The influence of exogenous DNA of different origin on the mitosis and chromosomes of irradiated meristematic cells ofVicia faba

In this paper we compare the influence of heterologous and isologous DNA on the radiation damage repair of primary root meristematic cells ofVicia faba. Roots, irradiated by exposure of 150 r were cultivated at different time intervals either in tap water, or in a solution of heterologous or isologous DNA. In comparing mitotic activity of meristematic cells it was found that both types of DNA studied enhance the recovery of irradiated cells. The frequency of postmetaphase chromosomal aberrations of irradiated cells was influenced also by post-irradiation action of exogenous DNA. While heterologous DNA exhibited synergical effect with radiation in the sense that it increased the post-irradiation incidence of aberrations in all time intervals studied, isologous DNA had a strong repair effect—the application caused a significant decrease of the percentage of post-metaphase aberrations. Both kinds of DNA caused changes in the relation of chromosome to chromatid aberrations; a higher percentage of chromatid aberrations was registered. The study of the distribution of aberrations between large and small chromosomes ofVicia faba showed that the post-irradiation application of heterologous DNA increases damage of small chromosomes while isologous DNA caused an increased repair ability in this chromosomal group.     

Aberrations Involving Chromosome 1 as a Possible Predictor of Odds Ratio for Colon Cancer - Results from the Krakow Case-Control Study

Background

There is still an open question how to predict colorectal cancer risk before any morphological changes appear in the colon.

Objective

The purpose was to investigate aberrations in chromosomes 1, 2 and 4 in peripheral blood lymphocytes analyzed by fluorescence in situ hybridization technique as a tool to assess the likelihood of colorectal cancer.

Methods

A hospital-based case-control study included 20 colon cancer patients and 18 hospital-based controls. Information about potential covariates was collected by interview. The frequency of stable and unstable chromosome aberrations in chromosome 1, 2 and 4 was assessed by fluorescence in situ hybridization technique.

Results

Colorectal cancer patients, as compared to controls, had a relatively higher frequency of chromosome 1 translocations (median: 3.5 versus 1.0 /1000 cells, p = 0.006), stable aberrations (3.8 versus 1.0 /1000 cells, p = 0.007) and total aberrations (p = 0.009). There were no differences observed for chromosomes 2 and 4. Our results showed an increase in the odds of having colon cancer by about 50–80% associated with an increase by 1/1000 cells in the number of chromosome 1 aberrations.

Conclusions

The results revealed that the frequency of chromosomal aberrations, especially translocations in chromosome 1, seems to be a promising method to show a colon cancer risk. Additionally, our study suggests the reasonableness of use of biomarkers such as chromosome 1 aberrations in peripheral blood lymphocytes in screening prevention programs for individuals at higher colon cancer risk to identify those who are at increased risk and require more frequent investigations, e.g. by sigmoidoscopy.     

Chromosomal aberrations in peripheral lymphocytes of patients treated with radium-224 for ankylosing spondylitis

The aim of this study was to investigate the in vivo frequency of chromosomal aberrations (primarily dicentric chromosomes and chromatid breaks) potentially induced by ²²⁴Ra -radiation in peripheral lymphocytes. The study was designed to serve as a cytogenetic analysis along with the therapeutic procedure of ankylosing spondylitis patients who were undergoing a treatment with ²²⁴Ra-chloride. The total administered activity was 10 MBq, and the treatment followed a schedule of 10 i.v. injections per week, each with a dose of 1 MBq of ²²⁴Ra. The calculation of absorbed doses delivered to the blood used the models suggested by the ICRP and yielded a value of 4.7 mGy/MBq. The frequency of chromosomal aberrations observed during the course of therapy was related to the blood dose. The frequency of dicentric chromosomes induced in vivo was found to agree well with the corresponding value of dicentrics induced in vitro. However—given that peripheral lymphocytes are in the cell cycles G₀ stage—an unexpected increase with dose in the yield of chromatid breaks was observed, with about 95% of them occurring in cells without any other chromosome-type aberrations. Reasons for the production of chromatid breaks are discussed.     

The human leukocyte test system

Summary Leukocyte cultures were set up with X-irradiated whole blood (200 R). Cells starting with their DNA synthesis between 25 and 35 h after culture initiation (early replicating cells) were pulse-labeled with tritiated thymidine ([³H] TdR). Mitoses were collected with colcemid in adjacent intervals from 36 up to 72 h after culture initiation. At fixation times of 50, 56, 62, and 72 h enough mitoses for a determination of the frequencies of chromosomal aberrations (dicentric and ring chromosomes) were found. After that the preparations were processed for autoradiography. All mitoses analyzed for chromosomal aberrations were re-analyzed for labeling, and the frequencies of chromosomal aberrations in labeled (=early replicating cells) and unlabeled (=late replicating cells) mitoses were compared. At all fixation times, higher frequencies of dicentric chromosomes were found in labeled as compared to unlabeled mitoses, indicating a higher sensitivity of early replicating cells to X-irradiation in the G₀ stage of the cell cycle.     

