Canine Genetic Primer .
by Susan Thorpe-Vargas, M.S., PhD . The purpose of this article is to provide a mini-course
in genetics that will serve the reader well during upcoming articles
on open registries and the Canine Genome Project, and
why they are so important to the future of the breeds we love.
Both the American Kennel Club and United Kennel Club are moving in the direction of genetic identification and registry. . Pull a few hairs out of your dog's tail. At the bottom
of a few of those hairs you will find a tiny root. That root contains
about 40,000 cells. Within each of those cells is about
6 feet of genetic material called DNA--your dog's entire genome. The information encoded inside the nucleus of that cell
is a unique "blueprint" of what makes up your dog. This blueprint
is absolutely specific to your particular animal, and
thus identifies your dog unconditionally among all other living things,
animal and vegetable. . Have you ever had a litter sired from more than one
male? Have you ever been disappointed in the results of a mating
between your bitch and the "top" stud dog? Well, the technology
is available now to positively and inexpensively identify
the sire of all your puppies, but parentage identification
is just a small part of what is possible. . In the not-so-distant future we will be able to compare
the genetic similarity between two prospective breeding pairs.
Imagine being able to see how closely related two dogs are
before you breed them. It is possible that what you thought
was a tight line-breeding, when looking at just the respective
pedigrees, would actually be a greater outcross for a particular
trait. . What if you could screen your dog for all sorts of
genetic diseases, or double up on the probabilities of a trait's expression
such as herding instinct or scenting? Even though the technology
has not yet reached this stage, it is coming. With the
human, canine, porcine, mouse and other genome projects under way,
the breeding game has now progressed to the next level. . Are you mystified by the genetic code? Do you blanch
at words such as allele, dominant, intron & exon? Do you
think of microsatellites as small orbs circling the Earth?
The time is coming when such words will be part of the everyday
vernacular. The study of genetics has previously been the domain
of specialists, but it is rapidly becoming part of the responsible
dog breeder's repertoire. . The study of genetics is much like learning a foreign
language. It really isn't all that difficult to become conversational
once you master the rules and become comfortable with a new
vocabulary. . Genetics 101 . Each cell within the body is composed of cytoplasm,
a jellylike layer of material that surrounds the nucleus. Within
the nucleus are a number of threadlike chromosomes that are almost
entirely made up of two kinds of chemical substances, proteins
and nucleic acids. . Nucleic acids have at least two functions: to pass
on hereditary characteristics and to trigger the manufacturing of
specific proteins. The two classes of nucleic acids are the
deoxyribonucleic acids (DNA) and the ribonucleic acids (RNA). . DNA, the genetic building block, is made up of substances
called nucleotides, each of which consists of a phosphate,
a sugar known as deoxyribose and any one of four nitrogen-containing bases. These four nitrogenous bases are adenine (A),
thymine (T), cytosine (C) and guanine (G). Canine DNA is about
6 billion nucleotide pairs long. . In mammals, the DNA molecule appears as two complementary strands that are wrapped around each other like the
railings of a spiral ladder, known more formally as the double
helix of Crick and Watson. The strands (sides of the ladder) are
composed of alternating phosphate and sugar molecules. The
nitrogen bases, joining in pairs, serve as the rungs. . Each base is attached to a sugar molecule that is
linked by a hydrogen bond to a complementary base on the opposite
strand. These bases are complementary because only adenine
pairs up with thymine, and only cytosine pairs up with guanine;
thus the pairs are AT and CG. For example, if one were looking
along a strand of DNA, that is reading DNA linearly, looking
down the two strands, the first segment pair might look
like this: .
STRAND A 5' . A . T . T . C . C . G . T . T . A . 3'
STRAND B 3' . T . A . A . G . G . C . A . A . T . 5'
. When DNA is duplicated during cell division, the chain
of nucleotides is synthesized from the 5' (5 prime) end
to the 3' (3 prime) end. This terminology should be considered
as a way to spatially orient oneself along the DNA strand.
