Top PDF Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia

Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia

Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia

In the present investigation, a cell free system for protein synthesis has been developed from human reticulocytes and this latter hypothesis tested further by comparing the capacity for[r]

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Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments

Easy and fast method for expression and native extraction of Plasmodium vivax Duffy binding protein fragments

An in-depth understanding of parasite-host interactions is important to comprehend the complex machinery involved in some microorganisms’ invasion of their tar- get cells and thus establish appropriate control methods. Concerning P. vivax, parasite protein interactions with their target cells (reticulocytes) has been studied with molecules obtained from eukaryote (i.e. Sf insect cells [7, 20], COS-7 cells [21], or wheat germ cell-free sys- tem [22]) or prokaryote (mainly E. coli) [23, 24] systems; the latter has been most used due to its methodological and economic advantages. In spite of the forgoing, the greatest challenge in using the E. coli system lies in the difficulty in obtaining functional molecules. An easy- to-use, fast and economic technique for producing and extracting two controls which would be useful regarding proteincell interaction techniques was thus standard- ized here; it was based on screening a molecule which is important for P. vivax binding to human reticulocytes.
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Synthetic Biology Goes Cell-Free

Synthetic Biology Goes Cell-Free

Molecular research has highlighted the importance of proteinprotein interactions and the resulting complexes that these interactions can generate. Whether it is for the biophysical study of these complexes or as vehicles for new therapeutic delivery (e.g., virus-like scaffolds for vaccines), there is a growing need for developing robust tools aimed at synthesis of such complexes. As in the case of membrane proteins, CFS have also demonstrated higher yields, compared to in vivo strategies, in the pro- duction of macromolecular assemblies such as virus-like particles (VLPs) [109]. Groundbreaking work by the Swartz group, demonstrating the cell-free expression of hepatitis B core antigen VLP (2 subunits) [91] in an E. coli-based cell-free system, opened the door to other re- searchers expressing a variety of macromolecular assem- blies including the E. coli RNA polymerase (5 subunits) [118] and an ATP synthase (25 subunits) [113]. Earlier work with reticulocyte lysate had also demonstrated cell- free expression of the human T-cell receptor (7 sub- units) [119]. Remarkably, a number of bacteriophages have now also been successfully expressed in CFS, in- cluding the T4 phage, which structurally contains 1500 proteins from 50 genes [56, 102–104] (Fig. 3).
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Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

Defect in messenger RNA for human hemoglobin synthesis in beta thalassemia

reticulocyte lysates of patients with homozygous beta thalassemia and sickle cell anemia. The messenger RNA stimulated the synthesis of human globin chains by a cell-free system derived from Krebs mouse ascites cells. In the presence of beta thalassemia messenger RNA, the system synthesized much less beta chain than alpha chain whereas in the presence of sickle cell anemia messenger RNA, nearly equal amounts of alpha and beta chains were synthesized. The beta/alpha synthetic ratios obtained in the cell-free system were similar to those obtained by incubating intact beta thalassemia and sickle cell anemia reticulocytes in the presence of radioactive leucine. The experiments provide direct
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Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell free in vitro translation RNA replication system by viral protein 3CDpro

Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell free in vitro translation RNA replication system by viral protein 3CDpro

made viral RNAs. The involvement of VPg in encapsida- tion has been previously proposed by Reuer et al. [15] who observed that some lethal VPg mutations still permit near normal minus and plus strand RNA synthesis in vivo. It has been known for some time that complementation between viral proteins is more efficient in the in vitro translation-RNA replication system than in vivo. This is most likely due to relatively large local concentrations of viral proteins that are translated from the input viral RNA template used in the in vitro reactions. The results described in this paper show that at least two functions of 3CD pro are complementable in vitro. One is in RNA syn-
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LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

The reduction in the rate of in viuo protein synthesis observed in all of these strains, together with the observed alterations in antibiotic sensitivity or ribosome assembly found[r]

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Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani

Ribosome Profiling Reveals HSP90 Inhibitor Effects on Stage-Specific Protein Synthesis in Leishmania donovani

