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Hagelstrom R.T.,Colorado State University | Hagelstrom R.T.,Translational Genomics Research Institute | Blagoev K.B.,National Science Foundation | Blagoev K.B.,University of Cambridge | And 3 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Werner syndrome and Bloom syndrome result from defects in the RecQ helicases Werner (WRN) and Bloom (BLM), respectively, and display premature aging phenotypes. Similarly, XFE progeroid syndrome results from defects in the ERCC1-XPF DNA repair endonuclease. To gain insight into the origin of cellular senescence and human aging, we analyzed the dependence of sister chromatid exchange (SCE) frequencies on location [i.e., genomic (G-SCE) vs. telomeric (T-SCE) DNA] in primary human fibroblasts deficient in WRN, BLM, or ERCC1-XPF. Consistent with our other studies, we found evidence of elevated T-SCE in telomerase-negative but not telomerasepositive backgrounds. In telomerase-negative WRN-deficient cells, T-SCE - but not G-SCE - frequencies were significantly increased compared with controls. In contrast, SCE frequencies were significantly elevated in BLM-deficient cells irrespective of genome location. In ERCC1-XPF-deficient cells, neither T- nor G-SCE frequencies differed from controls. A theoretical model was developed that allowed an in silico investigation into the cellular consequences of increased T-SCE frequency. The model predicts that in cells with increased T-SCE, the onset of replicative senescence is dramatically accelerated even though the average rate of telomere loss has not changed. Premature cellular senescence may act as a powerful tumor-suppressor mechanism in telomerase-deficient cells with mutations that cause T-SCE levels to rise. Furthermore, T-SCE-driven premature cellular senescencemay be a factor contributing to accelerated aging in Werner and Bloom syndromes, but not XFE progeroid syndrome.


Blagoev K.B.,National Science Foundation | Goodwin E.H.,Kromatid | Bailey S.M.,Colorado State University
Aging | Year: 2010

Telomeres are a hotspot for sister chromatid exchange (T-SCE). Any biological consequence of this form of instability remained obscure until quantitative modeling revealed a link between elevated T-SCE rates and accelerated cellular replicative senescence. This work strongly suggests that progressive telomere erosion is not the only determinant of replicative capacity; instead, T-SCE need to be considered as an independent factor controlling colony growth and senescence. Additionally high T-SCE rates have been observed in cells with deficiencies in WRN and BLM, the genes that are defective in Werner's and Bloom's syndromes, implying a connection to premature aging. In this Research Perspective we will explore some of the implications this recent work has for human health. © Blagoev et al.


Ray F.A.,Colorado State University | Robinson E.,Kromatid | McKenna M.,Kromatid | Hada M.,Universities Space Research Association | And 6 more authors.
Radiation and Environmental Biophysics | Year: 2014

Chromosome aberrations in blood lymphocytes provide a useful measure of past exposure to ionizing radiation. Despite the widespread and successful use of the dicentric assay for retrospective biodosimetry, the approach suffers substantial drawbacks, including the fact that dicentrics in circulating blood have a rather short halflife (roughly 1-2 years by most estimates). So-called symmetrical aberrations such as translocations are far more stable in that regard, but their high background frequency, which increases with age, also makes them less than ideal for biodosimetry. We developed a cytogenetic assay for potential use in retrospective biodosimetry that is based on the detection of chromosomal inversions, another symmetrical aberration whose transmissibility (stability) is also ostensibly high. Many of the well-known difficulties associated with inversion detection were circumvented through the use of directional genomic hybridization, a method of molecular cytogenetics that is less labor intensive and better able to detect small chromosomal inversions than other currently available approaches. Here, we report the dose-dependent induction of inversions following exposure to radiations with vastly different ionization densities [i.e., linear energy transfer (LET)]. Our results show a dramatic dose-dependent difference in the yields of inversions induced by low-LET gamma rays, as compared to more damaging high-LET charged particles similar to those encountered in deep space. © Springer-Verlag Berlin Heidelberg 2014.


Ray F.A.,Colorado State University | Ray F.A.,Kromatid | Zimmerman E.,Kromatid | Robinson B.,Kromatid | And 7 more authors.
Chromosome Research | Year: 2013

Chromosomal rearrangements are a source of structural variation within the genome that figure prominently in human disease, where the importance of translocations and deletions is well recognized. In principle, inversions - reversals in the orientation of DNA sequences within a chromosome - should have similar detrimental potential. However, the study of inversions has been hampered by traditional approaches used for their detection, which are not particularly robust. Even with significant advances in whole genome approaches, changes in the absolute orientation of DNA remain difficult to detect routinely. Consequently, our understanding of inversions is still surprisingly limited, as is our appreciation for their frequency and involvement in human disease. Here, we introduce the directional genomic hybridization methodology of chromatid painting - a whole new way of looking at structural features of the genome - that can be employed with high resolution on a cell-by-cell basis, and demonstrate its basic capabilities for genome-wide discovery and targeted detection of inversions. Bioinformatics enabled development of sequence- and strand-specific directional probe sets, which when coupled with single-stranded hybridization, greatly improved the resolution and ease of inversion detection. We highlight examples of the far-ranging applicability of this cytogenomics-based approach, which include confirmation of the alignment of the human genome database and evidence that individuals themselves share similar sequence directionality, as well as use in comparative and evolutionary studies for any species whose genome has been sequenced. In addition to applications related to basic mechanistic studies, the information obtainable with strand-specific hybridization strategies may ultimately enable novel gene discovery, thereby benefitting the diagnosis and treatment of a variety of human disease states and disorders including cancer, autism, and idiopathic infertility. © 2013 The Author(s).


