Heat Shock Promoter Research Articles

The Medaka zic1/zic4 Mutant Provides Molecular Insights into Teleost Caudal Fin Evolution

Teleosts have an asymmetrical caudal fin skeleton formed by the upward bending of the caudal-most portion of the body axis, the ural region. This homocercal type of caudal fin ensures powerful and complex locomotion and is regarded as one of the most important innovations for teleosts during adaptive radiation in an aquatic environment. However, the mechanisms that create asymmetric caudal fin remain largely unknown. The spontaneous medaka (teleost fish) mutant, Double anal fin (Da), exhibits a unique symmetrical caudal skeleton that resembles the diphycercal type seen in Polypterus and Coelacanth. We performed a detailed analysis of the Da mutant to obtain molecular insight into caudal fin morphogenesis. We first demonstrate that a large transposon, inserted into the enhancer region of the zic1 and zic4 genes (zic1/zic4) in Da, is associated with the mesoderm-specific loss of their transcription. We then show that zic1/zic4 are strongly expressed in the dorsal part of the ural mesenchyme and thereby induce asymmetric caudal fin development in wild-type embryos, whereas their expression is lost in Da. Comparative analysis further indicates that the dorsal mesoderm expression of zic1/zic4 is conserved in teleosts, highlighting the crucial role of zic1/zic4 in caudal fin morphogenesis.

Novel high-throughput RNAi vectors for plant biotechnology

With 2 figures and 1 table Abstract The knockdown of genes through RNA interference (RNAi) inducing vectors with one promoter regulating a sense and antisense sequence of the target gene separated by a spacer/intron region has been rampant in plant kingdom. Those constructs are difficult to clone because of long homologies. This study is the first to successfully use RNAi constructs with two promoters in inverse orientation flanking a DNA sequence of the target gene in plants. Different double-promoter constructs containing a β-d-glucuronidase gene–targeting sequence with constitutive 35S promoters or inducible heat shock promoters GmHSP17.5-E (Glycine max) were tested. We were able to knock down the β-d-glucuronidase expression in transgenic Nicotiana tabacum after transformation with these new RNAi constructs. The change in gene expression was tested with molecular, histochemical and fluorimetric methods. The results showed a very high and reliable knockdown rate for constitutive promoters. Owing to their consistent knockdown and the uncomplicated cloning, these new RNAi vectors are of outstanding importance for genomics and plant biotechnology.

Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model

Osteoclasts are macrophage-related bone resorbing cells of hematopoietic origin. Factors that regulate osteoclastogenesis are of great interest for investigating the pathology and treatment of bone diseases such as osteoporosis. In mammals, receptor activator of NF-κB ligand (Rankl) is a regulator of osteoclast formation and activation: its misexpression causes osteoclast stimulation and osteoporotic bone loss. Here, we report an osteoporotic phenotype that is induced by overexpression of Rankl in the medaka model. We generated transgenic medaka lines that express GFP under control of the cathepsin K promoter in osteoclasts starting at 12 days post-fertilization (dpf), or Rankl together with CFP under control of a bi-directional heat-shock promoter. Using long-term confocal time-lapse imaging of double and triple transgenic larvae, we monitored in vivo formation and activation of osteoclasts, as well as their interaction with osteoblasts. Upon Rankl induction, GFP-positive osteoclasts are first observed in the intervertebral regions and then quickly migrate to the surface of mineralized neural and haemal arches, as well as to the centra of the vertebral bodies. These osteoclasts are TRAP (tartrate-resistant acid phosphatase) and cathepsin K positive, mononuclear and highly mobile with dynamically extending protrusions. They are exclusively found in tight contact with mineralized matrix. Rankl-induced osteoclast formation resulted in severe degradation of the mineralized matrix in vertebral bodies and arches. In conclusion, our in vivo imaging approach confirms a conserved role of Rankl in osteoclastogenesis in teleost fish and provides new insight into the cellular interactions during bone resorption in an animal model that is useful for genetic and chemical screening.

