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DOE OSTI2025

Methodology for Extracting High-Molecular-Weight DNA from Field Collections of Macrofungi

Many macrofungi are impractical or impossible to culture. Consequently, DNA for long-read sequencing required for the assembly of high-quality genomes must be isolated from samples taken from the environment. Collection is often in remote locations, limiting the options for stabilising samples to methods that do not require refrigeration. Fungi contain species-specific arrays of metabolites that may complicate purification techniques and call for judgement to be made to apply appropriate modifications to the DNA extraction protocol in specific cases. The protocols and commentary we describe are informed by the preparation of DNA from a range of Australasian ectomycorrhizal and saprotrophic macrofungi. We collect samples into isopropanol at ambient temperature and employ a strategy of chromatin isolation followed by the sequential removal of unwanted molecular components to purify DNA.

Burgoyne, Leigh A↗

NASA NTRS1983

The disposition of DNA in Prochloron (Prochlorophyta)

The discovery of both chlorophyll a and b in the prokaryote Prochloron Lewin, a trait otherwise unique to eukaryotic photosynthetic organisms, has stimulated speculation on the possible endosymbiont origins of the plastids of eukaryotic cells. The arrangement of DNA in Prochloron was therefore dyed in situ with the fluorochrome dye DAPI and compared with the plastid DNA of various eukaryote cells. The DNA of Prochloron is found to be clearly different in arrangement and locale from that of blue-green algae. In the great size of its nucleoids and their apparently high DNA content, Prochloron also differs from the plastids of any eukaryotes, with the possible exception of dinoflagellates. Prochloron remains an evolutionary puzzle.

Coleman, A. W.↗

NASA NTRS1994

Heavy ion induced DNA-DSB in yeast and mammalian cells

Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

Loebrich, M.↗

NASA NTRS1996

Mobility of Electron in DNA Crystals by Laser Radiation

The mobility of electrons in laser radiated DNA is closed to the energy transfer and energy migration of a biological molecule. Arrhenius has studied the conductivity of the electrons in a biological molecule. But his result is far from the experimental result and meanwhile the relation between some parameters in his theory and the micro-quantities in DNA is not very clear. In this paper, we propose a new phonon model of electron mobility in DNA and use Lippman-Schwinger equation and S-matrix theory to study the mobility of electrons in DNA crystal. The result is relatively close to the experiment result and some parameters in Arrhenius theory are explained in our work.

Zhang, Kaixi↗

NASA NTRS1996

Specialization of the DNA-Cleaving Activity of a Group I Ribozyme Through In Vitro Evolution

In an earlier study, an in vitro evolution procedure was applied to a large population of variants of the Tetrahymena group 1 ribozyme to obtain individuals with a 10(exp 5)-fold improved ability to cleave a target single-stranded DNA substrate under simulated physiological conditions. The evolved ribozymes also showed a twofold improvement, compared to the wild-type, in their ability to cleave a single-stranded RNA substrate. Here, we report continuation of the in vitro evolution process using a new selection strategy to achieve both enhanced DNA and diminished RNA-cleavage activity. Our strategy combines a positive selection for DNA cleavage with a negative selection against RNA binding. After 36 "generations" of in vitro evolution, the evolved population showed an approx. 100-fold increase in the ratio of DNA to RNA-cleavage activity. Site-directed mutagenesis experiment confirmed the selective advantage of two covarying mutations within the catalytic core of ribozyme that are largely responsible for this modified behavior. The population of ribozymes has now undergone a total of 63 successive generations of evolution, resulting in an average 28 mutations relative to the wild-type that are responsible for the altered phenotype.

Tsang, Joyce↗

NASA NTRS1999

A Novel Technique for Performing Space Based Radiation Dosimetry Using DNA: Results from GRaDEx-I and the Design of GRaDEx-II

