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91.
Korolev S Knyazhanskaya E Anisenko A Tashlitskii V Zatsepin TS Gottikh M Agapkina J 《Nucleosides, nucleotides & nucleic acids》2011,30(7-8):651-666
Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates. 相似文献
92.
Mikheikin AL Surzhikov SA Zasedateleva OA Vasiliskov VA Pan'kov S Grechishnikova IV Kisselev LL Zasedatelev AS 《BioTechniques》2008,44(1):77-83
To analyze RNA interactions with RNA binding molecules an RNA microchip containing immobilized oligoribonucleotides with protective groups [t-butyldimethylsilyl (tBDMS)] at 2'-O- positions was developed. The oligonucleotides were immobilized within three-dimensional (3-D) hydrogel pads fixed on a glass support. The protective groups preserved the oligoribonucleodes from degradation and were suitable to be removed directly on the microchip when needed, right before its use. These immobilized, deprotected oligoribonucleotides were tested for their interaction with afluorescently labeled oligodeoxyribonucleotide and analyzed for their availability to be cleaved enzymatically by the RNase binase. Stability of tBDMS-protected immobilized oligoribonucleotides after 2.5 years of storage as well as after direct RNase action was also tested. Melting curves of short RNA/DNA hybrids that had formed into gel pads of the microchip were found to exhibit clearly defined S-like shapes, with the melting temperatures in full accordance with those theoretically predicted for the same ionic strength. This approach, based on keeping the protective groups attached to oligoribonucleotides, can be applied for manufacturing any RNA microchips containing immobilized oligoribonucleotides, including microchips with two-dimensional (2-D) features. These RNA microchips can be used to measure thermodynamic parameters of RNA/RNA or RNA/DNA duplexes as well as to study ligand- or protein-RNA interactions. 相似文献
93.
Ublinskaya AA Samsonov VV Mashko SV Stoynova NV 《Journal of microbiological methods》2012,89(3):167-173
The genetic manipulation of cells is the most promising strategy for designing microorganisms with desired traits. The most widely used approaches for integrating specific DNA-fragments into the Escherichia coli genome are based on bacteriophage site-specific and Red/ET-mediated homologous recombination systems. Specifically, the recently developed Dual In/Out integration strategy enables the integration of DNA fragments directly into specific chromosomal loci (Minaeva et al., 2008). To develop this strategy further, we designed a method for the precise cloning of any long DNA fragments from the E. coli chromosome and their targeted insertion into the genome that does not require PCR. In this method, the region of interest is flanked by I-SceI rare-cutting restriction sites, and the I-SceI-bracketed region is cloned into the unique I-SceI site of an integrative plasmid vector that then enables its targeted insertion into the E. coli chromosome via bacteriophage φ80 Int-mediated specialized recombination. This approach allows any long specific DNA fragment from the E. coli genome to be cloned without a PCR amplification step and reproducibly inserted into any chosen chromosomal locus. The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry. 相似文献
94.
Vasily N. Danilevich Andrey L. Mulyukin Andrey V. Machulin Vladimir V. Sorokin Sergey A. Kozlov 《Journal of biomolecular structure & dynamics》2019,37(4):918-930
Our previous studies demonstrated the formation of structurally diverse DNA-containing microparticles (DNA MPs) in PCR with Mg-pyrophosphate (MgPPi) as the structure-forming component. These DNA MPs were referred to major structural types: microdisks (2D MPs) with nanometer thickness and 3D MPs with sophisticated morphology and constructed from intersecting disks and their segments. Little is known about factors that influence both the morphology and size of DNA MPs, and the present study was aimed at fulfilling this gap. We showed that the addition of Mn2+ cations to PCR mixtures caused the profound changes in MPs morphology, depending on DNA polymerase used (KlenTaq or Taq). Asymmetric PCR with 20-fold decrease in the concentration of one of two primers facilitated the predominant formation of microdisks with unusual structure. The addition of 1 mM Na-pyrophosphate to PCR mixtures with synthesized DNA and subsequent thermal cycling (10–15 cycles) were optimal to produce microdisks or nanometer 3D particles. Using electron microscopy, we studied also the structure of inorganic micro- and nanoparticles from MgPPi, formed during multiple heating and cooling cycles of a mixture of Mg2+ and Na-pyrophosphate in various regimes. Also, we found the conditions to yield planar (Mg·Mn)PPi nanocrystals (diameter ~100 nm and thickness ~10 nm) which efficiently adsorbed exogenous DNA. These inorganic nanoparticles are promising for DNA delivery in transfection studies. Mechanisms to be involved in structural modifications of MPs and perspectives of their practical application are discussed. 相似文献
95.
