Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: MELANOCORTIN-4 RECEPTOR GENE AND USE AS A GENETIC
MARKER FOR FAT CONTENT, WEIGHT GAIN, AND/OR FEED --
CONSUMPTION OF ANIMALS
CROSS-REFE HTTCE TO F.LATED APPL.ICATIONy
This application claims the benefit of U.S. Patent No. 5,935,784 which was
filed on March 6, 1997.
GRANT RFFF FNC'F ('_ .A[ JS .
This invention was supported at least in part by grants from the United States
Department of Agriculture through the Iowa AgriculUue and Home Economics
Experiment
Station (IaHees) and Project Number IOW03148 (Hatch Funds). The United States
government may have certain rights in this invention.
FIEi .D OF THE INVENTION
The present invention relates to a method of genetically evaluating animals by
assaying for the presence of at least one genetic marker which is indicative
of one or more
of the traits of fat content, growth rate, and feed consumption. In
particular, the method
analyzes for variation in the melanocortin-0 receptor (MC4R) gene which is
indicative of
these traits. Even more particularly, the method analyzes for a polymorphism
in the MC4R
gene.
$ACKGROUND OF THE INVENTION
There is an increasing consumer demand for meat products having low fat
content.
This demand is fueled by accumulating evidence in the scientific literature
that a high
consumption of animal fat, especially fat with a high proportion of saturated
fatty acids,
represents a significant health hazard, including risk for cardiovascular
disease. Other
health concems associated with high fat meats include
their high content of cholesterol and the addition of relatively high amounts
of salt which
are added to improve the binding characteristics since salt aids in extracting
the native
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water binding component myosin from the meat. Furthermore, an increasing
number of
consumers find meat products containing chemical additives such as phosphates,
emulsifying additives, and anti-oxidants less acceptable.
Faced with consumers who seek a healthier meat product, meat producers are
continually pressed to offer cheaper and healthier products.
Cheaper products, of course, come from lowering costs of production. Producers
are always interested in improving the growth rate and feed conversion of
their animals.
Lower production costs come from the shorter time to market and lower costs of
feeding an
animal. This can increase the profit margin in the livestock industry and/or
result in lower
prices to the consumer.
By being able to select for animals which have the aforementioned traits,
producers
can raise animals with these desirable characteristics. Selection for
desirable traits has
traditionally been done using breeding techniques.
Genetic differences exist among individual meat producing animals as well as
among breeds which can be exploited by breeding techniques to achieve animals
with these
desirable characteristics. For example, Chinese breeds are known for reaching
puberty at
an early age and for their large litter size, while American breeds are known
for their
greater growth rates and leanness. Thus, it would be desirable to combine the
best
characteristics of both types of these breeds, thereby improving pork
production.
Often, however, heritability for desired traits is low, for example,
heritability for
litter size is around l0%-15%. Standard breeding methods which select
individuals based
upon phenotypic variations do not take fully into account genetic variability
or complex
gene interactions which exist. Therefore, there is a need for an approach that
deals with
selection for leanness, growth rate, and feed consumption at the cellular or
DNA level.
This method will provide a method for genetically evaluating animals to enable
breeders to
more accurately select those animals which not only phenotypically express
desirable traits
but those which express favorable underlying genetic criteria. This has
largely been
accomplished to date by marker assisted selection.
Restriction fragment length polymorphism (RFLP) analysis has been used by
several groups to study pig DNA. Jung et al., Theor. ppl. Genet_, 77:271-274
(1989),
discloses the use of RFLP techniques to show genetic
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variability between two pig breeds. Polymorphism was ciemonstrated for
awine leukocyte antigen (SLA) Class I genes in these breeds. Hoganson et al.,
Abetract for AnnyaLMeetin of i Midw stern Section of theAmerican Societv of
Animal Science. March 26-28, 1990, reports
on the polymorphism of swine major histocompatibility complex (MfflC) genes
for Chinese pigs, also demonstrated by RFLP analysis. Jung et al., Animal
Gengtics. 26:79-91 (1989), reports on RFLP
analysis of SLA ClasH I genes in cxrtain boars. The authors state that the
results suggest that there may be an association between ewine SY.AlMHC
i0 Claea I genes and production and performance traits. They further state
that
the use of SLA Class [ restriction fragments, as genetic markers, may have
potential in the future for improving pig growth performance.
The ability to follow a specific favorable genetic allule involves a novel
and lengthy process of the identiiScation of a DNA molecular marker for a
major effect gene. The marker may be linked to a single gene with a major
effect or linked to a number of genes with additive effects. DNA markers have
several advantages; sc:gregation is easy to measure and is unambiguous, and
DN4 markers are co-clominant, i.e., heterozygous and honiozygoua animal8
can be distinctively identified. Onoe a marker eystem i8 established selection
;zo decisions can be diatinctivel,j- identified. Once a marker syetem is
established
selection decisions could be made very easily, since DNA markers can be
asaayed any time after a tissue or blood sample caabe collected from the
individual infant aniataL
The use of een,e tic differences in receptor genes has become a valuable
-is marker system for seltiction. For example, United States Patents 5,550,024
and 5,374,526 issued to Rothschild et al. disclose a polymorphism in the pig
eatzogen receptor gene which is associated with larger littar size, the
disclosure of which is incorporated herein by refer-a=.
United States patent number 5,935,784 discloses polymorphic markers in
the pig prolactin receptor gene which are associated with larger litter size
and overall
reproductive efficiency.
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1338-= +40 89 222:3:394465 : It 9
13-09-2000 US 009916862
WO 00/06777 PCTIUS99/16862
WO-A-97/47316 discloses that mutations in the MC4R protein exist in
extremely obese human patients and that a predisposition to body weight
disorders can be ascertained by testing for mutations in i.he MC4R gene.
However, Gotoda et ill., Diabetologia 40 (1997) 976 disclose a lack of
correlation between tl particular point mutation in the MC4R gene (VaII03Ile)
and obesity in white human males. There was therefore no indication in the
art of a correlation bE-tween the MC4R gene and a means for selecting animals
with improved metabolic traits.
It can be seen irom the foregoing that a need exists for a method for
t0 selecting animals with the improved metabolic traits regarding fat content,
growth rate, and feed consumption.
3/A
AMENDED SHEET
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SUMMARY OF THE INVENTION
An object of the present invention is to provide a genetic marker based on or
within -
the MC4R gene which is indicative of fat content, growth rate, and/or feed
consumption.
Another object of the invention is to provide an assay for determining the
presence
of this genetic marker.
A further object of the invention is to provide a method of evaluating animals
that
increases accuracy of selection and breeding methods for the desired traits.
Yet another object of the invention is to provide a PCR amplification test
which
will greatly expedite the determination of presence of the marker.
An additional object of the invention is to provide a kit for evaluating a
sample of
animal DNA for the identified genetic marker.
These and other objects, features, and advantages will become apparent after
review
of the following description and claims of the invention which follow.
This invention relates to the discovery of a polymorphism within the
melanocortin-
4 receptor (MC4R) gene which is associated with fat content, growth rate, and
feed
conversion traits in animals. According to the invention, the association of
the MC4R
polymorphism with the trait(s) enables genetic markers to be identified for
specific breeds
or genetic lines. The TaqI restriction pattern which identifies the
polymorphism is used to
assay for the presence or absence of markers associated with the desirable
metabolic traits.
