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Past Issue in 2017
Volume: 7, Issue: 19
Biochemistry
Protein Expression and Purification of the Hsp90-Cdc37-Cdk4 Kinase Complex from Saccharomyces cerevisiae
Interactions between Hsp90, its co-chaperone Cdc37 and kinases have been biochemically studied for over three decades and have been shown to be functionally important in organisms from yeast to humans. However, formation of a stable complex for structural studies has been elusive. In this protocol we describe expression and purification of Hsp90-Cdc37-Cdk4 kinase protein complex from Saccharomyces cerevisiae utilizing the viral 2A sequences to titrate the three proteins at similar levels.
Cell Biology
DQ-Red BSA Trafficking Assay in Cultured Cells to Assess Cargo Delivery to Lysosomes
Lysosomes are the terminal end of the endocytic pathway having acidic environment required for active hydrolases that degrade the cargo delivered to these compartments. This process of cargo delivery and degradation by endo-lysosomes is a tightly regulated process and important for maintaining cellular homeostasis. Cargos like EGF (Epidermal Growth Factor), Dil-LDL (3,3’-Dioctadecylindocarbocyanine-Low Density Lipoprotein), Dextran, DQ-BSA (Dye Quenched-Bovine Serum Albumin) etc., are routinely used by researchers to analyze the role of various proteins in endocytic pathway. Trafficking of DQ-BSA in cells depleted of or over-expressing the gene of interest is a useful assay for identifying the role of various proteins in endocytic trafficking pathway. The protocol describes the DQ-Red BSA trafficking assay that can be used to study endocytic trafficking in various cell types.
Scanning Electron Microscopy of Motile Male Gametes of Land Plants
The only motile cells produced in land plants are male gametes (spermatozoids), which are reduced to non-flagellated cells in flowering plants and most gymnosperms. Although a coiled architecture is universal, the complexity of land plant flagellated cells varies from biflagellated in bryophytes to thousands of flagella per gametes in the seed plants Ginkgo and cycads. This wide diversity in number of flagella is associated with vast differences in cell size and shape. Scanning electron microscopy (SEM) has played an important role in characterizing the external form, including cell shape and arrangement of flagella, across the varied motile gametes of land plants. Because of the size and scarcity of released swimming sperm, it is difficult to concentrate them and prepare them for observation in the SEM. Here we detail an SEM preparation technique that yields good preservation of sperms cells across plant groups.
Microbiology
Quantification of Densities of Bacterial Endosymbionts of Insects by Real-time PCR
Increased attention has been paid to the endosymbiotic bacteria of insects. Because most insect endosymbionts are uncultivable, quantitative PCR (qPCR) is a practical and convenient method to quantify endosymbiont titers. Here we report a protocol for real-time qPCR based on SYBR Green I fluorescence as well as some tips to prevent possible pitfalls.
A Flow-assay for Farnesol Removal from Adherent Candida albicans Cultures
Here, we describe a protocol for a continuous flow system for C. albicans cultures growing adherent to a plastic surface. The protocol was adapted from a previous method established to simulate blood flow on endothelial cells (Wilson and Hube, 2010). The adapted protocol was used by us for the removal of molecules in C. albicans supernatants, especially farnesol, which accumulate over the time course of incubation and cannot be specifically depleted. The system used, however, allows various applications including the simulation of physiological flow conditions. Several example applications are given on the manufacturer’s website (https://ibidi.com/perfusion-system/112-ibidi-pump-system.html).
Molecular Biology
Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection
Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny.
Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues.
Protocol for Construction of a Tunable CRISPR Interference (tCRISPRi) Strain for Escherichia coli
We present a protocol for construction of tunable CRISPR interference (tCRISPRi) strains for Escherichia coli. The tCRISPRi system alleviates most of the known problems of plasmid-based expression methods, and can be immediately used to construct libraries of sgRNAs that can complement the Keio collection by targeting both essential and nonessential genes. Most importantly from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step oligo recombineering. Additional advantages of tCRISPRi over other existing CRISPRi methods include: (1) tCRISPRi shows significantly less than 10% leaky repression; (2) tCRISPRi uses a tunable arabinose operon promoter and modifications in transporter genes to allow a wide dynamic range with graded control by arabinose inducer; (3) tCRISPRi is plasmid free and the entire system is integrated into the chromosome; (4) tCRISPRi strains show desirable physiological properties.
