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.     

Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R-and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.     

Chromosome mapping of the human cytidine-5′-triphosphate synthetase (CTPS) gene to band 1p34.1–p34.3 by fluorescence in situ hybridization

Summary The human cytidine-5-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1–34.3 of the short arm of chromosome 1; 1p34.1–p34.3. Simple procedures for the detection of R-bands are described.     

R-banding produced by DNase I digestion of chromomycin-stained chromosomes

A distinct reverse (R-) banding pattern was produced on human chromosomes by digesting chromosome spreads with pancreatic deoxyribonuclease I (DNase I) in the presence of an excess of chromomycin A3 (CMA), followed by staining with Giemsa. The banding pattern corresponds with that obtained by chromomycin A3 fluorescence, and bands which fluorescence brightly with chromomycin appear darkly with Giemsa. The same relationship was observed in two plants, Scilla siberica and Ornithogalum caudatum, which have contrasting types of heterochromatin. Chromomycin bright C-bands stained darkly with the CMA/DNase I technique, whereas chromomycin negative C-bands appeared lightly stained. The digestion patterns are thought to reflect the variation in chromomycin binding capacity along the chromosome with R-bands and dark C-bands being sites which preferentially bind the antibiotic.     

DNA denaturation for ultrastructural banding and the mechanism underlying the fluorochrome-photolysis-Giemsa technique studied with anti-5-bromodeoxyuridine antibodies

G- and R-bands produced by an immunochemical approach were studied by electron microscopy (EM) to evaluate the role of DNA denaturation on banding quality. Excelent banding was observed only after adequate denaturation by HCl, NaOH and formamide, used in appropriate concentrations to provide uniform 5-bromodeoxyuridine (BrdUrd) exposure by generating single-stranded DNA. Formamide treatment resulted in less intercellular variability. High temperature and high concentrations of NaOH and HCl altered chromosomal morphology. Besides formamide, Hoechst 33258 prestaining which does not interfere with the binding of the anti-BrdUrd antibody and UV irradiation associated with formamide also produced high quality banding. On the other hand, consecutive Hoechst and UV treatment completely inhibited the immunochemical banding. The data indicate that Hoechst and UV act synergistically to disintegrate BrdUrd-substituted chromatin from which DNA is then extracted, leaving only the unsubstituted DNA stainable with Giemsa.     

Banding Human Chromosomes Using a Combined C-Banding-Fluorochrome Staining Technique

Slides pretreated for C-banding and stained with DAPI or CMA3 show different banding patterns in human metaphase chromosomes compared to those obtained with either standard Giemsa C-banding or fluorochrome staining alone. Human chromosomes show C-plus DA-DAPI banding after C-banding plus DAPI and enhanced R-banding after C-banding plus Chromomycin A3 staining. If C-banding preferentially removes certain classes of DNA and proteins from different chromosome domains, C-banding pre-treatment may cause a differential DNA extraction from G- and R-bands in human chromosomes, resulting in a preferential extraction of DNA included in G-bands. This hypothesis is partially supported by the selective cleavage and removal of DNA from R-bands of restriction endonuclease HaeIII with C-banding combined with DAPI or Chromomycin A3 staining. Structural factors relating to regional differences in DNA and/or proteins could also explain these results.     

Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts

Polyacrylamide slab gel electrophoresis and [35S]methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.     

Analysis of high-resolution R-bands, obtained by heat-denaturation and Giemsa staining, on human prophase chromosomes

RHG-bands (heat-denatured Giemsa R-bands) of human prophase chromosomes were analyzed at high resolution, and the banding patterns at prophase and metaphase are presented. The bands were compared with those of the International Standard Cytogenetic Nomenclature idiograms and of the G-band idiograms proposed by J. J. Yunis. The number, size, and position of the RHG-bands correspond rather well with their equivalent G-negative bands, but some differences were noted in the zones of preferential stretching, the juxtacentromeric regions, and the telomeres. Variations in the centromere index and the banding pattern in heterochromatin were also discussed.     

Chromosome banding analysis by slit-scan flow cytometry

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers.     

