Controlled gene expression using acute phase response elements

Previous work in our laboratory involved the creation of an inducible gene expression system based on the promoter of the major acute phase protein in humans, C-reactive protein (CRP). The aims of this project were to further characterise the CRP based expression vector and to modify it for use in pigs. The green fluorescent protein (GFP) was also evaluated as a reporter of transient inducible gene expression. The 30kb fragment containing the human CRP gene was sequenced and analysed for the presence of elements that may be responsible for the low basal levels of expression of the gene and for sequences that are responsible for the sexually dimorphic pattern of expression of the human CRP gene in transgenic mice. It was planned to use the information from these analyses to modify the expression vector for use in pigs. However, because it is not currently known how the CRP- based acute phase expression vector would behave in other species, the promoter of the major acute phase protein in pigs, ITIH4, was isolated. Due to the absence of homology between the pig ITIH4 promoter and the human CRP promoter, the pig ITIH4 promoter was further characterised. The investigations focused on the inducibility of the promoter in response to cytokine stimulation and the effect modification of the promoter would have on promoter activity and inducibility. It was found that the promoter was induced by IL-6 but not IL-1 (except at low concentrations). A combination of IL-1 and IL-6 was shown to result in a decrease in the inducibility of the construct by IL-6. Mutation of the promoter in order to decrease the basal level of expression and enhance inducibility was unsuccessful. In order to develop a system that will facilitate studies of inducible gene expression in vitro a destabilised variant of GFP was evaluated as a reporter gene. Although small increases in fluorescence intensity could be detected following stimulation, the analyses suggest that GFP is not as sensitive as other reporter genes for studying inducible gene expression

Isolation and Characterization of the Promoter and Partial Enhancer Region of the Porcine Inter-α-Trypsin Inhibitor Heavy Chain 4 Gene

A porcine genomic library was screened for clones containing the promoter of the major acute-phase protein in pigs, inter-α-trypsin heavy chain 4 (ITIH4). Following isolation of the promoter, a functional analysis was performed with Hep3B cells. The promoter was induced by interleukin-6 (IL-6) but not by IL-1β. However, IL-1β was shown to inhibit the IL-6-induced activation of the porcine ITIH4 promoter

Mapping the Transcription Start Points of the Staphylococcus aureus eap, emp, and vwb Promoters Reveals a Conserved Octanucleotide Sequence That Is Essential for Expression of These Genes▿ †

Mapping the transcription start points of the eap, emp, and vwb promoters revealed a conserved octanucleotide sequence (COS). Deleting this sequence abolished the expression of eap, emp, and vwb. However, electrophoretic mobility shift assays gave no evidence that this sequence was a binding site for SarA or SaeR, known regulators of eap and emp

Advances in in vitro and in vivo models for studying the staphylococcal factors involved in implant infections

Implant infections due to staphylococci are one of the greatest threats facing patients receiving implant devices. For many years researchers have sought to understand the mechanisms involved in the adherence of the bacterium to the implanted device and the formation of the unique structure, the biofilm, which protects the indwelling bacteria from the host defence and renders them resistant to antibiotic treatment. A major goal has been to develop in vitro and in vivo models that adequately reflect the real-life situation. From the simple microtiter plate assay and scanning electron microscopy, tools for studying adherence and biofilm formation have since evolved to include specialised equipment for studying adherence, flow cell systems, real-time analysis of biofilm formation using reporter gene assays both in vitro and in vivo, and a wide variety of animal models. In this article, we discuss advances in the last few years in selected in vitro and in vivo models as well as future developments in the study of adherence and biofilm formation by the staphylococci

Identification of the portions of MAR 1–68 that contribute to the anti-silencing and transcriptional effects.

