DNA is in the nucleus in discrete linear chromosomes, associated with proteins

Eukaryotic cells are distinguished from those of prokaryotes by possessing a nucleus, separated from the rest of the cell by a membrane, and containing the cell's genetic material. There is also a small amount of DNA in the mitochondria, discussed in Chapter 13. Nuclear DNA is organised in linear chromosomes rather than in a single circular one. The number and size of chromosomes varies considerably from one species to another (Table 1.1). There is a limited correlation between the total DNA content of a species and its evolutionary position. Many higher plants, amphibia and fish have a larger genome than humans and other mammals.

The nuclear DNA of eukaryotes exists as chromatin, associated with a number of proteins that are only found in the nucleus. The best known are the five major classes of histones (Table 6.1), which are among the most abundant cellular proteins. They are comparatively small proteins with an excess of positively charged amino acids (arginine and lysine) over the negatively charged ones. They are ideally suited for binding to negatively charged nucleic acids by multiple ionic interactions. Spermatozoa are exceptional in containing protamines, a group of even smaller highly positively charged proteins, in place of histones. They presumably aid in packaging the DNA into the small volume of the head of the sperm.

There has been an explosive growth in our detailed knowledge of genetics at the molecular level over the last few years, and it is likely that accretion of new knowledge will occur at an ever increasing rate. It is therefore very difficult even for the specialist to keep abreast of all the latest ideas which rapidly progress from hypothesis to theory to accepted dogma. In the time that it takes to write a comprehensive textbook it is inevitable that new ideas will be generated and many problems in the field elucidated so that such a book will certainly be out of date before the writing is finished, let alone published. Even during the writing of this small book, over the course of a little more than a year, much new information has come to light so that were it to be re-written in the next few months, appreciable differences would appear. It does not therefore claim to be a complete guide to the subject under review; nevertheless it attempts to present ideas that are reasonably well established and at the same time to cover a fairly wide field, albeit mostly not in great depth. The selection of topics as examples of our knowledge is somewhat arbitrary and conditioned by the author's own interests and expertise.

I believe that it should be a useful book for medical students who wish to become familiar with recent ideas and techniques in molecular biology to help in understanding further advances when they arrive.

Mitochondria and chloroplasts contain single circular chromosomes that direct the synthesis of a number of proteins, and also some of the RNAs that are required for the process. There is good evidence that these genomes are descended from separate lines of endosymbiotic bacteria (azotobacter and cyanobacteria). However, many of the genes that were originally present in these hypothetical ancestors have been translocated to the nucleus, so that their products must be transported into these organelles.

Yeast mitochondrial genome

This is about 78 kbp long, encoding two rRNA molecules, a complete set of tRNAs, and mRNAs directing the synthesis of at least nine proteins (Fig. 13.1 and Table 13.1). The polymerases for the synthesis of mitochondrial DNA and RNA, all the tRNA synthetases and the majority of the mitochondrial proteins (e.g. the enzymes of the citrate cycle and electron transport chain) are encoded by nuclear genes and synthesised on cytoplasmic ribosomes. All these proteins contain amino acid sequences to target them for translocation into the mitochondria.

The mitochondrial rRNAs (21S, 3200 nt; and 15S, 1660 nt) are somewhat smaller than prokaryotic rRNAs, and there is no rRNA corresponding to the 5.8S rRNA of the cytoplasmic ribosomes. The two rRNAs are encoded on widely separated parts of the genome, and are not transcribed together. There are genes for 24 tRNAs, some of which are clustered, but others occur singly.

In this rapidly advancing field there have been many discoveries in the six years since the second edition was written, so the time is ripe for a new edition to incorporate some of this new information. In order to keep the book within a reasonable length, and make it accessible to students living on ever decreasing grants, some material and topics of the earlier editions have been omitted or drastically shortened. The new edition has expanded coverage of both prokaryotic and eukaryotic replication and transcription, and post-transcriptional modifications of RNA. Elsewhere, other topics have been brought up to date. Inevitably, their selection has been largely influenced by my own interests and expertise. As there are several excellent accounts of genetic engineering available, Chapters 3 and 4 of the previous edition on methodology and vectors have been combined and shortened.

