The research presented here seeks to advance the understanding of the chemotaxis system in Escherichia coli through the use of a novel surface-templating approach. The chemosensory system in E. coli is an example of a two-component pathway and is common in many prokaryotes and is also found in eukaryotes. The system allows the cell to constantly sample the surrounding chemical environment, swimming toward attractants and away from repellants. The mechanism of signal propagation across the membrane to the kinase is not well understood. For many years a ligand-induced shift or rotation within a receptor dimer was thought to be alone responsible for signal propagation across the membrane bilayer. However, evidence for receptor clustering has mounted during the past decade from this lab and others. Inter-dimer interactions, more broadly, receptor clustering, has been observed making it possible for a signal to be propagated laterally as well as vertically through the membrane thus revealing a new avenue for communication and information processing within the cell. Receptor clustering is now believed responsible, in part or wholly, for the remarkable sensitivity, due to large gain, and also the system's ability to respond to a wide variety of environmental stimuli including pH, temperature, and many chemical signals. Reconstitution of this cluster, or 'brain' as referred to in some instances, is paramount in elucidating how this system functions and is the main goal of the research presented here. Reconstitution of membrane proteins has proved difficult, thus we have developed a new method to assemble a 2D array of activating cytoplasmic fragments (CFs) of the aspartate receptor of E. coli onto a lipid membrane. Histidine-tagged CFs of the Tar receptor were assembled on the surface of sonicated and extruded unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, the protein kinase CheA bound to and was activated up to 475-fold by vesicle-bound CF. Surface-assembled CF was also found to serve as a substrate of the receptor methyltransferase, CheR. Since neither significant CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it was concluded that surface-templating generates the organization among CF subunits required for biochemical activity. This novel reconstitution method will be generally applicable to other systems where signaling occurs at or near the membrane surface and also for the generation of new anchoring chemistries for the marriage of proteins to surfaces.

U14 is an evolutionary conserved small nucleolar RNA required for 18S ribosomal RNA production in the yeast Saccharomyces cerevisiae. This study attempts to identify cellular components that interact with U14 RNA with the aim of gaining insight into U14 function and identifying strategies for characterizing this activity. The objectives include: (1) development of a hypothetical secondary folding model for U14 RNA; (2) biochemical characterization of the U14 small nuclear ribonucleoprotein particle (snRNP); (3) identification of U14 binding proteins through biochemical and genetic approaches, and; (4) a genetic test to determine if U14 interacts with 18S RNA through an essential, complementary segment in U14 RNA, i.e. domain 18S-A. Two U14 consensus secondary structures were established by phylogenetic folding analysis. These structures are supported by a variety of genetic observations, but are only partially consistent with biochemical probing data developed by others. Additional research is required to determine the actual folding properties of U14 in vivo. Biochemical characterization showed that natural U14 RNA does not contain a trimethylguanosine (TMG) cap, while U14 synthesized from the GAL1 promoter does. Natural U14 occurs in two classes of RNP complexes: (i) a 10S RNP presumed to be a free snRNP and, (ii) a polydisperse, larger complex containing fibrillarin. The direct association of fibrillarin with in vitro synthesized U14 was not detected using a variety of assays. Analysis of the higher order structures sedimenting at approximately 90S revealed the presence of over one dozen snRNAs, two-thirds of which appear to be associated with fibrillarin. Several extragenic suppressor mutations were collected that relieve a lethal mutation in the box C sequence. One of these was identified as a putative sugar transport protein. It seems unlikely that this protein is directly related to U14 function per se, but may play a role in the regulation of U14 activity, possibly by influencing proteins that interact with U14 RNA or DNA. Mutations in a domain A-related sequence of 18S rDNA resulted in under-accumulation of 18S RNA, demonstrating the importance of this region for 18S RNA stability. This effect was not influenced by complementary changes in domain A of U14 RNA. The significance of these results are discussed in the context of U14 function and ribosome biogenesis.

