Dissertations / Theses on the topic 'Inducibility'

Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

Wong Wei-yan.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (leaves 221-242).
Abstracts in English and Chinese.
Thesis committee --- p.i
Declaration --- p.ii
Acknowledgements --- p.iii
Abstract in Chinese --- p.iv
Abstract in English --- p.vi
List of abbreviations --- p.viii
List of figures --- p.xi
List of tables --- p.xv
Content: --- p.xvi
General introduction --- p.1
Aldehyde dehydrogenase superfamily --- p.3
Background of antiquitin --- p.5
Plant antiqutins (ALDH7B) --- p.5
Animal antiquitins (ALDH7A) --- p.8
Human antiquitin information on NCBI --- p.14
Rationale of studying the inducibility of annquitin and overexpression of it in HEK293 and HepG2 cells --- p.16
Flowchart 1 Procedure of antiquitin expression studies in the HEK293 and HepG2 cells under stress --- p.19
Flowchart 2 Procedure to study antiquitin expression in the HEK293 and HepG2 cells after in silico promoter search --- p.20
Flowchart 3 Procedure to study antiquitin overexpressed HEK293 and HepG2 cells --- p.21
Chapter Chapter 1 --- Inducibility of antiquitin in the HEK293 and HepG2 cells under hyperosmotic stress
Chapter 1.1 --- Introduction --- p.22
Chapter 1.1.1 --- Cellular response to hyperosmotic stress --- p.22
Chapter 1.1.2 --- Methods to study the responses of cells under hyperosmotic stress --- p.24
Chapter 1.2 --- Materials --- p.26
Chapter 1.2.1 --- Cell culture media --- p.26
Chapter 1.2.2 --- Buffers for RNA use --- p.26
Chapter 1.2.3 --- Buffers for DNA use --- p.27
Chapter 1.2.4 --- Other chemicals --- p.27
Chapter 1.3 --- Methods --- p.28
Chapter 1.3.1 --- Culture of HEK293 and HepG2 cells --- p.28
Chapter 1.3.2 --- Hyperosmotic stress on HEK293 and HepG2 cells --- p.29
Chapter 1.3.3 --- MTT assay --- p.29
Chapter 1.3.4 --- Total RNA extraction --- p.30
Chapter 1.3.5 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.30
Chapter 1.3.6 --- Polymerase chain reaction (PCR) --- p.31
Chapter 1.3.7 --- Quantification of PCR products --- p.31
Chapter 1.3.8 --- Statistical analysis --- p.33
Chapter 1.4 --- Results --- p.34
Chapter 1.4.1 --- Viability of HEK293 and HepG2 cells under hyperosmotic stress --- p.34
Chapter 1.4.2 --- Validation of RNA quality --- p.34
Chapter 1.4.3 --- Validation and determination of PCR conditions --- p.40
Chapter 1.4.4 --- Inducibility of antiquitin in HEK293 cells under hyperosmotic stress
Chapter 1.4.5 --- Inducibility of antiquitin in HepG2 cells under hyperosmotic stress --- p.43
Chapter 1.4.6 --- Inducibility of aldose reductase under hyperosmotic stress --- p.43
Chapter Chapter 2 --- "In silico studies of human antiquitin promoter, genomics sequences and open reading frame" --- p.54
Chapter 2.1 --- Introduction --- p.54
Chapter 2.1.1 --- Eukaryotic promoters --- p.55
Chapter 2.1.2 --- Key events in transcriptional initiation --- p.55
Chapter 2.1.3 --- Alternative splicing of mRNA --- p.57
Chapter 2.1.4 --- Bipartite nuclear localization signal (NLS) --- p.57
Chapter 2.2 --- Methods --- p.60
Chapter 2.2.1 --- Putative promoter studies of human antiquitin --- p.60
Chapter 2.2.2 --- Putative promoter studies of Arabidopsis thaliana antiquitin --- p.60
Chapter 2.2.3 --- Analysis for the alternative splicing of human antiquitin mRNA --- p.60
Chapter 2.2.4 --- Analysis for the nuclear localization signal (NLS) of human antiquitin amino acid sequence --- p.61
