by Pui-Ki Kwan. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 134-143). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Table of content --- p.v / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- The formation of body axes --- p.1 / Chapter 1.2 --- Maternal genes are essential for development --- p.8 / Chapter 1.3 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.9 / Chapter 1.4 --- Establishment of an anterior to posterior bcd protein gradient --- p.12 / Chapter 1.5 --- The bcd protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.12 / Chapter 1.6 --- bcd protein acts as transcriptional regulators --- p.14 / Chapter 1.7 --- The anterior localization of bcd mRNA --- p.17 / Chapter 1.8 --- Components required for the localization of bcd mRNA --- p.17 / Chapter 1.8.1 --- Cis-acting elements --- p.17 / Chapter 1.8.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.19 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.8.2.1 --- "exuperantia, swallow and staufen are necessary for localization of bcd mRNA" --- p.21 / Chapter 1.8.2.2 --- exu protein is an absolute requirement for the localization --- p.24 / Chapter 1.8.2.3 --- Potential functions of exu based on the coding sequence --- p.25 / Chapter 1.8.2.4 --- Microtubules dependence of the localization --- p.26 / Chapter 1.8.2.5 --- Microtubules polarity directs localization of bcd RNAs --- p.27 / Chapter 1.9 --- Functions of exu in localization of bcd mRNA --- p.27 / Chapter CHAPTER 2 --- Characterization of deletion mutants of exu / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Construction of deletion mutants of exu --- p.31 / Chapter 2.2.1 --- Materials and Methods --- p.31 / Chapter 2.2.2 --- Results --- p.33 / Chapter 2.3 --- Analysis of exu protein in deletion mutants --- p.35 / Chapter 2.3.1 --- Materials and Methods --- p.35 / Chapter 2.3.1.1 --- Preparation of total ovary protein from the transgenic flies --- p.35 / Chapter 2.3.1.2 --- Analysis of protein content by SDS Polyacrylamide Gel Electrophoresis and immunoblotting --- p.35 / Chapter 2.3.2 --- Results --- p.36 / Chapter 2.4 --- Localization of bcd mRNA and exu protein in oogenesis --- p.39 / Chapter 2.4.1 --- Introduction --- p.39 / Chapter 2.4.2 --- Spatial and temporal distribution of exu protein in the deletion mutants --- p.41 / Chapter 2.4.2.1 --- Materials and Methods --- p.41 / Chapter 2.4.2.2 --- Results --- p.43 / Chapter 2.4.3 --- Spatial and temporal distribution of bcd mRNA in the deletion mutants --- p.56 / Chapter 2.4.3.1 --- Materials and Methods --- p.56 / Chapter 2.4.3.1.1 --- Principles of DIG-labeling and in situ hybridization --- p.56 / Chapter 2.4.3.1.2 --- Synthesis of DIG-labeled bcd DNA probe --- p.59 / Chapter 2.4.3.1.3 --- in situ hybridization of bcd mRNA in egg chambers using DIG-labeled DNA probe --- p.59 / Chapter 2.4.3.2 --- Results --- p.62 / Chapter 2.5 --- Discussion --- p.70 / Chapter CHAPTER 3 --- Determination of the interactions between exu and microtubules / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Localization of bcd mRNA and exu protein in the presence of drugs which destabilize cytoskeleton --- p.81 / Chapter 3.2.1 --- Materials and Methods --- p.81 / Chapter 3.2.2 --- Results --- p.82 / Chapter 3.3 --- Analysis of interactions between exu and microtubules by immunoprecipitation --- p.88 / Chapter 3.3.1 --- Materials and Methods --- p.88 / Chapter 3.3.1.1 --- Immunoprecipitation of exu protein and binding of microtubules --- p.88 / Chapter 3.3.1.2 --- Purification of tubulin from bovine or rat brains --- p.89 / Chapter 3.3.1.3 --- Determination of protein concentration of the tubulin stock by Folin-Lowry method --- p.90 / Chapter 3.3.1.4 --- Taxol-stabilized microtubules --- p.90 / Chapter 3.3.2 --- Results --- p.91 / Chapter 3.4 --- Analysis of interactions between exu and microtubules by cosedimentation --- p.94 / Chapter 3.4.1 --- Materials and Methods --- p.94 / Chapter 3.4.2 --- Results --- p.97 / Chapter 3.5 --- Analysis of interactions between exu and microtubules using detergent extracted ovary extract for co sedimentation --- p.100 / Chapter 3.5.1 --- Materials and Methods --- p.100 / Chapter 3.5.2 --- Results --- p.101 / Chapter 3.6 --- Analysis of intracellular distribution of exu protein and Release of exu protein by sodium carbonate treatment for cosedimentation with microtubules --- p.104 / Chapter 3.6.1 --- Materials and Methods --- p.104 / Chapter 3.6.1.1 --- Subcellular fractionation of ovary extracts --- p.104 / Chapter 3.6.1.2 --- Release of contents from fractions by sodium carbonate treatment --- p.105 / Chapter 3.6.1.3 --- Co sedimentation of exu protein with microtubules --- p.105 / Chapter 3.6.2 --- Results --- p.108 / Chapter 3.6.2.1 --- Intracellular distribution of exu protein --- p.108 / Chapter 3.6.2.2 --- Cosedimentation of exu protein with microtubules using Na2CO3 released extracts --- p.108 / Chapter 3.7 --- Cosedimentation of exu protein and microtubules in high ATP concentration --- p.113 / Chapter 3.7.1 --- Materials and Methods --- p.113 / Chapter 3.7.1.1 --- Preparation of ovary extracts and microtubules sedimentation --- p.113 / Chapter 3.7.1.2 --- Western blot using a chemiluminescent detection system --- p.114 / Chapter 3.7.2 --- Results --- p.115 / Chapter 3.8 --- Discussion --- p.122 / Chapter CHAPTER 4 --- Future Prospects --- p.125 / Appendix A Supplementary protocols --- p.126 / Appendix B Reagents --- p.131 / References --- p.134

