Insights Into the Function of the FATC DOMAIN of Saccharomyces Cerevisiae TRA1 Via Mutation and Suppresor Analysis PDF Download
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Author: Samantha A. Pillon
Publisher:
ISBN:
Category :
Languages : en
Pages : 206
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Book Description
The regulation of transcription is an important cellular function because it is the first step in gene regulation. In Saccharomyces cerevisiae, two protein complexes, SAGA and NuA4, act as regulators of transcription. A common protein shared between these two complexes, called Tra1, regulates transcriptional activation through its interaction with gene specific transcriptional activators. Tra1 is a member of the PIKK family of proteins, which are characterized by FAT, PI3K and FATC domains. The FATC domain encompasses the terminal 33-35 residues of the protein. Two mutations within the FATC domain, tra1-L3733A and tra1-F3744A, result in slow growth under stress conditions. Partially dominant mutations in the gene encoding Tti2, a 421 residues component of the TTT chaperone complex, suppressed these phenotypes. My goal was to further characterize the role of the FATC domain of Tra1 by determining which residues are important for function, and characterize how these relate to the TRA1-TTI2 interaction. I created alleles of tra1 which convert the terminal residue to serine (tra1-F3744S) or arginine (tra1-F3744R), an allele with the two terminal residues inverted (tra1-WF-FW), and alleles which resulted in deletions of one or two residues (tra1-1and tra1-2). tra1-WF-FW supported cell viability, whereas tra1-F3744S supported cell viability in the presence of the tti2- F328S suppressor. Slow growth at 37°C resulting from Tra1-WF-FW was also suppressed by tti2. Tra1-F3744S grew slowly at 37°C and on 6% ethanol in the presence of the suppressor. tra1-F3744R, tra1-1, and tra1-2 would not support viability in the presence or absence of the suppressor. To better understand the structure and function of Tti2, truncation mutations were created to identify essential regions of Tti2. These truncation mutations were assessed by analyzing cell viability, protein expression levels, and interaction with Tti1 and Tel2, two other members of the TTT complex. Only Tti2-53-421 (containing residues 53-421) supported viability and retained its ability to interact with Tel2 and Tti1 at near wildtype levels. Tti2-53-238 and Tti2-1-238 interact with Tel2 but do not support viability. All of the other mutations did not support viability and showed minimal binding affinity to Tti1 and Tel2 A terminal mutation of another PIKK family member, Mec1 (mec1-W2368A) also results in slow growth at 37°C. Interestingly, the tti2-F328S suppressor does not suppress the mec1- W2368A phenotype. Using bioinformatics approaches, I identified rpn3-L140P, encoding a component of the proteasomal cap, as a suppressor of mec1-W2368A.
Author: Samantha A. Pillon
Publisher:
ISBN:
Category :
Languages : en
Pages : 206
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Book Description
The regulation of transcription is an important cellular function because it is the first step in gene regulation. In Saccharomyces cerevisiae, two protein complexes, SAGA and NuA4, act as regulators of transcription. A common protein shared between these two complexes, called Tra1, regulates transcriptional activation through its interaction with gene specific transcriptional activators. Tra1 is a member of the PIKK family of proteins, which are characterized by FAT, PI3K and FATC domains. The FATC domain encompasses the terminal 33-35 residues of the protein. Two mutations within the FATC domain, tra1-L3733A and tra1-F3744A, result in slow growth under stress conditions. Partially dominant mutations in the gene encoding Tti2, a 421 residues component of the TTT chaperone complex, suppressed these phenotypes. My goal was to further characterize the role of the FATC domain of Tra1 by determining which residues are important for function, and characterize how these relate to the TRA1-TTI2 interaction. I created alleles of tra1 which convert the terminal residue to serine (tra1-F3744S) or arginine (tra1-F3744R), an allele with the two terminal residues inverted (tra1-WF-FW), and alleles which resulted in deletions of one or two residues (tra1-1and tra1-2). tra1-WF-FW supported cell viability, whereas tra1-F3744S supported cell viability in the presence of the tti2- F328S suppressor. Slow growth at 37°C resulting from Tra1-WF-FW was also suppressed by tti2. Tra1-F3744S grew slowly at 37°C and on 6% ethanol in the presence of the suppressor. tra1-F3744R, tra1-1, and tra1-2 would not support viability in the presence or absence of the suppressor. To better understand the structure and function of Tti2, truncation mutations were created to identify essential regions of Tti2. These truncation mutations were assessed by analyzing cell viability, protein expression levels, and interaction with Tti1 and Tel2, two other members of the TTT complex. Only Tti2-53-421 (containing residues 53-421) supported viability and retained its ability to interact with Tel2 and Tti1 at near wildtype levels. Tti2-53-238 and Tti2-1-238 interact with Tel2 but do not support viability. All of the other mutations did not support viability and showed minimal binding affinity to Tti1 and Tel2 A terminal mutation of another PIKK family member, Mec1 (mec1-W2368A) also results in slow growth at 37°C. Interestingly, the tti2-F328S suppressor does not suppress the mec1- W2368A phenotype. Using bioinformatics approaches, I identified rpn3-L140P, encoding a component of the proteasomal cap, as a suppressor of mec1-W2368A.
