The embryonic cell cycle of Xenopus laevis consists of rapid oscillations between S and M phase occurring in the absence of gap phases and their associated regulatory checkpoints. The end of the 12th cell cycle marks the onset of the midblastula transition (MBT) when the cell cycle lengthens establishing gap phases, their associated checkpoint pathways, and the initiation of zygotic transcription. During cell cycles 2-12, cyclins A and B are translated and expressed once per cell cycle until the MBT when their protein levels decrease due to a newly transcribed zygotic factor that leads to the deadenylation and subsequent loss of their mRNAs. In contrast, cyclin E is expressed at a constitutively high level during cell cycles 2-12. Furthermore, cyclin E levels are terminally lost coincident with initiation of the MBT in the continued presence of its adenylated mRNA. Terminal disappearance of cyclin E appears to be maternally directed and is not affected by zygotic transcription, translation, replication, or the nuclear to cytoplasmic ratio. This has led to the hypothesis that cyclin E is part of an autonomous maternally directed timer that determines the timing of the MBT. To investigate this possibility we have used antisense oligonucleotides to knockdown cyclin E and assess the affects on the timing of the MBT. Premature knockdown of cyclin E did not affect the timing of the MBT indicating it is not part of the maternal timer. Furthermore, prior to the MBT cyclin E protein has an unusually long half life. However, despite an increased stability the constitutively high levels of cyclin E require a low level of translation. We have also determined that the stable pattern of adenylation observed for cyclin E1 is specified by three cis-acting elements in its' 3' UTR. Deletion of the NPS, eCPE/ARE3, and ARE2 abolished adenylation. Additionally, a putative stem loop in ARE2 is targeted by ElrA the Xenopus homolog of HuR and a member of the ELAV gene family. Loss of adenylation required disruption of ElrA binding. These findings demonstrate ElrA functions in the correct adenylation of cyclin E1 mRNA.

The innate immune system provides nonspecific defenses against pathogens. Many diseases occur because of malfunctions in the innate immune system. In the present thesis, I have investigated two independent mechanisms of innate immunity in the lung and liver. Both mechanisms involve responses to bacterial infection and/or components of the bacterial wall (lipopolysaccharide). In the first model, I studied the role of submucosal glands in lung innate immunity through the use of tracheal xenograft airways with and without glands. This work provides evidence that submucosal glands are a major source of antibacterials that are critical for maintaining sterile airways. In the second model, I studied host responses to a gram-negative bacterial cell wall component (lipopolysaccharide) and how the liver coordinates cytokine responses that lead to endotoxic shock. This work examined how hepatic induction of NFkappaB and TNFalpha influenced survival in this lethal murine model of endotoxemic shock. My findings suggest that during the course of lethal endotoxic shock, NFkappaB activation has a predominantly pro-inflammatory effect in the liver through the induction of TNFalpha, and that TNFalpha influences the role of NFkappaB as an anti-apoptotic factor in the liver. In conclusion, my thesis suggests that maintaining a homeostatic balance in response to pathogens is an important function of the complex innate immune system.

