U.S. patent application number 12/652807 was filed with the patent office on 2010-10-14 for identification of fat and lean phenotypes in chickens using molecular markers.
This patent application is currently assigned to University of Delaware. Invention is credited to Wilfrid G. Carre, Larry A. Cogburn, Xiaofei Wang.
Application Number | 20100261173 12/652807 |
Document ID | / |
Family ID | 34990423 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100261173 |
Kind Code |
A1 |
Cogburn; Larry A. ; et
al. |
October 14, 2010 |
Identification Of Fat And Lean Phenotypes In Chickens Using
Molecular Markers
Abstract
The present invention provides methods of screening chickens to
determine those more likely to have a lean or fat phenotype. The
invention also provides methods of screening chickens to identify a
polymorphism associated with a fat or lean phenotype.
Inventors: |
Cogburn; Larry A.; (New
London, PA) ; Carre; Wilfrid G.; (Edinburgh, GB)
; Wang; Xiaofei; (Nashville, TN) |
Correspondence
Address: |
RATNERPRESTIA
P.O. BOX 1596
WILMINGTON
DE
19899
US
|
Assignee: |
University of Delaware
Newark
DE
|
Family ID: |
34990423 |
Appl. No.: |
12/652807 |
Filed: |
January 6, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11013546 |
Dec 16, 2004 |
7666590 |
|
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12652807 |
|
|
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60530051 |
Dec 16, 2003 |
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Current U.S.
Class: |
435/6.1 ;
435/6.12 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 2600/156 20130101; C12Q 1/6888 20130101; C12Q 2600/124
20130101; C12Q 2600/172 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
REFERENCE TO U.S. GOVERNMENT SUPPORT
[0002] This work is supported by a grant from the USDA-IFAFS,
Animal Genome Program (Award Number 00-52100-9614). The United
States government has certain rights in the invention.
Claims
1.-8. (canceled)
9. A method for screening chickens to identify chickens more likely
to have a lean phenotype or a fat phenotype, comprising identifying
in a sample of genetic material obtained from a chicken the
presence or absence of the nucleic acid sequence ATAGATGGC at
position 261-269 of SEQ ID NO:1, wherein the presence or absence of
the nucleic acid sequence correlates with a fat phenotype or a lean
phenotype.
10. The method of claim 9, wherein the identifying step comprises
amplifying at least a portion of SEQ ID NO:1, wherein the portion
contains position 261-269.
11. The method of claim 10, wherein the portion is amplified using
a primer that hybridizes to a region of SEQ ID NO: 1 upstream of
position 261 and a primer that hybridizes to a region of SEQ ID
NO:1 downstream of position 269.
12. The method of claim 10, wherein the portion is amplified using
a pair of primers selected from the group consisting of SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:24, and SEQ ID NO:25.
13. The method of claim 9, wherein the genetic material is obtained
from cells or tissues isolated from the chicken.
14. The method of claim 13, wherein the cells or tissues are
isolated from the abdominal fat or liver of the chicken.
15. The method of claim 13, wherein the cells are isolated from the
blood of the chicken.
Description
[0001] This application claims the benefit of provisional
application Ser. No. 60/530,051 filed Dec. 16, 2003, which is
hereby incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to method for identifying the
phenotype of a chicken using a genetic polymorphism associated with
a fat or lean phenotype. More particularly the invention relates to
method of identifying a fat or lean chicken phenotype by
determining the presence of an insertion/deletion associated with a
fat or lean phenotype in one or both of the duplicated chicken Spot
14 genes, also referred to as thyroid hormone responsive Spot 14
protein (THRSP .alpha. and THRSP .beta.) paralogs.
BACKGROUND OF THE INVENTION
[0004] Over the last decades intensive selection on growth rate has
been done in broiler chicken strains developed for meat production.
However, fatness has also been increased, leading to excessive
adiposity. By reducing feed efficiency and lean meat yield, this
excess of fat tissue is a major drawback for production.
[0005] In order to decipher the metabolic and genetic mechanisms
involved in the regulation of fatness in the chicken, some
investigators have developed experimental models of adiposity. Lean
and fat chicken lines have been divergently selected from adipose
tissue weight (Leclerq et al., 1980) and for very low density
lipoprotein (VLDL) plasma concentration (Whitehead, C. C., Griffin,
H. D., 1984). Studies performed in lean and fat lines developed by
Leclercq et al (1980) indicate that the difference in adiposity
between lines was not the result of a difference in food
consumption or in nutrient utilization. Stearoyl-Co-A desaturase
activity and plasma VLDL concentration were found to be higher in
the fat line (Legrand, P. and Hermier, D., 1992), suggesting a
higher lipogenesis rate in this line.
[0006] In the chicken, lipogenesis occurs essentially in the liver,
the adipose tissue being only a storage tissue (O'Hea, E. K. and
Leveille, G. A., 1968; Griffin et al., 1992).
[0007] The Spot 14 gene, also referred to as thyroid hormone
responsive Spot 14 protein (THRSP), encodes a small acidic protein
that was discovered in earlier studies of thyroid hormone action on
hepatocytes (Seelig et al., 1981; Jump et al., 1984; Liaw and
Towle, 1984). Although the exact molecular mechanism is not clear,
THRSP is strongly implicated as a transcription factor that
controls expression of major lipogenic enzymes. For instance, THRSP
is only expressed in lipogenic tissue such as liver, fat and the
mammary gland (Liaw and Towle, 1984; Jump and Oppenheimer, 1985).
THRSP mRNA levels are greatly increased by carbohydrate feeding or
insulin-injection and decreased by high plasma glucagon levels or
by feeding a diet rich in polyunsaturated fatty acids (Jump et al.,
1993). Hepatocytes transfected with a THRSP antisense
oligonucleotide express decreased mRNA levels in enzymes involved
in the lipogenic pathway [i.e., ATP-citrate lyase (ACLY), fatty
acid synthase (FAS) and malic enzyme (ME)] (Kinlaw et al., 1995;
Brown et al., 1997). Although an increase in lipogenesis was
observed in the THRSP knockout mouse, this contradiction could be
due to incomplete gene deletion or overcompensation by alternative
pathways (Zhu et al., 2001). Homodimers of THRSP interact with and
activate chicken ovalbumin upstream promoter-transcription factor 1
(COUP-TF1) in promoting transcription of L-type pyruvate kinase
(L-PK) through an interaction with specificity protein 1 (Sp1)
(Compe et al., 2001). Furthermore, the THRSP promoter region
contains three thyroid response elements (TREs) that work
synergistically and interact with far upstream region (FUR)
elements to maximize triiodothyronine (T.sub.3) responses in
hepatocytes (Liu and Towle, 1994). Apparently, the human THRSP
promoter responds more robustly to T.sub.3 than glucose, while the
rat THRSP promoter region is more responsive to glucose than
T.sub.3 (Campbell et al, 2003).
[0008] Many common diseases and conditions are not caused by a
genetic variation within a single gene, but are influenced by
complex interactions among multiple genes as well as environmental
and lifestyle factors. Genetic predisposition is the potential of
an individual to develop a disease or condition based on genes and
hereditary factors. Although both environmental and lifestyle
factors add tremendously to the uncertainty of developing a
disease, it is currently difficult to measure and evaluate their
overall effect on a disease process. By studying changes within a
gene that have been found to be associated with a disease trait,
researchers may begin to reveal relevant genes associated with a
disease. Polymorphisms can thus serve as biological markers for a
disease or trait associated with a disease. Therefore, it is
desirable to find polymorphism(s) which can be used for the
diagnosis of a disease (including metabolic diseases such as
obesity) and/or identification of a trait, such as polymorphisms
associated with a fat or lean chicken phenotype.
SUMMARY OF THE INVENTION
[0009] The invention provides methods of screening chickens to
determine those more likely to have a lean or fat phenotype
comprising the steps of obtaining a sample of genetic material from
a chicken; and identifying in the genetic material the presence of
at least one insertion or deletion of nucleotides associated with a
fat phenotype or a lean phenotype in the sequence encoding one or
both of the chicken thyroid hormone responsive Spot 14 protein
(THRSP) paralogs, THRSP.alpha. (SEQ ID NO: 1) and THRSP .beta. (SEQ
ID NO: 3).
[0010] The invention also provides methods of screening chickens to
identify a polymorphism associated with a fat or lean phenotype
comprising obtaining a sample of genetic material from a chicken;
and identifying in the genetic material the presence of at least
one insertion or deletion of nucleotides in the sequence encoding
one or both of the chicken thyroid hormone responsive Spot 14
protein (THRSP) paralogs, THRSP.alpha. (SEQ ID NO: 1) and THRSP
.beta. (SEQ ID NO: 3), that is associated with a fat phenotype or a
lean phenotype.
[0011] Preferably, the insertion or deletion is the insertion or
deletion of the sequence ATAGATGGC in THRSP .alpha. (bases 261-269
of the sequence shown in FIG. 1A) and/or the insertion or deletion
of the sequence GCCGAC in THRSP .beta. (bases 228-233 of the
sequence shown in FIG. 1B). The polymorphisms found in THRSP
.alpha. and THRSP .beta. involve a region of nucleotide sequence
known as variable number of tandem repeats (VNTRs) For example, the
sequence ATAGATGGC is repeated twice in THRSP .alpha., (bases
261-279 of the sequence shown in FIG. 1A) and the sequence GCCGAC
is repeated three times in THRSP .beta. (bases 228-245 of the
sequence shown in FIG. 1B).
[0012] The insertion/deletion of bases in THRSP .alpha. (FIG. 1A)
(SEQ ID NO: 1) and THRSP .beta. (FIG. 1B) (SEQ ID NO: 3) is
enclosed in a box. In the insertion alleles of THRSP .alpha. and
THRSP .beta., the boxed bases are present. In the deletion alleles
of THRSP .alpha. and THRSP .beta., the boxed bases are absent.
[0013] Preferably, the step of identifying the presence of the
polymorphism comprises the steps of: amplifying at least one
portion of the nucleotide sequence encoding THRSP .alpha. (SEQ ID
NO: 1) or THRSP (SEQ ID NO: 3) or both, in which the region
contains an insertion or deletion that is associated with a fat
phenotype or lean phenotype, and detecting the insertion or
deletion in the at least one amplified portion.
[0014] These and other,aspects of the invention are set out in the
following Detailed Description and in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the cDNA sequence and predicted protein
sequence of the chicken THRSP paralogs. (A) THRSP .alpha. cDNA (SEQ
ID NO: 1) and its predicted protein sequence (SEQ ID NO: 2). Primer
sequences used for PCR are indicated by the bold underlined
letters. The predicted leucine zipper motif is shown in bold
letters and the poly(A) signal is underlined. The boxes represent
the missing nt and aa residues in the deletion allele
(.alpha..sub.2). Sequence encoded by the 5`-UTR and 3'-UTR (exon 2)
is shown in lower case letters. The asterisk shows the stop codon.
