Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DIFFERENTIALLY EXPRESSED GENES
ASSOCIATED WITH OBESITY AND TYPE 2 DIABETES
FIELD OF THE INVENTION
The present invention relates generally to a nucleic acid molecule which
expressed in at
least the stomach, hypothalamus or liver identified using differential display
techniques
under differing physiological conditions. It is proposed that the nucleic acid
molecules
encode expression products associated with the modulation of obesity,
anorexia, weight
maintenance, diabetes and/or metabolic energy levels. More particularly, the
present
invention is directed to nucleic acid molecules and expression products
produced by
recombinant means from the nucleic acid molecule or isolated from natural
sources and
their use in therapeutic and diagnostic protocols for conditions such as
obesity, anorexia,
weight maintenance, diabetes and/or energy imbalance. The subject nucleic acid
molecule
and expression products and their derivatives, homologs, analogs and mimetics
are
proposed to be useful, therefore, as therapeutic and diagnostic agents for
obesity, anorexia,
weight maintenance, diabetes and/or energy imbalance or as targets for the
design and/or
identification of modulators of their activity and/or function.
BACKGROUND OF THE INVENTION
Bibliographic details of references provided in the subject specification are
listed at the end
of the specification.
Reference to any prior art in this specification is not, and should not be
taken as, an
acknowledgment or any form of suggestion that this prior art forms part of the
common
general knowledge in any country.
The increasing sophistication of recombinant DNA technology is greatly
facilitating
research and development in the medical, veterinary and allied human and
animal health
fields. This is particularly the case in the investigation of the genetic
bases involved in the
etiology of certain disease conditions. One particularly significant condition
from the stand
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point of morbidity and mortality is obesity and its association with type 2
diabetes
(formerly non-insulin-dependent diabetes mellitus or NIDDM) and cardiovascular
disease.
Obesity is defined as a pathological excess of body fat and is the result of
an imbalance
between energy intake and energy expenditure for a sustained period of time.
Obesity is
the most common metabolic disease found in affluent nations. The prevalence of
obesity in
these nations is alarmingly high, ranging from 10% to upwards of 50% in some
subpopulations (Bouchard, The genetics of obesity. Boca Raton: CRC Press,
1994). Of
particular concern is the fact that the prevalence of obesity appears to be
rising consistently
in affluent societies and is now increasing rapidly in less prosperous nations
as they
become more affluent and/or adopt cultural practices from the more affluent
countries
(Zimmet, Diabetes Cage 1 S(2): 232-247, 1992).
In 1995 in Australia, for example, 19% of the adult population were obese
(BMI>30). On
average, women in 1995 weighed 4.8 kg more than their counterparts in 1980
while men
weighed 3.6 kg more (Australian Institute of Health and Welfare, Heart, Stroke
and
Vascular diseases, Australian facts. AIHW Cat. No. CVD 7 Canberra: AIHW and
the
Heart Foundation of Australia, 1999). More recently, the AusDiab Study
conducted
between the years 1999 and 2000 showed that 65% of males and 45% of females
aged 25-
64 years were considered overweight (de Looper and Bhatia, Australia's Health
Trends
2001. Australian Institute of Health and Welfare (AIHW) Cat. No. PHE 24.
Canberra:
AIHW, 2001). The prevalence of obesity in the United States also increased
substantially
between 1991 and 1998, rising from 12% to 18% in Americans during this period
(Mokdad
et al., JAMA. 282(16): 1519-1522, 1999).
The high and increasing prevalence of obesity has serious health implications
for both
individuals and society as a whole. Obesity is a complex and heterogeneous
disorder and
has been identified as a key risk indicator of preventable morbidity and
mortality since
obesity increases the risk of a number of other metabolic conditions including
type 2
diabetes mellitus and cardiovascular disease (Must et al., JAMA. 282(16): 1523-
1529,
1999; Kopelman, Natuy~e 404: 635-643, 2000). Alongside obesity, the prevalence
of
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diabetes continues to increase rapidly. It has been estimated that there were
about 700,000
persons with diabetes in Australia in 1995 while in the US, diabetes
prevalence increased
from 4.9% in 1990 to 6.9% in 1999 (Molcdad, Diabetes Ca~~e 24(2): 412, 2001).