Because of the large amounts of cosmic radiation in the space environment relative to that on earth, the effects of radiation on the physiology of astronauts is of major concern. Doses of radiation which can cause acute or chronic biological effects are to be avoided, therefore determination of the amount of radiation exposure encountered during space flight and assessment of its impact on biological systems is critical. Quantifying the radiation dosage and damage to biological systems, especially to humans during repetitive high altitude flight and during long duration space flight is important for several reasons. Radiation can cause altered biosynthesis and long term genotoxicity resulting in cancer and birth defects, etc. Radiation damage to biological systems depends in a complex way on incident radiation species and their energy spectra. Typically non-biological, i.e. film or electronic monitoring systems with narrow energy band sensitivity are used to perform dosimetry and then results are extrapolated to biological models. For this reason it may be desirable to perform radiation dosimetry by using biological molecules e.g. DNA or RNA strands as passive sensors. A lightweight genotoxicology experiment was constructed to determine the degree to which in-vitro naked DNA extracted from tissues of a variety of vertebrate organisms is damaged by exposure to radiation in a space environment. The DNA is assayed by means of agarose gel electrophoresis to determine damage such as strand breakage caused by high momentum particles and photons, and base oxidation caused by free radicals. The length distribution of DNA fragments is directly correlated with the radiation dose. It is hoped that a low mass, low cost, passive biological system to determine dose-response relationship (increase in strand breaks with increase in exposure) can be developed to perform radiation dosimetry in support of long duration space flight, and to predict negative effects on biological systems (e.g. astronauts and greenhouses) in space. The payload was flown in a 2.5 cubic foot Get Away Special (GAS) container through NASA's GAS program. It was subjected to the environment of the space shuttle cargo bay for the duration of the STS-91 mission (9 days). Results of the genotoxicology and radiation dosimetry experiment (GRaDEx-I) as well as the design of an improved follow on payload are presented.

Ritter, Joe↗

NASA NTRS1999

A Novel Technique for Performing Space Based Radiation Dosimetry Using DNA-Results from GRaDEx-I and the Design of GRaDEx-II

Because of the large amounts of cosmic radiation in the space environment relative to that on earth, the effects of radiation on the physiology of astronauts is of major concern. Doses of radiation which can cause acute or chronic biological effects are to be avoided, therefore determination of the amount of radiation exposure encountered during space flight and assessment of its impact on biological systems is critical. Quantifying the radiation dosage and damage to biological systems, especially to humans during repetitive high altitude flight and during long duration space flight is important for several reasons. Radiation can cause altered biosynthesis and long term genotoxicity resulting in cancer and birth defects etc. Radiation damage to biological systems depends in a complex way on incident radiation species and their energy spectra. Typically non-biological, i.e. film or electronic monitoring systems with narrow energy band sensitivity are used to perform dosimetry and then results are extrapolated to biological models. For this reason it may be desirable to perform radiation dosimetry by using biological molecules e.g. DNA or RNA strands as passive sensors. A lightweight genotoxicology experiment was constructed to determine the degree to which in vitro naked DNA extracted from tissues of a variety of vertebrate organisms is damaged by exposure to radiation in a space environment. The DNA is assayed by means of agarose gel electrophoresis to determine damage such as strand breakage caused by high momentum particles and photons, and base oxidation caused by free radicals. The length distribution of DNA fragments is directly correlated with the radiation dose. It is hoped that a low mass, low cost, passive biological system to determine dose response relationship (increase in strand breaks with increase in exposure) can be developed to perform radiation dosimetry in support of long duration space flight, and to predict negative effects on biological systems (e.g. astronauts and greenhouses) in space. The payload was flown in a 2.5 cubic foot Get Away Special (GAS) container through NASA's GAS program. It was subjected to the environment of the space shuttle cargo bay for the duration of the STS-91 mission (9 days). Results of the genotoxicology and radiation dosimetry experiment (GRaDEx-1) as well as the design of an improved follow on payload are presented.

Ritter, Joe↗

NASA NTRS1999

DNA Repair-Protein Relocalization After Heavy Ion Exposure

Ionizing radiation is good at making DNA double strand breaks, and high linear energy transfer (LET) radiations such as heavy ion particles are particularly efficient. For this reason, the proteins belonging to repair systems that deal with double strand breaks are of particular interest. One such protein is Ku, a component in the non-homologous recombination repair system. The Ku protein is an abundant, heterodimeric DNA end-binding complex, composed of one 70 and one 86 kDa subunit. Ku protein binds to DNA ends, nicks, gaps, and regions of transition between single and double-stranded structure. These binding properties suggest an important role in DNA repair. The Ku antigen is important in this study because it is present in relatively large copy numbers and it is part of a double-strand-break repair system. More importantly, we consistently measure an apparent upregulation in situ that is not verified by whole-cell-lysate immunoblot measurements. This apparent upregulation is triggered by very low doses of radiation, thus showing a potentially useful high sensitivity. However, elucidation of the mechanism underlying this phenomenon is still to be done.

