Browsing by Author "Fox, George E."
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Item A phylogenetic analysis of the genus Bacillus using comparative 16S rRNA sequencing(1988) Lin, Chuzhao; Fox, George E.; Tu, Shiao-Chun; Oró, John F.; Jurtshuk, Peter, Jr.Inconsistencies between phenetic and genetic data have been known in the genus Bacillus for a number of years. In order to develop an evolutionarily reasonable classification of this genus, a large scale study has been undertaken to examine all the recognized species of the genus with a single molecular method, primer directed partial reverse transcriptase sequencing of 16S rRNA. In the work described here partial sequences exceeding 1100 nucleotides each are reported for B. circulans ATCC 4513, B. circulans NRS 746, B. azotefixans strain Hino, B. alvei ATCC 6344, and B. macquariensis ATCC 23464 . The validity of the partial sequence data was verified where possible with the known secondary structure features of B. subtilis 16S rRNA and in the case of B. alvei with 16S rRNA oligonucleotide cataloging data. This new data was analyzed in the context of 14 other strains of Bacillus whose 16S rRNA sequence is available as well as 21 other complete 16S rRNA sequences from a variety of procaryotes. Phylogenetic trees were constructed by three methods; unweighted pair-group method with arithmetic mean (UPGMA), Fitch and Margoliash and neighbor-joining. [...]Item A structural and phylogenetic analysis of the 16S rRNA of the Chlorobiaceae(1987) McGill, Ted John; Fox, George E.; Nelson, Daniel A.; Hecht, Ralph M.; Jurtshuk, Peter, Jr.Partial 16S rRNA sequences of 1212 and 943 nucleotides were determined for the green sulfur bacteria Chlorobium limicola and Pelodictvon luteolum by utilization of primer directed dideoxy sequencing with reverse transcriptase. The validity of the partial sequence data was verified, where possible, by the secondary structural model of eubacterial 16S rRNA, resulting in a relatively high degree of integrity for single strand sequencing. The previously available 16S ribosomal RNA catalogs for the green sulfur bacteria: Chlorobium limicola. Chlorobium vibrioforme, and Chioroherpeton thalassium were correctly aligned and in addition to aiding in verification of partial sequence data will in the future contribute to the continuing analysis of 16S rRNA structure. A secondary structure for the two green sulfur bacteria 16S rRNAs can be constructed that is essentially the same as that known for Escherichia coli. By proper alignment with an existing data base of 16S rRNAs a phylogenetic tree confirming the placement of the green sulfur bacteria as a unique group of photosynthetic organisms has been constructed, giving results that are in complete agreement with the catalog data for these organisms. The high degree of homology (95.9%) between the two partial sequences establishes the Chlorobiaceae as a closely knit, phylogenetically shallow, group of photosynthetic organisms.Item A survey of the incidence and function of plasmids in marine Vibrio SPP(1981) Hada, Howard Stanley; Sizemore, Ronald K.; Evans, John E.; Bennett, Edward O.; Fox, George E.Presumptive marine Vibrio spp. were collected from an operational oil field, control site, and an inshore site located in the Northwestern Gulf of Mexico. Of 523 isolates tested for plasmids using the cleared lysate and agarose gel techniques, 27% showed distinct plasmid bands. [...]Item A theoretical study of the effects of intrahelical and interhelical interactions on the helix-coil transitions of various single polypetides in water(1987) Ulrich, Charles D., II; McCammon, J. Andrew; Dyckes, Douglas F.; Kouri, Donald J.; Wentworth, Wayne E.; Fox, George E.The biological activity of a protein molecule strictly depends on its conformation. The helix-coil transition of a single chain polypeptide segment is an important factor in the determination of a protein molecule's conformation and has been implicated in a variety of physiologically important processes. In this work, a modified form of Dr.J. A. McCammon's simplified polypeptide model is utilized tostudy the importance of polypeptide hydrophobic content, alpha-helical amphipathicity, intrahelical electrostatic interactions, interhelical potential interactions andsurface binding to various fixed alpha-helical polypeptides of differing hydrophobic content to the coil-helix transition of various single chain polypeptide homopolymers and heteropolymers. This work demonstrates that in the absence of a site available for binding, as the hydrophobic content of a polypeptide chain increases, so does the