Protein-protein interactions are vital to every living organism, and it is thought that most, if not all proteins interact in some way with other proteins for purposes including for cellular metabolism, signal transduction and DNA replication. These protein complexes can range in stability from permanent to transient, and they are driven by interactions at the protein-protein interfaces including hydrophobicity, hydrogen bonding, electrostatic interactions, van der Waals interactions and covalent disulfide bonding. Many complexes, such as transient complexes of sequential enzymes called metabolons, are poorly understood. In recent years, there have been many efforts to mimic nature and engineer new protein complexes with defined spatial arrangements with increased stability and more efficient transport of the enzymatic reaction intermediates. There is much to be understood in these complexes, including the role of substrate channeling. In this dissertation, we study a natural metabolon and engineer new protein complexes. In our first study, we construct designed protein aggregates of the single enzyme small laccase (SLAC). SLAC is a multi-copper oxidase that can be easily genetically modified and is used as an oxygen-reduction catalyst on enzymatic bio-cathodes. A new dimeric interface is introduced, which, in combination with the threefold symmetry of the naturally trimeric SLAC, drives the self assembly of SLAC with two disulfide bonds in an oxidative environment. These enzymatically active aggregates form upon the addition of cupric ions to the purified protein, and electron microscopy shows the symmetry of the aggregates to be consistent with the design. We demonstrate improvements over the non-complexed enzyme including an increased resistance to permanent thermal denaturation and a lower reaction overpotential and increased current density when employed on an oxygen-reduction bio-cathode with single-walled carbon nanotubes incorporated into the enzyme aggregates. In our next line of work, we study a natural tricarboxylic acid (TCA) cycle metabolon, focusing on two enzymes: mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS). These enzymes have long been proposed to form a spatially organized complex that facilitates substrate channeling, a process in which a reaction intermediate is transferred directly from one enzyme active site to the next without first diffusing into the bulk through mechanisms such as electrostatic interactions. Structural evidence has been difficult to obtain due to the transient nature of many of these complexes. In Chapter 3, we examine the in vitro complex structure of the recombinant enzymes and find that it is similar to the recently proposed in vivo complex structure. Furthermore, there is evidence of a positively charged electrostatic channel connecting the enzyme active sites along which the oppositely charged reaction intermediate can travel by bounded diffusion. Site-directed mutagenesis along the channel on CS results in inhibited substrate channeling. Finally, we develop a platform to study substrate channeling in engineered multi-enzyme complexes. Efforts to engineer multi-enzyme complexes in recent years have made use of protein and nucleic acid-based scaffolds. Many of these complexes exhibit increased coupled enzymatic activities, but there is a question of what effects are due to substrate channeling and how to apply these strategies to any enzyme pair. In this work, we attach CS and the non-channeling cytosolic malate dehydrogenase to DNA and engineered protein cage scaffolds. These assemblies retain their enzymatic activities, and these methods can be used to study substrate channeling in many enzyme pairs including the naturally channeling and inhibited channeling TCA cycle enzymes.

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As a study organism, annual killifish (Austrofundulus limnaeus) provide a well suited study system for examining the effects of environmental temperature fluctuations at the cellular level. A. limnaeus persist in the harsh high desert climate of the Maracaibo Basin, Venezuela where they live in small, ephemeral freshwater pools. Temperatures in these waters can vary as much as 20 degrees C daily and reach maximums of over 40 degrees C due to the semi-arid climate. Previous cDNA microarray studies on killifish revealed the mRNA pattern for High Mobility Group Protein 1 (HMGB1) to be strongly affected by temperature perturbations. Specifically, peaks in hmgb1 transcript abundance were negatively correlated with temperature during temperature cycling, and experienced over a 10 fold difference in expression in response to the temperature cycle. Using the same temperature cycling experimental setup, this study's aim was three-fold: (1) to characterize the total amount of HMGB1 protein in adult male killifish livers, (2) to describe the subcellular localization of the HMGB1 protein in adult male killifish livers and (3) to sequence the 5' upstream region of the hmgb1 gene to identify possible stress responsive elements. We detected no significant difference in total HMGB1 protein levels as a consequence of temperature cycling. The data for subcellular localization of HGMB1 protein do not support a strong change in subcellular localization of the protein in response to temperature cycling; most of the HMGB1 protein is found in the cytoplasmic compartment in liver tissue. Although overall patterns of subcellular localization did not change significantly, we found a significant difference between nuclear HMGB1 protein levels in temperature cycled fish versus control (constant temperature) fish. This could suggest a muting of the natural translocation of HMGB1 into the nucleus observed in control fish at around 9:00 at night. Finally, the upstream region of the hgmb1 gene does reveal a number of putative stress responsive transcription factor binding sites.

