Springer protocols feed by Genetics/Genomics 
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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.Immunocytochemical Fluorescent In Situ Visualization of Proteins In Arabidopsis
The understanding of cellular and subcellular functions often relies on the ability to visualize proteins as close as possible to their endogenous locations. A number of immunocytochemical techniques have been developed to detect proteins in situ using specific antibodies raised against proteins of interest. Here, we describe in detail two protocols commonly, successfully employed in Arabidopsis research. The first allows for immunolocalization of proteins in whole-mount Arabidopsis roots without the need for physical sectioning. The second allows for immunolocalization of proteins on semi-thin microtome sections of wax-em...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Fluorescence Microscopy
Optical microscopy has developed as an indispensable tool for Arabidopsis cell biology. This is due to the high sensitivity, good spatial resolution, minimal invasiveness, and availability of autofluorescent proteins, which can be specifically fused to a distinct protein of interest. In this chapter, we introduce the theoretical concepts of fluorescence emission necessary to accomplish quantitative and functional cell biology using optical microscopy. The main focus lies on spectroscopic techniques, which, in addition to intensity-based studies, provide functional insight into cellular processes. (Source: Springer protocol...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Highly Reproducible ChIP-on-Chip Analysis to Identify Genome-Wide Protein Binding and Chromatin Status in Arabidopsis thaliana
Gene activity is regulated via chromatin dynamics in eukaryotes. In plants, alterations of histone modifications are correlated with gene regulation for development, vernalization, and abiotic stress responses. Using ChIP, ChIP-on-chip, and ChIP-seq analyses, the direct binding regions of transcription factors and alterations of histone modifications can be identified on a genome-wide level. We have established reliable and reproducible ChIP and ChIP-on-chip methods that have been optimized for the Arabidopsis model system. These methods are not only useful for identifying the direct binding of transcription factors and ch...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Forward Chemical Genetic Screening
Chemical genetics utilizes small molecules to perturb biological processes. Unlike conventional genetics methods, which involve the alteration of genetic information mostly with lasting effects, chemical genetics allows temporary and reversible alterations of biological processes. Furthermore, it enables the alteration of biological processes in a dose-dependent manner, providing an advantage over conventional genetics. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Gene Expression Profiling Using DNA Microarrays
We describe the steps, from RNA purification to data analysis, that are involved in obtaining data from DNA microarrays. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
A Pipeline for 15N Metabolic Labeling and Phosphoproteome Analysis in Arabidopsis thaliana
Within the past two decades, the biological application of mass spectrometric technology has seen great advances in terms of innovations in hardware, software, and reagents. Concurrently, the burgeoning field of proteomics has followed closely (Yates et al., Annu Rev Biomed Eng 11:49–79, 2009)—and with it, importantly, the ability to globally assay altered levels of posttranslational modifications in response to a variety of stimuli. Though many posttranslational modifications have been described, a major focus of these efforts has been protein-level phosphorylation of serine, threonine, and tyrosine residues (...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Chemical Fingerprinting of Arabidopsis Using Fourier Transform Infrared (FT-IR) Spectroscopic Approaches
Fourier transform infrared (FT-IR) spectroscopy is a fast, sensitive, inexpensive, and nondestructive technique for chemical profiling of plant materials. In this chapter we discuss the instrumental setup, the basic principles of analysis, and the possibilities for and limitations of obtaining qualitative and semiquantitative information by FT-IR spectroscopy. We provide detailed protocols for four fully customizable techniques: (1) Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS): a sensitive and high-throughput technique for powders; (2) attenuated total reflectance (ATR) spectroscopy: a technique tha...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Next-Generation Mapping of Genetic Mutations Using Bulk Population Sequencing
Next-generation sequencing platforms have made it possible to very rapidly map genetic mutations in Arabidopsis using whole-genome resequencing against pooled members of an F2 mapping population. In the case of recessive mutations, all individuals expressing the phenotype will be homozygous for the mutant genome at the locus responsible for the phenotype, while all other loci segregate roughly equally for both parental lines due to recombination. Importantly, genomic regions flanking the recessive mutation will be in linkage disequilibrium and therefore also be homozygous due to genetic hitchhiking. This information can be...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
iTILLING: Personalized Mutation Screening
One powerful approach to studying gene function is to analyze the phenotype of an organism carrying a mutant allele of a gene of interest. In order to use this experimental approach, one must have the ability to easily isolate individual organisms carrying desired mutations. A widely used method for accomplishing this task in plants and other organisms is a procedure called TILLING. A traditional TILLING project has at its foundation an ordered mutant population produced by treating seeds with a chemical mutagen. From this mutagenized seed, thousands of individual mutant lines are produced, and corresponding DNA samples ar...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Agrobacterium tumefaciens-Mediated Transient Transformation of Arabidopsis thaliana Leaves
Transient assays provide a convenient alternative to stable transformation. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Nevertheless, at difference of tobacco and other plant species, Arabidopsis thaliana remains recalcitrant to routine transient assays. In this chapter, we describe a transient expression assay using simple infiltration of intact Arabidopsis leaves with Agrobacterium tumefaciens carrying a plasmid expressing a reporter fluorescent protein. In this protocol, Agrobacterium aggressiveness was increased by a ...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Grafting in Arabidopsis
Grafting provides a simple way to generate chimeric plants with regions of different genotypes and thus to assess the cell autonomy of gene action. The technique of grafting has been widely used in other species, but in Arabidopsis, its small size makes the process rather more demanding. However, there are now several well-established grafting procedures available, which we described here, and their use has already contributed greatly to understanding of such processes as shoot branching control, flowering, disease resistance, and systemic silencing. (Source: Springer protocols feed by Genetics/Genomics)
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Exploiting Natural Variation in Arabidopsis
Natural variation for many traits is present within the species Arabidopsis thaliana. This chapter describes the use of natural variation to elucidate genes underlying the regulation of quantitative traits. It deals with the development and use of mapping populations, the detection and handling of genetic markers, the phenotyping of quantitative traits, and, finally, QTL analyses. The focus of the chapter is on the use and development of recombinant inbred lines, but other types of segregating populations, including genome-wide association mapping in natural populations, are also discussed. (Source: Springer protocols feed...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Global DNA Methylation Analysis Using Methyl-Sensitive Amplification Polymorphism (MSAP)
DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylati...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Arabidopsis Transformation with Large Bacterial Artificial Chromosomes
The study of a gene’s function requires, in many cases, the ability to reintroduce the gene of interest or its modified version back into the organism of choice. One potential caveat of this approach is that not only the coding region but also the regulatory sequences of a gene should be included in the corresponding transgenic construct. Even in species with well-annotated genomes, such as Arabidopsis, it is nearly impossible to predict which sequences are responsible for the proper expression of a gene. One way to circumvent this problem is to utilize a large fragment of genomic DNA that contains the coding region ...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
Identification of EMS-Induced Causal Mutations in Arabidopsis thaliana by Next-Generation Sequencing
Emerging next-generation sequencing (NGS) technologies are powerful tools for the identification of causal mutations underlying phenotypes of interest in Arabidopsis thaliana. Based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing a mutant to a different polymorphic accession and are analyzed for single nucleotide polymorphisms (SNPs). Then, a genome region spanning the causal mutation site is narrowed down by linkage analysis of SNPs in the accessions used to produce the F1 generation. Next, candidate SNPs for the causative mutation ...
Source: Springer protocols feed by Genetics/Genomics - September 26, 2013 Category: Genetics & Stem Cells Source Type: news