A new chromosome fluorescence banding technique combining DAPI staining with image analysis in plants

In this study, a new chromosome fluorescence banding technique was developed in plants. The technique combined 4,6-diamidino-2-phenylindole (DAPI) staining with software analysis including three-dimensional imaging after deconvolution. Clear multiple and adjacent DAPI bands like G-bands were obtained by this technique in the tested species including Hordeum vulgare L., Oryza officinalis, Wall & Watt, Triticum aestivum L., Lilium brownii, Brown, and Vicia faba L. During mitotic metaphase, the numbers of bands for the haploid genomes of these species were about 185, 141, 309, 456 and 194, respectively. Reproducibility analysis demonstrated that banding patterns within a species were stable at the same mitotic stage and they could be used for identifying specific chromosomes and chromosome regions. The band number fluctuated: the earlier the mitotic stage, the greater the number of bands. The technique enables genes to be mapped onto specific band regions of the chromosomes by only one fluorescence in situ hybridisation (FISH) step with no chemical banding treatments. In this study, the 45S and 5S rDNAs of some tested species were located on specific band regions of specific chromosomes and they were all positioned at the interbands with the new technique. Because no chemical banding treatment was used, the banding patterns displayed by the technique should reflect the natural conformational features of chromatin. Thus it could be expected that this technique should be suitable for all eukaryotes and would have widespread utility in chromosomal structure analysis and physical mapping of genes.     

Chromosome organisation in the Australian plague locust Chortoicetes terminifera

In Chortoicetes terminifera 45 independently-occurring B-chromosomes were tested and 23 distinct banding variants were detected with either G- or C-banding; six types were found more than once. In particular the Type I banding morph was detected 12 times indicating that individuals carrying this type may be under a different regime of selection compared with individuals bearing other types of banding morph; or the Type I may be subjected to a higher rate of meiotic drive in either or both sexes than other types. Also the Type I appeared to be obviously related to four other banding morphs whereas most types were not obviously related to any other banding morphs, but a few were similar in banding pattern to one or two other types. Three types of B-chromosomes were found in three or more different populations. A relatively high frequency of the Type I banding morph was found in one population, which was probably mainly composed of non-migratory individuals, and also in a laboratory-raised population. The most likely mechanisms for small changes in the banding sequence of the B-chromosomes are three-break insertions which are often indistinguishable from inversions. Rearrangements which add or delete bands, or sequences of bands, to or from B-chromosomes are probably the result of exchanges which are now known to take place in rare individuals with two B-chromosomes. The most distal region of all the banding morphs of the B-chromosome in C. terminifera, plus a short interstitial region in some types, is not late-replicating and has the banding characteristics of euchromatin. The rest of the chromatin of the B-chromosomes is heterochromatic and is the latest replicating heterochromatin in the whole genome. It consists of G-bands, which are also deeply stained with C-banding, and alternating G-interbands, which in turn are stained grey with C-banding. Both of these staining combinations are seen in heterochromatin of the normal complement. The heterochromatin of the B-chromosomes is condensed throughout 1st meiotic prophase in the male and in all somatic interphase nuclei where it can be quickly detected using the G-banding technique. The B-chromosome has a relatively constant, acrocentric morphology with a tendency to increase of length of the long arm as band numbers increase. Isochromosomes of the long arm have been seen only in laboratoryraised embryos. From egg pods with significantly fewer than expected B-chromosomes it is strongly suggested that more than one male may fertilize the eggs in a single pod.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

Different reactivity of Z-DNA antibodies with human chromosomes modified by actinomycin D and 5-bromodeoxyuridine