In
Australia, the annual costs of obesity associated with diabetes and other
disease conditions
has been conservatively estimated to be AU$810 million for 1992-3 (National
Health and
Medical Research Council, Acting on Australia's weight: A strategy for the
prevention of
overweight and obesity.Canberra: National Health and Medical Research Council,
1996).
In the U.S., the National Health Interview Survey (NHIS) estimated the
economic cost of
obesity in 1995 as approximately US$99 billion, thereby representing 5.7% of
total health
costs in the U.S. at that time (Wolf and Colditz, Obes Res. 6: 97-106, 1998).
A genetic basis for the etiology of obesity is indicated ihteY alia from
studies in twins,
adoption studies and population-based analyses which suggest that genetic
effects account
for 25-80% of the variation in body weight in the general population
(Bouchard, 1994,
supf~a; Kopelinan et al., Iyat J ~besity 18: 188-191, 1994; Ravussin,
Metabolisf~a 44(Suppl
3): 12-14, 1995). It is considered that genes determine the possible range of
body weight in
an individual and then the environment influences the point within this range
where the
individual is located at any given time (Bouchard, 1994, supra). However,
despite
numerous studies into genes thought to be involved in the pathogenesis of
obesity, there
have been surprisingly few significant findings in this area. In addition,
genome-wide
scans in various population groups have not produced definitive evidence of
the
chromosomal regions having a major effect on obesity.
A number of organs/tissues have been implicated in the pathophysiology of
obesity and
type 2 diabetes, and of particular interest are the hypothalamus, stomach and
liver. The
hypothalamus has long been recognized as a key brain area in the regulation of
energy
intake (Stellar, Psychol Rev 61: 5-22, 1954) and it is now widely accepted
that the
hypothalamus plays a central role in energy homeostasis, integrating and co-
ordinating a
large number of factors produced by and/or acting on the hypothalamus. A
number of these
factors have been investigated for their role in energy balance and body
weight regulation,
including neuropeptide Y, corticotropin-releasing hormone, melanin-
concentrating
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hormone, leptin and insulin. It has been proposed that genetic alterations
perturbing the
metabolic pathways regulating energy balance in the hypothalamus could
contribute to the
development of obesity, and subsequently diabetes. Thus, an important step in
understanding the function of the hypothalamus in regulating the metabolism of
an animal
requires the identification of the targets of these hormones. Such targets may
be whole
organs, and genes whose expression is regulated by the presence of these
hormones.
The role of the stomach in regulating food intake is thought to involve two
types of
signals: the degree of distension of the stomach and the activation of
chemoreceptors in the
gastric or intestinal wall (I~oopmans, Experimental studies on the control of
food intake.
In: Handbook of Obesity, Eds. GA Bray, C Bouchard, WPT James pp 273-312,
1995). The
gut is the largest endocrine organ in the body and after a meal a large number
of
gastrointestinal hormones are released. Some examples are gastrin,
somatostatin,
cholecystolcinin, gastric inhibitory polypeptide and neurotensin. Despite
general agreement
that the stomach provides part of the signal that restricts food intake during
a single meal,
the nature of this signal or how it is transmitted to the brain remains to be
determined.
Most likely the information relating to the degree of distension of the
stomach or the
presence of nutrients in the gastrointestinal walls is transmitted to the
brain through either
nerves or hormones. The role of the gut hormones identified to date in the
regulation of
food intake remains to be equivocally determined.
The liver also plays a significant role in a number of important physiological
pathways. It
has a major role in the regulation of metabolism of glucose, amino acids and
fat. In
addition the liver is the only organ (other than the gut) that comes into
direct contact with a
large volume of ingested, absorbed food via the portal vein and, therefore,
the liver is able
to "sense" or monitor the level of nutrients entering the body, particularly
the amounts of
protein and carbohydrate. It has been proposed that the liver may also have a
role in the
regulation of food intake through the transmission of unidentified signals
relaying
information to the brain about nutrient absorption from the gut and metabolic
changes
throughout the body (Russek, Nature 200: 176, 1963). The liver also plays a
crucial role in
maintaining circulating glucose concentrations by regulating pathways such as
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gluconeogenesis and glycogenolysis. Alterations in glucose homeostasis are
important
factors in the pathophysiology of impaired glucose tolerance and the
development of type
2 diabetes mellitus.
