Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Environmental Health Sciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pharmacology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005. / "December 2005" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

CP1 is a sequence specific DNA-binding protein of the yeast Saccharomyces cerevisiae which recognizes the highly conserved centromere DNA element I (CDEI) of yeast centromeres. The gene encoding CP1, which was designated CEP1 for centromere protein 1, was cloned and sequenced. CEP1 encodes a highly acidic protein of molecular weight 39,400. CEP1 was mapped to a position 4.6 centiMorgans centromere distal to SUP4 on the right arm of chromosome X. Phenotypic analysis of cep1 mutants demonstrated that yeast strains lacking CP1 are viable but have a 35% increase in cell doubling time, a ninefold increase in the rate of mitotic chromosome loss, and are methionine auxotrophs. Detailed analysis of the mitotic chromosome-loss phenotype showed that the loss is primarily due to chromosome nondisjunction (2:0 segregation). During meiosis cep1 null mutants exhibited aberrant segregation of centromere containing plasmids, chromosome fragments, and chromosomes. The predominant missegregation event observed was precocious sister segregation. The mutants also displayed a nonrandom 20% decrease in spore viability. Missegregation of chromosomes accounted for some but not all of this decreased spore viability, the remainder of which is presumed to be related to the pleiotropic consequences of the cep1 mutation. Together with the observed mitotic missegregation phenotype the results are interpreted as suggesting that CP1 promotes sister chromatid-kinetochore adhesion. The following conclusions are based on my mutational analysis of CP1: (1) CP1 is normally present in functional excess, (2) the C-terminal 143 amino acids are sufficient for full CP1 function in chromosome segregation and methionine metabolism, and (3) while DNA binding is apparently necessary for function, DNA binding per se is not sufficient. All of the mutations which caused an observable phenotype affected both centromere function and methionine metabolism. In addition, a direct correlation was observed in the degree to which both phenotypes were affected by different mutations. None of the mutant proteins displayed trans-dominant effects in a wild type background; however, two nonfunctional DNA binding-competent mutants exerted a dominant negative effect on the ability of PHO4 to suppress cep1 methionine auxotrophy. The data are consistent with a model in which CP1 performs a similar function at centromeres and promoters.

The regulation of protein function through oligomerization is a common theme in biological systems. In this work, I have focused on the effects of the oligomeric states of the human Rad52 protein on activities related to DNA binding. HsRad52, a member of the RAD52 epistasis group, is thought to play an important and as yet undefined role in homologous recombination. HsRad52 preferentially binds to ssDNA over dsDNA and stimulates HsRad51-mediated strand exchange (Benson et al., 1998). In either the presence or absence of DNA, HsRad52 has been observed to form both 10 nm ring-like structures as well as higher order oligomers consisting of multiple 10 nm rings (Van Dyck et al., 1998; Van Dyck et al., 1999). Earlier protein-protein interaction studies mapped the domain responsible for HsRad52 self-association in the N-terminus (residues 85-159) (Shen et al., 1996). Data presented here identifies a novel self-association domain in the C-terminus of HsRad52 that is responsible for the formation of higher order oligomers. VanDyck et al. observed DNA ending binding complexes consisting of multiple rings (Van Dyck et al., 1999). They proposed that these higher order oligomers may be functionally relevant. In this work, we demonstrate that DNA binding depends on neither ring shaped oligomers nor higher order oligomers but that activities of HsRad52 that require simultaneous interaction with more than one DNA molecule depend on the formation of higher order oligomers consisting of multiple HsRad52 rings. Early studies of HsRad52 proposed that the DNA binding domain resides in the highly conserved N-terminus of the protein (Park et al., 1996). A series of studies using truncation mutants of HsRad52 have provided evidence that supports this hypothesis. For example, we demonstrated that a truncation mutant containing only the first 85 residues of the protein is still able to bind DNA (Lloyd, submitted 2002). In this study, we demonstrate that aromatic (Y65, F79 and Y81) and hydrophobic (L43, I52 and I66) residues within the N-terminus contribute to DNA binding by either directly contacting the DNA or by stabilizing the structure of the protein. In summary, through the work presented in this dissertation, we have determined that the formation of 10 nm rings is mediated by a self-association domain in the N-terminus and that the formation of higher order oligomers consisting of multiple HsRad52 rings is mediated by an additional self-association domain in the C-terminus. We have correlated the oligomeric properties of HsRad52 with its biochemical functions related to DNA binding. Additionally, we have demonstrated that aromatic and hydrophobic residues contribute to DNA binding. Further studies will differentiate between the contribution of these residues to the DNA binding by stabilizing the overall structure of the protein versus making specific DNA contacts. Additional studies will also address how the oligomeric state of HsRad52 contributes to its role in HsRad51-mediated strand exchange.

