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MOLECULAR STUDIES OF TWO GENES,
AGOUTI AND EXTENSION,
WHICH DETERMINE PIGMENT PRODUCTION AND PATTERNING
IN TtlE DOMESTIC DOG
by
J Michael Newton
Copyright © J Michael Newton 1998
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF CELL BIOLOGY AND ANATOMY
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
19 9 8
UMI Number: 9829349
Copyright 1998 byNewton, J Michael
All rights reserved.
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THE UNIVERSITY OF ARIZONA <&
GRADUATE COLLEGE
As members of the Final Examination Committee, we certify that we have
read the dissertation prepared by
entitled
Michael Newton
Molecular studies of two genes, agouti and extension,
which determine pigment production and patterning in
the donestic dog
and recommend that it be accepted as fulfilling the dissertation
requirement for the Degree of
Doctor of Philosophy
3> A/ fr
~3o^
1
Date
Kaymond Kunyan a
Paul St. John
Herman Gordon
Date
h..i L ^
/-)
jjzhf;
,—,
g
Daniel Brower
Va- h r
Date
Final approval and acceptance of this dissertation is contingent upon
the candidate's submission of the final copy of the dissertation to the
Graduate College.
I hereby certify that I have read this dissertation prepared under my
direction and recommend that it be accepted as fulfilling the dissertation
requirement.
Diss^tation Director Joseph T. Bagnara
Date
3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial
fulfillment of requirements for an advanced degree at The
University of Arizona and is deposited in the University
Library to be made available to borrowers under rules of the
Library.
Brief quotations from this dissertation are allowable
without special permission, provided that accurate
acknowledgement of source is made. Requests for permission
for extended quotation from or reproduction of this
manuscript in whole or in part may be granted by the
copyright holder.
4
ACKNOWLEDGEMENTS
First, I would like to thank the people who provided
sairples for this project: Ms. Cindy Rivera (Tucson, AZ) for
the Doberman tail and placenta tissues, Dr. Ian Jackson
(Edinbiirgh, UK) for the Newfoundland DNA sanples. Dr.
Emmanuel Mignot (Stanford University) for Doberman DNA
samples, and Dr. Danika Metallinos (UC Davis) for whole blood
sairples of every other breed tested in these studies.
Secondly, I would like to thank all the members of the
Barsh lab, past and present, who provided everything from
reagents and advice to moral support. My time in their
presence enriched my life both as a scientist and as a
person. The memorable experiences are far too numerous to
mention but will always be carried with me.
A very special mention belongs to my extended family.
They have provided me with a support base beyond all
reasonable consideration. No graduate student has ever had
the kind of encouragement and honest desire to provide
opportrinities as I have been given by everyone in my wife's
family. They have also served as excellent examples of how
hard work, dedication, and love for ones craft can bring
about great things. Thank you.
None of the work presented here would have been possible
without the generosity and support of Dr. Greg Barsh. In his
laboratory at Stanford University I not only learned the
basic techniques and logic of molecular biology but I also
learned a great deal about academic science. Even though I
had a rather unique position in Greg's lab, being a student
at the University of Arizona, I received every benefit of
being a Barsh Lab Member including superb training in
methodologies as well as scientific approach. I will take his
example with me for the rest of my career.
Finally, I want to acknowledge my advisor. Dr. Joseph T.
Bagnara. My years in his laboratory, prior to my stay at
Stanford, were some of the most enjoyable of rry life. Under
his direction I was given the opportunity to learn classic
embryology and endocrinology in addition to all the current
research projects ongoing in his lab at the time. His true
love for scientific knowledge simply for the knowledge itself
was a tremendous example to set for a new graduate student.
Especially when it meant my taking a project dear to his
heart out of his lab and continuing its endeavor at another
university. I will cherish his philosophy and adhere to it as
much as possible as I approach my own future research
projects. Thank you Dr. Bagnara.
To Tara.
Your sacrifices on my behalf were extraordinary
and your love and support made this possible.
I Love you.
6
TABLE OF CONTENTS
LIST OF FIGURES AND TABLES
7
LIST OF ABBREVIATIONS
8
ABSTRACT
10
INTRODUCTION
Development of pigment cells
Mammalian pigment synthesis
Canine pigmentation
12
12
14
24
CHAPTER 1: Cloning of the a-MSH receptor from the domestic
dog and identification of sequence polymorphisms which
correlate with changes in coat color
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
28
28
30
33
48
CHAPTER 2: Cloning of the Doberman agouti cDNA and
demonstration that the agouti gene plays a conserved
role in mammalian pigmentation patterning
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
53
53
54
59
71
CONCLUSIONS
Future directions
74
76
REFERENCES
79
7
LIST OF FIGURES AND TABLES
Figure 1 Melanin synthesis pathway
15
Figure 1.1 Doberman Mclr genomic DNA sequence
36
Figure 1.2 Doberroan Mclr protein sequence
37
Figure 1.3 Conservation of the canine Mclr
39
Figure 1.4 Expression of the Doberman Mclr in dorsal and
ventral skin
42
Figure 2.1 Conplementary DNA. and deduced amino acid
sequence of the Doberman agouti gene
60
Figure 2.2 Ventral-specific expression of the Doberman
agouti mRNA
63
Figure 2.3 Structure of the Doberman agouti locus and
exon 1 transgene
65
Figure 2.4 Expression of the exon 1 transgene in E13.5
embryos
70
Table 1.1 Amino acid substitutions identified in
various dog breeds
45
Table 1.2 Carboxy-terminal trixncation of the Mclr
correlates with recessive yellow coat color
47
8
LIST OF ABBREVIATIONS
a-MSH
alpha-melanocyte stimulating hormone
ACTH
adrenocorticotropic hormone
cAMP
cyclic adenosine monophosphate
CDNA
conplementary deoxyribonucleic acid
DHI
5, 6-dihydoxi'indole
DHICA
5,6-dihydoxyindole-2-carboxylic acid
DMA
deoxyribonucleic acid
DOPA
3,4-dihydroxyphenyl-alanine
GPCR
G-protein coupled receptor
Khp
kilobase pairs
LacZ
bacterial p-galactosidase
Mclr
a-MSH receptor
mRNA
messenger ribonucleic acid
ORF
open reading frame
PGR
polymerase chain reaction
POMC
pro-opiomelanocortin
RT
reverse transcription
TRP
Tyrosinase-Related Protein
UTR
untranslated region
Nucleotides
A
C
G
I
T
N
Adenosine
Cytosine
Guanine
Inosine
Thymidine
undet ermined
9
LIST OF ABBREVIATIONS CONTINUED
Amino Acids
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
Alanine
Cysteine
Aspartic acid
Glutamic acid
Phenylalanine
Glycine
Histidine
Isoleucine
Lysine
Leucine
Methionine
Asparagine
Proline
Glutamine
Arginine
Serine
Threonine
Valine
Tryptophan
Tyrosine
10
ABSTRACT
Most of our current knowledge about mammalian
pigmentation and pigment synthesis has come from the mouse.
Despite the wealth of information still to be gained from the
mouse system, more could be learned by extending mouse
genetics into other mammals. An attractive model for
pigmentation studies is the domestic dog. The wide variety of
pigment patterns in the many breeds provide a deep source for
molecular and genetic investigation. The studies presented
here include molecular characterization of two genes which
play important roles in the pigmentation and pigment
patterning of domestic dogs. These results are supported by
coitparison with similar results from the mouse and other
mammalian species.
The two genes studied here are the melanocyte receptor
for a-MSH, encoded by the extension locus, and the fiinctional
antagonist of this receptor, the agouti protein, encoded by
the agouti locus. Studies in the mouse system have
demonstrated that the a-MSH receptor is required for the
production of black pigment (eumelanin) and that the action
of the agouti protein is to cause a switch from the synthesis
of black pigment to the production of yellow pigment
(pheomelanin). Various alleles at the extension locus result
in varying amounts of black pigment synthesis by melanocytes
in the hair follicle. Molecular characterization of these
alleles has identified missense mutations which alter
11
receptor activity and correlate with changes in coat color.
In the studies presented here I have identified sequence
changes in the gene for the a-MSH receptor in domestic dogs.
These changes include four missense mutations which correlate
with the dominantly inherited coat color of certain breeds as
well as a truncation in the receptor protein which correlates
with the recessive yellow coat color.
The key regulator of mammalian coat color patterning is
the agouti locus. Regional and ternporal patterns of agouti
expression in the mouse correlate with the production of
yellow pigment. Here I present evidence that the domestic dog
also expresses agouti and that its expression correlates with
pheomelanogenesis. Furthermore, I provide evidence that gene
regulatory elements which control the ventral-specific
expression of the agouti gene are conserved between canines
and mice.
12
INTRODUCTION
Nature provides us with fascinating images in almost
every conceivable color. Some of the most vivid examples of
Nature's palette are seen in vertebrate pigmentation. The
most common of all vertebrate pigmentation patterns is one in
which the animal has a dark dorsal surface and a pale or
white ventrum. However, often superimposed on this simple
pattern are colorful and cortplex patterns such a spots or
stripes. Colors are generated by a variety of mechanisms. For
example, the iridescent blues of some birds, are generated by
light-scatter through elements in the feathers and thus they
have been called structural colors. Other colors in nature
can arise from siirple deposition of colored dietary conpoxinds
in the skin. In contrast to these mechanisms is the system by
which almost every vertebrate animal generates color, via
pigment synthesis within highly specialized cells. These
pigment cells are derivatives of a well studied population of
cells in the embryo, the neural crest.
Development of pigment cells
Cells of the neural crest migrate from atop the neural
tube out into the developing embryo. Taking cues from their
local environment, neural crest cells differentiate into a
diverse array of cell types such as sensory neurons, cells of
the adrenal medulla, cells in the outflow tract of the heart.
13
and pigment cells, among others (LeDouarin, 1982; BronnerFraser, 1995). The complex developmental history of the
pigment cell which includes delamination of the neural crest
from the neuroepithelivmi, determination of specific cell
fates, migration to specific locations within the embryo,
communication with the environment, and ultimately
differentiation in the skin, requires the activity of many
genes at specific times. This complexity is evidenced by the
greater than fifty independent loci in the mouse which
influence coat color (Silvers, 1979). Some of these genes
affect the pigment cell itself, such as the enzymes of
pigment synthesis. Other genes affect the development of
pigment cell progenitors or the activity of pigment cells in
the skin. Unfortunately, only a small number of these genes
have been cloned and characterized. Further studies of these
loci will tell us not only about pigment cells but also about
the neural crest itself and general mechanisms of
development, as many genes which affect pigment cells are
also required for normal development of other neural crest
derivatives.
Unlike lower vertebrates which utilize three varieties
of pigment cells (Bagnara and Hadley, 1973), mammals contain
a single pigment cell type, the melanocyte. Melanocytes
produce two pigments; the first is eumelanin, which is black
or brown, and the other is a yellow or red pigment called
pheomelanin. The coat color of all mammals depends on the
14
uptake of these two pigments by hair follicle keratinocytes
which are siobsequently incorporated into growing hairs
(Russell, 1949; Galbraith, 1964).