h1 accordance with the present invention, genetic sequences were sought which
are
differentially expressed in particular vertebrate aumal tissues or organs
between either
fed, re-fed or fasting conditions, or between diabetic and non-diabetic
conditions. Novel
genes are identified which are differentially expressed at least in the
stomach, liver and/or
hypothalamus under one or both of the above-mentioned conditions. In
accordance with
the present invention, the inventors have isolated genes which are proposed to
be
associated with one or more biological functions associated with disease
conditions such as
but not limited to obesity, anorexia, diabetes or energy balance.
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SUMMARY OF THE INVENTION
Throughout this specification, unless the context requires otherwise, the word
"comprise",
or variations such as "comprises" or "comprising", will be understood to imply
the
inclusion of a stated element or integer or group of elements or integers but
not the
exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier
number (SEQ
ff~ NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers
<400>1
(SEQ ID NO:1), <q.00>2 (SEQ ID N0:2), etc. A summary of the sequence
identifiers is
provided in Table 1. A sequence listing is provided after the claims.
Analysis of genetic material from stomach, hypothalamus and liver tissue were
used to
identify candidate genetic sequences associated with a healthy state or with
physiological
conditions such as obesity, anorexia, weight maintenance, diabetes and/or
metabolic
energy levels. An animal model was employed comprising the Israeli Sand Rat
(Psammomys obesus). Three groups of animals were used designated Groups A, B
and C
based on metabolic phenotype as follows:-
Group A: lean animals (normoglycemic; normoinsulinemic);
Group B: obese, non-diabetic animals (normoglycemic; hyperinsulinemic); and
Group C: obese, diabetic animals (hyperglycemic; hyperinsulinemic).
Techniques including differential display PCR analysis, suppression
substractive
hybridization (SSH) and amplified fragment length polymorphism analysis of
mRNA from
stomach, liver or hypothalamus tissue were used to identify genetic sequences
differentially expressed in fed, re-fed and fasted mammals or in diabetic and
non-diabetic
mammals. The Israeli Sand Rat (Psamnaomys obesus) was found to be particularly
useful
for this analysis. In a preferred embodiment, seven differentially expressed
sequences were
identified designated herein AGT 119 [SEQ m NO:1], AGT 120 [SEQ ID N0:2], AGT
121 [SEQ ID N0:3], AGT 122 [SEQ ID NO:S], AGT 422 [SEQ ID N0:6], AGT 123 [SEQ
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)D N0:7] and AGT 504 [SEQ ID N0:8 and SEQ >D N0:9]. SEQ ID N0:9 is a genomic
form of AGT-504 and is also represented as SEQ ID N0:8.
AGT 119 was detected initially in stomach tissue using differential display
PCR and its
expression was elevated in fed animals compared ~to fasted or re-fed animals.
AGT 120 was
initially detected in stomach tissue using differential display PCR and its
expression was
elevated in fed animals compared to fasted or re-fed animals. AGT 121 was
initially
identified in the hypothalamus using differential display PCR and its
expression levels
were elevated in fasted diabetic, non-diabetic and lean animals compared to
fed animals
when separated by genotype. AGT 122 was initially identified in the liver
using differential
display PCR and was shown to have elevated expression levels in fasted
compared to fed
diabetic or non-diabetic animals. AGT 422 was identified suppression
subtractive
hybridization in liver tissue and its expression was elevated in fed lean
animals compared
to fed diabetic animals and elevated in fed, lean animals compared to fasted
lean animals,
in fed non-diabetic animals compared to fasted non-diabetic animals and fed
diabetic
animals compared to fasted diabetic animals. AGT 123 was identified in the
hypothalamus
tissue using differential display PCR and its expression was found to be
elevated in fasted
lean, non-diabetic animals and diabetic animals compared to fed animals. AGT
504 was
identified using amplified fragment length polymorphism analysis in genomic
DNA and its
expression in liver tissue was elevated in diabetic animals compared to lean
or non-diabetic
animals. A summary of the AGT genes is provided in Table 1.
