Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium ...falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.
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•mCherry tagged MsmUdgX (mChUdgX) has been developed as a specific probe to detect uracil sites in DNA.•mChUdgX overexpressing E. coli cell-lysate is as good in uracil detection as the pure protein.•Gel retardation assays, confocal microscopy and other assays can be performed using mChUdgX to detect uracil sites in DNA.•The techniques are robust, highly specific and reliable for uracil detection in DNA.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally ...modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking.
•We have generated biologically active tagged WNT1, PORCN, and WLS variants.•WNT1-Fc is useful for click chemistry based palmitoylation assays.•Co-expression of PORCN does not alter the subcellular localization of WNT1.•Co-expression of WLS causes a dramatic redistribution of WNT1.•We have directly visualized WNT1 on the cell surface and in filopodia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Fluorescent proteins are widely used molecular reporters in studying gene expression and subcellular protein localization. To enable the monitoring of transient cellular events in the model yeast ...Saccharomyces cerevisiae, destabilized green and cyan fluorescent proteins have been constructed. However, their co-utilization is limited by an overlap in their excitation and emission spectra. Although red fluorescent protein is compatible with both green and cyan fluorescent proteins with respect to spectra resolution, a destabilized red fluorescent protein is yet to be constructed for applications in S. cerevisiae. To realize this, we adopted a degron-fusion strategy to prompt destabilization of red fluorescent protein. Specifically, we fused two degrons derived from Cln2, a G1-specific cyclin that mediates cell cycle transition, to the N- or C-terminus of mCherry to generate four destabilized fluorescent proteins that are soluble and functional in S. cerevisiae. Importantly, the four mCherry fluorescent proteins are highly differential with regards to fluorescence half-life and intensity, which provides a greater choice of tools available for the study of dynamic gene expression and transient cellular processes in the model yeast.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
has emerged as the most virulent species in the
complex, accounting for sporotrichosis. Albeit the new insights into the understanding of host-pathogen interactions and comparative genomics of this ...fungi, the lack of genetic tools has hindered significant advances in this field of research. Here, we established an Agrobacterium tumefaciens
mediated transformation (ATMT) system to transform different strains of
. We report parameters that account for a transformation efficiency of 3,179 ± 1,171 transformants/co-cultivation, which include the use of A. tumefaciens AGL-1 in a 2:1 ratio (bacteria:fungi) during 72 h at 26°C. Our data show that a single-copy transgene is transferred to
that is mitotically stable in 99% of cells after 10 generations without selective pressure. In addition, we created a plasmid toolkit that allows the establishment of fusion proteins of any
gene of interest with sGFP or mCherry under the control of the GAPDH or H2A endogenous promoters. These modules allow different levels of expression of the desired fusion. Moreover, we successfully targeted these fluorescent proteins to the nucleus and used fluorescence-tagged strains to assess phagocytosis. Overall, our data show that the ATMT system is an easy-to-use and efficient genetic toolbox for studies on recombinant expression and gene function in
.
Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has recently become a public health concern. Although immunocompetent hosts are also prone to sporotrichosis, immunodeficient hosts often develop a more severe and disseminated form of disease. To date, the Rio de Janeiro state in Brazil is the most significant feline zoonotic transmission epicenter in the world, with more than 4,000 human and feline diagnosed cases. Cats play an essential role in the
infection due to their high susceptibility and transmissibility to other felines and humans.
is the most virulent etiological agent of sporotrichosis, causing the most severe clinical manifestations. Despite the increasing incidence of sporotrichosis, the identification of virulence traits important for disease establishment, development, and severity has been lacking. In this work, we established an efficient genetic toolbox to manipulate
that will guide future studies to define new virulence mechanisms and a better understanding of host-pathogen interactions from a molecular perspective.
Mitophagy is a lysosomal degradation pathway that selectively removes damaged, aged and dysfunctional mitochondria. Recent advances in understanding mitophagy highlight its importance in various ...physiological and pathological conditions including liver diseases. However, reliable quantitative assays to monitor mitophagy in cultured cells and in tissues are still scarce.
We describe a detailed protocol for monitoring mitophagy in primary cultured hepatocytes and mouse livers using cytochrome C oxidase subunit 8 (Cox8)-enhanced green fluorescent protein (EGFP)-mCherry, a dual color fluorescence based-imaging method.