Chapter 2.2.5 --- Nucleotide / amino acid sequence analyses --- p.61
Chapter 2.3 --- Results --- p.62
Chapter 2.3.1 --- Computer search for the putative cis-acting elements on human antiquitin promoter --- p.62
Chapter 2.3.2 --- Comparison of cis-acting elements found on human antiquitin promoter with those on Arabidopsis thaliana antiquitin promoter --- p.62
Chapter 2.3.3 --- Possibilities of alternative splicing isoforms of human antiquitin
Chapter 2.3.4 --- Possibilities of bipartite nuclear localization signals on human antiquitin protein --- p.83
Chapter Chapter 3 --- Overexpression of antiquitin in HEK293 and HepG2 cells and their characterization
Chapter 3.1 --- Introduction --- p.86
Chapter 3.1.1 --- Cell cycle of a human somatic cell --- p.88
Chapter 3.1.2 --- Detection of changes in the transcriptome --- p.90
Chapter 3.1.3 --- Human genome U133 Plus 2.0 array --- p.95
Chapter 3.1.4 --- Detection of changes in the proteome --- p.96
Chapter 3.1.5 --- MALDI-TOF MS --- p.97
Chapter 3.2 --- Materials --- p.99
Chapter 3.2.1 --- Solutions for cell culture use --- p.99
Chapter 3.2.2 --- Solutions for cloning --- p.99
Chapter 3.2.3 --- Buffers for cell cycle analysis --- p.99
Chapter 3.2.4 --- Buffers for two-dimensional (2D) electrophoresis --- p.100
Chapter 3.2.5 --- Solutions for silver staining --- p.101
Chapter 3.2.6 --- Solutions for Coomassie blue protein staining --- p.102
Chapter 3.2.7 --- Solutions for Western blotting --- p.102
Chapter 3.2.8 --- Solutions for mass spectrometry --- p.103
Chapter 3.3 --- Methods --- p.104
Chapter 3.3.1 --- Hypoosmotic stress --- p.104
Chapter 3.3.2 --- Heat shock --- p.104
Chapter 3.3.3 --- Oxidative stress treatment
Chapter 3.3.4 --- Chemical hypoxia --- p.104
Chapter 3.3.5 --- Treatment of forskolin --- p.106
Chapter 3.3.6 --- Culture of SHSY5Y cells and its differentiation --- p.106
Chapter 3.3.7 --- Cloning of pBUDCE4.1/ATQ --- p.106
Chapter 3.3.8 --- PCR product purification --- p.107
Chapter 3.3.9 --- Preparation of pEGFP.N1 vector for co-transfection --- p.109
Chapter 3.3.10 --- Transfection of HEK293 and HepG2 cells --- p.109
Chapter 3.3.11 --- Assays to characterize transient transfected HEK293 and HepG2 cells --- p.110
Chapter 3.3.11.1 --- Transfection efficiency monitoring --- p.110
Chapter 3.3.11.2 --- Cell cycle analysis --- p.112
Chapter 3.3.11.3 --- Cell doubling time measurement --- p.112
Chapter 3.3.11.4 --- Stress responsiveness --- p.113
Chapter 3.3.11.5 --- Oligonucleotide array analysis --- p.113
Chapter 3.3.11.5.1 --- Total RNA extraction --- p.113
Chapter 3.3.11.5.2 --- Oligonucleotide array preparations --- p.113
Chapter 3.3.11.5.3 --- Data analysis --- p.114
Chapter 3.3.11.6 --- Two-dimensional (2D) electrophoresis --- p.115
Chapter 3.3.11.6.1 --- Total protein extraction --- p.115
Chapter 3.3.11.6.2 --- Protein quantification --- p.115
Chapter 3.3.11.6.3 --- First dimension electrophoresis: isoelectric focusing (IEF) --- p.115
Chapter 3.3.11.6.4 --- Second dimension electrophoresis: SDS- --- p.116
Chapter 3.3.11.6.5 --- Silver staining --- p.116
Chapter 3.3.11.6.6 --- Spots detection --- p.117
Chapter 3.3.11.7 --- Preparations of samples for MALDI-TOF MS --- p.117
Chapter 3.3.11.7.1 --- Silver de-staining --- p.117
Chapter 3.3.11.7.2 --- In-gel tryptic digestion --- p.118
Chapter 3.3.11.7.3 --- Peptide extraction --- p.118
Chapter 3.3.11.7.4 --- ZipTip® samples desalting and concentrating --- p.119