The breed-dependent marker genotype (i.e., a marker in some breeds and a
nonmarker in
others) consists of a polymorphism within MC4R, a guanine to adenine
transition at
position 678 of the PCR product (a missense mutation of aspartic acid codon
(GAU) into
asparagine codon (AAU) at position 298 amino acid of the MC4R protein). The
invention
includes assays for detection of the marker as well as the sequence
characterization of the
polymorphism and includes novel sequences in the MC4R gene which may be used
to
design amplification primers for such an assay. Additionally, the invention
includes a
method for using the assay in breeding programs for animal selection and a kit
for
performing the assay.
Definitions
As used herein, "low fat content" or "leanness" means a biologically
significant
decrease in body fat relative to the mean of a given population.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the sequence listing for MC4R in pigs (SEQ ID NO:1). "X"
represents
the site of the polymorphism.
Figure 2 represents a comparison of the DNA sequence between the human (SEQ
ID NO:2) and the porcine (SEQ ID NO:3) MC4R gene.
Figure 3 represents a comparison of the amino acid sequence between the human
(SEQ ID NO:4) and the porcine (SEQ ID NO:5) MC4R gene.
Figures 4a, 4b, and 4c are linkage reports for MC4R from CRI-MAP.
Figure 5 depicts partial nucleotide and amino acid sequences (SEQ ID NO: 12)
of
the porcine MC4R gene. The amino acid translation shows an amino acid
substitution at
codon 298.
Figure 6 is an electrophoresis gel of TaqI digestion of the PCR product.
Molecular
marker (M) and MC4R genotypes are indicated at the top of each lane.
Figure 7 depicts multiple-alignments of the putative seventh transmembrane
domain of porcine MC4R with other MCRs and GPCRs. The "*" represents the
predicted
sequence positions for porcine MC4R. The other amino acid sequences were
obtained
from the GenBank database (accession numbers P32245, P70596, P41983, P56451,
P34974, P41968, P33033, Q01718, Q01726, Q28031, AF011466, P21554, P18089,
P30680, P47211). The missense variant in porcine MC4R substituted amino acid N
for D
in the position marked with an arrow. The Asp (D) residue is highly conserved
among
MCRs, and the Asn (N) residue is well conserved in most other GPCRs.
DETAILED DESCRIPTION OF THE PREFEIZRFD EMBODI--ENT
Obesity is a disease affecting energy balance. The control of energy
metabolism is
simple: store excessive energy as fat and manage the energy to avoid
superfluous energy
storage, i.e., obesity. Although several genes and signaling systems have been
implicated
in obesity, there has been little known about the interconnection of energy
homeostatic
mechanism and genetic polymorphism. The melanocortin-4 receptor (MC4R) has
been
3 0 shown to be an important mediator of long term weight homeostasis. MC4R
antagonists
can increase food intake and body weight during chronic administration.
Skuladottir, G.V.,
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et al., "Long term orexigenic effect of a novel melanocortin 4 receptor
selective
antagonist", British J. of Pharm., 126(l):27-34 (1999).
Lu et al. (1994) suggested that the melanocortin receptors are involved in
controlling food intake and energy balance through studying its antagonism to
the agouti
obesity syndrome. Huszar et al. (1997) found that inactivation of the
melanocortin-4
receptor gene (MC4R) resulted in a maturity onset obesity syndrome in mice and
demonstrated a major role of MC4R protein in the regulation of energy balance
related to
the agouti obesity syndrome. In addition, the MC4R protein mediates the
effects of leptin,
one of the important signaling molecules in energy homeostasis (Seeley et al.
1997).
According to the present invention, a variant or polymorphism in the MC4R gene
has been located, and this genetic variability is associated with phenotypic
differences in
the metabolic traits of fat content, growth rate, and/or feed consumption.
In one embodiment of the invention, an assay is provided for detection of
presence
of a desirable genotype. The assay involves amplifying the genomic DNA
purified from
blood, tissue, semen, or other convenient source of genetic material by the
use of primers
and standard techniques, such as the polymerase chain reaction (PCR), then
digesting the
DNA with a restriction enzyme (e.g., Taq 1) so as to yield gene fragments of
varying
lengths, and separating at least some of the fragments from others (e.g.,
using
electrophoresis).
The fragments may also be detected by hybridizing with a nucleotide probe
(e.g.,
radio-labeled cDNA probes) that contains all or at least a portion of the MC4R
gene cDNA
sequence to the separated fragments and comparing the results of the
hybridization with
assay results for a gene sequence known to have the marker or a sequence known
to not
have the marker. Selection and use of probes for detection of MC4R sequences
based on
the known and disclosed MC4R sequences is generally known to those skilled in
the art.
The probe may be any sequence which will hybridize to the separated digestion
products
and allow for detection.
Another embodiment of the invention provides a kit for assaying the presence
in a
MC4R gene sequence of a genetic marker. The marker being indicative of
inheritable traits
of fat content, growth rate, and/or feed consumption. The kit in a preferred
embodiment
also includes novel PCR primers comprising 4-30 contiguous bases on either
side of the
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polymorphism to provide an amplification system allowing for detection of the
Taq I
polymorphism by PCR and Taq I digestion of PCR products. The preferred primers
are
SEQ ID NO:8 and SEQ ID NO:9.
A further embodiment comprises a breeding method whereby an assay of the above
type is conducted on a plurality of gene sequences from different animals or
animal
embryos to be selected from and based on the results, certain animals are
either selected or
dropped out of the breeding program.
According to the invention, the polymorphism in the MC4R gene, identifiable by
the Taq I restriction pattern, is disclosed. As is known in the art,
restriction patterns are not
exact determinants of the size of fragments and are only approximate. The
polymorphism
is identifiable by three bands from a Taq I digestion of the PCR product, 466,
225, and 76
base pairs (bp) for one homozygous genotype (allele 1); two bands, 542 and 225
bp for
another homozygous genotype (allele 2); and four bands for the heterozygous
genotype
(542, 466, 225, and 76 bp). The marker for leanness and lower feed intake is
identifiable
by the 466/225/76 bands, except for the Chinese pigs, where the Chinese pigs'
marker for
leanness is the 542/225 bands. The marker for faster rate of gain is
identifiable by the
542/225 bands.
In addition, the polymorphism associated with the pattem has been identified
at the
nucleotide level. The polymorphic Taq I site was sequenced along with the
general
surrounding area. See SEQ ID NO: 1. The sequences surrounding the polymorphism
have
facilitated the development of a PCR test in which a primer of about 4-30
contiguous bases
taken from the sequence immediately adjacent to the polymorphism is used in
connection
with a polymerase chain reaction to greatly amplify the region before
treatment with the
Taq I restriction enzyme. The primers need not be the exact complement;
substantially
equivalent sequences are acceptable.
From sequence data, it was observed that in allele 2 the guanine is
substituted with
an adenine at position 678 of the PCR product or at position 298 amino acid of
the MC4R
protein changing the aspartic acid codon (GAU) into an asparagine codon (AAU).
The
PCR test for the polymorphism used a forward primer of 5'-TGG CAA TAG CCA AGA
3 0 ACA AG-3' (SEQ. ID NO: 6) and a reverse primer of 5'-CAG GGG ATA GCA ACA
GAT GA-3' (SEQ. ID NO: 7). Pig specific primers used were a forward primer of
5'-TTA
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AGT GGA GGA AGA AGG-3' (SEQ. ID NO: 8) and a reverse primer of 5'-CAT TAT
GAC AGT TAA GCG G-3' (SEQ ID NO:9). The resulting amplified product of about
7SO -
bp, when digested with Taq I, results in allelic fragments of 466, 225, and 76
bp (allele 1)
or 542 and 225 bp (allele 2).