The .sub.junction between exons 1 and 2 is indicated by the
inverted solid triangle. (B) THRSP .beta. cDNA (SEQ ID NO: 3) and
its predicted protein sequence (SEQ ID NO: 4). Primer sequences
used for PCR are indicated by the bold underlined letters. The
predicted leucine zipper motif is shown in bold letters and the
poly(A) signal is underlined. The boxes represent the missing nt
and aa residues in the deletion allele (.beta..sub.2). Sequence
encoded by the 5'-UTR is shown in lower case letters and the 3'-UTR
(exon 2) is shown in uppercase letters. The asterisk shows the stop
codon.
[0016] FIG. 2 shows protein sequence alignment of the Spot 14
family members: the THRSPs, gastrulation specific [zebrafish]G12
proteins , and the hypothetical [human] STRAIT11499 proteins.
Protein sequences for chicken [c] THRSP.alpha. (UD CAP3
Contig.sub.--8452.1) (SEQ ID NO: 2) and THRSP.beta. (UD CAP3
Contig.sub.--8452.2) (SEQ ID NO: 4), human [h] THRSP (AAH31989)
((SEQ ID NO: 5), mouse [m] THRSP (Q62264) (SEQ ID NO: 6), rat [r]
THRSP (P04143) (SEQ ID NO: 7) and zebrafish [z] (TC192887) (SEQ ID
NO: 10) THRSP were aligned using ClustalW with default parameters
and BLOSUM62 scoring matrix. This alignment includes two
structurally related proteins: gastrulation-specific protein G12
from zebrafish (P47805) and an apparently duplicated G12 protein
(zTC194742)(SEQ ID NO: 9) found in the database of the Institute
for Genomic Research (TIGR) (TIGR.org) which show a high degree of
structural similarity to the hypothetical [human] hSTRAIT11499
protein (AAH19332) (SEQ ID NO: 11), mSTRAIT11499 (Q9CQ20) (SEQ ID
NO: 12), cSTRAIT11499 (derived from UD CAP3 Contig.sub.--22252.1)
(SEQ ID NO: 13). Identical amino acid (aa) residues are shown
black, similar (positive) amino acid (aa) residues are shown in
gray and the hyphens denote gaps.
[0017] FIG. 3 shows a dendrogram of the phylogenetic, relationship
among Spot 14 family members: the THRSPs, the gastrulation-specific
[zebrafish] G12 proteins, and hypothetical [human] STRAIT11499
proteins. The phylogenetic tree was created using the ClustalW
program with default settings and the BLOSUM62 scoring matrix.
[0018] FIG. 4 shows the genomic organization of the chicken THRSP
paralogs. (A) Southern blot analysis of the THRSP gene. Genomic DNA
was digested to completion with restriction enzymes and hybridized
with a probe (pgf2n.pk005.j11) common to both THRSP .alpha. and
THRSP .beta. cDNAs. Two restriction fragments were expected after
PstI digestion. The darker band represents THRSP .alpha. because it
corresponds to the full-length probe, while only 230 by of the
probe corresponds to the THRSP .beta. cDNA (lighter band). (B)
Putative restriction map of genomic DNA harboring the THRSP
paralogs. The direction of transcription is indicated by the
arrows. The exact distance between THRSP .alpha. and THRSP.beta. is
unknown (dashed line). Open boxes represent location of the probe
used in the Southern blot (A) above. [Abbreviations used: H,
HindIII; B, BamHI; and P, PstI.] (C) The genomic structure of THRSP
.alpha., which includes a TATA box. Exon 1 represents the short
5'-UTR and the protein coding region, while exon 2 represents the
3'-UTR.
[0019] FIG. 5 shows the identification of a synteny group in
chicken genomic DNA that includes THRSP and two flanking genes
[NADH dehydrogenase (NDUFC2) and glucosyltransferase (ALG8)]. This
presence of this synteny group in chicken genomic DNA was by PCR
amplification of all four genes in two THRSP-positive BAC clones
(65J23 and 94A1) that were identified earlier by Cane et al (2001),
where only PCR products amplified from chicken BAC clone #65J23 are
shown. This synteny group is conserved in chickens [cChr1q41-44],
humans [11q13.5], rat [rChr1q32-33] and mouse [mChr7D3-E1].
[0020] FIG. 6 shows expression of THRSP transcripts in chicken
tissues. Total RNA (40 ng per reaction) was analyzed by real-time
qRT-PCR (Applied Biosystems (ABI)) using TaqMan by a universal
QuantiTech Sybr Green qRT-PCR kit (Qiagen). Primers were designed
using Primer Express 2.0 software (Applied Biosystems (ABI)). (A)
Expression of total THRSP in 11 tissues using common primers
(32F/93R). Values represent the mean.+-.SEM of duplicate
determinations in arbitrary units (AU). RNA from most tissues was
isolated from 5-week-old broiler chickens. RNA was extracted from
the thymus and epiphyseal growth plate of 3-week-old broiler
chickens. Testes and ovary RNA was isolated from 8-week-old Leghorn
chickens; RNA was also collected from the ovary of an adult (1 year
old) Leghorn hen. (B) Expression of THRSP .alpha. and THRSP .beta.
in fat and liver of 5-week-old broiler chickens. (C) Expression of
THRSP mRNAs in the liver during the peri-hatch period [Day 20
embryos (e20) and 1 day old (1 da) chicks]. Each value represents
the mean.+-.SEM of four embryos and four chicks. (D) The response
of hepatic THRSP .alpha. and THRSP .beta. mRNAs to changes in
nutritional state. Liver samples were collected from a fast-growing
strain of French (INRA) broiler chickens at six weeks of age after
a 48 h fast (S48) and at 4 h post re-feeding (RF4) following the 48
h fast (Beccavin et al, 2001). Each value represents the
mean.+-.SEM of four birds.
[0021] FIG. 7 shows evidence of polymorphisms in THRSP .alpha. and
THRSP .beta. genes in a group of stock chickens from the Iowa
Growth and Composition Resource Population (IGCRP). Genomic DNA (40
ng) from 16 chickens of mixed sexes, randomly chosen from
contemporary pure founder lines, was amplified by PCR with specific
primers for either THRSP .alpha. (DeletionF/DeletionR) or THRSP
.beta. (ParalogF/ParalogR). The PCR products for THRSP .alpha.
(Allele .alpha.1=136 bp; Allele .alpha.2=127 bp) were labeled with
.sup.32P-dCTP, separated in native polyacrylamide gel (8%), exposed
to a phosphorimager screen overnight and visualized with a
PhosphorImager (Storm 840, Molecular Dynamics). The PCR products
for THRSP .beta. (Allele .beta.1=151 bp; Allele .beta.2=145 bp)
were amplified with ThermalAce (Invitrogen) and separated in a 3%
agarose gel.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The methods of the invention are useful for identifying
individual chickens or groups of chickens that have a
predisposition for a lean or fat phenotype. Identification of birds
having a lean or fat phenotype is of interest to chicken breeders
and growers for use in marker assisted selection (MAS) breeding
programs. Insertions/deletions in one or both of the genes encoding
the THRSP paralogs (THRSP .alpha. and THRSP .beta.), also known as
Spot 14, are useful as a genetic markers for MAS programs in
poultry breeding. A chicken's phenotype (lean or fat) can be
determined from tissue or blood samples even before the chick is
hatched, without the need for raising potential breeder chickens to
adult age for measurement of the phenotype.
[0023] Applicants have discovered that THRSP, sometimes referred to
as Spot 14, has two forms, .alpha. and .beta. paralogs, in chickens
and that an insertion/deletion in one or more of the paralogs is
correlated with a fat or lean phenotype. Chicken Spot 14 (THRSP)
was first identified as a differentially-expressed EST
(pat.pk0032.c9.f) from microarray analysis of livers from chickens
divergently selected for fast or slow growth rate (Cogburn et al.,
2000; Cogburn et al., 2003a). An EST was discovered by differential
mRNA display in liver of genetically fat and lean chickens and
subsequently mapped to chicken Chr1q41-44 (Cane et al., 2001). This
EST was identified as chicken THRSP from alignment with an
annotated EST (pat.pk0072.c10.f) in the University of Delaware (UD)
chicken EST database. This chromosomal region in chickens also
harbors quantitative trait loci (QTL) for skin fatness (Ikeobi et
al., 2002) and abdominal fatness (Lagarrigue et al., 2003).
[0024] One aspect of the invention therefore provides a method of
screening chickens to determine those more likely to have a lean or
fat phenotype comprising the steps of: obtaining a sample of
genetic material from a chicken; and identifying the presence of
insertions or deletions of bases in the nucleotide sequence
encoding the duplicated chicken thyroid hormone responsive Spot 14
protein (i.e., the THRSP .alpha. and THRSP .beta. paralogs), which
sequences are set out in FIG. 1A (SEQ ID NO: 1) and 1B (SEQ ID NO:
3), that are associated with a fat phenotype or a lean phenotype.
Preferably, the methods of the invention detect an
insertion/deletion of a nine base sequence in the THRSP .alpha.
nucleotide sequence shown in FIG. 1A (SEQ ID NO: 1), wherein the
nine base VNTR sequence is ATAGATGGC (bases 261-269 of the sequence
shown in FIG. 1A (SEQ ID NO: 1)) and/or an insertion/deletion of
six to twelve bases in the THRSP .beta. nucleotide sequence shown
in FIG. 1B (SEQ ID NO: 3), wherein the six base VNTR sequence is
GCCGAC in THRSP .beta. (bases 228-233 of the sequence shown in FIG.
1B (SEQ ID NO: 3)).
[0025] Another aspect of the invention provides a method of
screening chickens to identify a polymorphism associated with a fat
or lean phenotype comprising the steps: of obtaining a sample of
genetic material from a chicken; and identifying the presence of
one or more insertions or deletions of nucleotides associated with
a fat phenotype or a lean phenotype in the sequence encoding one or
both of the THRSP paralogs. The nucleotide sequence encoding THRSP
.alpha. is set out in SEQ ID NO: 1 in FIG. 1A and the nucleotide
sequence encoding THRSP .beta. is set out in SEQ I NO: 3 in FIG.
1B. Preferably, the methods of the invention identify an
insertion/deletion of a nine base sequence in the sequence encoding
THRSP, wherein the nine base VNTR sequence is ATAGATGGC (bases
261-269 of the sequence shown in FIG. 1A (SEQ ID NO: 1)) and/or an
insertion/deletion of six to twelve bases in the THRSP .beta.
nucleotide sequence shown in. FIG. 1B, wherein the six base VNTR
sequence is GCCGAC in THRSP .beta. (bases 228-233 of the sequence
shown in FIG. 1B (SEQ ID NO: 3)).
