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Wong Ka Shing = 兩個二級蛋白質結構組中的氨基酸序列的統計特性 / 黃嘉誠. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 88-91). / Text in English; abstracts in English and Chinese. / Wong Ka Shing = Liang ge er ji dan bai zhi jie gou zu zhong de an ji suan xu lie de tong ji te xing / Huang Jiacheng. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Predicting tertiary structures of proteins --- p.2 / Chapter 1.2 --- Introduction to secondary structures of protein --- p.5 / Chapter 1.3 --- Prediction of secondary structural classes --- p.9 / Chapter 1.3.1 --- Multi-dimensional space representation --- p.10 / Chapter 2 --- Method of analysis --- p.12 / Chapter 2.1 --- Data sets --- p.12 / Chapter 2.2 --- Basic statistics of the data sets --- p.13 / Chapter 2.3 --- Method of analysis --- p.17 / Chapter 2.4 --- An interesting sum rule --- p.20 / Chapter 3 --- Results of analysis of C-words --- p.22 / Chapter 3.1 --- A first insight --- p.22 / Chapter 3.2 --- Distributions of 〈C(j)〉 --- p.24 / Chapter 3.2.1 --- "Comparing distributions for different m, s and secondary structural classes" --- p.29 / Chapter 3.3 --- Features of each secondary structural class --- p.33 / Chapter 3.3.1 --- C-words with the largest or the smallest values of 〈C(j)〉 in the all-a class --- p.33 / Chapter 3.3.2 --- C-words with the largest or the smallest values of 〈C(j)〉 in the all-β class --- p.35 / Chapter 4 --- Results of analysis of H-words --- p.37 / Chapter 4.1 --- 〈C(j)〉 for each H-word --- p.38 / Chapter 4.1.1 --- "When m increases, 〈C(j)〉 deviates more from 1" --- p.39 / Chapter 4.1.2 --- "When spacing parameter s is small, 〈C(j)〉 deviates more from 1" --- p.40 / Chapter 4.2 --- The mean over all possible H-words --- p.41 / Chapter 4.3 --- Comparing 〈C(j)〉 of different H-words --- p.43 / Chapter 4.3.1 --- A few plots --- p.43 / Chapter 4.3.2 --- Results --- p.44 / Chapter 4.4 --- Features of each secondary structural class --- p.47 / Chapter 4.4.1 --- A first insight --- p.47 / Chapter 4.4.2 --- The H-words with the largest values of 〈C(j)〉 in the all-α class with particular number of letter m --- p.54 / Chapter 4.4.3 --- The H-words with the smallest values of 〈C(j) in the all-α class with particular number of letter m --- p.57 / Chapter 4.4.4 --- The H-words with the largest values of 〈C(j) in the all-β class with particular number of letter m --- p.60 / Chapter 4.4.5 --- The H-words with the smallest values of 〈C(j) in the all-β class with particular number of letter m --- p.63 / Chapter 4.4.6 --- A summary --- p.66 / Chapter 4.5 --- Possible indicators to predict secondary structural class --- p.67 / Chapter 4.5.1 --- H-words with the largest values of --- p.68 / Chapter 4.5.2 --- H-words with the largest magnitude of --- p.71 / Chapter 4.5.3 --- Discussion --- p.71 / Chapter 5 --- Prediction using patterns found in H-words --- p.72 / Chapter 6 --- Conclusion --- p.86 / Bibliography --- p.88

Xia Lixin. / "August 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 210-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Hon, Ching Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-157). