6,577 research outputs found

    Aberrant phase separation and nucleolar dysfunction in rare genetic diseases

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    Thousands of genetic variants in protein-coding genes have been linked to disease. However, the functional impact of most variants is unknown as they occur within intrinsically disordered protein regions that have poorly defined functions1-3. Intrinsically disordered regions can mediate phase separation and the formation of biomolecular condensates, such as the nucleolus4,5. This suggests that mutations in disordered proteins may alter condensate properties and function6-8. Here we show that a subset of disease-associated variants in disordered regions alter phase separation, cause mispartitioning into the nucleolus and disrupt nucleolar function. We discover de novo frameshift variants in HMGB1 that cause brachyphalangy, polydactyly and tibial aplasia syndrome, a rare complex malformation syndrome. The frameshifts replace the intrinsically disordered acidic tail of HMGB1 with an arginine-rich basic tail. The mutant tail alters HMGB1 phase separation, enhances its partitioning into the nucleolus and causes nucleolar dysfunction. We built a catalogue of more than 200,000 variants in disordered carboxy-terminal tails and identified more than 600 frameshifts that create arginine-rich basic tails in transcription factors and other proteins. For 12 out of the 13 disease-associated variants tested, the mutation enhanced partitioning into the nucleolus, and several variants altered rRNA biogenesis. These data identify the cause of a rare complex syndrome and suggest that a large number of genetic variants may dysregulate nucleoli and other biomolecular condensates in humans.© 2023. The Author(s)

    Studies on genetic and epigenetic regulation of gene expression dynamics

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    The information required to build an organism is contained in its genome and the first biochemical process that activates the genetic information stored in DNA is transcription. Cell type specific gene expression shapes cellular functional diversity and dysregulation of transcription is a central tenet of human disease. Therefore, understanding transcriptional regulation is central to understanding biology in health and disease. Transcription is a dynamic process, occurring in discrete bursts of activity that can be characterized by two kinetic parameters; burst frequency describing how often genes burst and burst size describing how many transcripts are generated in each burst. Genes are under strict regulatory control by distinct sequences in the genome as well as epigenetic modifications. To properly study how genetic and epigenetic factors affect transcription, it needs to be treated as the dynamic cellular process it is. In this thesis, I present the development of methods that allow identification of newly induced gene expression over short timescales, as well as inference of kinetic parameters describing how frequently genes burst and how many transcripts each burst give rise to. The work is presented through four papers: In paper I, I describe the development of a novel method for profiling newly transcribed RNA molecules. We use this method to show that therapeutic compounds affecting different epigenetic enzymes elicit distinct, compound specific responses mediated by different sets of transcription factors already after one hour of treatment that can only be detected when measuring newly transcribed RNA. The goal of paper II is to determine how genetic variation shapes transcriptional bursting. To this end, we infer transcriptome-wide burst kinetics parameters from genetically distinct donors and find variation that selectively affects burst sizes and frequencies. Paper III describes a method for inferring transcriptional kinetics transcriptome-wide using single-cell RNA-sequencing. We use this method to describe how the regulation of transcriptional bursting is encoded in the genome. Our findings show that gene specific burst sizes are dependent on core promoter architecture and that enhancers affect burst frequencies. Furthermore, cell type specific differential gene expression is regulated by cell type specific burst frequencies. Lastly, Paper IV shows how transcription shapes cell types. We collect data on cellular morphologies, electrophysiological characteristics, and measure gene expression in the same neurons collected from the mouse motor cortex. Our findings show that cells belonging to the same, distinct transcriptomic families have distinct and non-overlapping morpho-electric characteristics. Within families, there is continuous and correlated variation in all modalities, challenging the notion of cell types as discrete entities

    Molecular Research in Rice: Agronomically Important Traits 2.0

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    This volume presents recent research achievements concerning the molecular genetic basis of agronomic traits in rice. Rice (Oryza sativa L.) is the most important food crop in the world, being a staple food for more than half of the world’s population. Recent improvements in living standards have increased the worldwide demand for high-yielding and high-quality rice cultivars. To develop novel cultivars with superior agronomic performance, we need to understand the molecular basis of agronomically important traits related to grain yield, grain quality, disease resistance, and abiotic stress tolerance. Decoding the whole rice genome sequence revealed that ,while there are more than 37,000 genes in the ~400 Mbp rice genome, there are only about 3000 genes whose molecular functions are characterized in detail. We collected in this volume the continued research efforts of scholars that elucidate genetic networks and the molecular mechanisms controlling agronomically important traits in rice

