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171.
Gilad Fuchs Yoav Voichek Sima Benjamin Shlomit Gilad Ido Amit Moshe Oren 《Genome biology》2014,15(5):1-11
Background
Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.Results
RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.Conclusions
This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly. 相似文献172.
173.
Gilad Yaakov Alba Duch María García-Rubio Josep Clotet Javier Jimenez Andrés Aguilera Francesc Posas 《Molecular biology of the cell》2009,20(15):3572-3582
Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication. 相似文献
174.
175.
Gilad Ofek Krisha McKee Yongping Yang Zhi-Yong Yang Jeff Skinner F. Javier Guenaga Richard Wyatt Michael B. Zwick Gary J. Nabel John R. Mascola Peter D. Kwong 《Journal of virology》2010,84(6):2955-2962
The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.The membrane-proximal external region (MPER) of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane glycoprotein is the target of three broadly neutralizing anti-HIV-1 antibodies, 2F5, Z13e, and 4E10, and is thus a potential site of HIV-1 vulnerability to the humoral immune response (21, 24, 27, 48). The MPER encompasses ∼25 residues at the carboxyl-terminal end of the predicted gp41 ectodomain, just before the transmembrane region, and is rich in aromatic residues, typical of bilayer-interfacial regions of membrane proteins (26, 36, 40). Mutation of selected MPER tryptophans abrogates gp41-mediated fusion of the viral and target cell membranes, indicating that this region is crucial for HIV-1 infectivity (23, 28). Structural studies of unbound forms of the gp41 MPER both in solution and in lipid contexts have demonstrated that it adopts a number of conformations, many of which are α-helical, and electron-paramagnetic resonance measurements have indicated lipid bilayer immersion depths for MPER residues that range from acyl to phospholipid headgroup regions (4, 7, 8, 19, 32, 37). The binding of neutralizing antibodies, such as 2F5, to the MPER must therefore account for the membrane milieu in which the epitope is found.The 2F5 antibody has been shown to exhibit ∼100-fold-enhanced binding to its epitope on uncleaved gp140s when presented in the context of lipid proteoliposomes (11, 25), and other studies have shown that 2F5 can contact phospholipids directly in the absence of gp41 (1, 3, 12, 22, 29, 30). The latter finding has led to the suggestion that 2F5 might be autoreactive (12), although passive transfusion of 2F5 does not appear to have deleterious effects (38) and 2F5 failed to react in some clinically based assays for autoreactive lipid antibodies (31, 39). The crystal structures of the 2F5 antibody in complex with its gp41 MPER epitope revealed that, despite the 22-residue length of the 2F5 heavy chain third complementarity-determining region (CDR H3) loop, contacts with the gp41 MPER peptide are made predominantly at the loop base. In some crystal structures, the tip of the loop protrudes away from gp41, while in others, it is disordered (9, 14, 25). A unique feature of the tip of the CDR H3 loop is that it contains a patch of hydrophobic residues, including residues L100A, F100B, V100D, and I100F (Kabat numbering), which, with the exception of I100F, do not contact gp41 (9, 10, 14, 25) (Fig. (Fig.1).1). While a prior study revealed the importance of residue F100B of the CDR H3 loop in 2F5-neutralizing activity, nonconservative residue substitutions at this position also appeared to diminish 2F5 binding to the immobilized MPER peptide and gp41 in enzyme-linked immunosorbent assay (ELISA) formats (47). Conversely, a more recent study has shown that alanine mutations in the 2F5 CDR H3 loop can affect neutralization without affecting gp41 binding (2).Open in a separate windowFIG. 1.2F5 CDR H3 loop mutagenesis. (A) Structure of 2F5 Fab (blue and gray) in complex with a gp41 peptide (red). The 2F5 CDR H3 (purple) contacts gp41 only at its base, while the tip extends away from the peptide. (B) Close-up view of the 2F5 CDR H3 loop, with hydrophobic residues at the loop tip shown in stick representation and colored green. (C) Mutations introduced into the tip of the 2F5 CDR H3 (100A to 100F) are defined, along with a plot of the Wimley-White predicted free energies of transfer to a lipid bilayer interface (black) or to octanol (gray) for each of the mutations.In this study, we sought to examine the role of the chemical nature of residues at the tip of the 2F5 CDR H3 loop in neutralization of HIV-1. Mutations were introduced into the 2F5 CDR H3 loop that altered its hydrophobicity, and the resulting 2F5 mutants were tested both for binding to a gp41 epitope peptide and for neutralization of HIV-1. The results showed that the tip of the 2F5 CDR H3 loop, and specifically its hydrophobic nature, is required for 2F5-mediated neutralization of HIV-1 by means that appear to be independent both of gp41 affinity and of isolate-specific sensitivity to neutralization by 2F5. 相似文献
176.
Sunesen M de Carvalho LP Dufresne V Grailhe R Savatier-Duclert N Gibor G Peretz A Attali B Changeux JP Paas Y 《The Journal of biological chemistry》2006,281(21):14875-14881
To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClbeta receptor of Caenorhabditis elegans and the vertebrate cationic alpha7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClbeta receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClbeta receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the P(Cl)/P(Na) ratios, but the GluClbeta receptor's mutants retained high capacity to select Cl(-) over Na(+). These results indicate that, unlike the proposed case of anionic Gly- and gamma-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClbeta receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClbeta receptor's channel, Cl(-) interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore. 相似文献
177.
The emerging field of genomics examines the relationship between genetic and phenotypic variation by describing and analyzing patterns of natural variation on a genome-wide scale. In this endeavor, an important tool is the use of microarrays, which enable simultaneous screening of thousands of assays. Microarrays were originally designed for the detection of differences between samples and are thus ideally suited to high-throughput studies of natural variation. Novel microarray platforms enable the high throughput survey of variation at multiple levels, including DNA sequences, gene expression, protein binding, and methylation. However, most microarray data analysis tools, notably normalization methods, were developed for experiments in which only few features differed between samples. In studies of natural variation, this assumption does not always hold, raising a number of new challenges. 相似文献
178.
We employ complementary field and laboratory‐based incubation techniques to explore the geochemical environment where siderite concretions are actively forming and growing, including solid‐phase analysis of the sediment, concretion, and associated pore fluid chemistry. These recently formed siderite concretions allow us to explore the geochemical processes that lead to the formation of this less common carbonate mineral. We conclude that there are two phases of siderite concretion growth within the sediment, as there are distinct changes in the carbon isotopic composition and mineralogy across the concretions. Incubated sediment samples allow us to explore the stability of siderite over a range of geochemical conditions. Our incubation results suggest that the formation of siderite can be very rapid (about two weeks or within 400 hr) when there is a substantial source of iron, either from microbial iron reduction or from steel material; however, a source of dissolved iron is not enough to induce siderite precipitation. We suggest that sufficient alkalinity is the limiting factor for siderite precipitation during microbial iron reduction while the lack of dissolved iron is the limiting factor for siderite formation if microbial sulfate reduction is the dominant microbial metabolism. We show that siderite can form via heated transformation (at temperature 100°C for 48 hr) of calcite and monohydrocalcite seeds in the presence of dissolved iron. Our transformation experiments suggest that the formation of siderite is promoted when carbonate seeds are present. 相似文献
179.
180.
Isaac R. Galatzer-Levy Yael Ankri Sara Freedman Yossi Israeli-Shalev Pablo Roitman Moran Gilad Arieh Y. Shalev 《PloS one》2013,8(8)