No description available.

Powerful mechanisms of genetic interference in both unicellular and multicellular organisms are based on the sequence-directed targeting of DNA or RNA by small effector RNAs. In many bacteria and almost all archaea, RNAs derived from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci are involved in an adaptable and heritable gene-silencing pathway. Resistance to phage infection is conferred by the incorporation of short invading DNA sequences into the prokaryotic genome as CRISPR spacer elements separated by short repeat sequences. A central aspect to this pathway is the processing of a long primary transcript (pre-crRNA) containing these repeats by crRNA endonucleases to generate the mature effector RNAs that interfere with phage or plasmid gene expression. Here we describe a structural and functional analysis of the CasE endonuclease of T. thermophilus a member of the Ecoli CRISPR sub-type. High resolution X-ray structures of CasE bound to repeat RNAs model both the pre-and post-cleavage complexes associated with processing the pre-crRNA. These structures establish the molecular basis of a specific CRISPR RNA recognition and suggest the mechanism for generation of effector RNAs responsible for gene-silencing.

Thesis (Ph.D.)--Syracuse University, 2008. / "Publication number: AAT 3323087."

Thesis (M.S.)--Georgia Southern University, 2009. / "A thesis submitted to the Graduate Faculty of Georgia Southern University in partial fulfillment of the requirements for the degree Master of Science." Directed by Laura B. Regassa. ETD. Includes bibliographical references (p. 64-69) and appendices.

<p> The higher taxonomic groups within Prokaryotes are presently distinguished mainly on the basis of their branching in phylogenetic trees. In most cases, no molecular, biochemical or physiological characteristics are known that are uniquely shared by species from these groups. Comparative genomic analyses are leading to discovery of molecular characteristics that are specific for different groups of Bacteria and Archaea. These markers include conserved inserts and deletions in universal proteins and lineagespecific proteins, which provide novel means for identifying and circumscribing these groups of prokaryotes in clear molecular terms and for understanding their evolution. Because of their taxa specificities, further studies on these newly discovered molecular characteristics should lead to discovery of novel biochemical and physiological characteristics that are unique to different groups of microbes. The focus of my project was phylogenomic studies for two large prokaryotic group: Actinobacteria and Archaea. My goals were to a) identify molecular markers that are specific to Actinobacteria and Archaea at different taxonomic levels, which will help to understand the phylogenetic relationship within these two major groups; b) understand the functional significance of Actinobacteria-specific proteins. By comparative genomics approach, a number of conserved indels in various proteins (viz. Coxl, GluRS, CTPsyn, Gft, GlyRS, TrmD, Gyrase A, SahH and SHMT) have been identified that are specific for all Actinobacteria and additional indels were found to be unique to its major subgroups, such as Corynebacterineae, Bifidobacteriaceae, etc. In parallel, a large number of proteins were discovered to be restricted to Actinobacteria at different phylogenetic depths. These identified conserved indels and proteins for the first time provide useful markers for defining and circumscribing the Actinobacteria phylum or its subgroups in clear molecular terms. Similar comparative genomic studies have been carried out on Archaea and a vast number of proteins have been identified that are unique to Archaea or its various lineages. Lastly, I have performed functional studies on one of the Actinobacteria-specific proteins (ASPl). The structure of ASPl was determined and structural comparison indicates that the function of this protein might be novel since it does not match any known protein with or without known function. </p> / Thesis / Doctor of Philosophy (PhD)

Prokaryotic genomes evolve via horizontal gene transfer (HGT), mutations, and rearrangements. HGT is a mechanism that plays a significant role in prokaryotic evolution and leads to biodiversity in nature. One of the important components of HGT is the genomic island (GI) which is a subsequence of the genome created by HGT. This research aims to identify the structures of the prokaryotic GIs that have a fundamental role in the adoption of prokaryotes and the impact of the species on the environment. Previous computational biology research has focused on developing tools that detect GIs in prokaryotic genomes, while there is little research investigating GI structure. This research introduces a novel idea that has not yet been addressed intensively, which is identifying additional structures of the GIs in prokaryotes. There are two main directions in this research used to study the prokaryotic GIs structure from each different perspective. In the first direction, the aim is to investigate GI patterns and the existence of biological connections across bacterial phyla in terms of GIs on a large scale. This direction mainly aims to pursue the novel idea of connecting GIs across prokaryotic and phage genomes via patterns of protein families across many species. A pattern is a sequence of protein families that is found to frequently occur in the genomes of a number of species. Here the large data set available from the IslandViewer4 database and protein families from the Pfam database have been combined. Furthermore, implementing a comprehensive strategy to identify patterns that makes use of HMMER, BLAST, and MUSCLE; also implement Python programs that link the analysis into a single pipeline. Research results demonstrate that related GIs often exist in multiple species that are not evolutionarily related and indeed may be from multiple bacterial phyla. Analysis of the discovered patterns led to the identification of biological connections among prokaryotes and phages through their GIs. A connection is an HGT relation represented as a pattern that exists in a phage and a number of prokaryotic species. These discovered connections suggest quite broad HGT connections across the bacterial kingdom and its associated phages. In addition, these connections provide the basis for additional analysis of the breadth of HGT and the identification of individual HGT events that span bacterial phyla. Moreover, these patterns can suggest the basis for discovering the specific patterns in pathogenic GIs that could play a crucial role in antibiotic resistance. The second direction aims to identify the structure of the GIs in terms of their location within the genome. Prokaryotic GIs have been analyzed according to the genome structure that they are located in, whether it be a circular or a linear genome. The analysis is performed to study the GIs' location in relation to the oriC, investigating the nature of the distances between the GIs, and determining the distribution of GIs in the genome. The analysis has been performed on all of the GIs in the data set. Moreover, the GIs in one genome from each species and the GIs of the most frequent species are in the data set, in order to avoid bias. Overall, the results showed that there are preferable sites for the GIs in the genome. In the linear genomes, they are usually located in the origin of replication area and terminus, and in the circular genomes they are located in the terminus. / Doctor of Philosophy / Prokaryotes are one of the most abundant species on earth that play an essential role in naturally shaping the planet and its life. This research aims to identify the structure of a component in these species that has a fundamental role in the adoption of prokaryotes and the impact of the species on the environment. This component is a part of the genome named the genomic island (GI). This dissertation aims to identify the structure of the GIs in two different ways that have not yet been addressed extensively. The first direction aims to discover patterns in the GIs and then use them to bring to light biological connections between prokaryotic and bacteriophages. In this direction, a comprehensive strategy has been utilized to identify patterns and connections. This strategy uses several tools such as BLAST, HMMER, and MUSCLE. Furthermore, Python programs that link the analysis into a single pipeline have been implemented. In the second direction, an investigation has been performed to understand the nature of the GIs' locations within the genome. This direction addresses three different analysis techniques to achieve its target. The three analyses are studying the GIs' location in relation to the origin of replication, investigating the nature of the distances between the GIs, and discovering the location distribution of GIs in the genome. The analysis is performed on linear genomes and circular genomes separately. In each group of GIs, the data set has been utilized to see the results from different perspectives. The overall analysis in both directions relived several findings. In the first direction, the discovered patterns merit deep investigation based on the possibility that they are related to diseases. In addition, in prokaryotic genomes, there are specific sites where the GIs can be frequently seen that need further search to understand the relation between the GIs' location and the content of the GI in terms of proteins.

