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alfalfa mosaic virus coat protein bridges rna and rna-dependent rna polymerase in vitro.alfalfa mosaic virus (amv) rna replication requires the viral coat protein (cp). amv cp is an integral component of the viral replicase; moreover, it binds to the viral rna 3'-termini and induces the formation of multiple new base pairs that organize the rna conformation. the results described here suggest that amv coat protein binding defines template selection by organizing the 3'-terminal rna conformation and by positioning the rna-dependent rna polymerase (rdrp) at the initiation site for mi ...200717400272
mn2+ suppressor mutations and biochemical communication between ty1 reverse transcriptase and rnase h domains.ty1 reverse transcriptase/rnase h (rt/rh) is exquisitely sensitive to manganese concentrations. elevated intracellular free mn(2+) inhibits ty1 retrotransposition and in vitro ty1 rt-polymerizing activity. furthermore, mn(2+) inhibition is not limited to the ty1 rt, as this ion similarly inhibits the activities of both avian myeloblastosis virus and human immunodeficiency virus type 1 rts. to further characterize mn(2+) inhibition, we generated rt/rh suppressor mutants capable of increased ty1 t ...200717537863
the promoter regions of the myb-regulated adora2b and mcm4 genes co-localize with origins of dna replication.the retroviral oncogene v-myb encodes a transcription factor (v-myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (amv). v-myb is thought to exert its biological effects by deregulating the expression of specific target genes. we have recently demonstrated that the chicken gas41 gene, whose promoter co-localizes with an origin of dna replication, is a bona fide myb target gene. because of this finding we have asked whether other myb-regulated ...200717822556
a chimeric plum pox virus shows reduced spread and cannot compete with its parental wild-type viruses in a mixed infection.the effect of a recombination event in the genomic 3' end on the biological properties and competitiveness of plum pox virus (ppv) was investigated. therefore, a fragment spanning the coat protein (cp) coding region and a part of the 3' non-translated region of a non-aphid-transmissible strain of ppv (ppv-nat) was replaced by the corresponding region of a ppv sour cherry isolate (ppv-soc). the resulting chimera (ppv-nat/soc) caused severe symptoms in nicotiana benthamiana, resembling those of pp ...200717872539
trans-lesion synthesis and rnaseh activity by reverse transcriptases on a true abasic rna template.while much is known about abasic dna, the biological impact of abasic rna is largely unexplored. to test the mutagenic potential of this rna lesion in the context of retroviruses, we synthesized a 31-mer oligoribonucleotide containing an abasic (ras) site and used it as a template for studying dna primer extension by hiv-1, avian myeloblastosis virus (amv) and moloney murine leukemia virus (mmlv) reversed transcriptases (rt). we found that trans-lesion synthesis readily takes place with hiv-1 rt ...200717932068
[study on evaluation of human immunodeficiency virus reverse transcriptase inhibitor by elisa].to establish an elisa method for detecting the activity of reverse transcriptase.200717940565
a proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts.v-myb oncogene of avian myeloblastosis virus (amv) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. the transforming effect of the v-myb can be suppressed using phorbol ester (tpa) or histone deacetylase inhibitor trichostatin a (tsa), the inducers of cell differentiation that are in clinical trials. in this study, we used proteomics-based approach to identify proteins with variable expression in differentiated bm2 cells. proteome variations induced by tpa ...200716930693
oncogenic point mutations in the myb dna-binding domain alter the dna-binding properties of myb at a physiological target gene.the oncoprotein v-myb of avian myeloblastosis virus (amv) transforms myelomonocytic cells by deregulating specific target genes. previous work has shown that the oncogenic potential of v-myb was activated by truncation of n- and c-terminal sequences of c-myb and was further increased by amino acid substitutions in the dna-binding domain and other parts of the protein. we have analyzed the activation of the chicken lysozyme gene which is strongly activated by c-myb but not by its oncogenic counte ...200717959653