The marker may be identified by any method known to one of ordinary skill in
the
art which identifies the presence or absence of the marker, including for
example, single-
strand conformation polymorphism analysis (SSCP), RFLP analysis, heteroduplex
analysis, denaturing gradient gel electrophoresis, and temperature gradient
electrophoresis,
ligase chain reaction or even direct sequencing of the MC4R gene and
examination for the
Taq I RFLP recognition pattern.
One or more additional restriction enzymes and/or probes and/or primers can be
used. Additional enzymes, constructed probes, and primers can be determined by
routine
experimentation by those of ordinary skill in the art.
Other possible techniques include non-gel systems such as TaqManTM (Perkin
Elmer). In this system, oligonucleotide PCR primers are designed that flank
the mutation
in question and allow PCR amplification of the region. A third oligonucleotide
probe is
then designed to hybridize to the region containing the base subject to change
between
different alleles of the gene. This probe is labeled with fluorescent dyes at
both the 5' and
3' ends. These dyes are chosen such that while in this proximity to each other
the
fluorescence of one of them is quenched by the other and cannot be detected.
Extension by
Taq DNA polymerase from the PCR primer positioned 5' on the template relative
to the
probe leads to the cleavage of the dye attached to the 5' end of the annealed
probe through
the 5' nuclease activity of the Taq DNA polymerase. This removes the quenching
effect
allowing detection of the fluorescence from the dye at the 3' end of the
probe. The
discrimination between different DNA sequences arises through the fact that if
the
hybridization of the probe to the template molecule is not complete, i.e.,
there is a
mismatch of some form, the cleavage of the dye does not take place. Thus, only
if the
nucleotide sequence of the oligonucleotide probe is completely complementary
to the
template molecule to which it is bound will quenching be removed. A reaction
mix can
contain two different probe sequences each designed against different alleles
that might be
present, thus, allowing the detection of both alleles in on reaction.
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Though the use of RFLPs is one method of detecting the polymorphism, other
methods known to one of ordinary skill in the art may be used. Such methods
include ongs
that analyze the polymorphic gene product and detect polymorphisms by
detecting the
resulting differences in the gene product.
Though the preferred method of separating restriction fragments is gel
electrophoresis, other alternative methods known to one skilled in the art may
be used to
separate and determine the size of the restriction fragments.
It is possible to indirectly select for the polymorphism with alternative DNA
markers. It is possible to establish a linkage between specific alleles of
alternative DNA
markers and alleles of DNA markers known to be associated with the MC4R gene
which
have previously been shown to be associated with a particular trait. Examples
of markers
on the published PiGMaP chromosome map which are linked to the MC4R gene
include
S0331, BHT0433, and S0313.
The reagents suitable for applying the methods of the present invention may be
packaged into convenient kits. The kits provide the necessary materials,
packaged into
suitable containers. At a minimum, the kit contains a reagent that identifies
the
polymorphism in the MC4R gene that is associated with the traits of interest,
fat content,
growth rate, and feed consumption. Preferably, the reagent that identifies the
polymorphism is a PCR set (a set of primers, DNA polymerase, and four
nucleoside
triphosphates) that hybridize with the MC4R gene or a fragment thereof.
Preferably, the
PCR set and restriction enzyme that cleaves the MC4R gene in at least one
place are
included in the kit. Preferably, the kit further comprises additional means,
such as
reagents, for detecting or measuring the detectable entity or providing a
control. Other
reagents used for hybridization, prehybridization, DNA extraction,
visualization, and
similar purposes may also be included, if desired.
The genetic markers, methods, and kits of the invention are useful in a
breeding
program to improve fat content, growth rate, and feed consumption
characteristics in a
breed, line, or population of animals. Continuous selection and breeding of
animals that
are at least heterozygous and preferably homozygous for the desired
polymorphism
associated with the particular trait would lead to a breed, line, or
population having those
desired traits. Thus, the marker is a selection tool.
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The following examples are offered to illustrate, but not limit the invention.
EXAMPLE 1
Melanocortin 4 Receptor PCR-RFLP Test - TaqI polymorphism and Genetic Linkage
Mapping of MC4R Gene
Primers:
Primers were designed from homologous regions of human and rat MC4R
sequences (Genbank Accession No. s77415 and u67863, respectively). These
primers were
used to amplify a 750-bp sequence of the porcine MC4R gene.
MC4R1: 5' TGG CAA TAG CCA AGA ACA AG 3' (SEQ ID NO:6)
MC4R4: 5' CAG GGG ATA GCA ACA GAT GA 3' (SEQ ID NO:7)
PCR Conditions:
Mix 1: l OX Promega Buffer 1.0 L
mM MgCl2 0.6 L
dNTPs mix (2.5mM each) 0.5 L
25 pmoV L MC4R1 0.1 L
25 pmol/ L MC4R4 0.1 L
dd sterile H20 7.5 L
Taq Polymerase (5 U/ L) 0.07 L
Genomic DNA (12.5 ng/ L) 1.0 L
Ten L of Mix 1 and DNA were combined in reaction tube, then overlaid with
mineral oil. The following PCR program was run: 94 C for 2 min.; 35 cycles of
94 C for
20 30 sec.; 58 C 1 min., and 72 C 1 min. 30 sec.; followed by a final
extension at 72 C for 15
min.
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Five l of the PCR reaction product was checked on a standard 1% agarose gel
to
confirm amplification success and clean negative control. Product size is
approximately ,
750 base pairs. Digestion was performed by the following procedure.
TaqI Digestion Reaction 10 IaL reaction
PCR product 5.0 L
l OX TaqI NE Buffer 1.0 L
BSA (10mg/ml) 0.1 L
Taql enzyme (20 U/ L) 0.5 L
dd sterile H20 3.4 L
A cocktail of the buffer, enzyme, BSA, and water was made. Five L was added
to
each reaction tube containing the DNA. The mixture was then incubated at 65 C
for at
least 4 hours to overnight. Loading dye was mixed with the digestion reaction
and the total
volume was loaded on a 3% agarose gel. The major bands for allele I are about
466, 225,
and 76 bp. The allele 2 genotype bands are 542 and 225 bp. The heterozygote
genotype
has both allele I and allele 2.
R t
The amplified PCR product is about 750 bp. The sequence of the PCR product
confirmed that the PCR product is MC4R gene with 97.6%, and 92.2% identities
at the
amino acid and DNA level, respectively, to corresponding human sequences. (see
Figs. 2
and 3).
The Taql digestion of the PCR product produced allelic fragments of 466, 225,
and
76 bp (allele 1), or 542 and 225 bp (allele 2). The heterozygote genotype has
both types of
alleles. Mendelian inheritance was observed in three three-generation
international
reference families, which were used to map this gene by linkage analysis.
The polymorphism between allele 1 and allele 2 resulting from a G-* A
transition
at position 678 of the PCR product revealed a missense mutation of Aspartic
acid codon
(GAU) into Asparagine codon (AAU) at position 298 amino acid of MC4R protein.
(See
Figure 1, SEQ ID NO:1).
