Cse4p is a centromere-specific chromatin protein with a histone-fold domain that is greater than 60% identical to histone H3 and the mammalian centromere protein, CENP-A. Cse4p has similar biochemical properties to H3 and is believed to replace H3 in centromere-specific nucleosomes in yeast. To identify residues in the histone-fold domain of Cse4p that function in specifying centromere structure and function, amino adds that differ between Cse4p and H3 were systematically changed to analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The severity of the phenotypes exhibited by the histone-fold domain mutants correlates directly with the number of putative DNA contact sites changed or alterations in regions implicated in histone interactions. These results indicate that the histone-fold domain of Cse4p functions through a cooperative mechanism in which residues throughout the histone-fold domain recognize centromere DNA. To investigate the relationship between the histone-fold domain of Cse4p and centromere DNA, chromosome loss rates were measured in double mutants carrying both a mutation in Cse4p and chromosomes with mutant centromere DNA. The Cse4p mutants had conserved changes in which analogously positioned residues in H3 were substituted for Cse4p residues at sites in the histone-fold domain that are in close proximity to the DNA. Mutations throughout the histone-fold domain caused significant increases in chromosome loss rates of chromosomes carrying conserved centromere DNA elements CDE I and II but showed no effect with CDE III mutant centromeres. This genetic evidence strongly supports direct interactions between Cse4p and CDE I and CDE II centromere DNA elements. The results are discussed in the context of the known structure of H3 and models are proposed which best describe the path of the centromere DNA around a Cse4p variant nucleosome that relies on critical contacts between CDE I and II, but not CDE III.

As availability of clean water resources decreases globally, evaluating water quality sensitively and reliably is becoming critical. Emerging contaminants, especially those categorized as endocrine disruptors, are of concern because of limited knowledge regarding non-target effects. Quantifying changes in the expression of vitellogenin, a gene normally expressed only in female fish but known to be induced in male fish exposed to estrogens, is a sensitive biomarker of estrogenic contamination in water. However, the utility and limitations of vitellogenin-gene-induction as a reliable bioassay are poorly defined. The work described here characterizes the assay by examining its sensitivity to experimental variables. After exposure to low levels of estradiol, two species demonstrated robust time- and concentration-dependent induction of vitellogenin mRNA. Exposure to a concentration gradient of environmental estrogens demonstrated that the Japanese medaka bioassay detects low, environmentally relevant levels of ethynylestradiol in a short laboratory assay, but does not detect low concentrations of a weakly estrogenic contaminant, bisphenol A. Although the expression of other genes was altered, the changes were not as robust as the vitellogenin response. The robustness of the vitellogenin bioassay was further investigated by manipulating the volume of test-water, the length of time between exposure and assessment, housing conditions, and access to food. The volume of test-water had no effect on the bioassay as all groups exposed to 100pM of estradiol expressed similar levels of vitellogenin. Furthermore, estradiol-induced vitellogenin levels remained high even after 72 hours of depuration in clean water and despite 72 hours of food deprivation while confined in field cages. These results demonstrate that the vitellogenin response is robust, reliable, and resistant to stresses associated with field applications. The effects of diclofenac, a pharmaceutical frequently detected in the environment, were assessed with the vitellogenin bioassay and cDNA microarray. Analyses revealed no robust differential regulation of gene expression and suggested that previous reports as to mechanisms of the drugs pathology are likely misleading. The information gained from this work forms a foundation of knowledge on gene expression bioassays in fish that will help guide implementation of the assays and interpretation of their data in a useful and appropriate way.

In this study, a transition state analogue (TSA) approach was employed for the isolation of catalytic antibodies with esterase or amidase activity. The TSA used in this study, phosphonamidate, mimics the transition state of a corresponding amide or ester hydrolysis reaction. We applied hybridoma technology to immortalize the germinal center B cells that were induced by the TSA, and antibodies and their genes were isolated from more than a hundred hybridoma cell lines. Degenerate primers were designed for “universal” amplification of mouse Ig genes. By using these degenerate primers, we have achieved a 100% amplification rate for more than a hundred Vκ and VH mouse Ig genes we have sampled. The germinal center (GC) diversity in response to the TSA was addressed by sequence analysis of 63 Vκ and 54 VH Ig genes isolate from GC derived and TSA positive hybridoma cell lines. Germline genes for all the Vκ and for some of the VH cDNAs were identified by aligning them to Vκ and VH germline databases. It was found that multiple germline families of Ig genes were involved in the immune response to the TSA. Somatic hypermutations were introduced into those Ig genes isolated from the hybridoma cell lines which had undergone isotype switching. Canonical conformations of the CDR for the sequenced antibodies were predicted by the available software. Preliminary data on the kinetic study on several TSA positive antibodies had showed that moderate esterase activities with kcat/Km up to 1,000M have been detected. Our results showed that the immune response to a specific TSA immunization was highly diverse, demonstrated by the multiple Ig germline gene involvement, unrestricted Vκ and VH combination to form an antibody, and extensive somatic hypermutations on the class switched antibodies. Thus, GC B cells provide an diverse repertoire of antibody molecules and the limitation of isolating catalytic antibodies by the TSA approach is how to efficiently screen this huge immune repertoire.

