Filming seismograms and related materials at the California Institute of Technology

NASA Astrophysics Data System (ADS)

Goodstein, Judith R.; Roberts, Paul

As part of the worldwide effort to create an international earthquake data bank, the seismology archive of the California Institute of Technology (Caltech) has been organized, labeled, described, and microfilmed. It includes a wide variety of original records, documents, and printed materials relating to local and distant earthquakes. The single largest and most complex component of the task has been the preparation and microfilming of Caltech's vast collection of original seismograms. The original proposal envisioned a modest project in which a selected number of seismographic records at Caltech could be made more generally available to the scientific community. These single-copy records are stored at Kresge Laboratory and comprise thousands of individual photographic sheets, each 30×92 cm. In the end, we microfilmed both the Pasadena station records and those written at the six original stations in the Caltech network. This task got underway in June 1981 and was completed in January 1985. In the course of the project, the staff sorted, arranged, inventoried, copied, and refiled more than 276,000 records written between January 10, 1923 and December 31, 1962. The microfilm edition of the earthquake records at the Seismological Laboratory at Pasadena and at auxiliary stations at Mount Wilson, Riverside, Santa Barbara, La Jolla, Tinemaha, and Haiwee (the latter two in the Owens Valley) consists of 461 reels of film. The film archive is cataloged and available to researchers in Caltech's Millikan Library in Pasadena, at the U.S. Geological Survey in Menlo Park, Calif, and at the World Data Center (National Oceanic and Atmospheric Administration) in Boulder, Colo.

A Conversation with Robert F. Christy Part II

NASA Astrophysics Data System (ADS)

Lippincott, Sara

2006-12-01

Robert F. Christy, Institute Professor of Theoretical Physics Emeritus at Caltech, recalls his wartime work at Los Alamos on the critical assembly for the plutonium bomb (“the Christy bomb”); the Alamogordo test, July 16, 1945; the postwar concerns of ALAS (Association of Los Alamos Scientists); his brief return to the University of Chicago and move to Caltech; friendship with and later alienation from Edward Teller; work with Charles and Tommy Lauritsen and William A. Fowler in Caltech’s Kellogg Radiation Laboratory; Freeman Dyson’s Orion Project; work on the meson and RR Lyrae stars; fellowship at Cambridge University; 1950s Vista Project at Caltech; his opposition to the Strategic Defense Initiative; and his post-retirement work for the National Research Council’s Committee on Dosimetry and on inertial-confinement fusion.

Regulation of yeast DNA polymerase δ-mediated strand displacement synthesis by 5′-flaps

PubMed Central

Koc, Katrina N.; Stodola, Joseph L.; Burgers, Peter M.; Galletto, Roberto

2015-01-01

The strand displacement activity of DNA polymerase δ is strongly stimulated by its interaction with proliferating cell nuclear antigen (PCNA). However, inactivation of the 3′–5′ exonuclease activity is sufficient to allow the polymerase to carry out strand displacement even in the absence of PCNA. We have examined in vitro the basic biochemical properties that allow Pol δ-exo− to carry out strand displacement synthesis and discovered that it is regulated by the 5′-flaps in the DNA strand to be displaced. Under conditions where Pol δ carries out strand displacement synthesis, the presence of long 5′-flaps or addition in trans of ssDNA suppress this activity. This suggests the presence of a secondary DNA binding site on the enzyme that is responsible for modulation of strand displacement activity. The inhibitory effect of a long 5′-flap can be suppressed by its interaction with single-stranded DNA binding proteins. However, this relief of flap-inhibition does not simply originate from binding of Replication Protein A to the flap and sequestering it. Interaction of Pol δ with PCNA eliminates flap-mediated inhibition of strand displacement synthesis by masking the secondary DNA site on the polymerase. These data suggest that in addition to enhancing the processivity of the polymerase PCNA is an allosteric modulator of other Pol δ activities. PMID:25813050

Effects of mass transfer between Martian satellites on surface geology

DTIC Science & Technology

2015-12-21

University Affiliated Research Center (UARC). Thanks to Bill Folkner (JPL/Caltech) for high-fidelity long-term Phobos/Deimos SPICE orbit propagations, and...created by JPL/Caltech to SPICE ephemeris information from NASA’s Navigation and Ancillary Information Facility (naif.jpl.nasa.gov) (Acton et al., 2002...References Acton, C. et al., 2002. Extending NASA’s SPICE ancillary information system to meet future mission needs. In: 2002 AIAA Space Operations

Obituary: James Adolph Westphal, 1930-2004

NASA Astrophysics Data System (ADS)

Danielson, G. Edward

2004-12-01

James A. Westphal died September 8, 2004. He had battled a neurological disease related to Alzheimer's for the past year. He was 74. James A. Westphal was born in Dubuque, Iowa, on June 13, 1930. He was raised in Tulsa, Oklahoma, and in Little Rock, Arkansas. Westphal earned his bachelor's degree in physics from the University of Tulsa in 1954, a year after he went to work as geophysical research group leader at Sinclair Research Lab in Tulsa. Westphal first got into the business of scientific instrumentation right after high school, when he did well-logging in Texas and Gulf Coast oil fields. In fact, his work at Sinclair Research Labs involved devising unorthodox methods for oil discovery; one of his discoveries of a new way of processing seismic data first brought him to the attention of Caltech professor Hewitt Dix, who is often considered the father of exploration geophysics. Westphal arrived at Caltech initially on a four-month leave of absence to devise a data processor for Dix, but never left. He discovered that the academic freedom individual professors enjoy was amenable to his own predilections, so he soon began branching out to other areas of scientific investigation at Caltech. Before long, he had teamed up with Bruce Murray to do thermal infrared scans of the moon in order to see if humans could even walk on the lunar surface without sinking into the dusty soil. Westphal and Murray's work showed that rocky areas could be identified with the thermal imaging, which in turn led to the inference that the Apollo astronauts could safely walk on the soil without sinking. Westphal and Murray also teamed up to do the first infrared imaging of Venus and Jupiter. Other projects at Caltech led to Westphal's being hired on permanently by Bob Sharp, who at the time was the geology division chairman. In the following years, Westphal involved himself in novel ways of studying volcanism in Hawaii and Mount St. Helens. He invented a simple and very sensitive tilt meter that allowed them to measure the volcano's expansion and help predict upcoming eruptions. Westphal also designed a way of creating a high-pressure aquarium for studies of deep-ocean animals as well as instruments for tracking glacial ice flows and capturing starlight. Caltech astronomers were pleased to discover that Westphal had an idea for an infrared camera for the historic 200-inch Hale Telescope--at the time the largest optical telescope in the world--that could measure the radiance of galaxies with greater precision and capture fainter galaxies than possible up to that time. He built various other instruments for the Hale Telescope, including a Silicon Intensified Target camera, which was a sort of transitional device between the photographic emulsion plates of the day and modern charged-coupled devices (CCDs) and which produced digital pictures with unparalleled clarity. The instrument is now in the Smithsonian's National Air and Space Museum's collection. Westphal and Jim Gunn (now at Princeton) recognized early that CCDs would revolutionize astronomy, and in the process of obtaining them for Palomar, Westphal and Gunn decided to put a team of astronomers together and design a CCD camera for use on the Hubble Space Telescope. Westphal was named principal investigator of the proposal and Jim Gunn was his deputy. They teamed with JPL to design the main camera for Hubble Space Telescope. (The Wide-Field / Planetary Camera), which proved to be an enormously successful part of the telescope's scientific mission. Westphal's research came up with a CCD coating that absorbed the UV photons and fluoresced back in the visible thus allowing the instrument to be used in the UV (The engineers dubbed the coating "mouse milk"). He didn't reveal the composition of the coating until NASA announced that the Caltech/JPL proposal was selected to provide the WF/PC Investigation. During the Hubble first light operations Jim had to diagnose the spherical aberration in the main 2.4 meter mirror, which caused the Hubble's initial focusing problems, while being photographed in real time by all the press cameras. A straight forward fix was devised for the WFPCII camera to bring the images into sharp focus. Once the repaired camera and the Hubble Space Telescope were safely in orbit, Westphal and his collaborators began receiving data on a regular basis. One of the early images of distant galaxies provided especially compelling evidence for the phenomenon known as gravitational lensing. "When this picture came in," Westphal said in a 1995 interview for Caltech's Engineering and Science magazine, "I put it under [Caltech physicist] Kip Thorne's door with a note saying, if you ever have any doubt about gravitational lenses, here's your proof." He was a member of the American Astronomical Society and served as the chairman of the Division for Planetary Sciences in 1979-1980. In 1991 Westphal received a MacArthur Fellowship. Already a tenured faculty member and the author of scores of refereed journal articles, and the creator of 15 patented inventions, Westphal was named director of Palomar Observatory in 1995 and served for three years. This assignment included being Caltech's representative to the Cara Board (which managed the KECK telescope). After lowering one of his custom-designed instruments into Old Faithful at Yellowstone National Park to study the geyser's cycling mechanism, he published a paper with Caltech graduate, Sue Kieffer, in 1997. The work, which received a good deal of media attention, confirmed previous assumptions about the geyser and also uncovered new details about the eruption cycle. Westphal took Caltech emeritus status in 1998, but remained active in research endeavors until his bout with this illness. Surviving Jim are his wife, Jean Westphal of Altadena; a son, Andrew Westphal, a daughter-in-law, Kim Taylor, and two granddaughters, Theresa and Laura Westphal, all of Richmond, California; two stepdaughters, Robin Stroll of Agoura Hills, California and Susan Stroll of Eagle Rock, California; and an uncle, Eddy Westphal of Indiana. A portion of this obituary was taken from a tribute by Robert Tindol published September 14, 2004 in an electronic Caltech Newsletter.

Image Halftoning and Inverse Halftoning for Optimized Dot Diffusion

DTIC Science & Technology

1998-01-01

systems.caltech.edu, ppvnath@sys.caltech.edu ABSTRACT The dot diffusion method for digital halftoning has the advantage of parallelism unlike the error ... halftoning : ordered dither [3], error diffusion [4], neural-net based methods [2], and more recently direct binary search (DBS) [10]. Ordered dithering is a...patterns. On the other hand error diffused halftones do not suffer from periodicity and offer blue noise characteristic [11] which is found to be

AirMSPI Level 1B2 V006 New Data for the NASA/JPL/Caltech ImPACT-PM Campaign

Atmospheric Science Data Center

2018-05-17

AirMSPI Level 1B2 V006 New Data for the NASA/JPL/Caltech ImPACT-PM Campaign ImPACT-PM Wednesday, May 16, 2018 The NASA Langley Atmospheric Science Data Center (ASDC) and Jet Propulsion ... flight campaign.   AirMSPI flies in the nose of NASA's high-altitude ER-2 aircraft. The instrument was built by JPL and the ...

Compound Separation

NASA Technical Reports Server (NTRS)

1981-01-01

Jet Propulsion Laboratory developed a new one-step liquid-liquid extraction technique which cuts processing time, reduces costs and eliminates much of the equipment required. Technique employs disposable extraction columns, originally developed as an aid to the Los Angeles Police Department, which allow more rapid detection of drugs as part of the department's drug abuse program. Applications include medical treatment, pharmaceutical preparation and forensic chemistry. NASA waived title to Caltech, and Analytichem International is producing Extubes under Caltech license.

Retention of Women in Geoscience Undergraduate and Graduate Education at Caltech

NASA Astrophysics Data System (ADS)

Alexander, C. J.

2001-12-01

Institutional barriers encountered by women in undergraduate and graduate schools may take many forms, but can also be as simple as a lack of community support. In the 1990's the California Institute of Technology (Caltech) made a commitment to the retention of women in their graduate and undergraduate schools. Their program included mentoring, focussed tutoring, self-esteem support groups, and other retention efforts. Under this program, the attrition rate of women has dramatically slowed. In this paper, we will discuss recent data from the American Geological Institude chronicling the enrollment and successes of women in the geosciences, the program instituted by Caltech, possible causes of attrition among women in the geosciences, as well as potential programs to address these problems. We will also present, from the nationwide study, data on geoscience departments which have been relatively successful at retaining and graduating women in Earth and Space Sciences.

Viscoelastic coupling of nanoelectromechanical resonators.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simonson, Robert Joseph; Staton, Alan W.

2009-09-01

This report summarizes work to date on a new collaboration between Sandia National Laboratories and the California Institute of Technology (Caltech) to utilize nanoelectromechanical resonators designed at Caltech as platforms to measure the mechanical properties of polymeric materials at length scales on the order of 10-50 nm. Caltech has succeeded in reproducibly building cantilever resonators having major dimensions on the order of 2-5 microns. These devices are fabricated in pairs, with free ends separated by reproducible gaps having dimensions on the order of 10-50 nm. By controlled placement of materials that bridge the very small gap between resonators, the mechanicalmore » devices become coupled through the test material, and the transmission of energy between the devices can be monitored. This should allow for measurements of viscoelastic properties of polymeric materials at high frequency over short distances. Our work to date has been directed toward establishing this measurement capability at Sandia.« less

Fogwater Inputs to a Cloud Forest in Puerto Rico

NASA Astrophysics Data System (ADS)

Eugster, W.; Burkard, R.; Holwerda, F.; Bruijnzeel, S.; Scatena, F. N.; Siegwolf, R.

2002-12-01

Fog is highly persistent at upper elevations of humid tropical mountains and is an important pathway for water and nutrient inputs to mountain forest ecosystems. Measurements of fogwater fluxes were performed in the Luquillo mountains of Puerto Rico using the eddy covariance approach and a Caltech-type active strand cloudwater collector. Rainfall and throughfall were collected between 25 June--7 August 2002. Samples of fog, rain, stemflow and throughfall were analyzed for inorganic ion and stable isotope concentrations (δ18O and δD). Initial results indicate that fog inputs can occur during periods without rain and last for up to several days. The isotope ratios in rainwater and fogwater are rather similar, indicative of the proximity of the Carribbean Sea and the close interrelation between the origins of fog and rain at our experimental site. Largest differences in isotope ratios for fog were found between daytime convective and nighttime stable conditions. Throughfall was always exceeding rainfall, indicating (a) the relevance of fogwater inputs and (b) the potentially significant undersampling of rainfall due to relatively high wind speeds (5.7 m/s mean) and the exposition of our field site close to a mountain ridge. Our size-resolved measurements of cloud droplets (40 size bins between 2 and 50 μm aerodynamic diameter) indicate that the liquid water content of fog in the Luquillo mountains is 5 times higher than previously assumed, and thus does not differ from the values reported from other mountain ranges in other climate zones. Average deposition rates are 0.88 mm and 6.5 mm per day for fog and rain, respectively.

VizieR Online Data Catalog: Star formation in active and normal galaxies (Tsai+, 2015)

NASA Astrophysics Data System (ADS)

Tsai, M.; Hwang, C.-Y.

2015-11-01

We selected 104 active galaxies from the lists of Melendez et al. (2010MNRAS.406..493M), Condon et al. 1991 (cat. J/ApJ/378/65), and Ho & Ulvestad 2001 (cat. J/ApJS/133/77). Most of the sources are identified as Active Galactic Nuclei (AGNs), and a few of them are classified as Luminous InfraRed Galaxies (LIRGs). We obtained 3.6 and 8μm infrared images of these galaxies from the Spitzer Archive (http://sha.ipac.caltech.edu/applications/Spitzer/SHA/) and 8GHz images from the VLA archive (http://archive.nrao.edu/archive/archiveimage.html). We also selected a nearby AGN sub-sample containing 21 radio-selected AGNs for further spatial analysis. We selected 25 nearby AGNs exhibiting no detected radio emission in order to compare with the results of the radio-selected sources. For comparison, we also selected normal galaxies with distances less than 15Mpc from the catalog of Tully 1994 (see cat. VII/145). We only selected the galaxies that have Spitzer archive data and are not identified as AGNs in either the Veron-Cetty & Veron 2006 (see cat. VII/258) AGN catalog or in the NED database (http://ned.ipac.caltech.edu/). Our results for the radio-selected and the non-radio-selected active galaxies are listed in Table1, and those for the normal galaxies are listed in Table2. (2 data files).

The second Caltech-Jodrell Bank VLBI survey. 1: Observations of 91 of 193 sources

NASA Technical Reports Server (NTRS)

Taylor, G. B.; Vermeulen, R. C.; Pearson, T. J.; Readhead, A. C. S.; Henstock, D. R.; Browne, I. W. A.; Wilkinson, P. N.

1994-01-01

We define the sample for the second Caltech-Jodrell Bank very long base interferometry (VLBI) survey. This is a sample of 193 flat- or gigahertz-peaked-spectrum sources selected at 4850 MHz. This paper presents images of 91 sources with a resolution of approximately 1 mas, obtained using VLBI observations at 4992 MHz with a global array. The remaining images and the integrated radio spectra will be presented in a forthcoming paper by Henstock et al.

Planetary submillimeter spectroscopy

NASA Technical Reports Server (NTRS)

Klein, M. J.

1988-01-01

The aim is to develop a comprehensive observational and analytical program to study solar system physics and meterology by measuring molecular lines in the millimeter and submillimeter spectra of planets and comets. A primary objective is to conduct observations with new JPL and Caltech submillimeter receivers at the Caltech Submillimeter Observatory (CSO) on Mauna Kea, Hawaii. A secondary objective is to continue to monitor the time variable planetary phenomena (e.g., Jupiter and Uranus) at centimeter wavelength using the NASA antennas of the Deep Space Network (DSN).

Low concentration of arsenite exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin Xujun; Department of Toxicology, Fourth Military Medical University, Xi'an, Shaanxi, 710032; Hudson, Laurie G.

2008-10-01

Epidemiological studies have associated arsenic exposure with many types of human cancers. Arsenic has also been shown to act as a co-carcinogen even at low concentrations. However, the precise mechanism of its co-carcinogenic action is unknown. Recent studies indicate that arsenic can interfere with DNA-repair processes. Poly(ADP-ribose) polymerase (PARP)-1 is a zinc-finger DNA-repair protein, which can promptly sense DNA strand breaks and initiate DNA-repair pathways. In the present study, we tested the hypothesis that low concentrations of arsenic could inhibit PAPR-1 activity and so exacerbate levels of ultraviolet radiation (UVR)-induced DNA strand breaks. HaCat cells were treated with arsenite and/ormore » UVR, and then DNA strand breaks were assessed by comet assay. Low concentrations of arsenite ({<=} 2 {mu}M) alone did not induce significant DNA strand breaks, but greatly enhanced the DNA strand breaks induced by UVR. Further studies showed that 2 {mu}M arsenite effectively inhibited PARP-1 activity. Zinc supplementation of arsenite-treated cells restored PARP-1 activity and significantly diminished the exacerbating effect of arsenite on UVR-induced DNA strand breaks. Importantly, neither arsenite treatment, nor zinc supplementation changed UVR-triggered reactive oxygen species (ROS) formation, suggesting that their effects upon UVR-induced DNA strand breaks are not through a direct free radical mechanism. Combination treatments of arsenite with PARP-1 inhibitor 3-aminobenzamide or PARP-1 siRNA demonstrate that PARP-1 is the target of arsenite. Together, these findings show that arsenite at low concentration exacerbates UVR-induced DNA strand breaks by inhibiting PARP-1 activity, which may represent an important mechanism underlying the co-carcinogenicity of arsenic.« less

A neural network-based approach to noise identification of interferometric GW antennas: the case of the 40 m Caltech laser interferometer

NASA Astrophysics Data System (ADS)

Acernese, F.; Barone, F.; de Rosa, M.; De Rosa, R.; Eleuteri, A.; Milano, L.; Tagliaferri, R.

2002-06-01

In this paper, a neural network-based approach is presented for the real time noise identification of a GW laser interferometric antenna. The 40 m Caltech laser interferometer output data provide a realistic test bed for noise identification algorithms because of the presence of many relevant effects: violin resonances in the suspensions, main power harmonics, ring-down noise from servo control systems, electronic noises, glitches and so on. These effects can be assumed to be present in all the first interferometric long baseline GW antennas such as VIRGO, LIGO, GEO and TAMA. For noise identification, we used the Caltech-40 m laser interferometer data. The results we obtained are pretty good notwithstanding the high initial computational cost. The algorithm we propose is general and robust, taking into account that it does not require a priori information on the data, nor a precise model, and it constitutes a powerful tool for time series data analysis.

DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

PubMed

Ribeck, Noah; Saleh, Omar A

2013-01-01

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

ZTF Undergraduate Astronomy Institute at Caltech and Pomona College

NASA Astrophysics Data System (ADS)

Penprase, Bryan Edward; Bellm, Eric Christopher

2017-01-01

From the new Zwicky Transient Facility (ZTF), an NSF funded project based at Caltech, comes a new initiative for undergraduate research known as the Summer Undergraduate Astronomy Institute. The Institute brings together 15-20 students from across the world for an immersive experience in astronomy techniques before they begin their summer research projects. The students are primarly based at Caltech in their SURF program but also includes a large cohort of students enrolled in research internships at Pomona College in nearby Claremont CA. The program is intended to introduce students to research techniques in astronomy, laboratory and computational technologies, and to observational astronomy. Since many of the students are previously computer science or physics majors with little astronomy experience, this immersive experience has been extremely helpful for enabling students to learn about the terminologies, techniques and technologies of astronomy. The field trips to the Mount Wilson and Palomar telescopes deepen their knowledge and excitement about astronomy. Lectures about astronomical research from Caltech staff scientists and graduate students also provide context for the student research. Perhaps more importantly, the creation of a cohort of like-minded students, and the chance to reflect about careers in astronomy and research, give these students opportunities to consider themselves as future research scientists and help them immensely as they move forward in their careers. We discuss some of the social and intercultural aspects of the experience as well, as our cohorts typically include international students from many countries and several students from under-represented groups in science.

Gauging the influence of increased search effort on reporting rates of bottlenose dolphin (Tursiops truncatus) strandings following the deepwater horizon oil spill.

PubMed

Pitchford, Jonathan L; Garcia, Michael; Pulis, Eric E; Ambert, Ashley Millan; Heaton, Andrew J; Solangi, Moby

2018-01-01

The co-occurrence of the Deepwater Horizon oil spill and the northern Gulf of Mexico cetacean Unusual Mortality Event have raised questions about the stability of inshore bottlenose dolphin (Tursiops truncatus) populations throughout the region. Several factors could have contributed to the ongoing event, but little attention has been paid to the potential effects of increased search effort and reporting of strandings associated with oil spill response activities, which were widespread for an extended period. This study quantified the influence of increased search effort by estimating the number of bottlenose dolphin strandings reported by oil spill responders and comparing monthly stranding rates with and without response-related records. Results showed that response teams reported an estimated 58% of strandings during the Active Response period within the study area. Comparison of Poisson rates tests showed that when responder-influenced stranding records were removed, the monthly stranding rates from the Active Response period (May 2010 -April 2014) were similar to the Post-Removal Actions Deemed Complete period (May 2013 -March 2015) (e.g., p = 0.83 for remote areas in Louisiana). Further, analyses using the Getis-Ord Gi* spatial statistic showed that when response-related stranding reports were removed from the Active Response period, significant spatial clustering of strandings (p < 0.05) was reduced by 48% in coastal Louisiana. Collectively, these results suggest that increased search effort resulting from the Deepwater Horizon oil spill response throughout remote portions of the Unusual Mortality Event geographic region had the capacity to increase reporting and recovery of marine mammal strandings to unusually high levels. To better understand how stranding data relates to actual mortality, more work is needed to quantify dolphin population size, population trends, and carcass detection rates including the role of search effort. This is vital for understanding the status of a protected species within the northern Gulf of Mexico.

An ultrasensitive electrochemical biosensor for polynucleotide kinase assay based on gold nanoparticle-mediated lambda exonuclease cleavage-induced signal amplification.

PubMed

Cui, Lin; Li, Yueying; Lu, Mengfei; Tang, Bo; Zhang, Chun-Yang

2018-01-15

Polynucleotide kinase (PNK) plays an essential role in cellular nucleic acid metabolism and the cellular response to DNA damage. However, conventional methods for PNK assay suffer from low sensitivity and involve multiple steps. Herein, we develop a simply electrochemical method for sensitive detection of PNK activity on the basis of Au nanoparticle (AuNP)-mediated lambda exonuclease cleavage-induced signal amplification. We use [Ru(NH 3 ) 6 ] 3+ as the electrochemically active indicator and design two DNA strands (i.e., strand 1 and strand 2) to sense PNK. The assembly of strand 2 on the AuNP surface leads to the formation of AuNP-strand 2 conjugates which can be subsequently immobilized on the gold electrode through the hybridization of strand 1 with strand 2 for the generation of a high electrochemical signal. The presence of PNK induces the phosphorylation of the strand 2-strand 1 hybrid and the subsequent cleavage of double-stranded DNA (dsDNA) by lambda exonuclease, resulting in the release of AuNP-strand 2 conjugates and [Ru(NH 3 ) 6 ] 3+ from the gold electrode surface and consequently the decrease of electrochemical signal. The PNK activity can be simply monitored by the measurement of [Ru(NH 3 ) 6 ] 3+ peak current signal. This assay is very sensitive with a detection limit of as low as 7.762 × 10 -4 UmL -1 and exhibits a large dynamic range from 0.001 to 10UmL -1 . Moreover, this method can be used to screen the PNK inhibitors, and it shows excellent performance in real sample analysis, thus holding great potential for further applications in biological researches and clinic diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Strand displacement by DNA polymerase III occurs through a tau-psi-chi link to single-stranded DNA-binding protein coating the lagging strand template.

PubMed

Yuan, Quan; McHenry, Charles S

2009-11-13

In addition to the well characterized processive replication reaction catalyzed by the DNA polymerase III holoenzyme on single-stranded DNA templates, the enzyme possesses an intrinsic strand displacement activity on flapped templates. The strand displacement activity is distinguished from the single-stranded DNA-templated reaction by a high dependence upon single-stranded DNA binding protein and an inability of gamma-complex to support the reaction in the absence of tau. However, if gamma-complex is present to load beta(2), a truncated tau protein containing only domains III-V will suffice. This truncated protein is sufficient to bind both the alpha subunit of DNA polymerase (Pol) III and chipsi. This is reminiscent of the minimal requirements for Pol III to replicate short single-stranded DNA-binding protein (SSB)-coated templates where tau is only required to serve as a scaffold to hold Pol III and chi in the same complex (Glover, B., and McHenry, C. (1998) J. Biol. Chem. 273, 23476-23484). We propose a model in which strand displacement by DNA polymerase III holoenzyme depends upon a Pol III-tau-psi-chi-SSB binding network, where SSB is bound to the displaced strand, stabilizing the Pol III-template interaction. The same interaction network is probably important for stabilizing the leading strand polymerase interactions with authentic replication forks. The specificity constant (k(cat)/K(m)) for the strand displacement reaction is approximately 300-fold less favorable than reactions on single-stranded templates and proceeds with a slower rate (150 nucleotides/s) and only moderate processivity (approximately 300 nucleotides). PriA, the initiator of replication restart on collapsed or misassembled replication forks, blocks the strand displacement reaction, even if added to an ongoing reaction.

Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.

PubMed

Hutinet, Geoffrey; Besle, Arthur; Son, Olivier; McGovern, Stephen; Guerois, Raphaël; Petit, Marie-Agnès; Ochsenbein, Françoise; Lecointe, François

2018-01-01

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo , but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro , Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo , though Sak4 and its SSB are unable to promote strand exchange in vitro . Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.

Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein

PubMed Central

Hutinet, Geoffrey; Besle, Arthur; Son, Olivier; McGovern, Stephen; Guerois, Raphaël; Petit, Marie-Agnès; Ochsenbein, Françoise; Lecointe, François

2018-01-01

Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves. PMID:29740405

Optical Properties of Laminarin Using Terahertz Time-Domain Spectroscopy (abstract)

NASA Astrophysics Data System (ADS)

Shin, Hee Jun; Maeng, Inhee; Oh, Seung Jae; Kim, Sung In; Kim, Ha Won; Son, Joo-Hiuk

2009-04-01

Terahertz spectroscopy is important in the study of biomolecular structure because the vibration and rotation energy of large molecules such as DNA, proteins, and polysaccharides are laid in terahertz regions. Terahertz time-domain spectroscopy (THz-TDS), using terahertz pulses generated and detected by femto-second pulses laser, has been used in the study of biomolecular dynamics, as well as carrier dynamics of semiconductors. Laminarin is a polysaccharide of glucose in brown algae. It is made up of β(1-3)-glucan and β(1-6)-glucan. β-glucan is an anticancer material that activates the immune reaction of human cells and inhibits proliferation of cancer cells. β-glucan with a single-strand structure has been reported to activate the immune reaction to a greater extent than β-glucan with a triple-strand helix structure. We used THz-TDS to characterize the difference between single-strand and triple-strand β-glucan. We obtained single-strand β-glucan by chemical treatment of triple-strand β-glucan. We measured the frequency dependent optical constants of Laminarin using THz-TDS. Power absorption of the triple-strand helix is larger than the single-strand helix in terahertz regions. The refractive index of the triple-strand helix is also larger than that of the single-strand helix.

Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

PubMed Central

Sun, Fei; Huang, Li

2013-01-01

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation. PMID:23821667

Size-resolved Chemical Composition of Cloud and Rain Water Collected during the Puerto Rico African Dust and Clouds Study (PRADACS) Campaign

NASA Astrophysics Data System (ADS)

Torres, E.; Valle Diaz, C. J.; Zurcher, F.; Lee, T.; Collett, J. L.; Fitzgerald, E.; Cuadra, L.; Prather, K. A.; Mayol-Bracero, O. L.

2011-12-01

The underlying physico-chemical processes of dust-aerosol interactions are poorly understood; even less understood is how aging impacts cloud properties and climate as the particles travel from Africa to the Caribbean region. Caribbean landmasses have tropical montane cloud forests (TMCFs) that are tightly coupled to the atmospheric hydrologic cycle. Small-scale shifts in temperature and precipitation could have serious ecological consequences. Therefore, this makes TMCFs an interesting ecosystem to see the effects African Dust (AD) might have on cloud formation and precipitation. As part of the Puerto Rico African Dust and Clouds Study (PRADACS) cloud and rain water samples for subsequent chemical analysis were collected at Pico del Este (PE) station in Luquillo, PR (1051 masl) during summer 2011. At PE, two cloud collectors (i.e., single stage (Aluminum version) and 2-stage (Teflon version) Caltech Active Strand Cloudwater Collector (CASCC)), and a rainwater collector were operated. Measurements such as the liquid water content (LWC), pH, conductivity., and composition of single particles using an aerosol time of flight mass spectrometer (ATOFMS) were performed. Preliminary results showed that days with the influence of African dust (AD), had LWC values that ranged from 300 to 500 mg/m3, pH values up to 5.7,, and conductivity up to 180 μS/cm. The ATOFMS showed titanium and iron ions, suggesting the presence of AD as well as, occasionally, sulfate and nitrate ions suggesting the influence of anthropogenic pollution. Results on the chemical composition and the physical properties of cloud, rainwater, and aerosol for the inorganic as well as the organic fraction and how these properties change for the different air masses observed will also be presented.

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans.

PubMed

Steiner, Florian A; Okihara, Kristy L; Hoogstrate, Suzanne W; Sijen, Titia; Ketting, René F

2009-02-01

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.

Classifying Saturn's F Ring Strands

NASA Astrophysics Data System (ADS)

Albers, Nicole; Sremcevic, M.; Esposito, L. W.; Colwell, J. E.

2009-09-01

The Cassini Ultraviolet Imaging Spectrograph (UVIS) High Speed Photometer (HSP) has recorded more than 113 stellar occultations by Saturn's F ring providing measurements with ring plane resolutions of a few dozen meters and better. Inner and outer F ring strands have been seen throughout the Cassini mission where they revealed themselves as non-continuous, azimuthally and temporally highly variable structures. In the light of a more accurate orbit description of the F ring core we find evidence for a ring that becomes dynamically more active as the system approaches anti-apse alignment with Prometheus. This is consistent with the observed increased strand activity. A recent strand that morphologically resembles the core is the strongest seen to date and points to the intricate relation between core and strands indicating the strands' violent creation. Using more than 150 identifications of various strands, we trace their kinematics and infer dynamical timescales and photometric properties. Implications for the dynamical evolution of the F ring will be discussed. This research was supported by the Cassini Project.

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

PubMed Central

Datta, Simanti; Costantino, Nina; Zhou, Xiaomei; Court, Donald L.

2008-01-01

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria. PMID:18230724

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

PubMed Central

Huang, Chu-Chun; Grubb, Jennifer; Thacker, Drew; Lee, Chih-Ying; Dresser, Michael E.; Hunter, Neil; Bishop, Douglas K.

2013-01-01

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1's strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51's strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1's chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1's dominance in promoting strand exchange between homologs involves repression of Rad51's strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination. PMID:24367271

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

PubMed Central

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

PubMed

Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

2015-01-01

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

miRNA*: a passenger stranded in RNA-induced silencing complex?

PubMed

Mah, S M; Buske, C; Humphries, R K; Kuchenbauer, F

2010-01-01

Processing of the pre-microRNA (pre-miRNA) through Dicer1 generates a miRNA duplex, consisting of a miRNA and miRNA* strand (also termed guide strand and passenger strand, respectively). Despite the general consensus that miRNA*s have no regulatory activity, recent publications have provided evidence that the abundance, possible function, and physiological relevance of miRNA*s have been underestimated. This review provides an account of our current understanding of miRNA* origination and activity, mounting evidence for their unique functions and regulatory mechanisms, and examples of specific miRNA*s from the literature.

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair

PubMed Central

Pluciennik, Anna; Dzantiev, Leonid; Iyer, Ravi R.; Constantin, Nicoleta; Kadyrov, Farid A.; Modrich, Paul

2010-01-01

MutLα (MLH1–PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion. PMID:20713735

Transferring Technology to Industry

NASA Technical Reports Server (NTRS)

Wolfenbarger, J. Ken

2006-01-01

This slide presentation reviews the technology transfer processes in which JPL has been involved to assist in transferring the technology derived from aerospace research and development to industry. California Institute of Technology (CalTech), the organization that runs JPL, is the leading institute in patents for all U.S. universities. There are several mechanisms that are available to JPL to inform industry of these technological advances: (1) a dedicated organization at JPL, National Space Technology Applications (NSTA), (2) Tech Brief Magazine, (3) Spinoff magazine, and (4) JPL publications. There have also been many start-up organizations and businesses from CalTech.

Bioconversion study conducted by JPL

NASA Technical Reports Server (NTRS)

Kalvinskas, J.

1978-01-01

The Jet Propulsion Laboratory (JPL) of Caltech conducted a study of bioconversion as a means of identifying the role of biomass for meeting the national energy fuel and chemical requirements and the role and means for JPL-Caltech involvement in bioconversion. The bioconversion study included the following categories; biomass sources, chemicals from biomass, thermochemical conversion of biomass to fuels, biological conversion of biomass to fuels and chemicals, and basic bioconversion sciences. A detailed review is included of the bioconversion fields cited with specific conclusions and recommendations given for future research and development and overall biomass system engineering and economic studies.

Initial results from the Caltech/DRSI balloon-borne isotope experiment

NASA Technical Reports Server (NTRS)

Schindler, S. M.; Buffington, A.; Christian, E. C.; Grove, J. E.; Lau, K. H.; Stone, E. C.; Rasmussen, I. L.; Laursen, S.

1985-01-01

The Caltech/DSRI balloonborne High Energy Isotope Spectrometer Telescope (HEIST) was flown successfully from Palestine, Texas on 14 May, 1984. The experiment was designed to measure cosmic ray isotopic abundances from neon through iron, with incident particle energies from approx. 1.5 to 2.2 GeV/nucleon depending on the element. During approximately 38 hours at float altitude, 100,000 events were recorded with Z or = 6 and incident energies approx. 1.5 GeV/nucleon. We present results from the ongoing data analysis associated with both the preflight Bevalac calibration and the flight data.

Initial results from the Caltech/DSRI balloon-borne isotope experiment

NASA Technical Reports Server (NTRS)

Schindler, S. M.; Buffington, A.; Christian, E. C.; Grove, J. E.; Lau, K. H.; Stone, E. C.; Rasmussen, I. L.; Laursen, S.

1985-01-01

The Caltech/DSRI balloon-borne High Energy Isotope Spectrometer Telescope (HEIST) was flown successfully from Palestine, Texas on 14 May 1984. The experiment was designed to measure cosmic ray isotopic abundances from neon through iron, with incident particle energies from approximately 1.5 to 2.2 GeV/nucleon, depending on the element. During approximately 38 hours at float altitude, 10 to the 5th events were recorded with Z or = 6 and incident energies 1.5 GeV/nucleon. We present results from the ongoing data analysis associated with both the pre-flight Bevalac calibration and the flight data.

Book Review - Panofsky on Physics, Politics, and Peace: Pief Remembers

NASA Astrophysics Data System (ADS)

Loew, Gregory

The following sections are included: * Introduction: Genesis of the Book * Nature and Nurture: Pief's Early Life * This Review * High School in Hamburg; University at Princeton and Caltech * Pief and the Bomb * Accelerators and Physics at UCRL * Events Leading up to the Loyalty Oath * Stanford, the Microwave Lab and HEPL * The Rise of SLAC * Building SLAC * Physics Research at SLAC in the First Ten Years * Other Accelerator Activities under Pief * Science Advising and International Science * Arms Control (1981-2007): The Unfinished Business

The Antiaircraft Journal. Volume 92, Number 6, November-December 1949

DTIC Science & Technology

1949-12-01

nation’s peacetime history . Strategic plans for the nation’s defense are far advanced beyond those of any previous peacetime year . The Ameri- can people...From April through October of that year , activities of the OSRD Group such as Caltech were largely absorbed by the permanent teChnical organization of...with only four years ’ service is los - ing as many credits as a nonveteran might earn in twenty years of reserve component service. Title III, Public

A Smart DNA Tweezer for Detection of Human Telomerase Activity.

PubMed

Xu, Xiaowen; Wang, Lei; Li, Kan; Huang, Qihong; Jiang, Wei

2018-03-06

Reliable and accurate detection of telomerase activity is crucial to better understand its role in cancer cells and to further explore its function in cancer diagnosis and treatment. Here, we construct a smart DNA tweezer (DT) for detection of telomerase activity. The DT is assembled by three specially designed single-stranded oligonucleotides: a central strand dually labeled with donor/acceptor fluorophores and two arm strands containing overhangs complementary to telomerase reaction products (TRPs). It can get closed through hybridization with TRPs and get reopen through strand displacement reaction by TRPs' complementary sequences. First, under the action of telomerase, telomerase binding substrates (TS) are elongated to generate TRPs ended with telomeric repeats (TTAGGG) n . TRPs hybridize with the two arm overhangs cooperatively and strain DT to closed state, inducing an increased fluorescence resonance energy transfer (FRET) efficiency, which is utilized for telomerase activity detection. Second, upon introduction of a removal strand (RS) complementary to TRPs, the closed DT is relaxed to open state via the toehold-mediated strand displacement, inducing a decreased FRET efficiency, which is utilized for determination of TRP length distribution. The detection limit of telomerase activity is equivalent to 141 cells/μL for HeLa cells, and telomerase-active cellular extracts can be differentiated from telomerase-inactive cellular extracts. Furthermore, TRPs owning 1, 2, 3, 4, and ≥5 telomeric repeats are identified to account for 25.6%, 20.5%, 15.7%, 12.5%, and 25.7%, respectively. The proposed strategy will offer a new approach for reliable, accurate detection of telomerase activity and product length distribution for deeper studying its role and function in cancer.

Structure of C3PO and Mechanism of Human RISC Activation

PubMed Central

Ye, Xuecheng; Huang, Nian; Liu, Ying; Paroo, Zain; Huerta, Carlos; Li, Peng; Chen, She; Liu, Qinghua; Zhang, Hong

2011-01-01

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide/passenger) siRNA onto and removing the passenger strand from Argonaute (Ago2). Ago2 contributes critically to RISC activation by nicking the passenger strand. Here, we reconstituted duplex siRNA-initiated RISC activity using recombinant human (h)Ago2 and C3PO, indicating a critical role for C3PO in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six Translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as a novel endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation: 1) Ago2 directly binds duplex siRNA and nicks the passenger strand; 2) C3PO activates RISC by degrading Ago2-nicked passenger strand. PMID:21552258

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication.

PubMed

Herzig, Eytan; Voronin, Nickolay; Kucherenko, Nataly; Hizi, Amnon

2015-08-01

The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. Reverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

DOE PAGES

Su, Fengtao; Mukherjee, Shibani; Yang, Yanyong; ...

2014-11-20

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRNmore » to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.« less

Two, Three, Many Strands of Activity Theory!

ERIC Educational Resources Information Center

Levant, Alex

2018-01-01

This piece critically follows David Bakhurst's 2009 article "Reflections on Activity Theory" in light of recent developments in the field. It sketches three directions for further research. First, it examines his identification of "two strands" of activity theory (AT)--the philosophical, which he associates with E.V. Ilyenkov…

Role of CREB in CML

DTIC Science & Technology

2007-02-01

antisense RNA for suppressing gene expression in nematode worms (Caenorhabditis elegans) 2. This was followed by the introduction of dsRNA into worms...When single-stranded antisense RNA and double stranded RNA was introduced into worms, they found that dsRNA was more effective than either strand...RISC ( RNA -induced silencing complex), which contains helicase activity that unwinds the two strands 3 of RNA molecules, allowing the antisense

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Venema, J.; van Hoffen, A.; Karcagi, V.

1991-08-01

The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5{prime} part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimersmore » removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3{prime} part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.« less

A portable FTS measurements during GOSAT and OCO-2 joint campaign in Western US 2017

NASA Astrophysics Data System (ADS)

Shiomi, K.; Kikuchi, N.; Kuze, A.; Hashimoto, M.; Suto, H.; Kataoka, F.; Kasai, K.; Arai, T.; Oshio, H.; Ohyama, H.; Morino, I.; Hori, A.; Hedelius, J.; Parker, H. A.; Roehl, C. M.; Wennberg, P. O.; Leifer, I.; Iraci, L. T.; Bruegge, C. J.; Schwandner, F. M.; Osterman, G. B.; Crisp, D.

2017-12-01

The column-average dry air mole fractions of carbon dioxide (XCO2), methane (XCH4) and carbon monoxide (XCO) were measured from the surface using direct sunlight at near-IR wavelengths. Simultaneous detection of CO is helpful to characterize CO2 source type. We measured XCO along with XCO2 and XCH4 using an upgraded portable Fourier transform spectrometer (FTS), EM27/SUN, in western US field experiments at 1) Caltech and JPL, in Pasadena, a northern Los Angeles suburb, 2) Railroad Valley (RRV), a desert playa in Nevada, and 3) Salton Sea, a terminal high-salinity lake in southern California's Imperial Valley. These measurements were conducted during the GOSAT/OCO-2 joint campaign in the early summer of 2017. Before and after US shipments, the JAXA EM27 was compared with the NIES EM27 and Tsukuba TCCON station to quantify the measurement bias and to identify any unanticipated changes. After US shipment, the JAXA EM27 was compared with the Caltech EM27 and the Caltech TCCON station with the NASA-Dryden TCCON station, which is now located at JPL for OCO-3 pre-flight test comparisons. At RRV, we obtained not only the EM27 data but also atmospheric profile of pressure, temperature and humidity by radiosonde and aerosol optical thickness (AOT) by Microtops. Then, we deployed the EM27 on the south side of the Salton Sea, where agriculture, active volcanism and geothermal power plants might produce CO2 and CH4 as sources. A large CO enhancement originating from wildfires was serendipitously measured in conjunction with a large AOT measured by the Microtops. The JAXA EM27 measurements at Pasadena, RRV and Salton Sea were taken in conjunction with GOSAT and OCO-2 overpass targets and are being used to assess the value of the EM27 as a portable transfer standard for validating space based measurements of greenhouse gases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Xuecheng; Huang, Nian; Liu, Ying

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide-passenger) siRNA onto Argonaute (Ago2) and removing the passenger strand. Ago2 contributes critically to RISC activation by nicking the passenger strand. Here we reconstituted duplex siRNA-initiated RISC activity using recombinant human Ago2 (hAgo2) and C3PO, indicating that C3PO has a critical role in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as anmore » endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation, in which Ago2 directly binds duplex siRNA and nicks the passenger strand, and then C3PO activates RISC by degrading the Ago2-nicked passenger strand.« less

Nucleoside Triphosphate Phosphohydrolase I (NPH I) Functions as a 5′ to 3′ Translocase in Transcription Termination of Vaccinia Early Genes*

PubMed Central

Hindman, Ryan; Gollnick, Paul

2016-01-01

Vaccinia virus early genes are transcribed immediately upon infection. Nucleoside triphosphate phosphohydrolase I (NPH I) is an essential component of the early gene transcription complex. NPH I hydrolyzes ATP to release transcripts during transcription termination. The ATPase activity of NPH I requires single-stranded (ss) DNA as a cofactor; however, the source of this cofactor within the transcription complex is not known. Based on available structures of transcription complexes it has been hypothesized that the ssDNA cofactor is obtained from the unpaired non-template strand within the transcription bubble. In vitro transcription on templates that lack portions of the non-template strand within the transcription bubble showed that the upstream portion of the transcription bubble is required for efficient NPH I-mediated transcript release. Complementarity between the template and non-template strands in this region is also required for NPH I-mediated transcript release. This observation complicates locating the source of the ssDNA cofactor within the transcription complex because removal of the non-template strand also disrupts transcription bubble reannealing. Prior studies have shown that ssRNA binds to NPH I, but it does not activate ATPase activity. Chimeric transcription templates with RNA in the non-template strand confirm that the source of the ssDNA cofactor for NPH I is the upstream portion of the non-template strand in the transcription bubble. Consistent with this conclusion we also show that isolated NPH I acts as a 5′ to 3′ translocase on single-stranded DNA. PMID:27189950

TALE activators regulate gene expression in a position- and strand-dependent manner in mammalian cells.

PubMed

Uhde-Stone, Claudia; Cheung, Edna; Lu, Biao

2014-01-24

Transcription activator-like effectors (TALEs) are a class of transcription factors that are readily programmable to regulate gene expression. Despite their growing popularity, little is known about binding site parameters that influence TALE-mediated gene activation in mammalian cells. We demonstrate that TALE activators modulate gene expression in mammalian cells in a position- and strand-dependent manner. To study the effects of binding site location, we engineered TALEs customized to recognize specific DNA sequences located in either the promoter or the transcribed region of reporter genes. We found that TALE activators robustly activated reporter genes when their binding sites were located within the promoter region. In contrast, TALE activators inhibited the expression of reporter genes when their binding sites were located on the sense strand of the transcribed region. Notably, this repression was independent of the effector domain utilized, suggesting a simple blockage mechanism. We conclude that TALE activators in mammalian cells regulate genes in a position- and strand-dependent manner that is substantially different from gene activation by native TALEs in plants. These findings have implications for optimizing the design of custom TALEs for genetic manipulation in mammalian cells. Copyright © 2013 Elsevier Inc. All rights reserved.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

PubMed

Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

2015-10-16

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

A cognitive operating system (COGNOSYS) for JPL's robot, phase 1 report

NASA Technical Reports Server (NTRS)

Mathur, F. P.

1972-01-01

The most important software requirement for any robot development is the COGNitive Operating SYStem (COGNOSYS). This report describes the Stanford University Artificial Intelligence Laboratory's hand eye software system from the point of view of developing a cognitive operating system for JPL's robot. In this, the Phase 1 of the JPL robot COGNOSYS task the installation of a SAIL compiler and a FAIL assembler on Caltech's PDP-10 have been accomplished and guidelines have been prepared for the implementation of a Stanford University type hand eye software system on JPL-Caltech's computing facility. The alternatives offered by using RAND-USC's PDP-10 Tenex operating sytem are also considered.

Data transfer nodes and demonstration of 100-400 Gbps wide area throughput using the Caltech SDN testbed

NASA Astrophysics Data System (ADS)

Mughal, A.; Newman, H.

2017-10-01

We review and demonstrate the design of efficient data transfer nodes (DTNs), from the perspective of the highest throughput over both local and wide area networks, as well as the highest performance per unit cost. A careful system-level design is required for the hardware, firmware, OS and software components. Furthermore, additional tuning of these components, and the identification and elimination of any remaining bottlenecks is needed once the system is assembled and commissioned, in order to obtain optimal performance. For high throughput data transfers, specialized software is used to overcome the traditional limits in performance caused by the OS, file system, file structures used, etc. Concretely, we will discuss and present the latest results using Fast Data Transfer (FDT), developed by Caltech. We present and discuss the design choices for three generations of Caltech DTNs. Their transfer capabilities range from 40 Gbps to 400 Gbps. Disk throughput is still the biggest challenge in the current generation of available hardware. However, new NVME drives combined with RDMA and a new NVME network fabric are expected to improve the overall data-transfer throughput and simultaneously reduce the CPU load on the end nodes.

Statistical analysis of the Nb3Sn strand production for the ITER toroidal field coils

NASA Astrophysics Data System (ADS)

Vostner, A.; Jewell, M.; Pong, I.; Sullivan, N.; Devred, A.; Bessette, D.; Bevillard, G.; Mitchell, N.; Romano, G.; Zhou, C.

2017-04-01

The ITER toroidal field (TF) strand procurement initiated the largest Nb3Sn superconducting strand production hitherto. The industrial-scale production started in Japan in 2008 and finished in summer 2015. Six ITER partners (so-called Domestic Agencies, or DAs) are in charge of the procurement and involved eight different strand suppliers all over the world, of which four are using the bronze route (BR) process and four the internal-tin (IT) process. In total more than 500 tons have been produced including excess material covering losses during the conductor manufacturing process, in particular the cabling. The procurement is based on a functional specification where the main strand requirements like critical current, hysteresis losses, Cu ratio and residual resistance ratio are specified but not the strand production process or layout. This paper presents the analysis on the data acquired during the quality control (QC) process that was carried out to ensure the same conductor performance requirements are met by the different strand suppliers regardless of strand design. The strand QC is based on 100% billet testing and on applying statistical process control (SPC) limits. Throughout the production, samples adjacent to the strand pieces tested by the suppliers are cross-checked (‘verified’) by their respective DAs reference labs. The level of verification was lowered from 100% at the beginning of the procurement progressively to approximately 25% during the final phase of production. Based on the complete dataset of the TF strand production, an analysis of the SPC limits of the critical strand parameters is made and the related process capability indices are calculated. In view of the large-scale production and costs, key manufacturing parameters such as billet yield, number of breakages and piece-length distribution are also discussed. The results are compared among all the strand suppliers, focusing on the difference between BR and IT processes. Following the completion of the largest Nb3Sn strand production, our experience gained from monitoring the execution of the QC activities and from auditing the results from the measurements is summarised for future superconducting strand material procurement activities.

