Introduction

In essence, all properties of organisms depend on the sum of their genes. There are two broad categories of genes: structural and regulatory. Structural genes encode for amino acid sequences of proteins which, as enzymes, determine the biochemical capabilities of the organism by catalysing particular synthetic or catabolic reactions or, alternatively, play more static roles as components of cellular structures. In contrast, the regulatory genes control the expression of the structural genes by determining the rate of production of their protein products in response to intra- or extracellular signals. The derivation of these principles has been achieved using well-known genetic techniques which will not be considered further here.

The seminal studies of Watson and Crick and others in the early 1950s led to the construction of the double-helix model depicting the molecular structure of DNA and subsequent hypotheses on its implications for the understanding of gene replication. Since then there has been a spectacular unravelling of the complex interactions required to express the coded chemical information of the DNA molecule into cellular and organismal expression. Changes in the DNA molecule making up the genetic complement of an organism is the means by which organisms evolve and adapt themselves to new environments.

Introduction

Globally, agriculture and food production are challenged to produce, in a sustainable way, sufficient, healthy and safe food for the further growing world population. It is estimated that nearly eight billion people will be living on this planet by 2020, with 3.5 billion living in urban areas. To feed this world population there will need to be substantial increases in the production of the staple food commodities, namely cereals (40% increase), meat (63%) and roots and tubers (40%). At least 80% of this food will need to be produced in developing countries, yet only about 6% of new virgin soil can be brought into cultivation. Mankind must, somehow, raise yields from areas planted with cereals (two-thirds of all energy in the human diet) to approximately double the present value. Consequently, there can be no alternative other than to plan, with modern scientific inputs, new agricultural systems that are sustainable yet intensive. Whereas in the last great ‘green revolution’ in agriculture in the 1960s to 1970s, the environment was adapted to the plant by increased use of fertilisers, biocides, irrigation, etc., modern sustainable agriculture must increasingly adapt the plant to the environment, breeding high-yielding crops that can grow in places deficient in nitrogen or water, and where plant diseases and pests prevail. Many aspects of modern biotechnology are, and will increasingly be, applied to agriculture.

A biomass strategy

It has been estimated that the annual net yield of plant biomass arising from photosynthesis is at least 120 billion tonnes of dry matter on land and about 50 billion tonnes from the world's oceans. Of the land-produced biomass, approximately 50% occurs in the complex form of lignocellulose.

The highest proportion of land-based biomass (44%) is produced as forest (Table 2.1). It is surprising to note that while agricultural crops account for only 6% of the primary photosynthetic productivity, from this amount is derived a major portion of food for humans and animals as well as many essential structural materials, textiles and paper products (Table 2.2). Many traditional agricultural products may well be further exploited with the increasing awareness of biotechnology. In particular, new technological approaches will undoubtedly be able to utilise the large volume of waste material from conventional food processing that presently finds little use.

Biomass agriculture, aquaculture and forestry may hold great economic potential for many national economies, particularly in tropical and subtropical regions (Fig. 2.1). Indeed, the development of biotechnological processes in developing areas where plant growth excels could well bring about a change in the balance of economic power. world has drawn heavily on fossil fuels that took millions of years to form beneath the beds of the oceans or in the depths of the earth. Furthermore, it is a very unequal pattern of usage.

Introduction

Major events in human history have, to a large extent, been driven by technology. Improved awareness of agriculture and metalworking brought mankind out of the Stone Age, while in the nineteenth century, the Industrial Revolution created a multitude of machinery together with ever-increasingly larger cities. The twentieth century was undoubtedly the age of chemistry and physics, spawning huge industrial activities such as petrochemicals, pharmaceuticals, fertilisers, the atom bomb, transmitters, the laser and microchips. However, there can be little doubt that the huge understanding of the fundamentals of life processes achieved in the latter part of the twentieth century will ensure that the twenty-first century will be dominated by biology and the associated technologies.

Societal changes are increasingly driven by science and technology. Currently, the impact of new biological developments must be absorbed not just by a minority (the scientists) but also by large numbers (the general public). If this does not happen the majority will be alienated. It is increasingly important to ensure a broad understanding of what bioscience and its related technologies will involve, and especially what the consequences will be of accepting or rejecting the new technical innovations.

The following chapters will examine how the new biotechnologists are: developing new therapies and cures for many human and animal diseases; designing diagnostic tests for increasing disease prevention and pollution control; improving many aspects of plant and animal agriculture; cleaning and improving the environment; and designing clean industrial manufacturing processes.

In this final chapter of Part II, the various techniques that can be used to identify cloned genes will be described. As with previous chapters, the basis of techniques that are perhaps not so widely used today will be included, to illustrate the principles of gene identification and characterisation. This will lead into the final section of the book, where various applications of the technology will be covered, and where we get a look at some of the more advanced developments in gene manipulation.

