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1.
Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1 h of denaturation with 4 N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1 h of denaturation with 4 N HCl in this study. After this treatment, DAPI (4′,6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained. 相似文献
2.
Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA 总被引:2,自引:0,他引:2
Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20 degrees C were examined for human promyelocytic leukemia cells (HL-60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti-BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double-stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10-20 min at 20 degrees C allowed denaturation of more than 80% of DNA and the maximum BrdUrd-linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small. 相似文献
3.
Gene expression can be altered by small molecules that target DNA; sequence as well as shape selectivities are both extremely important for DNA recognition by intercalating and groove‐binding ligands. We have characterized a carbohydrate scaffold (1) exhibiting DNA “shape readout” properties. Thermodynamic studies with 1 and model duplex DNAs demonstrate the molecule's high affinity and selectivity towards B* form (continuous AT‐rich) DNA. Isothermal titration calorimetry (ITC), circular dichroism (CD) titration, ultraviolet (UV) thermal denaturation, and Differential Scanning Calorimetry were used to characterize the binding of 1 with a B* form AT‐rich DNA duplex d[5′‐G2A6T6C2‐3′]. The binding constant was determined using ITC at various temperatures, salt concentrations, and pH. ITC titrations were fit using a two‐binding site model. The first binding event was shown to have a 1:1 binding stoichiometry and was predominantly entropy‐driven with a binding constant of approximately 108 M?1. ITC‐derived binding enthalpies were used to obtain the binding‐induced change in heat capacity (ΔCp) of ?225 ± 19 cal/mol·K. The ionic strength dependence of the binding constant indicated a significant electrolytic contribution in ligand:DNA binding, with approximately four to five ion pairs involved in binding. Ligand 1 displayed a significantly higher affinity towards AT‐tract DNA over sequences containing GC inserts, and binding experiments revealed the order of binding affinity for 1 with DNA duplexes: contiguous B* form AT‐rich DNA (d[5′‐G2A6T6C2‐3′]) >B form alternate AT‐rich DNA (d[5′‐G2(AT)6C2‐3′]) > A form GC‐rich DNA (d[5′‐A2G6C6T2‐3′]), demonstrating the preference of ligand 1 for B* form DNA. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 720–732, 2014. 相似文献
4.
Immunohistological optimization of detection of bromodeoxyuridine-labeled cells in decalcified tissue 总被引:1,自引:0,他引:1
Mice were injected with a range of bromodeoxyuridine (BrdU) concentrations from 0.01 mg to 10 mg, and their jaws were fixed in buffered formalin or modified Carnoy. After EDTA or formic acid decalcification, a range of DNA denaturation schedules was assessed and immunohistological detection of BrdU-containing nuclei was performed using the Sera Lab anti-BrdU antibody MAS 250b. For Carnoy-fixed tissue, denaturation in 1 N HCl for 8 min at 60 degrees C was capable of adequately detecting an injected dose of 0.05 mg but not a dose of 0.01 mg BrdU, whereas pepsin/HCl treatment gave only weak staining after injection of 1 mg BrdU. In comparison, formalin fixation required pre-treatment with 0.2-0.4% pepsin/HCl at 37 degrees C for comparable staining intensity, but could still not adequately detect a dose of 0.1 mg BrdU. There was little detectable difference in staining between EDTA- and formic acid-decalcified tissues after injection of 10 mg BrdU. 相似文献
5.
We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak. 相似文献
6.
Thermal denaturation studies show that 10-15% of the calf thymus DNA in the heat denatured (Tyr-Gly-Tyr-Gly-Tyr)-DNA complex renatures spontaneously after colling. The double-strandness of this DNA was verified by its resistance to single-strand Neurospora endonuclease and by its elution profile on hydroxypatite columns. The renatured DNA isolated by the latter technique was found to contain 56% GC compared to the 41% GC content of the whole thymus DNA. Alternating tryptophanyl-glycyl and histidyl-glycyl peptides also catalyze the same renaturation. A linear correlation was found between the thermal stabilization afforded to the DNA by the various peptides and their ability to "catalyze" DNA strand renaturation. 相似文献
7.