Mammalian pigment synthesis
The biochemistry of melanin synthesis is largely
understood (see Figure 1)(reviewed in Hearing and Tsukamoto,
1991; Hearing and King, 1993). The synthesis of both
eumelanin and pheomelanin begins with the amino acid tyrosine
and can proceed spontaneously at physiological pH. However,
the distinctive coat colors of mice and other mammals depends
on the combined action of at least three enzymes which are
related both structurally and evolutionarily. This family
includes the trifunctional enzyme Tyrosinase and the
Tyrosinase Related Proteins 1 and 2 (TRP-1, -2), encoded by
the albino, brown, and slaty loci, respectively. Tyrosinase
catalyzes the conversion of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) as well as the siabsequent oxidation of DOPA to
DOPAquinone. High concentrations of sulfhydryl groups foiind
in glutathione or cysteine can convert DOPAquinone to
cysteinylDOPA (Jara et al., 1988; Prota, 1980) which proceeds
through uncharacterized reactions to generate pheomelanin. In
the absence of such sulfhydryls, DOPAquinone rapidly cyclizes
to leucoDOPAchrome which converts spontaneously to
DOPAchrome. DOPAchrome is the pivotal intermediate in the
determination of melanin quality. In the absence of TRP-2,
15
Tyrosine
Tyrosinase
(albino)
DOPA
Tyrosinase
(albino)
DOPAquinone
glucachione or
cysteine
cysteinylDOPA
leucoDOPAchrome
DOPAchrome
1,4-Benzotjiiazinyl-alanine
DHICA
DHI
TRP-1
(brown)
Indole-5,6-quinone carboxylic acid
Tyrosinase
(albino)
PHEOMELANIN
Indole-5,6-quinone
EUMELANIN
Figure 1
Melanin synthesis pathway. This scheme outlines the
conversion of the amino acid tyrosine to the pigmented
polymers examelanin and pheomelanin as it occurs in mammalian
melanosomes (based on Hearing and Tsukamoto, 1991) .
16
DOPAchrome is converted to 5,6-dihydroxyindole (DHI) which
serves as the sxibstrate for the third activity of the
Tyrosinase enzyme, resulting in the production of Indole-5,6quinone. Ehamelanin formed by polymerization of this
intermediate results in slaty coloration. TRP-2 possesses
DOPAchrome tautomerase activity {Tsukamoto et al., 1992)
which produces 5,6-dihydroxyindole-2-carboxylic acid (DHICA).
Without further modification DHICA-melanins ara brownish and
relatively soliible. However, DHICA can also serve as the
substrate for the third enzyme in the Tyrosinase family, TRP1, which oxidizes DHICA, producing Indole-5,6-quinonecarboxylic acid (Jimenez-Cervantes et al., 1994).
Polymerization of this intermediate along with DHICA results
in black eumelanins.
Mammalian pigmentation patterns ultimately depend on the
relative distribution of pheomelanin. Thus knowledge of
intracellular conditions that promote the synthesis of
pheomelanin over eumelanin or visa versa is crucial to our
iinderstanding of mammalian pigmentation patterning. Insight
into the regulation of pigment production has come from the
mouse, where molecular, genetic, and biochemical studies have
identified two genes, extension and agouti, which have
profound effects on the synthesis of both pigments as well as
pigmentation patterning in mammals of multiple phylogenetic
orders.
11
Genetic studies in the mouse have revealed an
interesting interaction between the agouti and extension
loci. Namely, various alleles at either locus can overcome
the effect of some alleles at the other locus, i.e. they are
epistatic to one another (Silvers, 1979). Gain-of-function
alleles of the agouti locus on most backgrounds result in
yellow mice through the synthesis of pheomelanin instead of
eumelanin within melanocytes. Loss-of-function alleles at the
agouti locus in mice carrying normal extension alleles result
in black mice. The opposite is true for the extension locus.
Here, gain-of-function alleles result in black mice whereas
mice carrying loss-of-function alleles at the extension locus
are yellow. This last observation places the product of the
extension locus downstream of the agouti protein in a genetic
pathway because regardless of the agouti genotype, mice
lacking the extension gene product do not synthesize
eumelanin in hair follicle melanocytes but instead,
synthesize only pheomelanin. Skin transplantation experiments
between animals of various extension and agouti genotypes
demonstrated that the product of the extension locus acted
within melanocytes and that agouti acted in a non-melanocyte
autonomous fashion suggesting that it may act within the hair
follicle environment to regulate pigment synthesis (Lamoreux
and Mayer, 1975; Poole and Silvers, 1976). This
interpretation was both supported and extended by the cloning
and characterization of various alleles at both loci.
18
The extension locus (e) encodes the melanocyte receptor,
designated Mclr, for alpha-Melanocyte Stimulating Hormone (aMSH). The Mclr protein is one of a five-membered family
within the seven transmembrane, G-protein coupled receptor
superfamily (Robbins et al., 1993). Its ligand, a-MSH, is
cleaved from a large precursor protein, pro-opiomelanocortin
(POMC), and secreted by the pituitary gland (O'Donohue and
Dorsa, 1982). a-MSH stimulates the eumelanogenic activity of
mammalian melanocytes (Geshwind, 1966; Geshwind and Husby,
1972) and also effects the activity of all three pigment cell
types of arrphibians (Bagnara et al., 1979). Ligand binding by
this receptor elicits a rise in intracellular cyclicadenosine monophosphate (cAMP) through activation of adenylyl
cyclase. The cellular response to a-MSH can be mimicked by
cholera toxin, dibutyryl-cAMP or forskolin, a direct
activator of adenylyl cyclase, indicating that the Mclr is
coupled to Gs (Tamate and Takeuchi, 1983; Takeuchi et al.,
1989).
It is clear that a-MSH stimulates both tyrosinase
activity and eumelanin production at the expense of
pheomelanin but the mechanism by which this occurs is still
not fully understood. Aroca and colleagues (1993)
demonstrated that a-MSH stimulated transcription of
tyrosinase mRNA. and subsequent accumulation of tyrosinase
protein in cultured JB/MS melanoma cells whereas transcript
and protein levels of TRP-1 and -2 were unaffected by this
19
treatment. This suggests that etamelanin quantity is regulated
at the level of tyrosinase gene transcription and that the
other members of the tyrosinase family regulate melanin
quality. Under pheomelanogenic conditions, expression of TRP1 and -2 fall below detectable levels and tyrosinase activity
is also decreased, demonstrating that they are not required
for or may actually inhibit pheomelanin production (Kobayashi
et al., 1995). This is supported by biochemical studies of
Mclr mutations which alter the receptor's activity or
regulation. Mutations which create hyperactive or
constitutively active melanocortin receptors lead to more
dramatic increases in cAMP or higher baseline levels of cAMP,
respectively (Robbins et al., 1993). Mice or other mammals
carrying these hyperactive or constitutively active Mclr
alleles are black and do not synthesize pheomelanin. On the
other hand, mammals homozygous for truncated, and therefore
thought to be non-fixnctional, receptors synthesize
pheomelanin rather than eumelanin and are yellow or red.
While the Mclr controls the amount of eumelanin
synthesized and its absence leads to uniform yellow
coloration, the most important locus for mammalian
pigmentation patterning is that of the agouti gene. In fact,
unlike most loci in which mutations can alter pigmentation
(e.g. the dominant spotting series) , the agouti gene's only
known function is to determine the synthesis of pheomelanin
in precise patterns. At least 25 alleles of agouti have been
20
identified making it one of the richest sources for genetic
studies in the mouse (Siracusa, 1991). The murine allelic
series ranges from the top dominant allele, lethal yellow
(A^), which produces a completely yellow coat to the bottom
recessive allele, extreme non-agouti (a®), in which no
pheomelanin is produced at all. The phenotypic variation of
the various alleles lead to the speculation that the agouti
locus contained more than one gene (Silvers, 1979) . It has
since been determined that the agouti locus contains a single
gene which owes its phenotypic coitplexity to a variety of
molecular lesions which predominantly affect the expression
pattern of the intact protein-coding exons.
The human, mouse, and cow agouti proteins consist of
approximately 130 amino acid residues (Wilson et al., 1995;
Bultman et al., 1992; Miller et al., 1993; Oulmouden et al.,
1996) and contain a signal sequence for secretion, a central
basic region, and a carboxy-terminal cysteine-rich domain in
which 10 of 46 residues are cysteine with conserved spacing.
Genetic studies had already suggested that the fvinction of
the agouti protein was to cause a switch from the synthesis
of eumelanin to that of pheomelanin. Expression studies of
agouti mRNA demonstrated regional and temporal expression
differences which correlated with the synthesis of
pheomelanin providing insight into how agouti modulates
pigmentation patterns (Vrieling et al., 1994). RT-PCR
experiments identified two different classes of agouti mRNA
21
isoforms (Vrieling et al., 1994), one specific to expression
in ventral skin and the other expressed in both dorsal and
ventral skin, but only during the mid-portion of the hairgrowth cycle. In each of these classes there are three
protein-coding exons preceded by one or two alternative 5'
untranslated exons. Ventral-specific isoforms contain exon lA
and occasionally an alternatively spliced exon lA' (which lie
approximately 100 Kbp away from the protein-coding exons)
while hair cycle-specific isoforms carry exon IB or IC. The
two different mRNA species result from the use of alternative
transcription initiation sites within the agouti locus.
Genomic alterations which affect the activity of the two
different promoters have dramatic effects on mouse
pigmentation patterns. Molecular studies of four agouti
alleles and their effect on mice which carry them are
particularly illustrative of this point. These alleles.
White-bellied agouti (A"), Agouti (A), black-and-tan (a") , and
non-agouti (a) all code for the identical protein and differ
only in the fianctionality of the two different promoters. The
white-bellied agouti phenotype, considered to be the wildtype pattern, has a white or pale yellow ventrum and banded
dorsal hairs. The agouti mouse has only banded hairs on both
dorsal and ventral surfaces while the black-and-tan agouti
mouse has uniformly yellow hairs on the ventral surface and
solid black hairs dorsally. Finally, the non-agouti mouse is
black except for some yellow on the ears, mammaries, and
22
perinetun. Northern analysis of skin from each pigmentation
phenotype shows e3<pression of the exon lA-containing isoform
in the ventral skin of A" and a"^ mice throughout the hairgrowth cycle resulting in unhanded yellow hairs and
expression of IB and IC-containing isoforms in both dorsal
and ventral skin producing banded hairs in agouti and whitebellied agouti mice but not in black-and-tan variants
(Vrieling et al., 1994). RT-PCR of non-agouti skin failed to
detect any agouti mRNA (Bultman et al., 1994). Genomic
characterization of the four alleles has identified precise
mechanisms by which ventral-specific or hair cycle-specific
promoters have been disrupted (Bultman et al, 1994; Chen et
al., 1996; Sandulache et al., 1994).
The genetic interaction of agouti and extension suggest
that the two genes acted in opposition to determine pigment
color. Cloning of both genes lead to the hypothesis that the
agouti protein could directly inhibit the function of the
Mclr. The mechanism by which this occurs has been an area of
intense investigation. Lu and colleagues (1994) demonstrated
that partially purified recombinant mouse agouti protein
could reduce the percentage of radiolabeled ligand binding
and subsequent activation of adenylyl cyclase. The nature of
Mclr antagonism by the agouti protein was further
characterized by Blanchard et al. (1995). This group
demonstrated that agouti is a competitive antagonist of the
Mclr by the nature of its ability to competitively inhibit
23
the binding of radiolabeled a-MSH as well as cAMP
accxintulation following exposiire to a-MSH, desacetyl-MSH, pMSH, and ACTH.