Asaf Zviran Nofar Mor Yoach Rais Hila Gingold Shani Peles Elad Chomsky Sergey Viukov Jason D. Buenrostro Roberta Scognamiglio Leehee Weinberger Yair S. Manor Vladislav Krupalnik Mirie Zerbib Hadas Hezroni Diego Adhemar Jaitin David Larastiaso Shlomit Gilad Sima Benjamin Jacob H. Hanna 《Cell Stem Cell》2019,24(2):328-341.e9
96.
Gareth A. Palidwor Sergey Shcherbinin Matthew R. Huska Tamas Rasko Ulrich Stelzl Anup Arumughan Raphaele Foulle Pablo Porras Luis Sanchez-Pulido Erich E. Wanker Miguel A. Andrade-Navarro 《PLoS computational biology》2009,5(3)
A growing number of solved protein structures display an elongated structural
domain, denoted here as alpha-rod, composed of stacked pairs of anti-parallel
alpha-helices. Alpha-rods are flexible and expose a large surface, which makes
them suitable for protein interaction. Although most likely originating by
tandem duplication of a two-helix unit, their detection using sequence
similarity between repeats is poor. Here, we show that alpha-rod repeats can be
detected using a neural network. The network detects more repeats than are
identified by domain databases using multiple profiles, with a low level of
false positives (<10%). We identify alpha-rod repeats in
approximately 0.4% of proteins in eukaryotic genomes. We then
investigate the results for all human proteins, identifying alpha-rod repeats
for the first time in six protein families, including proteins STAG1-3, SERAC1,
and PSMD1-2 & 5. We also characterize a short version of these repeats
in eight protein families of Archaeal, Bacterial, and Fungal species. Finally,
we demonstrate the utility of these predictions in directing experimental work
to demarcate three alpha-rods in huntingtin, a protein mutated in
Huntington''s disease. Using yeast two hybrid analysis and an
immunoprecipitation technique, we show that the huntingtin fragments containing
alpha-rods associate with each other. This is the first definition of domains in
huntingtin and the first validation of predicted interactions between fragments
of huntingtin, which sets up directions toward functional characterization of
this protein. An implementation of the repeat detection algorithm is available
as a Web server with a simple graphical output: http://www.ogic.ca/projects/ard. This can be further visualized
using BiasViz, a graphic tool for representation of multiple sequence
alignments. 相似文献
97.
98.
Sergey Malchenko Jianping Xie Maria de Fatima Bonaldo Elio F. Vanin Bula J. Bhattacharyya Abdelhak Belmadani Guifa Xi Vasily Galat William Goossens Richard E.B. Seftor Tadanori Tomita John Crispino Richard J. Miller Martha C. Bohn Mary J.C. Hendrix Marcelo B. Soares 《Gene》2014
In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice. 相似文献
99.
Jocelyn Charlton Richard D Williams Mark Weeks Neil J Sebire Sergey Popov Gordan Vujanic William Mifsud Marisa Alcaide-German Lee M Butcher Stephan Beck Kathy Pritchard-Jones 《Genome biology》2014,15(8)
Background
Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.Results
Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.Conclusions
These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users. 相似文献100.
Nadezda Shershakova Elena Bashkatova Alexander Babakhin Sergey Andreev Alexandra Nikonova Igor Shilovsky Oleg Kamyshnikov Andrey Buzuk Olga Elisyutina Elena Fedenko Musa Khaitov 《PloS one》2015,10(8)
Atopic dermatitis (AD) is a widespread and difficult to treat allergic skin disease and is a tough challenge for healthcare. In this study, we investigated whether allergen-specific immunotherapy (ASIT) with a monomeric allergoid obtained by succinylation of ovalbumin (sOVA) is effective in a mouse model of atopic dermatitis. An experimental model of AD was reproduced by epicutaneous sensitization with ovalbumin (OVA). ASIT was performed with subcutaneous (SC) administration of increasing doses of OVA or sOVA. The levels of anti-OVA antibodies, as well as cytokines, were detected by ELISA. Skin samples from patch areas were taken for histologic examination. ASIT with either OVA or sOVA resulted in a reduction of both the anti-OVA IgE level and the IgG1/IgG2a ratio. Moreover, ASIT with sOVA increased the IFN-γ level in supernatants after splenocyte stimulation with OVA. Histologic analysis of skin samples from the sites of allergen application showed that ASIT improved the histologic picture by decreasing allergic inflammation in comparison with untreated mice. These data suggest that ASIT with a succinylated allergen represents promising approach for the treatment of AD. 相似文献