* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download module three
Mitochondrial DNA wikipedia , lookup
DNA paternity testing wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Gene therapy wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Public health genomics wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
SNP genotyping wikipedia , lookup
Metagenomics wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Human genome wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
DNA vaccination wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genome evolution wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Epigenomics wikipedia , lookup
Molecular cloning wikipedia , lookup
DNA supercoil wikipedia , lookup
Primary transcript wikipedia , lookup
Genomic library wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Genetic engineering wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Microsatellite wikipedia , lookup
Genome (book) wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Point mutation wikipedia , lookup
Non-coding DNA wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genome editing wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Designer baby wikipedia , lookup
Helitron (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Horse Genetics Module Three Equine Molecular Genetics Night Owl Education and Equestrian Night Owl Education and Equestrian Module three: Equine Molecular Genetics Lesson seven - An introduction to molecular genetics Introduction The structure of DNA DNA and chromosome structure Genes and proteins The genetic code What is a gene? Redundant DNA The Horse Genome Summary Lesson eight - Finding and characterising genes for a particular phenotype Introduction Physical mapping of the equine SCID gene Heterohybridoma panels Fluorescence in situ hybridisation (FISH) Comparative mapping Replicating DNA in a tube DNA microarrays and chips Summary References Lesson nine - molecular genetics testing Introduction Impressive: a tale of triumph and tragedy Genetic Testing Allele specific PCR: genetic testing the modern way Linkage testing PyroSequencing DNA microarrays and chips Fingerprinting Example of fingerprinting: conservation of the Przewalski horse Summary References Module three assignment Night Owl Education and Equestrian Seven: An introduction to molecular genetics Introduction Molecular genetics provides explanations for the various kinds of inheritance we have observed, including, for example, how dominance, pleiotropy and epistasis work. Understanding some basic molecular genetic concepts will also help you to appreciate and better grasp some of the very many applications that have resulted from the molecular revolution. From conservation and evolution, to colour and medical genetics, molecular genetics is making a big impact. The scientists involved with the horse genome project described at the end of this lesson were, and are, mainly interested in the diseases and disorders of horses. They realised that they could better and more speedily address their current and future research if more information were available on the horse genome. As a result many genetic tests now exist to help breeders, and many more are likely to become available over the next few years. In addition researchers are making, and will continue to make, progress in various areas of horse medicine. We touch on this in this lesson but explore it in more depth in following lessons. First though, and in order to understand the exciting developments of late, we must have a basic knowledge of the structures and functions of DNA, genes, proteins and the genome at the molecular level. Note: Before reading this lesson please be somewhere where you can access the internet, if at all possible. In many parts of the lesson I refer you to short animated video clips from YouTube, which I’d like you to view before moving on. Although they don’t contain any essential new information these animations may help you to a better and more visual understanding of the concepts presented here. 1 Night Owl Education and Equestrian The structure of DNA We’ve already learned that genetic material is made up of deoxyribonucleic acid (DNA). In contrast to other cell constituents DNA is not metabolised. It remains stable and intact as a large macromolecule. The basic building blocks of DNA are called nucleotides. A nucleotide contains one of four possible organic bases, one deoxyribose sugar unit and one phosphate group. The four bases in DNA are adenine (A) and guanine (G) (the purine bases) and thymine (T) and cytosine (C) (the pyrimidine bases). Nucleotides are linked together into long chains called polynucleotides by a process called polymerisation. The process is in a specific chemical orientation so that the chains which are formed have a direction, and can only be added to at one end. In 1953 James Watson and Francis Crick were the first to work out the 3D structure of DNA, for which they were awarded a Nobel Prize for Medicine. They worked out how the polynucleotides were organised within the DNA molecule, taking into account the findings of Erwin Chargaff (1949). Chargaff’s had found that: 1. The number of purine bases (A + G) = the number of pyrimidine bases (T + C). 2. The number of cytosine bases = the number of guanine bases (i.e. ratio of G:C = 1:1). 3. The number of adenine bases = the number of thymine bases (i.e. ratio of A:T = 1:1). From this Watson and Crick’s realised that adenine was always paired with thymine, and guanine was always paired with cytosine. This can only be achieved if DNA consists of two strands held together by specific base pairing. Here is a summary of the key features of the structure of DNA worked out by Watson and Crick: 1. The DNA molecule is a double helix, made up of two interlocked polynucleotide chains coiled around the same axis. They can only be separated by untwisting - not simply pulling apart sideways. 2. The sugar-phosphate groups are on the outside “backbone” of DNA, while the bases are on the inside. The molecule can be thought of as a ladder in which the base pairs are the rungs and the sugar-phosphate backbones represent the two sides. The ladder 2 Night Owl Education and Equestrian is then twisted into a double helix. 3. The chains are held together by hydrogen bonding between specific pairs of bases, according to Chargaff’s rules. Thymine pairs with adenine (hence the 1:1 ratio of A:T). Guanine pairs with cytosine (hence the 1:1 ratio of G:C). 4.The specific base pairing means that the sequence of nucleotides in one strand determines the sequence in the other strand. The two strands are said to be complementary. 5. The bases in the two strands will only fit together if the sugar molecules to which they are attached point in opposite directions. The strands are said to be anti-parallel. As homework I’d like you to watch a couple of short clips (about a minute each) on DNA structure on YouTube, if it’s at all possible for you. Please do so before going any further in the lesson. http://uk.youtube.com/watch?v=l-hrLs03KjY&feature=related http://uk.youtube.com/watch? v=qy8dk5iS1f0&feature=PlayList&p=6667B09F73CF7FE4&playnext=1&index=23 DNA molecules are extremely long and can be composed of hundreds of millions of nucleotides. Each chromatid of a horse chromosome contains one continuous molecule of DNA double helix running throughout its length. The arrangement of bases in the Watson and Crick model of DNA structure has two important features: 1. Nucleotides can occur along one of the polynucleotide strands in any order. The sequence of bases is important for storing and encoding the genetic information. 2. Complementary base pairing means that for any given sequence of bases in one of the strands, the sequence in the other one is determined. This gives a mechanism by which DNA can self-replicate to make more identical copies of itself. The two strands unwind from one another and each one then serves as a template for the synthesis of a new complementary strand. An enzyme called DNA-polymerase “zips up” the bases during the synthesis of new polynucleotide chains. This enzyme can be used to direct the synthesis of new molecules of DNA in a test-tube. 3 Night Owl Education and Equestrian As homework I’d like you to watch a couple of short clips (about a minute each) on DNA replication on YouTube, if it’s at all possible for you. Please do so before going any further. http://uk.youtube.com/watch?v=AGUuX4PGlCc&feature=related http://uk.youtube.com/watch?v=4jtmOZaIvS0&feature=related DNA and chromosome structure As we’ve seen a horses cell nucleus contains 32 pairs of chromosomes. Each chromosome is made up of a complex of protein and DNA, known as nucleoprotein or chromatin. The DNA carries the genetic information while the proteins are mainly concerned with chromosome “packaging“. The DNA is “packaged” and not present in a fully extended form in the chromosome. Various “levels” of packaging ensure the chromosomes stay intact and don’t become tangled. This organisation allows the DNA to be replicated when necessary, to be partially unravelled for gene activity and to be tightly packed and shortened when the chromosomes are moved during cell division. As homework I’d like you to watch a short clip on chromosome structure on YouTube, if it’s at all possible for you. Please do so before going any further. The clip shows how DNA is wrapped around proteins (called histones) and then further coiled and wrapped to make up chromosomes. http://uk.youtube.com/watch?v=AF2wwMReTf8&NR=1 It is also interesting to have a quick look at these beautiful horse chromosome photos, which show chromosomes with various levels of packing. The photo top right shows all the chromosomes in a cell, in tissue that has been especially prepared for the purpose. http://www.nzetc.org/etexts/Bio12Tuat02/Bio12Tuat02_095a(h280).jpg 4 Night Owl Education and Equestrian Remember • DNA is a long molecule made up of two polynucleotide chains twisted together into a double helix. • Polynucleotides contain one of four kinds of organic bases - adenine, thymine, cytosine and guanine - linked together in a long chain by their attachment to sugarphosphate backbones. • Hydrogen bonding between specific pairs of bases holds the polynucleotides together and also provides the mechanism for their self-replication. • The sequence of bases along a polynucleotide encodes the genetic information. • Horse chromosomes are composed of nucleoprotein fibres that are a complex of DNA and protein. • The organisation of horse chromosomes is complicated but in essence each chromatid consists of one molecule of double helix running through its length. Genes and proteins Proteins are organic compounds composed of amino acids linked together in long chains called polypeptides. Enzymes are proteins that do jobs. Twenty different amino acid subunits are commonly found in proteins. Each polypeptide has a specific sequence, which is essential to its structure and function. This sequence gives the protein molecule its primary structure. There is virtually an infinite number of ways in which 20 amino acids can be assembled to make up a polypeptide, giving a potentially limitless number of possible proteins. Amino acids in the chain interact in various ways to give a secondary structure, while disulphide bonds between sulphur-containing amino acids stabilise the molecule and give a three-dimensional or tertiary structure. Many proteins contain more than one polypeptide which interact to give a quaternary structure. The correct organisation of a protein depends on the sequence in the primary chain. In enzymes it is therefore also essential to its function. A change in amino acid sequence can lead to a loss, reduction or change of function of the protein. 