Cell walls of Bacillus subtilis macrofibers have been biochemically analyzed to determine the contribution of various surface polymers in the twist regulation. Helix hand inversion was induced by a variation in either the growth temperature or the nutritional composition of the culture medium. Initial experiments had demonstrated a fivefold difference in the sensitivity of right- and left-handed forms to muramidases indicating modifications of peptidoglycan as a possible mechanism underlaying inversion. An examination of lysozyme susceptibility of purified cell walls and whole cells derived from the two structural forms, however, exhibited no significant difference suggesting loss of the relevant component(s), perhaps biomechanical in nature, during disintegration of macrofibers. The effect of various twist modulators such as trypsin, ammonium sulfate and D-alanine on the development of helical twist in both switchable and "fixed" mutants were studied. The interaction matrices have established D-alanine as the most potent of right-factors. Intestinal alkaline phosphatase is reported as a newly discovered antagonist to the development of leftward twist. Heat inactivation and protein purification experiments strongly indicated that twist modulation was due to the phosphatase activity rather than minor protease contaminants. The chemical composition of cell walls purified from right- and left-handed structures was determined. No twist correlated differences in the overall content of peptidoglycan, teichoic acid and teichuronic acid were detected. Evidence is presented for the absence of correlation between the extent of ester-linked alanine substitution and twist state. These findings suggest that gross changes in wall composition is perhaps not the mechanism for hand inversion. From the profiles of the wall associated proteins, a 200 Kdal band has been identified whose presence is strongly correlated with the development of leftward twist. This polypeptide was found to be highly sensitive to trypsin; a property it shares with a previously proposed left-twist protein. Preliminary evidence for isolation of left-hand specific polyclonal antibodies is also presented. FJ7, a switchable mutant, was successfully transformed with a plasmid containing the Streptococcus transposon Tn917. A small bank of insertional mutants has been constructed for the isolation of mutants impaired in helix hand inversion.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

Thesis (M.S.)--Bioengineering, Georgia Institute of Technology, 2007. / Zhu, Cheng, Committee Chair ; Selvaraj, Periasamy, Committee Member ; Orville, Allen, Committee Member ; Butera, Robert, Committee Member.

CRISPR-Cas is an adaptive immune system unique to prokaryotes, which prevents infection by foreign genetic elements. Key to the function of CRISPR-Cas immunity is the ability to adapt to new threats in incorporating short segments, termed spacers, of invading DNA into the clustered regularly interspaced short palindromic repeat (CRISPR) array of the host. Spacers constitute immunological memories, used by CRISPR-associated (Cas) proteins to mount a sequence-specific attack on subsequent infections. The immunisation of the host is called CRISPR adaption. Adaption requires the integration of new spacers at a precise site in the CRISPR array. Two proteins, Cas1 and Cas2, are essential for adaptation; however, the mechanisms of spacer integration remain poorly understood. The work described here focused on understanding adaptation in Sulfolobus solfataricus. Using biochemical assays, I aimed to characterise the activity of the Cas1 and Cas2 proteins in this organism in order to understand their role in the insertion of new spacers. Additionally, I aimed to investigate how the expression of CRISPR-Cas components is regulated in this organism in response to viral infection. The results presented here show that expression of Cas1 was strongly upregulated in response to infection. A Csa3 protein from S. solfataricus was found to bind to the promoter for transcription of cas1, implying a role in the regulation observed. I reconstituted in vitro both the integration reaction performed by Cas1 and Cas2 proteins of S. solfataricus and the reverse of this reaction, disintegration. Cas1 was shown to impose sequence specificity on these reactions, selecting sites similar to the leader-repeat junction of the CRISPR locus. Finally, I demonstrated that, in addition to the intrinsic specificity of Cas1, there was a requirement for an additional host factor for site-specific integration in S. solfataricus.