Cytokinesis, the process of physically separating cells for division, requires the precise orchestration of numerous physical, mechanical, chemical and biological processes. For these processes to function well, complex coordination of various proteins, with crosstalk between them, either as signaling molecules or as just plain structural components that contribute to the physical separation must exist. Actin, a structural polymer and myosin, a motor are two proteins that contribute to this process significantly. Both proteins are assembled in the contractile ring and together are responsible for the process of constriction. A thorough understanding of the behavior of these proteins, in the contractile ring as well as outside in a cell undergoing cytokinesis is therefore important to prevent possible defects that might lead to deleterious diseases. In this dissertation research, a combination of techniques are made use of, that involve live imaging of fluorescently labeled proteins in cells undergoing cytokinesis along with the use of drugs that either disrupt the structure (integrity) or function of cytokinetic proteins. I generated two LLCPK1 (pig epithelial cell lines); one that stably expresses GFP-actin and the other that stably expresses Tandemn Dimer RFP-myosin regulatory light chain (TDRFP-MRLC). Live imaging and analysis of cells expressing GFP-actin shows that actin in the contractile is highly dynamic and need to be dynamic. Evidence is presented for new roles of Myosin II, in addition to generating the force for cytokinesis. Myosin not only contributes to disassembly of actin in the contractile ring but is also required to maintain actin in the equatorial region. Live imaging of the cell lines that expresses TDRFP-MRLC or GFP-actin helped in the better understanding of the role of microtubules in simultaneously regulating actin and myosin dynamics, not only in the contractile ring to allow ingression, but also in preventing contractile activity outside in the contractile ring. Cytokinesis involves other proteins besides actin and myosin, which help in their recruitment, assembly, ingression and subsequent disassembly. Decreasing the accumulation of actin in the contractile ring, by treatment with Latrunculin B facilitated the examination of spatial and temporal events involved in building the ring. Actin, myosin and other proteins organized as nodes that coalesce during ingression, similar to the fission yeast. We conclude that this mode of cytokinesis a highly conserved feature of cytokinesis.

Dissimilatory Fe(III) and Mn(IV) reduction is an anaerobic respiratory process common to many environments. A large number of bacteria that utilize these metals as terminal electron acceptors have been isolated and identified for the past decade. However, the molecular mechanisms of metal reduction remain unclear despite intensive research in this area. There are no genes or proteins identified, which are directly involved in metal reduction. The major goal of this work is to identify and isolate genes related to Fe(III) and Mn(IV) reduction as well as to determine the specific functions of their products. The organism used in this work is the facultatively anaerobic metal reducer S. putrefaciens MR-1. Transposon mutagenesis was used to generate mutants solely deficient in Fe(III) and Mn(IV) reduction. Analysis of the interrupted regions in two such mutants, SR-8 and SR-21, led to the identification of three genes, designated mtrC, mtrA and mtrB. The deduced amino acid sequence of mtrC and mtrA revealed that these genes encode deca-heme c-type cytochromes. The third gene, mtrB, was shown to encode an outer membrane protein of 679 amino acids. mRNA and Western blot analyses indicated that the three genes are organized in a single operon, mtrCAB, which is expressed constitutively. To elucidate the role of each protein in metal reduction, gene replacement was used to generate strains lacking either mtrC, mtrA or mtrB. Whole-cell suspensions of mtrC−A+B+, mirC+A−B+ and mtrC+A+B− mutants resulted in 4.6-fold, 31-fold and 70-fold decrease in iron reduction rates, respectively. In contrast to whole cells, the levels of iron reductase activity in the crude cell extracts of the mutants were similar to that observed in the wild type. These findings strongly suggest that the products of mtrC, mtrA and mtrB play an important role in metal reduction, however, they may not constitute the terminal metal reductase complex. Analysis of the DNA region upstream of mtrCAB revealed a presence of another multicytochrome gene cluster. Although the identified genes, mtrD, mtrE, mtrF and omcA, exhibited high degree of similarity to the mtrCAB operon, there was no evidence indicating their involvement in Fe(III) and Mn(IV) reduction in S. putrefaciens.

