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EQUINE VETERINARY EDUCATION / AE / MAY 2018


the horse genome (Bailey 2014; Hestand et al. 2015). The immense variety of proteins found biologically has been found to be largely driven by changes to the primary mRNA molecule after it comes off the genome, and before it is translated into a polypeptide, through the process of alternative splicing. Protein coding genes are made up of sequence regions


that are either expressed in the protein product and intervening sequences that do not code the protein. Sequences that are expressed in the protein product are termed exons, and intervening sequences are termed introns. What is an intron and what is an exon is defined by the final product. Figure 2 is a diagram that depicts a primary, unedited mRNA sequence. In this diagrammatic example, the primary mRNA transcript is made up of five exons represented by coloured rectangles. The introns are depicted by thin black connecting lines. This diagram depicts several mature mRNA transcripts that could be made from this one strand of primary RNA by splicing out different parts of the primary sequence. It is a great system – so creative and economical – a bit like our alphabet, but unidirectional. Different proteins can be made from the same gene – although they may be related, they may have actually different functions. The elegant process of alternative splicing enables the generation of mRNA transcript variants (or isoforms). Alternative splicing is the powerhouse behind the incredible complexity of proteins from only 20,000 or so protein coding genes. It is estimated that mRNA transcripts from ~95% of protein coding genes undergo this alternative splicing (Pan et al. 2008). This blossoming arena of science is illuminating many areas of previously poorly understood biological nuance in all fields of medicine and biology alike. Less than 3% of the genome actually codes for proteins.


The remaining 97% used to be termed ‘junk DNA’. Given that vertebrate protein coding genes show significant conservation in sequence and that the number of genes is not dramatically different between many vertebrate species, it should come as no surprise that the nonprotein coding DNA


Exons Primary messenger RNA Introns


is critically important. The differences between the protein coding sequences for the human genome and that of a chimpanzee is only ~4% (Varki and Altheide 2005). Most of the proteins are homologous and yet there are substantial differences between chimpanzees and man. These differences can be largely accounted for by differences in the regulation of the genetic code. Nonprotein coding DNA is responsible for a vast array of regulatory elements that dictate how and when the genome is used. These elements range from nontranscribed sequences in the DNA itself, such as promoter and enhancer sequences, to regions used as templates for noncoding RNA molecules (further discussion in the subsection Epigenetics).


DNA replication, fidelity and mutations


Chemically, we appreciate that the stability of DNA and the fidelity of Watson-Crick base pairing allowing it to be reliably passed from parent to offspring. The process of DNA replication in the cell cycle is a process with amazing fidelity. Replicative enzymes have a DNA repair capacity and a proof-reading capability, akin to checking the distance between railroad tracks for example, as an insurance policy against errors. In spite of these features, faithfully replicating 2700 million base pairs over and over again in all nucleated cells, over a lifetime, in the presence of mutagenic agents is a pretty tall order and mistakes are made. As such, it is easy to see how genetic variation occurs through generations, and how mutations can develop in an organism within its own lifetime, as is the case with many neoplastic diseases. Generation of de novo genome mutations occurs as a result of cellular replication errors; a few of these variations presently help to map the equine genome and track down regions correlated with clinical diseases.


Single nucleotide polymorphisms


As the name implies, a single nucleotide polymorphism (SNP: most people pronounce this ‘snip’)isdefined as a single nucleotide that differs between the same region on a chromosome with a population frequency of >1%. It may be in a protein-coding gene or in the intergenic region. In the example below two gene alleles are shown. For example:


Allele 1 for Gene X: CGTCATCGTCGT Allele 2 for Gene X: CGTCACCGTCGT


In this example you can see that the sixth nucleotide in


Selection of possible mRNA transcripts made by alternative splicing of the original primary mRNA transcript


Fig 2: Showing that a primary messenger RNA transcript can be processed into many different mature transcripts that code for different polypeptides. Exons are depicted as coloured rectangles and introns are depicted as black lines.


© 2016 EVJ Ltd


sequence is a T in Allele 1 and a C in the Allele 2. This is a T/C SNP. If this was, for example, located on Chromosome 1 at the 8,446,725 nucleotide position of the reference genome (that of the Thoroughbred mare Twilight) it would be annotated Chr1: g.8446725T>C. Some horses will have two copies of Allele 1, some two copies of Allele 2 and some will have one copy of each allele. The frequency of the SNP will vary depending on the population. For example, it may have different frequencies between Arabian and Thoroughbred horses. SNPs are distributed throughout the genome but tend to be more frequent in the noncoding regions. This is probably because the coding regions are under a greater selection pressure. SNPs are used as markers. They may happen to occur in a coding region and may affect how ‘fit’ an animal is, potentially conferring an advantage or disadvantage.


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