Zhang Junjun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-160). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement from Author --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xvi / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Literature Review --- p.2 / Chapter 1.2.1 --- Rice as a Model Monocot Cereal --- p.2 / Chapter 1.2.1.1 --- Genome Size --- p.3 / Chapter 1.2.1.2 --- Synteny --- p.3 / Chapter 1.2.1.3 --- Well-mapped Genome --- p.4 / Chapter 1.2.1.4 --- Amenable to Transformation --- p.4 / Chapter 1.2.2 --- Rice Genome Sequencing --- p.4 / Chapter 1.2.3 --- Rice Functional Genomics --- p.6 / Chapter 1.2.3.1 --- International Collaboration on Rice Functional Genomics --- p.6 / Chapter 1.2.3.2 --- Recent Progress on Rice Functional Genomics in China --- p.7 / Chapter 1.2.4 --- Large-scale Expressed Sequenced Tag (EST) Analysis --- p.8 / Chapter 1.2.4.1 --- Introduction --- p.8 / Chapter 1.2.4.2 --- Large-scale EST Analysis vs. SAGE and MPSS: Advantages and Shortcomings --- p.12 / Chapter 1.2.4.3 --- Large-scale Rice EST analysis --- p.14 / Chapter 1.2.4.4 --- Seed Specific Large-scale EST Analysis --- p.16 / Chapter 1.2.4.5 --- Bioinformatics Tools Involved --- p.17 / Chapter 1.2.5 --- Profiling Gene Expression using cDNA Microarray --- p.20 / Chapter 1.2.5.1 --- Introduction --- p.20 / Chapter 1.2.5.2 --- Advantages of cDNA Microarray in Gene Expression Profiling --- p.22 / Chapter 1.2.5.3 --- Profiling Gene Expression by cDNA Microarrays --- p.24 / Chapter 1.2.5.4 --- Profiling Seed-specific Gene Expression --- p.26 / Chapter 1.2.6 --- Overview of Current Information on the Biosynthesis and Gene Regulation of Major Components during Rice Seed Formation --- p.30 / Chapter 1.2.6.1 --- Starch --- p.30 / Chapter 1.2.6.2 --- Storage Proteins --- p.38 / Chapter 1.2.6.3 --- Lipids --- p.42 / Chapter Chapter 2 --- Materials and Methods --- p.44 / Chapter 2.1 --- Materials --- p.44 / Chapter 2.1.1 --- Chemicals --- p.44 / Chapter 2.1.2 --- Facilities and Instruments --- p.45 / Chapter 2.1.3 --- Commercial Kits --- p.46 / Chapter 2.1.4 --- Oligonucleotides --- p.47 / Chapter 2.1.5 --- Plant Materials --- p.50 / Chapter 2.1.6 --- Bacterial Strains --- p.52 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- mRNA Isolation --- p.52 / Chapter 2.2.2 --- Construction of cDNA Library --- p.53 / Chapter 2.2.2.1 --- Construction of cDNA Library in Phage --- p.53 / Chapter 2.2.2.2 --- Construction of cDNA Library in E.coli --- p.56 / Chapter 2.2.3 --- Screening of cDNA Library --- p.62 / Chapter 2.2.3.1 --- In situ Hybridization --- p.62 / Chapter 2.2.3.2 --- Probe Synthesis --- p.63 / Chapter 2.2.3.3 --- Hybridization and Detection --- p.64 / Chapter 2.2.4 --- Single-pass Sequencing of cDNAs --- p.65 / Chapter 2.2.5 --- Sequence Analysis --- p.67 / Chapter 2.2.6 --- BLAST Search --- p.71 / Chapter 2.2.7 --- Contig Analysis --- p.71 / Chapter 2.2.8 --- Database Management --- p.72 / Chapter 2.2.9 --- Selection Criteria for ESTs in Different Pathways --- p.72 / Chapter 2.2.10 --- Construction of Super Hybrid Rice Seed cDNA Microarray --- p.75 / Chapter 2.2.11 --- "Probe Synthesis, Microarray Hybridization and Detection" --- p.75 / Chapter 2.2.12 --- Quality Test of External Control --- p.78 / Chapter 2.2.13 --- Scanning of Microarray --- p.79 / Chapter 2.2.14 --- Analysis of Microarray Data --- p.79 / Chapter 2.2.15 --- Normalization --- p.79 / Chapter 2.2.16 --- Northern Blot Analysis --- p.80 / Chapter Chapter 3 --- Results and Discussion --- p.82 / Chapter 3.1 --- cDNA Libraries --- p.82 / Chapter 3.1.1 --- cDNA Library in Phage --- p.82 / Chapter 3.1.2 --- cDNA Library in E.coli --- p.84 / Chapter 3.2 --- Statistic Data of Screening and Single-pass Sequencing --- p.87 / Chapter 3.3 --- Genes Expressing in Developing Rice Seeds --- p.89 / Chapter 3.4 --- Novel ESTs in the Seed EST Set --- p.94 / Chapter 3.5 --- Unique Genes in the EST Set from the Phage cDNA Library --- p.94 / Chapter 3.6 --- Mapping ESTs onto Rice Genome --- p.95 / Chapter 3.7 --- The Most Abundant Transcripts Reflected by EST Redundancy --- p.98 / Chapter 3.8 --- ESTs and Representive Genes in Different Pathways --- p.102 / Chapter 3.9 --- ESTs Involved in Carbon Flows --- p.106 / Chapter 3.10 --- Microarray Configuration and Contents --- p.109 / Chapter 3.11 --- Quantification of Total RNA from Seeds at 5 Developing Stages --- p.110 / Chapter 3.12 --- Quality Test of External Control --- p.111 / Chapter 3.13 --- Scanning of Array Hybridization --- p.113 / Chapter 3.14 --- Reproducibility of Technical Duplicates --- p.116 / Chapter 3.15 --- Summary of Gene Expression Profiles in Developing Super Hybrid Rice Seeds --- p.117 / Chapter 3.16 --- Expression Profiles of Major Storage Compounds in Rice Seeds --- p.119 / Chapter 3.16.1 --- Expression profiles of storage protein genes --- p.119 / Chapter 3.16.2 --- Expression Profiles of Starch Synthesis Related Genes --- p.122 / Chapter 3.16.3 --- Expression Profiles of Lipid Synthesis Related Genes --- p.124 / Chapter 3.16.4 --- General Expression Patterns of Major Storage Compounds --- p.126 / Chapter 3.18 --- General Discussion --- p.131 / Chapter 3.18.1 --- Cautions when Interpreting the EST Data --- p.131 / Chapter 3.18.2 --- Comparison of EST Data Sets of Developing Seeds between Arabidopsis and Rice --- p.131 / Chapter 3.18.3 --- Comparison of cDNA Library Construction by Two Methods --- p.135 / Chapter 3.18.4 --- Comparison of Different Normalization Methods --- p.138 / Chapter 3.18.5 --- Comparison between Microarray and Northern Data --- p.140 / Chapter Chapter 4 --- Conclusion --- p.142 / Chapter Chapter 5 --- Future Perspectives --- p.145 / Reference Lists --- p.147