[0026] Fat phenotype refers to a phenotype wherein abdominal fat is
about 3-4% (or greater) of body weight. Lean phenotype refers to a
phenotype wherein abdominal fat is about 1 to 1.2% (or less) of
body weight. Abdominal fat is measured by measuring the live body
weight (in g or kg), killing the bird, careful dissection of the
abdominal fat pad including that surrounding the ventriculus
(gizzard) and that surrounding the cloaca (rectum), then measuring
the weight of the dissected abdominal fat pad, and is expressed as
percent of body weight (% BW). However, Whitehead, C. C., Griffin,
H. D. (1984), have divergently selected lean and fat lines of
chickens based on low or high plasma very low density lipoprotein
(VLDL) levels, respectively. These fat and lean lines of chickens
differ in their abdominal fat content (g/kg BW) by only 49%. Thus,
the degree of leanness or fatness selected in a given population of
chickens could vary depending on the genetic background and the
selection criteria. Therefore, the definition of leanness or
fatness should be based on a phenotypic difference in the average
abdominal fat content (% BW) with a difference of least two
standard error units.
[0027] Genetic material used in the methods of the invention may be
isolated from cells, tissues, blood or other samples according to
standard methodologies, such as the methods in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (1989). In certain
embodiments, analysis is performed on whole cell or tissue
homogenates or biological fluid samples without substantial
purification of the template nucleic acid. The genetic material may
be DNA or RNA. Where RNA is used, it may be desired to first
convert the RNA to a complementary DNA. A preferred source of
genetic material is blood. Chickens have nucleated red blood cells
which makes blood a convenient source of genetic material (i.e.,
genomic DNA).
[0028] The polymorphism indicative of a fat or lean phenotype
described herein can be identified by any method known in the art
that can be used for detecting insertions or deletions within a
nucleic acid sequence. A preferred method is a polymerase chain
reaction (PCR)-based assay followed by separation of the
amplification products by gel electrophoresis. Another preferred
method is a PCR-based assay using TaqMan.RTM. or molecular beacon
probes to detect the amplified target region.
[0029] A number of template dependent processes are available to
amplify the oligonucleotide sequences present in a given template
sample. One of the best-known amplification methods is the
polymerase chain reaction (referred to as PCR) which is described
in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159,
each of which is incorporated herein by reference in their
entirety.
[0030] A reverse transcriptase PCR (RT-PCR) amplification procedure
may be performed to quantify the amount of mRNA amplified. Methods
of reverse transcribing RNA into cDNA are well known and described
in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).
Alternative methods for reverse transcription utilize thermostable
DNA polymerases. These methods are described in WO 90/07641.
Polymerase chain reaction methodologies are well known in the art.
Representative methods of RT-PCR are described in U.S. Pat. Nos.
5,882,864, 5,673,517 and 5,561,058.
[0031] After amplification, the insertion/deletion can be detected
by methods known in the art such as separation of the amplification
products by gel electrophoresis, sequencing of the amplification
products, or hybridization with a nucleic acid probe.
[0032] Any sequencing method known to a person skilled in the art
may be employed. In particular, it is advantageous to use an
automated DNA sequencer. The sequencing is preferably carried out
with a double-stranded template by means of the chain-termination
method using fluorescent primers. An appropriate kit for this
purpose is provided from PE Applied Biosystems (PE Applied
Biosystems, Norwalk, Conn., USA).
[0033] Methods of gel electrophoresis are well known in the art.
The number of bases in the separated amplification products can be
determined by reference to markers of known nucleotide length.
[0034] The invention also provides primers and probes for use in
the assays to detect the insertion/deletion. The primers and probes
are based on and selected from the nucleotide sequence of THRSP
.alpha. set out in FIG. 1A (SEQ ID NO: 1) and of THRSP .beta. set
out in FIG. 1B (SEQ ID NO: 3), and will typically span the region
of THRSP .alpha. or THRSP .beta. sequence upstream or downstream of
the insertion/deletion sites, or span the insertion/deletion site
in the case of a probe and will have a length appropriate for the
particular detection method. One aspect of the invention thus
provides oligonucleotides comprising from about 10 to about 30
contiguous bases of the nucleotide sequence encoding THRSP .alpha.
(FIG. 1A) and/or of THRSP .beta. (FIG. 1B) or the complementary
sequence for use as probes or primers. Primers that will be used in
assays to quantitiate Spot 14 mRNA can be selected from any portion
of the THRSP .alpha. or THRSP .beta. nucleotide sequence shown in
FIGS. 1A and 1B that will provide reliable amplification of Spot 14
paralog nucleic acid. Presently preferred primers include the
primers set out in Table 1 (THRSP .alpha., THRSP .beta. and total
THRSP primers). The length of the oligonucleotide primers are
commonly in the range of 10 to 30 nucleotides in length, preferably
in the range of 18 to 25 nucleotides in length.
[0035] Probes can be any length suitable for specific hybridization
to the target nucleic acid sequence. The most appropriate length of
the probe may vary depending upon the hybridization method in which
it is being used; for example, particular lengths may be more
appropriate for use in microfabricated arrays (microarrays), while
other lengths may be more suitable for use in classical
hybridization methods. Such optimizations are known to the skilled
artisan. Suitable probes can range from about 5 nucleotides to
about 30 nucleotides in length. Additionally, a probe can be a
genomic fragment that can range in size from about 25 to about
2,500 nucleotides in length. The probe preferably overlaps at least
one polymorphic site occupied by any of the possible variant
nucleotides. The nucleotide sequence of the probe can correspond to
the coding sequence of the allele or to the complement of the
coding sequence of the allele.
[0036] Preferably, the PCR probes are TaqMan.RTM. probes which are
labeled at the 5'end with a fluorophore, and at the 3'-end with a
quencher or a minor groove binder and a quencher (for minor groove
binding assays), or molecular beacon probes. TaqMan probes,
suitable fluorophores and quenchers for use with TaqMan.RTM. probes
and PCR methods employing TaqMan probes are disclosed in U.S. Pat.
Nos. 5,210,015, 5,804,375, 5,487,792 and 6,214,979.
[0037] Hybridizations can be performed under stringent conditions,
e.g., at a salt concentration of no more than 1 M and a temperature
of at least 25.degree. C. For example, conditions of 5.times.SSPE
(750 mM NaCl, 50 mM Na-Phosphate, 5 mM EDTA, pH 7.4) and a
temperature of 25-30.degree. C., or equivalent conditions, are
suitable for allele-specific probe hybridizations. Equivalent
conditions can be determined by varying one or more of the
parameters given as an example, as known in the art, while
maintaining a similar degree of identity or similarity between the
target nucleotide sequence and the primer or probe used.
[0038] The reaction mixture for amplifying the DNA comprises 4
deoxynucleotide phosphates (dATP, dGTP, dCTP, dTTP) and heat stable
DNA polymerase (such as Taq polymerase), which are all known to the
skilled person in the art.
[0039] The oligonucleotide primers and probes can be synthesized by
any technique known to a person skilled in the art, based on the
structure of the nucleotide sequence of THRSP or its
complement.
[0040] The term "isolated" oligonucleotide refers to an
oligonucleotide that is found in a condition other than its native
environment. In a preferred form, the oligonucleotide is
substantially free from other nucleic acid sequences, such as other
chromosomal and extrachromosomal DNA and RNA, that normally
accompany or interact with it as found in its naturally occurring
environment. The term "isolated" oligonucleotide also embraces
recombinant oligonucleotides and chemically synthesized
oligonucleotides.
[0041] The invention further provides kits comprising at least one
set of primers for amplifying a region of the nucleotide sequence
of THRSP .alpha. and/or THRSP .beta. that span the
insertion/deletion sites. The assay kit can further comprise the
four deoxynucleotide phosphates (dATP, dGTP, dCTP, dTTP) and an
effective amount of a nucleic acid polymerizing enzyme. A number of
enzymes are known in the art which are useful as polymerizing
agents. These include, but are not limited to E. coli DNA
polymerase I, Klenow fragment, bacteriophage T7 RNA polymerase,
reverse transcriptase, and polymerases derived from thermophilic
bacteria, such as Thermus aquaticus. The latter polymerases are
known for their high temperature stability, and include, for
example, the Taq DNA polymerase I. Other enzymes such as
Ribonuclease H can be included in the assay kit for regenerating
the template DNA. Other optional additional components of the kit
include, for example, means used to label a probe and/or primer
(such as a fluorophore, quencher, chromogen, etc.), and the
appropriate buffers for reverse transcription, PCR, or
hybridization reactions. Usually, the kit also contains
instructions for carrying out the methods.
[0042] Synthetic chemistry techniques can be used to synthesize the
oligonucleotides of the invention.
[0043] All patents and patent applications cited in the present
application are expressly incorporated herein by reference for all
purposes. The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples, which are provided
for purposes of illustration only and are not intended to limit the
scope of the invention.
Examples
[0044] Abbreviations: THRSP, thyroid hormone-responsive Spot 14
protein; aa, amino acid; bp, base pair; NDUFC2, NADH dehydrogenase;
ALG8, glucosyltransferase; ACLY, ATP-citrate lyase; FAS, fatty acid
synthase; ME, malic enzyme; COUP-TF1, chicken ovalbumin upstream
promoter-transcription factor 1; L-PK, L-type pyruvate kinase; Sp1,
specificity protein 1; TRE, thyroid response elements, FUR, far
upstream region; T.sub.3, triiodothyronine; QTL, quantitative trait
loci; CAP3, contig assembly program 3; UD, University of Delaware;
CR1, chicken repeat 1; EST, expressed sequence tag; SSC, sodium
chloride, sodium citrate; BAC, bacterial artificial chromosome;
qRT-PCR, quantitative reverse transcriptase polymerase chain
reaction; IGCRP, Iowa Growth and Composition Resources Population;
BBSRC, British Biotechnology and Biological Sciences Research
Council; UTR, untranslated region; indel, insertion/deletion; kDa,
kilo Dalton; pI, isoelectric point; G12, gastrulation-specific
protein; STRAIT11499, hypothetical human protein; SRE, sterol
response element; SREBP1c, sterol response element binding protein
1c; ChoRE, carbohydrate response element.
[0045] 1. Materials and Methods
[0046] 1.1 Chicken EST Assembly and DNA Sequence Analyses
[0047] The in silico cDNA sequence of THRSP was assembled from
chicken EST sequences generated from two international chicken EST
projects (Boardman et al., 2002) (Cogburn et al., 2003b). and those
found in public databases (GenBank). Contigs were assembled using
CAP3 (Huang and Madan, 1999) with 40 by overlap and 90% identity;
the CAP3 assemblies and a chicken gene index are available from the
University of Delaware, Chicken Gene Index (Larry A. Cogburn).
