Differential sensitivity of muntjac lymphocyte chromosomes to mitomycin C,bromodeoxyuridine and hydroxylamine at different cell-cylce stages

Quantitative and qualitative analyses were made of aberrations induced by 3 hitherto well-known mutagens, mitomycin C (MC), 5-bromodeoxyuridine (BUdR and hydroxylamine hydrocholride (HA), in muntjac chromosomes, during different stages of the cell cycle. The sensitivity ro MC was increased in G₁, reached its maximum in early S and was considerably decreased in late S and G₂ stage treated cells. BUdR induced maximal aberrations when given during the synthetic phase and the cells in G₁ and G₂ were least affected. The sensitivity of the cells to HA in terms of induced chromosomal aberrations increased as they moved through the cell cycle, i.e. more damage was observed in cells treated in late S and G₂ stages than in those treated at G₁ and early S stages. While there were defined patterns of cell-cylce stage-dependent sensitivity for all 3 chemicals, the chromosomal sites being preferentially affected by each were found to be specific and invariant at different stages. Thus, it is presumed that the functional state of such “preferred sites” at one or other stage of the cell cycle is the factor responsible for the stage-dependent sensitivity of a cell towards these chemicals.     

Nonrandom distribution of chromosomal aberrations induced by three chemicals

The G-band locations of 3244 breakpoints induced by cis-platinum (II) diamminedichloride (PDD), 1460 breakpoints induced by cytosine arabinoside (ara-C), and 1257 breakpoints induced by triethylenemelamine (TEM) in human lymphocyte chromosomes were identified. The breakpoints induced by each of these chemicals demonstrated a significantly nonrandom distribution within the human karyotype. The overall pattern of the interarm distribution was dependent upon the chemical used, but certain chromosomes arms exhibited similar responses to all 3 chemicals. Comparison of the frequencies of breakpoints within individual G-bands indicated that (1) certain bands were susceptible to damage induced by all 3 chemicals; (2) certain bands were resistant to damage by all 3 chemicals; (3) certain bands demonstrated variable susceptibility to induced damage dependent upon the chemical agent; and (4) other bands demonstrated near expected frequencies of damage (by length) to all 3 agents.     

Patterns of DNA replication of human chromosomes. II. Replication map and replication model.

Combining higher resolution chromosome analysis and bromodeoxyuridine (BrdU) incorporation, our study demonstrates that: (1) Human chromosomes synthesize DNA in a segmental but highly coordinated fashion. Each chromosome replicates according to its innate pattern of chromosome structure (banding). (2) R-positive bands are demonstrated as the initiation sites of DNA synthesis in all human chromosomes, including late-replicating chromosomes such as the LX and Y. (3) Replication is clearly biphasic in the sense that late-replicating elements, such as G-bands, the Yh, C-bands, and the entire LX, initiate replication after it has been completed in the autosomal R-bands (euchromatin) with minimal or no overlap. The chronological priority of R-band replication followed by G-bands is also retained in the facultative heterochromatin or late-replicating X chromosome (LX). Therefore, the inclusion of G-bands as a truly late-replicating chromatin type or G(Q)-heterochromatin is suggested. (4) Lateral asymmetry (LA) in the Y chromosome can be detected after less than half-cycle in 5-bromodeoxyuridine (BrdUrd), and the presence of at least two regions of LA in this chromosome is confirmed. (5) Finally, the replicational map of human chromosomes is presented, and a model of replication chronology is suggested. Based on this model, a system of nomenclature is proposed to place individual mitoses (or chromosomes) within S-phase, according to their pattern of replication banding. Potential applications of this methodology in clinical and theoretical cytogenetics are suggested.     

Zebrafish chromosome banding.

Banding techniques were carried out on metaphase chromosomes of zebrafish (Danio rerio) embryos. The karyotypes with the longest chromosomes consist of 12 metacentrics, 26 submetacentrics, and 12 subtelocentrics (2n = 50). All centromeres are C-band positive. Eight chromosomes have a pericentric C-band in each arm and 22 chromosomes have one in the longest arm. Two chromosomes have a slightly heterochromatic long arm and five chromosomes have an Ag-NOR at the terminal end of the long arm. Other banding patterns and sex chromosomes could not be revealed.     

Specific modification of Q and R bands by 5-bromodeoxyuridine and actinomycin D]

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.     

Modification specifique des bandes Q et R par le 5-bromodeoxyuridine et l'actinomycine D

When introduced into human lymphocyte culture, 5-bromodeoxyuridine (BUdR) and actinomycin D (AMD) induce chromosome differentiation by lack of condensation of segments corresponding to Q-bands (BUdR) and R-bands (BUdR and AMD). The total amount of DNA per cell is not modified by these treatments. The non-condensed segments partly keep their properties of R- or Q-banding after heat treatment or staining with quinacrine mustard. On the other hand, they lose their properties after ASG treatment (G-bands), and emit modified fluorescence after staining with acridine orange. With heat treatment or QM staining, it seems that BUdR or AMD elongate the R or Q segments in several ways—homogeneous repartition or fragmentation of various types. On the other hand, this elongation seems homogeneous after Feulgen staining. This suggests that the relation between Feulgen-revealed DNA and substratum of the R- and Q-bands might not be direct.     