The KpnI, 1.2 and 1.5 kb families of interspersed repetitive DNAs from the African green monkey genome were isolated and characterized. Each family contains three populations of segments based on their sequence lengths and susceptibility to cleavage by the restriction enzymes KpnI and RsaI. The first population contains the smallest segments which are susceptible to both KpnI and RsaI cleavage and have fragment lengths of 1.2 kb (1.2 kb family) and 1.5 kb (1.5 kb family) respectively. The members in this population are referred to as KpnI-sensitive segments. The second population contains longer segments (> 2 kb) which represent fusions of members from different families. The fusion sequences are cleaved by KpnI at their termini but lack internal KpnI sites at the junctions that join the individual component members. The third population contains members from each family that are cleaved occasionally by KpnI (KpnI-resistant segments) and remained linked to the bulk of the high molecular weight DNA. KpnI 1.2 kb, 1.5 kb and KpnI-resistant populations were isolated and analyzed for the presence of internal RsaI sites. All members from both populations were cleaved by RsaI into a simple series of low molecular weight fragments. Some members from both the KpnI-sensitive and the KpnI-resistant populations were found to contain internal RsaI sites. Other members from both populations lacked internal RsaI sites. Genomic KpnI 1.2 kb segments were cloned and two recombinants pBK(1.2)14 and pBK(1.2)39 identified. The partial nucleotide sequence of clone Kpn(1.2)14 was determined. The sequence content of KpnI 1.2 and 1.5 kb families in DNA fragments that anchor DNA loops to the nuclear matrix (att-DNA) was also studied. The relative sequence content of both 1.2 and 1.5 kb families was found to be impoverished when compared to their content in total nuclear DNA. However, members in each family were found to be present in detectable amounts. The association of KpnI 1.2 and 1.5 kb family sequences with the nuclear matrix was also demonstrated by metrizamide gradient centrifugation of nuclear matrix complexes. The results suggest that some KpnI 1.2 and 1.5 kb segments are differentially associated with nuclear proteins.

Mutagenesis of the defective transducing bacteriophage lambda fus2 was used to isolate a collection of temperature-sensitive mutants of E. coli in the major ribosomal protein gene cluster. Four mutants were examined in detail. Two of the mutants were resistant to the ribosomal antibiotics neamine and spectinomycin. Another mutant was defective in 50S ribosomal subunit assembly at 42(DEGREES)C. The 30S subunit proteins S17 and S19 were changed in two different mutants. Each protein migrated as a more basic species in two-dimensional gels of ribosomal proteins. Ribosomes from each of the four mutants examined showed a temperature-dependent reduction in translational activity in cell-free assays. The kinetic assays showed declines in both the rate and extent of translation at three temperatures. Ribosomes from three of the four mutants were also found to have an increased rate of heat inactivation at 45(DEGREES)C compared to control particles. Mixed subunit assays idendtified a t.s. subunit in each mutant. A defect in reassociation at high temperature was found for the subunits from one mutant. Another mutant showed significantly high levels of misreading at 32(DEGREES)C and 42(DEGREES)C. Two mutants showed a decreased ability to bind 14C-phenylalanine tRNA at the two temperatures tested. The increased efficiency and utility of this mutagenesis method for the isolation of protein synthesis mutants is discussed.

Thesis (Ph. D.)--University of Michigan.

Thesis (Ph. D.)--University of Michigan.