Recent results have also suggested that agouti may have
a more coirplicated role than sirrple antagonism of a-MSH
binding. In fact, it has been shown that in the absence of
melanocortin agonist, the treatment of normal murine
melanocytes with purified recombinant agouti can inhibit
eumelanin production and the expression of the melanogenic
enzymes in a time- and dose-dependent manner (Sakai et al.,
1997). Furthermore, it has been shown that hioman agouti
protein could block the stimulatory effects of both forskolin
and dibutyryl cAMP on both tyrosinase activity and cell
proliferation in cultured hiiman melanocytes (Suzuki et al.,
1997). This is different than the results of Blanchard et al.
(1995) who found that forskolin activated adenylyl cyclase in
the presence of agouti. Furthermore, even though mice
homozygous for the recessive yellow extension allele (e/e)
are a pale yellow in color, extrafollicular melanocytes of
these mice still synthesize eumelanin (Lamoreux and Mayer,
1975). This suggests that the hair follicle environment
harbors an activity which promotes pheomelanin synthesis in
these receptor-deficient cells. The most obvious candidate
for this activity is the agouti protein which is normally
expressed exclusively by the dermal papilla cells within hair
follicles (Millar et al., 1995). The mechanism underlying
24
this aspect of the agouti protein is still unclear but it is
consistent with agouti having an effect beyond the inhibition
of a-MSH binding. This includes binding of the agouti protein
to a receptor on the melanocyte plasma membrane and eliciting
a reduced adenylyl cyclase activity through a G^-coupled
mechanism. Attempts to identify an "agouti receptor" have so
far been unproductive.
Canine pigmentation
Since their domestication nearly 15,000 years ago, dogs
have been an important part of many human societies.
Selective breeding practices have established a staggering
number of individual dog breeds. Many of these breeds were
established for specific tasks including the packing of
supplies by the Roman armies, herding livestock, or for
hunting a variety of game. The dog has proven particularly
amenable to selection for specific traits which has allowed
for the propagation of breeds carrying particular
characteristics such as higher intelligence, endurance,
strength, and loyalty. Concomitant with the selection for
particular characteristics, dog breeders also succeeded in
segregating a variety of alleles with coat color phenotypes.
Thus, like the mouse, the domestic dog is a rich source of
genetic variation for coat color loci. For many murine coat
color loci, a phenocopy can be found within at least one, and
25
often several, dog breeds. This begs the question of whether
the phenocopies are the result of homologous gene activity.
Like other mammals, the domestic dog is thought to carry
a series of alleles at both the extension and agouti loci
(Searle, 1968; Little, 1957; Willis, 1989). Unfortxanately,
the nomenclature for the various alleles at each locus is
very different from those of the mouse which can make the
understanding of scientific papers unnecessarily confusing.
For exairple, the top dominant allele in the extension series
proposed by Little (1957) and assumed by Willis (1989) does
not produce black dogs, but merely an increase in the
relative amount of black pigment in some areas, such as on
the face. Instead the dominantly inherited trait resulting in
all black dogs is assigned to the agouti series. While it is
certainly possible that domestic dogs do not have an allele
of extension which results in the all black phenotype, it is
more difficult to imagine a dominant agouti allele which
either stimulated the Mclr rather than antagonized it or
acted to displace functional agouti protein in a dominant
negative fashion. A more corrplete analysis of agouti alleles
from various breeds will be required to answer this question.
I will present data supporting the presence of three
extension alleles, a dominant black allele (E*), an allele
with behaviors similar to the wild-type extension allele of
the mouse (E), and a loss of function allele which correlates
with yellow coloration (e).
26
Other than the dominant black allele, the canine allelic
series for agouti proposed by Little is very similar to that
described for other mammals. They include a dominant yellow
allele, an allele which correlates with a pale ventrum and
banded dorsal hairs, and a recessive allele associated with
black-and-tan bicolor patterning. More coitplete understanding
of functional domains with any given protein can be obtained
through the molecular characterization of multiple alleles
within a species as well as allele cortparisons between
species. The phenotypic variation of coat colors in the
domestic dog make it an exciting new source of allelic
variation for several coat color loci, especially agouti and
extension.
In the studies presented here, I tested the hypothesis
that sequence variations with potential fionctional relevance
exist in the Mclr protein between dog breeds of various
colors and that domestic dogs express agouti in a pattern
which correlates with the synthesis of pheomelanin. I have
analyzed the Mclr gene from five pure-bred lines and
identified polymorphisms which correlate with the dominant
black and recessive yellow coat color phenotypes. I have also
cloned the Doberman agouti cDNA and demonstrated that the
agouti mRNA expression correlates with pheomelanogenesis in
Doberman skin. In addition I also identified a fragment of
Doberman genomic DNA which contains enhancer elements for the
regulated ventral-specific expression of the canine agouti
protein. Together these results provide evidence for the
contribution of both the Mclr and agouti proteins in the
production and patterning of pigmentation in the domestic
dog.
28
Chapter 1
Cloning of the a-MSH receptor from the domestic dog and
identification of sequence polymorphisms which correlate with
changes in coat color
Introduction
Many mammalian species contain a series of alleles at
the extension locus, including both gain-of-fimction and
loss-of-fionction alleles (Searle, 1968). The product of the
extension gene, the a-MSH receptor, stimulates an increase in
the synthesis of eumelanin upon ligand binding. Dominant
alleles correlate with increased activity and black
coloration while recessive alleles correlate with yellow or
red coloration. Sequence analyses of the Mclr from humans
(Valverde, et al., 1995), mice (Robbins, et al., 1993), foxes
(Vige, et al., 1997), horses (Marklund, et al., 1996), cattle
(Klungland, et al., 1995), and chickens (Takeuchi, et al.,
1996) have determined amino acid substitutions which
correlate with particular coat color phenotypes. In the cases
of mice and foxes, in vitro studies of cells expressing
receptor variants have gone on to demonstrate that gain-offxonction, dominant black alleles result in increased baseline
or higher half-maximal cAMP levels in response to ligand
binding (Robbins, et al., 1993; V5.ge, et al., 1997). These
activating mutations in the Mclr from all mammals so far
29
analyzed lie in a short section of the protein between
residues 65 and 100, a region which includes the second
transmembrane domain. The only exception to this grouping was
fovind in the fox, in which a cysteine to arginine
siibstitution at residue 125 is suspected to be the
functionally relevant mutation in the
allele of the Alaska
Silver Fox (V&ge, et al., 1997). Inactivating alleles usually
result from frameshift mutations which prematurely truncate
the receptor in a variety of locations across mammalian
species. One interesting exception to this is seen in the
horse, where it was found that a serine to phenylalanine
substitution in the second transmembrane domain rather than a
truncation correlated with the Chestnut phenotype (Marklund,
et al., 1996).
Like other mammals, the domestic dog carries an allelic
series which contains dominant alleles which increase the
amount of black pigment as well as recessive yellow alleles
similar in phenotype to those characterized in mice (Little,
1957; Willis, 1989; Searle, 1968). It is also proposed
(Little, 1957; Willis, 1989) that some breeds owe their black
color to a dominant allele in the agouti series, A^. In this
study I tested the hypothesis that sequence variations in the
Mclr protein from domestic dog breeds would correlate with
changes in coat color. Sequence analysis of the Mclr gene
from five breeds identified sequence polymorphisms. These
polymorphisms did, in fact, correlate with changes in coat
30
color. I have designated the wild-type allele as E, the
dominant black allele as E*, and the loss-of-function allele
as e.
Materials and Methods
Cloning of the Dobermem Mclr Doberman tails were
obtained during routine docking procedures in a
veterinarian's office. Total RNA was purified from the skin
of these sanples using the Acid Guanidinium Thiocyanide
procedure (Chomczynski and Sacchi, 1987). Total RNA was used
for gene-specific reverse transcription with the degenerate
primer A (5'C(G/T)IA{A/G)(C/T)TC(C/T)TG(G/A/T/C)C(A/T)IC(G/T) {A/G)AA-3')
according to manufacturer's guidelines (superscript, Gibco).
A portion of the Mclr cDNA was aiiplified from this pool using
the Polymerase Chain Reaction (PGR) and degenerate primers
designed from human and mouse sequences. In the first round
of aitplification primer A was used in combination with primer
B (5'-AT(T/C/A)GCIGTIGA(T/C)(C/A)GITA(C/T)AT-3') with
denaturing at 94° for 30 sec, annealing at 40° for 1 min, and
extending at 72° for 30 sec for 40 cycles. DNAs of the
expected size were gel purified using the GlassMax DNA
Isolation Kit (Gibco) and used as template in a hemi-nested
PGR using primers B and C (5'GAAGAAGGGCCA(G/A)CAIA(G/A)(G/A)AA(G/A)A-3') . The 402 bp
31
product which includes codons 138 to 272 from the Mclr open
reading frame was cloned into the T/A cloning vector pCR2.1
(InVitrogen) for sequence analysis.
The partial cDNA. was used as a probe to screen a
Doberman cosmid library. The cosmid library was generated by
partial Sau3A digestion of genomic DNA and ligation into the
SuperCos vector (Stratagene). Four independent cosmid clones
were isolated from -160,000 transformants. The Mclr gene was
then subcloned into pBluescript KS (Stratagene) for sequence
analysis. All sequencing was carried out using dye-terminator
chemistry and the ABI automated sequencing apparatus (Applied
Biosystems).
Northern analysis Thirty micrograms of total RNA from
dorsal and ventral Doberman tail skin was fractionated on a
formaldehyde/agarose gel and transferred to a nitrocellulose
filter (MagnaGraph, MSI). The immobilized RNA was then
hybridized to a ^"P-labeled Mclr partial cDNA (described
above) using standard procedures (Sambrook, Fritsch, and
Maniatis, 1989).
Mclr seczuence polymorphism detection
Genomic DNA
was purified from whole blood sartples or buccal swabs. A 1.3
Kbp anplicon was aitplified by PGR using the Advantage Tth
genomic DNA PGR kit (Promega) with the following conditions:
-200 ng DNA, 100 nmol each of primers D (5'GGTCATTGCTGAGCTGACAC-3') and E (5'-GAGATGCTGTCCAGTAGTCCC-3') ,
30 sec denaturing at 94°, 1 min annealing at 60°, and 2 min
32
extending at 12° for 40 cycles. Anplified Mclr products were
gel purified (GlassMax, Gibco) and directly sequenced with
the ABI system. The correct Mclr cDNA sequence for each breed
was determined from a consensus of multiple individuals from
each breed: 5 Dobermans, 4 Newfoundlands, 4 Black Labrador
Retrievers, 3 Yellow Labrador Retrievers, and 3 Golden
Retrievers. In all cases, sequence variation within a breed
was due to sequencing errors and not to polymorphisms,
whereas sequence differences between breeds are polymorphic.
