DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

Detection of DNA strand breakage in a marine amphipod by agarose gel electrophoresis: exposure to X-rays and copper

This article describes the leading steps to develop an assay of DNA damage for the marine amphipod Gammarus locusta, using agarose gel electrophoresis (AGE). To test the sensitivity and feasibility of the AGE technique, X-ray assays were performed with naked DNA and with live amphipods. These positive controls demonstrated the effectiveness of the AGE technique to not only discriminate distinct levels of DNA strand breakage in a dose-dependent manner, but also to identify and quantify the type of strand breakage induced. It was also shown that it is possible to detect DNA damage using whole-body DNA extracts from amphipods. To explore the potential of this technique for use in ecotoxicological studies with amphipods, a 96-h waterborne-copper toxicity test was performed. Copper-induced DNA strand breakage was first observed after 24 h of exposure, and was recorded again at 96 h, at a copper concentration of 20 μg l -1 . The absence of strand breakage after 48 h of exposure is discussed in the light of the underlying mechanisms of copper toxicity and DNA repair. These studies demonstrated the feasibility of including DNA damage as a biomarker in ecotoxicological studies with amphipods. Information gained from the use of this biomarker would help with the interpretation of chronic toxicity tests and would contribute to our understanding of the impact of genotoxic insult in marine invertebrates, particularly crustaceans.     

Overcoming cloning problems by staining agarose gels with crystal violet instead of ethidium bromide in lactate dehydrogenase gene from Plasmodium vivax and Plasmodium falciparum

In this study, lactate dehydrogenase gene from Plasmodium vivax has been tried to subclone into an expression vector. Some of the Plasmodium falciparum lactate dehydrogenase mutant genes have also been tried to clone and subclone into a vector, but we failed to clone or subclone either of the genes. DNA visualisation in electrophoretic gels typically requires UV radiation and the fluorecent dye ethidium bromide. A crystal violet-stained gel was run instead of an ethidium bromide gel and so avoided the use of UV radiation. This enabled us to clone or subclone both Plasmodium vivax lactate dehydrogenase gene and Plasmodium falciparum lactate dehydrogenase mutant genes into any desired vector.     

Two-dimensional restriction mapping by digestion with restriction endonucleases of DNA in agarose and polyacrylamide gels

We have studied with a number of bacterial restriction enzymes the conditions for digestion of DNA in agarose and polyacrylamide gels. The restriction endonucleases HpaII, MspI, HaeIII, HindIII, TaqI, HhaI, AluI, BamHI, EcoRI and SalI are capable of digesting DNA in agarose gels of low electroendosmosis and low sulfate concentration. All enzymes, except BamHI, are also capable of digesting DNA in polyacrylamide gels. With this method, rapid two-dimensional restriction mapping of genomes with low and high sequence complexity is possible.     

Regulation of mating in the budding yeast Saccharomyces cerevisiae by the zinc cluster proteins Sut1 and Sut2

The zinc cluster proteins Sut1 and Sut2 play a role in sterol uptake and filamentous growth in the budding yeast Saccharomyces cerevisiae. In this study, we show that they are also involved in mating. Cells that lack both SUT1 and SUT2 were defective in mating. The expression of the genes NCE102 and PRR2 was increased in the sut1 sut2 double deletion mutant suggesting that Sut1 and Sut2 both repress the expression of NCE102 and PRR2. Consistent with these data, overexpression of either SUT1 or SUT2 led to lower expression of NCE102 and PRR2. Furthermore, expression levels of NCE102, PRR2 and RHO5, another target gene of Sut1 and Sut2, decreased in response to pheromone. Prr2 has been identified as a mating inhibitor before. Here we show that overexpression of NCE102 and RHO5 also reduced mating. Our results suggest that Sut1 and Sut2 positively regulate mating by repressing the expression of the mating inhibitors NCE102, PRR2 and RHO5 in response to pheromone.     

Heterogeneity in Expression of Lipopolysaccharide and Major Outer-Membrane Proteins by Strains of Escherichia coli O157 with Different H-Serotypyes

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long-chain lipopolysaccharide (LPS) and major outer-membrane proteins (OMPs) by means of SDS-PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga-like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze-substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H-serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H-serotype. These data support the existence of heterogeneous groups of O157 strains.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Current applications of orcein in histochemistry. A brief review with some new observations concerning influence of dye batch variation and aging of dye solutions on staining

Current uses of orcein to demonstrate elastic fibers and, following permanganate oxidation (Shikata's modification), hepatitis B surface antigen, copper associated protein, and sulfated mucins, are reviewed. Variations in staining performance with batch of dye and age of dye solution is also discussed. Additional experimental findings support the view that the orcein stain for elastic tissue and Shikata's modification produces consistent, high quality results as long as appropriate controls and suitable dye batches, e.g., Biological Stain Commission certified dyes, are used.     