75 FR 4537 - Notice of Public Hearings for the Draft Environmental Impact Statement for the Silver Strand...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-28

... training activities within the Navy's Silver Strand Training Complex (SSTC) and southern nearshore areas of... activities, introduction of new platforms and equipment for training, and increased access and availability... readiness using the SSTC to support and conduct current, emerging, and future training activities. A Notice...

Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

PubMed

Farb, Joshua N; Morrical, Scott W

2009-01-16

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

Yeast Pif1 Accelerates Annealing of Complementary DNA Strands

PubMed Central

2015-01-01

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg2+. Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3′-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1. PMID:25393406

Yeast Pif1 accelerates annealing of complementary DNA strands.

PubMed

Ramanagoudr-Bhojappa, Ramanagouda; Byrd, Alicia K; Dahl, Christopher; Raney, Kevin D

2014-12-09

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg(2+). Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3'-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

Bypass of a psoralen DNA interstrand cross-link by DNA polymerases beta, iota, and kappa in vitro

PubMed Central

Smith, Leigh A.; Makarova, Alena V.; Samson, Laura; Thiesen, Katherine E.; Dhar, Alok; Bessho, Tadayoshi

2012-01-01

Repair of DNA inter-strand cross-links in mammalian cells involves several biochemically distinctive processes, including the release of one of the cross-linked strands and translesion DNA synthesis (TLS). In this report, we investigated in vitro TLS activity of psoralen DNA inter-strand cross-link by three DNA repair polymerases, DNA polymerase beta, kappa and iota. DNA polymerase beta is capable of bypassing a psoralen cross-link with a low efficiency. Cell extracts prepared from DNA polymerase beta knockout mouse embryonic fibroblast showed a reduced bypass activity of the psoralen cross-link and purified DNA polymerase beta restored the bypass activity. In addition, DNA polymerase iota mis-incorporated thymine across the psoralen cross-link and DNA polymerase kappa extended these mis-paired primer ends, suggesting that DNA polymerase iota may serve as an inserter and DNA polymerase kappa may play a role as an extender in the repair of psoralen DNA inter-strand cross-links. The results demonstrated here indicate that multiple DNA polymerases could participate in TLS steps in mammalian DNA inter-strand cross-link repair. PMID:23106263

Cryptic MCAT enhancer regulation in fibroblasts and smooth muscle cells. Suppression of TEF-1 mediated activation by the single-stranded DNA-binding proteins, Pur alpha, Pur beta, and MSY1.

PubMed

Carlini, Leslie E; Getz, Michael J; Strauch, Arthur R; Kelm, Robert J

2002-03-08

An asymmetric polypurine-polypyrimidine cis-element located in the 5' region of the mouse vascular smooth muscle alpha-actin gene serves as a binding site for multiple proteins with specific affinity for either single- or double-stranded DNA. Here, we test the hypothesis that single-stranded DNA-binding proteins are responsible for preventing a cryptic MCAT enhancer centered within this element from cooperating with a nearby serum response factor-interacting CArG motif to trans-activate the minimal promoter in fibroblasts and smooth muscle cells. DNA binding studies revealed that the core MCAT sequence mediates binding of transcription enhancer factor-1 to the double-stranded polypurine-polypyrimidine element while flanking nucleotides account for interaction of Pur alpha and Pur beta with the purine-rich strand and MSY1 with the complementary pyrimidine-rich strand. Mutations that selectively impaired high affinity single-stranded DNA binding by fibroblast or smooth muscle cell-derived Pur alpha, Pur beta, and MSY1 in vitro, released the cryptic MCAT enhancer from repression in transfected cells. Additional experiments indicated that Pur alpha, Pur beta, and MSY1 also interact specifically, albeit weakly, with double-stranded DNA and with transcription enhancer factor-1. These results are consistent with two plausible models of cryptic MCAT enhancer regulation by Pur alpha, Pur beta, and MSY1 involving either competitive single-stranded DNA binding or masking of MCAT-bound transcription enhancer factor-1.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

PubMed

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

2012-06-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Dynamics of Leading-strand Lesion Skipping by the Replisome

PubMed Central

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

SCDC Spanish Curricula Units. Language Arts, Unit 6, Grade 2, Teacher's Guide.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Language arts instructional and assessment activities for grade two are described in this teacher's guide to the language arts strand of unit six. Focus, objective and materials for each activity are given in English and Spanish, with teacher instructions only in Spanish. This strand includes a continuation of the basic activities of past units:…

SCDC Spanish Curricula Units. Science/Math Strand, Unit 7, Grade 3, Teacher's Guide--Multi-Ethnic Edition.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Instructional and assessment activities for unit seven of a science/math strand for Spanish-speaking students in grade three focus on the extended community. Activities support four spiraling questions on the elements, wants and needs, change occurrence, and results of change. For each activity, the focus, objective, and materials needed are…

5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

PubMed Central

Delagoutte, Emmanuelle; Baldacci, Giuseppe

2011-01-01

Instability of repetitive sequences originates from strand misalignment during repair or replicative DNA synthesis. To investigate the activity of reconstituted T4 replisomes across trinucleotide repeats (TNRs) during leading strand DNA synthesis, we developed a method to build replication miniforks containing a TNR unit of defined sequence and length. Each minifork consists of three strands, primer, leading strand template, and lagging strand template with a 5′ single-stranded (ss) tail. Each strand is prepared independently, and the minifork is assembled by hybridization of the three strands. Using these miniforks and a minimal reconstituted T4 replisome, we show that during leading strand DNA synthesis, the dNTP concentration dictates which strand of the structure-forming 5′CAG/5′CTG repeat creates the strongest impediment to the minimal replication complex. We discuss this result in the light of the known fluctuation of dNTP concentration during the cell cycle and cell growth and the known concentration balance among individual dNTPs. PMID:22096622

Investigations into Gravitational Wave Emission from Compact Body Inspiral into Massive Black Holes

NASA Technical Reports Server (NTRS)

Hughes, Scott A.

2005-01-01

In contrast to year 1 (when much of the activity associated with this grant focused upon developing our group at MIT), year 2 was a period of very focused attention on research problems. We made significant progress developing relativistic waveforms for the extreme mass ratio inspiral problem; we have pushed forward a formalism our group developed for mapping the spacetimes of massive compact objects; and, in collaboration with the Caltech group, we began to develop a framework for addressing issues in LISA data analysis for extreme mass ratio systems.

Ground-Based Calibration Support for Two Approved HST Programs

NASA Technical Reports Server (NTRS)

Stauffer, John R.

1998-01-01

This final report is a summary of the study on ground-based calibration support for two approved HST programs. A large set of new rotational periods for low mass stars in the Pleiades open cluster have been published and used to help interpret chromospheric and coronal activity indicators for low mass stars in the cluster. The Caltech/TJC/NASA Keck telescope in Hawaii has also been used to obtain spectra of brown dwarf candidates in the Pleiades. Those spectra help to derive an accurate and precise new age for that fiducial open cluster.

A critical role for topoisomerase IIb and DNA double strand breaks in transcription

PubMed Central

Calderwood, Stuart K.

2016-01-01

ABSTRACT Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb. PMID:27100743

A critical role for topoisomerase IIb and DNA double strand breaks in transcription.

PubMed

Calderwood, Stuart K

2016-05-26

Recent studies have indicated a novel role for topoisomerase IIb in transcription. Transcription of heat shock genes, serum-induced immediate early genes and nuclear receptor-activated genes, each required DNA double strands generated by topoisomerase IIb. Such strand breaks seemed both necessary and sufficient for transcriptional activation. In addition, such transcription was associated with initiation of the DNA damage response pathways, including the activation of the enzymes: ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase and poly (ADP ribose) polymerase 1. DNA damage response signaling was involved both in transcription and in repair of DNA breaks generated by topoisomerase IIb.

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Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.

PubMed

Gardner, Andrew F; Prangishvili, David; Jack, William E

2011-09-01

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Modelling toehold-mediated RNA strand displacement.

PubMed

Šulc, Petr; Ouldridge, Thomas E; Romano, Flavio; Doye, Jonathan P K; Louis, Ard A

2015-03-10

We study the thermodynamics and kinetics of an RNA toehold-mediated strand displacement reaction with a recently developed coarse-grained model of RNA. Strand displacement, during which a single strand displaces a different strand previously bound to a complementary substrate strand, is an essential mechanism in active nucleic acid nanotechnology and has also been hypothesized to occur in vivo. We study the rate of displacement reactions as a function of the length of the toehold and temperature and make two experimentally testable predictions: that the displacement is faster if the toehold is placed at the 5' end of the substrate; and that the displacement slows down with increasing temperature for longer toeholds. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Development of Scanning Ultrafast Electron Microscope Capability.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, Kimberlee Chiyoko; Talin, Albert Alec; Chandler, David W.

Modern semiconductor devices rely on the transport of minority charge carriers. Direct examination of minority carrier lifetimes in real devices with nanometer-scale features requires a measurement method with simultaneously high spatial and temporal resolutions. Achieving nanometer spatial resolutions at sub-nanosecond temporal resolution is possible with pump-probe methods that utilize electrons as probes. Recently, a stroboscopic scanning electron microscope was developed at Caltech, and used to study carrier transport across a Si p-n junction [ 1 , 2 , 3 ] . In this report, we detail our development of a prototype scanning ultrafast electron microscope system at Sandia National Laboratoriesmore » based on the original Caltech design. This effort represents Sandia's first exploration into ultrafast electron microscopy.« less

Shifting Plasma

NASA Image and Video Library

2017-05-09

Strands of plasma at the sun edge shifted and twisted back and forth over a 22-hour period, May 2-3, 2017. In this close-up from NASA Solar Dynamics Observatory, the strands are being manipulated by strong magnetic forces associated with active region. This kind of activity is not at all uncommon, but best viewed in profile. The images were taken in a wavelength of extreme ultraviolet light. To give a sense of scale, the strands hover above the sun more than several times the size of Earth. Movies are available at https://photojournal.jpl.nasa.gov/catalog/PIA21632

Development And Application Of The Ion Microprobe For Analysis Of Extraterrestrial Materials

NASA Technical Reports Server (NTRS)

Wasserburg, G. J.

2001-01-01

This report covers the work carried out under NASA Grant NAG5-4083. The research was directed toward analyses of early solar system material, of presolar grains preserved in meteorites, and toward theoretical studies of nucleosynthesis in stars related to the chemical evolution of the galaxy and the formation of the solar system. The work was carried out over the time period 15 February 1998 - 31 May 2001 and involved the participation of the following individuals: M. Busso, Visiting Associate, Professor of Astrophysics, Perugia University, Italy; B.-G. Choi, research fellow, now Associate Professor at Seoul National University, Korea; H. C. Connolly, research fellow, now at Kingsborough Community College, CUNY; R. Gallino, Visiting Associate, Professor of Astrophysics, University of Torino; Y. Guan, Smithsonian Institution; C. Hohenberg, Professor of Physics, Washington University, St. Louis; M. Heinrich, electronics and systems engineer, Caltech; W. Hsu, research fellow, Caltech; T. LaTourrette, research fellow, now at Rand Corporation; G. R. Huss, Senior Research Scientist, now at Arizona State University; N. Krestina, research fellow in geochemistry, Caltech; G. J. MacPherson, Smithsonian Institution; K. Nollett, research fellow in astrophysics; Y.-Z. Qian, Professor of Physics, University of Minnesota; G. Srinivasan, research fellow, now Research Scientist, Physical Research Laboratory, Ahmedabad, India.

HTTP as a Data Access Protocol: Trials with XrootD in CMS’s AAA Project

NASA Astrophysics Data System (ADS)

Balcas, J.; Bockelman, B. P.; Kcira, D.; Newman, H.; Vlimant, J.; Hendricks, T. W.; CMS Collaboration

2017-10-01

The main goal of the project to demonstrate the ability of using HTTP data federations in a manner analogous to the existing AAA infrastructure of the CMS experiment. An initial testbed at Caltech has been built and changes in the CMS software (CMSSW) are being implemented in order to improve HTTP support. The testbed consists of a set of machines at the Caltech Tier2 that improve the support infrastructure for data federations at CMS. As a first step, we are building systems that produce and ingest network data transfers up to 80 Gbps. In collaboration with AAA, HTTP support is enabled at the US redirector and the Caltech testbed. A plugin for CMSSW is being developed for HTTP access based on the DaviX software. It will replace the present fork/exec or curl for HTTP access. In addition, extensions to the XRootD HTTP implementation are being developed to add functionality to it, such as client-based monitoring identifiers. In the future, patches will be developed to better integrate HTTP-over-XRootD with the Open Science Grid (OSG) distribution. First results of the transfer tests using HTTP are presented in this paper together with details about the initial setup.

Early Days of SIS Receivers

NASA Astrophysics Data System (ADS)

Woody, D. P.

2009-12-01

The modern era of millimeter and submillimeter spectral line observations and interferometry started at end of the 1979 with the invention of the Superconductor-Insulator-Superconductor (SIS) mixer. Tom Phillips co-invented this device while working at Bell Telephone Labs (BTL) in Murray Hill, NJ. His group built the first astronomically useful SIS heterodyne receiver which was deployed on the Leighton 10.4 m telescope at the Caltech Owens Valley Radio Observatory (OVRO) in the same year. Tom Phillips joined the Caltech faculty in the early 1980s where his group continues to lead the way in developing state-of-the-art SIS receivers throughout the millimeter and submillimeter wavelength bands. The rapid progress in millimeter and submillimeter astronomy during 1980s required developments on many fronts including the theoretical understanding of the device physics, advances in device fabrication, microwave and radio frequency (RF) circuit design, mixer block construction, development of wideband low-noise intermediate frequency (IF) amplifiers and the telescopes used for making the observations. Many groups around the world made important contributions to this field but the groups at Caltech and the Jet Propulsion Laboratory (JPL) under the leadership of Tom Phillips made major contributions in all of these areas. The end-to-end understanding and developments from the theoretical device physics to the astronomical observations and interpretation has made this group uniquely productive.

Replicase activity of purified recombinant protein P2 of double-stranded RNA bacteriophage phi6.

PubMed

Makeyev, E V; Bamford, D H

2000-01-04

In nature, synthesis of both minus- and plus-sense RNA strands of all the known double-stranded RNA viruses occurs in the interior of a large protein assembly referred to as the polymerase complex. In addition to other proteins, the complex contains a putative polymerase possessing characteristic sequence motifs. However, none of the previous studies has shown template-dependent RNA synthesis directly with an isolated putative polymerase protein. In this report, recombinant protein P2 of double-stranded RNA bacteriophage phi6 was purified and demonstrated in an in vitro enzymatic assay to act as the replicase. The enzyme efficiently utilizes phage-specific, positive-sense RNA substrates to produce double-stranded RNA molecules, which are formed by newly synthesized, full-length minus-strands base paired with the plus-strand templates. P2-catalyzed replication is also shown to be very effective with a broad range of heterologous single-stranded RNA templates. The importance and implications of these results are discussed.

Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks

NASA Astrophysics Data System (ADS)

Singleton, Martin R.; Dillingham, Mark S.; Gaudier, Martin; Kowalczykowski, Stephen C.; Wigley, Dale B.

2004-11-01

RecBCD is a multi-functional enzyme complex that processes DNA ends resulting from a double-strand break. RecBCD is a bipolar helicase that splits the duplex into its component strands and digests them until encountering a recombinational hotspot (Chi site). The nuclease activity is then attenuated and RecBCD loads RecA onto the 3' tail of the DNA. Here we present the crystal structure of RecBCD bound to a DNA substrate. In this initiation complex, the DNA duplex has been split across the RecC subunit to create a fork with the separated strands each heading towards different helicase motor subunits. The strands pass along tunnels within the complex, both emerging adjacent to the nuclease domain of RecB. Passage of the 3' tail through one of these tunnels provides a mechanism for the recognition of a Chi sequence by RecC within the context of double-stranded DNA. Gating of this tunnel suggests how nuclease activity might be regulated.

Deletions at short direct repeats and base substitutions are characteristic mutations for bleomycin-induced double- and single-strand breaks, respectively, in a human shuttle vector system

NASA Technical Reports Server (NTRS)

Dar, M. E.; Jorgensen, T. J.

1995-01-01

Using the radiomimetic drug, bleomycin, we have determined the mutagenic potential of DNA strand breaks in the shuttle vector pZ189 in human fibroblasts. The bleomycin treatment conditions used produce strand breaks with 3'-phosphoglycolate termini as > 95% of the detectable dose-dependent lesions. Breaks with this end group represent 50% of the strand break damage produced by ionizing radiation. We report that such strand breaks are mutagenic lesions. The type of mutation produced is largely determined by the type of strand break on the plasmid (i.e. single versus double). Mutagenesis studies with purified DNA forms showed that nicked plasmids (i.e. those containing single-strand breaks) predominantly produce base substitutions, the majority of which are multiples, which presumably originate from error-prone polymerase activity at strand break sites. In contrast, repair of linear plasmids (i.e. those containing double-strand breaks) mainly results in deletions at short direct repeat sequences, indicating the involvement of illegitimate recombination. The data characterize the nature of mutations produced by single- and double-strand breaks in human cells, and suggests that deletions at direct repeats may be a 'signature' mutation for the processing of DNA double-strand breaks.

SCDC Spanish Curricula Units. Science/Math Strand, Unit 5, Grade 2, Teacher's Guide--Multi-Ethnic Edition.

ERIC Educational Resources Information Center

Operations Research Society of America, Arlington, VA.

Continuing the theme of the school as a community, the teacher's guide to the science/math strand of unit five presents both instructional and assessment activities for kits 17-20. Focus, materials and objective for each activity are in Spanish and English; teacher instructions are in Spanish only. In kit 17 the science activities deal with the…

BIOMECHANICS AND HISTOLOGICAL ANALYSIS IN RABBIT FLEXOR TENDONS REPAIRED USING THREE SUTURE TECHNIQUES (FOUR AND SIX STRANDS) WITH EARLY ACTIVE MOBILIZATION

PubMed Central

Severo, Antônio Lourenço; Arenhart, Rodrigo; Silveira, Daniela; Ávila, Aluísio Otávio Vargas; Berral, Francisco José; Lemos, Marcelo Barreto; Piluski, Paulo César Faiad; Lech, Osvandré Luís Canfield; Fukushima, Walter Yoshinori

2015-01-01

Objective: Analyzing suture time, biomechanics (deformity between the stumps) and the histology of three groups of tendinous surgical repair: Brazil-2 (4-strands) which the end knot (core) is located outside the tendon, Indiana (4-strands) and Tsai (6-strands) with sutures technique which the end knot (core) is inner of the tendon, associated with early active mobilization. Methods: The right calcaneal tendons (plantar flexor of the hind paw) of 36 rabbits of the New Zealand breed (Oryctolagus cuniculus) were used in the analysis. This sample presents similar size to human flexor tendon that has approximately 4.5 mm (varying from 2mm). The selected sample showed the same mass (2.5 to 3kg) and were male or female adults (from 8 ½ months). For the flexor tendons of the hind paws, sterile and driven techniques were used in accordance to the Committee on Animal Research and Ethics (CETEA) of the University of the State of Santa Catarina (UDESC), municipality of Lages, in Brazil (protocol # 1.33.09). Results: In the biomechanical analysis (deformity) carried out between tendinous stumps, there was no statistically significant difference (p>0.01). There was no statistical difference in relation to surgical time in all three suture techniques with a mean of 6.0 minutes for Tsai (6- strands), 5.7 minutes for Indiana (4-strands) and 5.6 minutes for Brazil (4-strands) (p>0.01). With the early active mobility, there was qualitative and quantitative evidence of thickening of collagen in 38.9% on the 15th day and in 66.7% on the 30th day, making the biological tissue stronger and more resistant (p=0.095). Conclusion: This study demonstrated that there was no histological difference between the results achieved with an inside or outside end knot with respect to the repaired tendon and the number of strands did not affect healing, vascularization or sliding of the tendon in the osteofibrous tunnel, which are associated with early active mobility, with the repair techniques applied. PMID:27027087

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Clinical Results of Flexor Tendon Repair in Zone II Using a six Strand Double Loop Technique.

PubMed

Savvidou, Christiana; Tsai, Tsu-Min

2015-06-01

The purpose of this study is to report the clinical results after repair of flexor tendon zone II injuries utilizing a 6-strand double-loop technique and early post-operative active rehabilitation. We retrospectively reviewed 22 patients involving 51 cases with zone II flexor tendon repair using a six strand double loop technique from September 1996 to December 2012. Most common mechanism of injuries was sharp lacerations (86.5 %). Tendon injuries occurred equally in manual and non-manual workers and were work-related in 33 % of the cases. The Strickland score for active range of motion (ROM) postoperatively was excellent and good in the majority of the cases (81 %). The rupture rate was 1.9 %. The six strand double loop technique for Zone II flexor tendon repair leads to good and excellent motion in the majority of patients and low re- rupture rate. It is clinically effective and allows for early postoperative active rehabilitation.

Labeled nucleotide phosphate (NP) probes

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2009-02-03

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Composition for nucleic acid sequencing

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-08-26

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-06-06

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Method for sequencing nucleic acid molecules

DOEpatents

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

2006-05-30

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Influence of oxidized purine processing on strand directionality of mismatch repair.

PubMed

Repmann, Simone; Olivera-Harris, Maite; Jiricny, Josef

2015-04-17

Replicative DNA polymerases are high fidelity enzymes that misincorporate nucleotides into nascent DNA with a frequency lower than [1/10(5)], and this precision is improved to about [1/10(7)] by their proofreading activity. Because this fidelity is insufficient to replicate most genomes without error, nature evolved postreplicative mismatch repair (MMR), which improves the fidelity of DNA replication by up to 3 orders of magnitude through correcting biosynthetic errors that escaped proofreading. MMR must be able to recognize non-Watson-Crick base pairs and excise the misincorporated nucleotides from the nascent DNA strand, which carries by definition the erroneous genetic information. In eukaryotes, MMR is believed to be directed to the nascent strand by preexisting discontinuities such as gaps between Okazaki fragments in the lagging strand or breaks in the leading strand generated by the mismatch-activated endonuclease of the MutL homologs PMS1 in yeast and PMS2 in vertebrates. We recently demonstrated that the eukaryotic MMR machinery can make use also of strand breaks arising during excision of uracils or ribonucleotides from DNA. We now show that intermediates of MutY homolog-dependent excision of adenines mispaired with 8-oxoguanine (G(O)) also act as MMR initiation sites in extracts of human cells or Xenopus laevis eggs. Unexpectedly, G(O)/C pairs were not processed in these extracts and failed to affect MMR directionality, but extracts supplemented with exogenous 8-oxoguanine DNA glycosylase (OGG1) did so. Because OGG1-mediated excision of G(O) might misdirect MMR to the template strand, our findings suggest that OGG1 activity might be inhibited during MMR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Double-stranded RNA virus in the human pathogenic fungus Blastomyces dermatitidis.

PubMed Central

Kohno, S; Fujimura, T; Rulong, S; Kwon-Chung, K J

1994-01-01

Double-stranded RNA viruses were detected in a strain of Blastomyces dermatitidis isolated from a patient in Uganda. The viral particles are spherical (mostly 44 to 50 nm in diameter) and consist of about 25% double-stranded RNA (5 kb) and 75% protein (90 kDa). The virus contains transcriptional RNA polymerase activity; it synthesized single-stranded RNA in vitro in a conservative manner. The newly synthesized single-stranded RNA was a full-length strand, and the rate of chain elongation was approximately 170 nucleotides per min. The virus-containing strain shows no morphological difference from virus-free strains in the mycelial phase. Although the association with the presence of the virus is unclear, the virus-infected strain converts to the yeast form at 37 degrees C, but the yeast cells fail to multiply at that temperature. Images PMID:7933142

Quaternary Activity of the Erciyes Fault Southeast of the Kayseri Basin, Turkey

NASA Astrophysics Data System (ADS)

Okumura, K.; Hayakawa, Y. S.; Kontani, R.; Fikri, K.

2016-12-01

The Erciyes fault in SE of the Kayseri basin is one of the most active Quaternary faults in Central Anatolia. Emre et al. (2011) mapped about 100 km long faults including a section runs across the Erciyes volcano. A M 7+ earthquake from the fault would be a big threat for the 1.5 million people in Kayseri basin, but little has been know about its activity and earthquake potential. We studied Plio-Pleistocene volacanics, Quaternary sediments, and UAV-SfM topography in southeast of the Kayseri basin and recognized significant dip-slip separation as well as sinistral slip in Late Quaternary. The Incesu ignimbrite (IC) of 2.52±0.49 Ma (Aydar et al., 2012) is a very distinctive densely welded ignimbrite layer in and around Kayseri basin. The Plinian pumice fall deposits from the Erciyes in Late Pleistocene (Sen et al. 2003) at Gesi Bagpnar (GBP) is another key-bed. There are two strands and one group of faults. The NE strike frontal strand separates the basin floor and the upland in SW extending from Kayseri city to more than 50 km NE. The Gesi Guney strand runs parallel to the frontal strand at 3 to 4 km away from the basin floor for 20 km from Ali Dag. The NS trending fault group is observed both inside and outside of the basin under IC. These NS faults are swarm of normal Pliocene faults. The Gesi Guney strand offsets IC around 120 m vertically. There is no information to infer the initiation of its activity, but the normal offset of an alluvial fan and unconsolidated fresh talus deposits indicate Late Quaternary activities. Near the SW end of the frontal strand, IC is vertically offset around 40 m. 15 km NE from the SW end, sand and gravel layers that intercalates GBP (0.11-0.14 Ma) are tilted to NW for 30 to 40 m and truncated by a sub-vertical sinistral faults. Most of frontal strand deformation occurred in Late Pleistocene because the offset of IC and GBP are similar. Estimated slip-rate of 0.3 to 0.4 mm/yr is significant for Central Anatolia.

SCDC Spanish Curricula Units. Language Arts Strand, Unit 9, Grade 3, Teacher's Guide.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Instructional and assessment activities are presented in this teacher's guide to unit nine's language arts strand for third graders. Activities in reading, language analysis, and creative expression, both oral and written, are formulated in kits 33-36; they center around the basic theme of the nation as a community. Focus, objectives, and…

SCDC Spanish Curricula Units. Fine Arts, Unit 4, Grade 2, Supplements & Ditto Packet.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The supplement and ditto packet to the unit four fine arts strand for second graders contains visual materials to aid the teacher in carrying out the instructional and assessment activities of this strand. Seatwork for individual students is featured along with materials intended for class participation activities. Illustrations, provided to help…

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

PubMed Central

Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

2015-01-01

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks*

PubMed Central

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

2013-01-01

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome

PubMed Central

Lewis, Jacob S.; Spenkelink, Lisanne M.; Schauer, Grant D.; Hill, Flynn R.; Georgescu, Roxanna E.; O’Donnell, Michael E.; van Oijen, Antoine M.

2017-01-01

The replisome, the multiprotein system responsible for genome duplication, is a highly dynamic complex displaying a large number of different enzyme activities. Recently, the Saccharomyces cerevisiae minimal replication reaction has been successfully reconstituted in vitro. This provided an opportunity to uncover the enzymatic activities of many of the components in a eukaryotic system. Their dynamic behavior and interactions in the context of the replisome, however, remain unclear. We use a tethered-bead assay to provide real-time visualization of leading-strand synthesis by the S. cerevisiae replisome at the single-molecule level. The minimal reconstituted leading-strand replisome requires 24 proteins, forming the CMG helicase, the Pol ε DNA polymerase, the RFC clamp loader, the PCNA sliding clamp, and the RPA single-stranded DNA binding protein. We observe rates and product lengths similar to those obtained from ensemble biochemical experiments. At the single-molecule level, we probe the behavior of two components of the replication progression complex and characterize their interaction with active leading-strand replisomes. The Minichromosome maintenance protein 10 (Mcm10), an important player in CMG activation, increases the number of productive replication events in our assay. Furthermore, we show that the fork protection complex Mrc1–Tof1–Csm3 (MTC) enhances the rate of the leading-strand replisome threefold. The introduction of periods of fast replication by MTC leads to an average rate enhancement of a factor of 2, similar to observations in cellular studies. We observe that the MTC complex acts in a dynamic fashion with the moving replisome, leading to alternating phases of slow and fast replication. PMID:28923950

Offshore Earthquakes Do Not Influence Marine Mammal Stranding Risk on the Washington and Oregon Coasts.

PubMed

Grant, Rachel A; Savirina, Anna; Hoppitt, Will

2018-01-26

The causes of marine mammals stranding on coastal beaches are not well understood, but may relate to topography, currents, wind, water temperature, disease, toxic algal blooms, and anthropogenic activity. Offshore earthquakes are a source of intense sound and disturbance and could be a contributing factor to stranding probability. We tested the hypothesis that the probability of marine mammal stranding events on the coasts of Washington and Oregon, USA is increased by the occurrence of offshore earthquakes in the nearby Cascadia subduction zone. The analysis carried out here indicated that earthquakes are at most, a very minor predictor of either single, or large (six or more animals) stranding events, at least for the study period and location. We also tested whether earthquakes inhibit stranding and again, there was no link. Although we did not find a substantial association of earthquakes with strandings in this study, it is likely that there are many factors influencing stranding of marine mammals and a single cause is unlikely to be responsible. Analysis of a subset of data for which detailed descriptions were available showed that most live stranded animals were pups, calves, or juveniles, and in the case of dead stranded mammals, the commonest cause of death was trauma, disease, and emaciation.

Improved silencing properties using small internally segmented interfering RNAs

PubMed Central

Bramsen, Jesper B.; Laursen, Maria B.; Damgaard, Christian K.; Lena, Suzy W.; Ravindra Babu, B.; Wengel, Jesper; Kjems, Jørgen

2007-01-01

RNA interference is mediated by small interfering RNAs (siRNAs) that upon incorporation into the RNA-induced silencing complex (RISC) can target complementary mRNA for degradation. Standard siRNA design usually feature a 19–27 base pair contiguous double-stranded region that is believed to be important for RISC incorporation. Here, we describe a novel siRNA design composed of an intact antisense strand complemented with two shorter 10–12 nt sense strands. This three-stranded construct, termed small internally segmented interfering RNA (sisiRNA), is highly functional demonstrating that an intact sense strand is not a prerequisite for RNA interference. Moreover, when using the sisiRNA design only the antisense strand is functional in activated RISC thereby completely eliminating unintended mRNA targeting by the sense strand. Interestingly, the sisiRNA design supports the function of chemically modified antisense strands, which are non-functional within the context of standard siRNA designs. This suggests that the sisiRNA design has a clear potential of improving the pharmacokinetic properties of siRNA in vivo. PMID:17726057

Are solar activity and sperm whale Physeter macrocephalus strandings around the North Sea related?

NASA Astrophysics Data System (ADS)

Vanselow, Klaus Heinrich; Ricklefs, Klaus

2005-04-01

In the final decades of the last century, an increasing number of strandings of male sperm whales ( Physeter macrocephalus) around the North Sea led to an increase in public interest. Anthropogenic influences (such as contaminants or intensive sound disturbances) are supposed to be the main causes, but natural environmental effects may also explain the disorientation of the animals. We compared the documented sperm whale strandings in the period from 1712 to 2003 with solar activity, especially with sun spot number periodicity and found that 90% of 97 sperm whale stranding events around the North Sea took place when the smoothed sun spot period length was below the mean value of 11 years, while only 10% happened during periods of longer sun spot cycles. The relation becomes even more pronounced (94% to 6%, n = 70) if a smaller time window from November to March is used (which seems to be the main southward migration period of male sperm whales). Adequate chi-square tests of the data give a significance of 1% error probability that sperm whale strandings can depend on solar activity. As an alternative explanation, we suggest that variations of the earth's magnetic field, due to variable energy fluxes from the sun to the earth, may cause a temporary disorientation of migrating animals.

C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation.

PubMed

Liu, Ying; Ye, Xuecheng; Jiang, Feng; Liang, Chunyang; Chen, Dongmei; Peng, Junmin; Kinch, Lisa N; Grishin, Nick V; Liu, Qinghua

2009-08-07

The catalytic engine of RNA interference (RNAi) is the RNA-induced silencing complex (RISC), wherein the endoribonuclease Argonaute and single-stranded small interfering RNA (siRNA) direct target mRNA cleavage. We reconstituted long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We used this core reconstitution system to purify an RNAi regulator that we term C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a Mg2+-dependent endoribonuclease that promotes RISC activation by removing siRNA passenger strand cleavage products. These studies establish an in vitro RNAi reconstitution system and identify C3PO as a key activator of the core RNAi machinery.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

PubMed Central

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Chemical Speciation of Water Soluble Ions and Metals of Cloud and Rain Water During the Puerto Rico African Dust and Clouds Study (PRADACS) Campaigns

NASA Astrophysics Data System (ADS)

Torres, E.; Valle Diaz, C. J.; Lee, T.; Collett, J. L.; Fitzgerald, E.; Cuadra-Rodriguez, L. A.; Prather, K. A.; Sánchez, M.; McDowell, W. H.; Mayol-Bracero, O. L.

2013-05-01

The underlying physico-chemical processes of dust particles interactions are poorly understood; even less understood is how aging impacts cloud properties and climate as the particles travel from Africa to the Caribbean region. Caribbean landmasses have tropical montane cloud forests (TMCFs) that are tightly coupled to the atmospheric hydrologic cycle. TMCFs are ecosystems to study the effects African Dust (AD) on cloud formation and precipitation as these are very sensitive ecosystems that respond to small changes in climate. As part of the Puerto Rico African Dust and Clouds Study (PRADACS), chemical analyses were performed on cloud and rain water samples collected at Pico del Este (PE) station in Luquillo, PR (1051 masl) during campaigns held from 2010 to 2012. At PE, two cloud collectors (i.e., single stage (Aluminum version), a 2-stage (Teflon version) Caltech Active Strand Cloudwater Collector (CASCC)), a rainwater collector, and anAerosol Time-Of-Flight Mass Spectrometer (ATOFMS) were operated. Chemical analyses performed on collected samples include pH, conductivity, ion chromatography (IC), and inductive coupled plasma (ICP). Results from these campaigns showed that on days that had air masses with the influence of AD, cloud water samples had higher conductivity and pH values on average (up to 5.7 and 180μS/cm, respectively) than those with air masses without AD influence. An increase in the concentrations of water-soluble ions like non-sea salt calcium and magnesium, and metals like magnesium, calcium and aluminum was observed and the appearance of iron was seen on ICP analyses. The ATOFMS, showed an increase on the amount of particles during AD influence with composition of aluminum, silicates, potassium, iron and titanium aerosols. The increase on the aforementioned species was constant in the three years of sampling, which give us confidence in the identification of the chemical species that are present during the influence of AD.

Re-Assessing the Measurement of Fogwater Inputs to a Tropical Ecosystem

NASA Astrophysics Data System (ADS)

Burkard, R.; Eugster, W.; Holwerda, F.; Bruijnzeel, S.; Scatena, F.; Siegwolf, R.

2002-12-01

For several years the hydrological importance of the fog- and cloudwater deposition to ecosystems in the tropics has been of great interest. In earlier studies carried out in the humid tropics the amount of deposited cloudwater was estimated by indirect methods based on the physical characteristics of the utilized cloudwater collector. In the temperate climatic zone of central Europe most of the studies dealing with cloudwater focus on the additional chemical input due to cloudwater in relation to the amount of deposited rainwater. During our experiment in the Luquillo mountains of Puerto Rico the different aspects of the chemical and hydrological impacts of cloudwater deposition have been investigated. During 43 days, cloudwater fluxes were measured with an eddy covariance setup consisting of a Solent ultrasonic anemometer and a size-resolving cloud droplet spectrometer. Cloudwater samples were taken with a Caltech-type active strand cloudwater collector. Additionally, measurements of rain, throughfall and stemflow were performed. Samples of fog, rain, throughfall and stemflow were analyzed for inorganic ion and stabile isotope concentrations (δ18O and δ2H). First analysis of the hydrological input show that there exist some significant differences in the deposited amount of cloudwater as measured with our instruments in comparison with previous studies carried out at the same location: Mean liquid water content was 78.6 mg m-3 during situations with a visibility below 1000 m (84% of the entire field campaign). The deposition rate of cloudwater was 0.88 mm d-1. A mismatch was found regarding the water balance. We conclude from this that the rainfall amount and therefore also the chemical input by rain is strongly underestimated due to wind-driven rain, which is not measured by standard rain gauges. Depending on the reference value, we have to conclude that the deposition of cloudwater accounts for 6--11% of wet deposition.

The occurrence of antibodies against single-stranded DNA in the sera of patients with acute and chronic leukaemia.

PubMed Central

Izui, S; Lambert, P H; Carpentier, N; Miescher, P A

1976-01-01

One hundred and seventy-five sera from thirty-three patients with acute myeloid leukaemia, forty-two patients with chronic myeloid leukaemia and twelve patients with acute lymphatic leukaemia were examined by a radioimmunological technique for the presence of antibodies against single-stranded and double-stranded DNA. The levels of single-stranded DNA binding activity was significantly higher in all three types of leukaemia compared to those of healthy controls. In contrast, none of these sera exhibited a positive reaction with double-stranded DNA. In some cases the level of serum anti-DNA antibodies increased after the decrease of the leucocyte count. The presence of anti-DNA antibodies in leukaemic patients may have some biological significance. PMID:780020

Strand displacement synthesis by yeast DNA polymerase ε

PubMed Central

Ganai, Rais A.; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-01-01

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. PMID:27325747

"The awe in which biologists hold physicists": Frits Went's first phytotron at Caltech, and an experimental definition of the biological environment.

PubMed

Munns, David P D

2014-01-01

After Darwin, experimental biology sought to unravel organisms. By the early twentieth century, organisms were broadly conceived as the product of their heredity and their environment. Much historical work has explored the scientific attack on the genotype, particularly through the new science of genetics. This article explores the tandem efforts to assert experimental control over the environment in which plants grew and developed. The case described here concerns the creation of the first phytotron at Caltech by botanist and plant physiologist Frits Went. Opening in 1949, the phytotron was a plant laboratory that, across a series of rooms and chambers, kept genes constant while regulating and maintaining defined ranges of known environments. This article details the context in which the phytotron emerged, how the phytotron gained its sobriquet, and how it served to cement the "environment" as a category of biological knowledge. Describing the institutional context of Caltech, its interdisciplinary culture, and its encouragement of adopting technology into biological science, I argue that the phytotron and the commensurate category of the "environment", were the product of the familiar movement to integrate the physical and biological sciences. In addition, however, the creation of the phytotron was also a broader story of plant physiologists establishing a definition of the "environment" in both physical and technological terms.

Nucleic acid analysis using terminal-phosphate-labeled nucleotides

DOEpatents

Korlach, Jonas [Ithaca, NY; Webb, Watt W [Ithaca, NY; Levene, Michael [Ithaca, NY; Turner, Stephen [Ithaca, NY; Craighead, Harold G [Ithaca, NY; Foquet, Mathieu [Ithaca, NY

2008-04-22

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

Site-Specific RNase A Activity Was Dramatically Reduced in Serum from Multiple Types of Cancer Patients

PubMed Central

Huang, Weiyan; Zhao, Mei; Wei, Na; Wang, Xiaoxia; Cao, Huqing; Du, Quan; Liang, Zicai

2014-01-01

Potent RNase activities were found in the serum of mammals but the physiological function of the RNases was never well illustrated, largely due to the caveats in methods of RNase activity measurement. None of the existing methods can distinguish between RNases with different target specificities. A systematic study was recently carried out in our lab to investigate the site-specificity of serum RNases on double-stranded RNA substrates, and found that serum RNases cleave double-stranded RNAs predominantly at 5′-U/A-3′ and 5′-C/A-3′ dinucleotide sites, in a manner closely resembling RNase A. Based on this finding, a FRET assay was developed in the current study to measure this site-specific serum RNase activity in human samples using a double stranded RNA substrate. We demonstrated that the method has a dynamic range of 10−5 mg/ml- 10−1 mg/ml using serial dilution of RNase A. The sera of 303 cancer patients were subjected to comparison with 128 healthy controls, and it was found that serum RNase activities visualized with this site-specific double stranded probe were found to be significantly reduced in patients with gastric cancer, liver cancer, pancreatic cancer, esophageal cancer, ovary cancer, cervical cancer, bladder cancer, kidney cancer and lung cancer, while only minor changes were found in breast and colon cancer patients. This is the first report using double stranded RNA as probe to quantify site-specific activities of RNase A in a serum. The results illustrated that RNase A might be further evaluated to determine if it can serve as a new class of biomarkers for certain cancer types. PMID:24805924

Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

PubMed Central

Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie; Chen, Yuling; Rabadan, Raul; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

2017-01-01

SUMMARY The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development. PMID:28431250

SCDC Spanish Curricula Units. Science/Math Strand, Unit 4, Grade Two, Teacher's Guide.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The teacher's guide for unit four of a Spanish science/math strand for second graders contains instructional and assessment activities for kits 13-16. Each designed for a two- to three-week teaching period, the kits' activities are geared toward guiding the child to discover correct answers through methods provided and, by putting materials in his…

SCDC Spanish Curricula Units. Fine Arts Strand, Unit 9, Grade 3, Supplement & Ditto Packet.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The supplement and ditto packet to the fine arts strand of unit nine for third graders contains illustrations, ditto masters, and four songbooks to help the teacher carry out the fine arts activities of this unit. Materials for both group activities and seatwork are included. Kit 33 uses the songbook "Canciones de Aqui y de Alla" and…

Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon

2012-03-16

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosenmore » DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.« less

Both DNA Polymerases δ and ε Contact Active and Stalled Replication Forks Differently

PubMed Central

Yu, Chuanhe; Gan, Haiyun

2017-01-01

ABSTRACT Three DNA polymerases, polymerases α, δ, and ε (Pol α, Pol δ, and Pol ε), are responsible for eukaryotic genome duplication. When DNA replication stress is encountered, DNA synthesis stalls until the stress is ameliorated. However, it is not known whether there is a difference in the association of each polymerase with active and stalled replication forks. Here, we show that each DNA polymerase has a distinct pattern of association with active and stalled replication forks. Pol α is enriched at extending Okazaki fragments of active and stalled forks. In contrast, although Pol δ contacts the nascent lagging strands of active and stalled forks, it binds to only the matured (and not elongating) Okazaki fragments of stalled forks. Pol ε has greater contact with the nascent single-stranded DNA (ssDNA) of the leading strand on active forks than on stalled forks. We propose that the configuration of DNA polymerases at stalled forks facilitates the resumption of DNA synthesis after stress removal. PMID:28784720

The Impact of Turtle Excluder Devices and Fisheries Closures on Loggerhead and Kemp's Ridley Strandings in the Western Gulf of Mexico

USGS Publications Warehouse

Lewison, R.L.; Crowder, L.B.; Shaver, D.J.

2003-01-01

The Sea Turtle Stranding and Salvage Network has been monitoring turtle strandings for more than 20 years in the United States. High numbers of strandings in the early to mid-1980s prompted regulations to require turtle excluder devices (TEDs) on shrimping vessels (trawlers). Following year-round TED implementation in 1991, however, stranding levels in the Gulf of Mexico increased. We evaluated the efficacy of TEDs and other management actions (e.g., fisheries closures) on loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempii) turtle populations by analyzing a long-term, stranding data set from the western Gulf of Mexico. Our analyses suggest that both sea turtle population growth and shrimping activity have contributed to the observed increase in strandings. Compliance with regulations requiring turtle excluder devices was a significant factor in accounting for annual stranding variability: low compliance was correlated with high levels of strandings. Our projections suggest that improved compliance with TED regulations will reduce strandings to levels that, in conjunction with other protective measures, should promote population recoveries for loggerhead and Kemp's ridley turtles. Local, seasonal fisheries closures, concurrent with TED enforcement, could reduce strandings to even lower levels. A seasonal closure adjacent to a recently established Kemp's ridley nesting beach may also reduce mortality of nesting adults and thus promote long-term population persistence by fostering the establishment of a robust secondary nesting site.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

PubMed

Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

PubMed

Wang, Guoping; Ding, Xiong; Hu, Jiumei; Wu, Wenshuai; Sun, Jingjing; Mu, Ying

2017-10-24

Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences. In contrast to current methods, UIMA is simple to product multimeric DNA, due to the independent of multiple primers and rolling circle structures. Through assaying the synthesized single-stranded DNA targets, UIMA performs high sensitivity and specificity, as well as the universality. In addition, a plausible mechanism of UIMA is proposed, involving short DNA bending, mismatch extension, and template slippage. UIMA is a good explanation for why nonspecific amplification easily happens in existing INAAs. As the simplest INAA till now, UIMA provides a new insight for deeply understanding INAA and opens a new avenue for thoroughly addressing nonspecific amplification.

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures

PubMed Central

Kang, Young-Hoon; Munashingha, Palinda Ruvan; Lee, Chul-Hwan; Nguyen, Tuan Anh; Seo, Yeon-Soo

2012-01-01

Saccharomyces cerevisiae Mph1 is a 3–5′ DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3′ to 5′ directionality, Mph1 displaced 3′-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5′-flap strand more efficiently than the 3′ flap strand from double-flap substrates, which is not expected for a 3–5′ DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5′ single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway. PMID:22090425

Exoplanet Orbit Database | Exoplanet Data Explorer

Science.gov Websites

, Strasbourg, France, NASA's Astrophysics Data System, the NASA Exoplanet Archive (and, formerly, the NASA/IPAC /Caltech. This research received generous funding from NASA and the NSF.

Internet-Based Solutions for a Secure and Efficient Seismic Network

NASA Astrophysics Data System (ADS)

Bhadha, R.; Black, M.; Bruton, C.; Hauksson, E.; Stubailo, I.; Watkins, M.; Alvarez, M.; Thomas, V.

2017-12-01

The Southern California Seismic Network (SCSN), operated by Caltech and USGS, leverages modern Internet-based computing technologies to provide timely earthquake early warning for damage reduction, event notification, ShakeMap, and other data products. Here we present recent and ongoing innovations in telemetry, security, cloud computing, virtualization, and data analysis that have allowed us to develop a network that runs securely and efficiently.Earthquake early warning systems must process seismic data within seconds of being recorded, and SCSN maintains a robust and resilient network of more than 350 digital strong motion and broadband seismic stations to achieve this goal. We have continued to improve the path diversity and fault tolerance within our network, and have also developed new tools for latency monitoring and archiving.Cyberattacks are in the news almost daily, and with most of our seismic data streams running over the Internet, it is only a matter of time before SCSN is targeted. To ensure system integrity and availability across our network, we have implemented strong security, including encryption and Virtual Private Networks (VPNs).SCSN operates its own data center at Caltech, but we have also installed real-time servers on Amazon Web Services (AWS), to provide an additional level of redundancy, and eventually to allow full off-site operations continuity for our network. Our AWS systems receive data from Caltech-based import servers and directly from field locations, and are able to process the seismic data, calculate earthquake locations and magnitudes, and distribute earthquake alerts, directly from the cloud.We have also begun a virtualization project at our Caltech data center, allowing us to serve data from Virtual Machines (VMs), making efficient use of high-performance hardware and increasing flexibility and scalability of our data processing systems.Finally, we have developed new monitoring of station average noise levels at most stations. Noise monitoring is effective at identifying anthropogenic noise sources and malfunctioning acquisition equipment. We have built a dynamic display of results with sorting and mapping capabilities that allow us to quickly identify problematic sites and areas with elevated noise.

Strand displacement synthesis by yeast DNA polymerase ε.

PubMed

Ganai, Rais A; Zhang, Xiao-Ping; Heyer, Wolf-Dietrich; Johansson, Erik

2016-09-30

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3'-5' exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3'-5' exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5' end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mutually Exclusive Formation of G-Quadruplex and i-Motif Is a General Phenomenon Governed by Steric Hindrance in Duplex DNA.

PubMed

Cui, Yunxi; Kong, Deming; Ghimire, Chiran; Xu, Cuixia; Mao, Hanbin

2016-04-19

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to possible offset arrangements between G-quadruplex and i-motif in opposite strands. The mutual exclusivity revealed here sets a molecular basis for cells to efficiently coordinate opposite biological activities of G-quadruplex and i-motif at the same dsDNA location.

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex.

PubMed

Xu, Ning; Gkountela, Sofia; Saeed, Khalid; Akusjärvi, Göran

2009-11-01

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

The influence of sequence context and length on the kinetics of DNA duplex formation from complementary hairpins possessing (CNG) repeats.

PubMed

Paiva, Anthony M; Sheardy, Richard D

2005-04-20

The formation of unusual structures during DNA replication has been invoked for gene expansion in genomes possessing triplet repeat sequences, CNG, where N = A, C, G, or T. In particular, it has been suggested that the daughter strand of the leading strand partially dissociates from the parent strand and forms a hairpin. The equilibrium between the fully duplexed parent:daugter species and the parent:hairpin species is dependent upon their relative stabilities and the rates of reannealing of the daughter strand back to the parent. These stabilities and rates are ultimately influenced by the sequence context of the DNA and its length. Previous work has demonstrated that longer strands are more stable than shorter strands and that the identity of N also influences the thermal stability [Paiva, A. M.; Sheardy, R. D. Biochemistry 2004, 43, 14218-14227]. Here, we show that the rate of duplex formation from complementary hairpins is also sequence context and length dependent. In particular, longer duplexes have higher activation energies than shorter duplexes of the same sequence context. Further, [(CCG):(GGC)] duplexes have lower activation energies than corresponding [(CAG):(GTC)] duplexes of the same length. Hence, hairpins formed from long CNG sequences are more thermodynamically stable and have slower kinetics for reannealing to their complement than shorter analogues. Gene expansion can now be explained in terms of thermodynamics and kinetics.

Single slit interference made easy with a strand of hair and a laser

NASA Astrophysics Data System (ADS)

Messer, Rebecca

2018-01-01

Students can easily measure the width of a strand of their own hair with a monochromatic light source such as a laser. This inexpensive activity engages students in an application of single slit diffraction using Babinet's principle.

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

PubMed Central

Schauer, Grant D.; O’Donnell, Michael E.

2017-01-01

The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand. PMID:28069954

Molecular interactions between single-stranded DNA-binding proteins associated with an essential MCAT element in the mouse smooth muscle alpha-actin promoter.

PubMed

Kelm, R J; Cogan, J G; Elder, P K; Strauch, A R; Getz, M J

1999-05-14

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Chlorella virus DNA ligase: nick recognition and mutational analysis.