Polyreactive immunoglobulins (Ig) exhibit a capacity to recognise multiple, structurally dissimilar antigens through a single combining site. This characteristic differentiates these Igs from monoreactive Igs which bind to a single antigen, usually with high specificity and affinity. Chronic B lymphocytic leukaemia (B CLL) is a malignancy identified by the incessant accumulation, in the peripheral circulation, of B lymphocytes of a mature and resting morphology. B CLL malignant cells generally express both surface IgM and the pan T cell antigen CD5. Moreover, the IgM on the surface of these CD5 positive B CLL cells is frequently polyreactive. This thesis examines the structural diversity found in the combining sites of B CLL derived Igs in an attempt to elucidate the structural basis of polyreactive antigen binding displayed by a significant proportion of human Igs. The genes encoding the variable (V) domains of five B CLL derived IgM antibodies (Bel, Tre, Yar, Hod and Jak) were cloned and sequenced (Chapter Two). When the light chain V domain genes were aligned with the closest germline VL and JL coding DNA sequences it was determined that there was either a complete absence of somatic mutation (Tre, Yar and Jak) or a minimal number of mutations (Bel and Hod) present in the rearranged VL domain genes. A remarkable fidelity in the splicing of VL to JL genes was noted suggesting that the diversity, normally introduced through variability of splicing VL to JL, is reduced in Igs expressed by B CLL cells. Furthermore, the markedly reduced primary structural diversity was highlighted when two of the VL domain genes (Yar and Hod) were found to be different in sequence by only four nucleotides and two amino acids. The heavy chain V domain genes of the same five Igs were sequenced in another study (Brock, 1995), however, it was interesting to analyse the sequences of the VH domain genes and compare them with the VL domain genes. The naive or gerrnline nature of the B CLL antibodies was reflected in the VH genes by either an absence or a low frequency of mutations within these sequences compared with germline immunoglobulin gene sequences. No obvious conserved motif, which could be related to polyreactivity, was observed when the primary protein sequence was analysed for distribution of identical or similar amino acids. Thus, homology modelling was used to construct three-dimensional models of the Fv (VL-VH) portions of the five B CLL IgM molecules to examine the structures of the combining sites of these Igs (Chapter Three). Framework regions were constructed using X-ray coordinates taken from highly hon~ologous human variable domain structures. Complementarity determining regions (CDR) were predicted by grafting loops, taken from known Ig structures, onto the Fv framework models. The CDR templates were selected, where possible, to be of the same length and of high residue identity or similarity. If a single template CDR was not appropriate to model a particular CDR the loop was built from loop sterns of known conformation, followed by chain closure with a p-turn. Template models were refined using standard molecular mechanics simulations. The binding sites were either relatively flat or contained a deep cavity at the VL-VH domain interface. Further differences in topology were the result of some CDR loops protruding into the solvent. Examination of the electrostatic molecular surface did not reveal a common structural feature within the binding sites of the five polyreactive Fv. While two of the binding cavities were positively charged the other three structures displayed either negatively charged or predominantly hydrophobic combining sites. These findings suggested that a diversity of structural mechanisms are involved in polyreactive antigen binding. Rcsidues within CDRs which have aromatic side-chains and are partially exposed to solvent were distributed across large regions of the combining sites. It is possible that these aromatic residues are responsible for the conserved binding to mouse Igs observed (Chapter Two) for the B CLL derived polyreactive IgM molecules. Two Fv molecules (Be1 and Tre) were cloned as dicistronic constructs, into the bacterial expression vector pFLAG. The expression of the Fvs was fully characterised and unfortunately the VL and VH of Be1 and Tre Igs did not associate in an appropriate manner to yield large quantities of purified Fv (Chapter Four). Expression of correctly folded and stabilised fragments of human polyreactive immunoglobulins would enable the structural basis for the polyreactive binding phenomenon to be fully explored using protein crystallography.