Renal improvement by zinc in diabetic mice is associated with glucose metabolism signaling mediated by metallothionein and Akt,but not Akt2

Human epidemiological and animal studies have shown the beneficial effect of zinc supplementation on mitigating diabetic nephropathy. However, the mechanism by which zinc protects the kidney from diabetes remains unknown. Here we demonstrate the therapeutic effects of zinc on diabetes-induced renal pathological and functional changes. These abnormalities were found in both transgenic OVE26 and Akt2-KO diabetic mouse models, accompanied by significant changes in glucose-metabolism-related regulators. The changes included significantly decreased phosphorylation of Akt and GSK-3β, increased phosphorylation of renal glycogen synthase, decreased expression of hexokinase II and PGC-1α, and increased expression of the Akt negative regulators PTEN, PTP1B, and TRB3. All of these were significantly prevented by zinc treatment for 3 months. Furthermore, zinc-stimulated changes in glucose metabolism mediated by Akt were actually found to be metallothionein dependent, but not Akt2 dependent. These results suggest that the therapeutic effects of zinc in diabetic nephropathy are mediated, in part, by the preservation of glucose-metabolism-related pathways via the prevention of diabetes-induced upregulation of Akt negative regulators. Given that zinc deficiency is very common in diabetics, this finding implies that regularly monitoring zinc levels in diabetic patients, as well as supplementing if low, is important in mitigating the development of diabetic nephropathy.     

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5-dithiobis(2-nitrobenzoic acid). Cys¹²², Cys¹²⁴ and Cys¹³² are placed near two catalytic sites, Lys¹⁹⁹ and Lys²⁵², and coordinate a zinc ion, referred to as a proximal zinc ion, and Cys²²³ is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as a distal zinc ion, with His¹³¹ . When the wild-type enzymes C122A (Cys¹²²Ala), C124A (Cys¹²⁴Ala), C132A (Cys¹³²Ala) and C223A (Cys²²³Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys¹²², Cys¹²⁴ and Cys¹³² under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys¹²², Cys¹²⁴ and Cys¹³² was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.     

16q22.1 microdeletion detected by array-CGH in a family with mental retardation and lobular breast cancer

We describe the case of a boy with psychomotor delay and dysmorphic features, with a germline 16q22.1 microdeletion identified by array-CGH. The deletion spans 0.24Mb and encompasses three genes (ZFP90, CDH3 and CDH1). The deletion has been demonstrated to be inherited from his mother who was affected by lobular breast cancer (LBC) without any other apparently phenotypic features. We suppose that the microdeletion, in particular ZFP90 which is cerebrally expressed, is causative for the boy's phenotype. Mental retardation in the affected boy can recognize several mechanisms such as variable expressivity, non-penetrance, multifactorial/polygenic inheritance, recessive inheritance, a second rearrangement event and epigenetics. Furthermore, we suggest that the deletion of the CDH1, a tumor suppressor gene, involved in hereditary diffuse gastric cancer (HDGC) and LBC predisposed the mother to the carcinoma.     

Triplet-triplet energy transfer in Peridinin-Chlorophyll a-protein reconstituted with Chl a and Chl d as revealed by optically detected magnetic resonance and pulse EPR: Comparison with the native PCP complex from Amphidinium carterae

The triplet state of the carotenoid peridinin, populated by triplet-triplet energy transfer from photoexcited chlorophyll triplet state, in the reconstituted Peridinin-Chlorophyll a-protein, has been investigated by ODMR (Optically detected magnetic resonance), and pulse EPR spectroscopies. The properties of peridinins associated with the triplet state formation in complexes reconstituted with Chl a and Chl d have been compared to those of the main-form peridinin-chlorophyll protein (MFPCP) isolated from Amphidinium carterae. In the reconstituted samples no signals due to the presence of chlorophyll triplet states have been detected, during either steady state illumination or laser-pulse excitation. This demonstrates that reconstituted complexes conserve total quenching of chlorophyll triplet states, despite the biochemical treatment and reconstitution with the non-native Chl d pigment. Zero field splitting parameters of the peridinin triplet states are the same in the two reconstituted samples and slightly smaller than in native MFPCP. Analysis of the initial polarization of the photoinduced Electron-Spin-Echo detected spectra and their time evolution, shows that, in the reconstituted complexes, the triplet state is probably localized on the same peridinin as in native MFPCP although, when Chl d replaces Chl a, a local rearrangement of the pigments is likely to occur. Substitution of Chl d for Chl a identifies previously unassigned bands at ∼ 620 and ∼ 640 nm in the Triplet-minus-Singlet (T − S) spectrum of PCP detected at cryogenic temperature, as belonging to peridinin.