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Allele frequencies were determined by genotyping of DNA samples from a small
number of animals from different breeds (Table 1). Allele I was observed with
a
frequency of 1 in Meishan, but was not observed or observed at very low
frequency in
Hampshire and Yorkshire. The frequencies of allele 1 in Landrace and Chester
White were
0.5, respectively.
Figures 2 and 3 illustrate the differences between the DNA and amino acid
sequences of the human and porcine MC4R gene (SEQ ID NOS:2-5).
TABLE 1
The Frequencv of Allele 1 in Different Pig Breeds
Breed # Animals Freq. Allele 1
Meishan 8 1
Large White 8 0.56
Yorkshire 6 0.08
Hampshire 5 0
Landrace 4 0.5
Chester White 4 0.5
Minzu 2 1
Wild Boar 2 1
Linkage Analyses
Two-point and multi-point linkage analyses were performed on the genotypes of
international reference families. See Figs. 4a-4c. The data were analyzed by
using the
CRI-MAP program. MC4R was significantly linked to several markers on porcine
chromosome (SSC) 1. The most closely linked markers (recombination fraction
and LOD
score in parentheses) are S0331 (0.02, 21.97), BHT0433 (0.02, 21.32), and
S0313 (0.00,
17.76) by two-point linkage analysis. A multi-point linkage analysis produced
the best
map order of markers and MC4R (with distance in Kosambi cM): KGF-5.8-CAPN3-2.5-
MEF2A-6.1-MC4R-5.6-S0313.
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Somatic cell hybrid panel of pig and rodent was used to assign MC4R to a
cytogenetic region. PCR products from pig specific primers were amplified in
clones 7, $_
16, 18, and 19. MC4R was localized to SSCIq 22-27.
EXAMPLE 2
MC4R Receptor PCR-RFLP Test using Pig Specific Primers and Genetic Linkage
Mapping of the Porcine MC4R Gene
PigSnecific Primer Sequences
Forward primer: 5'-TTA AGT GGA GGA AGA AGG-3' (SEQ ID NO:8)
Reverse primer: 5'-CAT TAT GAC AGT TAA GCG G-3' (SEQ ID NO:9)
Method of Detection
The PCR reaction was performed using
Porcine genomic DNA 12.5 ng
1 x PCR buffer
MgCIZ 1.5 mM
dNTP 0.125 mM
Forward primer 0.3 M
Reverse primer 0.3 M
Taq DNA polymerase (Promega) 0.35 U
in a 10 L final volume. The PCR profile included 2 min. at 94 C; 35 cycles of
30 sec. at
94 C, 1 min. at 56 C, 1 min. 30 sec. at 72 C; and 15 min. at 72 C in a
Robocycler
(Statagene, La Jolla, CA). A 5.0 L aliquot of the PCR products was digested
in a total
volume of 10 L with 10 U of Taql incubated overnight at 65 C. The digestion
products
were electrophoresed on a 3% agarose gel.
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Description of Pol,ymornhism
The TaqI digestion of the PCR product produced fragments of 466, 225, and 76
bR -
in allele I and 542 and 225 bp in allele 2. The heterozygous genotype has
fragments of
both allele 1 and allele 2.
Pattern of Inheritance
Autosomal segregation of Mendelian inheritance was observed in three three-
generation European PiGMaP families (Archibald et al., 1995).
Allele Frequencies
Allele frequencies were determined by genotyping the grandparental animals of
the
European PiGMaP families and unrelated animals from ISU reference families.
Allele 1
was observed with the following frequencies.
TABLE2
The Frequency of Allele I in Different Pig Breeds
Breed # Animals Freq. Allele 1
Meishan 8 1
Large White 8 0.56
Yorkshire 10 0.15
Hampshire 12 0
Landrace 8 0.56
Chester White 8 0.56
Minzu 2 1
Wild Boar 2 1
Chromosomal Location
Two-point and multi-point linkage analysis were performed on the genotypes of
three PiGMaP families using the CRI-MAP program (Green et al. 1990). MC4R was
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significantly linked to several markers on porcine chromosome 1(SSC 1). The
most
closely linked markers (recombination fraction and LOD score in parentheses)
are S0331 -
(0.02, 21.97), BHT0433 (0.02, 21.32), and S0313 (0.00, 17.76) according to two-
point
linkage analysis. The best map order of MC4R with respect to other linked
markers
produced by multi-point linkage analysis is as follows (with distance in
Kosambi cM):
KGF-5.8-CAPN3-2.5 -MEF2A-6.1-MC4R-5.6-SO313.
Comments
The Melanocortin-4 Receptor is a G protein-coupled, seven-transmembrane
receptor expressed in the brain. Huszar et al. (1997) found that inactivation
of MC4R gene
resulted in a maturity onset obesity syndrome in mice and demonstrated a major
role of
MC4R protein in the regulation of energy balance. The MC4R gene has been
mapped to
human chromosome 18q21.3 (Gantz et al., 1993). The localization of MC4R gene
to SSC
1 is consistent with previous chromosome painting data indicating synteny
between this
chromosome and HSA 18 and 15 (Goureau et al., 1996). However, the gene order
of
several genes previously mapped from HSA 18 and 15 to SSC 1, including CAPN3,
KGF,
and MEF2A, is not conserved with MC4R. Therefore, mapping of MC4R to SSC 1 may
identify an evolutionary breakpoint between HSA 18 and 15 in relation to SSC
1.
EXAMPLE 3
Association of Marker with Enhanced Metabolic Characteristics
In a preliminary study to determine which allele is associated with which
trait and
in which breeds, the genotypes of several lines of animals were correlated
with days to 110
kg, backfat measurements, daily gains, and average daily feed intake. The pigs
used in the
study were from lines from Pig Improvement Company (PIC).
Data was accumulated using the PCR test described supra for the I and 2 allele
of
the MC4R gene. The collected data is summarized in Tables 3-8 below.
According to the results, allele 1 is the significantly leaner allele (see P2
backfat
measurements) in all lines except in Chinese pigs where it is the fat allele.
Allele 2 is
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associated with significantly faster rate of gain (test daily gain) in the
tested commercial
lines. Overall allele 1 is associated with lower feed intake.