The epidermal growth factor receptor (EGFR) plays important roles in the control of many fundamental cellular processes including cell cycle, cell migration, cell metabolism and survival, cell proliferation and differentiation, as well as regulation of oocyte maturation and embryonic development. In the first part of this work I studied the EGFR expression and activation in cumulus cells (CCs). CCs are special cells immediately surrounding the oocyte. It has been shown that CCs in the isolated cumulus cell/oocyte complexes (COCs) exhibit both a slow rise in intracellular calcium concentration ([Ca 2+]i) and plasma membrane permeabilization in response to epidermal growth factor (EGF) stimulation. But cultured individual bovine CCs rarely showed a [Ca2+]i increase. The lack of response was confirmed to be due to a decrease of expression of endogenous EGFRs after dissociation. After CCs were reconstituted EGFR expression they showed robust, prolonged, EGF-stimulated [Ca2+] i elevations characteristic of CC responses in intact COCs followed by CC permeabilization and death. These responses were also confirmed being mediated by the IP3 signaling pathway. This EGFR activated Ca 2+ response in CCs followed by cell death may play an important role in the regulation of oocyte maturation. In the second part of this work, I identified an EGFR intramolecular regulation mechanism through study of the tyrosine phosphorylation in the EGFR regulatory domain (RD). EGFR signaling is partly controlled by tyrosine phosphorylation on the RD. There are 5 major tyrosine phosphorylation residues (992, 1068, 1086, 1148 and 1173) whose phosphorylation functions as the main platform for recruitment of downstream components. In order to understand the effect of intramolecular interactions among EGFR RD tyrosine residues, we constructed a series of single site mutant RDs. Each one replaces one major tyrosine phosphorylation residue with phenylalanine. After in vitro phosphorylation, the phosphorylation degree of each major tyrosine residue was quantitatively compared between mutant RDs and wild type RD using LC-ESI Ion Trap mass spectrometry. Our results indicate that Y1068 increases the phosphorylation of Y1148 and Y1173, Y1086 inhibits the phosphorylation of Y1068, Y1148 inhibits the phosphorylation of Y992 and Y1086, and Y1173 inhibits the phosphorylation of Y1068. Thus the EGFR exhibits extensively intramolecular interaction among its major tyrosine phosphorylation sites. Such intramolecular interaction may increase the complexity of EGFR signal transduction as well as modulate its efficacy.

Genetic, biochemical, and biophysical methods have been adopted to characterize the interactions between ligand and transmembrane receptor, between receptor and cytoplasmic proteins, and between cytoplasmic proteins in the bacterial chemosensory system in order to elucidate the signaling mechanism at the molecular level. Ligand binding to the serine receptor (Tsr) of Escherichia coli has been studied using Isothermal Titration Calorimetry (ITC) to determine the number of moles of ligand bound per mole of receptor (n), the binding constant $(K\sb{\rm a}),$ and the enthalpy of binding $(\Delta H)$ of serine to the receptor. The n-value for serine binding to octyl-$\beta$-D-glucopyranoside (OG)-solubilized Tsr (n = 0.5) was consistent with one molecule of serine binding to a receptor dimer. The values for $K\sb{\rm a}$ and $\Delta H$ were equivalent for the membrane and OG-solubilized samples and were found to be $\rm 3.6\times 10\sp4\ M\sp{-1}$ and $-18$ kcal/mol at 27$\sp\circ$C. Covalent modification of Tsr at the sites of methylation was found to have only a modest effect on serine binding. The interaction between Tsr and the methyltransferase (CheR) was also studied using ITC. Tsr bound to CheR with 1 to 1 ratio and a dissociation constant of 2 $\mu$M at 29$\sp\circ$C. Ligand-binding or modifications at the methylation sites of Tsr did not affect CheR binding. Truncation of Tar cytoplasmic-fragment at the C-terminus by 16 amino acids (aa) completely abolished CheR binding. This result led to the identification of the CheR binding site which is located at the C-terminal 5-aa segment remote from the methylation regions. The C-terminal truncated and crosslinkable Tsr mutant ($\Delta$34, D36C) was then constructed, which was found to be a poor substrate for CheR by itself. Coexpression of this binding site deleted Tsr with intact Tsr rescued its methylation activity even in the crosslinked form. The mechanism of interdimer methylation was established through this study. The autophosphorylating kinase CheA donates a phosphoryl group to either of two response regulator proteins, CheY or CheB. With ITC, it was demonstrated that CheA and a CheA fragment composed of aa residues 1 to 233 (CheA$\sb{1-233}$ bound to CheY with similar dissociation constants of 2.0 and 1.2 $\mu$M at 28$\sp\circ$C, respectively, indicating that the CheY binding site is wholly within the 1-233 aa locus. CheB bound to CheA$\sb{1-233}$ with a $K\sb{\rm d}$ of 3.2 $\mu$M, and also bound to intact CheA with the same affinity. CheY was found to compete with CheB for binding to CheA$\sb{1-233}.$ Phosphorylated CheY, in the presence of 6 mM Mg$\sp{2+},$ has a significantly lower affinity for CheA (20% of unphosphorylated form), but mutations at the active site of CheY have little effect on CheY-CheA interaction, a mutation remote from the active site (A103V) produced a 10-fold reduction in $K\sb{\rm a},$ indicating the separation of the binding site from the active site and a significant conformational change upon phosphorylation.