likelihood of both post-nucleation alpha-helical chain state propagation and random coil to total alpha-helix transition. The most favorable state is the all random coil state independent of the residue type content of the polypeptide, in this circumstance. Depending on the relative positions of oppositely charged residues in a polypeptide chain, salt-bridge formation tends to increase the probability of post nucleation chain state propagation and shift K1 toward 1H, as defined by the equilibrium state model. In the presence of a site available for binding, R, the likelihood of both hydrophobic and hydrophobically amphipathic ligand types undergoing 1 to R binding, as well as coil to helix transition is almost completely dependent on the overall hydrophobicity of R. The nucleation of 1 is more likely to occur in the bound state. As the hydrophobic content of the binding site increases, so does the favorability of the coil-helix transition of an amphipathic polypeptide. Based on the calculations drawn from this study, the factors effecting the coil to helix transition of the polypeptide chains ofinterest in this work may indeed play significant roles in a variety of physiologically important processes.Item Academia Arcana: A Gamified and Game-based Review Tool(2016-05) Thangadurai Rajendran, Mayur 1990-; Deng, Zhigang; Fox, George E.; Shi, Weidong; Rizk, NouhadOne of the main issues faced by students in college courses is the lack of motivation towards reviewing the subject material. While there have been many advances in the fields of gamification (of educational material) and game-based learning, that may help, each of these processes are different in their implementations and goals. However, it is believed that when combined, both of these different fields can complement each other in a way that creates a more powerful learning environment. In this thesis, a preliminary research study of how game-based learning and gamification can be effectively combined to provide an engaging tool for review that college students can use during an actual semester is shown. Specifically, this project is split into two parts. The first is the development of a novel education tool to help and motivate college students to better review course materials by engaging them in the form of a game. The tool is designed for the Computer Organization and Programming course and will allow students to take on the role of a character in order to progress through the game. As they advance, their character will gain levels and change along with their comprehension of the material they are studying. If students cannot answer questions to prove their understanding on a particular topic, they will be stuck in that part of the game until they are able to do so. Second is a study based on student's use of the game during regular college semesters. Based on the outcome of the study, it is seen that our tool can be used as an additional effective form of review of course materials. While it can effectually augment traditional teaching methods, it is not meant to replace them.Item Analysis of factors influencing a species' life history tactics : computer simulation of a temperate lizard model(1981) Filiatreau, Jay Charles; Jameson, David L.; Bryant, Edwin H.; Fox, George E.; Klass, Michael R.A model based on temperate lizards is developed to examine life history tactics (LHT). Parameters included in the model are: adult size, hatchling size, clutch size, individual growth rate, incubation period, time required to produce a clutch, length of the growing/reproductive season, winter mortality, egg mortality, and both non-specific and size-specific mortality during the growing/reproductive season. The population growth rate resulting from a particular combination of parameters is determined by computer simulation of each individual in the population. An optimal LHT is determined from the relative population growth rates for a given environment. The environmental parameter with the greatest influence on the optimal LHT is size-specific mortality. When there is greater mortality for small in-- dividuals then the population with large adult size will be the optimal LHT. When there is a reduced individual growth rate the optimal LHT will oe a population where less individual growth is required to reach maturity. A shorter length of the growing/reproductive season produces an optimal LHT with a larger adult size. Changes in non-specific mortality either during the growing/reproductive season or during the winter have no effect on the optimal LHT.Item Applications of metabolic profiling to tissue analysis : shrimp mariculture and neurological tumors(1979) Hines, Harry B.; Middleditch, Brian S.; Oro, John F.; Lawrence, Addison Lee; Kimball, Bruce A.; Fox, George E.The technique of metabolic profiling which was formerly restricted largely to the investigation of human disease by examination of body fluids has been applied to problems in shrimp mariculture and human cancer in the present study. In the first instance, a failure of captive Penaeus setiferus to reproduce prompted an investigation into the sterol constitution of these animals to determine whether a dietary deficiency of these compounds existed. [...]Item Aptamers and Phage as Tools for the Development of Novel and Sensitive Point-of-Care Diagnostics(2014-08) Adhikari, Meena 1979-; Willson, Richard C.; Fox, George E.; Gao, Xiaolian; Williams, Cecilia M.; Bikram, MalavosklishImmunochromatographic lateral flow assays (LFAs) represent a well-established point-of-care diagnostic analytical method for the primary testing of diverse samples. The sensitivity of conventional LFAs that use the standard colored nanoparticles as reporters lags behind the more elaborate laboratory techniques, such as plaque counting, PCR or ELISA. To address this issue, we have introduced an innovative approach that utilizes functionalized M13 bacteriophage nanoparticles as reporters in the lateral flow diagnostic system. M13 phage offers a multitude of binding sites on its major coat protein pVIII for reporter enzymes, and for affinity agents specific for the target of interest. Furthermore we employed SAM-AviTag phage, derivatives of phage M13 in which the N-terminus of the tail protein pIII is replaced by the enzymatically-biotinylatable AviTag peptide (GLNDIFEAQKIEWHE). The lysine residue (K) in the AviTag is a substrate for biotinylation by the E. coli biotin ligase (birA) enzyme. Using streptavidin or Neutravidin, any biotinylated affinity agent can then easily be linked to these enzymatically-biotinylated phage particles. Viral nanoparticles were functionalized with target-specific antibodies and multiple copies of an enzymatic reporter (horseradish peroxidase). These particles were successfully integrated into a lateral-flow immunochromatographic assay detecting MS2 virus, a model for viral pathogens. The limit of detection of the assay was 10^4 MS2/mL, 1000-fold more sensitive than the conventional gold nanoparticle lateral flow assays, and results could easily be evaluated, even without advanced lab instruments. Aptamers are short, library-selected nucleic acid molecules that can recognize and bind to pre-selected targets with high affinity and selectivity. They have been used as recognition elements in a variety of applications. We screened for DNA aptamers specific to the murine anti-lysozyme antibody, HyHEL-5. During the validation process, however, the originally selected aptamers did not show successful binding. As an alternative, literature DNA aptamers were employed to construct phage displaying aptamers and HRP, which were used as LFA reporters. We describe the first modification of bacteriophage particles with aptamers as bio-recognition elements, and demonstrate their use in ultrasensitive lateral flow assays detecting IgE and PBP2a (Penicillin binding protein) of Staphylococcus aureus.Item Bacillus pumilus SAFR-032 Genome Revisited: Sequence Update and Re-Annotation(PLoS One, 6/28/2016) Stepanov, Victor G.; Tirumalai, Madhan R.; Montazari, Saied; Checinska, Aleksandra; Venkateswaran, Kasthuri; Fox, George E.Bacillus pumilus strain SAFR-032 is a non-pathogenic spore-forming bacterium exhibiting an anomalously high persistence in bactericidal environments. In its dormant state, it is capable of withstanding doses of ultraviolet (UV) radiation or hydrogen peroxide, which are lethal for the vast majority of microorganisms. This unusual resistance profile has made SAFR-032 a reference strain for studies of bacterial spore resistance. The complete genome sequence of B. pumilus SAFR-032 was published in 2007 early in the genomics era. Since then, the SAFR-032 strain has frequently been used as a source of genetic/genomic information that was regarded as representative of the entire B. pumilus species group. Recently, our ongoing studies of conservation of gene distribution patterns in the complete genomes of various B. pumilus strains revealed indications of misassembly in the B. pumilus SAFR-032 genome. Synteny-driven local genome resequencing confirmed that the original SAFR-032 sequence contained assembly errors associated with long sequence repeats. The genome sequence was corrected according to the new findings. In