Chapter 3.3.11.7.5 --- MALDI-TOF MS --- p.119
Chapter 3.3.11.8 --- Western blotting --- p.119
Chapter 3.3.11.8.1 --- Antibodies probing --- p.120
Chapter 3.3.11.8.2 --- Enhanced chemiluminescence's (ECL) assay --- p.121
Chapter 3.4 --- Results --- p.122
Chapter 3.4.1 --- Inducibility of antiquitin in HEK293 cells under xenobiotic stimulus --- p.122
Chapter 3.4.2 --- Inducibility of antiquitin in HEK293 and HepG2 cells under chemical hypoxia --- p.122
Chapter 3.4.3 --- Inducibility of antiquitin in HEK293 and HepG2 cells under hypoosmotic stress --- p.122
Chapter 3.4.4 --- Inducibility of antiquitin in HEK293 and HepG2 cells under heat shock --- p.122
Chapter 3.4.5 --- Inducibility of antiquitin in HEK293 and HepG2 cells under forskolin challenge --- p.128
Chapter 3.4.6 --- Expression of antiquitin in differentiating SHSY5Y cells by retinoic acid and N2 supplement --- p.128
Chapter 3.4.7 --- Overexpression of antiquitin in HEK293 and HepG2 cells --- p.128
Chapter 3.4.8 --- Viability of transfected HEK293 and HepG2 cells under hyperosmotic stress --- p.136
Chapter 3.4.9 --- Cell doubling times of transfected HEK293 and HepG2 cells --- p.143
Chapter 3.4.10 --- Cell cycle analysis of transfected HEK293 and HepG2 cells --- p.143
Chapter 3.4.11 --- "Western blot analysis of cyclin D, cyclin A and cyclin B of transfected HEK293 and HepG2 cells" --- p.148
Chapter 3.4.12 --- RNA quality control tests for oligonucleotide array analysis --- p.148
Chapter 3.4.13 --- Oligonucleotide array analysis on transfected HEK293 and HepG2 cells --- p.155
Chapter 3.4.14 --- Two-dimensional electrophoresis of transfected HEK293 and HepG2 cells --- p.169
Chapter 3.4.15 --- MALDI-TOF MS of transfected HEK293 and HepG2 cells --- p.169
Chapter 3.4.16 --- Genes and proteins upregulnted in the antiquitin transfected HEK293 and HepG2 cells --- p.190
Discussion --- p.197
Reference --- p.221
Appendix Materials used in the project --- p.243

碩士
國立清華大學
生命科學系
85
Proliferating Cell Nuclear Antigen (PCNA) can function as an auxiliary factor of DNA polymerase delta and epsilon, is required for DNA synthesis andDNA repair. After serum stimulation of quiescent cells, the level of PCNA mRNAincrease and reaches its peak before onset of DNA synthesis. In the present study, a rat PCNA promoter with chloramphicol acetyltransferase (CAT) as reportwas stably transfected into CHO.K1 cells. The promoter named d693 containingsequences between -693 and +125 in reference to the transcription initiation aite has been previously shown to be serum responsive in transient expression study. The CAT activity in the stable transfectants named d693-pCAT.K1 cells also display the same character of serum-responsiveness. In addition, UV irradiation on d693-pCAT.K1 cells induced rat PCNA promoter activity in dose-dependence manner. Such UV inducibility was seen with quiescent cells and growing cells. Furthermore, the promoter region of rat PCNA promoter that isresponsible for UV induction are delimited within the region from -70 to +125,which contains an AP-1 and ATF/CRE site and has been shown to be serum responsive and E1A oncoprotein responsive in the previous studies. Furthermore,the UV inducibility of PCNA promoter dose not require the activities of p53 and E2F, as both p53 and E2F inhibited PCNA promoter activity when they were overexpressed in d693-pCAT.K1 cells. Thus, UV inducibility of the rat PCNA promoter dose not require p53 and E2F.

