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991.
Charles Q. Cai Yannick Doyon W. Michael Ainley Jeffrey C. Miller Russell C. DeKelver Erica A. Moehle Jeremy M. Rock Ya-Li Lee Robbi Garrison Lisa Schulenberg Ryan Blue Andrew Worden Lisa Baker Farhoud Faraji Lei Zhang Michael C. Holmes Edward J. Rebar Trevor N. Collingwood Beth Rubin-Wilson Philip D. Gregory Fyodor D. Urnov Joseph F. Petolino 《Plant molecular biology》2009,69(6):699-709
Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here
it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native
gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco
cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase
gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted,
homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize
a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait
stacking in plants. 相似文献
992.
Erica Novo Lorenzo Valfrè di Bonzo Stefania Cannito Sebastiano Colombatto Maurizio Parola 《The international journal of biochemistry & cell biology》2009,41(11):2089-2093
Hepatic myofibroblasts constitute a heterogenous population of highly proliferative, pro-fibrogenic, pro-inflammatory, pro-angiogenic and contractile cells that sustain liver fibrogenesis and then fibrotic progression of chronic liver diseases of different aetiology to the common advanced-stage of cirrhosis. These α-smooth muscle actin-positive myofibroblast-like cells, according to current literature, mainly originate by a process of activation and trans-differentiation that involves either hepatic stellate cells or fibroblasts of portal areas. Hepatic myofibroblasts can also originate from bone marrow-derived cells, including mesenchymal stem cells or circulating fibrocytes able to engraft chronically injured liver, as well as, in certain conditions, by a process of epithelial to mesenchymal transition involving hepatocytes and cholangiocytes. Hepatic myofibroblasts may have also additional crucial roles in modulating immune response and in the cross talk with hepatic progenitor (stem) cells as well as with malignant cells of either primary hepatocellular carcinomas or of metastatic cancers. 相似文献
993.
Erica A. H. Smithwick Daniel M. Kashian Michael G. Ryan Monica G. Turner 《Ecosystems》2009,12(5):792-806
Long-term, landscape patterns in inorganic nitrogen (N) availability and N stocks following infrequent, stand-replacing fire
are unknown but are important for interpreting the effect of disturbances on ecosystem function. Here, we present results
from a replicated chronosequence study in the Greater Yellowstone Ecosystem (Wyoming, USA) directed at measuring inorganic
N availability (ion-exchange resin bags) and ecosystem N pools among 77 lodgepole pine stands that varied in age and density.
Inorganic N availability ranged from 0.07 to 3.20 μN bag−1 d−1 and nitrate (NO3−) was, on average, 65% of total resin-sorbed N. Total ecosystem N stocks (live + detrital + soil) averaged 109.9 ± 3.0 g N m−2 (range = 63.7–185.8 g N m−2). Live N was 14%, detrital N was 29%, and soil N was 57% of total stocks. Soil NO3−, total ecosystem N, live N, and detrital N generally increased with stand age, but soil N stocks decreased. Models (AICc) to predict soil N availability and N stocks included soil P, soil Ca, bulk density, and pH in addition to age (adj R
2 ranged from 0.18 to 0.53) and density was included only for live N stocks. Patterns of N stocks and N availability with density
were strongest for young stands (<20 years) regenerating from extensive fire in 1988; for example, litterfall N stocks increased
with density (adj R
2 = 0.86, P < 0.001) but inorganic N availability declined (adj R
2 = 0.47, P < 0.003). Across the complex Yellowstone landscape, we conclude that N stocks and N availability are best predicted by a
combination of local soil characteristics in addition to factors that vary at landscape scales (stand density and age). Overall,
total ecosystem N stocks were recovered quickly following stand-replacing fire, suggesting that moderate increases in fire
frequency will not affect long-term landscape N storage in Greater Yellowstone.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Author contributions EAHS, MGT, and MGR conceived the study; DMK performed field research; EAHS and DMK oversaw laboratory analyses and analyzed
data; EAHS wrote the paper. 相似文献
994.