A zebrafish transgenic model of Ewing’s sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis

SUMMARYEwing’s sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing’s sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing’s sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing’s sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing’s sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

Simple, Economical Heat-Shock Devices for Zebrafish Housing Racks

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.

Kinetics of Doubletime Kinase-dependent Degradation of the Drosophila Period Protein

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT(S)), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT(S) in cell culture resembles that with wild-type DBT and posits that, in flies DBT(S) likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation.

MosSCI and Gateway Compatible Plasmid Toolkit for Constitutive and Inducible Expression of Transgenes in the C. elegans Germline

Here we describe a toolkit for the production of fluorescently tagged proteins in the C. elegans germline and early embryo using Mos1-mediated single copy insertion (MosSCI) transformation. We have generated promoter and 3′UTR fusions to sequences of different fluorescent proteins yielding constructs for germline expression that are compatible with MosSCI MultiSite Gateway vectors. These vectors allow tagged transgene constructs to be inserted as single copies into known sites in the C. elegans genome using MosSCI. We also show that two C. elegans heat shock promoters (Phsp-16.2 and Phsp-16.41) can be used to induce transgene expression in the germline when inserted via MosSCI transformation. This flexible set of new vectors, available to the research community in a plasmid repository, should facilitate research focused on the C. elegans germline and early embryo.

Pogostick: A New Versatile piggyBac Vector for Inducible Gene Over-Expression and Down-Regulation in Emerging Model Systems

BackgroundNon-traditional model systems need new tools that will enable them to enter the field of functional genetics. These tools should enable the exploration of gene function, via knock-downs of endogenous genes, as well as over-expression and ectopic expression of transgenes.MethodologyWe constructed a new vector called Pogostick that can be used to over-express or down-regulate genes in organisms amenable to germ line transformation by the piggyBac transposable element. Pogostick can be found at www.addgene.org, a non-profit plasmid repository. The vector currently uses the heat-shock promoter Hsp70 from Drosophila to drive transgene expression and, as such, will have immediate applicability to organisms that can correctly interpret this promotor sequence. We detail how to clone candidate genes into this vector and test its functionality in Drosophila by targeting a gene coding for the fluorescent protein DsRed. By cloning a single DsRed copy into the vector, and generating transgenic lines, we show that DsRed mRNA and protein levels are elevated following heat-shock. When cloning a second copy of DsRed in reverse orientation into a flanking site, and transforming flies constitutively expressing DsRed in the eyes, we show that endogenous mRNA and protein levels drop following heat-shock. We then test the over-expression vector, containing the complete cDNA of Ultrabithorax (Ubx) gene, in an emerging model system, Bicyclus anynana. We produce a transgenic line and show that levels of Ubx mRNA expression rise significantly following a heat-shock. Finally, we show how to obtain genomic sequence adjacent to the Pogostick insertion site and to estimate transgene copy number in genomes of transformed individuals.SignificanceThis new vector will allow emerging model systems to enter the field of functional genetics with few hurdles.

An Approach to the Production of Soluble Protein from a Fungal Gene Encoding an Aggregation-Prone Xylanase in Escherichia coli

The development of new procedures and protocols that allow researchers to obtain recombinant proteins is of fundamental importance in the biotechnology field. A strategy was explored to overcome inclusion-body formation observed when expressing an aggregation-prone fungal xylanase in Escherichia coli. pHsh is an expression plasmid that uses a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ32). A derivative of pHsh was constructed by fusing a signal peptide to xynA2 gene to facilitate export of the recombinant protein to the periplasm. The xylanase was produced in a soluble form. Three factors were essential to achieving such soluble expression of the xylanase: 1) the target gene was under the control of the Hsh promoter, 2) the gene product was exported into the periplasm, and 3) gene expression was induced by a temperature upshift. For the first time we report the expression of periplasmic proteins under the control of an Hsh promoter regulated by σ32. One unique feature of this approach was that over 200 copies of the Hsh promoter in an E. coli cell significantly increased the concentration of σ32. The growth inhibition of the recombinant cells corresponded to an increase in the levels of soluble periplasmic protein. Therefore, an alternative protocol was designed to induce gene expression from pHsh-ex to obtain high levels of active soluble enzymes.

