Experimental Manipulation of Gene Expression
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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.
Table of Contents
Contributors
Preface
1. Use of Phage λ Regulatory Signals to Obtain Efficient Expression of Genes in Escherichia Coli
I. Introduction
II. Expression of Prokaryotic Gene Products
III. Expression of Eukaryotic Genes
References
2. 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
References
3. Molecular Cloning in Bacillus Subtilis
I. Introduction
II. Plasmid Transformation
III. Plasmid Vectors
IV. Cloning Stratagems
V. Expression of Cloned Genes
VI. Conclusions
References
4. 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
References
5. 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
References
6. Genetic Engineering of Plants by Novel Approaches
I. Introduction
II. Novel Approaches to Creating Genetic Diversity
III. Concluding Remarks
References
7. XSV2, a Plasmid Cloning Vector that Can Be Stably Integrated in Escherichia Coli
I. Introduction
II. Materials and Methods
III. Results
IV. Discussion
References
8. 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
References
9. 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
References
10. 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
References
11. 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
References
12. 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
References
13. 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
References
14. Commercial Production of Recombinant DNA-Derived Products
I. Introduction
II. Production of Biosynthetic Human Insulin
III. Other Pharmaceutical Applications of Recombinant DNA
IV. Conclusion
References
Appendix 1. Two-Dimensional DNA Electrophoretic Methods Utilizing in Situ Enzymatic Digestions
I. Introduction
II. Experimental Procedures
III. Examples
IV. Conclusion
References
Appendix 2. Site-Specific Mutagenesis Using Synthetic Oligodeoxyribonucleotides as Mutagens
I. Introduction
II. Experimental Procedures
III. Example
IV. Conclusion
References
Index
Product details
- No. of pages: 330
- Language: English
- Copyright: © Academic Press 1983
- Published: October 28, 1983
- Imprint: Academic Press
- eBook ISBN: 9781483273976
About the Editor
Masayori Inouye
Affiliations and Expertise
Robert Johnson Medical School, Picataway, New Jersey, U.S.A.
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