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TABLE 1
Sutzznzafy of Differentially Expressed Geszes
GENE SEQ ID TISSUE CHARACTERISTICS DETECTION
NO: METHOD
AGT 119 1 stomach elevated expressiondifferential
in
fed animals compareddisplay PCR
to
fasted or re-fed
animals
AGT 120 2 stomach elevated expressiondifferential
in
fed animals compareddisplay PCR
to
fasted or re-fed
animals
AGT 121 3 and hypothalamuselevated expressiondifferential
4 in
fasted diabetic, display PCR
non-
diabetic and lean
animals
compared to fed
animals
when separated by
genotype.
AGT 1 5 liver elevated expressiondifferential
~2 in
fasted compared display PCR
to fed
diabetic and non-diabetic
animals
AGT 422 6 liver elevated expressionsuppression
in
fed lean animals subtractive
compared to fed hybridization
diabetic
animals and elevated(representational
expression in fed difference
lean
animals compared analysis)
to
fasted lean animals,
in
fed non-diabetic
animals
compared to fasted
non-
diabetic animals
and fed
diabetic animals
compared to fasted
diabetic animals
AGT 123 7 hypothalamuselevated expressiondifferential
in
fasted lean, non-diabeticdisplay PCR
and diabetic animals
compared to fed
animals
AGT 504 ~ liver elevated expressionamplified
in
(genomic) diabetic animals fragment length
and 9 compared to lean polymorphism
or non-
(cDNA) diabetic animals analysis
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The present invention provides, therefore, a nucleic acid molecule comprising
a sequence
of nucleotides encoding or complementary to a sequence encoding an expression
product
or a derivative, homolog, analog or mimetic thereof wherein said nucleic acid
molecule is
differentially expressed in one or more of stomach, liver or hypothalamus
tissue under fed
or unfed or diabetic or non-diabetic conditions.
The present invention further provides a nucleic acid molecule comprising a
nucleotide
sequence encoding or complementary to a sequence encoding an expression
product or a
derivative, homolog, analog or mimetic thereof wherein the nucleotide sequence
is as
substantially set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID
NO:S
or SEQ ID N0:6 or SEQ ID N0:7 or SEQ ID NO:B or SEQ ID N0:9 or a nucleotide
sequence having at least about 30% similarity to all or part of SEQ ID NO:1 or
SEQ ID
N0:2 or SEQ ID N0:3 or SEQ ID NO:4 or SEQ ID NO:S or SEQ ID NO:6 or SEQ ~
N0:7 or SEQ ID NO:8 or SEQ ID N0:9 and/or is capable of hybridizing to one or
more of
SEQ ID NO:1 or SEQ ID N0:2 or SEQ ID N0:3 or SEQ ID N0:4 or SEQ ID NO:S or
SEQ ID N0:6 or SEQ ID N0:7 or SEQ ID NO:8 or SEQ ID N0:9 or their
complementary
forms under low stringency conditions at 42°C and wherein the nucleic
acid molecule is
differentially expressed in one or more of stomach, liver or hypothalamus
tissue under fed
or unfed or diabetic or non-diabetic conditions.
The present invention also provides an isolated expression product or a
derivative,
homolog, analog or mimetic thereof which expression product is encoded by a
nucleotide
sequence which is differentially expressed in one or more of stomach, liver or
hypothalamus tissue under fed or unfed or diabetic or non-diabetic conditions.
More particularly, the present invention is directed to an isolated expression
product or a
derivative, homolog, analog or mimetic thereof wherein the expression product
is encoded
by a nucleotide sequence substantially as set forth in SEQ ID NO:1, SEQ ID
NO:2 or SEQ
ID N0:3 or SEQ ID NO:S or SEQ ID N0:6 or SEQ TD N0:7 or SEQ ID N0:8 or SEQ ID
N0:9 or a nucleotide sequence having at least 30% similarity to all or part of
SEQ ID
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NO:1, SEQ m N0:2 or SEQ ID N0:3 or SEQ m NO:S or SEQ m N0:6 or SEQ m N0:7
or SEQ m N0:8 or SEQ m N0:9 andlor is capable of hybridizing to SEQ m NO:l,
SEQ
m N0:2 or SEQ ID NO:3 or SEQ m NO:S or SEQ m N0:6 or SEQ m NO:7 or SEQ ID
NO:8 or SEQ ID N0:9 or their complementary forms under low stringency
conditions at
42°C.
