scholarly journals Shuttle Vector Expression in Thermococcus kodakaraensis: Contributions of cis Elements to Protein Synthesis in a Hyperthermophilic Archaeon

2008 ◽  
Vol 74 (10) ◽  
pp. 3099-3104 ◽  
Author(s):  
Thomas J. Santangelo ◽  
L'ubomíra Čuboňová ◽  
John N. Reeve
Keyword(s):  
Translation Initiation ◽  
Affinity Purification ◽  
Shuttle Vector ◽  
Selectable Markers ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Ribosome Binding ◽  
Translation Initiation Codon ◽  
Binding Sequence

ABSTRACT Shuttle vectors that replicate stably and express selectable phenotypes in both Thermococcus kodakaraensis and Escherichia coli have been constructed. Plasmid pTN1 from Thermococcus nautilis was ligated to the commercial vector pCR2.1-TOPO, and selectable markers were added so that T. kodakaraensis transformants could be selected by ΔtrpE complementation and/or mevinolin resistance. Based on Western blot measurements, shuttle vector expression of RpoL-HA, a hemagglutinin (HA) epitope-tagged subunit of T. kodakaraensis RNA polymerase (RNAP), was ∼8-fold higher than chromosome expression. An idealized ribosome binding sequence (5′-AGGTGG) was incorporated for RpoL-HA expression, and changes to this sequence reduced expression. Changing the translation initiation codon from AUG to GUG did not reduce RpoL-HA expression, but replacing AUG with UUG dramatically reduced RpoL-HA synthesis. When functioning as translation initiation codons, AUG, GUG, and UUG all directed the incorporation of methionine as the N-terminal residue of RpoL-HA synthesized in T. kodakaraensis. Affinity purification confirmed that an HA- plus six-histidine-tagged RpoL subunit (RpoL-HA-his6) synthesized ectopically from a shuttle vector was assembled in vivo into RNAP holoenzymes that were active and could be purified directly from T. kodakaraensis cell lysates by Ni2+ binding and imidazole elution.

scholarly journals Use of an Endogenous Plasmid Locus for Stable intransComplementation in Borrelia burgdorferi

2014 ◽  
Vol 81 (3) ◽  
pp. 1038-1046 ◽  
Author(s):  
Irene N. Kasumba ◽  
Aaron Bestor ◽  
Kit Tilly ◽  
Patricia A. Rosa
Keyword(s):  
Borrelia Burgdorferi ◽  
Shuttle Vector ◽  
Targeted Mutagenesis ◽  
Multiple Cloning Site ◽  
Stable Gene ◽  
Content Type ◽  
Shuttle Vectors ◽  
Stable Maintenance

ABSTRACTTargeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirocheteBorrelia burgdorferi. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers thanB. burgdorferiplasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.B. burgdorferihas over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochetein vivobut relatively unstable duringin vitrocultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number andin vivostability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into thebbe02locus, a site on lp25 that was previously shown to be nonessential during bothin vitroandin vivogrowth. We demonstrate the functional utility of this strategy by restoring infectivity to anospCmutant through complementation at this site on lp25 and stable maintenance of theospCgene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation inB. burgdorferi.

scholarly journals Mice Lacking the 68-Amino-Acid, Mammal-Specific N-Terminal Extension of WT1 Develop Normally and Are Fertile

2003 ◽  
Vol 23 (7) ◽  
pp. 2608-2613 ◽  
Author(s):  
Colin G. Miles ◽  
Joan Slight ◽  
Lee Spraggon ◽  
Maureen O'Sullivan ◽  
Charles Patek ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Histological Analysis ◽  
Normal Development ◽  
Wilms Tumor ◽  
Protein Isoforms ◽  
Translation Initiation Codon ◽  
Developing Kidney ◽  
Wt1 Protein

ABSTRACT Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis—consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.