<p>The AT core extended region of the MAR 1–68, as well as a series of sub-fragments of the 5′ and 3′ flanking regions, were cloned upstream of the EGFP reporter gene in both orientation and analyzed for their effects on GFP expression levels. Constructs containing the full-length MAR 1–68 or a control spacer DNA cloned upstream of the EGFP reporter gene were also transfected as controls. GFP fluorescence was measured by flow-cytometry on polyclonal cell pools obtained after 2 weeks of antibiotic selection following transfection, and the proportion of silent and of high expressor cells were scored as illustrated in Fig. 1B. Results illustrate the mean and standard deviation of 3 independent experiments. Significant differences relative to the corresponding control construct containing spacer DNA of the same size, as illustrated in Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079262#pone.0079262.s003" target="_blank">Fig. S3</a>, are indicated by stars above each bar, whereas line-associated stars indicate significant differences with constructs containing the full length MAR 1–68 or its extended core (Student test, P<0.05).</p

Schematic representation of MAR 1–68 subdomains and illustration of its anti-silencing and transcriptional effects.

<p>(A) Schematic diagram representing the full-length human MAR 1–68 and its series of sub-fragments, cloned upstream of a minimal SV40 promoter and EGFP reporter gene. The 3.6 kb MAR 1–68 was subdivided into three regions: The MAR 1–68 “extended AT core” region encompassing the AT dinucleotide-rich sequence (yellow box, labelled A), its 5′ (blue, labelled B) and 3′ (green, labelled C) adjacent regions. Putative transcription factor binding sites for the SATB1, NMP4, CEBP, Fast and Hox transcription factors are illustrated by ellipses. The 5′ and 3′ flanking regions were further divided in portions comprising nt 1–910 (labelled D), nt 864–1652 (E), nt 2444–3000 (F) and nt 3020–3628 (G). (B) A typical flow cytometry profile of CHO DG44 cells stably co-transfected with the GFP expression vector containing full-length human MAR 1–68 (black line) or control spacer DNA (no MAR, red line) and with a neomycin resistance plasmid. 10⁵ cells were subjected to flow cytometry analysis for GFP expression after 2 weeks of nemomycin selection. Cells displaying background fluorescence (silent cells) or high GFP expression levels are as indicated.</p

Relative contribution of MAR AT-rich cores and flanking sequences to the anti-silencing and transcriptional effects.

<p>The contribution of the AT rich DNA sequences of MAR 1–68 and X-29 alone (A), or combinations of the MAR 1–68 core with portions of its flanking sequences (B), were assessed for their anti-silencing and transcriptional augmentation activities as described in the legend to Fig. 2. An oligomeric form of the X-29 AT-rich region, consisting of three tandem repeats, was also analyzed. Results represent the mean±SD of 3 independent experiments and the statistical analysis are as for Fig. 2.</p

Association of human MARs with a specific chromatin pattern.

<p>A) 1683 predicted human MAR genomic locations were aligned using the central positions of their AT rich cores. ChiP-Seq profiles were calculated over the MAR collection for the histone modifications H3K4me3, H3K27me3, H3K36me3 and for RNA Polymerase II. (B) 25000 RefSeq promoters were aligned at their respective TSS positions and oriented according to the direction of transcription. ChiP-Seq profiles were calculated over the promoter collection for indicated histone modification, and for the RNA Pol II. Tag counts were normalized globally and they are expressed as a fold change over the non-precipitated input DNA profile.</p

The adhesive and immunomodulating properties of the multifunctional Staphylococcus aureus protein Eap

Adherence of Staphylococcus aureus to the host tissue is an important step in the initiation of pathogenesis. At least 10 adhesins produced by S. aureus have been described and it is becoming clear that the expression of these adhesins and their interactions with eukaryotic cells involve complex processes. Some of these, such as the fibronectin-binding proteins (FnBPs) and Clumping Factor A, are well characterized. However, in the last 10 years a number of novel S. aureus adhesins have been described. Functional analyses of these proteins, one of which is Eap (extracellular adherence protein, also known as Map and p70), are revealing important information on the pathogenesis of staphylococcal disease. More than 10 years after the first report of Eap, we are beginning to understand that this protein, which has a broad spectrum of functions, may be a critical factor in the pathogenesis of S. aureus. This review will focus on the interactions of Eap with eukaryotic cells, plasma proteins and the extracellular matrix as well as on the recently recognized role of Eap as an important mediator in the immune response to staphylococcal infection