Heat-inducible Cre-lox system for marker excision in transgenic rice

The present study assessed the efficacy of a heat-inducible cre gene for conditional removal of the marker gene from a rice genome via Cre-lox recombination. A cre gene controlled by the soybean heat-shock promoter was introduced into the rice genome along with the recombination target (lox) construct. Cre-mediated recombination was expected to remove the marker gene and activate the promoter-less GUS gene. Six transgenic lines displayed well-regulated heat-inducible Cre activity in the callus. However, only one line that contained a single copy of the cre gene maintained this property in the regenerated plants and their progeny. Marker-free progeny were obtained from the plant that was heat-treated at the seedling stage, indicating the inheritance of the recombination 'footprint'. The presence of the 'footprint' was verified by polymerase chain reaction and Southern analysis. Therefore, the cre gene controlled by the soybean heat-shock promoter is an effective tool for conditional removal of the marker gene in rice.

SIRT1 Deacetylase Activity and the Maintenance of Protein Homeostasis in Response to Stress: An Overview

The present review intends to summarize the, yet preliminary, but very important emerging data underlining the functions exerted by the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase SIRT1 on protein homeostasis. The main focus of the discussion is the cooperation between SIRT1 and the heat shock factor 1 (HSF1) responsible for activating the transcription of molecular chaperones, the protein-protective factors that resolve damaged/misfolded and aggregated proteins generated by heat stress or metabolism. SIRT1, a mammalian ortholog of the yeast silent information regulator 2, is a stress activated protein deacetylase that contributes to life-span extension by regulating different cell survival pathways, including replicative senescence, inflammation and resistance to hypoxic and heat stress. Phosphorylation is the major mechanism controlling the level and function of SIRT1 required for normal cell cycle progression and cell survival under stress conditions. Phosphorylated SIRT1 deacetylates and coactivates different substrates, including HSF1. Deacetylated HSF1 binds to the heat shock promoter element found upstream of genes encoding molecular chaperones. Overexpression of SIRT1 in cultured cells also helps them to survive exposure to heat stress. Conversely, its down-regulation accelerates the attenuation of the heat shock response promoting the release of HSF1 from its cognate promoter element. Very recently, in a mouse model for Alzheimer's disease, SIRT1 deacetylase activity was also found activating the transcription of α-secretase, the enzyme responsible for inhibiting the formation of aggregates of neuronal β-amyloid plaques. How SIRT1 activity protects cells from the deleterious effects of damaged/misfolded proteins and the implication of these findings on age-related pathologies are discussed.

Gene Switches for Deliberate Regulation of Transgene Expression: Recent Advances in System Development and Uses

It will be desirable in genetic therapies to exert temporal and/or spatial control over the expression of therapeutic genes. Temporal regulation may be achieved by placing the gene of interest under the control of one of several wellknown gene switches. These gene switches comprise at least two elements, i.e., a ligand-dependent transactivator (or transinhibitor) and a transactivator responsive promoter that is functionally linked to the gene of interest. The systems respond to doxycycline, insect steroid hormones, steroid hormone antagonists, rapalogs, or various nonnatural ligands. Deliberate spatial regulation may be attained by the use of promoters that are activated by physical forces which can be directed to an area in need of therapy. Best known are heat shock promoters and radiationinduced promoters. More complex gene switches are capable of both spatially and temporally regulating genes of interest. The present article reviews improvements and further refinements of the latter gene switches as well as exemplifies uses of these gene switches in research and experimental therapy. It further reports on more recently developed gene switches, including complex systems involving multiple transactivators (or transinhibitors) and/or regulating gene product functionality at the transcriptional, posttranscriptional and/or activity level.