The preferred genetic sequence of the present invention are referred to herein
as AGT 119,
AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504. The expression
products
encoded by AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504
are referred to herein as AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123
and AGT-504, respectively. The expression product may be an RNA (e.g. mRNA) or
a
protein. Where the expression product is an RNA, the present invention extends
to RNA-
related molecules associated thereto such as RNAi or intron or exon sequences
therefrom.
Even yet another aspect of the present invention relates to a composition
comprising AGT-
119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or its
derivatives, homologs, analogs or mimetics or agonists or antagonists of AGT-
119, AGT-
120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 together with one or
more pharmaceutically acceptable carriers and/or diluents.
Another aspect of the present invention contemplates a method for treating a
subject
comprising administering to said subject a treatment effective amount of AGT-
119, AGT-
120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or a derivative,
homolog,
analog or mimetic thereof or a genetic sequence encoding same or an agonist or
antagonist
of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504
activity or AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and/or AGT
504
gene expression for a time and under conditions sufficient to effect
treatment.
In accordance with this and other aspects of the present invention, treatments
contemplated
herein include but are not limited to obesity, anorexia, weight maintenance,
energy
imbalance and diabetes. Treatment may be by the administration of a
pharmaceutical
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composition or genetic sequences via gene therapy. Treatment is contemplated
for human
subjects as well as animals such as animals important to livestocl~ industry.
A fuxther aspect of the present invention is directed to a diagnostic agent
for use in
monitoring or diagnosing conditions such as but not limited to obesity,
anorexia, weight
maintenance, energy imbalance and/or diabetes, said diagnostic agent selected
from an
antibody to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504
or its derivatives, homologs, analogs or mimetics and a genetic sequence
comprising or
capable of annealing to a nucleotide strand associated with AGT 119, AGT 120,
AGT 121,
AGT 122, AGT 422, AGT 123 or AGT 504 useful i~2te~~ alia in PCR,
hybridization, RFLP
analysis or AFLP analysis.
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A summary of sequence identifiers used throughout the subject specification is
provided in
Table 2.
TABLE 2
SEQUENCE ID NO: DESCRTPTION
1 Nucleotide sequence of AGT 119
2 Nucleotide sequence of AGT 120
3 Nucleotide sequence of AGT 121
4 Corresponding amino acid of SEQ m N0:3
Nucleotide sequence of AGT 122
6 Nucleotide sequence of AGT=422
7 Nucleotide sequence of AGT 123
8 Nucleotide sequence of AGT 504 (genomic)
9 Nucleotide sequence of AGT 504 (cDNA)
primer
11 primer
12 AGT 119 (set 1) forward primer
13 AGT 119 (set 1) reverse primer
14 AGT 119 (set 2) forward primer
AGT 119 (set 2) reverse primer
16 AGT 120 forward primer
17 AGT 120 reverse primer
18 AGT 121 forward (insertion) primer
19 AGT 121 forward (deletion) primer
AGT 121 reverse primer
21 AGT 122 forward primer
22 AGT 122 reverse primer
23 AGT 422 forward primer
24 AGT 422 reverse primer
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SEQUENCE ID NO: DESCRIPTION
25 AGT 123 forward primer
26 AGT 123 reverse primer
27 AGT 504 forward primer
28 AGT 504 reverse primer
29 (3-actin forward primer
30 [3-actin reverse primer
31 (3-actin probe
32 Cyclophilin forward primer
33 Cyclophilin reverse primer
34 Cyclophilin probe
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphical representation showing AGT 119 stomach gene expression
in fed,
fasted and re-fed Psamrnoryays obesus.
Figure 2 is a graphical representation of AGT 120 stomach gene expression in
fasted, fed
and re-fed Psammof~ays obesus.
Figure 3 is a graphical representation showing the distribution of tissue in
which AGT 121
is expressed.
Figure 4 is a photographic representation showing Northern analysis of AGT 121
expression in (1) heart; (2) brain; (3) placenta; (4) lung; (5) liver; (6)
skeletal muscle); (7)
kidney and (8) pancreas.