Patent
Kromatid and Colorado State University | Date: 2011-05-13

Methods, compositions, and assays are described which are useful in identifying point mutations, identifying cancer cells, and diagnosing cancer.


Patent
Colorado State University and Kromatid | Date: 2011-10-11

A method and a kit for the identification of chromosomal inversions are described. Chromosomal inversions are difficult to detect unless they are quite large. The improved ability to detect chromosomal inversions is important to a number of medical applications, such as cancer and birth defects, as examples. Reporter species are attached to oligonucleotide strands designed such that they may hybridize to portions of only one of a pair of single-stranded sister chromatids prepared by the CO-FISH procedure, as an example. If an inversion has occurred, these marker probes will be detected on the sister chromatid at the same location as the inversion on the first chromatid.


Patent
Kromatid and Colorado State University | Date: 2015-07-27

A method and a kit for the identification of chromosomal inversions are described. Chromosomal inversions are difficult to detect unless they are quite large. The improved ability to detect chromosomal inversions is important to a number of medical applications, such as cancer and birth defects, as examples. Reporter species are attached to oligonucleotide strands designed such that they may hybridize to portions of only one of a pair of single-stranded sister chromatids prepared by the CO-FISH procedure, as an example. If an inversion has occurred, these marker probes will be detected on the sister chromatid at the same location as the inversion on the first chromatid.


Grant
Agency: National Aeronautics and Space Administration | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2009

We propose a novel approach to the detection of chromosomal inversions. Transmissible chromosome aberrations (translocations and inversions) have profound genetic effects, such as disrupting regulatory sequences that control gene expression, or creating genetic chimeras. These chromosome aberrations play a causative role in cancer, and they are induced by radiation. As such, chromosome aberrations are relevant to three NASA needs, biodosimetry, analysis of astronaut lymphocytes for cumulative radiation damage, and space radiation risk modeling. Of all structural chromosomal anomalies, inversions – a reversal of orientation of material within a chromosome – are the most difficult to detect. This is especially true of small inversions, most of which are invisible to all current cytogenetic techniques. Yet small inversions are likely the most transmissible (nonlethal) form of chromosomal damage, so they persist, a feature which lends credence to their use in retrospective biodosimetry. This Phase 1 project is intended to provide a proof-of-principle demonstration of a new method of molecular cytogenetics that will permit highly sensitive inversion detection. The project will help us to perfect our bioinformatics strategy for probe design, optimize probe labeling reactions, refine hybridization conditions, and establish a procedure for cost analysis. In Phase 2, we will scale-up probe production to make whole chromosome analysis possible. This next step, although conceptually simple, relies entirely on the processes devised and tested in Phase 1. Moreover efficient, cost-effective probe-making will be essential to commercialization (Phase 3). The technology readiness level at the end of the Phase 1 contract is expected to be 4-5, i.e. validated in laboratory and relevant environments.


Grant
Agency: National Aeronautics and Space Administration | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 600.00K | Year: 2010

We propose the continued development of a novel approach to the detection of chromosomal inversions. Transmissible chromosome aberrations (translocations and inversions) have profound genetic effects, such as disrupting regulatory sequences that control gene expression, or creating genetic chimeras. These chromosome aberrations play a causative role in cancer, and ionizing radiation is one of the most efficient agents known to induce them. As such, chromosome aberrations are relevant to three NASA needs, biodosimetry, analysis of astronaut lymphocytes for cumulative radiation damage, and space radiation risk modeling. Of all structural chromosomal anomalies, inversions – a reversal of orientation of material within a chromosome – are the most difficult to detect. This is especially true of small inversions, most of which are invisible to all current cytogenetic techniques. Yet small inversions are likely the most transmissible (nonlethal) form of chromosomal damage, so they persist for long periods. This is a useful feature for retrospective biodosimetry, and may also prove to be useful as an indicator of radiation quality.. In Phase 1 we demonstrated the use of a human chromosome 3, partial chromatid paint to detect a known inversion. During Phase 2, we will continue to improve the efficiency of the technology, an essential goal for commercialization (Phase 3) ultimately creating an improved and complete chromatid paint for chromosome 3. Finally, we will test the chromosome 3 'chromatid paint's' ability to detect radiation-induced inversions, and establish their frequency. The technology readiness level at the end of the Phase 2 contract is expected to be 5, i.e. validated in laboratory and relevant environments.


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