Author: Ann Marie Elizabeth Faust
Publisher:
ISBN:
Category :
Languages : en
Pages : 160
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Book Description
Mitosis is the process by which eukaryotic cells segregate their chromosomes before division. A critical stage of mitosis is anaphase, when the microtubule-based spindle segregates chromosomes into mother and daughter cells prior to cytokinesis. My dissertation research aimed to provide a better understanding of the regulation of anaphase progression and spindle function during mitosis in Saccharomyces cerevisiae. My research focused on two microtubule-associated proteins, Slk19 (CENP-F homolog) and Stu1 (CLASP homolog). These proteins play fundamental roles in anaphase progression, Stu1 in microtubule spindle stability and Slk19 in spindle midzone organization and Cdc14 phosphatase regulation. I found that Stu1 and Slk19 physically interact and that Slk19 regulates Stu1 localization during anaphase. In addition to its interaction with Slk19, I identified a number of other physical and genetic interactors of Stu1 through mass spectrometry, yeast two-hybrid and synthetic genetic analyses. These interactors provide insight into the role of Stu1 at kinetochores. I also investigated Stu1 function through a protein truncation analysis and purification of full-length Stu1 protein. My truncation analysis revealed that the Stu1 C-terminus is dispensable for viability but is necessary for proper protein localization. The N-terminus, however, is essential for viability. My attempts to purify Stu1 from insect cells were partially successful; the protein is extremely sensitive to proteolytic degradation, and under conditions that limit proteolysis, the protein appears to aggregate or oligomerize in solution. I also investigated the role of Slk19 sumoylation in anaphase progression. I determined that the Cdc14 early anaphase release (FEAR) network protein Slk19 is sumoylated in vivo and that sumoylation is important for restricting Cdc14 phosphatase localization to the nucleus at the end of anaphase. A slk19 sumoylation mutant causes premature Cdc14 movement from the nucleus to the bud neck, which affects mitotic exit, as this slk19 sumoylation mutant can partially rescue the spindle disassembly defect of the mitotic exit network mutant cdc15-2. This slk19 mutant also has aberrant spindle elongation dynamics, which might be due to a change in Cdc14 function during anaphase. In conclusion, my dissertation research has uncovered a number of previously unrecognized interactions among mitotic proteins and has revealed a novel function of sumoylation in the regulation of Cdc14 function during anaphase through the FEAR network protein Slk19.
Author: Olivier Binda
Publisher: Academic Press
ISBN: 012802609X
Category : Science
Languages : en
Pages : 468
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Book Description
Chromatin Signaling and Diseases covers the molecular mechanisms that regulate gene expression, which govern everything from embryonic development, growth, and human pathologies associated with aging, such as cancer. This book helps researchers learn about or keep up with the quickly expanding field of chromatin signaling. After reading this book, clinicians will be more capable of explaining the mechanisms of gene expression regulation to their patients to reassure them about new drug developments that target chromatin signaling mechanisms. For example, several epigenetic drugs that act on chromatin signaling factors are in clinical trials or even approved for usage in cancer treatments, Alzheimer's, and Huntington's diseases. Other epigenetic drugs are in development to regulate various class of chromatin signaling factors. To keep up with this changing landscape, clinicians and doctors will need to stay familiar with genetic advances that translate to clinical practice, such as chromatin signaling. Although sequencing of the human genome was completed over a decade ago and its structure investigated for nearly half a century, molecular mechanisms that regulate gene expression remain largely misunderstood. An emerging concept called chromatin signaling proposes that small protein domains recognize chemical modifications on the genome scaffolding histone proteins, facilitating the nucleation of enzymatic complexes at specific loci that then open up or shut down the access to genetic information, thereby regulating gene expression. The addition and removal of chemical modifications on histones, as well as the proteins that specifically recognize these, is reviewed in Chromatin Signaling and Diseases. Finally, the impact of gene expression defects associated with malfunctioning chromatin signaling is also explored. - Explains molecular mechanisms that regulate gene expression, which governs everything from embryonic development, growth, and human pathologies associated with aging - Educates clinicians and researchers about chromatin signaling, a molecular mechanism that is changing our understanding of human pathology - Explores the addition and removal of chemical modifications on histones, the proteins that specifically recognize these, and the impact of gene expression defects associated with malfunctioning chromatin signaling - Helps researchers learn about the quickly expanding field of chromatin signaling
Author: Jerry L. Workman
Publisher: Springer Science & Business Media
ISBN: 1461486246
Category : Medical
Languages : en
Pages : 594
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Book Description
While there has been an increasing number of books on various aspects of epigenetics, there has been a gap over the years in books that provide a comprehensive understanding of the fundamentals of chromatin. Chromatin is the combination of DNA and proteins that make up the genetic material of chromosomes. Its primary function is to package DNA to fit into the cell, to strengthen the DNA to prevent damage, to allow mitosis and meiosis, and to control the expression of genes and DNA replication. The audience for this book is mainly newly established scientists and graduate students. Rather than going into the more specific areas of recent research on chromatin the chapters in this book give a strong, updated groundwork about the topic. Some the fundamentals that this book will cover include the structure of chromatin and biochemistry and the enzyme complexes that manage it.