Neuroscience
Preparation of Teased Nerve Fibers from Rat Sciatic Nerve
Compared to tissue sectioning techniques, the technique of teasing single nerve fibers provides a better way to understand the structures of myelin sheaths and axons of the peripheral myelinated nerves. This protocol describes a method for preparation of teased single nerve fibers from rat sciatic nerve. In this protocol, fixed nerves are teased into single individual fibers and arranged onto adhesion microscope slides for further immuno-staining.
Plant Science
Xanthomonas oryzae pv. oryzae Inoculation and Growth Rate on Rice by Leaf Clipping Method
Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious bacterial diseases and a major impediment to the increase of rice yield. Appropriate methods for inoculation of Xoo and disease scoring are necessary to investigate the nature of the disease and the mechanism of plant resistance to the pathogen. As the most-widely grown crop in the worldwide, rice yield plays an important role in food security. Uncovering mechanisms of plant-pathogen interaction of rice and Xoo will help develop rice plants that are more resistant to disease caused by Xoo. Here we describe our validated and efficient methods for inoculation of Xoo and disease scoring.
Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells
Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of crop species. In this protocol, we adapted a previously published account that describes the use of Arabidopsis thaliana roots to investigate the interaction of A. thaliana and Agrobacterium tumefaciens. In our laboratory, we routinely use A. thaliana root explants to examine the factors that enhance the utility of EMT. In addition, the E-ART protocol can be used to study the transcriptional response of E. adhaerens and host plant following exposure to explant tissue, the transformability of different Ensifer adhaerens strains/mutants as well as testing the susceptibility of A. thaliana mutant lines as a means to decipher the mechanisms underpinning EMT.
Large-scale Maize Seedling Infection with Exserohilum turcicum in the Greenhouse
Northern corn leaf blight (NCLB) is a serious foliar disease of maize (Zea mays) worldwide and breeding for resistance is of primary importance for maize crop protection. Phenotyping for NCLB resistance is well established in the field, but such experiments depend on suitable environmental conditions and are seasonal. Here we describe a greenhouse seedling approach that is suitable for testing thousands of seedling plants in a single experiment with a duration of 37 days. Three scoring methods were used to quantify the disease severity: the area under the disease progress curve (AUDPC), the primary diseased leaf area of the inoculated leaves at 16 days post inoculation (PrimDLA at 16 dpi) and the incubation period (IP) that was determined as days from inoculation to symptom appearance. By testing a diverse panel of maize genotypes, a high correlation between the three different methods was observed (81.9% to 94.1%), indicating that each of scoring methods can be applied for disease quantification. Thus, the seedling assay developed served as a relatively simple and high-throughput method for phenotyping NCLB disease resistance under greenhouse condition.
Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants
Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is known about the diversity of plant male gametes was derived from detailed transmission electron microscopic studies. Because the process of spermatogenesis results in complete transformation of the shape and organization of these cells, TEM studies have yielded a wealth of information on cellular differentiation. Because green algal progenitor groups contain centrioles and a variety of motile cells, land plant spermatozoids also provide a plethora of opportunities to examine the evolution and eventual loss of centrioles and locomotory apparatus during land colonization. Here we provide a brief overview of the studies and methodologies we have conducted over the past 20 years that have elucidated not only the structural diversity of these cells but also the development of microtubule organizing centers, the de novo origin of centrioles and the ontogeny of structurally complex motile cells.
Scanning Electron Microscope (SEM) Imaging to Determine Inflorescence Initiation and Development in Olive
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Here we present a protocol that describes how to image the structure of the olive axillary bud meristem with a scanning electron microscope (SEM) in order to characterize its identity and developmental stage. Briefly, the specimen is fixed with glutaraldehyde, saturated with ethanol, dried in a critical point dryer (CPD) system, dissected, coated with a conducting material and imaged with a scanning electron microscopy (SEM).
Analysis of Xyloglucan Composition in Arabidopsis Leaves
Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks unsubstituted glucose residues in the backbone. The identities and relative amounts of released xyloglucan fragments are then determined using MALDI-TOF mass spectrometry.