CP1 (encoded by the gene CEP1) is a sequence-specific DNA-binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 are viable but exhibit defects in growth, chromosome segregation, and methionine biosynthesis. To investigate the basis of the methionine requirement, a YEp24-based yeast genomic DNA library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. PHO4 encodes a factor which positively regulates transcription of genes involved in phosphate metabolism via an interaction with CDEI-like elements within the promoters of these genes. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80, and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was dependent on a second transcriptional regulatory protein encoded by PHO2. Spontaneously arising extragenic suppressors of the cep1 methionine auxotrophy were also isolated; approximately one-third of the them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low-Pi growth deficiency of a pho84 mutant. The results suggest that CP1 functions as a transcriptional regulator of MET genes, and that activation of PHO4 restores expression to those genes transcriptionally-disabled by the cep1mutation. The results also suggest the existence of a network that cross-regulates transcription of genes involved in methionine biosynthesis and phosphate metabolism. A direct molecular approach to investigate CP1's role in MET gene expression was also taken. CDEI sites are associated with the promoter regions of most MET genes, but only MET16, the gene encoding PAPS reductase, has been shown to require CP1 for expression; both PAPS reductase activity, and MET16 mRNA are absent in cep1 mutants. Results of the present study demonstrate that CP1 participates in two systems which regulate expression of MET16, one triggered by methionine starvation and requiring the transactivator MET4 (pathway-specific control), and the other triggered by starvation for many different amino acids and requiring GCN4 (general control). CP1 was shown to mediate its regulatory function through the upstream CDEI site, and to act directly or indirectly to modulate the chromatin structure of the MET16 promoter. In addition, the pho80 mutation was found to partially restore MET16 expression to the cep1 strain, confirming the proposed nature of PHO4 suppression. A second methionine biosynthetic gene MET25, was also analyzed. Like MET16, MET25 was found to be regulated by both pathway-specific and general control mechanisms, but in contrast to MET16, CP1 only participated in the pathway-specific response of this gene. The results demonstrate that CP1, possibly by modulating changes in chromatin structure, assists the regulatory proteins MET4 and GCN4 in activating transcription of MET genes.

Salicylic acid (SA) is essential to the establishment of both local and systemic acquired resistance (SAR) against a wide range of phytopathogens. Isochorismate synthase 1 (ICS1) is the key enzyme involved in the synthesis of SA and it is transcriptionally activated by the regulatory proteins SAR deficient 1 (SARD1) and Calmodulin binding protein 60g (CBP60g). It has been demonstrated previously that the loss-of-function mutant, S-nitrosogluthione reductase 1-3 (gsnor1-3), increased cellular levels of S-nitrosylation. Significantly, accumulation of both free SA and its storage form SA-glucoside (SAG), were substantially reduced, disabling multiple SA-dependent immune responses. However, the molecular mechanism underlying this observation remains to be established. Our data suggests that the transcription of ICS1 and it regulators, SARD and CBP60g, are reduced in the gsnor1-3 mutant, implying that increased cellular S-nitrosylation blunts the expression of ICS1 by reducing the transcription of its activators. We demonstrated that SARD1 is S-nitrosylated in vitro resulting in inhibition of its DNA binding activity. Further, Cys438 of SARD1 was found to be the site of S-nitrosylation, demonstrated by the observation that the SARD1 C438S mutant was insensitive to NO regulation in regard to DNA binding activity.

abstract: Many therapeutics administered for some of the most devastating illnesses can be toxic and result in unwanted side effects. Recent developments have been made in an alternative treatment method, called gene therapy. Gene therapy has potential to rectify the genetic defects that cause a broad range of diseases. Many diseases, such as cancer, cystic fibrosis, and acquired immunodeficiency (AIDS) already have gene therapy protocols that are currently in clinical trials. Finding a non-toxic and efficient gene transfer method has been a challenge. Viral vectors are effective at transgene delivery however potential for insertion mutagenesis and activation of immune responses raises concern. For this reason, non-viral vectors have been investigated as a safer alternative to viral-mediated gene delivery. Non-viral vectors are also easy to prepare and scalable, but are limited by low transgene delivery efficacies and high cytotoxicity at effective therapeutic dosages. Thus, there is a need for a non-toxic non-viral vector with high transgene efficacies. In addition to the hurdles in finding a material for gene delivery, large-scale production of pharmaceutical grade DNA for gene therapy is needed. Current methods can be labor intensive, time consuming, and use toxic chemicals. For this reason, an efficient and safe method to collect DNA is needed. One material that is currently being explored is the hydrogel. Hydrogels are a useful subclass of biomaterials, with a wide variety of applications. This class of biomaterials can carry up to a thousand times their weight in water, and are biocompatible. At smaller dimensions, referred to as micro- and nanogels, they are very useful for many biomedical applications because of their size and ability to swell. Based on a previously synthesized hydrogel, and due to the advantages of smaller dimension in biomedical applications, we have synthesized aminoglycoside antibiotic based nanogels and microgels. Microgels and nanogels were synthesized following a ring opening polymerization of epoxide-containing crosslinkers and polyamine-containing monomers. The nanogels were screened for their cytocompatibilities and transfection efficacies, and were compared to polyethylenimine (PEI), a current standard for polymer-mediated transgene delivery. Nanogels demonstrated minimal to no toxicity to the cell line used in the study even at high concentrations. Due to the emerging need for large-scale production of DNA, microgels were evaluated for their binding capacity to plasmid DNA. Future work with the aminoglycoside antibiotic-based nanogels and microgels developed in this study will involve optimization of nanogels and microgels to facilitate in better transgene delivery and plasmid DNA binding, respectively. / Dissertation/Thesis / M.S. Chemical Engineering 2014