PubMed

Sriskanda, V; Shuman, S

1998-01-15

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

PubMed Central

Georgescu, Roxana E; Schauer, Grant D; Yao, Nina Y; Langston, Lance D; Yurieva, Olga; Zhang, Dan; Finkelstein, Jeff; O'Donnell, Mike E

2015-01-01

We have reconstituted a eukaryotic leading/lagging strand replisome comprising 31 distinct polypeptides. This study identifies a process unprecedented in bacterial replisomes. While bacteria and phage simply recruit polymerases to the fork, we find that suppression mechanisms are used to position the distinct eukaryotic polymerases on their respective strands. Hence, Pol ε is active with CMG on the leading strand, but it is unable to function on the lagging strand, even when Pol δ is not present. Conversely, Pol δ-PCNA is the only enzyme capable of extending Okazaki fragments in the presence of Pols ε and α. We have shown earlier that Pol δ-PCNA is suppressed on the leading strand with CMG (Georgescu et al., 2014). We propose that CMG, the 11-subunit helicase, is responsible for one or both of these suppression mechanisms that spatially control polymerase occupancy at the fork. DOI: http://dx.doi.org/10.7554/eLife.04988.001 PMID:25871847

Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference

PubMed Central

Angart, Phillip A.; Carlson, Rebecca J.; Adu-Berchie, Kwasi

2016-01-01

Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5′ terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5′ terminus (Nucleotides: 1–2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)–specific activity was found to be improved by lower hybridization stability in the 5′ terminus (Nucleotides: 3–4) of the loaded siRNA strand and greater hybridization stability toward the 3′ terminus (Nucleotides: 17–18). Concomitantly, specific recognition of the 5′ terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand. PMID:27399870

Monitor and Control of the Deep-Space network via Secure Web

NASA Technical Reports Server (NTRS)

Lamarra, N.

1997-01-01

(view graph) NASA lead center for robotic space exploration. Operating division of Caltech/Jet Propulsion Laboratory. Current missions, Voyagers, Galileo, Pathfinder, Global Surveyor. Upcoming missions, Cassini, Mars and New Millennium.

Glass Stronger than Steel

DOE R&D Accomplishments Database

Yarris, Lynn

2011-03-28

A new type of damage-tolerant metallic glass, demonstrating a strength and toughness beyond that of steel or any other known material, has been developed and tested by a collaboration of researchers from Berkeley Lab and Caltech.

VizieR Online Data Catalog: NIR albedos of main-belt asteroids (Masiero+, 2014)

NASA Astrophysics Data System (ADS)

Masiero, J. R.; Grav, T.; Mainzer, A. K.; Nugent, C. R.; Bauer, J. M.; Stevenson, R.; Sonnett, S.

2017-04-01

To fit for NIR albedos of main-belt asteroids, we use data from the WISE/NEOWISE all-sky single exposure source table, which are available for download from the Infrared Science Archive (IRSA, http://irsa.ipac.caltech.edu ; Cutri et al. 2012, xplanatory Supplement to the WISE All-Sky Data Release Products, http://wise2.ipac.caltech.edu/docs/release/allsky/expsup/index.html). We extract photometric measurements of all asteroids observed by WISE following the technique described in Masiero et al. (2011, J/ApJ/741/68) and Mainzer et al. (2011ApJ...731...53M). In particular, we use the NEOWISE observations reported to the MPC and included in the MPC's minor planet observation database as the final validated list of reliable NEOWISE detections of solar system objects. (1 data file).

The Telecommunications and Data Acquisition Report

NASA Technical Reports Server (NTRS)

Yuen, Joseph H. (Editor)

1994-01-01

This quarterly publication provides archival reports on developments in programs managed by JPL's Office of Telecommunications and Data Acquisition (TDA). In space communications, radio navigation, radio science, and ground-based radio and radar astronomy, it reports on activities of the Deep Space Network (DSN) in planning, supporting research and technology, implementation, and operations. Also included are standards activity at JPL for space data and information systems and reimbursable DSN work performed for other space agencies through NASA. The preceding work is all performed for NASA's Office of Space Communications (OSC). The TDA Office also performs work funded by other NASA program offices through and with the cooperation of OSC. Finally, tasks funded under the JPL Director's Discretionary Fund and the Caltech President's Fund that involve the TDA Office are included.

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3'-end.

PubMed

Merkiene, Egle; Gaidamaviciute, Edita; Riauba, Laurynas; Janulaitis, Arvydas; Lagunavicius, Arunas

2010-08-01

We improved the target RNA-primed RCA technique for direct detection and analysis of RNA in vitro and in situ. Previously we showed that the 3' --> 5' single-stranded RNA exonucleolytic activity of Phi29 DNA polymerase converts the target RNA into a primer and uses it for RCA initiation. However, in some cases, the single-stranded RNA exoribonucleolytic activity of the polymerase is hindered by strong double-stranded structures at the 3'-end of target RNAs. We demonstrate that in such hampered cases, the double-stranded RNA-specific Escherichia coli RNase III efficiently assists Phi29 DNA polymerase in converting the target RNA into a primer. These observations extend the target RNA-primed RCA possibilities to test RNA sequences distanced far from the 3'-end and customize this technique for the inner RNA sequence analysis.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase

PubMed Central

Malik, Omri; Khamis, Hadeel; Rudnizky, Sergei; Marx, Ailie

2017-01-01

Abstract Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors. PMID:28973474

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

PubMed

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Reduction of spontaneous somatic mutation frequency by a low-dose X irradiation of Drosophila larvae and possible involvement of DNA single-strand damage repair.

PubMed

Koana, Takao; Takahashi, Takashi; Tsujimura, Hidenobu

2012-03-01

The third instar larvae of Drosophila were irradiated with X rays, and the somatic mutation frequency in their wings was measured after their eclosion. In the flies with normal DNA repair and apoptosis functions, 0.2 Gy irradiation at 0.05 Gy/min reduced the frequency of the so-called small spot (mutant cell clone with reduced reproductive activity) compared with that in the sham-irradiated flies. When apoptosis was suppressed using the baculovirus p35 gene, the small spot frequency increased four times in the sham-irradiated control group, but the reduction by the 0.2-Gy irradiation was still evident. In a non-homologous end joining-deficient mutant, the small spot frequency was also reduced by 0.2 Gy radiation. In a mutant deficient in single-strand break repair, no reduction in the small spot frequency by 0.2 Gy radiation was observed, and the small spot frequency increased with the radiation dose. Large spot (mutant cell clone with normal reproductive activity) frequency was not affected by suppression of apoptosis and increased monotonically with radiation dose in wild-type larvae and in mutants for single- or double-strand break repair. It is hypothesized that some of the small spots resulted from single-strand damage and, in wild-type larvae, 0.2 Gy radiation activated the normal single-strand break repair gene, which reduced the background somatic mutation frequency.

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

PubMed Central

Petojevic, Tatjana; Pesavento, James J.; Costa, Alessandro; Liang, Jingdan; Wang, Zhijun; Berger, James M.; Botchan, Michael R.

2015-01-01

DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2–7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2–7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2–7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel. PMID:25561522

Late Quaternary slip history of the Mill Creek strand of the San Andreas fault in San Gorgonio Pass, southern California: The role of a subsidiary left-lateral fault in strand switching

USGS Publications Warehouse

Kendrick, Katherine J.; Matti, Jonathan; Mahan, Shannon

2015-01-01

The fault history of the Mill Creek strand of the San Andreas fault (SAF) in the San Gorgonio Pass region, along with the reconstructed geomorphology surrounding this fault strand, reveals the important role of the left-lateral Pinto Mountain fault in the regional fault strand switching. The Mill Creek strand has 7.1–8.7 km total slip. Following this displacement, the Pinto Mountain fault offset the Mill Creek strand 1–1.25 km, as SAF slip transferred to the San Bernardino, Banning, and Garnet Hill strands. An alluvial complex within the Mission Creek watershed can be linked to palinspastic reconstruction of drainage segments to constrain slip history of the Mill Creek strand. We investigated surface remnants through detailed geologic mapping, morphometric and stratigraphic analysis, geochronology, and pedogenic analysis. The degree of soil development constrains the duration of surface stability when correlated to other regional, independently dated pedons. This correlation indicates that the oldest surfaces are significantly older than 500 ka. Luminescence dates of 106 ka and 95 ka from (respectively) 5 and 4 m beneath a younger fan surface are consistent with age estimates based on soil-profile development. Offset of the Mill Creek strand by the Pinto Mountain fault suggests a short-term slip rate of ∼10–12.5 mm/yr for the Pinto Mountain fault, and a lower long-term slip rate. Uplift of the Yucaipa Ridge block during the period of Mill Creek strand activity is consistent with thermochronologic modeled uplift estimates.

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLaughlin, K. J.; Nash, R. P.; Redinbo, M. R.

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. Inmore » this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.« less

Possible Causes of a Harbour Porpoise Mass Stranding in Danish Waters in 2005

PubMed Central

Wright, Andrew J.; Maar, Marie; Mohn, Christian; Nabe-Nielsen, Jacob; Siebert, Ursula; Jensen, Lasse Fast; Baagøe, Hans J.; Teilmann, Jonas

2013-01-01

An unprecedented 85 harbour porpoises stranded freshly dead along approximately 100 km of Danish coastline from 7–15 April, 2005. This total is considerably above the mean weekly stranding rate for the whole of Denmark, both for any time of year, 1.23 animals/week (ranging from 0 to 20 during 2003–2008, excluding April 2005), and specifically in April, 0.65 animals/week (0 to 4, same period). Bycatch was established as the cause of death for most of the individuals through typical indications of fisheries interactions, including net markings in the skin and around the flippers, and loss of tail flukes. Local fishermen confirmed unusually large porpoise bycatch in nets set for lumpfish (Cyclopterus lumpus) and the strandings were attributed to an early lumpfish season. However, lumpfish catches for 2005 were not unusual in terms of season onset, peak or total catch, when compared to 2003–2008. Consequently, human activity was combined with environmental factors and the variation in Danish fisheries landings (determined through a principal component analysis) in a two-part statistical model to assess the correlation of these factors with both the presence of fresh strandings and the numbers of strandings on the Danish west coast. The final statistical model (which was forward selected using Akaike information criterion; AIC) indicated that naval presence is correlated with higher rates of porpoise strandings, particularly in combination with certain fisheries, although it is not correlated with the actual presence of strandings. Military vessels from various countries were confirmed in the area from the 7th April, en route to the largest naval exercise in Danish waters to date (Loyal Mariner 2005, 11–28 April). Although sonar usage cannot be confirmed, it is likely that ships were testing various equipment prior to the main exercise. Thus naval activity cannot be ruled out as a possible contributing factor. PMID:23460787

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Exploiting Tumor-Activated Testes Proteins to Enhance Efficacy of First-Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2015-10-01

TERMS Cancer Testis Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF...Cancer Testis Antigen (CTA) o Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What

DNA secondary structure of the released strand stimulates WRN helicase action on forked duplexes without coordinate action of WRN exonuclease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Byungchan, E-mail: bbccahn@mail.ulsan.ac.kr; Bohr, Vilhelm A.

2011-08-12

Highlights: {yields} In this study, we investigated the effect of a DNA secondary structure on the two WRN activities. {yields} We found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. {yields} These results imply that WRN helicase and exonuclease activities can act independently. -- Abstract: Werner syndrome (WS) is an autosomal recessive premature aging disorder characterized by aging-related phenotypes and genomic instability. WS is caused by mutations in a gene encoding a nuclear protein, Werner syndrome protein (WRN), a member of the RecQ helicase family, that interestingly possessesmore » both helicase and exonuclease activities. Previous studies have shown that the two activities act in concert on a single substrate. We investigated the effect of a DNA secondary structure on the two WRN activities and found that a DNA secondary structure of the displaced strand during unwinding stimulates WRN helicase without coordinate action of WRN exonuclease. These results imply that WRN helicase and exonuclease activities can act independently, and we propose that the uncoordinated action may be relevant to the in vivo activity of WRN.« less

Dna2 initiates resection at clean DNA double-strand breaks

PubMed Central

Paudyal, Sharad C.; Li, Shan; Yan, Hong; Hunter, Tony

2017-01-01

Abstract Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms. PMID:28981724

A difference in the pattern of repair in a large genomic region in UV-irradiated normal human and Cockayne syndrome cells.

PubMed

Shanower, G A; Kantor, G J

1997-11-01

Xeroderma pigmentosum group C cells repair DNA damaged by ultraviolet radiation in an unusual pattern throughout the genome. They remove cyclobutane pyrimidine dimers only from the DNA of transcriptionally active chromatin regions and only from the strand that contains the transcribed strand. The repair proceeds in a manner that creates damage-free islands which are in some cases much larger than the active gene associated with them. For example, the small transcriptionally active beta-actin gene (3.5 kb) is repaired as part of a 50 kb single-stranded region. The repair responsible for creating these islands requires active transcription, suggesting that the two activities are coupled. A preferential repair pathway in normal human cells promotes repair of actively transcribed DNA strands and is coupled to transcription. It is not known if similar large islands, referred to as repair domains, are preferentially created as a result of the coupling. Data are presented showing that in normal cells, preferential repair in the beta-actin region is associated with the creation of a large, completely repaired region in the partially repaired genome. Repair at other genomic locations which contain inactive genes (insulin, 754) does not create similar large regions as quickly. In contrast, repair in Cockayne syndrome cells, which are defective in the preferential repair pathway but not in genome-overall repair, proceeds in the beta-actin region by a mechanism which does not create preferentially a large repaired region. Thus a correlation between the activity required to preferentially repair active genes and that required to create repaired domains is detected. We propose an involvement of the transcription-repair coupling factor in a coordinated repair pathway for removing DNA damage from entire transcription units.

Offshore Earthquakes Do Not Influence Marine Mammal Stranding Risk on the Washington and Oregon Coasts

PubMed Central

Grant, Rachel A.; Savirina, Anna

2018-01-01

Simple Summary Marine mammals stranding on coastal beaches is not unusual. However, there appears to be no single cause for this, with several causes being probable, such as starvation, contact with humans (for example boat strike or entanglement with fishing gear), disease, and parasitism. We evaluated marine mammal stranding off the Washington and Oregon coasts and looked at offshore earthquakes as a possible contributing factor. Our analysis showed that offshore earthquakes did not make marine mammals more likely to strand. We also analysed a subset of data from the north of Washington State and found that non-adult animals made up a large proportion of stranded animals, and for dead animals the commonest cause of death was disease, traumatic injury, or starvation. Abstract The causes of marine mammals stranding on coastal beaches are not well understood, but may relate to topography, currents, wind, water temperature, disease, toxic algal blooms, and anthropogenic activity. Offshore earthquakes are a source of intense sound and disturbance and could be a contributing factor to stranding probability. We tested the hypothesis that the probability of marine mammal stranding events on the coasts of Washington and Oregon, USA is increased by the occurrence of offshore earthquakes in the nearby Cascadia subduction zone. The analysis carried out here indicated that earthquakes are at most, a very minor predictor of either single, or large (six or more animals) stranding events, at least for the study period and location. We also tested whether earthquakes inhibit stranding and again, there was no link. Although we did not find a substantial association of earthquakes with strandings in this study, it is likely that there are many factors influencing stranding of marine mammals and a single cause is unlikely to be responsible. Analysis of a subset of data for which detailed descriptions were available showed that most live stranded animals were pups, calves, or juveniles, and in the case of dead stranded mammals, the commonest cause of death was trauma, disease, and emaciation. PMID:29373509

Biochemical characterization of an exonuclease from Arabidopsis thaliana reveals similarities to the DNA exonuclease of the human Werner syndrome protein.

PubMed

Plchova, Helena; Hartung, Frank; Puchta, Holger

2003-11-07

The human Werner syndrome protein (hWRN-p) possessing DNA helicase and exonuclease activities is essential for genome stability. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small open reading frame (ORF) (AtWRNexo) with homology to the exonuclease domain of hWRN-p. Expression of this ORF in Escherichia coli revealed an exonuclease activity for AtWRN-exo-p with similarities but also some significant differences to hWRN-p. The protein digests recessed strands of DNA duplexes in the 3' --> 5' direction but hardly single-stranded DNA or blunt-ended duplexes. In contrast to the Werner exonuclease, AtWRNexo-p is also able to digest 3'-protruding strands. DNA with recessed 3'-PO4 and 3'-OH termini is degraded to a similar extent. AtWRNexo-p hydrolyzes the 3'-recessed strand termini of duplexes containing mismatched bases. AtWRNexo-p needs the divalent cation Mg2+ for activity, which can be replaced by Mn2+. Apurinic sites, cholesterol adducts, and oxidative DNA damage (such as 8-oxoadenine and 8-oxoguanine) inhibit or block the enzyme. Other DNA modifications, including uracil, hypoxanthine and ethenoadenine, did not inhibit AtWRNexo-p. A mutation of a conserved residue within the exonuclease domain (E135A) completely abolished the exonucleolytic activity. Our results indicate that a type of WRN-like exonuclease activity seems to be a common feature of the DNA metabolism of animals and plants.

JPL-20170926-TECHf-0001-Robot Descends into Alaska Moulin

NASA Image and Video Library

2017-09-26

JPL engineer Andy Klesh lowers a robotic submersible into a moulin. Klesh and JPL's John Leichty used robots and probes to explore the Matanuska Glacier in Alaska this past July. Image Credit: NASA/JPL-Caltech

The Caltech Concurrent Computation Program - Project description

NASA Technical Reports Server (NTRS)

Fox, G.; Otto, S.; Lyzenga, G.; Rogstad, D.

1985-01-01

The Caltech Concurrent Computation Program wwhich studies basic issues in computational science is described. The research builds on initial work where novel concurrent hardware, the necessary systems software to use it and twenty significant scientific implementations running on the initial 32, 64, and 128 node hypercube machines have been constructed. A major goal of the program will be to extend this work into new disciplines and more complex algorithms including general packages that decompose arbitrary problems in major application areas. New high-performance concurrent processors with up to 1024-nodes, over a gigabyte of memory and multigigaflop performance are being constructed. The implementations cover a wide range of problems in areas such as high energy and astrophysics, condensed matter, chemical reactions, plasma physics, applied mathematics, geophysics, simulation, CAD for VLSI, graphics and image processing. The products of the research program include the concurrent algorithms, hardware, systems software, and complete program implementations.

Leon Knopoff (1926-2011)

NASA Astrophysics Data System (ADS)

Davis, Paul; Jackson, David; Gilbert, Freeman

2011-06-01

Leon Knopoff died at his home in Sherman Oaks, Calif., on 20 January 2011 at the age of 85. A man of wide-ranging talents, he had the rare distinction of being simultaneously a professor of physics, a professor of geophysics, and a research musicologist at the University of California, Los Angeles (UCLA). As an undergraduate he studied electrical engineering and obtained his Ph.D. in physics and mathematics at the California Institute of Technology (Caltech) in 1949. He was recruited to the Institute of Geophysics (now the Institute of Geophysics and Planetary Physics) at UCLA in 1950 by Louis Slichter, where he became a professor of geophysics in 1957 and of geophysics and physics in 1961. He became a research musicologist in the UCLA Institute of Ethnomusicology soon after it was formed in 1960. Other appointments included faculty positions at Miami University in Ohio (1948-1950) and Caltech (1962-1963) and visiting appointments at Cambridge, Karlsruhe, Harvard, Santiago, Trieste, and Venice.

An observation of the Galactic center hard X-ray source, 1E 1740.7-2942, with the Caltech coded-aperture telescope

NASA Technical Reports Server (NTRS)

Heindl, William A.; Cook, Walter R.; Grunsfeld, John M.; Palmer, David M.; Prince, Thomas A.; Schindler, Stephen M.; Stone, Edward C.

1993-01-01

The Galactic center region hard X-ray source IE 1740.7-2942 has been observed with the Caltech Gamma-Ray Imaging Payload (GRIP) from Alice Springs, Australia, on 1988 April 12 and on 1989 April 3 and 4. We report here results from the 1989 measurements based on 14 hr of observation of the Galactic center region. The observations showed IE 1740.7-2942 to be in its normal state, having a spectrum between 35 and 200 keV characterized by a power law with an exponent of -2.2 +/- 0.3 and flux at 100 keV of (7.0 +/- 0.7) x 10 exp -5 sq cm s keV. No flux was detected above 200 keV. A search for time variability in the spectrum of IE 1740.7-2942 on one hour time scales showed no evidence for variability.

The MKID Camera

NASA Astrophysics Data System (ADS)

Maloney, P. R.; Czakon, N. G.; Day, P. K.; Duan, R.; Gao, J.; Glenn, J.; Golwala, S.; Hollister, M.; LeDuc, H. G.; Mazin, B.; Noroozian, O.; Nguyen, H. T.; Sayers, J.; Schlaerth, J.; Vaillancourt, J. E.; Vayonakis, A.; Wilson, P.; Zmuidzinas, J.

2009-12-01

The MKID Camera project is a collaborative effort of Caltech, JPL, the University of Colorado, and UC Santa Barbara to develop a large-format, multi-color millimeter and submillimeter-wavelength camera for astronomy using microwave kinetic inductance detectors (MKIDs). These are superconducting, micro-resonators fabricated from thin aluminum and niobium films. We couple the MKIDs to multi-slot antennas and measure the change in surface impedance produced by photon-induced breaking of Cooper pairs. The readout is almost entirely at room temperature and can be highly multiplexed; in principle hundreds or even thousands of resonators could be read out on a single feedline. The camera will have 576 spatial pixels that image simultaneously in four bands at 750, 850, 1100 and 1300 microns. It is scheduled for deployment at the Caltech Submillimeter Observatory in the summer of 2010. We present an overview of the camera design and readout and describe the current status of testing and fabrication.

NASA/IPAC Infrared Archive's General Image Cutouts Service

NASA Astrophysics Data System (ADS)

Alexov, A.; Good, J. C.

2006-07-01

The NASA/IPAC Infrared Archive (IRSA) ``Cutouts" Service (http://irsa.ipac.caltech.edu/applications/Cutouts) is a general tool for creating small ``cutout" FITS images and JPEGs from collections of data archived at IRSA. This service is a companion to IRSA's Atlas tool (http://irsa.ipac.caltech.edu/applications/Atlas/), which currently serves over 25 different data collections of various sizes and complexity and returns entire images for a user-defined region of the sky. The Cutouts Services sits on top of Atlas and extends the Atlas functionality by generating subimages at locations and sizes requested by the user from images already identified by Atlas. These results can be downloaded individually, in batch mode (using the program wget), or as a tar file. Cutouts re-uses IRSA's software architecture along with the publicly available Montage mosaicking tools. The advantages and disadvantages of this approach to generic cutout serving will be discussed.

Robust nonlinear control of vectored thrust aircraft

NASA Technical Reports Server (NTRS)

Doyle, John C.; Murray, Richard; Morris, John

1993-01-01

An interdisciplinary program in robust control for nonlinear systems with applications to a variety of engineering problems is outlined. Major emphasis will be placed on flight control, with both experimental and analytical studies. This program builds on recent new results in control theory for stability, stabilization, robust stability, robust performance, synthesis, and model reduction in a unified framework using Linear Fractional Transformations (LFT's), Linear Matrix Inequalities (LMI's), and the structured singular value micron. Most of these new advances have been accomplished by the Caltech controls group independently or in collaboration with researchers in other institutions. These recent results offer a new and remarkably unified framework for all aspects of robust control, but what is particularly important for this program is that they also have important implications for system identification and control of nonlinear systems. This combines well with Caltech's expertise in nonlinear control theory, both in geometric methods and methods for systems with constraints and saturations.

Supreme Court Hears Privacy Case Between NASA and Jet Propulsion Laboratory Scientists

NASA Astrophysics Data System (ADS)

Showstack, Randy

2010-10-01

After NASA put into practice the 2004 Homeland Security Presidential Directive-12, known as HSPD-12, Dennis Byrnes talked to then-NASA administrator Michael Griffin. Byrnes recalls that Griffin told him in 2007 that if he didn’t like the agency's implementation of HSPD-12, he should go to court. That's exactly what Byrnes, an employee of the California Institute of Technology (Caltech) working as a senior engineer at NASA's Jet Propulsion Laboratory (JPL) in Pasadena, Calif., did. Concerned about prying and open-ended background investigations of federal contractors through NASA's implementation of HSPD-12, he, along with lead plaintiff Robert Nelson and 26 other Caltech employees working at JPL, sued NASA. Following several lower court decisions, including an injunction issued by a U.S. federal appeals court in response to a plaintiff motion, the case made it all the way to the U.S. Supreme Court, which heard oral arguments on 5 October.

Investigations into the metabolic diversity of microorganisms as part of microbial diversity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leadbetter, Jared

DOE funds supported a key portion of the MBL Microbial Diversity (Woods Hole) program across 6 complete summers. The initial 4 years of the funded period were overseen by two co-Directors, Daniel Buckley (Cornell) and Steve Zinder (Cornell), who then completed their term. The final 2 summers were overseen by 2 new co-Directors, Jared R. Leadbetter (Caltech) and Dianne Newman (Caltech). The 6 funded summer iterations of the course included the incorporation of new themes such as single cell approaches applied to natural microbial communities (cell separation and sorting, genome amplification from single cells, and the use of Nano-SIMS tomore » examine assimilation of carbon and nitrogen from isotopically labeled substrates into single cells), genetics and genomics on bacteria freshly isolated during the course of the programs, quantitative systems biology, and modern quantitative light microscopy.« less

Transcriptional requirements of the distal heavy-strand promoter of mtDNA

PubMed Central

Zollo, Ornella; Tiranti, Valeria; Sondheimer, Neal

2012-01-01

The heavy strand of mtDNA contains two promoters with nonoverlapping functions. The role of the minor heavy-strand promoter (HSP2) is controversial, because the promoter has been difficult to activate in an in vitro system. We have isolated HSP2 by excluding its interaction with the more powerful HSP1 promoter, and we find that it is transcribed efficiently by recombinant mtRNA polymerase and mitochondrial transcription factor B2. The mitochondrial transcription factor A is not required for initiation, but it has the ability to alternatively activate and repress the HSP2 transcriptional unit depending on the ratio between mitochondrial transcription factor A and other transcription factors. The positioning of transcriptional initiation agrees with our current understanding of HSP2 activity in vivo. Serial deletion of HSP2 shows that only proximal sequences are required. Several mutations, including the disruption of a polycytosine track upstream of the HSP2 initiation site, influence transcriptional activity. Transcription from HSP2 is also observed when HeLa cell mitochondrial extract is used as the source of mitochondrial polymerase, and this transcription is maintained when HSP2 is provided in proper spacing and context to the HSP1 promoter. Studies of the linked heavy-strand promoters show that they are differentially regulated by ATP dosage. We conclude that HSP2 is transcribed and has features that allow it to regulate mitochondrial mRNA synthesis. PMID:22454497

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90.

PubMed

Iki, Taichiro; Yoshikawa, Manabu; Nishikiori, Masaki; Jaudal, Mauren C; Matsumoto-Yokoyama, Eiko; Mitsuhara, Ichiro; Meshi, Tetsuo; Ishikawa, Masayuki

2010-07-30

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed. Copyright 2010 Elsevier Inc. All rights reserved.

Stabilised DNA secondary structures with increasing transcription localise hypermutable bases for somatic hypermutation in IGHV3-23.

PubMed

Duvvuri, Bhargavi; Duvvuri, Venkata R; Wu, Jianhong; Wu, Gillian E

2012-07-01

Somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID) is a transcription-coupled mechanism most responsible for generating high affinity antibodies. An issue remaining enigmatic in SHM is how AID is preferentially targeted during transcription to hypermutable bases in its substrates (WRC motifs) on both DNA strands. AID targets only single stranded DNA. By modelling the dynamical behaviour of IGHV3-23 DNA, a commonly used human variable gene segment, we observed that hypermutable bases on the non-transcribed strand are paired whereas those on transcribed strand are mostly unpaired. Hypermutable bases (both paired and unpaired) are made accessible to AID in stabilised secondary structures formed with increasing transcription levels. This observation provides a rationale for the hypermutable bases on both the strands of DNA being targeted to a similar extent despite having differences in unpairedness. We propose that increasing transcription and RNAP II stalling resulting in the formation and stabilisation of stem-loop structures with AID hotspots in negatively supercoiled region can localise the hypermutable bases of both strands of DNA, to AID-mediated SHM.

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine.

PubMed

Shafirovich, V; Dourandin, A; Luneva, N P; Singh, C; Kirigin, F; Geacintov, N E

1999-03-01

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.

Detection of negative and positive RNA strand of poliovirus Sabin 1 and echovirus E19 by a stem-loop reverse transcription PCR.

PubMed

Fikatas, A; Dimitriou, T G; Kyriakopoulou, Z; Moschonas, G D; Amoutzias, G D; Mossialos, D; Gartzonika, C; Levidiotou-Stefanou, S; Markoulatos, P

2017-09-01

In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 -2 CCID 50 of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 5 and 1 CCID 50 respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 5 CCID 50 . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses. Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses. © 2017 The Society for Applied Microbiology.

The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

PubMed

Biswas, N; Weller, S K

2001-05-18

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.

The cryomechanical design of MUSIC: a novel imaging instrument for millimeter-wave astrophysics at the Caltech Submillimeter Observatory

NASA Astrophysics Data System (ADS)

Hollister, Matthew I.; Czakon, Nicole G.; Day, Peter K.; Downes, Thomas P.; Duan, Ran; Gao, Jiansong; Glenn, Jason; Golwala, Sunil R.; LeDuc, Henry G.; Maloney, Philip R.; Mazin, Benjamin A.; Nguyen, Hien Trong; Noroozian, Omid; Sayers, Jack; Schlaerth, James; Siegel, Seth; Vaillancourt, John E.; Vayonakis, Anastasios; Wilson, Philip; Zmuidzinas, Jonas

2010-07-01

MUSIC (Multicolor Submillimeter kinetic Inductance Camera) is a new facility instrument for the Caltech Submillimeter Observatory (Mauna Kea, Hawaii) developed as a collaborative effect of Caltech, JPL, the University of Colorado at Boulder and UC Santa Barbara, and is due for initial commissioning in early 2011. MUSIC utilizes a new class of superconducting photon detectors known as microwave kinetic inductance detectors (MKIDs), an emergent technology that offers considerable advantages over current types of detectors for submillimeter and millimeter direct detection. MUSIC will operate a focal plane of 576 spatial pixels, where each pixel is a slot line antenna coupled to multiple detectors through on-chip, lumped-element filters, allowing simultaneously imaging in four bands at 0.86, 1.02, 1.33 and 2.00 mm. The MUSIC instrument is designed for closed-cycle operation, combining a pulse tube cooler with a two-stage Helium-3 adsorption refrigerator, providing a focal plane temperature of 0.25 K with intermediate temperature stages at approximately 50, 4 and 0.4 K for buffering heat loads and heat sinking of optical filters. Detector readout is achieved using semi-rigid coaxial cables from room temperature to the focal plane, with cryogenic HEMT amplifiers operating at 4 K. Several hundred detectors may be multiplexed in frequency space through one signal line and amplifier. This paper discusses the design of the instrument cryogenic hardware, including a number of features unique to the implementation of superconducting detectors. Predicted performance data for the instrument system will also be presented and discussed.

Exploiting Tumor Activated Testes Proteins To Enhance Efficacy of First Line Chemotherapeutics in NSCLC

DTIC Science & Technology

2016-10-01

Antigen (CTA), Fanconia- Anemia (FA), DNA Damage, Genomic Instability, DNA Double Strand Break (DSB) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...Fanconia- Anemia (FA) o DNA Damage o Genomic Instability o DNA Double Strand Break (DSB) 3. Accomplishments • What were the major goals and objectives of

Australian Curriculum Linked Lessons: The Language of Chance

ERIC Educational Resources Information Center

Hurrell, Derek

2015-01-01

In providing a continued focus on tasks and activities that help to illustrate key ideas embedded in the "Australian Curriculum," this issue focuses on the Statistics and probability strand and the sub-strand of Chance. In the Australian Curriculum (ACARA, 2015), students are not asked to list outcomes of chance experiments and represent…

Australian Curriculum Linked Lessons

ERIC Educational Resources Information Center

Hurrell, Derek; O'Neil, Jennifer

2011-01-01

In providing a continued focus on tasks and activities that help to illustrate key ideas embedded in the new Australian Curriculum, this issue the authors focus, on Geometry in the Measurement and Geometry strand with strong links for an integrated focus on the Statistics and Probability strand. The small unit of work on the sorting and…

Mlh1-Mlh3, a Meiotic Crossover and DNA Mismatch Repair Factor, Is a Msh2-Msh3-stimulated Endonuclease*

PubMed Central

Rogacheva, Maria V.; Manhart, Carol M.; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

2014-01-01

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair. PMID:24403070

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Mlh1-Mlh3, a meiotic crossover and DNA mismatch repair factor, is a Msh2-Msh3-stimulated endonuclease.

PubMed

Rogacheva, Maria V; Manhart, Carol M; Chen, Cheng; Guarne, Alba; Surtees, Jennifer; Alani, Eric

2014-02-28

Crossing over between homologous chromosomes is initiated in meiotic prophase in most sexually reproducing organisms by the appearance of programmed double strand breaks throughout the genome. In Saccharomyces cerevisiae the double-strand breaks are resected to form three prime single-strand tails that primarily invade complementary sequences in unbroken homologs. These invasion intermediates are converted into double Holliday junctions and then resolved into crossovers that facilitate homolog segregation during Meiosis I. Work in yeast suggests that Msh4-Msh5 stabilizes invasion intermediates and double Holliday junctions, which are resolved into crossovers in steps requiring Sgs1 helicase, Exo1, and a putative endonuclease activity encoded by the DNA mismatch repair factor Mlh1-Mlh3. We purified Mlh1-Mlh3 and showed that it is a metal-dependent and Msh2-Msh3-stimulated endonuclease that makes single-strand breaks in supercoiled DNA. These observations support a direct role for an Mlh1-Mlh3 endonuclease activity in resolving recombination intermediates and in DNA mismatch repair.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism.

PubMed

Awate, Sanket; Brosh, Robert M

2017-06-08

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies.

Interactive Roles of DNA Helicases and Translocases with the Single-Stranded DNA Binding Protein RPA in Nucleic Acid Metabolism

PubMed Central

Awate, Sanket; Brosh, Robert M.

2017-01-01

Helicases and translocases use the energy of nucleoside triphosphate binding and hydrolysis to unwind/resolve structured nucleic acids or move along a single-stranded or double-stranded polynucleotide chain, respectively. These molecular motors facilitate a variety of transactions including replication, DNA repair, recombination, and transcription. A key partner of eukaryotic DNA helicases/translocases is the single-stranded DNA binding protein Replication Protein A (RPA). Biochemical, genetic, and cell biological assays have demonstrated that RPA interacts with these human molecular motors physically and functionally, and their association is enriched in cells undergoing replication stress. The roles of DNA helicases/translocases are orchestrated with RPA in pathways of nucleic acid metabolism. RPA stimulates helicase-catalyzed DNA unwinding, enlists translocases to sites of action, and modulates their activities in DNA repair, fork remodeling, checkpoint activation, and telomere maintenance. The dynamic interplay between DNA helicases/translocases and RPA is just beginning to be understood at the molecular and cellular levels, and there is still much to be learned, which may inform potential therapeutic strategies. PMID:28594346

Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

PubMed Central

Gomatos, Peter J.

1970-01-01

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn++ was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus. PMID:5483438

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction

NASA Astrophysics Data System (ADS)

Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

2018-02-01

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Universal, colorimetric microRNA detection strategy based on target-catalyzed toehold-mediated strand displacement reaction.

PubMed

Park, Yeonkyung; Lee, Chang Yeol; Kang, Shinyoung; Kim, Hansol; Park, Ki Soo; Park, Hyun Gyu

2018-02-23

In this work, we developed a novel, label-free, and enzyme-free strategy for the colorimetric detection of microRNA (miRNA), which relies on a target-catalyzed toehold-mediated strand displacement (TMSD) reaction. The system employs a detection probe that specifically binds to the target miRNA and sequentially releases a catalyst strand (CS) intended to trigger the subsequent TMSD reaction. Thus, the presence of target miRNA releases the CS that mediates the formation of an active G-quadruplex DNAzyme which is initially caged and inactivated by a blocker strand. In addition, a fuel strand that is supplemented for the recycling of the CS promotes another TMSD reaction, consequently generating a large number of active G-quadruplex DNAzymes. As a result, a distinct colorimetric signal is produced by the ABTS oxidation promoted by the peroxidase mimicking activity of the released G-quadruplex DNAzymes. Based on this novel strategy, we successfully detected miR-141, a promising biomarker for human prostate cancer, with high selectivity. The diagnostic capability of this system was also demonstrated by reliably determining target miR-141 in human serum, showing its great potential towards real clinical applications. Importantly, the proposed approach is composed of separate target recognition and signal transduction modules. Thus, it could be extended to analyze different target miRNAs by simply redesigning the detection probe while keeping the same signal transduction module as a universal signal amplification unit, which was successfully demonstrated by analyzing another target miRNA, let-7d.

Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

PubMed

Sidstedt, Maja; Hedman, Johannes; Romsos, Erica L; Waitara, Leticia; Wadsö, Lars; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter

2018-04-01

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. Graphical abstract PCR inhibition mechanisms of hemoglobin and immunoglobulin G (IgG). Cq quantification cycle, dsDNA double-stranded DNA, ssDNA single-stranded DNA.

Changes in RNA polymerase II progression influence somatic hypermutation of Ig-related genes by AID

PubMed Central

Kodgire, Prashant; Mukkawar, Priyanka; Ratnam, Sarayu; Martin, Terence E.

2013-01-01

Somatic hypermutation (SHM) of Ig genes is initiated by the activation-induced cytidine deaminase (AID), and requires target gene transcription. We previously proposed that AID may associate with the RNA polymerase II (Pol). Here, to determine aspects of the transcription process required for SHM, we knocked-in a transcription terminator into an Ig gene variable region in DT40 chicken B cell line. We found that the human β-globin terminator was an efficient inhibitor of downstream transcription in these cells. The terminator reduced mutations downstream of the poly(A) signal, suggesting that the process of transcription is essential for efficient SHM and that AID has better access to its target when Pol is in the elongating rather than terminating mode. Mutations upstream of the poly(A) site were almost doubled in the active terminator clones compared with an inactivated terminator, and this region showed more single-stranded DNA, indicating that Pol pausing assists SHM. Moreover, the nontranscribed DNA strand was the preferred SHM target upstream of the active terminator. Pol pausing during poly(A) site recognition may facilitate persistence of negative supercoils, exposing the coding single strand and possibly allowing the nascent RNA intermittent reannealing with the template strand, for prolonged access of AID. PMID:23752228

Strand exchange of telomeric DNA catalyzed by the Werner syndrome protein (WRN) is specifically stimulated by TRF2

PubMed Central

Edwards, Deanna N.; Orren, David K.; Machwe, Amrita

2014-01-01

Werner syndrome (WS), caused by loss of function of the RecQ helicase WRN, is a hereditary disease characterized by premature aging and elevated cancer incidence. WRN has DNA binding, exonuclease, ATPase, helicase and strand annealing activities, suggesting possible roles in recombination-related processes. Evidence indicates that WRN deficiency causes telomeric abnormalities that likely underlie early onset of aging phenotypes in WS. Furthermore, TRF2, a protein essential for telomere protection, interacts with WRN and influences its basic helicase and exonuclease activities. However, these studies provided little insight into WRN's specific function at telomeres. Here, we explored the possibility that WRN and TRF2 cooperate during telomeric recombination processes. Our results indicate that TRF2, through its interactions with both WRN and telomeric DNA, stimulates WRN-mediated strand exchange specifically between telomeric substrates; TRF2's basic domain is particularly important for this stimulation. Although TRF1 binds telomeric DNA with similar affinity, it has minimal effects on WRN-mediated strand exchange of telomeric DNA. Moreover, TRF2 is displaced from telomeric DNA by WRN, independent of its ATPase and helicase activities. Together, these results suggest that TRF2 and WRN act coordinately during telomeric recombination processes, consistent with certain telomeric abnormalities associated with alteration of WRN function. PMID:24880691

Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

PubMed

Fujimura, Tsutomu; Esteban, Rosa

2016-10-01

The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. © 2016 John Wiley & Sons Ltd.

Two distinct mechanisms ensure transcriptional polarity in double-stranded RNA bacteriophages.

PubMed

Yang, Hongyan; Makeyev, Eugene V; Butcher, Sarah J; Gaidelyte, Ausra; Bamford, Dennis H

2003-01-01

In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component. Only plus- but not minus-sense copies of genomic segments are produced during this reaction. In the case of phi6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity. Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, phi8 and phi13. While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn(2+) to produce minus-sense copies on phi13 dsRNA templates. Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including phi13 dsRNA) are activated by denaturation before the reaction. Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form.

Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

PubMed Central

Yang, Hongyan; Makeyev, Eugene V.; Bamford, Dennis H.

2001-01-01

The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription in Cystoviridae is controlled by the template specificity of the polymerase subunit. PMID:11602748

Two Distinct Mechanisms Ensure Transcriptional Polarity in Double-Stranded RNA Bacteriophages

PubMed Central

Yang, Hongyan; Makeyev, Eugene V.; Butcher, Sarah J.; Gaidelyte·, Aušra; Bamford, Dennis H.

2003-01-01

In most double-stranded RNA (dsRNA) viruses, RNA transcription occurs inside a polymerase (Pol) complex particle, which contains an RNA-dependent RNA Pol subunit as a minor component. Only plus- but not minus-sense copies of genomic segments are produced during this reaction. In the case of φ6, a dsRNA bacteriophage from the Cystoviridae family, isolated Pol synthesizes predominantly plus strands using virus-specific dsRNAs in vitro, thus suggesting that Pol template preferences determine the transcriptional polarity. Here, we dissect transcription reactions catalyzed by Pol complexes and Pol subunits of two other cystoviruses, φ8 and φ13. While both Pol complexes synthesize exclusively plus strands over a wide range of conditions, isolated Pol subunits can be stimulated by Mn2+ to produce minus-sense copies on φ13 dsRNA templates. Importantly, all three Pol subunits become more prone to the native-like plus-strand synthesis when the dsRNA templates (including φ13 dsRNA) are activated by denaturation before the reaction. Based on these and earlier observations, we propose a model of transcriptional polarity in Cystoviridae controlled on two independent levels: Pol affinity to plus-strand initiation sites and accessibility of these sites to the Pol in a single-stranded form. PMID:12502836

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

Unique helicase determinants in the essential conjugative TraI factor from Salmonella enterica serovar Typhimurium plasmid pCU1.

PubMed

McLaughlin, Krystle J; Nash, Rebekah P; Redinbo, Mathew R

2014-09-01

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

IP Network Design and Implementation for the Caltech-USGS Element of TriNet

NASA Astrophysics Data System (ADS)

Johnson, M. L.; Busby, R.; Watkins, M.; Schwarz, S.; Hauksson, E.

2001-12-01

The new seismic network IP numbering scheme for the Caltech-USGS element of TriNet is designed to provide emergency response plans for computer outages and/or telemetry circuit failures so that data acquisition may continue with minimal interruption. IP numbers from the seismic stations through the Caltech acquisition machines are numbered using private, non-routable IP addresses, which allows the network administrator to create redundancy in the network design, more freedom in choosing IP numbers, and uniformity in the LAN and WAN network addressing. The network scheme used by the Caltech-USGS element of TriNet is designed to create redundancy and load sharing over three or more T1 circuits. A T1 circuit can support 80 dataloggers sending data at a design rate of 19.2 kbps or 120 dataloggers transmitting at a nominal rate of 12.8 kbps. During a circuit detour, the 80 dataloggers on the failed T1 are equally divided between the remaining two circuits. This increases the loads on the remaining two circuits to 120 dataloggers, which is the maximum load each T1 can handle at the nominal rate. Each T1 circuit has a router interface onto a LAN at Caltech with an independent subnet address. Some devices, such as Solaris computers, allow a single interface to be numbered with several IP addresses, a so called "multinetted" interface. This allows the central acquisition computers to appear with distinct addresses that are routable via different T1 circuits, but simplifies the physical cables between devices. We identify these T1 circuits as T1-1, T1-2, and T1-3. At the remote end, each Frame Relay Access Device (FRAD) and connected datalogger(s) is a subnetted LAN. The numbering is arranged so the second octet in the LAN IP address of the FRAD and datalogger identify the datalogger's primary and alternate T1 circuits. For example; a LAN with an IP address of 10.12.0.0/24 has T1-1 as its primary T1, and T1-2 as its alternate circuit. Stations with this number scheme are sometimes referred to as group "12". LANs with IP addresses of 10.23.0.0/24 have T1-2 as the primary circuit, and T1-3 as the alternate circuit. Static routes on the acquisition system are used to direct traffic through the primary T1. The network can operate in one of three modes. The most common and desirable mode is "normal", where all three T1's are operational and Caltech has both a primary and secondary central acquisition system running. The second mode is a "failover", where the primary acquisition system is down (due to maintenance or failure) and the secondary acquisition system assumes the primary role. This includes sending acknowledgments to dataloggers and multicasts to the rest of the network. The third mode is a circuit detour. The port numbers on the central acquisition system for the dataloggers on the failed T1 are changed to match the auxiliary ports on the dataloggers. This allows for the auxiliary ports on the dataloggers to receive acknowledgements from the acquiring system through the detoured circuit. The static routes on the system are changed to go through the detoured circuit as specified by the group's IP numbers. At this point the two working T1's will be running at full capacity but the data acquisition will continue with minimal interruption while the third T1 is being restored. The primary acquisition computer continues to listen for data on the failed T1 should things improve spontaneously.

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

NASA Astrophysics Data System (ADS)

Gaspar, Miguel; Shenk, Thomas

2006-02-01

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks

PubMed Central

George, Tresa; Wen, Qin; Griffiths, Richard; Ganesh, Anil; Meuth, Mark; Sanders, Cyril M.

2009-01-01

Pif-1 proteins are 5′→3′ superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3′ ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity. PMID:19700773

A conserved MCM single-stranded DNA binding element is essential for replication initiation.

PubMed

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-04-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001.

A conserved MCM single-stranded DNA binding element is essential for replication initiation

PubMed Central

Froelich, Clifford A; Kang, Sukhyun; Epling, Leslie B; Bell, Stephen P; Enemark, Eric J

2014-01-01

The ring-shaped MCM helicase is essential to all phases of DNA replication. The complex loads at replication origins as an inactive double-hexamer encircling duplex DNA. Helicase activation converts this species to two active single hexamers that encircle single-stranded DNA (ssDNA). The molecular details of MCM DNA interactions during these events are unknown. We determined the crystal structure of the Pyrococcus furiosus MCM N-terminal domain hexamer bound to ssDNA and define a conserved MCM-ssDNA binding motif (MSSB). Intriguingly, ssDNA binds the MCM ring interior perpendicular to the central channel with defined polarity. In eukaryotes, the MSSB is conserved in several Mcm2-7 subunits, and MSSB mutant combinations in S. cerevisiae Mcm2-7 are not viable. Mutant Mcm2-7 complexes assemble and are recruited to replication origins, but are defective in helicase loading and activation. Our findings identify an important MCM-ssDNA interaction and suggest it functions during helicase activation to select the strand for translocation. DOI: http://dx.doi.org/10.7554/eLife.01993.001 PMID:24692448

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

PubMed

Abdullah, Ummi B; McGouran, Joanna F; Brolih, Sanja; Ptchelkine, Denis; El-Sagheer, Afaf H; Brown, Tom; McHugh, Peter J

2017-07-14

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo . © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Binding of radiation-induced phenylalanine radicals to DNA: influence on the biological activity of the DNA and on its sensitivity to the induction of breaks by gamma rays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vanderschans, G.P.; Vanrijn, C.J.S.; Bleichrodt, J.F.

1975-11-01

When an aqueous solution of double-stranded deoxyribonucleic acid (DNA) of bacteriophage PM2 containing phenylalanine and saturated with N2O is irradiated with gamma rays, radiation induced phenylalanine radicals are bound covalently. Under the conditions used about 25 phenylalanine molecules may be bound per lethal hit. Also for single-stranded PM2 DNA most of the phenylalanine radicals bound are nonlethal. Evidence is presented that in double-stranded DNA an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. Theremore » are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentration of these compounds in the vicinity of the negatively charged DNA molecules. (Author) (GRA)« less

Tn552 transposase purification and in vitro activities.

PubMed Central

Rowland, S J; Sherratt, D J; Stark, W M; Boocock, M R

1995-01-01

The Staphylococcus aureus transposon Tn552 encodes a protein (p480) containing the 'D,D(35)E' motif common to retroviral integrases and the transposases of a number of bacterial elements, including phage Mu, the integron-containing element Tn5090, Tn7 and IS3. p480 and a histidine-tagged derivative were overexpressed in Escherichia coli and purified by methods involving denaturation and renaturation. DNase I footprinting and gel binding assays demonstrated that p480 binds to two adjacent, directly repeated 23 bp motifs at each end of Tn552. Although donor strand cleavage by p480 was not detected, in vitro conditions were defined for strand transfer activity with transposon end fragments having pre-cleaved 3' termini. Strand transfer was Mn(2+)-dependent and appeared to join a single left or right end fragment to target DNA. The importance of the terminal dinucleotide CA-3' was demonstrated by mutation. The in vitro activities of p480 are consistent with its proposed function as the Tn552 transposase. Images PMID:7828593

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

PubMed

Fernández-Sierra, Mónica; Quiñones, Edwin

2015-03-15

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect. Copyright © 2015 Elsevier Inc. All rights reserved.

The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination†

PubMed Central

Besmer, Eva; Market, Eleonora; Papavasiliou, F. Nina

2006-01-01

Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter. PMID:16705187

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated genetic recombination.

PubMed

Yadav, Tribhuwan; Carrasco, Begoña; Serrano, Ester; Alonso, Juan C

2014-10-03

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP · Mg(2+)-bound form (RecA · ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA · ATP. This work demonstrates that RecA · ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA · dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Imaging denatured collagen strands in vivo and ex vivo via photo-triggered hybridization of caged collagen mimetic peptides.

PubMed

Li, Yang; Foss, Catherine A; Pomper, Martin G; Yu, S Michael

2014-01-31

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i) to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Memotain: A CAD/CAM nickel-titanium lingual retainer.

