Introduction

In preceding chapters, we have followed some of the seismic shifts in the balance between public and private sector activities in plant breeding over the past few decades. While some developments have been positive, others have fuelled concerns about the overall direction of plant breeding research, particularly its future capacity to deliver on the primary mission of crop improvement. Two key priorities should be the revitalisation of the public sector and the re-empowerment of plant breeding as a valued and socially necessary scientific discipline. To achieve this, we must seek to re-establish those structural balances in plant research that have gone so seriously awry over the past few decades. For example, we should restore the balance between the following: the public and private sectors as they relate to agriculture; transgenic methods and non-transgenic variation enhancement plant breeding strategies; academic research and applied R&D in the plant sciences; and between pragmatic, public-good crop improvement, especially for developing countries, and the more inward-looking topical issues (such as the debate on GM crops) that currently preoccupy many richer industrialised nations. In this chapter, we will begin by discussing how to revitalise our much depleted public sector, and re-establish some of the balances within plant science in general.

Introduction

For the past three hundred years, crop breeders have had access to a considerable amount of existing variation resulting from spontaneous mutation, and also from the various genetic manipulations, such as hybridisation, that they learned to perform as they improved their understanding of plant reproduction. By collecting varieties and landraces from around the world, breeders were also able to exploit a great deal of the variation present in the gene pool of the crop species itself. Breeders had learned how to force crops to hybridise with some of their wild relatives, and even with other more distantly related species. Not even the genus barrier could withstand their assault as the first experimental manmade intergenic hybrids were produced in the mid nineteenth century. At this stage, many of these achievements were only successful in glasshouses or field plots and had not yet resulted in the production of any new varieties of the major staple crops. To a great extent, farmers at the beginning of the twentieth century were still reliant on traditional crop landraces. Organised systems for the dissemination of improved seed stocks from the new scientific breeding programmes were just beginning to be established in a few countries, but were still far from effective.

Introduction

In this chapter, we will examine how we might progress beyond the present unsatisfactory state of plant breeding. In doing so, we should free up breeders to use the best of modern technology and scientific knowledge, while using such tools to address the key important challenges confronting twenty-first century agriculture. From our previous analysis, we can identify three serious issues relating to the evolution of plant breeding R&D over the past few decades. First, there is the withdrawal of the public sector from most aspects of practical research in many major countries, and the consequent academisation of much of its work. The dire straits of practical plant research and breeding are seen most acutely in Europe and Australasia. Although the problem is less marked in the USA, where a somewhat reduced, but still relatively vigorous and effective, practically orientated public sector continues to function, things could be greatly improved here as well to harness the full potential of researchers. Indeed, across the world, public sector bodies need to adopt a much more practical and outward-looking attitude towards plant breeding and crop improvement.

The second issue is the gap between an increasingly academically inclined public sector and a rather uncertain commercial private sector that appears to be in transition from its current seed-based, input trait dominated business models.

Introduction

By the mid-1940s, agricultural research and breeding centres had already been operating successfully for more than half a century in most industrialised countries. The benefits of this public sector led approach were universally obvious. Yields of each of the major commercial crops increased to the extent that food surpluses were generated in many of the main producer countries. Even tiny Britain, with its relative dearth of useful arable land, and a large, rapidly growing, urban/industrial population, was able to produce over three quarters of its food requirements. This achievement was largely thanks to a combination of assiduous attention to quality breeding and the introduction of intensive farming practices. By this time, the yield benefits arising from farming mechanisation (e.g. tractors and harvesters), that had been so prominent in the 1920s and 1930s, were being outstripped by gains conferred by biological improvements, i.e. from plant breeding. By harnessing plant genetics, breeders could also design crops that were specifically suited to the new high-input, fertiliser/pesticide regimes, and were also adapted to mechanised cultivation and harvesting. Improved breeding practices also extended further the prospects for even greater gains in yield and productivity.

Introduction

So far, we have concentrated on the techniques for manipulating the genome of E. coli. However, it is very likely that we will need to work with other organisms, too. We might be interested in some aspect of the biology of another species and want to study the effect of modifying it in some way. We might want to make genetically altered versions of commercially important species, such as crop plants, to improve their value. Or we might want to produce a protein in a form that requires some post-translational modification that E. coli is unable to accomplish. The principles that we have seen for E. coli apply in exactly the same way. We need to have suitable vectors, a means of getting the DNA into the organism and ways to select transformants. We may also need to take steps to increase expression. Often, we first clone DNA from one organism in E. coli and identify a recombinant containing a particular gene of interest. We then transfer that gene into some other host species to alter the host's properties. An organism containing a gene derived from elsewhere is said to be transgenic.

In this chapter we will look at the vectors and transformation systems available for a range of other organisms. We will look first at bacteria, and then we will consider fungi, plants (including algae) and animals.