TABLE 3
Number of observations
MC4R L02 L03 L19 L65 Overall L95
genotype
11 88 30 32 150 20
12 57 54 56 74 241 67
22 12 31 254 33 330 37
Total 721
MC4R genotype:
11 = homozygous allele 1
12 = heterozygous
1 o 22 = homozygous allele 2
TABLE 4
Number of observations (males/femalesl
MC4R L02 L03 L19 L65 Overall L95
genotype
11 9/79 12/18 15/17 36/114 0/20
12 9/48 37/17 12/44 44/30 102/139 0/67
22 3/9 28/3 89/165 21/12 141/189 0/37
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TABLE 5
DaXs to 110kg
MC4R L02 L03 L19 L65 Overall L95
genotype
11 169.7 172.4 169.6 168.5 219.1
12 170.2 171.5 165.0 171.2 168.7 212.2
22 165.3 173.4 162.9 170.3 167.1 211.4
P<.23 P<.75 P<.15 P<.76 P<.31 P<.27
TABLE 6
P2 backfat (mm)
MC4R L02 L03 L19 L65 Overall L95
Genotype
11 10.8 11.9 9.7 11.1 22.8
12 11.3 12.5 12.2 10.5 11.8 21.5
22 12.1 12.7 12.6 10.7 12.1 20.3
P<.10 P<.43 P<.34 P<.17 P<.006 P<.17
TABLE 7
Test daily gain l,gm/dl
MC4R L02 L03 L19 L65 Overall L95
genotype
11 882.2 811.0 881.8 871.9 688.8
12 891.2 820.5 875.6 873.0 876.3 676.2
22 969.1 819.5 906.7 906.2 906.9 692.5
P<.01 P<.96 P<.05 P<.24 P<.006 P<.66
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TABLE 8
Average daily feed intake (k~/d), bo r only. except L95 which was eilts only
MC4R L02 L03 L19 L65 Overall L95
genotype
11 2.31 1.78 1.75 1.89 2.05
12 2.11 1.90 1.97 1.90 1.96 2.03
22 2.15 1.97 2.00 1.97 2.02 2.08
P<.84 P<.14 P<.56 P<.14 P<.16 P<.36
EXAMPLE 4
A Missense Variant of the Porcine Melanocortin-4 Receptor (MC4R) Gene is
Associated with
Fatness, Growth, and Feed Intake Traits
To determine if there was an association of this MC4R polymorphism with
phenotypic variation the mutation was tested in a large number of individual
animals from
several different pig lines. Analyses of growth and performance test records
showed
significant associations of MC4R genotypes with backfat, growth rate and feed
intake in a
number of lines. It is probable that the variant amino acid residue of the
MC4R mutation
causes a significant change of the MC4R function. These results support the
functional
significance of a pig MC4R missense mutation and suggest that comparative
genomics
based on model species may be equally important for application to farm
animals as they
are for human medicine.
Identification of mutations in the leptin and the leptin receptor has provided
some
information on genetic components involved in the regulation of energy balance
(Zhang et
al. 1994; Tartaglia et al. 1995). Genetic studies using animal models have
facilitated the
identification of major genetic causes of obesity (Andersson 1996; Pomp 1997;
Giridharan
1998). Furthermore, several other genes involved in the neural signaling
pathway of
energy homeostasis have been identified (Flier and Maratos-Flier 1998;
Schwartz et al.
1999). Of particular interest among candidate signaling molecules involved in
the
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regulation of energy homeostasis is the melanocortin-4 receptor (MC4R). The
MC4R
response to leptin signaling is a link between food intake and body weight
(Seeley et al. 0
-
1997; Marsh et al. 1999). Neuropeptide Y (NPY) signaling in the central
nervous system
is also mediated by the MC4R protein (Kask et al. 1998). Several mutations in
MC4R
including frameshift and nonsense mutations are associated with dominantly
inherited
obesity in humans (Vaisse et al. 1998; Yeo et al. 1998). Some other MC4R
missense
mutations in humans have also been identified (Gotoda et al. 1997; Hinney et
al. 1999) but
the functional significance of these mutations has not been characterized.
Selection based on growth characteristics has been of great importance to the
pig
1 o industry because of costs associated with feeding and consumer preference
for lean meat.
Efficient genetic improvement in these quantitative traits may be augmented
through the
use of marker assisted selection (MAS) using high density genetic maps
(Dekkers and van
Arendonk 1998; Rothschild and Plastow 1999). An important tool in this process
is
comparative mapping using the well-developed human and mouse gene maps, which
assist
in the identification of corresponding genomic regions or major genes
controlling growth
and performance traits in the pig. Biological understanding of complex traits
in human or
model species offers an alternative approach to identify genes responsible for
the traits of
economic interest in livestock. Several quantitative trait loci (QTL) linkage
scans using
phenotypically divergent breeds and candidate gene analyses have been
successfully
conducted for fatness and growth traits (Yu et al. 1995; Casas-Carrillo et al.
1997; Knorr et
al. 1997; Knott et al. 1998; Rohrer et al. 1998; Wang et al. 1998; Paszek et
al. 1999), but
no individual genes with major effects on growth and perfonnance traits have
yet been
established for commercial populations. The role of MC4R in feed intake and
obesity
suggests it may be an important genetic marker for the growth-related traits
in the pig.
Materials and Methods
Animals. Pigs were raised under normal production conditions under the care of
PIC employees in nucleus farms in the United States and Europe. Pigs were put
on the
performance test at approximately 70 days of age and taken off test after 13
weeks. At the
end of the trial backfat was measured ultrasonically in real time (B mode) at
the 10' rib 2
cm from the centerline. Average daily gain (growth) over the test period was
calculated as
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weight gained divided by days on test. Days to 110 kg market weight was
estimated using
standard procedures and feed intake was measured using individual electronic
measurement equipment.
PCR amplification of a nig MC4R PnP fragment, Primers were designed from
homologous regions of human and rat MC4R sequences (GenBank accession no.
s77415
and u67863, respectively). The primers were: forward primer: 5'-TGG CAA TAG
CCA
AGA ACA AG-3' (SEQ. ID NO:6) and reverse primer: 5'-CAG GGG ATA GCA ACA
GAT GA-3' (SEQ. ID NO:7). The PCR reaction was performed using 12.5 ng of
porcine
genomic DNA, lx PCR buffer, 1.5 mM MgC12, 0.125 mM dNTPs, 0.3 mM of each
primer,
and 0.35 U Taq DNA polymerase (Promega) in.a lO L final volume. The conditions
for
PCR were as follows: 2 min at 94 C; 35 cycles of 30 s at 94 C, 1 min at 56 C,
1 min 30 s
at 92 C, and a final 15 min extension at 72 C in a Robocycler (Stratagene, La
Jolla, CA).
Sequencing and mutation detection. Sequencing of the PCR products from several
individual pigs of different breeds was conducted and the sequences were
compared to
detect any nucleotide change. Sequencing was performed on an ABI sequencer 377
(Applied Biosystems). The porcine MC4R sequence has been submitted to GenBank,
and
has accession number AF087937. The sequence analysis revealed one nucleotide
substitution situated within a Taql restriction enzyme recognition site (Kim
et al. 1999). A
set of primers was then designed to generate a smaller MC4R gene fragment,
which
contained only one informative TaqI restriction site to specify the
polymorphic site and to
facilitate the PCR-RFLP test. These primers were: forward 5'-TAC CCT GAC CAT
CTT
GAT TG-3' (SEQ. ID NO:10) and reverse: 5'-ATA GCA ACA GAT GAT CTC TTT G-3'
(SEQ. ID NO:11).
Statistical analysis. Analysis of variance procedures were used with a mixed
model
that accounted for the fixed effects of farm, test period, sex of the animal,
the MC4R
genotype and site (random). All animals in lines of American/European descent
(Lines A-
D) were pooled for the overall analysis and in this analysis line of origin
was included.
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Mean effects were estimated for each genotype and are presented in Tables 9-
15. Overall F
tests were used to determine level of significance.
Results
Identification of a missense mutation in the pjg MC4R gene. The MC4R gene
consists of approximately 1 kb of coding sequence contained within a single
exon. About
750 bp of a pig MC4R gene fragment was produced by PCR (Kim et al. 1999). The
sequence of the PCR product confirmed that the PCR product is the MC4R gene
with
92.2% and 97.6% identities at nucleotide and the amino acid levels,
respectively, to the
human MC4R sequence. Multiple alignments of the sequences from individual
animals of
several breeds identified a single nucleotide substitution (G->A; Fig. 5). The
polymorphism revealed a missense mutation that replaces aspartic acid (GAU)
with
asparagine (AAU) at the position identical to amino acid 298 of human MC4R
protein. To
confirm this base change, we designed pig-specific primers flanking the
polymorphic site
and analyzed the polymorphism as a TaqI PCR-RFLP gel (Fig. 6). Figure 6 shows
a Taql
digestion of the PCR product analyzed by agarose-gel electrophoresis. Allele I
produced
156 and 70 bp fragments and allele 2 produced a 226 bp fragment as the PCR-
RFLP. The
heterozygote has both allele I and 2 fragments. Molecular marker (M) and MC4R
genotypes are indicated at the top of each lane.