comparison of the thermal stabilities of reverse transcriptases from avian myeloblastosis virus and moloney murine leukaemia virus.reverse transcriptases (rts) from avian myeloblastosis virus (amv) and moloney murine leukaemia virus (mmlv) have been most extensively used as a tool for conversion of rna to dna. in this study, we compared the thermal stabilities of amv rt and mmlv rt by observing their irreversible thermal inactivation. the temperatures reducing initial activity by 50% in 10-min incubation, t(50), of amv rt were 47 degrees c without the template-primer (t/p), poly(ra)-p(dt)(12-18), and 52 degrees c with the t ...200818006517
novel fret-based assay to detect reverse transcriptase activity using modified dutp analogues.we have developed a novel continuous assay to measure reverse transcriptase (rt) polymerase activity. the assay uses fluorescence energy transfer measurements to detect the incorporation of complementary pairs of fluorescently labeled deoxyuridine into cdna product. the fluorescently labeled dutp substrates were prepared using commercially available reagents with a simple coupling reaction. the fluorescent dye pairs have significant spectral overlap which allows fret interaction between dyes inc ...200818163534
v-myb suppresses phorbol ester- and modifies retinoic acid-induced differentiation of human promonocytic u937 cells.the c-myb protooncogene as well as its transforming derivate, the v-myb oncogene code for transcription factors. they regulate transcription of specific target genes thus controlling proliferation, differentiation and apoptosis of hematopoietic cells. up-regulation of the c-myb expression or rearrangement/amplification of the myb locus are often involved in leukemogenesis. enforced myb expression blocks differentiation of various leukemic cell lines. human promonocytes u937 can be induced to dif ...200818505338
avian myeoloblastosis virus (amv): only one side of the coin.for many years, scientists and suppliers have refered to amv-rt as the reverse transcriptase produced by the avian myelobalstosis virus. this manuscript briefly reviews the molecular basis for biological dependence of amv for the envelope and rt proteins that are produced by its natural helper the myeloblastosis associated virus (mav). because the wide use of the term <<amv rt>> obscures scientific facts, it is worthwhile to clarify this issue for the scientific community, especially for younger ...200818558010
a dual activation mechanism for myb-responsive genes in myelomonocytic cells.the retroviral oncogene v-myb encodes a transcription factor (v-myb) which is responsible for the transformation of myelomonocytic cells by avian myeloblastosis virus (amv). v-myb is thought to exert its biological effects by deregulating the expression of specific target genes. here we have used dnasei hypersensitive site mapping and reporter gene assays to study the activation of three myb target genes--mim-1, the lysozyme gene and the c/ebpbeta gene--all of which are activated by myb in myelo ...200817950008
rna-dependent dna polymerases.reverse transcriptases (rts) are multifunctional enzymes, but are mainly used as rna-directed dna polymerases in first-strand cdna synthesis. specifically, oligodeoxynucleotides are used as primers for extension on rna templates. the dna synthesized from an rna template is referred to as complementary dna (cdna) and is often used as a template for pcr or converted to dsdna for cloning. this unit describes appropriate reaction conditions for rts from moloney murine leukemia virus (mmlv) and avian ...200818972389
amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site.the specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the p1, p3, and p4 positions of a naturally occurring type 1 cleavage site (val-ser-gln-asn-tyr downward arrowpro-ile-val-gln) in human immunodeficiency virus type 1 (hiv-1). previously, the substrate specificity of the p2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the ...200818701588
fbxw7 acts as an e3 ubiquitin ligase that targets c-myb for nemo-like kinase (nlk)-induced degradation.the c-myb proto-oncogene product (c-myb) is degraded in response to wnt-1 signaling via a pathway involving tak1 (transforming growth factor-beta-activated kinase 1), hipk2 (homeodomain-interacting protein kinase 2), and nlk (nemo-like kinase). nlk directly binds to c-myb, which results in the phosphorylation of c-myb at multiple sites, and induces its ubiquitination and proteasome-dependent degradation. here, we report that fbxw7, the f-box protein of an scf complex, targets c-myb for degradati ...200818765672
characterization of moloney murine leukaemia virus/avian myeloblastosis virus chimeric reverse transcriptases.reverse transcriptases (rts) from moloney murine leukaemia virus (mmlv) and avian myeloblastosis virus (amv) contain all the fingers, palm, thumb, connection and rnase h domains. the fingers, palm and thumb domains are thought to be involved in the reverse transcription activity, and the rnase h domain is in the rnase h activity. in this study, we characterized four chimeric rts which comprise one of the fingers, palm, thumb and rnase h domains originated from amv rt and the other three and conn ...200919060310