The PsbL, PsbM and PsbT subunits of photosystem II (PSII) are single-helix membrane-spanning proteins found at the monomer-monomer interface that may stabilize the dimeric complex. This study has characterised strains of Synechocystis sp. PCC 6803 where psbL, psbM and psbT have been interrupted by the insertion of antibiotic-resistance cassettes. The [Delta]PsbL strain exhibited slowed growth that correlated with a disruption in PSII assembly leading to an accumulation of CP43-less PSII monomers. Moreover, the [Delta]PsbL:[Delta]PsbM and [Delta]PsbL:[Delta]PsbT double mutants were not photoautotrophic. In contrast, the [Delta]PsbM and [Delta]PsbT strains grew photoautotrophically and supported oxygen evolution, albeit at reduced rates compared to wild type. S-state analyses showed that the removal of PsbM or PsbT did not affect the donor side reactions of PSII, which includes the oxidation of water, however, the removal of PsbT impaired electron flow between Q[A] and Q[B] on the acceptor side of PSII. Blue-Native PAGE revealed that removal of either PsbM or PsbT was insufficient to entirely disrupt dimer formation; however, the combined removal of PsbM and PsbT resulted in the predominance of monomeric forms of PSII in the [Delta]PsbM:[Delta]PsbT strain. Under high light (2 mE m⁻� s⁻� at 30�C), [Delta]PsbM and [Delta]PsbT cells were considerably more susceptible to photoinactivation than wild type; however, they were able to fully recover in a protein synthesis-dependent manner when returned to moderate light levels (0.03 mE m⁻� s⁻�). A requirement for Psb27 was found in the protein-synthesis-dependent recovery of photoinactivated [Delta]PsbT cells. More significantly, an absolute functional requirement was found for Psb27 in the [Delta]PsbM strain, where functional PSII complexes are not assembled in the absence of Psb27. These results suggest that Psb27 is critical for PSII assembly in the absence of PsbM, and also for the protein-synthesis-dependent recovery of PSII in the absence of PsbT. Moreover, in addition to Psb27, the PsbU subunit of the oxygen-evolving complex was also found to be an absolute functional requirement in the [Delta]PsbM strain, where functional PSII centres are not assembled when both PsbM and PsbU are absent. It appears, therefore, that PsbM has crucial functional interactions with specific extrinsic proteins located in the vicinity of the oxygen-evolving complex. Interestingly, the [Delta]PsbM strain was also found to have a high susceptibility to suppressor mutations, indicating it has important functional roles in the cyanobacterial cell.