Introduction

So far, we have seen how to clone particular sequences and identify them. In Chapter 8 we will look at how these clones can be put to use directly at the DNA level or to direct the synthesis of RNA or protein. However, it is often the case that we need to modify sequences before using them. Here are just three of the many situations in which we may need to do this:

1. (a) We are trying to identify promoters and regulatory sequences, and need to make a mutation in a putative promoter or regulatory sequence to see whether that actually affects the efficiency of transcription initiation.

2. (b) We are interested in how the primary and higher order structures of a protein determine its function. It might, therefore, be necessary to modify the codon for an amino acid we believe to be at the active site of an enzyme and then assess the effects of that change on catalytic activity. Directed alteration of particular parts of proteins as a way of probing the relationship between structure and function or altering the function in a controlled way is often termed protein engineering.

3. (c) Genes are often cloned without our fully understanding the role that the proteins they encode have in the cell. Assessing that role may be possible by inactivating the endogenous gene in an organism to generate a mutant strain. This approach is often called reverse genetics, to emphasize the contrast with the traditional approach whereby a strain carrying a mutation with specific effects is characterized first and the relevant gene cloned and analysed subsequently.

The basic technique

The method

Later chapters will describe the techniques for the amplification of DNA sequences by propagation inside cells. However, it is often possible to amplify specific sequences more simply and quickly by a direct enzymatic process called the polymerase chain reaction or PCR. The basic procedure is outlined in Figure 2.1. In the simplest case, PCR amplification requires that we know a small amount of nucleotide sequence at each end of the region to be amplified. Oligonucleotides complementary to that sequence are synthesized, typically 20 or so nucleotides long. These oligonucleotides are used as primers for enzymatic amplification.

A reaction mixture is set up containing a sample of DNA that includes the region to be amplified, the primers in large molar excess, deoxynucleoside triphosphates (dNTPs) and a heat-stable DNA polymerase. The most common enzyme for this purpose is the Taq polymerase, which is a DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus, which can be grown routinely in the laboratory at 75°C or more. This enzyme, which the bacterium uses for cellular DNA synthesis, has a temperature optimum of at least 80°C and is not readily denatured by the repeated heating and cooling cycles that we shall see are needed in the amplification process. There are many other thermophilic bacteria, and their polymerases can also be used, as discussed below.

Introduction

So far, we have considered the use of small plasmids as cloning vectors for E. coli. However, these are not the only molecules able to replicate inside bacterial cells. For example, the E. coli F (fertility) factor is a large plasmid that can replicate independently of the bacterial chromosome, like other plasmids (although it can also insert itself into the bacterial chromosome). The F factor is used as the basis of bacterial artificial chromosome (BAC) vectors, for cloning very large pieces of DNA. Bacteriophage viruses are also able to replicate inside the bacterial cell, and a number of them have been developed for use as cloning vectors. These include the phages M13, lambda, Mu and P1. The M13 vectors have a lot in common with the pUC vectors we looked at in Chapter 3. They can be used for generating single-stranded DNA and they have been particularly useful in DNA sequence determination, although they are now less widely used for this. The lambda vectors are used for more general cloning purposes. Phage Mu is often exploited for its ability to act as a transposable genetic element, and Phage P1 is particularly useful for the production of phage artificial chromosomes (PACs). Like BACs, these can be exploited for the cloning of very large pieces of DNA. There are also a selection of vectors that are hybrids between plasmids and phages.

Introduction

Cloning and manipulating genes requires the ability to cut, modify and join genetic material (usually DNA, but sometimes RNA) and check the parameters of the molecules, such as size, that are being manipulated. We will assume knowledge of the structure of the materials involved (DNA, RNA and so on) and start by describing the tools available for manipulating them. Many of the tools involved are enzymes that have important physiological roles in cells. To understand why they are useful for our purposes, we should be aware of their normal roles, too.

The choice of which enzyme is used for a particular purpose depends mainly on two considerations:

1. How easy (i.e. inexpensive) is it to purify? This will be determined by its abundance in the cell and by how easy it is to separate it from other undesirable activities.

2. How well does it do the job? This will depend upon its specificity (‘accuracy’) and specific activity (‘speed’) and upon the details of the reaction which it catalyses.

Other factors, such as stability, are also important.

Techniques of genetic manipulation can be applied to the production of the enzymes for genetic manipulation itself. It is possible to use cloned genes to prepare large quantities of these enzymes more easily, as well as to modify the genes to ‘improve’ their function, perhaps by slightly altering the properties of the enzymes they encode.

Introduction

So far, we have seen how libraries can be constructed using plasmids, phages and other vectors in E. coli. But it is not enough simply to be able to clone DNA at random. It is usually necessary to go on to identify members of a library that contain a piece of DNA with particular properties. Finding those members of the library is called screening. Most often, we screen libraries for sequences with a particular coding function to find the gene or cDNA for a particular protein. There are many different strategies for doing that, and we will look at those first. Sometimes libraries are screened for DNA with a particular function other than for coding, such as the ability to initiate or terminate transcription. We will look at those techniques too.

