[PDF] Functional Analysis Of The C Elegans Genome Using Rna Interference eBook

Author :
Publisher : Academic Press
Page : 0 pages
File Size : 27,67 MB
Release : 2013-09-04
Category : Science
ISBN : 9780123969682

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This new volume of Current Topics in Developmental Biology covers developmental timing, with contributions from an international board of authors. The chapters provide a comprehensive set of reviews covering such topics as the timing of developmental programs in Drosophila, temporal patterning of neural progenitors, and environmental modulation of developmental timing.

Author : Jay Mahesh Maniar
Publisher : Stanford University
Page : 321 pages
File Size : 26,75 MB
Release : 2010
Category :
ISBN :

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The development of the germline in Caenorhabditis elegans is a complex process involving the regulation of thousands of genes in a coordinated manner. These genes must direct the regulation of cell proliferation, meiosis, and sex determination, as well as gamete formation and fertilization. Several genes required for small RNA biogenesis and function are also required for the proper organization and development of the germline. EGO-1 is a putative RNA-directed RNA polymerase (RdRP) that is required for C. elegans germ-line development and efficient RNAi of germ-line expressed genes. Additionally, ego-1 mutants have been shown to exhibit defects in heterochromatin assembly on unpaired DNA and proper chromosome segregation during meiosis. Despite our understanding of the morphology of ego-1 mutant worms, it has remained unclear how the loss of a putative RdRP can cause such dramatic phenotypes in the C. elegans germline. That is, what role does EGO-1 play in promoting the development of the germline? There is strong evidence for the requirement of the C. elegans RdRP RRF-3 in producing endogenous small RNAs that target mRNA produced from specific genes. Additionally, there is evidence that EGO-1 may interact directly with chromatin. The questions become what target (or perhaps targets) is being disrupted in the ego-1 mutant and are they being disrupted at the chromatin or RNA level? Using high-throughput small RNA and messenger RNA sequencing we found that EGO-1 is required to produce small RNAs antisense to a number of germline-expressed genes through several developmental stages. We found that these genes fall into several classes including genes required for kinetochore (klp-7) and nuclear pore (npp-3) assembly, as well as the production of histone-modifying (set-21) and centromeric proteins (hcp-3). We also found several RNAi-related genes to be targets of EGO-1 (csr-1, mut-14, mut-16, prg-1, tsn-1). Finally, we show a strong correlation between the loss of small RNAs and the rise of mRNA levels in ego-1(-) animals.

Author : Harrison Wren Gabel
Publisher :
Page : 482 pages
File Size : 45,48 MB
Release : 2008
Category :
ISBN :

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The second screen employs an enhanced RNAi strain to more sensitively identify candidate RNAi genes. Integration of the genes identified in these screens with other RNAi screens and proteomic analysis for components of RNA silencing pathways in C. elegans generates a high-confidence list of shared small RNA pathway candidates. Loss-of-function analysis in mutants for some of the genes identified confirms their activity in RNAi. The exonuclease ERI-1 negatively regulates some RNAi pathways in worms and fungi and is required for the production of endogenous, small-interfering RNA (siRNA) in worms. I present data demonstrating that ERI-1 is a conserved rRNA processing component that mediates 3' end maturation of the 5.8S ribosomal RNA (rRNA) in both C. elegans and S. pombe. I show that one ERI-1 protein isoform, ERI-1b, mediates endogenous siRNA production and the association of the C. elegans Dicer ortholog, DCR-1, with a large complex that co-fractionates with the ribosome, Lastly, I identify the 7SL RNA as a second structural RNA substrate of ERI-1. Together these results indicate that ERI-1 plays a conserved dual role in the RNAi and structural RNA processing pathways.

Author : Jonatha M. Gott
Publisher : Springer Science & Business Media
Page : 437 pages
File Size : 37,65 MB
Release : 2008-02-05
Category : Science
ISBN : 1592597750

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This volume presents a comprehensive collection of cuttingedge methods for elucidating the function of new genes and altering gene expression. These readily reproducible techniques can be used either in transient and stable gene splicing applied to worms, flies, trypanosomes, mammals, and plants, or in studying RNA editing mechanisms in a wide range of organisms, including systems that involve the conversion of one base to another and insertion/deletion editing. Topics of interest include stable and transient RNA interference, gene silencing, RNA editing, bioinformatics, small noncoding RNAs, and RNomics. Special attention is given to methods for the identification and characterization of small RNAs involved in RNA interference or modification. Readily reproducible protocols for discovering new genes or altering gene expression.

