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Comparative Genomics of the Dictyostelids
Time of Update: 2021-02-18
The complete genomes ofDictyostelium discoideum ,Dictyostelium purpureum ,Polysphondylium pallidum andDictyostelium fasciculatum have been sequenced.
The proteins predicted to be encoded by the genes in each species have been compared to each other as well as to the complete compilation of nonredundant proteins from bacteria, plants, fungi, and animals.
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Antibody Phage Display: Overview of a Powerful Technology that Has Quickly Translated to the Clinic
Time of Update: 2021-02-18
A diverse library of recombinant antibodies is generated in this way and can then be exposed or panned on the antigen of interest, typically, this being a molecule associated with a particular pathological condition.
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Quick and reliable method to analyze meiotic segregation patterns in Coprinus cinereus using the polymerase chain reaction
Time of Update: 2021-02-18
The following components were added to the indicated concentrations after the first denaturation step for PCR-amplification (final volume 100 microliters): 67 mM Tris-HCl (pH 8.8), 16 mM (NH4)2SO4, 10 mM 2-mercaptoethanol, 3 mM MgCl2, 2 glycerol, 1 micromole of each primer (see below), dATP, dCTP, dGTP (each 200 uM), dUTP (500 uM), and 2.5 units of Amplitaq DNA Polymerase (Perkin Elmer Cetus).
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Vesicular Stomatitis Virus Pseudotypes of Retroviruses
Time of Update: 2021-02-18
Pseudotype viruses are phenotypically mixed virions containing the genome or nucleocapsid of one enveloped virus and the surface or envelope ( env ) glycoproteins of another. This chapter will conce
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Methods to Determine Proteolytic Activity of Lactic Acid Bacteria
Time of Update: 2021-02-18
Lactic acid bacteria (LAB) are considered weakly proleolytic when compared with many other groups of bacteria (e.g.,Bacillus, Proteus, Pseudomonas ). However, most strains of LAB rely on a complex p
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Construction of Unmarked Deletion Mutants in Mycobacteria
Time of Update: 2021-02-18
Site-specific recombinases such as theSaccharomyces cerevisiae Flp and the P1 phage Cre proteins have been increasingly used for the construction of unmarked deletions in bacteria.
In this chapter, we describe strategies and methods of how to use sequence-specific recombination mediated by Flp and Cre to construct mutants ofMycobacterium smegmatis ,Mycobacterium bovis BCG, andMycobacterium tuberculosis .
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Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages
Time of Update: 2021-02-18
Two basic methodologies have been used to generate mutant libraries in both fast- and slow-growing mycobacteria: chemical mutagenesis and transposon mutagenesis.
A detailed chemical mutagenesis protocol for the generation of mutant libraries in the fast-growing mycobacteria can be found in the previous volume of this manual ( 5 ).
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Detection of Hepatitis A Virus and Rotavirus Using Nucleic Acid Sequence-Based Amplification
Time of Update: 2021-02-18
Three viruses—noroviruses, rotavirus, and hepatitis A virus (HAV)—are implicated frequently in food-borne illness and have been ranked among the top 10 causes of food-borne disease over the past 10 years.
Molecular approaches based on the amplification of viral RNA have been proposed for the specific and ultrasensitive detection of enteric viruses in foods.
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Molecular Identification of Nematode Worms From Seafood (Anisakis spp. and Pseudoterranova spp.) and Meat (Trichinella spp.)
Time of Update: 2021-02-18
PCR amplification of ITS1 and ITS2 regions, followed by restriction fragment length polymorphism (RFLP), allows for the distinction among species of the generaAnisakis andPseudoterranova .
ForTrichinella worms, a multiplex PCR analysis can be used to distinguish among the eight recognized species and four genotypes ( Trichinella T6 and three populations ofT.
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Phage Replication Technology for Diagnosis and Drug Susceptibility Testing
Time of Update: 2021-02-18
Of the world’s infectious diseases, tuberculosis remains the leading cause of mortality and it has been estimated that of the eight million new cases that occur each year 95% are found in the less developed countries ( 1 , 2 ).
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Two-Step Strategy for Alteration of Immunoglobulin Specificity by In Vitro Mutagenesis
Time of Update: 2021-02-18
A two-step strategy for changing the specificity of antibodies (Abs) is presented, which we have used to change the specificity of an Ab from 11-deoxycortisol (11-DOC) to cortisol (CS). Two kinds of
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Vaccinia Virus as an Expression Vector
Time of Update: 2021-02-18
Vaccinia virus (Vv) is a member of the genus Orthopoxvirus, one of seven genera included in the family Poxviridae.
Most of these viruses infect vertebrates (Orthopoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Parapoxvirus), but one genus, Entomopoxvirus, infects insects.
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Production and Release Testing of Ovine Atadenovirus Vectors
Time of Update: 2021-02-18
New GDEPT vectors based on ovine atadenovirus andEscherichia coli purine nucleoside phosphorylase (PNP) have been developed for first time use in humans in a phase I trial for the treatment of prostate cancer.
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The Use of Peptide Nucleic Acids in Surface Plasmon Resonance for Detection of Red Tide Algae
Time of Update: 2021-02-18
There is a need for low-cost, rapid, and accurate detection of harmful organisms.
We have developed a method for RNA detection ofAlexandrium using a portable surface plasmon resonance biosensing instrument and peptide nucleic acid probes.
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Regulation of Murine Interferon Regulatory Factor Gene Expression in the Central Nervous System Determined by Multiprobe RNase Protection Assay
Time of Update: 2021-02-18
We sought to examine the expression and regulation of the IRF genes in a number of different murine models for immune- or virally induced central nervous system disease.
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Rapid Detection of Atypical Respiratory Bacterial Pathogens by Real-Time PCR
Time of Update: 2021-02-18
Here we describe a multiplex real-timePCR assay that has been validated for the detection of the rarely identified atypical bacterial pathogens:Legionella pneumophila, Mycoplasma pneumoniae , andChlamydophila pneumoniae .
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An Expression Vector for Multiple Ribozymes
Time of Update: 2021-02-18
Ribozymes are recognized as useful tools for the manipulation of genes because of their high specificity and the fact that they act without influencing the expression of genes that are unrelated targets ( 1 , 2 ).
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Viral Glycoprotein-Mediated Cell Fusion Assays Using Vaccinia Virus Vectors
Time of Update: 2021-02-18
This chapter will detail the methods of the vaccinia virus-based reporter-gene fusion assay and how it may be used to characterize the fusion mediated by the BSL-4-classified Hendra and Nipah viruses.
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Study of Adenovirus and CAR Axonal Transport in Primary Neurons
Time of Update: 2021-02-18
Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals.
Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.
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Derivation of Murine Dendritic Cells from Thymic Precursors
Time of Update: 2021-02-18
A combination of density centrifugation and immunomagnetic bead depletion successfully removes mature and immature thymocytes and non-T-lineage cells.
Note, it is important to deplete non-T-lineage cells, including erythrocytes, macrophages, and DC, which may otherwise contaminate the precursor preparation.
