Synthesis of an attached autosome,C(3)RM,inDrosophila pseudoobscura

Drosophila pseudoobscura has three acrocentric autosomes. In the experiments reported, homologous arms of the third chromosome were attached to the same centromere. This is a reversed metacentric compound third chromosome, denoted by C(3)RM. This compound chromosome is relatively fertile in within-strain crosses (ca. 50% egg hatch) but sterile when outcrossed to a normal karyotype. When constructing translocations for this experiment, the behavior of the Y-autosome translocations suggested that this species can tolerate more Y chromosome deficiency while retaining fertility than canDrosophila melanogaster. Finally, there were no Robertsonian exchanges observed among the 96 autosome-autosome translocations analyzed cytologically.     

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.     

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.     

Cytological studies of meiotic recombination in human males

We combined immunostaining and fluorescence in situ hybridization (FISH) methodology to directly examine meiotic exchanges in over 2,000 pachytene stage spermatocytes from 25 individuals. Our results indicate that, on average, there are about 50 exchanges per cell and that, with the exception of the acrocentric chromosomes, all chromosome arms harbor at least one exchange. We also identified significant among-individual variation in the mean number of exchanges, with an approximate 20% difference between individuals with "low" and those with "high" exchange frequencies.     

High incidence of sister chromatid exchanges and chromatid interchanges in the conditions of lowered activity of poly(ADP-ribose)polymerase

The effect of lowering the activity of poly(ADP-ribose)polymerase on chromosome stability has been examined. Chinese hamster ovary cells, CHO-Kl grown in a nicotinamide-free medium exhibited an increased frequency of sister chromatid exchanges in a time-dependent manner. Furthermore, addition of m-aminobenzamide which is known to be a strong inhibitor of poly(ADP-ribose)polymerase caused a manyfold increase in the frequency of both sister chromatid exchanges and non-sister chromatid interchanges. These results suggest that appropriate levels of NAD and the activity of poly(ADP-ribose)polymerase are required for maintaining chromosome stability.     

Giemsa C-banding of rye meiotic chromosomes and the nature of “terminal” chiasmata

The pattern of homologous chromosome association at metaphase I of meiosis in rye has been analysed and interpreted by means of the Giemsa C-banding procedure. The rationale for this approach stems from the unexpected coincidence of terminal Giemsa-bands in most chromosome arms and distal chiasma localisation which characterises this species. Analysis of banded metaphase I and anaphase I configurations suggests that meiotic exchanges occur proximal to the terminal Giemsa-bands, that is sub-terminally. The apparently terminal appearance of many chiasmata at metaphase I has been analysed by Giemsa-banding and shown to be more likely to result from bivalent distortion due to contraction and/or stretching (pseudoterminalisation) than from chiasma terminalisation in the accepted sense.     

Analysis of the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodomys ordii)

The frequency of sister chromatid exchanges (SCEs) has been determined for C band and non-C band regions of chromosomes of the kangaroo rat after staining with the fluorescence plus giemsa (FPG) technique. After one complete round of DNA synthesis in the presence of bromodeoxyuridine (BrdU) staining of the C band regions revealed simple or complex asymmetries between chromatids. After two complete rounds of DNA synthesis in the presence of BrdU harlequin chromosomes were observed. Analysis of the distribution of SCE in chromosomes at their 1st and 2nd mitosis showed that relatively few exchanges occur within C band regions, although the frequency of SCEs is high at the junction between C band and non-C band chromosome regions.     

Molecular genetics of radiation-induced chromosome breaks in a gene area in Drosophila: "position" effect of gene mutation?

On the sample of 43 gamma-ray and neutron-induced inversion or translocation exchanges with the vestigial (vg) phenotype, the molecular cytogenetic analysis of distribution of exchange breakpoints on the molecular map of Drosophila vg region (subsection 49D3-4 on the polytene chromosome 2R) was performed using hybridisation in situ technique. Simultaneously, PCR-assay of DNA alterations in all exons and introns (except for intron 4) of the vg gene for 18 mutants with exchange breakpoints outside of the gene was carried out. The results obtained by these molecular genetic techniques have shown that 1) radiation-induced breaks under chromosome exchanges with the vg phenotype were regularly located inside of the vg gene (19 cases out of 43 studied ones or 44.2%) passing through the large introns; 2) breakpoints were frequently flanked by deletions of the gene as whole (3 exchanges) or of its major part (3 exchanges); 3) many of the breaks (18/43 or 41.8%) are situated outside (distal or proximal) of the gene although such mutants have got the vg phenotype; 4) 2/3 (12/18 or 66.7%) vg mutants with the breakpoint outside of gene show the intragenic DNA lesions (microdeletions, microinversions) occurring obviously independently and simultaneously with the neighbor chromosome breaks; 5) only each third vg mutant with break outside of the gene (6/18 or 33.3%) have the unchanged gene subregions under study and presents obviously the result of "position effect" which appear to manifest itself for a distance of 2-30 kb (more near and farther locations of the proximal and distal breakpoints, respectively, relative to the vg gene). Our findings showing regular induction of the multiple genetic lesions (chromosome breaks and mutations of the adjacent genes) on the both ends of chromosome exchange induced by single track produced by gamma-rays or neutrons were discussed as a scientific basis for the conceptually new approaches to the assessment of both genetic damage numbers in the cell genome with chromosome exchange (the multiple genetic lesions) and radiation genetic risk (our molecular genetic approach showing the need for an increase of risk levels at least on a factor of 3 for the heritable chromosome alterations detected by the ordinary cytogenetic monitoring).     

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.     

Intrachanges as part of complex chromosome-type exchange aberrations

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.     

The database for analysis of quantitative characteristics of chromosome aberration frequencies in the culture of human peripheral blood lymphocytes]

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330,000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 +/- 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.     

The Database for Analysis of Quantitative Characteristics of Chromosome Aberration Frequencies in the Culture of Human Peripheral Blood Lymphocytes

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330 000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 ± 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.     

Distribution of Mitomycin C-induced damage in human chromosomes with special reference to regions of repetitive DNA

The distribution of gaps, breaks, exchanges and other effects induced by Mitomycin C on human chromosomes was analysed to examine the possibility of a correlation between chromosome lesions and regions of repetitive DNA. Homologous and non-homologous exchanges between chromosomes Nos. 1, 9 and 16, and also between and with the acrocentric chromosomes were more frequent than expected, but breaks and gaps appeared to be located randomly with regard to chromosome light and dark bands.     

Possible mechanisms of the occurrence of chromosome restructurings. IV. Chromosome restructurings in spontaneous mutagenesis

An attempt was undertaken to modify the spontaneous mutation process by varying its conditions in somatic cells of different species and tissues. The rate of chromosome aberrations and their types were studied in anaphase and metaphase. Under normal conditions, chromosome breaks were only found to occur. Breakage of chromosomes occurs during interphase, and as a result, acentric fragments are located outside the equatorial plate during metaphase. This process of chromosome breakage leads to elimination of some genetic material, without concomitant exchanges, and therefore, it has been named "elimination" process. Spontaneous chromosome mutagenesis manifesting itself at cytogenetic level was concluded to be an elimination process directed to elimination of a portion of chromatin from chromosomes. When the conditions of spontaneous mutagenesis are altered, in particular, by cardiovascular diseases in man, by partial inhibition of DNA repair in mice and pea cells, by transformation of Chinese hamster cells, upon ageing of pea seeds-qualitative changes in the chromosomal aberrations are registered, connected with the appearance of chromosome exchanges and acentric fragments situated within the equatorial plate during metaphase. These two types of chromosome aberrations are proposed to be considered as new criteria of pathology. A system of processes was suggested to exist, preventing the appearance of aberrations during mitosis, and it is supposed to be one of the most significant homeostatic systems.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Association of inter- and intrachromosomal exchanges with the distribution of low- and high-LET radiation-induced breaks in chromosomes