碩士
逢甲大學
化學工程學系
88
Expression vector appears to be a dispensable tool for genetic enginee-ring. The advance of DNA recombinant technology allows a versatile type of expression vectors to be developed by many research groups. However, many vectors developed to date are not completely suitable for engineering purpose. For instance, the leakiness of promoters and costly way of induc-tion are common problems, thereby leading to the limited use of these vectors for industrial application. Therefore, in this study we have attem-pted to develop three kinds of expression vectors with characteristic of tight regulation and economic way of induction. (1)The expression system based on the runaway plasmid containing T7 A1 promoter: To characterize this expression system, lacZ was fused to T7 A1 Pro- moter on a runaway plasmid. The strain harboring the recombinant plasmid was found to produce -galactosidase with 40,000 to 45,000 Miller uints upon thermal and chemical induction. In contrast, only 60 to 90 Miller units was produced at an uninduced state. Overall, it accounts for an induction ratio ranging from 350- to 570-fold, indicating the tightness of this expression system. (2)The expression system based on the runaway plasmid containing thermally regulated promoter: As shown above, the expression system based on runaway plasmid carrying a controllable promoter is particularly useful for tight regulation of cloned genes. However, the requirement for chemical induction calls for further improvement for this type of expression system. Accordingly, the expression system was further improved by incorporating the promoter subject to thermal control. As a consequence, the cell carrying the composite vector was capable of producing Pck with 13.15 mol/min/mg upon solely thermal induction. In sharp contrast, Pck yield was hardly detectable at an uninduced state. (3)The expression system based on the promoter under thermal regulation: Expression vectors derived from lac-type promoters are most commonly used. To make these systems more feasible use, lacI gene was mutated by site-directed mutagenesis to become thermally sensitive. Among four types of lacI gene thus mutated, the one with Ala241→Thr exhibited most thermal stability at low temperature (ranging 30 to 37℃), but it lost stability at high temperature (above 39℃). The vector carrying mutant lacI (Ala241→Thr) was constructed to overexpress lacZ, and the result showed that the recombinant strain yielded -galactosidase with 53, 000 Miller uints upon thermal induction. In sharp contrast, the uninduced level accounting for only 130 Miller units was generated. In conclusion, the expression systems developed in this study are characterized by stringent regulation. Moreover, the induction method by using temperature change for these vectors indicates an economic and easy way for controlling the expression of cloned genes in Escherichia coli. The successful demonstration of potential use of these vectors for high produ-ction of recombinant proteins has further suggested their promise in indu- strial application.

博士
國立清華大學
生命科學系
88
ABSTRACT Proliferating cell nuclear antigen (PCNA), also known as a processivity factor of DNA polymerase delta and epsilon, is required for eukaryotic cell DNA synthesis and repair. Expression of PCNA gene is growth-regulated and stress-responsive. This thesis is composed of four chapters. In chapter 1, the general introduction of PCNA is provided. Previously, the rat PCNA promoter responsive to UV irradiation and serum stimulation has been localized to nucleotides -70 and it contains an ATF (nucleotides -51 to -44) and AP-1 sites (nucleotides -64 to -58). In chapter 2, the rat PCNA promoter, and the endogenous PCNA mRNA and protein are demonstrated to be UV inducible in serum or serum-free recovery, suggesting that this UV inducibility is ubiquitous in cycling and non-cycling cells but not an artifact. Similarly, cells synchronized at all stages show that the UV inducibility of rat PCNA promoter is increased in dose-dependent manner. Hence, the PCNA gene is firstly reported to be UV inducible in all cell stages. Subsequently, the regulating mechanisms for UV inducibility of PCNA gene are addressed. Concerned with the DNA damage induced by UV irradiation, the DNA-dependent protein kinase (DNA-PK) mutant cells (xrs-6) were found to be UV inducible as well as its parental CHO.K1 cells. Moreover, the DNA-PK down-stream pathways are known to involve the p53 and DNA-PK can phosphorylate the p53. However, UV inducibility of rat and human PCNA expression is found to be independent on the UV induced p53 expression (chapter 2 and 3, respectively). These observations suggest that DNA damage checkpoint pathways is not essential for UV inducibility of PCNA gene. Therefore, the cell signal pathways except the DNA damage are investigated in the followings. Given the thiol-rich nature of PCNA and its role in proliferation, PCNA might be affected by