Selected strains of Streptococcus equi subsp. zooepidemicus produce a bacteriocin-like inhibitory substance called zoocin A. Zoocin A has bacteriolytic activity against closely related species of streptococci, including the etiological agents of dental caries and Streptococcus pyogenes. Zoocin A functions to hydrolyse the peptidoglycan of bacterial cell walls. Producer-cell immunity to zoocin A is encoded by zif, the zoocin A immunity factor. The zooA-zif gene locus is chromosomally encoded and flanked by two transposon-like sequences with significant similarity to the lss-lif locus encoding lysostaphin in Staphylococcus simulans. This suggested the possibility that the zooA-zif locus was acquired by S. equi subsp. zooepidemicus through a horizontal gene transfer event. Studies conducted to examine this hypothesis were unable to establish the mechanism by which zooA-zif was acquired. Examination of 24 S. equi subsp. zooepidemicus strains using random amplified polymorphic DNA analysis and pulse field gel electrophoresis showed that the species was highly heterogeneous. Examination of the 24 strains using Southern hybridisation revealed only three S. equi subsp. zooepidemicus strains contained the zooA and zif genes; however the partial open reading frames (flaR and rgg-like) flanking zooA and zif appeared to be conserved in all tested strains. Long template PCR was used to examine this chromosomal area containing the zooA and zif genes in 24 S. equi subsp. zooepidemicus strains. These results, along with comparison of sequence data from two zoocin A producing strains, and analysis of the sequenced S. equi subsp. zooepidemicus H70 and S. equi subsp. equi 4047 genomes showed the area surrounding the zooA-zif locus to be one of considerable genetic variation substantiating the hypothesis that zooA-zif were acquired via horizontal gene transfer. Studies on zoocin A production suggested that zooA expression was under carbon catabolite regulation, (CCR). In the Gram-positive bacteria CCR, is mediated by the carbon catabolite control protein (CcpA). CcpA is a DNA binding protein that can negatively regulate transcription of its target gene by binding to a catabolite-responsive element (CRE). This study reports the regulation of zif and zooA expression in response to cellular growth on different carbon sources, the presence of a ccpA-like gene in the genome of S. equi subsp. zooepidemicus, the presence of a CRE sequence in the promoter region of zooA, and the effect of a ccpA deletion on zooA expression. Zoocin A activity, zooA and zif mRNA transcript levels, and zooA and zif reporter gene activities were monitored during growth of S. equi subsp. zooepidemicus 4881 in a pH-controlled batch culture system. Zoocin A titres were maximal when the bacteria were grown on a starch-based medium and minimal when grown on a glucose-based medium. Northern hybridization showed that zif was constitutively expressed during growth regardless of the carbon source available. In contrast, zooA was transcribed at low levels in cells grown on a glucose-based medium but at much higher levels in cells grown on a starch-based medium. Use of a zooA promoter-chloramphenicol acetyl transferase (CAT) reporter demonstrated that zooA expression increased 17-fold in cells grown on a starch-based medium. Use of a zif promoter-CAT reporter showed that in cells grown on a starch-based medium zif was transcribed approximately 4 times as strongly as in cells grown on glucose. Transcription of zif was always much less than that of zooA. A ccpA deletion mutant appeared to exhibit complete de-repression of zooA under all growth conditions. Together these data showed that zoocin A expression was regulated in response to the carbon source on which the bacteria were grown.

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

No description available.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Babatunde Ogunnaike and Jeremy S. Edwards, Dept. of Chemical Engineering. Includes bibliographical references.

This dissertation explores the genetic basis of gene expression in Fundulus heteroclitus by focusing on the role of the environment and its effects on gene expression and by making direct estimates of heritability using cDNA microarrays. The second chapter describes the utility of F. heteroclitus cDNA microarrays for studies of F. heteroclitus which seek to understand the genetic variation in gene expression. Measurements of mRNA fluorescence and concentration as well as differences in sample preparation and sampling of blood from a single individual over time demonstrate that F. heteroclitus cDNA microarrays are quantitative, reproducible and consistent. The third chapter examines the effect of the environment and genetic factors on the variation of gene expression. F. heteroclitus cDNA microarrays are used to determine whether a genetic component of gene expression can describe the variation in gene expression between inbred and outbred individuals from the same population. The results show that variation in mRNA expression is related to the genetic variation among individuals within a group. While chapter three reveals that there is a genetic component of variation in gene expression, the percentage of genes that are significantly heritable was not known. In the fourth chapter, the heritability of the variation in gene expression is estimated to determine the genetic basis of gene expression in F1 individuals from natural, outbred populations of F. heteroclitus. The data presented in chapter 4 are the first to formally estimate the genetic component of gene expression in F. heteroclitus. The estimates of heritability range from 0.25 to 0.86 depending on the estimation method with approximately 6.5% of genes having significant heritability. The results presented in this dissertation support the concept that genetic variation affects variation in mRNA expression among natural populations of F. heteroclitus. Natural, heritable variation in gene expression is important for understanding evolutionary adaptation and the role of natural selection in evolutionary processes.

Thesis (M.S.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Karen Katula; submitted to the Dept. of Biology. Includes bibliographical references (p. 59-61).

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.