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Author: Ching-Ti Liu
Publisher:
ISBN: 9781109897388
Category :
Languages : en
Pages : 202
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Book Description
Characterization of protein-protein interaction has become a key step in understanding the cellular mechanism because most biological processes cannot be carried out without protein-protein interaction. This dissertation focuses on a set of well-validated protein complex data from MIPS. It is commonly believed that functionally related genes tend to have similar expression patterns. However, we found notable contradictory evidence. Indeed, except for a few large protein complexes such as cytoplasmic or mitochondrial ribosomal proteins, the co-expression level of genes from the same complex is on the average not much higher than that from a pair of randomly selected genes. We then demonstrate the impact of these six protein complexes on other genes' regulation. The ways of incorporating the ChIP-on-Chip data is also studied by defining a pairwise co-regulation rate is defined for protein complexes. With this pairwise rate of co-regulation, we successfully link co-regulation and co-expression. To extend the concept of networks, we also study the relationship between protein complexes rather than proteins or genes alone. Specifically, we treat one complex or one group of genes as one node rather single gene. Liquid association is then applied to investigate the dynamic relationship between protein complexes.
Author: Ching-Ti Liu
Publisher:
ISBN: 9781109897388
Category :
Languages : en
Pages : 202
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Book Description
Characterization of protein-protein interaction has become a key step in understanding the cellular mechanism because most biological processes cannot be carried out without protein-protein interaction. This dissertation focuses on a set of well-validated protein complex data from MIPS. It is commonly believed that functionally related genes tend to have similar expression patterns. However, we found notable contradictory evidence. Indeed, except for a few large protein complexes such as cytoplasmic or mitochondrial ribosomal proteins, the co-expression level of genes from the same complex is on the average not much higher than that from a pair of randomly selected genes. We then demonstrate the impact of these six protein complexes on other genes' regulation. The ways of incorporating the ChIP-on-Chip data is also studied by defining a pairwise co-regulation rate is defined for protein complexes. With this pairwise rate of co-regulation, we successfully link co-regulation and co-expression. To extend the concept of networks, we also study the relationship between protein complexes rather than proteins or genes alone. Specifically, we treat one complex or one group of genes as one node rather single gene. Liquid association is then applied to investigate the dynamic relationship between protein complexes.
Author: Christina Smolke
Publisher: John Wiley & Sons
ISBN: 3527688099
Category : Science
Languages : en
Pages : 532
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Book Description
A review of the interdisciplinary field of synthetic biology, from genome design to spatial engineering. Written by an international panel of experts, Synthetic Biology draws from various areas of research in biology and engineering and explores the current applications to provide an authoritative overview of this burgeoning field. The text reviews the synthesis of DNA and genome engineering and offers a discussion of the parts and devices that control protein expression and activity. The authors include information on the devices that support spatial engineering, RNA switches and explore the early applications of synthetic biology in protein synthesis, generation of pathway libraries, and immunotherapy. Filled with the most recent research, compelling discussions, and unique perspectives, Synthetic Biology offers an important resource for understanding how this new branch of science can improve on applications for industry or biological research.