Author : Jerry J. Buccafusco
Publisher : CRC Press
Page : 341 pages
File Size : 23,58 MB
Release : 2000-08-29
Category : Medical
ISBN : 1420041819

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Using the most well-studied behavioral analyses of animal subjects to promote a better understanding of the effects of disease and the effects of new therapeutic treatments on human cognition, Methods of Behavior Analysis in Neuroscience provides a reference manual for molecular and cellular research scientists in both academia and the pharmaceutic

Author : Ian A. Hope
Publisher : OUP Oxford
Page : 306 pages
File Size : 22,27 MB
Release : 1999-12-09
Category : Science
ISBN : 019159198X

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Caenorhabditis Elegans has been a popular model organism for biological research for over thirty years and has been used to investigate many aspects of animal development, for example apoptosis, the Hox genes, signal transduction pathways, and the development of the nervous system. It has recently taken on new importance with the publication of the entire genome sequence in 1998. The first chapter gives all the basic information on C. elegans required to use it: it's natural history, anatomy, life cycle, development, and evolution. Information on how to obtain, grow, and maintain C. elegans for use as a model system is given in Chapter 4. Chapters 2 and 3 describe the genome project and show how to use genome sequence information by searching the database for homologues using different search methods and then how to analyse the search data. The next chapter gives the essential practical details of transformation and common uses for the technique. Chapter 6 covers reverse genetics and describes strategies for gene inactivation that are known to work in C elegans: epigenetic inactivation and mutational germ line inactivation. Chapter 7 is designed to help the user analyse phenotype by microscopy and includes Normaski, fluorescence, 4-dimensional, and electron microscopy. Techniques for studying the neurobiology of C. elegans are given in chapter 8. Chapter 9 describes the three commonly used approaches for studying gene expression and Chapter 10 deals with the common methods of molecular biology essential for gene characterization. C. elegans is not the ideal organism for biochemical studies, but chapter 11 describes several procedures for producing biochemically useful quantities of pure tissues. The final chapter is about conventional genetics and details the standard procedures for selfing and crossing; mutagenesis and mutant screening; characterization of mutants; gene mapping; temperature-shift experiments and mosaic analysis. Caenorhabditis Elegans: A Practical Approach will therefore provide all the background information necessary for use of C. elegans as a model system.

Author : Kevin Strange
Publisher : Springer Science & Business Media
Page : 292 pages
File Size : 47,11 MB
Release : 2008-02-05
Category : Medical
ISBN : 1597451517

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Molecular biology has driven a powerful reductionist, or “molecule-c- tric,” approach to biological research in the last half of the 20th century. Red- tionism is the attempt to explain complex phenomena by defining the functional properties of the individual components of the system. Bloom (1) has referred to the post-genome sequencing era as the end of “naïve reductionism. ” Red- tionist methods will continue to be an essential element of all biological research efforts, but “naïve reductionism,” the belief that reductionism alone can lead to a complete understanding of living organisms, is not tenable. Organisms are clearly much more than the sum of their parts, and the behavior of complex physiological processes cannot be understood simply by knowing how the parts work in isolation. Systems biology has emerged in the wake of genome sequencing as the s- cessor to reductionism (2–5). The “systems” of systems biology are defined over a wide span of complexity ranging from two macromolecules that interact to carry out a specific task to whole organisms. Systems biology is integrative and seeks to understand and predict the behavior or “emergent” properties of complex, multicomponent biological processes. A systems-level characteri- tion of a biological process addresses the following three main questions: (1) What are the parts of the system (i. e.

Author : Zhaozhao Qin
Publisher :
Page : 164 pages
File Size : 30,1 MB
Release : 2016
Category :
ISBN :

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For over 30 years, researchers have taken advantage of genetic balancers and forward genetic screens to isolate lethal mutations, which have been studied to identify essential genes in C. elegans. Using traditional genetic methods, such as genetic mapping, complementation tests, and transgenic rescue assays, many essential genes have been successfully identified. However, to pinpoint a specific essential gene the involved experiments are usually labor intensive and time consuming. Nowadays, genetic methods combined with whole genome sequencing (WGS) and bioinformatics analysis provide an effective approach for the molecular identification of essential genes. In my thesis I successfully identified 64 new essential genes with 107 lethal mutations in genomic regions of C. elegans of around 14 Mb from Chromosome III(mid) and Chromosome V(left), by combining genetic mapping, Illumina sequencing, bioinformatics analyses, and experimental validation. Most of these genes have multiple recovered mutant alleles. Of these 64 genes 5 have new alleles identified, which had not been previously studied by RNA interference depletion. Furthermore, by investigating the locations of lethal missense mutations in essential genes, I have identified five novel protein functional domains. Functional characterization of the identified essential genes shows that most of them are enzymes, including helicases, tRNA synthetase, and kinases. There are also ribosomal proteins. Gene Ontology functional annotation also indicates that essential genes tend to execute enzyme and nucleic acid binding activities during fundamental processes, such as intracellular protein synthesis. Essential gene analysis shows that compared to non-essential genes, essential genes have fewer paralogs, and encode proteins that are in protein interaction hubs. Essential genes are also more abundantly and consistently expressed over all developmental stages than non-essential genes. All these essential genes traits in C. elegans are consistent with those of human disease genes. Unsurprisingly, most (90%) human orthologs of essential genes in this study are related to human diseases. Therefore, functional characterization of essential genes underlines their importance as proxies for understanding the biological functions of human disease genes.