Banding patterns in newt chromosomes by the giemsa stain

Specific banding patterns can be produced on the mitotic chromosomes of the newt species Triturus vulgaris meridionalis and T. italicus by using the Giemsa stain technique. These bands are most useful cytogenetic markers in karyotyping, since they facilitate identification of the individual elements of the complements. Evaluation of the shape of chromosomes as well as of the banding patterns produced by the Giemsa stain indicates that the karyotypes of T. vulgaris meridionalis and T. italicus are differentiated: hence the specific distinction of the two Salamandrids, still debated by taxonomists, appears supported by chromosome evidence. — Most of the bands seem to correspond to the heterochromatic tracts observable on mitotic chromosomes from embryos and larvae either untreated or submitted to cold treatment. Besides, the comparison of mitotic karyotypes and lampbrush maps shows that the bands located near the centromeric regions of mitotic chromosomes probably correspond to the so-called bars visible on either side of centromeres of lampbrush chromosomes, while some of the subterminal bands may correspond to the sphere.This work was financially supported by C. N. R., Roma.     

Studies of Chromosome Banding with Giemsa in Vicia faba and Allium cepa

Giemsa dye is a complex mixture containing methylene blue, its oxidation products-azure Ⅰ, Ⅱ, Ⅲ, and their eosinate. The results of our experiments have demonstrated that staining with methylene blue alone can give a faint trace of banding as well as azure Ⅰ, Ⅱ. No bands are obtained with eosin. Nevertheless, good chromosome bandings can be often produced by staining with methylene blue-eosinate or azure Ⅱ-eosinate. These data indicate that eosinate has an important effect for the formation of C-banding on plant chromosomes. In our experiments, the treatments of chromosomes with trypsin or papain have also resulted in good C-banding pattern when slides are stained with Giemsa. We found that the slides untreated with proteinase showed homogeneous intense chromosome staining and, on the contrary, the slides treated with proteinase led to palestaining chromosomes and presenting bandings. It has shown that proteinase, especially trypsin, not only can remove a large amount of chromosomal protein but also can remove DNA and results in C-bandings. Treated properly with trypsin and followed by the Feulgen staining, chromosomes can also produce the C-bandings, but chromosomes treated overtime with trypsin are stained more palely in Feulgen reaction or lead to colourlessness. The above results have further proved that trypsin technique removes large amounts of chromosome DNA and removes less from the C-band regions than from the non-band regions. In this paper we mainly discussed the effects of protein on mechanism of plant chromosome banding. We consider that the production of plant C-banding is probably due to the differential accessibility of nucleoprotein between euehromatin and heteroehromatin regions. It brings about selective removal of nucleoprotein from the chromosome arms. We have compared the effect of trypsin with papain and pepsin on producing bands. Good bands are produced by Giemsa staining chromosomes with trypsin, but no bands are obtained by staining chromosomes treated with pepsin. So the results have expressed that histones are possibly playing more important role in C-bandings.     

Restriction endonuclease banding of rainbow trout chromosomes

Rainbow trout chromosomes were treated with nine restriction endonucleases, stained with Giemsa, and examined for banding patterns. The enzymes AluI, MboI, HaeIII, HinfI (recognizing four base sequences), and PvuII (recognizing a six base sequence) revealed banding patterns similar to the C-bands produced by treatment with barium hydroxide. The PvuII recognition sequence contains an internal sequence of 4 bp identical to the recognition sequence of AluI. Both enzymes produced centromeric and telomeric banding patterns but the interstitial regions stained less intensely after AluI treatment. After digestion with AluI, silver grains were distributed on chromosomes labeled with [³H]thymidine in a pattern like that seen after AluI-digested chromosomes are stained with Giemsa. Similarly, acridine orange (a dye specific for DNA) stained chromosomes digested with AluI or PvuII in patterns resembling those produced with Giemsa stain. These results support the theory that restriction endonucleases produce bands by cutting the DNA at specific base pairs and the subsequent removal of the fragments results in diminished staining by Giemsa. This technique is simple, reproducible, and in rainbow trout produces a more distinct pattern than that obtained with conventional C-banding methods.     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

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.     