Success in any cloning experiment depends on being able to identify the desired gene sequence among the many different recombinants that may be produced. Given that a large genomic library may contain a million or more cloned sequences, whic h are not readily distinguishable from each other by simple analytical methods, it is clear that identification of the target gene is potentially the most difficult part of the cloning process. Fortunately there are several selection/identification methods that can be used to overcome most of the problems that arise.

There are two terms that require definition before we proceed, these being selection and screening. Selection is where some sort of pressure (e.g. the presence of an antibiotic) is applied during the growth of host cells containing recombinant DNA. The cells with the desired characteristics are therefore selected by their ability to survive. This approach ranges in sophistication, from simple selection for the presence of a vector, up to direct selection of cloned genes by complementation of defined mutations.

Before examining some of the specific techniques used in gene manipulation, it is useful to consider the basic methods required for handling, quantifying and analysing nucleic acid molecules. It is often difficult to make the link between theoretical and practical aspects of a subject, and an appreciation of the methods used in routine work with nucleic acids may be of help when the more detailed techniques of gene cloning and analysis are described.

Isolation of DNA and RNA

Every gene manipulation experiment requires a source of nucleic acid, in the form of either DNA or RNA. It is therefore important that reliable methods are available for isolating these components from cells. There are three basic requirements: (i) opening the cells in the sample to expose the nucleic acids for further processing,(ii) separation of the nucleic acids from other cell components, and (iii) recovery of the nucleic acid in purified form. A variety of techniques may be used, ranging from simple procedures with few steps, up to more complex purifications involving several different stages. These days, most biological supply companies sell kits that enable purification of nucleic acids from a range of sources.

The first step in any isolation protocol is disruption of the starting material, which may be viral, bacterial, plant or animal. The method used to open cells should be as gentle as possible, preferably utilising enzymatic degradation of cell wall material (if present) and detergent lysis of cell membranes.

On 5th July 1996 a lamb was born at the Roslin Research Institute near Edinburgh. It was an apparently normal event, yet it marked the achievement of a milestone in biological science. The lamb was a clone, and was named Dolly. She was the first organism to be cloned from adult differentiated cells, which is what makes the achievement such a ground-breaking event. In this chapter we will look briefly at this area of genetic technology.

In this book so far, we have been considering the topic of molecular cloning, where the aim of an experimental process is to isolate a gene sequence for further analysis and use. In organismal cloning, the aim is to generate an organism from a cell that carries a complete set of genetic instructions. We have looked at the methods for generating transgenic organisms in Chapter 12, and a discussion of organismal cloning is a natural extension to this, although transgenic organisms are not necessarily (and at present are not usually) clones. In a similar way, a clone need not necessarily be transgenic. Thus, although not strictly part of gene manipulation technology, organismal cloning has become a major part of genetics in a broader sense. The public have latched on to cloning as an issue for concern, and thus a discussion of the topic is essential even in a book where the primary goal is to illustrate the techniques of gene manipulation.

Early thoughts and experiments

The announcement of the birth of Dolly in a paper in the journal Nature in February 1997 rocked the scientific community.

Advances in genetics continue to be made at an ever increasing rate, which makes writing an introductory text somewhat difficult. In the few years since the first edition was published, many new applications of gene manipulation technology have been developed, covering a diverse range of disciplines. The temptation in preparing this second edition was to concentrate on the applications, and ignore the fundamental principles of the technology. However, I wished to retain many of the features of the first edition, in which a basic technical introduction to the subject was the main aim of the text. Thus some of the original methods used in gene manipulation have been kept as examples of how the technology developed, even though some of these have become little used or even obsolete. From the educational point of view, this should help the reader cope with more advanced information about the subject – a sound grasp of the basic principles is an important part of any introduction to genetic engineering. I have been gratified by the many positive comments about the first edition, and I hope that this new edition is as well received.

In trying to strike a balance between the methodology and the applications of gene manipulation, I have divided the text into three sections. Part I deals with basic molecular biology, Part II with the methods used to manipulate genes, and Part III with the applications.

This final chapter is short. It does not answer any questions, but simply raises them for consideration. There are no ‘correct’ answers to these questions, as each must be addressed from the perspective of the individual, family, society, race or nation that is facing up to the situation. There are no diagrams or photographs, and very little factual information. However, the topics discussed are probably the most important that a student of genetic engineering can consider. In practical terms, relatively few people will ever go on to work in science and technology, but we will all have to cope with the consequences of gene-based research and its applications. Informed and vigorous debate is the only way that the developments of gene manipulation technology can become accepted and established.

Is science ethically and morally neutral?

It is often said that science per se is neither ‘good’ nor ‘bad’, and that it is therefore ethically and morally neutral. Whilst this may be true of science as a process, it is the developments and applications that arise from the scientific process that pose the ethical questions. The example that is often quoted is the development of the atomic bomb – the science was interesting and novel, and of itself ethically neutral, but the application (i.e. use of the devices in conflict) posed a completely different set of moral and ethical questions. Also, science is, of course, carried out by scientists, who are most definitely not ethically and morally neutral, as they demonstrate the same breadth and range of opinion as the rest of the human race.