addition, a significantly improved annotation is now available. Gene orders were compared and portions of the genome arrangement were found to be similar in a wide spectrum of Bacillus strains.Item Bacillus safensis FO-36b and Bacillus pumilusSAFR-032: a whole genome comparison of two spacecraft assembly facility isolates(BMC Microbiology, 6/8/2018) Tirumalai, Madhan R.; Stepanov, Victor G.; Wünsche, Andrea; Montazari, Saied; Gonzalez, Tacquel O.; Venkateswaran, Kasthuri; Fox, George E.Bacillus strains producing highly resistant spores have been isolated from cleanrooms and space craft assembly facilities. Organisms that can survive such conditions merit planetary protection concern and if that resistance can be transferred to other organisms, a health concern too. To further efforts to understand these resistances, the complete genome of Bacillus safensis strain FO-36b, which produces spores resistant to peroxide and radiation was determined. The genome was compared to the complete genome of B. pumilus SAFR-032, and the draft genomes of B. safensis JPL-MERTA-8-2 and the type strain B. pumilusATCC7061T. Additional comparisons were made to 61 draft genomes that have been mostly identified as strains of B. pumilus or B. safensis.Item Bacterial genotyping by 16S rRNA mass cataloging(BMC Bioinformatics, 2006-06) Jackson, George W.; McNichols, Roger J.; Fox, George E.; Willson, Richard C.Background: It has recently been demonstrated that organism identifications can be recovered from mass spectra using various methods including base-specific fragmentation of nucleic acids. Because mass spectrometry is extremely rapid and widely available such techniques offer significant advantages in some applications. A key element in favor of mass spectrometric analysis of RNA fragmentation patterns is that a reference database for analysis of the results can be generated from sequence information. In contrast to hybridization approaches, the genetic affinity of any unknown isolate can in principle be determined within the context of all previously sequenced 16S rRNAs without prior knowledge of what the organism is. In contrast to the original RNase T1 cataloging method, when digestion products are analyzed by mass spectrometry, products with the same base composition cannot be distinguished. Hence, it is possible that organisms that are not closely related (having different underlying sequences) might be falsely identified by mass spectral coincidence. We present a convenient spectral coincidence function for expressing the degree of similarity (or distance) between any two mass-spectra. Trees constructed using this function are consistent with those produced by direct comparison of primary sequences, demonstrating that the inherent degeneracy in mass spectrometric analysis of RNA fragments does not preclude correct organism identification.Results: Neighbor-joining trees for important bacterial pathogens were generated using distances based on mass spectrometric observables and the spectral coincidence function. These trees demonstrate that most pathogens will be readily distinguished using mass spectrometric analyses of RNA digestion products. A more detailed, genus-level analysis of pathogens and near relatives was also performed, and it was found that assignments of genetic affinity were consistent with those obtained by direct sequence comparisons. Finally, typical values of the coincidence between organisms were also examined with regard to phylogenetic level and sequence variability. Conclusion: Cluster analysis based on comparison of mass spectrometric observables using the spectral coincidence function is an extremely useful tool for determining the genetic affinity of an unknown bacterium. Additionally, fragmentation patterns can determine within hours if an unknown isolate is potentially a known pathogen among thousands of possible organisms, and if so, which one.Item Biochemical mechanism of action of certain glucoside and nucleoside dialdehyde inhibitors(1985) Kalbag, Suraj S.; Kimball, Aubrey P.; Geanangel, Russell A.; OroÌ�, John F.; Nelson, Daniel A.; Fox, George E.The periodate-oxidation products of 4-methyl umbell- iferyl 7-[beta]-D-glucoside (MUGox), n-dodecyl [beta]-D-glucoside (DGox), n-amyl-[beta]-D-glucoside (AGox), 6,7 dihydroxy 6-[beta]-D- glucoside (esculinox), 4-hydroxy phenyl [beta]-D-glucoside (arbutinox), salicyladehyde-B-D-glucoside (helicinox), indoxyl-[beta]-D-glucoside (IGox) and [beta]-Naphthyl [delta]-D-glucoside (N[alpha] Gox) were synthesized and screened for activity against L1210 leukemia in-vitro. MUGox, DGox, Arbutinox and Helicinox