Comparison of the effects of radiation and deoxyribonucleic acids on the mitosis in roots ofVicia faba

Differences as well as similarities in the action of ionizing radiation and deoxyribonucleic acids from various sources on mitosis in root cells ofVicia faba were established. The time course of occurrence of aberrations were examined. Whereas in irradiated broad beant the maximum percentage of aberrations was observed immediately after irradiation, the aps plication of non-isologous DNA was followed by maximum aberrations after 8–16 hours. As all the time-intervals studied, an incraasad number of aberrations was found during metaphase-as compared with anaphases, both after irradiation and after application of DNA. A comparison of isologous, homologous and heterologous DNA as inductors of chromosomal aberrations supported our previous findings and showed that the efficiency of DNA depends on the genetic difference between donor and acceptor. During a study of distribution of aberrations between large and small chromosomes of meristematic cells ofVicia faba, at various time-intervals it was obsarved that after irradiation the distribution of aberrations between individual chromosomes is proportional to their total length, whereas the effect of heterologous DNA is mostly in the damage to small chromosomes. It was also found that aftar irradiation mostly chromatid aberrations are formed at shorter time-intervals and only later chromosomal aberrations will appear. On the other hand, heterologous DNA brings about in all time-intervals a predominance of chromatid aberrations.     

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed ^{1, 2}. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope ^{3, 4}. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies ^{5, 6}. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes ^7-10.The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (http://www.spectral-imaging.com/).SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia ^11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models ¹⁴. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).     

An Integrative CGH,MSI and Candidate Genes Methylation Analysis of Colorectal Tumors

Background

Different DNA aberrations processes can cause colorectal cancer (CRC). Herein, we conducted a comprehensive molecular characterization of 27 CRCs from Iranian patients.

Materials and Methods

Array CGH was performed. The MSI phenotype and the methylation status of 15 genes was established using MSP. The CGH data was compared to two established lists of 41 and 68 cancer genes, respectively, and to CGH data from African Americans. A maximum parsimony cladogram based on global aberrations was established.

Results

The number of aberrations seem to depend on the MSI status. MSI-H tumors displayed the lowest number of aberrations. MSP revealed that most markers were methylated, except RNF182 gene. P16 and MLH1 genes were primarily methylated in MSI-H tumors. Seven markers with moderate to high frequency of methylation (SYNE1, MMP2, CD109, EVL, RET, LGR and PTPRD) had very low levels of chromosomal aberrations. All chromosomes were targeted by aberrations with deletions more frequent than amplifications. The most amplified markers were CD248, ERCC6, ERGIC3, GNAS, MMP2, NF1, P2RX7, SFRS6, SLC29A1 and TBX22. Most deletions were noted for ADAM29, CHL1, CSMD3, FBXW7, GALNS, MMP2, NF1, PRKD1, SMAD4 and TP53. Aberrations targeting chromosome X were primarily amplifications in male patients and deletions in female patients. A finding similar to what we reported for African American CRC patients.

Conclusion

This first comprehensive analysis of CRC Iranian tumors reveals a high MSI rate. The MSI tumors displayed the lowest level of chromosomal aberrations but high frequency of methylation. The MSI-L were predominantly targeted with chromosomal instability in a way similar to the MSS tumors. The global chromosomal aberration profiles showed many similarities with other populations but also differences that might allow a better understanding of CRC''s clinico-pathological specifics in this population.     

Influence of exogenous DNA on Ypenyl-treated chromosomes ofVicia faba L.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0·01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.     

Sex chromosomal dimorphisms narrated by X-chromosome translocation in a spiny frog (<Emphasis Type="Italic">Quasipaa boulengeri</Emphasis>)

Background

In the general model of sex chromosome evolution for diploid dioecious organisms, the Y (or W) chromosome is derived, while the homogametic sex presumably represents the ancestral condition. However, in the frog species Quasipaa boulengeri, heteromorphisms caused by a translocation between chromosomes 1 and 6 are not related to sex, because the same heteromorphic chromosomes are found both in males and females at the cytological level. To confirm whether those heteromorphisms are unrelated to sex, a sex-linked locus was mapped at the chromosomal level and sequenced to identify any haplotype difference between sexes.