This dissertation is in two parts. The first part investigates the role of lipid signalling pathways in an intracellular phenomenon i.e. the formation of the nuclear envelope. The second part is a study of lipid signalling pathways in extracellular phenomenon, i.e. HeLa cell adhesion and spreading on a gelatin substrate. The disassembly and formation of the nuclear envelope are crucial steps in the progression of mitosis. Nuclear envelope dynamics involve many steps and since vesicular transport, binding and fusion of vesicles are essential for the formation of the nuclear envelope it was our interest to explore whether there were any parallel pathways, such as found in the secretary pathways, that were also used in the formation of the nuclear envelope. There is little information on the proteins of membrane vesicles that reconstitute the nuclear envelope and practically none about their lipid composition. It was therefore important to determine their lipid structure in order to proceed with investigating whether, during the formation of the nuclear envelope, similar signalling pathways to those in the vesicle trafficking operate. Cytoplasmic membrane vesicle fractions (MVs) from sea urchin eggs which, contribute to the nuclear envelope were studied. The phospholipid composition of the membrane vesicles, MV1, MV2 and MV3 was determined using a novel approach for direct quantification of phospholipids from two-dimensional 31 P-1H nuclear magnetic resonance spectroscopy with isotropic mixing. MV2 and MV3 have similar compositions typical of the ER and the Golgi membranes. However, MV1 is mainly composed of phosphatidylinositol with phosphatidylcholine being the minor phospholipid present in MV1. Furthermore, we determined that phosphatidylinositol specific phospholipase C (PI-PLC) promoted nuclear envelope formation. However, in the absence of MV1, PI-PLC, did not induce nuclear envelope formation. Inhibition of membrane vesicle fusion in a dose dependent manner, by wortmannin (a specific inhibitor of the PI-3kinase pathway) suggested that the PI-3 kinase branch of the phosphatidylinositol pathway may be involved in the formation of the nuclear envelope. These experiments indicate that the inositol phospholipid pathways, as in constitutive membrane trafficking, are also involved in the formation of the nuclear envelope. Furthermore, it is the first time that a biological membrane, MV1, with such an unusual composition has been reported and that it has been demonstrated the phosphatidylinositol hydrolysis is involved in the formation of the nuclear envelope. In the second part the goal was to determine which lipoxygenase metabolites were involved in facilitating HeLa cell adhesion and spreading to a gelatin substrate. Reverse phase HPLC methods demonstrated that HeLa cell homogenates converted arachidonic acid into 12-, 15- and 5-hydroperoxyeicosatetraenoic (HETEs), indicating that 12, 15 and 5 lipoxygensases are active in HeLa cells. In order to investigate which lipoxygenase pathway are required to induce cell spreading the lipoxygenase pathway was inhibited by 25uM nordihydroguaiaretic acid (NDGA), a specific inhibitor of the lipoxygenases and MK866, a specific inhibitor of leukotriene biosynthesis. The effect of the different enantiomers of 12-BETE and 15-HETE on the reversal of NDGA inhibition was determined. The leukotrienes that overcome the inhibition of cell spreading by NDGA and MK866 were also determined. It can be concluded that 12,15 and 5 lipoxygenase enzymes are present and active in HeLa cells; and 12-HETE, 15-HETE, leukotrienes LTB4, LTD4 and LTC4 play an active role in HeLa cell spreading on a gelatin substrate.