Mitochondria are visualized in yellow fluorescence due to the merged EGFP and mCherry signal. In contrast, autolysosome enclosed mitochondria are shown as red puncta due to quenching of EGFP green fluorescence in acidic compartments. Quantifying the number of red-only puncta in each cell can obtain a quantitative measure for mitophagy.
Cox8-EGFP-mCherry assay can specifically target to mitochondria and be used to monitor mitophagy in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic
and
studies. During the last years, a few plasmid vectors have been developed aimed ...at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species
, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter P
or P
from the pneumococcal
gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying P
also harbor the
gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.
Neurons emit axons, which form synapses, the fundamental unit of the nervous system. Neuroscientists use genetic anterograde tracing methods to label the synaptic output of specific neuronal ...subpopulations, but the resulting data sets are too large for manual analysis, and current automated methods have significant limitations in cost and quality. In this paper, we describe a pipeline optimized to identify anterogradely labeled presynaptic boutons in brain tissue sections. Our histologic pipeline labels boutons with high sensitivity and low background. To automatically detect labeled boutons in slide-scanned tissue sections, we developed BoutonNet. This detector uses a two-step approach: an intensity-based method proposes possible boutons, which are checked by a neural network-based confirmation step. BoutonNet was compared to expert annotation on a separate validation data set and achieved a result within human inter-rater variance. This open-source technique will allow quantitative analysis of the fundamental unit of the brain on a whole-brain scale.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
59.
Fluorescent reporters for Staphylococcus aureus Malone, Cheryl L.; Boles, Blaise R.; Lauderdale, Katherine J. ...
Journal of microbiological methods,
06/2009, Volume:
77, Issue:
3
Journal Article
Peer reviewed
Open access
With the emergence of
Staphylococcus aureus as a prominent pathogen in community and healthcare settings, there is a growing need for effective reporter tools to facilitate physiology and ...pathogenesis studies. Fluorescent proteins are ideal as reporters for their convenience in monitoring gene expression, performing host interaction studies, and monitoring biofilm growth. We have developed a suite of fluorescent reporter plasmids for labeling
S. aureus cells. These plasmids encode either green fluorescent protein (GFP) or higher wavelength reporter variants for yellow (YFP) and red (mCherry) labeling. The reporters were placed under control of characterized promoters to enable constitutive or inducible expression. Additionally, plasmids were assembled with fluorescent reporters under control of the
agr quorum-sensing and sigma factor B promoters, and the fluorescent response with wildtype and relevant mutant strains was characterized. Interestingly, reporter expression displayed a strong dependence on ribosome binding site (RBS) sequence, with the superoxide dismutase RBS displaying the strongest expression kinetics of the sequences examined. To test the robustness of the reporter plasmids, cell imaging was performed with fluorescence microscopy and cell populations were separated using florescence-activated cell sorting (FACS), demonstrating the possibilities of simultaneous monitoring of multiple
S. aureus properties. Finally, a constitutive YFP reporter displayed stable, robust labeling of biofilm growth in a flow-cell apparatus. This toolbox of fluorescent reporter plasmids will facilitate cell labeling for a variety of different experimental applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
GFP-binding protein (or GBP) has been recently developed in various systems and organisms as an efficient tool to purify GFP-fusion proteins. Due to the high affinity between GBP and GFP or GFP ...variants, this GBP-based approach is also ideally suited to alter the localization of functional proteins in live cells. In order to facilitate the wide use of the GBP-targeting approach in the fission yeast
, we developed a set of pFA6a-, pJK148- and pUC119-based vectors containing GBP- or GBP-mCherry-coding sequences and variants of inducible
or constitutive
promoters that result in different levels of expression. The GBP or GBP-mCherry fragments can serve as cassettes for N- or C-terminal genomic tagging of genes of interest. We illustrated the application of these vectors in the construction of yeast strains with Dma1 or Cdc7 tagged with GBP-mCherry and efficient targeting of Dma1- or Cdc7-GBP-mCherry to the spindle pole body by Sid4-GFP. This series of vectors should help to facilitate the application of the GBP-targeting approach in manipulating protein localization and the analysis of gene function in fission yeast, at the level of single genes, as well as at a systematic scale.
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