transient protein expression in three pisum sativum (green pea) varieties.the expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. expression vectors based on a tobacco mosaic virus (tmv) are the most commonly utilized and the primary plant used, nicotiana benthamiana, has demonstrated the abili ...200919156736
evaluation of different rt enzyme standards for quantitation of retroviruses using the single-tube fluorescent product-enhanced reverse transcriptase assay.pcr-based reverse transcriptase (rt) assays are highly sensitive for broad detection of retroviruses. these assays are currently used for demonstrating the absence of retroviral contamination in vaccines and can also be applied to clinical and laboratory research to investigate low-virus replication. a single-tube fluorescent product-enhanced reverse transcriptase assay (stf-pert) has been published that was highly sensitive for retrovirus detection (<10 virions), with enhanced reproducibility a ...200919186191
explorations into neolignan biosynthesis: concise total syntheses of helicterin b, helisorin, and helisterculin a from a common intermediate.helicterins a and b (1 and 2), helisorin (3), and helisterculin a (4) are structurally unique natural products with the ability to combat the avian myeloblastosis virus. biogenetically, their architectures are considered to be products of seemingly straightforward diels-alder, radical-based, or acid-induced dimerizations of common, simpler precursors. yet, the pursuit of such blueprints in the laboratory has failed thus far in enabling their successful synthesis. herein, we describe the first to ...200919191699
evidence for variation in the optimal translation initiation complex: plant eif4b, eif4f, and eif(iso)4f differentially promote translation of mrnas.eukaryotic initiation factor (eif) 4b is known to interact with multiple initiation factors, mrna, rrna, and poly(a) binding protein (pabp). to gain a better understanding of the function of eif4b, the two isoforms from arabidopsis (arabidopsis thaliana) were expressed and analyzed using biophysical and biochemical methods. plant eif4b was found by ultracentrifugation and light scattering analysis to most likely be a monomer with an extended structure. an extended structure would facilitate the ...200919493973
higher random oligo concentration improves reverse transcription yield of cdna from bioptic tissues and quantitative rt-pcr reliability.real time quantitative reverse transcription-pcr (qrt-pcr) is the most sensitive technique for detection and quantification of mrna targets. reliable quantification of gene expression in formalin-fixed, paraffin-embedded tissues (ffpe), however, has been subjected to serious limitations so far, mainly due to the fragmentation of rna transcripts. we tried to improve the sensitivity and reliability of mrna quantification in ffpe by boosting the reverse transcription (rt) step, that is neglected in ...200919619529
myb-induced chromatin remodeling at a dual enhancer/promoter element involves non-coding rna transcription and is disrupted by oncogenic mutations of v-myb.the oncogene v-myb of avian myeloblastosis virus (amv) encodes a transcription factor (v-myb) that transforms myelomonocytic cells by deregulating the expression of specific target genes. v-myb has acquired its oncogenic potential by truncation as well as by a number of point mutations of its cellular progenitor c-myb. as a result of these changes, the target gene spectrum v-myb differs from that of c-myb. we recently showed that the chicken mim-1 gene, a c-myb target gene that is not activated ...200919841477
complete genome amplification of equine influenza virus subtype 2.this work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (h3n8). a thermoscript reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or moloney murine leukemia virus reverse transcriptase was used. this enzyme has demonstrated higher thermal stability and is described as suitable to make long cdna with a complex secondary structure. the product obtained by this method can be cloned, used in later sequencing rea ...200920085182
polymerase incorporation and miscoding properties of 5-chlorouracil.inflammation-mediated hypochlorous acid (hocl) can damage dna, dna precursors, and other biological molecules, thereby producing an array of damage products such as 5-chlorouracil (clu). in this study, we prepared and studied 5-chloro-2'-deoxyuridine (cldu) and clu-containing oligonucleotide templates. we demonstrate that human k-562 cells grown in culture with 10 mum cldu incorporate substantial amounts of cldu without significant toxicity. when in the template, clu residues pair with datp but ...201020104909
