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  • 1
    Publication Date: 2015-12-10
    Description: The maize B centromere has been used as a model for centromere epigenetics and as the basis for building artificial chromosomes. However, there are no sequence resources for this important centromere. Here we used transposon display for the centromere-specific retroelement CRM2 to identify a collection of 40 sequence tags that flank CRM2 insertion points on the B chromosome. These were confirmed to lie within the centromere by assaying deletion breakpoints from centromere misdivision derivatives (intracentromere breakages caused by centromere fission). Markers were grouped together on the basis of their association with other markers in the misdivision series and assembled into a pseudocontig containing 10.1 kb of sequence. To identify sequences that interact directly with centromere proteins, we carried out chromatin immunoprecipitation using antibodies to centromeric histone H3 (CENH3), a defining feature of functional centromeric sequences. The CENH3 chromatin immunoprecipitation map was interpreted relative to the known transmission rates of centromere misdivision derivatives to identify a centromere core domain spanning 33 markers. A subset of seven markers was mapped in additional B centromere misdivision derivatives with the use of unique primer pairs. A derivative previously shown to have no canonical centromere sequences (Telo3-3) lacks these core markers. Our results provide a molecular map of the B chromosome centromere and identify key sequences within the map that interact directly with centromeric histone H3.
    Electronic ISSN: 2160-1836
    Topics: Biology
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  • 2
    Publication Date: 2014-08-12
    Description: Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of RNA-guided adaptive immune systems that protect bacteria and archaea from viruses and plasmids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble into a 405-kilodalton multisubunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Here we present the 3.24 angstrom resolution x-ray crystal structure of Cascade. Eleven proteins and a 61-nucleotide crRNA assemble into a seahorse-shaped architecture that binds double-stranded DNA targets complementary to the crRNA-guide sequence. Conserved sequences on the 3' and 5' ends of the crRNA are anchored by proteins at opposite ends of the complex, whereas the guide sequence is displayed along a helical assembly of six interwoven subunits that present five-nucleotide segments of the crRNA in pseudo-A-form configuration. The structure of Cascade suggests a mechanism for assembly and provides insights into the mechanisms of target recognition.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188430/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉   〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4188430/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Jackson, Ryan N -- Golden, Sarah M -- van Erp, Paul B G -- Carter, Joshua -- Westra, Edze R -- Brouns, Stan J J -- van der Oost, John -- Terwilliger, Thomas C -- Read, Randy J -- Wiedenheft, Blake -- 082961/Wellcome Trust/United Kingdom -- 082961/Z/07/Z/Wellcome Trust/United Kingdom -- 100140/Wellcome Trust/United Kingdom -- 52006931/Howard Hughes Medical Institute/ -- F32 GM108436/GM/NIGMS NIH HHS/ -- GM063210/GM/NIGMS NIH HHS/ -- P01 GM063210/GM/NIGMS NIH HHS/ -- P20GM103500/GM/NIGMS NIH HHS/ -- P41GM103393/GM/NIGMS NIH HHS/ -- R01 GM108888/GM/NIGMS NIH HHS/ -- R01GM108888/GM/NIGMS NIH HHS/ -- Y1-GM-1104/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2014 Sep 19;345(6203):1473-9. doi: 10.1126/science.1256328. Epub 2014 Aug 7.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA. ; Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, Netherlands. ; Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA. ; Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. ; Department of Microbiology and Immunology, Montana State University, Bozeman, MT 59717, USA. bwiedenheft@gmail.com.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25103409" target="_blank"〉PubMed〈/a〉
    Keywords: CRISPR-Associated Proteins/*chemistry ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Crystallography, X-Ray ; Escherichia coli/*genetics ; Escherichia coli Proteins/*chemistry ; RNA Editing ; RNA, Bacterial/*chemistry ; RNA, Guide/*chemistry
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Bulletin of environmental contamination and toxicology 51 (1993), S. 703-709 
    ISSN: 1432-0800
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Medicine
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Bulletin of environmental contamination and toxicology 64 (2000), S. 258-262 
    ISSN: 1432-0800
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Medicine
    Type of Medium: Electronic Resource
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  • 5
    Publication Date: 2015-09-30
    Description: In bacteria and archaea, short fragments of foreign DNA are integrated into Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) loci, providing a molecular memory of previous encounters with foreign genetic elements. In Escherichia coli , short CRISPR-derived RNAs are incorporated into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Recent structures of Cascade capture snapshots of this seahorse-shaped RNA-guided surveillance complex before and after binding to a DNA target. Here we determine a 3.2 Å x-ray crystal structure of Cascade in a new crystal form that provides insight into the mechanism of double-stranded DNA binding. Molecular dynamic simulations performed using available structures reveal functional roles for residues in the tail, backbone and belly subunits of Cascade that are critical for binding double-stranded DNA. Structural comparisons are used to make functional predictions and these predictions are tested in vivo and in vitro . Collectively, the results in this study reveal underlying mechanisms involved in target-induced conformational changes and highlight residues important in DNA binding and protospacer adjacent motif recognition.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 6
