Denise C. Monteleone

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.

Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA.

We have built an electronic imaging system based on a modified charge-coupled-device television camera that directly quantitates the distribution of fluorescence from electrophoretic gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike the photographic system that it replaces, the response of the camera is directly proportional to the intensity of incident fluorescence, and image data are digitized and stored in computer memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand breaks in DNA.

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: Analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks☆

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.

Quantifying DNA damage by gel electrophoresis, electronic imaging and number‐average length analysis

DNA damages that can be converted to single‐ or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge‐coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled DNA.

Quantifying Double-Strand Breaks and Clustered Damages in DNA by Single-Molecule Laser Fluorescence Sizing

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.

Microbial community dynamics in uranium contaminated subsurface sediments under biostimulated conditions with high nitrate and nickel pressure

Background, aim, and scopeThe subsurface at the Oak Ridge Field Research Center represents an extreme and diverse geochemical environment that places different stresses on the endogenous microbial communities, including low pH, elevated nitrate concentrations, and the occurrence of heavy metals and radionuclides, including hexavalent uranium [U(VI)]. The in situ immobilization of U(VI) in the aquifer can be achieved through microbial reduction to relatively insoluble U(IV). However, a high redox potential due to the presence of nitrate and the toxicity of heavy metals will impede this process. Our aim is to test biostimulation of the endogenous microbial communities to improve nitrate reduction and subsequent U(VI) reduction under conditions of elevated heavy metals.Materials and methodsColumn experiments were used to test the possibility of using biostimulation via the addition of ethanol as a carbon source to improve nitrate reduction in the presence of elevated aqueous nickel. We subsequently analyzed the composition of the microbial communities that became established and their potential for U(VI) reduction and its in situ immobilization.ResultsPhylogenetic analysis revealed that the microbial population changed from heavy metal sensitive members of the actinobacteria, α- and γ-proteobacteria to a community dominated by heavy metal resistant (nickel, cadmium, zinc, and cobalt resistant), nitrate reducing β- and γ-proteobacteria, and sulfate reducing Clostridiaceae. Coincidentally, synchrotron X-ray absorption spectroscopy analyses indicated that the resulting redox conditions favored U(VI) reduction transformation to insoluble U(IV) species associated with soil minerals and biomass.DiscussionThis study shows that the necessary genetic information to adapt to the implemented nickel stress resides in the endogenous microbial population present at the Oak Ridge FRC site, which changed from a community generally found under oligotrophic conditions to a community able to withstand the stress imposed by heavy metals, while efficiently reducing nitrate as electron donor. Once nitrate was reduced efficient reduction and in situ immobilization of uranium was observed.ConclusionsThis study provides evidence that stimulating the metabolism of the endogenous bacterial population at the Oak Ridge FRC site by adding ethanol, a suitable carbon source, results in efficient nitrate reduction under conditions of elevated nickel, and a decrease of the redox potential such that sulfate and iron reducing bacteria are able to thrive and create conditions favorable for the reduction and in situ immobilization of uranium. Since we have found that the remediation potential resides within the endogenous microbial community, we believe it will be feasible to conduct field tests.Recommendations and perspectivesBiostimulation of endogenous bacteria provides an efficient tool for the successful in situ remediation of mixed-waste sites, particularly those co-contaminated with heavy metals, nitrate and radionuclides, as found in the United States and other countries as environmental legacies of the nuclear age.

Computer network for data acquisition, storage and analysis.

Modern scientific instruments can produce huge quantities of data, usually in digital form. However, data acquisition is only one of three important functions. To be useful, data must also be stored and analyzed. Fortunately, the same computer-based technologies that facilitate the generation of large data-sets provide tools to accomplish these tasks. We describe a data system based on computers connected to a network, developed for this purpose.

Fluorescence of matrix isolated guanine and 7-methylguanine.

We have prepared argon and nitrogen matrices containing guanine and 7-methylguanine, and measured their absorption, fluorescence excitation and fluorescence emission spectra. The fluorescence excitation spectrum of guanine shows four well-resolved bands in the range from 170 to 290 nm; excitation at the wavelengths of each of these bands results in a fluorescence emission with maximum intensity near 350 nm and a single-exponential decay with a lifetime of about 10 ns. There are significant differences between the fluorescent excitation and emission spectra of guanine and of 7-methylguanine, suggesting that the fluorescence observed from the guanine sample does not arise from a minority tautomer.

Time-resolved fluorescence using synchrotron radiation excitation: A powered fourth-harmonic cavity improves pulse stability

The pulsed nature or ‘‘time structure’’ of synchrotron radiation from electron storage rings is used to measure the kinetics of the decay of electronically excited states and is particularly useful because the wavelength of excitation can be chosen at will. However, changes in the length of the pulses of radiation from a storage ring resulting from the gradual decrease of current circulating in the ring during the course of a ‘‘fill’’ limit the duration of data collection, and hence photometric sensitivity. A fourth‐harmonic cavity that was recently added to the vacuum ultraviolet (VUV) storage ring at the National Synchrotron Light Source slows the loss of current during a fill, and thus increases the total number of photons produced. When operated in a passive (unpowered) mode, however, the fourth‐harmonic cavity increases both the average width of the photon pulses and the changes in width that occur during a fill, thus reducing the usefulness of the VUV ring in timing experiments. We demonstrate that ...

Soft x-ray circular dichroism and scattering using a modulated elliptically polarizing wiggler and double synchronous detection

We have constructed an experimental station (beamline) at the National Synchrotron Light Source to measure circular dichroism (CD) using soft x-rays (250 less than or equal to hv less than or equal to 900 eV) from a time modulated elliptically polarizing wiggler. The polarization of the soft x-ray beam switches periodically between two opposite polarizations, hence permitting the use of phase-sensitive (lock-in) detection. While the wiggler can be modulated at frequencies up to 100 Hz, switching transients limit the actual practical frequency to approximately equals 25 Hz. With analog detection, switching transients are blocked by a chopper synchronized to the frequency and phase of the wiggler. The CD is obtained from the ratio of the signal recovered at the frequency of polarization modulation, f, to the average beam intensity, which is recovered by synchronous detection at frequency 2f.