Contig and unassembled singlet sequences were used in BlastN and
BlastX searches for identification of chicken genes.
[0048] The in silico cDNA sequence of chicken THRSP was also used
in BlastN searches against the GenBank chicken genome trace archive
deposited by the Washington University Genome Center. The sequences
of the Blast hits and their mate pairs were retrieved and used to
build genomic contigs, which were then used in subsequent BlastN
searches. This in silico chromosome walking procedure was repeated
five times. The final genomic contigs and singlets were used to
blast against our CAP3 database to identify genes in the vicinity
of THRSP .alpha. and THRSP .beta.. To avoid multi-locus chicken
repeat 1 (CR1) repetitive sequences, genomic regions containing CR1
sequences were carefully inspected.
[0049] For Southern blot analysis, chicken genomic DNA was
extracted from liver and digested with restriction enzymes in
buffer supplied by the manufacturer (Promega, Madison, Wis.). The
digested DNA (25 .mu.g) was then precipitated with ethanol and
resuspended in water. Restriction fragments were then separated in
1% agarose gel and transferred onto a nylon membrane. A chicken
THRSP probe was labeled with .sup.32P-dCTP by PCR amplification of
insert in a UD Spot 14 EST clone (pgf2n.pk005.j11) using 32F and
DeletionR primers (see Table 1). Hybridization was carried out at
42.degree. C. overnight in Dig Easy Hyb buffer (Roche;
Indianapolis, Ind.) with the .sup.32P-labeled probe
(1.times.10.sup.6 dpm/ml). After hybridization, the filter was
sequentially washed in 1.times.SSC, 0.2.times.SSC and 0.1.times.SSC
supplemented with 0:1% SDS at 62.degree. C. for 15 min each. The
membrane was exposed to a phosphor screen overnight and scanned
with a phosphorImager (Storm 840, Molecular Dynamics).
[0050] 1.2 Analysis of Two Chicken BAC Clones
[0051] Two chicken BAC clones (65J23 and 94A1) which were positive
for chicken THRSP (Cane et al, 2001) were obtained from the Texas
A&M University BAC Center. The BAC DNA was prepared using the
Large Construct Kit (Qiagen, Valencia, Calif.). The primers for
chicken NADH dehydrogenase (NDUFC2) and glucosyltransferase (ALG8)
were designed from in silico cDNA sequences (UD CAP3
Contig.sub.--7797.2 and Contig.sub.--3078.1, respectively) which
correspond to these chicken genes (see Table 1).
[0052] 1.3 RNA Isolation and Real-Time Quantitative RT-PCR
[0053] Tissues of interest were taken immediately after cervical
dislocation, snap frozen in liquid nitrogen and stored at
-80.degree. C. until extraction of RNA. Total RNA was extracted
using a RNeasy midi kit (Qiagen; Valencia, Calif.) and its
concentration determined by reading the optical density at 260 nm.
Samples were diluted in RNase free water to a concentration of 20
ng/.mu.l and stored in a 96-well plate at -80.degree. C. Real-time
quantitative RT-PCR (qRT-PCR) was performed with a 7900HT Sequence
Detection System (TaqMan) (Applied Biosystems (ABI), Foster City,
Calif.) using the TaqMan Master Mix Kit and gene-specific molecular
beacon probes (Applied Biosystems (ABI)) for 18S and total THRSP
(Table 1). Primers were designed using Primer Express 2.0 software
(Applied Biosystems (ABI)). For the remaining four genes, the
QuantiTech SYBR green RT-PCRkit (Qiagen, Valencia, Calif.) and
gene-specific PCR primers (see Table 1) were used in 20 .mu.l per
reaction following protocols recommended by the manufacturer. The
concentration of total RNA in each sample was ensured by analyzing
18S RNA by qRT-PCR, which showed no significant difference between
samples. A standard curve and conversion factor between primer sets
32F/93R (detects both THRSP.alpha. and .beta.) and
DeletionF/DeletionR (.alpha. specific) were generated using a
plasmid from a THRSP EST clone (pgf2n.pk005.j11) as template, which
was diluted to the optimal concentration range (4.26.times.10.sup.4
to 1.75.times.10.sup.8 copies per .mu.l) in water containing 20
ng/.mu.l yeast RNA. The template was then amplified following a
standard TaqMan qRT-PCR protocol (Applied Biosystems (ABI)). The
expression of THRSP .beta. in chicken tissue was calculated by
taking the difference between total THRSP (32F/93R primers) and
THRSP .alpha.-specific (DeletionF/DeletionR primers)
measurements.
[0054] 1.4 Genotyping and Trait Association Analysis
[0055] The Iowa Growth and Composition Resources Population (IGCRP)
were used to study the association of the THRSP .alpha. and THRSP
.beta. polymorphisms with abdominal fat traits. This population was
established by crossing a broiler sire (from a commercial broiler
breeder male line) with dams from two unrelated highly-inbred lines
(Leghorn G-B2 and Fayoumi M15.2). These two inbred lines are more
than 99% inbred (Zhou and Lamont, 1999). Two F.sub.1 male offspring
of the same sire, one from each genetic cross (F.sub.1 Leghorn and
F.sub.1 Fayoumi) were randomly selected and each rooster mated with
20 half-sib F.sub.1 females, producing about 720 F.sub.2 offspring
in three hatches. Abdominal fat weight (Fat) was measured and also
expressed as a percentage of body weight at 8 weeks of age (%Fat).
For genotyping of THRSP .alpha., genomic DNA samples (40 ng) were
amplified by PCR using fluorescence forward primer 6FAM-DeletionF
and reverse primer DeletionR at 0.2 .mu.M each with 0.2 U Taq DNA
polymerase and 1.5 mM MgCl.sub.2 in 20 PCR was performed for 35
cycles of 45 sec at 94.degree. C., 45 sec at 55.degree. C., and 60
sec at 72.degree. C. after denaturation at 95.degree. C. for 2 min.
Final extension was carried out for 5 min. The 6FAM-DeletionF and
reverse primer DeletionR produce a 127 or 136 by amplicon as
described in Table 1. The 136 by amplicon is representative of
THRSP .alpha.1 which is the THRSP .alpha. insertion. The 127 by
amplicon is representative of the THRSP .alpha.2 which is the THRSP
.alpha. deletion.
[0056] PCR genotyping of the THRSP .beta. polymorphism was
performed using ThermalAce PCR kit (Invitrogen; Carlsbad, Calif.),
which is specifically designed to amplify very GC rich regions of
DNA; and the ParalogF/ParalogR primers (Table 1). Thermal cycles
were essentially the same as used in typing THRSP .alpha., except
that denaturation was at 98.degree. C. The ParalogF/ParalogR
primers produce a 145 or 151 by amplicon as shown in Table 1. The
151 by amplicon is representative of the THRSP .beta.1 which is the
THRSP .beta. insertion. The 145 by amplicon is representative of
THRSP .beta.2 which is the THRSP .beta. deletion.
[0057] The JMP.RTM. program (SAS Institute; Cary, N.C.) (Sall and
Lehman, 1996) was used to conduct the general linear model test for
association between genotype and fat traits based on model for the
whole F.sub.2 population:
Y=.mu.+G+Sex+Dam.sub.random(Cross)+Hatch.sub.randome. Where Y is
the dependent variable, .mu. is population mean, G is genotype, and
e is the random error. For analysis of each genetic cross, the
statistical model was the same except that Dam.sub.random was
substituted for Dam.sub.random(Cross), because the crosses were
analyzed separately.
[0058] 2. Results
[0059] 2.1. Identification of THRSP.alpha. and THRSP .beta.
Genes.
[0060] The in silico cDNA sequence (UD CAP3 Contig.sub.--8452.1) of
chicken THRSP .alpha. (FIG. 1) was assembled from a total of 61
ESTs found in the University of Delaware (UD) chicken EST database
(http://www.chickest.udel.edu/), the British Biotechnology and
Biological Sciences Research Council (BBSRC) chick EST database
(http://www.chick.umist.ac.uk/) (Boardman et al., 2002), and
GenBank. The THRSP .alpha. contig sequence is 874 by and it
includes two closely located poly(A) signals in the 3'-UTR and a
poly(A) tail. No additional sequence was found at the 5'-end of
THRSP .alpha. by 5'-RACE analysis (Invitrogen). Northern blot
analysis showed the THRSP .alpha. transcript is 1.1 kb (data not
shown). The predicted size of the THRSP.alpha. peptide is either
129 or 132 aa (due to the 9-bp indel polymorphism in coding region)
with a molecular weight of 14.471 or 14.185 kDa and a pI of 4.61 or
4.53 (FIG. 1A). As predicated by the PSORT II program (University
of Tokyo, Japan) (http://psort.ims.u-tokyo.ac.jp/), this peptide is
localized in the nucleus and has a leucine zipper motif in the C
terminus. The predicted chicken THRSP.alpha. peptide (FIG. 1A) has
a low similarity (29% identities; 46% positives) to the human THRSP
aa sequence (Grillasca et al., 1997) and to a gastrulation specific
protein, G12 (33% identities; 45% positives) found in zebrafish
(Conway, 1995). When a BlastX search of 1630 chicken protein
sequences, derived from complete open reading frames in the UD CAP3
chicken EST assemblies, was made against the non-redundant human
protein set in GenBank, the similarity of THRSP.alpha. was among
the weakest 2%.
[0061] The chicken THRSP .beta. (UD CAP3 Contig.sub.--8452.2) was
identified by searching the chicken UD CAP3 contig database, using
chicken THRSP .alpha. cDNA as an "electronic" probe. The THRSP
.beta. iii silico cDNA was assembled from eight ESTs found in the
BBSRC collection (adult liver, 5 ESTs; adult adipose tissue, 2
ESTs; adult heart, 1 EST); it is 670 by long with a typical poly(A)
signal sequence. The THRSP .beta. cDNA is almost identical to THRSP
.alpha. isoform in the first 230 nt at the 5'-end, which encodes a
nearly identical N-terminus. The overall similarity of the chicken
THRSP .alpha. and THRSP .beta. paralogs is 70% identical and 79%
positive (FIG. 1B). The THRSP .beta. cDNA is extremely GC-rich in
the 3'-end, which makes it a difficult target for cloning and PCR
amplification. Similar to the THRSP.alpha. isoform, the predicted
THRSP.beta. protein is acidic (pI 5.1 or 4.96) with a molecular
weight of 14.470 or 14.656 kDa and a leucine zipper motif in the
C-terminus.
