Chromosome banding and identification. Fluorescence.
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Acridine Orange↗
Biomedical subjects
A T Sumner
Publications and source records attributed to A T Sumner.
Functional aspects of the longitudinal differentiation of chromosomes.
The discovery of chromosome banding techniques over 20 years ago has revealed extensive longitudinal differentiation of chromosomes. This longitudinal differentiation can be classified into four types: heterochromatin, euchromatic bands, nucleolar organisers (NORs) and kinetochores. The telomeres, at the ends of chromosomes, cannot be detected by banding methods, but are clearly shown by in situ hybridisation. The functions of nucleolar organisers, kinetochores, and telomeres are reasonably well known, but the reasons for the differentiation of the greater part of the chromatin into heterochromatin and euchromatic segments remains uncertain. The function of heterochromatin may be sought in its centrometric location, where part of it is associated with the kinetochores, and another part appears to hold the sister chromatids together until anaphase. It appears that highly conserved nucleotide sequences are not required for these functions, but highly repeated sequences may be necessary. Nevertheless, these functions cannot explain the whole of heterochromatin. G-banding and other methods for euchromatic banding have shown that the euchromatic parts of chromosomes are divided into two major compartments, one gene-rich and the other gene-poor, which also differ in many other properties. The reason for this, which seems to be a fundamental property of chromosome organisation in eukaryotes, is totally obscure. Nevertheless, the observations that the greatest concentrations of genes tend to be found near the ends of chromosomes, and that the telomeres are often located at the nuclear envelope, suggest that a mechanism may have evolved to ensure that active genes are close to the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The distribution of genes on chromosomes: a cytological approach.
Studies during the last 20 years have shown that the chromosomes of many organisms, especially those of higher vertebrates, consist of a series of segments having different properties. These can be recognized as, for example, G- and R-bands. Recent studies have indicated that genes tend to lie in the R-bands rather than in the G-bands, although the number of genes that has been mapped with high precision is, as yet, only a very small proportion of the total, probably much less than 1%. We have therefore sought to study the distribution of genes on chromosomes using a cytological approach in conjunction with "universal" markers for genes. Such markers include mRNA and the gene-rich, G+C-rich H3 fraction of DNA, both of which can be localized using in situ hybridization, and DNase I hypersensitivity, and digestion by restriction enzymes known to show selectivity for the CpG islands associated with active genes, both of which can be detected using in situ nick translation. We have chosen to use the approaches involving in situ nick translation and have shown that the patterns of DNase I hypersensitivity and of CpG islands on human chromosomes show a strict correspondence to R-banding patterns: Deviations from R-banding patterns reported by previous investigators who have made similar studies appear to be attributable to excessive digestion.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Micronuclei in neonatal lymphocytes treated with the topoisomerase II inhibitors amsacrine and etoposide.
It has been suggested that the enzyme topoisomerase II may be important in chromosome segregation due to the role played by the enzyme in decatenating the intertwined DNA molecules that result from DNA replication. Inhibition of the enzyme has been found by some workers to inhibit chromatid separation in mammalian cells, while others have reported that the passage of cells through mitosis is unaffected. Inhibition of the enzyme with topoisomerase II inhibiting drugs also results in the formation of micronuclei as a consequence of DNA damage. We have used the micronucleus assay with CREST staining to investigate whether the micronuclei formed in neonatal lymphocytes after inhibition of topoisomerase II are formed from whole chromosomes, implying non-disjunction, or acentric fragments. We found that treatment with both amasacrine and etoposide caused a dose-related increase in the number of CREST negative micronuclei, with only a very small increase in the number of CREST positive micronuclei at high concentrations of the compounds. Although we cannot conclude from our experiments that treatment with topoisomerase II inhibitors does not affect the segregation of neonatal lymphocytes, the production of CREST negative micronuclei suggests that segregation abnormalities are less important than other mechanisms which may cause cytotoxicity from exposure to these compounds.
Amsacrine↗
The distribution of genes on human chromosomes as studied by in situ nick translation.
We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme--nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.
