Experimental Manipulation of Gene Expression

1st Edition - October 28, 1983

Editor: Masayori Inouye

eBook ISBN:

9 7 8 - 1 - 4 8 3 2 - 7 3 9 7 - 6

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… Read more

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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.

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