Metting, N. F.↗

NASA NTRS1999

Global Mapping of Underwater UV Irradiances and DNA-Weighted Exposures using TOMS and SeaWiFS Data Products

The global stratospheric ozone-layer depletion results In an increase in biologically harmful ultraviolet (UV) radiation reaching the surface and penetrating to ecologically significant depths in natural waters. Such an increase can be estimated on a global scale by combining satellite estimates of UV irradiance at the ocean surface from the Total Ozone Mapping Spectrometer (TOMS) satellite instrument with the SeaWIFS satellite ocean-color measurements in the visible spectral region. In this paper we propose a model of seawater optical properties in the UV spectral region based on the Case I water model in the visible range. The inputs to the model are standard monthly SeaWiFS products: chlorophyll concentration and the diffuse attenuation coefficient at 490nm. Penetration of solar UV radiation to different depths in open ocean waters is calculated using the RT (radiative transfer) quasi-single scattering approximation (QSSA). The accuracy of the QSSA approximation in the water is tested using more accurate codes. The sensitivity study of the underwater UV irradiance to atmospheric and oceanic optical properties have shown that the main environmental parameters controlling the absolute levels of the UVB (280-320nm) and DNA-weighted irradiance underwater are: solar-zenith angle, cloud transmittance, water optical properties, and total ozone. Weekly maps of underwater UV irradiance and DNA-weighted exposure are calculated using monthly-mean SeaWiFS chlorophyll and diffuse attenuation coefficient products, daily SeaWiFS cloud fraction data, and the TOMS-derived surface UV irradiance daily maps. The final products include global maps of weekly-average UVB irradiance and DNA-weighted daily exposures at 3m and 10m, and depths where the UVB irradiance and DNA-weighted dose rate at local noon are equal to 10% of their surface values.

Vasilkov, Alexander↗

NASA NTRS2001

Crystallization of bFGF-DNA Aptamer Complexes Using a Sparse Matrix Designed for Protein-Nucleic Acid Complexes

The Sparse Matrix approach for obtaining lead crystallization conditions has proven to be very fruitful for the crystallization of proteins and nucleic acids. Here we report a Sparse Matrix developed specifically for the crystallization of protein-DNA complexes. This method is rapid and economical, typically requiring 2.5 mg of complex to test 48 conditions. The method was originally developed to crystallize basic fibroblast growth factor (bFGF) complexed with DNA sequences identified through in vitro selection, or SELEX, methods. Two DNA aptamers that bind with approximately nanomolar affinity and inhibit the angiogenic properties of bFGF were selected for co-crystallization. The Sparse Matrix produced lead crystallization conditions for both bFGF-DNA complexes.

Cannone, Jaime J.↗

NASA NTRS2002

RNA-Cleaving DNA Enzymes with Altered Regio- or Enantioselectivity

In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5' - phosphodiester following a wibonucleotide or a 3',5' -phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5' -phosphodiester exhibits a k(sub cat) of approx. 0.01/ min and catalytic efficiency, k(sub cat)/k(sub m) of approx. 10(exp 5)/ M min. The enzyme that cleaves an L-ribonudeotide is about 10-fold slower and has a catalytic efficiency of approx. 4 x 10(exp 5)/ M min. Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 C. In a comparison of each enzyme s activity with either its corresponding substrate that contains an unnatural ribonudeotide or a substrate that instead contains a standard ribonucleotide, the 2',5' -phosphodiester-deaving DNA enzyme exhibited a regioselectivity of 6000- fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 50-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.

Ordoukhanian, Phillip↗

NASA NTRS2001

Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

Non-NASA Center↗

NASA NTRS1993

Finite-size effects on long-range correlations: implications for analyzing DNA sequences

We analyze the fluctuations in the correlation exponents obtained for noncoding DNA sequences. We find prominent sample-to-sample variations as well as variations within a single sample in the scaling exponent. To determine if these fluctuations may result from finite system size, we generate correlated random sequences of comparable length and study the fluctuations in this control system. We find that the DNA exponent fluctuations are consistent with those obtained from the control sequences having long-range power-law correlations. Finally, we compare our exponents for the DNA sequences with the exponents obtained from power-spectrum analysis and correlation-function techniques, and demonstrate that the original "DNA-walk" method is intrinsically more accurate due to reduced noise.