Allele Specific Oligo Hybridization A 122 bp PGR
product covering nucleotides 1596 to 1718 from the Mclr
region was aitplified from genomic DNA with the following
conditions: 200 ng genomic DNA, 100 pmol each of primers F
{5'-GTGGAAGTCGATCATTGACC-3') and G {5'-GTGGGGAGCAGCCTGGGGTG3'), 30 sec denaturing at 94°, 45 sec annealing at 55°, and
30 sec extending at 72° for 40 cycles. Samples were
immobilized on nylon filters (Hybond-N+, Amersham) and
prepared for hybridization using a slot blot apparatus
according to Dyson (1991). Oligos specific for the truncated
allele {5'-AGGAGGTGTGAAAGACTC-3') or the non-truncated allele
(5'-AGGAGGTGCGAAAGAGTC-3') were phosphoirylated with '~P by
Polynucleotide Kinase and hybridized to the immobilized PGR
products.
33
Results
A portion of the Doberman Mclr cDNA was amplified bydegenerate RT-PCR (see Materials and Methods) and then used
as a probe for screening a Doberman cosmid library. Four
overlapping cosmids were isolated, from which the Mclr was
subcloned into plasmids for sequencing. The sequence of the
Doberman Mclr gene and the deduced amino acid sequence is
shown in Figures 1.1 and 1.2, respectively. In genomic DNA a
suboptimal TATA box is found 610 nucletides upstream of the
translational start site (position 123 in Figure 1.1) and the
polyadenylation signal is found 148 nucleotides 3' of the
stop codon (position 1835). If these are both utilized during
transcription and mRNA processing, the predicted mRNA would
be approximately 1800 nucleotides with the actual length
dependent on the length of the polyadenylate tail. The
Doberman Mclr, considered as the wild-type allele, is well
conserved with other Mclr genes so far identified (see Figure
1.3). At the amino acid level it is 96% identical to fox, 85%
identical to horse and cattle, approximately 75% identical to
hxjman and mouse, and 58% identical to chicken proteins.
Within the protein itself, potential glycosylation sites are
conserved among mammalian Mclr proteins, the amino-terminus
possesses the characteristics of a signal sequence for import
into the endoplasmic reticulum and the C-terminal
palmitoylation site found in other G-protein coupled receptor
34
(GPCR) family members is also present, all indicating that
the canine Mclr is oriented correctly with respect to the
plasma membrane.
The melanocyte receptor for a-MSH is a member of a
family within the GPCRs. In hiomans and mice, each of the five
family members has varying affinity for the ligand a-MSH as
well as specific tissue distributions (Tsigos et al., 1995);
only the Mclr is expressed in the melanocytes of the skin. To
verify that the cloned gene was indeed the melanocytespecific receptor I used the same probe as was used to
identify the cosmid clones in Northern analysis of Doberman
skin. As is shown in Figure 1.4, this probe identifies an
mRNA species extracted from both dorsal (black) and ventral
(yellow) skin. Although the RNA samples were partially
degraded and not equally loaded between lanes, the length of
the mRNA transcript appears similar to that of the 18S rRNA
subunit which has not been characterized in the dog. However,
in humans and mice this rRNA is approximately 1.9 Kbp. In
addition to sequence similarity to Mclr genes from other
species, the cloned gene is also expressed in the appropriate
tissue to function as the melanocyte receptor for a-MSH.
Mutations in the Mclr have been shown to have coat color
phenotypes in several mammals including humans, mice, horses,
and foxes. With the rich variety of coat colors and patterns
in the domestic dog, I wanted to determine whether some coat
35
Figrore 1.1
Doberman Mclr genomic DNA sequence.
Two overlapping fragments of Doberman genomic DNA were
subcloned from a cosmid library and partially sequenced to
identify the Mclr sequence. These two restriction fragments,
a 6.5 Kbp Hindlll and a 2.2 Kbp PstI fragment, overlap at the
3' end of the open reading frame (ORF); a PstI site lies 2 bp
3' of the translation-terminating TGA codon. The 951 bp ORF
is shown in capital letters with flanking DNA in lower case.
As with other members of the melanocortin receptor family,
the Doberman Mclr is found on a single exon. A sxiboptimal
TATA box (TATAT instead of TATAA) is underlined and the first
polyadenlylation signal following the TGA codon is double
underlined.
gaggtagggagttagggttcaagttgggatgagtgtcagcagccagtgac 50
tgtatttaggcaggtggcatcaggtgggtgttcagagatcagacagttcc 100
caaaaaaaaatcmtatttacaatatatttaaaaaaccccacataaacaaa 150
cantgggagcggagccaggtgagggggcacacatccagntgagccccagc 200
gtccaangtccttggagccaggatagccctgggtgccatttgtgctgcct 250
ggagggtgcagtggttctntcngatgcatgcatcccgggtgcacgcccat 300
cacgtggccacctcaggaggaggggctccggagcctttaaagatgtggag 350
aaaggcttcattctttctggagtgggacctcagccccctccaggcatggg 400
aaagcgggagccctgagccgccatgaggcagcaagaagatagaaacgtac 450
gtctgaacctgagcaactgcccctccatggaagaggtggggaggtgggct 500
gagggtcgaggggtccaaagagantagagggttggggtccgggctgggaa 550
gcgactgctntgtcaggaagctggactctctctggctgggtcattgctga 600
gctgacacttgtacagaccgggagagggcaaatgtgagggcggccctgga 650
ggacagacaggccctgntggtggcaccatgagctgagcaagacacctgag 700
agcgaggacc tgctc tgc tccc tgc tgggaccATGTCTGGGCAGGGCCCC 750
CAGAGAAGGCTGCTGGGCTCTCTCAATGGCACCTCCCCAGCCACCCCTCA 800
CTTCGAGCTGGCTGCCAACCAGACCGGGCCCCGGTGCCTGGAGGTGTCCA 850
TTCCCGACGGGCTGTTCCTCAGCCTGGGGCTGGTGAGCGTAGTGGAAAAT 900
GTGCTGGTGGTGGCCGCCATTGCCAAGAACCGCAACCTGCACTCACCCAT 950
GTATTACTTCATCGGTTGCCTGGCTGTGTCCGACCTGCTGGTGAGCGTGA 1000
GCAATGTGCTGGAGACAGCCGTCATGCTGCTGGTGGTGGCAGGCGCCTTG 1050
GCTGCTCAGGCTGCAGTGGTGCAGCAGCTGGACGACATCATTGACATGCT 1100
CATCTGTGGTTCCATGGTATCCAGCCTCTGCTTCCTGGGCGCCATTGCCG 1150
TGGACCGCTACATCTCCATCTTCTACGCGCTGCGATACCACAGCATCGTC 1200
ACACTCCCGCGGGCGTGGCGGGCCATCTCCGCTATCTGGGTGGCTAGCGT 1250
CCTCTCCAGCACGCTCCTCATTGCCTACTACAATCACACGGCCGTCCTGC 1300
TTTGTCTTGTCAGCTTCTTTGTAGCCATGCTGGTGCTCATGGCAGTGCTG 1350
TACGTCCACATGCTTGCCCGCGCCTGCCAGCACGCCCGAGGTATTGCCCG 1400
GCTCCATAAGAAGCAGCACTTCATCCCCCAGGGCTTTGGCCTCAAGGGCG 1450
CTGCCACACTCACTATCCTGCTGGGCATTTTCTTCCTTTGCTGGGGCCCC 1500
TTCTTCTTGCACCTCTCACTCGTGGTCCTCTGCCCTCAACACCCCATCTG 1550
TGGCTGCGTCTTTCAGAACTTCAACTTCTTCCTCACCCTCATCATCTGCA 1600
ACTCCATCATTGACCCCTTCATCTACGCCTTCCGCAGCCAGGAGCTCCGA 1650
AAGACTCTCCAAGAGGTAGTGCTATGTTCCTGGTGAggctgcaggc ttga 1700
ggcc agggtgctgggc agaggggggtggtgattgatacccatgtgactgg 1750
ggcagtcncttgcagaaaaggacagatgagctgatctgtggtgtggtgga 1800
tqcatagaccctctgaaqccaqanaaaggaataaacanaaatctccagga 1850
gttgctgtgganaatggagcaggctggggagatggtggggccncanacac 1900
nanagccaggtccgggactactggacagcatctctggctgctcctgnana 1950
gt tc ct tc tc cacccagggacc aggcaaggcctgcacacacccacc11tg 2000
ctgccangtc tggatatttgggcccctgcccgttgggatgtgaaatc tea 2050
gtgttggtancatgacaagtnctgctctcgggaagcctccangaaaagga 2100
ctttgtgaccacccantcctcacaggactgcttcgggaaaaantgaactc 2150
ctgt tgcaggcacagggactgtc tacnggaactgcantggaaggtagggc 2200
tgatctaaagtccactggtaaccaaananctggtgacccanctgttgtgc 2250
ccttgcnccccntnttccccccaaanctttgttggtcnntgcctgctccn 2300
ggccnagg 2308
37
MSGQGPQRRLLGSLNGTSPATPHFELAANQTGPRCLEVSIPDGLFLSLQL
VSWENVLWAAIAKNmJLHSPMYYFIGCLA.VSDLLVSVSNVLETAVMLL
VAAGAIAAOAAWOOLDDIIDVLICGSMLSSLCFLGAIAVDRYLSIFYAIi
RYHSIVTLPRAWRAISAIWVASVLSSTLFIAYYMHTAVLLCLVSFFVAML
VLMAVLYVHMLARACOHARGIARLHKROHFIPOGFGLKGAATLTILLGIF
FLCWGPFFLHLSLMVLCPOHPICGCVFONFNLFLTLIICNSIIDPFIYAF
RSQELRKTLQEVVLC.SW 317
50
100
150
200
250
300
Figure 1.2
Doberman Mclr protein sequence.
Conceptual translation of the ORF shown in Figure 1.1a yields
the 317 amino acid Doberman Mclr protein. This protein is
very well conserved with other a-MSH receptors and contains
all the hallmarks of the melanocortin receptor family. These
include seven hydrophobic domains capable of traversing the
membrane (underlined), a consensus sequence for asparaginelinked glycosylation at residue 184 (bold faced), and a site
for carboxy-terminal palimitoylation at residue 315
(underlined and bold faced). This sequence designates the
wild-type (E) allele.
38
Figure 1.3
Conservation of the canine Mclr.
The deduced amino acid sequence of the Doberman Mclr (Figure
1.2) was aligned to the wild-type Mclr sequences determined
for the fox, horse, cow, human, mouse, and chicken. Amino
acid residues which are identical to those of the Doberman
are boxed.
39
20
Do zrna n
Fo»c
HurTLa.
n
Mods«
Chii.cz Keen
Do
toe rTn£kri
FOJC
Ho
irse
Cow
Humeri
L
B I PI wl c 1:^1 Y I V
M V t,
NfTriL*
M^
L-nriu
I L>i A W CfQ <3 ~
Mo\ase
Ch i c teen
Do t»e rmekn
Fosc
Hor*se
L.
Cow
L L> V s rc T HumcLn
Mouse
s DD t ML II ML. I V
V S ^7
Ch
i c Ken
L
L .. HHSS EP >MMVVVYFF '* I
I
I ^ H S P M Y Y F X
H S P M Y ^1 F I
L.
L. H S PP/Tl
M Y
F* I
Y Y
Y F
L. L.
Do be irm^ n
Fox
Ho
Cowxrse
It I
A. G
,^1 Ll
Rl A.
"vi
Grrri
L, VV I xr R^ri
H i G I VI l I V ^r* R rTTr
Human
Mouse
ChicJcen
RR YV LL,
Do toe trm«a n
Fojc
Ho
Cowxrse
Hurrwin
Mouse
Chiiclcen
Pt M I L.
