Genome-wide Analysis of Transcriptional Expression Programs, Regulatory Networks and Cis-regulatory Sequences in Saccharomyces Cerevisiae PDF Download
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Author: Christopher T. Harbison
Publisher:
ISBN:
Category :
Languages : en
Pages : 456
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Historically, knowledge of gene-specific transcription has been accumulated by the study of the individual genetic and physical interactions between transcriptional regulators and the genes they regulate, often requiring considerable time and effort. Microarray technology now enables investigation of gene expression at the level of the entire genome, allowing researchers access to rich datasets and promising new levels of depth in the understanding of transcriptional regulation. Our lab has made use of these technologies both to measure the levels of all mRNA transcripts within a population of cells, as well as to locate the regions within the genome that are bound by transcriptional regulators. Such studies not only allow for the functional annotation of both genes and regulators, but can also provide clues about the identity of the regulatory regions within DNA, the structure of global regulatory networks and the regulation of DNA-binding proteins. These and other insights are presented here based on our genome-wide studies of transcriptional regulation in the yeast Saccharomyces cerevisiae.
Author: Christopher T. Harbison
Publisher:
ISBN:
Category :
Languages : en
Pages : 456
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Book Description
Historically, knowledge of gene-specific transcription has been accumulated by the study of the individual genetic and physical interactions between transcriptional regulators and the genes they regulate, often requiring considerable time and effort. Microarray technology now enables investigation of gene expression at the level of the entire genome, allowing researchers access to rich datasets and promising new levels of depth in the understanding of transcriptional regulation. Our lab has made use of these technologies both to measure the levels of all mRNA transcripts within a population of cells, as well as to locate the regions within the genome that are bound by transcriptional regulators. Such studies not only allow for the functional annotation of both genes and regulators, but can also provide clues about the identity of the regulatory regions within DNA, the structure of global regulatory networks and the regulation of DNA-binding proteins. These and other insights are presented here based on our genome-wide studies of transcriptional regulation in the yeast Saccharomyces cerevisiae.
Author: Ezra Gray Jennings
Publisher:
ISBN:
Category :
Languages : en
Pages : 500
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Author: Justin Daniel Smith
Publisher:
ISBN:
Category :
Languages : en
Pages :
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This thesis is organized into two primary areas of research: cis-regulatory evolution and genome engineering. It is now known that noncoding regions comprise the vast majority of genomic regions under selective constraint in mammals. Despite this, the ability to detect natural selection on noncoding regions has lagged behind the ability to detect selection on coding regions. In Chapter 1, we introduce a new test to detect selection on cis-regulatory elements, and demonstrate its utility on three mammalian transcriptional enhancers. In Chapter 2, we investigate the evolution of resistance to the mycotoxin citrinin by comparing two closely related species of budding yeast, Saccharomyces paradoxus and Saccharomyces cerevisiae. Applying a genome-wide test for selection on cis-regulation, we identified five genes involved in resistance in S paradoxus, four of which are necessary for resistance and increase resistance in S cerevisiae when over-expressed. In the second half of this dissertation, I discuss my work with genome engineering and CRISPR-Cas9. In Chapter 2, we build an improved CRISPR activator (CRISPRa) to simultaneously induce overexpression (in S cerevisiae) of the four genes identified to confirm their role in citrinin resistance. In Chapter 3, we build an inducible CRISPR interference (CRISPRi) system and and designed gRNA libraries to determine design rules for CRISPRi gRNA design in S cerevisiae. We determined that gRNAs targeted to a region with low nucleosome occupancy and high chromatin accessibility within a window of 0 to 200bp upstream of the transcription start site (TSS) are most likely to be effective. In Chapter 4 we confirm and refine these rules in a much larger library consisting of ~9000 unique strains. Additionally, we present a novel method for parsing complex oligonucleotide libraries into single, sequence verified DNA sequences using high throughput sequencing and yeast synthetic biology. We utilize this technology to create and characterize a collection of ~9000 individual inducible CRISPRi strains to the vast majority of essential and respiratory essential genes in S cerevisiae. In Chapter 5, we compare mismatch tolerance for Cas9 in vitro and in vivo (in S cerevisiae) and test a variety of truncated and full-length gRNAs (with 17, 18, and 20 nucleotides of complementarity sequence). We observed notable differences between in vitro and in vivo Cas9 cleavage specificity profiles, with in vivo cleavage being more sensitive/less tolerant to mismatches.