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vi / Table of Contents --- p.viii / List of Tables --- p.xiv / List of Figures --- p.xv / List of Abbreviations --- p.xvii / List of Chemicals --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Mycobacterium tuberculosis --- p.1 / Chapter 1.2 --- Tuberculosis --- p.2 / Chapter 1.3 --- Pathogenesis of TB --- p.3 / Chapter 1.3.1 --- Mycobacterial entry to the host macrophage --- p.3 / Chapter 1.3.2 --- Modulation of the host endocytic pathway --- p.4 / Chapter 1.3.2.1 --- The fusion between lysosome and mycobacterial phagosomeis blocked --- p.4 / Chapter 1.3.2.2 --- The endosomal pH of mycobacterial phagosome is preserved --- p.5 / Chapter 1.3.2.3 --- Mtb successfully mediates host cell apoptosis inhibition --- p.6 / Chapter 1.3.3 --- Cell migration and granuloma formation --- p.7 / Chapter 1.4 --- Insights from the complete genome sequence of Mtb H37Rv --- p.9 / Chapter 1.4.1 --- PE protein family --- p.9 / Chapter 1.4.2 --- PPE protein family --- p.10 / Chapter 1.5 --- Interaction between PE and PPE proteins --- p.11 / Chapter 1.5.1 --- Integrated bioinformatics prediction --- p.11 / Chapter 1.5.2 --- In vitro interaction studies of PE25/PPE41 protein complex --- p.12 / Chapter 1.5.3 --- Structural characterization of PE/PPE complex as revealed by PE25/PPE41 crystal structure --- p.14 / Chapter 1.6 --- Biological roles of PE and PPE proteins --- p.15 / Chapter 1.6.1 --- Immunological roles of PE family proteins --- p.15 / Chapter 1.6.2 --- Immunological roles of PPE family proteins --- p.16 / Chapter 1.6.3 --- Immunological roles of PE/PPE protein complex --- p.16 / Chapter 1.7 --- Sub-cellular localization of PE and PPE proteins --- p.17 / Chapter 1.8 --- PE and PPE as exported proteins --- p.18 / Chapter 1.8.1 --- Association of Mtb secreted proteins to pathogenesis of tuberculosis --- p.18 / Chapter 1.8.2 --- Exported PE and PPE proteins --- p.19 / Chapter 1.9 --- Differential expression of PE and PPE genes --- p.20 / Chapter 1.10 --- Objective of project --- p.21 / Chapter CHAPTER 2 --- "CLONING, EXPRESSION, PURIFICATION AND VERIFICATION OF PE15 AND PPE20 AS COMPLEX" --- p.24 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Bacterial expression plasmids for expression of PE or PPE proteins --- p.24 / Chapter 2.1.2 --- E. coli strains --- p.27 / Chapter 2.1.3 --- Reagents and buffers for molecular cloning --- p.27 / Chapter 2.1.4 --- Reagents and buffers for E. coli protein expression --- p.29 / Chapter 2.1.5 --- Buffers for protein purification --- p.30 / Chapter 2.2 --- Methods --- p.31 / Chapter 2.2.1 --- Molecular cloning --- p.31 / Chapter 2.2.1.1 --- Primers --- p.31 / Chapter 2.2.1.2 --- Gene Amplification by Polymerase Chain Reaction (PCR) --- p.33 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.34 / Chapter 2.2.1.4 --- Extraction and purification of DNA from agarose gel by QIAquick Gel Extraction Kit --- p.34 / Chapter 2.2.1.5 --- Restriction digestion of DNA --- p.35 / Chapter 2.2.1.6 --- Purification of DNA by QIAquick PCR Purification Kit --- p.36 / Chapter 2.2.1.7 --- Ligation of DNA and expression vector to produce recombinant plasmid --- p.37 / Chapter 2.2.1.8 --- Transformation of plasmid into E. coli competent cell --- p.38 / Chapter 2.2.1.9 --- PCR Screening of recombinant clones --- p.39 / Chapter 2.2.1.10 --- Plasmid DNA purification using the QIAprep Spin Miniprep Kit --- p.40 / Chapter 2.2.1.11 --- DNA sequencing --- p.41 / Chapter 2.2.2 --- Expression of PE15 and PPE20 proteins --- p.41 / Chapter 2.2.2.1 --- Small scale protein expression of PE 15 and PPE20 proteins --- p.41 / Chapter 2.2.2.2 --- Small scale co-expression of PE 15 and PPE20 proteins --- p.42 / Chapter 2.2.2.3 --- Protein solubility analysis --- p.43 / Chapter 2.2.2.4 --- Large scale expression of PE and PPE proteins --- p.43 / Chapter 