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 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.     

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.     

Simple differential Giemsa staining of sister chromatids after treatment with photosensitive dyes and exposure to light and the mechanism of staining

The essential steps of the 33258 Hoechst-Giemsa method for differential chromatid staining consist of (1) 33258 Hoechst treatment, (2) exposure to light, and (3) Giemsa staining. The staining was shown to be a function of the concentration of 33258 Hoechst and the light exposure. The dye was successfully replaced by various metachromatic dyes such as thionine. Two simple methods are proposed. Failure of the pale stained chromatids to restore Giemsa affinity with urea and trypsin and the diminished Feulgen reaction after light exposure suggest that not masking proteins but photolysis of the BrdU-incorporation chromatid components in the present of photosensitive dyes play a role in the differential staining.     

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 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.     

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).     

On the mechanism of differential giemsa staining of BrdU-substituted chromatids

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocyte spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with Giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.     

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.     

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.     

Further studies on the mechanism involved in differential staining of BUdR-substituted Vicia faba chromosomes

In a preceding publication we reported that photolysis of BUdR-substituted Vicia faba chromatids occurs during observation with a fluorescence microscope when chromosomes were mounted in a solution containing trypsin and a photosensitive dye (Hoechst 33258 or acridine orange). The present investigations support the hypothesis that the rapid dissolving of the double BUdR-substituted (BB) chromatids observed with our method is due to single-strand breaks induced by a photosensitive dye-visible light system. The agents cysteamine and potassium iodide which reduce BUdR radicals and in this way may inhibit single-strand breaks modify the rate of chromosomes showing differential staining. It was totally suppressed by high cysteamine concentrations and markedly reduced by potassium iodide. Several acridine dyes were tested concerning their ability to induce differential staining. Some of them, e.g. aurophosphine and coriphosphine O, yield good results, others, e.g. acriflavine and acridine yellow, give poor differential staining. In an experiment in which the trypsin concentration was varied to induce approximately optimum and non-optimum digestion conditions the necessity of trypsin treatment in our method was confirmed.     

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.