redox change. N-acetyl-L-cysteine (NAC), a GSH precursor, was used and inhibited this UV inducibility of PCNA promoter, suggesting that the UV inducibility of PCNA involves the oxidative stress. For comparison to rat PCNA expression as shown in chapter 3, the role of p53 in human PCNA gene expression is studied by infection of the human papillomavirus type 16 E6 oncogene into human fibroblasts (HF). The introduction of E6 to human fibroblasts abrogates the function of cellular p53 and p21WAF1 and the cell growth control in this system. For serum stimulation, the confluent E6-infected cells (E6-HF) but not the parental cells exhibited a marked induction of PCNA. For UV exposure, both parental and E6-infected cells show the PCNA induction at a modest level in the recovery of serum or serum-free containing medium. These observations suggest that p53 may be involved in serum stimulation but not in UV induction of human PCNA gene expression. Therefore, the UV inducibility of both rat and human PCNA expression shows the same characteristic of p53 independence. To define the transcription factors responsive to UV inducibility and serum responsiveness of PCNA promoter, the gel mobility shift assays (EMSA) were used in chapter 4. In serum stimulated or UV irradiated cells, nuclear extracts incubated with probe containing ATF and AP-1 sites also show the increasing formation of the DNA:protein complexes. Similarly, the AP-1 DNA binding ability is enhanced by serum stimulation or UV irradiation. Consisting with the finding, serum responsiveness and UV inducibility of rat PCNA promoter were shown to be AP-1 related by CAT assay previously. This is firstly reported that the DNA:protein interactions require the simultaneous involvement of ATF/CRE and AP-1 sites as either element can abrogate the complexes in the competition experiment, suggesting there are cross talk between these two sites when cells were serum-stimulated or UV-irradiated. Moreover, both the distance and the sequence are essential to complex formation in serum-stimulated cells. ATF-1, but not ATF-2 and CREB, in association with other factors is involved in regulating the serum stimulation of the rat PCNA promoter activity via the proximal ATF and AP-1 sites. In conclusion, this thesis establishes the UV inducibility of rat and human PCNA gene and provides the mechanisms for understanding the serum stimulation and UV induction of PCNA response. The most important contribution is to demonstrate that the oxidative stress, but not the DNA damage checkpoint pathways including DNA-PK and p53, is one of the factors for regulating the UV inducibility of PCNA gene.

碩士
國立臺灣大學
職業醫學與工業衛生研究所
97
Polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-furans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) are all important endocrine disrupting chemicals and also persistent organic pollutants. Among them, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause most significant health effects in humans. The PCDFs have similar structure and toxicity as PCDDs. Approximately 2000 people were exposed to rice oil contaminated with polychlorinated biphenyls (PCBs) and their heat-degradation products, mainly polychlorinated dibenzofurans (PCDFs) in central Taiwan in 1979. CYP1A2 activity was found to be induced by such exposure in exposed individuals. The aryl hydrocarbon receptor (AhR) pathway was known to be critical in mediating such enzyme induction. Aryl hydrocarbon receptor nuclear translocator (ARNT) and AhR repressor (AhRR) played important roles in transcriptional regulation of AhR. In order to determine whether the ARNT and AhRR polymorphisms are associated with individual inducibility of CYP1A2. We examined the people exposed to PCBs and PCDFs and their community controls, 173 previously participated in a study on CYP1A2 activity as measured by caffeine metabolic rate, which was in turn found to be positively associated with serum 2,3,7,8-TCDD toxic equivalency (TEQ). Genotyping was done for the following SNPs：AhRR(rs2292596)(C/G), ARNT(rs7517566)(A/G), ARNT(rs3820541)(C/G), ARNT(rs3768016)(C/T), and ARNT(rs2228099)(C/G). The allele frequencies of AhRR (rs2292596) were 0.62 for allele C, 0.38 for allele G; those for ARNT (rs7517566) were 0.79 for allele A, and 0.21 for allele G, ARNT (rs3820541) 0.82 for allele C and 0.18 for allele G, ARNT (rs3768016) 0.58 for allele C and 0.42 for allele T, and ARNT (rs2228099) 0.59 for allele C and 0.42 for allele G. At a similar TEQ dose range, those subjects with AhRR GG genotype had the highest induced CYP1A2 activity, followed by those with CG genotype. Those with CC genotypes had lowest inducibility (p<0.05). ARNT(rs3768016) CC genotype had highest inducibility, followed by CT, and TT genotypes. ARNT (rs2228099) GG genotype had highest inducibility, followed by CG, and CC genotypes. We conclude that AhRR and ARNT genotypes might interact with PCBs and PCDFs in cytochrome P450 enzyme induction effects.