The 5' end is referred to as upstream and the 3' inch end is referred
to as downstream. The two strands are held together by weak
electrical bonds between the bases on each strand, thus forming
basepairs (bp). Each strand has its own polarity opposite of
the other. Thus if you turned the strands upside down, the picture
would not change. An easy way to visualize the opposite
polarity aspect of the two chains is to think of two identical
snakes intertwined around each other but facing opposite
directions (head to tail and tail to head). Thus each half of
the double helix can serve as a genetic template of its complementary
half. . Before a cell can express a particular gene, it must
first transcribe that specific part of the DNA into messenger ribonucleic
acid (mRNA). This is similar to the formation of a complementary strand of DNA during the division of the double helix,
except that RNA contains uracil (U) instead of thymine as
one of its four nucleotide bases. In the process of transcribing DNA
into mRNA, all the T bases are converted to U bases. These
bases--C,G, A, and U-- are the alphabet of the genetic code. A sequence
of AGATC in the coding strand of the DNA produces a sequence
UCUAG in the mRNA. . When a cell is expressing a particular gene, that
means it is producing either a specific protein or polypeptide
(a short sequence of amino acids). It is also able to do this by translating
a codon composed of three bases. For example, CUU stands for
the amino acid leucine. CUA, CUG, and CUC also "code"
for leucine, so there is some redundancy in the system. Notice
in this example that only the base is different (A vs. G vs.
C). The term degeneracy is used when a change in a base does not
affect the amino acid being added to the peptide. . Breaking Down the Gene . So much for DNA and RNA. What is a gene? A gene is
the basic unit of inheritance. Each one carries a set of directions
for producing either a protein or a polypeptide. If all
goes well, a complete set of genes--one half from each parent--is
inherited. If the two copies of each gene are exactly alike, the
progeny are homozygous at that locus. If the gene inherited from
one parent is different from the gene inherited from the other,
the progeny are heterozygous. Different forms of the same gene are
called alleles. . In the dog, the various genes are located among 78
different chromosomes. What we don't know is how many genes
exist, although a rough estimate has been made that there
may be about 100,000. We also don't know where on the various chromosomes specific genes are located. In fact, we
have just recently karyotyped the canine. This means that we
are able to differentiate between specific chromosomes. This will
be valuable information when we finally are able to map the canine chromosome. Such a genetic map will now only allow
us to determine the position of genes relative to each other,
but also will tell us their approximate distance apart
on the helix. . At the molecular level, a gene is that portion of
DNA that codes for a specific polypeptide. It also includes regions
preceding and following (known as the leader and trailer) as
well as noncoded regions within the gene called introns that
act like spacers between the coding sequences known as exons.
Between the genes are long stretches of noncoding areas, and
it is in these sections that Mother Nature has given us a gift
to help map the canine genome. . Interspersed along the entire length of the genome
are regions called microsatellites. These areas of DNA consist
of tandem repeats (identical or nearly so) of a short basic
repeating unit, such as TGTGTGTGTGTGTG...,ATTATTATTATTATT..., etc. They can be mono-, di-, tri- or tetranucleotide
blocks, and are referred to as short tandem repeat polymorphic
(STRP) markers. Considered in evolutionary terms, these regions tend to show a higher percentage of variations, so
even closely related individuals will exhibit differences. These
variations can be as simple as a change of one basepair, called
a point mutation, or as different as the deletion or addition
of basepairs. For example, these repeats usually appear in blocks
that vary from 10 to 30 units long. A puppy could inherit a
(TG)10 from its dam and a (TG)14 from its sire. If the pup carries
enough of these parental type alleles, it is possible to ascertain
parentage. However, further variations in markers would be necessary
to differentiate between siblings. (Even though these
regions are not considered genes in that they do not code for
polypeptides, different forms of these areas also are called alleles.) . Identifying Genetic Markers . It has been suggested that it will require about 1,000
microsatellites to saturate the canine genome, so that there will
be a marker about every 3 megabases (a megabase is 1 million basepairs).
This will ensure that once these markers have been identifies,
at least one of them will be associated with, and inherited
along with, a specific gene. Once a marker has become linked to
a particular gene that has been characterized for a specific trait
or disease, it then could be used as a diagnostic tool to screen
for a desired characteristic or to identify a carrier (or an affected
individual) of a genetically transmitted disease. This would be
extremely valuable information, as many inherited diseases are
of the late onset type. This usually means the disease does not
become evident until the dog is well past the age where it
might have been used for breeding. . The use of simple sequence repeats in identifying
canine polymorphic markers has been a fairly recent innovation. Prior
to this, a technique called restriction fragment length polymorphism (RFLP)
markers were used to construct gene maps. Using special enzymes
that recognize basepair sequences, it is possible to cut
DNA into various lengths. These segments can be separated by gel electrophoresis because DNA carries an overall negative electric molecular
charge. Under the influence of an electric field, the different
fragments migrate toward the positive charge at a speed that corresponds
to their molecular weight. Since the shorter fragments travel
faster than the longer pieces, it is possible by using this technique
to differentiate between segments that differ by as little as one nucleotide. . RFLP thus provides the basis for a technique called
DNA fingerprinting that also can establish a parent-progeny
relationship. The chief disadvantage of this procedure is that it
is extremely labor intensive (read expensive) and requires a great deal
of genetic material. Tandem repeat markers have an advantage
over RFLP because they can be assayed by polymerase chain reaction
(PCR) and have a higher polymorphic information content
(PIC). . PCR is a technique that increases a specific section
of DNA about 1 million times. Since it is an automated procedure,
the reaction can be repeated as many times as needed to
obtain ample DNA for that area being investigated. The DNA is then
separated using gel electrophoresis, and because the variations
in length correspond to those of the repeat sequence, it is
possible to recognize individual differences. The main drawback
of this procedure is that the primers used in PCR amplification for a dog are not always the same for other mammals,
so unique markers must be developed for every species. . The term PIC is a little more complex. If a marker
is to be useful, it must be unique. As the number of variations within
each marker increases, it becomes more and more individualized
and therefore has a higher polymorphic information content.
