Translocations of chromosome 12

Summary Human chromosome 12 has been used as a model for studying the distributions of sites of induced and spontaneous breaks. The breakpoints were determined from (1) translocations involving chromosome 12, (2) spontaneous breaks in untreated cultures, (3) radiation-induced breaks, and (4) spontaneous breaks in Fanconi's anaemia.Statistical analysis showed discordance in the results both between the eleven individual bands and between the four assessments. Also, the distribution of breaks for all bands was significantly diferent from random in each assessment. Certain bands added considerable bias to the results, and when analysed individually, only four bands (p11.1, q13, q24, and p13) showed distributions over the four assessments that were significantly different from random. These four bands are Giemsa-negative bands, and two (p13 and q24) are adjacent to telomeres, while p11.4 is adjacent to the centromere. The fourth band, q13, is a known fragile site.It is concluded that bands adjacent to centromeres, which are not C-banded, are peculiarly sensitive to breakage. Telomeric bands are variable in their response to different conditions of breakage, and both the physical structure of the telomere and the specific gene sequences of individual telomeres are probably of importance in determining this response. The fragile site q13 responds as if breakage at this site is due to the base composition of the DNA.     

Fault bars timing and duration: the power of studying feather fault bars and growth bands together

Growth bands and fault bars, widespread features of feathers that form during regeneration, have largely been studied independently. Growth bands result from normal regeneration: each pair of dark/light bands forms every 24 h. Fault bars are a response to stress during regeneration, creating a translucent line that can break the feather. We studied the relative position and width of these two structures in feathers of nestling and adult white storks Ciconia ciconia. We first confirmed that one growth band represents 24 h of feather regeneration. Fault bars did not occur at random within growth bands: 65.7% (in nestlings) and 45.6% (in adults) of them occurred in one out of six defined segments within a growth band, namely that segment corresponding to the first one‐third of night time hours. The width of fault bars relative to growth bands suggested that fault bars were produced during a median (range) of 7.0 h (2.7–27.0) in nestlings and 3.7 h (1.8–7.9) in adults. Fault bars were concentrated at feather tips in nestlings, but at central locations in adult feathers. Our results suggest that, in general, fault bars are a discrete event of a finite duration occurring mainly during the night (particularly in nestlings). This, along with current knowledge, suggests that acute stressors, rather than chronic ones, are responsible for fault bar formation. Thus, such acute punctual stressors (a matter of minutes) can have long‐lasting (months?years) physiological effects due to the wing load increase from feather breakage caused by fault bars.     

Structural chromosomal rearrangements in HpaII-treated human lymphocytes.

Restriction endonucleases have been shown to induce chromosome damage in a variety of cultured cells. We recently reported the coincidence between MspI-induced breakage and the location of common fragile sites. We have extended our study to HpaII, which induced a 4.5-fold increase in total breakage compared to controls. It appeared that a major contribution was given by stable chromosome rearrangements, which were present at a 14-fold increased frequency in comparison to the spontaneous levels. Moreover, several chromosome bands were involved in rearrangements in different cultures from different donors. Notably, HpaII-induced breakage occurred in the same bands where breakpoints of constitutional and neoplastic rearrangements are located.     

Bands involved in primary chromosome rearrangements in sarcomas are not constitutionally liable to breakage in sarcoma patients

Summary The localization of breakpoints in spontaneous chromosome aberrations, i.e., chromatid and chromosome gaps, breaks, and exchanges, has been studied in cultured skin fibroblasts from 34 untreated patients with musculoskeletal sarcoma and 38 controls. A total of 325 aberrations in the sarcoma group and 251 in the control group could be assigned to particular bands. The distribution was non-random (P<0.001) in both groups. Twenty-one bands in the sarcoma group and 20 in the control group appeared as hot spots, with 11 represented in both groups. Only three hot spots, all of which were present among both patients and controls, coincided with bands involved in primary sarcoma-associated chromosome rearrangements. The results indicate that the chromosome breakage pattern of non-malignant cells is similar in sarcoma patients and controls. Hence, the occurrence of primary structural rearrangements in sarcomas cannot be accounted for by any constitutional proneness to chromosome breakage at these bands.     

Congenital chromosome breakage clusters within Giemsa-light bands and identifies sites of chromatin instability

Analysis of the distribution of published chromosome breaks in cells with constitutional chromosome aberrations showed a nonrandom distribution of breaks among chromosomes and chromosome regions. A significant amount of breakage occurred at Giemsa-negative bands. In addition, chromosome sites associated with a number of fragile sites and cellular oncogene sites were affected nonrandomly. The data are consistent with the hypothesis that chromosome breakage occurs in somatic or germ cells as a result of recombinational errors involving actively transcribing chromatin regions or regions of unstable DNA sequence structure placed in proximity during interphase.     

Nonrandom distribution of exchange points in patients with structural rearrangements

High resolution studies of structural rearrangements were carried out using the G-band technique. A total of 220 breakage points were identified within individual bands from 117 unrelated cases born with a structural rearrangement. Breakage points were not evenly distributed along chromosomes in terms of G-band patterns. There was an excess involvement of light bands and a striking lack of dark bands in both reciprocal translocations and inversions. In reciprocal translocations, the middle part of a chromosome arm has less chance of being the site of an exchange than the terminal and centromeric parts. The implications of these results are briefly discussed.     

Cytoprotective effect of honey against chromosomal breakage in fanconi anemia patients in vitro

BACKGROUND:

Natural honey is widely used all over the world as a complementary and alternative medicine in various disorders including Fanconi anemia (FA). FA is a rare genetic chromosomal instability syndrome caused by impairment of DNA repair and reactive oxygen species (ROS) imbalance. This disease is also related to bone marrow failure and cancer. The aim of this study was to evaluate the cytoprotective effect of honey on mitomycin C (MMC-) induced chromosomal damage in peripheral lymphocytes from FA patients.

MATERIALS AND METHODS:

Treatment of these complications with alkylation agents MMC may enhance chromosomal breakage. We have evaluated the effect of honey on MMC- induced chromosomal breakage in FA blood cells using chromosomal breakage assay. The basal chromosomal breakage count was higher among FA patients than healthy subjects.

RESULTS:

The addition of MMC alone gave a significantly higher of chromosomal breakage in FA patients than control group (P < 0.0001). Pre- treatment with honey significantly inhibited breakage induced by MMC in FA patients by its antioxidant effect.

CONCLUSION:

Honey can prevent MMC- induced chromosomal breakage by its antioxidant effect.     

Raman spectroscopic study of DNA after photosensitive damage caused by hypocrellins A and B

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Cartographic study: Breakpoints in 1574 families carrying human reciprocal translocations

Reciprocal translocations (rcp) are among the most common constitutional chromosomal aberrations in man. Using a European database of 1574 families carrying autosomal rcp, a cartographic study was done on the breakpoints involved. The breakpoints are non-randomly distributed along the different chromosomes, indicating “hot spots”. Breakpoints of rcp that result in descendants that are unbalanced chromosomally at birth are more frequent in a distal position on chromosomal arms, and 65% of them are localised in R-bands. Among the R-bands, bands rich in GC islands and poor in Alu repetitive sequences are more frequently the site of breakpoints, as well as bands that include a fragile site. This result suggests that the variation in degree of methylation in GC islands could be involved in chromosomal breakage and hence in chromosomal rearrangements. Received: 10 April 1995 / Revised: 1 July 1995     

Inhalation exposure-rate of ethylene oxide affects the level of DNA breakage and unscheduled DNA synthesis in spermiogenic stages of the mouse

The effect of ethylene oxide (EtO) inhalation-exposure rate on the induction of DNA breakage in late spermatids and on unscheduled DNA synthesis (UDS) in early spermatids was studied. The exposures were 450 parts per million (ppm) for 4 h, 900 ppm for 2 h, and 1800 ppm for 1 h. Thus, the total exposure was always 1800 ppm-h. Both DNA breakage and UDS were found to increase by a factor of approximately 3 in going from the low to high EtO concentration, suggesting that the molecular dose of EtO to the testis had increased by a similar factor. Our results are consistent with the EtO exposure-rate effect found by Generoso et al. (1986) for induction of dominant-lethal mutations in late spermatids and early spermatozoa.     