Author: Stefan Hohmann
Publisher: Springer Science & Business Media
ISBN: 3540456112
Category : Science
Languages : en
Pages : 398
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Book Description
Every cell has developed mechanisms to respond to changes in its environment and to adapt its growth and metabolism to unfavorable conditions. The unicellular eukaryote yeast has long proven as a particularly useful model system for the analysis of cellular stress responses, and the completion of the yeast genome sequence has only added to its power This volume comprehensively reviews both the basic features of the yeast genral stress response and the specific adapations to different stress types (nutrient depletion, osmotic and heat shock as well as salt and oxidative stress). It includes the latest findings in the field and discusses the implications for the analysis of stress response mechanisms in higher eukaryotes as well.
Author: David E. Fisher
Publisher: Springer Science & Business Media
ISBN: 1592592309
Category : Medical
Languages : en
Pages : 441
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Book Description
David Fisher, MD, PhD, and an authoritative panel of academic, cutting-edge researchers review and summarize the current state of the field. Describing the broad roles of tumor suppressors from a perspective based in molecular biology and genetics, the authors detail the major suppressors and the pathways they regulate, including cell cycle progression, stress responses, apoptosis, and responses to DNA damage. Leading-edge and forward-looking, Tumor Suppressor Genes in Human Cancer illuminates what is currently known of tumor suppressor genes and their regulation, work that is already beginning to revolutionize cancer target elucidation, drug discovery, and treatment design.
Author: Christine Guthrie
Publisher:
ISBN: 9780123106704
Category : Molecular biology
Languages : en
Pages : 933
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Book Description
Guide to Yeast Genetics and Molecular Biology presents, for the first time, a comprehensive compilation of the protocols and procedures that have made Saccharomyces cerevisiae such a facile system for all researchers in molecular and cell biology. Whether you are an established yeast biologist or a newcomer to the field, this volume contains all the up-to-date methods you will need to study "Your Favorite Gene" in yeast. Basic Methods in Yeast Genetics**Physical and genetic mapping**Making and recovering mutants**Cloning and Recombinant DNA Methods**High-efficiency transformation**Preparation of yeast artificial chromosome vectors**Basic Methods of Cell Biology**Immunomicroscopy**Protein targeting assays**Biochemistry of Gene Expression**Vectors for regulated expression**Isolation of labeled and unlabeled DNA, RNA, and protein
Author: Horst Feldmann
Publisher: John Wiley & Sons
ISBN: 3527644865
Category : Science
Languages : en
Pages : 349
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Book Description
Yeast is one of the oldest domesticated organisms and has both industrial and domestic applications. In addition, it is very widely used as a eukaryotic model organism in biological research and has offered valuable knowledge of genetics and basic cellular processes. In fact, studies in yeast have offered insight in mechanisms underlying ageing and diseases such as Alzheimers, Parkinsons and cancer. Yeast is also widely used in the lab as a tool for many technologies such as two-hybrid analysis, high throughput protein purification and localization and gene expression profiling. The broad range of uses and applications of this organism undoubtedly shows that it is invalubale in research, technology and industry. Written by one of the world's experts in yeast, this book offers insight in yeast biology and its use in studying cellular mechanisms.
Author: Suzanne Peterson
Publisher: Academic Press
ISBN: 0123854741
Category : Science
Languages : en
Pages : 652
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Book Description
This manual is a comprehensive compilation of "methods that work" for deriving, characterizing, and differentiating hPSCs, written by the researchers who developed and tested the methods and use them every day in their laboratories. The manual is much more than a collection of recipes; it is intended to spark the interest of scientists in areas of stem cell biology that they may not have considered to be important to their work. The second edition of the Human Stem Cell Manual is an extraordinary laboratory guide for both experienced stem cell researchers and those just beginning to use stem cells in their work. - Offers a comprehensive guide for medical and biology researchers who want to use stem cells for basic research, disease modeling, drug development, and cell therapy applications - Provides a cohesive global view of the current state of stem cell research, with chapters written by pioneering stem cell researchers in Asia, Europe, and North America - Includes new chapters devoted to recently developed methods, such as iPSC technology, written by the scientists who made these breakthroughs
Author: Vincenzo E. A. Russo
Publisher:
ISBN:
Category : Medical
Languages : en
Pages : 716
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Book Description
Many inheritable changes in gene function are not explained by changes in the DNA sequence. Such epigenetic mechanisms are known to influence gene function in most complex organisms and include effects such as transposon function, chromosome imprinting, yeast mating type switching and telomeric silencing. In recent years, epigenetic effects have become a major focus of research activity. This monograph, edited by three well-known biologists from different specialties, is the first to review and synthesize what is known about these effects across all species, particularly from a molecular perspective, and will be of interest to everyone in the fields of molecular biology and genetics.