The MC4R missense mutation is within a highly conserved region a_mong
melanocortin recentors (MCR). The MCR is a subfamily of G-protein coupled
receptors
(GPCR) containing certain conserved structural elements common to most other
GPCRs,
but overall amino acid identities between MCR and other GPCRs are low (Tatro
1996). A
multiple-alignment of the predicted amino acid sequences of the pig MC4R with
MC4R
proteins from other species, other MCR proteins, or representative GPCRs
showed that the
aspartic acid found at position 298 of the seventh transmembrane domain is
very highly
conserved in the MCR proteins (Fig. 7). It is interesting to note, however,
that this position
is occupied by asparagine in most other GPCRs. The MCR proteins show 40-80%
amino
acid identity with each other (Tatro 1996), but the second intracytoplasmic
loop and the
seventh transmembrane domain are highly conserved among MCR proteins (Gantz et
al.
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1993). Some of the relationships between MCR structure and function have been
discovered by the studies of natural and experimental mutations in humans and
mice .
(Robbins et al. 1993; Valverde et al. 1995; Frandberg et al. 1998). These
studies indicate
that some mutations in highly conserved regions cause structural changes and
alter the
function of the receptor. The Asp298Asn substitution mutation could have an
effect on the
function of the receptor. However, this will require further testing but it is
known that
change of the homologous residue in MC1R (Asp294His) is associated with fair
skin and
red hair in humans (Valverde et al. 1995).
The MC4R missense mutation is associated yvith obesitv-zelatPd traits To
investigate the effects of the missense mutation, the relationship of MC4R
genotypes was
analyzed for the effects on variation in growth rate, backfat, and feed
consumed in over
1,800 animals from several commercial pig lines from PIC, an international pig
breeding
company. The animals were from closed commercial lines of European/American
breeds
(Lines A-D) together with a line originating from a cross between a European
and a
Chinese breed (Line E). In lines A-D significant associations of the MC4R
genotypes were
found for all perfonmance traits. The animals homozygous for allele 1 had on
average
significantly less backfat (P <.001), lower daily gain (P <.001), and lower
feed intake(P <
.01) than those of the homozygous 22 genotype animals (Tables 11, 13, & 15).
Overall,
pigs with the 11 genotype had approximately 9% less backfat than pigs with the
22
genotype (Table 11), whereas pigs with the 22 genotype grow significantly
faster (37g/day)
than pigs with the 11 genotype (Table 13). These results appear to be a
function of appetite
because the 22 genotype animals consume considerably more feed (Table 15). The
association between the missense variant of the MC4R gene and related
performance traits
is clearly established in European/American breeds. Although the number of
tested
animals is much smaller, these results were not seen in the considerably
fatter Chinese
crossed line (Line E). Interestingly, line E shows a trend for backfat in the
opposite
direction to that observed in the other lines (Table 11).
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Discussion
The present study clearly demonstrates that the porcine MC4R missense mutation-
is -
significantly associated with several performance traits in pigs. Allele 1
representing
Asp298, the well conserved amino acid within other MCR subtypes and other
species
MC4R, was associated with less backfat thickness, slower growth rate, and
lower feed
intake and allele 2 representing Asn298 was associated with fatter, higher
feed intake, and
faster growing animals. As the highly conserved residues in the melanocortin
receptor
proteins have important roles for ligand binding or intracellular signal
transmission (Tatro
1996), the MC4R variants might exert functionally distinct abilities in the
regulation of
food intake and body weight. Further testing of this hypothesis will provide
important
insights into the structural basis of MCR function and a molecular target for
the treatment
of human obesity.
Allele I was associated with the fattest animals in Line E, which was derived
by
crossing a Chinese Large White breed with a line of Meishan origin. This is
surprising
given that the mutation causes a significant amino acid change in a well-
conserved region.
The result may be due to sampling. However, if we assume that this result will
be
significant when more results are added there are several possible
explanations. One
possibility could be the difference in the background gene effects
(epistasis). As growth
and fatness are complex polygenic traits, it is certainly possible that the
Chinese breed has
some distinct allelic interactions derived from several hundred years of
isolation and these
putative interaction(s) might create variation in polygenic traits within
crosses between
widely different lines (Frankel and Schork 1996). Several QTL analyses have
been
conducted for fatness and growth traits using divergent lines (Cases-Carrillo
et al. 1997;
Knott et al. 1998; Rohrer et al. 1998; Wang et al. 1998; Paszek et al. 1999),
but QTL have
not been reported near the C4R locus, which maps to chromosome 1 at
approximately 80
cm on the linkage map (data not shown). It may mean that the epistatic effects
of the
MC4R alleles suggested in Line E have made it difficult to observe the MC4R
locus in
most QTL experiments which have involved crosses between Chinese and
European/American lines. It is likely that the effect of some alleles will be
variable in the
different backgrounds and hard to detect in QTL experiments involving
genetically
divergent breeds.
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The effect of MC4R variant will possibly be explained by further studies on
the
biological effect caused by this mutation in other pig breeds and lines.
However, given the -
strong relationship of MC4R variants to leanness, growth and feed intake, this
mutation
could be used immediately for merker assisted selection (Meuwissen and Goddard
1996) to
develop lines of pigs to satisfy particular customer requirements. For
instance, in sow lines
where appetite is normally decreased after farrowing, selection for the MC4R 2
allele could
help improve feed intake. Furthermore, in some lines deemed to be too fat,
selection for
allele 1 could be employed and in lines that were considered too slow growth
allele 2
selection could be also employed. Therefore, genotyping for the MC4R mutation
in pig
breeding lines will improve the selection efficiency of feed related
production traits
including growth and leanness. The candidate gene approach has also been used
for
investigating the role of the porcine leptin gene (Jiang and Gibson 1999).
However, in the
leptin case, although there was evidence for an association between a leptin
polymorphism
and backfat depth in a cross between a commercial breed and an unimproved
line, there
was no clear association in the different commercial lines tested (Jiang and
Gibson 1999).
Therefore, it should not be assumed that since one fmds a gene that one can
assume a
relationship exists. In contrast, with MC4R we have determined that variation
in this
candidate gene can explain significant variation for backfat, growth rate, and
feed intake in
commercial lines of pigs. These results with MC4R illustrate the potential
value of
comparative genetic analyses using candidate genes in livestock genomics.