[0062] 2.2 Sequence Alignment and Structural Analysis of Spot 14
Protein Family
[0063] A protein database search has revealed that THRSP family has
three structurally related members in chickens and zebrafish,
whereas mammals (i.e., human, mouse or rat) have only two members.
A sequence comparison shows the structural similarity among Spot 14
(THRSP), the zebrafish gastrulation-specific protein (G12), and the
hypothetical human protein (STRAIT11499) for chicken, human, mouse,
rat and zebrafish (FIG. 2). The Spot 14 protein family shares three
conserved domains: a highly hydrophobic aa sequence (PSLLRDV) near
N-terminus, a second hydrophobic region in the middle and the
leucine zipper motif in the carboxyl terminus.
[0064] A phylogenetic analysis shows that a common ancestor of
birds, fishes and mammals could have two genes that encode
structurally related proteins (FIG. 3). The THRSP protein is found
in chickens, humans, rodents and zebrafish (zTC192887). The second
member of the THRSP protein family found among these animals is the
hypothetical [human] STRAIT11499 proteins, which includes the two
zebrafish orthologs (G12 and zTC194742). The THRSP gene is
duplicated in chickens, whereas the gastrulation-specific G12 gene
is duplicated in zebrafish. The zG12 and zTC194742-derived proteins
found in zebrafish are similar in aa sequence (57% identity; 73%
positive). In contrast, the zebrafish THRSP protein (derived from
zTC192887) is different from both G12 (40% identical; 58% positive)
and zTC194742 (47% identical; 60% positive) proteins.
[0065] 2.3. Genomic Organization
[0066] To gain some insight into the genomic organization of the
two chicken genes, Southern blot analysis was performed using a
probe that hybridizes to both genes, although the hybridization
signal was stronger with a isoform (FIG. 4A). Genomic sequence of
15 kb that includes the THRSP .alpha. gene and its flanking regions
was assembled from raw chicken genome trace files in GenBank
(http://www.ncbi.nih.gov/Traces/trace.cgi). Alignment of THRSP
.alpha. cDNA sequence with chicken genomic sequence shows that this
gene contains two exons and one intron (FIGS. 1A and 4C). Similar
to the human gene, the first exon encodes the entire cTHRSP
protein, while exon 2 represents the 3'-UTR. Analysis of about 800
by in the 5'-flanking region of the THRSP .alpha. gene shows a TATA
box that is 39 by upstream of the transcription start site (FIG.
4C).
[0067] Genes in the vicinity were searched using the strategy
described in section 2.1. Genomic sequence (ssi42g12.b1, GenBank GI
no. 253911732) for THRSP .beta. was identified in one end of a
genomic clone. The other end of the same genomic clone
(ssi42g12.g1, GenBank GI no. 253911843) contained the THRSP .alpha.
gene. Therefore, the chicken THRSP paralogs are closely linked,
probably within a few kb, (see FIG. 4B) and are transcribed from
the same direction. Coding sequences for chicken orthologs of human
hypothetical protein (MGC2376; GenBank accession no.
XP.sub.--133614), NADH dehydrogenase (NDUFC2) and were also found
to flank Spot 14 (THRSP .alpha.) by in silico chromosomal walking.
PCR analysis of two previously identified (Carre et al., 2001)
THRSP-positive BAC clones (65J23 and 94A1) demonstrates the
presence of THRSP .alpha.-.beta., NDUFC2 and glucosyltransferase
(ALG8) from this synteny group in chicken genomic DNA (FIG. 5).
[0068] 2.4. Expression of THRSP Genes
[0069] The expression of the chicken THRSP genes was examined by
qRT-PCR using two primers (32F/93R) that are common to both THRSP
.alpha. and THRSP .beta. (Table 1; FIGS. 1A and 1B). Among 11
tissues examined, liver had the highest expression level of THRSP
mRNA, with fat, thymus and ovary expressing lower amounts (FIG.
6A). Thus, the THRSP genes appear to be predominantly expressed in
lipogenic tissue in the chicken. Direct measurement of THRSP .beta.
was not possible by TaqMan analysis (qRT-PCR) because the unique
region in THRSP .beta. cDNA is very GC-rich. Therefore, an indirect
method was used to examine THRSP .beta. expression in liver and fat
tissue (FIG. 6B). First, we obtained the total THRSP mRNA level by
using 32F/93R primer pairs; then, the THRSP .alpha. mRNA level was
determined using the specific DeletionF/DeletionR primer set. The
relative abundance of THRSP .beta. was calculated from the
difference between total THRSP and specific THRSP .alpha. mRNA
levels (FIGS. 6B, -C and -D). The relative abundance of THRSP
.alpha. and THRSP .beta. was examined in liver and abdominal fat of
five-week-old broiler chickens (4), where the abundance of THRSP
.alpha. was 2- to 3-times greater than that of THRSP .beta.,
respectively (FIG. 6B). Previously, a dramatic increase in chicken
total THRSP mRNA levels in liver of 1 day old chicks was found when
compared to late embryos (e16, e18 and e20) (Cogburn et al.,
2003b). Therefore, we examined whether the expression of THRSP
.alpha. and THRSP .beta. was differentially regulated during this
period. A dramatic increase of 13- to 20-fold was detected in THRSP
.alpha. and THRSP .beta. (FIG. 6C) mRNA levels (respectively) at 1
day post-hatching. Since the expression of THRSP responds rapidly
to nutritional factors, we also examined whether prolonged fasting
and re-feeding (Beccavin et al., 2001) would differentially
regulate expression of the hepatic THRSP paralogs. Both THRSP
.alpha. and THRSP .beta. mRNA levels were down-regulated after a 48
hr fast and up-regulated at 4 hr after re-feeding (FIG. 6D),
although the re-feeding response of THRSP .beta. was slightly
higher (8-fold increase) than that of THRSP .alpha. (5-fold
increase). Therefore, the transcription of THRSP .alpha. and THRSP
.beta. appears to respond similarly to developmental and
nutritional factors.
[0070] 2.5. THRSP .alpha. and THRSP .beta. Polymorphisms
(Haplotypes) and Their Association with Abdominal Fat Traits
[0071] Both THRSP .alpha. and THRSP .beta. genes are polymorphic
which involves a number of iterations of short repeats [9 by
(ATAGATGGC) in THRSP .alpha. and 6 by (ACGCCG) in THRSP .beta.]
located in the middle of protein coding region near leucine zipper
motif These polymorphisms result in the insertion or deletion of
three aa in THRSP.alpha. and two aa in THRSP.beta. protein. These
polymorphisms represent a haplotype which is a set of linked
alleles from linked genes on one chromosome. The F.sub.2 generation
from the broiler.times.Leghorn cross represented in the IGCRP (Deeb
and Lamont, 2002) were genotyped for THRSP .alpha. and THRSP .beta.
alleles or haplotypes (FIG. 7; Table 2) to determine if the THRSP
haplotypes are associated with deposition of abdominal fat in
chickens from this resource .sub.po.sub.pulation. In FIG. 7 and
Table 2, allele .alpha.1 represents the THRSP .alpha. insertion.
Allele .alpha.2 represents the THRSP a deletion. Allele .beta.1
represents the THRSP .beta. insertion. Allele .beta.2 represents
the THRSP .beta. deletion.
[0072] The THRSP .alpha.1.beta.1/.alpha.2.beta.2 genotype was
associated with the lowest abdominal fat content [Fat (g) and %
Fat] in the broiler.times.Leghorn cross (Table 2). In contrast, the
greatest amount of abdominal fat [Fat (g) and % Fat (% BW) traits]
was associated with the THRSP .alpha.1.beta.2/.alpha.2.beta.2
genotype. This represents a difference of about 7.5 g of body fat
between the two genotypes. There are four possible haplotypes,
.alpha.1.beta.1, .alpha.1.beta.2, .alpha.2.beta.1 and
.alpha.2.beta.2. In the present example, only three of the
haplotypes were found (.alpha.1.beta.1, .alpha.2.beta.2 and
.alpha.1.beta.2). The leanest haplotype in this example was
.alpha.1.beta.1 and the fattest .alpha.1.beta.2. We predict from
these data that the leanest haplotype would be .alpha.2.beta.1,
although it was not found in the present population.
[0073] 3. Discussion
[0074] The discovery of duplicated, but distinct, Spot 14 (THRSP
.alpha. and THRSP .beta.) genes and the insertion/deletion
polymorphisms in THRSP .alpha. and THRSP .beta. that is associated
with abdominal fat traits are described. A search of our CAP3
database of chicken EST assemblies has also revealed a third
structurally-related member of the THRSP protein family in chickens
(cSTRAIT11499). A search for orthologs of this protein family in
human, mouse, rat and zebrafish, where extensive EST data are
available, has revealed three members of the THRSP protein family
in chickens (THRSP.alpha., THRSP.beta. and cSTRAIT11499) and
zebrafish [zTHRSP (zTC192887), zG12 and zTC194742]. In contrast,
there are only two family members found in mammals (THRSP and
STRAIT11499). All members of this protein family have three
conserved domains which could be of functional importance (FIG. 2).
Another chicken EST (a singlet) found in the BBSRC database
(GenBank accession no. BU440998) has an exceptionally high homology
(99% nucleotide sequence identity) to bovine THRSP; however, this
probably represents a contaminating bovine cDNA sequence.
[0075] A synteny group, containing an ortholog of MGC2376, NADH
dehydrogenase (NDUFC2) and glucosyltransferase (ALG8), flanks the
chicken THRSP .alpha. and THRSP .beta. genes and is highly
conserved among chickens, rats, mice and humans, where they are
located on cChr1q41-44, rChr1q32-33, mChr7D3-E1 and hChr11q13.5,
respectively. Our study clearly shows that one of the THRSP genes
appears after the divergence of mammals and birds. This finding
suggests that a chromosomal duplication event has occurred in the
chicken. Gene duplication is a common process in genome evolution
(Tatusov et al., 1997), where each copy of the duplicated genes
acquires different mutations that could lead to altered function.
One copy of a duplicated gene usually shows a faster rate of
evolution (Zhang et al., 2003). THRSP.beta. is more similar in aa
sequence to mammalian THRSP. However, chicken THRSP .beta. has an
unusually high GC content, a feature that is not found in either
human or mouse orthologs. We have shown that expression of the
THRSP paralogs is coordinately regulated in liver and fat, during
post-hatching development and by re-feeding. It is interesting to
note that the flanking NDUFC2 gene in chicken is also highly
polymorphic. Alignment of 22 ESTs and chicken genomic trace
sequence reveals two alternatively-spliced isoforms and two
polymorphic sites in chicken NDUFC2. One site is located in the
5`-UTR and involves a gcc repeat, whereas the other polymorphic
site is located in the 3'-UTR and involves four by (ataa).
