Differential Giemsa staining of kinetochores in meiotic chromosomes of two higher plants

Differential Giemsa staining techniques have been used to stain kinetochores in meiotic chromosomes of two higher plants. Using these techniques it has been possible to follow changes in kinetochore behavior and appearance through meiosis.     

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 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.     

History of modern chromosomal analysis. Differential staining of plant chromosomes

Differential staining methods found extensive use in karyotype studies of many plant and animal species and provide for reliable identification of all chromosomes of the organism. Below we describe the most widespread methods and history of their advent. In addition, we discuss specific structure of the chromosomes and possible mechanisms responsible for differential segmentation.     

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.     

Differential fluorescent staining of Drosophila chromosomes with quinacrine mustard

The polytene and mitotic chromosomes of D. melanogaster, D. simulans, D. ananassae, and D. virilis were stained with the fluorescent dye, quinacrine mustard (QM). In all these species except D. ananassae, we have detected species-specific chromosomal loci which exhibit an extremely brilliant fluorescence. Most, but not all, of the brilliantly fluorescing areas are located in heterochromatic chromosome regions. Cytochemical and chemical methods have been employed to demonstrate that the brilliant fluorescence represents regions of acid labile non-covalent binding between DNA and QM whereas the moderate overall fluorescence is primarily due to covalent bonding (by alkylation) of the QM to DNA. The exact mode of binding of QM in the brilliant areas and the nature of the DNA in these areas are not known. The possible biological significance of the DNA in the brilliant regions is discussed.This study was supported by a Research Grant (GM 10499) from the National Institutes of Health, U.S. Public Health Service.This paper is dedicated with respect and affection to Professor Berwind P. Kaufmann on the occasion of his 74th birthday, April 23, 1971.     

Differential staining of plant chromosomes after hydrochloric acid treatments (Hy bands)

Differentiation of preferentially staining heterochromatic segments was achieved in somatic chromosomes of five monocotyledoneous species, when acetic-ethanol fixed meristems were subjected to 0.1 or 0.2 N HCl at temperatures between 60° and 80 °C, and stained with aceto-carmine. Suitable incubation time was temperature dependent and afforded 30 to 5 minutes. Several variations of the procedure were tested. The following plants were investigated:Allium cepa, A. flavum, A. carinatum, Scilla Sibirica, Fritillaria meleagris. InAllium carinatum besides heavily staining bands there appearently exist also bands even more labile against the HCl-treatment than euchromatin. The banding patterns do not reveal in all the species mentioned the total heterochromatin present in the form of chromocenters in the interphase nuclei, and do not always coincide with preferentially Giemsastaining segments as found by previous authors. The technique presented here and its variations seem to be a valuable and simple instrument for recognition and discrimination of heterochromatin. With the aid of these methods a high degree of structural heterozygosity was stated inAllium carinatum, A. flavum, andScilla sibirica. For the resp. heavily and weakly staining bands the abbreviations Hy+bands and Hy–bands are suggested.     

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.     

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 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.     

On the Quinacrine fluorescence and Giemsa staining patterns of the chromosomes of Vicia faba

Abstract

A comparison has been made between the Quinacrine fluorescence bands and the bands obtained with a denaturating-reannealing-Giemsa technique in Vicia faba. The results show that some of the bands, particularly on the M and, proximally, on the S chromosomes are visible with both techniques. A complex pattern of bands on the S chromosomes is revealed with the Giemsa technique. Both the similarities and the differences between the banding patterns obtained with the two methods in Vicia faba may indicate various degrees of DNA repetitiousness and other physico-chemical properties in the chromosome segments involved.     

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.