A. UU R
Do toe trm<xn
Fo>c
Hojrse
Cow
HurriAn
Mouse
Cbi
i G Jcen
H
H
" ^ r rHi T L>rgn R
Do be r*tn<cir
Fo>c
Hofse
Cow
Hurrwin
Mouse
Chi
1 clcen
K A.
V/ Vn LL. LL. CCZ L.
HH II vl
L. VV
PT-R
L.LJ A.
R A. C a
TA R A C Q Hi V rRH s
FfTTl r_ I A. O H
2&0
wW G
-r-F"P L T
L.
G
I
F FFp cL.
L Cc
LLj G
F
C
G I VI F F L. C W
W
LL ,GG II FF FF LL. CC WW
L. GI V i F Fprn C W
Dotoer-m^n
Fox
Ho
Cowtrse
Hum^n
Mouse
Ch i.c Ken
K ro] p
y
F
FF
F
FF
y L ^
F L, H
F
F L.
L. HH
FF LL HH
F| £»' I H
L 5
L S
U S
L.
S
L.|
LLi TIL,[I
2-^0
V L 4 P •s-fn
toe inn^i n
Q H Do
F03C
Q HH Hoirse
PF _Q
Cow
HumAn
P
Mouse
-vr("*Tn
!-> ''
H
TT Chi ic Ken
L I
2 8 0
F nn L
FF M L
M
"n FF
I r D
X [ V| D
II VT DD
T
V V1
Vl D
D
p
P
P
P
P
P
FL
LLa
L.
L>
II
IX
IX
Y
Y
Y
Y
Y
Y
AA
AA.
AA
F
F
F
F
F
F
Do toe rrma.n
Fox
Horse
Cow
HumAn
Mouse
Chx
i cKen
Do toe xrm^n
FoxITS e
Ho
Cow
HumAn
Mouse
Ch i cKen
Decoirac l.on * Deco iTAC 1 on # 3. '
exactly.
Box 2res±dues chiAC rriACchi DotoeinnA.n
40
color variants were the result of mutations in the Mclr, and
if so, were they related to the mutations found in the fox
(V^ge, et al., 1997). To answer these questions I directly
sequenced a 1.3 kb PGR product of the Mclr gene from genomic
DNA from two black breeds (Newfoundland and Black Labrador
Retrievers) and two yellow or red breeds (Yellow Labradors
and Golden Retrievers) and conpared the deduced amino acid
sequences to those of the Doberman. The sequence variants
thus identified are shown in Table 1.1. Interestingly,
although the Doberman and fox Mclr genes are 96 and 98%
identical at the protein and nucleotide levels, respectively,
the domestic dog appears to have different mutations
correlating with coat color phenotypes than that fovind in the
fox (Vige, et al., 1997).
In previously identified activating mutations, the
phenotype has been attributable to a single amino acid
substitution (e.g. Robbins et al., 1993). However, in the dog
I found four identical substitutions occurring in the Black
Labrador and the Newfoxindland conpared to the normal allele
of the Doberman. Two of these substitutions, serine to
glycine at residue 90 (S90G) and alanine to threonine at
residue 105 (A105T), occur within or near the second
transmembrane domain, close to activating mutations
identified in mice and cattle. Another substitution, leucine
to valine at residue 129 (L129V) occurs in transmembrane
domain three, near the activating mutation in foxes. And
41
Figvire 1.4
Expression, of the Doberman Mclr in dorsal and ventral skin.
Total RNA was extracted from black dorsal skin and yellow
ventral skin of docked Doberman tails and sxibjected to
formaldehyde/agarose gel electrophoresis. Immobilized RNA. was
hybridized to a 32P-labeled partial Mclr cDNA probe using
standard procedures. The Mclr signal was visualized in both
dorsal and ventral skin samples running close to the 18S rRMA
band (indicated with arrow). The Mclr is expressed at
comparable levels in dorsal and ventral skin. To verify
approximately equal loading in each lane, the filter was also
hybridized to a partial GAPDH cDNA probe (bottom panel).
42
CO
2
o
c
<D
Q
28S
18S
>'•.''':.v i<'
GAPDH
43
finally, the proline to glutamine siibstitution at residue 159
(P159Q) change lies in the second cytoplasmic loop. It is
still unknown if any one of these substitutions could alter
receptor function or if some combination of changes is
required.
The Mclr from Yellow Labradors is identical to that of
the Black Labrador and Newfoundland except that it is
prematurely truncated by a C->T transition in codon 306. This
mutation shortens the cytoplasmic C-terminal tail by twelve
amino acids, removing the palmitoylation site. The Golden
Retriever carries a third Mclr variant which includes the
identical C-terminal truncation but only two of the four
amino acid substitutions, L129V and P159Q. The L129V and
P159Q substitutions can be ruled out as inactivating
mutations because they are also found in the E' allele. This
suggests that the short truncation inactivates the receptor.
To test if this truncation was found in other yellow or
red colored dogs, I employed an allele specific oligo
hybridization protocol to screen a larger number of
individual dogs. An oligo spanning the C->T transition with
sequences identical to either the tiruncated or non-truncated
allele (see Materials and Methods) was hybridized to
immobilized PGR products amplified from genomic DNA from 48
dogs encompassing 21 breeds and several pigmentation
patterns. The hybridization pattern was consistent with the
truncation being responsible for the yellow or red coat color
44
in that the truncation was not found in the homozygous state
in any non-yellow dogs (see Table 2.2). I was unable to
conclude that the truncated receptor was found in all
yellow/red breeds; two individual Red Chow Chow sattples
carried the non-truncated allele suggesting that the coat
color of this breed must be due to another Mclr variant or
another gene entirely, such as the agouti gene.
45
Residue #
Breed
Doberman
Golden Retriever
Yellow Labrador
Black Labrador
Newfoundland
90
105
129
159
306
S
s
G
G
G
A
A
T
T
T
L
V
V
V
V
P
Q
Q
Q
Q
R
STOP
STOP
R
R
Table 1.1
Amino acid suibstitutions identified in various dog breeds.
Sequence analysis of Mclr PGR products from the five breeds
tested identified sequence polymorphisms which resulted in
amino acid substitutions in the Mclr protein. The Doberman
represents the wild-type (E) allele in dogs. Identical
substitutions relative to the Doberman sequence were found in
two breeds with solid black coloration (Black Labradors and
Newfoundlands). I have designated this allele as E* to
indicate its dominance to E. The two yellow variants (Yellow
Labradors and Golden Retrievers) share a common tiruncation at
codon 306. This truncation is likely to result in the loss of
receptor function. This is reflected in the designation of
the trvincated receptor as the e allele.
46
Table 1.2
Carboxy-terminal truncation of the Mclr correlates with
recessive yellow coat color.
A 122 bp PGR product which spans codon 306 was artplified from
genomic DNAs of 21 breeds and immobilized on duplicate
nitrocellulose filters. The immobilized DNAs were hybridized
to oligos specific for either the tiruncated or non-truncated
allele. The hybridization of each oligo under standard
conditions differed between breeds of differing genotype. As
controls, several individual dogs from a Labrador pedigree
were included because they are known to be segregating the E'
and e alleles. From this it was possible to determine the
genotype of each individual dog with respect to this
trioncation of the Mclr. All non-yellow dogs carried at least
one copy of the non-truncated allele. Furthermore, all dogs
homozygous for the truncation were yellow. This is suggestive
that the truncation results in loss of receptor fxinction.
47
ASOH Pattern
Breed
Doberman
Golden.
Retriever
Yellow Labrador
Black/Chocolate
Labrador
Newfoundland
Buff Spaniel
Black Spaniel
Samoved
English
Shepherd
Akita
Rottweiler
Bernese
Mountain Dog
Black-and-tan
Terrier
Australian
Shepherd
Black Giant
Schnauzer
Dalmatian
Black Scottie
Red Chow Chow
Airedale
Border Collie
Black Flat
Coated
Retriever
homozygous
full
length
3/3
heterozygous
homozygous
for
truncation
4/4
9/9
1/3
2/3
3/3
2/2
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
4/4
2/2
2/2
2/2
1/1
1/1
48
Discussion
As with other mammals, a variety of coat colors for the
domestic dog have been assigned to variant alleles at the
extension locus (Searle, 1968; Little, 1957; Willis, 1989).
In mice it has been demonstrated that self black coat color
which is inherited in a dominant fashion is due to gain-offunction alleles at the extension locus and black coloration
inherited as a recessive trait is due to loss-of-function at
the agouti locus. This rule has not been contradicted in
other mammalian species to date. In horses, cattle, and
foxes, both recessive and dominant coat color traits have
been correlated with amino acid substitutions in the Mclr
protein. I wanted to determine if similar amino acid
substitutions could be identified in the Mclr of domestic
dogs and if these substitutions would correlate with changes
in coat color. To test this hypothesis I cloned the gene for
the wild-type receptor of the Dobeirman breed and then
corrpared it to the Mclr gene of breeds with different coat
color phenotypes.
The Doberman Mclr (E) is very well conserved with
respect to other cloned Mclr genes. The similarity extends
from the first transmembrane domain through the carboxyterminus. Alignment of all cloned a-MSH receptors highlights
136 invariant residues, all of which are also conserved in
49
the dog Mclr. The sequencing of the Mclr from a variety of
breeds elucidated several polymorphisms which correlate with
coat color phenotypes. Two black breeds (Newfoundland and
Labrador Retriever) share the same allele (E*) which carries
four amino acid substitutions compared to the Doberman E
allele. Functional tests of the various substitutions, both
alone and in combination, will be required to determine their
role in receptor activity. All four lie in regions of the
protein previously implicated in receptor activity in other
studies. Two alleles of the mouse (E^° and E®°'"), the E° allele
in bovines, as well as a polymorphism in the chicken Mclr
cluster near residue 100 at the transmembrane/extracellular
loop junction, further focusing attention to this region for
fxjinctional relevance. These residues are likely to be
important in maintaining proper receptor conformation. It is
possible that ligand binding by the receptor induces a
similar alteration in conformation as caused by amino acid
substitutions leading to guanine nucleotide exchange on the
linked heterotrimeric G-protein. Alternatively, it is
possible that the gain-of-function alleles result in the
receptor's inability to interact with the agouti paracrine
signaling molecule or to be inactivated by intracellular
regulatory mechanisms, such as receptor internalization.
Sequencing of the Mclr from yellow Labrador Retrievers
and Golden Retrievers identified two additional alleles, both
proposed to be loss-of-fimction. All yellow Labradors tested
50
were found to be homozygous for an allele identical to the E*
allele of its black counterpart except for a short truncation
of the cytoplasmic carboxy-terminal tail. This same
truncation was also found in Golden Retrievers. Unlike yellow
Labradors, the Golden Retriever shares only two sxibstitut ions
with the E" allele, L129V and P159Q, which can be ruled out as
inactivating mutations by virtue of their occurrence in the
gain-of-fvinction allele. This leaves the truncation as the
likely explanation for the recessive yellow coat in these
breeds. Allele specific oligo hybridization further
demonstrated that the truncation was also found in Samoyeds
and buff American Spaniels but not in black American Spaniels
or any other pigmentation phenotype including self black
(Scottie. Giant Schnauzer, and Flat Coated Retriever), blackand-tan (Rottweiler, Airedale, Terrier, and Bernese Mountain
Dog), or in more corrplicated patterns such as blue merle,
tricolor, or spotted (Australian and English Shepherds and
Dalmatian).
