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丁香实验推荐阅读
Construction of Phage Antibody Repertoires from the Blood of West Nile Virus-Infected Donors

A method for the construction of West Nile virus immune donor antibody repertoires is described. B cells are harvested from a suitable donor and the antibody variable genes are amplified using polymerase chain reaction (PCR). The PCR fragments are cloned in a phage display vector to construct a r ...

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Affinity Isolation of Antigen-Specific Circulating B Cells for Generation of Phage Display-Derived Human Monoclonal Antibodies

A method is described for affinity isolation of antigen-specific circulating B cells of interest for subsequent generation of immune antibody phage display libraries. This approach should overcome the problem of low yields of monoclonal antibodies of interest in the libraries gene ...

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Studying Adaptation and Homeostatic Behaviors of Kinetic Networks by Using MATLAB

Organisms have the ability to counteract environmental perturbations and keep certain components within a cell homeostatically regulated. Closely related to homeostasis is the behavior of perfect adaptation where an organism responds to a step-wise perturbation by regulati ...

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Stochastic Analysis of Gene Expression

In this chapter, stochasticity in gene expression is investigated using -expansion technique. Two theoretical models are considered here, one concern the stochastic fluctuations in a single-gene network with negative feedback regulation, and the other the additivity of noise pr ...

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Clustering Change Patterns Using Fourier Transformation with Time-Course Gene Expression Data

To understand the behavior of genes, it is important to explore how the patterns of gene expression change over a period of time because biologically related gene groups can share the same change patterns. In this study, the problem of finding similar change patterns is induced to clustering with t ...

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Measuring In Vivo Signaling Kinetics in a Mitogen-Activated Kinase Pathway Using Dynamic Input Stimulation

Determining the in vivo kinetics of a signaling pathway is a challenging task. We can measure a property we termed pathway bandwidth to put in vivo bounds on the kinetics of the mitogen-activated protein kinase (MAPk) signaling cascade in Saccharomyces cerevisiae that responds to hyperosm ...

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Linearizer Gene Circuits with Negative Feedback Regulation

Gene functional studies consist of phenotyping cells with altered gene expression. Improving the precision of current gene expression control techniques would enable more detailed studies of gene function. Here, we provide protocols for building synthetic gene constructs for t ...

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Luminescence as a Continuous Real-Time Reporter of Promoter Activity in Yeast Undergoing Respiratory Oscillations or Cell Division Rhythms

This chapter describes a method for generating yeast respiratory oscillations in continuous culture and monitoring rhythmic promoter activity of the culture by automated real-time recording of luminescence. These techniques chiefly require the use of a strain of Saccharomyces ...

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Construction of cis-Regulatory Input Functions of Yeast Promoters

Promoters contain a large number of binding sites for transcriptional factors transmitting signals from a variety of cellular pathways. The promoter processes these input signals and sets the level of gene expression, the output of the gene. Here, we describe how to design genetic construc ...

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Genomic-Wide Methods to Evaluate Transcription Rates in Yeast

Gene transcription is a dynamic process in which the desired amount of an mRNA is obtained by the equilibrium between its transcription (TR) and degradation (DR) rates. The control mechanism at the RNA polymerase level primarily causes changes in TR. Despite their importance, TRs have been rar ...

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Global Estimation of mRNA Stability in Yeast

Turnover of mRNA is an important level of gene regulation. Individual mRNAs have different intrinsic stabilities. Moreover, mRNA stability changes dynamically with conditions such as hormonal stimulation or cellular stress. While accurate methods exist to measure the half-life ...

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Biochemical Systems Analysis of Signaling Pathways to Understand Fungal Pathogenicity

Over the past decade, researchers have recognized the need to study biological systems as integrated systems. While the reductionist approaches of the past century have made remarkable advances of our understanding of life, the next phase of understanding comes from systems-level inv ...

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Functional Mapping of Expression Quantitative Trait Loci that Regulate Oscillatory Gene Expression

Genetic networks underlying many biological processes, such as vertebrate somitogenesis, cell cycle, hormonal signaling, and circadian rhythms, are characterized by oscillations in gene expression. It has been recognized that the frequency and amplitude of gene expression os ...

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Finding Modulators of Stochasticity Levels by Quantitative Genetics

Although bakers and wine makers constantly select, compare, and hunt for new wild strains of Saccharomyces cerevisiae, yeast geneticists have long focused on a few “standard” strains to ensure reproducibility and easiness of experimentation. And so far, the wonderful natural resource ...

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Evolutionary Aspects of a Genetic Network: Studying the Lactose/Galactose Regulon of Kluyveromyces lactis

The budding yeast Kluyveromyces lactis has diverged from the Saccharomyces lineage before the whole-genome duplication and its genome sequence reveals lower redundancy of many genes. Moreover, it shows lower preference for fermentative carbon metabolism and a broader substra ...

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Analysis of Subtelomeric Silencing in Candida glabrata

Analysis of gene function often involves detailed studies of when a given gene is expressed or silenced. Transposon mutagenesis is a powerful tool to generate insertional mutations that provide with a selectable marker and a reporter gene that can be used to analyze the transcriptional act ...

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Morphological and Molecular Genetic Analysis of Epigenetic Switching of the Human Fungal Pathogen Candida albicans

Candida albicans is a pleiomorphic fungal pathogen whose morphogenetic plasticity has long been considered as a major virulence factor. In addition to the yeast-filament transition, C. albicans cells also have the unique ability to switch between two epigenetic phases referred to as wh ...

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Quantitation of Cellular Components in Cryptococcus neoformans for System Biology Analysis

Methods and procedures in molecular biology used to study fungal pathogenesis have significantly improved during the last decade. In this chapter, we provide step-by-step procedures for performing genetics and biochemical studies in the human pathogenic fungal microorganism C ...

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Isolation of H. pylori Genomic DNA and Restriction Analysis

Since Helicobacter pylori was first described in 1983 (1), the study of genomic DNA has been central to the development of its microbiology and molecular genetics. For instance, DNA base composition estimation (mol% G+C) was crucial in demonstrating affinities of the microorganism to the ge ...

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Detection of Mutations in Mycobacteria by PCR-SSCP (Single-Strand Conformation Polymorphism)

There are a number of techniques available for the detection and characterization of mutations that take advantage of the flexibility and power of polymerase chain reaction (PCR) (Table 1). Sequencing remains the gold standard, but it is not very efficient as a screening tool, in particular wh ...

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