5 Night Owl Education and Equestrian As early as 1909 the English physician Archibald Garrod suggested that genes work through their control over the production of enzymes. Then in 1941 Beadle and Tatum showed that a mutation in a single gene resulted in a change in the activity of a single enzyme. They realised that all biochemical processes are under genetic control. These processes progress through a series of steps, with each step being controlled by a single enzyme, in turn coded for by a single gene. They supposed that genes might act by determining the structure of enzymes. With some minor exceptions Beadle and Tatum’s ideas have been shown to be essentially correct. Enzymes are only one sort of protein. There are others that also play important roles in structure and metabolism, including structural proteins, antibody proteins of the immune system, some hormones (e.g. insulin) and tubulin, which is concerned with moving chromosomes move during cell division. The structure of all of these proteins, and not just the enzymes, is encoded in the genes of the DNA: each gene encodes a polypeptide that either makes up a protein, or combines with other polypeptides to make a protein. The genetic code Once it was realised that genes specify the amino acid sequence of polypeptide chains it became obvious that their information must be carried in the sequence of bases in the DNA. The way in which the genetic information is encoded in DNA is referred to as the genetic code. It’s now known that triplets of bases code each amino acid. The triplets are known as codons, and the code of DNA has been completely known for some time. In horses the DNA is in the nucleus, while the assembly of amino acids into proteins occurs outside the nucleus, in the cytoplasm. Information is transferred from the site from the nucleus by another kind of nucleic acid known as ribonucleic acid (RNA). RNA molecules are actively engaged in the manufacture of proteins. Like DNA, RNA is 6 Night Owl Education and Equestrian composed of nucleotides polymerised into polynucleotide chains, although there are some slight differences in the compositions of RNA and DNA. RNA is a single-stranded molecule, folded into various forms containing some double-stranded regions. Three different types of RNA molecules play key roles in the biosynthesis of proteins. Messenger RNA (mRNA) carries the genetic message from the DNA to the site of protein synthesis in the cytoplasm. The DNA double helix unwinds in the region of the gene being expressed. A strand of mRNA is made that is complementary to one of the DNA strands, known as the template, in a process known as transcription (copying). An enzyme called RNA polymerase catalyses transcription. The mRNA polynucleotide is unzipped from the DNA template as it’s made. The completed mRNA molecules are then transported to the site of protein synthesis, which occurs on structures called ribosomes. These are made up of protein and ribosomal RNA and are vital to proper polypeptide synthesis. Ribosomes can be thought of as polypeptide “factories”. Transfer RNA (tRNA) pick up amino acids and carry them to the ribosomes so that they can be joined together into polypeptides. There are at least 20 different tRNA molecules, one for each amino acid. One end of each tRNA contains a triplet of exposed nucleotides, known as the anticodon, which is complementary to one (or more) of the codons carried in mRNA. The other end has a site for attachment to a specific amino acid. Each tRNA picks up a particular amino acid and matches its anticodon with the complementary codon in mRNA. This ensures the amino acids can be assembled in the correct sequence. The amino acids are then linked to form polypeptides. There are special stop signals that end polypeptide synthesis. The tRNAs that pair with their codons do not carry an amino acid. The decoding of mRNA into a polypeptide chain is called translation. Several ribosomes may attach to a mRNA molecule, one behind another, so that several polypeptides are made from each mRNA molecule. Once synthesised polypeptides dissociate from the ribosome and are released into the cytoplasm where they may undergo posttranslational modification to form a functional protein. As homework I’d like you to watch a short clip (about 4 minutes) on DNA transcription and translation on YouTube, if it’s at all possible for you. Please do so before going any further. 7 Night Owl Education and Equestrian http://uk.youtube.com/watch?v=41_Ne5mS2ls&NR=1 What is a gene? So far we have thought of a gene as being a unit of heredity. Now that we know about the structure of DNA we can add that genes are sequences of nucleotide pairs along a DNA molecule which code for polypeptide products. However transfer and ribosomal RNA molecules are also coded for by genes, made directly by transcription from the DNA, in the same way as mRNA. So, in molecular terms, a gene is a sequence of nucleotide pairs along a DNA molecule which codes for polypeptide or RNA products. Redundant DNA Most horse genes have far more DNA in them than is actually needed to code for the amino acids in their polypeptide products. Within the coding DNA, known as exons, are stretches of non-coding DNA, called introns. Many genes are composed mostly of introns. When transcription occurs all of the DNA bases are copied into the mRNA transcript. An enzyme then snips out the introns to form the mature mRNA. In addition there are large regions of the chromosomes that don’t contain any genes at all. Some of these are composed of repetitive DNA where short base sequences are repeated millions of times. This repetitive DNA has no known function. Some of it is transcribed, but we have no idea why. The function of redundant DNA is not known, but quite possibly it doesn‘t have one. It may be a product of genome evolution that is of no benefit to the horse, but exists for its own sake. The Horse Genome The total DNA content of a cell is called its genome. The nuclear genome is the total DNA 8 Night Owl Education and Equestrian content of the haploid nucleus. Some cell organelles, such as mitochondria, have their own genomes. Genomes are species specific, compared to genotypes that are specific to individuals within a species. The Horse Genome Project was started in 1995. It is an international cooperative project involving over a hundred scientists in twenty countries. Initially the goal of the Horse Genome Project was to make a genetic map for the horse. The 32 pairs of chromosomes were characterised and genetic “landmarks” identified on each chromosome. In this way points of reference were established to relate the horse genome to the human genome sequence. Information from the human genome could then be used without the great expense of sequencing the horse genome. The map now includes the positions of both genes and non-coding “marker” sequences. It will also help in the identification of areas of the genome contributing to multigenic (or quantitative) traits, such as behavioural and performance traits and disease susceptibility. Information on genetic variability in horses, will help to address issues about the relatedness of different breeds and the evolution of horses. During the first ten years the scientists used “genomics” to address important health issues of horses, including the finding, isolation, characterisation and sequence determination of some important genes like that for equine severe combined immune disorder, which is discussed elsewhere in the course. Scientists believe that genome information will help them gain a better understanding of inherited medical problems, and also of disease organisms. They expect it will help them make progress in the prevention and diagnosis of diseases and disorders, and the development of vaccines, therapies and treatments. Sequencing of the horse genome began in February 2006, using techniques developed for, and information gained from, the human genome project. By July the entire genome had been chopped into 30,000,000 pieces and the DNA sequence of every piece was determined! Re-assembling the sequences into the correct order was not a trivial task and took until January 2007. The sequence was published on public databases for use by biomedical and veterinary http://genome.ucsc.edu/. 