共查询到20条相似文献,搜索用时 15 毫秒
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《遗传学报》2016,(2)
正CRISPR(clustered regularly interspaced short palindromic repeats)-Cas9-based genome editing has revolutionized functional genomics in many biological research fields.The specificity and potency of CRISPR-Cas9 genome editing make it ideal for investigating the function of genes in vivo(Hsu 相似文献
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The Human Genome Project has generated nucleotide sequences from an estimated 80,000 to 100,000 genes, only a small fraction of which have a known role. Nucleotide sequence information alone is insufficient to predict gene function. One of the most powerful ways of revealing gene function, as demonstrated in bacteria, worms, yeast, and flies, is to generate mutations and characterize them at both the phenotypic and the molecular levels. Given the physiological and anatomical parallels between mouse and human, genotype–phenotype relationships established in mice can be extrapolated to human syndromes. A new method is described for functional genetic analyses in the mouse that uses loxP/Cre engineering to generate coat color-tagged large deletions. The haploid regions can then be dissected by mutagenesis withN-ethyl-N-nitrosourea in phenotype-driven screens to obtain functional information on genes in any desired region of the mouse genome. 相似文献
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A new method employing extremely high frequencies (EHFs) is proposed for the selective isolation of actinomycetes from soil. The pretreatment of soil suspensions with EHF wavelengths of 5.6 and 7.1 mm led to a nonselective isolation of actinomycetes. At the same time, the irradiation of soil suspensions within wavelength bands of 3.8–5.8 and 8–11.5 mm considerably augmented the total number of isolated actinomycetes and increased the fraction of the isolated rare genera by 2 and 7 times, respectively. The rare actinomycete genera were represented by Actinomadura, Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia, Saccharotrix, and Streptosporangium. 相似文献
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S. Takashima A. Casaleggio F. Giuliano M. Morando P. Arrigo S. Ridella 《Biophysical journal》1986,49(5):1003-1008
The high frequency dielectric constant of poly-adenine (poly-A) was measured between 1 MHz and 1 GHz. The purpose of these experiments was to investigate the state of water molecules that are bound to the charged groups of the poly-A molecule. Analysis of the data using the Maxwell's mixture equation revealed the dielectric constant of bound water higher than we expected. Using Onsager's internal field in Debye's equation, we calculated the dielectric constant of water in the vicinity of a charged ion. The result of this computation demonstrates that the dielectric constant of bound water is much smaller than the normal value only in the immediate proximity of charged ions (within 2 Å). The dielectric constant increases rapidly to the normal value as the distance increases from 2 to 4 Å. This observation indicates that charged sites of polyions have only short range interactions with the surrounding water molecules. However, this conclusion pertains only to rotary diffusion of bound water since dielectric measurement is unable to detect translational diffusion. 相似文献
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John M. Szymanski Quentin Jallerat Adam W. Feinberg 《Journal of visualized experiments : JoVE》2014,(86)
The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo. 相似文献
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Guohong Liu Christopher Q. Weston Long K. Pham Shannon Waltz Helen Barnes Paula King Dan Sphar Robert T. Yamamoto R. Allyn Forsyth 《PloS one》2016,11(1)
We describe continuing work to develop restriction endonucleases as tools to enrich targeted genomes of interest from diverse populations. Two approaches were developed in parallel to segregate genomic DNA based on cytosine methylation. First, the methyl-sensitive endonuclease HpaII was used to bind non-CG methylated DNA. Second, a truncated fragment of McrB was used to bind CpG methylated DNA. Enrichment levels of microbial genomes can exceed 100-fold with HpaII allowing improved genomic detection and coverage of otherwise trace microbial genomes from sputum. Additionally, we observe interesting enrichment results that correlate with the methylation states not only of bacteria, but of fungi, viruses, a protist and plants. The methods presented here offer promise for testing biological samples for pathogens and global analysis of population methylomes. 相似文献
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Mariarosaria De Falco Federico Catalano Mosè Rossi Maria Ciaramella Mariarita De Felice 《PloS one》2015,10(11)
The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing. 相似文献
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Caitlin A. Kowalsky Matthew S. Faber Aritro Nath Hailey E. Dann Vince W. Kelly Li Liu Purva Shanker Ellen K. Wagner Jennifer A. Maynard Christina Chan Timothy A. Whitehead 《The Journal of biological chemistry》2015,290(44):26457-26470
Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. 相似文献
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Linlin Yin Lisette A. Maddison Mingyu Li Nergis Kara Matthew C. LaFave Gaurav K. Varshney Shawn M. Burgess James G. Patton Wenbiao Chen 《Genetics》2015,200(2):431-441
Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. 相似文献
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Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants 总被引:2,自引:1,他引:2
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Martin Dominik Vollmer Helga Hoier Hans-Jürgen Hecht Ursula Schell Janosch Grning Adrian Goldman Michael Schlmann 《Applied microbiology》1998,64(9):3290-3299
Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other. 相似文献