Therefore, this chromosomal region in the chicken appears to be a
hot spot for genomic reorganization.
[0076] Expression of the murine THRSP has been extensively studied
in liver and adipose tissue, where nutritional and hormonal factors
intricately regulate its expression (Clarke et al., 1990; Jump et
al., 1994; Liu and Towle, 1994). Enhanced long-chain fatty acid
synthesis occurs in lipogenic breast cancer, where THRSP is
necessary for tumor growth. Therefore, amplification of the THRSP
gene is a prognosticator of lipogenic breast cancer in humans
(Moncur et al., 1998). In the present study, we have demonstrated
the association of the THRSP .alpha. and THRSP .beta. polymorphisms
with abdominal fat traits in a broiler.times.Leghorn cross. There
is a clear association of the THRSP .alpha. and THRSP .beta.
haplotypes with fat traits. The insertion/deletion polymorphisms in
THRSP .alpha. and explains about 14% of the variation in abdominal
fat, which correlates well with the estimate of about 21 polygenes
that control expression of the % Fat trait in this population (Deeb
and Lamont, 2002). It is particularly interesting that the
insertion/deletion polymorphisms in the THRSP .alpha. and THRSP
.beta. paralogs involve aspartic acid near the leucine zipper
motif, which is critical for homodimerization of THRSP and
subsequent transcriptional control of lipogenic enzymes (Cunningham
et al., 1997). This could add additional complexity to dimerization
of this acidic transcriptional activator in chickens. In some
chickens, there are four different isoforms of THRSP that could
form different dimmer combinations. If these isoforms act
differently in controlling fat deposition, it could be much more
complicated to determine the effect of each individual allele.
[0077] Our initial transcriptional profiling studies have shown
that the expression of THRSP in liver is up-regulated by
metabolically-active T.sub.3, post-hatching development and
re-feeding after a prolonged period of fasting. In chickens, this
thyroid hormone-regulated putative transcription factor (THRSP)
appears to play a key role in regulating the expression of six
enzymes in the lipogenic pathway (see FIG. 4 in Cogburn et al.,
2003b). As a homodimer, THRSP interacts with nuclear receptors
(i.e., COUP-TF1) in the transcriptional control of lipogenic
enzymes (Cunningham et al., 1997; Cunningham et al., 1998; Compe et
al., 2001). Furthermore, the mammalian THRSP promoter contains
multiple response elements that respond to thyroid hormone (TRE)
(Liu and Towle, 1994), carbohydrates (ChoRE) (Koo and Towle, 2000)
and sterols (SRE, sterol response element), particularly SREBP-1c
(Jump et al., 2001). These multiple response elements exert THRSP's
control over the expression of key lipogenic, glycolytic and
gluconeogenic enzymes in a tissue-specific and fuel-dependent
manner (Brown et al., 1997). In chickens, the expression of THRSP
mRNA increases dramatically in the liver of newly-hatched chicks as
they begin to synthesize and deposit abdominal fat. It has been
consistently found that THRSP responds to metabolic perturbations
and it is found in clusters of functionally-related genes (i.e.,
enzymes and transcription factors) that control metabolism and fat
deposition in the chicken (Cogburn et al., 2003a; Cogburn et al.,
2003b).
[0078] 4. Conclusions
[0079] Duplicated paralogs of Spot 14 in the chicken, THRSP .alpha.
and THRSP .beta. were identified by sequence analysis of contigs
assembled from our chicken EST collection and those in public
databases (>309,000 ESTs). A computational analysis of THRSP
proteins has reveal three highly-conserved domains in two
structurally-related proteins from the THRSP family (THRSP and
STRAIT11499, a hypothetical human protein) across a number of
vertebrates (chicken, zebrafish, rat, mouse and human).
Transcription of THRSP .alpha. and THRSP .beta. mRNA in lipogenic
tissues appears to be controlled by developmental, hormonal and
nutritional factors. Polymorphic alleles involving tandem repeats
(of either 9 or 6 bp) were found in the putative protein coding
region of the chicken THRSP .alpha. (a 9 by indel) and THRSP .beta.
(a 6 by indel) genes. Our study shows that the THRSP .alpha. and
THRSP .beta. loci are associated with abdominal fat traits in a
broiler.times.Leghorn resource population. Furthermore, assembly of
THRSP-positive chicken genomic sequences has revealed a synteny
group of THRSP and its flanking genes [NADH dehydrogenase (NDUFC2)
and glucosyltransferase (ALG8)] that is highly conserved in
chickens, humans, mice and rats. The chicken THRSP genes are
located on Chr1q41-44 near QTL for fatness. These observations
support a role of THRSP in control of lipogenesis and expression of
abdominal fat traits in the domestic chicken.
TABLE-US-00001 TABLE 1 Quantitative RT-PCR (TaqMan) and PCR Primers
SEQ Primer ID Amplicon Gene Sequence NO: Size (bp) 18S RNA* Forward
GTGCATTTATCAG 14 76 ACCAAAACCAA Reverse GCGATCGGCTCG 15 AGGTTA
THRSP .alpha.* DeletionF GCCTCCGTCAC 16 127 or 136 CGATCAG
DeletionR CGGTCAGAACCT 17 GCTGCAA THRSP .beta. ParalogF GCGTCCTTCAC
18 145 or 151 CGAGCG ParalogR TGGCTGAGGATCT 19 GCTGCAG NDUFC2 465F
CGTGTGGATGGCAA 20 151 GATGTT 615R CAACTCCAGGCTT 21 GCTGCAT ALG8
1053F GCCTTGTTGTTTG 22 460 TGCGTTG 1203R AAATGCCCTGTGGT 23 TGTCAGA
Total THRSP* 32F TTCTCGGCCACG 24 71 CAGAAG 93R AAGACCCCTCGC 25
AGCAGG *These primer sets were used in TaqMan real-time qRT-PCR
analysis.
TABLE-US-00002 TABLE 2 Association of chicken THRSP.alpha. and
THRSP.beta. alleles (haplotypes) with fat traits in the Iowa Growth
and Composition Resource Population (IGCRP) Abdominal Fat Genotype
Number of Birds Weight (g) Fat (% BW)
.alpha.1.beta.1/.alpha.2.beta.2 28 47.43 .+-. 2.92.sup.a 2.88 .+-.
0.17.sup.a .alpha.2.beta.2/.alpha.2.beta.2 43 52.11 .+-.
2.30.sup.ab 3.27 .+-. 0.13.sup.ab .alpha.1.beta.2/.alpha.1.beta.1
39 50.44 .+-. 2.58.sup.ab 3.27 .+-. 0.15.sup.ab
.alpha.1.beta.2/.alpha.2.beta.2 156 50.97 .+-. 1.19.sup.ab 3.23
.+-. 0.07.sup.ab .alpha.1.beta.2/.alpha.1.beta.2 106 54.96 .+-.
2.30.sup.b 3.45 .+-. 0.08.sup.b Note: The traits used were
abdominal fat weight (g) and abdominal fat expressed as a percent
of body weight (% BW). Values .+-. SEM that possess a different
superscript letter are significantly (P < 0.05) different.
REFERENCES
[0080] Beccavin et al., 2001, Insulin-like growth factors and body
growth in chickens divergently selected for high or low growth
rate. J. Endocrinol. 168, 297-306. [0081] Boardman et al., 2002, A
comprehensive collection of chicken cDNAs. Curr. Biol. 12,
1965-1969. [0082] Brown et al. 1997. "Spot 14" protein functions at
the pretranslational level in the regulation of hepatic metabolism
by thyroid hormone and glucose. J. Biol. Chem. 272, 2163-2166.
[0083] Cane et al. 2001. Development of 112 unique expressed
sequence tags from chicken liver using an arbitrarily primed
reverse transcriptase-polymerase chain reaction and single strand
conformation gel purification method. Anim. Genet. 32, 289-297.
[0084] Clarke et al., 1990. Nutritional control of rat liver fatty
acid synthase and S14 mRNA abundance. J. Nun. 120, 218-224. [0085]
Cogburn et al., 2000. DNA microarray analysis of gene expression in
the liver of broiler chickens divergently selected for growth rate.
Poult. Sci. 79 (suppl.1), 72. [0086] Cogburn et al., 2003a.
Expressed sequence tags, DNA chip technology and gene expression
profiling. In: Muir, M. W. and Aggrey, S. E. (Eds.), Poultry
Genetics, Breeding and Biotechnology. CABI Publishing, Wallingford,
Oxon, UK, pp. 629-646. [0087] Cogburn et al., 2003b. Systems-wide
chicken DNA microarrays, gene expression profiling and discovery of
functional genes. Poult. Sci. 82, 6378-6383.
[0088] Compe et al., 2001. Spot 14 protein interacts and
co-operates with chicken ovalbumin upstream promoter-transcription
factor 1. in the transcription of the L-type pyruvate kinase gene
through a specificity protein 1 (Sp1) binding site. Biochem. J.
358,175-183. [0089] Conway, G., 1995. A novel gene expressed during
zebrafish gastrulation identified by differential RNA display.
Mech. Dev. 52, 383-391. [0090] Cunningham et al., 1997. Spot 14
protein-protein interactions: evidence for both homo- and
heterodimer formation in vivo. Endocrinology 138, 5184-5188. [0091]
Cunningham et al., 1998. "Spot 14" protein: a metabolic integrator
in normal and neoplastic cells. Thyroid 8, 815-825. [0092] Deeb,
N., Lamont, S. J., 2002. Genetic architecture of growth and body
composition in unique chicken populations. J. Hered. 93, 107-18.
[0093] Griffin et al., 1992. "Adipose tissue lipogenesis and fat
deposition in leaner broiler chickens", J. Nutr. 122,363-368.
[0094] Grillasca et al., 1997. Cloning and initial characterization
of human and mouse Spot 14 genes. FEBS Lett. 401, 38-42. [0095]
Huang, X., Madan, A., 1999. CAP3: A DNA sequence assembly program.
Genome Res. 9, 868-877. [0096] Ikeobi et al., 2002. Quantitative
trait loci affecting fatness in the chicken. Anim. Genet, 33,
428-435. [0097] Jump et al., 1984. Rapid effects of
triiodothyronine on hepatic gene expression. Hybridization analysis
of tissue-specific triiodothyronine regulation of mRNAS14. J. Biol.
Chem. 259, 2789-2797. [0098] Jump, D. B., Oppenheimer, J. H., 1985.