9 researchers. It is now available online at Night Owl Education and Equestrian The DNA sequence of the horse genome consists of about 2.7 billion DNA base pairs. The DNA used for sequencing was from a Thoroughbred mare at Cornell University College of Veterinary Medicine, US. Researchers are working to improve the accuracy and resolution of the horse genome sequence, and also to look at variation. Many scientists are now working on diverse aspects of horse inheritance, disease and medical biochemistry, based on the data provided from the horse genome project. A list of these is given at http://www.uky.edu/Ag/Horsemap/hgpprojects.html. A useful spin-off of the horse genome project was the discovery of the genetic basis for many simple genetic traits in horses, including coat colour and several hereditary diseases. Molecular tests have been developed and are now commercially available to horse breeders, a list and some details of these is given in another lesson. Genomics has shown that there are only around 20,000 genes in mammals, not the 100,000 or more that was once imagined, representing only about 2% of chromosomal DNA. The rest of the genome does not encode genes, as we‘ve discussed already. Remember • genes act by determining the structure of proteins, including enzymes • one gene is responsible for specifying the amino acids sequence of one polypeptide chain • DNA is encoded so that one triplet of bases carries the information specifying one amino acid • DNA is transcribed into a molecule of single-stranded messenger RNA • the introns of mRNA are removed • mRNA is translated into protein through the involvement of ribosomal and transfer RNA • ribosomal and transfer RNA is transcribed directly from DNA • a gene is a sequence of nucleotides of DNA that codes for an RNA or protein product • 10 there is a large amount of non-coding DNA, much of which may have no function Night Owl Education and Equestrian Summary DNA is a long molecule made up of two polynucleotide chains twisted together into a double helix. Polynucleotides contain one of four kinds of organic bases - adenine, thymine, cytosine and guanine - linked together in a long chain by their attachment to sugar-phosphate backbones. Hydrogen bonding between specific pairs of bases holds the polynucleotides together and also provides the mechanism for their self-replication. The sequence of bases along a polynucleotide encodes the genetic information. Horse chromosomes are composed of nucleoprotein fibres that are a complex of DNA and protein. The organisation of eukaryote chromosomes is complicated but in essence each chromatid consists of one molecule of double helix running through its length. Genes act by determining the structure of proteins, including enzymes. One gene is responsible for specifying the amino acids sequence of one polypeptide chain, a polypeptide being a protein or part of it. DNA is encoded so that one triplet of bases carries the information specifying one amino acid. DNA is transcribed into a molecule of singlestranded messenger RNA, with the introns being removed after transcription. The processed mRNA is translated into protein through the involvement of ribosomal and transfer RNA, both of which are themselves transcribed directly from DNA. From a molecular point of view a gene can be defined as a sequence of nucleotides of DNA that codes for an RNA or protein product. Besides genes chromosomes contain a large amount of non-coding redundant DNA, much of which may have no function. 11 Night Owl Education and Equestrian Lesson eight: Finding and characterising genes for a particular phenotype Introduction This chapter gives an insight into how molecular genetics research proceeds, including some techniques used for finding and characterising genes. The linkage of equine severe combined immunodeficiency (equine SCID) to molecular markers was discussed in the lesson on linkage (lesson five), and this example is expanded on here. Having found two genetic markers linked to the gene for equine SCID researchers were in a position to locate the approximate position of the gene in the genome. Establishing the genetic linkage of a gene of interest to markers whose approximate location is known (or can easily be found) is called linkage mapping. Mapping a gene is the first essential step before it can be isolated and characterized. Once the sequence of a gene and it’s mutant alleles are known it’s possible to develop diagnostic kits, in order to genotype potential carriers, for example. It also becomes possible to start to determine the structure, action and role of the gene product, and how it influences the phenotype. This is particularly important for genes of medical significance: understanding a disease or disorder is essential for developing preventative and management strategies, and ultimately working towards cures. Often cross species comparisons can be made, so that advances in one species can benefit research in another. The completion of the sequencing of the equine genome, and the sequencing of the genomes of various other species, has lead to an explosion of new research, and a speeding up of research which would have previously progressed more slowly. Physical mapping of the equine SCID gene Physically mapping a gene (or marker) is the method by which it is located at a particular chromosomal location. We’ll look at two common methods which have both 12 Night Owl Education and Equestrian been used to locate horse genes and genetic markers, including those used for equine SCID. These are the use of heterohybridoma panels and fluorescence in situ hybridisation (FISH). Mapping in one organism often makes use of knowledge gained from others, as we’ll see in the case of equine SCID. Similar conditions exist in humans and mice and these have been studied at the molecular level, although they involve different mutant genes in the different animals. In humans the condition called ADA-SCID - adenosine deaminase deficient severe combined immunodeficiency disease – is a very serious disorder. Until recently children suffering from this condition were kept in germ-free bubbles, and couldn’t lead a normal life. Such children often died from illnesses considered minor in other children. Sufferers of ADA-SCID lack the enzyme adenosine deaminase, causing toxic levels of deoxyadenosine (the ADA substrate) to build up. The toxin kills the T lymphocytes (white blood cells) of the immune system. The gene for ADA-SCID was one of first human disease genes to be cloned and characterised. Now that the molecular basis of ADA-SCID is understood, children with the condition are given weekly injections of PEG-ADA, to prevent the build up of toxin (PEG is polyethylene glycol, which stabilises the ADA and protects the enzyme from being broken down in the body). Initially it was thought that the ADA gene might also be affected in equine SCID foals. However biochemical tests showed that ADA levels in equine SCID foals are normal and it was concluded that a different gene must be affected in humans and horses. A similar condition in mice is caused by a deficiency of DNA-protein kinase (DNA-PK), due to a mutant DNA-PK gene. This knowledge prompted further biochemical tests which revealed that SCID affected foals are also DNA-PK deficient. This suggests that the gene for either DNA-PK, or for a cofactor of DNA-PK, is defective in horses. Researchers reasoned that if DNA-PK is linked to HTG4 & HTG8 (the two markers the SCID disease gene was shown to be linked to) then it’s likely that SCID in horses is also caused by a mutation in the DNA-PK gene. This was tested using somatic cell genetic techniques involving the use of heterohybridoma panels. 13 Night Owl Education and Equestrian Heterohybridoma panels Hybridomas are cells made by the fusion between tumour cells (myeloma cells, which are essentially immortal) and other somatic cells (usually white blood cells). Heterohybridomas are hybridomas where the fused cells are from different species. Such cell lines are perpetuated in culture and used for genetic mapping and other genetics and immunological research – they are sometimes called somatic cell hybrids, although this broader term includes other kinds of cell hybrids, e.g. between plant cells. Heterohybridoma panels are made up of a series of clones of somatic hybrid cells from which some of the chromosomes of one of the species have been lost. For equine studies somatic hybrid cells are made by fusing horse white blood cells (B or T lymphocytes) to rodent tumour cells, in the case of equine SCID mouse myeloma cells were used. First horse and mouse cell cultures are mixed together. The cells are fused either by incubating them with a particular virus (a sendai virus) which forms bridges between cells, or with the chemical polyethylene glycol (PEG), which induces the fusion of cell membranes. The cell mixture that results includes some unfused cells (parental cells). There are also some fusions between cells of the same type (i.e. between 2 horse cells or 2 mouse cells). These are called homokaryotic cells. There are a few heterokaryotic cells – fusions between horse and mouse cells (the word heterokaryotic refers to there being “different nuclei”). Heterokaryotic cells must be selected from the mixture. The selection process makes use of biochemical complementation. Each cell line is mutant for one enzyme required for growth on a particular growth medium. The horse and mouse cell lines are deficient for different enzymes essential for cell survival and growth. Once the cell mixture is transferred to the growth medium only the hybrid cells grow – the deficiency in the mouse cell line is complemented by the normal enzyme in the horse cell line, and viceversa. 14 Night Owl Education and Equestrian One commonly used system involves the use of a medium containing hypoxanthine aminopterin thymidine (HAT). The aminopterin in the medium blocks the normal synthesis of DNA, and therefore the cells can‘t divide and form colonies. An alternative “salvage” pathway for DNA synthesis can occur in cells able to use the hypoxanthine and thymidine in the HAT medium. For this working copies of two particular genes are required to provide the enzymes needed for the salvage pathway. Each type of cell has one enzyme that the other doesn’t have. Only heterokaryotic cells have both enzymes. As the hybrid cells divide by mitosis their nuclei are unstable (the cells have been forcibly combined and haven’t evolved to contain all those chromosomes of such diverse origin!). The horse chromosomes are lost at random resulting in cells with different numbers of different horse chromosomes (it‘s not known why the horse chromosomes are lost in this case, but not the mouse ones). Eventually each small group of heterokaryotic cells have one or a few different horse chromosomes (the chromosome containing the gene allowing the cell to grow on the selective medium is, of course, always maintained). The hybrid cells are transferred to a growth medium that helps to slow or stop further chromosome loss. A panel of cell lines are established, each with a different genotype with regard to which horse chromosomes are present and absent. A heterohybridoma panel is established such that each chromosome can be uniquely identified. A simplified example for an organism with 5 chromosomes illustrates the how heterohybridoma panel work. The clone panel consists of 4 cell lines A-D. There are 5 chromosomes (numbered), each of which can be uniquely identified by its presence or absence in clones A-D. Chromosome 1 appears in all cell lines, and is the one carrying the essential complementation gene. Chromosome 2 appears in only cell lines A and B, which isn’t true for any of the other chromosomes. Look at the table carefully and you will see that no two chromosomes are present in the same way in the clones. 