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A novel metal-inducible and amplifiable metallothionein (MT) expression system, pNK, was firstly optimised and characterised for the production of a reporter protein, human growth hormone (hGH) in a suspension CHO cell line grown in a serum-free media. The pNK-based hGH production was demonstrated in cadmium-free condition under various fermentation modes (batch, fed-batch and perfusion) and scales (flask to bench-top bioreactor). Improvement of specific productivity of recombinant protein from pNK was shown to be possible by addition of butyrate or substrate substitution of glutamine by glutamate. Combination of fed-batch and butyrate addition strategies resulted in more than one gram per litre of hGH being obtained from the pNK expression system in a bioreactor. In the second part of the project, based on a statistical approach suggested by Plackett-Burman (P-B), a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support a Super-CHO cell line, C2.8-325, to grow as a single-cell suspension culture with comparable growth rate and viable cell number as observed in a commercial medium containing undefined additives. Using Dulbecco's Modified Eagle's Medium/Ham's F12 1:1 mixture (DMEM/F12) as the basal medium, a P-B design matrix screened 10 nutritional components. Components shown potentially beneficial for cell growth rate and viable cell number were supplemented to DMEM/F12 to formulate the SPF medium. Finally, the pNK expression system and the Super-CHO cell line were applied simultaneously in an attempt to express a humanised anti-CD48 monoclonal antibody (MAb), IgG1-N2A (N2A-MAb). This aimed to test C2.8-SPF grown in newly developed SPF medium for transfection, clone development and recombinant protein production. A stable and N2A-MAb expressing C2.8-SPF cell line was successfully constructed, and N2A MAb expression was subsequently amplified and demonstrated in various cultivation scales (flask and bioreactor). This project demonstrated that the novel metal-inducible and - amplifiable mammalian expression system, pNK, and the novel mammalian host cell-line, Super-CHO C2.8-SPF, capable of growing as a single-cell suspension culture in a chemically-defined protein-free medium, SPF, could be utilised in combination to provide a new, low-cost, and regulatory-compliant recombinant protein expression platform, suitable for the biopharmaceutical industry to use in the manufacture of therapeutic recombinant proteins.

LepA is a highly conserved GTP-binding protein of unknown function. Its amino acid sequence reveals that it is a GTPase with homology to elongation factor G (EF-G). Previous data led to the hypothesis that LepA negatively regulates a posttranslational process such as protein folding. To examine this possibility, two sets of strains carrying mutated alleles encoding molecular chaperones in E. coli were transformed with a lepA expression vector. LepA had a dominant negative effect specifically in a dnaK25 strain whose product exhibits a 20-fold lower ATPase activity compared to wild-type DnaK. The expression of DnaK and other heat-shock proteins is repressed following temperature downshift. Aptly, it was found that temperature shift from 37 degrees Celcius to 15 degrees Celcius in cells harboring a lepA expression vector led to the induction of lepA and downstream lepB. Furthermore, like cold-shock genes, lepA and lepB are induced by sublethal doses of chloramphenicol, although it appears that lep operon induction is related to the antibiotic's action on the 50S ribosome. Due to LepA's insolubility, it could not be confirmed whether it interacts with DnaK, DnaJ or which other proteins it interacts with. Two-dimensional gel electrophoretic analysis revealed the absence of an isoform of OmpA in two lepA deletion strains. It is possible that LepA is involved in a folding pathway that is responsible for the conformation of this isoform. Phylogenetic analysis showed that while LepA is extremely well conserved and has been identified in all completed Bacterial and Eukaryal genomes, it is not present in the completed genomes of any Archaea. Sequence analysis revealed the existence of N-terminus mitochondrial import sequences in Eukaryal LepA orthologues. Additionally, A. thaliana contains a second LepA orthologue that clusters phylogenetically with Synechocystis LepA and has a chloroplastic import sequence. This indicates that plastidal LepA was acquired in A. thaliana (and probably in all plants) through endosymbiosis of an ancestral cyanobacterium. In constrast, mitochondrial LepA are not closely related to those of a- proteobacteria, believed to be the precursors of mitochondria. These findings imply that in sharp contrast to mitochondrial LepA, chloroplastic LepA is under strong evolutionary pressure to remain conserved.