Modern protein catalysts are often viewed as possessing exquisite specificities for their cognate physiological substrates. In contrast, primordial catalysts are thought to have possessed much broader substrate specificities, a characteristic that likely afforded the survival of their host organisms under a plethora of diverse environmental conditions. Recent experimental work suggests that present day enzymes often retain the ability to recognize and transform a variety of natural and unnatural compounds that are structurally distinct from their target substrate. The widespread existence of such promiscuity could prove generally useful both in the natural and directed evolution of new proteins. To probe the persistence of enzyme promiscuity in modern proteomes we studied the model organism Escherichia coli due to its rapid growth, ease of genetic manipulation and many years of prior research on this organism which have generated abundant knowledge on its metabolism. The first exploration into uncovering enzyme promiscuity, described in chapter two, examines the proton transfer reaction catalyzed by triosephosphate isomerase (TIM). Triosephosphate isomerase catalyzes the interconversion of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the E. coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding for a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of L-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate. In chapter three we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a Kd value of 5.8 ± 0.2 µM in the absence of NADPH and a Kd value of 11 ± 1.3 µM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the L-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K+ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K+ channels with hemoprotein-like, O2-sensing properties. To date the molecular origin of O2 sensing by voltage-gated K+ channels is unknown and the results presented herein suggest a role for heme in this process. In chapter four we probe the network of genes within E. coli that can provide resistance to the nonnatural toxin bromoacetate. Microbial niches contain toxic chemicals that are capable of forcing organisms into periods of intense natural selection to afford survival. Elucidating the mechanisms by which microbes evade environmental threats has direct relevance for understanding and combating the rise of antibiotic resistance. In this study we used a toxic small-molecule, bromoacetate, to model the selective pressures imposed by antibiotics and anthropogenic toxins. We report the results of genetic selection experiments that identify nine genes from Escherichia coli whose overexpression affords survival following exposure to a lethal concentration of bromoacetate. Eight of these genes encode putative transporters or transmembrane proteins, while one encodes the essential peptidoglycan biosynthetic enzyme, UDP-N-acetylglucosamine enolpyruvoyl transferase (MurA). Biochemical studies demonstrate that the primary physiological target of bromoacetate is MurA, which becomes irreversibly inactivated via alkylation of a critical active-site cysteine. Genetic experiments also identify 63 single-gene mutants of E. coli that display increased susceptibility to bromoacetate. One hypersensitive bacterium lacks yliJ, a gene that encodes a glutathione transferase capable of catalyzing the detoxification of bromoacetate with a kcat/Km value of 5.4 × 103 M-1 s-1. The catalytic proficiency of YliJ, which exceeds 5 orders of magnitude, is particularly noteworthy considering the enzyme is unlikely to have previously encountered bromoacetate. In total, our results indicate that nearly 2% of the E. coli proteome contributes to, or can be recruited to provide, bromoacetate resistance. This illustrates the wealth of intrinsic survival mechanisms that can be exploited by bacteria when they are challenged with toxins. The work described here illuminates the vast metabolic and functional plasticity of protein function harbored within bacteria. Their ability to recruit latent and weakly active proteins for novel functions enables survival under diverse nutritional and environmental challenges. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2010. / June 14, 2010. / Enzyme Function, Antibiotic Resistance, Glutathione Transferase / Includes bibliographical references. / Brian Miller, Professor Directing Dissertation; Hank Bass, University Representative; Hong Li, Committee Member; Lei Zhu, Committee Member; M. Elizabeth Stroupe, Committee Member.

This dissertation presents the concept, development, and characterization of a new methodology for both qualitative and quantitative analysis of protein digests in solution. Two beam fluorescence cross correlation spectroscopy is used to characterize the migration rates of fluorescently labeled peptides present in a poly (methyl methacrylate) (PMMA) microfluidic system. To achieve ultimate sensitivity, a two beam confocal microscope is employed to allow low background, single molecule detection. Two spatially separated laser beams are focused to near-diffraction limited spots and then positioned a few microns apart within a narrow region of a PMMA microdevice. Mobility measurements of the protein fragments are determined by the transit time for a single peptide to traverse through both detection volumes. Cross correlation of the fluorescence intensity signals from each confocal volume is used characterize the distribution of transit times. Electrophoresis conditions are employed and each peptide in a mixture will migrate at a characteristic velocity that depends on its size and charge. The cross correlation analysis yields a distribution of velocities reminiscent of an electropherogram in that each peak is evidence of an individual peptide. For a specific peptide digest, one can generate a fingerprint spectrum from the cross correlation data. The fingerprint could then be matched to a library of individual protein spectra allowing the rapid identification of the protein from whence the peptide mixture was derived. Our proposed method eliminates some of the shortcomings associated with current microfluidic technology. For example, analytes are monitored in free solution without actually separating the mixture; this eliminates the need for generating an analyte plug or migration over long distances. Also, since single molecule fluorescence is utilized it is possible to analyze multiple complex species at sub-nanomolar concentrations, in turn minimizing sample consumption. The two-beam fluorescence cross correlation method has the potential to be a high speed, highly sensitive alternative approach for protein and peptide analysis. / A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2006. / May 3, 2006. / Electrophoresis, Proteomics, Microfluidics, Single Molecule, Fluorescence / Includes bibliographical references. / Kenneth D. Weston, Professor Directing Dissertation; Peter G. Fajer, Outside Committee Member; Joseph B. Schlenoff, Committee Member; Oliver Steinbock, Committee Member.

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