PubMed

Kravitz, Neal D; Grauer, Dan; Schumacher, Pascal; Jo, Yong-Min

2017-04-01

Approximately 1/2 of maxillary and 1/5 of mandibular multi-stranded lingual retainers fail during retention in some form, either bond failure or wire breakage. Memotain is a new CAD/CAM fabricated lingual retainer wire made of custom-cut nickel-titanium, as an alternative to multi-stranded lingual retainers. It offers numerous perceived advantages to the traditional multi-stranded stainless steel wire, including precision fit, avoidance of interferences, corrosion resistance and even the potential for minor tooth movement as an active lingual retainer. Copyright © 2017 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

SCDC Spanish Curricula Units. Spanish SL, Unit 6, Grade 2, Teacher's Guide.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Spanish for second graders is explored in the instructional and assessment activities of the unit six Spanish as a Second Language strand. The function of the strand is to help provide the English-dominant child with the structures and vocabulary needed for effective communication in a bilingual environment. Focus, objective, and materials for…

Characterization of a periplasmic S1-like nuclease coded by the Mesorhizobium loti symbiosis island

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pimkin, Maxim; Miller, C. Glenn; Blakesley, Lauryn

DNA sequences encoding hypothetical proteins homologous to S1 nuclease from Aspergillus oryzae are found in many organisms including fungi, plants, pathogenic bacteria, and eukaryotic parasites. One of these is the M1 nuclease of Mesorhizobium loti which we demonstrate herein to be an enzymatically active, soluble, and stable S1 homolog that lacks the extensive mannosyl-glycosylation found in eukaryotic S1 nuclease homologs. We have expressed the cloned M1 protein in M. loti and purified recombinant native M1 to near homogeneity and have also isolated a homogeneous M1 carboxy-terminal hexahistidine tag fusion protein. Mass spectrometry and N-terminal Edman degradation sequencing confirmed the proteinmore » identity. The enzymatic properties of the purified M1 nuclease are similar to those of S1. At acidic pH M1 is 25 times more active on single-stranded DNA than on double-stranded DNA and 3 times more active on single-stranded DNA than on single-stranded RNA. At neutral pH the RNase activity of M1 exceeds the DNase activity. M1 nicks supercoiled RF-I plasmid DNA and rapidly cuts the phosphodiester bond across from the nick in the resultant relaxed RF-II plasmid DNA. Therefore, M1 represents an active bacterial S1 homolog in spite of great sequence divergence. The biochemical characterization of M1 nuclease supports our sequence alignment that reveals the minimal 21 amino acid residues that are necessarily conserved for the structure and functions of this enzyme family. The ability of M1 to degrade RNA at neutral pH implies previously unappreciated roles of these nucleases in biological systems.« less

SNMIB/Apollo protects leading-strand telomeres against NHEJ-mediated repair.

PubMed

Lam, Yung C; Akhter, Shamima; Gu, Peili; Ye, Jing; Poulet, Anaïs; Giraud-Panis, Marie-Josèphe; Bailey, Susan M; Gilson, Eric; Legerski, Randy J; Chang, Sandy

2010-07-07

Progressive telomere attrition or deficiency of the protective shelterin complex elicits a DNA damage response as a result of a cell's inability to distinguish dysfunctional telomeric ends from DNA double-strand breaks. SNMIB/Apollo is a shelterin-associated protein and a member of the SMN1/PSO2 nuclease family that localizes to telomeres through its interaction with TRF2. Here, we generated SNMIB/Apollo knockout mouse embryo fibroblasts (MEFs) to probe the function of SNMIB/Apollo at mammalian telomeres. SNMIB/Apollo null MEFs exhibit an increased incidence of G2 chromatid-type fusions involving telomeres created by leading-strand DNA synthesis, reflective of a failure to protect these telomeres after DNA replication. Mutations within SNMIB/Apollo's conserved nuclease domain failed to suppress this phenotype, suggesting that its nuclease activity is required to protect leading-strand telomeres. SNMIB/Apollo(-/-)ATM(-/-) MEFs display robust telomere fusions when Trf2 is depleted, indicating that ATM is dispensable for repair of uncapped telomeres in this setting. Our data implicate the 5'-3' exonuclease function of SNM1B/Apollo in the generation of 3' single-stranded overhangs at newly replicated leading-strand telomeres to protect them from engaging the non-homologous end-joining pathway.

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

PubMed

Ihara, Makoto; Takeshita, Satoshi; Okaichi, Kumio; Okumura, Yutaka; Ohnishi, Takeo

2014-03-01

From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

Caltech campus executive LDRD.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shepodd, Timothy J.; Knudsen, Tamara

2013-01-01

The environment most brain systems of humans and other animals are almost constantly confronted with is complex and continuously changing, with each time step updating a potentially bewildering set of opportunities and demands for action. Far from the controlled, discrete trials used in most neuro- and psychological investigations, behavior outside the lab at Caltech is a seamless and continuous process of monitoring (and error correction) of ongoing action, and of evaluating persistence in the current activity with respect to opportunities to switch tasks as alternatives become available. Prior work on frontopolar and prefrontal task switching, use tasks within the samemore » modality (View a stream of symbols on a screen and perform certain response mappings depending on task rules). However, in these task switches the effector is constant: only the mapping of visual symbols to the specific button changes. In this task, the subjects are choosing what kinds of future action decisions they want to perform, where they can control either which body part will act, or which direction they will orient an instructed body action. An effector choice task presents a single target and the subject selects which effector to use to reach the target (eye or hand). While the techniques available for humans can be less spatially resolved compared to non-human primate neural data, they do allow for experimentation on multiple brain areas with relative ease. Thus, we address a broader network of areas involved in motor decisions. We aim to resolve a current dispute regarding the specific functional roles of brain areas that are often co-activated in studies of decision tasks, dorsal premotor cortex(PMd) and posterior parietal cortex(PPC). In one model, the PPC distinctly drives intentions for action selection, whereas PMd stimulation results in complex multi-joint movements without any awareness of, nor subjective feeling of, willing the elicited movement, thus seems to merely help execute the chosen action.« less

Ionic modulation of QPX stability as a nano-switch regulating gene expression in neurons

NASA Astrophysics Data System (ADS)

Baghaee Ravari, Soodeh

G-quadruplexes (G-QPX) have been the subject of intense research due to their unique structural configuration and potential applications, particularly their functionality in biological process as a novel type of nano--switch. They have been found in critical regions of the human genome such as telomeres, promoter regions, and untranslated regions of RNA. About 50% of human DNA in promoters has G-rich regions with the potential to form G-QPX structures. A G-QPX might act mechanistically as an ON/OFF switch, regulating gene expression, meaning that the formation of G-QPX in a single strand of DNA disrupts double stranded DNA, prevents the binding of transcription factors (TF) to their recognition sites, resulting in gene down-regulation. Although there are numerous studies on biological roles of G-QPXs in oncogenes, their potential formation in neuronal cells, in particular upstream of transcription start sites, is poorly investigated. The main focus of this research is to identify stable G-QPXs in the 97bp active promoter region of the choline acetyltransferase (ChAT) gene, the terminal enzyme involved in synthesis of the neurotransmitter acetylcholine, and to clarify ionic modulation of G-QPX nanostructures through the mechanism of neural action potentials. Different bioinformatics analyses (in silico), including the QGRS, quadparser and G4-Calculator programs, have been used to predict stable G-QPX in the active promoter region of the human ChAT gene, located 1000bp upstream from the TATA box. The results of computational studies (using those three different algorithms) led to the identification of three consecutive intramolecular G-QPX structures in the negative strand (ChAT G17-2, ChAT G17, and ChAT G29) and one intramolecular G-QPX structure in the positive strand (ChAT G30). Also, the results suggest the possibility that nearby G-runs in opposed DNA strands with a short distance of each other may be able to form a stable intermolecular G-QPX involving two DNA complementary strands (ds ChAT G21). Formation of G-QPX structures, by blocking the availability of the transcription factor binding site (TFBS) on double stranded DNA, can interfere with transcriptional activation. This suggests that there is competition between TFBS binding to dsDNA and the conversion to high order non-B form secondary structures (G-QPXs) in the active promoter region. TFBS mapping analysis of the active promoter region of the human ChAT gene revealed that it contains multiple consensus AP-2alpha and Sp1 binding sites and consensus sites for other TF, including multiple sites for GR-alpha, Pax-5, p53 and GC box proteins. (Abstract shortened by ProQuest.).

A Summer Research Program of NASA/Faculty Fellowships at the Jet Propulsion Laboratory

NASA Technical Reports Server (NTRS)

Albee, Arden

2004-01-01

The NASA Faculty Fellowship Program (NFFP) is designed to give college and university faculty members a rewarding personal as well as enriching professional experience. Fellowships are awarded to engineering and science faculty for work on collaborative research projects of mutual interest to the fellow and his or her JPL host colleague. The Jet Propulsion Laboratory (JPL) and the California Institute of Technology (Caltech) have participated in the NASA Faculty Fellowship Program for more than 25 years. Administrative offices are maintained both at the Caltech Campus and at JPL; however, most of the activity takes place at JPL. The Campus handles all fiscal matters. The duration of the program is ten continuous weeks. Fellows are required to conduct their research on-site. To be eligible to participate in the program, fellows must be a U.S. citizen and hold a teaching or research appointment at a U.S. university or college. The American Society of Engineering Education (ASEE) contracts with NASA and manages program recruitment. Over the past several years, we have made attempts to increase the diversity of the participants in the NFFP Program. A great deal of attention has been given to candidates from minority-serving institutions. There were approximately 100 applicants for the 34 positions in 2002. JPL was the first-choice location for more than half of them. Faculty from 16 minority-serving institutions participated as well as four women. The summer began with an orientation meeting that included introduction of key program personnel, and introduction of the fellows to each other. During this welcome, the fellows were briefed on their obligations to the program and to their JPL colleagues. They were also given a short historical perspective on JPL and its relationship to Caltech and NASA. All fellows received a package, which included information on administrative procedures, roster of fellows, seminar program, housing questionnaire, directions to JPL, maps of the local area, and a copy of the JPL Universe (a JPL newsletter). A calendar of events for the 2002 NFFP Program was designed to expose the fellows to the full range of JPL activities, seminars, tours, and trips to NASA Dryden, Goldstone, and Palomar Observatory. Weekly brown-bag lunches were also scheduled. The lunches provided a time for airing problems that may have arisen during the previous week, soliciting suggestions for program enhancement, announcements, and general socializing. Professor and Mrs. Albee also hosted the annual Summer Faculty Welcome Party at their home. During their ten-week tenure at JPL, the visiting faculty carried out projects in a wide variety of JPL's science, engineering, and technology disciplines, including communication, planetary science, materials research, reliability and quality assurance, astronomy, guidance and control, and micro-sensors. At the end of the NFFP Program, all fellows were required to complete a one-page summary of their summer s work. This was in addition to any documentation required by their host organization. Distribution of the final paycheck was dependent upon submission of this one-page summary and completion of NASA's NFFP evaluation in the EdCATS system. Fellows were also asked to complete a questionnaire for JPL, which enables the program administrators to make any appropriate changes to make the program more beneficial and effective for all involved. The 2002 NFFP Program at JPUCaltech was considered unanimously highly successful by both fellows and JPL colleagues. It provided a significant experience to most faculty members and fresh ideas to JPL researchers. Each year, suggestions for improvement include expansion of the program, longer terms, larger stipends, funds to support graduate students, and funds to continue collaborative research. The NASA Faculty Fellowship Program continues to occupy a significant place in JPL programs and serves to strengthen the ties between NASA, JPL, Caltech, and t academic community. This program is an important part of NASA's commitment to education. No inventions or patents were created during this program.

Corrosion of post-tensioning strands in ungrouted ducts - unstressed condition

NASA Astrophysics Data System (ADS)

Hutchison, Michael

Recent failures and severe corrosion distress of post-tensioned (PT) bridges in Florida have revealed corrosion of the 7-wire strands in tendons. Post-tensioned duct assemblies are fitted with multiple 7-wire steel strands and ducts are subsequently filled with grout. During construction, the length of time from the moment in which the strands have been inserted into the ducts, until the ducts are grouted, is referred to as the `ungrouted' period. During this phase, the steel strands are vulnerable to corrosion and consequently the length of this period is restricted (typically to 7 days) by construction guidelines. This investigation focuses on determining the extent of corrosion that may take place during that period, but limited to strands that were in the unstressed condition. Visual inspections and tensile testing were used to identify trends in corrosion development. Corrosion induced cracking mechanisms were also investigated via wire bending and metallographic cross section evaluation. Corrosion damage on unstressed strands during ungrouted periods of durations in the order of those otherwise currently prescribed did not appear to seriously degrade mechanical performance as measured by standardized tests. However the presence of stress in the ungrouted period, as is normally the case, may activate other mechanisms (e.g., EAC) that require further investigation. As expected in the unstressed condition, no evidence of transverse cracking was observed.

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

PubMed Central

Fornander, Louise H.; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

2014-01-01

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction. PMID:24304898

Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament.

PubMed

Fornander, Louise H; Renodon-Cornière, Axelle; Kuwabara, Naoyuki; Ito, Kentaro; Tsutsui, Yasuhiro; Shimizu, Toshiyuki; Iwasaki, Hiroshi; Nordén, Bengt; Takahashi, Masayuki

2014-02-01

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

Einstein and Millikan

NASA Astrophysics Data System (ADS)

Erwin, Charlotte

2005-03-01

Albert Einstein traveled to America by boat during the great depression to consult with scientists at the California Institute of Technology. He was a theoretical physicist, a Nobel Prize winner, and a 20th century folk hero. Few members of the general public understood his theories, but they idolized him all the same. The invitation came from physicist Robert Millikan, who had initiated a visiting-scholars program at Caltech shortly after he became head of the school in 1921. Einstein's visits to the campus in 1931, 1932, and 1933 capped Millikan's campaign to make Caltech one of the physics capitals of the world. Mount Wilson astronomer Edwin Hubble's discovery that redshifts are proportional to their distances from the observer challenged Einstein's cosmological picture of a static universe. The big question at Caltech in 1931 was whether Einstein would give up his cosmological constant and accept the idea of an expanding universe. By day, Einstein discussed his theory and its interpretation at length with Richard Tolman, Hubble, and the other scientists on the campus. By night, Einstein filled his travel diary with his personal impressions. During his third visit, Einstein sidestepped as long as possible the question of whether conditions in Germany might prevent his return there. After the January 30 announcement that Hitler had become chancellor of Germany, the question could no longer be evaded. He postponed his return trip for a few weeks and then went to Belgium for several months instead of to Berlin. In the fall of 1933, Albert Einstein returned to the United States as an emigre and became a charter member of Abraham Flexner's new Institute for Advanced Study in Princeton, New Jersey. Why did Einstein go to Princeton and not Pasadena?

Turbulent Fogwater Flux Measurements Above A Forest

NASA Astrophysics Data System (ADS)

Burkard, R.; Eugster, W.; Buetzberger, P.; Siegwolf, R.

Many forest ecosystems in elevated regions receive a significant fraction of their wa- ter and nutrient input by the interception of fogwater. Recently, several studies have demonstrated the suitability of the eddy covariance technique for the direct measure- ment of turbulent liquid water fluxes. Since summer 2001 a fogwater flux measure- ment equipment has been running at a montane site above a mixed forest canopy in Switzerland. The measurement equipment consists of a high-speed size-resolving droplet spectrometer and a three-dimensional ultrasonic anemometer. The chemical composition of the fogwater was determined from samples collected with a modified Caltech active strand collector. The deposition of nutrients by fog (occult deposition) was calculated by multiplying the total fogwater flux (total of measured turbulent and calculated gravitational flux) during each fog event by the ionic concentrations found in the collected fogwater. Several uncertainties still exist as far as the accuracy of the measurements is con- cerned. Although there is no universal statistical approach for testing the quality of the liquid water flux data directly, results of independent data quality checks of the two time series involved in the flux computation and accordingly the two instruments (ultrasonic anemometer and the droplet spectrometer) are presented. Within the measurement period, over 80 fog events with a duration longer than 2.5 hours were analyzed. An enormous physical and chemical heterogeneity among these fog events was found. We assume that some of this heterogeneity is due to the fact that fog or cloud droplets are not conservative entities: the turbulent flux of fog droplets, which can be referred to as the liquid water flux, is affected by phase change processes and coagulation. The measured coexistence of upward fluxes of small fog droplets (di- ameter < 10 µm) with the downward transport of larger droplets indicates the influ- ence of such processes. With the aid of the measured data we will present an approach which is based on the evolution of the stable isotope ratios during a fog event, on how to deal with the complexity of the evaporation and condensation processes during a fog event.

Measurements of Mercury in Rain and Fog Water from the Central Coast of California Measurements of Mercury in Rain and Fog Water from the Central Coast of California

NASA Astrophysics Data System (ADS)

Flegal, A. R.; Weiss-Penzias, P. S.; Ortiz, C.; Acosta, P.; Ryan, J. P.; Collett, J. L.

2011-12-01

Mercury (Hg) is a toxic element that can bioaccumulate in higher trophic level aquatic organisms and poses a health risk to humans and wildlife who consume those organisms. This widespread problem is exemplified by a recent survey of game fish from 152 California Lakes, which found that at least one species in 74% of the lakes sampled exceeded the lowest health threshold for methylmercury. The atmosphere is known to be an important pathway for transport of anthropogenic and natural Hg emissions sources. In this study, we investigated wet deposition of Hg through the precipitation of fog and rain water on the Central Coast of California. Fog (or marine stratus) is common on the California Central Coast and is a significant contributor to the hydrologic cycle, yet concentrations of Hg in fog have not previously been measured in this region. Our samples were collected from a small boat in the Monterey Bay, at the harbor in Moss Landing, and from a rooftop on the University of California, Santa Cruz campus, during June - July 2011 using a Caltech Active Strand Cloud Water Collector-2 that has been used previously for collection of Hg samples. Aqueous samples were analyzed for total Hg using EPA method 1631. Rainwater samples were also collected in Santa Cruz between March and June 2011. Hg concentrations ranged from 1-19 ng/L in fog and from 1-3 ng/L in rain. A previous study in Santa Cruz found a wider range of 2-18 ng/L Hg in rain, and previous studies of Hg in fog from the U.S. and Canada reported concentrations of 2-430 ng/L. Thus, our results are consistent with previous findings that Hg concentrations in fog water are at least as high, if not higher than Hg concentrations in rain. This suggests that in environments where fog is an important contributor to total precipitation, like coastal California, a significant fraction of Hg wet deposition may be occurring via fog precipitation.

Methylmercury and other chemical constituents in Pacific coastal fog water from seven sites in Central/Northern California (FogNet) during the summer of 2014

NASA Astrophysics Data System (ADS)

Weiss-Penzias, P. S.; Heim, W. A.; Fernandez, D.; Coale, K. H.; Oliphant, A. J.; Dann, D.; Porter, M.; Hoskins, D.; Dodge, C.

2014-12-01

This project investigates the mercury content in summertime Pacific coastal fog in California and whether fog could be an important vector for ocean emissions of mercury to be deposited via fog drip to upland coastal ecosystems. Efforts began in early 2014 with the building of 7 active-strand fog collectors based on the Colorado State University Caltech CASCC design. The new UCSC CASCC includes doors sealing the collector which open under microcomputer control based on environmental sensing (relative humidity). Seven sites spanning from Trinidad in the north to Marina in the south have collected samples June-August 2014 under a project called FogNet. Fog conditions were favorable for collecting large water volumes (> 250 mL) at many sites. Fog samplers were cleaned with soap and deionized water daily and field blanks taken immediately following cleaning. Fog water samples were collected overnight, split into an aliquot for anion and DOC/DIC analysis and the remaining sample was acidified. Monomethyl mercury (MMHg) concentrations in samples and field blanks for 3 sites in FogNet are shown in the accompanying figure. The range of MMHg concentrations from 10 fog water samples > 100 mL in volume was 0.9-9.3 ng/L (4.5-46.4 pM). Elevated MMHg concentrations (> 5 ng/L, 25 pM) were observed at 2 sites: UC Santa Cruz and Bodega Bay. The field blanks produced MMHg concentrations of 0.08-0.4 ng/L (0.4-2.0 pM), which was on average < 10% of the sample concentration and suggests the artifact due to sampling was small. The observed MMHg concentrations in fog water observed is this study are 1-2 orders of magnitude greater than MMHg concentrations seen previously in rain water samples from the California coast suggesting an additional source of MMHg to fog. Shipboard measurements of dimethyl mercury (DMHg) in coastal California seawater during the time period of FogNet operations (summer 2014) reveal surface waters that were supersaturated in DMHg which represents a potential source of organic mercury to the overlying fog bank.

SCDC Spanish Curricula Units. Science/Math Strand, Unit 6, Grade 2, Supplement & Ditto Packet.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The supplement and ditto packet to the science-math strand of unit six for second graders features visual materials to help the teacher carry out the instructional and assessment activities for the unit's four kits. Illustrations are provided to help stimulate oral language and conceptual development. Some are designed to be cut out for…

Archaeal ribonuclease P proteins have potential for biotechnological applications where precise hybridization of nucleic acids is needed.

PubMed

Miyanoshita, Mitsuru; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto

2015-01-01

Fluorescence resonance energy transfer-based assay showed that archaeal ribonuclease P (RNase P) proteins significantly promoted DNA annealing and strand displacement. Moreover, we found that archaeal RNase P proteins could discriminate nucleotide exchanges in DNA chains via their activity accelerating DNA strand displacement, suggesting that they have potential for biotechnological application to genetic diagnosis.

SCDC Spanish Curricula Units. Social Science Strand, Unit 8, Grade 3, Teacher's Guide--Multi-Ethnic Edition.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Communities in the nation is the theme of this social science strand for Spanish-speaking third graders. Content sources in kits 29-32 are stories, games, interviews, visual aids, and the students' own experiences. Focus, objective, and materials for each learning and assessment activity are given in English and Spanish; detailed teacher…

SCDC Spanish Curricula Units. Social Science Strand, Unit 6, Grade 2, Supplement & Ditto Packet.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The supplement and ditto packet to the social science strand of unit six for second graders contains materials for both group activities and seatwork for individual students. Based on the theme of the neighborhood as a community, the visual materials can be used to help stimulate oral language and conceptual development. Pictures of businesses,…

North American oriented strand board markets, arbitrage activity, and market price dynamics: A smooth transition approach

Treesearch

Barry Goodwin; Matthew Holt; Jeffrey P. Prestemon

2011-01-01

Price dynamics for North American oriented strand board markets are examined. The role of transactions costs are explored vis-à-vis the law of one price. Nonlinearities induced by unobservable transactions costs are modeled by estimating time-varying smooth transition autoregressions (TV-STARs). Results indicate that nonlinearity and structural change are important...

Trifluorothymidine exhibits potent antitumor activity via the induction of DNA double-strand breaks.

PubMed

Suzuki, Norihiko; Nakagawa, Fumio; Nukatsuka, Mamoru; Fukushima, Masakazu

2011-05-01

TAS-102 is an oral anticancer drug composed of trifluorothymidine (TFT) and TPI (an inhibitor of thymidine phosphorylase that strongly inhibits the biodegradation of TFT). Similar to 5-fluorouracil (5FU) and 5-fluoro-2'-deoxyuridine (FdUrd), TFT also inhibits thymidylate synthase (TS), a rate-limiting enzyme of DNA biosynthesis, and is incorporated into DNA. TFT exhibits an anticancer effect on colorectal cancer cells that have acquired 5FU and/or FdUrd resistance as a result of the overexpression of TS. Therefore, we examined the mode of action of TFT-induced DNA damage after its incorporation into DNA. When HeLa cells were treated with TFT, the number of ring-open aldehyde forms at apurinic/apyrimidinic sites increased in a dose-dependent manner, although we previously reported that no detectable excisions of TFT paired to adenine were observed using uracil DNA glycosylases, thymine DNA glycosylase or methyl-CpG binding domain 4 and HeLa whole cell extracts. To investigate the functional mechanism of TFT-induced DNA damage, we measured the phosphorylation of ATR, ATM, BRCA2, chk1 and chk2 in nuclear extracts of HeLa cells after 0, 24, 48 or 72 h of exposure to an IC(50) concentration of TFT, FdUrd or 5FU using Western blot analysis or an enzyme-linked immunosorbent assay (ELISA). Unlike FdUrd and 5FU, TFT resulted in an earlier phosphorylation of ATR and chk1 proteins after only 24 h of exposure, while phosphorylated ATM, BRCA2 and chk2 proteins were detected after more than 48 h of exposure to TFT. These results suggest that TFT causes single-strand breaks followed by double-strand breaks in the DNA of TFT-treated cells. TFT (as TAS-102) showed a more potent antitumor activity than oral 5FU on CO-3 colon cancer xenografts in mice, and such antitumor potency was supported by the increased number of double-strand breaks occurring after single-strand breaks in the DNA of the TFT-treated tumors. These results suggest that TFT causes single-strand breaks after its incorporation into DNA followed by double-strand breaks, resulting in DNA damage. This effect of TFT on DNA may explain its potent anticancer activity in cancer therapy.

Trifluorothymidine exhibits potent antitumor activity via the induction of DNA double-strand breaks

PubMed Central

SUZUKI, NORIHIKO; NAKAGAWA, FUMIO; NUKATSUKA, MAMORU; FUKUSHIMA, MASAKAZU

2011-01-01

TAS-102 is an oral anticancer drug composed of trifluorothymidine (TFT) and TPI (an inhibitor of thymidine phosphorylase that strongly inhibits the biodegradation of TFT). Similar to 5-fluorouracil (5FU) and 5-fluoro-2′-deoxyuridine (FdUrd), TFT also inhibits thymidylate synthase (TS), a rate-limiting enzyme of DNA biosynthesis, and is incorporated into DNA. TFT exhibits an anticancer effect on colorectal cancer cells that have acquired 5FU and/or FdUrd resistance as a result of the overexpression of TS. Therefore, we examined the mode of action of TFT-induced DNA damage after its incorporation into DNA. When HeLa cells were treated with TFT, the number of ring-open aldehyde forms at apurinic/apyrimidinic sites increased in a dose-dependent manner, although we previously reported that no detectable excisions of TFT paired to adenine were observed using uracil DNA glycosylases, thymine DNA glycosylase or methyl-CpG binding domain 4 and HeLa whole cell extracts. To investigate the functional mechanism of TFT-induced DNA damage, we measured the phosphorylation of ATR, ATM, BRCA2, chk1 and chk2 in nuclear extracts of HeLa cells after 0, 24, 48 or 72 h of exposure to an IC50 concentration of TFT, FdUrd or 5FU using Western blot analysis or an enzyme-linked immunosorbent assay (ELISA). Unlike FdUrd and 5FU, TFT resulted in an earlier phosphorylation of ATR and chk1 proteins after only 24 h of exposure, while phosphorylated ATM, BRCA2 and chk2 proteins were detected after more than 48 h of exposure to TFT. These results suggest that TFT causes single-strand breaks followed by double-strand breaks in the DNA of TFT-treated cells. TFT (as TAS-102) showed a more potent antitumor activity than oral 5FU on CO-3 colon cancer xenografts in mice, and such antitumor potency was supported by the increased number of double-strand breaks occurring after single-strand breaks in the DNA of the TFT-treated tumors. These results suggest that TFT causes single-strand breaks after its incorporation into DNA followed by double-strand breaks, resulting in DNA damage. This effect of TFT on DNA may explain its potent anticancer activity in cancer therapy. PMID:22977515

Asymmetric Regulation of Bipolar Single-stranded DNA Translocation by the Two Motors within Escherichia coli RecBCD Helicase*

PubMed Central

Xie, Fuqian; Wu, Colin G.; Weiland, Elizabeth; Lohman, Timothy M.

2013-01-01

Repair of double-stranded DNA breaks in Escherichia coli is initiated by the RecBCD helicase that possesses two superfamily-1 motors, RecB (3′ to 5′ translocase) and RecD (5′ to 3′ translocase), that operate on the complementary DNA strands to unwind duplex DNA. However, it is not known whether the RecB and RecD motors act independently or are functionally coupled. Here we show by directly monitoring ATP-driven single-stranded DNA translocation of RecBCD that the 5′ to 3′ rate is always faster than the 3′ to 5′ rate on DNA without a crossover hotspot instigator site and that the translocation rates are coupled asymmetrically. That is, RecB regulates both 3′ to 5′ and 5′ to 3′ translocation, whereas RecD only regulates 5′ to 3′ translocation. We show that the recently identified RecBC secondary translocase activity functions within RecBCD and that this contributes to the coupling. This coupling has implications for how RecBCD activity is regulated after it recognizes a crossover hotspot instigator sequence during DNA unwinding. PMID:23192341

Modelling nanoflares in active regions and implications for coronal heating mechanisms

PubMed Central

Cargill, P. J.; Warren, H. P.; Bradshaw, S. J.

2015-01-01

Recent observations from the Hinode and Solar Dynamics Observatory spacecraft have provided major advances in understanding the heating of solar active regions (ARs). For ARs comprising many magnetic strands or sub-loops heated by small, impulsive events (nanoflares), it is suggested that (i) the time between individual nanoflares in a magnetic strand is 500–2000 s, (ii) a weak ‘hot’ component (more than 106.6 K) is present, and (iii) nanoflare energies may be as low as a few 1023 ergs. These imply small heating events in a stressed coronal magnetic field, where the time between individual nanoflares on a strand is of order the cooling time. Modelling suggests that the observed properties are incompatible with nanoflare models that require long energy build-up (over 10 s of thousands of seconds) and with steady heating. PMID:25897093

Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes

PubMed Central

Kim, Kevin; Lee, Young Sik; Carthew, Richard W.

2007-01-01

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA. PMID:17123955

Quantitative, non-invasive imaging of radiation-induced DNA double strand breaks in vivo

PubMed Central

Li, Wenrong; Li, Fang; Huang, Qian; Shen, Jingping; Wolf, Frank; He, Yujun; Liu, Xinjian; Hu, Y. Angela; Bedford, Joel. S.; Li, Chuan-Yuan

2011-01-01

DNA double strand breaks is a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA double strand breaks is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here we report a novel approach to image and quantify DNA double strand breaks in live mammalian cells through bi-fragment luciferase reconstitution. N- and C- terminal fragments of firefly luciferase gene were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DNA double strand breaks, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C- luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, non-invasive quantification of DNA double strand breaks in cells irradiated with x-rays and 56Fe ions. Furthermore, it allowed for the evaluation of DNA double strand breaks (DSBs) non-invasively in vivo in irradiated tumors over two weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DNA double strand break repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time. PMID:21527553

Research in cosmic and gamma ray astrophysics: Cosmic physics portion

NASA Technical Reports Server (NTRS)

Stone, Edward C.; Mewaldt, Richard A.; Schindler, Stephen

1993-01-01

Research in particle astrophysics at the Space Radiation Laboratory (SRL) of the California Institute of Technology is supported under NASA Grant NAGW-1919. A three-year proposal for continuation of support was submitted a year ago and put into effect 1 October 1992. This report is the combined progress report and continuation application called for under the Federal Demonstration Project. Gamma-ray Astrophysics at SRL is separately supported under NAGW-1919 and will be separately summarized and proposed. This report will document progress and plans for our particle spectroscopy activities and for related data analysis, calibration, and community service activities. A bibliography and a budget will be attached as appendices. The Caltech SRL research program includes a heavy emphasis on elemental and isotopic spectroscopy of energetic particles in the cosmic radiation; in solar, interplanetary, and anomalous 'cosmic' radiation; and in planetary magnetospheres as discussed.

Concurrent Image Processing Executive (CIPE). Volume 2: Programmer's guide

NASA Technical Reports Server (NTRS)

Williams, Winifred I.

1990-01-01

This manual is intended as a guide for application programmers using the Concurrent Image Processing Executive (CIPE). CIPE is intended to become the support system software for a prototype high performance science analysis workstation. In its current configuration CIPE utilizes a JPL/Caltech Mark 3fp Hypercube with a Sun-4 host. CIPE's design is capable of incorporating other concurrent architectures as well. CIPE provides a programming environment to applications' programmers to shield them from various user interfaces, file transactions, and architectural complexities. A programmer may choose to write applications to use only the Sun-4 or to use the Sun-4 with the hypercube. A hypercube program will use the hypercube's data processors and optionally the Weitek floating point accelerators. The CIPE programming environment provides a simple set of subroutines to activate user interface functions, specify data distributions, activate hypercube resident applications, and to communicate parameters to and from the hypercube.

Dynamic Resource Allocation in Conservation Planning

DTIC Science & Technology

2011-08-01

0932392 and IIS-0953413, the Caltech Center for the Mathematics of Information, and by the US Fish and Wildlife Service. We thank J. Bakker, J. Bush ...Moilanen, A.; Pakkala, T.; and Kuussaari, M. 1996. The quantitative incidence function model and persistence of an endangered butterfly metapopulation

Lunar and planetary studies

NASA Technical Reports Server (NTRS)

Muhleman, Duane O.; Goldreich, P.; Ingersoll, A. P.; Westphal, J. A.

1988-01-01

This grant supports the core program in planetary astronomy at Caltech. The research includes observations in the IR, sub-mm, mm and cm wavelengths at national and Caltech observatories with a strong emphasis on integrating the observations with spacecraft data and with models of atmospheric structure, dynamics and chemistry. Muhleman's group made extensive observations of Saturn, Uranus and Neptune which are being interpreted in terms of deep atmospheric structures which are obvious in the 2 and 6 cm maps of Saturn and Uranus. The microwave measurements are one of the few sources of information below the 2 bar level. Goldreich is investigating the dynamics of narrow rings with postdoctoral fellow, Pierre-Yves Longaretti. Their work has focused on the role of collisional stresses on the precession of the rings, since the Voyager radio science results imply that the previous model based on the ring's self-gravity is not the entire story. In addition Borderies, Goldreich and Tremaine have completed an investigation of the dynamics of the Encke division in Saturn's A ring.

Mycobacterium leprae RecA is structurally analogous but functionally distinct from Mycobacterium tuberculosis RecA protein.

PubMed

Patil, K Neelakanteshwar; Singh, Pawan; Harsha, Sri; Muniyappa, K

2011-12-01

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein. Copyright © 2011 Elsevier B.V. All rights reserved.

Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.

PubMed

Joseph, Prem Raj B; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M; Garofalo, Roberto P; Rajarathnam, Krishna

2013-09-17

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Using dual-polarization interferometry to study surface-initiated DNA hybridization chain reactions in real time.

PubMed

Huang, Fujian; Xu, Pingping; Liang, Haojun

2014-01-15

In this study we used dual-polarization interferometry to investigate DNA hybridization chain reactions (HCRs) at solid-liquid interfaces. We monitored the effects of variations in mass, thickness, and density of the immobilized initiator on the subsequent HCRs at various salt concentrations. At low salt concentrations, the single-stranded DNA (ssDNA) initiator was attached uniformly to the chip surface. At high salt concentrations, it lay on the surface at the onset of the immobilization process, but the approaching ssDNA forced the pre-immobilized ssDNA strands to extend into solution as a result of increased electrostatic repulsion between the pre-adsorbed and approaching ssDNA chains. Injection of a mixture of H1 and H2 increased the mass and thickness of the films initially, but thereafter the thickness decreased. These changes indicate that the long double-stranded DNA that formed lay on the surface, rather than extended into the solution, thereby suppressing the subsequent initiation activity of the released single-strand parts of H1 and H2. Increasing the salt concentration increased the HCR efficiency and reaction rate. The HCR efficiency of the initiator ssDNA immobilized on its 5' end was higher than that immobilized on its 3' end, suggesting that the released single-strand parts of H1 and H2 close to the chip surface decreased the initiation activity relative to those of the ones extending into solution. © 2013 Elsevier B.V. All rights reserved.

Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices.

PubMed

Robertson, Neil M; Hizir, Mustafa Salih; Balcioglu, Mustafa; Wang, Rui; Yavuz, Mustafa Selman; Yumak, Hasan; Ozturk, Birol; Sheng, Jia; Yigit, Mehmet V

2015-09-15

In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR-10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

Overview of the Inland California Translational Consortium

NASA Astrophysics Data System (ADS)

Malkas, Linda H.

2017-05-01

The mission of the Inland California Translational Consortium (ICTC), an independent research consortium comprising a unique hub of regional institutions (City of Hope [COH], California Institute of Technology [Caltech], Jet Propulsion Laboratory [JPL], University of California Riverside [UCR], and Claremont Colleges Keck Graduate Institute [KGI], is to institute a new paradigm within the academic culture to accelerate translation of innovative biomedical discoveries into clinical applications that positively affect human health and life. The ICTC actively supports clinical translational research as well as the implementation and advancement of novel education and training models for the translation of basic discoveries into workable products and practices that preserve and improve human health while training and educating at all levels of the workforce using innovative forward-thinking approaches.

A Study of Stranding of Juvenile Salmon by Ship Wakes Along the Lower Columbia River Using a Before-and-After Design: Before-Phase Results

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pearson, Walter H.; Skalski, J R.; Sobocinski, Kathryn L.

2006-02-01

Ship wakes produced by deep-draft vessels transiting the lower Columbia River have been observed to cause stranding of juvenile salmon. Proposed deepening of the Columbia River navigation channel has raised concerns about the potential impact of the deepening project on juvenile salmon stranding. The Portland District of the U.S. Army Corps of Engineers requested that the Pacific Northwest National Laboratory design and conduct a study to assess stranding impacts that may be associated with channel deepening. The basic study design was a multivariate analysis of covariance of field observations and measurements under a statistical design for a before and aftermore » impact comparison. We have summarized field activities and statistical analyses for the ?before? component of the study here. Stranding occurred at all three sampling sites and during all three sampling seasons (Summer 2004, Winter 2005, and Spring 2005), for a total of 46 stranding events during 126 observed vessel passages. The highest occurrence of stranding occurred at Barlow Point, WA, where 53% of the observed events resulted in stranding. Other sites included Sauvie Island, OR (37%) and County Line Park, WA (15%). To develop an appropriate impact assessment model that accounted for relevant covariates, regression analyses were conducted to determine the relationships between stranding probability and other factors. Nineteen independent variables were considered as potential factors affecting the incidence of juvenile salmon stranding, including tidal stage, tidal height, river flow, current velocity, ship type, ship direction, ship condition (loaded/unloaded), ship speed, ship size, and a proxy variable for ship kinetic energy. In addition to the ambient and ship characteristics listed above, site, season, and fish density were also considered. Although no single factor appears as the primary factor for stranding, statistical analyses of the covariates resulted in the following equations: (1) Stranding Probability {approx} Location + Kinetic Energy Proxy + Tidal Height + Salmonid Density + Kinetic energy proxy ? Tidal Height + Tidal Height x Salmonid Density. (2) Stranding Probability {approx} Location + Total Wave Distance + Salmonid Density Index. (3) Log(Total Wave Height) {approx} Ship Block + Tidal Height + Location + Ship Speed. (4) Log(Total Wave Excursion Across the Beach) {approx} Location + Kinetic Energy Proxy + Tidal Height The above equations form the basis for a conceptual model of the factors leading to salmon stranding. The equations also form the basis for an approach for assessing impacts of dredging under the before/after study design.« less

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

PubMed Central

Kuzminov, Andrei

1999-01-01

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

Development of novel decoy oligonucleotides: advantages of circular dumb-bell decoy.

PubMed

Tomita, Naruya; Tomita, Tetsuya; Yuyama, Kazuhiko; Tougan, Takahiro; Tajima, Tsuyoshi; Ogihara, Toshio; Morishita, Ryuichi

2003-04-01

The inhibition of specific transcription regulatory proteins is a novel approach to regulate gene expression. The transcriptional activities of DNA binding proteins can be inhibited by the use of double-stranded oligonucleotides (ODNs) that compete for binding to their specific target sequences in promoters and enhancers. Transfection of this cis-element double-stranded ODN, referred to as decoy ODN, has been reported to be a powerful tool that provides a new class of anti-gene strategies to gene therapy and permits examination of specific gene regulation. We have demonstrated the usefulness of this decoy ODN strategy in animal models of restenosis, myocardial infarction, glomerulonephritis and rheumatoid arthritis. However, one of the major limitations of decoy ODN technology is the rapid degradation of phosphodiester ODNs by intracellular nucleases. To date, several different types of double-stranded decoy ODNs have been developed to overcome this issue. Circular dumb-bell (CD) double-stranded decoy ODNs that were developed to resolve this issue have attracted a high level of interest. In this review, the applications of decoy ODN strategy and the advantages of modified CD double-stranded decoy ODNs will be discussed.

BLM helicase facilitates telomere replication during leading strand synthesis of telomeres

PubMed Central

Kosiyatrakul, Settapong T.

2015-01-01

Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome–associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, we found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, our findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis. PMID:26195664

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

High-resolution physical and functional mapping of the template adjacent DNA binding site in catalytically active telomerase.

PubMed

Romi, Erez; Baran, Nava; Gantman, Marina; Shmoish, Michael; Min, Bosun; Collins, Kathleen; Manor, Haim

2007-05-22

Telomerase is a cellular reverse transcriptase, which utilizes an integral RNA template to extend single-stranded telomeric DNA. We used site-specific photocrosslinking to map interactions between DNA primers and the catalytic protein subunit (tTERT) of Tetrahymena thermophila telomerase in functional enzyme complexes. Our assays reveal contact of the single-stranded DNA adjacent to the primer-template hybrid and tTERT residue W187 at the periphery of the N-terminal domain. This contact was detected in complexes with three different registers of template in the active site, suggesting that it is maintained throughout synthesis of a complete telomeric repeat. Substitution of nearby residue Q168, but not W187, alters the K(m) for primer elongation, implying that it plays a role in the DNA recognition. These findings are the first to directly demonstrate the physical location of TERT-DNA contacts in catalytically active telomerase and to identify amino acid determinants of DNA binding affinity. Our data also suggest a movement of the TERT active site relative to the template-adjacent single-stranded DNA binding site within a cycle of repeat synthesis.

Reovirus-induced Ribonucleic Acid Polymerase

PubMed Central

Watanabe, Y.; Gauntt, C. J.; Graham, A. F.

1968-01-01

A virus-induced ribonucleic acid (RNA) polymerase activity was found in L cells infected with type 3 reovirus. Most of the enzyme is associated with the “large particle” fraction of the infected cells. The enzyme first appeared at 3 to 5 hr after infection and increased in amount until 7 to 9 hr. All four ribonucleoside triphosphates are incorporated in vitro into an acid-insoluble form by the enzyme. The major part of the product formed in vitro is a double-stranded RNA indistinguishable from viral RNA by electrophoresis on polyacrylamide gel. Approximately 40% of the product is a single-stranded RNA of relatively small molecular weight. More than 95% of the nucleotides incorporated into double-stranded RNA by the enzyme are bound in internal 3′-5′-phosphodiester linkages extending back from both 3′- and 5′-termini of the RNA strands. PMID:5725319

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanli; Sheng, Gang; Juranek, Stefan

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas twomore » critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.« less

XPD-dependent activation of apoptosis in response to triplex-induced DNA damage

PubMed Central

Kaushik Tiwari, Meetu; Rogers, Faye A.

2013-01-01

DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of γH2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with γH2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation. PMID:23913414

Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways

PubMed Central

Leitner, Wolfgang W.; Hwang, Leroy N.; Deveer, Michael J.; Zhou, Aimin; Silverman, Robert H.; Williams, Bryan R.G.; Dubensky, Thomas W.; Ying, Han; Restifo, Nicholas P.

2006-01-01

Cancer vaccines targeting ‘self’ antigens that are expressed at consistently high levels by tumor cells are potentially useful in immunotherapy, but immunological tolerance may block their function. Here, we describe a novel, naked DNA vaccine encoding an alphavirus replicon (self-replicating mRNA) and the self/tumor antigen tyrosinase-related protein-1. Unlike conventional DNA vaccines, this vaccine can break tolerance and provide immunity to melanoma. The vaccine mediates production of double-stranded RNA, as evidenced by the autophosphorylation of protein kinase R. Double-stranded RNA is critical to vaccine function because both the immunogenicity and the anti-tumor activity of the vaccine are blocked in mice deficient for the RNase L enzyme, a key component of the 2′,5′-linked oligoadenylate synthetase antiviral pathway involved in double-stranded RNA recognition. This study shows for the first time that alphaviral replicon-encoding DNA vaccines activate innate immune pathways known to drive antiviral immune responses, and points the way to strategies for improving the efficacy of immunization with naked DNA. PMID:12496961

Optically Controlled Signal Amplification for DNA Computation.

PubMed

Prokup, Alexander; Hemphill, James; Liu, Qingyang; Deiters, Alexander

2015-10-16

The hybridization chain reaction (HCR) and fuel-catalyst cycles have been applied to address the problem of signal amplification in DNA-based computation circuits. While they function efficiently, these signal amplifiers cannot be switched ON or OFF quickly and noninvasively. To overcome these limitations, a light-activated initiator strand for the HCR, which enabled fast optical OFF → ON switching, was developed. Similarly, when a light-activated version of the catalyst strand or the inhibitor strand of a fuel-catalyst cycle was applied, the cycle could be optically switched from OFF → ON or ON → OFF, respectively. To move the capabilities of these devices beyond solution-based operations, the components were embedded in agarose gels. Irradiation with customizable light patterns and at different time points demonstrated both spatial and temporal control. The addition of a translator gate enabled a spatially activated signal to travel along a predefined path, akin to a chemical wire. Overall, the addition of small light-cleavable photocaging groups to DNA signal amplification circuits enabled conditional control as well as fast photocontrol of signal amplification.

Histone Variant Regulates DNA Repair via Chromatin Condensation | Center for Cancer Research

Cancer.gov

Activating the appropriate DNA repair pathway is essential for maintaining the stability of the genome after a break in both strands of DNA. How a pathway is selected, however, is not well understood. Since these double strand breaks (DSBs) occur while DNA is packaged as chromatin, changes in its organization are necessary for repair to take place. Numerous alterations have

SCDC Spanish Curricula Units. Spanish SL Strand, Unit 6, Grade 2, Supplement & Ditto Packet.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

The supplement and ditto packet to the Spanish as a Second Language strand of unit six for second graders contains activities for both seatwork and group participation. Unit six is oriented toward the basic theme of the neighborhood as a community; Kits 21-24 reflect the spiraling questions of what the elements are, how wants and needs are…

SCDC Spanish Curricula Units. Science/Math Strand, Unit 8, Grade 3, Teacher's Guide--Multi-Ethnic Edition.

ERIC Educational Resources Information Center

Spanish Curricula Development Center, Miami Beach, FL.

Built around a theme of communities in the nation, this teacher's guide for unit eight of a science/math strand for Spanish-speaking children in grade three contains both learning and assessment activities. This unit addresses what the elements are, how needs and wants are satisfied, how change occurs, and what the results of change are. The guide…

An Analysis of Enzyme Kinetics Data for Mitochondrial DNA Strand Termination by Nucleoside Reverse Transcription Inhibitors

PubMed Central

Wendelsdorf, Katherine V.; Song, Zhuo; Cao, Yang; Samuels, David C.

2009-01-01

Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-γ hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-γ) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-γ with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication. PMID:19132079

The fission yeast meiosis-specific Dmc1 recombinase mediates formation and branch migration of Holliday junctions by preferentially promoting strand exchange in a direction opposite to that of Rad51

PubMed Central

Murayama, Yasuto; Tsutsui, Yasuhiro; Iwasaki, Hiroshi

2011-01-01

Homologous recombination proceeds via the formation of several intermediates including Holliday junctions (HJs), which are important for creating crossover products. DNA strand exchange is a core reaction that produces these intermediates that is directly catalyzed by RecA family recombinases, of which there are two types in eukaryotes: universal Rad51 and meiosis-specific Dmc1. We demonstrated previously that Rad51 promotes four-strand exchange, mimicking the formation and branch migration of HJs. Here we show that Dmc1 from fission yeast has a similar activity, which requires ATP hydrolysis and is independent of an absolute requirement for the Swi5–Sfr1 complex. These features are critically different from three-strand exchange mediated by Dmc1, but similar to those of four-strand exchange mediated by Rad51, suggesting that strand exchange reactions between duplex–duplex and single-duplex DNAs are mechanistically different. Interestingly, despite similarities in protein structure and in reaction features, the preferential polarities of Dmc1 and Rad51 strand exchange are different (Dmc1 promotes exchange in the 5′-to-3′ direction and Rad51 promotes exchange in the 3′-to-5′ direction relative to the ssDNA region of the DNA substrate). The significance of the Dmc1 polarity is discussed within the context of the necessity for crossover production. PMID:21363965

Non-encapsidation Activities of the Capsid Proteins of Positive-strand RNA Viruses

PubMed Central

Ni, Peng; Kao, C. Cheng

2013-01-01

Viral capsid proteins (CPs) are characterized by their role in forming protective shells around viral genomes. However, CPs have additional and important roles in the virus infection cycles and in the cellular response to infection. These activities involve CP binding to RNAs in both sequence-specific and nonspecific manners as well as association with other proteins. This review focuses on CPs of both plant and animal-infecting viruses with positive-strand RNA genomes. We summarize the structural features of CPs and describe their modulatory roles in viral translation, RNA-dependent RNA synthesis, and host defense responses. PMID:24074574

Refolding of β-Stranded Class I Chitinases of Hippophae rhamnoides Enhances the Antifreeze Activity during Cold Acclimation

PubMed Central

Gupta, Ravi; Deswal, Renu

2014-01-01

Class I chitinases hydrolyse the β-1,4-linkage of chitin and also acquire antifreeze activity in some of the overwintering plants during cold stress. Two chitinases, HrCHT1a of 31 kDa and HrCHT1b of 34 kDa, were purified from cold acclimated and non-acclimated seabuckthorn seedlings using chitin affinity chromatography. 2-D gels of HrCHT1a and HrCHT1b showed single spots with pIs 7.0 and 4.6 respectively. N-terminal sequence of HrCHT1b matched with the class I chitinase of rice and antifreeze proteins while HrCHT1a could not be sequenced as it was N-terminally blocked. Unlike previous reports, where antifreeze activity of chitinase was cold inducible, our results showed that antifreeze activity is constitutive property of class I chitinase as both HrCHT1a and HrCHT1b isolated even from non-acclimated seedlings, exhibited antifreeze activity. Interestingly, HrCHT1a and HrCHT1b purified from cold acclimated seedlings, exhibited 4 and 2 times higher antifreeze activities than those purified from non-acclimated seedlings, suggesting that antifreeze activity increased during cold acclimation. HrCHT1b exhibited 23–33% higher hydrolytic activity and 2–4 times lower antifreeze activity than HrCHT1a did. HrCHT1b was found to be a glycoprotein; however, its antifreeze activity was independent of glycosylation as even deglycosylated HrCHT1b exhibited antifreeze activity. Circular dichroism (CD) analysis showed that both these chitinases were rich in unusual β-stranded conformation (36–43%) and the content of β-strand increased (∼11%) during cold acclimation. Surprisingly, calcium decreased both the activities of HrCHT1b while in case of HrCHT1a, a decrease in the hydrolytic activity and enhancement in its antifreeze activity was observed. CD results showed that addition of calcium also increased the β-stranded conformation of HrCHT1a and HrCHT1b. This is the first report, which shows that antifreeze activity is constitutive property of class I chitinase and cold acclimation and calcium regulate these activities of chitinases by changing the secondary structure. PMID:24626216

Refolding of β-stranded class I chitinases of Hippophae rhamnoides enhances the antifreeze activity during cold acclimation.