Alkoxy-auxins Are Selective Inhibitors of Auxin Transport Mediated by PIN, ABCB, and AUX1 Transporters

Polar auxin movement is a primary regulator of programmed and plastic plant development. Auxin transport is highly regulated at the cellular level and is mediated by coordinated transport activity of plasma membrane-localized PIN, ABCB, and AUX1/LAX transporters. The activity of these transporters has been extensively analyzed using a combination of pharmacological inhibitors, synthetic auxins, and knock-out mutants in Arabidopsis. However, efforts to analyze auxin-dependent growth in other species that are less tractable to genetic manipulation require more selective inhibitors than are currently available. In this report, we characterize the inhibitory activity of 5-alkoxy derivatives of indole 3-acetic acid and 7-alkoxy derivatives of naphthalene 1-acetic acid, finding that the hexyloxy and benzyloxy derivatives act as potent inhibitors of auxin action in plants. These alkoxy-auxin analogs inhibit polar auxin transport and tropic responses associated with asymmetric auxin distribution in Arabidopsis and maize. The alkoxy-auxin analogs inhibit auxin transport mediated by AUX1, PIN, and ABCB proteins expressed in yeast. However, these analogs did not inhibit or activate SCF(TIR1) auxin signaling and had no effect on the subcellular trafficking of PIN proteins. Together these results indicate that alkoxy-auxins are inactive auxin analogs for auxin signaling, but are recognized by PIN, ABCB, and AUX1 auxin transport proteins. Alkoxy-auxins are powerful new tools for analyses of auxin-dependent development.

Epithelial-Connective Tissue Interactions Induced by Thyroid Hormone Receptor Are Essential for Adult Stem Cell Development in the Xenopus laevis Intestine

In the amphibian intestine during metamorphosis, stem cells appear and generate the adult absorptive epithelium, analogous to the mammalian one, under the control of thyroid hormone (TH). We have previously shown that the adult stem cells originate from differentiated larval epithelial cells in the Xenopus laevis intestine. To clarify whether TH signaling in the epithelium alone is sufficient for inducing the stem cells, we have now performed tissue recombinant culture experiments using transgenic X. laevis tadpoles that express a dominant-positive TH receptor (dpTR) under a control of heat shock promoter. Wild-type (Wt) or dpTR transgenic (Tg) larval epithelium (Ep) was isolated from the tadpole intestine, recombined with homologous or heterologous nonepithelial tissues (non-Ep), and then cultivated in the absence of TH with daily heat shocks to induce transgenic dpTR expression. Adult epithelial progenitor cells expressing sonic hedgehog became detectable on day 5 in both the recombinant intestine of Tg Ep and Tg non-Ep (Tg/Tg) and that of Tg Ep and Wt non-Ep (Tg/Wt). However, in Tg/Wt intestine, they did not express other stem cell markers such as Musashi-1 and never generated the adult epithelium expressing a marker for absorptive epithelial cells. Our results indicate that, while it is unclear why some larval epithelial cells dedifferentiate into adult progenitor/stem cells, TR-mediated gene expression in the surrounding tissues other than the epithelium is required for them to develop into adult stem cells, suggesting the importance of TH-inducible epithelial-connective tissue interactions in establishment of the stem cell niche in the amphibian intestine.

The UNC-4 homeobox protein represses mab-9 expression in DA motor neurons in Caenorhabditis elegans

The T-box transcription factor mab-9 has been shown to be required for the correct fate of the male-specific blast cells B and F, normal posterior hypodermal morphogenesis, and for the correct axon migration of motor neurons that project circumferential commissures to dorsal muscles. In this study, an RNAi screen designed to identify upstream transcriptional regulators of mab-9 showed that silencing of unc-4 (encoding a paired-class homeodomain protein) increases mab-9::gfp expression in the nervous system, specifically in posterior DA motor neurons. Over-expression of unc-4 from a heat-shock promoter has the opposite effect, causing repression of mab-9 in various cells. We find that mab-9 expression in unc-37 mutants is also elevated in DA motor neurons, consistent with known roles for UNC-37 as a co-repressor with UNC-4. These results identify mab-9 as a novel target of the UNC-4/UNC-37 repressor complex in motor neurons, and suggest that mis-expression of mab-9 may contribute to the neuronal wiring defects in unc-4 and unc-37 mutants.