Analysis of different banding patterns and late replicating regions in chromosomes of Allium cepa,A. sativum and A. nigrum

Different banding procedures and preferential Giemsa staining of late replicating DNA-rich regions were carried out in metaphase chromosomes of three species belonging to different sections of the genus Allium (A. cepa, A. sativum and A. nigrum). The banding, as well as the late replicating patterns were species-specific. The late replicating pattern proved to be, in all cases, the more detailed, and represented the highest percentage of the karyotype differentially stained. Lower percents of the karyotype positively stained were accounted for by C-banding, by modified C-banding and by N-banding. In A. cepa interphase nuclei the pattern of constitutive heterochromatin fitted well with that of late replicating DNA-rich regions, but the coincidence with that revealed by C-banding was only partial. This supports the suggestion that late replicating regions may be considered to be a special category of heterochromatin. On the other hand, it seems that not all C-banded material replicates at the end of the S phase. By the modified C-banding, stained centromere dots or small bands, as well as bands at the NORs are observed.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

Giemsa banding,quinacrine fluorescence and DNA-replication in chromosomes of cattle (Bos taurus)

Almost all the 30 chromosome pairs of cattle can be identified by their banding patterns made be visible by a Giemsa staining technique described previously. The banding pattern of the X chromosome shows striking similarities with the banding pattern of the human X chromosome. — The centromeric region of the acrocentric autosomes contains a highly condensed DNA. This DNA is removed by the Giemsa staining procedure as can be shown by interference microscopic studies. If the chromosomes are stained with quinacrine dihydrochloride these centromeric regions are only slightly fluorescent. — Autoradiographic studies with ³H-thymidine show that the DNA at the centromeric regions starts and finishes its replication later than in the other parts of the chromosomes.     

C banding in polytene chromosomes of Simulium ornatipes and S. melatum (Diptera: Simuliidae)

Polytene and mitotic chromosomes of Simulium ornatipes and S. melatum were subjected to C banding procedures. In both species polytene chromosomes consistently show C banding of centromere regions, telomeres, nucleolar organiser and, unexpectedly, numerous interstitial sites. The interstitial C banding sites correspond to morphologically single polytene bands. Their response is graded and independent of band size. Interstitial C bands in S. ornatipes are scattered throughout the complement, whereas in S. melatum they are clustered. Supernumerary heterochromatic segments in S. ornatipes also exhibit strong C banding and inverted segments can differ from standard in C banding pattern. — Mitotic chromosomes of both species show a single centric C band with indications of two weak interstitial bands in S. ornatipes, suggesting that many C band regions, detectable in polytene chromosomes, are not resolved by present techniques in mitotic chromosomes. — Contrary to current opinion that C banding is diagnostic for constitutive heterochromatin, the interstitial C band sites of polytene chromosomes are regarded as euchromatic. Conversely, the heterochromatic pericentric regions of S. ornatipes are not C banded. — It appears that polytene chromosomes offer a promising system for the elucidation of C banding mechanisms.     

Hoechst 33258 fluorescent staining of Drosophila chromosomes

Metaphase chromosomes of D. melanogaster, D. virilis and D. eopydei were sequentilly stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophia's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation by the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differtiated by a combination of quinacrine and Hoechst staining. The distal block is always Q-H- while the proximal block is, for the various autosomes, either Q-H-, Q+H- or Q+H+. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H+ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H- heterochromatin. This affinity of fluorochrome similar heterochromatin was been seen in colcemide induced multiple somatic non-disjunctions where H+ chromosomes were distributed to one rosette and H- chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q+ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be "the most important factor responsible for quinacrine brightness.' All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single a single DNA molecule which is one heptanucleotide repeated two million times.     

Karyomorphology of six taxa in Chrysanthemum sensu lato (Anthemideae) in Egypt and their genetic relationships by Giemsa C‐banding

Abstract Giemsa C‐banding was applied to the chromosome complements of six diploid species belonging to six genera in Chrysanthemum sensu lato (Anthemideae) distributed in Egypt. Four types of C‐banding distribution were observed in the taxa as follows: (i) negative C‐banding in Anacyclus monanthos (L.) Thell.; (ii) all bands in terminal regions in Achillea fragrantissima (Forssk.) Sch. Bip, which showed 32 bands on 18 chromosomes; (iii) all eight bands at centromeric regions on eight chromosomes in Matricaria recutita L.; and (iv) bands at terminal and centromeric regions in Brocchia cinerea Vis. (12 terminal and six centromeric bands on 12 chromosomes), Cotula barbata DC. (four terminal, six centromeric, and eight short arm bands on 16 chromosomes), and Glebionis coronaria (L.) Cass. ex Spach. (eight terminal on the short arms and four large bands in centromeric regions on 12 chromosomes).     