PubMed

Gupta, Ravi; Deswal, Renu

2014-01-01

Class I chitinases hydrolyse the β-1,4-linkage of chitin and also acquire antifreeze activity in some of the overwintering plants during cold stress. Two chitinases, HrCHT1a of 31 kDa and HrCHT1b of 34 kDa, were purified from cold acclimated and non-acclimated seabuckthorn seedlings using chitin affinity chromatography. 2-D gels of HrCHT1a and HrCHT1b showed single spots with pIs 7.0 and 4.6 respectively. N-terminal sequence of HrCHT1b matched with the class I chitinase of rice and antifreeze proteins while HrCHT1a could not be sequenced as it was N-terminally blocked. Unlike previous reports, where antifreeze activity of chitinase was cold inducible, our results showed that antifreeze activity is constitutive property of class I chitinase as both HrCHT1a and HrCHT1b isolated even from non-acclimated seedlings, exhibited antifreeze activity. Interestingly, HrCHT1a and HrCHT1b purified from cold acclimated seedlings, exhibited 4 and 2 times higher antifreeze activities than those purified from non-acclimated seedlings, suggesting that antifreeze activity increased during cold acclimation. HrCHT1b exhibited 23-33% higher hydrolytic activity and 2-4 times lower antifreeze activity than HrCHT1a did. HrCHT1b was found to be a glycoprotein; however, its antifreeze activity was independent of glycosylation as even deglycosylated HrCHT1b exhibited antifreeze activity. Circular dichroism (CD) analysis showed that both these chitinases were rich in unusual β-stranded conformation (36-43%) and the content of β-strand increased (∼11%) during cold acclimation. Surprisingly, calcium decreased both the activities of HrCHT1b while in case of HrCHT1a, a decrease in the hydrolytic activity and enhancement in its antifreeze activity was observed. CD results showed that addition of calcium also increased the β-stranded conformation of HrCHT1a and HrCHT1b. This is the first report, which shows that antifreeze activity is constitutive property of class I chitinase and cold acclimation and calcium regulate these activities of chitinases by changing the secondary structure.

Inhibition of APOBEC3G activity impedes double-stranded DNA repair.

PubMed

Prabhu, Ponnandy; Shandilya, Shivender M D; Britan-Rosich, Elena; Nagler, Adi; Schiffer, Celia A; Kotler, Moshe

2016-01-01

The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to ionizing radiation. In the current study, we show that additional peptides derived from Vif, A3G, and APOBEC3F, which contain the LYYF motif, inhibit deamination activity. Each residue in the Vif25-39 sequence moderately contributes to the inhibitory effect, whereas replacing a single residue in the LYYF motif completely abrogates inhibition of deamination. Treatment of A3G-expressing lymphoma cells exposed to ionizing radiation with the new inhibitory peptides reduces double-strand break repair after irradiation. Incubation of cultured irradiated lymphoma cells with peptides that inhibit double-strand break repair halts their propagation. These results suggest that A3G may be a potential therapeutic target that is amenable to peptide and peptidomimetic inhibition. © 2015 FEBS.

Quencher-free fluorescence strategy for detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification.

PubMed

Liu, Haisheng; Ma, Changbei; Zhou, Meijuan; Chen, Hanchun; He, Hailun; Wang, Kemin

2016-11-01

This work demonstrates a novel method for DNA methyltransferase (MTase) activity detection with a quencher-free molecular beacon (MB) probe based on exonuclease (Exo) III-assisted signal amplification. In the presence of Dam MTase and DpnI endonuclease, the elaborately designed hairpin substrate (MB1) was cleaved into two parts (part A and part B). Exo III can then digest part A and release a single-stranded target of the 2-aminopurine-labeled MB (MB2). Subsequently, the MB2 can hybridize with its target to form a double-stranded structure with a protruding 3'-terminus and then trigger the digestion of MB2 by Exo III. During the digestion of MB2, the 2-aminopurine is separated from the DNA strands and released free in solution, inducing an increase of the fluorescent signal. Owing to the presence of a recessed 3'-terminus in the formed double-stranded DNA, Exo III-assisted recyclable cleavage of MB2 was achieved. Therefore, an amplified fluorescence signal was observed. Under the optimized conditions, Dam MTase can be detected in the range of 0.2-40 units/mL with a limit of detection of 0.2 units/mL and good selectivity. Furthermore, the present assay can be used for screening potential DNA MTase inhibitors. Graphical Abstract A quencher-free fluorescence assay for sensitive detection of DNA methyltransferase activity based on exonuclease III-assisted signal amplification is reported.

Mixed Dimensional Van der Waals Heterostructures for Opto-Electronics.

NASA Astrophysics Data System (ADS)

Jariwala, Deep

The isolation of a growing number of two-dimensional (2D) materials has inspired worldwide efforts to integrate distinct 2D materials into van der Waals (vdW) heterostructures. While a tremendous amount of research activity has occurred in assembling disparate 2D materials into ``all-2D'' van der Waals heterostructures, this concept is not limited to 2D materials alone. Given that any passivated, dangling bond-free surface will interact with another via vdW forces, the vdW heterostructure concept can be extended to include the integration of 2D materials with non-2D materials that adhere primarily through noncovalent interactions. In the first part of this talk I will present our work on emerging mixed-dimensional (2D + nD, where n is 0, 1 or 3) heterostructure devices performed at Northwestern University. I will present two distinct examples of gate-tunable p-n heterojunctions 1. Single layer n-type MoS2\\ (2D) combined with p-type semiconducting single walled carbon nanotubes (1D) and 2. Single layer MoS2 combined with 0D molecular semiconductor, pentacene. I will present the unique electrical properties, underlying charge transport mechanisms and photocurrent responses in both the above systems using a variety of scanning probe microscopy techniques as well as computational analysis. This work shows that van der Waals interactions are robust across different dimensionalities of materials and can allow fabrication of semiconductor devices with unique geometries and properties unforeseen in bulk semiconductors. Finally, I will briefly discuss our recent work from Caltech on near-unity absorption in atomically-thin photovoltaic devices. This work is supported by the Materials Research Center at Northwestern University, funded by the National Science Foundation (NSF DMR-1121262) and the Resnick Sustainability Institute at Caltech.

The NuSTAR Education and Public Outreach Program

NASA Astrophysics Data System (ADS)

Cominsky, Lynn R.; McLin, K. M.; NuSTAR Team

2010-01-01

The Nuclear Spectroscopic Telescope Array (NuSTAR) is a NASA Small Explorer mission led by Caltech, managed by JPL, and implemented by an international team of scientists and engineers, under the direction of CalTech Professor Fiona Harrison, principal investigator. NuSTAR is a pathfinder mission that will open the high-energy X-ray sky for sensitive study for the first time. By focusing X-rays at higher energies (up to 79 keV) NuSTAR will answer fundamental questions about the Universe: How are black holes distributed through the cosmos? How were the elements that compose our bodies and the Earth forged in the explosions of massive stars? What powers the most extreme active galaxies? Perhaps most exciting is the opportunity to fill a blank map with wonders we have not yet dreamed of: NuSTAR offers the opportunity to explore our Universe in an entirely new way. The purpose of the NuSTAR E/PO program is to increase student and public understanding of the science of the high-energy Universe, by capitalizing on the synergy of existing high-energy astrophysics E/PO programs to support the mission's objectives. Our goals are to: facilitate understanding of the nature of collapsed objects, develop awareness of the role of supernovae in creating the chemical elements and to facilitate understanding of the physical properties of the extreme Universe. We will do this through a program that includes educator workshops through NASA's Astrophysics Educator Ambassador program, by writing articles for Physics Teacher and Science Scope magazines to reach a broader community of educators, and by working with informal educators through museums and planetaria to develop an exhibit that includes a model of NuSTAR and describes the mission's science objectives. We will also develop printed materials such as a mission factsheet that describes the mission.

The NuSTAR Education and Public Outreach Program

NASA Astrophysics Data System (ADS)

Cominsky, Lynn R.; McLin, K. M.; NuSTAR Team

2010-03-01

NuSTAR is a NASA Small Explorer mission led by Caltech, managed by JPL, and implemented by an international team of scientists and engineers, under the direction of CalTech Professor Fiona Harrison, principal investigator. NuSTAR is a pathfinder mission that will open the high-energy X-ray sky for sensitive study for the first time. By focusing X-rays at higher energies (up to 79 keV) NuSTAR will answer fundamental questions about the Universe: How are black holes distributed through the cosmos? How were the elements that compose our bodies and the Earth forged in the explosions of massive stars? What powers the most extreme active galaxies? Perhaps most exciting is the opportunity to fill a blank map with wonders we have not yet dreamed of: NuSTAR offers the opportunity to explore our Universe in an entirely new way. The purpose of the NuSTAR E/PO program is to increase student and public understanding of the science of the high-energy Universe, by capitalizing on the synergy of existing high-energy astrophysics E/PO programs to support the mission's objectives. Our goals are to: facilitate understanding of the nature of collapsed objects, develop awareness of the role of supernovae in creating the chemical elements and to facilitate understanding of the physical properties of the extreme Universe. We will do this through a program that includes educator workshops through NASA's Astrophysics Educator Ambassador program, by writing articles for Physics Teacher and Science Scope magazines to reach a broader community of educators, and by working with informal educators through museums and planetaria to develop an exhibit that includes a model of NuSTAR and describes the mission's science objectives. We will also develop printed materials such as a mission factsheet that describes the mission.

Forward modeling transient brightenings and microflares around an active region observed with Hi-C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobelski, Adam R.; McKenzie, David E., E-mail: kobelski@solar.physics.montana.edu

Small-scale flare-like brightenings around active regions are among the smallest and most fundamental of energetic transient events in the corona, providing a testbed for models of heating and active region dynamics. In a previous study, we modeled a large collection of these microflares observed with Hinode/X-Ray Telescope (XRT) using EBTEL and found that they required multiple heating events, but could not distinguish between multiple heating events on a single strand, or multiple strands each experiencing a single heating event. We present here a similar study, but with extreme-ultraviolet data of Active Region 11520 from the High Resolution Coronal Imager (Hi-C)more » sounding rocket. Hi-C provides an order of magnitude improvement to the spatial resolution of XRT, and a cooler temperature sensitivity, which combine to provide significant improvements to our ability to detect and model microflare activity around active regions. We have found that at the spatial resolution of Hi-C (≈0.''3), the events occur much more frequently than expected (57 events detected, only 1 or 2 expected), and are most likely made from strands of the order of 100 km wide, each of which is impulsively heated with multiple heating events. These findings tend to support bursty reconnection as the cause of the energy release responsible for the brightenings.« less

Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA

NASA Technical Reports Server (NTRS)

Opitz, J. G.; Ciglic, M. I.; Haugg, M.; Trautwein-Fritz, K.; Raillard, S. A.; Jermann, T. M.; Benner, S. A.

1998-01-01

Bovine seminal ribonuclease (RNase) binds, melts, and (in the case of RNA) catalyzes the hydrolysis of double-stranded nucleic acid 30-fold better under physiological conditions than its pancreatic homologue, the well-known RNase A. Reported here are site-directed mutagenesis experiments that identify the sequence determinants of this enhanced catalytic activity. These experiments have been guided in part by experimental reconstructions of ancestral RNases from extinct organisms that were intermediates in the evolution of the RNase superfamily. It is shown that the enhanced interactions between bovine seminal RNase and double-stranded nucleic acid do not arise from the increased number of basic residues carried by the seminal enzyme. Rather, a combination of a dimeric structure and the introduction of two glycine residues at positions 38 and 111 on the periphery of the active site confers the full catalytic activity of bovine seminal RNase against duplex RNA. A structural model is presented to explain these data, the use of evolutionary reconstructions to guide protein engineering experiments is discussed, and a new variant of RNase A, A(Q28L K31C S32C D38G E111G), which contains all of the elements identified in these experiments as being important for duplex activity, is prepared. This is the most powerful catalyst within this subfamily yet observed, some 46-fold more active against duplex RNA than RNase A.

RNA chaperone activity of human La protein is mediated by variant RNA recognition motif.

PubMed

Naeeni, Amir R; Conte, Maria R; Bayfield, Mark A

2012-02-17

La proteins are conserved factors in eukaryotes that bind and protect the 3' trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3'OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3'OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets.

Dispersion, Mixing, and Combustion in Turbulent and High-Speed Flows, Air-Breathing Propulsion, and Hypersonic Flight

DTIC Science & Technology

2010-03-31

postdoctoral research of Antonino Ferrante (currently Assistant Professor at the University of Washington). The fluid dynamics video "LES of an inclined jet...Northrop Professor of Aeronautics and Professor of Applied Physics, Caltech, and ChiefTechnologist, JPL. Ferrante, Antonino : Postdoctoral Scholar in

JPL Non-NASA Programs

NASA Technical Reports Server (NTRS)

Cox, Robert S.

2006-01-01

A viewgraph presentation describing JPL's non-NASA Programs is shown. The contents include: 1) JPL/Caltech: National Security Heritage; 2) Organization and Portfolio; 3) Synergistic Areas of Interest; 4) Business Environment; 5) National Space Community; 6) New Business Environment; 7) Technology Transfer Techniques; 8) Innovative Partnership Program (IPP); and 9) JPL's Track Record.

Micro Power

DTIC Science & Technology

2004-02-01

GENERATOR ENERGIZED BY A MEMS BASED CHEMICAL- THERMAL REACTOR” 5-6 PI: Professor Robert F. Savinell Co-I(s): Professors C. C. Liu, M. H . Litt, and J. S...January 8, 2002 PI Chih-Ming Ho UCLA Yu-Chong Tai Caltech Siegfried Janson Henry Helvajian Aerospace Corporation H.Q. Yang Andrzej J. Przekwasa CFDRC Co

RNA polymerase activity is associated with viral particles isolated from Leishmania braziliensis subsp. guyanensis.

PubMed Central

Widmer, G; Keenan, M C; Patterson, J L

1990-01-01

Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found. Images PMID:2370680

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

PubMed Central

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress. PMID:26765540

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks.

PubMed

Federico, María Belén; Vallerga, María Belén; Radl, Analía; Paviolo, Natalia Soledad; Bocco, José Luis; Di Giorgio, Marina; Soria, Gastón; Gottifredi, Vanesa

2016-01-01

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles.

PubMed

Molina-Sánchez, Maria D; García-Rodríguez, Fernando M; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3' end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro . The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods.

Functionality of In vitro Reconstituted Group II Intron RmInt1-Derived Ribonucleoprotein Particles

PubMed Central

Molina-Sánchez, Maria D.; García-Rodríguez, Fernando M.; Toro, Nicolás

2016-01-01

The functional unit of mobile group II introns is a ribonucleoprotein particle (RNP) consisting of the intron-encoded protein (IEP) and the excised intron RNA. The IEP has reverse transcriptase activity but also promotes RNA splicing, and the RNA-protein complex triggers site-specific DNA insertion by reverse splicing, in a process called retrohoming. In vitro reconstituted ribonucleoprotein complexes from the Lactococcus lactis group II intron Ll.LtrB, which produce a double strand break, have recently been studied as a means of developing group II intron-based gene targeting methods for higher organisms. The Sinorhizobium meliloti group II intron RmInt1 is an efficient mobile retroelement, the dispersal of which appears to be linked to transient single-stranded DNA during replication. The RmInt1IEP lacks the endonuclease domain (En) and cannot cut the bottom strand to generate the 3′ end to initiate reverse transcription. We used an Escherichia coli expression system to produce soluble and active RmInt1 IEP and reconstituted RNPs with purified components in vitro. The RNPs generated were functional and reverse-spliced into a single-stranded DNA target. This work constitutes the starting point for the use of group II introns lacking DNA endonuclease domain-derived RNPs for highly specific gene targeting methods. PMID:27730127

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Horwitz, M.S.; Friefeld, B.R.; Keiser, H.D.

1982-12-01

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60/sup 0/C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases ..cap alpha.., BETA, ..gamma.. and had no antibody to the 72,000-daltonmore » DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.« less

Alterations in ATR in nasal NK/T-cell lymphoma and chronic active Epstein-Barr virus infection.

PubMed

Liu, Angen; Takakuwa, Tetsuya; Luo, Wen-Juan; Fujita, Shigeki; Aozasa, Katsuyuki

2006-07-01

Nasal natural killer (NK)/T-cell lymphoma (NKTCL) and chronic active Epstein-Barr virus infection (CAEBV) are relatively frequent, especially in Asia, and are poor in prognosis. Both diseases are proliferative diseases of NK/T cells that show highly complicated karyotypes, suggesting the involvement of chromosomal instability. ATR is an important gene for DNA damage response and chromosomal stability. To evaluate the role of ATR gene alterations in the pathogenesis of NKTCL and CAEBV, the whole coding region of the ATR gene was examined in cell lines derived from NKTCL and CAEBV, as well as tumor samples from patients. ATR alterations were detected in two of eight NKTCL and in one of three CAEBV lines. Most aberrant transcripts observed were deletions resulting from aberrant splicing. ATR alterations were also detected in four of 10 NKTCL clinical samples. Both NKTCL and CAEBV cell lines with ATR alterations showed a delay or abrogation in repair of ionizing radiation-induced DNA double-strand breaks and ultraviolet-induced DNA single-strand breaks, and both exhibited a defect in p53 accumulation. These findings show that alterations in the ATR gene result in an abnormal response to DNA double-strand break and single-strand break repair, suggesting a role for ATR gene alterations in NKTCL lymphomagenesis.

A Comparison of Two Single-Stranded DNA Binding Models by Mutational Analysis of APOBEC3G

PubMed Central

Shindo, Keisuke; Li, Ming; Gross, Phillip J.; Brown, William L.; Harjes, Elena; Lu, Yongjian; Matsuo, Hiroshi; Harris, Reuben S.

2012-01-01

APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between “Brim” and “Kink” models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G. PMID:24832226

RNA packaging device of double-stranded RNA bacteriophages, possibly as simple as hexamer of P4 protein.

PubMed

Kainov, Denis E; Pirttimaa, Markus; Tuma, Roman; Butcher, Sarah J; Thomas, George J; Bamford, Dennis H; Makeyev, Eugene V

2003-11-28

Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

PubMed

Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

2015-11-15

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

Annealing of Complementary DNA Sequences During Double-Strand Break Repair in Drosophila Is Mediated by the Ortholog of SMARCAL1.

PubMed

Holsclaw, Julie Korda; Sekelsky, Jeff

2017-05-01

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. We hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. We used unique genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP-binding activity of Marcal1 also reduced annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP-binding defect mutant showed reduced end joining, shedding light on the interaction between SDSA and end-joining pathways. Copyright © 2017 by the Genetics Society of America.

Mechanism of action of chromogranin A on catecholamine release: molecular modeling of the catestatin region reveals a β-strand/loop/β-strand structure secured by hydrophobic interactions and predictive of activity

PubMed Central

Tsigelny, Igor; Mahata, Sushil K.; Taupenot, Laurent; Preece, Nicholas E.; Mahata, Manjula; Khan, Imran; Parmer, Robert J.; O’Connor, Daniel T.

2009-01-01

A novel fragment of chromogranin A, known as ‘catestatin’ (bovine chromogranin A344–364), inhibits catecholamine release from chromaffin cells and noradrenergic neurons by acting as a non-competitive nicotinic cholinergic antagonist, and may therefore constitute an endogenous autocrine feedback regulator of sympathoadrenal activity. To characterize how this activity depends on the peptide’s structure, we searched for common 3-dimensional motifs for this primary structure or its homologs. Catestatin’s primary structure bore significant (29–35.5% identity, general alignment score 44–57) sequence homology to fragment sequences within three homologs of known 3-dimensional structures, based on solved X-ray crystals: 8FAB, 1PKM, and 2IG2. Each of these sequences exists in nature as a β-strand/loop/β-strand structure, stabilized by hydrophobic interactions between the β-strands. The catestatin structure was stable during molecular dynamics simulations. The catestatin loop contains three Arg residues, whose electropositive side chains form the terminus of the structure, and give rise to substantial uncompensated charge asymmetry in the molecule. A hydrophobic moment plot revealed that catestatin is the only segment of chromogranin A predicted to contain amphiphilic β-strand. Circular dichroism in the far ultraviolet showed substantial (63%) β-sheet structure, especially in a hydrophobic environment. Alanine-substitution mutants of catestatin established a crucial role for the three central arginine residues in the loop (Arg351, Arg353, and Arg358), though not for two arginine residues in the strand region toward the amino-terminus. [125I]Catestatin bound to Torpedo membranes at a site other than the nicotinic agonist binding site. When the catestatin structure was ‘docked’ with the extracellular domain of the Torpedo nicotinic cholinergic receptor, it interacted principally with the β and δ subunits, in a relatively hydrophobic region of the cation pore extracellular orifice, and the complex of ligand and receptor largely occluded the cation pore, providing a structural basis for the non-competitive nicotinic cholinergic antagonist properties of the peptide. We conclude that a homology model of catestatin correctly predicts actual features of the peptide, both physical and biological. The model suggests particular spatial and charge features of the peptide which may serve as starting points in the development of non-peptide mimetics of this endogenous nicotinic cholinergic antagonist. PMID:9809795

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

NASA Technical Reports Server (NTRS)

Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

2003-01-01

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin.

PubMed

Cotmore, S F; Tattersall, P

1998-11-01

Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses, such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays to study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yanli; Juranek, Stefan; Li, Haitao

Here we report on a 3.0 {angstrom} crystal structure of a ternary complex of wild-type Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide DNA and a 20-nucleotide target RNA containing cleavage-preventing mismatches at the 10-11 step. The seed segment (positions 2 to 8) adopts an A-helical-like Watson-Crick paired duplex, with both ends of the guide strand anchored in the complex. An arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in an inactive conformation, is released on ternary complex formation. The nucleic-acid-binding channel between the PAZ- and PIWI-containing lobes of argonaute widens on formationmore » of a more open ternary complex. The relationship of structure to function was established by determining cleavage activity of ternary complexes containing position-dependent base mismatch, bulge and 2'-O-methyl modifications. Consistent with the geometry of the ternary complex, bulges residing in the seed segments of the target, but not the guide strand, were better accommodated and their complexes were catalytically active.« less

Recognition of β-Strand Motifs by RseB Is Required for σE Activity in Escherichia coli ▿

PubMed Central

Kulp, Adam; Kuehn, Meta J.

2011-01-01

Gram-negative bacteria react to misfolded proteins in the envelope through a myriad of different stress response pathways. This cohort of pathways allows the bacteria to specifically respond to different types of damage, and many of these have been discovered to have key roles in the virulence of bacterial pathogens. Misfolded outer membrane proteins (OMPs) are typically recognized by the σE pathway, a highly conserved envelope stress response pathway. We examined the features of misfolded OMPs with respect to their ability to generate envelope stress responses. We determined that the secondary structure, particularly the potential to form β strands, is critical to inducing the σE response in an RseB-dependent manner. The sequence of the potential β-strand motif modulates the strength of the σE response generated by the constructs. By understanding the details of how such stress response pathways are activated, we can gain a greater understanding of how bacteria survive in harsh environments. PMID:21908666

Studies of Xenopus laevis mitochondrial DNA: D-loop mapping and characterization of DNA-binding proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cairns, S.S.

1987-01-01

In X. laevis oocytes, mitochondrial DNA accumulates to 10/sup 5/ times the somatic cell complement, and is characterized by a high frequency of a triple-stranded displacement hoop structure at the origin of replication. To map the termini of the single strands, it was necessary to correct the nucleotide sequence of the D-loop region. The revised sequence of 2458 nucleotides contains 54 discrepancies in comparison to a previously published sequence. Radiolabeling of the nascent strands of the D-loop structure either at the 5' end or at the 3' end identifies a major species with a length of 1670 nucleotides. Cleavage ofmore » the 5' labeled strands reveals two families of ends located near several matches to an element, designated CSB-1, that is conserved in this location in several vertebrate genomes. Cleavage of 3' labeled strands produced one fragment. The unique 3' end maps to about 15 nucleotides preceding the tRNA/sup Pro/ gene. A search for proteins which may bind to mtDNA in this region to regulate nucleic acid synthesis has identified three activities in lysates of X. laevis mitochondria. The DNA-binding proteins were assayed by monitoring their ability to retard the migration of labeled double- or single-stranded DNA fragments in polyacrylamide gels. The DNA binding preference was determined by competition with an excess of either ds- or ssDNA.« less

Structure-based design of novel chemical modification of the 3'-overhang for optimization of short interfering RNA performance.

PubMed

Xu, Lexing; Wang, Xin; He, Hongwei; Zhou, Jinming; Li, Xiaoyu; Ma, Hongtao; Li, Zelin; Zeng, Yi; Shao, Rongguang; Cen, Shan; Wang, Yucheng

2015-02-10

Short interfering RNAs (siRNAs) are broadly used to manipulate gene expression in mammalian cells. Although chemical modification is useful for increasing the potency of siRNAs in vivo, rational optimization of siRNA performance through chemical modification is still a challenge. In this work, we designed and synthesized a set of siRNAs containing modified two-nucleotide 3'-overhangs with the aim of strengthening the interaction between the 3'-end of the siRNA strand and the PAZ domain of Ago2. Their efficiency of binding to the PAZ domain was calculated using a computer modeling program, followed by measurement of RNA-Ago2 interaction in a surface plasmon resonance biochemical assay. The results suggest that increasing the level of binding of the 3'-end of the guiding strand with the PAZ domain, and/or reducing the level of binding of the sense strand through modifying the two-nucleotide 3'-overhangs, affects preferential strand selection and improves siRNA activity, while we cannot exclude the possibility that the modifications at the 3'-end of the sense strand may also affect the recognition of the 5'-end of the guiding strand by the MID domain. Taken together, our work presents a strategy for optimizing siRNA performance through asymmetric chemical modification of 3'-overhangs and also helps to develop the computer modeling method for rational siRNA design.

Characterization of the interaction of yeast enolase with polynucleotides.

PubMed

al-Giery, A G; Brewer, J M

1992-09-23

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Design and synthesis of target-responsive hydrogel for portable visual quantitative detection of uranium with a microfluidic distance-based readout device.

PubMed

Huang, Yishun; Fang, Luting; Zhu, Zhi; Ma, Yanli; Zhou, Leiji; Chen, Xi; Xu, Dunming; Yang, Chaoyong

2016-11-15

Due to uranium's increasing exploitation in nuclear energy and its toxicity to human health, it is of great significance to detect uranium contamination. In particular, development of a rapid, sensitive and portable method is important for personal health care for those who frequently come into contact with uranium ore mining or who investigate leaks at nuclear power plants. The most stable form of uranium in water is uranyl ion (UO2(2+)). In this work, a UO2(2+) responsive smart hydrogel was designed and synthesized for rapid, portable, sensitive detection of UO2(2+). A UO2(2+) dependent DNAzyme complex composed of substrate strand and enzyme strand was utilized to crosslink DNA-grafted polyacrylamide chains to form a DNA hydrogel. Colorimetric analysis was achieved by encapsulating gold nanoparticles (AuNPs) in the DNAzyme-crosslinked hydrogel to indicate the concentration of UO2(2+). Without UO2(2+), the enzyme strand is not active. The presence of UO2(2+) in the sample activates the enzyme strand and triggers the cleavage of the substrate strand from the enzyme strand, thereby decreasing the density of crosslinkers and destabilizing the hydrogel, which then releases the encapsulated AuNPs. As low as 100nM UO2(2+) was visually detected by the naked eye. The target-responsive hydrogel was also demonstrated to be applicable in natural water spiked with UO2(2+). Furthermore, to avoid the visual errors caused by naked eye observation, a previously developed volumetric bar-chart chip (V-Chip) was used to quantitatively detect UO2(2+) concentrations in water by encapsulating Au-Pt nanoparticles in the hydrogel. The UO2(2+) concentrations were visually quantified from the travelling distance of ink-bar on the V-Chip. The method can be used for portable and quantitative detection of uranium in field applications without skilled operators and sophisticated instruments. Copyright © 2016 Elsevier B.V. All rights reserved.

The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

PubMed

Lan, Susan; Kamel, Wael; Punga, Tanel; Akusjärvi, Göran

2017-02-28

The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Single-stranded DNA condensed with poly-L-lysine results in nanometric particles that are significantly smaller, more stable in physiological ionic strength fluids and afford higher efficiency of gene delivery than their double-stranded counterparts.

PubMed

Molas, M; Bartrons, R; Perales, J C

2002-08-15

Nonviral gene transfer vectors have been actively studied in the past years in order to obtain structural entities with minimum size and defined shape. The final size of a gene transfer vector, which is compacted into unimolecular complexes, is directly proportional to the mass of the nucleic acid to be compacted. Thus, the purpose of this study was to assess the possibility of producing ssDNA vectors and their biophysical and biological characterization. We have obtained ssDNA/poly-L-lysine complexes that are significantly smaller than their double-stranded counterparts. We have also identified a lesser aggregative behavior of compacted single-stranded vs. double-stranded DNA vectors in the presence of physiological NaCl concentrations. Expression of compacted ssDNA is observed in hepatoma cell lines. Moreover, we have successfully delivered galactosylated ssDNA complexes into cells that express the asialoglycoprotein receptor via receptor-mediated endocytosis. The reduced size and biophysical behavior of ssDNA vectors may provide an advantage for transfection of eukaryotic cells.

Human Immunodeficiency Virus Integration Protein Expressed in Escherichia Coli Possesses Selective DNA Cleaving Activity

NASA Astrophysics Data System (ADS)

Sherman, Paula A.; Fyfe, James A.

1990-07-01

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.

Interstrand cross-linking of DNA by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas.

PubMed

Kohn, K W

1977-05-01

Bifunctional alkylating agents are known to cross-link DNA by simultaneously alkylating two guanine residues located on opposite strands. Despite this apparent requirement for bifunctionality, 1-(2-chloroethyl)-1-nitrosoureas bearing a single alkylating function were found to cross-link DNA in vitro. Cross-linking was demonstrated by showing inhibition of alkali-induced strand separation. Extensive cross-linking was observed in DNA treated with 1-(2-chloroethyl)-1-nitrosourea, 1,3-bis-(2-chloroethyl)-1-nitrosourea, and 1-(2-chloroethyl(-3-cyclohexyl-1-nitrosourea. The reaction occurs in two steps, an intital binding followed by a second step which can proceed after removal of unbound drug. It is suggested that the first step is chloroethylation of a nucleophilic site on one strand and that the second step involves displacement of Cl- by a nucleophilic site on the opposite strand, resulting in an ethyl bridge between the strands. Consistent with this possibility, 1-(2-fluoroethyl)-3-cyclohexyl-1-nitrosourea produced much less cross-linking, as expected from the known low activity of F-, compared with Cl-, as leaving group. 1-Methyl-1-nitrosourea, which is known to depurinate DNA, produced no detectable cross-linking.

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

Transcription coupled repair deficiency protects against human mutagenesis and carcinogenesis: Personal Reflections on the 50th anniversary of the discovery of xeroderma pigmentosum.

PubMed

Cleaver, James E

2017-10-01

Xeroderma pigmentosum (XP) patients who lack the main damage recognition protein for global genome repair (GGR), XPC, have greatly increased skin cancer rates and elevated mutation frequencies originating from unrepaired ultraviolet photoproducts in the nontranscribed regions of the genome and in nontranscribed strands of expressed genes. But they show no increased mutations in transcribed strands. In contrast, cancer is absent from Cockayne syndrome (CS) patients that have defective transcription coupled repair (TCR) despite severe photosensitivity, CS patients remarkably show no elevation of UV induced mutagenesis implying that defective TCR may be protective against mutagenesis and carcinogenesis. Mutation avoidance in CS is postulated to occur through arrested transcription that generates a tripled stranded R loop consisting of DNA double strands and a nascent mRNA strand. R loops result in S phase apoptosis or activation of ATM kinase that causes a delay in DNA replication until TCR, or transcript cleavage by TFIIS or RNAaseH, relieves the transcription block. Resumption of replication then occurs on repaired DNA without concomitant mutagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of the 1906 near-surface rupture zone of the San Andreas Fault, San Francisco Peninsula segment, near Woodside, California

USGS Publications Warehouse

Rosa, C.M.; Catchings, R.D.; Rymer, M.J.; Grove, Karen; Goldman, M.R.

2016-07-08

High-resolution seismic-reflection and refraction images of the 1906 surface rupture zone of the San Andreas Fault near Woodside, California reveal evidence for one or more additional near-surface (within about 3 meters [m] depth) fault strands within about 25 m of the 1906 surface rupture. The 1906 surface rupture above the groundwater table (vadose zone) has been observed in paleoseismic trenches that coincide with our seismic profile and is seismically characterized by a discrete zone of low P-wave velocities (Vp), low S-wave velocities (Vs), high Vp/Vs ratios, and high Poisson’s ratios. A second near-surface fault strand, located about 17 m to the southwest of the 1906 surface rupture, is inferred by similar seismic anomalies. Between these two near-surface fault strands and below 5 m depth, we observed a near-vertical fault strand characterized by a zone of high Vp, low Vs, high Vp/Vs ratios, and high Poisson’s ratios on refraction tomography images and near-vertical diffractions on seismic-reflection images. This prominent subsurface zone of seismic anomalies is laterally offset from the 1906 surface rupture by about 8 m and likely represents the active main (long-term) strand of the San Andreas Fault at 5 to 10 m depth. Geometries of the near-surface and subsurface (about 5 to 10 m depth) fault zone suggest that the 1906 surface rupture dips southwestward to join the main strand of the San Andreas Fault at about 5 to 10 m below the surface. The 1906 surface rupture forms a prominent groundwater barrier in the upper 3 to 5 m, but our interpreted secondary near-surface fault strand to the southwest forms a weaker barrier, suggesting that there has been less or less-recent near-surface slip on that strand. At about 6 m depth, the main strand of the San Andreas Fault consists of water-saturated blue clay (collected from a hand-augered borehole), which is similar to deeply weathered serpentinite observed within the main strand of the San Andreas Fault at nearby sites. Multiple fault strands in the area of the 1906 surface rupture may account for variations in geologic slip rates calculated from several paleoseismic sites along the Peninsula segment of the San Andreas Fault.t.

Properties of Native High-Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles.

PubMed

McMahon, Kaylin M; Plebanek, Michael P; Thaxton, C Shad

2016-11-15

Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High-density lipoproteins (HDL) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) Load single-stranded RNA; 2) Are anionic, which requires charge reconciliation between the RNA and HDL, and 3) Actively target scavenger receptor type B-1 (SR-B1) to deliver RNA. Emphasizing these particular parameters, we employed templated lipoprotein particles (TLP), mimics of spherical HDLs, and self-assembled them with single-stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA-TLPs actively target SR-B1 to potently reduce androgen receptor (AR) and enhancer of zeste homolog 2 (EZH2) proteins in multiple cancer cell lines. Systemic administration of siRNA-TLPs demonstrated no off-target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single-stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR-B1.

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

Sweeping Jet Actuators - A New Design Tool for High Lift Generation

NASA Technical Reports Server (NTRS)

Graff, Emilio; Seele, Roman; Lin, John C.; Wygnanski, Israel

2013-01-01

Active Flow Control (AFC) experiments performed at the Caltech Lucas Wind Tunnel on a generic airplane vertical tail model proved the effectiveness of sweeping jets in improving the control authority of a rudder. The results indicated that a momentum coefficient (C(sub u)) of approximately 2% increased the side force in excess of 50% at the maximum conventional rudder deflection angle in the absence of yaw. However, sparsely distributed actuators providing a collective C(sub u) approx. = 0.1% were able to increase the side force in excess of 20%. This result is achieved by reducing the spanwise flow along the swept back rudder and its success is attributed to the large sweep back angle of the vertical tail. This current effort was sponsored by the NASA Environmentally Responsible Aviation (ERA) project.

The AstroVR Collaboratory, an On-line Multi-User Environment for Research in Astrophysics

NASA Astrophysics Data System (ADS)

van Buren, D.; Curtis, P.; Nichols, D. A.; Brundage, M.

We describe our experiment with an on-line collaborative environment where users share the execution of programs and communicate via audio, video, and typed text. Collaborative environments represent the next step in computer-mediated conferencing, combining powerful compute engines, data persistence, shared applications, and teleconferencing tools. As proof of concept, we have implemented a shared image analysis tool, allowing geographically distinct users to analyze FITS images together. We anticipate that \\htmllink{AstroVR}{http://astrovr.ipac.caltech.edu:8888} and similar systems will become an important part of collaborative work in the next decade, including with applications in remote observing, spacecraft operations, on-line meetings, as well as and day-to-day research activities. The technology is generic and promises to find uses in business, medicine, government, and education.

Path to Collagenolysis

PubMed Central

Prior, Stephen H.; Byrne, Todd S.; Tokmina-Roszyk, Dorota; Fields, Gregg B.

2016-01-01

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å2 of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling. PMID:26887942

Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

PubMed

Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

2012-10-19

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels*

PubMed Central

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P.; Khun, Joelle; Vos, Marten H.; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-01-01

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5′ and 3′ flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction. PMID:22431731

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels.

PubMed

Creze, Christophe; Ligabue, Alessio; Laurent, Sébastien; Lestini, Roxane; Laptenok, Sergey P; Khun, Joelle; Vos, Marten H; Czjzek, Mirjam; Myllykallio, Hannu; Flament, Didier

2012-05-04

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Effect of Molecular Crowding and Ionic Strength on the Isothermal Hybridization of Oligonucleotides

PubMed Central

Markarian, Marie Z.; Schlenoff, Joseph B.

2010-01-01

The isothermal hybridization of complimentary oligonucleotides, 15-mer, 25-mer, 35-mer, and a molecular beacon, was investigated under varying conditions of molecular crowding and ionic strength, using hypochromicity to follow strand pairing and polyethylene glycol as a crowding agent. Thermodynamic analysis of the results revealed the addition of counterions to the oligonucleotide backbones, Δψ, to be dependent on the strand G-C content and the molecular crowding. A decrease in Δψ was observed with both increasing GC% and solution PEG content. In contrast, the number of bound water molecules depended on the activity of Na+, where two regimes were observed. At aNa+⟨0.05 and increasing molecular crowding, water molecules were released into the DNA solutions and oligonucleotide pairing was favored with both increasing hydrophobic forces, while at aNa+≥0.05, water molecules were bound to the strands and the extent of double strand formation decreased with increasing PEG wt%. PMID:20701389

Electrochemical product detection of an asymmetric convective polymerase chain reaction.

PubMed

Duwensee, Heiko; Mix, Maren; Stubbe, Marco; Gimsa, Jan; Adler, Marcel; Flechsig, Gerd-Uwe

2009-10-15

For the first time, we describe the application of heated microwires for an asymmetric convective polymerase chain reaction (PCR) in a modified PCR tube in a small volume. The partly single-stranded product was labeled with the electrochemically active compound osmium tetroxide bipyridine using a partially complementary protective strand with five mismatches compared to the single-stranded product. The labeled product could be successfully detected at a gold electrode modified with a complementary single-stranded capture probe immobilized via a thiol-linker. Our simple thermo-convective PCR yielded electrochemically detectable products after only 5-10 min. A significant discrimination between complementary and non-complementary target was possible using different immobilized capture probes. The total product yield was approx. half the amount of the classical thermocycler PCR. Numerical simulations describing the thermally driven convective PCR explain the received data. Discrimination between complementary capture probes and non-complementary capture probes was performed using square-wave voltammetry. The coupling of asymmetric thermo-convective PCR with electrochemical detection is very promising for future compact DNA sensor devices.

Food Production, Population Growth, and Environmental Quality. Caltech Population Program Occasional Papers, Series 1, Number 7.

ERIC Educational Resources Information Center

Groth, Edward, III

This paper, one in a series of occasional publications, discusses trends in food production and population growth, emphasizing how environmental quality will be affected. The series is intended to increase understanding of the interrelationships between population growth and socioeconomic and cultural patterns throughout the world, and to…

China's Population Program at the Grassroots Level: Report on a Field Trip, Summer 1972.

ERIC Educational Resources Information Center

Chen, Pi-chao

The goals of the Caltech Population Program are to increase understanding of the interrelationships between population growth and socioeconomic and cultural patterns throughout the world and to communicate this understanding. This series of occasional papers is one step in the process of communicating research results. The papers deal primarily…

Genesis of "Biochemistry: A Problems Approach"

ERIC Educational Resources Information Center

Wood, William B.

2002-01-01

When the author began teaching as a young assistant professor at Caltech in 1966, his assignment was to take over the undergraduate biochemistry course taught for many years by Henry Borsook, who was about to retire. Students dreaded this course. Having delighted in biochemistry during his graduate training at Stanford, he was determined to put…

Visual word ambiguity.

PubMed

van Gemert, Jan C; Veenman, Cor J; Smeulders, Arnold W M; Geusebroek, Jan-Mark

2010-07-01

This paper studies automatic image classification by modeling soft assignment in the popular codebook model. The codebook model describes an image as a bag of discrete visual words selected from a vocabulary, where the frequency distributions of visual words in an image allow classification. One inherent component of the codebook model is the assignment of discrete visual words to continuous image features. Despite the clear mismatch of this hard assignment with the nature of continuous features, the approach has been successfully applied for some years. In this paper, we investigate four types of soft assignment of visual words to image features. We demonstrate that explicitly modeling visual word assignment ambiguity improves classification performance compared to the hard assignment of the traditional codebook model. The traditional codebook model is compared against our method for five well-known data sets: 15 natural scenes, Caltech-101, Caltech-256, and Pascal VOC 2007/2008. We demonstrate that large codebook vocabulary sizes completely deteriorate the performance of the traditional model, whereas the proposed model performs consistently. Moreover, we show that our method profits in high-dimensional feature spaces and reaps higher benefits when increasing the number of image categories.

Vertical columns of NO2, HONO, HCHO, CHOCHO and aerosol extinction: diurnal and seasonal variations in context of CalNex and CARES

NASA Astrophysics Data System (ADS)

Ortega, I.; Coburn, S.; Oetjen, H.; Sinreich, R.; Thalman, R. M.; Waxman, E.; Volkamer, R.

2011-12-01

We present results from two ground-based University of Colorado Multi Axis Differential Optical Absorption Spectroscopy (CU-MAX-DOAS) instruments that were deployed during the CALNEX and CARES 2010 field campaigns. Ground based CU-MAX-DOAS measurements were carried out through Dec 2010, and measured vertical column abundances of nitrogen dioxide (NO2), nitrous acid (HONO), formaldehyde (HCHO), glyoxal (CHOCHO), and aerosol extinction, which is determined indirectly from observing the oxygen dimers (O4). The measurements were acquired on the top of Millikan library at Caltech, Pasadena, CA, at the Fontana Arrows site located 60 Km east of Caltech, and for a limited period also downwind of Sacramento at T1 site during CARES. In the South Coast Air Basin, the MAX-DOAS instruments at both sites collected an extended time series of use to test satellites, and atmospheric chemistry models. We determine the state of the planetary boundary layer by comparing the columns observations with in-situ sensors, and place the CALNEX and CARES measurements intensive into seasonal context.

Tier-scalable reconnaissance: the future in autonomous C4ISR systems has arrived: progress towards an outdoor testbed

NASA Astrophysics Data System (ADS)

Fink, Wolfgang; Brooks, Alexander J.-W.; Tarbell, Mark A.; Dohm, James M.

2017-05-01

Autonomous reconnaissance missions are called for in extreme environments, as well as in potentially hazardous (e.g., the theatre, disaster-stricken areas, etc.) or inaccessible operational areas (e.g., planetary surfaces, space). Such future missions will require increasing degrees of operational autonomy, especially when following up on transient events. Operational autonomy encompasses: (1) Automatic characterization of operational areas from different vantages (i.e., spaceborne, airborne, surface, subsurface); (2) automatic sensor deployment and data gathering; (3) automatic feature extraction including anomaly detection and region-of-interest identification; (4) automatic target prediction and prioritization; (5) and subsequent automatic (re-)deployment and navigation of robotic agents. This paper reports on progress towards several aspects of autonomous C4ISR systems, including: Caltech-patented and NASA award-winning multi-tiered mission paradigm, robotic platform development (air, ground, water-based), robotic behavior motifs as the building blocks for autonomous tele-commanding, and autonomous decision making based on a Caltech-patented framework comprising sensor-data-fusion (feature-vectors), anomaly detection (clustering and principal component analysis), and target prioritization (hypothetical probing).

Suboptimal Doses of Raltegravir Cause Aberrant HIV Integrations | Center for Cancer Research

Cancer.gov

When a cell is infected with HIV, a DNA copy of the HIV genome is inserted into that cell’s chromosomal DNA. This insertion reaction is carried out by the viral enzyme integrase (IN) and involves two distinct steps: removal of two nucleotides from each 3’ end of the viral DNA, followed by the strand transfer reaction, in which the viral DNA ends are inserted into the host chromosomal DNA. Integration is essential for viral replication, making it an important target for antiviral therapy. Raltegravir, and the other approved integrase inhibitor, Elvitegravir, are called integrase strand transfer inhibitors (INSTIs), because they bind to the active site of IN and block the strand transfer reaction.      

Discovery of 2-Pyridinone Aminals: A Prodrug Strategy to Advance a Second Generation of HIV-1 Integrase Strand Transfer Inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raheem, Izzat T.; Walji, Abbas M.; Klein, Daniel

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore required for HIV integrase strand transfer inhibition represents a vibrant area of research within drug discovery. Here we present the discovery of a new class of HIV integrase strand transfer inhibitors based on the 2-pyridinone core of MK-0536. These efforts led to the identification of two lead compounds with excellent antiviral activity and preclinical pharmacokinetic profiles to support a once-daily human dose prediction. Dose escalating PK studies in dog revealed significant issues with limited oral absorption and required an innovative prodrug strategy to enhance the high-dose plasmamore » exposures of the parent molecules.« less

BLM and RMI1 alleviate RPA inhibition of TopoIIIα decatenase activity.

PubMed

Yang, Jay; Bachrati, Csanad Z; Hickson, Ian D; Brown, Grant W

2012-01-01

RPA is a single-stranded DNA binding protein that physically associates with the BLM complex. RPA stimulates BLM helicase activity as well as the double Holliday junction dissolution activity of the BLM-topoisomerase IIIα complex. We investigated the effect of RPA on the ssDNA decatenase activity of topoisomerase IIIα. We found that RPA and other ssDNA binding proteins inhibit decatenation by topoisomerase IIIα. Complex formation between BLM, TopoIIIα, and RMI1 ablates inhibition of decatenation by ssDNA binding proteins. Together, these data indicate that inhibition by RPA does not involve species-specific interactions between RPA and BLM-TopoIIIα-RMI1, which contrasts with RPA modulation of double Holliday junction dissolution. We propose that topoisomerase IIIα and RPA compete to bind to single-stranded regions of catenanes. Interactions with BLM and RMI1 enhance toposiomerase IIIα activity, promoting decatenation in the presence of RPA.

RNA Chaperone Activity of Human La Protein Is Mediated by Variant RNA Recognition Motif*

PubMed Central

Naeeni, Amir R.; Conte, Maria R.; Bayfield, Mark A.

2012-01-01

La proteins are conserved factors in eukaryotes that bind and protect the 3′ trailers of pre-tRNAs from exonuclease digestion via sequence-specific recognition of UUU-3′OH. La has also been hypothesized to assist pre-tRNAs in attaining their native fold through RNA chaperone activity. In addition to binding polymerase III transcripts, human La has also been shown to enhance the translation of several internal ribosome entry sites and upstream ORF-containing mRNA targets, also potentially through RNA chaperone activity. Using in vitro FRET-based assays, we show that human and Schizosaccharomyces pombe La proteins harbor RNA chaperone activity by enhancing RNA strand annealing and strand dissociation. We use various RNA substrates and La mutants to show that UUU-3′OH-dependent La-RNA binding is not required for this function, and we map RNA chaperone activity to its RRM1 motif including a noncanonical α3-helix. We validate the importance of this α3-helix by appending it to the RRM of the unrelated U1A protein and show that this fusion protein acquires significant strand annealing activity. Finally, we show that residues required for La-mediated RNA chaperone activity in vitro are required for La-dependent rescue of tRNA-mediated suppression via a mutated suppressor tRNA in vivo. This work delineates the structural elements required for La-mediated RNA chaperone activity and provides a basis for understanding how La can enhance the folding of its various RNA targets. PMID:22203678

Slicer function of Drosophila Argonautes and its involvement in RISC formation

PubMed Central

Miyoshi, Keita; Tsukumo, Hiroko; Nagami, Tomoko; Siomi, Haruhiko; Siomi, Mikiko C.

2005-01-01

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage (“Slicer”) activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as “Slicer” of the passenger strand of the siRNA duplex. PMID:16287716

Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer.

PubMed

Seamon, Kyle J; Bumpus, Namandjé N; Stivers, James T

2016-11-08

Sterile alpha motif and HD domain protein 1 (SAMHD1) is a unique enzyme that plays important roles in nucleic acid metabolism, viral restriction, and the pathogenesis of autoimmune diseases and cancer. Although much attention has been focused on its dNTP triphosphohydrolase activity in viral restriction and disease, SAMHD1 also binds to single-stranded RNA and DNA. Here we utilize a UV cross-linking method using 5-bromodeoxyuridine-substituted oligonucleotides coupled with high-resolution mass spectrometry to identify the binding site for single-stranded nucleic acids (ssNAs) on SAMHD1. Mapping cross-linked amino acids on the surface of existing crystal structures demonstrated that the ssNA binding site lies largely along the dimer-dimer interface, sterically blocking the formation of the homotetramer required for dNTPase activity. Surprisingly, the disordered C-terminus of SAMHD1 (residues 583-626) was also implicated in ssNA binding. An interaction between this region and ssNA was confirmed in binding studies using the purified SAMHD1 583-626 peptide. Despite a recent report that SAMHD1 possesses polyribonucleotide phosphorylase activity, we did not detect any such activity in the presence of inorganic phosphate, indicating that nucleic acid binding is unrelated to this proposed activity. These data suggest an antagonistic regulatory mechanism in which the mutually exclusive oligomeric state requirements for ssNA binding and dNTP hydrolase activity modulate these two functions of SAMHD1 within the cell.

Activation energies for dissociation of double strand oligonucleotide anions: evidence for watson-crick base pairing in vacuo.

PubMed

Schnier, P D; Klassen, J S; Strittmatter, E F; Williams, E R

1998-09-23

The dissociation kinetics of a series of complementary and noncomplementary DNA duplexes, (TGCA)(2) (3-), (CCGG)(2) (3-), (AATTAAT)(2) (3-), (CCGGCCG)(2) (3-), A(7)*T(7) (3-), A(7)*A(7) (3-), T(7)*T(7) (3-), and A(7)*C(7) (3-) were investigated using blackbody infrared radiative dissociation in a Fourier transform mass spectrometer. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained. Activation energies range from 1.2 to 1.7 eV, and preexponential factors range from 10(13) to 10(19) s(-1). Dissociation of the duplexes results in cleavage of the noncovalent bonds and/or cleavage of covalent bonds leading to loss of a neutral nucleobase followed by backbone cleavage producing sequence-specific (a - base) and w ions. Four pieces of evidence are presented which indicate that Watson-Crick (WC) base pairing is preserved in complementary DNA duplexes in the gas phase: i. the activation energy for dissociation of the complementary dimer, A(7)*T(7) (3-), to the single strands is significantly higher than that for the related noncomplementary A(7)*A(7) (3-) and T(7)*T(7) (3-) dimers, indicating a stronger interaction between strands with a specific base sequence, ii. extensive loss of neutral adenine occurs for A(7)*A(7) (3-) and A(7)*C(7) (3-) but not for A(7)*T(7) (3-) consistent with this process being shut down by WC hydrogen bonding, iii. a correlation is observed between the measured activation energy for dissociation to single strands and the dimerization enthalpy (-DeltaH(d)) in solution, and iv. molecular dynamics carried out at 300 and 400 K indicate that WC base pairing is preserved for A(7)*T(7) (3-) duplex, although the helical structure is essentially lost. In combination, these results provide strong evidence that WC base pairing can exist in the complete absence of solvent.

