John Trunk

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.

ALFALFA SEEDLINGS GROWN OUTDOORS ARE MORE RESISTANT TO UV-INDUCED DNA DAMAGE THAN PLANTS GROWN IN A UV-FREE ENVIRONMENTAL CHAMBER

Abstract The relative UV sensitivities of alfalfa seedlings grown outdoors versus plants grown in a growth chamber under UV‐filtered cool white fluorescent bulbs have been determined using three criteria: (1) level of endogenous DNA damage as sites for the UV endonuclease from Micrococcus luteus. (2) susceptibility to pyrimidine dimer induction by a UV challenge exposure and (3) ability to repair UV‐induced damage. We find that outdoor‐grown plants contain approximately equal frequencies of endogenous DNA damages, are less susceptible to dimer induction by a challenge exposure of broad‐spectrum UV and photorepair dimers more rapidly than plants grown in an environmental chamber under cool white fluorescent lamps plus a filter that removes most UV radiation. These data suggest that plants grown in a natural environment would be less sensitive to UVB‐induced damage than would be predicted on the basis of studies on plants grown under minimum UV.

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.

Simultaneous resolution of spectral and temporal properties of UV and visible fluorescence using single-photon counting with a position-sensitive detector

A new fluorescence spectrometer has been assembled at the U9B beamline of the National Synchrotron Light Source to allow simultaneous multiwavelength and time‐resolved fluorescence detection, as well as spatial imaging of the sample fluorescence. The spectrometer employs monochromatized, tunable UV and visible excitation light from a synchrotron bending magnet and an imaging spectrograph equipped with a single‐photon sensitive emission detector. The detector is comprised of microchannel plates in series, with a resistive anode for encoding the position of the photon‐derived current. The centroid position of the photon‐induced electron cascade is derived in a position analyzer from the four signals measured at the corners of the resistive anode. Spectral information is obtained by dispersing the fluorescence spectrum across one dimension of the detector photocathode. Timing information is obtained by monitoring the voltage divider circuit at the last MCP detector. The signal from the MCP is used as a ‘‘sta...

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 polarization measurements for entire emission spectra with a resistive-anode, single-photon-counting detector: The Fluorescence Omnilyzer

We report a fluorescence analyzer that records simultaneously the temporal profiles for both orthogonal linear polarizations for all wavelengths in a fluorescence emission spectrum. The Analyzer combines a resistive-anode single-photon-counting photomultiplier, imaging spectrograph, Wollaston polarizer, multiparameter analyzer with histograming memory, and standard timing electronics. The spectrograph disperses the fluorescence spectrum across the photocathode of the photomultiplier, and the Wollaston polarizer separates the spectra of the two polarizations in opposite directions from the center of the photocathode perpendicular to the direction of spectral dispersion. The locations at which each photon reaches the photocathode is determined by the ratios of the charges read from the four corners of the resistive anode. One of the two address coordinates that determine where in histogramming memory each photon is recorded is obtained by measuring the time of arrival of the photon at the detector relative ...

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.