The truncation is short by most standards and it is difficult
to speculate how this truncation might result in a loss of
receptor activity. The issue is also complicated by the fact
that the melanocortin subfamily of GPCRs is not well
conserved with other members of the superfamily (Tsigos,
1995), that the length of the carboxy-terminal tail varies
widely within the GPCRs, and by the various effector coupling
systems and mechanisms of receptor regulation. In several
51
other studies, truncations of the cytoplasmic tail which
remove consensus sites for Serine/Threonine phosphorylation
lead to hyperactive receptors in vitro (Grady et al., 1997).
The Mclr must be regulated by another mechanism as it
contains no such Serine/Threonine phosphorylation sites. This
is consistent with the truncation in the e alleles not
leading to hyperactivity.
It is still unclear how this novel truncation could
inactivate the receptor. Two possibilities are evident, the
loss of localization signals and sxibsequent misrouting of the
protein away from the cell surface, or from the inability to
couple to downstream effectors. The short cytoplasmic tail
does contain a conserved palmitoylation site at residue 315.
The functional relevance of Mclr palmitoylation is unknown
but studies of the human arginine vasopressin V2 receptor,
another GPCR, demonstrated that alteration of the putative
palmitoylation sites by site-directed mutagenesis affected
receptor trafficking (Schulein et al., 1996). It has also
been shown that for the human B2-adrenergic receptor, sitedirected mutagenesis of the palmitoylation site drastically
reduced agonist-mediated activation of adenylyl cyclase
(O'Dowd et al., 1989). Palmitoylation might also play a role
in G-protein coupling. Heterotrimeric G-proteins have been
shown to interact with the third cytoplasmic loop, between
transmembrane domains 5 and 6, and the C-terminal tail (e.g.
Kobilka et al., 1988).
52
Future in vitro studies of ligand binding and effector
coupling will further our imderstanding of the mechanisms
underlying the behavior of both mutant alleles identified in
this study. It will be very interesting to determine if the
truncation results in a loss-of-function. If it is indeed the
case, understanding the mechanism by which this truncation
inactivates the receptor will likely provide insight into Gprotein coupling or intracellular trafficking of the
melanocortin receptor family.
53
Chapter
2
Cloning of the Doberman agouti cDNA and demonstration that
the agouti gene plays a conserved role in mammalian
pigmentation patterning
Introduction
Like the Mclr, the agouti locus dramatically affects
coat colors in mice. For example, ubiquitous expression of
the agouti protein in mice carrying the lethal yellow allele
(A^) causes conpletely yellow pigmentation (Silvers, 1979;
Bultman et al, 1992; Miller et al., 1993). The
allele is
also associated with several pleiotropic effects such as
obesity and increased tumor susceptibility due to the ectopic
expression of the agouti protein and its interaction with
other melanocortin receptors, pheno types which are mimicked
by the ubiquitous expression of the normal agouti protein in
trcinsgenic mice (Klebig et al., 1995; Millar et al., 1995;
Wilson et al., 1995). Mice lacking the agouti protein (e.g.
those carrying the recessive alleles non-agouti (a) or
extreme non-agouti (a®) ) have little or no pheomelanin and are
black (Bultman et al., 1994; Hustad et al., 1995). The
specific pigmentation patterning in many strains of mice are
the results of alternative agouti expression patterns
(Vrieling et al., 1994), which arise from the use of two
different promoters. The first generates a ventral-specific
54
mRNA isoform which produces the pale ventrum phenotype. The
other promoter is utilized for a hair cycle-specific isofonn
which results in a s\ibapical band of pheomelanin in an
otherwise black hair. The two mRNA isoforms code for the
identical agouti protein and differ from one another only in
the 5' untranslated exons (Vrieling et al., 1994).
Like other mammals, the domestic dog is suspected to
carry a variety of alleles at the agouti locus (Searle, 1968;
Little, 1957; Willis, 1989). The canine allelic series
consists of four alleles, including a dominant yellow allele,
an allele which correlates with a pale ventrum and banded
dorsal hairs, and a recessive allele associated with blackand-tan bicolor patterning. The large number of canine
phenocopies of murine pigmentation patterns suggests that the
domestic dog possesses an agouti gene which regulates
expression of pheomelanin by a mechanism similar to that of
the mouse. Here I report the cloning of the canine agouti
cDNA from the Doberman and present arguments for its role in
the dorsal/ventral pigmentation patterning of this breed.
Materials and Methods
Cloning of the Doberman agouti cDNA: Doberman tails
were collected during routine tail docking procedures at a
veterinarian's office. Total RNA was extracted from the
yellow skin on the ventral side of the tail samples using the
55
Acid Guanidiniiam Thiocyanide protocol (Chomczynski and
Sacchi, 1987). A partial agouti cDNA was obtained via Reverse
Transcription (RT) using a degenerate primer designed from
the mouse cDNA, reverse primer A (5'A(A/G)NACNC{G/T) {G/A)CANGT(G/A)CA-3') and the manufacturer's
guidelines (Superscript, Gibco). This partial cDNA was
aitplified by the Polymerase Chain Reaction (PGR) using primer
A in combination with a degenerate primer based on human,
mouse, and bovine agouti sequences, forward primer B (5'G(G/T)ITTC(C/T)TITG(C/T)TT(C/T)TT(C/T)ACIG-3' ). PGR
conditions included 50 pmols of each primer, 30 sec at 94° to
denatxire, 1 min at 55° to anneal, and 30 sec at 72° to
extend, repeated for 40 cycles. The resultant products were
used as tenplate for a hemi-nested PGR using the same
conditions with primer B and reverse primer C (5'AA(G/A)AANC(T/G)(G/A)CA(T/C)TG(G/A)CA-3') . The cDNA, which
contains codons 13-119, was cloned into the T/A cloning
vector pCR2.1 (InVitrogen) for sequence analysis.
The 5' and 3' ends of the canine agouti cDNA were
identified using a Rapid Amplification of cDNA Ends (RAGE)
kit (Gibco). Initial RT from yellow tail skin RNA enployed
reverse primer D (5'-AGTTGGAGGTGTTGCGAGTG-3', nucleotides
421-440 in Figure 2.1a). PGR anplification of the 5'RACE
product was obtained with the same PGR conditions as above
using the forward "anchor primer" and reverse primer E (5'AGGGAGGTTCTTCATGGAAG-3 ', nucleotides 366-385). A hybridizing
56
band was gel purified using the Geneclean kit (Bio 101) and
used as tenplate for a hemi-nested reamplification using the
"anchor primer" in combination with reverse primer F (5'CTTTTCCGCCTCTTTTCTGC-3', nucleotides 324-343). The 5'RACE
product, covering nucleotides 1-343 was gel purified and
cloned into the pCR-Script vector (Stratagene) for sequence
analysis.
To determine the 3' end, reverse transcription was
carried out using yellow tail skin RNA. and the oligo-dT
"adapter primer". PGR amplification of the 3'RAGE product was
obtained using the supplied "anplification primer" and
forward primer G (5'-AAGGATGACAGGAGCCTAAGG-3', nucleotides
227-247) using the same PGR conditions as above except
primers were annealed for 1 min at 50°. An aliquot of this
reaction was used as tenplate for a hemi-nested PGR using the
"anplification primer" and forward primer H (5'TTGGATTTCCGTTGTGTGTG-3', nucleotides 269-288). The 3'RAGE
product covering nucleotides 269-786 was gel purified and
cloned into the pGR2.1 vector for sequence analysis.
Mapping of axons in genomic DNA: In Southern
hybridization experiments, a probe containing exon 1 and part
of exon 2 (nucleotides 1-173 of the cDNA) detected two
restriction fragments in canine genomic DNA cut with a
variety of restriction enzymes. To clone these fragments,
Doberman genomic DNA was digested with Nhel and fractionated
by agarose gel electrophoresis. Fractions which hybridized to
57
the probe were ligated to Xbal-digested Lambdaphage arms
(LambdaGEM-11, Promega) to generate two size-selected
libraries. Phage clones corresponding to each Nhel fragment
were isolated from each library and subcloned into plasmids
for sequence analysis and mapping studies.
Each Nhel fragment siibclone was digested with a variety
of restriction enzymes, fractionated by agarose gel
electrophoresis and analyzed by Southern hybridizations to
determine the relative location of exons 1, 2, and 3 (exon 4
was not contained on either fragment, see Results) . Mapping
experiments eirployed the following ^"P-labeled oligo probes:
exon 1 (5'-CTGCTACTGACGATTCCTGG-3', nucleotides 87-106); exon
2 {5'-GCCTCCAAGGATGAATATCTTCCG-3', nucleotides 130-153); and
exon 3 (5'-AAAGATCAGC:AGAAAAGAGGC-3', nucleotides 316-335).
Data from hybridization patterns and sequence information was
used to generate the map sho\^7n in Figure 2.3.
Northern analysis: Total RNA was purified from black
dorsal and yellow ventral tail skin as above. RNA was
fractionated by formaldehyde/agarose gel electrophoresis and
transferred to a nitrocellulose filter (MagnaGraph, MSI)
according to standard protocols (Sambrook, Fritsch, and
Maniatis, 1989). Total RNA samples used in this analysis had
undergone partial degradation which limited the signal
quality and made it difficult to load equivalent amounts in
each lane. Immobilized RNA was hybridized to a ^^P-labeled
probed which contained exon 2 sequence (nucleotides 130-288).
58
The hybridization signal was visualized following a seven dayexposure of BioMax x-ray film (Kodak).
Tremsgenic analysis: The transgenic reporter
construct was kindly provided by Dr. Alex Joyner (Mount Sinai
Research Institute, Toronto, Canada). The transgene reporter
construct has been previously described (Kothary et al.,
1989). Briefly, the reporter construct includes the hsp68
promoter, the full length E. coli p-galactosidase gene, and a
3' sequence from SV40 which contains one intron and the
polyadenylation signal to promote transcript stability (see
Figure 2.3b). A 14 Kbp Nhel fragment carrying exon 1 and an
8.3 Kbp Nhel fragment carrying exons 2 and 3, described
above, were subcloned from the phage isolates into transgene
constructs using the Xhol restriction site. Plasmids were
linearized and gel purified with the Qiaex II DNA Isolation
kit (Qiagen) for microinjection. Microinjections and animal
care of host mice was performed at the Stanford University
Transgenic Mouse Facility (Stanford, CA). Two different host
strains (FVB and B6CBA) were used with identical results. On
embryonic day 13.5 {E13.5), when endogenous embryonic agouti
expression is readily detected by in situ analysis (Millar et
al., 1995), host mothers were euthanized and founder embryos
were harvested for analysis of transgene expression. Embryos
were fixed in 4% paraformaldehyde, washed three times in
0.02% Nonidet P-40/0.01% deoxycholate for 15 minutes each.
59
and stained for two hours to overnight in an X-gal staining
solution to visualize the blue reaction product.
Transgenic embryos were identified with Southern
analysis. Genomic DMA was purified from yolk sacs and
digested with BamHI. Saitiples were then fractionated by
agarose gel electrophoresis and transferred to nylon filters
(Hybond-N*, Amersham) according to standard protocols.