Chromosome Mapping↗
A microchromosome derived from chromosome 11 in a patient with the CREST syndrome of scleroderma.
A patient with the CREST syndrome of scleroderma was found to carry a mosaicism for a supernumerary microchromosome. The microchromosome was approximately 1 micron in size and present in over half of the lymphocyte metaphases examined. It bound centromeric proteins specifically recognized by CREST autoimmune sera (including the patient's serum). In situ hybridization with a panel of chromosome-specific alpha-satellite probes showed that the microchromosome was derived from chromosome 11, most or all of its chromatin consisting of the chromosome 11 subset of alpha-satellite DNA. It had no detectable telomeric sequences. Microchromosomes observed by electron microscopy had no visible free ends. The chromatin looked exactly the same as it did in normal chromosomes. Although we have no direct evidence for a circular structure, we conclude that the microchromosome originated by an interstitial deletion including the alpha-satellite DNA sequences and subsequent ring formation. The newly formed chromosomal element proved to be relatively stable somatically and was transmitted through meiosis. Since it possesses at least some structural and functional features of a centromeric region, the microchromosome can be thought of as an isolated centromere.
Centromere↗
Heterochromatin characterization of sex chromosomes in Triturus marmoratus (Urodela, Salamandridae).
The sex chromosomes of the Iberian marbled newt, Triturus marmoratus, were studied using various banding techniques, including restriction enzyme/nick translation (RE/NT) procedures. Four types of heterochromatin on the sex chromosomes could be distinguished: (1) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT negative, and HaeIII/NT and HinfI/NT positive; (2) distamycin A/DAPI and chromomycin A3/distamycin A positive, but RE/NT negative; (3) AT rich, but RE/NT negative; and (4) distamycin A/DAPI and chromomycin A3/distamycin A positive, EcoRI/NT and HinfI/NT negative, but HaeIII/NT positive. These data suggest a common origin for the terminal heterochromatic domains of both the X and Y chromosomes in this species.
Animals↗
Inhibitors of topoisomerases do not block the passage of human lymphocyte chromosomes through mitosis.
Cultured human lymphocytes have been treated with a number of topoisomerase inhibitors, to see whether topoisomerase II is involved in the process of chromosome segregation at anaphase. Results were assessed by examination of cytogenetical preparations of spread chromosomes. Four effects were observed, although no inhibitor produced all four effects. These effects were: inhibition of entry into mitosis; chromosome breakage and rearrangement; inhibition of chromosome condensation; and inhibition of chromosome segregation. Evidence for the last was ambiguous. Although there was evidence that separation of chromatids was affected when cells were treated with colchicine as well as topoisomerase II inhibitors (most notably with nalidixic acid, which resulted in complete fusion of the chromatids), no evidence was obtained to show that, in the absence of colchicine, cells treated with inhibitors could not proceed through anaphase normally. The topoisomerase I inhibitor, camptothecin, differed from the topoisomerase II inhibitors in not showing any effect on chromosome condensation or any significant effect on segregation.
Amsacrine↗
Scanning electron microscopy of mammalian chromosomes from prophase to telophase.
Changes in the morphology of human and murine chromosomes during the different stages of mitosis have been examined by scanning electron microscopy. Two important findings have emerged from this study. The first is that prophase chromosomes do not become split into pairs of chromatids until late prophase or early metaphase. This entails two distinct processes of condensation, the earlier one starting as condensations of chromosomes into chromomeres which then fuse to form a cylindrical body. After this cylindrical body has split in two longitudinally, further condensation occurs by mechanisms that probably include coiling of the chromatids as well as other processes. The second finding is that the centromeric heterochromatin does not split in two at the same time as the rest of the chromosome, but remains undivided until anaphase. It is proposed that the function of centromeric heterochromatin is to hold the chromatids together until anaphase, when they are separated by the concerted action of topoisomerase II acting on numerous similar sites provided by the repetitive nature of the satellite DNA in the heterochromatin. A lower limit to the size of blocks of centromeric heterochromatin is placed by the need for adequate mechanical strength to hold the chromatids together, and a higher limit by the necessity for rapid splitting of the heterochromatin at anaphase. Beyond these limits malsegregation will occur, leading to aneuploidy. Because the centromere remains undivided until anaphase, it cannot undergo the later stage of condensation found in the chromosome arms after separation into chromatids, and therefore the centromere remains as a constriction.
