Differential giemsa staining of sister chromatids after extraction with acids

Chromosomes of Chinese hamster strain cells were air-dried on slides after BrdU substitution for two or three rounds of replication. The preparations were treated with 20% PCA at 55 degrees C for 20-30 min, or 5N HCl at 55 degrees C for 15-20 min. After staining with Giemsa, unifilarly BrdU-substituted chromatids stained faintly and bifilarly substituted chromatids stained darkly. Such a pattern of sister chromatid differential staining was confirmed by the examination of metaphase cells grown with BrdU for three rounds of replication.     

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.     

Interaction of chromosomal proteins with BrdU substituted DNA as determined by chromatin-DNA competition.

Chromatin-DNA competition has been utilized to examine the general nature of chromosomal proteins interacting more strongly with BrdU substituted DNA. When chromatin is incubated with an excess of purified DNA, a portion of the chromosomal proteins will exchange to the purified DNA. These two complexes can then be separated on Metrizamide gradients due to their differing protein/DNA ratios. Using this technique we observe that most nonhistone chromosomal proteins will exchange to a competitor DNA, the extent of exchange being directly dependent upon the competitor DNA being present in excess. While essentially the same proteins will migrate to either unsubstituted or BrdU substituted DNA, the substituted DNA is found to be a quantitatively better competitor and its effectiveness as a competitor is directly related to the level of BrdU substitution.     

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

Estimating transition probabilities of singly and doubly stochastic matrices

Vector quadratic programming techniques, based on the classical theory of vector quadratic forms, subjected to linear restrictions, minimization techniques are applied to the estimation of the transition probabilities of singly and doubly stochastic matrices.     

Characterization of doubly substituted polysaccharide derivatives

Derivatives of cellulose, amylose and chitosan, bearing simultaneously 10-undecenoyl and arylaminocarbonyl or benzoyl groups were characterized by the combined use of 1H NMR and elemental analysis. The mathematical manipulation of elemental analysis data permits the calculation of the degree of substitution for each kind of substituent. The method was validated and is applicable to other derivatives.     

Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation

Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2'-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.     

Different effects of 33258 Hoechst and DAPI in fluorescent staining of sister chromatids differentially substituted with bromodeoxyuridine

Summary After substitution with 5-bromodeoxyuridine (BrdUrd) for two rounds of replication, chromosomes in cytological preparations stained with 33258 Hoechst show upon epiluminescence an immediate differential sister chromatid fluorescence. When stained with DAPI, however, which has a structural resemblance to part of the 33258 Hoechst molecule, such a differential pattern of fluorescence was only induced after some delay. Upon restaining with the same dye the differential fluorescence appeared instantly. In preparations double stained with ethidium bromide and 33258 Hoechst the induction of a differential staining of sister chromatids with 33258 Hoechst was not accompanied by a differential staining with ethidium bromide. Once a differential staining was obtained with DAPI in preparations double stained with ethidium bromide and DAPI, the ethidium bromide pattern also appeared to be differential upon subsequent observation. No differentiation could be obtained with ethidium bromide alone. The observations described in the case of 33258 Hoechst staining are in agreement with a molecular quenching by BrdUrd without gross structural consequences for the DNA. In the case of DAPI staining, however, there occurs a differential photolysis of BrdUrd-substituted DNA. Besides the nature, most likely the size, of the fluorochrome molecules themselves, the state of the fixed chromatin appeared also to play a role in determining the mechanism of the sister chromatid differentiation: after prolonged incubation in buffer, BrdUrd-containing chromosomes stained with 33258 Hoechst showed a differential staining evidently caused by photolysis, indicating that they had become more susceptible to light.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.     

Chromosomal differentiation in two species of Aedes and their hybrids revealed by giemsa C-banding

Giemsa C-banding patterns in two species of mosquitoes, Aedes aegypti and Aedes mascarensis, their hybrids and backcross progeny revealed differences in the sex chromosome pair. In A. aegypti, the female determining or the m chromosome in both males and females shows a conspicuous band in the centromere region and another band in one arm. The male determining or the M chromosome is devoid of any bands. Progeny of crosses involving A. aegypti females and A. mascarensis males showed interesting albeit unexpected results. The intercalary band was suppressed in both sons and daughters. When such F₁ sons were backcrossed to A. aegypti females, a proportion of males developed into intersexes. These intersex progeny differed from the normal males in terms of their banding pattern. In the reciprocal cross (A. mascarensis female × A. aegypti male), the F₁ and the backcross progeny yielded the expected C-banding patterns. The implications of the reversible expression of the intercalary band on the A. aegypti m chromosome and its relevance to genetic regulation are discussed.     

Increased carbonic anhydrase activity in Friend erythroleukemia cells during DMSO-stimulated erythroid differentiation and its inhibition by BrdU.

Carbonic anhydrase activity is increased in Friend erythroleukemia (FL) cells during the enhancement of erythroid differentiation in the presence of dimethylsulfoxide (DMSO) or butyric acid. Untreated FL cells show an increase in enzyme activity associated with logarithmic growth. The increase in the specific activity of carbonic anhydrase in the differentiating treated cells, however, appears to be due to at least two additional general mechanisms: (1) an induction of carbonic anhydrase paralleling the stimulation of hemoglobin synthesis and (2) the stability and/or retention of active carbonic anhydrase as compared to most of the other cell proteins. The stimulation of carbonic anhydrase activity in the treated cells is inhibited by 5-bromo-2'-deoxyuridine (BrdU). This is the first demonstration of BrdU inhibition of a DMSO induced product not directly related to hemoglobin.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

The interaction of substituted and rigidly linked diquinolines with DNA

Viscometric measurements with circular and sonicated rodlike DNA fragments were used to explore whether ring substituents or conformationally restricted linkers promote bifunctional intercalation amongst a series of binuclear 4-aminoquinolines bridged via their 4-amino group. We find that ligands comprising unsubstituted quinolines and piperazine or pyrazole linkages bisintercalate. Quinoline-substituted alkyl-linked dimers intercalate in either a mixed monofunctional-bifunctional mode or bind with only one of their chromophores intercalated depending on the nature of the substituents. Equilibrium dialysis measurements show that the binding affinity for calf thymus DNA of the compounds studied ranges from (1.2-12) . 10(4) M-1 in buffer of ionic strength 0.1. Both co-operative and antico-operative binding isotherms were obtained and there is evidence for a second binding mode for the piperazine-linked diquinoline at saturating binding levels. For this compound the high-affinity association constant decreases with increasing ionic strength, 3.4 cations being released per bound ligand molecule. Partition dialysis measurements with DNAs of differing base composition indicate that the compounds studied are either AT selective or sequence neutral depending on ligand structure. For example, the pyrazole linker imparts a marked specificity for binding to AT-rich DNA, whereas the piperazine linker does not. Kinetic measurements using the surfactant-sequestration method reveal that DNA-diquinoline complexes dissociate very rapidly by complex mechanisms with rate constants greater than 100 s-1 in buffer of ionic strength 0.1.     

Interactions of nuclear proteins with DNA,during sperm differentiation in the ram

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nuleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous knobby fibers that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30–200 Å region.