15 Night Owl Education and Equestrian Chromosome 1 2 3 4 5 line Clone A + + - - + Clone B + + - + - Clone C + - + + - Clone D + - + - - number: Which chromosomes are present in any particular clone might be identified in various ways. One technique is called karyotyping - where the individual chromosomes are identified according to their particular features. The features include their relative sizes and structures, and the particular and unique light and dark bands that form when they are stained with certain chemicals (chromosome banding). With any particular stain each chromosome has a banding pattern specific to itself, by which it can be identified. Computers are sometimes used to help with the comparison and recognition of chromosomes. Another common technique for identifying which chromosomes are present is by using chromosome specific molecular marker probes. The sequence of the markers probes is known, and their chromosome location has been previously established. The probes have the ability to stick to DNA with which it has a complementary sequence (called hybridisation). The probe is visualised, either by attaching radioactive labels or dye molecules to it. Un-hybridised probe is washed away, if there’s any probe left sticking to a chromosome it indicates it is the probes particular chromosome. Central resources of clone panels have been established and maintained, to prevent researchers from having to repeat the process of clone panel formation every time they wish to look for a particular gene. Gene locations are tested by looking for the presence of molecular markers that have already been shown to be linked to the gene of interest. The process is the same for using marker probes, only the probes are made with the marker 16 Night Owl Education and Equestrian sequence. In the example in the table, if the marker probes always hybridised to DNA of clones B and C but never to that of A and D one would conclude that the markers, and the gene of interest, are located on chromosome 4. It isn’t always necessary to know exactly which chromosomes are which in order to get some useful information about the location of a gene. In the case of equine SCID the researchers didn’t know the identity of all the chromosomes in their clone panel. Nevertheless in a large panel of clones HTG4 and HTG8 were always with the DNAPK gene. This they tested using marker probes for HTG4 and HTG8, and a biochemical test for DNA-PK. This showed that DNA-PK was linked to the markers HTG4 and HTG8. Given the connection of DNA-PK and SCID in mice, and the lack of DNA-PK in equine SCID foals, this evidence of linkage makes DNA-PK a likely equine SCID candidate gene. In other words it looked as though the disease gene was DNA-PK. The linkage was confirmed, and chromosome location finally demonstrated, using a technique called fluorescence in situ hybridisation (FISH). (Actually two different research groups worked on the problem at once, one using heterohybridoma panels, the other using FISH.) Fluorescence in situ hybridisation (FISH) FISH is the direct visualization of the chromosome location of a gene or molecular marker using the hybridisation of probes with attached fluorescent molecules. Special treatments are used that make chromosomes easy to visualise. Appropriately treated cells are put on glass slides, stained and viewed using a microscope. The chromosomal DNA is denatured in situ (in place) by dipping the slide in alkali (the chromosomes essentially stay where they are but the strands of the DNA helices disassociate from one another). In this state the DNA will hybridise with complementary DNA probes that are added to the preparation. The site of the hybridisation is visualised by attaching fluorescent dye “labels” to the 17 Night Owl Education and Equestrian probes. Using different dye molecules the order and relative positions of various genes and markers can be established. The chromosomes themselves are identified using a chromosome banding technique, as discussed earlier for the clone panel method. In the equine SCID study the HTG4 and HTG8 markers were used as probes, leading to the discovery that the markers - and therefore the equine SCID gene - were on the short arm of chromosome 9. Comparative mapping Many genes are the same in different animals. Accurate predictions about horse genomes can often be made by comparison to the genomes of other species. This speeds up mapping and gene isolation. As an example the gene for hyperkalemic periodic paralysis disease in horses is the same as one in humans. In the case of equine SCID the human DNA-PK was used to make a FISH probe. This technique of using cross-species probes is called zoo-FISH. In this way DNA-PK was localised to the short arm of chromosome 9, band 12 (symbolised 9p12; p stands for petite, from the French for small). We’ve already seen that the equine SCID gene must also be in this location, more evidence that the equine SCID gene is the DNA-PK gene itself. This completes our story of how the equine SCID gene was mapped and a likely candidate gene identified. The mapping information was used in order to isolate and sequence the normal and disease alleles of the gene. Remember • Mapping is the first essential step before a gene can be isolated and characterized. • Linkage analysis can determine which molecular markers a gene is close to, the chromosome location of these markers can easily found using physical 18 Night Owl Education and Equestrian mapping. There are various methods of physical mapping, including the use of • heterohybridoma panels and fluorescence in situ hybridisation (FISH). Heterohybridoma panels are made up of a series of clones of somatic hybrid • cells from which some of the chromosomes of one of the species have been lost. In heterohybridoma panels each chromosome can be uniquely associated • with a particular clone of cells. FISH is the direct visualization of the chromosome location of a gene or • molecular marker using the hybridisation of probes with attached fluorescent molecules. • Accurate predictions about horse genomes can often be made by comparison to the genomes of other species, e.g. using zoo-FISH. Replicating DNA in a tube Whole genomic DNA can be extracted from blood, hair follicle cells, meat, bones, teeth, semen and urine. Researchers are usually interested in studying a particular bit of DNA. It might be that they want to sequence the DNA in a particular region mapped to be associated with a particular phenotype, such as a genetic disease like equine SCID, or a colour or pattern. Similarly genetic tests, discussed in the next lesson, assay only one or a few genes, sometimes even only part of a gene, which is a tiny fraction of the genome. In either case whole DNA is extracted first, and then a technique is used to preferentially replicate the bit of DNA which is of interest, so there are lots of copies of it. Most genetic tests available to horse breeders involve extracting whole DNA from hair follicle cells, which are at the base of hairs pulled from the mane or tail. Usually pulling 20-40 hairs is all that’s necessary to conduct one or several of these tests. The technique of preferential replication was invented by Kary Mullis, and is known as the polymerase chain reaction (PCR). PCR can be used to amplify even tiny amounts of DNA, so it can be used on fossil samples as well as fresh ones. The development of this 19 Night Owl Education and Equestrian technique was hugely important to molecular geneticists, and is now routinely used for most molecular genetics research and applications. PCR requires that short sequences of DNA are known on either side of the piece to be copied. Copies of these short sequences are made and are known as primers. The DNA is heated up to separate the two strands of the DNA helices (a process called denaturation). The primers bind to this denatured DNA and provide “starting places” for DNA replication. Often primers can be used across species: those developed to work with one species will also work with others. Other ingredients are required for successful DNA replication, including bases (raw materials) and an enzyme called Taq polymerase (the enzyme is from a heat tolerant microbe called Thermophilus aquaticus). PCR results in the exponential increase in the number of copies of DNA between the primers, as illustrated: Exponential increase in DNA: start 1 1 cycle 2 2 cycles 4 3 cycles 8 4 cycles 16 x starting amount of DNA ... 10 cycles 1024 DNA Sequencing DNA Sequencing is done to determine the order of nucleotides within it. It is the nucleotide sequence that makes up the genetic code. As we saw in the introductory molecular genetics lesson the nucleotide sequence of a gene determines the amino acid sequence of the protein produced from it. By knowing the sequence of a gene we can 20 Night Owl Education and Equestrian learn about the protein it produces and the biochemical processes with which this is involved. This can be made easier if the gene turns out to be similar to one that has already been characterised in other organisms. In the case of equine SCID sequencing revealed a five base pair deletion in part of the gene for a DNA-PK subunit. A DNA test is now available through Vetgen to identify carriers of equine SCID, and is discussed further in the next lesson on genetic testing. There are various ways of sequencing DNA, and the process is usually automated. Just one, relatively new technique, will be briefly described here. I chose to describe this technique as one genetic testing company told me they were looking at the technique as a possible basis for doing some of their molecular equine tests in the future. The technique is called pyrosequencing. Pyrosequencing is based on "sequencing by synthesis”, and involves synthesizing the complementary strand of DNA to be sequenced (Ronaghi et al, 1998). The template DNA is immobilized and its complementary strand is synthesised one base pair at a time. Solutions of the four possible nucleotides are added and removed sequentially (i.e. one at a time), along with the necessary enzymes for DNA synthesis and chemi-luminescent signal molecules. If the nucleotide complements the first unpaired base of the DNA the DNA is extended and light is given off. A camera records the flash of light. If it does not then nothing happens and apyrase degrades the free nucleotides in the mixture so that the next nucleotide can be tried. The light signals show which nucleotide bases have been added, and in what order, so determining the DNA sequence as it is synthesised. DNA microarrays and chips Microarrays are a way of performing lots of DNA or RNA assays all at once. Depending on the application testing can be against tens, hundreds, thousands, or even hundreds of thousands of sequences. An array contains these sequences fixed to a surface. Common surfaces include glass and silicon chips. When silicon chips are used 21 Night Owl Education and Equestrian the microarray might be referred to as a DNA chip. Each sequence is fixed in a particular known position on the array (either by spotting or printing technique): a tiny spot of DNA among many, all