Animals↗
Selective digestion of mouse chromosomes with restriction endonucleases. II. X-ray microanalysis of HaeIII-treated chromosomes.
We used X-ray microanalysis to study the changes induced in mouse metaphase chromosomes as a result of digestion with the restriction endonuclease HaeIII. The phosphorus X-ray signal was used as a marker for DNA and the sulfur signal for protein. Calcium, iron, copper, and zinc were also detected. HaeIII induced a loss of phosphorus from both the centromeres and chromosome arms, but the losses in the arms were much greater. These changes were accompanied by an increase in the electron density of the centromeres and a reduction in that of the arms. No reduction in the sulfur signal in either arms or centromeres occurred as a result of HaeIII digestion. Except for calcium, which showed only a moderate reduction, the inorganic ions exhibited very large losses as a result of HaeIII digestion. The differentiation of chromosome arms and centromeres as a result of HaeIII digestion is therefore not simply due to differential loss of DNA but also involves structural reorganization of the chromatin, as shown by electron microscopy. This reorganization does not involve loss of proteins but may be correlated with changes in the amounts of inorganic ions known to be involved in chromatin condensation.
Animals↗
Patterns of digestion of human chromosomes by restriction endonucleases demonstrated by in situ nick translation.
A restriction enzyme-nick translation procedure has been developed for localizing sites of restriction endonuclease action on chromosomes. This method involves digestion of fixed chromosome preparations with a restriction enzyme, nick translation with DNA polymerase I in the presence of biotinylated-dUTP, detection of the incorporated biotin label with streptavidinalkaline phosphatase, and finally staining for alkaline phosphatase. Results obtained obtained on human chromosomes using a wide variety of restriction enzymes are described, and compared with results of Giemsa and Feulgen staining after restriction enzyme digestion. Results of nick translation are not in general the opposite of those obtained with Giemsa staining, as might have been expected. Although the nick translation procedure is believed to give a more accurate picture of the distribution of restriction enzyme recognition sites on chromosomes than Giemsa staining, it is clear that the results of the nick translation experiments are affected by accessibility to the enzymes of the chromosomal DNA, as well as by the extractability of the DNA.
Alkaline Phosphatase↗
Electron microscopy and biochemical analysis of mouse metaphase chromosomes after digestion with restriction endonucleases.
Electron microscopy (EM) of whole mounted mouse chromosomes, light microscopy (LM), and agarose gel electrophoresis of DNA were used to investigate the cytological effect on chromosomes of digestion with the restriction endonucleases (REs) AluI, HinfI, HaeIII and HpaII. Treatment with AluI produces C-banding as seen by LM, cuts DNA into small fragments, and reduces the density of centromeres and disperses the chromatin of the arms as determined by EM. Treatment with HinfI produces C-banding, cuts DNA into slightly larger fragments than does AluI and increases the density of centromeres and disperses the fibres in the chromosomal arms. Exposure to HaeIII produces G- + C-banding, cuts the DNA into large fragments, and results in greater density of centromeres and reduced density of arms. Finally HpaII digestion produces G-like bands, cuts the DNA into the largest fragments found and results in greater density of centromeres and the best preservation of chromosomal arms detected by EM. These results provide evidence for: (1) REs producing identical effects in the LM (AluI and HinfI) produce different effects in the EM. (2) All enzymes appear to affect C-bands but while REs such as AluI reduce the density of these regions, other enzymes such as HpaII, HaeIII or HinfI increase their density. Conformational changes in the chromatin could explain this phenomenon. (3) The appearance of chromosomes in the EM is related to the action of REs on isolated DNA. The more the DNA is cut by the enzyme, the greater the alteration of the chromosomal ultrastructure.