Interleukin-2 is one of the lymphokines secreted by T helper type 1 cells upon activation mediated by T-cell receptor (TCR) and accessory molecules. The ability to express IL-2 is correlated with T-lineage commitment and is regulated during T cell development and differentiation. Understanding the molecular mechanism of how IL-2 gene inducibility is controlled at each transition and each differentiation process of T-cell development is to understand one aspect of T-cell development. In the present study, we first attempted to elucidate the molecular basis for the developmental changes of IL-2 gene inducibility. We showed that IL-2 gene inducibility is acquired early in immature CD4- CD8-TCR- thymocytes prior to TCR gene rearrangement. Similar to mature T cells, a complete set of transcription factors can be induced at this early stage to activate IL-2 gene expression. The progression of these cells to cortical CD4^+CD8^+TCR^(1o) cells is accompanied by the loss of IL-2 gene inducibility. We demonstrated that DNA binding activities of two transcription factors AP-1 and NF-AT are reduced in cells at this stage. Further, the loss of factor binding, especially AP-1, is attributable to the reduced ability to activate expression of three potential components of AP-1 and NF-AT, including c-Fos, FosB, and Fra-2. We next examined the interaction of transcription factors and the IL-2 promoter in vivo by using the EL4 T cell line and two non-T cell lines. We showed an all-or-none phenomenon regarding the factor-DNA interaction, i.e., in activated T cells, the IL-2 promoter is occupied by sequence-specific transcription factors when all the transcription factors are available; in resting T cells or non-T cells, no specific protein-DNA interaction is observed when only a subset of factors are present in the nuclei. Purposefully reducing a particular set of factor binding activities in stimulated T cells using pharmacological agents cyclosporin A or forskolin also abolished all interactions. The results suggest that a combinatorial and coordinated protein-DNA interaction is required for IL-2 gene activation. The thymocyte experiments clearly illustrated that multiple transcription factors are regulated during intrathymic T-cell development, and this regulation in tum controls the inducibility of the lineage-specific IL-2 gene. The in vivo study of protein-DNA interaction stressed the combinatorial action of transcription factors to stably occupy the IL-2 promoter and to initiate its transcription, and provided a molecular mechanism for changes in IL-2 gene inducibility in T cells undergoing integration of multiple environmental signals.

碩士
國立清華大學
生命科學系
85
Proliferating cell nuclear antigen (PCNA), anauxiliary factor of DNA polymerase D, is required in DNA replication and excision repair. Previous studies have shown that the rat PCNA promoter possesses the serum responsiveness and UV inducibility.In this thesis, the role of AP-1 site (at -64/-58) in rat PCNA promoter in the aspects of serum responsiveness and UV inducibility was studied. The site-specific AP-1 mutant of rat PCNA promoter was constructed using overlap PCR method. The AP-1 mutant promoter isremarkably less active in comparison with itsrespective wild type promoter. Furthermore, the AP-1 mutant partially loses the serum responsiveness and UVinducibility in the experiments in which the mutant promoter was stably transfected into cells. Resultsof this thesis study suggest that AP-1 site (at -64/-58) is necessary for basal activity and plays significant role serum stimulation and UV induction of the rat PCNA promoter.

