Experimental Manipulation of Gene Expression discusses a wide range of host systems in which to clone and express a gene of interest. The aims are for readers to quickly learn the versatility of the systems and obtain an overview of the technology involved in the manipulation of gene expression. Furthermore, it is hoped that the reader will learn enough from the various approaches to be able to develop systems and to arrange for a gene of particular interest to express in a particular system. The book opens with a chapter on the design and construction of a plasmid vector system used to achieve high-level expression of a particular phage regulatory protein normally found in minute amounts in a phage-infected bacterial cell. This is followed by separate chapters on topics such as high-level expression vectors that utilize efficient Escherichia coli lipoprotein promoter as well as various other portions of the lipoprotein gene Ipp; DNA cloning systems for streptomycetes; and the design and application of vectors for high-level, inducible synthesis of the product of a cloned gene in yeast.

ContributorsPreface1. Use of Phage ¿ Regulatory Signals to Obtain Efficient Expression of Genes in Escherichia Coli I. Introduction II. Expression of Prokaryotic Gene Products III. Expression of Eukaryotic Genes References2. Multipurpose Expression Cloning Vehicles in Escherichia Coli I. Introduction II. pIN-I Vectors III. pIN-II Vectors IV. pIN-III Vectors V. pIM Vectors: High-Copy-Number Vectors VI. pIC Vectors: Hybrid Expression Vectors VII. Promoter-Proving Vectors VIII. General Cloning Strategy IX. Summary References3. Molecular Cloning in Bacillus Subtilis I. Introduction II. Plasmid Transformation III. Plasmid Vectors IV. Cloning Stratagems V. Expression of Cloned Genes VI. Conclusions References4. Developments in Streptomyces Cloning I. Introduction II. Vectors III. Use of Tn5 in Relation to Streptomyces DNA IV. Applications of DNA Cloning in Streptomyces V. Concluding Remarks References5. Vectors for High-Level, Inducible Expression of Cloned Genes in Yeast I. Introduction II. Materials and Methods III. Results and Discussion IV. Summary Appendix: Plasmid Construction References6. Genetic Engineering of Plants by Novel Approaches I. Introduction II. Novel Approaches to Creating Genetic Diversity III. Concluding Remarks References7. XSV2, a Plasmid Cloning Vector that Can Be Stably Integrated in Escherichia Coli I. Introduction II. Materials and Methods III. Results IV. Discussion References8. Construction of Highly Transmissible Mammalian Cloning Vehicles Derived from Murine Retroviruses I. Introduction II. General Strategy III. Construction of a Prototype Retrovirus Vector IV. Rescue of Recombinant Genomes as Infectious Virus V. Characteristics of Retrovirus-Mediated Transformation VI. Useful Derivative Vectors VII. Conclusions and Prospects References9. Use of Retro virus-Derived Vectors to Introduce and Express Genes in Mammalian Cells I. Introduction II. Organization of the M-MuLV Genome III. Use of Retrovirus Vectors to Study the Mechanism of Gene Expression of the M-MuLV Genome IV. A General Transduction System Derived from the M-MuLV Genome V. Summary and Prospects References10. Production of Posttranslationally Modified Proteins in the SV40-Monkey Cell System I. Introduction II. SV40 Late-Replacement Vectors III. Human Growth Hormone IV. Hepatitis B Surface Antigen V. Conclusions and Prospects References11. Adenovirus Type 5 Region-EIA Transcriptional Control Sequences I. Introduction II. Deletion Mutations in the 5'-Flanking Sequences of Ad5 Region E1A III. Analysis of Mutagenized Templates in Cell-Free Transcription Extracts IV. Analysis of Cytoplasmic E1A mRNAs Found In Vivo after Infection with Deletion Mutants V. 5'-End Analyses of E1A mRNAs Synthesized In Vivo after Infection with Deletion Mutants VI. E1A Transcriptional Control Region and Comparison to Other Eukaryotic Control Regions References12. Expression of Proteins on the Cell Surface Using Mammalian Vectors I. How Proteins Are Normally Expressed on Mammalian Cell Surfaces II. Why It Would Be Useful to Express Proteins on the Surface of the Mammalian Cell III. Hemagglutinin of Influenza Virus Is the Best-Characterized Integral Membrane Protein IV. The Gene Coding for Hemagglutinin Is of Simple Structure V. Vector Systems VI. Hemagglutinin Is Efficiently Expressed from Both the Early and Late SV40 Promoters VII. Small-t Intron Leads to Genetic Instability of the Early-Replacement, Vector VIII. Hemagglutinin Synthesized by SV40-HA Recombinants is Biologically Active IX. Removing the C-Terminal Hydrophobie Sequence Converts Hemagglutinin from an Integral Membrane Protein to a Secreted Protein X. Prospects References13. Expression of Human Interferon-7 in Heterologous Systems I. Introduction II. Structure of the Human Interferon-7 cDNA III. Heterologous Expression in Escherichia Coli IV. Expression in the Yeast Saccharomyces Cerevisiae V. Conclusion References14. Commercial Production of Recombinant DNA-Derived Products I. Introduction II. Production of Biosynthetic Human Insulin III. Other Pharmaceutical Applications of Recombinant DNA IV. Conclusion ReferencesAppendix 1. Two-Dimensional DNA Electrophoretic Methods Utilizing in Situ Enzymatic Digestions I. Introduction II. Experimental Procedures III. Examples IV. Conclusion ReferencesAppendix 2. Site-Specific Mutagenesis Using Synthetic Oligodeoxyribonucleotides as Mutagens I. Introduction II. Experimental Procedures III. Example IV. Conclusion ReferencesIndex