The Leishmania HSP families comprise members that are expressed constitutively during both life cycle stages, e.g., HSP70 and HSP90 (30), and others whose expression increases during the conversion into the amastigote stage (21, 33, 34). The in vitro conversion from elongated, flagellated L. donovani promastigotes to ovoid, aflagellated, so-called axenic amastigotes can be achieved by the elevation of the culture temperature to 37°C and the acidification of the growth medium (35). The same morphological differ- entiation can be observed when L. donovani is treated with the HSP90-specific inhibitors geldanamycin (GA) or radicicol (RAD), which both target the special ATPase domain of HSP90 chaperones. These parasites also show an amastigote-like morphology and an increased expression of the amastigote-specific A2 protein family (14). This points to a central role for HSP90 in the parasite’s life cycle and stage conversion. In Leishmania, HSP90 (synonymously called HSP83) is encoded by multiple tandemly arranged gene copies (27, 36), and is a highly abundant, constitutively expressed protein in Leishmania spp. (30). It interacts with chaperones such as HSP70 and various cochaperones to form so-called foldosome complexes (37). Both GA and RAD bind HSP90 and inhibit its ATPase domain, thereby abrogating foldosome activity and causing cell growth arrest
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Cell Structure and History (1).ppt

Cell Structure and History (1).ppt

Nucleolus Nucleus Nuclear envelope Ribosome (attached) Ribosome (free) Cell Membrane Rough endoplasmic reticulum Golgi apparatus Mitochondrion Smooth endoplasmic reticulum Centrioles.[r]

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Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Protein Synthesis in Cell-Free Systems: an Effect of Interferon

Stimulation of amino acid incorporation by EMC RNA with untreated "74S" ribosomes from control chick embryo fibroblasts CEF, U and CEF which had been exposed to 35 0 and 140 A units of i[r]

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Alphacoronavirus Transmissible Gastroenteritis Virus nsp1 Protein Suppresses Protein Translation in Mammalian Cells and in Cell-Free HeLa Cell Extracts but Not in Rabbit Reticulocyte Lysate

Alphacoronavirus Transmissible Gastroenteritis Virus nsp1 Protein Suppresses Protein Translation in Mammalian Cells and in Cell-Free HeLa Cell Extracts but Not in Rabbit Reticulocyte Lysate

While translation initiation mediated by the cricket paralysis virus (CrPV) IRES requires only the 40S and 60S ribosomes (13, 25), both cap-dependent and CrPV IRES-mediated trans- lation require the same factors for the subsequent steps after translation initiation. Incubation of Ren-CrPV-FF (10), a bi- cistronic RNA carrying the CrPV IRES between the upstream rLuc gene and the downstream fLuc gene, with TGEV nsp1 in HeLa cell extract resulted in the suppression of cap-dependent translation but not CrPV-mediated translation (Fig. 3D). These data suggest that TGEV nsp1 suppressed translation at the initiation step but did not affect the postinitiation steps in HeLa cell extract. Similar experiments using dicistronic RNA carrying the hepatitis C virus (HCV) IRES showed that TGEV nsp1 suppressed HCV IRES-mediated translation but less ef- ficiently than cap-dependent translation (Fig. 3E). Based on assigning the values obtained for HCV IRES-driven fLuc and cap-dependent rLuc activities in the presence of GST as 100%, SCoV nsp1 inhibited both fLuc and rLuc activities by ⬃ 99%. TGEV nsp1 inhibited rLuc activity by ⬃ 97% and fLuc activity by ⬃ 70%. In contrast, TGEV nsp1 suppressed EMCV IRES- mediated translation as efficiently as it suppressed cap-depen- dent translation (Fig. 3F).
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Glial and Neuronal Functions of the Drosophila Homolog of the Human SWI/SNF Gene ATR-X (DATR-X) and the jing Zinc-Finger Gene Specify the Lateral Positioning of Longitudinal Glia and Axons

Glial and Neuronal Functions of the Drosophila Homolog of the Human SWI/SNF Gene ATR-X (DATR-X) and the jing Zinc-Finger Gene Specify the Lateral Positioning of Longitudinal Glia and Axons

EP(3)3084 contains a transposon in proximity to a novel gene known by its FlyBase transcript identifier as CG15507. Despite strong effects of EP(3)3084 expres- sion in the eye these were specifically strongly enhanced after coexpression with jing, DAtx2, and JIGR1. Further- more, each gene specifically interacted with each other but not with randomly chosen EP lines, suggesting a functional relationship between the four genes. The EP elements in these lines are located in the 59 untranslated region of the downstream genes, suggesting they may result in overexpression (BDGP). Given the regulatory role of MADF domains, it is possible that JIGR1 regu- lates gene expression with Jing and DATR-X (E ngland et al. 1992; B haskar and C ourey 2002). Alternatively, JIGR1 may regulate the expression of a Jing/DATR-X target gene. Likewise, DAtx2 may be involved in regu- lating the translation of a protein that is an essential component of a Jing/DATR-X/JIGR1 complex or a down- stream target of these genes (S atterfield et al. 2002; C iosk et al. 2004). A role for the orthologs of transla- tional regulators in MR has been shown for the Dro- sophila ortholog of fragile-X MR 1 (Dfmr1). Dfmr1 regulates the MAP1B homolog of Futsch to control synaptic structure and function in the embryonic Drosophila CNS (Z hang et al. 2001). Therefore, genetic screening and phenotypic analysis in Drosophila have the power to decipher pathways and the cellular bases of MR genes.
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DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