High basal expression and 3,5,3'-triiodothyronine regulation of
messenger ribonucleic acid S14 in lipogenic tissues. Endocrinology
117, 2259-2266. [0099] Jump et al., 1993. Polyunsaturated fatty
acids inhibit S14 gene transcription in rat liver and cultured
hepatocytes. Proc. Natl. Acad. Sci. 90, 8454-8458. [0100] Jump et
al., 1994. Coordinate regulation of glycolytic and lipogenic gene
expression by polyunsaturated fatty acids. J. Lipid Res. 35,
1076-1084. [0101] Jump et al., 2001. Functional interaction between
sterol regulatory element-binding protein-1c, nuclear factor Y, and
3,5,3'-triiodothyronine nuclear receptors. J. Biol. Chem. 276,
34419-34427. [0102] Kinlaw et al., 1995. Direct evidence for a role
of the "spot 14" protein in the regulation of lipid synthesis. J.
Biol. Chem. 270, 16615-16618. [0103] Koo, S. H., Towle, H. C.,
2000. Glucose regulation of mouse S(14) gene expression in
hepatocytes. Involvement of a novel transcription factor complex.
J. Biol. Chem. 275, 5200-5207. [0104] Lagarrigue et al., 2003. "An
initial QTL scan for abdominal fatness and breast muscle weight in
broiler chickens.", Plant & Animal Genome XI Conference, San
Diego, Calif., 2003, pp. 595. [0105] Leclerq et al., 1980.
"Selecting broilers for low or high abdominal fat: initial
observations" Br. Poul. Sci. 21, 107-113. [0106] Legrand, P. and
Hermier, D., 1992. "Hepatic D9 desaturation and plasma VLDL in
genetically lean and fat chickens." Int. J. Obesity 16, 289-294.
[0107] Liaw, C. W., Towle, H. C., 1984. Characterization of a
thyroid hormone-responsive gene from rat. J. Biol. Chem. 259,
7253-7260. [0108] Liu, H. C., Towle, H. C., 1994. Functional
synergism between multiple thyroid hormone response elements
regulates hepatic expression of the rat S14 gene. Mol. Endocrinol.
8, 1021-1037. [0109] Moncur et al., 1998. The "Spot 14" gene
resides on the telomeric end of the 11q13 amplicon and is expressed
in lipogenic breast cancers: implications for control of tumor
metabolism. Proc. Natl. Acad. Sci. 95, 6989-6994. [0110] O'Hea, E.
K. and Leveille, G. A., 1968. "Lipogenesis in isolated adipose
tissue of the domestic chick (Gallus domesticus)" Comp. Biochem.
Physiol. 26, 111-120. [0111] Sall, J., Lehman, A., 1996. JMP Start
Statistics: A guide to statistical and data analysis using JMP and
JMP IN software. Duxbury Press, Wadsworth Publishing Company,
Belmont, Calif. [0112] Seelig et al., 1981. Thyroid hormone
attenuates and augments hepatic gene expression at a
pretranslational level. Proc. Natl. Acad. Sci. 78, 4733-4737.
[0113] Tatusov et al., 1997. A genomic perspective on protein
families. Science 278, 631-637. [0114] Whitehead, C. C., Griffin,
H. D., 1984. "Development of divergent lines of lean and fat
broilers using plasma very low density lipoprotein concentration as
selection criterion: the first three generations". Br. Poult. Sci.
25, 573-582. [0115] Zhang et al., 2003. Different evolutionary
patterns between young duplicate genes in the human genome. Genome
Biol. 4, R56. [0116] Zhou, H., Lamont, S. J., 1999. Genetic
characterization of biodiversity in highly inbred chicken lines by
microsatellite markers. Anim. Genet. 30, 256-264. [0117] Zhu et
al., 2001. Spot 14 gene deletion increases hepatic de novo
lipogenesis. Endocrinology 142, 4363-4370.
Sequence CWU 1
1
251823DNAGallus gallus 1gaggagaggc aaggagtggg gccatggagc agtacttctc
ggccacgcag aagatggagc 60aggaggtgat gttccccagc ctgctgcgag gggtcttccc
gcaggacggg gccgacccag 120ccaccggcgg ccccgcagac ctctacgagc
actaccagct cctcaaggcc atcaagcccg 180tggtggagcg aggcctggcc
tccgtcaccg atcagagccc caccagcaat gccgacgccg 240acacggcccc
atatgatggc atagatggca tagatgggaa tctggaggag cggctgtccc
300accacatgaa tggcttgcag caggttctga ccgacctcac caaaaacacc
aaagctctca 360cccggaggta cagccagatc ctggaggaga tcaacctcgg
tgaaggtcag agcagctcat 420gagcctgcac acggagactc caagggtgat
ctgacgtttg cagcccagcg gcagctttat 480tcctgtgcca agtcccacca
aggaatgtct tctgcacaga ccagcacaga ggcttggctg 540taccatccaa
gctgccacat ggccaatcct cccggcaaac ctcactgctt tgtttcacct
600ccattcccgg ggattgcctt gcagtaggca gggcagaaat gagcgttcgc
tgttattgct 660tcctagaagg catctgtaac cttgaaacaa tgcttttgtt
ctgcatgtgc ctagaccacc 720tccccactag tttctttgat aatgtcacca
gttcccaaag tcaatgatct gaaataaaat 780gcaataataa aatgaaaaaa
aaaactcagt gcggaatttg aaa 8232132PRTGallus gallus 2Met Glu Gln Tyr
Phe Ser Ala Thr Gln Lys Met Glu Gln Glu Val Met1 5 10 15Phe Pro Ser
Leu Leu Arg Gly Val Phe Pro Gln Asp Gly Ala Asp Pro 20 25 30Ala Thr
Gly Gly Pro Ala Asp Leu Tyr Glu His Tyr Gln Leu Leu Lys 35 40 45Ala
Ile Lys Pro Val Val Glu Arg Gly Leu Ala Ser Val Thr Asp Gln 50 55
60Ser Pro Thr Ser Asn Ala Asp Ala Asp Thr Ala Pro Tyr Asp Gly Ile65
70 75 80Asp Gly Ile Asp Gly Asn Leu Glu Glu Arg Leu Ser His His Met
Asn 85 90 95Gly Leu Gln Gln Val Leu Thr Asp Leu Thr Lys Asn Thr Lys
Ala Leu 100 105 110Thr Arg Arg Tyr Ser Gln Ile Leu Glu Glu Ile Asn
Leu Gly Glu Gly 115 120 125Gln Ser Ser Ser 1303684DNAGallus gallus
3gcgaggcgaa gcgcggggcc atggagcggt acttctcggc cacgcagaag atggagcagg
60aggtgatgtt ccccagcctg ctgcgagggg tcttcccgca ggacggggcc gacccagccg
120ccgacggccc cgcggacctc tacgagcgct accagctcct caaggccatc
aagcccgtgg 180tggagcgagg cctggcgtcc ttcaccgagc gcagctccgc
cggccacgcc gacgccgacg 240ccgacgccga ggacgcggcg gccgcagccg
acggggcggc cggcagcctg gagcagcggc 300tgtgccacca cctggccggg
ctgcagcaga tcctcagcca cctgaccagg gacaccgccg 360ccctgacgcg
ccgctacagc cagatcctgg agcggatcag ccccggcgac gcgcagccca
420gctggtgacc ccgcgcggct ccgctcagcg ccgcgggacg gggcggcctc
cgagcggcgc 480cggcagagcc gcggagccgt ctgcggggcc gtcccgcggt
gccccgcggc tccgccgtgc 540gctccgtcct cggagagcgc cgcgctgccg
cgctgggctc ggacggagcc gtgcggcgcc 600ggcgccttcg ggctggatcc
cggagccgcg cagcgctgcc ctctctcgtg ttttctaata 660aaactcgtgt
ttttccgcaa aaaa 6844135PRTGallus gallus 4Met Glu Arg Tyr Phe Ser
Ala Thr Gln Lys Met Glu Gln Glu Val Met1 5 10 15Phe Pro Ser Leu Leu
Arg Gly Val Phe Pro Gln Asp Gly Ala Asp Pro 20 25 30Ala Ala Asp Gly
Pro Ala Asp Leu Tyr Glu Arg Tyr Gln Leu Leu Lys 35 40 45Ala Ile Lys
Pro Val Val Glu Arg Gly Leu Ala Ser Phe Thr Glu Arg 50 55 60Ser Ser
Ala Gly His Ala Asp Ala Asp Ala Asp Ala Glu Asp Ala Ala65 70 75
80Ala Ala Ala Asp Gly Ala Ala Gly Ser Leu Glu Gln Arg Leu Cys His
85 90 95His Leu Ala Gly Leu Gln Gln Ile Leu Ser His Leu Thr Arg Asp
Thr 100 105 110Ala Ala Leu Thr Arg Arg Tyr Ser Gln Ile Leu Glu Arg
Ile Ser Pro 115 120 125Gly Asp Ala Gln Pro Ser Trp 130
1355146PRTHomo sapiens 5Met Gln Val Leu Thr Lys Arg Tyr Pro Lys Asn
Cys Leu Leu Thr Val1 5 10 15Met Asp Arg Tyr Ala Ala Glu Val His Asn
Met Glu Gln Val Val Met 20 25 30Ile Pro Ser Leu Leu Arg Asp Val Gln
Leu Ser Gly Pro Gly Gly Gln 35 40 45Ala Gln Ala Glu Ala Pro Asp Leu
Tyr Thr Tyr Phe Thr Met Leu Lys 50 55 60Ala Ile Cys Val Asp Val Asp
His Gly Leu Leu Pro Arg Glu Glu Trp65 70 75 80Gln Ala Lys Val Ala
Gly Ser Glu Glu Asn Gly Thr Ala Glu Thr Glu 85 90 95Glu Val Glu Asp