Constitutive MEK1 Activation Rescues Anthrax Lethal Toxin-Induced Vascular Effects In Vivo

Anthrax lethal toxin (LT) increases vascular leakage in a number of mammalian models and in human anthrax disease. Using a zebrafish model, we determined that vascular delivery of LT increased permeability, which was phenocopied by treatment with a selective chemical inhibitor of MEK1 and MEK2 (also known as mitogen-activated protein kinase [MAPK] kinase, MEK, or MKK). Here we investigate further the role of MEK1/phospho-ERK (pERK) in the action of LT. Overexpression of wild-type zebrafish MEK1 at high levels did not induce detrimental effects. However, a constitutively activated version, MEK1(S219D,S223D) (MEK1DD), induced early defects in embryonic development that correlated with increased ERK/MAPK phosphorylation. To bypass these early developmental defects and to provide a genetic tool for examining the action of lethal factor (LF), we generated inducible transgenic zebrafish lines expressing either wild-type or activated MEK1 under the control of a heat shock promoter. Remarkably, induction of MEK1DD transgene expression prior to LT delivery prevented vascular damage, while the wild-type MEK1 line did not. In the presence of both LT and MEK1DD transgene expression, cardiovascular development and function proceeded normally in most embryos. The resistance to microsphere leakage in transgenic animals demonstrated a protective role against LT-induced vascular permeability. A consistent increase in ERK phosphorylation among LT-resistant MEK1DD transgenic animals provided additional confirmation of transgene activation. These findings provide a novel genetic approach to examine mechanism of action of LT in vivo through one of its known targets. This approach may be generally applied to investigate additional pathogen-host interactions and to provide mechanistic insights into host signaling pathways affected by pathogen entry.

FLOUR BEETLES JOIN THE GAL4/UAS CLUB

Being able to control gene expression in defined parts of an organism's body and at distinct developmental stages allows researchers to determine the functional significance of gene expression patterns. Unfortunately, the ability to do this has been limited to a few genetic model organisms. Johannes Schinko and colleagues at the Georg-August-Universität Göttingen recently tackled the task of adding a new species, the red flour beetle (Tribolium castaneum, which, as a beetle, represents the most species-rich metazoan taxon) to the list of model organisms in which spatial and temporal control of gene expression is possible. The team published their work in BMC Developmental Biology.The team focused on adapting existing technology to their animal. The GAL4/UAS system is a widely used technique for controlling gene expression in fruit flies. It is composed of a yeast protein (GAL4) that specifically binds to a sequence of DNA known as the upstream activator sequence (UAS), triggering the expression of any gene inserted immediately behind the UAS. The choice of gene is up to the researcher. By themselves, GAL4 and UAS do nothing in a non-yeast cell, but bring them together in the same cell and together they function as an ‘on’ switch that activates expression of the chosen gene. Researchers then control GAL4 expression so that they can selectively switch their chosen gene on and off in specific locations and at specific times.First, Schinko and his colleagues tried to directly translate all of these Drosophila techniques into beetles. They incorporated GAL4 (under the control of fruit fly promoters) into flour beetle tissues. They then put ‘reporter genes’ under UAS control in the same tissues. Reporter genes encode proteins (e.g. green fluorescent protein) that allow researchers to visualise when and where GAL4 successfully initiates gene expression. But all of this turned out to be a dead end. Under conditions that lead to robust GAL4 activity in flies, the researchers saw no evidence of reporter gene expression in beetles. Fruit-fly-based GAL4/UAS appeared not to work in beetles.The team reasoned that the use of native beetle promoters might solve the problem, so they swapped a fruit fly heat shock promoter, which switches on the insect's heat shock response, for an equivalent beetle promoter. When these beetles were heat shocked, they showed robust reporter gene expression in multiple body areas, indicating that the GAL4/UAS system was functioning. The team then went on to assay how quickly they could induce this reporter gene expression in embryos. They found evidence of reporter gene activity within 2–4 hours of heat shock. This response time is only a fraction of the time needed for embryogenesis (72 h), allowing researchers to use the GAL4/UAS system for fine analyses of gene function in T. castaneum embryos during development. Finally, the team tested a second beetle promoter ‘Tc-hairy’ and found reporter gene expression confined primarily to the central nervous system of the animal. This suggests that GAL4/UAS can also be used to spatially restrict the activity of selected genes in beetles.Overall, the work of Schinko and colleagues lays the methodological foundations for a new era of powerful developmental and genetic experiments in flour beetles. The experiments presented are not flashy, but they are carefully done with an eye on the long-term future. Unfortunately, the importance of (and the amount of work that goes into) this type of methods publication is often under-appreciated. Ironically though, in the long run, these types of understated ‘tool-building’ efforts often end up being the real engines driving innovation in biological research.