Immobilized DNA was hybridized to a 0.8 Kbp BamHI/BstEII
fragment containing the hsp68 portion of the transgene which
identified an endogenous doublet near 7 Kbp in all lanes and
a 4.2 Kbp band in transgenic embryos.
Results
The canine agouti cDMA sequence (Figure 2.1a) was
generated using Reverse Transcription followed by the
Polymerase Chain Reaction (RT-PCR) or 5'- and 3'-Rapid
Aitplification of cDNA Ends procedures (RACE). The 0.8 Kbp
cDNA contains a 133 codon open reading frame (ORF) beginning
at nucleotide 140. The initiator methionine codon for this
ORF lies in the sequence AGGATGAAT which does not match the
Kozak consensus sequence for eukaryotic translation (Kozak,
1989) however, it is in-frame with three stop codons present
in the 5' untranslated region (UTR) and aligns very well with
the cDNA sequences of the fox, human, and mouse. The canine
60
a.
cttggagaatcattgatctaagaacaacattttgatttaagaacccttct
tttgatctggaattatctgttattgaccacagagagctgctactgacgat
tcctgggacagggccc tgtatagtgagaggcctccaaggATGAATATCTT
CCGCCTACTCCTGGCCACCCTACTGGTCTCCCTGTGCTTCCTCACTGCCT
ACAGCCACCTGGCTGAGGAAAAGCCCAAGGATGACAGGAGCCTAAGGAGC
AACTCCTCTGTGAACCTTTTGGATTTCCCTTCTGTCTCTATTGTAGCACT
GAACAAGAAATCCAAAAAAATCAGCAGAAAAGAGGCGGAAAAGAAGAGAT
CTTCTAAGAAAAAGGCTTCGATGAAGAACGTGGCTCGTCCCCGGCCCCCG
CCACCCACCCCCTGCGTGGCCACTCGCAACAGCTGCAAGTCCCCGGCGCC
CGCCTGCTGTGACCCCTGCGCCTCCTGCCAGTGCCGCTTCTTCCGCAGCG
CCTGCACCTGCCGCGTTCTCAGTCCCAGATGCTGAacgcgcccagcggcc
tccagggggt tggctga11atc taagaagtacggc111ggggatccc tgg
gtggctcagcggtttgacgcctgcttttggcccaaggtgcgatcctggag
tgccgggatcgaatcccatgtcaggctccggcgtggaacctgcttctccc
tctgcctgtgtctctgcctccctctctctttctctctatgtctatcatga
ataaataaataanatc 11taaaaaaaaaaaaaaaaa 786
50
100
150
200
250
300
3 50
400
450
500
550
600
650
700
750
b.
MNIFRT.T.T,ATLLVSLCFLTAYSHLAEEKPKDDRSLRSNSSVNLLDFPSV 5 0
SIVALNKKSKKISRKEAEKKRSSKKKASMKNVARPRPPPPTPCVATRNS 100
CKSPAPACCDPCASCQCRFFRSACTCRVLSPRC 13 3
Figure 2.1
Coirplementary DNA and deduced amino acid sequence of the
Doberman agouti.
(a) The Doberman agouti cDNA was determined from sequence
analysis of overlapping RT-PCR and RACE products isolated
from the yellow skin of combined tail samples. The cDNA
sequence is well conserved with other cloned agouti sequences
and is likely to represent the conplete mRNA transcript. The
133 codon open reading frame (ORF) is indicated in capital
letters and the 5' and 3' untranslated regions are in lower
case, (b) Conceptual translation of the Doberman agouti cDNA
ORF provides the amino acid sequence of the agouti protein of
the domestic dog. This sequence is very well conserved with
that of the fox as well as with both human and mouse agouti
proteins. It contains a hydrophobic amino terminus for its
entry into the secretory pathway and all ten invariant,
carboxy-terminal cysteine residues. This suggests that the
domestic dog does possess a functional agouti protein.
61
agouti protein sequence (Figure 2.1b) obtained by conceptual
translation of this ORF is highly conserved with other cloned
agouti genes and contains a hydrophobic amino-terminus, a
highly basic central region, and a cysteine-rich carboxyterminal domain. The cysteine-rich domain has been shown to
contain the a-MSH antagonizing activity (Kiefer et al., 1997)
and each of the ten invariant cysteines are required for full
activity of the mouse agouti protein (Perry et al., 1996).
The most highly conserved portion of the canine agouti
protein is in this carboxy-terminal region (96% over the
final 46 residues versus ~80% for the entire protein) and all
ten cysteines are present and correctly spaced. These results
suggest that the canine agouti gene codes for a functional
protein.
Cortparison of the 5'UTR to the Genbank database detected
homology to the mouse agouti exon lA' and flanking genomic
sequence. In the mouse, exon lA' is preceded by exon lA which
together make up the 5'UTR in ventral-specific transcripts
(Vrieling et al., 1994). By contrast the dog agouti first
exon contains 130 non-protein coding nucleotides present
without interruption by an intron in canine genomic DNA (see
Figure 2.3). Northern analysis of Doberman tail skin RNA
using a partial cDNA probe (see Materials and Methods)
demonstrates expression of a single agouti isoform in ventral
(pheomelanic) but not dorsal (eumelanic) skin (Figure 2.2).
The sequence similarity between the Doberman first exon and
62
Figxire 2.2
Ventral-specific expression of the Doberman agouti mRNA.
Total RNA was extracted from black dorsal skin and yellow
ventral skin of docked Doberman tails and sxibjected to
formaldehyde/agarose gel electrophoresis. Immobilized RNA was
hybridized to a 32P-labeled probe corresponding to exon 2 of
the agouti mRNA. using standard procedures. The agouti signal
was detected only in RNA extracted from yellow ventral skin
(indicated with arrow). This demonstrates that Doberman
agouti expression correlates with the synthesis of
pheomelanin in canine skin. The relative amount of total RNA
in each lane was determined by hybridization to a partial
GAPDH cDNA probe (bottom panel).
Dorsal
Ventral
0
>
"D
hO
00
00
O)
O)
D
1
Ol
lAi
64
murine exon lA', and the expression of the canine agouti mRNA
in ventrally located pheomelanic skin supports the view that
the cDNA in Figure 2.la is the canine equivalent of the
ventral-specific agouti mRNA isoform of mice.
To further characterize the canine agouti locus I
isolated large pieces of genomic DNA containing exon 1 or
exons 2 and 3. In cases where the genomic structure has been
determined, the agouti gene contains three protein-coding
exons and one or two non-protein-coding 5' exons (a similar
genomic structure has also been recently been found for the
highly homologous human and mouse agouti-related protein,
also called ART (Ollmann et al., 1997; Shutter et al.,
1997)). Sequence and mapping data from the cloned DNA
fragments demonstrated that the dog has a similar genomic
structure for exons 2 and 3 as previously determined for
hximan and mouse loci. As in humans and mice, the hydrophobic
and basic regions lie on exons 2 and 3 with the cysteine-rich
domain contained on a single, fourth exon. Although the
canine exon 4 was not contained on the cloned fragments, the
conservation of cDNA sequence and genomic structure for the
other exons argues against there being more than a single
exon for the essential cysteine-rich region previously
discussed.
Fragments carrying either exon 1 or exons 2 and 3 were
cloned into a transgene construct to test for the presence of
ventral-specific enhancers in transgenic mice. I found that
N
K
P RI
a.
J
P
RI
-II-
1
Sa
B
Nc S
N
J
I
u
Ha
1
2
3
2 Kbp
N
b.
P RI
1_1_L
K
J
P
RI
J_L
hsp68 LacZ SV40pA
Figure 2,3
Structure of the Doberman agouti locus and exon 1 transgene.
(a) Two Nhel fragments of genomic DNA were isolated from Doberman genomic DNA
using directed Lambdaphage libraries. These two fragments carried either exon 1 or
exons 2 and 3. Sequence analysis of subcloned fragments combined with Southern
hybridization patterns of exon-specific oligos was used to determine the structure
presented above. Exon 4 was not contained on the cloned DNAs. (b) The 14 Kbp
fragment carrying exon 1 was subcloned directly from the phage isolate into the
transgene plasmid. This plasmid consists of a 4.2 Kbp reporter cassette inserted
into a standard cloning vector (pBluscript KS). Linearization of the completed
plasmid produces a 21.2 Kbp transgene which was used for the generation of
transgenic mice. Restriction enzymes: B: BamHI; K: Kpnl; N: Nhel; Nc: Ncol; P;
PstI; RI: EcoRI; S: Smal; and Sa: Sad.
a\
ui
66
only the fragment carrying exon 1 was able to direct
e3<pression of the LacZ reporter in embryonic day 13.5
{E13 .5)(Figvire 2.4). Embryos transgenic for the exon 1
transgene in which the transcriptional direction of exon 1
points toward the reporter gene expressed p-galactosidase
activity widely.
From four foiander embryos with this
transgene one expressed reporter activity almost ubiquitously
producing a conpletely blue transgenic embryo following the
staining procedure. One expressed the reporter strongly in
the skin of the ventral trunk including the shoulder area and
on the proximal portions of the limbs (Figure 2.4b). The two
other foiinders with this construct expressed the reporter
very weakly or not at all, which probably results from the
proximity of silencing elements near the transgene insertion
site.
Embryos transgenic for this same 14 Kbp Nhel fragment
subcloned in the opposite orientation expressed the pgalactosidase reporter in a pattern more consistent with the
hypothesis that this fragment carries ventral-specific
regulatory elements (see Figure 2.4 c-h). With this
transgene, two of six founders did not express reporter
activity, and one embryo expressed only weakly. However, the
expression pattern found in the remaining three more closely
matched the distribution of yellow coloration in the Doberman
breed. All three showed high levels of expression on the
ventral/lateral neck and up toward the pinnae and on the
67
distal limbs. Limb expression was highest in the distal
portions and on the ventral side where the limbs meet the
body trunk. These embryos also show lower levels of
expression in the ventral trunk itself. This may indicate the
presence of a more general ventral enhcincer element in this
region, which has reduced activity in this orientation. It is
also possible that this transgene does not carry all of the
regulatory elements of ventral-specific agouti expression,
both enhancers and repressors, and that another genomic DMA
fragment which partially overlaps the one analyzed here may
more conpletely recapitulate the Doberman agouti mRNA
expression pattern. However, this fragment does appear to
contain at least most of the Doberman regulatory elements.
The high level of expression found on the ventral side of the
neck and in the limbs is very similar to the distribution of
yellow hairs in the Doberman and to the embryonic expression
of mouse agouti (Millar et al., 1995) Interestingly, I also
found that one embryo carrying this transgene (Figure 2.4 g
and h) expressed LacZ in a thin, longitudinal stripe on the
belly between the fore- and hind-limbs. This pattern is also
seen in mice transgenic for a similar construct containing
mouse exons lA and lA' (Y. Chen and G.S. Barsh, unpioblished
results). Sequence conparison of these fragments between the
mouse and the dog is still incorrplete. However, a 35 bp
stretch located -0.6 Kbp upstream of dog exon 1 is 82%
identical to a similarly located segment in the mouse agouti
68
locus. Analysis of this region using the Signal Scan
algorithm
{http:/ /www.dna.affrc.go.jp/htdocs /sigscan/signal.html)
detects the presence of an E-box (CANNTG) for potential
interaction with helix-loop-helix transcription factors. More
sequence conparison and analysis of this region will be
required to determine if these 35 bp play any fxinctional role
in agouti expression. It is, however, noteworthy that of -1.5
Kbp of genomic sequence flanking the dog exon 1, this is the
only stretch with sequence homology to the mouse.