Autoreactive, immature T cells (thymocytes) are deleted from the thymus during development by the process of negative selection. This mechanism occurs when the thymocyte, T cell receptor (TCR) recognizes self-antigen, causing the cell to die by an apoptotic pathway. This mechanism results in the deletion of autoreactive T cells. Thymocyte development proceeds through several stages, determined by the differential expression of the T cell co-receptor molecules CD4 and CD8. The developmental stage where negative selection occurs is one in which thymocytes are expressing a functional TCR on the cell surface along with both CD4 and CD8, termed the double positive stage. These thymocytes are in a quiescent, G0 state and make up greater than 80% of the total population. The demonstration that cell cycle progression plays a role in the apoptotic process of several quiescent cell types, as well as the requirement for mature T cells to be in the late G1 phase during activation induced cell death, suggested that thymocytes may also advance to the G1 stage of the cell cycle prior to apoptosis. It has been established that the early cell cycle genes c-fos, c-jun, and c-myc are induced in thymocytes after stimulation, indicating possible entry into the cell cycle. The hypothesis that thymocytes enter the cell cycle before undergoing apoptosis was tested by examining expression levels of the various G1 cyclins and cyclin dependent kinase inhibitors at the mRNA and protein levels. Several indications of an early G1 cell cycle transition occurring during thymocyte apoptosis were observed, such as the downregulation of p27KIP1 and p130, the upregulation of cyclin D3, and the phosphorylation of the retinoblastoma protein. Finally, the requirement for the activation of the cyclin-dependent kinase 2 (CDK2) in negative selection was examined. It was shown that the phosphorylation and expression of the TCR-mediated apoptosis-related transcription factors Nur77 and Egr1 are downstream of CDK2 activation. In addition, a protein associated with the transcription factor Egr-1 was identified as a possible target of CDK2 kinase activity.

Recent clinical evidence indicates that thyroid hormone plays an essential role in fetal brain development. However, the mechanism by which thyroid hormone affects development has been largely unexplored. Because thyroid hormone receptors (TRs) are ligand-activated transcription factors, the TR-mediated effects of thyroid hormone in the fetal brain will necessarily be manifested first by changes in gene expression. Therefore, I used differential display to identify genes expressed in the fetal brain that are affected by acute thyroxine administration to the dam before the onset of fetal thyroid function. I identified 11 putative thyroid hormone-regulated genes using differential display. Eight of these genes are selectively expressed in areas of the gestational day (G) 16 brain that contain TRs, indicating that these genes may be directly regulated by maternal thyroid hormone. Next, the distributions of three of these genes, neuroendocrine-specific protein (NSP), Oct-1, and a known thyroid hormone-regulated gene, RC3/neurogranin, were characterized. All mRNAs are expressed from at least G14 until adulthood in brain areas that contain TRs and their regulation by maternal thyroid hormone was confirmed using in situ hybridization in the G16 cortex. Additionally, I examined the effects of thyroid hormone on NSP and Oct-1 in the adult brain. I demonstrated that NSP and Oct-1 are expressed in the adult brain and are regulated by thyroid hormone. These studies provide the first evidence that maternal thyroid hormone directly affects fetal brain development by regulating the expression of specific genes in vivo. These data support the concept that maternal thyroid hormone exerts a direct action on the expression of genes that are important for normal neurological development of the fetus. Collectively, these data have clinical importance because thyroid hormone affects NSP, Oct-1, and RC3/neurogranin expression in brain regions affected in cretinism and congenital hypothyroidism. These three genes are regulated by thyroid hormone from at least G14 to adulthood and are expressed in brain areas known to be affected by hypothyroidism. These areas include the cortex, hippocampus, and cerebellum. The data presented in this dissertation provide experimental evidence that NSP, Oct-1, and RC3/neurogranin may be partially responsible for the detrimental effects of hypothyroidism in developing brain and support several recent clinical studies indicating that untreated fetomaternal hypothyoidism adversely affects fetal brain development.

