Differential giemsa staining of kinetochores and nucleolus organizer heterochromatin in mitotic chromosomes of higher plants

Differential Giemsa staining techniques have been used to stain kinetochores and nucleolus organizer heterochromatin in four species of higher plants. Using these techniques it has been possible to follow developmental changes of kinetochores through mitosis. In addition, these same techniques also have allowed the determination of the number and sites of nucleolus organizers in the various chromosome complements studied.     

Differential Giemsa staining in plants

Interference contrast and scanning electron microscopy studies of BSG treated Tulipa somatic chromosomes revealed transverse ridges coincident with the Giemsa positive areas. No cytological differentiation of the Giemsa banded regions was observed either prior to staining, or following destaining in methanol. Thus, the ridges observed must be interpreted as a result of an accumulation of dye molecules rather than a differential distribution of chromosomal material either before or after staining.     

Differential Giemsa staining in plants

Various modifications of reported banding techniques were performed using several cultivars of the genus Tulipa. Banding was obtained with Giemsa using a modified BSG technique and is reported for three cultivars. The chromosome banding noted in all cultivars was confined to terminal and interstitial regions; no banding was observed at the centromere. Complete banding patterns were established for two of the cultivars examined. The amount of banding per total chromosome complement of these cultivars was approximately 40% and 28%. The results demonstrated the existence of a wide range in the amount of constitutive heterochromatin as measured by the amount of banding between cultivars of similar and different species origins. The banding obtained is discussed with respect to the nature of the heterochromatin exhibited.     

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.     

Giemsa banding of meiotic chromosomes

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.     

Differential Giemsa staining in plants : III. DNA base composition

DNAs isolated from three cultivars of Tulipa displaying a range of constitutive heterochromatin (<10% to 40%), showed very little or no difference in DNA base composition as determined from buoyant densities and thermal transition profiles. Four possible explanations for the interactions of the Giemsa dye and the chromatin are discussed with reference to the mechanism of Giemsa banding. A method for the rapid isolation of higher plant DNAs is described.     

Differential staining of interspecific chromosomes in somatic cell hybrids by alkaline Giemsa stain.

Staining of chromosome preparations of Chinese hamster-human hybrid cells and mouse-chimpanzee hybrids with alkaline Giemsa has yielded color differentiation of the interspecific chromosomes. Bicolor chromosomes, indicating apparent translocations also are observed for each of these hybrids. The specific color differences observed provide a rapid means of recognizing and aiding in the identification of the interspecific chromosomes and apparent translocations in these somatic cell hybrids.     

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes

Summary Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.     

Considerations on the mechanism of differential Giemsa staining of BrdU-substituted chromosomes

Summary The staining properties of unifilarly bromodeoxyuridine (BrdU)-substituted chromatids were compared using fluorescent-plus-Giemsa (FPG) staining methods. It was found that the staining intensity of chromatids which had incorporated BrdU in the next to last S-phase is less than that of chromatids whose BrdU-containing strand came from the last cell cycle. Thus, FPG-staining is not a function of the number of BrdU-substituted DNA strands alone. These findings lead to the conclusion that the primary point of action of PFG staining leading to sister chromatid differentiation (SCD) are chromosomal proteins which have been altered in the replication of BrdU-substituted DNA and that the demonstration of the SCD and replication patterns with the same staining procedure is based on different mechanisms.     

Giemsa staining of the sites replicating DNA early in human lymphocyte chromosomes.

A timetable for the initiation of DNA replication in human lymphocyte chromosomes has been established by a technique which allows detection of areas of chromosomes replicating at a given interval of the S-phase. The resolution of the method, using 33258 Hoechst-Giemsa staining, is more refined than that obtained with 3H-thymidine autoradiography. Early replicating regions coincide with R-bands. The timetable is rather coarse since replication may start asynchronously in the same region of homologous autosomes of the same metaphase and since even the sequence of bands appearing on individual chromosomes sometimes deviates from the rule.     

Differential expression of a phosphoepitope at the kinetochores of moving chromosomes

A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.     

Differential silver staining of chromatin in metaphase chromosomes

The silver techniques used to demonstrate nucleolar organizer regions and cores in chromosomes can also differentially stain chromatin within chromosomes. Direct silver staining of mouse and human chromosomes resulted in preferential staining of centromeric regions and non-nucleolar secondary constrictions, both of which are composed of constitutive heterochromatin. After C-banding, these regions were no longer silver-stainable, suggesting that the biochemical constituents (presumably non-histone proteins) which contain the reaction sites for silver are extracted during the banding treatment. Light and electron microscopy of chromosomes G-banded with trypsin and then silver-stained revealed heavier deposits of silver over the condensed aggregates of chromatin within the band regions than over the more dispersed interband chromatin. At the ultrastructural level, chromatin fibres were covered with silver grains, indicating that there are many reaction sites for this metal along the fibres. These results suggest that the degree of silver staining in any region of the chromosome may be contingent upon the concentration of chromatin in that region. This finding may have important implications concerning the nature of the silver-stained core-like structure in chromosomes. If a preferential dispersion of chromatin fibres occurs at the periphery of the chromosome during slide preparation, leaving the central region of each chromatid relatively undispersed, this difference in the concentration of chromatin may account for the differential silver staining of these regions and the consequent appearance of a core-like structure.     

Silver staining of meiotic chromosomes in the fungus,Coprinus cinereus

We have taken advantage of the synchronous meiotic process in the basidiomycete Coprinus cinereus to develop a simple and rapid method to selectively stain meiotic chromosomes and nucleoli in this fungus without prior removal of the cell wall. Electron microscopic examination of these silver-stained chromosomes indicated that the lateral elements of the synaptonemal complexes were prominently stained, and terminal attachment plaques were apparent. We found that a translocation quadrivalent could be recognized easily in the light microscope using these methods. The procedures appear suitable for the characterization of chromosome rearrangements in this small genome, and should facilitate cytogenetic analysis in this fungus.     

Light microscope analysis of meiotic prophase chromosomes by silver staining

A method is described for the silver staining of the synaptonemal complex in surface-spread mammalian spermatocytes for light microscope examination. The method is quick, reliable, of broad applicability, and provides a means of making karyotype analysis at meiotic prophase. Many hundreds of suitable cells can be examined in an average preparation in a relatively short space of time. It has so far been applied only to mammalian spermatocytes, but could be used for karyotype analysis in oocytes of mammals and also applied to gonocytes of non-mammalian species.     

Combined silver staining of the nucleolus organizing regions and Giemsa banding in human chromosomes

Summary A combination of the silver-staining method of the nucleolus organizer regions (NORs) with a Giemsa-banding method is deccribed. This double staining allows a rapid identification of the NOR-bearing chromosomes.Supported by the Deutsche Forschungsgemeinschaft (Za 32/14).