69
Figiore 2.4
Expression of the exon 1 transgene in E13.5 embryonic mice.
Expression of the bacterial p-galactosidase gene is directed
to specific regions of transgenic mice by enhancer elements
from the Doberman agouti locus. In each case, enzyme activity
is detected by enzymatic cleavage of the 4-chloro-5-bromo-eindoyl-p-D-galactopyranoside substrate producing an insolxjble
blue product (shown here as black regions in the embryonic
mice). (a) Non-transgenic embryo with some non-specific
staining in the eye. (b) Exon 1 transgene-carrying embryo in
which the exon 1 sequence is in the same transcriptional
orientation as the reporter gene. In this orientation the
transgene is widely expressed but highest levels of
expression are detected in the limbs and ventral/lateral
trunk as well as the ventral neck and pinnae, (c-h) exon 1
transgene-carrying embryos with the opposite orientation as
in panel b. Three transgenic embryos are shown from lateral
and frontal viewpoints. All three embryos show high levels of
expression in the distal limbs and lateral neck/pinnae
region. There is some lower level expression in the lateral
trunk seen in panels c and e. This pattern is similar to the
distribution of yellow pigmentation in the Doberman.
Surprisingly, the embryo shown in panels g and h also shows
expression of the reporter gene activity in a thin line
between the fore- and hindlimbs. This is not a region of
yellow pigmentation in the Doberman but is detected in E13 .5
embryos expressing an identical reporter construct driven by
ventral-specific transcriptional enhancer elements from the
murine agouti locus (Y. Chen and G.S. Barsh, unpublished
results).
71
DISCUSSION
Data from the mouse have shown that the synthesis of the
yellow pigment pheomelanin results from the expression of the
agouti protein, which antagonizes signaling through the a-MSH
receptor, Mclr. Decreased Mclr signaling promotes a switch
from the synthesis of eumelanin to that of pheomelanin.
Regional or tenporal differences in agouti expression
resulting in coordinated inhibition of the Mclr are
responsible for pigmentation patterns. Expression is
regulated by the use of alternative promoters which give rise
to different agouti mRNA. isoforms, a ventral-specific isoform
or a hair cycle-specific isoform. I have cloned the agouti
cDNA from the Doberman breed of domestic dogs and foiind that
it is likely to be responsible for the pigmentation
patterning of this breed and others as well.
The canine agouti protein is well conserved with those
cloned from other species. Features which have been
iirplicated in proper agouti function in the mouse, including
the signal sequence for its secretion and the ten invariant
cysteines in the carboxy-terminal domain, are present in the
Doberman protein. And consistent with the Doberman agouti
first exon being highly homologous to the mouse ventralspecific exon lA', the Doberman agouti mRNA was detected by
Northern analysis in the yellow ventral skin but not in the
black dorsal skin of the tail. The sequence conservation of
72
the protein and its expression in pheomelanic skin are
consistent with it playing the same role in pigmentation
patterning in the domestic dog as in mice.
Like the black-and-tan agouti mouse, the Doberman has
yellow hairs on the ventral side of the ears, the ventral
neck and muzzle, on the distal limbs, and the ventrum. Also
like the black-and-tan mouse. Northern analysis using
protein-coding sequence from the cDNA as a probe likely to
detect any agouti mRNA isoforms demonstrated that the
Doberman expresses only a single agouti mRNA isoform. It will
be interesting to test for other canine agouti mRNA isoforms
in other breeds, especially those with banded hairs such as
the German Shepherd or Shetland Sheepdog, as they might
express a hair cycle-specific mRNA isoform in addition to the
ventral-specific isoform identified in the Doberman.
Toward identification of the upstream regulators of
agouti expression, I analyzed fragments of genomic DNA for
the presence of ventral-specific agouti enhancer elements
using transgenic mice. I found that a large fragment
containing 2.2 Kbp of sequence 5' of the Doberman first exon
and -12.8 Kbp of the first intron was able to direct
expression of a LacZ reporter gene to the skin of embryonic
mice in a pattern very similar to the distribution of yellow
hairs of both the Doberman and the black-and-tan agouti
mouse. This suggests that the regulatory elements for at
least the ventral-specific agouti promoter have been
73
conserved between carnivores and rodents. Limited sequence
analysis of this transgene has identified a short, 35 bp
stretch of sequence conservation between the Doberman and
mouse agouti 5'-flanking genomic DNA. The presence of an Ebox for potential interaction with helix-loop-helixcontaining transcription factors in the middle of this
stretch makes this an attractive region for future studies.
This is not the only E-box located 5' of the agouti first
exon and more sequence data will surely identify more sites
for potential transcription factor binding as well as
additional regions of conservation. Future experiments of
this work could include a functional test of the canine
agouti protein in transgenic mice similar to experiments with
the human agouti (Wilson et al., 1995) or murine agouti
variants (Perry et al., 1995 and 1996). It would also be
interesting to identify hair-cycle specific mRNA isoforms and
to obtain more sequence information of genomic DNA flanking
exon 1.
74
CONCLUSIONS
Studies of mammalian pigmentation have been instrxomental
in extending our knowledge of development, molecular
evolution, cell biology, and genetics as well as providing
insight into the mechanisms by which mammals and other
vertebrates generate colorful and cortplex patterns. Although
much is known about the biochemistry of pigment production
and embryonic development of pigment cells, relatively little
is known about cellular events which ultimately determine the
type of pigment (eumelanin versus pheomelanin) synthesized
within those pigment cells. In all vertebrates, the
pituitary-derived hormone a-MSH stimulates eumelanin
production via increased signaling through its receptor,
Mclr, the product of the extension locus. In mammals, the
production of eumelanin can be switched to that of
pheomelanin through the activity of the agouti protein, a
functional antagonist of the Mclr. Several pigmentation
patterns of mice are generated by the combined action of
these two proteins. The studies presented here provide
molecular evidence that the extension and agouti loci work
together to generate pigmentation patterns in the domestic
dog as well. I have cloned the corrplete agouti cDNA from the
Doberman breed and demonstrated that it contains all the
major characteristics of the well-studied mouse ortholog and
that is expressed only in regions of the skin which
75
synthesize pheomelanin. In addition, I have demonstrated the
presence of transcription enhancer elements which promote the
ventral-specific expression of the Doberman agouti mRNA. This
provides evidence that the domestic dog possesses a
functional agouti gene which can regulate the regional
expression of yellow coat color.
I have also identified sequence polymorphisms in the
Mclr gene which correlate with inherited changes in coat
color. Specifically I have identified four identical amino
acid sxibstitutions in two breeds which generate a dominantly
inherited black coat and have identified a carboxy-terminal
truncation of the Mclr which correlates very highly with the
recessive yellow coat color phenotype. Although not a
universal trait in yellow or red dogs, this truncation was
not present in the homozygous state in any non-yellow dog.
This suggests that the truncation is sufficient to inactivate
the Mclr protein. Biochemical studies of Mclr variants which
carry various combinations of the amino acid substitutions
foxind in the E* allele relative to the E allele will be
required to determine what role the individual residues play
in Mclr fxinction. The non-conservative amino acid
substitutions found in the E* allele all lie in regions of the
Mclr protein previously iiiplicated in wild-type hormone
responsiveness. Thus, it is teirpting to speculate that one or
more of these substitutions creates a constitutively active
receptor much like the mouse sombre mutations (E®° and E®°*").
76
Futxire directions
These studies provide evidence for the conseirvation of
agouti and extension action in pigmentation patterning of the
domestic dog. The large variations in pigmentation between
breeds suggests that the domestic dog is an exciting new
source of allelic variation for molecular and genetic
studies. It is probably too ambitious to imagine large
breeding colonies and positional cloning of pigment genes as
has been done in the mouse. However, the rapid progress in
the genetics and molecular biology of murine pigmentation,
along with future data from the Mouse Genome Project has and
will continue to identify important genes which will sirtplify
their identification in the dog. Just as I have shown with
the canine agouti and extension genes, analysis of other
canine pigment genes will provide further insight into the
biology of these genetic loci and their protein products.
Several important experiments are suggested by the data
generated in the dog Mclr project. Firstly, the amino acid
siobstitutions which occur between the wild-type (Doberman, E)
allele and the suspected gain-of-f\inction (E*) should be
tested individually and in combination for functionality.
This could be accomplished by expressing the variant Mclr
proteins in cell culture and measuring baseline and ligandinduced changes in intracellular cAMP. This has been achieved
for both the murine and fox Mclr variants. The suspected
loss-of-function (truncated, e) allele could be tested in the
77
same system. In this case, I would expect that the trnincated
protein would not elicit any rise in intracellular cAMP in
response to ligand exposure. This result could then be
followed up with subcellular localization of the truncated
Mclr protein to determine if this mutant receptor reaches the
plasma membrane. Such subcellular localization may require
the use of an epitope-tagged Mclr protein because it has so
far been very difficult to raise anti-Mclr antisera. If the
truncated Mclr is indeed directed to the plasma membrane, it
would be interesting to study its interaction, or lack
thereof, with the heterotrimeric G-proteins.
The canine agouti project also lends itself to future
experiments. I would be very interested in experiments
directed at the identification of other agouti mRNA isoforms.
The banding of hairs is several breeds suggests that, like
the mouse, the domestic dog possesses hair cycle-dependent
expression of the agouti protein. It is likely that this hair
cycle-specific expression is directed by a hair cycle
promoter. Identification of alternative 5' untranslated exons
could be achieved by 5'RACE procedures on RNA isolated from
the midportion of the hair-growth cycle in the appropriate
breed. These exons could then be mapped in relation to those
identified in this study to coitplete the characterization of
the canine agouti genomic structure.
The studies presented here demonstrate the conservation
of ventral-specific regulatory elements. Limited sequence
78
analysis suggests that the regulatory protein may be a member
of the helix-loop-helix family of transcription factors.
Before characterization of this E-box element is undertaken
in earnest, the 14 Kbp transgene should be sequenced in its
entirety. The more genomic DNA sequence to conpare between
the mouse and dog, the more significant any conserved
stretches will become. If no other elements are conserved,
the focus should then be brought back to the 35 bp E-boxcontaining element. The iirportance of this element could be
tested in two ways. Firstly, the 14 Kbp transgene
specifically lacking this 35 bp would be a very direct test
of this element. Likewise, a multimer of this 35 bp could be
tested in transgenic mice for the ventral-specific enhancer
activity. Other blocks of conserved sequence could be tested
in the same manner, were they to be identified in future
sequence comparisons.
Finally, the cDNA and deduced amino acid sequence
presented here comes from a single, siitply-patterned breed.
It is conceivable that sequence variations within the
protein-coding exons could be identified in other breeds.
Sequence polymorphisms which correlate with changes in agouti
patterning would then be open to future work as outlined
above for the Mclr protein.
I had a lot of fun completing this work and I wish those
who follow me in this endeavor the best of luck. I'll be
watching for their progress.
79
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