Glu Ser Ala Ser Gly Glu Leu Asp Leu Glu Ala Gln 100 105 110Phe His
Leu His Phe Ser Ser Leu His His Ile Leu Met His Leu Thr 115 120
125Glu Lys Ala Gln Glu Val Thr Arg Lys Tyr Gln Glu Met Thr Gly Gln
130 135 140Val Trp1456150PRTMus musculus 6Met Gln Val Leu Thr Lys
Arg Tyr Pro Lys Asn Cys Leu Leu Thr Val1 5 10 15Met Asp Arg Tyr Ser
Ala Val Val Arg Asn Met Glu Gln Val Val Met 20 25 30Ile Pro Ser Leu
Leu Arg Asp Val Gln Leu Ser Gly Pro Gly Gly Ser 35 40 45Val Gln Asp
Gly Ala Pro Asp Leu Tyr Thr Tyr Phe Thr Met Leu Lys 50 55 60Ser Ile
Cys Val Glu Val Asp His Gly Leu Leu Pro Arg Glu Glu Trp65 70 75
80Gln Ala Lys Val Ala Gly Asn Glu Thr Ser Glu Ala Glu Asn Asp Ala
85 90 95Ala Glu Thr Glu Glu Ala Glu Glu Asp Arg Ile Ser Glu Glu Leu
Asp 100 105 110Leu Glu Ala Gln Phe His Leu His Phe Cys Ser Leu His
His Ile Leu 115 120 125Thr His Leu Thr Arg Lys Ala Gln Glu Val Thr
Arg Lys Tyr Gln Glu 130 135 140Met Thr Gly Gln Val Leu145
1507150PRTRattus norvegicus 7Met Gln Val Leu Thr Lys Arg Tyr Pro
Lys Asn Cys Leu Leu Lys Val1 5 10 15Met Asp Arg Tyr Ser Ala Val Val
Arg Asn Met Glu Gln Val Val Met 20 25 30Ile Pro Ser Leu Leu Arg Asp
Val Glu Leu Met Gly Tyr Gly Gly Ser 35 40 45Val Gln Asp Gly Ala Pro
Asp Leu Tyr Thr Tyr Phe Thr Met Leu Lys 50 55 60Ser Ile Cys Val Glu
Val Asp His Gly Leu Leu Pro Arg Glu Glu Trp65 70 75 80Gln Ala Lys
Val Ala Gly Asn Glu Gly Ser Glu Ala Glu Asn Glu Ala 85 90 95Ala Glu
Thr Glu Glu Ala Glu Glu Asp Arg Leu Ser Glu Glu Leu Asp 100 105
110Leu Glu Ala Gln Phe His Leu His Phe Ser Ser Leu His His Ile Leu
115 120 125Thr His Leu Thr Gln Lys Ala Gln Glu Val Thr Gln Lys Tyr
Gln Glu 130 135 140Met Thr Gly Gln Val Leu145 1508147PRTDanio rerio
8Met Met Gln Ile Cys Asp Ser Tyr Asn Gln Lys Asn Ser Leu Phe Asn1 5
10 15Ala Met Asn Arg Phe Ile Gly Ala Val Asn Asn Met Asp Gln Thr
Val 20 25 30Met Val Pro Ser Leu Leu Arg Asp Val Pro Leu Asp Gln Glu
Glu Glu 35 40 45Lys Glu Val Thr Ser Phe Gln Asp Gly Asp Met Tyr Gly
Ser Tyr Val 50 55 60Leu Leu Lys Ser Ile Arg Asn Asp Ile Glu Trp Gly
Val Leu Gln Ala65 70 75 80Glu Glu Arg Arg Lys Glu Lys His Gly Val
Thr Thr Thr Ser Leu Glu 85 90 95Val Ser Arg Ile Glu Pro Asn Asp Lys
Asp Leu Glu Lys Leu Phe His 100 105 110Tyr His Leu Ser Gly Leu His
Thr Val Leu Ala Lys Leu Thr Arg Lys 115 120 125Ala Asn Thr Leu Thr
Asn Arg Tyr Lys Gln Glu Ile Gly Ile Gly Gly 130 135 140Cys Gly
Asn1459152PRTDanio rerio 9Met Gln Met Ser Glu Pro Leu Ser Gln Lys
Asn Ala Leu Tyr Thr Ala1 5 10 15Met Asn Arg Phe Leu Gly Ala Val Asn
Asn Met Asp Gln Thr Val Met 20 25 30Val Pro Ser Leu Leu Arg Asp Val
Pro Leu Asp Gln Glu Lys Glu Gln 35 40 45Gln Lys Leu Thr Asn Asp Pro
Gly Ser Tyr Leu Arg Glu Ala Glu Ala 50 55 60Asp Met Tyr Ser Tyr Tyr
Ser Gln Leu Lys Ser Ile Arg Asn Asn Ile65 70 75 80Glu Trp Gly Val
Ile Arg Ser Glu Asp Gln Arg Arg Lys Lys Asp Thr 85 90 95Ser Ala Ser
Glu Pro Val Arg Thr Glu Glu Glu Ser Asp Met Asp Leu 100 105 110Glu
Gln Leu Leu Gln Phe His Leu Lys Gly Leu His Gly Val Leu Ser 115 120
125Gln Leu Thr Ser Gln Ala Asn Asn Leu Thr Asn Arg Tyr Lys Gln Glu
130 135 140Ile Gly Ile Ser Gly Trp Gly Gln145 15010165PRTDanio
rerio 10Met Met Gln Leu Ser Asn Asp Ser His Cys Asn Lys His Ser Leu
Leu1 5 10 15Asn Val Met Asn Arg Phe Ile Ala Ala Ala Asn Asn Met Asp
Glu Thr 20 25 30Ile Met Val Pro Asn Leu Leu Arg Asp Val Pro Leu Glu
Asp Gln Glu 35 40 45Ser His Ala Ser Val Ser His Asn Asn Asn Asn Asn
Asn Glu Pro Ser 50 55 60Phe Pro Asn Lys Gln Arg Asp Met Tyr Glu His
Tyr Leu Leu Leu Lys65 70 75 80Ser Ile Lys Asn Asp Met Glu Trp Gly
Leu Leu Lys Arg Glu Met Ala 85 90 95Gly Gly Ala Ser Phe Leu Glu Met
Ala Val Lys Gln Glu Glu Leu Pro 100 105 110Gln Met Lys Gly Glu Ala
Val Glu Glu Gly Pro Asp Leu Glu Gly Gln 115 120 125Phe His Tyr His
Leu His Gly Leu Phe Ser Val Leu Ser Lys Leu Thr 130 135 140Val Gln
Ala Asp His Leu Thr Asn Arg Tyr Lys Arg Glu Ile Gly Gly145 150 155
160Gly Ser Leu Leu Arg 16511183PRTHomo sapiens 11Met Met Gln Ile
Cys Asp Thr Tyr Asn Gln Lys His Ser Leu Phe Asn1 5 10 15Ala Met Asn
Arg Phe Ile Gly Ala Val Asn Asn Met Asp Gln Thr Val 20 25 30Met Val
Pro Ser Leu Leu Arg Asp Val Pro Leu Ala Asp Pro Gly Leu 35 40 45Asp
Asn Asp Val Gly Val Glu Val Gly Gly Ser Gly Gly Cys Leu Glu 50 55
60Glu Arg Thr Pro Pro Val Pro Asp Ser Gly Ser Ala Asn Gly Ser Phe65
70 75 80Phe Ala Pro Ser Arg Asp Met Tyr Ser His Tyr Val Leu Leu Lys
Ser 85 90 95Ile Arg Asn Asp Ile Glu Trp Gly Val Leu His Gln Pro Pro
Pro Pro 100 105 110Ala Gly Ser Glu Glu Gly Ser Ala Trp Lys Ser Lys
Asp Ile Leu Val 115 120 125Asp Leu Gly His Leu Glu Gly Ala Asp Ala
Gly Glu Glu Asp Leu Glu 130 135 140Gln Gln Phe His Tyr His Leu Arg
Gly Leu His Thr Val Leu Ser Lys145 150 155 160Leu Thr Arg Lys Ala
Asn Ile Leu Thr Asn Arg Tyr Lys Gln Glu Ile 165 170 175Gly Phe Gly
Asn Trp Gly His 18012182PRTMus musculus 12Met Met Gln Ile Cys Asp
Thr Tyr Asn Gln Lys His Ser Leu Phe Asn1 5 10 15Ala Met Asn Arg Phe
Ile Gly Ala Val Asn Asn Met Asp Gln Thr Val 20 25 30Met Val Pro Ser
Leu Leu Arg Asp Val Pro Leu Ser Glu Pro Glu Ile 35 40 45Asp Glu Val
Ser Val Glu Val Gly Gly Ser Gly Gly Cys Leu Glu Glu 50 55 60Arg Thr
Thr Pro Ala Pro Ser Pro Gly Ser Ala Asn Glu Ser Phe Phe65 70 75
80Ala Pro Ser Arg Asp Met Tyr Ser His Tyr Val Leu Leu Lys Ser Ile
85 90 95Arg Asn Asp Ile Glu Trp Gly Val Leu His Gln Pro Ser Ser Pro
Pro 100 105 110Ala Gly Ser Glu Glu Ser Thr Trp Lys Pro Lys Asp Ile
Leu Val Gly 115 120 125Leu Ser His Leu Glu Ser Ala Asp Ala Gly Glu
Glu Asp Leu Glu Gln 130 135 140Gln Phe His Tyr His Leu Arg Gly Leu
His Thr Val Leu Ser Lys Leu145 150 155 160Thr Arg Lys Ala Asn Ile
Leu Thr Asn Arg Tyr Lys Gln Glu Ile Gly 165 170 175Phe Ser Asn Trp
Gly His 18013159PRTHomo sapiens 13Met Gln Ile Cys Asp Ser Tyr Ser
Gln Lys Tyr Ser Leu Phe Asn Ala1 5 10 15Met Asn Arg Phe Ile Gly Ala
Val Asn Asn Met Asp Gln Thr Val Met 20 25 30Val Pro Ser Leu Leu Arg
Asp Val Pro Leu Leu Leu Gly Glu Leu Asp 35 40 45Ala Ala Gly Ala Val
Cys Pro Glu Arg Glu Ala Ala Pro Gly Gly Ala 50 55 60Tyr Phe Ser Arg
Arg Asp Met Tyr Ser His Tyr Val Leu Leu Lys Ser65 70 75 80Ile Arg
Asn Asp Ile Glu Trp Gly Val Val Gln Gln Ala Ala Gly Glu 85 90 95Glu
Ala Ala Arg Lys Lys Asp Lys Leu Gly Gly Gly Pro Ala Glu Glu 100 105
110Ala Glu Ala Glu Glu Asp Leu Glu Gln Gln Phe His Tyr His Leu Ser
115 120 125Gly Leu His Thr Val Leu Ser Lys Leu Thr Arg Lys Ala Asn
Val Leu 130 135 140Thr Asn Arg Tyr Lys Gln Glu Ile Gly Phe Gly Ser
Trp Gly Gln145 150 1551424DNAGallus gallus 14gtgcatttat cagaccaaaa
ccaa 241518DNAGallus gallus 15gcgatcggct cgaggtta 181618DNAGallus
gallus 16gcctccgtca ccgatcag 181719DNAGallus gallus 17cggtcagaac
ctgctgcaa 191817DNAGallus gallus 18gcgtccttca ccgagcg
171920DNAGallus gallus 19tggctgagga tctgctgcag 202020DNAGallus
gallus 20cgtgtggatg gcaagatgtt 202120DNAGallus gallus 21caactccagg
cttgctgcat 202220DNAGallus gallus 22gccttgttgt ttgtgcgttg
202321DNAGallus gallus 23aaatgccctg tggttgtcag a 212418DNAGallus
gallus 24ttctcggcca cgcagaag 182518DNAGallus gallus 25aagacccctc
gcagcagg 18
* * * * *
References