    Patenting Genetic Information

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    The U.S. biotechnology industry got its start and grew to maturity over roughly three decades, beginning in the 1980s. During this period genes were patentable, and many gene patents were granted. University researchers performed basic research— often funded by the government—and then patented the genes they discovered with the encouragement of the Bayh-Dole Act, which sought to encourage practical applications of basic research by allowing patents on federally funded inventions and discoveries. At that time, when a researcher discovered the function of a gene, she could patent it such that no one else could work with that gene in the laboratory without a license. She had no right, however, to control genes in nature, including in human bodies. Universities licensed their researchers’ patents to industry, which brought in significant revenue for further research. University researchers also used gene patents as the basis for obtaining funding for start-up enterprises spun out of university labs. It was in this environment that many of today’s biotechnology companies started. In 2013, the Supreme Court held that naturally occurring genes could no longer be patented. This followed a 2012 decision that disallowed patents on many diagnostic processes. These decisions significantly changed the intellectual property protections in the biotechnology industry. Nevertheless, the industry has continued to grow and thrive. This Article investigates two questions. First, if some form of exclusive rights still applied to genes, would the biotech industry be even more robust, with more new entrants in addition to thriving, well-established companies? Second, does the current lack of protection for gene discoveries incentivize keeping such discoveries secret for the many years that it can take to develop a therapeutic based thereon—to the detriment of patients who could benefit from knowledge of the genetic associations, even before a treatment is developed? The Article concludes by analyzing what protection for discovering genetic associations, if any, will most increase social welfare

    The role of anillin and ESCRT proteins in the control of cytokinesis in eukaryotes

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    Cytokinesis is the final step of the cell division cycle, and the major events of the process are conserved from fungi to animal cells when cellular constituents are separated to produce two daughter cells. Cytokinesis by the fission yeast Schizosaccharomyces pombe is compromised by the loss of anillin/Mid1p and they assemble slowly into an abnormal contractile ring. Phosphorylation by aurora and polo kinase during different cell cycle steps regulates several aspects of Mid1p function. In this thesis, we focussed on understanding the genetic interactions between the mid1 and vps4 genes, the physical interactions between Mid1p and Vps4p proteins, and the mechanisms by which they work together to regulate one another during cytokinesis. In this thesis, we identified genetic interactions between mid1 phospho-mutants plo1-ts35, ark1-ts11, and vps4∆ genes using tetrad analysis. Synthetic viabilityand morphological studies identified potentially important Mid1p amino acid residues that are required for successful cytokinesis. We observed defective growth and morphology in cells with mutations in Mid1p at serine positions 332, 523, and 531. This analysis suggested a strong genetic interaction between mid1 and vps4 genes. Furthermore, Mid1p is phosphorylated by aurora ark1 and polo plo1 kinases, genetic interactions between mid1, ark1, and plo1 kinase genes is essential for cell viability, and it is also required for the correct cellular localization of Mid1p protein. Such analysis revealed serine residues S332, S523, and S531 to be required for the Mid1p function and its interaction with vps4, ark1, and plo1 genes. Pull-down assay was used to determine the physical interaction between the Nterminal domain, middle and C-terminal domains of Mid1p with Vps4p. We observed a positive physical interaction between the C-terminal domain and Vps4p, suggesting physical interaction between C-terminal and Vps4p. However, a negative physical interaction between N-terminal and Middle with Vps4p was observed. Bioimage analysis showed septation defects and mislocalization at serine positions S332, 523, and 531 of mid1 phospho-mutant indicating the importance of phosphorylation of Mid1p in cortical anchorage and nuclear localization during cell division. The mislocalization patterns observed present evidence that Mid1p is implicated in the recruitment of nodes and endosomal vesicle elements to drive cytokinesis processes. Combined, these data suggest a genetic and biochemical interaction between Mid1p and Vps4p is important for cytokinesis and further implied that phosphorylation of Mid1p by aurora and polo kinases is significant for this processes

    Targeting Fusion Proteins of HIV-1 and SARS-CoV-2

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    Viruses are disease-causing pathogenic agents that require host cells to replicate. Fusion of host and viral membranes is critical for the lifecycle of enveloped viruses. Studying viral fusion proteins can allow us to better understand how they shape immune responses and inform the design of therapeutics such as drugs, monoclonal antibodies, and vaccines. This thesis discusses two approaches to targeting two fusion proteins: Env from HIV-1 and S from SARS-CoV-2. The first chapter of this thesis is an introduction to viruses with a specific focus on HIV-1 CD4 mimetic drugs and antibodies against SARS-CoV-2. It discusses the architecture of these viruses and fusion proteins and how small molecules, peptides, and antibodies can target these proteins successfully to treat and prevent disease. In addition, a brief overview is included of the techniques involved in structural biology and how it has informed the study of viruses. For the interested reader, chapter 2 contains a review article that serves as a more in-depth introduction for both viruses as well as how the use of structural biology has informed the study of viral surface proteins and neutralizing antibody responses to them. The subsequent chapters provide a body of work divided into two parts. The first part in chapter 3 involves a study on conformational changes induced in the HIV-1 Env protein by CD4-mimemtic drugs using single particle cryo-EM. The second part encompassing chapters 4 and 5 includes two studies on antibodies isolated from convalescent COVID-19 donors. The former involves classification of antibody responses to the SARS-CoV-2 S receptor-binding domain (RBD). The latter discusses an anti-RBD antibody class that binds to a conserved epitope on the RBD and shows cross-binding and cross-neutralization to other coronaviruses in the sarbecovirus subgenus.</p
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