The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in p...
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The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy. Plants displaying phenotypes of interest are first isolated by screening an EMS mutant collection generated in the miniature cultivar Micro-Tom. A recombinant F2 population is then produced by crossing the mutant with a wild-type (WT; non-mutagenized) genotype, and F2 segregants displaying the same phenotype are subsequently pooled. Finally, whole-genome sequencing and analysis of allele distributions in the pools allow for the identification of the causal mutation. The whole process, from the isolation of the tomato mutant to the identification of the causal mutation, takes 6–12 months. This strategy overcomes many previous limitations, is simple to use and can be applied in most laboratories with limited facilities for plant culture and genotyping. less
DNA methylation is an important and potentially heritable component of the epigenetic machinery that has a major role in the control of gene expression and can be deregulated in many diseases. This modification of gen...
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DNA methylation is an important and potentially heritable component of the epigenetic machinery that has a major role in the control of gene expression and can be deregulated in many diseases. This modification of genomic DNA can be assessed using the methylated DNA immunoprecipitation (MeDIP) method, based on the quantification of methylated DNA fragments enriched using an antibody specific for methyl-cytosines. The relationship between the enrichment level and the real DNA methylation status is complex, and only a few methods have been developed to evaluate MeDIP enrichment measures to estimate the absolute or relative number of methyl-cytosines in a given sample. Two such methods are MEDME and BATMAN. This chapter focuses on the description and use of the former with a brief discussion of the latter. less
Although the number of sequenced insect genomes numbers in the hundreds, little is known about gene regulatory sequences in any species other than the well-studied Drosophila melanogaster. We provide here a detailed p...
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Although the number of sequenced insect genomes numbers in the hundreds, little is known about gene regulatory sequences in any species other than the well-studied Drosophila melanogaster. We provide here a detailed protocol for using SCRMshaw, a computational method for predicting cis-regulatory modules (CRMs, also “enhancers”) in sequenced insect genomes. SCRMshaw is effective for CRM discovery throughout the range of holometabolous insects and potentially in even more diverged species, with true-positive prediction rates of 75% or better. Minimal requirements for using SCRMshaw are a genome sequence and training data in the form of known Drosophila CRMs; a comprehensive set of the latter can be obtained from the SCRMshaw download site. For basic applications, a user with only modest computational know-how can run SCRMshaw on a desktop computer. SCRMshaw can be run with a single, narrow set of training data to predict CRMs regulating a specific pattern of gene expression, or with multiple sets of training data covering a broad range of CRM activities to provide an initial rough regulatory annotation of a complete, newly-sequenced genome. less
Pseudouridine (Ψ) is the first-discovered RNA modification abundantly present in many classes of RNAs, which plays a pivotal role in a series of biological processes. Accurately identifying the location of Ψ sites i...
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Pseudouridine (Ψ) is the first-discovered RNA modification abundantly present in many classes of RNAs, which plays a pivotal role in a series of biological processes. Accurately identifying the location of Ψ sites is helpful for relevant downstream researches. In this chapter, we introduce a website PIANO—for pseudouridine site (Ψ) identification and functional annotation, which enables researchers to predict human putative Ψ sites with a high-accuracy (average AUC of 0.955 under the full transcript model and 0.838 under the mature mRNA model when testing on six independent datasets). The posttranscriptional regulatory mechanisms of putative Ψ sites including miRNA-targets, RBP-binding regions, and splicing sites were also annotated. A comprehensive query database was also provided to deposit over 4300 human Ψ modifications, which is currently the most complete collection of experimental-derived Ψ sites. The PIANO website is freely accessible at: http://*** or http://180.208.58.19/Ψ-WHISTLE . less
With the gut microbiome being implicated in many diseases and even offering the potential for future therapies, its study has been gaining interest over the years. However, to gain more in-depth insight and understand...
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With the gut microbiome being implicated in many diseases and even offering the potential for future therapies, its study has been gaining interest over the years. However, to gain more in-depth insight and understanding of the impact of the microbiome on our health, it is critical to have accurate microbial data that is reproducible and comparable across studies. Variation in experimental techniques, particularly DNA extraction and sequencing methods, may result in variability and the inability to compare results. In this chapter, we describe in detail our microbiome analysis methodology focusing on stool samples. less
A primary goal in neuroscience is to understand how the brain gives rise to innate and learned behavior. One way to expand the contributions of zebrafish in this respect is to use a computerized system that can train ...
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A primary goal in neuroscience is to understand how the brain gives rise to innate and learned behavior. One way to expand the contributions of zebrafish in this respect is to use a computerized system that can train larval zebrafish in various behavior assays. This will allow more efficient genetic and chemical screens aimed at investigating mechanisms of brain function. In this chapter, we describe a simple, economical and efficient way to automate avoidance learning in larval zebrafish. A webcam is used to acquire images for custom-built software to identify the position of fish in real-time. Based on this information, the computer delivers training stimuli from a universal serial bus module board that connects to the testing tanks through a small electrical circuit board. The training protocol is pre-programmed and the entire process is automatic, so no experimenter needs to be present and no offline video analysis is required. less
Level - Beginner, but attendees should have some experience with a high-level programming *** tutorial is designed for those who have some familiarity with a programming language, but who are new to Ada. In the mornin...
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ISBN:
(纸本)9781605582740
Level - Beginner, but attendees should have some experience with a high-level programming *** tutorial is designed for those who have some familiarity with a programming language, but who are new to Ada. In the morning, we will discuss the basics of programming in Ada, including types, packages, syntax rules, and other Ada programming constructs. In the afternoon, we will cover Ada's object-oriented programming and concurrent-programming features. Many examples will be shown; freely downloadable Ada programming environments and tools will be demonstrated.
This chapter describes how mathematical models can be used to investigate the possible range of behaviors for mutant forms of a protein. A mathematical model of the RAS signaling network that has previously been devel...
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This chapter describes how mathematical models can be used to investigate the possible range of behaviors for mutant forms of a protein. A mathematical model of the RAS signaling network that has previously been developed and applied to specific RAS mutants will be adapted for the process of computational random mutagenesis. By using this model to computationally investigate the range of RAS signaling outputs that would be anticipated over a wide range of the relevant parameter space, one can gain intuition about the types of behaviors that would be demonstrated by biological RAS mutants. less
Physarum polycephalum is a protist slime mould that exhibits a high degree of responsiveness to its environment through a complex network of tubes and cytoskeletal components that coordinate behavior across its unicel...
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Physarum polycephalum is a protist slime mould that exhibits a high degree of responsiveness to its environment through a complex network of tubes and cytoskeletal components that coordinate behavior across its unicellular, multinucleated body. Physarum has been used to study decision making, problem solving, and mechanosensation in aneural biological systems. The robust generative and repair capacities of Physarum also enable the study of whole-body regeneration within a relatively simple model system. Here we describe methods for growing, imaging, quantifying, and sampling Physarum that are adapted for investigating regeneration and repair. less
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