A comprehensive phylogenetic study of the core genome of thirteen Bacillus and related species using known and novel techniques

Includes supplementary digital materials.Advisors: Mitrick A. Johns.Committee members: Melvin R. Duvall; Gordon D. Pusch; Thomas L. Sims; Wesley Swingley.A comprehensive study into the phylogeny of the Bacillus core genome was accomplished using two primary studies. The first study involved two previously studied methods, MUMmer and BSR, and one novel approach, RINC, to compare core genome phylogeny. BSR analysis revealed genomic rearrangements among the genomes of interest. MUMmer was used to confirm these genomic rearrangements and provide evidence that 76.2% of the inverted regions were statistically significant. Circular chromosome comparisons connecting homologous core genes revealed an ancestral inversion pivoted on the terminus in several species. The inversions were resolved, allowing for the identification of the location of the core genome before the inversion event. These analyses led to development of a novel approach to core genome phylogeny, RINC. Based on the tree produced, as well as agreement of groups of nodes with previous studies, the RINC approach was successful at comparing the core genome of Bacillus. RINC offers a simplistic yet powerful tool for core genome phylogeny.In the second study, core genomes of Bacillus and Eudicot were determined individually from whole genome sequences of members of each genus, along with closely related species using BSRs. The Eudicot core genome was used as a control for evidence that results observed in the Bacillus core genomes were not due to horizontal gene transfer. Each core genome was analyzed with MrBayes and BUCKy to test the robustness of both approaches using sequences from different domains of life. Three multiple sequence alignments, ClustalW2, MUSCLE, and T-COFFEE, were used as inputs for MrBayes. Over 75% of all genes studied had a resolved gene tree topology after one of ten million generations of MrBayes, regardless of MSA. BUCKy calculated concordance trees for both core genomes. BUCKy tree topology was not affected by the MSAs. In both core genomes, high concordance factors were found on interior nodes, defining the relationship between groups of species. Outer nodes between outgroups had lower concordance factors, leading to the conclusion that genomes of more species would need to be included in order to resolve the phylogeny from more distant relatives.Ph.D. (Doctor of Philosophy

Differentiated and exponentially growing HL60 cells exhibit different sensitivity to some genotoxic agents in the comet assay

The aim of this study was to investigate the effect of the cell differentiation status on the sensitivity to genotoxic insults. For this, we utilized the comet assay to test the DNA damage after treatment with 5 different substances with different mechanism of action in human promyelocytic HL60 cells with or without cell differentiation. A 4-hour MMS treatment induced a significant and concentration-dependent increase in DNA damage for both differentiated and undifferentiated cells, but the difference in sensitivity was only significant at the highest concentration. A 4-hour doxorubicin treatment did not induce DNA damage in differentiated HL60 cells, while it did in undifferentiated cells with its highest tested concentration. A one-hour etoposide treatment caused significant increase in DNA damage concentration dependently in both cell variants. This DNA damage was significantly higher in undifferentiated HL60 cells with several tested concentrations of etoposide. The treatment with the oxidizing substances hydrogen peroxide and potassium bromate yielded significant DNA damage induction in both undifferentiated and differentiated cells with no difference according to the differentiation status. Doxorubicin and etoposide are known to inhibit topoisomerase II. The activity of this enzyme has been shown to be higher in undifferentiated actively proliferating cells than in differentiated cells. This may be of relevance when exposures to topoisomerase-inhibiting compounds or the genotoxicity of compounds with unknown mechanism of action are assessed in routine testing

Exposure Setup and Dosimetry for a Study on Effects of Mobile Communication Signals on Human Hematopoietic Stem Cells in vitro

In this paper we describe the design of an exposure setup used to study possible non-thermal effects due to the exposure of human hematopoietic stem cells to GSM, UMTS and LTE mobile communication signals. The experiments are performed under fully blinded conditions in a TEM waveguide located inside an incubator to achieve defined environmental conditions as required for the living cells. Chamber slides containing the cells in culture medium are placed on the septum of the waveguide. The environmental and exposure parameters such as signal power, temperatures, relative humidity and CO2 content of the surrounding atmosphere are monitored permanently during the exposure experiment. The power of the exposure signals required to achieve specific absorption rates of 0.5, 1, 2 and 4 W kg−1 are determined by numerical calculation of the field distribution inside the cell culture medium at 900 MHz (GSM), 1950 MHz (UMTS) and 2535 MHz (LTE). The dosimetry is verified both with scattering parameter measurements on the waveguide with and without containers filled with cell culture medium and with temperature measurements with non-metallic probes in separate heating experiments

Systematic Analysis of Whole Exome Sequencing Determines RET G691S Polymorphism as Germline Variant in Melanoma

Abstract The RET proto-oncogene encodes a receptor tyrosine kinase that is activated by glial cell derived neutrotrophic factor (GDNF). Previous studies have found that a single nucleotide polymorphism (SNP), RETp (G691S), in the juxtamembrane domain enhances the signaling pathway and promotes tumor growth by GDNF in pancreatic and thyroid cancer in addition to melanoma. It is uncertain however whether this SNP is a germline variant or somatic mutation. A prior study reported that the RETp variant was a germline SNP in desmoplastic and non-desmoplastic melanomas. In the present study, we examined both melanoma tissue samples and matching peripheral blood DNA to determine if RETp was 1) a germline or somatic variant, 2) more frequent in certain melanoma subtypes, and 3) frequency in brain metastasis. We examined the peripheral blood of 197 melanoma patients whom had at least one matched tumor, and 42 patients with brain metastasis. RETp was present as a germline SNP in 33% of patients. There were no significant differences in RETp frequency among the different melanoma subtypes, and RETp was not correlated with brain metastasis

Systematic Analysis of Whole Exome Sequencing Determines RET G691S Polymorphism as Germline Variant in Melanoma

The RET proto-oncogene encodes a receptor tyrosine kinase that is activated by glial cell derived neutrotrophic factor (GDNF). Previous studies have found that a single nucleotide polymorphism (SNP), RETp (G691S), in the juxtamembrane domain enhances the signaling pathway and promotes tumor growth by GDNF in pancreatic and thyroid cancer in addition to melanoma. It is uncertain however whether this SNP is a germline variant or somatic mutation. A prior study reported that the RETp variant was a germline SNP in desmoplastic and non-desmoplastic melanomas. In the present study, we examined both melanoma tissue samples and matching peripheral blood DNA to determine if RETp was 1) a germline or somatic variant, 2) more frequent in certain melanoma subtypes, and 3) frequency in brain metastasis. We examined the peripheral blood of 197 melanoma patients whom had at least one matched tumor, and 42 patients with brain metastasis. RETp was present as a germline SNP in 33% of patients. There were no significant differences in RETp frequency among the different melanoma subtypes, and RETp was not correlated with brain metastasis

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The current assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n=14) containing 32,928 and 36,697 protein-coding genes, respectively. The Petunia lineage has experienced at least two rounds of paleohexaploidization, the older gamma hexaploidy event, which is shared with other Eudicots, and the more recent Solanaceae paleohexaploidy event that is shared with tomato and other Solanaceae species. Transcription factors that were targets of selection during the shift from bee- to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral color patterns and pollination systems. The high quality genome sequences will enhance the value of Petunia as a model system for basic and applied research on a variety of unique biological phenomena

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms