Experimental Manipulation of Gene Expression (eBook)

Masayori Inouye (Herausgeber)

eBook Download: EPUB

2014 | 1. Auflage
330 Seiten
Elsevier Science (Verlag)
978-1-4832-7397-6 (ISBN)

Infos

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.

Inhalt

Front Cover 1
Experimental Manipulation of Gene Expression 4
Copyright Page 5
Table of Contents 6
Contributors 12
Preface 14
CHAPTER 1. Use of Phage . Regulatory Signals to Obtain Efficient Expression of Genes in Escherichia 
16 
I. Introduction 17
II. Expression of Prokaryotic Gene Products 17
III. Expression of Eukaryotic Genes 23
Acknowledgment 29
References 29
CHAPTER 2. Multipurpose Expression 
30 
I. Introduction 30
II. pIN-I 
32 
III. plN-II 
36 
IV. pIN-lll Vectors 38
V. pIM Vectors: High-Copy-Number Vectors 39
VI. pIC Vectors: Hybrid Expression Vectors 41
VII. Promoter-Proving Vectors 43
VIII. General Cloning Strategy 45
IX. Summary 46
References 46
CHAPTER 3. Molecular Cloning in Bacillus 
48 
I. Introduction 48
II. Plasmid Transformation 49
III· Plasmid Vectors 51
IV. Cloning Stratagems 60
V. Expression of Cloned Genes 62
VI. Conclusions 63
Acknowledgments 63
References 64
CHAPTER 4. Developments in Streptomyces 
68 
I. Introduction 69
II. Vectors 69
III. Use of Tn5 in Relation to Streptomyces 
81 
IV. Applications of DNA Cloning in 
82 
V. Concluding Remarks 94
References 95
CHAPTER 5. Vectors for High-Level, Inducible Expression of Cloned Genes in 
98 
I. Introduction 99
II. Materials and Methods 100
III. Results and Discussion 102
IV. Summary 122
Appendix: Plasmid Constructions 122
References 130
CHAPTER 6. Genetic Engineering of Plants by Novel Approaches 134
I. Introduction 134
II. Novel Approaches to Creating Genetic 
136 
III. Concluding Remarks 148
References 149
CHAPTER 7. .SV2, a Plasmid Cloning Vector that Can Be Stably Integrated in 
152 
I· Introduction 153
II. Materials and Methods 153
III. Results 156
IV. Discussion 165
Acknowledgments 167
References 167
CHAPTER 8. Construction of Highly Transmissible Mammalian Cloning Vehicles Derived from Murine Retroviruses 170
I. Introduction 170
II. General Strategy 172
III. Construction of a Prototype Retrovirus 
174 
IV. Rescue of Recombinant Genomes as 
177 
V. Characteristics of Retrovirus-Mediated Transformation 179
VI. Useful Derivative Vectors 182
VII. Conclusions and Prospects 185
Acknowledgments 187
References 187
CHAPTER 9. Use of Retrovirus-Derived Vectors to Introduce and Express 
190 
I. Introduction 190
II. Organization of the M-MuLV Genome 191
III. Use of Retrovirus Vectors to Study the Mechanism of Gene Expression of the 
193 
IV. A General Transduction System Derived 
196 
V. Summary and Prospects 202
References 203
CHAPTER 10. Production of Posttranslationally Modified Proteins in the SV40–Monkey Cell System 206
I. Introduction 206
II. SV40 Late-Replacement Vectors 207
III. Human Growth Hormone 208
IV. Hepatitis B Surface Antigen 215
V. Conclusions and Prospects 222
References 224
CHAPTER 11. Adenovirus Type 5 Region-E1A Transcriptional Control Sequences 226
I. Introduction 226
II. Deletion Mutations in the 5'-Flanking 
228 
III. Analysis of Mutagenized Templates in 
229 
IV. Analysis of Cytoplasmic E1A mRNAs Found in Vivo after Infection with Deletion 
231 
V. 5'-End Analyses of E1A 
233 
VI. .1. 
234 
Acknowledgments 237
References 237
CHAPTER 12. Expression of Proteins on the Cell Surface Using Mammalian Vectors 240
I. How Proteins Are Normally Expressed on 
241 
II. Why It Would Be Useful to Express 
243 
III. Hemagglutinin of Influenza Virus Is the 
244 
IV. The Cene Coding for Hemagglutinin Is of 
247 
V. Vector Systems 248
VI. Hemagglutinin Is 
252 
VII. Small-t Intron Leads to Genetic 
253 
VIII. Hemagglutinin Synthesized by SV40-HA Recombinants Is Biologically 
255 
IX. Removing the C-Terminal Hydrophobie Sequence Converts Hemagglutinin from an 
257 
X. Prospects 258
References 259
CHAPTER 13. Expression of Human Interferon-. in Heterologous Systems 262
I. Introduction 262
II. Structure of the Human lnterferon-. 
263 
III. Heterologous Expression in 
264 
IV. Expression in the Yeast Saccharomyces 
266 
V. Conclusion 271
References 272
CHAPTER 14. Commercial Production of Recombinant DNA-Derived Products 274
I. Introduction 274
II. Production of Biosynthetic Human Insulin 276
III. Other Pharmaceutical Applications of 
287 
IV. Conclusion 291
References 292
APPENDIX 1: Two-Dimensional DNA Electrophoretic Methods Utilizing in Situ 
294 
I. Introduction 295
II. Experimental Procedures 295
III. Examples 298
IV. Conclusion 304
References 305
APPENDIX 2: Site-Specific Mutagenesis Using Synthetic Oligodeoxyribonucleotides 
306 
I. Introduction 307
II. Experimental Procedures 308
III. Example 313
IV. Conclusion 316
References 317
Index 320