Database screening

One of the easiest ways to find a clone for a particular sequence (usually a gene or a cDNA) is to exploit genomic analyses. For many organisms, partial or complete genome sequences are available electronically in genomic databases. There are also EST databases, which are sequences of large collections of cDNA clones, typically sequenced at random from libraries constructed from different tissue types or under different conditions. The clones used to build the genomic and EST databases are generally widely available. If such databases exist for the organism you are interested in, then it is a simple matter to search the database by computer. This kind of screening is often described as in silico.

The field of gene cloning and manipulation has changed dramatically since the first edition of this book appeared, and this development is reflected in the changes I have introduced in the second edition. The applications of PCR methods have expanded enormously, and “omics” and reverse genetic technologies are available across a wide range of organisms. Significant improvements have also been made in established areas, such as in the hosts and vectors for protein expression, and in the use of fluorescent proteins as reporter genes. As with the first edition, I have tried to stress the principles underlying the vectors we use, and avoid long and detailed lists (which would soon become out of date, anyway). Recognizing the necessity of being able to devise appropriate strategies for individual experimental situations, I have added a final chapter that gives examples and suggestions.

I am grateful to the members of my lab who waited patiently while the pressure of finishing this edition (which became known as my ‘big book of fun’!) delayed other things. I am particularly grateful to the people who helped directly in various ways, especially Mim Bower, Jon Burton, Ellen Nisbet, Saul Purton, Beatrix Schlarb-Ridley, and Petrus de Vries. I would also like to thank Katrina Halliday and Clare Georgy of Cambridge University Press, together with Peter Lewis and Rasika Mathur of Keyword Group for their technical expertise, patience and encouragement.

Use as DNA

Cloned DNA can be used directly in too many different ways to describe here. They include sequencing, blotting (Southern, northern, southwestern, etc.), transcript mapping, footprinting and bandshift (gel retardation) assaying. Details of these can be found in general molecular and cell biology textbooks. Cloned DNA can also be used to build microarrays, as described in Chapter 1. We will concentrate first in this chapter on the use of cloned DNA sequences for expression, i.e. for directing the synthesis of RNA or protein. As we shall see, many cloning vectors have been developed specifically for this purpose. They are called expression vectors. We will then look at the use of cloned genes as tools for studying the function of other sequences.

Synthesis of RNA

Why synthesize RNA?

It may be necessary to produce RNA for a number of reasons. We might be studying RNA processing events, such as splicing or cleavage, in vitro and, therefore, need to produce RNA of a single type in the presence of as few contaminating proteins as possible. Or the aim might be to make a protein in a radiolabelled form by translation in vitro of an appropriate RNA in the presence of radioactive amino acids.

Vector systems

One approach to making RNA might be to use a vector with a powerful promoter to direct transcription in a bacterial cell in vivo and then isolate the RNA. However, because the purification of RNA from bacterial cells is difficult, and the isolation of individual RNA species even more so, the production of RNA is usually done by transcription from cloned DNA in vitro.

Introduction

The aim of this chapter is to show you how the different techniques we have covered in the preceding chapters can be put together to study biological systems. Some examples of things you might want to do will be given, and strategies will be suggested for achieving those aims. It may be helpful to read the description of the problem first, design your solution and then compare it with the suggested one. It is important to realize that there is rarely a single ‘correct’ answer. There may be other equally suitable strategies in addition to the ones suggested here.

Scenario 1

(a) You are studying a bacterium that grows in a particular ecological niche. You cannot culture it in the laboratory, but you can isolate small quantities of cells that microscopic analysis indicates are not contaminated with other bacteria. You want to obtain ribosomal RNA gene sequence data to study the taxonomy of the bacterium.

You could use PCR with primers to regions of ribosomal RNA genes that are conserved across a wide range of bacteria to amplify the corresponding sequences from the bacterium of interest. If the PCR product looked sufficiently specific (i.e. it appeared to be a single band in gel electrophoresis) and was in sufficient quantity, then you could determine its DNA sequence directly.

Introduction

We encountered the concept of a library, a collection of random DNA clones, in Chapter 3. Having learned about cloning in bacteriophage viruses and their derivatives in Chapter 4, we are now in a position to look in more detail at making libraries.

Libraries can conveniently be divided into two categories: genomic libraries, which are made from the total genomic DNA of an organism, and cDNA libraries, which are made from DNA copies of its RNA sequences. We will look first at how these two types of library are made. Sometimes, though, it is useful to be able to make a more specialized genomic or cDNA library, enriched for particular sequences, so we will look at how they are made too. The library we looked at in Chapter 3 was a plasmid genomic library. It was represented by a large number of colonies on a plate, each containing a plasmid with a defined insert. We can also use a phage vector; handling large numbers of phages is often more convenient than handling large numbers of plasmids. When using phage, we get a collection of plaques on a lawn of bacterial cells, rather than colonies. Each plaque will contain a single type of phage with a defined DNA insert. With cosmids, fosmids, BACs and PACs, replicating as plasmids, the library will again be colonies on a plate.