were the most active, having IC50 values of 40, 48, 44 and 47 [micrometers], respectively. MUGox was found to inhibit DNA, RNA and protein synthesis significantly in L1210 cells and the cytotoxicity resulted possibly through a block in the G2+ M phase of the cell cycle. Its effects on the in-vitro activity of 2- cycle purified bovine brain tubulin were examined. MUGox. inhibited tubulin assembly only minimally at 100 [micrometers] but stabilized assembled microtubules from depolymerization at the same concentration. [...]Item Biochemical mechanism of action of Pseudouridinedi-carboxaldehyde(1982) Clark, Judith A.; Fox, George E.; Kimball, Aubrey P.; Middleditch, Brian S.; Oró, John F.; Bear, John L.This study was undertaken to determine the biochemical mechanism of action of Pseudouridinedicarboxaldehyde ( [psi]-OX). The systematic name for this compound is (2R,4S)-2-hydroxymethyl-4-(2',,4'-dioxopyrimidyl)-3-oxapentane-l,5-dial. In this study, a time-decay [ H3 ] colchicine-binding assay was done to determine whether [Pseudouridinedicarboxaldehyde] decreased the amount of tubulin dimers in L1210 cells. L1210 cells which were enriched in the G2 phase of the cell cycle were used as a control. Several experiments were done to study the possible conversion of [Pseudouridinedicarboxaldehyde] to a metabolite which could be competing with the colchicine-binding site of tubulin. Tubulin was polymerized in the presence of [Pseudouridinedicarboxaldehyde] to observe the effect of the drug on microtubule formation. Treadmilling studies were done to determine whether [Pseudouridinedicarboxaldehyde] was inhibiting the depolymerization of microtubules. Immunofluorescence studies were done to observe the effect of [Pseudouridinedicarboxaldehyde] on microtubules in L1210 cells. [...]Item Bioluminescent enzyme immunoassay for captopril(1988) Chen, Huey-Ju; Tu, Shiao-Chun; Gray, Horace B., Jr.; Fox, George E.; Faith, RobertCaptopril is an orally active antihypertension agent. A sensitive method for captopril quantitation in biological fluid is highly desired for clinical and diagnostic practices. The few assay methods available so far do not fully satisfy these needs. In this project, a bioluminescent enzyme immunoassay for captopril has been developed. Polyclonal anti-captopril antibody was induced with captopril-protein conjugate in rabbits. A captopril derivative bearing a thiopyridine was linked to an essential cysteinyl thiol of Vibrio harveyi luciferase through a mixed disulfide linkage. The luciferase is catalytically inactive in this conjugated form but can be released from the conjugate and activated by reduction with dithiothreitol. A fixed quantity of anti- captopril antibody was allowed to react with a constant level of the Captopril-Iuciferase conjugate and various amounts of captopril. An excess amount of immobilized goat anti-rabbit immunoglobulin was added subsequent to a period of incubation. After separating the immunoprecipitate from the soluble phase by centrifugation, the inactive luciferase in the precipitated Captopril-luciferase conjugate was reactivated by dithiothreitol and the in vitro bioluminescence activity was determined. The higher the amount of non-labeled captopril is in the original sample, the lower the luciferase activity that will be detected. [...]Item Centers of motion associated with EF-Tu binding to the ribosome(RNA Biology, 4/27/2016) Paci, Maxim; Fox, George E.Structural centers of motion (pivot points) in the ribosome have recently been identified by measurement of conformational changes in rRNA resulting from EF-G GTP hydrolysis. This series of measurements is extended here to the ribosome’s interactions with the cofactor EF-Tu. Four recent EF-Tu bound ribosome structures were compared to unbound structures. A total of 16 pivots were identified, of which 4 are unique to the EF-Tu interaction. Pivots in the GTPase associated center and the sarcin-ricin loop omitted previously, are found to be mobile in response to both EF-Tu and EF-G binding. Pivots in the intersubunit bridge rRNAs are found to be cofactor specific. Head swiveling motions in the small subunit are observed in the EF-Tu bound structures that were trapped post GTP hydrolysis. As in the case of pivots associated with EF-G, the additional pivots described here are associated with weak points in the rRNA structures such as non-canonical pairs and bulge loops. The combined set of pivots should be regarded as a minimal set. Only several states available to the ribosome have been presented in this work. Future, precise crystal structures in conjunction with experimental data