Dale_Molecular Genetics of Bacteria 4th ed

Preface

When the first edition of this book was published (1989), DNA sequencing was largely confined to the characterization of individual cloned genes; although some viruses had been completely sequenced, the concept of determining the complete sequence of a bacterial genome was still some way in the future. Even in the second edition (1994), the situation had only developed to the stage that ‘Genome-sequencing projects are under way for several bacterial species . . . ’ – the first complete genome sequences were to appear in 1995. In the third edition (1998), it was still possible to produce a list of the bacteria that had been completely sequenced, although with the caveat that it would be out of date very quickly. Now, it is no longer sensible even to attempt to produce such a list. The widespread use of automated, robotic methods means that new genome sequences are appearing weekly, if not daily. Coupled with this has been the advent of the polymerase chain reaction (PCR) – a ‘recent development’ in the first edition – and more recently microarrays and proteomics that exploit genome sequence data for global analysis of gene expression.

This technological explosion has largely relegated many of the classical techniques in bacterial genetics to the pages of history, and poses a considerable problem in writing, or updating, a book such as this. Excluding the older methods completely would mean we would lose all sense of how the subject developed to the stage we are now at. Furthermore, the limitations of the purely molecular approach should be appreciated. Sooner or later, in order to fully understand the roles that specific genes play in the biology of an organism, one has to return to studying the organism itself, rather than just analysing its DNA sequence on a computer. So the compromise approach that we have adopted has been to slim down the description of the classical methods, while hopefully still retaining enough to provide historical perspective. This allows space to provide a fuller introduction to the world of genomics and post-genomics (the study of genome sequences and the exploitation of that data for analysis of gene expression and other features), and also to expand the discussion of the ways in which our knowledge of the biological properties of the organisms themselves has developed through the study of bacterial genetics (whether by classical or molecular methods).

Of course this is a compromise, and some will find it unsatisfactory. Why have we included some topics and excluded others? The choice is a very personal one. It should be remembered that this book is not even trying to be a comprehensive textbook of bacterial genetics, but rather to provide a manageable-sized distillation of the subject primarily for those students for whom bacterial genetics is one of a wide number of courses taken and for whom the sheer size of most genetics textbooks presents a daunting obstacle. At the same time, we hope that some of you will find the topics introduced in this book sufficiently interesting and exciting (as indeed they are) that you will want to enquire further.

Jeremy Dale

Simon F. Park

hydrophobic bases are in the centre, shielded from the water and the hydrophilic phosphate groups are exposed.

1.1. 4 Different forms of the double helix

A full consideration of DNA structure would be extremely complex, and would have to take into account interactions with the surrounding water itself, as well as the influence of other solutes or solvents. The structure of DNA can therefore vary to some extent according to the conditions. In vitro, two main forms are found. The Watson and Crick structure refers to the B form, which is a righthanded helix with 10 base pairs per turn (Figure 1.4). Under certain conditions, isolated DNA can adopt an alternative form known as the A form, which is also a right-handed helix, but more compact with about 11 base pairs per turn. Within

Sugar-phosphate

backbone

Pairing of bases

Figure 1.4 Diagrammatic structure of B-form DNA. The two anti-parallel sugarphosphate chains form a right-handed helix with the bases in the centre, held together by hydrophobic interactions and hydrogen bonding

the cell, DNA resembles the B form more closely, but has about 10.4 base pairs per turn (it is underwound, see below)

Certain DNA sequences, notably those containing alternating G and C residues, tend to form a left-handed helix, known as the Z form (since the sugarphosphate backbone has a zig-zag structure rather than the regular curve shown in the B form). Although Z DNA was originally demonstrated using synthetic oligonucleotides, naturally occurring DNA within the cell can adopt a lefthanded structure, at least over a short distance or temporarily. The switch from leftto right-handed can have important influences on the expression of genes in that region.

1.1. 5 Supercoiling

Within the cell, the DNA helix is wound up into coils; this is known as supercoiling. Figure 1.5 shows a simple demonstration of supercoiling, which the reader can easily try out for him/herself by taking a strip of paper and twisting one end to introduce one complete turn (i.e. the same side of the paper is facing the reader at each end). It will now look like the illustration shown in Figure 1.5a. The two ends should then be brought towards each other so that the conformation will change to that shown in Figure 1.5b which is a simple form of supercoiling. Not only has the strip of paper become supercoiled, but also the degree of twisting appears to have changed (in this example it now appears not to be twisted at all). If both ends have been held firmly, the twist of the strip cannot have

(a)not 'Supercoiled'

(b)'Supercoiled'

B C

D A

Figure 1.5 Interaction between twisting and supercoiling. (a) A ribbon with a single complete twist, without supercoiling. (b) The same ribbon, allowed to form a supercoil; the ribbon is now not twisted