Author: Daechan Park
Publisher:
ISBN:
Category :
Languages : en
Pages : 248
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Book Description
Transcriptional regulation is a complicated process controlled by numerous factors such as transcription factors (TFs), chromatin remodeling enzymes, nucleosomes, post-transcriptional machineries, and cis-acting DNA sequence. I explored the complex transcriptional regulation in eukaryotes through three distinct studies to comprehensively understand the functional genomics at various steps. Although a variety of high throughput approaches have been developed to understand this complex system on a genome wide scale with high resolution, a lack of accurate and comprehensive annotation transcription start sites (TSS) and polyadenylation sites (PAS) has hindered precise analyses even in Saccharomyces cerevisiae, one of the simplest eukaryotes. We developed Simultaneous Mapping Of RNA Ends by sequencing (SMORE-seq) and identified the strongest TSS and PAS of over 90% of yeast genes with single nucleotide resolution. Owing to the high accuracy of TSS identified by SMORE-seq, we detected possibly mis-annotated 150 genes that have a TSS downstream of the annotated start codon. Furthermore, SMORE-seq showed that 5'-capped non-coding RNAs were highly transcribed divergently from TATA-less promoters in wild-type cells under normal conditions. Mapping of DNA-protein interactions is essential to understanding the role of TFs in transcriptional regulation. ChIP-seq is the most widely used method for this purpose. However, careful attention has not been given to technical bias reflected in final target calling due to many experimental steps of ChIP-seq including fixation and shearing of chromatin, immunoprecipitation, sequencing library construction, and computational analysis. While analyzing large-scale ChIP-seq data, we observed that unrelated proteins appeared to bind to the gene bodies of highly transcribed genes across datasets. Control experiments including input, IgG ChIP in untagged cells, and the Golgi factor Mnn10 ChIP also showed the strong binding at the same loci, indicating that the signals were obviously derived from bias that is devoid of biological meaning. In addition, the appearance of nucleosomal periodicity in ChIP-seq data for proteins localizing to gene bodies is another bias that can be mistaken for false interactions with nucleosomes. We alleviated these biases by correcting data with proper negative controls, but the biases could not be completely removed. Therefore, caution is warranted in interpreting the results from ChIP-seq. Nucleosome positioning is another critical mechanism of transcriptional regulation. Global mapping of nucleosome occupancy in S. cerevisiae strains deleted for chromatin remodeling complexes has elucidated the role of these complexes on a genome wide scale. In this study, loss of chromodomain helicase DNA binding protein 1 (Chd1) resulted in severe disorganization of nucleosome positioning. Despite the difficulties of performing ChIP-seq for chromatin remodeling complexes due to their transient and dynamic localization on chromatin, we successfully mapped the genome-wide occupancy of Chd1 and quantitatively showed that Chd1 co-localizes with early transcription elongation factors, but not late transcription elongation factors. Interestingly, Chd1 occupancy was independent of the methylation levels at H3K36, indicating the necessity of a new working model describing Chd1 localization.
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: Martha Beasley Arnaud
Publisher:
ISBN:
Category : Genetic transcription
Languages : en
Pages : 264
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Book Description
Author: Priyasri Shotton
Publisher:
ISBN:
Category :
Languages : en
Pages : 268
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Book Description
DNA is the storehouse of genetic information for the normal functioning of all cellular processes. This DNA is "read" by RNA Polymerases (RNAP) to carry out the transcription process, which is one of the most vital regulatory mechanisms within the cell for gene expression. There are three important RNA Polymerases available in the cell. RNAPI synthesizes ribosomal RNAs, while RNAPII makes mRNA precursors, snRNAs and most microRNAs, additionally RNAPIII takes care of tRNAs, small RNAs and 5S rRNAs that occur in the nucleus and the cytosol. The work in the author's lab focuses on the function of RNAPII transcription. It is not straightforward for RNAPII to transcribe the DNA, it needs to struggle with two major roadblocks that are in its path. First, is the nucleosomal barrier that is scattered across the coding regions of active genes. The mechanism to overcome this major obstacle is a very complex process that involves an extensive range of proteins which work collectively to help in the alteration of chromatin structure that ultimately facilitates transcriptional regulation. In the thesis, the author has attempted to take a look at this intricate network and has studied the function of Rad26p, in yeast (associated with CSB-Cockayne syndrome B, in humans), a transcription coupled DNA repair factor, having an additional role in transcription. The results reveal that Rad26p plays a novel role in the regulation of elongation by modulating histone H2A-H2B dimer occupancy.
Author: Kateland Simmons
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
We have formed the basis for a synthetic gene regulatory network in S. cerevisiae to assess the relative contribution of proteins and protein interactions to the function of transcriptional regulatory complexes. This gene network contains two synthetic transcriptional regulator (TR) fusion proteins that control transcriptional activation and repression of a reporter expressed by a synthetic promoter. This system can be modulated and quantified specifically by our manipulations since the transcriptional regulators (TRs) are only active when drug induced. In this thesis, I characterized and optimized my gene network to enable accurate measurements of transcriptional regulation. I demonstrated that my synthetic repressor interacts with a selected native transcriptional regulatory complex. Future mutation experiments can be performed to investigate the relative importance that the selected protein interaction plays on transcriptional regulation. New synthetic repressor fusion proteins can be created to study the relative importance of protein interactions between other TRs, making this tool versatile. This tool will allow us to quantify interactions and gain a deeper understanding of the complicated mechanisms controlling transcriptional regulation.