碩士
國立臺灣大學
免疫學研究所
94
Both peripheral and thymus NK1-CD4+CD8-CD44low T cells displayed an age-associated increase in Qa-2 expression, but the magnitude of the increase was relatively small for NK1-CD4+CD8-CD44low thymocytes and was much more pronounced for peripheral NK1-CD4+CD8-CD44low T cells. Qa-2 expression was uniformly high for the vast majority of NK1-CD4+CD8-CD44low T cells obtained from peripheral tissues and organs. On the other hand, Qa-2 expression was weaker and more heterogeneous for NK1-CD4+CD8-CD44low T cells from the thymus. An inverse correlation between the level of Qa-2 expression and IL-4 inducibility was found for both thymus and spleen NK1-CD4+CD8-CD44low T cells, although the level of IL-4 inducibility was significantly higher for cells from the thymus than the spleen. Similar enrichment of IL-4 inducibility in peripheral NK1-CD4+CD8-CD44low T cells was observed in mice carrying disrupted p59fyn or il4r genes. Isolation of the Vb(2/7/8)+ subset from spleen Qa-2lowNK1-CD4+CD8-CD44low T cells resulted in further enrichment in IL-4 inducibility. In the absence of exogenously added cytokines, TCR-stimulated IL-4 production by spleen Qa-2lowNK1-CD4+8-CD44low T cells promoted their own differentiation toward Th2 effectors in an IL-4-dependent manner. Under Th1 priming conditions, both Qa-2low and Qa-2hi subsets of spleen NK1-CD4+CD8-CD44low T cells differentiated into high level IFNg-producing Th1 effectors. Under Th2 priming conditions, both Qa-2low and Qa-2hi subsets of spleen NK1-CD4+CD8-CD44low T cells differentiated into high level IL-4-produccing Th2 effectors. Thus, the generally accepted low level IL-4 inducibility by naïve CD4+ T cells is due to high level IL-4 inducibility by a small subpopulation of peripheral CD4+ T cells and not due to low level IL-4 inducibility by all or most naïve CD4+ T cells. Furthermore, this small subset of Qa-2lowNK1-CD4+CD8-CD44low T cells, by virtue of its naïve phenotype, prompt IL-4 inducibility, and distribution in the periphery, is expected to play a significant role in the initial stages of immune response by rapidly releasing IL-4 which may in turn direct Th2 response and other IL-4-dependent processes.

碩士
國立清華大學
生命科學系
86
Proliferating cell nuclear antigen (PCNA), an auxiliary factor of DNA polymerase delta, is required in eukaryotic DNA replication and repair. Previous studies by series deletion have shown that the rat PCNA promoter possesses the serum responsiveness and UV inducibility in the region of nucleotide between -70 and +125 which contains one AP-1 site and one ATF/ CRE site. Furthermore, it was found that the AP-1 mutation partially loses the serum responsiveness and UV inducibility. In this thesis,we try to characterize the role of AP-1 site and proximal ATF/CRE site in rat PCNA promoter in the aspects of serum responsiveness and UV inducibility. Using megapriming PCR method, we constructed the site-specific ATF/CRE mutated promoter and the AP-1/ATF double mutated promoter. In comparison with the AP-1 mutant promoter, the ATF/CRE mutant promoter partially loses the serum responsiveness but represents more UV inducibility even than the wild type. Furthermore, when the AP-1/ATF double mutated promoter was transfected stably into CHO.K1 cells, the serum responsiveness and UV inducibility decreased remarkably. Results of this thesis suggest that the proximal AP-1 site and ATF/CRE site are both necessary for basal activity and play crucial roles in serum stimulation of rat PCNA promoter. On the other hand, the ATF/CRE site may be a negative regulator in UV induction of rat PCNA promoter. It remains for further investigation.

Cytochrome P450 enzymes (CYPs), found in almost all organisms, are involved in endobiotic metabolism and detoxification of xenobiotic compounds, such as drugs, pollutants, and insecticides. In insects, CYPs play a major role in conferring resistance to various insecticides including DDT. In Drosophila and other insects, DDT-resistant strains exhibit increased expression of multiple P450 genes; however, the mechanism of overexpression is unknown. Since many CYP genes including Cyp6a8 of Drosophila are induced by caffeine and other xenobiotics, these chemicals are used as tools to understand the regulation of these genes. Previously it was shown that the 0.8-kb (-1/-732) and 0.2-kb (-1/-170) upstream DNA of Cyp6a8 of the DDT-resistant 91-R strain support caffeine, DDT, and Phenobarbital induction in adult flies and S2 cells, the 0.2-kb DNA has many transcriptionally important sequence motifs. In the present investigation, site-directed mutagenesis was performed on the putative TATA box and CREB/AP-1 motifs located at the -97/-101, -57/-61, -43/-47, and -6/-10 regions of the 0.2- and 0.8 DNAs to determine their cis-regulatory role in caffeine and PB induction in S2 cells using luciferase reporter system. Results showed that all four deletions in 0.2- and 0.8-kb DNA decreased both basal and caffeine-induced activities, but maximum effect was seen with the -57/-61 deletion. Second, the TATA mutations greatly decreased basal activity, but they did not decrease caffeine-inducibility as much as the -57/-61 mutations. Third, the effects of other three deletions on basal activities were not as pronounced in the 0.8-kb environment as were seen in the 0.2-kb environment. Taken together these results suggest that of all four putative CREB/AP1 sites the one located at -57/-61 region is most important for both basal and caffeine-induced activities. The results also suggest that the additional 600 bases upstream of -1/-170 have distal elements that interact with the proximal promoter in the 0.2-kb DNA and boost basal transcription. A model suggesting interactions of all cis elements with the basal promoter for basal and induced transcription has been proposed.