Repair of cytokine-induced DNA damage in cultured rat islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1beta (IL-1beta), interferon gamma (IFN gamma) and tumour necrosis factor alpha (TNF alpha) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4 h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during,the cytokine treatment and islets are incubated for 4 h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4 h in the presence of hydroxyurea (HU) and 1-beta-D-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

A rapid method for the purification of fatty acid synthetase from the yeast Saccharomyces cerevisiae

A rapid method for the isolation and purification of small quantities of highly active fatty acid synthetase (FAS) from several strains of the yeast Saccharomyces cerevisiae, is presented. The purification procedure which is the shortest reported to this date (18 hr), involves the release of the enzyme by either cell wall digestion with Zymolyase 60000 or cell wall disruption by glass beads, followed by 35-50% ammonium sulfate fractionation, desalting by Sephadex G-25 chromatography, then calcium phosphate gel treatment, concentration by 50% ammonium sulfate precipitation, sedimentation of the enzyme in the ultracentrifuge and finally, column chromatography on DEAE Bio-Gel A. Fatty acid synthetase prepared by the cell breakage method, was found to be homogeneous according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-Tris-glycine disc gel electrophoresis and immunoelectrophoresis criteria. However, enzyme prepared from Zymolyase treated cells showed several proteolytic bands in addition to FAS bands, on SDS-PAGE. Enzyme obtained by both methods of cell breakage, showed a similar behavior throughout the purification procedure and gave a similar yield of enzyme of high specific activity (4800-7200 nmol/min/mg) that remained stable for several months at -85 degrees C.     

Gender differences and the interpretation of genetic instability in Alzheimer's disease.

The neuronal degeneration and death which characterize Alzheimer's disease (AD) may stem from a constitutive genetic instability related to DNA repair deficits. To test this hypothesis, we treated peripheral blood lymphocytes from persons with AD, age-matched controls, and young controls with two drugs that induce chromosome breakage. Bleomycin, a radiomimetic antineoplastic drug, causes single- and double-stranded DNA breaks through the generation of activated oxygen radicals. Methyl methane-sulfonate (MMS) is a monofunctional alkylating agent that binds covalently to DNA. Cells were grown in culture for 72 h, with drug treatments for 4 h (bleomycin) or 24 h (MMS) prior to harvest. Fifty cells per subject per drug were scored for chromosome breakage. Breakage rates for both drugs in AD women were significantly higher than those in age-matched control women. This was not the case in men, due to the very high induced breakage rates seen in the age-matched normal control men. Because the induced breakage rates in AD women and AD men are equivalent, it seems likely that an independent factor may be contributing to genetic instability in the normal control men. Our findings indicate that the interpretation of the response of AD lymphocyte chromosomes to DNA-damaging chemicals can be strongly confounded by the effects of gender ratio in the control population sampled. These findings have important implications for the design of future studies of Alzheimer's disease, as well as for the assessment of health risks in unaffected elderly populations.     

Repair of Cytokine-induced DNA Damage in Cultured Rat Islets of Langerhans

Treatment of cultured rat pancreatic islets of Langerhans with the combined cytokines interleukin-1β (IL-1β), interferon γ (IFN γ) and tumour necrosis factor α (TNF α) leads to DNA damage including strand breakage. We have investigated the nature of this damage and its repairability. When islets are further incubated for 4?h in fresh medium, the level of cytokine-induced strand breakage remains constant. If the nitric oxide synthase inhibitor N^G-monomethyl-l-arginine (NMMA) is present during cytokine treatment, then strand breakage is prevented. If NMMA is added following, rather than during, the cytokine treatment and islets are incubated for 4?h, further nitric oxide synthesis is prevented and most cytokine-induced strand breaks are no longer seen. To investigate DNA repair following cytokine treatment, cells were transferred to fresh medium and incubated for 4?h in the presence of hydroxyurea (HU) and 1-β-d-arabinosyl cytosine (AraC), as inhibitors of strand rejoining. In the presence of these inhibitors there was an accumulation of strand breaks that would otherwise have been repaired. However, when further nitric oxide synthesis was inhibited by NMMA, significantly less additional strand breakage was seen in the presence of HU and AraC. We interpret this, as indicating that excision repair of previously induced base damage did not contribute significantly to strand breakage. Levels of oxidised purines, as indicated by formamidopyrimidine glycosylase (Fpg) sensitive sites, were not increased in cytokine-treated islets. We conclude that in these primary insulin-secreting cells: (a) the DNA damage induced by an 18?h cytokine treatment is prevented by an inhibitor of nitric oxide synthase, (b) much of the damage is in the form of apparent strand breaks rather than altered bases such as oxidised purines, (c) substantial repair is ongoing during the cytokine treatment and this repair is not inhibited in the presence of nitric oxide.     

