Simultaneous observation of quinacrine bands and silver grains on radiolabeled metaphase chromosomes

A procedure is described for quinacrine banding of radiolabeled metaphase chromosomes for autoradiography. The chromosomes can be labeled either in vivo or by in situ hybridization. The banding procedure involves treating the slides with RNase and formamide and staining in quinacrine. The slides are then processed for autoradiography. After development of the photoemulsion, the chromosomes can be karyotyped with UV light by their fluorescent banding patterns and the silver grains overlaying the chromosomes can be demonstrated by the addition of tungsten light. It is possible by careful manipulation of the visible light to simultaneously observe both fluorescent bands and silver grains. This technique should significantly increase the accuracy of chromosome identification after autoradiography and decrease the time and effort required for such analysis.     

Procion brilliant orange, a counterstain for decalcified sections vitally labeled with procion brilliant red H-8 bs.

Procion brilliant red H-8 BS is a fluorescent dye that stains the organic matrix of bone supravitally. A procedure is described for counterstaining sections of such bone for visible light examination without interfering with the demonstration of sites of bound dye under UV illumination. Sections are brought to water, stained in Delafield's alum hematoxylin for 10 minutes, washed in tap water for 10 minutes, counterstained in 1% Procion brillant orange M-GS for 15 minutes and washed in distilled water for 10 minutes. After dehydration the sections were mounted in Eukitt.     

Proteins of metaphase chromosomes and interphase chromatin.

Metaphase chromosomal and interphase chromatin proteins from cells of two species have been compared by polyacrylamide gel electrophoresis. Consistent, common changes in the quantitative distribution of the nonhistone chromosomal proteins are observed in both species. Proteins of ca. 65,000 and 68,000 MW are enriched in interphase chromatin while proteins of ca. 50,000 and 200,000 are more prominent components of metaphase chromosomes. A group of proteins of 90,000-100,000 are also increased in metaphase chromosomes compared to interphase chromatin. By two dimensional gel analysis, the most abundant proteins from chromosomes of both cell types are similar, suggesting a structural role for these nonhistone proteins (1).     

Filter combinations and light sources for fluorescence microscopy of quinacrine mustard or quinacrine stained chromosomes

Summary The efficiency of various combinations of primary and secondary filters, and light sources for the fluorescence microscopy of chromosomes stained with quinacrine mustard or quinacrine has been studied quantitatively. Using epi-illumination, strong fluorescence could be obtained with a mercury or xenon lamp in combination with two KP 490 short-wave pass interference filters (tilted to an angle of 60° with the excitation beam) as primary filter, and a K 490 as a secondary filter. The combination of a mercury lamp and a narrow band interference filter with a maximal transmission at about 436 nm as a primary filter together with a K 490 secondary filter results in a good visual image contrast, sufficiently strong fluorescence, and a relatively slow rate of fading.     

Ethidium bromide: destruction and decontamination of solutions

Ethidium bromide in water, TBE buffer, Mops buffer, and cesium chloride solution may be completely degraded by reaction with sodium nitrite and hypophosphorous acid. Only non-mutagenic reaction mixtures were produced. Destruction was greater than 99.8% in all cases; the limit of detection was 0.5 micrograms ethidium bromide per milliliter of solution. Ethidium bromide also may be removed completely from the above solutions by using Amberlite XAD-16 resin. The limit of detection was 0.05 micrograms ethidium bromide per milliliter of solution (0.27 micrograms/ml when cesium chloride solution was used).     

Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A simple method for the extraction of Coomassie brilliant blue R from stained protein bands excised from polyacrylamide gels is described. Spectrophotometric measurement of the eluted dye forms the basis of a sensitive assay to quantitate proteins in gels in the range 0.5-10 micrograms. The method requires no unusual equipment and is suitable for measurement of multiple samples. The polypeptide is not extracted and remains available for further analysis. The technique has been applied to three proteins and gels of various acrylamide percentages.     

Structural features of nucleosomes in interphase and metaphase chromosomes

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Ethidium bromide enhancement of frameshift mutagenesis caused by photoactivatable ethidium analogs.

Ethidium azide analogs (3-amino-8-azido-ethidium monoazide and ethidium diazide) have been developed as photosensitive probes in order to analyze directly the reversible in vivo interactions of ethidium bromide. Our preliminary observations [11], relating the mutagenic potential of the monoazide analog of ethidium, have been extended and refined, using the highly purified ethidium azide analogs [5]. A number of physical-chemical studies indicate that the monoazide analog interaction with nucleic acids, prior to photolysis, resembles remarkably the interaction of the parent ethidium (unpublished). It was anticipated, therefore, that competition by ethidium for the ethidium monoazide mutagenic sites in Salmonella TA1538 would be observed when these drugs were used in combination. Previous results in fact showed a decreased production of frameshift mutants when ethidium bromide was added to the ethidium monoazide in the Ames assay [1]. However, more extensive investigations, reported here, have shown that this apparent competition was the result of neglecting the toxic effects of ethidium monoazide and its enhanced toxocity in the presence of ethidium bromide. Conversely, an enhancement of the azide mutagenesis and toxicity for both the mono- and diazide analogs was seen when ethidium bromide was used in combination with these analogs.     

Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.     

Identification and location of the Y-body in interphase by quinacrine and Giemsa

Summary In interphase nuclei the Y-body can be stained by Giemsa after treating the preparations with diluted trypsin solution. The Y-chromosome as studied by Giemsa technique and by the fluorescence technique is found to be preferentially close to the nucleolus.

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Zusammenfassung Der Y-Körper kann im Interphasekern nach Giemsa-Behandlung mit verdünnter Trypsinlösung gezeigt werden. Die nucleolusnahe Position des Y-Körpers läßt sich in Interphasekernen mit der Giemsa-Technik und im Fluorescenzbild demonstrieren.