Figure 5 is a graphical representation showing AGT 121 expression in energy
restricted
hypothalamus.
Figure 6 is a graphical representation showing expression of AGT 121 versus
level of
body weight.
Figure 7 is a graphical representation showing expression of AGT 121 versus
cha~lge in
glucose levels.
Figure 8 graphical representation showing expression of AGT 121 versus scap
fat.
Figure 9 is a graphical representation of AGT 122 hepatic gene expression in
Groups A, B
and C fed and fasted Psammonays obesus.
Figure 10 is a graphical representation of AGT 122 hepatic gene expression for
fed and
fasted Psammomys obesus(pooled data) .
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Figure 11 is a graphical representation of the association between AGT 122
hepatic gene
expression and body weight in fed Psantmontys obesus.
Figure 12 is a graphical representation of AGT 422 hepatic gene expression
data from
Groups A, B and C fed and fasted Psammomys obesus.
Figure 13 is a graphical representation of AGT 422 hepatic gene expression in
all fed and
fasted Psamnzotnys obesus (pooled data).
Figure 14 is a graphical representation of AGT 123 hypothalamic gene
expression in
Groups A, B and C fed and fasted PsamnZOnays obesus.
Figure 15 is a graphical representation of AGT 504 hepatic gene expression in
Groups A,
B and C Psantmomys obesus.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is predicated in part on the identification of novel
genes associated
if2te~ alia with regulation of obesity, anorexia, weight maintenance, diabetes
and/or
metabolic energy levels. The genes are identified following differential
screening of
mRNA from one or more of stomach, liver or hypothalamus tissue in fed, re-fed
and fasted
mammals or in diabetic and non-diabetic mammals. The selection of stomach,
liver and
hypothalamus is not intended to imply that differential expression does not
occur in other
tissue.
Accordingly, one aspect of the present invention provides a nucleic acid
molecule
comprising a sequence of nucleotides encoding or complementary to a sequence
encoding
an expression product or a derivative, homolog, analog or mimetic thereof
wherein said
nucleic acid molecule is differentially expressed in one or more of stomach,
liver or
hypothalamus tissue under fed (or re-fed) or unfed or diabetic or non-diabetic
conditions.
The term "differentially expressed" is used in its most general sense and
includes elevated
levels of an expression product such as mRNA or protein or a secondary product
such as
cDNA in one tissue compared to another tissue or in the same tissue but under
different
conditions. Examples of different conditions includes differential expression
in tissue from
fed, re-fed and fasting animals or diabetic and non-diabetic animals.
Differential
expression is conveniently determined by a range of techniques including
polymerase
chain reaction (PCR) such as real-time PCR. Other techniques include
suppression
subtractive hyridization (SSH) and amplified fragment length polymorphism
(AFLP)
analysis.
Conveniently, an animal model may be employed to study the differences in gene
expression in animal tissues under different conditions. In particular, the
present invention
is exemplified using the Psafranaonays obesus (the Israeli Sand Rat) animal
model of
dietary-induced obesity and type 2 diabetes. In their natural desert habitat,
an active
lifestyle and saltbush diet ensure that they remain lean and normoglycemic
(Shafrir and
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Gutman, J. Basic Clin. Physiol. Phanna. 4: 83-99, 1993). However, in a
laboratory setting
on a diet of ad libitum chow (on which many other animal species remain
healthy), a range
of pathophysiological responses are seen (Barnett et al., Diabetologia 37: 671-
676, 1994a;
Barnett et al., Int. J. Obesity 18: 789-794, 1994b; Barnett et al., Diabete
Nutn. Metab. 8:
42-47, 1995). By the age of 16 weeks, more than half of the animals become
obese and
approximately one third develop type 2 diabetes. Only hyperphagic animals go
on to
develop hyperglycemia, highlighting the importance of excessive energy intake
in the
pathophysiology of obesity and type 2 diabetes in Psamnaomys obesus (Collier
et al., Ann.
New Yonk Acad. Sci. 827: 50-63, 1997a; Walder et al., Obesity Res. 5: 193-200,
1997a).
Other phenotypes found include hyperinsulinemia, dyslipidemia and impaired
glucose
tolerance (Collier et al., 1997a, supy~a; Collier et al., Exp. Cliya.