Contributed Review: Absolute spectral radiance calibration of fiber-optic shock-temperature pyrometers using a coiled-coil irradiance standard lamp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fat’yanov, O. V., E-mail: fatyan1@gps.caltech.edu; Asimow, P. D., E-mail: asimow@gps.caltech.edu

2015-10-15

We describe an accurate and precise calibration procedure for multichannel optical pyrometers such as the 6-channel, 3-ns temporal resolution instrument used in the Caltech experimental geophysics laboratory. We begin with a review of calibration sources for shock temperatures in the 3000-30 000 K range. High-power, coiled tungsten halogen standards of spectral irradiance appear to be the only practical alternative to NIST-traceable tungsten ribbon lamps, which are no longer available with large enough calibrated area. However, non-uniform radiance complicates the use of such coiled lamps for reliable and reproducible calibration of pyrometers that employ imaging or relay optics. Careful analysis of documentedmore » methods of shock pyrometer calibration to coiled irradiance standard lamps shows that only one technique, not directly applicable in our case, is free of major radiometric errors. We provide a detailed description of the modified Caltech pyrometer instrument and a procedure for its absolute spectral radiance calibration, accurate to ±5%. We employ a designated central area of a 0.7× demagnified image of a coiled-coil tungsten halogen lamp filament, cross-calibrated against a NIST-traceable tungsten ribbon lamp. We give the results of the cross-calibration along with descriptions of the optical arrangement, data acquisition, and processing. We describe a procedure to characterize the difference between the static and dynamic response of amplified photodetectors, allowing time-dependent photodiode correction factors for spectral radiance histories from shock experiments. We validate correct operation of the modified Caltech pyrometer with actual shock temperature experiments on single-crystal NaCl and MgO and obtain very good agreement with the literature data for these substances. We conclude with a summary of the most essential requirements for error-free calibration of a fiber-optic shock-temperature pyrometer using a high-power coiled tungsten halogen irradiance standard lamp.« less

Developing an Education and Public Outreach (EPO) program for the Caltech Tectonics Observatory

NASA Astrophysics Data System (ADS)

Kovalenko, L.; Nadin, E.; Avouac, J.

2008-12-01

The Caltech Tectonics Observatory (TO) is an interdisciplinary center, focused on geological processes occurring at the boundaries of Earth's tectonic plates. The timescales of these processes span from a few tens of seconds (the typical duration of an earthquake) to tens of millions of years (the time it takes to build mountains). Over the past four years, the TO has brought together 15 Caltech faculty from different fields, several visiting scientists from around the globe, and a few tens of graduate students and postdoctoral students, collaborating on scientific projects. A major objective of the TO now is to develop an Education and Public Outreach (EPO) program. Our goals are to (1) stimulate the interest of students and the general public in Earth Sciences, particularly in the study of tectonic processes, (2) inform and educate the general public about TO discoveries and advancements, and (3) make available the data and techniques developed by the TO for use in classrooms of all levels. To this effect, we have been developing our website for accessibility by the general public and writing educational web articles on TO research. A recent well-visited example is "The science behind the recent 2008 earthquake in China." We distribute animations that illustrate the mechanisms of earthquakes and tsunamis, and the various techniques used by TO scientists in their scientific investigations. The TO website also provides access to geodetic data collected by TO instruments and to the source models of recent large earthquakes as analyzed by TO scientists. The TO hosts tours of its facilities for local elementary school students and is working on developing education modules for high school and undergraduate classes. We are now working on a plan to offer short courses over the summer for undergraduate and graduate students in other institutions, in order to train them to analyze a variety of data and use techniques developed by TO scientists.

In vitro synthesis of minus-strand RNA by an isolated cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3' end of the virus RNA.

PubMed

Osman, Toba A M; Coutts, Robert H A; Buck, Kenneth W

2006-11-01

Cereal yellow dwarf virus (CYDV) RNA has a 5'-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3'-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3'-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.

Tudor staphylococcal nuclease is a structure-specific ribonuclease that degrades RNA at unstructured regions during microRNA decay.

PubMed

Li, Chia-Lung; Yang, Wei-Zen; Shi, Zhonghao; Yuan, Hanna S

2018-05-01

Tudor staphylococcal nuclease (TSN) is an evolutionarily conserved ribonuclease in eukaryotes that is composed of five staphylococcal nuclease-like domains (SN1-SN5) and a Tudor domain. TSN degrades hyper-edited double-stranded RNA, including primary miRNA precursors containing multiple I•U and U•I pairs, and mature miRNA during miRNA decay. However, how TSN binds and degrades its RNA substrates remains unclear. Here, we show that the C. elegans TSN (cTSN) is a monomeric Ca 2+ -dependent ribonuclease, cleaving RNA chains at the 5'-side of the phosphodiester linkage to produce degraded fragments with 5'-hydroxyl and 3'-phosphate ends. cTSN degrades single-stranded RNA and double-stranded RNA containing mismatched base pairs, but is not restricted to those containing multiple I•U and U•I pairs. cTSN has at least two catalytic active sites located in the SN1 and SN3 domains, since mutations of the putative Ca 2+ -binding residues in these two domains strongly impaired its ribonuclease activity. We further show by small-angle X-ray scattering that rice osTSN has a flexible two-lobed structure with open to closed conformations, indicating that TSN may change its conformation upon RNA binding. We conclude that TSN is a structure-specific ribonuclease targeting not only single-stranded RNA, but also unstructured regions of double-stranded RNA. This study provides the molecular basis for how TSN cooperates with RNA editing to eliminate duplex RNA in cell defense, and how TSN selects and degrades RNA during microRNA decay. © 2018 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Chemical repair of base lesions, AP-sites, and strand breaks on plasmid DNA in dilute aqueous solution by ascorbic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hata, Kuniki; Advanced Science Research Center, Japan Atomic Energy Agency, 2-4 Shirakatashirane, Tokai-mura, Naka-gun, Ibaraki 319-1195; Urushibara, Ayumi

Highlights: •We report a novel mechanism of radiation protection of DNA by chemical activity of ascorbic acid. •The “chemical repair” of DNA damage was revealed using biochemical assay and chemical kinetics analysis. •We found that ascorbic acid significantly repairs precursors of nucleobase lesions and abasic sites. •However, ascorbic acid seldom repairs precursors of DNA-strand breaks. -- Abstract: We quantified the damage yields produced in plasmid DNA by γ-irradiation in the presence of low concentrations (10–100 μM) of ascorbic acid, which is a major antioxidant in living systems, to clarify whether it chemically repairs radiation damage in DNA. The yield ofmore » DNA single strand breaks induced by irradiation was analyzed with agarose gel electrophoresis as conformational changes in closed circular plasmids. Base lesions and abasic sites were also observed as additional conformational changes by treating irradiated samples with glycosylase proteins. By comparing the suppression efficiencies to the induction of each DNA lesion, in addition to scavenging of the OH radicals derived from water radiolysis, it was found that ascorbic acid promotes the chemical repair of precursors of AP-sites and base lesions more effectively than those of single strand breaks. We estimated the efficiency of the chemical repair of each lesion using a kinetic model. Approximately 50–60% of base lesions and AP-sites were repaired by 10 μM ascorbic acid, although strand breaks were largely unrepaired by ascorbic acid at low concentrations. The methods in this study will provide a route to understanding the mechanistic aspects of antioxidant activity in living systems.« less

Myofibroblasts Electrotonically Coupled to Cardiomyocytes Alter Conduction: Insights at the Cellular Level from a Detailed In silico Tissue Structure Model

PubMed Central

Jousset, Florian; Maguy, Ange; Rohr, Stephan; Kucera, Jan P.

2016-01-01

Fibrotic myocardial remodeling is typically accompanied by the appearance of myofibroblasts (MFBs). In vitro, MFBs were shown to slow conduction and precipitate ectopic activity following gap junctional coupling to cardiomyocytes (CMCs). To gain further mechanistic insights into this arrhythmogenic MFB-CMC crosstalk, we performed numerical simulations in cell-based high-resolution two-dimensional tissue models that replicated experimental conditions. Cell dimensions were determined using confocal microscopy of single and co-cultured neonatal rat ventricular CMCs and MFBs. Conduction was investigated as a function of MFB density in three distinct cellular tissue architectures: CMC strands with endogenous MFBs, CMC strands with coating MFBs of two different sizes, and CMC strands with MFB inserts. Simulations were performed to identify individual contributions of heterocellular gap junctional coupling and of the specific electrical phenotype of MFBs. With increasing MFB density, both endogenous and coating MFBs slowed conduction. At MFB densities of 5–30%, conduction slowing was most pronounced in strands with endogenous MFBs due to the MFB-dependent increase in axial resistance. At MFB densities >40%, very slow conduction and spontaneous activity was primarily due to MFB-induced CMC depolarization. Coating MFBs caused non-uniformities of resting membrane potential, which were more prominent with large than with small MFBs. In simulations of MFB inserts connecting two CMC strands, conduction delays increased with increasing insert lengths and block appeared for inserts >1.2 mm. Thus, electrophysiological properties of engineered CMC-MFB co-cultures depend on MFB density, MFB size and their specific positioning in respect to CMCs. These factors may influence conduction characteristics in the heterocellular myocardium. PMID:27833567

In Vitro Synthesis of Minus-Strand RNA by an Isolated Cereal Yellow Dwarf Virus RNA-Dependent RNA Polymerase Requires VPg and a Stem-Loop Structure at the 3′ End of the Virus RNA▿

PubMed Central

Osman, Toba A. M.; Coutts, Robert H. A.; Buck, Kenneth W.

2006-01-01

Cereal yellow dwarf virus (CYDV) RNA has a 5′-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3′-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3′ terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3′ end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3′-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg. PMID:16928757

Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

PubMed

Liu, Jie; Berger, Christopher L; Morrical, Scott W

2013-11-12

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

CTC1-STN1 coordinates G- and C-strand synthesis to regulate telomere length.

PubMed

Gu, Peili; Jia, Shuting; Takasugi, Taylor; Smith, Eric; Nandakumar, Jayakrishnan; Hendrickson, Eric; Chang, Sandy

2018-05-17

Coats plus (CP) is a rare autosomal recessive disorder caused by mutations in CTC1, a component of the CST (CTC1, STN1, and TEN1) complex important for telomere length maintenance. The molecular basis of how CP mutations impact upon telomere length remains unclear. The CP CTC1 L1142H mutation has been previously shown to disrupt telomere maintenance. In this study, we used CRISPR/Cas9 to engineer this mutation into both alleles of HCT116 and RPE cells to demonstrate that CTC1:STN1 interaction is required to repress telomerase activity. CTC1 L1142H interacts poorly with STN1, leading to telomerase-mediated telomere elongation. Impaired interaction between CTC1 L1142H :STN1 and DNA Pol-α results in increased telomerase recruitment to telomeres and further telomere elongation, revealing that C:S binding to DNA Pol-α is required to fully repress telomerase activity. CP CTC1 mutants that fail to interact with DNA Pol-α resulted in loss of C-strand maintenance and catastrophic telomere shortening. Our findings place the CST complex as an important regulator of both G-strand extensions by telomerase and C-strand synthesis by DNA Pol-α. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

PubMed Central

Tsabar, Michael; Mason, Jennifer M.; Chan, Yuen-Ling; Bishop, Douglas K.; Haber, James E.

2015-01-01

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1ATR/Tel1ATM-dependent DNA damage response or caffeine's inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2's Rad51 filament dismantling activity or Rad51's ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments. PMID:26019181

Fluorescence Cross-Correlation Spectroscopy Reveals Mechanistic Insights into the Effect of 2′-O-Methyl Modified siRNAs in Living Cells

PubMed Central

Ohrt, Thomas; Staroske, Wolfgang; Mütze, Jörg; Crell, Karin; Landthaler, Markus; Schwille, Petra

2011-01-01

RNA interference (RNAi) offers a powerful tool to specifically direct the degradation of complementary RNAs, and thus has great therapeutic potential for targeting diseases. Despite the reported preferences of RNAi, there is still a need for new techniques that will allow for a detailed mechanistic characterization of RNA-induced silencing complex (RISC) assembly and activity to further improve the biocompatibility of modified siRNAs. In contrast to previous reports, we investigated the effects of 2′-O-methyl (2′OMe) modifications introduced at specific positions within the siRNA at the early and late stages of RISC assembly, as well as their influence on target recognition and cleavage directly in living cells. We found that six to 10 2′OMe nucleotides on the 3′-end inhibit passenger-strand release as well as target-RNA cleavage without changing the affinity, strand asymmetry, or target recognition. 2′OMe modifications introduced at the 5′-end reduced activated RISC stability, whereas incorporations at the cleavage site showed only minor effects on passenger-strand release when present on the passenger strand. Our new fluorescence cross-correlation spectroscopy assays resolve different steps and stages of RISC assembly and target recognition with heretofore unresolved detail in living cells, which is needed to develop therapeutic siRNAs with optimized in vivo properties. PMID:21689532

Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

PubMed

Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

2005-12-06

Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

Scientific Integrity and Executive National Security Proclamations: A Conflict of the Modern Age

NASA Astrophysics Data System (ADS)

Nelson, R.; Banerdt, B.; Bell, J. L.; Byrnes, D. V.; Carlisle, G. L.; D'Addario, L. R.; Weissman, P. R.; Eisenhardt, P. R.; Foster, S. D.; Golombek, M. P.; Gorjian, V.; Gorjian, Z.; Hale, A. S.; Kulleck, J. G.; Laubach, S. L.; McElrath, T. P.; Penanen, K. I.; Satter, C.; Walker, W. J.

2010-12-01

In 2004, in response to the events of September, 11, 2001, President George W. Bush issued Homeland Security Presidential Directive #12, an executive order requiring a uniform means of identification (i.e. identification badge) for all employees and contractors at federal facilities. To comply with this directive NASA ordered that its contract employees at the Jet Propulsion Laboratory 'voluntarily' agree to an open ended, unrestricted, background investigation into the intimate details of their private lives. These employees do not have security clearances and do not work with classified material. Caltech, which employs the JPL personnel under a NASA management contract, informed the employees that if they did not ‘voluntarily’ consent to the background investigation, they would be assumed to have voluntarily resigned and therefore be denied access to JPL (i.e. they would be functionally terminated). In October 2007, twentyeight JPL employees filed suit in Federal District Court. After an initial dismissal by the lowest federal court, the Ninth Circuit Court of Appeals issued an injunction against Caltech and NASA, stopping the background investigations. The Appeals Court found that the investigations were not narrowly tailored to meet the specific needs of NASA and therefore violated the employee’s legitimate expectation of informational privacy. This injunction has been reviewed and upheld several times by various panels of the Ninth Circuit Court of Appeals. In November 2009, the United States Department of Justice petitioned the U.S. Supreme Court requesting that it overturn this injunction. The Supreme Court accepted the case for oral arguments and scheduled them for October 5, 2010. A decision is imminent (if it has not been made already). The case has opened the following questions regarding all research workers under government contract: 1. What impact would such intrusive investigations have on open scientific inquiry and scientific integrity? 2. How much can the Government reasonably ask about the backgrounds of its contract employees? 3. What rights do employees have to challenge the findings of their background investigations, and can the results of such investigations be contested in the courts? 4. What is the responsibility of a contract employer such as Caltech to protect the privacy of contract research workers like those at JPL? 5. What is the responsibility of professional societies in representing the interests of federal contract employees such as those at JPL? 6. What would constitute a reasonable background investigation of contract employees that would be narrowly tailored in order to protect informational privacy? This work is a private venture. Affiliations are listed for identification purposes only. No NASA, Caltech or JPL resources were expended in producing this abstract.

Fluorometric determination of nucleic acids based on the use of polydopamine nanotubes and target-induced strand displacement amplification.

PubMed

Ge, Jia; Bai, Dong-Mei; -Geng, Xin; Hu, Ya-Lei; Cai, Qi-Yong; Xing, Ke; Zhang, Lin; Li, Zhao-Hui

2018-01-10

The authors describe a fluorometric method for the quantitation of nucleic acids by combining (a) cycled strand displacement amplification, (b) the unique features of the DNA probe SYBR Green, and (c) polydopamine nanotubes. SYBR Green undergoes strong fluorescence enhancement upon intercalation into double-stranded DNA (dsDNA). The polydopamine nanotubes selectively adsorb single-stranded DNA (ssDNA) and molecular beacons. In the absence of target DNA, the molecular beacon, primer and SYBR Green are adsorbed on the surface of polydopamine nanotubes. This results in quenching of the fluorescence of SYBR Green, typically measured at excitation/emission wavelengths of 488/518 nm. Upon addition of analyte (target DNA) and polymerase, the stem of the molecular beacon is opened so that it can bind to the primer. This triggers target strand displacement polymerization, during which dsDNA is synthesized. The hybridized target is then displaced due to the strand displacement activity of the polymerase. The displaced target hybridizes with another molecular beacon. This triggers the next round of polymerization. Consequently, a large amount of dsDNA is formed which is detected by addition of SYBR Green. Thus, sensitive and selective fluorometric detection is realized. The fluorescent sensing strategy shows very good analytical performances towards DNA detection, such as a wide linear range from 0.05 to 25 nM with a low limit of detection of 20 pM. Graphical abstract Schematic of a fluorometric strategy for highly sensitive and selective determination of nucleic acids by combining strand displacement amplification and the unique features of SYBR Green I (SG) and polydopamine nanotubes.

Explanation for excessive DNA single-strand breaks and endogenous repair foci in pluripotent mouse embryonic stem cells.

PubMed

Banáth, J P; Bañuelos, C A; Klokov, D; MacPhail, S M; Lansdorp, P M; Olive, P L

2009-05-01

Pluripotent mouse embryonic stem cells (mES cells) exhibit approximately 100 large gammaH2AX repair foci in the absence of measurable numbers of DNA double-strand breaks. Many of these cells also show excessive numbers of DNA single-strand breaks (>10,000 per cell) when analyzed using the alkaline comet assay. To understand the reasons for these unexpected observations, various methods for detecting DNA strand breaks were applied to wild-type mES cells and to mES cells lacking H2AX, ATM, or DNA-PKcs. H2AX phosphorylation and expression of other repair complexes were measured using flow and image analysis of antibody-stained cells. Results indicate that high numbers of endogenous gammaH2AX foci and single-strand breaks in pluripotent mES cells do not require ATM or DNA-PK kinase activity and appear to be associated with global chromatin decondensation rather than pre-existing DNA damage. This will limit applications of gammaH2AX foci analysis in mES cells to relatively high levels of initial or residual DNA damage. Excessive numbers of single-strand breaks in the alkaline comet assay can be explained by the vulnerability of replicating chromatin in mES cells to osmotic shock. This suggests that caution is needed in interpreting results with the alkaline comet assay when applied to certain cell types or after treatment with agents that make chromatin vulnerable to osmotic changes. Differentiation of mES cells caused a reduction in histone acetylation, gammaH2AX foci intensity, and DNA single-strand breakage, providing a link between chromatin structural organization, excessive gammaH2AX foci, and sensitivity of replicating mES cell chromatin to osmotic shock.

SYNTHETIC STRANDS OF CARDIAC MUSCLE

PubMed Central

Purdy, Joyce E.; Lieberman, Melvyn; Roggeveen, Anne E.; Kirk, R. Gary

1972-01-01

Spontaneously active bundles of cardiac muscle (synthetic strands) were prepared from isolated cells of 11–13-day old embryonic chick hearts which were disaggregated with trypsin. Linear orientation of the cells was obtained by plating them on agar-coated culture dishes in which either grooves were cut in the agar film or a thin line of palladium was deposited over the agar. The influence of cell-to-cell and cell-to-substrate interactions was observed with time lapse cinematography and the formation of the synthetic strand was shown to involve both random and guided cell movements, enlargement of aggregates by accretion and coalescence, and the compact linear arrangement of cells along paths of preferential adhesion. Electron microscope investigations of these strands showed that a dispersed population of heart cells organized into an inner core of muscle cells and an outer sheath of fibroblast-like cells. The muscle cells contained well-developed, but widely spaced myofibrils, a developing sarcoplasmic reticulum associated in part with the myofibrils and in part with the sarcolemma, an abundance of nonmembrane bound ribosomes and glycogen, and a prominent Golgi complex. Numerous specialized contacts were observed between the muscle cells in the strand, e.g., fasciae adherentes, desmosomes, and nexuses. A distinct type of muscle cell characterized by its pale appearance was regularly observed in the strand and was noted to be similar to Purkinje cells described in the adult avian conduction system and in developing chick myocardium. The present findings were compared with other observations of the developing myocardium, in situ, and it was concluded that, by a number or criteria, the muscle cells of the strand were differentiating normally and suitably organized for electrophysiological studies. PMID:4656702

Mechanism for CCC DNA synthesis in hepadnaviruses.

PubMed

Sohn, Ji A; Litwin, Samuel; Seeger, Christoph

2009-11-30

Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

Marine mammal strandings and environmental changes: a 15-year study in the St. Lawrence ecosystem.

PubMed

Truchon, Marie-Hélène; Measures, Lena; L'Hérault, Vincent; Brêthes, Jean-Claude; Galbraith, Peter S; Harvey, Michel; Lessard, Sylvie; Starr, Michel; Lecomte, Nicolas

2013-01-01

Understanding the effects of climatic variability on marine mammals is challenging due to the complexity of ecological interactions. We used general linear models to analyze a 15-year database documenting marine mammal strandings (1994-2008; n = 1,193) and nine environmental parameters known to affect marine mammal survival, from regional (sea ice) to continental scales (North Atlantic Oscillation, NAO). Stranding events were more frequent during summer and fall than other seasons, and have increased since 1994. Poor ice conditions observed during the same period may have affected marine mammals either directly, by modulating the availability of habitat for feeding and breeding activities, or indirectly, through changes in water conditions and marine productivity (krill abundance). For most species (75%, n = 6 species), a low volume of ice was correlated with increasing frequency of stranding events (e.g. R(2)adj = 0.59, hooded seal, Cystophora cristata). This likely led to an increase in seal mortality during the breeding period, but also to increase habitat availability for seasonal migratory cetaceans using ice-free areas during winter. We also detected a high frequency of stranding events for mysticete species (minke whale, Balaenoptera acutorostrata) and resident species (beluga, Delphinapterus leucas), correlated with low krill abundance since 1994. Positive NAO indices were positively correlated with high frequencies of stranding events for resident and seasonal migratory cetaceans, as well as rare species (R(2)adj = 0.53, 0.81 and 0.34, respectively). This contrasts with seal mass stranding numbers, which were negatively correlated with a positive NAO index. In addition, an unusual multiple species mortality event (n = 114, 62% of total annual mortality) in 2008 was caused by a harmful algal bloom. Our findings provide an empirical baseline in understanding marine mammal survival when faced with climatic variability. This is a promising step in integrating stranding records to monitor the consequences of environmental changes in marine ecosystems over long time scales.

Marine Mammal Strandings and Environmental Changes: A 15-Year Study in the St. Lawrence Ecosystem

PubMed Central

Truchon, Marie-Hélène; Measures, Lena; L’Hérault, Vincent; Brêthes, Jean-Claude; Galbraith, Peter S.; Harvey, Michel; Lessard, Sylvie; Starr, Michel; Lecomte, Nicolas

2013-01-01

Understanding the effects of climatic variability on marine mammals is challenging due to the complexity of ecological interactions. We used general linear models to analyze a 15-year database documenting marine mammal strandings (1994–2008; n = 1,193) and nine environmental parameters known to affect marine mammal survival, from regional (sea ice) to continental scales (North Atlantic Oscillation, NAO). Stranding events were more frequent during summer and fall than other seasons, and have increased since 1994. Poor ice conditions observed during the same period may have affected marine mammals either directly, by modulating the availability of habitat for feeding and breeding activities, or indirectly, through changes in water conditions and marine productivity (krill abundance). For most species (75%, n = 6 species), a low volume of ice was correlated with increasing frequency of stranding events (e.g. R2 adj = 0.59, hooded seal, Cystophora cristata). This likely led to an increase in seal mortality during the breeding period, but also to increase habitat availability for seasonal migratory cetaceans using ice-free areas during winter. We also detected a high frequency of stranding events for mysticete species (minke whale, Balaenoptera acutorostrata) and resident species (beluga, Delphinapterus leucas), correlated with low krill abundance since 1994. Positive NAO indices were positively correlated with high frequencies of stranding events for resident and seasonal migratory cetaceans, as well as rare species (R2 adj = 0.53, 0.81 and 0.34, respectively). This contrasts with seal mass stranding numbers, which were negatively correlated with a positive NAO index. In addition, an unusual multiple species mortality event (n = 114, 62% of total annual mortality) in 2008 was caused by a harmful algal bloom. Our findings provide an empirical baseline in understanding marine mammal survival when faced with climatic variability. This is a promising step in integrating stranding records to monitor the consequences of environmental changes in marine ecosystems over long time scales. PMID:23544059

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

PubMed

Cochran, A G; Tong, R T; Starovasnik, M A; Park, E J; McDowell, R S; Theaker, J E; Skelton, N J

2001-01-31

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

The Guggenheim Aeronautics Laboratory at Caltech and the creation of the modern rocket motor (1936-1946): How the dynamics of rocket theory became reality

NASA Astrophysics Data System (ADS)

Zibit, Benjamin Seth

This thesis explores and unfolds the story of discovery in rocketry at The California Institute of Technology---specifically at Caltech's Guggenheim Aeronautics Laboratory---in the 1930s and 1940s. Caltech was home to a small group of engineering students and experimenters who, beginning in the winter of 1935--1936, formed a study and research team destined to change the face of rocket science in the United States. The group, known as the Guggenheim Aeronautics Laboratory (GALCIT, for short) Rocket Research Group, invented a new type of solid-rocket propellant, made distinct and influential discoveries in the theory of rocket combustion and design, founded the Jet Propulsion Laboratory, and incorporated the first American industrial concern devoted entirely to rocket motor production: The Aerojet Corporation. The theoretical work of team members, Frank Malina, Hsueh-shen Tsien, Homer J. Stewart, and Mark Mills, is examined in this thesis in detail. The author scrutinizes Frank Malina's doctoral thesis (both its assumptions and its mathematics), and finds that, although Malina's key assertions, his formulae, hold, his work is shown to make key assumptions about rocket dynamics which only stand the test of validity if certain approximations, rather than exact measurements, are accepted. Malina studied the important connection between motor-nozzle design and thrust; in his Ph.D. thesis, he developed mathematical statements which more precisely defined the design/thrust relation. One of Malina's colleagues on the Rocket Research Team, John Whiteside Parsons, created a new type of solid propellant in the winter of 1941--1942. This propellant, known as a composite propellant (because it simply was a relatively inert amalgam of propellant and oxidizer in non-powder form), became the forerunner of all modern solid propellants, and has become one of the seminal discoveries in the field of Twentieth Century rocketry. The latter chapters of this dissertation discuss the creation of the jet Propulsion Laboratory, the founding of the Aerojet Corporation, and emphasizes the issue of JPL's close relation to military development of the rocket becomes a core subject of this thesis. Cooperation between engineers in an academic setting and the military was not merely inevitable in the 1940s---it was actively fostered and proved quite profitable to all concerned. The deep relationship between the Guggenheim Aeronautics Laboratory and the Army Air Force was one model of the evolution of a permanent institutional edifice, weaving academic research and military end-use together. The dissertation concludes that what began as a modest effort to understand rocket theory in greater depth led within ten years to both research and development tracks which have profoundly altered the technological and military definition of modern history.

Origins Space Telescope: Community Participation

NASA Astrophysics Data System (ADS)

Carey, Sean J.; Origins Space Telescope Study Team

2017-01-01

The Origins Space Telescope (OST) is the mission concept for the Far-Infrared Surveyor, a study in development by NASA in preparation for the 2020 Astronomy and Astrophysics Decadal Survey. Origins is planned to be a large aperture, actively-cooled telescope covering a wide span of the mid- to far-infrared spectrum. Its imagers and spectrographs will enable a variety of surveys of the sky that will discover and characterize the most distant galaxies, Milky-Way, exoplanets, and the outer reaches of our Solar system. Origins will enable flagship-quality general observing programs led by the astronomical community in the 2030s. This poster will outline the ways in which the astronomical community can participate in the STDT activities and a summary of tools that are currently available or are planned for the community during the study. The Science and Technology Definition Team (STDT) would like to hear your science needs and ideas for this mission. The team can be contacted at firsurveyor_info@lists.ipac.caltech.edu.

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, wasmore » found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.« less

A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

PubMed

Gumpper, Ryan H; Li, Weike; Castañeda, Carlos H; Scuderi, M José; Bashkin, James K; Luo, Ming

2018-04-15

Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs. Copyright © 2018 American Society for Microbiology.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

PubMed

Drané, Pascal; Brault, Marie-Eve; Cui, Gaofeng; Meghani, Khyati; Chaubey, Shweta; Detappe, Alexandre; Parnandi, Nishita; He, Yizhou; Zheng, Xiao-Feng; Botuyan, Maria Victoria; Kalousi, Alkmini; Yewdell, William T; Münch, Christian; Harper, J Wade; Chaudhuri, Jayanta; Soutoglou, Evi; Mer, Georges; Chowdhury, Dipanjan

2017-03-09

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

Role of the Pepino mosaic virus 3'-untranslated region elements in negative-strand RNA synthesis in vitro.

PubMed

Osman, Toba A M; Olsthoorn, René C L; Livieratos, Ioannis C

2014-09-22

Pepino mosaic virus (PepMV) is a mechanically-transmitted positive-strand RNA potexvirus, with a 6410 nt long single-stranded (ss) RNA genome flanked by a 5'-methylguanosine cap and a 3' poly-A tail. Computer-assisted folding of the 64 nt long PepMV 3'-untranslated region (UTR) resulted in the prediction of three stem-loop structures (hp1, hp2, and hp3 in the 3'-5' direction). The importance of these structures and/or sequences for promotion of negative-strand RNA synthesis and binding to the RNA dependent RNA polymerase (RdRp) was tested in vitro using a specific RdRp assay. Hp1, which is highly variable among different PepMV isolates, appeared dispensable for negative-strand synthesis. Hp2, which is characterized by a large U-rich loop, tolerated base-pair changes in its stem as long as they maintained the stem integrity but was very sensitive to changes in the U-rich loop. Hp3, which harbours the conserved potexvirus ACUUAA hexamer motif, was essential for template activity. Template-RNA polymerase binding competition experiments showed that the ACUUAA sequence represents a high-affinity RdRp binding element. Copyright © 2014 Elsevier B.V. All rights reserved.

Charles F. Richter; an interview

USGS Publications Warehouse

Spall, H.

1980-01-01

Charles F. Richter, renowned seismologist, is a professor emeritus at the California Institue of techonology (Caltech). He is best known to the public for the Richter magnitude scale; but he is equally recognized in the scientific community for many other contributions to seismology including his books Elementary Seismology (1958) and Seismicity of the Earth (coauthored in 1954 with Beno Gutenberg). 

Deflection Measurements on Propeller 5503 in Ahead and Crashback

DTIC Science & Technology

2016-10-01

the dots on the blade that were visible for the run . Not all points could be determined for each picture during each run . One issue discovered with...Channel (LCC) in February and April of 2009. The deflection of the blades was measured using defocused particle image velocimetry. Comparisons were made... Blade Deflection Measurement CalTech

John Todd--Numerical Mathematics Pioneer

ERIC Educational Resources Information Center

Albers, Don

2007-01-01

John Todd, now in his mid-90s, began his career as a pure mathematician, but World War II interrupted that. In this interview, he talks about his education, the significant developments in his becoming a numerical analyst, and the journey that concluded at Caltech. Among the interesting stories are how he met his wife-to-be the mathematician Olga…

Demographic Trends in the Republic of Zaire. Caltech Population Program Occasional Papers, Series 1, Number 5.

ERIC Educational Resources Information Center

Boute, Joseph

This paper, one in a series of occasional publications, discusses population growth and trends in the African Republic of Zaire. The series is intended to increase understanding of the interrelationships between population growth and socioeconomic and cultural patterns throughout the world, and to communicate this understanding to scholars and…

Architecture of the Yeast RNA Polymerase II Open Complex and Regulation of Activity by TFIIF

PubMed Central

Fishburn, James

2012-01-01

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB–p-bromoacetamidobenzyl–EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain. PMID:22025674

Reversible Regulation of Catalytic Activity of Gold Nanoparticles with DNA Nanomachines

NASA Astrophysics Data System (ADS)

Zhou, Peipei; Jia, Sisi; Pan, Dun; Wang, Lihua; Gao, Jimin; Lu, Jianxin; Shi, Jiye; Tang, Zisheng; Liu, Huajie

2015-09-01

Reversible catalysis regulation has gained much attention and traditional strategies utilized reversible ligand coordination for switching catalyst’s conformations. However, it remains challenging to regulate the catalytic activity of metal nanoparticle-based catalysts. Herein, we report a new DNA nanomachine-driven reversible nano-shield strategy for circumventing this problem. The basic idea is based on the fact that the conformational change of surface-attached DNA nanomachines will cause the variation of the exposed surface active area on metal nanoparticles. As a proof-of-concept study, we immobilized G-rich DNA strands on gold nanoparticles (AuNPs) which have glucose oxidase (GOx) like activity. Through the reversible conformational change of the G-rich DNA between a flexible single-stranded form and a compact G-quadruplex form, the catalytic activity of AuNPs has been regulated reversibly for several cycles. This strategy is reliable and robust, which demonstrated the possibility of reversibly adjusting catalytic activity with external surface coverage switching, rather than coordination interactions.

Diastereoselective cyclization of an aminobenzoic acid derivative and chiroptical properties of triple-stranded helical bis(phenylethynyl)benzene.

PubMed

Yamakado, Ryohei; Matsuoka, Shin-ichi; Suzuki, Masato; Takeuchi, Daisuke; Masu, Hyuma; Azumaya, Isao; Takagi, Koji

2015-04-04

The diastereoselective cyclization of 2,5-dibromo-4-hexylaminobenzoic acid was achieved by the microwave-assisted condensation using SiCl4. Moreover, the triple-stranded helical structure of bis(phenylethynyl)benzene units embedded in the cyclic tri(benzamide) scaffold was obtained by a Sonogashira-Hagihara coupling reaction. Two optically active enantiomers that do not racemize even at the elevated temperature were separated by chiral HPLC. The chiral helical topology was revealed by the spectroscopic data and theoretical calculation.

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ*

PubMed Central

Xu, Yang; Wu, Xiling; Her, Chengtao

2015-01-01

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress. PMID:26055704

Thiophene antibacterials that allosterically stabilize DNA-cleavage complexes with DNA gyrase.

PubMed

Chan, Pan F; Germe, Thomas; Bax, Benjamin D; Huang, Jianzhong; Thalji, Reema K; Bacqué, Eric; Checchia, Anna; Chen, Dongzhao; Cui, Haifeng; Ding, Xiao; Ingraham, Karen; McCloskey, Lynn; Raha, Kaushik; Srikannathasan, Velupillai; Maxwell, Anthony; Stavenger, Robert A

2017-05-30

A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.

Unexpected sequences and structures of mtDNA required for efficient transcription from the first heavy-strand promoter

PubMed Central

Uchida, Akira; Murugesapillai, Divakaran; Kastner, Markus; Wang, Yao; Lodeiro, Maria F; Prabhakar, Shaan; Oliver, Guinevere V; Arnold, Jamie J; Maher, L James; Williams, Mark C; Cameron, Craig E

2017-01-01

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications. DOI: http://dx.doi.org/10.7554/eLife.27283.001 PMID:28745586

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

Three New Structures of Left-Handed RadA Helical Filaments: Structural Flexibility of N-Terminal Domain Is Critical for Recombinase Activity

PubMed Central

Chang, Yu-Wei; Ko, Tzu-Ping; Lee, Chien-Der; Chang, Yuan-Chih; Lin, Kuei-Ann; Chang, Chia-Seng; Wang, Andrew H.-J.; Wang, Ting-Fang

2009-01-01

RecA family proteins, including bacterial RecA, archaeal RadA, and eukaryotic Dmc1 and Rad51, mediate homologous recombination, a reaction essential for maintaining genome integrity. In the presence of ATP, these proteins bind a single-strand DNA to form a right-handed nucleoprotein filament, which catalyzes pairing and strand exchange with a homologous double-stranded DNA (dsDNA), by as-yet unknown mechanisms. We recently reported a structure of RadA left-handed helical filament, and here present three new structures of RadA left-handed helical filaments. Comparative structural analysis between different RadA/Rad51 helical filaments reveals that the N-terminal domain (NTD) of RadA/Rad51, implicated in dsDNA binding, is highly flexible. We identify a hinge region between NTD and polymerization motif as responsible for rigid body movement of NTD. Mutant analysis further confirms that structural flexibility of NTD is essential for RadA's recombinase activity. These results support our previous hypothesis that ATP-dependent axial rotation of RadA nucleoprotein helical filament promotes homologous recombination. PMID:19295907

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

PubMed

Wang, Mengke; Lin, Zihan; Liu, Qing; Jiang, Shan; Liu, Hua; Su, Xingguang

2018-07-05

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL -1 with a detection limit (LOD) of 0.039 U mL -1 . Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results. Copyright © 2018 Elsevier B.V. All rights reserved.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing.

PubMed

Koralewska, Natalia; Hoffmann, Weronika; Pokornowska, Maria; Milewski, Marek; Lipinska, Andrea; Bienkowska-Szewczyk, Krystyna; Figlerowicz, Marek; Kurzynska-Kokorniak, Anna

2016-01-01

Ribonuclease Dicer plays a pivotal role in RNA interference pathways by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. While details of Dicer regulation by a variety of proteins are being elucidated, less is known about non-protein factors, e.g. RNA molecules, that may influence this enzyme's activity. Therefore, we decided to investigate the question of whether the RNA molecules can function not only as Dicer substrates but also as its regulators. Our previous in vitro studies indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both. Those studies were carried out with commercial Dicer preparations. Nevertheless, such preparations are usually not homogeneous enough to carry out more detailed RNA-binding studies. Therefore, we have established our own system for the production of human Dicer in insect cells. In this manuscript, we characterize the RNA-binding and RNA-cleavage properties of the obtained preparation. We demonstrate that Dicer can efficiently bind single-stranded RNAs that are longer than ~20-nucleotides. Consequently, we revisit possible scenarios of Dicer regulation by single-stranded RNA species ranging from ~10- to ~60-nucleotides, in the context of their binding to this enzyme. Finally, we show that siRNA/miRNA-sized RNAs may affect miRNA production either by binding to Dicer or by participating in regulatory feedback-loops. Altogether, our studies suggest a broad regulatory role of short RNAs in Dicer functioning.

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription

PubMed Central

Ritchie, Shawn A.; Pasha, Mohammed K.; Batten, Danielle J. P.; Sharma, Rajendra K.; Olson, Douglas J. H.; Ross, Andrew R. S.; Bonham, Keith

2003-01-01

The human SRC gene encodes pp60c–src, a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes. PMID:12595559

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

PubMed

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

2014-06-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

PubMed Central

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

2014-01-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

RPA physically interacts with the human DNA glycosylase NEIL1 to regulate excision of oxidative DNA base damage in primer-template structures.

PubMed

Theriot, Corey A; Hegde, Muralidhar L; Hazra, Tapas K; Mitra, Sankar

2010-06-04

The human DNA glycosylase NEIL1, activated during the S-phase, has been shown to excise oxidized base lesions in single-strand DNA substrates. Furthermore, our previous work demonstrating functional interaction of NEIL1 with PCNA and flap endonuclease 1 (FEN1) suggested its involvement in replication-associated repair. Here we show interaction of NEIL1 with replication protein A (RPA), the heterotrimeric single-strand DNA binding protein that is essential for replication and other DNA transactions. The NEIL1 immunocomplex isolated from human cells contains RPA, and its abundance in the complex increases after exposure to oxidative stress. NEIL1 directly interacts with the large subunit of RPA (K(d) approximately 20 nM) via the common interacting interface (residues 312-349) in NEIL1's disordered C-terminal region. RPA inhibits the base excision activity of both wild-type NEIL1 (389 residues) and its C-terminal deletion CDelta78 mutant (lacking the interaction domain) for repairing 5-hydroxyuracil (5-OHU) in a primer-template structure mimicking the DNA replication fork. This inhibition is reduced when the damage is located near the primer-template junction. Contrarily, RPA moderately stimulates wild-type NEIL1 but not the CDelta78 mutant when 5-OHU is located within the duplex region. While NEIL1 is inhibited by both RPA and Escherichia coli single-strand DNA binding protein, only inhibition by RPA is relieved by PCNA. These results showing modulation of NEIL1's activity on single-stranded DNA substrate by RPA and PCNA support NEIL1's involvement in repairing the replicating genome. Copyright 2010 Elsevier B.V. All rights reserved.

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

PubMed Central

Hanaoka, Shingo; Nagadoi, Aritaka; Nishimura, Yoshifumi

2005-01-01

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2. PMID:15608118

Marine mammal strandings in the New Caledonia region, Southwest Pacific.

PubMed

Borsa, Philippe

2006-04-01

Four hundred twenty three marine mammals, in 72 stranding events, were recorded between 1877 and 2005 in New Caledonia, the Loyalty Islands, and Vanuatu in the southwest Pacific. Sixteen species were represented in this count, including: minke whale, Balaenoptera acutorostrata (1 single stranding), sei whale, B. borealis (1 single stranding), blue whale, B. musculus (1 single stranding), humpback whale, Megaptera novaeangliae (2 single strandings), giant sperm whale, Physeter macrocephalus (18 single strandings, 2 pair strandings), pygmy sperm whale, Kogia breviceps (5 single strandings), dwarf sperm whale, K. sima (2 single strandings, 1 triple stranding), Blainville's beaked whale, Mesoplodon densirostris (2 single strandings), short-finned pilot whale, Globicephala macrorhynchus (4 strandings, 56 individuals), melon-headed whale, Peponocephala electra (1 single stranding and 2 mass strandings totalling 231 individuals), common dolphin, Delphinus delphis (1 single stranding), spinner dolphin, Stenella longirostris (1 pair stranding and 2 mass strandings of groups of approximately 30 individuals each), Indian Ocean bottlenose dolphin, Tursiops aduncus (2 single strandings), dugong, Dugong dugon (14 single strandings), and New Zealand fur seal, Arctocephalus forsteri (3 single strandings). A stranded rorqual identified as an Antarctic minke whale (B. bonaerensis), with coloration patterns that did not match known descriptions, was also reported. Sei whale was recorded for the first time in the tropical Southwest Pacific region and Antarctic minke whale, melon-headed whale, and Indian Ocean bottlenose dolphin were recorded for the first time in New Caledonia. Strandings of sperm whales were most frequent in the spring, but also occurred in autumn months, suggesting a seasonal pattern of occurrence possibly related to seasonal migration. One stranded humpback whale bore the scars of a killer whale's attack and one dugong was injured by a shark. Scars left by propellers were noted on several stranded animals including one Antarctic minke whale, one pygmy sperm whale, one dwarf sperm whale, and four dugongs. Collisions with vessels were suspected to be a frequent cause of death for dugong.

The Effect of Dimethyl Sulfoxide on Supercoiled DNA Relaxation Catalyzed by Type I Topoisomerases

PubMed Central

Lv, Bei; Dai, Yunjia; Liu, Ju; Zhuge, Qiang; Li, Dawei

2015-01-01

The effects of dimethyl sulfoxide (DMSO) on supercoiled plasmid DNA relaxation catalyzed by two typical type I topoisomerases were investigated in our studies. It is shown that DMSO in a low concentration (less than 20%, v/v) can induce a dose-related enhancement of the relaxation efficiency of Escherichia coli topoisomerase I (type IA). Conversely, obvious inhibitory effect on the activity of calf thymus topoisomerase I (type IB) was observed when the same concentration of DMSO is used. In addition, our studies demonstrate that 20% DMSO has an ability to reduce the inhibitory effect on EcTopo I, which was induced by double-stranded oligodeoxyribonucleotides while the same effect cannot be found in the case of CtTopo I. Moreover, our AFM examinations suggested that DMSO can change the conformation of negatively supercoiled plasmid by creating some locally loose regions in DNA molecules. Combining all the lines of evidence, we proposed that DMSO enhanced EcTopo I relaxation activity by (1) increasing the single-stranded DNA regions for the activities of EcTopo I in the early and middle stages of the reaction and (2) preventing the formation of double-stranded DNA-enzyme complex in the later stage, which can elevate the effective concentration of the topoisomerase in the reaction solution. PMID:26682217

Slicing-independent RISC activation requires the argonaute PAZ domain.

PubMed

Gu, Shuo; Jin, Lan; Huang, Yong; Zhang, Feijie; Kay, Mark A

2012-08-21

Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood. The Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3' end of small RNAs. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells. We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs, PAZ-disrupted Agos bound duplex small interfering RNAs, but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.

Double-stranded Endonuclease Activity in Bacillus halodurans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas2 Protein*

PubMed Central

Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P.; Ke, Ailong

2012-01-01

The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5′-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg2+ or Mn2+), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1–α1 loop. PMID:22942283

Electron attachment-induced DNA single-strand breaks at the pyrimidine sites

PubMed Central

Gu, Jiande; Wang, Jing; Leszczynski, Jerzy

2010-01-01

To elucidate the contribution of pyrimidine in DNA strand breaks caused by low-energy electrons (LEEs), theoretical investigations of the LEE attachment-induced C3′–O3′, and C5′–O5′ σ bond as well as N-glycosidic bond breaking of 2′-deoxycytidine-3′,5′-diphosphate and 2′-deoxythymidine-3′,5′-diphosphate were performed using the B3LYP/DZP++ approach. The base-centered radical anions are electronically stable enough to assure that either the C–O or glycosidic bond breaking processes might compete with the electron detachment and yield corresponding radical fragments and anions. In the gas phase, the computed glycosidic bond breaking activation energy (24.1 kcal/mol) excludes the base release pathway. The low-energy barrier for the C3′–O3′ σ bond cleavage process (∼6.0 kcal/mol for both cytidine and thymidine) suggests that this reaction pathway is the most favorable one as compared to other possible pathways. On the other hand, the relatively low activation energy barrier (∼14 kcal/mol) for the C5′–O5′ σ bond cleavage process indicates that this bond breaking pathway could be possible, especially when the incident electrons have relatively high energy (a few electronvolts). The presence of the polarizable medium greatly increases the activation energies of either C–O σ bond cleavage processes or the N-glycosidic bond breaking process. The only possible pathway that dominates the LEE-induced DNA single strands in the presence of the polarizable surroundings (such as in an aqueous solution) is the C3′–O3′ σ bond cleavage (the relatively low activation energy barrier, ∼13.4 kcal/mol, has been predicted through a polarizable continuum model investigation). The qualitative agreement between the ratio for the bond breaks of C5′–O5′, C3′–O3′ and N-glycosidic bonds observed in the experiment of oligonucleotide tetramer CGAT and the theoretical sequence of the bond breaking reaction pathways have been found. This consistency between the theoretical predictions and the experimental observations provides strong supportive evidences for the base-centered radical anion mechanism of the LEE-induced single-strand bond breaking around the pyrimidine sites of the DNA single strands. PMID:20430827

A Fortunate Life in Astronomy

NASA Astrophysics Data System (ADS)

Osterbrock, Donald E.

I have had a very fortunate career in astronomy, benefiting greatly from numerous accidents of fate. I grew up in Cincinnati, Ohio, served in the US Army Air Force in World War II, and had all my further education at the University of Chicago, from PhB in the College to PhD in astronomy and astrophysics. There, as a postdoc at Princeton University, and as a young faculty member at Caltech and Mount Wilson and Palomar Observatories, I had excellent teachers and mentors. I have done research primarily on gaseous nebulae and active galactic nuclei, but also made a few early contributions on stellar interiors and the heating in the outer layers of the Sun. The major part of my scientific career was at the University of Wisconsin and Lick Observatory, but I also had three productive years at the Institute for Advanced Study.

In Vitro Antibacterial Activity of AZD0914, a New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-Positive, Fastidious Gram-Negative, and Atypical Bacteria

PubMed Central

Bradford, Patricia A.; Otterson, Linda G.; Basarab, Gregory S.; Kutschke, Amy C.; Giacobbe, Robert A.; Patey, Sara A.; Alm, Richard A.; Johnstone, Michele R.; Potter, Marie E.; Miller, Paul F.; Mueller, John P.

2014-01-01

AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development. PMID:25385112

Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

PubMed Central

Jin, Xin; Sun, Tingting; Zhao, Chuanke; Zheng, Yongxiang; Zhang, Yufan; Cai, Weijing; He, Qiuchen; Taira, Kaz; Zhang, Lihe; Zhou, Demin

2012-01-01

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA. PMID:22039150

Structure and function of the septum nasi and the underlying tension chord in crocodylians.

PubMed

Klenner, Sebastian; Witzel, Ulrich; Paris, Frank; Distler, Claudia

2016-01-01

A long rostrum has distinct advantages for prey capture in an aquatic or semi-aquatic environment but at the same time poses severe problems concerning stability during biting. We here investigate the role of the septum nasi of brevirostrine crocodilians for load-absorption during mastication. Histologically, both the septum nasi and the septum interorbitale consist of hyaline cartilage and therefore mainly resist compression. However, we identified a strand of tissue extending longitudinally below the septum nasi that is characterized by a high content of collagenous and elastic fibers and could therefore resist tensile stresses. This strand of tissue is connected with the m. pterygoideus anterior. Two-dimensional finite element modeling shows that minimization of bending in the crocodilian skull can only be achieved if tensile stresses are counteracted by a strand of tissue. We propose that the newly identified strand of tissue acts as an active tension chord necessary for stabilizing the long rostrum of crocodilians during biting by transforming the high bending stress of the rostrum into moderate compressive stress. © 2015 Anatomical Society.

Synthesis of 4'-C-aminoalkyl-2'-O-methyl modified RNA and their biological properties.

