Recombinant DNA and Genetic Experimentation

Proceedings of a Conference on Recombinant DNA, Jointly Organised by the Committee on Genetic Experimentation (COGENE) and the Royal Society of London, Held at Wye College, Kent, UK, 1-4 April, 1979

Joan Morgan, W. J. Whelan

• Publisher: Pergamon
• Year: 2013
• Print ISBN: 9780080244273
• eBook ISBN: 9781483147062
• Topic: Biological Sciences
• Subtopic: Evolution
• Index: Biological Sciences

Recombinant DNA and Genetic Experimentation contains papers from the Proceedings of a Conference on Recombinant DNA held in London on April 1-4, 1979. This books reviews recombinant DNA research and discusses advances in the application of recombinant DNA research and the regulations affecting such research. Part 1 of the book deals with recombinant DNA techniques that are useful in the biological perspective. These techniques include tests for rare gene exchanger and laboratory genetic manipulations. Part 2 addresses the achievements of recombinant DNA research such as the detection of homologous sequences and progress made in the research of animal viruses. Part 3 discusses the practical benefits of recombinant DNA research, covering topics such as the production of valuable proteins in alternate biological hosts. These proteins are shown as being valuable to society, besides being scientific curiosities. An important presentation is Part 4 of the symposium, which discusses the guidelines and legislations affecting recombinant DNA research such as prior restraint, prohibitions, risks, and approval of the conduct of such experiments. Part 5 concerns a review of the basic assumptions made in the symposium, while Part 6 tackles the question of what options are left open in the international arena, in the medical field, and in the eyes of the public. This collection of papers can prove beneficial for molecular biologists, DNA researchers, molecular geneticists, ecologists and endocrinologists, and pharmacologists.

ACHIEVEMENTS OF RECOMBINANT DNA RESEARCH

EXPERIMENTAL TECHNIQUES AND STRATEGIES FOR DNA CLONING

S.N. Cohen, Departments of Genetics and Medicine, Stanford University School of Medicine, Stanford, California 943O5, U.SA

Publisher Summary

This chapter discusses experimental techniques and strategies for DNA cloning. The requirements for cloning are (1) a replicon (cloning vehicle or vector) that is able to propagate itself in a recipient organism, (2) a method of joining another DNA segment to the vector, (3) a procedure for introducing the composite molecule into a biologically functional recipient cell, and (4) a method of selecting or identifying the cells that have acquired the hybrid DNA species. These are the in vivo tools for the recombinant DNA work. The earliest DNA cloning experiments utilized bacterial plasmids, which are circular extrachromosomal elements of DNA that are capable of autonomous replication. Most simply, a plasmid can be viewed as a replication system to which another segment of DNA has been linked by natural recombination mechanisms. The use of plasmids as vectors in DNA cloning experiments depends on the ability to introduce plasmid DNA molecules into bacterial cells by transformation. The development of a procedure for transformation of E. coli K-12 with plasmid DNA has made it possible to establish clones of bacterial cells that carry the progeny of a single plasmid DNA molecule. This, in turn, has enabled studies with plasmids in ways that previously had been practical only with bacteriophages.

The task I have been assigned is to review briefly the techniques that collectively are known as “recombinant DNA methodology”, and to describe some of the experimental strategies used in this research. Although recombinant DNA is commonly referred to as “gene splicing”, “gene cloning” would seem a more appropriate term. There are four general requirements: 1) a replicon (cloning vehicle or vector) able to propagate itself in a recipient organism, 2) a method of joining another DNA segment to the vector, 3) a procedure for introducing the composite molecule into a biologically functional recipient cell, and 4) a method of selecting or identifying those cells that have acquired the hybrid DNA species.

The earliest DNA cloning experiments utilized bacterial plasmids, which are circular extrachromosomal elements of DNA that are capable of autonomous replication. Most simply, a plasmid can be viewed as a replication system to which another segment of DNA has been linked by natural recombination mechanisms. For plasmids isolated from natural sources, the added segment commonly provides the host carrying the plasmid with a selective advantage, so that the plasmid is propagated in the bacterial population. Plasmids are widespread among bacteria and encode a variety of traits, including resistance to antibiotics and heavy metals; production of enterotoxins, virulence factors or antibiotics; fertility functions; production of restriction and modification enzymes; resistance to UV irradiation; a capacity to metabolize polycyclic hydrocarbons, such as carbon and octane; and tumorigenicity in plants.