To study the effects of low- and high-linear energy transfer (LET) radiation on break locations within a chromosome, we exposed human epithelial cells in vitro to (137)Cs γ rays at both low and high dose rates, secondary neutrons at a low dose rate, and 600 MeV/u iron ions at a high dose rate. Breakpoints were identified using multicolor banding in situ hybridization (mBAND), which paints chromosome 3 in 23 different colored bands. For all four radiation scenarios, breakpoint distributions were found to be different from the predicted distribution based on band width. Detailed analysis of chromosome fragment ends involved in inter- or intrachromosomal exchanges revealed that the distributions of fragment ends participating in interchromosomal exchanges were similar between the two low-LET radiation dose rates and between the two high-LET radiation types, but the distributions were less similar between low- and high-LET radiations. For fragment ends participating in intrachromosomal exchanges, the distributions for all four radiation scenarios were similar, with clusters of breaks found in three regions. Analysis of the locations of the two fragment ends in chromosome 3 that joined to form an intrachromosomal exchange demonstrated that two breaks with a greater genomic separation can be more likely to rejoin than two closer breaks, indicating that chromatin folding can play an important role in the rejoining of chromosome breaks. Comparison of the breakpoint distributions to the distributions of genes indicated that the gene-rich regions do not necessarily contain more breaks. In general, breakpoint distributions depend on whether a chromosome fragment joins with another fragment in the same chromosome or with a fragment from a different chromosome.     

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.     

A case of pure red cell aplasia with a high incidence of spontaneous chromosome breakage: A possible X-ray sensitive syndrome

Summary A patient with a pure red cell aplasia (Blackfan-Diamond Anemia), and with many congenital abnormalities and growth retardation, has been found to have a chromosome breakage syndrome. In this patient, the frequencies of spontaneous chromosome aberrations and micronuclei in PHA stimulated peripheral blood lymphocytes are elevated when compared to those in normal individuals. The frequency of sister chromatid exchanges is within normal range. The response to mitomycin C (MMC) in the micronucleus test, using lymphocytes, shows a similar increase in the patient's lymphocytes to that in normal individuals, indicating no increased sensitivity to MMC.The frequencies of X-ray induced dicentric chromosomes and micronuclei in the peripheral blood lymphocytes are elevated in the patient. But as the patient clinically does not have any signs of ataxia telangiectasia, this combination of clinical and laboratory findings of this case does not correspond with any of the other known chromosome breakage syndromes.     

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

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

Complex chromosome exchanges induced by gamma rays in human lymphocytes: an mFISH study

Loucas, B. D. and Cornforth, M. N. Complex Chromosome Exchanges Induced by Gamma Rays in Human Lymphocytes: An mFISH Study. Radiat. Res. 155, 660-671 (2001). Combinatorial multi-fluor fluorescence in situ hybridization (mFISH) allows the simultaneous painting of each pair of homologous chromosomes, thereby eliminating many of the difficulties previously associated with the analysis of complex rearrangements. We employed mFISH to visualize exchanges in human lymphocytes and found significant frequencies of these aberrations after gamma-ray doses of 2 and 4 Gy. At 4 Gy, roughly half of the cells contained at least one complex exchange that required anywhere from 3 to 11 initial chromosome breaks. At this dose, more than 40% of gross cytogenetic damage, as measured by the total number of exchange breakpoints, was complex in origin. Both simple and complex exchanges were found to have nonlinear dose responses, although the latter showed significantly more upward curvature. In many cases, it could be deduced that the initial breaks leading to a particular complex exchange were proximate, meaning that the resulting broken chromosome ends all must have been capable of interacting freely during the exchange process. For other complex exchanges, the rearrangement could just as well have resulted from two or more simpler exchanges that occurred sequentially. The results demonstrate the utility of mFISH in visualizing intricacies of the exchange process, but also highlight the various sources of ambiguity concerning cytogenetic analysis that remain despite the power of this approach.