Quantum Leaps in Biochemistry

L.A. Stocken, M.G. Ord, L.A. Stocken, M.G. Ord

• Publisher: Elsevier Science
• Year: 1996
• ISBN:
  9780080540061
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

This volume covers such quantum leaps in the field of biochemistry as the coding properties of DNA and the central dogma, manipulating DNA, extranuclear DNA, protein synthesis and the ribosome, and cell cycles.

Chapter 1

Introduction

In Volume 1, Early Adventures in Biochemistry, we described the experimental methods used in the elucidation of the main pathways of intermediary metabolism in animals. We drew attention to the “forgotten men of biochemistry” and their achievements, and tried to show younger biochemists how, in spite of very primitive equipment, certain fundamental concepts were advanced. These were that ATP was the primary energy source for chemical and physical work done by cells, that proteins were the workhorses of the cell and contributed significantly to the structures from which the cells are composed, and that events in cells were spatially and temporally organized.

Three further propositions that emerged during the 1950s were only touched on in the first volume—the role of DNA as the carrier of the inherited information of the cell, the metabolic activity of the different species of RNA, particularly the role of the ribosome in protein synthesis, and the ideas of Jacob and Monod regarding the regulation of expression from the genome. The first two of these are now considered in more detail in Chapters 2, 3, and 5 of this volume.

The exponential growth of molecular biology followed from the development of experimental techniques for analyzing the nucleotide sequences of DNA. The various procedures by which “foreign” DNA can be introduced into and expressed by host cells are reviewed in Chapter 3. We hope to consider regulation of expression of the genome in Volume 3.

The finding of extracellular DNAs in mitochondria and chloroplasts (Chapter 4), and the establishment of their probable endosymbiotic origins, was followed by the discovery, in plants and lower organisms, of the movement of DNA molecules between plastids, mitochondria, and nuclei. That mechanisms exist for interchange between plastid and nuclear DNA was another discovery of great evolutionary significance.

Other important experimental innovations include the application of nuclear magnetic resonance (NMR) to the study of protein structure. NMR has provided what is currently the most powerful method for examining protein interactions with macromolecules, substrates, and other solutes. The results of such studies, together with our present ability to deduce protein sequences and likely functions from genetic data, have led to a major change in our thinking about proteins. Up to 1960 attention was primarily focused on the properties of enzymes and their mechanisms of action. In the past 20 years many more proteins have been discovered, of which most occur only in small amounts in cells and probably have regulatory roles either in the nucleus or in processes whereby extracellular events at the cell surface lead to intracellular responses. Most of these proteins are not enzymes: instead their effects are exerted through contacts with other proteins or cell constituents. Genetic and structural analyses, aided by highly sophisticated computer techniques, now concentrate on protein domains (see Doolittle, 1995). The size and shape of these domains makes them analyzable by NMR (Chapter 6), which, along with X-ray crystallography, has been an important means by which these regions have been identified.

Glycobiology is a striking example of a branch of biochemical research whose existence has been almost totally dependent on the introduction of novel analytical methods (see Chapter 7). In the 1970s glycosylated molecules, usually complex mixtures of closely related compounds, were difficult to separate and whose precise composition defied analysis. These problems are now largely overcome, determinants for protein glycosylation are emerging, and its tissue and species diversity at different stages of normal or pathological development can now be examined.

The integration of the synthesis of proteins and their migration to the appropriate regions of the cell, or for export, is considered, inter alia, in Chapter 5. A further topic in cell biology—the way in which the behavior of the cell is directed successively towards growth, DNA replication, and cell division—is discussed in Chapter 8. Analysis of the cell cycle illustrates the way in which advances in biochemistry have utilized the full range of classical, genetic, and physical methods.

The first volume drew attention to the work of early biochemists who established metabolic pathways using very simple apparatus. This volume covers some of the phenomenal advances made since the 1950s, facilitated in large part by the expansion in the 1960s both in numbers of scientists and in available resources.

Since many of the above areas of research are still under active investigation, we have asked the contributors to focus on what appear to them to be the conceptually significant developments and how these were achieved, and not to attempt an up-to-the-minute coverage of each topic. Their long-term experience has produced authoritative accounts of the quantum leaps made in their fields.