caulimoviridae tubule-guided transport is dictated by movement protein properties.plant viruses move through plasmodesmata (pd) either as nucleoprotein complexes (npcs) or as tubule-guided encapsidated particles with the help of movement proteins (mps). to explore how and why mps specialize in one mechanism or the other, we tested the exchangeability of mps encoded by dna and rna virus genomes by means of an engineered alfalfa mosaic virus (amv) system. we show that caulimoviridae (dna genome virus) mps are competent for rna virus particle transport but are unable to mediate ...201020130061
duplex structural differences and not 2'-hydroxyls explain the more stable binding of hiv-reverse transcriptase to rna-dna versus dna-dna.human immunodeficiency virus reverse transcriptase (hiv-rt) binds more stably in binary complexes with rna-dna versus dna-dna. current results indicate that only the -2 and -4 rna nucleotides (-1 hybridized to the 3' recessed dna base) are required for stable binding to rna-dna, and even a single rna nucleotide conferred significantly greater stability than dna-dna. replacing 2'- hydroxyls on pivotal rna bases with 2'-o-methyls did not affect stability, indicating that interactions between hydro ...201020338878
optimisation of reverse transcription can improve the sensitivity of rt-pcr for detection of classical swine fever virus.classical swine fever is a highly contagious, notifiable disease of pigs and wild boars listed by the world organisation for animal health (oie). therefore, methods employed in the diagnosis of csf should be fast, sensitive and specific. the aim of this study was optimisation of the reverse transcription reaction to increase the sensitivity of real-time rt-pcr for the detection of classical swine fever virus, the aetiological agent of the disease. the efficiency of reverse transcription reaction ...201020460224
effects of organic solvents on the reverse transcription reaction catalyzed by reverse transcriptases from avian myeloblastosis virus and moloney murine leukemia virus.the use of certain organic chemicals has been found to improve yields and specificity in pcr. in this study, we examined the effects of dimethyl sulfoxide (dmso), formamide, and glycerol on the reverse transcription reaction catalyzed by reverse transcriptases (rts) from avian myeloblastosis virus (amv) and moloney murine leukemia virus (mmlv). at 42 °c, dmso at 24% v/v and formamide at 12-14% inhibited the cdna synthesis reaction, but dmso at 12% and formamide at 6-8% improved the efficiency of ...201020834159
in vitro and in vivo studies of the rna conformational switch in alfalfa mosaic virus.the 3' termini of alfalfa mosaic virus (amv) rnas adopt two mutually exclusive conformations, a coat protein binding (cpb) and a trna-like (tl) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. previously, switching between cpb and tl conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral rna (r. c. l. olsthoorn et al., embo j. 18:4856-4864, 1999). in the present study, ...201019923185
complete nucleotide sequence of a spanish isolate of alfalfa mosaic virus: evidence for additional genetic variability.alfalfa mosaic virus (amv) is a plant virus that is distributed worldwide and can induce necrosis and/or yellow mosaic on a large variety of plant species, including commercially important crops. it is the only virus of the genus alfamovirus in the family bromoviridae. amv isolates can be clustered into two genetic groups that correlate with their geographic origin. here, we report for the first time the complete nucleotide sequence of a spanish isolate of amv found infecting cape honeysuckle (t ...201121327783
comparison of the thermal stabilities of the αβ heterodimer and the α subunit of avian myeloblastosis virus reverse transcriptase.avian myeloblastosis virus reverse transcriptase (amv rt) is a heterodimer consisting of a 63-kda α subunit and a 95-kda β subunit. in this study, we explored the role of the interaction between the α and β subunits on amv rt stability. the recombinant amv rt α subunit was expressed in insect cells and purified. it exhibited lower thermal stability than the native amv rt αβ heterodimer. unlike the αβ heterodimer, the α subunit was not stabilized by template-primer. these results suggest that int ...201121821920
Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs.The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces f ...201122205995
Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates. In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most ...201122149499
mechanism of hiv reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity.several physiologically relevant cations including ca(2+), mn(2+), and zn(2+) have been shown to inhibit hiv reverse transcriptase (rt), presumably by competitively displacing one or more mg(2+) ions bound to rt. we analyzed the effects of zn(2+) on reverse transcription and compared them to ca(2+) and mn(2+). using nucleotide extension efficiency as a readout, zn(2+) showed significant inhibition of reactions with 2 mm mg(2+), even when present at only ∼5 μm. mn(2+) and ca(2+) were also inhibit ...201121953456
Molecular breeding of transgenic white clover (Trifolium repens L.) with field resistance to Alfalfa mosaic virus through the expression of its coat protein gene.Viral diseases, such as Alfalfa mosaic virus (AMV), cause significant reductions in the productivity and vegetative persistence of white clover plants in the field. Transgenic white clover plants ectopically expressing the viral coat protein gene encoded by the sub-genomic RNA4 of AMV were generated. Lines carrying a single copy of the transgene were analysed at the molecular, biochemical and phenotypic level under glasshouse and field conditions. Field resistance to AMV infection, as well as mi ...201121947755
Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification.ABSTRACT: For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both und ...201122185375
base-pairing preferences, physicochemical properties and mutational behaviour of the dna lesion 8-nitroguanine.8-nitro-2'-deoxyguanosine (8-nitrodg) is a relatively unstable, mutagenic lesion of dna that is increasingly believed to be associated with tissue inflammation. due to the lability of the glycosidic bond, 8-nitrodg cannot be incorporated into oligodeoxynucleotides (odns) by chemical dna synthesis and thus very little is known about its physicochemical properties and base-pairing preferences. here we describe the synthesis of 8-nitro-2'-o-methylguanosine, a ribonucleoside analogue of this lesion, ...201222965127
viral reverse transcriptases show selective high affinity binding to dna-dna primer-templates that resemble the polypurine tract.previous results using a selex (systematic evolution of ligands by exponential enrichment)-based approach that selected dna primer-template duplexes binding with high affinity to hiv reverse transcriptase (rt) showed that primers mimicking the 3' end, and in particular the six nt terminal g tract, of the rna polypurine tract (ppt; hiv ppt: 5'-aaaagaaaagggggg-3') were preferentially selected. in this report, two viral (moloney murine leukemia virus (mulv) and avian myeloblastosis virus (amv)) and ...201222848574
improving the thermal stability of avian myeloblastosis virus reverse transcriptase α-subunit by site-directed mutagenesis.avian myeloblastosis virus reverse transcriptase (amv rt) is a heterodimer consisting of a 63 kda α-subunit and a 95 kda β subunit. moloney murine leukaemia virus reverse transcriptase (mmlv rt) is a 75 kda monomer. these two rts are the most extensively used for conversion of rna to dna. we previously developed several mutations that increase the thermostability of mmlv rt and generated a highly stable mmlv rt variant e286r/e302k/l435r/d524a by combining three of them (glu286→arg, glu302→lys, a ...201222426840
kl-6 regulated the expression of hgf, collagen and myofibroblast differentiation.kl-6 is a pulmonary epithelial-derived mucin which is secreted mainly by type ii alveolar epithelial cells. the level of kl-6 in serum is closely correlated to the clinical activity of various interstitial lung diseases (ild) and acts as a prognostic factor for ild patients. previous studies have showed that kl-6 promoted chemotaxis, proliferation and inhibited the apoptosis of human lung fibroblasts. however, the underlying mechanism remains unknown. in this study, we investigated the function ...201324302189
microwave-assisted rapid enzymatic synthesis of nucleic acids.herein we report microwave-induced enhancement of the reactions catalyzed by escherichia coli dna polymerase i and avian myeloblastosis virus-reverse transcriptase. the reactions induced by microwaves result in a highly selective synthesis of nucleic acids in 10-50 seconds. in contrast, same reactions failed to give desired reaction products when carried out in the same time periods, but without microwave irradiation. each of the reactions was carried out for different duration of microwave expo ...201627159147
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