Dependence of heterochromatin differential staining on the time of its reduplication and the degree of condensation]

A comparison of the chromosomes banding pattern after G-and C-staining with the time of DNA reduplication and the degree of chromosome condensation, was carried out using Chinese hamster metaphase chromosomes. Chromosome condensation was studied under 5-bromodeoxyuridine and 5-bromodeoxycytidine treatment. All the chromosomal segments stained with C-technique are also stainable with G-technique, while only some G-positive segments are capable to be C-bands. C-bands are heterochromatic segments characterized by extremely late replication and great delay in condensation under the analog action, while G-bands are segments with earlier labelling and irregular decondensation. The data obtained suggest a close correlation between the capability of chromosomal region of G- and C- staining and the degree of its heterochromatinization.     

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study

Summary A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%–85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.     

Dynamic G- and R-banding of human chromosomes for electron microscopy

Synchronized human lymphocytes were exposed to 5-bromo-2-deoxyuridine (BrdUrd) for incorporation in either G-or R-bands. The substituted bands were revealed by monoclonal anti-BrdUrd antibodies disclosed with either gold-labeled antibodies or with the protein A-gold complex. Sharp G-or R-banding, specific for electron microscopy (EM), was obtained. These banding patterns, referred to as GB-AAu (G-bands by BrdUrd using Antibodies and gold [Au]) and RB-AAu (R-bands by BrdUrd using Antibodies and gold [Au]), resemble dynamic band patterns (GBG and RBG) much more than they do morphologic band patterns (GTG and RHG). The G- and R- band patterns allow accurate chromosome identification and karyotyping. An actual karyotype of human GB-AAu-banded chromosomes at the 750 band level, photographed in the EM, is presented. The method produces excellent band separation and band contrast. Variations in band staining intensities were noted and correlated with BrdUrd enrichment. The C-band regions were positively stained after GB-AAu banding while they were negatively stained after RB-AAu banding. Telomeres appeared heterogeneous after GB-AAu banding suggesting that part of the telomeric bands might be late replicating.     

On the induction of chromosome structural changes by hydroxylamine in Vicia faba

Primary roots of a new karyotype of Vicia faba with all chromosomes inter-distinguishable have been used to study the induction by hydroxylamine hydrochloride (HA) of chromatid aberrations and their intrachromosomal distribution. HA induced both chromatid intra- and interchanges of the delayed type. The effectiveness of HA increased with increasing temperature and was dependent on the pH during treatment (more aberrations at pH 7.5 as compared with 4.8). The frequency of incomplete reunion was markedly higher after HA treatment than after treatment with maleic hydrazide (MH) or ethanol. In combined treatments, HA reduced the reunion involvement in HA-induced aberrations of certain chromosome segments was found and compared with distribution patterns of chromatid aberrations after treatment with MH and ethanol. Data and hypotheses concerning possible modes of action of HA eventually resulting in chromosome structural changes are discussed. It is concluded that alterations of the cytosine moiety in chromosomal DNA are not responsible for chromosomal damage induced by HA.     

Replication banding patterns in the spined loach,Cobitis taenia L. (Pisces,Cobitidae)

The chromosomal complement of Cobitis taenia was analysed by replication banding techniques to determine whether there were specific patterns that could allow distinction of the different chromosomes. The diploid chromosome number of 2n = 48 is diagnostic of this species. In vivo 5-bromodeoxyuridine (5-BrdU) incorporation induced highly reproducible replication bands. Most of the chromosome pairs were distinguishable on the base of their banding patterns. The karyotype, consisting of five pairs of metacentrics, nine pairs of submetacentrics and 10 pairs of subtelocentrics and acrocentrics, was confirmed. C-banding and replication banding patterns were compared, and heterochromatin was both early and later replicating. C-positive heterochromatin in centromeric regions was mainly early replicating, but that located in pericentromeric regions was late replicating. Most of the late-replicating regions found interstitially were C-band negative. The results obtained so far for combined chromosomal staining methods of C. taenia and other Cobitis fish species are discussed.     

Inhibitory effect of ethidium bromide on mitotic chromosome condensation and its application to high-resolution chromosome banding

Ethidium bromide (EB) is known to intercalate between stacked base pairs without specific base-pair preference. Its use in cultured human lymphocytes and Burkitt's lymphoma cells resulted in the accumulation of cells in prophase and prometaphase stages. Inhibition of mitotic chromosome condensation as a possible mechanism involved in this phenomenon is discussed. A simple method for obtaining high-resolution banding patterns on elongated chromosomes was devised as follows: Human lymphocytes cultured for 3 days with phytohemagglutinin were exposed to EB (5-10 micrograms/ml) and Colcemid (0.02 micrograms/ml) simultaneously for 2 h and then routinely harvested for chromosome preparation. High-resolution G-bands were obtained by Giemsa staining following mild trypsin treatment.     

