EQUINE VETERINARY EDUCATION / AE / MAY 2018


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for during the process of domestication of the horse, as well as providing evidence that the Equus lineage originated approximately 4–4.5 million years ago (Orlando et al. 2013; Schubert et al. 2014). DNA is made up of four nucleotides that base pair: G to


C, and A to T. This spatial arrangement ensures that the two anti-parallel helical backbones stay a consistent distance apart – like train tracks. Genomic DNA is the template for all of the proteins and regulatory elements of that organisma. The length of the equine genome is about 2700 million base pairs (2.7 Gb) and similar in size to the human genome (about 3 Gb) (International Human Genome Sequencing Consortium 2004). However, the equine genome is packaged into 31 pairs of chromosomes (autosomes) and a pair of sex chromosomes (XX or XY). It is estimated that as a continual length, the human genome would be about 2 m in length. It is an astonishing thought that all cells, with just a few exceptions such as red blood cells, have this length tidily packed into their nuclei. This packaging is achieved by the genomic DNA being wrapped around histone proteins – like long hair wrapped around hair curlers where the DNA is the hair and the histones are the hair curlers. It is further astounding to think that this DNA is selectively ‘unzipped’ to open up units, such as protein coding genes, for use according to the cells’ needs and cell type.


Genes, RNA and protein coding


The genome is made up of protein coding genes and noncoding sequences, but on a primary sequence level, it appears as one huge ordered series of As, Cs, Gs and Ts. Francis Crick proposed the Central Dogma of molecular biology: one gene-one message-one polypeptide (Crick et al. 1961; Crick 1970) (Fig 1). Basically, a specific length of DNA sequence, a gene, is transcribed in to a ribose nucleic acid message (messenger RNA; mRNA), and that mRNA message is translated into a polypeptide by ribosomal machinery, thus synthesising protein. The ribosomal machinery translates the RNA sequence code in three nucleotide frames, a codon, for which a specific amino acid is selected. This concept was remarkable at the time, as there was still debate over which dictated which. It is now abundantly clear that DNA sequence dictates polypeptide sequence, and not vice versa. This Central Dogma has remained a fundamental tenant of molecular biology ever since, only now with some modifications. RNA differs in its chemical structure from DNA. The


presence of a hydroxyl group on the ribose sugar makes the RNA molecule less stable as it is prone to hydrolysis. Additionally, uracil (U) is used in place of T in RNA, distinguishing it from DNA and making it more unstable biochemically. RNA also exists as a single stranded molecule. These inherent properties, coupled with an abundance of degradative RNase enzymes found in the natural world, make the lifespan of mRNA molecules finite. Messenger RNAs can be considered the Post-it notes of nature. They are useful when they are needed, but must be regulated. For example, using my Post-it note analogy: if you left a note for your technician to order a case of penicillin because stocks were getting low, you would expect that note to be acted upon and then destroyed. One case of penicillin will be enough for a while. If your technician does not discard the note after acting upon it, every time they see that note they order


DNA DNA is transcribed to an RNA message mRNA


mRNA is translated into a protein at the ribosome


Protein


Fig 1: A schematic representation of the central dogma of molecular biology whereby one gene codes one messenger RNA, which codes one polypeptide. The directionality is of historical importance. The DNA dictates the sequence of the protein, not vice versa.


another case of penicillin. In short order your pharmacy will be overstocked with penicillin and it will be chaotic. So it is with mRNA. Both the rate of mRNA synthesis and the rate of degradation are important. Studies that look at the expression of genes are actually studying the steady state levels of mRNA in a given tissue or cell and using that as an estimation of how actively those genes are being transcribed (or expressed). It is only an estimate because it does not take into account adjustments that increase or decrease the lifespan of those mRNA molecules.


Genome structure conservation and the human genome project


An early and significant discovery from genome projects has been the great similarity of the structure and organisation among vertebrate genomes, an area of study called comparative genomics. As a consequence of this similarity, DNA sequences that help to characterise gene structure in one species can be used to predict gene structure for other genomes. The annotation of the horse genome benefited greatly from the extensive information developed in connection with the human genome project. Insulin, for example, is a highly conserved protein. Human insulin differs from porcine insulin and bovine insulin by just one and three amino acids, respectively. Additionally, sequences of genes that lie together on a chromosome in one species will probably be together on a chromosome in another species. For example, human chromosome 8 (HSA8 – from Homo sapiens) is homologous to equine chromosome 9 (ECA9 – Equus caballus) by virtue of the gene sequences located on it (Raudsepp et al. 1996; Bailey 2014). This is termed conserved synteny: conserved between species and synteny from Greek meaning ‘on the same band’. At the onset of the Human Genome Project in the very


early 1990s, the number of protein coding genes was significantly overestimated (www.genome.gov) (International Human Genome Sequencing Consortium 2004). These predictions were based on the sheer variety of proteins found by biochemists over the years. As the project progressed, it was clear that this number was going to be closer to 21,000– 22,000 protein-coding genes. The final number is still being argued, and it is similar in number to protein coding genes in


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