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1.
D Marie  D Vaulot    F Partensky 《Applied microbiology》1996,62(5):1649-1655
Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.  相似文献   

2.
A D Wolfe 《Biochemistry》1977,16(1):30-33
The cationic triphenylmethane dyes crystal violet, methyl green, and malachite green inhibited DNA synthesis catalyzed by Escherichia coli B polymerase I (polymerase I). Lower concentrations of the dyes inhibited DNA replication as a direct function of the proportion of AT to GC in the DNA of Clostridium perfringens, Escherichia coli, and Micrococcus lysodeikticus. When the intercalant proflavin was employed, the GC-rich micrococcal DNA was most severely inhibited. In addition, both the triphenylmethanes and proflavin inhibited product hydrolysis catalyzed by polymerase I.  相似文献   

3.
The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.  相似文献   

4.
Although evidence for the evolution of terrestrial species on islands continues to rapidly accumulate, little is known about the evolution of marine species in geographically isolated environments such as islands as ocean currents often facilitate gene flow among populations. In this study, we focused on marine lakes of the Palau Islands, which are considered to be true analogues of terrestrial islands for marine species. To examine evolutionary processes in marine lakes, we conducted population genetic analyses on marine lake and lagoon populations of the striped silverside, Atherinomorus endrachtensis, using two mitochondrial DNA markers differing in evolutionary rate, the cytochrome b gene and the control region. The analyses revealed that the amount of genetic diversity of marine lake populations is much lower than that of lagoon populations and high levels of genetic differentiation occur among marine lake and lagoon populations. The present study has shown that marine lake populations have been completely isolated and have differentiated from lagoon populations, and each marine lake population is experiencing different evolutionary processes. These findings clearly demonstrate that marine lakes are excellent environments for the evolutionary study of marine species.  相似文献   

5.
Two fluorescent dyes, TO-PRO-1 iodide and 5-CFDA-AM, were evaluated for LIVE/DEAD assessment of unicellular marine algae Brachiomonas submarina and Tetraselmis suecica. Epifluorescence microscopy was used to estimate cell viability in predetermined mixtures of viable and non-viable algal cells and validated using microplate growth assay as reference measurements. On average, 5-CFDA-AM underestimated live cell abundance by ~25% compared with viability estimated by the growth assay, whereas TO-PRO-1 iodide provided accurate viability estimates. Furthermore, viability estimates based on staining with TO-PRO-1 iodide were not affected by a storage period of up to one month in -80°C, making the assay a good candidate for routine assessment of phytoplankton populations in field and laboratory studies.  相似文献   

6.
An Escherichia coli virus T1-induced DNA methyltransferase was identified by activity gel analysis in homogenates of infected E. coli DNA-adenine-methylation-deficient strains. Although the Mr of this protein (31,000) is in the same range as that of the E. coli DNA adenine methyltransferase, the two proteins are not closely related; the E. coli dam gene does not hybridize with T1 DNA. Selective conditions for measurement of the T1 activity were developed, and the enzyme was purified to functional homogeneity, as shown by activity analysis in polyacrylamide gels. Requirements for optimal activity of the viral enzyme were determined to be pH 6.9, ionic strengths below 0.1 M KCl, and a temperature between 40 and 43 degrees C. The Km for S-adenosyl-L-methionine is 4.9 microM. The purified T1 DNA methyltransferase is capable of methylating adenine in 5'-GATC-3' sites in vitro.  相似文献   

7.
We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.  相似文献   

8.
Changes in mean cell size, DNA and cell density were monitored at 6-h intervals for 72 h in populations of six species (eight clones) of marine dinoflagellates to determine the temporal relationships between the cell cycle events of DNA replication and cytokinesis. Batch cultures were maintained at 15 or 20°C on a 12-h light: 12-h dark photoperiod. Cell densities and size frequency distributions were determined conductimetrically and the amount of DNA within populations was measured fluorometrically. A variety of intra- and interspecific relationships were observed, ranging from parallel phasing of cell cycle processes to variations which involved the temporal uncoupling of DNA synthesis from the phased pattern of cell division which is characteristic of dinoflagellate cell cycles. Daily growth rates of individual populations varied from 0.05 (Gymnodinium nelsoni) to 2.08 (Amphidinium carteri) cell divisions day-1 and DNA doubling rates ranged from 0 to 1.14 day-1. Mean doubling rates for DNA were usually 30–40% lower than those for cells. The degree of difference in these rates and the amount of variability evident in cell cycle sequences may be major factors in determining the rate and extent of development of dinoflagellate populations in nature.  相似文献   

9.
The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.  相似文献   

10.
A major taxon of obligate marine bacteria within the order Actinomycetales has been discovered from ocean sediments. Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems. In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth. Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (Escherichia coli numbering). Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family Micromonosporaceae and suggests novelty at the genus level. This is the first evidence for the existence of widespread populations of obligate marine actinomycetes. Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.  相似文献   

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