References

Doolittle, R.F. The multiplicity of domains in proteins. Annu. Rev. Biochem.. 1995;64:287–314.

Chapter 2

The Coding Properties of DNA and the Central Dogma

Margery G. Ord and Lloyd A. Stocken

Introduction 3

Information Storage and Transfer Before 1953 3

The Structure of DNA: Its Verification and Implications 5

The Discovery of the Code 7

The Central Dogma 11

Polymerases and Related Enzymes 17

Summary 23

Notes 23

References 23

Introduction

This chapter is concerned with observations prior to 1953 which indicated a role for DNA in information transfer, and the experiments (up to 1980) which validated the Watson and Crick structure for DNA and its consequences.

Information Storage and Transfer Before 1953

Nuclei, first isolated by Miescher in 1869, were found to contain a phosphorus-rich substance, nuclein. When similar material was analyzed from salmon sperm, two components were distinguished—an acidic phosphorus-containing nucleic acid and a basic protein, protamine. Thymonucleic acid from thymus glands contained phosphorus; the bases thymine, cytosine, adenine, and guanine; and the pentose sugar, 2-deoxyribose-DNA. The nucleic acid obtained from yeast, RNA, contained uracil, not thymine, and ribose rather than deoxyribose.

That DNA and protein were the major components of chromosomes became evident from cytochemical staining and UV microscopy in the 1920s and 1930s. The preparation of nucleic acids, free from traces of protein, was however extremely difficult. Both DNA and especially RNA were easily degraded during isolation, and methods for their analysis were extremely primitive. Determinations of the nitrogen and phosphorus contents of DNA were consistent with a nucleotide structure, and analyses of the bases indicated roughly equimolar proportions of purines and pyrimidines. By the 1930s a tetranucleotide structure for DNA had therefore been proposed by Levene. Since this did not appear to allow the range of protein diversity already apparent, it was supposed that inherited information was a property of the protein(s) of the chromosomes, not of the DNA (For refs., see Ord and Stocken, 1995).

The experiments of Griffiths (1928) on mice infected with pneumococci showed that information could be transferred between cells. Small numbers of living pneumococci type II (rough coated), which did not cause fatal bacteremia, were injected into mice together with a large inoculum of heat-inactivated (killed) type III (smooth coated) pneumococci. Blood from animals which subsequently died yielded pure cultures of type III, virulent, bacteria. Later experiments showed that cell-free extracts from the virulent strain could carry out the transformation. In 1944, Avery, McLeod, and McCarty established that extracts which had been virtually freed from protein by chloroform, and which contained neither detectable lipid nor serologically identifiable polysaccharide, brought about transformation. The transforming principle was resistant to hydrolysis by RNAase, trypsin, or chymotrypsin, but was destroyed by DNAase, i.e. it appeared to be DNA. Once transformed, the pneumococci could be propagated as the smooth, encapsulated strain without further exposure to the transforming principle.

In spite of this apparently clear-cut demonstration of the capacity of DNA to transform cells, the possible presence of small amounts of protein in the extract could not be excluded. With the limited knowledge of its structure then available, those who were unable to accept that DNA could carry the necessary information to cause transformation were still able to attribute the change to protein in the extract.

Explicit evidence for the ability of DNA to transform came from the neat experiments of Hershey⁶ and Chase (1952) using T_even bacteriophage grown in [³²P]P_i to label the DNA and ³⁵S-methionine to label the protein of the viral coat. The radioactive phage was then harvested and used to infect unlabeled E. coli. All the ³²P-labeled DNA entered the bacterium, but the ³⁵S-protein coat of the virus adhered to the outside of the cell and could be shaken off by agitation in a Waring blender. No labeled sulfur was detected in the new protein of the viral particle, which must therefore have been programmed by the entering DNA.

Amounts of DNA/cell showed that nuclei from different organisms contained different amounts of DNA/nucleus, and that in a given species the amount of DNA/diploid cell was twice that in a haploid.

There were also indications of a role for RNA in protein synthesis—the presence of DNA was not essential. In 1934, in experiments with Acetobularia, a photosynthetic marine organism, H...