NASA Discipline Cardiopulmonary↗

NASA NTRS1995

Comparison of DNA fragmentation and color thresholding for objective quantitation of apoptotic cells

Apoptosis is a process of cell death characterized by distinctive morphological changes and fragmentation of cellular DNA. Using video imaging and color thresholding techniques, we objectively quantitated the number of cultured CD4+ T-lymphoblastoid cells (HUT78 cells, RH9 subclone) displaying morphological signs of apoptosis before and after exposure to gamma-irradiation. The numbers of apoptotic cells measured by objective video imaging techniques were compared to numbers of apoptotic cells measured in the same samples by sensitive apoptotic assays that quantitate DNA fragmentation. DNA fragmentation assays gave consistently higher values compared with the video imaging assays that measured morphological changes associated with apoptosis. These results suggest that substantial DNA fragmentation can precede or occur in the absence of the morphological changes which are associated with apoptosis in gamma-irradiated RH9 cells.

Non-NASA Center↗

NASA NTRS2004

Novel Image Processing Interface to Relate DSB Spatial Distribution from Immunofluorescence Foci Experiments to the State-of-the-Art Models of DNA Breakage

A recently developed software (NASARadiationTrackImage) allows a quick and automatic segmentation of foci that indicate spatial localization of specific proteins that are visualized by immunofluorescence. Of interest are the spatial and temporal distribution of foci such as gammaH2AX, a signal of the phosphorylation of a variant of the histone H2A that has been shown to correspond to DSBs, or proteins involved in DSB processing, such as ATM, Rad51, and p53, following exposures of human cells to high charge and energy (HZE) ion irradiation. Experimental data are recorded as sets of two-dimensional images in color with cells and foci of gammaH2AX, ATM, Rad51 or others shown. Different cells, levels of radiation and timing after radiation were recorded. The software allows us to calculate the number of foci per cell, overall intensity of light in foci and their spatial organization. A simple statistical model allows for testing of foci overlap (eclipse). A more complex statistical model previously known as DNAbreak simulates track structure and random chromosome geometry. It has one adjustable parameter corresponding to an average intensity of DSB creation in cubic micrometers of DNA volume per particle track or unit dose. Its limitation is the low-resolution limit both in physical space and DSB's along DNA. It works adequately on the scale of a cell and provides further insights on how the geometry of tracks and DNA affects genomic damage of the cell and subsequent repair. Future developments of the model for the description of the time evolution of DNA damage response proteins, and more robust track structure models will be discussed.

Ponomarev, A. L.↗

NASA NTRS2007

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

Hu, Shaowen↗

NASA NTRS2011

DNA Damage Signals and Space Radiation Risk

Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

Cucinotta, Francis A.↗

NASA NTRS2012

Calculation of the Frequency Distribution of the Energy Deposition in DNA Volumes by Heavy Ions

Radiation quality effects are largely determined by energy deposition in small volumes of characteristic sizes less than 10 nm representative of short-segments of DNA, the DNA nucleosome, or molecules initiating oxidative stress in the nucleus, mitochondria, or extra-cellular matrix. On this scale, qualitatively distinct types of molecular damage are possible for high linear energy transfer (LET) radiation such as heavy ions compared to low LET radiation. Unique types of DNA lesions or oxidative damages are the likely outcome of the energy deposition. The frequency distribution for energy imparted to 1-20 nm targets per unit dose or particle fluence is a useful descriptor and can be evaluated as a function of impact parameter from an ions track. In this work, the simulation of 1-Gy irradiation of a cubic volume of 5 micron by: 1) 450 (1)H(+) ions, 300 MeV; 2) 10 (12)C(6+) ions, 290 MeV/amu and 3) (56)Fe(26+) ions, 1000 MeV/amu was done with the Monte-Carlo simulation code RITRACKS. Cylindrical targets are generated in the irradiated volume, with random orientation. The frequency distribution curves of the energy deposited in the targets is obtained. For small targets (i.e. <25 nm size), the probability of an ion to hit a target is very small; therefore a large number of tracks and targets as well as a large number of histories are necessary to obtain statistically significant results. This simulation is very time-consuming and is difficult to perform by using the original version of RITRACKS. Consequently, the code RITRACKS was adapted to use multiple CPU on a workstation or on a computer cluster. To validate the simulation results, similar calculations were performed using targets with fixed position and orientation, for which experimental data are available [5]. Since the probability of single- and double-strand breaks in DNA as function of energy deposited is well know, the results that were obtained can be used to estimate the yield of DSB, and can be extended to include other targeted or non-target effects.

Plante, Ianik↗