Author: M. Francis Shannon
Publisher: CRC Press
ISBN:
Category : Science
Languages : en
Pages : 156
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Book Description
While every cell of an organism has an identical genomic content, extremely complex networks exist to tailor the genomic output to the needs of that cell. This program of gene expression is different for every cell type and stage of development. In addition, the cell can respond to its environment by modulating its gene expression program in a fairly dramatic manner. For many decades gene transcription has been investigated in systems from bacteria to mammalian cells and along the way many landmark findings have set new paradigms that often apply across wide evolutionary distances. Studying individual genes, however, especially in mammalian systems has been a painstaking business and although we know the transcription activators and other complexes that control specific genes in minute detail, generalizing these findings has often proven to be difficult. It has become clear that transcription factors do not operate alone but form complex networks in the cell. If one component of this complexity is disturbed then there are repercussions across the entire network, but it has been impossible to study these networks until very recently. The advent of microarray technology within the last decade has revolutionized how we study gene transcription. There are several types of array technology that essentially screen for relative mRNA levels for many thousands of genes at once. We do not focus here on the technology as this has become routine and is available to many researchers. Microarray technology has given us the ability to measure the entire gene expression program of a cell in a single experiment and compare it to other cells thus allowing a global view of cell behaviour at the level of gene transcription. Expression profiling, as this endeavour has become known, is now a relatively simple undertaking and hundreds, probably thousands of papers have been published demonstrating the power of this technology. Expression profiling has been applied to many diverse biological problems and is also being developed as a method for disease diagnosis especially in the cancer classification field. There are constant improvements or modified uses of the technology that are allowing more and more high throughput experiments to be carried out.
Author: Markus Juhana Herrgard
Publisher:
ISBN:
Category :
Languages : en
Pages : 736
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Author: Tyson A. Clark
Publisher:
ISBN:
Category : Eukaryotic cells
Languages : en
Pages : 422
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Author: John Jason Wyrick
Publisher:
ISBN:
Category :
Languages : en
Pages : 496
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(Cont.) Telomere-proximal genes were derepressed over regions extending 20 kb from telomere ends, well beyond the extent of Sir protein binding and the effects resulting from loss of Sir function. These results indicate that histones make Sir-independent contributions to telomeric silencing, and that the role of histones located elsewhere in chromosomes is gene specific To map the in vivo binding sites of transcription factors, a new technique-genome-wide location analysis-was developed. Genome-wide location and expression analysis was used to identify the target genes of the yeast activator proteins Gal4 and Ste 12. All of the known targets for these two activators were confirmed, and new target genes in multiple functional pathways were identified.
Author: Ana Serra Barros
Publisher:
ISBN:
Category : Genomes
Languages : en
Pages : 442
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Author: Xueying C. Li
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 186
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The genetic architecture of phenotypic divergence is a central question in evolutionary biology. Genetic architecture is impacted by whether evolution occurs through accumulation of many small-effect or a few large-effect changes, the relative contribution of coding and cis-regulatory changes, and the prevalence of epistatic effects. Our empirical understanding of the genetic basis of evolutionary change remains incomplete, largely because reproductive barriers limit genetic analysis to those phenotypes that distinguish closely related species. In this dissertation, I use hybrid genetic analysis to examine the basis of thermal divergence between two post-zygotically isolated species, Saccharomyces cerevisiae and S. uvarum. S. cerevisiae is relatively heat tolerant, whereas S. uvarum is heat sensitive but outperforms S. cerevisiae at 4°C. Gene expression analysis with an S. cerevisiae and S. uvarum hybrid revealed a small set of 136 genes with temperature-dependent cis-acting differences, suggesting that the temperature divergence has not caused widespread cis-regulatory divergence. Using a genome-wide non-complementation screen, I found a single nuclear-encoded gene with a modest contribution to heat tolerance, and a large effect of the species' mitochondrial DNA (mitotype). Recombinant mitotypes and allele replacements indicate multiple mitochondria-encoded genes contribute to thermal divergence, with the coding sequence of COX1 showing a moderate effect on both heat and cold tolerance. The non-complementation approach also identified allele differences of CUP2, a copper-binding transcription factor, in copper resistance of S. cerevisiae and S. uvarum. Chimeric alleles showed that multiple changes underlie the resistance of S. cerevisiae CUP2, with cis-regulatory changes having a larger effect than coding changes. Taken together, my findings suggest that evolution of interspecific phenotypic differences often involves accumulation of small-to-medium effect changes, such as those in mitochondrial DNA and CUP2, and can occur through both coding and cis-regulatory changes.
Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 946
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