EFFECT OF MC4R GENOTYPE ON SEVERAL PRODUCTION TRAITS IN THE PIG
TABLE 9
Number of observations (males/females/totals) for Days to 110 kg and backfat
MC4 LINE A LINE B LINE C LINE D Total Line E
R
11 9/212/221 12/94/106 37/17/54 58/323/381 0/20/20
12 9/150/159 37/96/133 12/158/170 152/30/182 210/434/644 0/67/67
22 3/16/19 28/36/64 89/356/445 155/12/167 275/420/695 0/37/37
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TABLE 10
Days to 110kg
MC4 LINE A LINE B LINE C LINE D Total Line E
R
11 166.3+/-0.8 168.4+/-1.4 170.0+/-2.4 167.9+/-0.9 219.1+/-4.8
12 165.6+/-0.9 166. 8+/-1.1 163.9+/-1.0 170.2+/-1.8 166.9+/-0.8 212. 2+/-3 .4
22 162. 3+/-2.3 166. 8+/-1. 5 161. 5+/-0. 8 167. 0+/-1.9 164.6+/-0.9 211.4+/-
4.0
P <.24 P <.47 P <.007 P <.10 P <.001 P <.27
TABLE 11
10t' rib Backfat (mm)
MC4 LINE A LINE B LINE C LINE D Total Line E
R
11 10.7+/-0.2 12.1+/-0.2 9.8+/-0.5 11.1+/-0.2 22.8+/-1.2
12 11.2+/-0.2 12. 5+/-0.2 12.3+/-0.2 10.5+/-0.4 11.6+/-0.2 21.5+/-0.9
22 12. 5+/-0. 5 12. 6+/-0.3 12.7+/-0.2 10.9+/-0.4 12.0+/-0.2 20.3+/-1.0
P <.02 P <.31 P <.06 P <.05 P<.001 P <.17
TABLE 12
Number of observations (males/females/totals for Test dailyg~
MC4 1 LINE A LINE B LINE C LINE D Total Line E
R
11 9/105/114 12/38/50 37/17/54 58/160/218 0/20/20
12 9/65/74 37/35/72 12/97/109 152/30/182 210/227/437 0/67/67
22 3/13/15 28/15/43 89/225/314 155/12/167 275/265/539 0/37/37
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TABLE 13
Test daily gain ( dav)
MC4 LINE A LINE B LINE C LINE D Total Line E
R
11 892.6+/-10.4 841.7+/-13.8 882.2+/-18.4 871.9+/-10.2 688.8+/-24.5
12 913.3+/-11.6 868.4+/-12.1 882.2+/-12.9 883.7+/-14.3 885.1+/-8.9 676.2+/-
17.6
22 982.8+/-22.8 862.4+/-15.1 913.4+/-10.5 904.6+/-15.1 908.8+/-9.3 692.5+/-
20.4
P <.001 P <.28 P <.006 P <.20 P <.001 P <.66
TABLE 14
Number of observations (males/females/total) for average daily feed intake
MC4R LINE A LINE B LINE C LINE D Total Line E
11 7/0/7 11/0/11 13/0/13 31/0/31 0/18/18
12 8/0/8 31/0/31 9/0/9 34/0/34 82/0/82 0/63/63
22 3/0/3 25/0/25 74/0/74 16/0/16 118/0/118 0/32/32
TABLE 15
Average daily feed intake (kg/day). boars only excent LINE E which
was gilts onlv
MC4 LINE A LINE B LINE C LINE D Total Line E
R
11 2.31+/-0.2 1.78+/-0.09 1.75+/-0.06 1.94+/-0.07 2.05+/-0.10
12 2.11+/-0.3 1.90+/-0.07 1.97+/-0.10 1.90+/-0.07 2.03+/-0.06 2.03+/-0.07
22 2.15+/-0.4 1.97+/-0.06 2.00+/-0.07 1.97+/-0.08 2.11+/-0.06 2.08+/-0.08
P <.84 P <.14 P <.56 P <.14 P <.01 P <.36
Having described the invention with reference to particular compositions,
theories
of effectiveness, and the like, it will be apparent to those of skill in the
art that it is not
intended that the invention be limited by such illustrative embodiments or
mechanisms,
and that modifications can be made without departing from the scope or spirit
of the
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CA 02337495 2007-02-02
WO 00/06777 PCT/US99/16862
invention, as defined by the appended claims. It is intended that all such
obvious
modifications and variations be included within the scope of the present
invention as =-
defined in the appended claims. The claims are meant to cover the claimed
components
and steps in any sequence which is effective to meet the objectives there
intended, unless
the context specifically indicates to the contrary.
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. ~ '
SEQUENCE LISTING
<110> Iowa State University Research Foundation
<120> Melanocortin-4 Receptor Gene and Use as a Genetic
Marker for Fat Content, Weight Gain, and/or Feed
Consumption in Animals
<130> 2926/0004
<140> 2,337,495
<141> 1999-07-26
<150> PCT/US99/16862
<151> 1999-07-26
<150> 60/094,287
<151> 1998-07-27
<150> 60/116,196
<151> 1999-01-15
<160> 11
<170> PatentIn Ver. 2.0
<210> 1
<211> 746
<212> DNA
<213> porcine
<220>
<221> variation
<222> (678)
<223> G/A
<400> 1
acaagaatct gcattcaccc atgtactttt tcatctgtag cctggctgtg gctgatatgc 60
tggtgagcgt ttccaatggg tcagaaacca ttgtcatcac cctattaaac agcacggaca 120
cggacgcaca gagtttcaca gtgaatattg ataatgtcat tgactcagtg atctgtagct 180
ccttactcgc ctcaatttgc agcctgcttt cgattgcagt ggacaggtat tttactatct 240
tttatgctct ccagtaccat aacattatga cagttaagcg ggttggaatc atcatcagtt 300
gtatctgggc agtctgcacg gtgtcgggtg ttttgttcat catttactca gatagcagtg 360
ctgttattat ctgcctcata accgtgttct tcaccatgct ggctctcatg gcttctctct 420
atgtccacat gttcctcatg gccagactcc acattaagag gatcgccgtc ctcccaggca 480
ctggcaccat ccgccaaggt gccaacatga agggggcaat taccctgacc atcttgattg 540
gggtctttgt ggtctgctgg gcccccttct tcctccactt aatattctat atctcctgcc 600
cccagaatcc atactgtgtg tgcttcatgt ctcactttaa tttgtatctc atcctgatca 660
tgtgtaattc catcatcgat cccctgattt atgcactccg gagccaagaa ctgaggaaaa 720
ccttcaaaga gatcatctgt tgctat 746
<210> 2
<211> 840
<212> DNA
<213> Homo sapiens
<400> 2
atatcttagt gattgtggca atagccaaga acaagaatct gcattcaccc atgtactttt 60
tcatctgcag cttggctgtg gctgatatgc tggtgagcgt ttcaaatgga tcagaaacca 120
ttatcatcac cctattaaac agtacagata cggatgcaca gagtttcaca gtgaatattg 180
ataatgtcat tgactcggtg atctgtagct ccttgcttgc atccatttgc agcctgcttt 240
32