The use of plasmids as vectors in DNA cloning experiments depends on the ability to introduce plasmid DNA molecules into bacterial cells by transformation. The development of a procedure for transformation of E. coli K-12 with plasmid DNA has made it possible to establish clones of bacterial cells that carry the progeny of a single plasmid DNA molecule and this in turn has enabled studies with plasmids in ways that previously had been practical only with bacteriophages. Because transformation enables the cloning of individual plasmid DNA molecules, my colleagues and I reasoned some years ago that if a foreign DNA segment could be inserted within a plasmid DNA molecule while it was outside of the cell, the replication apparatus of the plasmid might be used to replicate the foreign DNA segment in the same way that the replication systems of naturally formed plasmids can replicate various segments that have been linked to them by natural biological processes. To do this, the exogenous DNA segment would have to be inserted at a site that does not interfere with the ability of the plasmid to replicate or with the expression of genes required for selection of transformant cells that had acquired the hybrid plasmid DNA molecule. Of course, it was not possible to know before the initial DNA cloning experiments were done whether linkage of a bacterial plasmid replicon to a foreign DNA segment would result in a viable combination. Could an E. coli plasmid replication system in fact propagate and amplify a piece of DNA that had been derived from a very dissimilar biological species?

The task of breaking plasmids open at specific sites was much simplified when the properties of Type II restriction enzymes were discovered. Such enzymes recognize specific DNA sequences that are identical in the 5’ to 3’ direction on each of the two DNA strands. One enzyme that was among the earliest to be studied (the EcoRI endonuclease) cleaves its recognition sequence asymmetrically so that projecting single-strand termini are formed. These “cohesive ended” termini thus formed can be rejoined by hydrogen bonding of the overlapping nucleotide bases and ligated to yield covalently linked DNA segments. Since the six base pair recognition sequence of the EcoRI enzyme is always cleaved identically by the endonuclease, identical cohesive ends are formed on DNA segments derived from different biological sources, and these segments can readily be linked. A number of other endonucleases with similar properties but different recognition sequences have been identified.

Alternatively a nucleotide “tail” consisting of a polymeric run of identical nucleotides (e.g., dA) can be added to one DNA species and a tail of complementary nucleotides (e.g., dT) can be added to another; the two DNA species can then be joined by hydrogen bonding of the complementary nucleotide bases and subsequent ligation. It has also become apparent that DNA termini with projecting ends are not required for joining; even blunt-ended DNA molecules can be linked using the ligase encoded by bacteriophage T4.

In the work that Chang, Boyer, Helling and I reported several years ago, EcoRI-generated segments of several different bacterial plasmids were inserted into the pSC101 vector, and the composite molecules were introduced into E. coli K-12 by transformation. Since the site of insertion (i.e., the EcoRI endonuclease cleavage site of pSC101) did not interfere with the capacity of the plasmid to replicate or express its tetracycline resistance gene, the vector could be used to clone the introduced DNA fragments. In subsequent experiments, Chang and I found that DNA derived from a plasmid indigenous to a quite unrelated bacterial species (Staphylococcus aureus) could be cloned in E. coli K-12 using similar procedures, and moreover that the bacterial genes derived from the Gram-positive coccus could be expressed in the Gram-negative species that served as the new host. It has since become apparent that heterospecific gene expression can also be accomplished between various other bacterial species combinations, but that phenotypic expression in some heterospecific gene transplants is a more complex matter.

Since I expect that Ken Murray will be discussing the use of bacteriophages as vectors in DNA cloning experiments, I need not cover this subject except to note that following the initial demonstration that a bacteriophage λ derivative (which had been mutated to remove most of the cleavage sites for the endonuclease being used) could be employed as a vector, a large number of recombinant DNA experiments have been carried out using phage vectors, and more recently plasmid-phage combinations.

It is worthwhile pointing out that the essential tools for recombinant DNA work (plasmid or phage replicons plus enzymes that cleave DNA at specific sites) are natural biological products. About two years ago, it was demonstrated that the EcoRI endonuclease and DNA ligase can accomplish in vivo the cleaving and joining processes mediated by these enzymes during in vitro recombinant DNA experiments. While such in vivo events occur at low frequency and require special experimental techniques for detection, they nevertheless show the qualitative similarity between the in vitro and in vivo processes. Moreover, from the considerations discussed earlier in this meeting by Bodmer and others, one would expect that low frequency events that provide a bacterial host with a natural biological advantage will be selected during the course of natural evolution, whereas combinations that occur at higher frequency but provide no selective advantage will be lost during evolution.

The transposable genetic elements discussed earlier by Peter Starlinger can also be used for carrying out “recombinant DNA” experiments in vivo. In experiments designed to study the process of transposition, my co-workers and I recen...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• PREFACE
• PARTICIPANTS
• INTRODUCTION AND WELCOME
• THE PURPOSE OF THE MEETING
• RECOMBINANT DNA TECHNIQUES PUT INTO BIOLOGICAL PERSPECTIVE
• ACHIEVEMENTS OF RECOMBINANT DNA RESEARCH
• PRACTICAL BENEFITS OF RECOMBINANT DNA RESEARCH
• GUIDELINES AND LEGISLATION
• RE-EXAMINATION OF BASIC ASSUMPTIONS
• WHAT OPTIONS ARE NOW OPEN?