The expression of genetic information carried in the DNA of living cells involves two major steps, transcription and translation. In eukaryotes, the initial transcription products need to be further processed before they can serve as templates for protein synthesis. One such process is splicing, which selectively removes the introns and connects the coding sequences (exons). Splicing also deposits a multiprotein complex, the exon junction complex (EJC), at a position approximately 20 nucleotides upstream of exon exon junctions of spliced mRNA to facilitate post-splicing RNA processing. Among eight identified EJC components, transcription factor Aly/REF and splicing factor UAP56 have been shown to stimulate the export of both spliced and unspliced mRNA. They provide links between these cellular events. During the export, two other components, Y14 and Mago will accompany mRNA into the cytoplasm and maintain association with mRNA until the first round of translation. They are considered core proteins in EJC. During the period of my study, I first crystallized and determined the structure of UAP56(Δ1–44) at 1.95 Å resolution by molecular replacement. The structure reveals that UAP56 belongs to the DEXD RNA helicase family and contains minimal helicase domains in an elongated shape similar to the well-studied protein eIF4A. I then crystallized and determined the crystal structure of Drosophila Y14 (DmY14) RRM domain (a.a. 47–156) in complex with full-length Mago nashi (Mago) at 1.85 Å resolution. The structure shows that the strong interaction maintained between these two proteins is the result of hydrophobic interaction between the RNP motifs of Y14 and Mago. The structure implies that Y14 can not interact with RNA in a manner similar to other RRM-containing proteins. The stable association between Y14/Mago and mRNA is either bridged by unknown protein factors or requires a folding rearrangement.

The cytoplasmic region (c-fragment) of the E. coli aspartate receptor plays an important role in modulating intracellular signaling cascade and adaptation. This study focuses on understanding the structural organization of the receptor and characterizing the interactions between the receptor and the methyltransferase. Monitored by circular dichroism (CD), thermal denaturation of the c-fragment was found to be reversible. The c-fragment derived from signaling mutants displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in gel filtration chromatography (GFC). An analysis of secondary structure indicated that the oligomers were more folded than monomers. A nondenaturing detergent, octylglucoside (OG), and an $\alpha$-helix promoting reagent, trifluoroethanol (TFE), were used to reveal perturbing influences on the secondary and tertiary structure of the c-fragment and were interpreted to influence the extent of coiled-coil in the c-fragment. OG was found to diminish the cooperativity of the thermal denaturation, which could be restored by the addition of glycerol and phospholipid. These conditions where the cooperatlve transition was observed correlated with the conditions where methylation of the intact receptor had been observed. Binding between the receptor and the methyltransferase was characterized using isothermal titration calorimetry (ITC), giving a complex of 1:1 stoichiometry. Both dimeric and monomeric protein behaved in the same way, suggesting that the interaction between the receptor and the methyltransferase was not influenced by an equilibrium between the monomer and dimer of the cytoplasmic domain. The c-fragment of the receptor proved to have thie same binding activity as did the full-length receptor; a deletion of either 36 or 16 amino acids from the carboxyl terminus led to a csmplete loss of the binding activity. Finally, a synthetic peptide corresponding to the last five amino acids (NWETF) was found to have all binding activity of the full-length receptor. Evidence that the NWETF binding site for the transferase, which is strictly conserved among the serine and aspartate receptors, has physiological significance was obtained in assays of the transferase activity in which excess peptide was able to completely block the receptor methylation. The binding site was thus identified with the carboxyl "tail" of the receptor, indicating the mechanism by which the transferase can modify all the sites of methylation which it is bound to the receptor. Also, methylation between receptors was deduced as a possible consequence of this mode of binding, by comparing the data obtained in this study with numerous observations on receptor methylation and adaptation found in literature.

A fluorescence microscope and a digital imaging system including a charge coupled device and a computer were used to address two lines of inquiry each using individual A431 cells as the subject. The ATP-induced calcium response was approached using ratio imaging techniques and fura-2, a fluorescent calcium ion indicator. We demonstrated that ATP induced a calcium response in a dose and calcium-dependent manner. The calcium stores in A431 cells were selectively emptied and refilled under experimental control. A431 cells were shown to be desensitized upon repeated stimulation with ATP. Calcium pool emptying and desensitization were found to be concurrent processes. The second pursuit was to develop and employ an approach for measuring the kinetics of ligand and receptor association and dissociation on individual cells using fluorescence microscopy and digital imaging techniques. The strategy was used to study EGF binding to its receptors on individual cells. Binding curves were fit to single and two site exponential functions which assumed first order kinetics binding to independent binding sites. The values for k$\sb{\rm on}$ and k$\sb{\rm off}$ were similar to those found in the literature. However the assumptions that the k$\sb{\rm off}$ rate is independent of (EGF) and that there are two independent populations of EGF-R in respect to affinity for EGF were challenged. EGF-receptor density was found to vary from cell to cell, while the on- and off-rates did to a lesser extent. This approach increased the temporal resolution of binding phenomenon and can be used to analyze a variety of ligand and receptor dynamics.

The requirement of DNA synthesis for the release of Ɣ-radiation-induced mitotic inhibition in mammalian cells has been studied. Mammalian cells in which DNA synthesis had been inhibited by treatment with fluorodeoxyuridine (FUdR) were not released from radiation-induced mitotic inhibition until the FUdR block was removed. After removal of the block, mitotic figures reappeared, but only after a time equivalent to the usual mitotic delay caused by the particular radiation dose employed. This suggests that repair of the mitotic inhibition lesion can not proceed unless the pathway for DNA synthesis is intact. Further evidence for the requirement of DNA synthesis in the release of mitotic inhibition came from the observation of radiation-induced synthesis of DNA during G₂, a stage in the cell cycle normally not associated with such synthesis. / Thesis / Master of Science (MSc)

Chloramphenicol has been used to study mitochondrial biogenesis in mammalian cells by examining its effect on: the incorporation of radioactive amino acids into protein by isolated mitochondria, the growth of L cells, the level of representative enzymes and cytochromes in the mitochondria and cytoplasm and the structure of mitochondria and L cells. A reversible inhibition of synthesis of cytochrome c oxidase was obtained by treating cells with D-threo-chloramphenicol for 90 hr. Recovery of cytochrome c oxidase activity was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis. Cycloheximide also reversibly inhibited cytochrome c oxidase formation in cells which were not treated with D-chloramphenicol. It is suggested that the mitochondria and the nucleus have a joint control in the formation of a functionally active cytochrome c oxidase enzyme. / Thesis / Doctor of Philosophy (PhD)

The understanding of basic cellular processes has been greatly facilitated through investigation of the behaviour of mutant forms. In a similar way the mechanisms of genetic recombination may be clarified by a study of strains which are known to show inherited differences in recombination behaviour at meiosis. The haploid fungus Neurospora crassa is particularly well suited to such an investigation since recombination frequency heterogeneity has been extensively reported in that organism, and the differences are believed to be, to a large extent, under genetic control. Strains showing recombination frequency heterogeneity over a marked genetic region have been extensively analysed in the present work and the mode of action of the factors controlling recombination frequency has been investigated by combining differing strains in heterokaryons. / Thesis / Doctor of Philosophy (PhD)