Mutagenized fragments of the 165 rRNA gene were placed within plasmid-borne rrnB operons and expressed under the control of the rrn promoters, P1P2. Several base changes within the conserved C1400 region led to reduced host-cell growth rate, and, notably, transition mutations at position 1395 or 1407, or the deletion of base 1400, appeared to be lethal. The latter mutations were investigated by transferring them to plasmids in which the P$\sb1$P$\sb2$ promoters had been replaced with the inducible leftward promoter of the phage lambda; it was found that host cells expressing the putative lethal mutations died within 3 to 4 generations. Analysis of the rRNA and ribosomes present at 3 generations after induction of the mutant operons demonstrated that the mutant rRNA was processed to 16S rRNA and that induction of the altered rRNA genes led to significant accumulation of free ribosomal subunits. Sequencing of the rRNA in 30S subunits and 70S ribosomes showed that mutant rRNA was assembled into subunits, but that the mutant subunits were assembled into 70S ribosomes at low frequency. Furthermore, subunits containing the altered 165 rRNA were less likely to associate with 50S subunits, in vitro, than wild-type subunits, suggesting that the lethal mutations led to defective subunit association, in vivo. In addition, the conserved nucleotide G1505 was mutagenized, and it was found that 3 point mutations at position 1505 could suppress the lethal phenotype associated with the U1395, U1407 or $\Delta$1400 mutations. The doubly-mutant rRNA appeared to be fully processed to 16S rRNA. Although the suppressed strains accumulated excess ribosomal subunits, 90% of the rRNA associated with both 30S and 70S ribosomal particles contained a mutant allele at position 1505 within 3 generations after induction. The latter observation is consistent with the ribosome-feedback model of rRNA transcription control, which posits that initiation-competent 70S ribosomes exert a negative feedback effect at the rrn P1P2 promoters. The data are interpreted as evidence that nonfunctional ribosomes carrying lethal mutations in the 1400 region can be rendered functional by second-site mutations at position 1505.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves xxx-xxx. Also available online.

The objective of the first series of experiments was to assess biochemical changes during in vitro maturation of oocytes collected from ovaries of adult cattle and calves (<6 mo old). Activity and/or concentrations of maturation-promoting factor, mitogen-activated protein kinase, and inositol 1,4,5-trisphosphate receptor were determined and they were significantly lower in calf oocytes. Developmental competence of parthenogenic embryos was studied after reciprocal transfer of metaphase II chromosomes between cow and calf oocytes and transfer of cumulus cells into cow and calf ooplasts. Development was higher in embryos originating from adult ooplasts. Collectively, these results support the hypothesis that lack of developmental competence of calf oocytes is due to their failure or inability to complete ooplasmic maturation. We hypothesized that the germinal vesicle (GV) stage oocyte was capable of inducing a meiotic like division of the somatic cell nucleus. To test this hypothesis, cumulus cells in G0, G1 or metaphase (M) were transferred to GV stage bovine oocytes. Denuded GV oocytes were fused by an electrical pulse and matured in vitro. Chromosome segregation was assessed after staining with Hoechst 33342. All cell stages had similar fusion and survival rates and were capable of undergoing a meiotic-like division on subsequent maturation. Human and mouse fibroblasts and bovine cumulus cells in G1 were transferred to GV or prometaphase I (PM I) stage bovine oocytes. Human and mouse fibroblasts were also transferred to PM 1 mouse oocytes. Meiotic maturation was assed by staining DNA with Hoechst 333342 and chromosome segregation by FISH analysis for bovine chromosomes 6 and 13 and for human chromosomes X and 18. Fusion and survival rates were better when PM I oocytes were used as recipients. Somatic cells were capable of undergoing a meiotic-like division. FISH analysis for reconstructed bovine oocytes revealed just 1 chromosome 6 and 13 per nuclei in 3 of 6 oocytes. After inter-specific nuclear transfer, chromosomes never completed meiotic segregation. These results support the hypothesis that haploidization of somatic cells can be induced after homologous transfer of nuclei into immature oocytes and is influenced by stage of oocyte development.

Thesis (Ph.D.)--Tufts University, 2004. / Adviser: Marc d'Alarcao. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 114-118). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.)--University of Minnesota, 1999. / Includes bibliographical references (leaves 74-98). Also available on the World Wide Web as a PDF file.