Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
2001-04-18
2004-09-07
Moran, Marjorie (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S019000, C702S127000, C435S006120, C435S018000, C435S183000, C204S456000
Reexamination Certificate
active
06789022
ABSTRACT:
BACKGROUND OF THE INVENTION
Ionizing radiation may produce cancer, death and loss of neural function in humans and animals, and induce killing, mutation and chromosomal aberrations in cells [Bissell et al., (1997)
Modeling Human Risk: Cell and Molecular Biology in Context,
Lawrence Berkeley National Laboratory, Univ. of California, Berkeley]. Humans are exposed to low doses of radiation during air travel, from radon in homes, during space travel or in areas of low-level contamination, including former nuclear weapon production sites. Nuclear energy production facility workers may encounter higher doses of ionizing radiation than others. In addition, humans encounter higher radiation doses during radiotherapy and humans, animals and plants encounter much higher radiation doses in contaminated areas such as Chernobyl and near the sites of other nuclear mishaps [Bissell, 1997; Yang et al.,
Radiation Res.
148 (Sup. 5): S17 (1997); Tucker et al.,
Radiation Res.
148: 216 (1997); Bigbee et al.,
Radiation Res.
147: 215 (1997); Fry et al., Radiation Res. 150: 695 (1998)].
Ionizing radiation induces many different types of DNA damages [Wallace,
Radiation Res.
150 (Sup. 5): S60 (1998)] and the identity of the specific lesion types that are responsible for the biological effects of radiation remains uncertain. Understanding the long term effects of low and high doses of ionizing radiation on living organisms requires identification of critical radiation-induced DNA lesions, assessment of their reparability and determination of the consequences of misrepaired or unrepaired, persistent lesions.
Lethal and mutagenic effects of ionizing radiation result principally from incompletely or incorrectly repaired DNA lesions [Ward,
Radiation Res.
104: S103 (1985) and
Int. J. Radiation Biol.
66: 427 (1994)]. Ionizing radiation induces high levels of isolated DNA lesions, including single strand breaks (SSBs), damaged bases and abasic sites that are located at a distance from other damages (Wallace, 1998). Such isolated damages are generally repaired efficiently, and their repair may be enhanced by priming ionizing radiation doses [Le et al.,
Science
280: 1066 (1998)]. Ionizing radiation also induces closely spaced lesions, including double strand breaks (DSBs) that result from two or more single strand breaks being induced on opposing DNA strands within 10-20 base pairs of one another [Van Der Schans,
Int. J. Radiation Biol.
33: 105 (1978);
Olive, Radiation Res.
150: S42 (1998)].
It has been postulated that ionizing radiation also produces other clustered DNA damages that are composed of other closely spaced lesions on opposing DNA strands [Ward,
Radiation Res.
86: 185 (1981)]. Although it has been postulated that such clustered DNA damages contribute significantly to the biological effects induced by radiation [Ward, 1985 and 1994; Goodhead,
Int. J. Radiation Biol.
65: 7 (1994); Ward,
Radiation Res.
86: 185 (1995)], it has not been possible to demonstrate their induction in genomic DNA following low doses of ionizing radiation. Therefore, it has not been possible to evaluate the biological impact of low doses of radiation by measuring DNA damage induction and repair.
Using model systems of oligonucleotides bearing defined lesions at specific relative spacings on opposing strands, it has been possible to examine model damage clusters [Chaudhry and Weinfeld,
J. Biol. Chem.
272: 15650 (1997) and
J. Mol. Biol.
249: 914 (1995); Harrison et al.,
Nucleic Acids Res.
26: 932 (1998)]. Such studies indicate that damage clusters may be non-repairable, highly repair-resistant, or pre-mutagenic damages. Because it has not been possible to measure damage clusters induced in genomic DNA by irradiation, it is not known whether low doses of ionizing radiation produce significant levels of clustered DNA damages in cells. In addition, the composition and frequency of damage clusters is unknown.
Ultraviolet (UV) radiation, which induces the formation of cyclobutane pyrimidine dimers in DNA, has been demonstrated to produce closely spaced cyclobutane pyrimidine dimers. UV-induced closely spaced dimers located on opposing strands of DNA have been demonstrated using an enzyme-based assay that converts the closely spaced cyclobutane pyrimidine dimers into closely spaced single strand nicks that are revealed as double strand breaks by centrifugation or electrophoretic separation techniques [Lam and Reynolds,
Radiation Res.
166: 187 (1986)]. Because ionizing radiation induces a heterogeneous variety of damages, it has been refractory to the use of such simple assay techniques to demonstrate the induction of clustered DNA damages by ionizing radiation, particularly by low doses of ionizing radiation.
Irradiation of plasmid DNA with 900-10,000 Gy of neutrons produced short DNA fragments consistent with clustered DSBs [Pang et al.,
Radiation Res.
150: 612 (1998)]. Thermal denaturation and S1 nuclease analysis of &ggr;-irradiated &lgr; DNA suggested production of bulky lesions [Martin-Bertram et al.,
Radiation Environ. Biophys.
27: 305 (1983)]. S1 nuclease and gamma endonuclease treatment of &lgr; DNA irradiated with 2,000-8,000 Gy of rays suggested the close proximity of unpaired DNA regions and base damages [Kohfeldt et al.,
Radiation Environ. Biophys.
27: 123 (1988)], and S1 analysis of human cells exposed to 100 Gy of &ggr;-rays indicated closely spaced damages, probably nicks and gaps [Legault et al.,
Mol. Cell. Biol.
17: 5437 (1997)]. At these high radiation doses, clustered DNA damages could include sites resulting from multiple independent radiation hits and therefore these model studies do not necessarily reveal damages that are induced with low doses of radiation (physiological doses that result in high cell survival).
Makrigiorgos et al. [
Int. J. Radiation Biol.
74: 99 (1998)] state that gel electrophoresis-based methods to detect clustered damages in DNA, such as are used in model DNA studies, cannot be applied to genomic DNAs or mixtures of DNAs of unknown DNA sizes. Therefore, as a model for the detection of ionizing radiation-induced clustered DNA damages, Makrigiorgos, et al. developed a fluorescence energy transfer method for detecting and quantifying closely spaced aldehyde-containing abasic sites that were artificially introduced into DNA by acid depurination. Based on the experiments reported below, the limit of sensitivity of the fluorescence energy transfer method has been calculated to be approximately 1 per 17,000 base pairs, which would correspond to high radiation doses (approximately 400 Gy, cf. FIG.
4
). Although the fluorescence energy transfer method could be applied to the detection and quantitation of closely spaced aldehyde-containing abasic sites that may be induced by high doses of ionizing radiation, the method suffers from lack of sensitivity and the inability to detect other forms of clustered DNA damages.
The development of a sensitive method for the detection and quantitation of a variety of types of clustered DNA damages would facilitate the evaluation of the biological impact of radiation exposure. Such an assay system would be additionally useful for monitoring damage resulting from exposure to physiological doses of ionizing radiation in the home and/or workplace and in monitoring the efficacy of radiation therapy protocols. In addition, such an assay system would provide a method for assessing radiation damage to humans, crops, livestock and wildlife following a nuclear mishap.
An assay for clustered DNA damages would be additionally useful for the detection and quantification of clustered damages in DNA from biological specimens following exposure of the specimens to chemical agents, including known and suspected DNA damaging agents and known or prospective chemotherapeutic agents.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a method for detecting and quantifying
Bogosian Margaret C.
Brookhaven Science Associates LLC
Clow Lori A.
Moran Marjorie
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