Textauszug

CHAPTER 1

Use of Phage λ Regulatory Signals to Obtain Efficient Expression of Genes in Escherichia coli

ALLAN SHATZMAN*, YEN-SEN HO* and MARTIN ROSENBERG*,     Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Publisher Summary

There are many gene products of biological interest that cannot be studied simply because they cannot be obtained in quantities sufficient for biochemical analysis. Recombinant technology provides some new approaches to the solution of this problem. This chapter describes the design and construction of a plasmid vector system used to achieve high-level expression of a particular phage regulatory protein normally found in only minute amounts in a phage-infected bacterial cell. The chapter describes the subsequent development of this plasmid into a vector system that offers the potential for efficiently expressing essentially any gene product in Escherichia coli. The cII protein of bacteriophage λ is a transcriptional activator that positively regulates expression from two different phage promoters. These two promoters are coordinately controlled by this effector, and this control is essential for normal integration of the phage into the host genome, that is, formation of a bacterial lysogen.

I Introduction

II Expression of Prokaryotic Gene Products

A Overproduction of the Phage λ Regulatory Protein cII

B Expression of Other Genes in pKC30

III Expression of Eukaryotic Genes

A Vector Construction

B Expression of lacZ in pAS1

C Expression of SV40 Small-t Antigen

D Expression of Functional Mammalian Metallothioneines

References

I. Introduction

There are many gene products of biological interest that cannot be studied simply because they cannot be obtained in quantities sufficient for biochemical analysis. Recombinant technology now provides us with some new approaches to the solution of this problem. This chapter describes the design and construction of a plasmid vector system used to achieve high-level expression of a particular phage regulatory protein normally found in only minute amounts in a phage-infected bacterial cell. We also describe the subsequent development of this plasmid into a vector system that offers the potential for efficiently expressing essentially any gene product in Escherichia coli.

II. Expression of Prokaryotic Gene Products

A. Overproduction of the Phage λ Regulatory Protein cII

The cII protein of bacteriophage λ is a transcriptional activator that positively regulates expression from two different phage promoters (Herskowitz and Hage, 1980; Shimatake and Rosenberg, 1981). These two promoters are coordinately controlled by this effector, and this control is essential for normal integration of the phage into the host genome (i.e., formation of a bacterial lysogen). In order to study cII and its role in transcriptional activation, it was necessary to express the gene product at levels sufficient to allow its purification and biochemical analysis. Because cII is made in only small amounts during a normal phage infection and presents the added difficulty of being rapidly turned over by the host, the protein could not be easily detected or obtained.