CA 02337495 2001-07-27
caattgcagt ggacaggtac tttactatct tctatgctct ccagtaccat aacattatga 300
cagttaagcg ggttgggatc agcataagtt gtatctgggc agcttgcacg gtttcaggca 360
ttttgttcat catttactca gatagtagtg ctgtcatcat ctgcctcatc accatgttct 420
tcaccatgct ggctctcatg gcttctctct atgtccacat gttcctgatg gccaggcttc 480
acattaagag gattgctgtc ctccccggca ctggtgccat ccgccaaggt gccaatatga 540
agggagcgat taccttgacc atcctgattg gcgtctttgt tgtctgctgg gccccattct 600
tcctccactt aatattctac atctcttgtc ctcagaatcc atattgtgtg tgcttcatgt 660
ctcactttaa cttgtatctc atactgatca tgtgtaattc aatcatcgat cctctgattt 720
atgcactccg gagtcaagaa ctgaggaaaa ccttcaaaga gatcatctgt tgctatcccc 780
tgggaggcct ttgtgacttg tctagcagat attaaatggg gacagagcac gcaatatagg 840
<210> 3
<211> 746
<212> DNA
<213> porcine
<400> 3
acaagaatct gcattcaccc atgtactttt tcatctgtag cctggctgtg gctgatatgc 60
tggtgagcgt ttccaatggg tcagaaacca ttgtcatcac cctattaaac agcacggaca 120
cggacgcaca gagtttcaca gtgaatattg ataatgtcat tgactcagtg atctgtagct 180
ccttactcgc ctcaatttgc agcctgcttt cgattgcagt ggacaggtat tttactatct 240
tttatgctct ccagtaccat aacattatga cagttaagcg ggttggaatc atcatcagtt 300
gtatctgggc agtctgcacg gtgtcgggtg ttttgttcat catttactca gatagcagtg 360
ctgttattat ctgcctcata accgtgttct tcaccatgct ggctctcatg gcttctctct 420
atgtccacat gttcctcatg gccagactcc acattaagag gatcgccgtc ctcccaggca 480
ctggcaccat ccgccaaggt gccaacatga agggggcaat taccctgacc atcttgattg 540
gggtctttgt ggtctgctgg gcccccttct tcctccactt aatat+tctat atctcctgcc 600
cccagaatcc atactgtgtg tgcttcatgt ctcactttaa tttgtatctc atcctgatca 660
tgtgtaattc catcatcaat cccctgattt atgcactccg gagccaagaa ctgaggaaaa 720
ccttcaaaga gatcatctgt tgctat 746
<210> 4
<211> 311
<212> PRT
<213> Homo sapiens
<220>
<221> miscfeature
<222> () . . ()
<223> "X" can be any amino acid
<400> 4
Gin Leu Phe Val Ser Pro Glu Val Phe Val Thr Leu G:Ly Val Ile Ser
1 5 10 15
Leu Leu Glu Asn Ile Leu Val Ile Val Ala Ile Ala Lys Asn Lys Asn
20 25 30
Leu His Ser Pro Met Tyr Phe Phe Ile Cys Ser Leu A1.a Val Ala Asp
35 40 45
Met Leu Val Ser Val Ser Asn Gly Ser Glu Thr Ile Ile Ile Thr Leu
50 55 60
Leu Asn Ser Thr Asp Thr Asp Ala Gin Ser Phe Thr Val Asn Ile Asp
65 70 75 80
Asn Val Ile Asp Ser Val Ile Cys Ser Ser Leu Leu A=La Ser Ile Cys
85 90 95
Ser Leu Leu Ser Ile Ala Val Asp Arg Tyr Phe Thr I}.e Phe Tyr Ala
33
CA 02337495 2001-07-27
100 105 110
Leu Gln Tyr His Asn Ile Met Thr Val Lys Arg Val G:1y Ile Ser Ile
115 120 1:25
Ser Cys Ile Trp Ala Ala Cys Thr Val Ser Gly Ile Leu Phe Ile Ile
130 135 140
Tyr Ser Asp Ser Ser Ala Val Ile Ile Cys Leu Ile Thr Met Phe Phe
145 150 155 160
Thr Met Leu Ala Leu Met Ala Ser Leu Tyr Val His Met Phe Leu Met
165 170 175
Ala Arg Leu His Ile Lys Arg Ile Ala Val Leu Pro G.1y Thr Gly Ala
180 185 190
Ile Arg Gln Gly Ala Asn Met Lys Gly Ala Ile Thr Leu Thr Ile Leu
195 200 205
Ile Gly Val Phe Val Val Cys Trp Ala Pro Phe Phe Leu His Leu Ile
210 215 220
Phe Tyr Ile Ser Cys Pro Gin Asn Pro Tyr Cys Val Cys Phe Met Ser
225 230 235 240
His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile Ile Asp
245 250 255
Pro Leu Ile Tyr Ala Leu Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys
260 265 270
Glu Ile Ile Cys Cys Tyr Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser
275 280 285
Arg Tyr Ala Pro Pro Glu Asn Asp Ile Xaa Val Ile Cys Asn Phe Ile
290 295 300
Asp Glu Asn Thr Ile Ala Leu
305 310
<210> 5
<211> 248
<212> PRT
<213> porcine
<400> 5
Lys Asn Leu His Ser Pro Met Tyr Phe Phe Ile Cys Ser Leu Ala Val
1 5 10 15
Ala Asp Met Leu Val Ser Val Ser Asn Gly Ser Glu Thr Ile Val Ile
20 25 30
Thr Leu Leu Asn Ser Thr Asp Thr Asp Ala Gln Ser Phe Thr Val Asn
35 40 4; 5
Ile Asp Asn Val Ile Asp Ser Val Ile Cys Ser Ser Leu Leu Ala Ser
50 55 60
Ile Cys Ser Leu Leu Ser Ile Ala Val Asp Arg Tyr Phe Thr Ile Phe
34
CA 02337495 2001-07-27
. ~ ,
65 70 75 80
Tyr Ala Leu Gln Tyr His Asn Ile Met Thr Val Lys A:rg Val Gly Ile
85 90 95
Ile Ile Ser Cys Ile Trp Ala Val Cys Thr Val Ser G:ly Val Leu Phe
100 105 110
Ile Ile Tyr Ser Asp Ser Ser Ala Val Ile Ile Cys Leu Ile Thr Val
115 120 125
Phe Phe Thr Met Leu Ala Leu Met Ala Ser Leu Tyr Val His Met Phe
130 135 140
Leu Met Ala Arg Leu His Ile Lys Arg Ile Ala Val Leu Pro Gly Thr
145 150 155 160
Gly Thr Ile Arg Gln Gly Ala Asn Met Lys Gly Ala I:1e Thr Leu Thr
165 170 175
Ile Leu Ile Gly Val Phe Val Val Cys Trp Ala Pro Phe Phe Leu His
180 185 190
Leu Ile Phe Tyr Ile Ser Cys Pro Gln Asn Pro Tyr Cys Val Cys Phe
195 200 205
Met Ser His Phe Asn Leu Tyr Leu Ile Leu Ile Met Cys Asn Ser Ile
210 215 220
Ile Asn Pro Leu Ile Tyr Ala Leu Arg Ser Gln Glu Leu Arg Lys Thr
225 230 235 240
Phe Lys Glu Ile Ile Cys Cys Tyr
245
<210> 6
<211> 20
<212> DNA
<213> porcine
<400> 6
tggcaatagc caagaacaag 20
<210> 7
<211> 20
<212> DNA
<213> porcine
<400> 7
caggggatag caacagatga 20
<210> 8
<211> 18
<212> DNA
<213> porcine
<400> 8
ttaagtggag gaagaagg 18
<210> 9
CA 02337495 2001-07-27
<211> 19
<212> DNA
<213> porcine
<400> 9
cattatgaca gttaagcgg 19
<210> 10
<211> 20
<212> DNA
<213> porcine
<400> 10
taccctgacc atcttgattg 20
<210> 11
<211> 22
<212> DNA
<213> porcine
<400> 11
atagcaacag atgatctctt tg 22
36