will likely show additional functional pivoting elements in the rRNA.Item Characterization of harveyicin and demonstration of competitive dominance in the bacteriocin producing strain of Beneckea harveyi sy(1981) Hoyt, Peter Robinson; Sizemore, Ronald K.; Howe, Nathan R.; Cowles, Joe R.; Fox, George E.Beneekea (Lucibacterium) harveyi, a marine, bioluminescent enteric bacterium, was the first marine bacterium shown to produce a bacteriocin-like substance mediated by a plasmid. Characterization of this bacteriocin-like substance, and determination of its activity under in vitro and simulated in situ conditions, were the objectives of this study. This harveyicin was shown to be proteinaceous by its sensitivity to certain proteolytic enzymes. The non-dialyzable nature of this bacteriocin allowed for its concentration under negative pressure. Fractional precipitation by ammonium sulfate also produced high recovery of bacteriocin. [...]Item Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces(Microbiome, 4/8/2019) Sielaff, Aleksandra Checinska; Urbaniak, Camilla; Mohan, Ganesh Babu Malli; Stepanov, Victor G.; Tran, Quyen; Wood, Jason M.; Minich, Jeremiah; McDonald, Daniel; Mayer, Teresa; Knight, Rob; Karouia, Fathi; Fox, George E.; Venkateswaran, KasthuriThe International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth.Item Chromatin structure and histone acetylation in the yeast Saccharomyces cerevisiae(1986) Alonso, William R.; Nelson, Daniel A.; Fox, George E.; Hecht, Ralph M.; Tu, Shiao-Chun; Wells, Dan E.The repeating unit of chromatin is the nucleosome, which consists of 200 base pairs of supercoiled DNA wrapped around an octamer comprised of two each of the histones H2A, H2B, H3 and H4. All eukaryotes enzymatically acetylate and deacetylate specific lysine residues within the aminoterminal regions of the histones, potentially altering the conformation of local domains within the chromatin. The nature of yeast chromatin and role of histone acetylation in modulating chromatin structure and gene expression were investigated in the yeast Saccharomyces cerevisiae. One specific goal was to ascertain the levels of histone acetylation in yeast cells during different phases of the growth curve. The histones in the mitotically active log phase yeast were strikingly maintained in a hyperacetylated form, unlike the histones in most higher eukaryotic cells which are predominantly mono- or nonacetylated. The levels of histone acetylation and rate of acetate incorporation were reduced in the stationary phase yeast cells, suggesting a general relationship between decreased genome activity and diminished histone acetylation. In the course of these studies a unique nuclear histone deacetylase was discovered. Subsequently, a high molecular weight deacetylase complex (10^6) was isolated and partially characterized. A striking feature of the yeast histone deacetylase was that it was not inhibited by n-butyrate, a potent inhibitor of all other known histone deacetylases. Finally, the solubility of polynucleosomes and nucleosome cores isolated from yeast and chicken erythrocytes were compared in buffers containing mono- and divalent cations. Yeast polynucleosomes are considerably more soluble than the chicken erythrocyte nucleohistone. Furthermore, yeast chromatin solubility was not affected by the extent of histone acetylation or by gene induction, parameters known to enhance the solubility of polynucleosomes isolated from chicken erythrocytes. It was concluded that the absence of a linker histone (H1) in yeast and the presence of specific yeast core histone sequences were the main contributors to the remarkable yeast polynucleosome solubility. A reasonable conclusion, based on the above studies, is that the structure of yeast chromatin is unique and particularly suited to the needs of this rapidly dividing, transcriptionally active organism.Item Clues to unraveling the coral species problem: distinguishing species from geographic variation in Porites across the Pacific with molecular markers and microskeletal traits(PeerJ, 2/3/2015) Forsman, Zac; Wellington, Gerrard M.; Fox, George E.; Toonen, Robert J.Morphological variation in the geographically widespread coral Porites lobata can make it difficult to distinguish from other massive congeneric species. This morphological variation could be attributed to geographic variability, phenotypic plasticity, or a combination of such factors. We examined genetic and microscopic morphological variability in P. lobata samples from the Galápagos, Easter Island, Tahiti, Fiji, Rarotonga, and Australia. Panamanian P. evermanni