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In Vivo Analyses of Viral RNA Translation
Time of Update: 2021-01-16
Positive-strand RNA viruses often use noncanonical strategies to usurp the host translational machinery for their own benefit.
Use of recombinant viral constructs containing the reporter luciferase gene allows us to discern whether a particular RNA sequence or secondary structure elicits an effect on initiation of translation or recoding.
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In Vitro Protein Import by Isolated Chloroplasts
Time of Update: 2021-01-16
The majority of chloroplast proteins are nuclear-encoded and synthesized in the cytosol; therefore, there must be some mechanism that allows proteins to cross the chloroplast envelope (the membrane that delineates the organelle) yet also discriminates between the plastid and other organelles, like mitochondria ( 2 ).
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Large-Scale Culture of Plant Cells
Time of Update: 2021-01-16
It was soon found, however, that although plant cell suspensions appear to be similar in many ways to microbial cultures, there are, in fact, key differences that can have a significant influence on large-scale cultivation.
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Probing Interactions Between Plant Virus Movement Proteins and Nucleic Acids
Time of Update: 2021-01-16
MPs are multifunctional proteins, and one of their functions is almost invariably binding to nucleic acids.
We also describe protocols for purification of recombinant viral MPs over-expressed in bacteria and production of different DNA and RNA probes for these binding assays.
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Development of an Efficient Inverse PCR Method for Isolating Gene Tags from T-DNA Insertional Mutants in Rice
Time of Update: 2021-01-16
The central goal of current genomics research in plants, as in other organisms, is to elucidate the functions of every gene.
Development of efficient methods for isolating the genomic sequences flanking insertion elements accelerates the systematic cataloging of insertional mutants, and thus allows functions to be assigned to uncharacterized genes via reverse genetic approaches.
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Simulating Labeling to Estimate Kinetic Parameters for Flux Control Analysis
Time of Update: 2021-01-16
In a top-down approach to model assembly, unknown kinetic parameters are calculated using experimental data such as metabolite pool concentrations and transient labeling patterns after supply of an isotopically labeled substrate.
This chapter describes a modeling approach to estimate kinetic parameters which are then used to perform metabolic control analysis.
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Mathematical Modeling of Isotope Labeling Experiments for Metabolic Flux Analysis
Time of Update: 2021-01-16
Toward this, this chapter provides background information and examples to enable the reader to (1) model metabolic networks, (2) simulate ILEs, and (3) understand the optimization and statistical methods commonly used for flux evaluation.
Furthermore, we briefly discuss methods to improve flux estimates.
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Genetic Transformation of Wheat via Agrobacterium-Mediated DNA Delivery
Time of Update: 2021-01-16
The method described involves an initial incubation of wheat immature embryos in a liquid culture ofAgrobacterium tumefaciens .
Tissue culture of the embryos on plant media with a correct balance of hormones allows embryogenic callus formation followed by regeneration of plantlets, and in the later stages of tissue culture a selectable marker (herbicide) is included to minimize the incidence of non-transformed plants.
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Fertilization In Vitro and Culture of Fertilized Ovules of Higher Plants
Time of Update: 2021-01-16
Sexual reproduction in higher plants consists of a series of complex processes, including pollination, double fertilization, embryo differentiation, and seed formation.
Development of methods for pollination and fertilization under controlled conditions in vitro has greatly facilitated the study of these processes ( 1 ).
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Production of a His-Tagged Canecystatin in Transgenic Sugarcane
Time of Update: 2021-01-16
In this chapter, we describe production of transgenic sugarcane expressing a His-tagged cystatin under the control of the maize ubiquitin promoter.
These studies demonstrate that sugarcane can be a viable expression system for recombinant protein production and that the His-tag purification strategy used to isolate the purified protein was effective.
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Enhancer Trapping in Plants
Time of Update: 2021-01-16
With improvements in plant transformation technologies, T- DNA and/or transposon-based gene and enhancer-tagged populations in various crop species are being developed to augment functional annotation of genes and also to help clone important genes.
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Screening Arabidopsis Genotypes for Drought Stress Resistance
Time of Update: 2021-01-16
A high throughput drought screen is described forArabidopsis that is based on a gravimetric method to monitor and control water content of the soil.
To screen for plant growth under mild drought conditions, 30% of field capacity can be used, which is equal to 2 g H 2 O/g dry soil.
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Electroporation of DNA into the Unicellular Green Alga Chlamydomonas reinhardtii
Time of Update: 2021-01-16
Recently developed techniques for efficient transformation ( 2 – 8 ) and insertional mutagenesis ( 9 ) ofChlamydomonas nuclear and organelle genomes promise to accelerate the genetic analysis of these and other complex cellular processes.
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Selection of Chloroplast Mutants
Time of Update: 2021-01-16
The chloroplast genome encodes a number of proteins, including thylakoid proteins and the large subunit of ribulose biphosphate carboxylase, associated with the structure and function of the chloropl
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Identification of Thioredoxin Targeted Proteins Using Thioredoxin Single Cysteine Mutant-immobilized Resin
Time of Update: 2021-01-16
A single-cysteine mutant capable of forming a stable mixed disulfide bond with target proteins was immobilized on resin and used to capture potential target proteins.
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Agroinoculation and Agroinfiltration: Simple Tools for Complex Gene Function Analyses
Time of Update: 2021-01-16
With the explosive growth of genomic information and the development of advanced vectors to dissect plant gene function, this reliable method of viral gene delivery in plants, has been recruited and morphed into a technique popularly known as agroinfiltration.
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Preparation of Immunogens and Production of Antibodies
Time of Update: 2021-01-16
The quality of reagents greatly affects the interpretation of serological tests.
Methods used in conventional viral purification and molecular cloning and expression of target viral proteins to obtain antigens for immunization are presented.
Immunization of rabbits, mice and chickens and isolation of immunoglobulin from immunized animals also are described.
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Movement Profiles: A Tool for Quantitative Analysis of Cell-to-Cell Movement of Plant Viral Movement Proteins
Time of Update: 2021-01-16
Expressing cells can be directly monitored for subcellular localization and cell-to-cell movement of the MP:GFP fusion protein into neighboring cells by confocal scanning microscopy.
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Chloramphenicol Acetyl Transferase Assay
Time of Update: 2021-01-16
The chloramphenicol acetyltransferase(cat) gene, isolated from transposon Tn9 ofEscherichia coli ( 1 ), is a convenient genetic marker for studies of transformation.
(For a detailed description of the CAT gene,see Chapter 1 .) In plants, the CAT system is generally used in transient, rather than in stable, gene expression studies.
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Separation of Plant Proteins by Electrophoresis
Time of Update: 2021-01-16
The present chapter describes methods for the extraction, electrophoresis, and detection of proteins, and for their transfer to membranes for microsequencing.
These can be used for the direct analysis of expressed proteins, or combined with Western blotting as described in the following chapter (Chapter 35 ).