Ling Xiong Xiaole L. Chen Hannah R. Silver Noreen T. Ahmed Erica S. Johnson 《Molecular biology of the cell》2009,20(4):1241-1251
Many Saccharomyces cerevisiae mutants defective in the SUMO pathway accumulate elevated levels of the native 2 μm circle plasmid (2 μm). Here we show that accumulation of 2 μm in the SUMO pathway mutants siz1Δ siz2Δ, slx5Δ, and slx8Δ is associated with formation of an aberrant high-molecular-weight (HMW) form of 2 μm. Characterization of this species from siz1Δ siz2Δ showed that it contains tandem copies of the 2 μm sequence as well as single-stranded DNA. Accumulation of this species requires both the 2 μm–encoded Flp recombinase and the cellular homologous recombination repair (HRR) pathway. Importantly, reduced SUMO attachment to Flp is sufficient to induce formation of this species. Our data suggest a model in which Flp that cannot be sumoylated causes DNA damage, whose repair via HRR produces an intermediate that generates tandem copies of the 2 μm sequence. This intermediate may be a rolling circle formed via break-induced replication (BIR), because mutants defective in BIR contain reduced levels of the HMW form. This work also illustrates the importance of using cir° strains when studying mutants that affect the yeast SUMO pathway, to avoid confusing direct functions of the SUMO pathway with secondary effects of 2 μm amplification. 相似文献
995.
Erica D. Dawson Amber W. Taylor James A. Smagala Kathy L. Rowlen 《Molecular biotechnology》2009,42(1):117-127
We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time
PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific
to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved
analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard
culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7
and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time
PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based
non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection. 相似文献
996.
997.
Lee Coffey Erica Owens Karen Tambling David O’Neill Laura O’Connor Catherine O’Reilly 《Antonie van Leeuwenhoek》2010,98(4):455-463
Nitriles are widespread in the environment as a result of biological and industrial activity. Nitrile hydratases catalyse
the hydration of nitriles to the corresponding amide and are often associated with amidases, which catalyze the conversion
of amides to the corresponding acids. Nitrile hydratases have potential as biocatalysts in bioremediation and biotransformation
applications, and several successful examples demonstrate the advantages. In this work a real-time PCR assay was designed
for the detection of Fe-type nitrile hydratase genes from environmental isolates purified from nitrile-enriched soils and
seaweeds. Specific PCR primers were also designed for amplification and sequencing of the genes. Identical or highly homologous
nitrile hydratase genes were detected from isolates of numerous genera from geographically diverse sites, as were numerous
novel genes. The genes were also detected from isolates of genera not previously reported to harbour nitrile hydratases. The
results provide further evidence that many bacteria have acquired the genes via horizontal gene transfer. The real-time PCR
assay should prove useful in searching for nitrile hydratases that could have novel substrate specificities and therefore
potential in industrial applications. 相似文献
998.
999.
Erica Andersen Namrata Asuri Matthew Clay Mary Halloran 《Journal of visualized experiments : JoVE》2010,(36)
The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.Open in a separate windowClick here to view.(58M, flv) 相似文献
1000.
Rosenblum EB Fisher MC James TY Stajich JE Longcore JE Gentry LR Poorten TJ 《Diseases of aquatic organisms》2010,92(2-3):131-147
Ten years after the first discovery of the chytrid pathogen Batrachochytrium dendrobatidis (Bd), the catastrophic effect of Bd on wild amphibian populations is indisputable. However, a number of persistent questions remain about Bd's origin and mechanisms of pathogenicity. Here we discuss the promise of genetic and genomic tools for answering these previously intractable questions about the biology and evolutionary history of Bd. Full genomes of 2 Bd strains have recently been sequenced, and Bd research on this species using population genetics, phylogenetics, proteomics, comparative genomics and functional genomics is already underway. We review some of the insights gleaned from the first studies using these genome-scale approaches focusing particularly on Bd's genomic architecture, patterns of global genetic variation, virulence factors and genetic interactions with hosts. Avenues of future research promise to be particularly fruitful and highlight the need for integrative studies that unite genetic, ecological and spatial data in both Bd and its amphibian hosts. 相似文献