This is a little like saying my house is on First Street,
then adding that it is on the corner of First Street and A Avenue. If
next I say it is on the northwest corner, it is easier to locate. Then
if I add that it is a white house with green shutters, etc., you can
see that each little bit of information increases the ability to find my
house. It is these characteristics that make markers useful for parentage
verification and for the purposes of positive identification. . The Genetic Crystal Ball . Given the advances in identification, the United Kennel
Club, wanting to avoid the obvious pitfalls in the American
Kennel Club's pedigree honor system, has contracted with
Zoogen of Davis, Calif., to use its genetic identification services
for registration purposes. The authors have reported previously
in DOG WORLD that the Canadian government will not allow importation of dogs under the age of 10 months for
resale if the claim that the dogs are purebred is supported only
by AKC registration. There are estimated to have been several
thousand bogus AKC registrations. No one really knows the extent
of the fraudulent registrations. . The AKC, in its February meeting, agreed to cooperate
with the Institute for Genetic Disease Control in Animals
in assembling a health and information database. The AKC-GDC plan
proposes that sometime in the next three to five years, the
LGDC would be placed under the aegis of the AKC, and its
work would parallel the AKC's registration program. In
an effort to strengthen the registry, the AKC hopes to announce
a pilot program sometime this spring to incorporate DNA testing
in an effort to support and expand its registration facility
and discourage fraud. . VetGen, a company associated with the University of
Michigan and Michigan State University, currently offers identification and pedigree validation services, and also is able
to test for several genetically transmitted diseases. An example
of such a program already in place is the progressive retinal
atrophy (PRA) screening under the auspices of the Irish Setter Genetic Registry through Purdue and Cornell universities.
This is just the beginning. In the future we can anticipate that even
polygenic diseases such as hip dysplasia ultimately will be
avoided by standard DNA testing. Prevention of genetic disease, drug design, therapy protocols, identification and parentage verification
are just a few of the many beneficial options that soon
will be available to breeders and pet owners. . It is fortunate for us that the dog is the ideal animal
for this type of genetic analysis. No other species has such
variations in body type, traits and behaviors. Compare the appearance
of a Newfoundland to a Tibetan Terrier, or the scenting
abilities of a Bloodhound to a Samoyed. Contrast the phlegmatic behavior
of the St. Bernard to the scrappiness of the whole Terrier
Group. For this reason, test matings between different breeds
are proving very useful for mapping studies. Information gleaned
from this canine genome research can be applied to the
humane genome and other mammalian genome projects because many genes have been highly conserved throughout evolution. . Exciting advances in the field of genetics are opening
up opportunities for the breeder that until now have
only been dreamed of in our attempts to produce the perfect
dog. For those interested in pursuing this subject of genome projects,
whether animal or vegetable, much information is available
through various sites on the Internet. The keyword "genome"
will provide plenty of sites to visit. Additionally, the
Department of Energy puts out "Human Genome Program: Primer on Molecular Genetics." There also is the very readable
"Exons, Introns, and Talking Genes" by Christopher Wills,
a professor at the University of California at San Diego. . CREDITS . The authors would like to thank the following for their time and
valuable assistance: Wazyl Malyj, University of California at Davis; Dr. Jasper Rine,
University of California at Berkeley; Dr. Elaine Ostrander, Fred Hutchinson Cancer
Institute, Seattle; and from the University of Oregon at Eugene, friend and
teacher Dr. Jim Long, and teachers Dr. George Sprague and Dr. Tom Stevens. . . .