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D C Monteleone

Publications and source records attributed to D C Monteleone.

12 recordsLinked to original sources
PubMed2001

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.

DNA↗
PubMed2001

The integrating ion imager: a device for determining heavy ion doses during irradiations.

We have designed and built an integrating ion imaging system (I3) that records the spatial distribution of the dose of heavy ions incident on samples irradiated at the radiobiology beamline of the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The images of dose are integrated over the duration of the exposure. Unlike the images formed on X-ray film, these images are linear with the incident dose. Heavy ions are incident on a phosphor that is located just behind the sample position. Visible light emitted from the phosphor is collected by a lens and focused onto a scientific grade charge coupled device (CCD) cooled to about -45 degrees C. The phosphor and CCD camera are integral parts of a modular sample holder designed for irradiating molecular samples, which is easily mounted on the sample platform of the beamline. The imager can be adapted to other types of samples. The present CCD image is digitized to 14 bits (16,384 intensity levels), but the dynamic range is extended by adjusting the aperture of the CCD camera lens. Digital images from the CCD are routinely transferred over the BNL local area network for archival storage on a UNIX server, from which they can be opened from any authorized computer with access to the Internet. Images obtained with no sample in place record the dose at all points on the target field. When a sample is in place, an image of the sample appears providing its exact location with respect to fiducial marks recorded for all images. Areas surrounding the image of the sample are used in comparison with companion no-sample images to get exact doses over the sample. The contrast mechanism responsible for image formation is the shift along the Bragg curve resulting from loss of energy of the ions as they pass through the sample--not from a change in ion flux reaching the phosphor. The sharpness of the images formed with the DNA samples we have recorded indicates that neither scattering of the incident heavy ions or the generation of secondary ions contribute significantly.

Elementary Particles↗
PubMed1997

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.

Computer Communication Networks↗
PubMed1996

A comparison of electrophoretic resolution for snapshot and finish-line imaging.

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland, Anal. Biochem. 210, 378-388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish-line imaging provided higher resolving power. The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%.

Bacteriophage T7↗
PubMed1994

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.

Argon↗
PubMed1992

Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure.

The ultraviolet circular dichroism of a protein can be used to estimate the net fraction of its amino acids in different classes of secondary structure. Recent advances in the accuracy of such calculations have resulted from improved computational techniques, as well as extension of the spectral region analyzed to wavelengths less than 180 nm, a wavelength range beyond the limit of most laboratory-based circular dichroism spectrometers. We describe a spectrometer that uses UV radiation from the National Synchrotron Light Source at the Brookhaven National Laboratory to record circular dichroism spectra of proteins (and other biologically important molecules) in aqueous solution over the optimum wavelength range required for calculation of secondary structures. This instrument is available for use by scientists from academic, commercial and research institutions.

Animals↗
PubMed1987

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.

DNA↗
PubMed1987

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.

DNA↗
PubMed1986

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.

DNA Damage↗
PubMed1984

Two-dimensional, computer-controlled film scanner: quantitation of fluorescence from ethidium bromide-stained DNA gels.

A two-dimensional scanner based on a digital plotter is described. The device is used to analyze photographic negatives of ethidium bromide-stained DNA-agarose gels. Scanning is controlled by and photometric data transferred to a computer for processing, storage, display, and analysis such as integration of the areas under bands and determination of the mean distances of migration of polydisperse samples. An integral light source and detector module designed for reading optical "bar-codes" is mounted in place of the pen of the plotter. Spatial resolution and reproducibility are about 0.2 and 0.005 mm, respectively. Photometric precision as good as one part per thousand is achieved by sinusoidal modulation of the intensity of the light source and synchronous, phase-sensitive detection of the signal from the detector by a lock-in amplifier. No part of the sensor assembly touches the surface of the negative. In contrast to a densitometer, the computer transforms photometric data to values directly proportional to the amount of DNA at given points on the original gel. The ability to move the sensor in two dimensions over the negative allows for the integration across the width of a lane correctly allowing for the nonuniform distribution of the DNA.

Computers↗