2.2.3 --- SDS-PAGE --- p.44 / Chapter 2.2.4 --- Bradford assay --- p.45 / Chapter 2.2.5 --- Protein purification of PE15 --- p.46 / Chapter 2.2.5.1 --- Sonication and extraction of proteins --- p.46 / Chapter 2.2.5.2 --- Protein purification of PE15 by gutathione-sepharose affinity chromatography --- p.46 / Chapter 2.2.5.3 --- Protein purification of PE15 by re-binding to glutathione-sepharose --- p.47 / Chapter 2.2.5.4 --- Protein purification of PE15 by size exclusion chromatography --- p.47 / Chapter 2.2.5.5 --- Protein purification of GST-PE15 --- p.48 / Chapter 2.2.6 --- Protein purification of PPE20(PPE) --- p.49 / Chapter 2.2.6.1 --- Sonication and extraction of proteins --- p.49 / Chapter 2.2.6.2 --- Protein purification of PPE20(PPE) by glutathione-sepharose affinity chromatography --- p.49 / Chapter 2.2.6.3 --- Protein purification of PPE20(PPE) by re-binding to glutathione-sepharose --- p.49 / Chapter 2.2.6.4 --- Protein purification of PPE20(PPE) by size exclusion chromatography --- p.50 / Chapter 2.2.6.5 --- Protein purification of GST-PPE20(PPE) --- p.50 / Chapter 2.2.7 --- Verification of PEPPE protein complex --- p.50 / Chapter 2.2.7.1 --- Size exclusion chromatography --- p.50 / Chapter 2.2.7.2 --- Cross-linking --- p.50 / Chapter 2.2.7.3 --- Pull-down assay --- p.51 / Chapter 2.3 --- Results --- p.52 / Chapter 2.3.1 --- Construction of bacterial expression plasmids for PE15 and PPE20 genes --- p.52 / Chapter 2.3.2 --- Expression of PE15 and PPE20 proteins --- p.54 / Chapter 2.3.2.1 --- Small scale protein expression of PE15 --- p.55 / Chapter 2.3.2.2 --- Small scale protein expression of PPE20 --- p.56 / Chapter 2.3.2.3 --- Small scale co-expression of PE15 and PPE20 proteins --- p.60 / Chapter 2.3.3 --- Large scale purification of PE15 and PPE20(PPE) proteins --- p.66 / Chapter 2.3.3.1 --- Large scale purification of PE15 --- p.66 / Chapter 2.3.3.2 --- Large scale purification of GST-PE15 --- p.68 / Chapter 2.3.3.3 --- Large scale purification of PPE20(PPE) --- p.69 / Chapter 2.3.3.4 --- Large scale purification of GST-PPE20(PPE) --- p.70 / Chapter 2.3.4 --- Verification of PE15/PPE20 complex --- p.71 / Chapter 2.3.4.1 --- Size exclusion chromatography --- p.71 / Chapter 2.3.4.2 --- Cross-linking study --- p.74 / Chapter 2.3.4.3 --- Pull-down assay --- p.77 / Chapter 2.4 --- Discussion --- p.79 / Chapter 2.4.1 --- Expression of PE15 and PPE20 proteins --- p.79 / Chapter 2.4.2 --- Co-expression of PE15 and PPE20 proteins --- p.81 / Chapter 2.4.3 --- Prediction of PE/PPE interaction --- p.82 / Chapter 2.4.4 --- Study ofPE15 and PPE20(PPE) interaction --- p.83 / Chapter CHAPTER 3 --- PRELIMINARY X-RAY ANALYSIS OF PPE20(PPE) PROTEIN CRYSTAL --- p.86 / Chapter 3.1 --- Materials --- p.86 / Chapter 3.1.1 --- Crystallization screening kits --- p.86 / Chapter 3.1.2 --- Crystallization chemicals --- p.86 / Chapter 3.2 --- Methods --- p.87 / Chapter 3.2.1 --- Crystallization screening using Phoenix´ёØ RE --- p.87 / Chapter 3.2.2 --- Optimization of PPE20(PPE) crystals by grid screening --- p.88 / Chapter 3.2.3 --- Optimization using Additive screen for PPE20(PPE) --- p.88 / Chapter 3.2.4 --- X-ray diffraction and data collection --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Crystallization screening --- p.89 / Chapter 3.3.2 --- Crystallization optimization --- p.90 / Chapter 3.3.3 --- Preliminary X-ray diffraction analysis --- p.92 / Chapter 3.3.4 --- Attempts to solve the phase by molecular replacement --- p.96 / Chapter 3.4 --- Discussion --- p.97 / Chapter CHAPTER 4 --- ISOLATION OF INTERACTING PARTNERS OF PE15 AND PPE20(PPE) FROM HUMAN MACROPHAGE U-937 --- p.99 / Chapter 4.1 --- Materials --- p.99 / Chapter 4.1.1 --- Mammalian cell line --- p.99 / Chapter 4.1.2 --- Mammalian cell growth medium --- p.99 / Chapter 4.1.3 --- Reagents and buffers for mammalian cell culture --- p.100 / Chapter 4.1.4 --- Reagents and buffers for mass spectrometry sample preparation --- p.100 / Chapter 4.2 --- Methods --- p.101 / Chapter 4.2.1 --- U-937 cell culturing --- p.101 / Chapter 4.2.1.1 --- Thawing U-937 cells --- p.101 / Chapter 4.2.1.2 --- Monitoring cell growth --- p.102 / Chapter 4.2.1.3 --- Cell differentiation --- p.102 / Chapter 4.2.1.4 --- Cell Harvesting --- p.103 / Chapter 4.2.2 --- In-vitro pull-down to identify interacting partners of PE15 or PPE20(PPE) --- p.103 / Chapter 4.2.2.1 --- Preparation of cellular proteins from U-937 cells --- p.103 / Chapter 4.2.2.2 --- Pre-clearing of U-937 supernatant --- p.104 / Chapter 4.2.2.3 --- Pull-down of U-937 cellular proteins with immobilized GST-PE15 --- p.104 / Chapter 4.2.2.4 --- Pull-down of U-937 cellular proteins with immobilized GST-PPE20(PPE) --- p.106 / Chapter 4.2.2.5 --- SDS-PAGE analysis --- p.106 / Chapter 4.2.2.6 --- Silver staining --- p.106 / Chapter 4.2.3 --- Mass- Spectrometry --- p.107 / Chapter 4.2.3.1 --- De-staining of silver stained gel spots --- p.107 / Chapter 4.2.3.2 --- Trypsin digestion --- p.108 / Chapter 4.2.3.3 --- Peptide Extraction --- p.108 / Chapter 4.2.3.4 --- Desalting and concentration of peptide mixture --- p.109 / Chapter 4.3 --- Results --- p.110 / Chapter 4.3.1 --- U-937 differentiation --- p.110 / Chapter 4.3.2 --- In-vitro pull-down --- p.113 / Chapter 4.3.2.1 --- Pull-down with immobilized GST-PE15 --- p.113 / Chapter 4.3.2.2 --- Pull-down with immobilized GST-PPE20(PPE) --- p.116 / Chapter 4.3.2.3 --- Mass spectrometry identification of protein --- p.120 / Chapter 4.4 --- Discussion --- p.122 / Chapter 4.4.1 --- Differentiation of U-937 --- p.122 / Chapter 4.4.2 --- Isolation of PE15 and PPE20(PPE) interacting partners from U-937 --- p.125 / Chapter CHAPTER 5 --- CONCLUSION AND FUTURE PERSPECTIVES --- p.133 / Chapter 5.1 --- Conclusion --- p.133 / Chapter 5.2 --- Future perspectives --- p.137

Call number: LD2668 .T4 1985 J54 / Master of Science

Call number: LD2668 .T4 1986 C58 / Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

Chloroplast and cytoplasmic ribosomes have been isolated from a number of species of the angiosperm genus Nicotiana. Ribosomal subunit and monosome proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Resultant two-dimensional electrophoretic patterns of chloroplast and cytoplasmic ribosomal proteins were processed by a computer algorithm, developed to formally compare different electrophoretic patterns by the construction of two-dimensional, conformal average electrophoretic mobility maps. The chloroplast ribosomal subunit of N. tabacum contains 22-24 distinct basic polypeptides (pI > 5) and 2-3 acidic proteins (pI < 5). The 50S chloroplast ribosomal subunit possesses at least 1 acidic and 33-35 basic proteins. 