Animals↗
Unfixed and fixed human chromosomes show different staining patterns after restriction endonuclease digestion.
Restriction endonucleases (REs) have been widely used to produce banding patterns on chromosomes, but it remains uncertain to what extent the patterns are due to the sequence specificity of the enzymes, and to what extent chromatin structure influences the pattern of digestion. To throw light on this question, we have digested with restriction endonucleases unfixed chromosomes prepared in two different ways (isolated, and whole metaphase cells spread with a cytocentrifuge) and compared the results with those obtained on conventionally fixed chromosomes. Unfixed isolated chromosomes are easily destroyed by REs; after fixation with cold methanol, which produced minimal alteration to the chromatin structure, the chromosomes are resistant to the action of REs, and conventional methanol-acetic acid fixation is required to permit the induction of banding patterns by REs. Unfixed cytocentrifuge preparations, in which the chromosomes are still surrounded by cytoplasm, are much more resistant to the action of REs, and again banding patterns were only induced after methanol-acetic acid fixation. We conclude that the action of restriction endonucleases on chromosomes is strongly influenced by chromatin organisation, and that methanol-acetic acid fixation is required to permit the induction of conventional banding patterns on chromosomes.
Chromosome Banding↗
Flow cytometry measurements of human chromosome kinetochore labeling.
A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.
Cells, Cultured↗
Mammalian chromosome banding--an expression of genome organization.
Banding of metaphase chromosomes is an invaluable aid to analysing the complex genomes of vertebrates, but the biochemical basis for this phenomenon is poorly understood. Advances in molecular biology are beginning to point to features of genome organization that may play roles in chromosome banding.
Animals↗
Analysis of human metaphase chromosomes using antibodies to double-stranded and single-stranded DNA: staining patterns are related to DNA conformation.
Fresh and 6-day-old fixed chromosome spreads, both untreated and treated with various banding techniques and nucleases, were stained using monoclonal antibodies to double-stranded and single-stranded DNA. DNA in fixed chromosome preparations became progressively denatured with ageing. The staining pattern of untreated chromosomes with anti-double-stranded DNA antibodies (which resembles G-banding) was determined by the conformation of the chromosomal DNA.
Antibodies, Monoclonal↗
The DNA content of Chinese hamster meiotic metaphase chromosomes.
DNA values of Chinese hamster male meiotic metaphase chromosomes were measured by slide-based Feulgen cytometry. All the autosomes were distinguishable on the basis of their DNA content. No significant differences were found between the autosomes of the two male animals studied, but a significant difference was found in the DNA content of the sex-chromosome pair between these two animals.
Animals↗
Factors affecting preparation of chromosomes for scanning electron microscopy using osmium impregnation.
Osmium impregnation techniques have become useful for imparting conductivity to tissue specimens for SEM, thereby avoiding coating with gold or other metals. Such techniques have been developed to produce aesthetically pleasing images of mammalian (particularly human) chromosomes prepared by standard cytogenetical methods which use methanol-acetic acid fixation. The present study was designed: (1) to examine changes in the appearance of chromosomes as a result of preparation by osmium impregnation techniques; (2) to assess the function and importance of the various stages of chromosome preparation; and (3) to identify the chemical groups responsible for osmium binding. Methanol-acetic acid fixed chromosomes are known to have lost many proteins during fixation, and appear to be flattened down on the substrate. Osmium impregnation swells these flattened chromosomes to a variable extent, but the result is inevitably an artefact, albeit a useful one, and not a true representation of the chromosome in vivo. The size of chromatin fibres, for example, is the consequence of the degree of protein extraction during fixation, the loss of material during pre-treatments (e.g. trypsin), and the amount of osmium uptake during impregnation. Trypsin pre-treatment removes a surface coating of protein from the chromosomes as well as exposing chemical groups which can react with osmium. The principal reactive site appears to be amino groups, which bind glutaraldehyde, which in turn binds thiocarbohydrazide, to which the osmium becomes attached. Pre-treatments other than trypsin can be used to extract chromosomal material and to reveal different aspects of chromosome structure.
Animals↗