Biological monitoring of non‐thermal effects of mobile phone radiation: recent approaches and challenges

This review describes recent developments in analysing the influence of radio-frequency electromagnetic fields (RF-EMFs ) on biological systems by monitoring the cellular stress response as well as overall gene expression. Recent data on the initiation and modulation of the classical cellular stress response by RF-EMFs, comprising expression of heat shock proteins and stimulation of stress-activated protein kinases, are summarised and evaluated. Since isothermic RF-EMF exposure is assumed rather than proven there are clear limitations in using the stress response to describe non-thermal effects of RF-EMFs. In particular, further experiments are needed to characterise better the threshold of the thermal heat shock response and the homogeneity of the cellular response in the whole sample for each biological system used. Before then, it is proposed that the absence of the classical stress response can define isothermal experimental conditions and qualifies other biological effects of RF-EMFs detected under these conditions to be of non-thermal origin. To minimise the probability that by making this assumption valuable insights into the nature of biological effects of RF-EMFs could be lost, proteotoxic non-thermal RF-EMF effects should also be monitored by measuring activities of labile intracellular enzymes and/or levels of their metabolites before the threshold for the heat shock response is reached. In addition, non-thermal induction of the stress response via promoter elements distinct from the heat shock element (HSE) should be analysed using HSE-mutated heat shock promoter reporter constructs. Screening for non-thermal RF-EMF effects in the absence of a classical stress response should be performed by transcriptomics and proteomics. Recent approaches demonstrate that due to their high-throughput characteristics, these methods inherently generate false positive results and require statistical evaluation based on quantitative expression analysis from a sufficient number of independent experiments with identical parameters. In future approaches, positive results must be confirmed by independent quantitative methods and should also be evaluated in vivo to prove possible non-thermal effects of RF-EMFs on living beings. If successful, this strategy should contribute to identification of new underlying molecular mechanisms of interaction between RF-EMFs and living beings distinct from absorption of thermal energy.

Sumoylation of Arabidopsis heat shock factor A2 (HsfA2) modifies its activity during acquired thermotholerance

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulate many cellular processes. In this work we show SUMOylation of the heat shock transcription factor, AtHsfA2, in connection with the plant's response to heat stress and acquired thermotolerance. Using the Yeast two hybrid and the bimolecular fluorescence complementation system, we have found that AtSUMO1 physically interacts with AtHsfA2. Further investigation allowed us to determine that Lys 315 of AtHsfA2 is the main SUMOylation site. Overexpression of AtSUMO1 led to a decrease in AtHsfA2 transcriptional activation of heat shock promoters. We have examined the effect of AtSUMO1 on AtHsfA2 during heat shock treatments. The phenotype of seedlings overexpressing AtSUMO1 resembled the phenotype of AtHsfA2 knock out seedlings, which were more sensitive than wild type seedlings to repeated heat treatment. Furthermore, AtSUMO1 overexpressing seedlings exhibited lower expression levels of small heat shock proteins as compared with wild type seedlings after heat treatment. Based on our findings, we suggest that AtSUMO1 is involved in the regulation of AtHsfA2 in acquired thermotolerance.