Author: Vince Harjono
Publisher:
ISBN:
Category :
Languages : en
Pages : 111
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Book Description
Post-transcriptional regulation represents a powerful and versatile mechanism to fine-tune gene expression to meet cellular and environmental demands. One important aspect of post-transcriptional regulation involves regulation of protein translation, the process of building proteins from a messenger RNA. In this dissertation, I use biochemical and molecular biology techniques to study how translation is mechanistically regulated by both mRNA and protein factors. In chapter 2, I discuss the development of a quantitative method in eukaryotes to measure ribosomal stalls of cis-mRNA factors on protein elongation. We find that different distributions of nonoptimal codons trigger different surveillance and rescue pathways despite similar levels of elongation delay. In chapter 3, I explore the relationship between translatability and mRNA localization during glucose starvation and investigate potential factors that influence this relationship. We find that a complex made from Rvb1 and Rvb2 is involved in promoter-directed cytoplasmic fate in a subset of stress response genes in glucose starvation. Furthermore, we use carefully designed reporters to interrogate how translatability determines cytoplasmic localization and find that active translation is linked to exclusion from stress-induced cytoplasmic granules. Finally in chapter 4, I discuss improvements on the method we have developed, possible future directions for the work described in this dissertation, and my concluding remarks.
Author: Gregory A. Cary
Publisher:
ISBN:
Category :
Languages : en
Pages : 129
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Book Description
Although gene expression begins with transcription, there are a variety of mechanisms that cells use to control and tune expression post-transcriptionally. Many post-transcriptional regulatory functions including translational regulation, transcript surveillance, intracellular RNA localization, and RNA decay occur in organelles known as RNA granules. RNA granules, such as processing (P)-bodies, are cytoplasmic accumulations of translationally repressed mRNA and associated proteins that are ubiquitous among eukaryotes. Much of what is known about RNA granule biology has been observed through genetic and cytological experimentation and very few biochemical enrichments of these structures have been reported. In this work I present an affinity enrichment strategy for Dhh1, a conserved core component of P-bodies, from the budding yeast Saccharomyces cerevisiae. We identify proteins co-enriching with Dhh1 using tandem mass spectrometry and show that many known RNA granule proteins are enriched by this approach. We go on to compare the association of proteins with the complex across two environmental conditions to examine the effect of stress induction on RNA granule assemblies. We find that metabolic enzymes and molecular chaperones are typically more abundant in the stress-induced P-body complex and demonstrate that one chaperone, YDJ1, is involved in the stress-induced aggregation of several P-body proteins into cytoplasmic foci. We also identify RNA co-enriching with Dhh1 and detect several classes of catalytic RNA as well as a strong enrichment for the mRNA encoding the P-body protein PAT1. Finally, I present and discuss the characterization of a yeast strain that exhibits sensitivity to the drug puromycin. The puromycin-sensitive strain incorporates the drug into nascent proteins in vivo and I discuss how this is a unique and useful approach for the detection of protein biosynthesis. The techniques developed and employed in this dissertation provide novel perspectives on post-transcriptional regulatory processes and enable further investigations into how these regulatory programs are executed within the cell.
Author: David Matthew Reynolds
Publisher:
ISBN:
Category : Cyclin-dependent kinases
Languages : en
Pages : 212
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Book Description
In this thesis the biochemical and genetic characterization of Fkh2p identifies it as a major component of SFF. It has been shown to bind DNA in an Mcm1p dependent manner and the Fkh2p DNA binding domain is essential for this interaction. The protein interaction domain of Mcm1p has been demonstrated to be essential for ternary complex formation. Fkh2p, along with a functionally redundant protein Fkh1p, has been show to control the periodic expression of the CLB2 cluster genes. The functional characterisation of the Fkh2p domains reveals an important role for both the Forkhead associated domain and the C-terminus. Ndd1p. another protein important for mitotic progression, is shown to be important for CLB2 cluster regulation by de-repressing Fkh2p and activating gene expression. The role of cdk activity is shown to act through the CLB2 cluster upstream activating sequences, possibly through Ndd1p.