arranged in a grid pattern. There are lots of uses of such arrays. Studies of gene expression can determine which gene sequences are turned on and off, for example in normal individuals and in those with a particular disease or genetic disorder. In this case it might be the mRNA from the test samples that are tested against the sequences on the microarray. Another application is to look at the genotype of an individual at many genes at once. This can be extended to compare individuals within a population, for example to assess genetic diversity and inbreeding, or to compare populations, for example to determine evolutionary relationships. Whatever the application if sequences from the samples of interest hybridize with the immobilized sequences on the array fluorescent molecules bind too, producing fluorescent signals. All un-hybridized DNA and unbound fluorescent molecules are then washed away. The fluorescent signals are then analyzed by a computer to see which sequences hybridize. Remember • The PCR technique is used to preferentially replicate the bit of DNA of interest in extracted whole genomic DNA, so there are lots of copies of it. • DNA Sequencing is done to determine the order of nucleotides within it. It is the nucleotide sequence that makes up the genetic code. • There are various ways of sequencing DNA, and the process is usually automated. • Pyrosequencing is based on "sequencing by synthesis", and may be used for equine molecular tests in the near future. • Microarrays are a way of performing lots of DNA or RNA assays all at once. • There are lots of uses of micro arrays, including studying gene expression, genotyping an individual at many genes at once, and assessing genetic diversity, 22 Night Owl Education and Equestrian inbreeding and evolutionary relationships. Summary There are now many and varied, ingenious and eloquent molecular techniques for all manner of research and practical applications, the study of which could easily fill a course or so of their own. Nevertheless you now have some idea of some of the key techniques involved in finding and characterising genes for a particular phenotype, and can appreciate the importance of their application to veterinary medicine. In the next lesson we consider genetic testing in more depth, and give you some idea of the range of applications for such tests. References Bailey, E., Graves, KT, Cothran, E.G., Reid, R., Lear, T.L. and Ennis, R.B. 1995. Synteny mapping horse microsatellite markers using a heterohybridoma panel. Animal Genetics 26 (3), 177-180. Bailey, E., Reid, R., Skow, L.C., Mathiason, K., Lear, T.L. and McGuire, T.C. 1997. Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; Synteny and FISH mapping to ECA9. Animal Genetics 28 (4), 268273. Metalinos, D.L., Bowling, A.T. Rine, J. 1998. A missense mutation in the endotheline-B receptor gene is associated with Lethal White Foal Syndrome: an equine version of Hirschsprung Disease. Mammalian Genome 9, 426-431. Pitra, C., Curson, A., Nurnberg, P., Krawczak, M. and Brown, S. 1996. An assessment of inbreeding in Asian wild horse (Equus przewalskii Poliakov 1881) populations using DNA fingerprinting. Arch. Tierzucht, Dummertorf 39 (6), 589-596. Ronaghi, M., Uhlén, M. and Nyrén, P. 1998. A sequencing method based on real-time pyrophosphate". 9705713. 23 Science 281: 363. doi:10.1126/science.281.5375.363. PMID Night Owl Education and Equestrian Shin EK, Perryman L, Meek K. A kinase negative mutation of DNA-PKcs in equine SCID results in defective coding and signal joint formation. The Journal of Immunology 158 (8), 3565-3569. Weiler R, Leber R, Moore BB, VanDyk LF, Perryman LE, Meek K. Equine severe combined immunodeficiency: A defect in V(D)J recombination and DNA-dependent protein kinase activity. Proc Natl Acad Sci USA 92, 1148-2249. Vetgen. SCID. http://www.vetgen.com/equine-scid-service.html 24 Night Owl Education and Equestrian Lesson nine: Molecular genetics testing Introduction This lesson looks at various types of genetic testing and their application. Such tests are usually based on knowing the sequence of a gene and it’s mutant alleles, as discussed in the last lesson. Sometimes though the sequence is not yet known, when linkage analysis can be useful. To give you an idea of how genetic disorders can be quickly spread through horse populations we start off by considering the story of the spread of hyperkalemic periodic paralysis. When you have read this you will appreciate that genetic testing can be a very important tool in reducing (and hopefully eliminating) such disorders. New ways of genetic testing are constantly being developed and a few techniques are briefly discussed which are likely to be used for horse genetic tests in the future. Another aspect of genetic testing does not look at genes at all, but instead uses several non-coding molecular markers to make “fingerprints”. Such fingerprints are highly variable between individuals and have various uses. They are especially useful tools in the conservation of wild and rare horses. Impressive: a tale of triumph and tragedy Before going on to discuss genetic testing in more detail it is interesting to see how genetic disorders can be spread through a horse population. From there we can realise the importance a simple genetic test can play in reducing (and hopefully one day eliminating) the disorder from that population. The tale of Impressive is a good one for breeders to remember, especially if it helps them to take care when planning their own breeding programmes. On the 15th April 1968 (and on the first birthday of my brother!) Impressive was born. He was a chestnut Appendix American Quarter Horse colt foal, with royal thoroughbred 25 Night Owl Education and Equestrian breeding on both sides of his pedigree. He became one of the most famous and successful Quarter Horses in history. Impressive changed hands a number of times, his price rising at each new sale. He was a successful halter horse, and also raced for a while. In 1974, at age six, he became the first World Champion Open Aged halter stallion, with 48 halter points. Apparently his owner was offered $300,000 for him but refused the offer, saying that there "ain't nobody in this world got enough money to buy this horse."! Impressive was in demand as a sire, popular for his muscular and refined form, he turned out one champion after another, siring almost 30 World Champions! Even though at one time his stud fee was a staggering $25,000 he eventually fathered about 2,250 foals, including Noble Tradition, a four-times World Champion halter stallion and a highly successful sire in his own right. In 1992 13 of the top 15 halter horses were descendants of this amazing horse. In 1993 he was estimated to have in excess of 55,000 living descendants, including Quarter Horses, Paints and Appaloosas. He died on the 20th March 1995, at the grand age of thirty-seven. Although he died, he is not forgotten, and is now estimated to have over 100,000 living descendants! It seemed that Impressive would go down in history as one hell of a horse: Impressive by name, impressive by nature! And then tragedy struck when Impressive was linked to something far less happy: his genetic legacy included a mutation recently implicated in the rare muscular disorder known as known as hyperkalemic periodic paralysis (HYPP). The disorder is inherited as a dominant condition. As such it requires only one parent to have and pass on the gene and the disease. Breeding of an affected mare or stallion to a normal horse will result in a 50% chance of an affected foal. If two affected horses are bred together then there’s a 75% chance of the foal being affected. The big problem with HYPP is that sometimes heterozygous animals (with one copy of the mutant HYPP gene) appear to be asymptomatic, as was Impressive himself: in genetics language HYPP is not fully penetrant. It also has variable expressivity (more severe in some animals than others). In other horses the disorder only shows itself later in life 26 Night Owl Education and Equestrian (again, in genetics language, HYPP is a late onset disorder). This can make it difficult to know whether a horse has, and will therefore pass, on the mutant gene. These characters are expected for a widespread dominant harmful mutation: if the health of every carrier was severely affected before breeding then few would be used for breeding and the mutation would become very rare. It gradually became evident that many descendants of Impressive were inflicted with the painful, alarming and often fatal disease. To my knowledge the disorder has never been observed in horses of other lineages. As Impressive ascended to the top of the sire's list owners of his foals began to notice a strange muscular twitching that often left their horses temporarily unable to move. These episodes, which varied widely in degree and duration, were usually mis-diagnosed as tying-up syndrome or colic, but they are now known to be caused by hyperkalemic periodic paralysis. At the time Impressive's descendants continued to make history in the show ring and as breeding stock, some making their owners large amounts of money. It hadn’t been proved that the disorder was exclusive to his line, and no one wanted to be the first to publicly implicate Impressive as the source of HYPP. Many owners of affected horses considered HYPP an inconvenience, not a reason to refrain from showing and breeding their valuable horses. Most quarter horse owners were still unaware of HYPP. AQHA and the University of California-Davis Equine Research Laboratory collaborated in research to learn more about the disorder. They found that a mutation disrupts sodium ion channels in the muscles, causing uncontrolled sodium influxes that alter the voltage current of muscle cells. This in turn causes uncontrolled muscle twitching, stiffness and profound muscle weakness. Horses with HYPP can experience unpredictable attacks of paralysis which, in severe cases, can lead to collapse and sudden death. The disorder is inherited as a dominant condition, but must involve interactions with other genes and/or the environment since not all horses with the mutation show symptoms, or if they do then the symptoms may occur intermittently. Foals homozygous for the mutant gene have respiratory problems and may not survive. In horses where HYPP has been 27 Night Owl Education and Equestrian diagnosed a combination of regular exercise and a diet low in potassium can help to keep the disease under control. The mutant HYPP gene occurs due to a single nucleotide change in the wild type HYPP gene. The DNA test for HYPP detects the presence or absence of this specific mutation in the HYPP gene. During the past few years the pressure built to reveal the extent and nature of HYPP, and to