Variation in human acrocentric chromosomes with acridine orange reverse banding.

Twenty-five normal subjects were studied by acridine orange reverse (RFA) banding in order to obtain a preliminary estimate of the type and frequency of variations in color and length. Color variations were classified into 1 of 6 colors and size variations into 1 of 5 levels. The same cells were also studied by Q banding. Acridine orange reverse banding was found to be more useful than Q banding for characterizing variations in chromosomes 14, 15, 21 and 22. In addition, it was found that there was no consistent relationship between pale or bright Q banding and the various colors observed with RFA banding. For the optimal characterization of a chromosomal variation, multiple banding technics, including RFA banding, are necessary.     

Additional observations on the preparation of R banded human chromosomes with acridine orange.

A number of technical factors which affect acridine orange R banding (RFA banding) were studied. These variables included age of slide, timing of fixation, details of incubation mounting and the use of sequential technics. Optimal RFA banding was obtained between 15 and 20 days but good or very good preparations were obtained between 7 days and 2 months. Improved results were obtained in slides that were 3-4 months old by refixing the slides in ethanol acetic acid. Intermittent movement of slides during incubation in buffer as well as the details of mounting and removal of cover slips were found to be important. The best sequential banding was obtained with the sequence of Q to R but good results were obtained with the sequence G to R using ASG banding. Satisfactory results with the sequence R to C were not obtained. With careful attention to these variables good RFA binding can be obtained over a period of several months.     

Fluorescence polarization of stretched polytene chromosomes stained with acridine orange

The molecules of the fluorescent dye acridine orange (AO) bind to DNA in such a way that the absorption and emission dipoles lie on a plane perpendicular to the DNA axis. For this reason, definite fluorescence polarization should correspond to each mode of spatial DNA packing. A chromosome, considered as an axially symmetrical ensemble of DNA, was characterized by two experimental parameters, P and P , i.e., by polarizations of fluorescence excited by light polarized parallel and perpendicular to the symmetry axis. In view of the sequential order in the packing levels of DNA fiber in a chromosome, it was suggested that, under mechanical stretching, the highest level is disrupted first, then the others, in the order of their sequence.Isolated chromosomes of Chironomus thummi were stained with AO and stretched with needles of a micromanipulator. From the changes of P and P measured during stretching it was concluded the polytene chromosome bands have three, at least, DNA packing levels, tentatively described as 100 å fiber, 250 å coil and chromomere.     

2-colored fluorescence of differentially condensed chromosomes stained with acridine orange]

Metaphase chromosomes of the Chinese hamster differentially-condensed under the influence of a) 5-bromdeoxyuridine, b) colcemide, and c) cold, were stained with acridine-orange (AO) in concentrations of 1.5 X 10(-7) to 3 X 10(-5) g/ml at pH 4.1 to 8.5. It was found that stretched chromosomal segments fluoresced in the orange/red part of the spectrum, whereas normally condensed ones--were green. The colour distribution along the chromosome depended mainly on the AO concentration and the exposure in the UV-light, and was independent of pH and molarity of the buffer. Apparently this phenomenon cannot be attributed to the uneven denaturation of the chromosomal DNA, but rather depends on structural and/or chemical differences between euchromatin and heterochromatin.     

Size variation polymorphisms of the short arm of human acrocentric chromosomes determined by R-banding by fluorescence using acridine orange (RFA)

Summary One hundred normal Caucasians were studied by the RFA technique to estimate the frequencies of size variation of the short arm of acrocentric chromosomes. Each size variation was classified into one of five levels. The most frequent size level (code) was 3; therefore, this was regarded as the average size. If one excludes the average size, the frequencies of size variation by RFA for chromosome 13, 14, 15, 21, and 22 were 22.5, 19.5, 14.5, 19, and 17% respectively. There was no significant difference for the overall frequencies of size variation between sexes. Furthermore, the RFA technique detects more variation in the size of human acrocentric chromosomes than any other method.     

Selective staining of pericentromeric heterochromatin regions in chromosomes of spontaneously dividing cells with the use of the acridine orange fluorochrome

A novel phenomenon of unusual selective acridine orange (AO) staining ofpericentromeric heterochromatin regions (HRs) in chromosomal preparations from tissue with known spontaneous mitotic activity (chorionic villi, placenta, embryonic tissues, bone marrow, and testes), as well as embryonic stem cells, is described. Staining with 0.01% AO in a citric-phosphate (pH 5.5) or sodium phosphate (pH 7.0) buffer solution allows the HRs of human chromosomes (1q12, 9q12, 13p11.2, 14p11.2, 15p11.2, 16q11.2, 21p11.2, 22p11.2, and Yq12) and pericentromeric HRs of mouse chromosomes to be reliably detected by the red fluorescence of AO. This method of AO staining does not require any pretreatment. Explanations for metachromatic AO staining of polymorphic pericentromeric HRs in chromosomes of spontaneously dividing cells are suggested. A high reproducibility of the specific AO staining makes it possible to suggest its use as a reliable quick method for detection of polymorphic HRs of human chromosomes in cytogenetic prenatal diagnosis and oncohematology.     

Binding of acridine orange by chromatin in human lymphocytes with different capabilities for adhesion]

A population of morphologically homogenous peripheral blood lymphocytes separated on a column with glass beads was studied microfluorimetrically. It was found that high adhesive lymphocytes bound acridine orange in a greater extent than did low adhesive cells.     

Cytochemical significance of acridine orange staining of human plasmodia with some comments on double-stranded DNA

Summary Air-dried blood smears and erythrocyte suspension from patients infected with Plasmodium falciparum, stained under optimal conditions with acridine orange, permit easy detection of plasmodia with fluorescence microscopy together with a clear cytochemical colour differentiation of nuclear DNA (green or green-yellow) and cytoplasmic RNA (orange-red fluorescence). Judging from fluorescence characteristics of nuclei (DNase sensitive and RNase resistant green or green-yellow), the plasmodial DNA appears to be double-stranded.