I am most grateful to Dr Tim Benton at CUP for his encouragement and gentle guidance; to Mrs Sarah Price for her meticulous and helpful copy editing; and, as always, to my wife for her support and also for help in the preparation of some of the figures. I have also been greatly helped by being allowed ready access to the libraries of the University of Hertfordshire and of my old College, the Medical College of St Bartholomew's Hospital.

Collagen

Collagen is the most abundant protein in the body, comprising approximately 25% of the body's protein. There are actually at least 19 distinct but related types. The molecules of each type consist of three long polypeptide chains tightly wound together to form a triple helix. Types I, II, III and V have long and fibrous molecules: the other types are non-fibrillar. In some cases there are two identical chains and one that is different, while in other types the three chains are identical. As far as is known the individual polypeptides in the various types are all encoded by separate genes. Most of the mature chains are about 1000 amino acids long and are characterised by the presence of a glycine residue at every third position through a good deal of their length. Its tiny side chain can be easily accommodated inside the tightly wound helix. They are also unusually rich in proline, and many prolyl residues (and also some lysyl ones) undergo post-translational hydroxylation. There are extensions at each end of the molecules (telo-peptides) that are removed after the chains are wound together to form the triple helix. In some types the regular repetitions of glycine followed by two other amino acids are interrupted at intervals so that the molecules may be kinked rather than straight. The exons of the genes of the fibrillar collagens tend to be rather short, and the gene for Type I contains no fewer than 54 exons that mostly contain 45 or 54 nt or multiples or sums of these numbers, beginning with complete glycine codons.

Termination of transcription

Almost all eukaryotic mRNAs, with the notable exception of those for his tones, have a sequence of adenyl residues [the poly(A) tail] added post-transcriptionally in the nucleus by the action of the enzyme poly(A) polymerase. Typically, this tail contains about 50–250 A residues that increase the stability of mRNAs by protecting them against degradation by ribonucleases.

Although signals in the DNA directing RNA polymerase to definite sites for initiation of transcription are known, the mechanism for ending transcription is not well understood. Downstream from the termination codon there is always a sequence of nucleotides that is transcribed and appears in the mRNA but is not translated. This is very variable in length and may, sometimes, exceed 1000 nt. The sequence AATAAA is present in nearly all animal protein-coding genes about 10–30 nt upstream from the site of poly(A) addition. Natural or artificial mutation of this sequence interferes with proper 3′-end processing and may also cause increased transcription past the normal region of termination. Thalassaemias (Chapter 10.2) can arise by mutation of AATAAA to AATAAG or AACAAA in the poly-adenylation signal of the β-globin gene.

The AAUAAA sequence in a pre-mRNA is a nucleation site where a protein complex is built up. This contains the Cleavage and Poly-adenylation Stimulatory Factor (CPSF) and the Cleavage Stimulatory Factor, both of which are oligomeric. The latter also contacts a sequence rich in U, or G and U residues a little way downstream.

Since the first edition of this book appeared there have been many advances in our understanding of the genome, so it is opportune to review some of these in this new edition.

New techniques continue to be invented, and those that have come into general use are described in Chapter 4. Chapter 7 on eukaryotic gene organisation and expression has been completely re-written as it is in these fields that some of the most dramatic advances have occurred in the past five years. The chapter on hormone genes has been transmuted into a chapter on gene families since much information is now available about their evolution. In Chapter 12 it has been possible to include new material on chloroplast genomes. Elsewhere there have been some re-arrangements and updating of material.

I am most grateful to Dr Adam Wilkins at CUP who asked me to prepare a new edition and to Dr Paul Lasko of the Department of Genetics at Cambridge University. They have both made some very helpful comments and suggestions after reading the first draft. Dr Robin Smith, also of CUP, saw the final version through the press and also made some valuable suggestions for which I am very thankful.

Finally, I should like to thank colleagues at Barts, especially Professor Gavin Vinson and Dr Ian Phillips, for much encouragement and helpful discussion, and, as ever, my wife for her forebearances while I have been working on the book.