PubMed

Koizumi, Kana; Maeda, Yusuke; Kano, Toshifumi; Yoshida, Hisae; Sakamoto, Taiichi; Yamagishi, Kenji; Ueno, Yoshihito

2018-05-17

In this paper, we describe the synthesis of 4'-C-aminoalkyl-2'-O-methylnucleosides and the properties of RNAs containing these analogs. Phosphoramidites of 4'-C-aminoethyl and 4'-C-aminopropyl-2'-O-methyluridines were prepared using glucose as starting material, and RNAs containing the analogs were synthesized using the phosphoramidites. Thermal denaturation studies revealed that these nucleoside analogs decreased the thermal stabilities of double-stranded RNAs (dsRNAs). Results of NMR, molecular modeling, and CD spectra measurements suggested that 4'-C-aminoalkyl-2'-O-methyluridine adopts an C2'-endo sugar puckering in dsRNA. The 4'-C-aminoalkyl modifications in the passenger strand and the guide strand outside the seed region were well tolerated for RNAi activity of siRNAs. Single-stranded RNAs (ssRNAs) and siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs showed high stability in buffer containing bovine serum. Thus, siRNAs containing the 4'-C-aminoethyl and 4'-C-aminopropyl analogs are good candidates for the development of therapeutic siRNA molecules. Copyright © 2018 Elsevier Ltd. All rights reserved.

High-pressure liquid-monopropellant strand combustion.

NASA Technical Reports Server (NTRS)

Faeth, G. M.

1972-01-01

Examination of the influence of dissolved gases on the state of the liquid surface during high-pressure liquid-monopropellant combustion through the use of a strand burning experiment. Liquid surface temperatures were measured, using fine-wire thermocouples, during the strand combustion of ethyl nitrate, normal propyl nitrate, and propylene glycol dinitrate at pressures up to 81 atm. These measurements were compared with the predictions of a variable-property gas-phase analysis assuming an infinite activation energy for the decomposition reaction. The state of the liquid surface was estimated using a conventional low-pressure phase equilibrium model, as well as a high-pressure version that considered the presence of dissolved combustion-product gases in the liquid phase. The high-pressure model was found to give a superior prediction of measured liquid surface temperatures. Computed total pressures required for the surface to reach its critical mixing point during strand combustion were found to be in the range from 2.15 to 4.62 times the critical pressure of the pure propellant. Computed dissolved gas concentrations at the liquid surface were in the range from 35 to 50% near the critical combustion condition.

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.

2012-10-25

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealedmore » that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.« less

A homogeneous nucleic acid hybridization assay based on strand displacement.

PubMed Central

Vary, C P

1987-01-01

A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal strand. Strand displacement, therefore, causes conversion of the RNA from double to single-stranded form. The single-strand specificity of polynucleotide phosphorylase (EC 2.7.7.8) allows discrimination between double-helical and single-stranded forms of the RNA signal strand. As displacement proceeds, free RNA signal strands are preferentially phosphorolyzed to component nucleoside diphosphates, including adenosine diphosphate. The latter nucleotide is converted to ATP by pyruvate kinase(EC 2.7.1.40). Luciferase catalyzed bioluminescence is employed to measure the ATP generated as a result of strand displacement. Images PMID:3309890

Structure-function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice.

PubMed Central

Chao, H.; Sönnichsen, F. D.; DeLuca, C. I.; Sykes, B. D.; Davies, P. L.

1994-01-01

Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction. PMID:7849594

Aromatic residues located close to the active center are essential for the catalytic reaction of flap endonuclease-1 from hyperthermophilic archaeon Pyrococcus horikoshii.

PubMed

Matsui, Eriko; Abe, Junko; Yokoyama, Hideshi; Matsui, Ikuo

2004-04-16

Flap endonuclease-1 (FEN-1) possessing 5'-flap endonuclease and 5'-->3' exonuclease activity plays important roles in DNA replication and repair. In this study, the kinetic parameters of mutants at highly conserved aromatic residues, Tyr33, Phe35, Phe79, and Phe278-Phe279, in the vicinity of the catalytic centers of FEN-1 were examined. The substitution of these aromatic residues with alanine led to a large reduction in kcat values, although these mutants retained Km values similar to that of the wild-type enzyme. Notably, the kcat of Y33A and F79A decreased 333-fold and 71-fold, respectively, compared with that of the wild-type enzyme. The aromatic residues Tyr33 and Phe79, and the aromatic cluster Phe278-Phe279 mainly contributed to the recognition of the substrates without the 3' projection of the upstream strand (the nick, 5'-recess-end, single-flap, and pseudo-Y substrates) for the both exo- and endo-activities, but played minor roles in recognizing the substrates with the 3' projection (the double flap substrate and the nick substrate with the 3' projection). The replacement of Tyr33, Phe79, and Phe278-Phe279, with non-charged aromatic residues, but not with aliphatic hydrophobic residues, recovered the kcat values almost fully for the substrates without the 3' projection of the upstream strand, suggesting that the aromatic groups of Tyr33, Phe79, and Phe278-Phe279 might be involved in the catalytic reaction, probably via multiple stacking interactions with nucleotide bases. The stacking interactions of Tyr33 and Phe79 might play important roles in fixing the template strand and the downstream strand, respectively, in close proximity to the active center to achieve the productive transient state leading to the hydrolysis.

DNA-binding and oxidative properties of cationic phthalocyanines and their dimeric complexes with anionic phthalocyanines covalently linked to oligonucleotides.

PubMed

Kuznetsova, A A; Lukyanets, E A; Solovyeva, L I; Knorre, D G; Fedorova, O S

2008-12-01

Design of chemically modified oligonucleotides for regulation of gene expression has attracted considerable attention over the past decades. One actively pursued approach involves antisense or antigene oligonucleotide constructs carrying reactive groups, many of these based on transition metal complexes. The complexes of Fe(II) and Co(II) with phthalocyanines are extremely good catalysts of oxidation of organic compounds with molecular oxygen and hydrogen peroxide. The binding of positively charged Fe(II) and Co(II) phthalocyanines with single- and double-stranded DNA was investigated. It was shown that these phthalocyanines interact with nucleic acids through an outside binding mode. The site-directed modification of single-stranded DNA by O2 and H2O2 in the presence of dimeric complexes of negatively and positively charged Fe(II) and Co(II) phthalocyanines was investigated. These complexes were formed directly on single-stranded DNA through interaction between negatively charged phthalocyanine in conjugate and positively charged phthalocyanine in solution. The resulting oppositely charged phthalocyanine complexes showed significant increase of catalytic activity compared with monomeric forms of phthalocyanines Fe(II) and Co(II). These complexes catalyzed the DNA oxidation with high efficacy and led to direct DNA strand cleavage. It was determined that oxidation of DNA by molecular oxygen catalyzed by complex of Fe(II)-phthalocyanines proceeds with higher rate than in the case of Co(II)-phthalocyanines but the latter led to a greater extent of target DNA modification.

Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

NASA Astrophysics Data System (ADS)

Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

2016-03-01

We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

Australian Curriculum Linked Lessons

ERIC Educational Resources Information Center

Hurrell, Derek

2014-01-01

In providing a continued focus on tasks and activities that help to illustrate key ideas embedded in the new Australian Curriculum, this issue will focus on Number in the Number and Algebra strand. In this article Derek Hurrell provides a few tried and proven activities to develop place value understanding. These activities are provided for…

Cloud chemistry in eastern China: Observations from Mt. Tai

NASA Astrophysics Data System (ADS)

Collett, J. L.; Shen, X.; Lee, T.; Wang, X.; Li, Y.; Wang, W.; Wang, T.

2010-07-01

Until recently, studies of fog and cloud chemistry in China have been rare - even though the fate of China’s large sulfur dioxide emissions depends, in part, on the ability of regional clouds to support rapid aqueous oxidation to sulfate. Sulfur dioxide oxidized in regional clouds is more likely to be removed by wet deposition while sulfur dioxide that undergoes slower gas phase oxidation is expected to survive longer in the atmosphere and be transported over a much broader spatial scale. Two 2008 field campaigns conducted at Mt. Tai, an isolated peak on the NE China plain, provide insight into the chemical composition of regional clouds and the importance of various aqueous phase sulfur oxidation pathways. Single and two-stage Caltech Active Strand Cloudwater Collectors were used to collect bulk and drop size-resolved samples of cloudwater. Collected cloudwater was analyzed for key species that influence in-cloud sulfate production, including pH, S(IV), H2O2, Fe and Mn. Other major cloud solutes, including inorganic ions, total organic carbon (TOC), formaldehyde, and organic acids were also analyzed, as were gas phase concentrations of SO2, O3, and H2O2. A wide range of cloud pH was observed, from below 3 to above 6. High concentrations of cloudwater sulfate were consistent with abundant sulfur dioxide emissions in the region. Sampled clouds were also found to contain high concentrations of ammonium, nitrate, and organic carbon. Peak TOC concentrations reached approximately 200 ppmC, among the highest concentrations ever measured in cloudwater. Hydrogen peroxide was found to be the dominant aqueous phase S(IV) oxidant when cloud pH was less than approximately 5.4. Despite its fast reaction with sulfur dioxide in cloud droplets, high concentrations of residual hydrogen peroxide were measured in some clouds implying a substantial additional capacity for sulfate production. Ozone was found to be an important S(IV) oxidant when cloud pH was high. Oxidation of S(IV) by oxygen, catalyzed by Fe (III) and Mn(II) was generally the second or third fastest pathway for sulfate production. Differences between the pH and trace metal concentrations of small and large cloud droplets were observed, giving rise to aqueous phase sulfate production rates that were drop size-dependent for the ozone and metal-catalyzed pathways.

Formation of template-switching artifacts by linear amplification.

PubMed

Chakravarti, Dhrubajyoti; Mailander, Paula C

2008-07-01

Linear amplification is a method of synthesizing single-stranded DNA from either a single-stranded DNA or one strand of a double-stranded DNA. In this protocol, molecules of a single primer DNA are extended by multiple rounds of DNA synthesis at high temperature using thermostable DNA polymerases. Although linear amplification generates the intended full-length single-stranded product, it is more efficient over single-stranded templates than double-stranded templates. We analyzed linear amplification over single- or double-stranded mouse H-ras DNA (exon 1-2 region). The single-stranded H-ras template yielded only the intended product. However, when the double-stranded template was used, additional artifact products were observed. Increasing the concentration of the double-stranded template produced relatively higher amounts of these artifact products. One of the artifact DNA bands could be mapped and analyzed by sequencing. It contained three template-switching products. These DNAs were formed by incomplete DNA strand extension over the template strand, followed by switching to the complementary strand at a specific Ade nucleotide within a putative hairpin sequence, from which DNA synthesis continued over the complementary strand.

Sequential self-assembly of DNA functionalized droplets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yin; McMullen, Angus; Pontani, Lea-Laetitia

Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Though biology relies on such schemes, they have not been available in materials science. We demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activatesmore » the next droplet in the sequence, akin to living polymerization. This strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.« less

Passenger strand loading in overexpression experiments using microRNA mimics.

PubMed

Søkilde, Rolf; Newie, Inga; Persson, Helena; Borg, Åke; Rovira, Carlos

2015-01-01

MicroRNAs (miRNAs) are important regulators of gene function and manipulation of miRNAs is a central component of basic research. Modulation of gene expression by miRNA gain-of-function can be based on different approaches including transfection with miRNA mimics; artificial, chemically modified miRNA-like small RNAs. These molecules are intended to mimic the function of a miRNA guide strand while bypassing the maturation steps of endogenous miRNAs. Due to easy accessibility through commercial providers this approach has gained popularity, and accuracy is often assumed without prior independent testing. Our in silico analysis of over-represented sequence motifs in microarray expression data and sequencing of AGO-associated small RNAs indicate, however, that miRNA mimics may be associated with considerable side-effects due to the unwanted activity of the miRNA mimic complementary strand.

Crystallization of the avian reovirus double-stranded RNA-binding and core protein σA

PubMed Central

Hermo-Parrado, X. Lois; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Fox, Gavin C.; Vazquez-Iglesias, Lorena; Martínez-Costas, José; Benavente, Javier; van Raaij, Mark J.

2007-01-01

The avian reovirus protein σA plays a dual role: it is a structural protein forming part of the transcriptionally active core, but it has also been implicated in the resistance of the virus to interferon by strongly binding double-stranded RNA and thus inhibiting the double-stranded RNA-dependent protein kinase. The σA protein has been crystallized from solutions containing ammonium sulfate at pH values around 6. Crystals belonging to space group P1, with unit-cell parameters a = 103.2, b = 129.9, c = 144.0 Å, α = 93.8, β = 105.1, γ = 98.2° were grown and a complete data set has been collected to 2.3 Å resolution. The self-rotation function suggests that σA may form symmetric arrangements in the crystals. PMID:17565188

DISE: A Seed-Dependent RNAi Off-Target Effect That Kills Cancer Cells.

PubMed

Putzbach, William; Gao, Quan Q; Patel, Monal; Haluck-Kangas, Ashley; Murmann, Andrea E; Peter, Marcus E

2018-01-01

Off-target effects (OTEs) represent a significant caveat for RNAi caused by substantial complementarity between siRNAs and unintended mRNAs. We now discuss the existence of three types of seed-dependent OTEs (sOTEs). Type I involves unintended targeting through the guide strand seed of an siRNA. Type II is caused by the activity of the seed on the designated siRNA passenger strand when loaded into the RNA-induced silencing complex (RISC). Both type I and II sOTEs will elicit unpredictable cellular responses. By contrast, in sOTE type III the guide strand seed preferentially targets essential survival genes resulting in death induced by survival gene elimination (DISE). In this Opinion article, we discuss DISE as a consequence of RNAi that may preferentially affect cancer cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Sequential self-assembly of DNA functionalized droplets

DOE PAGES

Zhang, Yin; McMullen, Angus; Pontani, Lea-Laetitia; ...

2017-06-16

Complex structures and devices, both natural and manmade, are often constructed sequentially. From crystallization to embryogenesis, a nucleus or seed is formed and built upon. Sequential assembly allows for initiation, signaling, and logical programming, which are necessary for making enclosed, hierarchical structures. Though biology relies on such schemes, they have not been available in materials science. We demonstrate programmed sequential self-assembly of DNA functionalized emulsions. The droplets are initially inert because the grafted DNA strands are pre-hybridized in pairs. Active strands on initiator droplets then displace one of the paired strands and thus release its complement, which in turn activatesmore » the next droplet in the sequence, akin to living polymerization. This strategy provides time and logic control during the self-assembly process, and offers a new perspective on the synthesis of materials.« less

The International GPS Network for Charting the Evolving Global Reference Frame

NASA Technical Reports Server (NTRS)

Zumberge, J. F.; Heflin, M. B.; Lindqwister, U. J.; Neilan, R. E.; Watkins, M. M.

1995-01-01

The Telecommunications and Engineering Division of Caltech's Jet Propulsion Laboratory is funded by the National Aeronautics and Space Administration to play a variety of roles in applying the Global Positioning System (GPS) to geodesy and geodynamics. Among these are the operation of dozens of globally-distributed, permanently-operating Earth fixed GPS stations. This, and other applications are described.

Impact Cratering Physics al Large Planetary Scales

NASA Astrophysics Data System (ADS)

Ahrens, Thomas J.

2007-06-01

Present understanding of the physics controlling formation of ˜10^3 km diameter, multi-ringed impact structures on planets were derived from the ideas of Scripps oceanographer, W. Van Dorn, University of London's, W, Murray, and, Caltech's, D. O'Keefe who modeled the vertical oscillations (gravity and elasticity restoring forces) of shock-induced melt and damaged rock within the transient crater immediately after the downward propagating hemispheric shock has processed rock (both lining, and substantially below, the transient cavity crater). The resulting very large surface wave displacements produce the characteristic concentric, multi-ringed basins, as stored energy is radiated away and also dissipated upon inducing further cracking. Initial calculational description, of the above oscillation scenario, has focused upon on properly predicting the resulting density of cracks, and, their orientations. A new numerical version of the Ashby--Sammis crack damage model is coupled to an existing shock hydrodynamics code to predict impact induced damage distributions in a series of 15--70 cm rock targets from high speed impact experiments for a range of impactor type and velocity. These are compared to results of crack damage distributions induced in crustal rocks with small arms impactors and mapped ultrasonically in recent Caltech experiments (Ai and Ahrens, 2006).

A Low Noise NbTiN-Based 850 GHz SIS Receiver for the Caltech Submillimeter Observatory

NASA Technical Reports Server (NTRS)

Kooi, J. W.; Kawamura, J.; Chen, J.; Chattopadhyay, G.; Pardo, J. R.; Zmuidzinas, J.; Phillips, T. G.; Bumble, B.; Stern, J.; LeDuc, H. G.

2000-01-01

We have developed a niobium titanium nitride (NbTiN) based superconductor- insulator-superconductor (SIS) receiver to cover the 350 micron atmospheric window. This frequency band lies entirely above the energy gap of niobium (700 GHz), a commonly used SIS superconductor. The instrument uses an open structure twin-slot SIS mixer that consists of two Nb/AlN/NbTiN tunnel junctions, NbTiN thin-film microstrip tuning elements, and a NbTiN ground plane. The optical configuration is very similar to the 850 GHz waveguide receiver that was installed at the Caltech Submillimeter Observatory (CSO) in 1997. To minimize front-end loss, we employed reflecting optics and a cooled beamsplitter at 4 K. The instrument has an uncorrected receiver noise temperature of 205K DSB at 800 GHz and 410K DSB at 900 GHz. The degradation in receiver sensitivity with frequency is primarily due to an increase in the mixer conversion loss, which is attributed to the mismatch between the SIS junction and the twin-slot antenna impedance. The overall system performance has been confirmed through its use at the telescope to detect a wealth of new spectroscopic lines.

The earthshine s spectra

NASA Astrophysics Data System (ADS)

Montanes Rodriguez, P.; Palle, E.; Goode, P.; Koonin, S.; Hickey, J.; Qiu, J.; Yurchysyn, V.

The Earthshine project, was run by California Institute of Technology (Caltech) between 1993 and 1995. Since 1998, it has been a collaborative effort between Caltech and Big Bear Solar Observatory (BBSO)/New Jersey Institute of Technology (NJIT). Our primary goal is the precise determination of a global and absolutely calibrated Earth's albedo and its synoptic, seasonal, and annual variability; as well as the measurement and investigation of the resolved reflected spectrum of the integrated Earth in the infrared region. The absorption in the infrared region, mainly due to rotational and vibrational transitions of the molecules, show the absorption bands of various telluric and solar components allowing the analysis of the Earth's spectrum such as it would be observed from the outer space. In this paper we present preliminary results of spectroscopic observations, made at Palomar Observatory with the 60-inch telescope's echelle spectrograph. They targeted the visible and near infrared region of the electromagnetic spectrum, and were performed in the spectral range (< 1μm) of the bands of Oxygen A, Oxygen B, water and Hydrogen alpha (H). The first three are typically terrestrial molecular bands. The fourth line, H, is a solar line, used mainly for spectral calibration.

Herschel Observations of C+ in the Vicinity of Star Forming Complexes in the Galactic Plane

NASA Astrophysics Data System (ADS)

Pineda, Jorge; Velusamy, T.; Langer, W.; Goldsmith, P.; Li, D.; Yorke, H.

2010-05-01

The CII fine-structure line at 158 um, is an excellent tracer of the warm diffuse gas and the hot, dense Photon Dominated Regions (PDRs). We can, therefore, use the CII emission as a probe to understand the effects of star formation on their interstellar environment. Here we present the first results from the Galactic Observations of Terahertz C+ (GOT C+), a Herschel Key Project study of CII fine structure emission in the vicinity of star forming complexes. In the Priority Science Phase of HIFI observations, the GOT C+ project collects data along a dozen lines of sight passing near star forming regions in the inner Galaxy from longitude 310 degrees to 25 degrees. We discuss our first results on the transition between dense and hot gas (traced by CII) and dense and cold gas (traced by 12CO and 13CO). This research was conducted at the Jet Propulsion Laboratory, California Institute of Technology under contract with the National Aeronautics and Space Administration. JLP was supported under the NASA Postdoctoral Program at JPL, Caltech, administered by Oak Ridge Associated Universities through a contract with NASA, and is currently supported as a Caltech-JPL Postdoc.

Numerical Study of High-Speed Droplet Impact on Surfaces and its Physical Cleaning Effects

NASA Astrophysics Data System (ADS)

Kondo, Tomoki; Ando, Keita

2015-11-01

Spurred by the demand for cleaning techniques of low environmental impact, one favors physical cleaning that does not rely on any chemicals. One of the promising candidates is based on water jets that often involve fission into droplet fragments and collide with target surfaces to which contaminant particles (often micron-sized or even smaller) stick. Hydrodynamic force (e.g., shearing and lifting) arising from the droplet impact will play a role to remove the particles, but its detailed mechanism is still unknown. To explore the role of high-speed droplet impact in physical cleaning, we solve compressible Navier-Stokes equations with a finite volume method that is designed to capture both shocks and material interfaces in accurate and robust manners. Water hammer and shear flow accompanied by high-speed droplet impact at a rigid wall is simulated to evaluate lifting force and rotating torque, which are relevant to the application of particle removal. For the simulation, we use the numerical code recently developed by Computational Flow Group lead by Tim Colonius at Caltech. The first author thanks Jomela Meng for her help in handling the code during his stay at Caltech.

Caltech water-ice dusty plasma: preliminary results

NASA Astrophysics Data System (ADS)

Bellan, Paul; Chai, Kilbyoung

2013-10-01

A water-ice dusty plasma laboratory experiment has begun operation at Caltech. As in Ref., a 1-5 watt parallel-plate 13.56 MHz rf discharge plasma has LN2-cooled electrodes that cool the neutral background gas to cryogenic temperatures. However, instead of creating water vapor by in-situ deuterium-oxygen bonding, here the neutral gas is argon and water vapor is added in a controlled fashion. Ice grains spontaneously form after a few seconds. Photography with a HeNe line filter of a sheet of HeNe laser light sheet illuminating a cross section of dust grains shows a large scale whorl pattern composed of concentric sub-whorls having wave-like spatially varying intensity. Each sub-whorl is composed of very evenly separated fine-scale stream-lines indicating that the ice grains move in self-organized lanes like automobiles on a multi-line highway. HeNe laser extinction together with an estimate of dust density from the intergrain spacing in photographs indicates a 5 micron nominal dust grain radius. HeNe laser diffraction patterns indicate the ice dust grains are large and ellipsoidal at low pressure (200 mT) but small and spheroidal at high pressure (>600 mT). Supported by USDOE.

Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats.

PubMed

Gabsalilow, Lilia; Schierling, Benno; Friedhoff, Peter; Pingoud, Alfred; Wende, Wolfgang

2013-04-01

Targeted genome engineering requires nucleases that introduce a highly specific double-strand break in the genome that is either processed by homology-directed repair in the presence of a homologous repair template or by non-homologous end-joining (NHEJ) that usually results in insertions or deletions. The error-prone NHEJ can be efficiently suppressed by 'nickases' that produce a single-strand break rather than a double-strand break. Highly specific nickases have been produced by engineering of homing endonucleases and more recently by modifying zinc finger nucleases (ZFNs) composed of a zinc finger array and the catalytic domain of the restriction endonuclease FokI. These ZF-nickases work as heterodimers in which one subunit has a catalytically inactive FokI domain. We present two different approaches to engineer highly specific nickases; both rely on the sequence-specific nicking activity of the DNA mismatch repair endonuclease MutH which we fused to a DNA-binding module, either a catalytically inactive variant of the homing endonuclease I-SceI or the DNA-binding domain of the TALE protein AvrBs4. The fusion proteins nick strand specifically a bipartite recognition sequence consisting of the MutH and the I-SceI or TALE recognition sequences, respectively, with a more than 1000-fold preference over a stand-alone MutH site. TALE-MutH is a programmable nickase.

Virtual screening on an α-helix to β-strand switchable region of the FGFR2 extracellular domain revealed positive and negative modulators.

PubMed

Diaz, Constantino; Corentin, Herbert; Thierry, Vermat; Chantal, Alcouffe; Tanguy, Bozec; David, Sibrac; Jean-Marc, Herbert; Pascual, Ferrara; Françoise, Bono; Edgardo, Ferran

2014-11-01

The secondary structure of some protein segments may vary between α-helix and β-strand. To predict these switchable segments, we have developed an algorithm, Switch-P, based solely on the protein sequence. This algorithm was used on the extracellular parts of FGF receptors. For FGFR2, it predicted that β4 and β5 strands of the third Ig-like domain were highly switchable. These two strands possess a high number of somatic mutations associated with cancer. Analysis of PDB structures of FGF receptors confirmed the switchability prediction for β5. We thus evaluated if compound-driven α-helix/β-strand switching of β5 could modulate FGFR2 signaling. We performed the virtual screening of a library containing 1.4 million of chemical compounds with two models of the third Ig-like domain of FGFR2 showing different secondary structures for β5, and we selected 32 compounds. Experimental testing using proliferation assays with FGF7-stimulated SNU-16 cells and a FGFR2-dependent Erk1/2 phosphorylation assay with FGFR2-transfected L6 cells, revealed activators and inhibitors of FGFR2. Our method for the identification of switchable proteinic regions, associated with our virtual screening approach, provides an opportunity to discover new generation of drugs with under-explored mechanism of action. © 2014 Wiley Periodicals, Inc.

A pinch of salt goes a long way in communicating astronomy

NASA Astrophysics Data System (ADS)

Manxoyi, S.

2008-06-01

The building of the Southern African Large Telescope not only revolutionised the methods of data collection in astronomy as a science in South Africa, but also changed the face, approach and impact of astronomy communication in our country. This presentation examines the various ways in which SALT has been supporting and continues to drive astronomy communication with the public. These include the following strands: learner activities, educator programmes, special events and national events as well general public programmes. The learner activities include SALT tours, space camps, stargazing, astronomy quiz, workshops, science clubs and job shadowing. The educators' strand includes workshops, projects, mini conferences, tours, team and co teaching. The public is catered for through special events, national events, exhibitions, star parties and festivals.

Direct DNA binding by Brca1.

PubMed

Paull, T T; Cortez, D; Bowers, B; Elledge, S J; Gellert, M

2001-05-22

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Electron attachment to DNA single strands: gas phase and aqueous solution.

PubMed

Gu, Jiande; Xie, Yaoming; Schaefer, Henry F

2007-01-01

The 2'-deoxyguanosine-3',5'-diphosphate, 2'-deoxyadenosine-3',5'-diphosphate, 2'-deoxycytidine-3',5'-diphosphate and 2'-deoxythymidine-3',5'-diphosphate systems are the smallest units of a DNA single strand. Exploring these comprehensive subunits with reliable density functional methods enables one to approach reasonable predictions of the properties of DNA single strands. With these models, DNA single strands are found to have a strong tendency to capture low-energy electrons. The vertical attachment energies (VEAs) predicted for 3',5'-dTDP (0.17 eV) and 3',5'-dGDP (0.14 eV) indicate that both the thymine-rich and the guanine-rich DNA single strands have the ability to capture electrons. The adiabatic electron affinities (AEAs) of the nucleotides considered here range from 0.22 to 0.52 eV and follow the order 3',5'-dTDP > 3',5'-dCDP > 3',5'-dGDP > 3',5'-dADP. A substantial increase in the AEA is observed compared to that of the corresponding nucleic acid bases and the corresponding nucleosides. Furthermore, aqueous solution simulations dramatically increase the electron attracting properties of the DNA single strands. The present investigation illustrates that in the gas phase, the excess electron is situated both on the nucleobase and on the phosphate moiety for DNA single strands. However, the distribution of the extra negative charge is uneven. The attached electron favors the base moiety for the pyrimidine, while it prefers the 3'-phosphate subunit for the purine DNA single strands. In contrast, the attached electron is tightly bound to the base fragment for the cytidine, thymidine and adenosine nucleotides, while it almost exclusively resides in the vicinity of the 3'-phosphate group for the guanosine nucleotides due to the solvent effects. The comparatively low vertical detachment energies (VDEs) predicted for 3',5'-dADP(-) (0.26 eV) and 3',5'-dGDP(-) (0.32 eV) indicate that electron detachment might compete with reactions having high activation barriers such as glycosidic bond breakage. However, the radical anions of the pyrimidine nucleotides with high VDE are expected to be electronically stable. Thus the base-centered radical anions of the pyrimidine nucleotides might be the possible intermediates for DNA single-strand breakage.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses.

PubMed

Son, Kyung-No; Liang, Zhiguo; Lipton, Howard L

2015-09-01

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation. An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Activation Energies for Dissociation of Double Strand Oligonucleotide Anions: Evidence for Watson–Crick Base Pairing in Vacuo

PubMed Central

Schnier, Paul D.; Klassen, John S.; Strittmatter, Eric F.; Williams*, Evan R.

2005-01-01

The dissociation kinetics of a series of complementary and noncomplementary DNA duplexes, (TGCA)23−, (CCGG)23−, (AATTAAT)23−, (CCGGCCG)23−, A7·T73−, A7·A73−, T7·T73−, and A7·C73− were investigated using blackbody infrared radiative dissociation in a Fourier transform mass spectrometer. From the temperature dependence of the unimolecular dissociation rate constants, Arrhenius activation parameters in the zero-pressure limit are obtained. Activation energies range from 1.2 to 1.7 eV, and preexponential factors range from 1013 to 1019 s−1. Dissociation of the duplexes results in cleavage of the noncovalent bonds and/or cleavage of covalent bonds leading to loss of a neutral nucleobase followed by backbone cleavage producing sequence-specific (a – base) and w ions. Four pieces of evidence are presented which indicate that Watson–Crick (WC) base pairing is preserved in complementary DNA duplexes in the gas phase: i. the activation energy for dissociation of the complementary dimer, A7·T73−, to the single strands is significantly higher than that for the related noncomplementary A7·A73− and T7·T73− dimers, indicating a stronger interaction between strands with a specific base sequence, ii. extensive loss of neutral adenine occurs for A7·A73− and A7·C73− but not for A7·T73− consistent with this process being shut down by WC hydrogen bonding, iii. a correlation is observed between the measured activation energy for dissociation to single strands and the dimerization enthalpy (−ΔHd) in solution, and iv. molecular dynamics carried out at 300 and 400 K indicate that WC base pairing is preserved for A7·T73− duplex, although the helical structure is essentially lost. In combination, these results provide strong evidence that WC base pairing can exist in the complete absence of solvent. PMID:16498487

Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

PubMed

Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

2013-07-31

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

SAMHD1 Sheds Moonlight on DNA Double-Strand Break Repair.

PubMed

Cabello-Lobato, Maria Jose; Wang, Siyue; Schmidt, Christine Katrin

2017-12-01

SAMHD1 (sterile α motif and histidine (H) aspartate (D) domain-containing protein 1) is known for its antiviral activity of hydrolysing deoxynucleotides required for virus replication. Daddacha et al. identify a hydrolase-independent, moonlighting function of SAMHD1 that facilitates homologous recombination of DNA double-strand breaks (DSBs) by promoting recruitment of C-terminal binding protein interacting protein (CTIP), a DNA-end resection factor, to damaged DNA. These findings could benefit anticancer treatment. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Splicing stimulates siRNA formation at Drosophila DNA double-strand breaks

PubMed Central

Merk, Karin; Breinig, Marco; Böttcher, Romy; Krebs, Stefan; Blum, Helmut; Boutros, Michael

2017-01-01

DNA double-strand breaks trigger the production of locus-derived siRNAs in fruit flies, human cells and plants. At least in flies, their biogenesis depends on active transcription running towards the break. Since siRNAs derive from a double-stranded RNA precursor, a major question is how broken DNA ends can generate matching sense and antisense transcripts. We performed a genome-wide RNAi-screen in cultured Drosophila cells, which revealed that in addition to DNA repair factors, many spliceosome components are required for efficient siRNA generation. We validated this observation through site-specific DNA cleavage with CRISPR-cas9 followed by deep sequencing of small RNAs. DNA breaks in intron-less genes or upstream of a gene’s first intron did not efficiently trigger siRNA production. When DNA double-strand breaks were induced downstream of an intron, however, this led to robust siRNA generation. Furthermore, a downstream break slowed down splicing of the upstream intron and a detailed analysis of siRNA coverage at the targeted locus revealed that unspliced pre-mRNA contributes the sense strand to the siRNA precursor. Since splicing factors are stimulating the response but unspliced transcripts are entering the siRNA biogenesis, the spliceosome is apparently stalled in a pre-catalytic state and serves as a signaling hub. We conclude that convergent transcription at DNA breaks is stimulated by a splicing dependent control process. The resulting double-stranded RNA is converted into siRNAs that instruct the degradation of cognate mRNAs. In addition to a potential role in DNA repair, the break-induced transcription may thus be a means to cull improper RNAs from the transcriptome of Drosophila melanogaster. Since the splicing factors identified in our screen also stimulated siRNA production from high copy transgenes, it is possible that this surveillance mechanism serves in genome defense beyond DNA double-strand breaks. PMID:28628606

DNA hairpins promote temperature controlled cargo encapsulation in a truncated octahedral nanocage structure family

NASA Astrophysics Data System (ADS)

Franch, Oskar; Iacovelli, Federico; Falconi, Mattia; Juul, Sissel; Ottaviani, Alessio; Benvenuti, Claudia; Biocca, Silvia; Ho, Yi-Ping; Knudsen, Birgitta R.; Desideri, Alessandro

2016-07-01

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr01806h

The NuSTAR Education and Public Outreach Program

NASA Astrophysics Data System (ADS)

Cominsky, Lynn R.; McLin, K. M.; Boggs, S.; Christensen, F.; Craig, W.; Hailey, C. J.; Harrison, F.; Stern, D.; Zhang, W.; NuSTAR Team

2013-01-01

NuSTAR is a NASA Small Explorer mission led by Caltech, managed by JPL, and implemented by an international team of scientists and engineers, under the direction of CalTech Professor Fiona Harrison, principal investigator. NuSTAR is a pathfinder mission that is opening the high-energy X-ray sky for sensitive study for the first time. By focusing X-rays at higher energies (up to 79 keV) NuSTAR will answer fundamental questions about the Universe: How are black holes distributed through the cosmos? How were the elements that compose our bodies and the Earth forged in the explosions of massive stars? What powers the most extreme active galaxies? Perhaps most exciting is the opportunity to fill a blank map with wonders we have not yet dreamed of: NuSTAR offers the opportunity to explore our Universe in an entirely new way. The purpose of the NuSTAR E/PO program is to increase understanding of the science of the high-energy Universe, by capitalizing on the synergy of existing high-energy astrophysics E/PO programs to support the mission’s objectives. Our goals are to: facilitate understanding of the nature of collapsed objects, develop awareness of the role of supernovae in creating the chemical elements and to facilitate understanding of the physical properties of the extreme Universe. We will do this through a program that includes educator workshops through NASA's Astrophysics Educator Ambassador program, a technology education unit for formal educators, articles for Physics Teacher and/or Science Scope magazines, and work with informal educators on a museum exhibit that includes a model of NuSTAR and describes the mission’s science objectives. Extensive outreach is also underway by members of the Science Team, who are working with high school students, undergraduates and graduate students. We are also developing printed materials that describe the mission and special workshops for girls at public libraries in order to improve the STEM pipeline.

The NuSTAR Education and Public Outreach Program

NASA Astrophysics Data System (ADS)

Cominsky, Lynn R.; McLin, K. M.; Boggs, S. E.; Christensen, F.; Hailey, C. J.; Harrison, F.; Stern, D.; Zhang, W.; NuSTAR Team

2013-04-01

NuSTAR is a NASA Small Explorer mission led by Caltech, managed by JPL, and implemented by an international team of scientists and engineers, under the direction of CalTech Professor Fiona Harrison, principal investigator. NuSTAR is a pathfinder mission that is opening the high-energy X-ray sky for sensitive study for the first time. By focusing X-rays at higher energies (up to 79 keV) NuSTAR will answer fundamental questions about the Universe: How are black holes distributed through the cosmos? How were the elements that compose our bodies and the Earth forged in the explosions of massive stars? What powers the most extreme active galaxies? Perhaps most exciting is the opportunity to fill a blank map with wonders we have not yet dreamed of: NuSTAR offers the opportunity to explore our Universe in an entirely new way. The purpose of the NuSTAR E/PO program is to increase understanding of the science of the high-energy Universe, by capitalizing on the synergy of existing high-energy astrophysics E/PO programs to support the mission’s objectives. Our goals are to: facilitate understanding of the nature of collapsed objects, develop awareness of the role of supernovae in creating the chemical elements and to facilitate understanding of the physical properties of the extreme Universe. We will do this through a program that includes educator workshops through NASA's Astrophysics Educator Ambassador program, a technology education unit for formal educators, articles for Physics Teacher and/or Science Scope magazines, and work with informal educators on a museum exhibit that includes a model of NuSTAR and describes the mission’s science objectives. Extensive outreach is also underway by members of the Science Team, who are working with high school students, undergraduates and graduate students. We are also developing printed materials that describe the mission and special workshops for girls at public libraries in order to improve the STEM pipeline.

The NuSTAR Education and Public Outreach Program

NASA Astrophysics Data System (ADS)

Cominsky, Lynn R.; McLin, K. M.; NuSTAR Science Team

2011-09-01

NuSTAR is a NASA Small Explorer mission led by Caltech, managed by JPL, and implemented by an international team under the direction of CalTech Professor Fiona Harrison. NuSTAR is a pathfinder mission that will open the high-energy X-ray sky for sensitive study for the first time. By focusing X-rays at energies up to 79 keV, NuSTAR will answer fundamental questions about the Universe: How are black holes distributed through the cosmos? How were the elements that compose our bodies and the Earth forged in the explosions of massive stars? What powers the most extreme active galaxies? Perhaps most exciting is the opportunity to fill a blank map with wonders we have not yet dreamed of: NuSTAR offers the opportunity to explore our Universe in an entirely new way. The purpose of the NuSTAR E/PO program is to increase understanding of the science of the high-energy Universe, by capitalizing on the synergy of existing high-energy astrophysics E/PO programs to support the mission's objectives. Our goals are to: facilitate understanding of the nature of collapsed objects, develop awareness of the role of supernovae in creating the chemical elements and to facilitate understanding of the physical properties of the extreme Universe. We will do this through a program that includes educator workshops through NASA's Astrophysics Educator Ambassador program, a technology education unit for formal educators, articles for Physics Teacher and Science Scope magazines, and work with informal educators on a museum exhibit that includes a model of NuSTAR and describes the mission's science objectives. Extensive outreach is also underway by members of the Science Team, who are working with high school students, undergraduates and graduate students. We will also develop printed materials that describe the mission, and help develop the STEM pipeline through local after-school programs.

The California-Arizona Minority Partnership for Astronomy Research and Education (CAMPARE): A New Model for Promoting Minority Participation in Astronomy Research and Education

NASA Astrophysics Data System (ADS)

Rudolph, Alexander L.; Impey, C. D.; Bieging, J. H.; Phillips, C. B.; Tieu, J.; Prather, E. E.; Povich, M. S.

2013-01-01

The California-Arizona Minority Partnership for Astronomy Research and Education (CAMPARE) program represents a new and innovative kind of research program for undergraduates: one that can effectively carry out the goal of recruiting qualified minority and female students to participate in Astronomy and Planetary Science research opportunities, while mentoring them in a way to maximize the chance that these students will persist in obtaining their undergraduate degrees in STEM fields, and potentially go on to obtain their PhDs or pursue careers in those fields. The members of CAMPARE comprise a network of comprehensive universities and community colleges in Southern California and Arizona (most of which are minority serving institutions), and four major research institutions (University of Arizona Steward Observatory, the SETI Institute, and JPL/Caltech). Most undergraduate research programs focus on a single research institution. By having multiple institutions, we significantly broaden the opportunities for students, both in terms of breadth of research topics and geographical location. In its first three years, the CAMPARE program has had 20 undergraduates from two CSU campuses, both Hispanic Serving Institutions, take part in research and educational activities at four research institutions, the University of Arizona Steward Observatory, the SETI Institute, and JPL/Caltech. Of the 20 participants, 9 are women and 11 are men, a much more even split than is typical in Astronomy research programs; 10 are Hispanic, 2 are African American, and 1 is part Native American, including 2 female Hispanic and 2 female African-American participants, an exceptionally high participation rate (65%) for students from underrepresented minority groups. Of the five participants who have graduated since the program began, two are in graduate programs in Physics or Astronomy, two are pursuing a K-12 teaching credential, and one has enlisted in the Nuclear Propulsion Officer Candidate (NUPOC) program of the U.S. Navy.

The central branch of the North Anatolian Fault In The Southern Marmara Sea: Evidence for a distributed, Holocene-active fault system

NASA Astrophysics Data System (ADS)

Barın, Burcu; Okay, Seda; Çifçi, Günay; Dondurur, Derman; Cormier, Marie Helene; Sorlien, Christopher; Meriç İlkimen, Elif

2015-04-01

The North Anatolian Fault (NAF) is a major right-lateral transform fault in northern Turkey that branches westward into several strands in the vicinity of the Sea of Marmara. The main northern branch bisects the Marmara Sea from east to west, and seismic reflection profiles acquired over the past 15 years have revealed its complex geometry. Further, the several basins that developed along that branch record stratigraphic sequences that provide the needed framework to interpret the relative timing of tectonic deformation in the Marmara Sea. In contrast, the central branch, which snakes across the shallow southern shelf of the Marmara Sea, has been much less investigated. Here, we analyze a comprehensive dataset of high-resolution multi-channel, sparker, and CHIRP seismic profiles, which were collected with the facilities of Seismic Laboratory (SeisLab) in the Institute of Marine Sciences and Technology and R/V K. Piri Reis belonging to Dokuz Eylül University, along the central branch in 2008 (TAMAM expedition) and in 2013-2014 (SoMAR expedition), within the framework of a bilateral TÜBİTAK - NSF project. In combination with other existing seismic profiles, these new data reveal that the Central Branch consists of multiple faults strands that are distributed across the broad southern shelf. They also reveal that many of these strands are Holocene-active, although they slip at slower rates than the northern branch and are associated with slower basin subsidence or local uplift. Lastly, seismic data image a system of half-grabens across the southern shelf that are associated with the strands of the central branch. Strata within these half-grabens are progressively tilted and consistently dip to the south. Further analysis will be conducted to determine whether the formation of these grabens are controlled by oblique slip on the strands of the central branch, or by slip on detachment faults beneath the southern shelf.

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

PubMed Central

Manhart, Carol M.; Ni, Xiaodan; White, Martin A.; Ortega, Joaquin; Surtees, Jennifer A.

2017-01-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA. PMID:28453523

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

PubMed

Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

2017-04-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

PubMed

Chan, Chi-Ping; Yuen, Chun-Kit; Cheung, Pak-Hin Hinson; Fung, Sin-Yee; Lui, Pak-Yin; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

2018-03-07

PACT is a double-stranded RNA-binding protein that has been implicated in host-influenza A virus (IAV) interaction. PACT facilitates the action of RIG-I in the activation of the type I IFN response, which is suppressed by the viral nonstructural protein NS1. PACT is also known to interact with the IAV RNA polymerase subunit PA. Exactly how PACT exerts its antiviral activity during IAV infection remains to be elucidated. In the current study, we demonstrated the interplay between PACT and IAV polymerase. Induction of IFN-β by the IAV RNP complex was most robust when both RIG-I and PACT were expressed. PACT-dependent activation of IFN-β production was suppressed by the IAV polymerase subunits, polymerase acidic protein, polymerase basic protein 1 (PB1), and PB2. PACT associated with PA, PB1, and PB2. Compromising PACT in IAV-infected A549 cells resulted in the augmentation of viral RNA (vRNA) transcription and replication and IFN-β production. Furthermore, vRNA replication was boosted by knockdown of PACT in both A549 cells and IFN-deficient Vero cells. Thus, the antiviral activity of PACT is mediated primarily via its interaction with and inhibition of IAV polymerase. Taken together, our findings reveal a new facet of the host-IAV interaction in which the interplay between PACT and IAV polymerase affects the outcome of viral infection and antiviral response.-Chan, C.-P., Yuen, C.-K., Cheung, P.-H. H., Fung, S.-Y., Lui, P.-Y., Chen, H., Kok, K.-H., Jin, D.-Y. Antiviral activity of double-stranded RNA-binding protein PACT against influenza A virus mediated via suppression of viral RNA polymerase.

Toehold-mediated strand displacement reaction-dependent fluorescent strategy for sensitive detection of uracil-DNA glycosylase activity.

PubMed

Wu, Yushu; Wang, Lei; Jiang, Wei

2017-03-15

Sensitive detection of uracil-DNA glycosylase (UDG) activity is beneficial for evaluating the repairing process of DNA lesions. Here, toehold-mediated strand displacement reaction (TSDR)-dependent fluorescent strategy was constructed for sensitive detection of UDG activity. A single-stranded DNA (ssDNA) probe with two uracil bases and a trigger sequence were designed. A hairpin probe with toehold domain was designed, and a reporter probe was also designed. Under the action of UDG, two uracil bases were removed from ssDNA probe, generating apurinic/apyrimidinic (AP) sites. Then, the AP sites could inhibit the TSDR between ssDNA probe and hairpin probe, leaving the trigger sequence in ssDNA probe still free. Subsequently, the trigger sequence was annealed with the reporter probe, initiating the polymerization and nicking amplification reaction. As a result, numerous G-quadruplex (G4) structures were formed, which could bind with N-methyl-mesoporphyrin IX (NMM) to generate enhanced fluorescent signal. In the absence of UDG, the ssDNA probe could hybridize with the toehold domain of the hairpin probe to initiate TSDR, blocking the trigger sequence, and then the subsequent amplification reaction would not occur. The proposed strategy was successfully implemented for detecting UDG activity with a detection limit of 2.7×10 -5 U/mL. Moreover, the strategy could distinguish UDG well from other interference enzymes. Furthermore, the strategy was also applied for detecting UDG activity in HeLa cells lysate with low effect of cellular components. These results indicated that the proposed strategy offered a promising tool for sensitive quantification of UDG activity in UDG-related function study and disease prognosis. Copyright © 2016 Elsevier B.V. All rights reserved.

CarD uses a minor groove wedge mechanism to stabilize the RNA polymerase open promoter complex.

PubMed

Bae, Brian; Chen, James; Davis, Elizabeth; Leon, Katherine; Darst, Seth A; Campbell, Elizabeth A

2015-09-08

A key point to regulate gene expression is at transcription initiation, and activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, is found in many bacteria, including Thermus species, but absent in Escherichia coli. To delineate the molecular mechanism of CarD, we determined crystal structures of Thermus transcription initiation complexes containing CarD. The structures show CarD interacts with the unique DNA topology presented by the upstream double-stranded/single-stranded DNA junction of the transcription bubble. We confirm that our structures correspond to functional activation complexes, and extend our understanding of the role of a conserved CarD Trp residue that serves as a minor groove wedge, preventing collapse of the transcription bubble to stabilize the transcription initiation complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter complexes, explaining the need for CarD.

Shape changing thin films powered by DNA hybridization

NASA Astrophysics Data System (ADS)

Shim, Tae Soup; Estephan, Zaki G.; Qian, Zhaoxia; Prosser, Jacob H.; Lee, Su Yeon; Chenoweth, David M.; Lee, Daeyeon; Park, So-Jung; Crocker, John C.

2017-01-01

Active materials that respond to physical and chemical stimuli can be used to build dynamic micromachines that lie at the interface between biological systems and engineered devices. In principle, the specific hybridization of DNA can be used to form a library of independent, chemically driven actuators for use in such microrobotic applications and could lead to device capabilities that are not possible with polymer- or metal-layer-based approaches. Here, we report shape changing films that are powered by DNA strand exchange reactions with two different domains that can respond to distinct chemical signals. The films are formed from DNA-grafted gold nanoparticles using a layer-by-layer deposition process. Films consisting of an active and a passive layer show rapid, reversible curling in response to stimulus DNA strands added to solution. Films consisting of two independently addressable active layers display a complex suite of repeatable transformations, involving eight mechanochemical states and incorporating self-righting behaviour.

DNA hybridization activity of single-stranded DNA-conjugated gold nanoparticles used as probes for DNA detection

NASA Astrophysics Data System (ADS)

Kira, Atsushi; Matsuo, Kosuke; Nakajima, Shin-ichiro

2016-02-01

Colloidal nanoparticles (NPs) have potential applications in bio-sensing technologies as labels or signal enhancers. In order to meet demands for a development of biomolecular assays by a quantitative understanding of single-molecule, it is necessary to regulate accuracy of the NPs probes modified with biomolecules to optimize the characteristics of NPs. However, to our knowledge, there is little information about the structural effect of conjugated biomolecules to the NPs. In this study, we investigated the contribution of a density of single-stranded DNA (ssDNA) conjugating gold NP to hybridization activity. Hybridization activity decreased in accordance with increases in the density of attached ssDNAs, likely due to electrostatic repulsion generated by negatively charged phosphate groups in the ssDNA backbone. These results highlight the importance of controlling the density of ssDNAs attached to the surface of NPs used as DNA detection probes.

Identification of a broad-spectrum inhibitor of virus RNA synthesis: validation of a prototype virus-based approach

PubMed Central

Filone, Claire Marie; Hodges, Erin N.; Honeyman, Brian; Bushkin, G. Guy; Boyd, Karla; Platt, Andrew; Ni, Feng; Strom, Kyle; Hensley, Lisa; Snyder, John K.; Connor, John H.

2013-01-01

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify new chemical scaffolds for development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity, and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses. PMID:23521799

Reuse, Repurposing and Learning Design--Lessons from the DART Project

ERIC Educational Resources Information Center

Bond, Stephen T.; Ingram, Caroline; Ryan, Steve

2008-01-01

Digital Anthropological Resources for Teaching (DART) is a major project examining ways in which the use of online learning activities and repositories can enhance the teaching of anthropology and, by extension, other disciplines. This paper reports on one strand of DART activity, the development of customisable learning activities that can be…

Colorimetric molecular diagnosis of the HIV gag gene using DNAzyme and a complementary DNA-extended primer.

PubMed

Kim, Seong U; Batule, Bhagwan S; Mun, Hyoyoung; Byun, Ju-Young; Shim, Won-Bo; Kim, Min-Gon

2018-02-07

We have developed a novel strategy for the colorimetric detection of PCR products by utilizing a target-specific primer modified at the 5'-end with an anti-DNAzyme sequence. A single-stranded DNAzyme sequence folds into a G-quadruplex structure with hemin and shows strong peroxidase activity. When the complementary strand binds to the DNAzyme sequence, it blocks the formation of the G-quadraduplex structure and loses its peroxidase activity. In the presence of the target gene, PCR amplification proceeds, and anti-DNAzyme sequence modified primers present in the reaction mixture form a double strand through primer extension. Therefore, it does not block the DNAzyme sequence. Further, a colorimetric signal is generated by the addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and H 2 O 2 at the end of the reaction. We have successfully detected a single copy of the HIV type 1 gag gene in buffer and 10 copies in human serum. The strategy developed could be used to detect DNA and RNA in complex biological samples by simple primer designing that includes DNAzyme and a DNA extended primer.

