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Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 55-64).

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A spontaneous deletion mutant of Bacillus phage φ29 (φ29Δ1) is characterized in the first part of this study. This mutant has a 1,112 base pair deletion, which covers the entire coding sequence of genes 14 and 15 including the early promoter, B2. While lysis is very delayed, the phage DNA synthesis and internal phage development appears to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in φ29Δ1. Results also suggest that genes 14 and 15 are dispensable for bacteriophage φ29 growth, and that the B2 promoter may also be despensable for the early functions of φ29. To further explore the function of gene 15, a DNA fragment of φ29 chromosome, encoding the entire sequence of this gene, has been cloned into the Escherichia coli expression vector pPLc245, under the control of the phage lambda major early leftward promoter, PL. Upon heat induction, a protein with an apparent size of 26 kdal was over-produced. This protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to the other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. This is the first lysozyme to be cloned and purified from a gram positive system. Bacteriophage φ29 lysozyme has been characterized in the last part of this study. Results show that this enzyme seems to more active than hen egg-white lysozyme against B. subtilis, E. coli, and M. lysodeikticus cells. Most of the characteristics of φ29 lysozyme appears to be similar to the P22 and T4 lysozymes, however, φ29 lysozyme seems to be about 2 times more thermostable than the other two lysozymes.

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by Yung Mei Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 130-135. / ACKNOWLEDEMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iii / ABBREVIATIONS --- p.viii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Chemistry and Structure of Trichosanthin --- p.1 / Chapter 1.1.1 --- Chemistry --- p.1 / Chapter 1.1.2 --- Primary Structure --- p.2 / Chapter 1.1.3 --- Three-dimensional Structure --- p.4 / Chapter 1.2 --- Biological Activities of Trichosanthin --- p.6 / Chapter 1.2.1 --- Abortifacient Properties --- p.6 / Chapter 1.2.1A --- Termination of Mid-term Gestation --- p.6 / Chapter 1.2.1B --- Inhibition of Early Pregnancy --- p.12 / Chapter 1.2.2 --- Immunological Properties --- p.13 / Chapter 1.2.2A --- Antigenicity and Allergenicity --- p.13 / Chapter 1.2.2B --- Immunosuppressive Effects --- p.14 / Chapter 1.2.3 --- Anti-tumour Activity --- p.15 / Chapter 1.2.4 --- Ribosome-inactivating Activity --- p.16 / Chapter 1.2.5 --- Human Immunodeficiency Virus (HIV) Inhibitory Activity --- p.20 / Chapter 1.3 --- Objectives and Strategy of Cloning the cDNA of Trichosanthin --- p.21 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- General Techniques --- p.24 / Chapter 2.1.1 --- Extraction with Phenol --- p.24 / Chapter 2.1.2 --- Ethanol Precipitation --- p.24 / Chapter 2.1.3 --- Spectrophotometry --- p.25 / Chapter 2.1.4 --- Restriction Digestion of DNA --- p.25 / Chapter 2.1.5 --- End-labelling DNA with Recessed 3'-ends --- p.25 / Chapter 2.1.6 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.1.7 --- Elution of DNA from Agarose Gel --- p.26 / Chapter 2.1.8 --- Minipreparation of Bacteriophage A DNA from Plate Lysates --- p.27 / Chapter 2.1.9 --- Minipreparation of Plasmid DNA --- p.28 / Chapter 2.1.10 --- Large-scale Preparation of Plasmid DNA --- p.29 / Chapter 2.1.10A --- By Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient --- p.29 / Chapter 2.1.10B --- By Using QIAGEN-pack 100 Cartridge --- p.31 / Chapter 2.1.11 --- Preparation and Transformation of Competent Escherichia coli --- p.32 / Chapter 2.1.12 --- Preparation of Nucleic Acid Probes --- p.34 / Chapter 2.1.12A --- By Nick Translation --- p.34 / Chapter 