scholarly journals A thermo-responsive plasmid for biconditional protein expression

2018 ◽  
Author(s):  
Agathe Lermant ◽  
Alicia Magnanon ◽  
Alexandra Silvain ◽  
Paul Lubrano ◽  
Marie Lhuissier ◽  
...  
Keyword(s):  
Heat Shock ◽  
Translational Control ◽  
Cold Shock ◽  
Regulatory Sequence ◽  
Temperature Threshold ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Lambda Phage ◽  
Single Plasmid ◽  
Temperature Ranges

AbstractHere we develop a temperature sensitive expression vector that allows the selective production of distinct proteins over different temperature ranges with a single plasmid. We use theE. colicold shock translational control system (the cspA 5’Untranslated Transcribed Region - UTR) to drive the expression of a desired protein below a certain temperature threshold, and the lambda phage pL/cI857 transcriptional repressor system to drive the expression of a different protein above a certain temperature threshold. In this developmental work we use the chromogenic reporter proteins amilCP (blue chromoprotein) and monomeric red fluorescent protein (mRFP) to assess the function of the thermo-sensitive regulatory sequences over the desired temperature ranges. Our results show temperature dependent response of the cold shock regulatory sequence. However, our sequence design for the heat shock regulatory sequence did not give the intended result. The integration of these two temperature sensitive elements into a single plasmid awaits the re-design of the heat shock sequence.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

scholarly journals Transcriptional Heat Shock Response in the Smallest Known Self-Replicating Cell, Mycoplasma genitalium

2006 ◽  
Vol 188 (8) ◽  
pp. 2845-2855 ◽  
Author(s):  
Oxana Musatovova ◽  
Subramanian Dhandayuthapani ◽  
Joel B. Baseman
Keyword(s):  
Heat Shock ◽  
Elevated Temperatures ◽  
Regulation Of Gene Expression ◽  
Bacterial Pathogen ◽  
Transcriptional Response ◽  
Mycoplasma Genitalium ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Promoter Regions ◽  
Transient Nature

ABSTRACT Mycoplasma genitalium is a human bacterial pathogen linked to urethritis and other sexually transmitted diseases as well as respiratory and joint pathologies. Though its complete genome sequence is available, little is understood about the regulation of gene expression in this smallest known, self-replicating cell, as its genome lacks orthologues for most of the conventional bacterial regulators. Still, the transcriptional repressor HrcA (heat regulation at CIRCE [controlling inverted repeat of chaperone expression]) is predicted in the M. genitalium genome as well as three copies of its corresponding regulatory sequence CIRCE. We investigated the transcriptional response of M. genitalium to elevated temperatures and detected the differential induction of four hsp genes. Three of the up-regulated genes, which encode DnaK, ClpB, and Lon, possess CIRCE within their promoter regions, suggesting that the HrcA-CIRCE regulatory mechanism is functional. Additionally, one of three DnaJ-encoding genes was up-regulated, even though no known regulatory sequences were found in the promoter region. Transcript levels returned to control values after 1 h of incubation at 37°C, reinforcing the transient nature of the heat shock transcriptional response. Interestingly, neither of the groESL operon genes, which encode the GroEL chaperone and its cochaperone GroES, responded to heat shock. These data suggest that M. genitalium selectively regulates a limited number of genes in response to heat shock.

scholarly journals Rox3 and Rts1 Function in the Global Stress Response Pathway in Baker's Yeast

Genetics ◽  
1996 ◽  
Vol 142 (4) ◽  
pp. 1083-1093 ◽  
Author(s):  
Carlos C Evangelista ◽  
Ana M Rodriguez Torres ◽  
M Paullin Limbach ◽  
Richard S Zitomer
Keyword(s):  
Stress Response ◽  
Heat Shock ◽  
Osmotic Stress ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Temperature Sensitive ◽  
Global Stress ◽  
Rna Accumulation

Abstract Yeast respond to a variety of stresses through a global stress response that is mediated by a number of signal transduction pathways and the cis-acting STRE DNA sequence. The CYC7 gene, encoding iso-2-cytochrome c, has been demonstrated to respond to heat shock, glucose starvation, approach-to-stationary phase, and, as we demonstrate here, to osmotic stress. This response was delayed in a the hogl-Δ1 strain implicating the Hog1 mitogen-activated protein kinase cascade, a known component of the global stress response. Deletion analysis of the CYC7 regulatory region suggested that three STRE elements were each capable of inducing the stress response. Mutations in the ROX3 gene prevented CYC7 RNA accumulation during heat shock and osmotic stress. ROX3 RNA levels were shown to be induced by stress through a novel regulatory element. A selection for high-copy suppressors of a ROX3 temperature-sensitive allele resulted in the isolation of RTS1, encoding a protein with homology to the B′ regulatory subunit of protein phosphatase 2A0. Deletion of RTS1 caused temperature and osmotic sensitivity and increased accumulation of CYC7 RNA under all conditions. Over-expression of this gene caused increased CYC7 RNA accumulation in rox3 mutants but not in wild-type cells.

scholarly journals Cell Cycle-Dependent Binding of Yeast Heat Shock Factor to Nucleosomes