40S and 60S cytoplasmic ribosomal subunits of the same species have 26-30 and 47-50 basic polypeptides, respectively. Molecular weights of chloroplast ribosomal proteins (ChRP) and cytoplasmic ribosomal proteins (CyRP) were estimated. There was little similarity between the 2D electrophoretic patterns of ChRP and CyRP of N. tabacum. However, 2D-PAGE patterns of N. tabacum ChRP and CyRP were qualitatively isomorphous with homologous patterns of Chlamydomonas reinhardi, pea and spinach. In terms of molecular weight and electrophoretic pattern N. tabacum ChRP were found to be more closely affiliated with prokaryotic ribosomal proteins than with CyRP from the same species. ChRP were isolated from N. gossei (an Australian species) and its reciprocol interspecies hybrids with N. tabacum (denoted by: T x G and G x T). Interspecies polymorphisms between homologous N. tabacum and N. gossei ChRP were delineated by computerized mobility mapping and co-electrophoresis of radiolabeled N. tabacum ChRP with a large molar excess of N. gossei ChRP. The inheritance mode (Mendelian vs. maternal) of a number of well-defined interspecies ChRP polymorphisms was determined by co-electrophoresis of radioiodinated N. tabacum ChRP with T x G and G x T hybrid ChRP. Results indicate that at least 4 30S ChRP and 3 50S ChRP are encoded by nuclear genes. 30S ChRP from an N. tabacum line carrying a maternally-inherited streptomycin-resistance mutation (SR-1) were compared to N. tabacum 30S ChRP by mobility mapping. Two differences were established between the SR-1 and wild-type 30S ChRP average mobility maps. These findings correlate with the reduced affinity of SR-1 30S chloroplast ribosomal subunits for ('3)H-dihydrostreptomycin, and show that at least one 30S ChRP is encoded by chloroplast DNA. Preparative 2D-PAGE and reverse high performance liquid chromatography (RPHPLC) separation techniques for complex ribosomal protein mixtures were developed. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI

Microvitellogenin is a female specific yolk protein from the tobacco hornworm moth Manduca sexta. A cDNA library was constructed from poly (A)⁺ RNA isolated from adult female fat body. cDNA clones of mRNA for microvitellogenin were isolated by screening the cDNA library with antiserum against microvitellogenin. The results of Northern blot analysis and hybrid selection indicated that the cDNA clone was specific for microvitellogenin. The complete nucleotide sequence of the 834 base pair cDNA insert has been determined by the dideoxy chain termination method. The deduced amino acid sequence was compared with the N-terminal sequence determined by Edman degradation, an amino terminal extension of 17 amino acids appeared to be a signal peptide. The cDNA sequence predicts that the mature microvitellogenin is a protein of 232 amino acids with a calculated molecular weight of 26,201. A comparison of the translated amino acid sequence with the sequences in National Biomedical Research Foundation protein library did not establish any sequence similarity with known proteins. The microvitellogenin gene begins to be expressed in the fat body on the first day of the wandering (prepupal) females as determined by using the cDNA insert as a probe to hybridize with the mRNA for microvitellogenin. The cDNA probe was also used to screen a genomic library of M. sexta, yielding three genomic clones for microvitellogenin. One of them was characterized and it contained the complete microvitellogenin gene. The gene sequence was determined. Comparison to the cDNA sequence showed that the microvitellogenin gene contains an intron near the 5'-end of the non-coding region. The 5'-flanking sequence of the gene has been compared to the same regions of yp genes of Drosophila and vitellogenin genes of locust, some similar sequences have been observed and discussed.