The ultimate means of limiting the influence of an individual cell on the physiology of a multicellular organism is to induce the death of that cell. Apoptosis is a genetically regulated form of cell death that removes cells that are malfunctioning, unnecessary or damaged. During development, cells are produced in excess and those that are not optimal in form, location or function are removed via apoptosis. In the adult organism, apoptosis allows for the turnover of cells that have carried out specialized functions and maintains tissue homeostasis. Negative selection is the developmental process by which immature T cells that have inappropriate reactivity to self antigen are induced to undergo apoptosis. During work in the lab confirming the requirement for the orphan nuclear hormone receptor, Nur77, for thymocyte apoptosis, an upregulation of the early growth response 1 gene (egr-1) was observed. This thesis investigates the requirement for transcriptional activation mediated by EGR-1 during the apoptosis of DO11.10, a cell line model of thymocyte negative selection. A dominant negative form of EGR-1, WT1EGR1, was expressed in DO11.10. The ability of these transfectants to undergo apoptosis in response to a variety of stimuli was measured. Another important function of apoptosis is to limit the life span of activated immune cells. The inception of the second part of this work was the clinical observation that exposure of non-healing wounds to hyperbaric oxygen (HBO), 100% oxygen at elevated atmospheric pressures, aids in the healing of these wounds. The hypothesis tested here is that HBO enhances the apoptosis of immune cells. Such an enhancement would promote the resolution of chronic inflammation and aid in wound healing. It is demonstrated that HBO enhances apoptosis of immune cells in response to stimuli relevant to both the regulation of the immune system and the application of HBO as an adjuvant to anti-cancer therapy. This study provides a new approach for studying the role of oxygen and its derivatives in apoptosis. The findings also support the continued investigation of expanding the clinical application of HBO.

Clostridial neurotoxins (BoNTs) are among the most toxic substances presently known. They are a unique group of metalloproteases which catalyze single site cleavage of specific proteins involved in the docking and fusion of synaptic vesicles with plasma membrane for neurotransmitter release. BoNTs are classified into seven distinct serotypes, designated as A to G. Botulinum neurotoxins type A (BoNT/A) is produced by Clostridium botulinum type A as a complex with a group of neurotoxin associated proteins (NAPs). Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. We have examined the structure of BoNT/A in aqueous solution, and found the structure in aqueous solution differs dramatically from that resolved by x-ray crystallography, both at secondary and quaternary levels. In terms of secondary structure, BoNT/A in aqueous solution has about 47% β-sheet structure as revealed by infrared spectroscopy, while x-ray crystallography revealed only 17% β-sheet structure. In terms of quaternary structure, BoNT/A exists as a dimer in aqueous solution, which contrasts with the reported monomeric structure of BoNT/A based on the x-ray crystallography. The dimeric form of BoNT/A can self-dissociate into monomeric form at a concentration lower than 50 nM. This concentration-dependent structural change has a significant impact on the endopeptidase activity of BoNT/A: the catalytic efficiency of the monomeric BoNT/A is about 4-fold higher than that of its dimeric form. This difference implies a sterically restricted catalytic site of BoNT/A in the dimeric form of BoNT/A. Reduction of disulfide bond between heavy chain and light chain of BoNTs is required for their enzymatic activity. We are investigating structural differences of BoNT/A under reducing and non-reducing conditions. Reduction of disulfide bond between heavy and light chains of BoNT/A induced a molten globule conformation at physiological temperature (37°C). This molten globule conformation of BoNT/A has been proven to be the enzymatically active structure.

Synaptic transmission between a neuron and its target is crucially dependent upon the precise spatial arrangement of proteins in the pre- and postsynaptic apparatus. PDZ domain-containing proteins such as the Drosophila tumor suppressor Discs-Large (DLG) play critical roles in synapse maturation by regulating the assembly of synaptic protein complexes. DLG is composed of a number of modular domains, including three PDZ-domains, an SH3 domain and an enzymatically inactive Guanylate Kinase-like (GUK) domain. Previous studies have shown that the PDZ domains of DLG mediate clustering of Shaker K+ channels and of the cell adhesion molecule Fasciclin II. However, the function of the GUK domain has been unclear. To understand the role of the GUK domain, we carried out a yeast-two hybrid screen for interacting partners of the DLG GUK domain. This screen lead to the identification of a novel synapse-associated protein, GUKHolder (GUKH). GUKH is a 1044 amino acid protein with a molecular weight of 110 kDa. Its sequence includes a GUK-holding domain, a region homologous to the C-terminal of the long isoform of Kelch, a WH1-like domain, and a PDZ-domain binding motif. These latter two features suggest that GUKH may interact not only with DLG, but also with other proteins, including proteins containing PDZ domains. GUKH is expressed at the larval neuromuscular junction and at epithelial cell borders in partial colocalization with DLG. Further, DLG can be co-immunoprecipitated with GUKH from Drosophila extracts, indicating an in vivo interaction between the two proteins. GUKH has also been shown to interact directly with SCRIBBLE (SCRIB), another synaptically expressed PDZ-domain protein known to have a genetic interaction with dlg in epithelial tissues. Synaptic SCRIB immunoreactivity is mislocalized in both gukh and dlg mutants. gukh, scrib, and dlg mutants all exhibit synaptic bouton defects at the ultrastructural level. These data indicate that all three proteins are required for proper synapse maturation, and support a model that the three proteins exist in a tripartite complex, with GUKH forming a link between the other two proteins, and further, between the protein scaffolds organized by the two proteins.