Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Generation and Analysis of Chromosomal Contact Maps of Yeast Species
Genome-wide derivatives of the chromosome conformation capture (3C) technique are now well-established approaches to study the multiscale average organization of chromosomes from bacteria to mammals. However, the experimental parameters of the protocol have to be optimized for different species, and the downstream experimental products (i.e., pair-end sequences) are influenced by these parameters. Here, we describe a complete pipeline to generate 3C-seq libraries and compute chromosomal contact maps of yeast species. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Systematic Determination of Transcription Factor DNA-Binding Specificities in Yeast
Understanding how genes are regulated, decoding their “regulome”, is one of the main challenges of the post-genomic era. Here, we describe the in vitro method we used to associate cis-regulatory sites with cognate trans-regulators by characterizing the DNA-binding specificity of the vast majority of yeast transcription factors using Protein Binding Microarrays. This approach can be implemented to any given organism. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
ChIPseq in Yeast Species: From Chromatin Immunoprecipitation to High-Throughput Sequencing and Bioinformatics Data Analyses
We present here a general protocol to perform ChIPseq analyses in yeast species. This protocol has been optimized to identify target genes of specific transcription factors but can be used for any other DNA binding proteins. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Genome-Wide Chromatin Immunoprecipitation in Candida albicans and Other Yeasts
Chromatin immunoprecipitation experiments are critical to investigating the interactions between DNA and a wide range of nuclear proteins within a cell or biological sample. In this chapter we outline an optimized protocol for genome-wide chromatin immunoprecipitation that has been used successfully for several distinct morphological forms of numerous yeast species, and include an optimized method for amplification of chromatin immunoprecipitated DNA samples and hybridization to a high-density oligonucleotide tiling microarray. We also provide detailed suggestions on how to analyze the complex data obtained from these expe...
Source: Springer protocols feed by Microbiology - October 21, 2015 Category: Microbiology Source Type: news
Preparation of Cultured Cells Using High-Pressure Freezing and Freeze Substitution for Subsequent 2D or 3D Visualization in the Transmission Electron Microscope
Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples and it is particularly useful when determining virus–host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast, and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the m...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Studying the Dynamics of Coronavirus Replicative Structures
Coronaviruses (CoVs) generate specialized membrane compartments, which consist of double membrane vesicles connected to convoluted membranes, the so-called replicative structures, where viral RNA synthesis takes place. These sites harbor the CoV replication–transcription complexes (RTCs): multi-protein complexes consisting of 16 nonstructural proteins (nsps), the CoV nucleocapsid protein (N) and presumably host proteins. To successfully establish functional membrane-bound RTCs all of the viral and host constituents need to be correctly spatiotemporally organized during viral infection. Few studies, however, have inve...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Quantification of Interferon Signaling in Avian Cells
Activation of the type I interferon (IFN) response is an essential defense mechanism against invading pathogens such as viruses. This chapter describes two protocols to quantify activation of the chicken IFN response through analysis of gene expression by real-time quantitative PCR and by quantification of bioactive IFN protein using a bioassay. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Transcriptome Analysis of Feline Infectious Peritonitis Virus Infection
Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal’s Z test statistical analysis is used to analyze the dif...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Investigation of the Functional Roles of Host Cell Proteins Involved in Coronavirus Infection Using Highly Specific and Scalable RNA Interference (RNAi) Approach
Since its identification in the 1990s, the RNA interference (RNAi) pathway has proven extremely useful in elucidating the function of proteins in the context of cells and even whole organisms. In particular, this sequence-specific and powerful loss-of-function approach has greatly simplified the study of the role of host cell factors implicated in the life cycle of viruses. Here, we detail the RNAi method we have developed and used to specifically knock down the expression of ezrin, an actin binding protein that was identified by yeast two-hybrid screening to interact with the Severe Acute Respiratory Syndrome Coronavirus ...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
A Field-Proven Yeast Two-Hybrid Protocol Used to Identify Coronavirus–Host Protein–Protein Interactions
Over the last 2 decades, yeast two-hybrid became an invaluable technique to decipher protein–protein interaction networks. In the field of virology, it has proven instrumental to identify virus–host interactions that are involved in viral embezzlement of cellular functions and inhibition of immune mechanisms. Here, we present a yeast two-hybrid protocol that has been used in our laboratory since 2006 to search for cellular partners of more than 300 viral proteins. Our aim was to develop a robust and straightforward pipeline, which minimizes false-positive interactions with a decent coverage of target cDNA libra...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Studying Coronavirus–Host Protein Interactions
To understand the molecular mechanisms of viral replication and pathogenesis, it is necessary to establish the virus–host protein interaction networks. The yeast two-hybrid system is a powerful proteomic approach to study protein–protein interactions. After the identification of specific cellular factors interacting with the target viral protein using the yeast two-hybrid screening system, co-immunoprecipitation and confocal microscopy analyses are often used to verify the virus–host protein interactions in cells. Identification of the cellular factors required for viral survival or eliminating virus infe...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Single Particle Tracking Assay to Study Coronavirus Membrane Fusion
We describe how to carry out single particle tracking of FECV fusion in this SLB platform to obtain fusion kinetics. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Identification of Protein Receptors for Coronaviruses by Mass Spectrometry
As obligate intracellular parasites, viruses need to cross the plasma membrane and deliver their genome inside the cell. This step is initiated by the recognition of receptors present on the host cell surface. Receptors can be major determinants of tropism, host range, and pathogenesis. Identifying virus receptors can give clues to these aspects and can lead to the design of intervention strategies. Interfering with receptor recognition is an attractive antiviral therapy, since it occurs before the viral genome has reached the relative safe haven within the cell. This chapter describes the use of an immunoprecipitation app...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Protein Histochemistry Using Coronaviral Spike Proteins: Studying Binding Profiles and Sialic Acid Requirements for Attachment to Tissues
Protein histochemistry is a tissue-based technique that enables the analysis of viral attachment patterns as well as the identification of specific viral and host determinants involved in the first step in the infection of a host cell by a virus. Applying recombinantly expressed spike proteins of infectious bronchitis virus onto formalin-fixed tissues allows us to profile the binding characteristics of these viral attachment proteins to tissues of various avian species. In particular, sialic acid-mediated tissue binding of spike proteins can be analyzed by pretreating tissues with various neuraminidases or by blocking the ...
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
Engineering Infectious cDNAs of Coronavirus as Bacterial Artificial Chromosomes
The large size of the coronavirus (CoV) genome (around 30 kb) and the instability in bacteria of plasmids carrying CoV replicase sequences represent serious restrictions for the development of CoV infectious clones using reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these problems, several approaches have been established in the last 13 years. Here we describe the engineering of CoV full-length cDNA clones as bacterial artificial chromosomes (BACs), using the Middle East respiratory syndrome CoV (MERS-CoV) as a model. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - February 28, 2015 Category: Microbiology Source Type: news