Relation between the SCE points and the DNA replication bands

A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication bands (=euchromatic segments=negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.     

Induction of a high incidence of damage to the X chromosomes of Rattus (Mastomys) natalensis by base analogues,viruses, and carcinogens

Cultured embryonic cells from Rattus (Mastomys) natalensis were treated with the base analogues 5-bromodeoxyuridine and 2-aminopurine, the viruses herpes simplex virus and adenovirus type 12, and the carcinogens 7,12-dimethylbenz(a)anthracene and urethan. Treatment with these agents, except urethan, causes an increase in the incidence of chromosome aberrations. The induced aberrations are not randomly distributed among the chromosomes or within a chromosome. The X chromosome, especially its long arm, is more sensitive to these agents than is any other chromosomes in the complement. — A possible relationship among the high incidence of damage, the positive heteropycnosis, and the late replication in the X chromosome of R. natalensis is discussed.     

Evidence for a highly differentiated sex chromosome heteromorphism in the salamander Necturus maculosus (Rafinesque)

The mitotic chromosomes of the neotenic (sensu Gould, 1977, and Alberch et al., 1979) salamander Necturus maculosus (Rafinesque) have been examined using a C-band technique to demonstrate the distribution of heterochromatin. The C-banded mitotic chromosomes provide evidence of a highly differentiated XY male/XX female sex chromosome heteromorphism, in which the X and Y chromosomes differ greatly in size and morphology, and in the amount and distribution of C-band heterochromatin. The X chromosome represents one of the largest biarmed chromosomes in the karyotype and is indistinguishable from similar sized autosomes on the basis of C-band heterochromatin. The Y chromosome, on the other hand, is diminutive, morphologically distinct from all other chromosomes of the karyotype, and is composed almost entirely of C-band heterochromatin. The discovery of an X/Y chromosome heteromorphism in this species is consistent with the observation by King (1912) of a heteromorphic spermatogenic bivalent. Karyological and phylogenetic implications are discussed.     

Harlequin banding and localisation of sister-chromatid exchanges.

Harlequin banding (HB) was standardised on Indian muntjac chromosomes by superimposing harlequin staining or sister-chromatid differentiation and G-banding after incorporation of bromodeoxyuridine (BrdU) or cholorodeoxyuridine (CldU), and after treatment with BrdU plus mitomycin C (MMC). SCEs were localized on these chromosomes with the aid of the G-band map. There were more SCEs in G-bands than in R-bands in BrdU-incorporated chromosomes. CldU-incorporated chromosomes, however, did not show a preferential localization of SCEs in either G- or R-bands. When BrdU + MMC-induced SCEs were localized in harlequin-banded chromosomes, there was a significantly greater number of SCEs in R-bands; and there was a concomitant reduction in the frequency of SCEs in G-bands, as compared to the SCEs observed in this region after BrdU incorporation alone. Centromeric regions of chromosomes 1 and X had preferred sites for occurrence of SCEs in BrdU-incorporated chromosomes, the preferred sites being more in G-bands after BrdU and CldU incorporation and in R-bands after treatment of BrdU-incorporated chromosomes with MMC. Thus the formation of SCEs is not restricted by structure per se as defined by euchromatin or heterochromatin, but depends on the site of lesion production, type of lesion and repair pathway followed.     

Mechanisms of chromosome banding

Photo-oxidation of mitotic human chromosomes has been used in conjunction with anti-cytosine and anti-adenosine antibodies to produce R-banding. To elucidate the mechanism of this banding procedure we have examined the effect of photo-oxidation alone on chromosomes and nuclei. With short exposures to light in the presence of dilute methylene blue, C-band areas on chromosomes 1, 9, 16 and the terminal segment of the Y stain poorly. We call this phenomena reverse C-banding. After 18 h of exposure to light the chromosomes are swollen and show very little staining with quinacrine or Giemsa. Quantitative autoradiography shows that their DNA is almost completely extracted. Cytophotometric measurements also confirm that nuclear DNA is progressively extracted according to the length of exposure to light. When chromosomes are exposed to dilute methylene blue alone, without light, G-banded chromosomes result. We suggest the following explanation for these observations. In dilute methylene blue, C-band regions take up the greatest amount of dye and after short periods of photo-oxidation the DNA of these regions is preferentially destroyed resulting in reverse C-banding. Autoradiography in photo-oxidized chromosomes suggested that this preferential destruction of C-segments occurred in our experiments. With more prolonged exposure the DNA of the G-bands regions is preferentially destroyed and staining the remaining DNA with sensitive fluorescent labeled anti-C antibodies results in R-banding.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.