Horse Genetics Module Three Equine Molecular Genetics · Night Owl Education and Equestrian Module three: Equine Molecular Genetics Lesson seven - An introduction to molecular genetics

Horse Genetics

Module Three

Equine Molecular Genetics

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Night Owl Education and Equestrian

Module three: Equine Molecular Genetics

Lesson seven - An introduction to molecular geneticsIntroductionThe structure of DNADNA and chromosome structure Genes and proteinsThe genetic codeWhat is a gene?Redundant DNAThe Horse GenomeSummary

Lesson eight - Finding and characterising genes for a particular phenotypeIntroductionPhysical mapping of the equine SCID gene Heterohybridoma panelsFluorescence in situ hybridisation (FISH)Comparative mappingReplicating DNA in a tubeDNA microarrays and chipsSummaryReferences

Lesson nine - molecular genetics testingIntroductionImpressive: a tale of triumph and tragedyGenetic TestingAllele specific PCR: genetic testing the modern wayLinkage testingPyroSequencing DNA microarrays and chipsFingerprintingExample of fingerprinting: conservation of the Przewalski horseSummaryReferences

Module three assignment


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.

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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


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.

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http://uk.youtube.com/watch?v=qy8dk5iS1f0&feature=PlayList&p=6667B09F73CF7FE4&playnext=1&index=23

http://uk.youtube.com/watch?v=qy8dk5iS1f0&feature=PlayList&p=6667B09F73CF7FE4&playnext=1&index=23

http://uk.youtube.com/watch?v=l-hrLs03KjY&feature=related


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

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http://www.nzetc.org/etexts/Bio12Tuat02/Bio12Tuat02_095a%5C(h280%5C).jpg

http://uk.youtube.com/watch?v=AF2wwMReTf8&NR=1

http://uk.youtube.com/watch?v=4jtmOZaIvS0&feature=related

http://uk.youtube.com/watch?v=AGUuX4PGlCc&feature=related


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 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 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.

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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

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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 post-translational 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.

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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

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http://uk.youtube.com/watch?v=41_Ne5mS2ls&NR=1


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 researchers. It is now available online at

http://genome.ucsc.edu/.

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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

• there is a large amount of non-coding DNA, much of which may have no function

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http://www.uky.edu/Ag/Horsemap/hgpprojects.html


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 single-

stranded 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.

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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

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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.

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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 vice-

versa.

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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.

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Chromosome number:

line

1 2 3 4 5

Clone A + + - - +Clone B + + - + -Clone C + - + + -Clone D + - + - -

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

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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 DNA-PK 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


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


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


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 11 cycle 22 cycles 43 cycles 84 cycles 16...10 cycles 1024

x starting amountof DNA

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


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


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


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), 268-

273.

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

9705713.23


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

http://www.vetgen.com/equine-scid-service.html


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


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


(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


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


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


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


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


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


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


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


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


• 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


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), 268-

273.

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

http://www.vetgen.com/equine-scid-service.html


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


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


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


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 [7,13]

Q8. Which best describes physical mapping? [1,14]

A. 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


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

lost. [1,16]

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 number:

line

1 2 3 4 5 6

Clone A + + - + - -Clone B + + - - + +Clone C - + + - + -Clone D - + + + - +

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


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


Q16. Which statement about DNA microarrays is false? [1,22]

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


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?

Disease allele

Normal allele

enzyme cutting site

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

Disease allele

Normal allele

enzyme cutting site

Cutting site destroyed by mutation





A B C[3,26]

page 8 of 13


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


dam 1 foal 1 dam2 foal 2 stallion 1 stallion 2— — — — — — —— — — — — — — — ——

— —— —

— ——

—— — —

— —

— —

— —— —

— —— —

—— —

——

— ———

— —

— — — —

— — —

page 10 of 13


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


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


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

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Horse Genetics Module Three Equine Molecular Genetics · Night Owl Education and Equestrian Module three: Equine Molecular Genetics Lesson seven - An introduction to molecular genetics