5430 research outputs found
Sort by
The Evolving Campus Exhibit, details
No full textDetails of the exhibit The Evolving Campus
Idea, design - Olga Nilova, Special Collections Librarian
Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1045/thumbnail.jp
The Rockefeller Institute Quarterly, 1959
Get PDFThe Rockefeller Institute Quarterly, Summer 1959
Full texthttps://digitalcommons.rockefeller.edu/the-evolving-campus/1049/thumbnail.jp
Sarah J. Schlesinger Oral History. Part 7: Clinical development
No full textInterview recorded in spring 2019. Part of The Rita and Frits Markus Library Oral History project.https://digitalcommons.rockefeller.edu/sarah-schlesinger/1006/thumbnail.jp
The Role of ZMYND8 in Immunoglobulin Class Switch Recombination
Get PDFClass switch Recombination (CSR) also known as Immunoglobulin (Ig) Class switching is a genomic recombination/deletion reaction that diversifies the effector component of the antibody response but preserves antigen specificity. CSR is initiated by the enzyme activation induced cytidine deaminase (AID), which produces nucleotide mismatches in actively transcribed immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3\u27 Regulatory Region (3\u27RR), a prototypical super-enhancer located at the 3\u27 of the Igh locus, is essential for acceptor switch region transcription, but the mechanism by which it regulates this process is not well defined. After targeting by AID, nearby mismatches in the donor switch region are processed into DNA double strand breaks (DSBs), translocated to DSBs in the acceptor switch region, and ligated through the DNA Damage Repair (DDR) pathway, non-homologous end-joining (NHEJ). Critical components of CSR are 53BP1 and its effector RIF1 because they inhibit end resection to promote NHEJ and oppose competing pathways in DDR. However, the mechanism by which RIF1 effects end-protection in CSR and binds to 53BP1 is still unknown In these studies, I identified a novel component of the RIF1 interactome, ZMYND8, a chromatin reader and transcriptional repressor that binds to RIF1 and facilitates effective CSR. Unexpectedly, ZMYND8 promotes CSR independently of RIF1. In B cells, ZMYND8 binds active promoters and super-enhancers, including the Igh enhancer the 3\u27RR. ZMYND8 controls 3\u27RR activity by regulating polymerase loading. In its absence there is increased 3\u27 RR polymerase loading and decreased acceptor region transcription and CSR. Thus, ZMYND8 controls CSR by regulating the activity of the 3\u27 Igh super enhancer
Functional Organization of Molecular Memories in the CRISPR-Cas Immune System
Get PDFCRISPR-Cas systems endow bacteria and archaea with adaptive immunity against foreign genetic threats, like phages and plasmids. These immune systems are comprised of CRISPR-associated (Cas) protein effectors and DNA-based storage of immunological memories in the CRISPR array. The CRISPR array is a series of direct repeats intercalated by variable spacer sequences (~30bp) of foreign origin. Upon infection, spacers are excised from the foreign genome and integrated into the array. The array is then transcribed and parsed into individual CRISPR RNAs, each containing a single spacer sequence, which are used by Cas nucleases to identify foreign nucleic acids for destruction. Thus, spacer sequences represent molecular memories that serve to define the specificity of the CRISPR immune response. New spacers are added invariably to the 5\u27 end of the array; therefore, the first spacer matches the most recent foreign invader. How this order is established and whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. In my thesis work, I showed that conserved nucleotides within the leader, a sequence located immediately upstream of the CRISPR array, specify the site of new spacer integration with high fidelity. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. To interrogate the importance of polarized spacer addition, I compared the immune responses generated by CRISPR systems containing wild type and mutant leader sequences. I showed that spacers added through polarized acquisition give rise to more robust immunity than spacers added to the middle of the array. This demonstrated that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the latest and most immediate threat to the host. Because addition of new spacers pushes existing spacers further downstream, each spacer added to the CRISPR array weakens the immunity provided by already existing spacers within the array. How CRISPR systems address this conundrum had not been explored. In this thesis work, I showed that CRISPR systems exhibit significant natural variation in the rates of spacer acquisition and thereby can modulate the lifespan of existing spacers in the array. Fast-adapting systems can respond quickly to new invaders, but existing spacers rapidly lose their potency. In contrast, slow-adapting systems preserve potency of existing spacers at the cost of reduced rates of spacer acquisition. I showed that bacteria have overcome these tradeoffs by harboring multiple CRISPR systems that acquire new spacers at different rates. I also found that leader-repeat junctions serve as a means for spacer acquisition complexes to discriminate between related CRISPR arrays. I propose a model whereby bacteria can harbor two related CRISPR systems as a means to form both shortand long-term immunological memories against foreign invaders. Bacteria were once thought to possess only primitive forms of innate immunity, but this notion was turned on its head by the discovery of the CRISPRCas immune system. My thesis work has revealed a deeper complexity of bacterial immunity and evolution by demonstrating that CRISPR systems functionally organize molecular memories of past invaders as a means to confer optimal immunity to the host
The Evolving Campus exhibit, details
No full textDetails of the exhibit The Evolving Campus
Idea, design - Olga Nilova, Special Collections Librarian
Photograph - Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1005/thumbnail.jp