Plants must be capable of detecting and responding to a wide variety of environmental stimuli, yet much remains to be determined about the mechanisms by which these processes occur. I describe herein a proof-of-concept mutant screen for identifying mutants aberrant in their responses to a variety of stimuli including heat, cold, touch, and darkness. Such mutants will undoubtedly serve as tools to investigate the molecular pathways by which plants detect and respond to their environment. In addition, I also describe a large-scale gene expression experiment utilizing Affymetrix ATH1 microarrays which focuses on the short-term responses of plants to touch and darkness. These chips enable an expression level survey of over 22,000 genes simultaneously. We identify 589 genes that have increased expression in plants 30 minutes after touch stimulation and 171 genes that have decreased expression. We also identify 461 genes that have increased expression in plants after 30 minutes of darkness treatment and 72 genes that have decreased expression. The identities of the regulated genes suggest that calcium and kinase signaling, cell wall modification, disease resistance and secondary transcriptional responses may be altered in plants subjected to mechanostimulation or darkness.

博士
國立臺灣大學
免疫學研究所
93
CD1d-restricted NKT cells respond to TCR-stimulation by prompt IL-4 production and were originally thought to play a major role in the initiation of IgE response. NK1-CD44lowCD4+CD8- thymocytes were also capable of TCR-stimulated prompt IL-4 inducibility in all common mouse strains examined. The property of IL-4 inducibility by these NK1-CD44lowCD4+CD8- thymocytes was found primarily in Va3.2+ and Vb2/7/8+ cells. Unlike NKT cells, the development of IL-4 inducibility+ NK1-CD44lowCD4+CD8- thymocytes was b2m-, CD1d-, and p59fyn-independent. NK1-CD44lowCD4+CD8- thymocytes also produced IL-5, IL-10, and IL-13, but very low levels of IFN-g in response to TCR stimulation. The IL-4 inducibility+ NK1-CD44lowCD4+CD8- thymocytes expressed highly diverse CDR3 in both TCR a and b chains. Va3.2 and Vb8 CDR3 from single IL-4 mRNA+ and IL-4 mRNA- cells were subcloned into full-length Va3.2 and Vb8 transgene constructs, and injected into fertilized eggs to produce TCR transgenic mice. A “P”-series of 8 independent TCR transgenic lines (4 each of TCR-a and -b) were produced from Va3.2 and Vb8 cloned from 4 randomly selected IL-4-producing single NK1-CD44lowCD4+CD8- thymocytes. Another “N”-series of 6 independent TCR transgenic lines (3 each of TCR-a and -b) were produced from Va3.2 and Vb8 cloned from 3 randomly selected IL-4-non-producing single NK1-CD44lowCD4+CD8- thymocytes. Single TCR-a and -b transgenic mice were crossed to generate double TCR-ab transgenic mice. All P-series TCR-ab transgenic mice displayed elevated IL-4 inducibility in Va3.2+Vb8+ thymocytes and increased serum IgE levels. All N-series TCR-ab transgenic mice, on the other hand, expressed low or negligible IL-4 inducibility in Va3.2+Vb8+ thymocytes and had normal serum IgE levels. Positive selection of P3 TCR-ab transgenic T cells was MHC-II-, but not MHC-I- or CD1d-dependent. Positive selection was necessary but not sufficient in conferring IL-4 inducibility which was dependent on the presence of TCR-gd T cells. These results demonstrate 1) the existence of a MHC-II-restricted CD4+ T cell subset capable of prompt IL-4 inducibility; 2) the critical roles played by CDR3 of both TCR-a and -b chains and by TCR-gd T cells in the acquisition of IL-4 inducibility and elevated serum IgE levels; and 3) the likelihood for the IL-4 inducibility+ NK1-CD44lowCD4+CD8- T cells to participate in immune response to a relatively large number of antigens due to their highly diverse TCR repertoire.