eliminate HYPP by selective breeding. A genetic test was made available to horse breeders in 1992, and can identify affected horses with virtual certainty. The simple blood test is available at the University of California at Davis School of Veterinary Medicine, Department of Medicine, or through the AQHA. Breeders were pressurised to show that their Impressive bred horses were negative for HYPP, or to remove them from breeding. The lucky ones protected their investments by advertising their negative test results along with their stud services and sales. If you are considering buying a horse with Impressive bloodlines, whether for breeding or not, you would be wise to ensure that the horse is HYPP negative before committing to buy. At the AQHA 2004 convention a motion was passed to set January 1st 2007 as the date after which foals testing homozygous for HYPP would no longer be registerable, with mandatory testing for HYPP for the descendants of Impressive. The Appaloosa Association followed suit and have disallowed the registry of homozygous foals from January 1st 2008. The American Palomino registries have gone all the way and passed rulings against both homozygous and heterozygous HYPP horses, beginning January 1st 2007. Part and half-blood registries need to follow suit and pass rulings on both testing and registration. If enough pressure is brought to bear it might eventually be possible to eliminate this awful disease from horse populations, so that Impressive may be remembered for his impressive legacy rather than as the founder of a genetic disorder. Genetic Testing The list of molecular genetic tests now available to horse-breeders is increasing all the 28 Night Owl Education and Equestrian time. The methods that underlie the tests are generally similar, even when the testing is for very different traits. As it happens the method used for testing for hyperkalemic periodic paralysis disease (HYPP) is the same as that used to test for the “red factor” gene. The red factor test distinguishes the alleles of the extension gene, which is useful information for people wanting to breed black horses. Black horses may be of genotype EE or Ee. Breeding together heterozygous blacks may therefore produce chestnut foals. The test is for black horses whose genotype at the extension locus is ambiguous. Researchers at the Swedish University of Agricultural Sciences found that the alleles producing black and red pigment differed by a single nucleotide that resulted in a single amino acid substitution. For both tests the part of the gene coding for the horse muscle sodium channel or the extension locus is amplified from whole blood samples using the polymerase chain reaction, discussed in the last lesson. The next part of the test relies on the fact that in both cases mutation has affected a site in gene called a host restriction endonuclease enzyme cutting site. Host restriction endonuclease enzymes are sequence specific DNA cutters: they recognise a specific base sequence and chop the DNA at that point. They occur naturally, with a purpose of destroying the DNA of pathogenic organisms. Different enzymes recognise different specific sequences. These enzymes have been put to use by molecular geneticists for many tasks where DNA has to be precisely cut, including so that it can be joined to other DNA, to make DNA molecules "to order". Sometimes alleles of a particular gene differ in whether they possess sequences for particular host restriction endonuclease enzymes. This is the case for HYPP and the extension locus, and provides a useful test. Some alleles will be cut into pieces by a particular enzyme or enzymes. Some won’t. The cut up alleles are therefore in smaller pieces of DNA. These pieces can by separated and visualised by a process called electrophoresis. The DNA pieces travel through a rectangular gel, under the influence of 29 Night Owl Education and Equestrian an electric current. The small bits travel farther than the big bits, so that the DNA pieces are separated according to their size. In this way, and by the use of dyes or radio-active labels, it is possible to "see" which alleles are present in DNA samples. The PCR fragments are then cut using a host restriction enzyme which recognizes and cuts the specific base sequences in the DNA. In both cases the mutation affects the host restriction enzyme cutting site. The mutant and normal wild-type alleles therefore yield a different number of fragments on cutting. Each of the three possible genotypes can be distinguished from one another according to the number and size of fragments observed after electrophoresis. Allele specific PCR: genetic testing the modern way For many genes the differences between alleles do not happen to be at the site of a restriction cutting enzyme. I contacted several genetic testing companies and they confirmed that the majority of horse genetic tests are now based on some variation of an allele specific PCR test. One variant of allele specific PCR is used for testing alleles that differ in length, due to deleted or added DNA. It is used for testing for equine SCID, discussed in the last lesson. A company called VetGen do the test for potential SCID carriers among Arabian horses and part-breds. A single pair of PCR primers are used to amplify both the normal and mutant alleles, but as the two differ in size by five base pairs they can be readily distinguished by electrophoresis. More direct allele specific PCR is used for other genes. The diagnostic test now available to identify horses at risk of producing lethal white overo (LWO) foals, is an example of this. LWO has been shown to be due to a mutation in the endotheline-B receptor gene, with there being a single nucleotide difference between the wild type and mutant genes. PCR primers are used that will recognise and attach to the mutant sequence, but which will not initiate replication of the wild-type allele. DNA amplification therefore only occurs in 30 Night Owl Education and Equestrian samples containing the mutant gene, i.e. in those from carriers of LWO. Fluorescent molecules are used for DNA replication and are incorporated into the PCR products, if there are any. This allows immediate genotyping, with fluorescence indicating that the mutation is present. Discrimination of alleles in a single tube can also be achieved using two pairs of allele specific primers, one for each allele. This method of genotyping is used for distinguishing single nucleotide polymorphisms of medical importance in humans, and may in the future be used for horse tests too. Depending on the genotype of the sample, a mixed signal of fluorescence is observed for a heterozygote while a single signal is observed for a homozygote. The newer methods for allele specific genotyping are relatively simple and cheap, with the advantage that (usually) no post-PCR manipulation is required. Linkage testing Sometimes the specific mutation causing a genetic disorder is not known, as is the case for cerebellar abiotrophy (CA), a recessively inherited neurological condition found almost exclusively in Arabian horses. If the gene has nevertheless been mapped to a particular region then tests can be carried out for a group of nearby genetic molecular markers instead. In the case of CA markers that are usually inherited with the disorder are used as a diagnostic tool, to identify affected foals and potential carriers of the disease. Arab breeders can test their horses before breeding in order to avoid breeding two suspected carriers together. The Veterinary Genetics Laboratory at the University of California Davis, US, also performs a dun test using linkage analysis as the gene for dun hasn’t yet been identified. 31 Night Owl Education and Equestrian PyroSequencing, DNA microarrays and chips Pyrosequencing, based on "sequencing by synthesis”, is discussed in the last lesson. Some companies are now considering the use of pyrosequencing for horse genetic testing. If such tests do become available it is possible that new allele variants might be identified that were previously unknown, if they exist. This includes the occurrence, or otherwise, of further agouti alleles. Students should note though that it is most likely that only small parts of the gene in the region of the known allele variations will actually be sequenced. Microarrays and DNA chips could be used to look at the genotype of an individual at many genes at once, as discussed in the last lesson. At least one horse genetic testing facility is looking into this possibility. It is noted that microarrays might be used to test for polygenic traits, allowing for selection for quantitative traits, as discussed in the lesson on complex traits and polygenic inheritance. Remember • Genetic tests can be used to determine the genotype of horses for a particular phenotype of interest. This can include colours and patterns, as well as genetic disorders and potentially other traits too. • Sometimes we accidentally breed genetic disorders into our horses without realising, sometimes they occur anyway. Genetic tests can help in the elimination of genetic disorders and diseases from horses. • The methods that underlie the tests are generally similar, even when the testing is for very different traits. • Sometimes alleles of a particular gene differ in whether they possess sequences for particular restriction cutting enzymes. This provides a useful test since some alleles are cut into pieces, while others aren‘t. Electrophoresis can be used to “see” which alleles are present in a sample. • The majority of horse genetic tests are now based on some variation of an allele 32 Night Owl Education and Equestrian specific PCR test. • Variations of allele specific PCR can distinguish alleles either according to their sizes or some aspect of sequence difference. • When the specific gene causing a phenotype is not known linkage analysis might still be used to infer genotype. • PyroSequencing, DNA microarrays and DNA chips may well be used for future equine genetics testing. • Microarrays might be used to test for polygenic traits, allowing for selection for quantitative polygenic traits. Fingerprinting Fingerprinting is a technique invented by UK scientist Alec Jeffrey’s in 1984, and famous for its application to forensic science, e.g. in the OJ Simpson case. The technique has been applied for many other purposes, including for studying horses and their relatives. Equine applications of fingerprinting include: Paternity and maternity analysis for stud books, conservation research and • maintaining breed purity. • Forensic testing, including for proving identity when trying to detect race doping. • The conservation and management of wild, feral and ancient horse breeds, and other equine species such as zebras. In