Detection of 5-bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: role of the DNA denaturation step 总被引:1,自引:0,他引:1
R Moran Z Darzynkiewicz L Staiano-Coico M R Melamed 《The journal of histochemistry and cytochemistry》1985,33(8):821-827
Immunochemical detection of cells that incorporate 5-bromodeoxyuridine (BrdUrd) requires prior denaturation of DNA in situ to make BrdUrd binding sites accessible to the antibodies. A technique is described in which the DNA denaturation step is facilitated by a) prior dissociation of histones from DNA and b) the use of low ionic strength buffer in which the cells are suspended during heating. Dissociation of histones is achieved by cell treatment with 0.08N HCl at 0 degree C, which a) increases accessibility of DNA to propidium iodide (and following the denaturation to the antibodies); b) lowers stability of DNA to thermal denaturation; c) decreases differences between various cell types due to variability in chromatin structure; and d) ensures more complete DNA denaturation. Cell heating (80-95 degrees C) at low ionic strength (1 mM Na+) eliminates the need for formamide and results in extensive and rapid DNA denaturation. The method was applied in Friend leukemia, L1210 and HL-60 cell lines, and to bone marrow, experimental animal tumor and primary human tumor cells. 相似文献
8.
Cervantes-Cervantes MP Calderón-Salinas JV Albores A Muñoz-Sánchez JL 《Biological trace element research》2005,103(3):229-248
Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS)
and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II)
greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O2) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC
and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus,
the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage
λ DNA or proteins using either hydrogen peroxide (H2O2) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks),
spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation
temperature [T
d], denaturation enthalpy [ΔH], and heat capacity [C
p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in
DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects
were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O2 and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins.
The physiological relevance of these structural effects should be further investigated. 相似文献
9.
The degree of chromosomal DNA (cDNA) denaturation and renaturation on polytene chromosomes has been measured by UV microspectrophotometry. Also DNA losses occurring upon denaturation have been quantified by Feulgen, gallocyanin-chromalum and UV. It has been observed that denaturation in alkali (0.07 N NaOH at room temperature) and formamide (90% formamide; 0.1 SSC, pH 7.2) at 65 °C removes about 30% of the DNA. Low DNA loss occurs upon denaturation in HCl (0.24 M) at room temperature and 60% formamide: 2 × 10?4 M EDTA (pH 8) at 55 °C. The presence of 4% formaldehyde in the denaturation buffer prevents DNA loss. After denaturation of chromosomes in 0.1 × SSC containing 4% formaldehyde at 100 °C for 30 sec, an hyperchromicity of 39 °C is observed. The denaturation efficiency varies with the denaturation treatment. The percentage reassociation was measured from the difference in the UV absorption of renatured chromosomes and that of denatured chromosomes from the same set. It seems that in our conditions DNA:DNA reassociation does not occur. The efficiency of hybridization is proportional to the denaturation extent of the DNA. However, the entire fraction of DNA which has been denatured is not available for hybridization. 相似文献
10.
The structural organization of mouse metaphase chromosomes 总被引:1,自引:0,他引:1
The binding of highly purified anti-nucleoside antibodies to mouse (Mus musculus) metaphase chromosomes was studied by an immunofluorescence technique. The chromosomal DNA was denatured by one of two selective denaturation procedures because these antibodies reacted with single stranded but not native DNA. After ultraviolet irradiation (UV), which produced single stranded regions primarily in AT rich DNA, the binding of antiadenosine (anti-A) produced a pattern of fluorescent bands similar to that produced by quinacrine (Q-bands). Additional foci of bright fluorescence were observed at the centrometric (C-band) regions, which are known to contain AT rich satellite DNA. After photooxidation, which produced single stranded regions in GC rich DNA, the binding of anti-A produced a fluorescent banding pattern similar to the R-banding pattern seen after thermal denaturation and staining with coriphosphine O. After photooxidation, R-band patterns were also obtained with anti-cytidine (anti-C) and anti-5-methylcytidine (anti-M). After either UV irradiation or photooxidation, anti-M, but not anti-C, showed intense binding to the C-band regions of mouse chromosomes. — These findings led to the following conclusions: (1) Antibody banding patterns reflect the presence of a class of AT rich, GC poor DNA in chromosome regions which show bright quinacrine fluorescence and in the regions that contain the AT rich satellite DNA. (2) The alternate, quinacrine dull regions contain a relatively GC rich class of DNA which appears to be more highly methylated than the AT rich DNA in the Q-bright bands, but not the AT rich satellite DNA in the Q-dull C-bands. (3) 5-Methylcytosine residues occur in a sequence of mouse satellite DNA that contains both adjacent pyrimidines and guanine residues. The basic repeating unit of mouse satellite DNA is known to contain the sequence 5-GAAAAATGA-3 (Biro et al., 1975). Therefore, assuming the antibodies used could detect single bases in denatured DNA, the methylated sequence in mouse satellite DNA
相似文献
11.