Once recombinant DNA molecules have been constructed in vitro, the desired sequence can be isolated. In some experiments hundreds of thousands of different DNA fragments may be produced, and the isolation of a particular sequence would seem to be an almost impossible task. It is a bit like looking for the proverbial needle in a haystack – with the added complication that the needle is made of the same material as the haystack! Fortunately the methods available provide a relatively simple way to isolate specific gene sequences.

Three things have to be done to isolate a gene from a collection of recombinant DNA sequences: (i) the individual recombinant molecules have to be physically separated from each other,(ii) the recombinant sequences have to be amplified to provide enough material for further analysis, and (iii) the specific fragment of interest has to be selected by some sort of sequence-dependent method. In this chapter I consider the first two of these requirements, which in essence represent the systems and techniques involved in genecloning. This is an essential part of most genetic manipulation programmes. Even if the desired result is a transgenic organism, the gene to be used must first be isolated and characterised, and therefore cloning systems are required. Methods for selecting specific sequences are described in Chapter 8.

The biology of gene cloning is concerned with the selection and use of a suitable carrier molecule or vector, and a living system or host in which the vector can be propagated.

Biotechnology is one of those difficult terms that can mean different things to different people. In essence, it is the use of an organism (usually a microorganism) or a biologically derived substance (usually an enzyme) in a production or conversion process. Thus brewing and wine-making, food processing and manufacture, the production of pharmaceuticals and even the treatment of sewage can all be classed as aspects of biotechnology. In many cases the organism or enzyme is used in its natural form, and is not modified apart from perhaps having been subjected to selection methods to enable the best strain or type of enzyme to be used for a particular application. However, despite its traditional roots, modern biotechnology is often associated with the use of genetically modified systems. In this chapter we will consider the impact that gene manipulation technology has had on some biotechnological applications, with particular reference to the production of useful proteins.

The products of biotechnological processes are destined for use in a variety of fields such as medicine, agriculture and scientific research. It is perhaps an arbitrary distinction to separate the production of a therapeutic protein from its clinical application, as both could be considered as ‘biotechnology’ in its broadest sense. In a similar way, the developing area of transgenic plants and animals is also part of biotechnology, and undoubtedly the information provided by genome sequencing will give rise to many more diverse biotechnological applications.

The production of a transgenic organism involves altering the genome so that a permanent change is effected. This is different from somatic cell gene therapy, in which the effects of the transgene are restricted to the individual who receives the treatment. In fact, the whole point of generating a transgenic organism is to alter the germ line, so that the genetic change is inherited in a stable pattern following reproduction. This is one area of genetic engineering that has caused great public concern, and there are many complex issues surrounding the development and use of transgenic organisms. In addition, the scientific and technical problems associated with genetic engineering in higher organisms are often difficult to overcome. This is partly due to the size and complexity of the genome, and partly due to the fact that the development of plants and animals is an extremely complex process that is still not yet fully understood at the molecular level. Despite these difficulties, methods for the generation of transgenic plants and animals are now well established, and the use of transgenic organisms has already had a major impact in a range of different disciplines. In this chapter we will consider the development and use of transgenic plants and animals.

Transgenic plants

All life on earth is dependent on the photosynthetic fixation of carbon dioxide by plants. We sometimes lose sight of this fact, as most people are removed from the actual process ogfenerating our food, and the supemrarket shelves have all sorts of exotic processed foods and pre-prepared meals that somehow swamp the vegetable section.

The genetic engineer needs to be able to cut and join DNA from different sources. In addition, cer tain modifications may have to be carried out to the DNA during the various steps required to produce, clone and identify recombinant DNA molecules. The tools that enable these manipulations to be performed are enzymes, which are purified from a wide range of organisms and can be bought from various suppliers. In this chapter I examine some of the important classes of enzymes that make up the genetic engineer's toolkit.

Restriction enzymes – cutting DNA

The restriction enzymes, which cut DNA at fidened sites, represent one of the most important groups of enzymes for the manipulation of DNA. These enzymes are found in bacterial cells, where they function as part of a protective mechanism called the restriction–modification system. In this system the restriction enzyme hydrolyses any exogenous DNA that appears in the cell. To prevent the enzyme acting on the host cell DNA, the modification enzyme of the system (a methylase) modifies the host DNA by methylation of particular bases in the recognition sequence, which prevents the restriction enzyme from cutting the DNA.

Restriction enzymes are of three types (I, II or III). Most of the enzymes used today are type II enzymes, which have the simplest mode of action. These enzymes are nucleases (see Section 4.2.1), and as they cut at an internal position in a DNA strand (as opposed to beginning degradation at one end) they are known as endonucleases.