particular assessing genetic diversity and maintaining it through inbreeding avoidance. • Establishing evolutionary relationships between equine species, and between equine and other species. Fingerprinting involves the use of non-coding molecular markers, which are usually assumed to have a neutral effect on the phenotype. These are used to describe patterns of variation in marker DNA that are unique, or nearly unique, to individuals. Markers commonly used are called variable number tandem repeats (VNTRs). Their useful feature is that they consist of a short DNA sequence, with different alleles varying 33 Night Owl Education and Equestrian according to the number of repeat units. For example, if AT is the sequence then 3 possible various alleles might "look" as follows (only one strand of DNA is shown, AT is said to be a dinucleotide repeat): AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT Different alleles are therefore distinguished by their different sizes. The difference is determined according to how far they travel when subjected to electrophoresis. There may be many different possible alleles at any particular locus. Several loci will be looked at, depending on what the study is for. The characteristic "bar-code" arrangement of different alleles on an electrophoresis gel is what’s called the fingerprint. The probability of two randomly chosen individuals sharing the same fingerprint is low even if only a few polymorphic loci are used. The fingerprint may be transferred to a nylon or nitro-cellulose membrane (called Southern hybridisation or Southern blotting, after its inventor Edward Southern), and photographed for more permanent records. Software is sometimes used to analyse complex fingerprints, but other fingerprints may be easy enough to analyse by eye. The relatedness of horses is determined according to the degree of band sharing observed in the fingerprints. The markers used for fingerprinting are carefully chosen. Population data is used to determine the likelihood of band sharing in unrelated individuals, and the number of bands used for any particular fingerprint depends on the marker diversity within the population. Example of fingerprinting: conservation of the Przewalski horse Initial attempts at saving the unique Przewalski’s were hampered with problems. Captive breeding programs had the aim of re-introduction into their former habitat. In an early attempt to allow captive-bred horses to return to a wild state five horses were released in 34 Night Owl Education and Equestrian to a French reserve. Although they survived the first winter four later died of congenital defects associated with inbreeding, (inbreeding leads to an increase in the occurrence of deleterious recessive disorders, as will be discussed in detail in another lesson). The fifth horse died of a heart-attack after being struck by lightning. To say this was unfortunate seems somewhat of an understatement. While nothing can be done about lightning, conservationists can make a reasonable attempt to avoid inbreeding. By incorporating genetic fingerprint tests into their stud-books, they can help to determine the relatedness of individuals. By carefully choosing unrelated mates they can minimize inbreeding and help to maintain the genetic diversity of the population, which is important for future evolution. Also since unrelated individuals are more likely to produce genetically healthy offspring there is more chance of them surviving re-introduction, and producing offspring of their own. Thirteen micro-satellites are used. DNA is replicated using PCR, with primers isolated from the domestic horse E. caballus. The genotype at the markers is then assessed using electrophoresis. Mostly there are different alleles present at each locus in the different species, E. caballus and E. przewalskii: in only 4 loci is the predominant allele the same in both species. These micro-satellites can therefore also be used as species specific markers, for example check that no crossing occurs between the two species (which could spoil the conservation effort). Remember • Fingerprinting involves the use of non-coding molecular markers, which are usually assumed to have a neutral effect on the phenotype. These are used to describe patterns of variation in marker DNA that are unique, or nearly unique, to individuals. • Applications of fingerprinting include paternity and maternity analysis, forensic testing, conservation and management, assessing genetic diversity and establishing evolutionary relationships. 35 Night Owl Education and Equestrian • Determining the relatedness of individuals is important to avoid inbreeding and maintain genetic diversity in rare species. Congenital defects are often associated with inbreeding, as they are in the Przewalski’s horse. Summary Genetic tests can be used to determine the genotype of horses for a particular phenotype of interest. This includes colours and patterns, genetic disorders and potentially other traits in the future. Sometimes genetic disorders are accidentally spread through horses by our breeding practices. Genetic tests can help to eliminate genetic disorders that have spread in this way. The methods that underlie the tests are generally similar, even when the testing is for very different traits. The majority of horse genetic tests are now based on some variation of an allele specific PCR test, which can distinguish alleles either according to their sizes or some aspect of sequence difference. When the specific gene causing a phenotype is not known linkage analysis might still be used to infer genotype. PyroSequencing, DNA microarrays and DNA chips may well be used for future equine genetics testing. Microarrays might be used to test for polygenic traits, allowing for selection for quantitative polygenic traits. Fingerprinting involves the use of non-coding molecular markers, which are usually assumed to have a neutral effect on the phenotype. These are used to describe patterns of variation in marker DNA that are unique, or nearly unique, to individuals. Applications of fingerprinting include paternity and maternity analysis, forensic testing, conservation and management, assessing genetic diversity and establishing evolutionary relationships. Determining the relatedness of individuals is important to avoid inbreeding and maintain genetic diversity in rare species. Serious congenital defects are often associated with inbreeding, as they are in the Przewalski’s horse. 36 Night Owl Education and Equestrian References Bailey, E., Graves, KT, Cothran, E.G., Reid, R., Lear, T.L. and Ennis, R.B. 1995. Synteny mapping horse microsatellite markers using a heterohybridoma panel. Animal Genetics 26 (3), 177-180. Bailey, E., Reid, R., Skow, L.C., Mathiason, K., Lear, T.L. and McGuire, T.C. 1997. Linkage of the gene for equine combined immunodeficiency disease to microsatellite markers HTG8 and HTG4; Synteny and FISH mapping to ECA9. Animal Genetics 28 (4), 268273. Metalinos, D.L., Bowling, A.T. Rine, J. 1998. A missense mutation in the endotheline-B receptor gene is associated with Lethal White Foal Syndrome: an equine version of Hirschsprung Disease. Mammalian Genome 9, 426-431. Pitra, C., Curson, A., Nurnberg, P., Krawczak, M. and Brown, S. 1996. An assessment of inbreeding in Asian wild horse (Equus przewalskii Poliakov 1881) populations using DNA fingerprinting. Arch. Tierzucht, Dummertorf 39 (6), 589-596. Ronaghi, M., Uhlén, M. and Nyrén, P. 1998. A sequencing method based on real-time pyrophosphate". Science 281: 363. doi:10.1126/science.281.5375.363. PMID 9705713. Shin EK, Perryman L, Meek K. A kinase negative mutation of DNA-PKcs in equine SCID results in defective coding and signal joint formation. The Journal of Immunology 158 (8), 3565-3569. Weiler R, Leber R, Moore BB, VanDyk LF, Perryman LE, Meek K. Equine severe combined immunodeficiency: A defect in V(D)J recombination and DNA-dependent protein kinase activity. Proc Natl Acad Sci USA 92, 1148-2249. Vetgen. SCID. http://www.vetgen.com/equine-scid-service.html 37 Night Owl Education and Equestrian Module three assignment Q1. Which of the following statements about DNA structure is false? A. DNA consists of organic bases, deoxyribose sugar units and phosphate groups. B. The basic building blocks of DNA are called nucleotides. C. The four bases in DNA are adenine, guanine, thymine and cytosine. D. The bases make up the backbone of DNA, being on the outside of the molecule. E. Specific base pairing means that the sequence of nucleotides in one strand of DNA determines the sequence in the other strand. [1,1] Q2. Which describes the important features about the arrangement of bases in the Watson and Crick model of DNA structure ? A. It allows the storage of genetic information and it gives a mechanism by which DNA can self-replicate to make more identical copies of itself. B. The number of cytosine bases equals the number of thymine bases. C. The bases in the two strands will only fit together if the sugar molecules to which they are attached point in the same direction. D. The base sequence in one strand is identical to that in the complementary strand, indicating how self-replication occurs. E. The arrangement of the bases could not be used to store genetic information, which is in the sugar-phosphate backbone of the DNA. [1,2] page 1 of 13 Night Owl Education and Equestrian Q3. Which is the best molecular description of genes? A. All genes code for a single enzyme. B. All genes code for a single polypeptide. C. Genes are sequences of nucleotide pairs along a DNA molecule which code for polypeptide products. D. Genes are sequences of nucleotide pairs along a DNA molecule which code for either polypeptide or RNA products. E. Genes are units of heredity. [1,3] Q4. Which of the following statements about transcription is false? A. During transcription mRNA is made that is complementary to the coding strand of the gene. B. Transcription occurs outside of the cell nucleus. C. Transcription is catalysed by an enzyme called RNA polymerase. D. The mRNA polynucleotide is unzipped from the DNA template as it’s made. E. mRNA molecules are transported from the site of transcription to the site of protein synthesis. [1,4] Q5. What is translation? A. The formation of mRNA by copying of DNA. B. The copying of new strands of DNA using the complementary strand as a template. C. The decoding of transfer RNA into a polypeptide chain. D. The decoding of ribosomal RNA into a polypeptide chain E. The decoding of messenger RNA into a polypeptide chain. [1,5] page 2 of 13 Night Owl Education and Equestrian Q6. Which best describes linkage mapping? A. Linkage mapping is the method by which a gene or marker is located to a specific chromosomal location. B. Linkage mapping is the technique