Direct Binding to Replication Protein A (RPA)-coated Single-stranded DNA Allows Recruitment of the ATR Activator TopBP1 to Sites of DNA Damage*

PubMed Central

Acevedo, Julyana; Yan, Shan; Michael, W. Matthew

2016-01-01

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes. PMID:27129245

Environmental Predictors of Seabird Wrecks in a Tropical Coastal Area

PubMed Central

Fulgencio de Moura, Jailson; Siciliano, Salvatore

2016-01-01

Beached bird surveys have been widely used to monitor the impact of oil pollution in the oceans. However, separating the combined effects of oil pollution, environmental variables and methodological aspects of beach monitoring on seabird stranding patterns is a challenging task. The effects of a comprehensive set of oceanographic and climatic variables and oil pollution on seabird strandings in a tropical area of Brazil were investigated herein, using two robust and innovative methods: Generalized Linear Mixed Models and Structural Equation Modeling. We assessed strandings of four resident seabird species along 480 km of beaches divided into 11 sampling areas, between November 2010 and September 2013. We found that increasing the distance from the nearest breeding island reduce the seabird stranding events. Storm activity and biological productivity were the most important factors affecting the stranding events of brown boobies Sula leucogaster, Cabot’s terns Thalasseus acuflavidus and kelp gulls Larus dominicanus. These species are also indirectly affected by warm tropical waters, which reduce chlorophyll-a concentrations. Beach surveys are, thus, useful to investigate the mortality rates of resident species near breeding sites, where individuals are more abundant and exposed to local factors associated with at-sea mortality. In contrast, conservation actions and monitoring programs for far-ranging seabird species are needed in more distant foraging areas. Furthermore, beach monitoring programs investigating the impact of oil pollution on seabirds need to account for the effects of environmental factors on stranding patterns. The present study also demonstrated that seabirds inhabiting tropical coastal waters are sensitive to climate conditions such as adverse weather, which are expected to increase in frequency and intensity in next decades. PMID:27992578

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model.

PubMed

Noguchi, Yasunori; Yuan, Zuanning; Bai, Lin; Schneider, Sarah; Zhao, Gongpu; Stillman, Bruce; Speck, Christian; Li, Huilin

2017-11-07

During replication initiation, the core component of the helicase-the Mcm2-7 hexamer-is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide-oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2-Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion. Copyright © 2017 the Author(s). Published by PNAS.

Detection and molecular characterization of Giardia and Cryptosporidium in common dolphins (Delphinus delphis) stranded along the Galician coast (Northwest Spain).

PubMed

Reboredo-Fernández, A; Gómez-Couso, H; Martínez-Cedeira, J A; Cacciò, S M; Ares-Mazás, E

2014-05-28

The ubiquitous protozoan parasites Giardia and Cryptosporidium have been detected from many species of captive and free-living wildlife, representing most mammalian orders. Twenty species of marine mammals have been reported to inhabit Galician waters and the region has one of the highest rates of stranding in Europe. Evidence from stranding, reported by-catches and sightings, suggests that the common dolphin (Delphinus delphis) is the most abundant cetacean on the Galician coast (Northwest Spain). The objective of this study was to detect and molecularly characterize isolates of Giardia and Cryptosporidium obtained from common dolphins stranded in this area. Between 2005 and 2012, sections of large intestine from 133 common dolphins stranded along the Galician coast were collected by the personnel of the Galician Stranding Network (Coordinadora para o Estudo dos Mamíferos Mariños, CEMMA). Using direct immunofluorescence antibody test (IFAT) and PCR amplification and sequencing of the SSU-rDNA, β-giardin genes and the ITS1-5.8S-ITS2 region, Giardia and Cryptosporidium were detected in 8 (6.0%) and 12 samples (9.0%), respectively. In two samples, co-infection by both parasites was observed. The molecular characterization revealed the presence of Giardia duodenalis assemblages A (genotypes A1 and A2) and B and Cryptosporidium parvum in these samples. This constitutes the first study in which the presence of Giardia and Cryptosporidium has been investigated in common dolphins on the European Atlantic coast, and it is also the first report of C. parvum in this host. Our findings indicate that these animals could act as reservoir of these waterborne parasites or could be victims of the contamination originated by anthropogenic activities. Copyright © 2014 Elsevier B.V. All rights reserved.

Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets.

PubMed

Indu, S; Kochat, V; Thakurela, S; Ramakrishnan, C; Varadarajan, Raghavan

2011-01-01

Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel β-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ¹ value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in T(m). All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG⁰ = -3.3 to -6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity. © 2010 Wiley-Liss, Inc.

Femtosecond near-infrared laser microirradiation reveals a crucial role for PARP signaling on factor assemblies at DNA damage sites

PubMed Central

Saquilabon Cruz, Gladys Mae; Kong, Xiangduo; Silva, Bárbara Alcaraz; Khatibzadeh, Nima; Thai, Ryan; Berns, Michael W.; Yokomori, Kyoko

2016-01-01

Laser microirradiation is a powerful tool for real-time single-cell analysis of the DNA damage response (DDR). It is often found, however, that factor recruitment or modification profiles vary depending on the laser system employed. This is likely due to an incomplete understanding of how laser conditions/dosages affect the amounts and types of damage and the DDR. We compared different irradiation conditions using a femtosecond near-infrared laser and found distinct damage site recruitment thresholds for 53BP1 and TRF2 correlating with the dose-dependent increase of strand breaks and damage complexity. Low input-power microirradiation that induces relatively simple strand breaks led to robust recruitment of 53BP1 but not TRF2. In contrast, increased strand breaks with complex damage including crosslinking and base damage generated by high input-power microirradiation resulted in TRF2 recruitment to damage sites with no 53BP1 clustering. We found that poly(ADP-ribose) polymerase (PARP) activation distinguishes between the two damage states and that PARP activation is essential for rapid TRF2 recruitment while suppressing 53BP1 accumulation at damage sites. Thus, our results reveal that careful titration of laser irradiation conditions allows induction of varying amounts and complexities of DNA damage that are gauged by differential PARP activation regulating protein assembly at the damage site. PMID:26424850

Structural analysis of nested neutralizing and non-neutralizing B cell epitopes on ricin toxin's enzymatic subunit.

PubMed

Rudolph, Michael J; Vance, David J; Cassidy, Michael S; Rong, Yinghui; Shoemaker, Charles B; Mantis, Nicholas J

2016-08-01

In this report, we describe the X-ray crystal structures of two single domain camelid antibodies (VH H), F5 and F8, each in complex with ricin toxin's enzymatic subunit (RTA). F5 has potent toxin-neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Å(2) in complex with RTA and made contact with three prominent secondary structural elements: α-helix B (Residues 98-106), β-strand h (Residues 113-117), and the C-terminus of α-helix D (Residues 154-156). F8 buried 1103 Å(2) in complex with RTA that was centered primarily on β-strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions CDRs were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β-strand within RTA's centrally located β-sheet. A comparison of the two structures reported here to several previously reported (RTA-VH H) structures identifies putative contact sites on RTA, particularly α-helix B, associated with potent toxin-neutralizing activity. This information has implications for rational design of RTA-based subunit vaccines for biodefense. Proteins 2016; 84:1162-1172. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Dynamic protein assembly by programmable DNA strand displacement.

PubMed

Chen, Rebecca P; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-04-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

PubMed

Pu, Xinzhu; Wang, Zemin; Klaunig, James E

2015-08-06

Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

Toehold-Mediated Displacement of an Adenosine-Binding Aptamer from a DNA Duplex by its Ligand.

PubMed

Monserud, Jon H; Macri, Katherine M; Schwartz, Daniel K

2016-10-24

DNA is increasingly used to engineer dynamic nanoscale circuits, structures, and motors, many of which rely on DNA strand-displacement reactions. The use of functional DNA sequences (e.g., aptamers, which bind to a wide range of ligands) in these reactions would potentially confer responsiveness on such devices, and integrate DNA computation with highly varied molecular stimuli. By using high-throughput single-molecule FRET methods, we compared the kinetics of a putative aptamer-ligand and aptamer-complement strand-displacement reaction. We found that the ligands actively disrupted the DNA duplex in the presence of a DNA toehold in a similar manner to complementary DNA, with kinetic details specific to the aptamer structure, thus suggesting that the DNA strand-displacement concept can be extended to functional DNA-ligand systems. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic protein assembly by programmable DNA strand displacement

NASA Astrophysics Data System (ADS)

Chen, Rebecca P.; Blackstock, Daniel; Sun, Qing; Chen, Wilfred

2018-03-01

Inspired by the remarkable ability of natural protein switches to sense and respond to a wide range of environmental queues, here we report a strategy to engineer synthetic protein switches by using DNA strand displacement to dynamically organize proteins with highly diverse and complex logic gate architectures. We show that DNA strand displacement can be used to dynamically control the spatial proximity and the corresponding fluorescence resonance energy transfer between two fluorescent proteins. Performing Boolean logic operations enabled the explicit control of protein proximity using multi-input, reversible and amplification architectures. We further demonstrate the power of this technology beyond sensing by achieving dynamic control of an enzyme cascade. Finally, we establish the utility of the approach as a synthetic computing platform that drives the dynamic reconstitution of a split enzyme for targeted prodrug activation based on the sensing of cancer-specific miRNAs.

Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction.

PubMed

Guo, Qiuping; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Li, Wei; Tang, Hongxing; Li, Huimin

2009-02-01

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 x 10(-15) M.

Exonuclease of human DNA polymerase gamma disengages its strand displacement function.

PubMed

He, Quan; Shumate, Christie K; White, Mark A; Molineux, Ian J; Yin, Y Whitney

2013-11-01

Pol γ, the only DNA polymerase found in human mitochondria, functions in both mtDNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo(-) (3'-5' exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo(-), is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB. © 2013. Published by Elsevier B.V.

DNA end-processing enzyme polynucleotide kinase as a potential target in the treatment of cancer.

PubMed

Allinson, Sarah L

2010-06-01

Pharmacological inhibition of DNA-repair pathways as an approach for the potentiation of chemo- and radio-therapeutic cancer treatments has attracted increasing levels of interest in recent years. Inhibitors of several enzymes involved in the repair of DNA strand breaks are currently at various stages of the drug development process. Polynucleotide kinase (PNK), a bifunctional DNA-repair enzyme that possesses both 3'-phosphatase and 5'-kinase activities, plays an important role in the repair of both single strand and double strand breaks and as a result, RNAi-mediated knockdown of PNK sensitizes cells to a range of DNA-damaging agents. Recently, a small molecule inhibitor of PNK has been developed that is able to sensitize cells to ionizing radiation and the topoisomerase I poison, camptothecin. Although still in the early stages of development, PNK inhibition represents a promising means of enhancing the efficacy of existing cancer treatments.

APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

PubMed

Nowarski, Roni; Kotler, Moshe

2013-06-15

High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zahn, Karl E.; Averill, April M.; Aller, Pierre

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contactsmore » to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.« less

Shifting Plasma

NASA Image and Video Library

2017-12-08

Strands of solar material at the sun's edge shifted and twisted back and forth over a 22-hour period in this footage captured May 2-3, 2017, by NASA’s Solar Dynamics Observatory. In this close-up, the strands are being manipulated by strong magnetic forces associated with active regions. To give a sense of scale, the strands that hover above the sun are more than several times the size of Earth. These images were taken in a wavelength of extreme ultraviolet light, which is typically invisible to our eyes, but was colorized here in red. go.nasa.gov/2qJzPD2 Credit: NASA/Goddard/SDO NASA image use policy. NASA Goddard Space Flight Center enables NASA’s mission through four scientific endeavors: Earth Science, Heliophysics, Solar System Exploration, and Astrophysics. Goddard plays a leading role in NASA’s accomplishments by contributing compelling scientific knowledge to advance the Agency’s mission. Follow us on Twitter Like us on Facebook Find us on Instagram

What Is Deep Vein Thrombosis?

MedlinePlus

... the blood to trigger the activity of the enzyme thrombin. Active thrombin then forms long protein strands that clump together with platelets and red blood cells to form clots. Read less Risk Factors Risk factors for VTE include a history of a previous VTE event; surgery; medical conditions ...

Image Halftoning Using Optimized Dot Diffusion

DTIC Science & Technology

1998-01-01

ppvnath@sys.caltech.edu ABSTRACT The dot diffusion method for digital halftoning has the advantage of parallelism unlike the error diffusion ...digital halftoning : ordered dither [1], error diffusion [2], neural-net based methods [8], and more recently direct binary search (DBS) [7]. Ordered...from periodic patterns. On the other hand error diffused halftones do not suffer from periodicity and offer blue noise characteristic [3] which is

(abstract) Absolute Flux Calibrations of Venus and Jupiter at 32 GHz

NASA Technical Reports Server (NTRS)

Gatti, Mark S.; Klein, Michael J.

1994-01-01

The microwave flux densities of Venus and Jupiter at 32 GHz have been measured using a calibration standard radio telescope system at the Owens Valley Radio Observatory (OVRO) during April and May of 1993. These measurements are part of a joint JPL/Caltech program to accurately calibrate a catalog of other radio sources using the two bright planets as flux standards.

Quantum Algorithms for Fermionic Quantum Field Theories

DTIC Science & Technology

2014-04-28

preskill@theory.caltech.edu 1 Report Documentation Page Form ApprovedOMB No. 0704-0188 Public reporting burden for the collection of information is...NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98...operators of momentum modes. (The choice between these forms of measurement depends on the application.) 2.3 Complexity In this section we bound the

A Treasure Trove of Planets Found

NASA Image and Video Library

2017-02-28

Announcement of the discovery of seven rocky planets orbiting TRAPPIST-1, a star 40 light years from Earth. Three of the planets are in the habitable zone, though all seven could have liquid water. Animation with interviews featuring Sean Carey, Manager, Spitzer Science Center, Caltech/IPAC; Nikole Lewis, James Webb Telescope Project Scientist, Space Telescope Science Institute; and MIchael Gillon, Principal Investigator, TRAPPIST, University of Liege, Belgium.

A Conversation with Robert F. Christy Part I

NASA Astrophysics Data System (ADS)

Lippincott, Sara

2006-09-01

Robert F. Christy, Institute Professor of Theoretical Physics Emeritus at Caltech, recalls his childhood in British Columbia; his undergraduate years at the University of British Columbia; his graduate work with J. Robert Oppenheimer at Berkeley; and his work on the Manhattan Project, first with Enrico Fermi at the Metallurgical Laboratory of the University of Chicago and then as a member of the Theoretical Division at Los Alamos.

Team RoboSimian

NASA Image and Video Library

2015-06-09

Many members of Team RoboSimian and a few guests gather with competition hardware at a "Meet the Robots" event during the DARPA Robotics Challenge Finals in Pomona, California, on June 6, 2015. The RoboSimian team at JPL is collaborating with partners at the University of California, Santa Barbara, and the California Institute of Technology in Pasadena. Caltech manages JPL for NASA. http://photojournal.jpl.nasa.gov/catalog/PIA19329

ASK Magazine. No. 9

NASA Technical Reports Server (NTRS)

Post, Todd (Editor)

2002-01-01

Most of this issue is about ACE (Advanced Composition Explorer). We've collected stories by four members of the ACE management team: Don Margolies, the mission manager from Goddard Space Flight Center; Frandsen, science payloads manager from the Caltech Jet Propulsion Laboratory; Mary Chin, project manager in charge of spacecraft development at the Johns Hopkins Applied Physics Laboratory; and Frank Snow, operations and ground systems manager at Goddard.

Synthesis, Biological Evaluation, and Structure–Activity Relationships of Novel Substituted N-Phenyl Ureidobenzenesulfonate Derivatives Blocking Cell Cycle Progression in S-Phase and Inducing DNA Double-Strand Breaks

PubMed Central

2012-01-01

Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents. PMID:22694057

Synthesis, biological evaluation, and structure-activity relationships of novel substituted N-phenyl ureidobenzenesulfonate derivatives blocking cell cycle progression in S-phase and inducing DNA double-strand breaks.

PubMed

Turcotte, Vanessa; Fortin, Sébastien; Vevey, Florence; Coulombe, Yan; Lacroix, Jacques; Côté, Marie-France; Masson, Jean-Yves; C-Gaudreault, René

2012-07-12

Twenty-eight new substituted N-phenyl ureidobenzenesulfonate (PUB-SO) and 18 N-phenylureidobenzenesulfonamide (PUB-SA) derivatives were prepared. Several PUB-SOs exhibited antiproliferative activity at the micromolar level against the HT-29, M21, and MCF-7 cell lines and blocked cell cycle progression in S-phase similarly to cisplatin. In addition, PUB-SOs induced histone H2AX (γH2AX) phosphorylation, indicating that these molecules induce DNA double-strand breaks. In contrast, PUB-SAs were less active than PUB-SOs and did not block cell cycle progression in S-phase. Finally, PUB-SOs 4 and 46 exhibited potent antitumor activity in HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membranes, which was similar to cisplatin and combretastatin A-4 and without significant toxicity toward chick embryos. These new compounds are members of a promising new class of anticancer agents.

Formaldehyde activation of mitoxantrone yields CpG and CpA specific DNA adducts

PubMed Central

Parker, Belinda S.; Cutts, Suzanne M.; Cullinane, Carleen; Phillips, Don R.

2000-01-01

Recently we have found that mitoxantrone, like Adriamycin, can be activated by formaldehyde and subsequently form adducts which stabilise double-stranded DNA in vitro. This activation by formaldehyde may be biologically relevant since formaldehyde levels are elevated in those tumours in which mitoxantrone is most cytotoxic. In vitro transcription analysis revealed that these adducts block the progression of RNA polymerase during transcription and cause truncated RNA transcripts. There was an absolute requirement for both mitoxantrone and formaldehyde in transcriptional blockage formation and the activated complex was found to exhibit site specificity, with blockage occurring prior to CpG and CpA sites in the DNA (non-template strand). The stability of the adduct at 37°C was site dependent. The half-lives ranged from 45 min to ~5 h and this was dependent on both the central 2 bp blockage site as well as flanking sequences. The CpG specificity of mitoxantrone adduct sites was also confirmed independently by a λ exonuclease digestion assay. PMID:10648792

RNA major groove modifications improve siRNA stability and biological activity

PubMed Central

Terrazas, Montserrat; Kool, Eric T.

2009-01-01

RNA 5-methyl and 5-propynyl pyrimidine analogs were substituted into short interfering RNAs (siRNAs) to probe major groove steric effects in the active RNA-induced silencing complex (RISC). Synthetic RNA guide strands containing varied combinations of propynyl and methyl substitution revealed that all C-5 substitutions increased the thermal stability of siRNA duplexes containing them. Cellular gene suppression experiments using luciferase targets in HeLa cells showed that the bulky 5-propynyl modification was detrimental to RNA interference activity, despite its stabilization of the helix. Detrimental effects of this substitution were greatest at the 5′-half of the guide strand, suggesting close steric approach of proteins in the RISC complex with that end of the siRNA/mRNA duplex. However, substitutions with the smaller 5-methyl group resulted in gene silencing activities comparable to or better than that of wild-type siRNA. The major groove modifications also increased the serum stability of siRNAs. PMID:19042976

Probing Gαi1 Protein Activation at Single Amino Acid Resolution

PubMed Central

Sun, Dawei; Maeda, Shoji; Matkovic, Milos; Mendieta, Sandro; Mayer, Daniel; Dawson, Roger; Schertler, Gebhard F.X.; Madan Babu, M.; Veprintsev, Dmitry B.

2016-01-01

We present comprehensive single amino acid resolution maps of the residues stabilising the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and of the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-6. Key residues in this cluster are Y320, crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the inter-domain interface and release of GDP. PMID:26258638

Analysis of the crystal structure of an active MCM hexamer.

PubMed

Miller, Justin M; Arachea, Buenafe T; Epling, Leslie B; Enemark, Eric J

2014-09-29

In a previous Research article (Froelich et al., 2014), we suggested an MCM helicase activation mechanism, but were limited in discussing the ATPase domain because it was absent from the crystal structure. Here we present the crystal structure of a nearly full-length MCM hexamer that is helicase-active and thus has all features essential for unwinding DNA. The structure is a chimera of Sulfolobus solfataricus N-terminal domain and Pyrococcus furiosus ATPase domain. We discuss three major findings: 1) a novel conformation for the A-subdomain that could play a role in MCM regulation; 2) interaction of a universally conserved glutamine in the N-terminal Allosteric Communication Loop with the AAA+ domain helix-2-insert (h2i); and 3) a recessed binding pocket for the MCM ssDNA-binding motif influenced by the h2i. We suggest that during helicase activation, the h2i clamps down on the leading strand to facilitate strand retention and regulate ATP hydrolysis.

Hubble tracks down a galaxy cluster's dark matter

NASA Astrophysics Data System (ADS)

2003-07-01

Unique mass map hi-res Size hi-res: 495 kb Credits: European Space Agency, NASA and Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, USA) Unique mass map This is a mass map of galaxy cluster Cl0024+1654 derived from an extensive Hubble Space Telescope campaign. The colour image is made from two images: a dark-matter map (the blue part of the image) and a 'luminous-matter' map determined from the galaxies in the cluster (the red part of the image). They were constructed by feeding Hubble and ground-based observations into advanced mathematical mass-mapping models. The map shows that dark matter is present where the galaxies clump together. The mass of the galaxies is shown in red, the mass of the dark matter in blue. The dark matter behaves like a 'glue', holding the cluster together. The dark-matter distribution in the cluster is not spherical. A secondary concentration of dark-matter mass is shown in blue to the upper right of the main concentration. Sky around galaxy cluster Cl0024+1654 hi-res Size hi-res: 3742 kb Credits: European Space Agency, NASA and Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, USA) Sky around galaxy cluster Cl0024+1654 This is a 2.5-degree field around galaxy cluster Cl0024+1654. The cluster galaxies are visible in the centre of the image in yellow. The image is a colour composite constructed from three Digitized Sky Survey 2 images: Blue (shown in blue), Red (shown in green), and Infrared (shown in red). HST observes shapes of more than 7000 faint background galaxies hi-res Size hi-res: 5593 kb Credits: European Space Agency, NASA and Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, USA) Hubble observes shapes of more than 7000 faint background galaxies Five days of observations produced the altogether 39 Hubble Wide Field and Planetary Camera 2 (WFPC2) images required to map the mass of the galaxy cluster Cl0024+1654. Each WFPC2 image has a size of about 1/150 the diameter of the full Moon. In total, the image measures 27 arc-minutes across, slightly smaller than the diameter of the Moon. The observed warped shapes of more than 7000 faint background galaxies have been converted into a unique map of the dark matter in the cluster. The images were taken through a red filter and have been reduced a factor of two in size. Ground-based image of the galaxy cluster C10024+1654 hi-res Size hi-res: 4699 kb Credits: European Space Agency, NASA and Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, USA) Ground-based image of the galaxy cluster C10024+1654 This is a colour image of the galaxy cluster C10024+1654 obtained with the CFHT12k camera at the Canada France Hawaii Telescope on Mauna Kea (Hawaii). The cluster clearly appears as a concentration of yellow galaxies in the centre of this image although cluster galaxies actually extend at least to the edge of this image. This image measures 21 x 21 arc-minutes. Clusters of galaxies are the largest stable systems in the Universe. They are like laboratories for studying the relationship between the distributions of dark and visible matter. In 1937, Fritz Zwicky realised that the visible component of a cluster (the thousands of millions of stars in each of the thousands of galaxies) represents only a tiny fraction of the total mass. About 80-85% of the matter is invisible, the so-called 'dark matter'. Although astronomers have known about the presence of dark matter for many decades, finding a technique to view its distribution is a much more recent development. Led by Drs Jean-Paul Kneib (from the Observatoire Midi-Pyrénées, France/Caltech, United States), Richard Ellis and Tommaso Treu (both Caltech, United States), the team used the NASA/ESA Hubble Space Telescope to reconstruct a unique 'mass map' of the galaxy cluster CL0024+1654. It enabled them to see for the first time on such large scales how mysterious dark matter is distributed with respect to galaxies. This comparison gives new clues on how such large clusters assemble and which role dark matter plays in cosmic evolution. Tracing dark matter is not an easy task because it does not shine. To make a map, astronomers must focus on much fainter, more distant galaxies behind the cluster. The shapes of these distant systems are distorted by the gravity of the foreground cluster. This distortion provides a measure of the cluster mass, a phenomenon known as 'weak gravitational lensing'. To map the dark matter of CL0024+1654, more than 120 hours observing time was dedicated to the team. This is the largest amount of Hubble time ever devoted to studying a galaxy cluster. Despite its distance of 4.5 thousand million light-years (about one third of the look-back time to the Big Bang) from Earth, this massive cluster is wide enough to equal the angular size of the full Moon. To make a mass map that covers the entire cluster required observations that probed 39 regions of the galaxy cluster. The investigation has resulted in the most comprehensive study of the distribution of dark matter in a galaxy cluster so far and extends more than 20 million light-years from its centre, much further than previous investigations. Many groups of researchers have tried to perform these types of measurements with ground-based telescopes. However, the technique relies heavily on finding the exact shapes of distant galaxies behind the cluster. The sharp vision of a space telescope such as NASA-ESA's Hubble is superior. The study reveals that the density of dark matter on large scales drops sharply with distance from the cluster centre. This confirms a picture that has emerged from recent detailed computer simulations. As Richard Ellis says: "Although theorists have predicted the form of dark matter in galaxy clusters from numerical simulations based on the effects of gravity alone, this is the first time we have convincing observations to back them up. Some astronomers had speculated clusters might contain large reservoirs of dark matter in their outermost regions. Assuming our cluster is representative, this is not the case." The team noticed that dark matter appears to clump together in their map. For example, they found concentrations of dark matter associated with galaxies known to be slowly falling into the system. Generally, the researchers found that the dark matter traces the cluster galaxies remarkably well and over an unprecedented range of physical scales. "When a cluster is being assembled, the dark matter will be smeared out between the galaxies where it acts like a glue," says Jean-Paul Kneib."The overall association of dark matter and 'glowing matter' is very convincing evidence that structures like CL0024+1654 grow by merging of smaller groups of galaxies that were already bound by their own dark matter components." Future investigations using Hubble's new camera, the Advanced Camera for Surveys (ACS), will extend this work when Hubble is trained on a second galaxy cluster later this year. ACS is 10 times more efficient than the Wide Field and Planetary Camera 2 used for this investigation, making it possible to study finer mass clumps in galaxy clusters and help work out how the clusters are assembled. Notes for editors The team is composed of Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, United States), Patrick Hudelot (Observatoire Midi-Pyrénées, France),Richard S. Ellis (Caltech, United States), Tommaso Treu (Caltech, United States), Graham P. Smith (Caltech, United States), Phil Marshall (MRAO, United Kingdom), Oliver Czoske (Institut für Astrophysik und Extraterrestrische Forschung, Germany), Ian Smail (University of Durham, United Kingdom) and Priya Natarajan (Yale University, United States). The ground-based observations were done with the Canada-France-Hawaii Telescope (CFHT) using the CFHT12k camera, the Keck telescopes, and the Hale 5-metre telescope at Palomar, United States, using the WIRC camera. The team will present their study at the General Assembly of the International Astronomical Union. They will also publish their results in a forthcoming issue of Astrophysical Journal. For broadcasters, animations of the discovery and general Hubble Space Telescope background footage is available from http://www.spacetelescope.org/video/releases.html Image credit: European Space Agency, NASA and Jean-Paul Kneib (Observatoire Midi-Pyrénées, France/Caltech, United States)

Monitoring the Transcriptional Activity of Human Endogenous Retroviral HERV-W Family Using PNA Strand Invasion into Double-Stranded DNA.

PubMed

Machnik, Grzegorz; Skudrzyk, Estera; Bułdak, Łukasz; Ruczyński, Jarosław; Kozłowska, Agnieszka; Mucha, Piotr; Rekowski, Piotr; Szkróbka, Witold; Basiak, Marcin; Bołdys, Aleksandra; Sławska, Helena; Okopień, Bogusław

2018-02-01

In the presented assay, we elaborated a method for distinguishing sequences that are genetically closely related to each other. This is particularly important in a situation where a fine balance of the allele abundance is a point of research interest. We developed a peptide nucleic acid (PNA) strand invasion technique for the differentiation between multiple sclerosis-associated retrovirus (MSRV) and ERVWE1 sequences, both molecularly similar, belonging to the human endogenous retrovirus HERV-W family. We have found that this method may support the PCR technique in screening for minor alleles which, in certain conditions, may be undetected by the standard PCR technique. We performed the analysis of different ERVWE1 and MSRV template mixtures ranging from 0 to 100% of ERVWE1 in the studied samples, finding the linear correlation between template composition and signal intensity of final reaction products. Using the PNA strand invasion assay, we were able to estimate the relative ERVWE1 expression level in human specimens such as U-87 MG, normal human astrocytes cell lines and placental tissue. The results remained in concordance with those obtained by semi-quantitative or quantitative PCR.

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

PubMed

Scalabrin, Matteo; Quintieri, Luigi; Palumbo, Manlio; Riccardi Sirtori, Federico; Gatto, Barbara

2017-02-20

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

Static and dynamic parasitic magnetizations and their control in superconducting accelerator dipoles

NASA Astrophysics Data System (ADS)

Collings, E. W.; Sumption, M. D.

2001-05-01

Long dipole magnets guide the particle beams in synchrotron-type high energy accelerators. In principal Cu-wound DC-excited dipoles could be designed to deliver a very uniform transverse bore field, i.e. with small or negligible harmonic (multipolar) distortion. But if the Cu is replaced by (a) superconducting strand that is (b) wound into a Rutherford cable carrying a time-varying transport current, extra magnetizations present within the windings cause distortions of the otherwise uniform field. The static (persistent-current) strand magnetization can be reduced by reducing the filament diameter, and the residue compensated or corrected by strategically placed active or passive components. The cable’s interstrand coupling currents can be controlled by increasing the interstrand contact resistance by: adjusting the level of native oxidation of the strand, coating it, or by inserting a ribbon-like core into the cable itself. Methods of locally compensating the magnetization of NbTi and Nb 3Sn strand and cable are discussed, progress in coupling-current suppression through the use of coatings and cores is reviewed, and a method of simultaneously reducing both the static and dynamic magnetizations of a NbTi cable by means of a thin Ni core is suggested.

A Submillimeter Survey of Dust Continuum Emission in Local Dust-Obscured Galaxies

NASA Astrophysics Data System (ADS)

Lee, Jong Chul; Hwang, Ho Seong; Lee, Gwang-Ho

2015-08-01

Dusty star-forming galaxies are responsible for the bulk of cosmic star formation at 1

Origins Space Telescope

NASA Astrophysics Data System (ADS)

Cooray, Asantha R.; Origins Space Telescope Study Team

2017-01-01

The Origins Space Telescope (OST) is the mission concept for the Far-Infrared Surveyor, a study in development by NASA in preparation for the 2020 Astronomy and Astrophysics Decadal Survey. Origins is planned to be a large aperture, actively-cooled telescope covering a wide span of the mid- to far-infrared spectrum. Its spectrographs will enable 3D surveys of the sky that will discover and characterize the most distant galaxies, Milky-Way, exoplanets, and the outer reaches of our Solar system. Origins will enable flagship-quality general observing programs led by the astronomical community in the 2030s. The Science and Technology Definition Team (STDT) would like to hear your science needs and ideas for this mission. The team can be contacted at firsurveyor_info@lists.ipac.caltech.edu. I will summarize the OST STDT, mission design and instruments, key science drivers, and the study plan over the next two years.

29 years of surprises from hotspots: A personal perspective

NASA Astrophysics Data System (ADS)

Students Of Eao, .; Okal, E. A.

2003-12-01

I arrived at Caltech on 26 August 1974, to begin my graduate studies at the Seismo Lab, then under the Directorship of Don L. Anderson. These were the days, among other topics, of Don's famous multilingual footnote on the "definition..., antecedents..., supporters and detractors" of the concept of "plume" [GSA Bull., 86, p. 1593, 1975], and even though I was not to set foot on a hotspot island until my first trip to Tahiti in December 1977 (those stopovers at Keflavik on the 199-dollar Loftleidir runs did not really count), I quickly acquired a mild form of Don's contagious fascination for the activity and structure of hotspots. As a tribute to Don, I have chosen to recap here a few surprising results obtained, with the help of my students, past and present, over several decades of work on the seismological sources and structures in the neighborhood of hotspot islands.

Multisite two-photon imaging of neurons on multielectrode arrays

NASA Astrophysics Data System (ADS)

Potter, Steve M.; Lukina, Natalia; Longmuir, Kenneth J.; Wu, Yan

2001-04-01

We wish to understand how neural systems store, recall, and process information. We are using cultured networks of cortical neurons grown on microelectrode arrays as a model system for studying the emergent properties of ensembles of living neurons. We have developed a 2-way communication interface between the cultured network and a computer- generated animal, the Neurally Controlled Animat. Neural activity is used to control the behavior of the Animat, and 2- photon time-lapse imaging is carried out in order to observe the morphological changes that might underlie changes in neural processing. The 2-photon microscope is ideal for repeated imaging over hours or days, with submicron resolution and little photodamage. We have designed a computer-controlled microscope stage that allows imaging several locations in sequence, in order to collect more image data. For the latest progress, see: http://www.caltech.edu/~pinelab/PotterGroup.htm.

Probing the interaction of archaeal DNA polymerases with deaminated bases using X-ray crystallography and non-hydrogen bonding isosteric base analogues.

PubMed

Killelea, Tom; Ghosh, Samantak; Tan, Samuel S; Heslop, Pauline; Firbank, Susan J; Kool, Eric T; Connolly, Bernard A

2010-07-13

Archaeal family-B DNA polymerases stall replication on encountering the pro-mutagenic bases uracil and hypoxanthine. This publication describes an X-ray crystal structure of Thermococcus gorgonarius polymerase in complex with a DNA containing hypoxanthine in the single-stranded region of the template, two bases ahead of the primer-template junction. Full details of the specific recognition of hypoxanthine are revealed, allowing a comparison with published data that describe uracil binding. The two bases are recognized by the same pocket, in the N-terminal domain, and make very similar protein-DNA interactions. Specificity for hypoxanthine (and uracil) arises from a combination of polymerase-base hydrogen bonds and shape fit between the deaminated bases and the pocket. The structure with hypoxanthine at position 2 explains the stimulation of the polymerase 3'-5' proofreading exonuclease, observed with deaminated bases at this location. A beta-hairpin element, involved in partitioning the primer strand between the polymerase and exonuclease active sites, inserts between the two template bases at the extreme end of the double-stranded DNA. This denatures the two complementary primer bases and directs the resulting 3' single-stranded extension toward the exonuclease active site. Finally, the relative importance of hydrogen bonding and shape fit in determining selectivity for deaminated bases has been examined using nonpolar isosteres. Affinity for both 2,4-difluorobenzene and fluorobenzimidazole, non-hydrogen bonding shape mimics of uracil and hypoxanthine, respectively, is strongly diminished, suggesting polar protein-base contacts are important. However, residual interaction with 2,4-difluorobenzene is seen, confirming a role for shape recognition.

Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

PubMed

Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

1997-04-15

The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other conserved loops that converge at the active site cleft. The catalytic loop (residues 166-171) and the Mg2+ positioning loop (residues 184-186) are a stable part of the large lobe and have low B-factors in all structures solved to date. The stability of the glycine-rich loop is highly dependent on the ligands that occupy the active site cleft with maximum stability achieved in the ternary complex containing Mg x ATP and the peptide inhibitor. In this ternary complex the gamma-phosphate is secured between both lobes by hydrogen bonds to the backbone amide of Ser 53 in the glycine-rich loop and the amino group of Lys 168 in the catalytic loop. In the adenosine ternary complex the water molecule replacing the gamma-phosphate hydrogen bonds between Lys 168 and Asp 166 and makes no contact with the small lobe. This glycine-rich loop is thus the most mobile component of the active site cleft, with the tip of the loop being highly sensitive to what occupies the gamma-subsite.

Sequence-Specific DNA Photosplitting of Crosslinked DNAs Containing the 3-Cyanovinylcarbazole Nucleoside by Using DNA Strand Displacement.

PubMed

Nakamura, Shigetaka; Kawabata, Hayato; Fujimoto, Kenzo

2016-08-17

An oligodeoxynucleotide (ODN) containing the ultrafast reversible 3-cyanovinylcarbazole ((CNV) K) photo-crosslinker was photo-crosslinked to a complementary strand upon exposure to 366 nm irradiation and photosplit by use of 312 nm irradiation. In this paper we report that the photoreaction of (CNV) K on irradiation at 366 nm involves a photostationary state and that its reaction can be controlled by temperature. Guided by this new insight, we proposed and have now demonstrated previously unknown photosplitting of (CNV) K aided by DNA strand displacement as an alternative to heating. The photo-crosslinked double-stranded DNA (dsDNA) underwent >80 % photosplitting aided by DNA strand displacement on irradiation at 366 nm without heating. In this photosplitting based on DNA strand displacement, the relative thermal stability of the invader strand with respect to the template strands plays an important role, and an invader strand/template strand system that is more stable than the passenger strand/template strand system induces photosplitting without heating. This new strand-displacement-aided photosplitting occurred in a sequence-specific manner through irradiation at 366 nm in the presence of an invader strand. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, T.

1998-09-29

The subject invention disclosed herein is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

Single-fluorophore monitoring of DNA hybridization for investigating the effect of secondary structure on the nucleation step.

PubMed

Jo, Joon-Jung; Kim, Min-Ji; Son, Jung-Tae; Kim, Jandi; Shin, Jong-Shik

2009-07-17

Nucleic acid hybridization is one of the essential biological processes involved in storage and transmission of genetic information. Here we quantitatively determined the effect of secondary structure on the hybridization activation energy using structurally defined oligonucleotides. It turned out that activation energy is linearly proportional to the length of a single-stranded region flanking a nucleation site, generating a 0.18 kcal/mol energy barrier per nucleotide. Based on this result, we propose that the presence of single-stranded segments available for non-productive base pairing with a nucleation counterpart extends the searching process for nucleation sites to find a perfect match. This result may provide insights into rational selection of a target mRNA site for siRNA and antisense gene silencing.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, Tuan

1998-01-01

The subject invention disclosed herein is a new gene probe biosensor and methods thereof based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed thereon. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays.

Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

PubMed

Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

2004-06-01

The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

Surface enhanced Raman gene probe and methods thereof

DOEpatents

Vo-Dinh, T.

1998-07-21

The subject invention disclosed is a new gene probe biosensor and methods based on surface enhanced Raman scattering (SERS) label detection. The SER gene probe biosensor comprises a support means, a SER gene probe having at least one oligonucleotide strand labeled with at least one SERS label, and a SERS active substrate disposed on the support means and having at least one of the SER gene probes adsorbed. Biotargets such as bacterial and viral DNA, RNA and PNA are detected using a SER gene probe via hybridization to oligonucleotide strands complementary to the SER gene probe. The support means supporting the SERS active substrate includes a fiberoptic probe, an array of fiberoptic probes for performance of multiple assays and a waveguide microsensor array with charge-coupled devices or photodiode arrays. 18 figs.

Development length of 0.6-inch prestressing strand in standard I-shaped pretensioned concrete beams

NASA Astrophysics Data System (ADS)

Barnes, Robert Wesley

The use of 0.6 in prestressing strand at a center-to-center spacing of 2 in allows for the optimal implementation of High Strength Concrete (HSC) in precast, prestressed concrete bridge superstructures. For this strand configuration, partial debonding of strands is a desirable alternative to the more traditional method of draping strands to alleviate extreme concrete stresses after prestress release. Recent experimental evidence suggests that existing code provisions addressing the anchorage of pretensioned strands do not adequately describe the behavior of these strands. In addition, the anchorage behavior of partially debonded strands is not fully understood. These uncertainties have combined to hinder the full exploitation of HSC in pretensioned concrete construction. A research study was conducted to determine the anchorage behavior of 0.6 in strands at 2 in spacing in full-size bridge members. The experimental program consisted of assessing transfer and development lengths in plant-cast AASHTO Type I I-beams. The influence of concrete compressive strengths ranging from 5700 to 14,700 psi was examined. In order to consider the full range of strand surface conditions found in practice, the prestressing strand featured either a bright mill finish or a rusted surface condition. The anchorage behavior of partially debonded strands was investigated by using a variety of strand debonding configurations---including debonded strand percentages as high as 75 percent. A limited investigation of the effect of horizontal web reinforcement on anchorage behavior was performed. Pull-out tests were performed in an attempt to correlate results with the bond quality of the strands used in the study. The correlation between strand draw-in and the anchorage behavior of prestressing strands was also examined. A review of the evolution and shortcomings of existing code provisions for the anchorage of prestressing strands is presented. Results of the experimental program are reported, along with recommended design procedures based on these results and those from other studies. The use of 0.6 in strand at 2 in spacing is concluded to be safe, and partial debonding of prestressing strands is shown to be an effective means of reducing stresses in the end regions of pretensioned girders.

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

PubMed

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein has dual RNA binding modes.

PubMed

Jeeva, Subbiah; Pador, Sean; Voss, Brittany; Ganaie, Safder Saieed; Mir, Mohammad Ayoub

2017-01-01

Crimean Congo hemorrhagic fever, a zoonotic viral disease, has high mortality rate in humans. There is currently no vaccine for Crimean Congo hemorrhagic fever virus (CCHFV) and chemical interventions are limited. The three negative sense genomic RNA segments of CCHFV are specifically encapsidated by the nucleocapsid protein into three ribonucleocapsids, which serve as templates for the viral RNA dependent RNA polymerase. Here we demonstrate that CCHFV nucleocapsid protein has two distinct binding modes for double and single strand RNA. In the double strand RNA binding mode, the nucleocapsid protein preferentially binds to the vRNA panhandle formed by the base pairing of complementary nucleotides at the 5' and 3' termini of viral genome. The CCHFV nucleocapsid protein does not have RNA helix unwinding activity and hence does not melt the duplex vRNA panhandle after binding. In the single strand RNA binding mode, the nucleocapsid protein does not discriminate between viral and non-viral RNA molecules. Binding of both vRNA panhandle and single strand RNA induce a conformational change in the nucleocapsid protein. Nucleocapsid protein remains in a unique conformational state due to simultaneously binding of structurally distinct vRNA panhandle and single strand RNA substrates. Although the role of dual RNA binding modes in the virus replication cycle is unknown, their involvement in the packaging of viral genome and regulation of CCHFV replication in conjunction with RdRp and host derived RNA regulators is highly likely.

Stability and free energy calculation of LNA modified quadruplex: a molecular dynamics study

NASA Astrophysics Data System (ADS)

Chaubey, Amit Kumar; Dubey, Kshatresh Dutta; Ojha, Rajendra Prasad

2012-03-01

Telomeric ends of chromosomes, which comprise noncoding repeat sequences of guanine-rich DNA, which are the fundamental in protecting the cell from recombination and degradation. Telomeric DNA sequences can form four stranded quadruplex structures, which are involved in the structure of telomere ends. The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadrulex as an attractive target for cancer therapeutic intervention. Molecular dynamic simulation offers the prospects of detailed description of the dynamical structure with ion and water at molecular level. In this work we have taken a oligomeric part of human telomeric DNA, d(TAGGGT) to form different monomeric quadruplex structures d(TAGGGT)4. Here we report the relative stabilities of these structures under K+ ion conditions and binding interaction between the strands, as determined by molecular dynamic simulations followed by energy calculation. We have taken locked nucleic acid (LNA) in this study. The free energy molecular mechanics Poission Boltzman surface area calculations are performed for the determination of most stable complex structure between all modified structures. We calculated binding free energy for the combination of different strands as the ligand and receptor for all structures. The energetic study shows that, a mixed hybrid type quadruplex conformation in which two parallel strands are bind with other two antiparallel strands, are more stable than other conformations. The possible mechanism for the inhibition of the cancerous growth has been discussed. Such studies may be helpful for the rational drug designing.

Kinetics of end-to-end collision in short single-stranded nucleic acids.

PubMed

Wang, Xiaojuan; Nau, Werner M

2004-01-28

A novel fluorescence-based method, which entails contact quenching of the long-lived fluorescent state of 2,3-diazabicyclo[2.2.2]-oct-2-ene (DBO), was employed to measure the kinetics of end-to-end collision in short single-stranded oligodeoxyribonucleotides of the type 5'-DBO-(X)n-dG with X = dA, dC, dT, or dU and n = 2 or 4. The fluorophore was covalently attached to the 5' end and dG was introduced as an efficient intrinsic quencher at the 3' terminus. The end-to-end collision rates, which can be directly related to the efficiency of intramolecular fluorescence quenching, ranged from 0.1 to 9.0 x 10(6) s(-1). They were strongly dependent on the strand length, the base sequence, as well as the temperature. Oligonucleotides containing dA in the backbone displayed much slower collision rates and significantly higher positive activation energies than strands composed of pyrimidine bases, suggesting a higher intrinsic rigidity of oligoadenylate. Comparison of the measured collision rates in short single-stranded oligodeoxyribonucleotides with the previously reported kinetics of hairpin formation indicates that the intramolecular collision is significantly faster than the nucleation step of hairpin closing. This is consistent with the configurational diffusion model suggested by Ansari et al. (Ansari, A.; Kuznetsov, S. V.; Shen, Y. Proc.Natl. Acad. Sci. USA 2001, 98, 7771-7776), in which the formation of misfolded loops is thought to slow hairpin formation.

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rakus,J.; Fedorov, A.; Fedorov, E.

2007-01-01

The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain andmore » a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only conserved residues in the enolase superfamily, establishing the primary functional importance of the Mg2+-assisted strategy for stabilizing the enolate anion intermediate.« less

Estimation of Prestress Force Distribution in Multi-Strand System of Prestressed Concrete Structures Using Field Data Measured by Electromagnetic Sensor

PubMed Central

Cho, Keunhee; Cho, Jeong-Rae; Kim, Sung Tae; Park, Sung Yong; Kim, Young-Jin; Park, Young-Hwan

2016-01-01

The recently developed smart strand can be used to measure the prestress force in the prestressed concrete (PSC) structure from the construction stage to the in-service stage. The higher cost of the smart strand compared to the conventional strand renders it unaffordable to replace all the strands by smart strands, and results in the application of only a limited number of smart strands in the PSC structure. However, the prestress forces developed in the strands of the multi-strand system frequently adopted in PSC structures differ from each other, which means that the prestress force in the multi-strand system cannot be obtained by simple proportional scaling using the measurement of the smart strand. Therefore, this study examines the prestress force distribution in the multi-strand system to find the correlation between the prestress force measured by the smart strand and the prestress force distribution in the multi-strand system. To that goal, the prestress force distribution was measured using electromagnetic sensors for various factors of the multi-strand system adopted on site in the fabrication of actual PSC girders. The results verified the possibility to assume normal distribution for the prestress force distribution per anchor head, and a method computing the mean and standard deviation defining the normal distribution is proposed. This paper presents a meaningful finding by proposing an estimation method of the prestress force based upon field-measured data of the prestress force distribution in the multi-strand system of actual PSC structures. PMID:27548172

The multiple personalities of Watson and Crick strands.

PubMed

Cartwright, Reed A; Graur, Dan

2011-02-08

In genetics it is customary to refer to double-stranded DNA as containing a "Watson strand" and a "Crick strand." However, there seems to be no consensus in the literature on the exact meaning of these two terms, and the many usages contradict one another as well as the original definition. Here, we review the history of the terminology and suggest retaining a single sense that is currently the most useful and consistent. The Saccharomyces Genome Database defines the Watson strand as the strand which has its 5'-end at the short-arm telomere and the Crick strand as its complement. The Watson strand is always used as the reference strand in their database. Using this as the basis of our standard, we recommend that Watson and Crick strand terminology only be used in the context of genomics. When possible, the centromere or other genomic feature should be used as a reference point, dividing the chromosome into two arms of unequal lengths. Under our proposal, the Watson strand is standardized as the strand whose 5'-end is on the short arm of the chromosome, and the Crick strand as the one whose 5'-end is on the long arm. Furthermore, the Watson strand should be retained as the reference (plus) strand in a genomic database. This usage not only makes the determination of Watson and Crick unambiguous, but also allows unambiguous selection of reference stands for genomics. This article was reviewed by John M. Logsdon, Igor B. Rogozin (nominated by Andrey Rzhetsky), and William Martin.

DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.

PubMed Central

Saunders, K; Lucy, A; Stanley, J

1991-01-01

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2). Images PMID:2041773

Origins Space Telescope: Galaxy and Black Hole Evolution over Cosmic Time

NASA Astrophysics Data System (ADS)

Pope, Alexandra; Origins Space Telescope Study Team

2017-01-01

The Origins Space Telescope (OST) is the mission concept for the Far-Infrared Surveyor, a study in development by NASA in preparation for the 2020 Astronomy and Astrophysics Decadal Survey. Origins is planned to be a large aperture, actively-cooled telescope covering a wide span of the mid- to far-infrared spectrum. Its imagers and spectrographs will enable a variety of surveys of the sky that will discover and characterize the most distant galaxies, Milky-Way, exoplanets, and the outer reaches of our Solar system. Origins will enable flagship-quality general observing programs led by the astronomical community in the 2030s. The Science and Technology Definition Team (STDT) would like to hear your science needs and ideas for this mission. The team can be contacted at firsurveyor_info@lists.ipac.caltech.edu. This presentation will provide a summary of the science case related to galaxy formation and evolution. Origins will investigate the connection between black hole growth and star formation, understand the role of feedback from supernovae and active galactic nuclei, probe the multiphase interstellar medium, and chart the rise of metals over cosmic time.

Origins Space Telescope: Study Plan

NASA Astrophysics Data System (ADS)

Nayyeri, Hooshang; Cooray, Asantha; Origins Space Telescope Study Team

2018-01-01

The Origins Space Telescope (OST) is the mission concept for the Far-Infrared Surveyor, a study in development by NASA in preparation for the 2020 Astronomy and Astrophysics Decadal Survey. Origins is planned to be a large aperture, actively-cooled telescope covering a wide span of the mid- to far-infrared spectrum. Its spectrographs will enable 3D surveys of the sky that will discover and characterize the most distant galaxies, Milky-Way, exoplanets, and the outer reaches of our Solar system. Origins will enable flagship-quality general observing programs led by the astronomical community in the 2030s. The Science and Technology Definition Team (STDT) would like to hear your science needs and ideas for this mission. The team can be contacted at firsurveyor_info@lists.ipac.caltech.edu. This presentation will provide a summary of the OST STDT, the OST Study Team based at NASA Goddard Space Flight Center, study partners, and the advisory panel to the study. This presentation will also summarize recent activities, including the process used to reach a decision on the mission architecture, the identification of key science drivers, and the key study milestones between 2017 and 2020.