specimens were used as a previously established distinct outgroup against which to test genetic and morphological methods of discrimination. We employed a molecular analysis of variance (AMOVA) based on ribosomal internal transcribed spacer region (ITS) sequence, principal component analysis (PCA) of skeletal landmarks, and Mantel tests to compare genetic and morphological variation. Both genetic and morphometric methods clearly distinguished P. lobata and P. evermanni, while significant genetic and morphological variance was attributed to differences among geographic regions for P. lobata. Mantel tests indicate a correlation between genetic and morphological variation for P. lobata across the Pacific. Here we highlight landmark morphometric measures that correlate well with genetic differences, showing promise for resolving species of Porites, one of the most ubiquitous yet challenging to identify architects of coral reefs.Item Computational Techniques Applied to RNA Structures and Bacterial Genomes(2019-08) Tran, Quyen Duc 1984-; Fox, George E.; Williams, Loren Dean; Briggs, James M.; Widger, William R.Modern molecular biology studies increasingly require customized computation support. This is especially true for studies associated with atomic resolution structures and/or NextGen sequencing. In this dissertation, three separate studies are described in which modern computational methodologies play a key role. The first case is a model system that seeks to understand how complexity might arise in a prebiotic RNA World in which RNA polymerization through an RNA replicase was not possible. The experimental system exposes an initial 20-mer seed RNA to 180 min of continuous cycles of synthesis and degradation. The resulting pools of RNA were characterized by NextGen sequencing. The seed RNA rapidly disappeared and was replaced by an increasing number and variety of both larger and smaller variants. The analysis made it possible to characterize the extent and manner in which sequence space was explored. The RNA products lacked large numbers of point mutations but instead incorporate additions and subtractions of fragments of the original RNAs. The system demonstrates that, if such equilibrium were established in a prebiotic world, it would result in significant exploration of RNA sequence space and likely increased complexity. No matter how much complexity can be generated by an RNA World alone, the ability to synthesize peptides would be a momentous discovery. In modern organisms, the PTC, where peptide bonds are fashioned, also serves as an entrance to an exit tunnel so that a growing peptide can leave. In essence, the PTC exit cavity is an evolved RNA nano-pore that accommodates the termini of the A and P-site tRNAs, which carry the activated amino acid or the nascent peptide, respectively. Is this pore unique? In examining various atomic resolution structures, it became clear that such pores can arise readily from the packing of RNA or RNA-like molecules as they increased in size. This hypothesis is supported by our finding of at least five additional pores in the 23S rRNA that range 1.0-1.5 nm in size. Examination of other RNAs identified 11 other RNA pores. Structurally, the PTC and other 23S rRNA pores are very similar, i.e. both are created from the packing of helices, but RNA bases in the latter do not protrude into the pore. Instead, these pores are lined by negatively charged backbone atoms. While the additional 23S rRNA pores seem to lack catalytic function, they illustrate that random RNAs would not have to be very large to create a variety of pores and voids. Thus, pores of the PTC type may have occurred with some frequency in an RNA World, thereby allowing the rapid emergence of primitive translation machinery. Finally, a detailed examination of structural conservation in a ribosomal RNA was undertaken. 5S ribosomal RNA is thought to assist communication between the PTC region of the ribosome and the decoding center on the small ribosomal subunit. This is facilitated by changes in the orientation of 5S rRNA. The canonical 5S rRNA secondary structure includes 30 non-standard base-base interactions. The initial expectation was that these interactions would change as the ribosome went through its synthesis cycle. Forty atomic resolution structures of 5S rRNAs from both different organisms and different stages in the translation cycle were examined. The set of non-standard base pairs continues to be conserved in essentially all of the structures. This strongly suggests that the 5S rRNA behaves as a rod with structural changes originating from the backbone.