Chiasma formation in duplicated segments of the haploid rye genome

In meiosis of haploid rye associations of two or more chromosomes are observed. In order to investigate whether these associations are chiasmate, metaphase I and anaphase I associations were analysed after Giemsa banding. — At anaphase I chromatid exchanges between differently marked chromosome arms were observed, which proved the presence of real chiasmata. The association between banded and unbanded arms shows that the heterochromatic telomeres do not act as secondary pairing sources. Different statistical approaches were used to test randomness of chiasma formation. It appeared to be non-random, which showed that the segments involved were non-randomly located and probably limited in number. The nature of these segments is discussed.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

The mechanism of G and C banding in mammalian metaphase chromosomes

Bands have been observed in the fixed mitotic chromosomes of Mus musculus L cell with no further treatment. These bands, which are very similar to G bands, can be seen by phase contrast microscopy, ultraviolet microscopy and Gold/Palladium shadowing. This indicates that the cause of banding is a differential distribution of chromatin. Alterations in chromatin morphology can be induced by post fixation treatments of the fixed chromosome. A model is constructed on this evidence which unifies the apparent variety in the techniques which are thought to induce G bands and explains the action of Giemsa stain. It is concluded that these treatments act by promoting the disruption of chromatin structure which is then reformed in the presence of the Giemsa stain, the Azure-B component of the stain acting in a manner similar to divalent cations. A new technique for inducing C bands is reported. The denaturation and differential reassociation of DNA is not a suitable explanation of the mechanism of this technique. The hypothesis put forward here, explaining G bands, also explains the induction of C bands as a result of a differential destruction of chromatin morphology. The differential distribution of chromatin that gives G bands is thought to be disrupted by a technique that maintains the more resistant morphology of the centromeric heterochromatin, C bands.     

Dynamic aspects of trypsin-Giemsa banding

Summary The trypsin-Giemsa banding procedure was adapted so that chromosomes could be observed through the microscope during treatment and staining. Trypsin treatment resulted only in a swelling of the chromatids. Chromosome bands which appear as raised structures with interference contrast optics emerged only after staining with Giemsa. These structures remain after Giemsa destaining, suggesting that an irreversable change in chromosome structure is induced by Giemsa.Observations of the stain flow indicate that the positioning of the chromosomes has an effect on the quality of band production. These studies also revealed that bands appear in a reproducible sequence on individual chromosomes, which suggests that alterations take place at different rates along the length of the chromosomes.     

Dynamic aspects of trypsin-giemsa banding.

The trypsin-Giemsa banding procedure was adapted so that chromosomes could be observed through the microscope during treatment and staining. Trypsin treatment resulted only in a swelling of the chromatids. Chromosome bands which appear as raised structures with interference contrast optics emerged only after staining with Giemsa. These structures remain after Giemsa destaining, suggesting that an irreversable change in chromosome structure is induced by Giemsa. Observations of the stain flow indicate that the positioning of the chromosomes has an effect on the quality of band production. These studies also revealed that bands appear in a reproducible sequence on individual chromosomes, which suggests that alterations take place at different rates along the length of the chromosomes.     

Banding differences between tiger salamander and axolotl chromosomes

The Hoechst 33258 - Giemsa banding patterns were compared on axolotl (Ambystoma mexicanum Shaw) and axolotl - tiger salamander (Ambystoma tigrinum Green) species hybrid prophase chromosomes. Approximately 369 bands per haploid chromosome set were seen in the axolotl and about 344 bands in the tiger salamander. In the haploid set of 14 chromosomes, chromosome 3 has a constant short or q-arm terminal constriction at the location of the nucleolar organizer. Chromosomes 14 Z and W carry the sex determinants, the female being the heterogametic sex (ZW). The banding patterns of chromosomes 1, 6, 11, and 14 Z of the two species are apparently indistinguishable by our banding method. In the axolotl, chromosome 9 has a small long or p-arm terminal deletion. In the tiger salamander, the remaining 10 chromosomes have terminal or internal deletions. No translocations or inversions seem to have occurred since the gene pool separation of the two closely related species.     

A relation between G-, C-, and N-band patterns as revealed by progressive oxidation of chromosomes and a note on the nature of N-bands

Near-ultraviolet irradiation of chromosome preparations mounted in a hydrogen peroxide solution resulted in an oxidative disintegration of the structure of fixed metaphase chromosomes with concomitant production of various band patterns appearing after staining with Giemsa. Neither irradiation nor hydrogen peroxide alone could produce banding. After irradiation in the presence of hydrogen peroxide the gradually increasing effect of oxidation on the chromosomes along the gradient of light intensities from the periphery of the slide towards the radiation focus in the centre of the slide became visible as G-, C-, and N-banding, respectively. Close to the centre only contours of chromosomes were left after this treatment. Although G-banding and differential DNA-extraction often went together, extraction of DNA was not an absolute requirement to obtain a G-band pattern. N-bands appeared to be the chromosomal regions that were most resistant to destruction. Staining methods specific for DNA failed to demonstrate these bands, although with Giemsa an intense staining reaction occurred. On the analogy of the staining behaviour of model protein preparations with Giemsa a phosphoprotein nature is suggested for the N-band material in the chromosomes.