Results

Chromosome 1 was assigned to the sex chromosome pair by mapping the sex-linked locus. X-chromosome translocation was demonstrated and confirmed by the karyotypes of the progeny. Translocation heteromorphisms were involved in normal and translocated X chromosomes in the rearranged populations. Based on phylogenetic inference using both male and female sex-linked haplotypes, recombination was suppressed not only between the Y and normal X chromosomes, respectively the Y and translocated X chromosomes, but also between the normal and translocated X chromosomes. Both males and females shared not only the same translocation heteromorphisms but also the X chromosomal dimorphisms in this frog.

Conclusions

The reverse of the typical situation, in which the X is derived and the Y has remained unchanged, is known to be very rare. In the present study, X-chromosome translocation has been known to cause sex chromosomal dimorphisms. The X chromosome has gone processes of genetic differentiation and/or structural changes by chance, which may facilitate sex chromosome differentiation. These sex chromosomal dimorphisms presenting in both sexes may represent the early stages of sex chromosome differentiation and aid in understanding sex chromosome evolution.

     

Sperm chromosome analysis of a man heterozygous for a pericentric inversion of chromosome 20

Summary The sperm chromosomes of a man heterozygous for inv(20)(p13q11.2) were analyzed. Twenty-six sperm chromosome complements were examined, of which fourteen contained the normal chromosome, and twelve the inverted chromosome. None of the sperm complements contained a recombinant chromosome 20. The frequency of structural chromosomal aberrations unrelated to the inversion was 11.5% (3/26). Numerical aberrations were not observed. The percentages of X- and Y-bearing sperm were 56% and 44%, respectively, which was similar to the expected 11 ratio.     

Bloom's syndrome and EM9 cells in BrdU-containing medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption

Bloom's syndrome (BS) and EM9 cells both display elevated frequencies of sister chromatid exchange (SCE) following growth for two rounds of DNA replication in bromodeoxyuridine (BrdU)-containing medium. To learn whether hyperresponsiveness to BrdU itself might play a role in causing the SCE elevation, the effects of BrdU on two other parameters, cellular proliferation and chromosome disruption, were examined, comparing the responses of BS and normal lymphoblastoid cells and of EM9 and CHO cells. BS and normal cells responded similarly with respect to growth for 4 days in BrdU-containing medium (0, 1, 3, and 5 g/ml). Chromosome aberrrations were increased only slightly in the BS and normal cells after 2 days in BrdU. CHO cells responded to growth in BrdU-containing medium like BS and normal cells; however, little growth of EM9 was detected at any of the BrdU concentrations employed. CHO and EM9 cells also exhibited strikingly different amounts of chromosome damage following growth in BrdU. After 2 days in 1, 3, and 5 g/ml BrdU 21%, 46%, and 50%, respectively, of the CHO cells had chromosome aberrations in contrast to 92%, 96%, and 98% of the EM9 cells. Most of the aberrations in the BrdU-treated CHO cells consisted of what appeared to be polycentric and ring chromosomes or chromosomes exhibiting telomere association. Acentric fragments were absent from most cells with polycentric and ring chromosomes, indicating either that the abnormal chromosomes were formed during an earlier cell cycle or that the abnormal chromosomes represent a form of association in which the telomeres are apposed so tightly that the juncture between chromosomes cannot be identified microscopically. EM9 cells treated with BrdU exhibited many chromatid and isochromatid gaps and breaks as well as numerous quadriradial, triradial, and complex interchange configurations. In addition, the types of aberrations present in CHO cells also were increased greatly in number. The different responses of BS and EM9 cells to growth in BrdU suggest that the molecular defects in the two cell types are different.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

Different hybrid effects in reciprocal crosses betweenChironomus thummi thummi andCh. th. piger including spontaneous chromsome aberrations and sterility

Hybrids from reciprocal erosses between twoChironomus thummi thummi andCh. th. piger laboratory stocks show four abnormalities in comparison to the parental stocks. One cross direction (Ch. th. thummi x Ch. th. piger ) is characterized by chromosome aberrations, reduced hatchability and malformations, whereas in reciprocal hybrids both sexes are sterile. Sterility is the consequence of rudimentary or non developed gonads.InCh. th. thummi x Ch. th. piger crosses chromosome aberrations were analysed in salivary gland nuclei. These aberrations are all somatic in origin, and they are induced during the first 40 h of embryonic development, prior to the onset of polytenization. The chromosomes of both subspecies are equally affected. In all four chromosomes breaks occur preferentially at specific regions. Reduced hatchability and malformations are presumably caused by chromosome mutations because within egg-masses a correlation exists between the rate of salivary gland chromosome aberrations and the rates of hatchability and malformations.