Genetic Engineering

Contributors

Preface

The Preparation and Screening of a cDNA Clone Bank

I Introduction

A What is a cDNA Clone Bank?

Β Why is a cDNA Clone Bank Useful?

C When Might it be Preferable to Use a Genomic Bank?

D Summary

II The Preparation of a cDNA Clone Bank

A General Considerations

Β The Enzymology of cDNA Cloning

C The Purification of Messenger RNA

D The Synthesis of cDNA

Ε The Synthesis of Double-Stranded cDNA

F Cleavage of the Hairpin Loop and Removal of Single-Stranded cDNA

G The Generation of Cohesive Ends on the Double-Stranded cDNA

Η The Basic Principles of Cloning DNA in Bacterial Plasmids

I Restriction Enzyme Cleavage of the Vector

J Generation of Suitable Cohesive Ends on the Vector and the Insertion of Double-Stranded cDNA

Κ The Relative Merits of Restriction Enzyme Linkers and Homopolymer Tails as Methods for the Insertion of Double-Stranded cDNA

L Transformation of E. coli and Storage of the cDNA Clones

III The Screening of a cDNA Clone Bank

A Primary Screening

Β Secondary Screening

IV The Future of cDNA Cloning

A Potential Methods of Screening of cDNA Clone Bank that do not Require Expression of the Cloned Sequence in E. coli

Β Potential Methods of Screening a cDNA Clone Bank that do Require Expression of the Cloned Sequence in E. coli

V References

DNA Analysis and the Antenatal Diagnosis of Hemoglobinopathies

I Introduction

A The Clinical Problem of the Hemoglobinopathies

II Globin Proteins

A Problems of Detection in the Fetus

Β The Inherited Hemoglobinopathies

C The Antenatal Diagnosis of Hemoglobinopathies — Why DNA?

III DNA Structural Analysis

A Techniques

Β Methodology

C Timing and Reliability

D DNA Structures and their Detection

IV Antenatal Diagnosis by the Direct Detection of Dysfunctional Genes

A The Detection of Gene Deletions

Β The Detection of Point Mutations — The Structural Variants

V Antenatal Diagnosis by the Indirect Detection of Dysfunctional Genes — The Use of Polymorphisms

A Allele-Specific Linked Polymorphisms

Β Linked Polymorphisms

C How Useful are Linked Polymorphisms in Antenatal Diagnosis?

VI The Application and Future Applications of DNA Analysis in Antenatal Diagnosis

A Genetic Counselling

Β The Implementation of the Techniques of DNA Analysis in a Clinical Setting

C Improvements in the Technique

D Extension to Other Genes

VII Conclusion

VIII Acknowledgements

IX References

Expression of Cloned Genes in Cell-Free Systems and in Microinjected Xenopus Oocytes

I Introduction

II Transcription of Isolated Genes by RNA Polymerase III

A Templates and Transcripts

Β Expression in Microinjected Oocytes

C Expression in Vitro

D Transcription Signals Encoded in the DNA Sequence

Ε Factors Which Confer Transcriptional Specificity

F In Vitro Analysis of the Developmental Regulation of 5s RNA Synthesis

G Post-Transcriptional Processing of Polymerase III Transcripts

III Transcription of Isolated Genes by RNA Polymerase II

A Templates and Transcripts

Β Transcription of Isolated Chromatin in Vitro

C The Endogenous Activities of Isolated Nuclei

D In Vitro Transcription in Cellular Extracts

Ε Transcription by Polymerase II in Microinjected Xenopus Oocytes

F Transcription Signals Encoded in the Template

G RNA Processing

Η Prospects for Reconstituting Regulation

IV Transcription of Isolated Genes by RNA Polymerase I

A Templates and Transcripts

Β Transcription of Ribosomal RNA Genes in Isolated Nuclei and Nucleoli

C Transcription of Isolated Genes by RNA Polymerase I

D Regulation of Polymerase I Transcription

V Conclusions and Prospects

A The Activities of Expression Systems

Β Analysis of Gene Regulation

VI Acknowledgements

VII References