Strukturverändernde Wirkung von FUdR auf polytäne Chromosomen und Beziehungen zwischen Replikationsdauer und Bruchhäufigkeit von Querscheiben

Larvae of Chironomus th. thummi at the age of 10 hours after hatching were treated for 20 hours with 10^–4 M FUdR. The salivary gland chromosomes were studied at fourth instar. FUdR induces chromosomal constrictions and partial breakage of various diameters ranging from 1/2 to less than 1/16 of the total cross section of the polytene chromosomes. Breaks were predominantly found in chromosome regions containing bands of high DNA content. By H³-thymidine-autoradiography it is demonstrated that bands which are frequently broken are late replicating. This is shown by histograms correlating the distribution of breaks over the chromosomes with labeling patterns obtained in late S.—As bands with a great amount of DNA do not only replicate late but also spend the longest time in DNA synthesis, it is assumed that they also represent the largest replicons. It is discussed if this is the reason why FUdR induces breaks preferentialy in bands of high DNA content.     

Evolutionary conserved chromosomal segments in the human karyotype are bounded by unstable chromosome bands

In this paper an ancestral karyotype for primates, defining for the first time the ancestral chromosome morphology and the banding patterns, is proposed, and the ancestral syntenic chromosomal segments are identified in the human karyotype. The chromosomal bands that are boundaries of ancestral segments are identified. We have analyzed from data published in the literature 35 different primate species from 19 genera, using the order Scandentia, as well as other published mammalian species as out-groups, and propose an ancestral chromosome number of 2n = 54 for primates, which includes the following chromosomal forms: 1(a+c(1)), 1(b+c(2)), 2a, 2b, 3/21, 4, 5, 6, 7a, 7b, 8, 9, 10a, 10b, 11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y. From this analysis, we have been able to point out the human chromosome bands more "prone" to breakage during the evolutionary pathways and/or pathology processes. We have observed that 89.09% of the human chromosome bands, which are boundaries for ancestral chromosome segments, contain common fragile sites and/or intrachromosomal telomeric-like sequences. A more in depth analysis of twelve different human chromosomes has allowed us to determine that 62.16% of the chromosomal bands implicated in inversions and 100% involved in fusions/fissions correspond to fragile sites, intrachromosomal telomeric-like sequences and/or bands significantly affected by X irradiation. In addition, 73% of the bands affected in pathological processes are co-localized in bands where fragile sites, intrachromosomal telomeric-like sequences, bands significantly affected by X irradiation and/or evolutionary chromosomal bands have been described. Our data also support the hypothesis that chromosomal breakages detected in pathological processes are not randomly distributed along the chromosomes, but rather concentrate in those important evolutionary chromosome bands which correspond to fragile sites and/or intrachromosomal telomeric-like sequences.     

Nano-fibers produced by viral infection of amoeba visualized by atomic force microscopy

In the course of an atomic force microscopy investigation of mimivirus, we observed that disrupted virions released masses of fibers that were several hundreds of nanometers in length and that could not be explained as nucleic acid or polysaccharide. The fibers exhibited a strong 7 nm periodicity along their lengths. They existed singly, and also as ribbons, cables, and in multi stranded coils. In the aggregate structures, the periodic bands of the individual fibers aligned laterally to produce ribbons and other superstructures having a corresponding pattern of 7 nm periodic transverse bands. We have not observed such fibers in studies of other virus and cellular systems. The fibers are mechanically flexible and resistant to breakage. Occasionally fibers were associated with toroidal protein complexes, assumed to be processive enzyme complexes, apparently in the act of modifying the fibers.     

Significance of Abnormal Protein Bands in Patients with Multiple Myeloma following Autologous Stem Cell Transplantation

Aim

We studied the characteristics of small abnormal protein bands (APB) (including oligoclonal bands and new apparent monoclonal bands) that are frequently detected by serum protein electrophoresis (SPEP) and isoelectric focusing (IEF) in the post-autologous stem cell transplant setting.