Premixing the labeled reticulocyte cell-free products with the same amount of infected-cell extract normally used in a DNA binding experiment, representing a great excess over that synth[r]

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Ligation free ribosome profiling of cell type specific translation in the brain

Ligation free ribosome profiling of cell type specific translation in the brain

We used ligation-free ribosome profiling to measure genome-wide translation in the forebrains of adult mice. Unlike fragments generated in RNA-Seq, ribosome foot- prints map to the transcriptome with a three-nucleotide periodicity due to the characteristic translocation inter- val of the ribosome as it translates codons [1]. To verify that the RNA libraries generated using our technique originate from ribosome footprints, we computed the power spectrum of the 5′ mapping positions of RNA fragments (Fig. 1b). As expected, the data are highly periodic with a characteristic frequency of ~0.33 nucleo- tides − 1 , similar to what has been observed for conven- tional ribosome profiling [1]. In addition to three- nucleotide periodicity, ribosome profiling also exhibits a characteristic gene body distribution. The majority of reads are expected to map to the coding sequences (CDSs) of transcripts, whereas relatively few should map to the untranslated regions (UTRs) [1]. Many genes have been shown to contain unannotated upstream ORFs (uORFs) and so we also expect that more reads will map to the 5′ UTRs than the 3′ UTRs, which are largely de- pleted of ribosomes. As shown in Fig. 1c, ligation-free ribosome profiling reads map to the transcriptome with the expected gene body distribution.
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Viral Proteins Formed in a Cell-Free RABBIT Reticulocyte System Programmed with RNA from a Temperature-Sensitive Mutant of Sindbis Virus

Viral Proteins Formed in a Cell-Free RABBIT Reticulocyte System Programmed with RNA from a Temperature-Sensitive Mutant of Sindbis Virus

We expected that in vitro translation of the mRNA from a temperature-sensitive mutant unable to make capsid or discrete envelope protein at the nonpermissive temperature would provide fu[r]

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Rhinovirus multistranded RNA: dependence of the replicative form on the presence of actinomycin D.

Rhinovirus multistranded RNA: dependence of the replicative form on the presence of actinomycin D.

Inhibition of protein synthesis in reticulocyte lysates by double-stranded and multistranded RNAs from rhinovirus-infected and noninfected HeLa cells 931 virus replication structure foun[r]

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Evidence that vesicular stomatitis virus produces double-stranded RNA that inhibits protein synthesis in a reticulocyte lysate.

Evidence that vesicular stomatitis virus produces double-stranded RNA that inhibits protein synthesis in a reticulocyte lysate.

Effect of melting and micrococcal nuclease on the residual fractional capacity of polyA+ RNA from VSV-infected HeLa cells and polyI:polyC to inhibit protein synthesis by reticulocyte lys[r]

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In Vitro Synthesis of Turnip Yellow Mosaic Virus Coat Protein in a Wheat Germ Cell-Free System

In Vitro Synthesis of Turnip Yellow Mosaic Virus Coat Protein in a Wheat Germ Cell-Free System

To analyze the in vitro synthesized products extract was prepared essentially as described by on sodium dodecyl sulfate SDS-polyacrylamide Davies and Kaesberg 5, with a few modifications[r]

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Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

The virus was propagated twice in suckling mouse brain, isolated, plaque purified four times on L929 cells, and then grown in Sac- cells at low multiplic10 Downloaded from http://jvi.asm[r]

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Hemoglobin synthesis in β thalassemia: the properties of the free α chains

Hemoglobin synthesis in β thalassemia: the properties of the free α chains

carboxymethylcellulose resin at pHs from 7.0 to 10.0, and do not elute with HbA. However, when chemically prepared hemoglobin H (Hbb 4 ) is added to the fresh hemolysates, the free a-chains are readily recovered in the HbA peak. This indicates that the free a-chains are able to combine normally with b-chains to form HbA. Freshly labeled hemolysates were also subjected to Sephadex G-100 chromatography. The free a-chains eluted as a broad peak migrating between myoglobin and hemoglobin, […]

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Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts.

Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts.

Cell-free translation of foot-and-mouth disease virus FMDV RNA in a rabbit reticulocyte system produces functional proteins, including viral protease 3C, which plays a major role in proc[r]

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