    Publication Date: 2014-12-17
    Description: Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3'-5' turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters in vitro activity in Mtr4 and TRAMP and causes slow growth phenotypes in vivo . We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
    Publication Date: 2016-09-03
    Description: Bacteria and archaea acquire resistance to foreign genetic elements by integrating fragments of foreign DNA into CRISPR (clustered regularly interspaced short palindromic repeats) loci. In Escherichia coli , CRISPR-derived RNAs (crRNAs) assemble with Cas proteins into a multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade recognizes DNA targets via protein-mediated recognition of a protospacer adjacent motif and complementary base pairing between the crRNA spacer and the DNA target. Previously determined structures of Cascade showed that the crRNA is stretched along an oligomeric protein assembly, leading us to ask how crRNA length impacts the assembly and function of this complex. We found that extending the spacer portion of the crRNA resulted in larger Cascade complexes with altered stoichiometry and preserved in vitro binding affinity for target DNA. Longer spacers also preserved the in vivo ability of Cascade to repress target gene expression and to recruit the Cas3 endonuclease for target degradation. Finally, longer spacers exhibited enhanced silencing at particular target locations and were sensitive to mismatches within the extended region. These findings demonstrate the flexibility of the Type I-E CRISPR machinery and suggest that spacer length can be modified to fine-tune Cascade activity.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 8
    Publication Date: 2015-11-13
    Description: The Canada Basin and the southern Alpha-Mendeleev ridge complex underlie a significant proportion of the Arctic Ocean, but the geology of this undrilled and mostly ice-covered frontier is poorly known. New information is encoded in seismic wide-angle reflections and refractions recorded with expendable sonobuoys between 2007 and 2011. Velocity–depth samples within the sedimentary succession are extracted from published analyses for 142 of these records obtained at irregularly spaced stations across an area of 1.9E + 06 km 2 . The samples are modelled at regional, subregional and station-specific scales using an exponential function of inverse velocity versus depth with regionally representative parameters determined through numerical regression. With this approach, smooth, non-oscillatory velocity–depth profiles can be generated for any desired location in the study area, even where the measurement density is low. Practical application is demonstrated with a map of sedimentary thickness, derived from seismic reflection horizons interpreted in the time domain and depth converted using the velocity–depth profiles for each seismic trace. A thickness of 12–13 km is present beneath both the upper Mackenzie fan and the middle slope off of Alaska, but the sedimentary prism thins more gradually outboard of the latter region. Mapping of the observed-to-predicted velocities reveals coherent geospatial trends associated with five subregions: the Mackenzie fan; the continental slopes beyond the Mackenzie fan; the abyssal plain; the southwestern Canada Basin; and, the Alpha-Mendeleev magnetic domain. Comparison of the subregional velocity–depth models with published borehole data, and interpretation of the station-specific best-fitting model parameters, suggests that sandstone is not a predominant lithology in any of the five subregions. However, the bulk sand-to-shale ratio likely increases towards the Mackenzie fan, and the model for this subregion compares favourably with borehole data for Miocene turbidites in the eastern Gulf of Mexico. The station-specific results also indicate that Quaternary sediments coarsen towards the Beaufort-Mackenzie and Banks Island margins in a manner that is consistent with the variable history of Laurentide Ice Sheet advance documented for these margins. Lithological factors do not fully account for the elevated velocity–depth trends that are associated with the southwestern Canada Basin and the Alpha-Mendeleev magnetic domain. Accelerated porosity reduction due to elevated palaeo-heat flow is inferred for these regions, which may be related to the underlying crustal types or possibly volcanic intrusion of the sedimentary succession. Beyond exploring the variation of an important physical property in the Arctic Ocean basin, this study provides comparative reference for global studies of seismic velocity, burial history, sedimentary compaction, seismic inversion and overpressure prediction, particularly in mudrock-dominated successions.
    Keywords: Marine Geosciences and Applied Geophysics
    Print ISSN: 0956-540X
    Electronic ISSN: 1365-246X
    Topics: Geosciences
    Published by Oxford University Press on behalf of The Deutsche Geophysikalische Gesellschaft (DGG) and the Royal Astronomical Society (RAS).
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  • 9
    Publication Date: 2000-02-01
    Print ISSN: 0007-4861
    Electronic ISSN: 1432-0800
    Topics: Energy, Environment Protection, Nuclear Power Engineering , Medicine
    Published by Springer
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