Unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying the circadian clock in cyanobacteria. Despite tremendous work over the last decade in identification of clock-related loci and elucidation of molecular mechanisms of the central oscillator, many details of the basic steps in generating circadian rhythms of biological processes remain unsolved and many components are still missing. A transposon-mediated mutagenesis and sequencing strategy has been adopted to disrupt essentially every locus in the genome so as to identify all of the loci that are involved in clock function. The complete genome sequence has been determined by a combination of shotgun sequences and transposon-mediated sequences. The S. elongatus PCC 7942 genome is 2,695,903 bp in length, and has a 55.5% GC content. Automated annotation identified 2,856 protein-coding genes and 51 RNA coding loci. A system for community refinement of the annotation was established. Organization and characteristic features of the genome are discussed in this dissertation. More than 95% of the PCC 7942 genome has been mutagenized and mutants affected in approximately 30% of loci have been screened for defects in circadian function. Approximately 70 new clock loci that belong to different functional categories have been discovered through a team effort. Additionally, functional analysis of insertion mutants revealed that the Type-IV pilus assembly protein PilN and the RNA chaperon Hfq are involved in transformation competence of S. elongatus cells. Functional analysis of an atypical short period kaiA insertional mutant showed that the short period phenotype is caused mainly by the truncation of KaiA by three amino acid residues. The interaction between KaiC and the truncated KaiA is weakened as shown by fluorescence anisotropy analysis. Deletion analysis of pANL, the large endogenous plasmid, implies that two toxin-antitoxin cassettes were responsible for inability to cure cells of this plasmid. In summary, the results indicate that this functional genomics project is very promising toward fulfilling our goal to assemble a comprehensive view of the cyanobacterial circadian clock. The mutagenesis reagents and dataset generated in this project will also benefit the greater scientific community.

The genetic defects that results in TAL1 oncogene activation are commonly seen in leukemic cells of the patient with T-cell Acute Lymphoblastic Leukemia ( T-ALL ). The ectopic expression of TAL1 oncoprotein perturbs the development of T-cell, hence promotes the formation of leukemia. TAL1 gene encodes proteins with basic helix-loop-helix ( bHLH ) domain, a protein dimerization and DNA binding domain. In T-ALL cells, two Tal1 proteins, pp42(1-331 amino acids) and pp22(176-331 amino acids) are produced that both contain bHLH domain. Both proteins interact with immunoglobulin gene enhancer binding protein, E12/E47 to form DNA-binding heterodimers, that can bind to consensus E-box DNA sequence AACAGATGGT. Phosphorylation of S122 residue modulates the trans-activation potential of Tal1 protein. In addition, S172 is an inducible c-AMP dependent protein kinase (PKA) phosphorylation site in vivo. The phosphorylation of TAL1 S172 upon stimulation by forskolin can increase the DNA binding of E12-Tal1 heterodimer. We used site-directed mutagenesis to investigate the effect of S194,S224 mutation on the function of truncated Tal1 oncoprotein.Mutant Tal1 and E12 proteins expression plasmids were constructed and introduced into COS-1 cells by cotransfection. Tal1 and E12 protein expression in transfected cell were evaluated by Western blotting. The protein-DNA interaction were evaluated by electrophorectic mobility shift assay. The mutation of S194 and S224 of Tal1 protein all resulted in the loss of DNA-binding complex formation. This data indicated that these serine residues are essential for bHLH function. However, the phosphorylation status of these two residues in vivo, and what kinase is responsible for the phosphorylation of these residues, await further investigation.

This dissertation describes several strategies used to create diversity in non-immune antibody libraries. Two of the strategies were used to create two separate peptide focused libraries. Both of these strategies used to create these antigen-class focused libraries used a single scaffold antibody gene that contained diversity only in the variable heavy region. The scaffold antibody gene one of the libraries, the M:anti-pep library, was chosen based on hypervariable loop canonical structures that are characteristic of other anti-peptide antibodies. Additionally, all of the contact residues of this antibody are commonly used contact residues in other anti-peptide antibodies. These positions and others were varied to incorporate the natural diversity of other anti-peptide antibodies. The second library, the Hu:anti-pep, is based on a widely used, unique combination of human germline antibody segments that express well in bacterial expression. Positions were chosen for variation based on their usage as contact residues in both anti-peptide and anti-protein antibodies. The diversity was less focused than with the M:anti-pep library, incorporating all 20 amino acids at "high usage" positions and only four amino acids at "low usage" positions. Both libraries were validated by phage display selections against the peptide angiotensin (AT) and neuropeptide Y (NPY). The M:anti-pep library yielded specific antibodies to both peptides with dissociation constants as low as 14 nM against AT and 18 nM against NPY. The Hu:anti-pep library yielded specific clones with higher dissociation constants: 49 nM against NPY and 11 [mu]M against AT. The final strategy used to introduce diversity is widely used for affinity maturation of low affinity, previously selected antibodies. Extremely high rates of mutagenesis (2.2% of the gene to 2.7%) were used to create two libraries of the anti-digoxin antibody 26-10. The libraries had been screened by others in an attempt to examine the effects of highrates of mutagenesis on the directed evolution of an antibody. A total of 91 isolated clones from both libraries were sequenced. Several consensus mutations were identified near the CDRH3 in the isolated clones, indicating that they had a positive, selectable effect. This study confirmed that high-error rate antibody libraries contain more active clones than expected. Combinations of the selected consensus mutations from these libraries provide moderate enhancements to the kinetics and expression of the wild-type antibody in a non-synergistic manner. / text

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In this thesis is described the identification and characterisation of two morphogenesis mutants of Arabidopsis thaliana. One, vertically challenged (vch1) exhibits much reduced cellular elongation. The second, altered suspensor fate (asf1) is embryonic- lethal. A thorough phenotypic analysis of both mutants is presented, as are the results of genetic analysis. Vch1 exhibits a severe reduction in cellular elongation throughout the plant, resulting in a dwarfed phenotype. Despite its stunted morphology, vch1 exhibits normal cellular patterning, demonstrating that cell morphogenesis can be uncoupled from correct cellular pattern formation, vch1 follows a normal life history by all parameters examined demonstrating that the timing of developmental events can be uncoupled from correct morphogenesis. The phenotype of vch1 cannot be rescued by the exogenous supply of a range of hormones, signalling inhibitors or growth conditions, although it can respond to each, in a proportionately similar manner to wild type seedlings. No defects in cell wall architecture nor in cytoskeletal organisation were detected during this study. Speculative models for the role of the VCH gene are proposed. In asf1, the embryo proper arrests at the transition stage of embryogenesis. The wild type suspensor is a single file of cells which serves to anchor the embryo proper to the maternal tissue and acts as a conduit for, and source of, nutrients to the developing embryo. In asf1, suspensor cells undergo inappropriate proliferation following the arrest of the embryo proper. Evidence is presented from cytological and ultrastructural examination, and expression of spatially restricted gus-fusion marker genes, that the ectopically divided suspensor cells take on aspects of embryo proper-like character. Models for the role of the ASF1 gene are proposed. It is likely that the mutant phenotype results from disruption of intercellular communication between the embryo proper and suspensor.

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Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph.D.)--Ohio University, November, 2007. / Title from PDF t.p. Includes bibliographical references.