Summary The thermal melting profile of purified Saccharomyces cerevisiae ribosomal DNA (rDNA) is biphasic indicating considerable intramolecular heterogeneity in base composition. The first phase of the transition, about 20% of the total hyperchromic shift, has a Tm of 80.6°C and the second phase has a Tm of 87.3°C, corresponding to GC contents of 28 and 44%, respectively. The Tm of the nonribosomal nuclear DNA, called DNA, is 85.7°C. This heterogeneity in GC distribution in the rDNA is also reflected in its denaturation map. A denaturation map of the 5.6×106 dalton rDNA SmaI restriction fragment, which represents monomer units of the rDNA, shows that specific regions of the repeating unit denature more readily than the remainder and apparently have a significantly higher AT content. By aligning the rDNA denaturation map with the restriction endonuclease map, we have been able to determine that the AT-rich segments are localized in the transcribed and nontranscribed spacer regions of the rDNA repeating unit. Buoyant density determinations of individual rDNA restriction fragments corroborate the locations of AT-rich regions.A denaturation map of the tandem repeating units in higher molecular weight rDNA has also been constructed and compared with the map of the SmaI fragment. The results show that the repeating units are uniform in size, that they are not separated by large heterogeneous regions, and that they are arranged in head-to-tail array. 相似文献
12.
Interaction of tyrosyl, histidyl, and tryptophanyl peptides with DNA: specificity and mechanism of the interaction 总被引:2,自引:2,他引:0
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In the Tyr-(Gly)1-4-Tyr series maximal thermal stabilization of calf thymus DNA (δTm=10°) occurred with the Tyr-(Gly)2-Tyr peptide, where three base pairs could separate the two tyrosyl residues. Tyr-Gly-Tyr-Gly-Tyr stabilized the DNA by 6°. The alternating Trp-Gly-Trp-Gly-Trp and His-Gly-His-Gly-His peptides were equally as effective as the Tyr-Gly-Tyr-Gly-Tyr peptide in stabilizing calf thymus DNA against thermal denaturation. But the alternating Phe-Gly-Phe-Gly-Phe peptide afforded little stabilization, suggesting that a sidechain possessing both a conjugated π-electron system and an electron donor atom is necessary for DNA stabilization. Introduction of electron withdrawing iodo or nitro group into the tyrosyl sidechains almost completely abolished the stabilizing effect. Although the tyrosyl peptides seem to be specific for GC-base pairs, no correlation was found in natural DNA between% GC and% thermal stabilization. Eukaryotic DNAs showed twice the stabilization of prokaryotic DNAs with the same GC content. 相似文献
13.
Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter asapp against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region. 相似文献
14.
Embryo DNA, isolated from ungerminated seeds of Lupinus angustifolius L., contains an exceptionally high amount of guanine-cytosine-rich satellite DNA. The thermal denaturation curve of total embryo DNA is biphasic with an inflexion point at 62% denaturation, indicating the presence of satellite DNA. The satellite fraction could be separated from the mainband DNA by three successive preparative CsCl-gradient centrifugations. The densities of the DNA fractions are 1.7045 g cm-3 and 1.6925 g cm-3, respectively. The percentages of guanine-cytosine calculated from these densities are comparable to the percentages of GC calculated from the melting temperatures. Finally, ressociation studies prove that foldback DNA and highly repeated sequences are much more frequent in the satellite DNA fraction than in the mainband DNA.Abbreviation
C
o
t
the product of the DNA concentration (mol nucleotides l-1) and the time (s) of incubation in a DNA reassociation reaction
- GC
guanine-cytosine
- np
nucleotide parirs
- T
temperature interval between 16 and 84% denaturation 相似文献
15.
A cytological technique based on heat denaturation of in situ chromosomal DNA followed by differential reassociation and staining with acridine orange was developed. Mouse nuclei and chromosomes in fixed cytological preparations show a red-orange fluorescence after thermal DNA denaturation (2–4 minutes at 100° C), and fluoresce green if denaturation is followed by a total DNA reassociation (two minutes or more at 65–66°C). — A reassociation time between a few and 60–90 seconds demonstrates the centromeric heterochromatin of chromosomes (which sometimes aggregate in the form of clusters) and the interphase chromocenters in green, the chromosomal arms fluorescing red-orange. Under the same conditions, the Y chromosome presents a pale green or yellow-green fluorescence along its chromatids, but its centromeric region fluoresces weakly. — The interpretation is suggested that the fast-reassociating chromosomal DNA (as detected by AO in centromeric heterochromatin and interphase chromocenters), represents repetitive DNA. 相似文献
16.
Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A3 (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed. 相似文献
17.
Z. Shu L. Shao H. -Y. Huang X. -Q. Zeng Z. -F. Lin G. -Y. Chen C. -L. Peng 《Photosynthetica》2009,47(4):548-558
In the present study, we investigated the antioxidative potential in leaves of the chromatic (CC) versus green (GC) Amaranthus tricolor L. under moderate high-temperature stress at 45°C. Before heat stress, CC had significantly higher levels of betacyanins
[about 3.2 mg g−1(FM)] than the green [1.8 mg g−1(FM) (p<0.01), while similar chlorophyll (Chl) content [about 2 mg g−1(FM)] was observed between both cultivars. After exposure to high temperature (45°C) for 6 days, betacyanins in leaves of
CC were remarkably increased (about 2 times of that in control samples grown at 30°C). In contrast, betacyanins in GC significantly
decreased by 56% in comparison with that of the control. Chl level in CC was higher than that in GC after heat stress for
6 days. Flavonoids and total phenolics in both cultivars were increased, but much more in CC. Significantly less H2O2 accumulation was observed in the leaves and stems of CC than in those of GC under heat stress. Interestingly, much stronger
circadian oscillation in fluorescence was observed in both cultivars after treatment at 45°C, which suggested that heat stress
stimulates endogenous rhythms of photosystem II (PSII). Under moderate high-temperature stress, Chl fluorescence parameters
Fv/Fm (maximum quantum yield of PSII), qP (coefficient of photochemical quenching), ΦPSII (effective PSII quantum yield), and ETR (electron transport rate) exhibited a gradual decrease, NPQ (nonphotochemical quenching)
showed a slight increase followed by a gradual decline, whereas Fo (minimum fluorescence of a dark-adapted leaf) increased continuously. In contrast to GC, after 120 h of high-temperature
treatment, CC exhibited significantly lower Fo level, and higher levels of Fv/Fm and NPQ. It is clear that PSII in CC was more stable than that in GC. The results indicate that betacyanins are an effective
antioxidant, and probably contribute greatly to the higher thermal stability of PSII and higher tolerance to heat stress. 相似文献
18.
The guanine plus cytosine (GC) content of the deoxyribonucleic acid (DNA) of 11 isolates of host-dependent (H-D) bdellovibrios and 18 host-independent (H-I) derivatives was determined from thermal denaturation curves and buoyant densities in CsCl. The H-D and respective H-I cultures have GC contents which are identical within the limits of experimental error. Most cultures of Bdellovibrio bacteriovorus, including the holotype culture, have 50.4 +/- 0.9 moles% GC in their DNA; two bdellovibrio isolates of presently uncertain nomenclatural status contain DNA of about 43% GC. Optical melting profiles of all the DNA from all of these organisms are particularly steep, indicating little compositional heterogeneity. Chromatography of acid hydrolysates of Bdellovibrio nucleic acids reveal no unusual components. The DNA content per cell of one H-I derivative is about one-third the amount per Escherichia coli cell growing at a comparable rate. 相似文献
19.
This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues. 相似文献
20.
C Stroobants L Sossountzov E Miginiac 《The journal of histochemistry and cytochemistry》1990,38(5):641-647
We describe a method for localizing replicating cells in detached tobacco leaves allowed to root. The proposed protocol has shown that formalin fixation and Spurr embedding of petiole bases can be used for demonstrating DNA synthesis after bromodeoxyuridine (BrdU) incorporation. The incorporated BrdU was immunologically visualized. After resin removal, different procedures of DNA denaturation and protease digestion were tested. Combined hydrolysis with 4 N HCl for 10 min at room temperature and digestion with 0.4% pepsin for 15 min at 37 degrees C led to the best reproducible results, with either the peroxidase or the gold detection system. The method is rapid and sensitive, with precise resolution. It can be used at the light and electron microscopic levels. Its potential application is to elucidate in the same organ the role of cytokinins, a class of plant growth regulators, in dividing cells and to define the chronology of their biosynthesis in roots in relation to DNA synthesis. 相似文献