2.1.12B --- By Random-primed Labelling --- p.34 / Chapter 2.1.13 --- Sephadex G-50 Spun-column Chromatography --- p.35 / Chapter 2.1.14 --- Monitoring the Progress of Probe Labelling Reactions --- p.37 / Chapter 2.1.15 --- Liquid Scintillation Counting --- p.37 / Chapter 2.1.16 --- Southern and Northern Hybridizations --- p.38 / Chapter 2.1.17 --- Autoradiography --- p.41 / Chapter 2.2 --- Techniques for Constructing a Complementary DNA (cDNA) Library --- p.42 / Chapter 2.2.1 --- Extraction and Purification of Plant RNA --- p.42 / Chapter 2.2.2 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.44 / Chapter 2.2.3 --- Ohgo(dT)-cellulose Column Chromatography --- p.46 / Chapter 2.2.4 --- In Vitro Translation in Rabbit Reticulocyte Lysate --- p.47 / Chapter 2.2.5 --- SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) of In Vitro Translation Products --- p.48 / Chapter 2.2.6 --- cDNA Synthesis --- p.50 / Chapter 2.2.6A --- First Strand Synthesis --- p.50 / Chapter 2.2.6B --- Second Strand Synthesis --- p.51 / Chapter 2.2.6C --- Purification of the Double-stranded cDNA (ds cDNA) --- p.51 / Chapter 2.2.7 --- Methylation of the cDNA with EcoRI Methylase --- p.52 / Chapter 2.2.8 --- Addition of EcoRl Cohesive Termini onto the cDNA --- p.52 / Chapter 2.2.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA --- p.53 / Chapter 2.2.10 --- Ligation of the cDNA with EcoRI-digested λgt10 --- p.55 / Chapter 2.2.11 --- In Vitro Packaging --- p.56 / Chapter 2.2.12 --- Titration of the λgt10 Library V --- p.56 / Chapter 2.3 --- Screening the cDNA Library in λgt10 with a Nucleic Acid Probe --- p.57 / Chapter 2.4 --- Subcloning DNA Fragments in pUC18 --- p.59 / Chapter 2.4.1 --- Dephosphorylation of Linearized pUC18 with Protruding 5' Termini --- p.59 / Chapter 2.4.2 --- Ligation of Foreign DNA with Linearized pUC18 with Cohesive Termini --- p.60 / Chapter 2.5 --- DNA Sequencing on Double-stranded Templates --- p.60 / Chapter 2.5.1 --- DNA Sequencing Reaction --- p.61 / Chapter 2.5.1A --- Alkaline Denaturation of Double-stranded Plasmid Template --- p.61 / Chapter 2.5.1B --- Labelling Reaction and Termination Reactions --- p.61 / Chapter 2.5.2 --- DNA Sequencing Electrophoresis --- p.62 / Chapter CHAPTER THREE --- CONSTRUCTION AND CLONAL SCREENING OF cDNA LIBRARY FROM ROOT TUBERS OF TRICHOSANTHES KIRILOWII MAXIMOWICZ --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Isolation of Total RNA from Root Tubers --- p.69 / Chapter 3.3 --- Purification of Poly(A)+ RNA from Total RNA --- p.70 / Chapter 3.4 --- Northern Blot Analysis of the Poly(A)+ RNA --- p.72 / Chapter 3.5 --- In Vitro Translation of the Poly(A)+ RNA --- p.75 / Chapter 3.6 --- cDNA Synthesis --- p.78 / Chapter 3.7 --- Southern Blot Analysis of the cDNA --- p.81 / Chapter 3.8 --- Addition of EcoRI Cohesive Termini to the cDNA --- p.81 / Chapter 3.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA vi --- p.84 / Chapter 3.10 --- Ligation of the Size Fractionated cDNA with EcoRI-digested λgt10 and In Vito Packaging of the A Hybrids --- p.85 / Chapter 3.11 --- Analysis of cDNA Inserts --- p.85 / Chapter 3.12 --- Clonal Screening of the cDNA Library --- p.88 / Chapter 3.13 --- Characterization of Positive Clones --- p.88 / Chapter 3.14 --- Subcloning Positive Inserts into pUC18 --- p.92 / Chapter 3.15 --- Discussion --- p.93 / Chapter CHAPTER FOUR --- DNA SEQUENCING OF POSITIVE CLONES --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Sequencing Strategies --- p.104 / Chapter 4.2.1 --- pTCS48210 Sequencing --- p.m / Chapter 4.2.2 --- pTCS5021 Sequencing --- p.107 / Chapter 4.3 --- Results and Discussion --- p.108 / Chapter 4.3.1 --- Nucleotide Sequence and Deduced Amino Acid Sequence --- p.108 / Chapter 4.3.2 --- Secondary Structure Predicted from the Amino Acid Sequence Encoded by the Trichosanthin cDNA --- p.116 / APPENDIX A --- p.124 / APPENDIX B --- p.126 / APPENDIX C --- p.129 / REFERENCES --- p.130