Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

Stockert, Juan C.; Lisanti, José A.

doi:10.1007/bf00284934

Cited by 63 publications

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“…For AO band ing, including selective staining of HRs, the sample or chromosome slide should be pretreated to denature or † Deceased. decondense chromatin (e.g., the RFA and RBA meth ods) [1,[5][6][7].Earlier, we found bright red fluorescence of the 1qh, 9qh, 16qh, and Yqh regions after AO staining of chorionic villus slides prepared by the direct method and not subjected to any additional treatment [8]. This unexpected finding stimulated our studies on the char acteristics of AO staining of specimens prepared by different methods from cells of different origins.…”

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confidence: 47%

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

et al. 2012

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369 † Various methods are used for detection of hetero chromatin regions (HRs) of chromosomes. The CBG method is the most versatile; it allows the staining of centromeric and pericentromeric HR of all chromo somes, as well as a HR in the long arm of the human Y chromosome. Different variants of C banding using Giemsa staining make it possible to image only active centromeric regions (Cd staining) or pericentromeric HRs of individual chromosomes (G11 staining) [1,2]. The fluorochromes D287/170, DAPI, and Hoechst 33258 in combination with contrasting agents allows the staining of only polymorphic HRs of chromo somes 1, 9, 15, 16, 22, and Y [3].The acridine orange (AO) fluorochrome has been used in human cytogenetics for a long time. AO is metachromatic due to the characteristics of its inter action with nucleic acids (NAs), proteins, and other biopolymers [4,5]. In a solution, AO may exist in either the monomeric form, which intercalates into double stranded NA and stains it green, or in the dimeric form, which electrostatically binds with sin gle stranded NA and stains it red. AO is known to stain chromosomes yellow-green, relatively homoge neously, in non pretreated specimens. For AO band ing, including selective staining of HRs, the sample or chromosome slide should be pretreated to denature or † Deceased. decondense chromatin (e.g., the RFA and RBA meth ods) [1,[5][6][7].Earlier, we found bright red fluorescence of the 1qh, 9qh, 16qh, and Yqh regions after AO staining of chorionic villus slides prepared by the direct method and not subjected to any additional treatment [8]. This unexpected finding stimulated our studies on the char acteristics of AO staining of specimens prepared by different methods from cells of different origins. MATERIALS AND METHODS Human metaphase chromosome preparations from samples of the chorion, placenta, and embryonic organs.Samples of chorionic villi (41 samples) and embryonic organs (the liver, kidney, brain, intestine, lung, and eye) of ten human embryos were obtained after thera peutic abortions for medical or social reasons at a ges tational age of 5-12 weeks, as well as samples of 12 placentas performed by Caesarian section at a ges tational age of 36-40 weeks.The "quick direct" method [9]. Chorionic villi or fragments of organs were put into a hypotonic solution containing colchicine at a final concentration of 0.8 µg/mL. The hypotonic solution was 0.9% sodium citrate. For three samples in parallel experiments, 1% KCl was used. Then, the samples were prefixed at Abstract-A novel phenomenon of unusual selective acridine orange (AO) staining of pericentromeric het erochromatin 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, 22...

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confidence: 47%

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

et al. 2012

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“…No green sphere-carrying filaments were observed. When acridine orange-stained cells are examined at 440 to 480 nm, it is generally assumed that double-stranded DNA fluoresces green (520 nm), while RNA or single-stranded DNA fluoresces orange-red (650 nm) (24)(25)(26). Thus, orange-red fluorescence in an exponentially growing cell may be regarded as a sign of high metabolic activity (i.e., cells producing more ribosomes), while yellow-green fluorescence may be a sign of dormant cells.…”

