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Description
V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By examining the dynamic fluorescence changes during the RAG-mediated cleavage reactions, and by manipulating the reaction conditions, I was able to characterize some fundamental properties of RAG-DNA interactions before and after cleavage. Firstly, Mg2+, known as a physiological cofactor at the excision step, also promotes the HP-CEs retention in the RAG complex after cleavage. Secondly, the structure of pre-cleavage complex may affect the subsequent collaborations with NHEJ for end resolution. Thirdly, the non-core region of RAG2 may have differential influences on the PCC retention of HP-CEs and SEs. Furthermore, I also provide the first evidence of RAG1-mediated regulation of RAG2. Our study provides important insights into the multilayered regulatory mechanisms, in modulating recombination events in developing lymphocytes and paves the way for possible development of detection and diagnotic markers for defective recombination events that are often associated immunodeficiency and/or lymphoid malignancy.
ContributorsWang, Guannan (Author) / Chang, Yung (Thesis advisor) / Levitus, Marcia (Committee member) / Misra, Rajeev (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2012
Description
Dengue virus infects millions of people every year. Yet there is still no vaccine available to prevent it. Here we use a neutralizing epitope determinant on the dengue envelope (E) protein as an immunogen to be vectored by a measles virus (MV) vaccine. However the domain III (DIII) of the dengue 2 E protein is too small to be immunogenic by itself. In order for it to be displayed on a larger particle, it was inserted into the amino terminus of small hepatitis B surface antigen (HBsAg, S) coding sequence. To generate the recombinant MV vector and verify the efficiency of this concept, a reverse genetics system was used where the MV vectors express one or two additional transcription units to direct the assembly of hybrid HBsAg particles. Two types of recombinant measles virus were produced: pB(+)MVvac2(DIII-S,S)P and pB(+)MVvac2(DIII-S)N. Virus recovered from pB(+)MVvac2(DIII-S,S)P was viable. An ELISA assay was performed to demonstrate the expression and secretion of HBsAg. Supernatant from MVvac2(DIII-S,S)P infected cells confirmed that hybrid HBsAg-domain III particles with a density similar to traditional HBsAg particles were released. Characteristics of the subviral particle have been analyzed for the successful incorporation of domain III. The replication fitness of the recombinant MV was evaluated using multi-step growth kinetics and showed reduced replication fitness when compared to the parental strain MVvac2. This demonstrates that viral replication is hindered by the addition of the two inserts into MV genome. Further analysis of MVvac2(DIII-S)N is needed to justify immune response studies in a small animal model using both of the generated recombinant vectors.
ContributorsHarahap, Indira Saridewi (Author) / Reyes del Valle, Jorge (Thesis director) / Hogue, Brenda (Committee member) / Misra, Rajeev (Committee member) / Barrett, The Honors College (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor) / School of Human Evolution and Social Change (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which was shown by HPLC and protein analysis. Although Δsll1951 was more susceptible to osmotic stress than the wild type, the general viability of the mutant remained unaffected. In a different study a combination of mutants having single or multiple deletions of putative carotenoid cleavage dioxygenase (CCD) genes was created. CCDs are presumed to play a role in the breakdown of carotenoids or apo-carotenoids. The carotenoid profiles of the mutants that were grown under conditions of increased reactive oxygen species were analyzed by HPLC. Pigment lifetimes of all strains were estimated by 13C-labeling. Carotenoid composition and metabolism were similar in all strains leading to the conclusion that the deleted CCDs do not affect carotenoid turnover in Synechocystis. The putative CCDs either do not fulfill this function in cyanobacteria or alternative pathways for carotenoid degradation exist. Finally, slr0941, a gene of unknown function but a conserved genome position in many cyanobacteria downstream of the δ-carotene desaturase, was disrupted. Initially, the mutant strain was impaired in growth but displayed a rather normal carotenoid content and composition, but an apparent second-site mutation occurred infrequently that restored growth rates and caused an accumulation of carotenoid isomers not found in the wild type. Based on the obtained data a role of the slr0941 gene in carotenoid binding/positioning for isomerization and further conversion to mature carotenoids is suggested.
ContributorsTrautner, Christoph (Author) / Vermaas, Willem Fj (Thesis advisor) / Chandler, Douglas E. (Committee member) / Misra, Rajeev (Committee member) / Bingham, Scott E (Committee member) / Arizona State University (Publisher)
Created2011
Description
Macromolecular structural biology advances the understanding of protein function through the structure-function relationship for applications to scientific challenges like energy and medicine. The proteins described in these studies have applications to medicine as targets for therapeutic drug design. By understanding the mechanisms and dynamics of these proteins, therapeutics can be designed and optimized based on their unique structural characteristics. This can create new, focused therapeutics for the treatment of diseases with increased specificity — which translates to greater efficacy and fewer off-target effects. Many of the structures generated for this purpose are “static” in nature, meaning the protein is observed like a still-frame photograph; however, the use of time-resolved techniques is allowing for greater understanding of the dynamic and flexible nature of proteins. This work advances understanding the dynamics of the medically relevant proteins NendoU and Taspase1 using serial crystallography to establish conditions for time-resolved, mix-and-inject crystallographic studies.
ContributorsJernigan, Rebecca Jeanne (Author) / Fromme, Petra (Thesis advisor) / Hansen, Debra (Thesis advisor) / Chiu, Po-Lin (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2022
Description
I studied the molecular mechanisms of ultraviolet radiation mitigation (UVR) in the terrestrial cyanobacterium Nostoc punctiforme ATCC 29133, which produces the indole-alkaloid sunscreen scytonemin and differentiates into motile filaments (hormogonia). While the early stages of scytonemin biosynthesis were known, the late stages were not. Gene deletion mutants were interrogated by metabolite analyses and confocal microscopy, demonstrating that the ebo gene cluster, was not only required for scytonemin biosynthesis, but was involved in the export of scytonemin monomers to the periplasm. Further, the product of gene scyE was also exported to the periplasm where it was responsible for terminal oxidative dimerization of the monomers. These results opened questions regarding the functional universality of the ebo cluster. To probe if it could play a similar role in organisms other than scytonemin producing cyanobacteria, I developed a bioinformatic pipeline (Functional Landscape And Neighbor Determining gEnomic Region Search; FLANDERS) and used it to scrutinize the neighboring regions of the ebo gene cluster in 90 different bacterial genomes for potentially informational features. Aside from the scytonemin operon and the edb cluster of Pseudomonas spp., responsible for nematode repellence, no known clusters were identified in genomic ebo neighbors, but many of the ebo adjacent regions were enriched in signal peptides for export, indicating a general functional connection between the ebo cluster and biosynthetic compartmentalization. Lastly, I investigated the regulatory span of the two-component regulator of the scytonemin operon (scyTCR) using RNAseq of scyTCR deletion mutants under UV induction. Surprisingly, the knockouts had decreased expression levels in many of the genes involved in hormogonia differentiation and in a putative multigene regulatory element, hcyA-D. This suggested that UV could be a cue for developmental motility responses in Nostoc, which I could confirm phenotypically. In fact, UV-A simultaneously elicited hormogonia differentiation and scytonemin production throughout a genetically homogenous population. I show through mutant analyses that the partner-switching mechanism coded for by hcyA-D acts as a hinge between the scytonemin and hormogonia based responses. Collectively, this dissertation contributes to the understanding of microbial adaptive responses to environmental stressors at the genetic and regulatory level, highlighting their phenomenological and mechanistic complexity.
ContributorsKlicki, Kevin (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Wilson, Melissa (Committee member) / Mukhopadhyay, Aindrila (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2021