In vivo evaluation of the context sequence of the translation initiation codon in plants

Plant Science ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Marcin Lukaszewicz ◽  
Marc Feuermann ◽  
Bénédicte Jérouville ◽  
Arnaud Stas ◽  
Marc Boutry
Keyword(s):  
Translation Initiation ◽  
Initiation Codon ◽  
In Vivo Evaluation ◽  
Translation Initiation Codon

scholarly journals Site-directed mutagenesis of a Saccharomyces cerevisiae mitochondrial translation initiation codon.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 659-668 ◽  
Author(s):  
L S Folley ◽  
T D Fox
Keyword(s):  
Translation Initiation ◽  
Structural Features ◽  
Gene Replacement ◽  
Site Directed Mutagenesis ◽  
Initiation Codon ◽  
Translation Initiation Site ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Translation Initiation Codon

Abstract We have used a generally applicable strategy for gene replacement in yeast mitochondria to mutate the translation initiation codon of the COX3 gene from AUG to AUA. The mutation, cox3-1, substantially reduced, but did not eliminate, translation of cytochrome c oxidase subunit III (coxIII). Strains bearing the mutation exhibited a leaky (partial) nonrespiratory growth phenotype and a reduced incorporation of radiolabeled amino acids into coxIII in vivo in the presence of cycloheximide. Hybridization experiments demonstrated that the mutation had little or no effect on levels of the COX3 mRNA. Residual translation of the cox3-1 mutant mRNA was dependent upon the three nuclearly coded mRNA-specific activators PET494, PET54 and PET122, known from previous studies to work through a site (or sites) upstream of the initiation codon to promote translation of the wild-type mRNA. Furthermore, respiratory growth of cox3-1 mutant strains was sensitive to decreased dosage of genes PET494 and PET122 in heterozygous mutant diploids, unlike the growth of strains carrying wild-type mtDNA. Some residual translation of the cox3-1 mRNA appeared to initiate at the mutant AUA codon, despite the fact that the 610-base 5'-mRNA leader contains numerous AUA triplets. We conclude that, while AUG is an important component of the COX3 translation initiation site, the site probably is also specified by other sequence or structural features.

scholarly journals Construction of a Shuttle Vector for, and Spheroplast Transformation of, the Hyperthermophilic Archaeon Pyrococcus abyssi

2002 ◽  
Vol 68 (11) ◽  
pp. 5528-5536 ◽  
Author(s):  
Soizick Lucas ◽  
Laurent Toffin ◽  
Yvan Zivanovic ◽  
Daniel Charlier ◽  
Hélène Moussard ◽  
...  
Keyword(s):  
Shuttle Vector ◽  
Low Frequency ◽  
Vector System ◽  
Hyperthermophilic Archaea ◽  
Gene Encoding ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Bacterial Vector ◽  
Genes Encoding ◽  
Pyrococcus Abyssi

ABSTRACT Our understanding of the genetics of species of the best-studied hyperthermophilic archaea, Pyrococcus spp., is presently limited by the lack of suitable genetic tools, such as a stable cloning vector and the ability to select individual transformants on plates. Here we describe the development of a reliable host-vector system for the hyperthermophilic archaeon Pyrococcus abyssi. Shuttle vectors were constructed based on the endogenous plasmid pGT5 from P. abyssi strain GE5 and the bacterial vector pLitmus38. As no antibiotic resistance marker is currently available for Pyrococcus spp., we generated a selectable auxotrophic marker. Uracil auxotrophs resistant to 5-fluoorotic acid were isolated from P. abyssi strain GE9 (devoid of pGT5). Genetic analysis of these mutants revealed mutations in the pyrE and/or pyrF genes, encoding key enzymes of the pyrimidine biosynthetic pathway. Two pyrE mutants exhibiting low reversion rates were retained for complementation experiments. For that purpose, the pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase) of the thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, was introduced into the pGT5-based vector, giving rise to pYS2. With a polyethylene glycol-spheroplast method, we could reproducibly transform P. abyssi GE9 pyrE mutants to prototrophy, though with low frequency (102 to 103 transformants per μg of pYS2 plasmid DNA). Transformants did grow as well as the wild type on minimal medium without uracil and showed comparable OPRTase activity. Vector pYS2 proved to be very stable and was maintained at high copy number under selective conditions in both Escherichia coli and P. abyssi.

scholarly journals Translation of the chloroplast psbC mRNA is controlled by interactions between its 5' leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii.