by which a gene of interest is established to be genetically linked to other known markers whose approximate chromosome location is already known. C. Linkage mapping involves sequencing genes linked to a gene of interest. D. Linkage mapping involves using heterohybridoma panels and zoo-FISH. E. Linkage mapping is a technique to preferentially replicate DNA of interest by linking it to a primer. [1,6] Q7. In a linkage study to determine the genetic linkage of a disorder to molecular markers a stallions offspring are genotyped for three markers, all known to be near possible candidate genes in other species. The stallion itself has the following genotype, for the dominantly inherited disorder (allele D): Dd M1m1 M2m2 M3m3. The dams all had the following genotype: dd m1m1 m2m2 m3m3. The offspring had the following genotypes, in roughly equal proportions: Dd M1m1 M2m2 M3m3 dd m1m1 M2m2 M3m3 Dd M1m1 m2m2 M3m3 dd m1m1 m2m2 M3m3 Dd M1m1 M2m2 m3m3 dd m1m1 M2m2 m3m3 Dd M1m1 m2m2 m3m3 dd m1m1 m2m2 m3m3 page 3 of 13 Night Owl Education and Equestrian Which marker allele is linked to disorder allele D? (Hint: first work out what has been inherited from the stallion in each genotype of foal. Then ask, for each marker in turn, “does this segregate independently of the disorder gene?”) A. m1 B. m2 C. m3 D. M1 E. M2 F. M3 Q8. Which best describes physical mapping? A. [7,13] [1,14] Physical mapping is the technique by which linkage relationships between genes and markers are established. B. Physical mapping involves sequencing a gene of interest. C. Physical mapping is the method by which a gene or marker is located to a particular chromosomal location. D. Physical mapping is the technique of using heterohybridoma panels. E. Physical mapping is a technique to preferentially replicate DNA of interest. Q9. Which of the following about SCID in Arabian horses is not true? A. A similar condition in mice is caused by a DNA-protein kinase deficiency. B. A biochemical test revealed that SCID affected foals are deficient in adenosine deaminase. C. The gene for equine-SCID is on horse chromosome 9. D. DNA sequencing revealed a five base pair deletion in part of the gene for a DNA-PK subunit. E. A test is now available through Vetgen to identify Arabian carriers of equine SCID. [1,15] page 4 of 13 Night Owl Education and Equestrian Q10. Which of the following best describes heterohybridoma panels? A. Heterohybridoma panels are made up of a series of clones of somatic hybrid cells from which some of the chromosomes of one of the species have been lost. B. Heterohybridoma panels are made by the fusion between tumour and other somatic cells of the same species. C. Heterohybridoma panels are made up of a series of homokaryotic cells. D. Heterohybridoma panels are made up of a series of homokaryotic and heterokaryotic cells. E. Heterohybridoma panels are made up of a series of clones of somatic hybrid cells from which all of the chromosomes of one of the species has been [1,16] lost. The table summarises part of a clone panel. Equine chromosomes 1-6 can be uniquely identified by their presence (+) or absence (-) in particular clones of the panel. Use the table to answer the questions 11-13. Chromosome 1 2 3 4 5 6 line Clone A + + - + - - Clone B + + - - + + Clone C - + + - + - Clone D - + + + - + number: Q11. On which equine chromosome do you think is the gene essential for cell survival on the growth medium? Briefly say why (a sentence should be enough). [1,17] page 5 of 13 Night Owl Education and Equestrian Q12. A biochemical assay revealed that clones B and C produced an enzyme associated with a particular genetic disease. The product was not produced in any of the other clones in the panel. On which chromosome do you think the enzyme for the gene is located? Briefly say why (a sentence should be enough). [1,18] Q13. Using the FISH technique markers known to be linked to a disease gene of interest were found to hybridise to horse chromosomes in clones A and D, but not in chromosomes in other clones. Which chromosome is the disease gene on? [1,19] Q14. Which statement about PCR is false? A. PCR is used to preferentially replicate DNA of interest, so there are lots of copies of it. B. PCR is now routinely used for many molecular genetics applications. C. PCR stands for the polymerase chain reaction. D. PCR results in a geometric increase in the number of copies of DNA between the primers E. PCR requires that short sequences of DNA are known on either side of the piece to be copied. [1,20] Q15. Which statement about DNA sequencing is false? A. DNA sequencing is done to determine the order of nucleotides within it. B. DNA sequencing revealed a five base pair deletion in part of a DNA-PK subunit gene responsible for equine SCID. C. Pyrosequencing is the only way of sequencing DNA in the laboratory. D. DNA sequencing might be used for equine genetic testing in the future. E. DNA sequencing can reveal the genetic code of a gene. [1,21] page 6 of 13 Night Owl Education and Equestrian [1,22] Q16. Which statement about DNA microarrays is false? A. Microarrays are a way of performing lots of DNA or RNA assays all at once. A. A DNA chip is a DNA microarray on a silicon chip. A. Microarrays can be used to compare gene expression in healthy and diseased patients. A. Microarrays can only be used to assay DNA. A. Microarrays can allow genotyping of individuals at many genes at once. Q17. What are the reasons that the dominant disorder HYPP could be spread so extensively through a horse population? A. It is not fully penetrant so that some heterozygotes appear to be asymptomatic. It shows variable expressivity between horses and may be late onset in some horses. Therefore heterozygous horses may be bred from without anyone realising that their foals may be affected, or carriers. A. All heterozygotes are asymptomatic, allowing the gene to be passed on to foals without anyone knowing. A. The disorder is always late onset, so that horses are bred before they are diagnosed with HYPP. A. All horses homozygous for HYPP are asymptomatic, allowing the gene to be passed on to foals without anyone knowing. A. Although HYPP is fully penetrant and early-onset it shows variable expressivity, so that heterozygous horses may be bred from without anyone realising they have HYPP. [1,23] page 7 of 13 Night Owl Education and Equestrian Q18. The allele for a recessively inherited genetic disorder has a mutation that destroys a host restriction endonuclease enzyme cutting site, as shown in the diagram. After PCR and cutting of the alleles from 3 horses an electrophoresis gel was made, as shown in the picture. The horses are labelled A-C. Which horse has the disorder? Which is a carrier of the disorder? Which horse is normal and not a carrier? enzyme cutting site Normal allele Disease allele Cutting site destroyed by mutation 3 DNA samples were loaded here on the electrophoresis gel an electric current moved DNA this way A B C [3,26] page 8 of 13 Night Owl Education and Equestrian Q19. Sarah has a patterned horse that is probably tobiano, but might also carry the overo gene (so called tovero). It’s difficult to be sure from the phenotype, and she isn’t sure about the exact breeding of her mare either. She wants to produce a nicely marked foal and has her eye on a particular overo stallion as a possible sire. She wants to know if she is at risk of producing a lethal white overo foal. Briefly say what you can tell her about the chances of a LWO foal, and advice you would give her, before using the stallion. [5,31] Q20. Andy has a field full of mares with which he’s run a favourite stallion. Unfortunately a young stallion got in with the mares afterwards. Andy isn’t certain if the youngster might’ve mated with one or two of the mares, who may not have gotten pregnant the first time. The following diagram shows a fingerprint from the two foals of uncertain sire, their dams and the two potential sires. For each foal say which stallion is the sire. [4,35] page 9 of 13 Night Owl Education and Equestrian dam 1 foal 1 dam2 foal 2 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — — stallion 2 — — — stallion 1 — — — — — — — — — — — page 10 of 13 Night Owl Education and Equestrian Q21. Conservationists are trying to decide the best potential mate for a particular mare. Their fingerprints at various markers are shown. Say which stallion you would choose, and briefly explain your decision. Do you think the mare is directly related to any of the stallions, or any of the stallions directly related to each other? If so how? mare stallion 1 — — — — — stallion 2 — — — — stallion 3 — — — — — — — — — — — — — — — — — — — — — — — — — — — — — [8,43] page 11 of 13 Night Owl Education and Equestrian Q22. Urine samples are often tested for prohibited substances among race horses (dope testing). When a horse is found "doping-positive" the result is sometimes contested ("the samples must have been mixed up", for example, "my horse was clean!"). Fingerprinting has been used to match horses to urine samples. The following shows a fingerprint from a race, U indicates the doped urine sample. Fiery Fred — Go-fast Glyn Racing Ronnie — — — — — — — — — — Tearaway Trevor U — — — — — — — — — — — — — — — — — — — — — — — — — — — — — Which horse was doped? Two of the horses are brothers, which two do you think and why? [5,48] page 12 of 13 Night Owl Education and Equestrian Q23. Which of the following statements about chromosomes is true? A. Chromosomal DNA molecules are really short and composed of only a few hundred nucleotides each. A. Each chromatid of a horse chromosome contains one continuous molecule of DNA double helix running throughout its length. A. Horse chromosomes are made up of nucleoprotein, which contains various types of protein but no DNA. A. Proteins carry genetic information, which is packaged into chromosomes by being wrapped up with DNA. A. If chromosomes were packaged then the DNA could never be replicated or unravelled for gene activity. [1,49] Q24. Which of the following statements about DNA is false? A. Most horse genes have far more DNA in them than is actually needed to code for the amino acids in their polypeptide products. A. Within genes in general there is coding DNA, known as exons, and stretches of non-coding DNA, called introns. Most horse genes don’t have any introns. A. Large regions of the chromosomes don’t contain any genes at all. A. Repetitive DNA, which may consist of a short base sequence repeated millions of times, has no known function. Some of it is transcribed, but we have no idea why. A. The function of redundant DNA is not known, but quite possibly it doesn‘t have one. It may be a product of genome evolution that is of no benefit to the horse, but exists for its own sake. [1,50] page 13 of 13