DNA sequences do not code for amino acids. The sections that code for amino acids are called exons. Most proteins are encoded by (i.e. transcribed and translated from) multiple exons separated by DNA sequences called introns. Together, exons and introns comprise what we have traditionally called genes. When DNA is transcribed into RNA, the entire section of introns and exons is made into RNA. Next, editing enzymes process the RNA strand, clipping out introns to make the final transcript. The final transcript, called messenger RNA (mRNA) is used for translation of the message into the amino acid sequence in the protein. For example, the gene for TRPM1 spans 103,840 bases on chromosome 1 when including all exons and introns. After processing, TRPM1, has five possible mRNA transcripts based on alternate splicing of 24–26 exons with the final transcript ranging in length from 5459 bases to 5630 bases. Fig. 4.3 illustrates a hypothetical intron-exon structure of a gene and the resulting mRNA following transcription and RNA processing to remove 3 introns separating 4 exons.
Fig. 4.3. This image illustrates a hypothetical gene with four exons and three introns. The introns can be very large and the exons very small. The entire exon/intron structure is transcribed then the introns are removed. Following transcription and mRNA processing, only the exons remain.
The most common signal to begin or conclude splicing out a section of DNA occurs at the beginning of the intron (bases GT) and the end of the intron (bases AG). A mutation at the first site will cause inclusion of the intron sequence in the processed mRNA while a mutation at the second will cause deletion of the next exon. Therefore, it is important to consider intron DNA sequences as well as exon DNA sequences when looking for genetic variants that affect genes. This type of variant is called alternate splicing and is implicated in some human diseases and has been identified as the cause of the sabino1 coat color variant in horses. In addition, alternate splicing appears to be normal in the function of many cells and tissues. Various regulatory factors can mask existing splice sites and cause alternate splicing for most genes. We are still learning how this occurs. However, we believe that the creation of proteins with slightly different domains creates proteins with slightly different functions.
In addition to introns and exons, there are large stretches of DNA between genes called, intergenic DNA that separate exon/intron regions. We do not know the role for DNA in these regions, however this DNA is probably important for maintaining special relationships among DNA chromosomal elements and much of it is actually transcribed and may play a role in regulating transcription.
Examples of DNA changes affecting genes
Nucleotide substitution changing an amino acid
These include variation in the coat color genes Extension or MC1R, aka black/bay/chestnut (Marklund et al., 1996; see Chapter 7), Cream dilution (Mariat et al., 2003; Chapter 8), and Champagne dilution (Cook et al., 2008; Chapter 8). These three coat color genes show genetic variation because of a SNP in the gene that changes an amino acid making up the protein. These would be called synonymous variants since they are found in exons. Changes in amino acids can alter receptor function or disable enzymatic function.
Deletion/loss of DNA resulting in loss of the codon reading frame
Lavender foal syndrome (Brooks et al., 2010) and severe combined immunodeficiency syndrome (Shin et al., 1997) both involve deletions in the coding sequence for a protein which destroys the function of that protein by shifting the reading frame used to translate the DNA sequence into the protein (Chapter 15).
Changes in gene expression (alternate splicing)
A single nucleotide base change in the intronic splice site of the KIT gene causes an unusual transcript causing the sabino1 phenotype. For this variant, an entire exon is absent from the resulting protein. In this case the variant is a SNP that destroys the signal for creating the splice. However, since it is not in an exon, it is neither synonymous nor non-synonymous (Brooks and Bailey, 2005; Chapter 9).
Chromosome inversions
The Tobiano mutation, which causes white spotting (Brooks et al., 2008; Chapter 9), occurs well outside the gene thought to be responsible for the tobiano color pattern. In this case, the mutation is a large chromosome rearrangement; approximately 50 megabases (50,000,000 bases) of DNA near the gene have an inverted order when compared with that of other horses. The precise effect of this mutation is not known but thought to affect the function of DNA sequences outside the gene that regulates expression of the gene during development.
Changes affecting gene expression
Mutation can alter splice sites for excision of introns or DNA sequences that serve as binding sites for regulatory molecules determining time and amount of expression. The variant affecting the gene for appaloosa (leopard spotting) is an insertion outside the coding portion of the gene that alters the amount of transcription by the TRPM1 gene (Bellone et al., 2010).
Where Are Genes Found?
Genes in the nucleus
Horse genes are packaged into 64 DNA molecules called chromosomes that are found in the nucleus of nearly every cell. Chromosomes can be seen with the aid of a microscope and dyes that bind to DNA or to the proteins associated with DNA. The genetic information of all domesticated horses is nearly identical and, not surprisingly, horses of all breeds have the same number, size, and shape of chromosomes.
Before a cell divides, chromosomal DNA is in an extended form and the areas active in transcription are exposed for action by polymerases. Electron micrographs show an image not unlike tangled spaghetti. When a cell starts the process of division into two daughter cells, chromosomes condense by supercoiling about chromatin proteins to form discrete rod-shaped bodies we recognize as the classical image of chromosomes. Careful cutting and matching of stained chromosome images obtained from microscopic examination of a cell in the process of division shows that the 64 chromosomes can be arranged as a series of 32 pairs of chromosome structures (Fig. 4.4). This array of paired and condensed chromosomes is visualized as a karyotype. The only distinguishing feature between most horse karyotypes is a difference between males and females seen in a single pair of chromosomes (the sex chromosomes), which are discussed in a later section.
Fig.