Summary Antibodies against Z-DNA react with fixed metaphase chromosomes of man and other mammals. Indirect immunofluorescence staining shows that chromosomal segments corresponding to R- and T-bands preferentially fix Z-DNA antibodies. In this work Z-DNA antibodies were used as a probe for DNA conformation in euchromatin of fixed human chromosomes whose condensation or staining were modified by actinomycin D (AMD) and by 5-bromodeoxyuridine (BrdU). Treatments with AMD and BrdU were performed to induce a G-banding by modification of chromosomal segments corresponding to R- and T-bands. Long BrdU treatments were used to induce asymmetrical and partially undercondensed chromosomes by substitution of thymidine in one or both DNA strand. Our results show a clear difference of Z-DNA antibodies reactivity after AMD or BrdU treatment. The G-banding obtained after AMD treatment is not reversed by Z-DNA antibodies staining since these antibodies bind very weakly to the undercondensed R-bands. On the other hand, the G-banding obtained by BrdU is completely reversed giving typical R-banding, as on untreated chromosomes. For asymmetrical chromosomes an R-, T-banding pattern is always observed but there is a decrease of the fluorescence intensity proportional to the degree of BrdU incorporation. We conclude that AMD treatment greatly disturbs Z-DNA antibodies binding suggesting a change in DNA conformation, whereas BrdU treatments do not suppress but only weaken the specific binding of Z-DNA antibodies on R- and T-bands. The direct involvement of thymidine substitution in DNA sequences recognized by Z-DNA antibodies is discussed.     

Chromosome banding and compaction

Summary We have described a characteristic substructure of mitotic chromosomes, the chromosomal unit fibre, with lengths about five times the length of the corresponding metaphase chromosomes and a uniform diameter of 0.4 m. In order to study the relationship of chromosome banding to chromosome compaction, methods have been devised to obtain banding patterns on chromosomal unit fibres, similar to G-band patterns of intact mitotic chromosomes. The total number of bands plus interbands per haploid human karyotype is estimated at about 3000. The banding pattern of chromosomal unit fibres indicates a certain resemblance to the normal G-banding pattern of human chromosomes even if the details indicate a short-range random distribution.     

水稻染色体G—带的研究

用改良的ASG法首次在籼稻(O.sativa subsp.indica)品种珍汕97和粳稻(O.subsp.iaponica)品种秀岭的有丝分裂染色体上显示了G-带,并作了相应的G-带核型分析。就同一材料来说,随着有丝分裂时期的推进,染色体上带纹数目逐渐减少。籼、粳亚种间相对应的同源染色体上G-带带纹特征彼此相似。讨论了水稻G-带带型与染色体不同区域分化的关系;G-带带型与籼、粳稻分歧的关系;以及G-显带的方法。     

Idiograms of horse chromosomes at prometaphase, early metaphase, and midmetaphase after R-banding by BrdU incorporation followed by the fluorochrome-photolysis-Giemsa technique

We present three idiograms of equine chromosomes, R-banded after BrdU incorporation and stained by the fluorochrome-photolysis-Giemsa technique. The haploid set of prometaphasic chromosomes shows 591 bands (range 7-38 per chromosome), the early metaphasic set 404 (range 5-26), and the midmetaphasic set 272 (range 3-18). Following cell synchronization with thymidine, more than twice as many R-bands were revealed on the resulting prometaphasic chromosomes, making possible the establishment of a very accurate and characteristic representation of this banding pattern in the domestic horse.     

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.     

Early and late replication patterns of increased resolution in human lymphocyte chromosomes

High resolution patterns of DNA replication in human lymphocyte chromosomes during early and late S-phases were studied by means of the BrdU-Hoechst-Giemsa technique. The late replicating bands were found to be identical with highly detailed G-bands. Between early replicating bands and R-bands subtile differences were observed. A possible correlation between a replication band seen on the chromosomal level and a replication cluster observed after fiber autoradiography is discussed. Dedicated to Professor Dr. Wolfgang Beermann on the occasion of his 60th birthday     

Electron microscopy of gold-labeled human and equine chromosomes

We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. The fusion appears to be owing to chromatin reorganization. Our results underline the value of using immunogold reagents, which are ideal probes for antigen localization on chromosomes.     

Genes and genomes: Chromosome bands – flavours to savour

The mammalian chromosome is longitudinally heterogeneous in structure and function and this is the basis for the specific banding patterns produced by various chromosome staining techniques. The two most frequently used techniques are G, or Giemsa banding and R, or reverse banding. Each type of stained band is characterised by variations in gene density, time of replication, base composition, density of repeat sequences, and chromatin packaging. It is increasingly apparent that R and G bands, which are complementary to each other, represent separate compartments of the euchromatic human genome, with R bands containing the vast majority of genes. R bands are also more GC-rich, contain a higher density of Alu repeats, and replicate earlier in S phase, than G bands. These properties may be interdependent and may have coevolved.