We have developed a simple and rapid system for the denaturation of nucleic acids and their subsequent analysis by gel electrophoresis. RNA and DNA are denatured in 1 M glyoxal (ethanedial) and 50% (vol/vol) dimethyl sulfoxide, at 50 degrees. The glyoxalated nucleic acids are then subjected to electrophoresis through either acrylamide or agarose gels in a 10 mM sodium phosphate buffer at pH 7.0. When glyoxalated DNA molecules of known molecular weights are used as standards, accurate molecular weights for RNA are obtained. Furthermore, we have employed the metachromatic stain acridine orange for visualization of nucleic acids in gels. This dye interacts differently with double- and single-stranded polynucleotides, fluorescing green and red, respectively. By using these techniques, native and denatured DNA and RNA molecules can be analyzed on the same slab gel.

https://www.pnas.org/content/pnas/74/11/4835.full.pdf

Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange

The deliberate and orderly removal of cells by programmed cell death is a common phenomenon during the development of metazoan animals. We have examined the distribution and ultrastructural appearance of cell deaths that occur during embryogenesis in Drosophila melanogaster. A large number of cells die during embryonic development in Drosophila. These cells display ultrastructural features that resemble apoptosis observed in vertebrate systems, including nuclear condensation, fragmentation and engulfment by macrophages. Programmed cell deaths can be rapidly and reliably visualized in living wild-type and mutant Drosophila embryos using the vital dyes acridine orange or nile blue. Acridine orange appears to selectively stain apoptotic forms of death in these preparations, since cells undergoing necrotic deaths were not significantly labelled. Likewise, toluidine blue staining of fixed tissues resulted in highly specific labelling of apoptotic cells, indicating that apoptosis leads to specific biochemical changes responsible for the selective affinity to these dyes. Cell death begins at stage 11 (approximately 7 hours) of embryogenesis and thereafter becomes widespread, affecting many different tissues and regions of the embryo. Although the distribution of dying cells changes drastically over time, the overall pattern of cell death is highly reproducible for any given developmental stage. Detailed analysis of cell death in the central nervous system of stage 16 embryos (13-16 hours) revealed asymmetries in the exact number and position of dying cells on either side of the midline, suggesting that the decision to die may not be strictly predetermined at this stage. This work provides the basis for further molecular genetic studies on the control and execution of programmed cell death in Drosophila.

Programmed cell death during Drosophila embryogenesis

Acridine Orange fluorescent staining was applied to the micronucleus test in mice and rats. Micronuclei emitted bright green fluorescence and were easily distinguished from micronucleus-like inclusions or contaminants. In rat bone-marrow cells, micronuclei with green fluorescence could be easily distinguished from granules accidentally dispersed from broken mast cells, which showed bright red fluorescence. Therefore, it is recommended that the Acridine Orange staining method be used to provide more reliable data in the micronucleus test.

An application of Acridine Orange fluorescent staining to the micronucleus test.

Publisher Summary The nucleolus is organized at the nucleolus-organizing region of the chromosomes, which are generally visible as secondary constriction regions in metaphase chromosomes. The chromatin within the constriction region is lost at interphase inside the nucleolar mass. The chromatin is highly extended at this stage. At the ultrastructural level, the nucleolus has at least three components: (1) a granular component consisting mainly of ribonucleoproteins (RNP) granules—pars granulosa, (2) a fibrillar component, consisting of RNP fibrils—pars fibrosa, and (3) chromatin elements. Chromatin elements may be present in three forms: (1) nucleolus-associated chromatin, which most likely does not take part in nucleolus formation; however, the possibility of its association with condensed inactive ribosomal cistrons, at least in some cells, cannot be overruled at present, (2) septalike intranucleolar chromatin, and (3) isolated or dispersed intranucleolar chromatin threads. Intranucleolar chromatin is often associated with the pars fibrosa. Identical components can also be found in isolated nucleoli. Studies on the nucleoli in giant chromosomes indicate that the intranucleolar chromatin in these nucleoli is present as puffs of different sizes. The nucleolar chromatin is not an autonomous structure of the nucleolus but is a continuous structure and part of the nucleolar chromosome.

The nucleolar structure.

Lists are presented of references to all known publications describing cell properties that serve to characterize (i) known strains of HeLa and purported human cell lines indicated as HeLa contaminants, (ii) strains of human cell lines contaminated with human but non-HeLa cells, and (iii) strains of cells contaminated by cells from one or more other species. Frequencies of cell cross-contaminations are cited and references are presented to relatively simple techniques that could serve to detect such contamination.

https://science.sciencemag.org/content/sci/212/4493/446.full.pdf

Cross-contamination of cells in culture

Publisher Summary This chapter summarizes the salient information available about dye contaminants in four basic dyes comrnonly employed in the histochemical detection of nucleic acids The fact that only four dyes, acridine orange (AO), basic fuchsin, methyl green, and the pyronins, are selected does not mean that there are no noteworthy contaminants in other dyes used in the staining of nucleic acids This chapter focuses on cytochemical studies with acridine orange and the influence of dye contaminants in the staining of nucleic acids Acridine orange is one of the more important diphenylmethane derivatives The dye is prepared in a manner similar to that employed in the synthesis of pyronin Y (G) AO is one of a number of dyes which exhibits the phenomenon of metachromasy This term refers to a property shared by many aniline dyes which undergo a major shift in their visible absorption bands to lower wavelengths following binding by many polyanions In high concentrations of nucleic acids, a new absorption band appeared at longer wavelengths than the monomer band Other basic dyes, like rosaniline, which do not polymerize in the free state, also interact with DNA to producc a spectral shift With rosaniline it is observed that as the ionic strength of the dye solution decreases, there is an increase in the number of binding sites on DNA

Frederick H. Kasten

Papers

Cytochemical studies with acridine orange and the influence of dye contaminants in the staining of nucleic acids.

Nucleic Acid Synthetic Patterns in Synchronized Mammalian Cells

Nucleolar and Cytoplasmic Ribonucleic Acid Inhibition by Excess Thymidine

Phase contrast time-lapse cinematography of cultivated normal and psoriatic adult human skin.