Section: Resultsmentioning

confidence: 99%

Low-Temperature Growth of Shewanella oneidensis MR-1

Abboud

Popa

Souza-Egipsy

et al. 2005

Appl Environ Microbiol

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Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of Ϸ35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (Ϸ22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of Ϸ67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.Shewanella oneidensis MR-1 was first isolated from sediments of Lake Oneida in New York State (20). The cells are gram-negative straight rods capable of moving by means of a single polar flagellum. S. oneidensis MR-1 is a mesophilic, facultative anaerobe having a respiratory type of metabolism, with an optimal growth temperature of Ϸ30°C. Lake Oneida is a shallow freshwater system that freezes over completely during the winter (from early to mid-December to mid-or late January, with complete ice-out in March or April), with ice thickness reaching up to 70 cm (19). During May, water temperatures begin to rise, reaching a maximum of 25°C in midsummer (19).Because of their ability to metabolically reduce metals such as U(VI), Tc(VII), and Cr(VI), organisms such as Shewanella are considered candidates for bioremediation of subsurface metal-contaminated areas. However, subsurface temperatures are often low, and predictions of the utility of Shewanella in such environments require an understanding of the effects of low temperatures on their metabolism and general ecology. Recently, it was shown that S. oneidensis MR-1, while capable of growth at low temperature, exhibited a growth transition at about 10°C, and below these temperatures showed a differential increase in rRNA synthesis compared to DNA synthesis (4). Here we report on other properties of MR-1 grown at low temperature (3°C), including a dramatically different phenotype with changes in morphology, growth rate, ultrastructure, and protein and lipid composition. MATERIALS AND METHODSCells of Shewanella oneidensis strain MR-1 (ATCC 700550) were grown overnight aerobically in batch cultures at 22°C in 150-ml flasks containing 50 ml of Luria-Bertani (LB) broth Miller (Difco) at 130 rpm. For growth measurements, new cultures were started by transferring 1.0 ml of the original culture to a 250-ml Erlenmeyer flask containing 100 ml of LB and incubated at 130 rpm at two temperatures (3 and 22°C). All incubations were done in triplicate. Cells analyzed for proteins and phospholipid fatty acids were harvested by centrifugation and washed with sterile phosphate-buffered saline buffer, wet weight was determined, and the cells were frozen at Ϫ70°C until analysis.The turbidity of the cultures was measured at 600 nm. For measuring cell ...

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“…Otherwise, the chromosomes fluoresce green immediately upon neutralization or after only a few seconds or minutes under annealing conditions. In some studies, the centromeres 324 Kurnit DNA and C-handing turned green first and the chromosome arms became green after more prolonged annealing (Stockert and Lisanti, 1972;Chapei.le et al, 1973;Comings et al, 1973).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, a series of indirect determinations of the helical status of the chromo somal DNA after C-banding treatment steps gave apparently contradic tory results. These ranged from the finding that most of the chromosomal DNA was denatured (Chamberlain and Walker, 1965;Von Borstel et al, 1966;Mac Innes and Uretz, 1967) to the opposite result that it was mostly double-stranded (R igi.er et al, 1969;Kernell and R ingertz, 1972; Stockert and Lisanti, 1972;Comings et al, 1973) after C-banding.…”

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confidence: 99%

DNA helical content during the C-banding procedure

Kurnit¹

1974

Cytogenet Genome Res

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DNA was isolated from chromosomes or nuclei scraped off slides after each step of the C-banding procedure. Analysis by buoyant density-gradient centrifuga-tion and digestion with nucleases specific for single-stranded nucleic acids showed: 80 % or more of the DNA remained double-stranded after fixation to slides and 70 % or more was single-stranded after the slides were treated with alkali, whether the slides were annealed afterwards or not. Furthermore, highly repeated DNAs remained single-stranded after annealing following denaturation. Since the annealing of slides after denaturation does not effect the reassociation of either bulk or highly repeated DNA sequences, C-banding apparently results from DNA-protein interactions rather than preferential strand reassociation of highly repeated DNAs.

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Acridine-orange differential fluorescence of fast- and slow-reassociating chromosomal DNA after in situ DNA denaturation and reassociation

Cited by 63 publications

References 39 publications

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

Low-Temperature Growth of Shewanella oneidensis MR-1

DNA helical content during the C-banding procedure

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