Bases de preparación y políticas contables

In the previous section, the importance of the primary sequence of proteins, and how it affects their catalytic activity – for the sake of the semi-permeable catalytic bag model – has been described. What hasn’t been described so far is how that primary sequence is determined. To understand this requires an investigation of the main features of the ‘genetic material’ or ‘nucleic acids’. It is worth briefly noting that the term ‘nucleic acid’ refers to 2 different acidic polymers found within a cell – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). These are fundamental to the variability of the catalytic properties of the cell, and will be explained in more detail in this section. The polymers are referred to as ‘nucleic acids’ as they were originally extracted from a central cell structure called the nucleus; however whilst not all cells have a nucleus, all living things and even viruses contain nucleic acids.

to produce proteins, which in turn facilitate life. DNA is a double-stranded, α-helical polymer made up of four unique base molecules – adenine (A), cytosine (C), guanine (G) and thymine (T) that are joined in a specific sequence. Like protein, DNA has a regular repeating backbone structure, where the bases are joined by negatively charged phospho-diester bonds, and the bases themselves are planar hydrophobic disks that stack on top of each other. The double-helix is an anti-parallel dimer, where one strand is the inverse of the other – in this case, where an A is present on the first strand, it will be hydrogen-bonded to a T on the other and vice-versa. The same is also true for C and G. The structure was first identified famously by Watson and Crick in 1953, work that lead to them being awarded the Nobel Prize in Physiology or Medicine along with Maurice Wilkins in 1962 (Watson and Crick, 1953). The sequence of all the DNA in a cell is referred to as the genome sequence. Within the genome sequence, the specific sequence – as with proteins – is vitally important.

From a conceptual point of view, DNA can be considered as the blueprints for the catalytic bag. The instructions for forming the complete list of every protein that can possibly be produced, under any condition, is found within the genome. The blueprints for an individual protein are referred to as a ‘gene’, and the collection of all protein-coding sequences within an organism is referred to as the ‘genome’. As mentioned above, however, the exact numbers and even the specific presence of any of the catalytic components is variable. Not all of the potential catalysts that the genome has blueprints for will be produced in the same quantities, in fact not even all the potential catalysts that can be produced will be under all conditions. This can be considered using a facile analogy of a factory producing cars – there will be a blueprint for a wheel, an axle, a door; however a standard car will require 4 wheels, 2 axles, between 3 and 5 doors, etc. depending on the model. In a cell, whilst the genome exists as a central repository for all the proteins that can be produced, a second nucleic acid, RNA, is responsible for actually converting those blueprints into reality.

Before protein is produced, selected parts of the DNA sequence must be ’transcribed’ into a substance called mRNA (mesenger RNA) by a protein called RNA polymerase. mRNA is a subset of RNA (ribonucleic acid) exclusively used for the transfer of information from DNA into protein (Brenner et al., 1961). RNA is a single-stranded polymer molecule with a highly variant structure depending on the sequence. Whilst RNA also has a wide variety of functions, including structural features and regulatory controls, these are diverse and fall outwith the scope of this review. The DNA bases are transcribed into equivalently named RNA bases – A, C and G; with the exception of thymine, which transcribes to uracil (U), carrying the sequence information from the DNA forward. This sequence of bases are read by a molecule called a ribosome, which translates the sequence into a protein. As described above, proteins act together to produce functions within a cell

1.3. KEY BIOLOGICAL PRINCIPLES 39 based on the genome sequence, and so the movement of information flows from DNA to RNA to protein. This is referred to as the central dogma of molecular biology (Crick, 1970).

This direct link between the genome and the catalytic capabilities of a cell has important consequences for engineering. Conceptually, by removing a gene from the genome, the capability for a cell to produce the protein it coded for has been removed as well. The inverse is also true, it is possible to add a gene or collection of genes to a genome and add functionality. This forms the fundamental principles of genetic engineering. This first methods for targeted genetic modification of an organism were developed by Cohen and Boyer in 1973 (Cohen et al., 1973), and 2 years later in 1975 the historic Asilomar conference on recombinant DNA molecules was called due to concerns arising over the engineering of life (Berg et al., 1975). Whilst finding a gene that relates to a specific protein can be done relatively easily, the principles behind rationally engineering a system are more complicated.

Production of mRNA is controlled by a complex series of feedback reactions, which enable the production of proteins in response to the specific requirements at the time. One of the major difficulties in understanding this is that a snap-shot of cellular response is heavily influenced by the previous state the cell was in, which can make understanding and engineering cells difficult. A modern cell cannot function without a comprehensive collection of all of the materials mentioned above in a prior state, which has been provided in the form of an unbroken chain of living organisms since the first origins of life around 4 billion years ago (Haldane, 1929).

When physically ‘translating’ mRNA into a protein sequence, there is a clear issue of disparity between the number of nucleic acid bases (4) and the number of commonly used amino acids (21). To get around this issue the bases of mRNA are read in groups of 3 bases at a time, these triplets of bases are also known as ’codons’. There are unique codons for each amino acid, as well as specific codons indicating the beginning and end of a protein coding sequence (start and stop codons, respectively). As there are 64 possible

codons (3 bases with 4 possible states per base, 43 _{= 64) and only 23 codon ‘states’;}

with 21 amino acids plus start and stop codons, the genetic code is degenerate (Crick, 1968). This degeneracy means that different codons code for the same amino acid. By measuring the occurence of codons within the translated genome a ‘codon usage table’ can be produced. This shows the rate of occurence of individual codons within specific organisms, which have been found to be biased towards certain codons in certain species; however comprehensive studies of this phenomenon only became possible in the post- genomic era (Duret, 2002).

level investigations. This simply refers to genome sequence being known, and also being run through computational tools to identify putative genes and proteins.