1. CLONING STRATEGIES

Initially, we attempted to increase the cellular levels of cII by cloning the gene onto a multicopy plasmid vector (i.e., a pBR322 derivative) such that it was transcribed from a “strong” bacterial promoter (Fig. 1). A DNA fragment (1.3 kb) carrying the cII gene and the N-terminal 80% of the phage O gene (Fig. 1A) was inserted downstream of different promoter signals that had been cloned onto various pBR322 derivatives (Fig. 1B). Stable transformants containing cII as part of a constitutive transcription unit could not be obtained (Fig. 1a). Instead, the only stable recombinants isolated were those carrying the gene in orientation opposite to the direction of transcription (Fig. 1b). Stable isolates were also obtained using vectors that did not provide for transcription of the cII gene (Fig. 1d,e) or by fragmenting the cII gene and cloning pieces into a transcription unit (Fig. 1c). These results suggested that increased expression of cII is an event lethal to the bacterial cell. We now know that this is a common phenomenon when gene products are overproduced in E. coli using recombinant procedures. Clearly, cII provides an excellent model system for attempting to obtain efficient expression of a lethal gene product that is relatively unstable in the bacterial cell, besides being an interesting transcriptional regulatory molecule.

Fig. 1 Scheme of the cloning strategy used initially to overproduce the phage λ cII protein. (A) A 1.3-kb HaeIII restriction fragment from phage λ that carries the entire cII gene and part of the O gene (for details see text; Shimatake and Rosenberg, 1981). (B) Cloning scheme for inserting the fragment shown in (A) into pBR322 derivatives that either do or do not carry a promoter signal P upstream of the site of insertion (see text for details).

In order to clone the cII gene onto a multicopy plasmid vector as part of an efficient transcriptional unit, we reasoned that the promoter signal used would have to be regulated. This would enable us to obtain the desired recombinant under conditions in which the cII gene is not expressed. Cells carrying this vector could then be grown to high density and subsequently induced for expression of cII. Because the cells were already at high density, the lethality of the product would be of little consequence. Moreover, if the induction and expression were efficient, the loss of product due to turnover would be minimized. For these reasons and others to be discussed later, we placed the cII gene under the transcriptional regulation of the bacteriophage promoter signal, PL. This “strong” promoter has been shown to be 8–10 times more efficient than the well-characterized promoter P1ac, which regulates the E. coli lactose operon. In fact, using a vector system designed specifically for comparing and characterizing transcriptional regulatory signals, PL was found to be as or more efficient than all other promoters tested (McKenney et al., 1981; Rosenberg et al., 1982b).

The PL promoter, contained on a 2.4-kb HindIII–BamHI restriction fragment derived from phage λ, was cloned between the HindIII and BamHI sites of pBR322 (Fig. 2). The resulting vector pKC30 was found to be highly unstable. In general, plasmids that carry strong promoters are unstable, presumably owing to detrimental effects on replication resulting from high levels of transcription. Our problem of instability was overcome by repressing PL transcription using bacterial hosts that contain an integrated copy of the λ genome (i.e., bacterial lysogens). In these cells, PL transcription is controlled by the phage-repressor protein (cI), a product synthesized continuously and regulated autogenously in the lysogen (Ptashne et al., 1976). It was demonstrated that certain lysogens synthesize repressor in amounts sufficient to inhibit PL expression completely on the multicopy vector. Thus the cells can be stably transformed and the vector maintained in these lysogenic hosts. Moreover, by using a lysogen carrying a temperature-sensitive mutation in the cI gene (cI857; Sussman and Jacob, 1962), PL-directed transcription can be activated at any time. Induction is accomplished by simply raising the temperature of the cell culture from 32 to 42°C. Thus cells carrying the vector can be grown to high density at 32°C without expression of the cloned gene and subsequently induced at 42°C to synthesize the product.

Fig. 2 Schematic diagram of plasmid pKC30 and the insertion of the HaeIII restriction fragment (see Fig. 1A) carrying the λ cII gene into the HpaI restriction site. pKC30 is a derivative of pBR322 and contains a 2.4-kb HindIII–BamHI restriction fragment derived from phage λ inserted between the HindIII and BamHI restriction sites within the tetracycline gene of pBR322 (R. N. Rao, unpublished). This insert contains the operator (OL) and a site for N recognition (NutL) (Franklin and Bennett, 1979). DNA fragments carrying the appropriate gene are cloned into the HpaI restriction site that occurs within the N-gene coding region. The fragment containing the cII gene also contains a site for N recognition...

Produkt-Details

• Erscheint lt. Verlag: 28.6.2014
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Evolution
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 1-4832-7397-0 / 1483273970
• ISBN-13: 978-1-4832-7397-6 / 9781483273976