Methods

In a retrospective analysis of patients with multiple myeloma undergoing transplantation, paraprotein identity and quantification were performed using standard immunofixation electrophoresis. The nature of any new bands was determined by IEF which distinguished between oligoclonal bands and apparent monoclonal bands.

Results

Of 49 myeloma cases, the median follow-up was 33.7 months (range, 5.6–97.5 months) and 24 patients had relapsed. Thirty six (73%) developed APB. 22 patients had more than one episode of APB and 6 patients had more than 2 episodes resulting in a total of 69 episodes of APB observed post-transplant. IEF demonstrated 54 of these APB were oligoclonal bands and 15 appeared to be monoclonal. Of the 15 episodes of apparent monoclonal bands, 10 had differing heavy or light chain restriction compared to the original myeloma paraprotein and 5 had the same heavy and light chain restriction but different band location in the SPEP lane. Ten of these apparent monoclonal bands resolved, 5 persisted, and only one represented true disease progression. The presence of APB impacted favourably on event-free survival (p=0.05).

Conclusion

Small APB are very frequent post-transplant for myeloma, and IEF can identify these APB as oligoclonal or monoclonal. Apparent monoclonal bands may represent relapsed disease, but in the vast majority of cases it does not, and most likely represents a transient phenomenon representing regeneration of a limited immune response.     

A selection model to estimate the interaction between food particles and the post-canine teeth in human mastication.

Food comminution during chewing is the composite result of selection and breakage. In the selection process, every food particle has a chance of being placed between the antagonistic post-canine teeth and being subjected to subsequent breakage. The selection chance, being the ratio between the number of selected and offered particles, has been mathematically described as a function of the number of particles offered, in terms of the number of breakage sites available on the teeth and particle affinity, i.e. the fraction of breakage sites occupied by one particle. The assumption has been made that particles are successively selected during a jaw-closing phase and that the selection chance of subsequent particles having the opportunity to occupy a breakage site proportionally decreases with the unoccupied fraction of the breakage sites left. The number of selected particles of a single size then asymptotically approaches the total number of breakage sites available for that size, when the number of particles offered increases. The critical particle number, derived from the measure of particle affinity, indicates the number of particles by which the breakage sites become saturated. The selection model for single particle sizes has been successfully applied to describe one-chew experiments, using various numbers and sizes of particles made of a silicone-rubber. After pseudo-chewing movements the subjects were unexpectedly instructed to carry out a real chew on particles (half-cubes). Undamaged, hence non-selected half-cubes could afterwards be distinguished from broken particles. The model has been extended to a particle mixture to describe the selection of particles of a certain size while other particles of different sizes are present. If a two-way competition between smaller and larger particles is assumed, the model predicts that the ratios of the selection chances between different particle sizes do not depend upon the numbers of the particles in the mixture.     

Oxygen toxicity in Treponema pallidum: deoxyribonucleic acid single-stranded breakage induced by low doses of hydrogen peroxide

The effect of hydrogen peroxide on Treponema pallidum was investigated. The in vitro loss of virulence (as measured by rabbit inoculation) of T. pallidum was accelerated by as low as 100 microM hydrogen peroxide in the complex maintenance medium used. Higher doses led to rapidly accelerated death with 500 microM hydrogen peroxide causing sterilization of the medium within 3 to 4 h. Since hydrogen peroxide is known to cause single-stranded breaks in DNA, the effect of hydrogen peroxide on the treponemal genome was examined. Extensive breakage was caused by 100 microM hydrogen peroxide as determined on alkaline sucrose gradients. A limit was reached at 250 microM and above. Single-stranded breaks could be demonstrated as early as 5-10 min after exposure to hydrogen peroxide when the treponemes were exposed to 250 microM hydrogen peroxide; accelerated death was evident by 2 h past exposure demonstrating that DNA breakage was preceding death. Treponemal death caused by penicillin did not result in DNA breakage. The repair-proficient bacterium Escherichia coli K-12 was compared with T. pallidum. It required 10-100 times more hydrogen peroxide to cause various levels of breakage. Escherichia coli K-12 rapidly repaired DNA breakage once hydrogen peroxide was removed by addition of catalase. Treponema pallidum, in comparison, showed little or no repair in vitro. Addition of catalase or dithiothreitol to the medium protected against all but a low level of breakage; this may reflect on the ability of catalase and reducing agents to protect T. pallidum against oxygen toxicity in vitro.