Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Jun and Fos are components of the AP1 family of transcription factors that bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. The specific protein-DNA interactions are driven by the binding of basic zipper (bZIP) domains of Jun and Fos to TPA response element (TRE) and cAMP response element (CRE) within the promoters of target genes. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry in conjunction with molecular modeling and semi-empirical analysis, I characterize AP1-DNA interactions in thermodynamic and structural terms. My data show that the binding of bZIP domains of Jun-Fos heterodimer to TRE and CRE are under enthalpic control accompanied by entropic penalty at physiological temperatures. This is in agreement with the notion that protein-DNA interactions are largely driven by electrostatic interactions and intermolecular hydrogen bonding. A larger than expected heat capacity change suggests that the basic regions within the bZIP domains are unstructured in the absence of DNA and interact in a coupled folding and binding manner. Further analysis demonstrates that Jun-Fos heterodimer can tolerate single nucleotide variants of the TRE consensus sequence and binds in the biologically relevant micromolar to submicromolar range. Of particular interest is the observation that the Jun-Fos heterodimer binds to specific variants in a preferred orientation. 3D atomic models reveal that such preference in orientation results from asymmetric binding and may in part be attributable to chemically distinct but structurally equivalent residues within the basic regions of Jun and Fos. I further demonstrate that binding of the biologically relevant Jun-Jun homodimer to TRE and CRE occurs with favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures in a manner akin to the binding of Jun-Fos heterodimer. However, anomalously large negative heat capacity changes provoke a model whereby Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. The data also reveal that the heterodimerization of leucine zippers is modulated by the basic regions and these regions may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain a-helical conformation in isolation and the formation of the native coiled coil a-helical dimer is attained through a coupled folding-dimerization mechanism. Taken together, this dissertation marks the first comprehensive thermodynamic analysis of an otherwise well-studied and vitally important transcription factor. My studies shed new light on the forces driving the AP1-DNA interaction in thermodynamic and structural terms. The implications of these novel findings on the development of novel therapies for the treatment of disease with greater efficacy coupled with low toxicity cannot be overemphasized.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Sequence-specific PCR markers were derived from one new and eight previously identified random amplified polymorphic DNA (RAPD) markers for the purpose of identifying grape (Vitis) rootstocks. The markers were developed because the RAPD assay was found to be inconsistent and the original RAPD markers unreliable. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In one case, bands of the same size generated by the same primer in different rootstocks -- normally scored as the same marker -- failed to cross-hybridize, implying a lack of homology between the bands. In another case, a band scored as absent based on ethidium bromide staining was detected by hybridization. One of nine RAPD bands was cloned in the present study. All nine were partially sequenced and sequence-specific pairs of primers were synthesized for each for use under stringent PCR conditions. Three of the primer pairs generated products only from the rootstocks from which the original RAPD bands had been cloned; three others produced products from additional rootstocks while still generating polymorphisms; two others generated apparent length variants from some accessions; and one primer pair resulted in a loss of polymorphism. Based on the eight polymorphic markers, five of nine rootstocks could be unambiguously distinguished. / Graduation date: 1996

Problem gambling is the subclinical form of pathological gambling and both are characterized by difficulties in the limiting of money and time spent on gambling. Genetic and personality factors have been implicated in gambling disorders (PG). As PG is classified as an impulse-control disorder, the serotonin (5-HT) system has been suggested to be involved. We sought to better understand the complex relationship between personality traits, PG, and 5-HT genes. We investigated ten 5-HT candidate genes for association with PG and personality traits. We also examined personality traits for association with PG. We found that MAOA and HTR3A haplotypes were associated with Agreeableness and Conscientiousness personality domains, PG was associated with high Neuroticism and low Conscientiousness scores, and the MAOA gene may play a role in PG. Our findings contribute to the better understanding of how 5-HT genes may be involved in the neurobiological mechanisms underlying PG and personality.

Problem gambling is the subclinical form of pathological gambling and both are characterized by difficulties in the limiting of money and time spent on gambling. Genetic and personality factors have been implicated in gambling disorders (PG). As PG is classified as an impulse-control disorder, the serotonin (5-HT) system has been suggested to be involved. We sought to better understand the complex relationship between personality traits, PG, and 5-HT genes. We investigated ten 5-HT candidate genes for association with PG and personality traits. We also examined personality traits for association with PG. We found that MAOA and HTR3A haplotypes were associated with Agreeableness and Conscientiousness personality domains, PG was associated with high Neuroticism and low Conscientiousness scores, and the MAOA gene may play a role in PG. Our findings contribute to the better understanding of how 5-HT genes may be involved in the neurobiological mechanisms underlying PG and personality.

No description available.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease. Genetic factors playing an important role in disease susceptibility have long been suggested. Transforming growth factor-beta (TGF-beta) regulates differentiation and proliferation of T cells. Therefore, it could be a candidate gene for the development of systemic lupus erythematosus. Allelic polymorphisms in TGF-beta promoter region (-988, -800, -509) and in exon 1 (codon 10 and codon 25) have been suggested to associate with SLE susceptibility. Allelic polymorphisms at positions -988, -800, -509, codon 10 and codon 25 on TGF-beta gene in 138 SLE patients and 182 healthy controls were analyzed in this study. TGF-beta polymorphisms were determined by PCR amplification and sequencing. With the previous polymorphic data of interleukin-4 (IL-4) -590 and interleukin-10 (IL-10) -819, associations of cytokine genotyoe and allele frequencies were analyzed. Results showed that there were differences in the genotype distribution of TGF-beta promoter region at position -509 and in the signal sequence at codon 10 (Leu¡÷Pro) between case and control groups in this study. However, no significant differences were found for all the TGF-beta polymorphisms. Allele frequency of IL-10 -819 was significantly associated with the susceptibility of SLE (p = 0.011). No significant associations were found between lupus nephritis with all the cytokine polymorphisms, but CNS involvement and lung involvement were associated with the polymorphisms studied in this research.

Rheumatoid arthritis (RA) is one kind of chronic inflammation disease. It affects not only joint, but often has the infringement outside the joint, such as internal organs. Besides the environmental factor, the heredity factor is involved in the pathogenesis of RA too. HLA-DR4 was found to play a role in RA pathogenesis in Taiwan. Because the DR4 positive patients actually are only one-half, other than HLA-DR gene may also be involved in RA pathogenesis. NF£eB plays an important role in immune inflammation. Activity of I£eB can affect NF£eB. I£eB£\ is a critical member in the I£eB protein family. Moreover, I£eBL is functionally similar to I£eB£\. Therefore we extrapolated polymorphisms of I£eBL gene and I£eB£\ gene of the RA patients and normal subjects. In this research, studies of these two genes from 79 RA patients and 81 normal subjects were divides into two parts. The first part used polymerase chain reaction, direct sequencing, special sequence polymerase chain reaction as well as limit fragment length polymorphism to study relations between I£eBL gene and HLA-DR4 and RA occurrence. The second part used polymerase chain reaction and limit fragment length polymorphism, to analyze relation between I£eB£\ gene and the RA occurrence. We found that there is significant increase of -421 -/A base deletion polymorphism in I£eBL gene from DR4 positive RA patients and -262 T/C polymorphism in I£eBL gene from DR4 negative RA patients. A significant reduction of -519 C/T polymorphism in I£eB£\ gene from DR4 negative RA patients was found too. In conclusion, polymorphisms of -421 -/A base deletion as well as -262 T/C in I£eBL gene may be involved in the pathogenesis of RA by alteration of I£eBL activity and thereafter binding of I£eBL to NF£eB.

No description available.