The gardens north of Caspary Auditorium in fall
No full textThe gardens north of Caspary Auditorium in fall. Photo by Lubosh Stepanekhttps://digitalcommons.rockefeller.edu/the-evolving-campus/1051/thumbnail.jp
The Rockefeller Institute: 1960-1989
No full texthttps://digitalcommons.rockefeller.edu/the-evolving-campus/1053/thumbnail.jp
Non-Coding RNA Features Critical to the Replication of HIV-1
Get PDFThe HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication including, but not limited to transcription, splicing, nuclear export, translation, packaging and reverse transcription. Despite this extensive existing catalogue of RNA sequences that are critical to its replication, chemical probing and targeting mutagenesis studies suggest that the HIV-1 genome may contain many more RNA elements of unknown important function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we conducted a global synonymous mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice sites. This data from the Group 2 mutants indicates complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication. In silico analysis of the Group 3 mutants revealed that our mutagenesis had significantly increased the frequency of CG dinucleotides in sections of the viral genome to that of random sequence. This is important due to the remarkable CG suppression in both the HIV-1 and human genomes, and we had therefore disrupted the dinucleotide congruence that exists between HIV-1 and the genome of its host. We recoded these mutants to selectively remove either only the CG dinucleotides or only remove the mutations that did not encode a CG dinucleotide. Analysis of these mutants clearly demonstrated that the addition of CG dinucleotides were the causative mutations entirely responsible for the observed replication defects. qPCR analysis and smFISH microscopy revealed that the addition of CG dinucleotides to HIV-1 resulted in a depletion of the cytoplasmic mRNA molecules where the CG-dinucleotides were encoded as exons. A targeted siRNA screen for proteins that destabilize cytoplasmic RNA identified the Zinc-finger Antiviral Protein (ZAP) as responsible for the restriction of the CG-high HIV-1, specifically by targeting CGhigh viral RNA. CLIP-Seq experiments demonstrate that ZAP binds directly to CG dinucleotides in both cellular and viral RNA. Collectively these studies implicate ZAP as a cellular protein that can recognize CG-high viral RNA and is possibly a cellular mechanism for determining self from non-self RNA based on the CG composition. TRIM25 has previously been identified as a cofactor for two cytosolic RNA binding proteins that have antiviral functions, RIG-I where it is an essential cofactor, and ZAP where it functions as an enhancing cofactor. The mechanism by which TRIM25 enhances the antiviral activity of ZAP currently remains unclear. Through CLIP-Seq experiments in cells knocked out for TRIM25, we determined that ZAP does not require TRIM25 to recognize CG-high RNA. Using full length mutants of TRIM25 that are deficient for either RNA binding, E3 ligase activity, or formation of higher order multimers, our data suggest that the key biological activity required for TRIM25 to enhance ZAP is the formation of higher order multimers. Analyzing the replication of CG-high HIV-1 in different cell lines indicates that ZAP is not equally potent across all cell lines. The degree of potency ZAP possess against CG-high HIV-1 does not correlate with TRIM25 expression, suggesting the possibility of an additional ZAP cofactor that is heterogeneously expressed in varying cell lines. siRNA screens have been used in an attempt to identify a yet undiscovered cofactor, but so far these experiments have not yielded any such factor
Neural Mechanisms that Control an Innate Foraging Behavior in Caenorhabditis Elegans
Get PDFThe ability to efficiently locate food is critical for survival. Thus, animals modify their foraging patterns based on recent experience and current conditions to increase their likelihood of finding food. One highly conserved foraging strategy is local search, an intensive exploration over several minutes of the region where food resources were last encountered. As time since the last food encounter passes, animals transition to global search strategies to explore distant areas. The local-to-global search foraging pattern has been observed in fish, reptiles, insects, birds, and mammals, yet few studies ask how an animal\u27s brain generates this ancient behavior. Here, I ask this question in the nematode Caenorhabditis elegans. In Chapter 1, I characterize the behavior in wildtype animals and find that local search is a food memory that is regulated by food history and by internal satiety states. In addition, I describe the behavior in individual animals and find that although the behavior is reliable at a population level, there is large variability between individuals. In Chapter 2, I conduct a candidate genetic screen to first find a gene important for local search, and then define a circuit for local search behavior. The circuit consists of two parallel multimodal circuit modules that control local search. In each module, chemosensory or mechanosensory glutamatergic neurons that detect food-related cues trigger local search by inhibiting separate integrating neurons through a metabotropic glutamate receptor, MGL-1. The chemosensory and mechanosensory modules are separate and redundant, as glutamate release from either can drive the full behavior. In addition, the ability of the sensory modules to control local search is gated by the internal nutritional state of the animal. In Chapter 3, I characterize neuronal activity within the chemosensory module. Spontaneous activity patterns in the chemosensory module encode information about the time since the last food encounter and correlate with the foraging behavior. Glutamate acts within the module to shape activity patterns at various time scales. Taken together, these experiments reveal a circuit configuration that allows for the robust control of an innate adaptive behavior