphocyte cultures versus MAM AC contraction. The lower concentration was less than the TD50 dose. Compared to control cultures, the lower concentration caused a higher incidence of sister chromatid exchange in eight of nine individuals. The cumulative mean value for all control cultures was 5.32 exchanges per cell while that for cultures treated with the higher concentration was 10.73.     

Driving chromosome segregation: lessons from the human and Drosophila centromere-kinetochore machinery

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.