2000 ◽  
Vol 20 (17) ◽  
pp. 6435-6448 ◽  
Author(s):  
Christina Bourgeois Venturi ◽  
Alexander M. Erkine ◽  
David S. Gross
Keyword(s):  
Heat Shock ◽  
Nuclear Dna ◽  
Heat Shock Factor ◽  
Binding Activity ◽  
S Phase ◽  
Regulatory Sequences ◽  
Prevailing Paradigm ◽  
Cycle Dependent ◽  
Gain Access

ABSTRACT In the nucleus, transcription factors must contend with the presence of chromatin in order to gain access to their cognate regulatory sequences. As most nuclear DNA is assembled into nucleosomes, activators must either invade a stable, preassembled nucleosome or preempt the formation of nucleosomes on newly replicated DNA, which is transiently free of histones. We have investigated the mechanism by which heat shock factor (HSF) binds to target nucleosomal heat shock elements (HSEs), using as our model a dinucleosomal heat shock promoter (hsp82-ΔHSE1). We find that activated HSF cannot bind a stable, sequence-positioned nucleosome in G1-arrested cells. It can do so readily, however, following release from G1 arrest or after the imposition of either an early S- or late G2-phase arrest. Surprisingly, despite the S-phase requirement, HSF nucleosomal binding activity is restored in the absence of hsp82 replication. These results contrast with the prevailing paradigm for activator-nucleosome interactions and implicate a nonreplicative, S-phase-specific event as a prerequisite for HSF binding to nucleosomal sites in vivo.

Role for mRNA localization in translational activation but not spatial restriction of nanos RNA

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 659-669 ◽  
Author(s):  
S.E. Bergsten ◽  
E.R. Gavis
Keyword(s):  
Translational Control ◽  
Rna Localization ◽  
Early Embryo ◽  
Posterior Pole ◽  
Translational Repression ◽  
Control Element ◽  
Regulatory Sequences ◽  
Cis Acting ◽  
Body Patterning ◽  
Localization Signal

Patterning of the anterior-posterior body axis during Drosophila development depends on the restriction of Nanos protein to the posterior of the early embryo. Synthesis of Nanos occurs only when maternally provided nanos RNA is localized to the posterior pole by a large, cis-acting signal in the nanos 3′ untranslated region (3′UTR); translation of unlocalized nanos RNA is repressed by a 90 nucleotide Translational Control Element (TCE), also in the 3′UTR. We now show quantitatively that the majority of nanos RNA in the embryo is not localized to the posterior pole but is distributed throughout the cytoplasm, indicating that translational repression is the primary mechanism for restricting production of Nanos protein to the posterior. Through an analysis of transgenes bearing multiple copies of nanos 3′UTR regulatory sequences, we provide evidence that localization of nanos RNA by components of the posteriorly localized germ plasm activates its translation by preventing interaction of nanos RNA with translational repressors. This mutually exclusive relationship between translational repression and RNA localization is mediated by a 180 nucleotide region of the nanos localization signal, containing the TCE. These studies suggest that the ability of RNA localization to direct wild-type body patterning also requires recognition of multiple, unique elements within the nanos localization signal by novel factors. Finally, we propose that differences in the efficiencies with which different RNAs are localized result from the use of temporally distinct localization pathways during oogenesis.

Ubiquitin gene expression in Dictyostelium is induced by heat and cold shock, cadmium, and inhibitors of protein synthesis

1988 ◽  
Vol 90 (1) ◽  
pp. 51-58 ◽  
Author(s):  
A. Muller-Taubenberger ◽  
J. Hagmann ◽  
A. Noegel ◽  
G. Gerisch
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Heat Shock ◽  
Differential Expression ◽  
Dictyostelium Discoideum ◽  
Heat Shock Response ◽  
Cold Shock ◽  
Multifunctional Protein ◽  
Cadmium Treatment ◽  
Shock Response

Ubiquitin is a highly conserved, multifunctional protein, which is implicated in the heat-shock response of eukaryotes. The differential expression of the multiple ubiquitin genes in Dictyostelium discoideum was investigated under various stress conditions. Growing D. discoideum cells express four major ubiquitin transcripts of sizes varying from 0.6 to 1.9 kb. Upon heat shock three additional ubiquitin mRNAs of 0.9, 1.2 and 1.4 kb accumulate within 30 min. The same three transcripts are expressed in response to cold shock or cadmium treatment. Inhibition of protein synthesis by cycloheximide leads to a particularly strong accumulation of the larger ubiquitin transcripts, which code for polyubiquitins. Possible mechanisms regulating the expression of ubiquitin transcripts upon heat shock and other stresses are discussed.

Increased production of genetic mosaics in Habrobracon juglandis by cold shock of newly oviposited eggs

Development ◽  
1976 ◽  
Vol 36 (1) ◽  
pp. 127-131
Author(s):  
R. M. Petters ◽  
D. S. Grosch
Keyword(s):  
Heat Shock ◽  
Cold Shock ◽  
Differential Mortality ◽  
Wild Type ◽  
Cleavage Stage ◽  
Genetic Mosaics

Eggs from an ebony stock exposed to 5·5°C prior to syngamy exhibited increased production of genetic mosaics in comparison with untreated eggs from the same females. No increase in mosaic production occurred for cold-shocked cleavage-stage embryos from the ebony stock or from pre-cleavage cold-shocked eggs from a wild-type stock. Heat shock of pre-syngamy eggs also failed to increase the production of genetic mosaics. These findings are onsistent with predictions based on the post-cleavage fertilization theory of mosaic origin in Habrobracon or with a hypothesis of differential mortality.

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