1997 ◽  
Vol 17 (6) ◽  
pp. 3440-3448 ◽  
Author(s):  
W Zerges ◽  
J Girard-Bascou ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Translation Initiation ◽  
Translational Control ◽  
Reporter Genes ◽  
Activation Function ◽  
Wild Type ◽  
Cis Acting ◽  
Translation Initiation Codon ◽  
Nuclear Loci

Translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5' untranslated region (5' UTR) and the functions encoded by two nuclear loci, which we name here TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC 5' UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1 function. These data demonstrate that sequences located in the middle of the psbC 5' UTR, TBC1, and TBC3 functionally interact to control the translation of the psbC mRNA.

scholarly journals Naturally Occurring Adenines within mRNA Coding Sequences Affect Ribosome Binding and Expression in Escherichia coli

2006 ◽  
Vol 189 (2) ◽  
pp. 501-510 ◽  
Author(s):  
Jay E. Brock ◽  
Robert L. Paz ◽  
Patrick Cottle ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Start Codon ◽  
Coding Sequences ◽  
E Coli ◽  
Ribosome Binding ◽  
Naturally Occurring ◽  
In Vivo Expression

ABSTRACT Translation initiation requires the precise positioning of a ribosome at the start codon. The major signals of bacterial mRNA that direct the ribosome to a translational start site are the Shine-Dalgarno (SD) sequence within the untranslated leader and the start codon. Evidence for the presence of many non-SD-led genes in prokaryotes provides a motive for studying additional interactions between ribosomes and mRNA that contribute to translation initiation. A high incidence of adenines has been reported downstream of the start codon for many Escherichia coli genes, and addition of downstream adenine-rich sequences increases expression from several genes in E. coli. Here we describe site-directed mutagenesis of the E. coli aroL, pncB, and cysJ coding sequences that was used to assess the contribution of naturally occurring adenines to in vivo expression and in vitro ribosome binding from mRNAs with different SD-containing untranslated leaders. Base substitutions that decreased the downstream adenines by one or two nucleotides decreased expression significantly from aroL-, pncB-, and cysJ-lacZ fusions; mutations that increased downstream adenines by one or two nucleotides increased expression significantly from aroL- and cysJ-lacZ fusions. Using primer extension inhibition (toeprint) and filter binding assays to measure ribosome binding, the changes in in vivo expression correlated closely with changes in in vitro ribosome binding strength. Our data are consistent with a model in which downstream adenines influence expression through their effects on the mRNA-ribosome association rate and the amount of ternary complex formed. This work provides evidence that adenine-rich sequence motifs might serve as a general enhancer of E. coli translation.

scholarly journals Defining Components of the Chromosomal Origin of Replication of the Hyperthermophilic Archaeon Pyrococcus furiosus Needed for Construction of a Stable Replicating Shuttle Vector

2011 ◽  
Vol 77 (18) ◽  
pp. 6343-6349 ◽  
Author(s):  
Joel Farkas ◽  
Daehwan Chung ◽  
Megan DeBarry ◽  
Michael W. W. Adams ◽  
Janet Westpheling
Keyword(s):  
Pyrococcus Furiosus ◽  
Shuttle Vector ◽  
Plasmid Replication ◽  
Origin Of Replication ◽  
Binding Studies ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Shuttle Vectors ◽  
Chromosomal Origin ◽  
Functional Components

ABSTRACTWe report the construction of a series of replicating shuttle vectors that consist of a low-copy-number cloning vector forEscherichia coliand functional components of the origin of replication (oriC) of the chromosome of the hyperthermophilic archaeonPyrococcus furiosus. In the process of identifying the minimum replication origin sequence required for autonomous plasmid replication inP. furiosus, we discovered that several features of the origin predicted by bioinformatic analysis andin vitrobinding studies were not essential for stable autonomous plasmid replication. A minimum region required to promote plasmid DNA replication was identified, and plasmids based on this sequence readily transformedP. furiosus. The plasmids replicated autonomously and existed in a single copy. In contrast to shuttle vectors based on a plasmid from the closely related hyperthermophilePyrococcus abyssifor use inP. furiosus, plasmids based on theP. furiosuschromosomal origin were structurally unchanged after transformation and were stable without selection for more than 100 generations.

scholarly journals USP9X-mediated KDM4C deubiquitination promotes lung cancer radioresistance by epigenetically inducing TGF-β2 transcription

2021 ◽  
Author(s):  
Xiaohua Jie ◽  
William Pat Fong ◽  
Rui Zhou ◽  
Ye Zhao ◽  
Yingchao Zhao ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Affinity Purification ◽  
Smad Signaling ◽  
Lung Cancer Patients ◽  
Lysine Specific Demethylase ◽  
Underlying Mechanisms ◽  
H3k9me3 Level ◽  
Critical Binding

AbstractRadioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

scholarly journals Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samir Sissaoui ◽  
Stuart Egginton ◽  
Ling Ting ◽  
Asif Ahmed ◽  
Peter W. Hewett
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Akt Activation ◽  
Forkhead Box ◽  
Pi3k Activity ◽  
Binding Sequence

AbstractPlacenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

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