Endocninol. Diabetes 105:
36-37, 1997b). Psamnzomys obesus exhibit a range of bodyweight and blood
glucose and
insulin levels which form a continuous curve that closely resembles the
patterns found in
human populations, including the inverted U-shaped relationship between blood
glucose
and insulin levels known as "Starling's curve of the pancreas" (Barnett et
al., 1994a,
supra). It is the heterogeneity of the phenotypic response of Psanamomys
obesus which
makes it an ideal model to study the etiology and pathophysiology of obesity
and type 2
diabetes.
The animals are conveniently classified into three groups designated Groups A,
B and C:
Group A: animals are lean;
Group B: animals are obese and non-diabetic; and
Group C: animals are obese and diabetic.
In accordance with the present invention, a number of differentially expressed
genetic
sequences were identified in stomach, liver or hypothalamus tissue in
Psanunomys obesus
under different feeding regimes (i.e. fed, re-fed or fasting) or under
diabetic or non-
diabetic conditions.
Another aspect of the present invention provides a nucleic acid molecule
comprising a
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nucleotide sequence encoding or complementary to a sequence encoding an
expression
product or a derivative, homolog, analog or mimetic thereof wherein said
nucleotide
sequence is as substantially set forth in SEQ ID NO:1 (AGT-119) or SEQ ID N0:2
(AGT-
120) or SEQ ID N0:3 (AGT-121) or SEQ ID NO:S (AGT-122) or SEQ ID NO:6 (AGT-
422) or SEQ ID N0:7 (AGT-123) or SEQ ID N0:8 (AGT-504 genomic) or SEQ ID NO:9
(AGT-504 cDNA) or a nucleotide sequence having at least about 30% similarity
to all or
part of SEQ ID NO:1 or SEQ ID N0:2 or SEQ ID N0:3 or SEQ ID NO:S or SEQ ID
N0:6
or SEQ ID N0:7 or SEQ ID N0:8 or SEQ ID N0:9 and/or is capable of hybridizing
to one
or more of SEQ ID NO:1 or SEQ ID N0:2 or SEQ ID N0:3 or SEQ ID N0:4 or SEQ ID
NO:S or SEQ ID N0:6 or SEQ ID N0:7 or SEQ ID NO:B or SEQ ID N0:9 or their
complementary forms under low stringency conditions at 42°C and wherein
said nucleic
acid molecule is differentially expressed in one or more of stomach, liver or
hypothalamus
tissue under fed or unfed or diabetic or non-diabetic conditions.
Higher similarities are also contemplated by the present invention such as
greater than
about 40% or 50% or 60% or 70% or 80% or 90% or 95% or 96% or 97% or 98% or
99%
or above.
An expression product includes an RNA molecule such as an mRNA transcript as
well as a
protein. Some genes are non-protein encoding genes and produce mRNA or other
RNA
molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein
interaction. The RNA (e.g. mRNA) may act directly or via the induction of
other
molecules such as RNAi or via products mediated from splicing events (e.g.
exons or
introns). Other genes encode mRNA transcripts which are then translated into
proteins. A
protein includes a polypeptide. The differentially expressed nucleic acid
molecules,
therefore, may encode mRNAs only or, in addition, proteins. Both mRNAs and
proteins
are forms of "expression products".
Reference herein to similarity is generally at a level of comparison of at
least 15
consecutive or substantially consecutive nucleotides. It is particularly
convenient,
however, to determine similarity by comparing a total or complete sequence,
after optimal
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alignment.
The term "similarity" as used herein includes exact identity between compared
sequences
at the nucleotide level. Where there is non-identity at the nucleotide level,
"similarity"
S includes differences between sequences which may encode different amino
acids that are
nevertheless related to each other at the structural, functional, biochemical
and/or
conformational levels. In a particularly preferred embodiment, nucleotide
sequence
comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more
polynucleotides
include "reference sequence", "comparison window", "sequence similarity",
"sequence
identity", "percentage of sequence similarity", "percentage of sequence
identity",
"substantially similar" and "substantial identity". A "reference sequence" is
at least 12 but
frequently 15 to 18 and often at least 25 or above, such as 30 monomer units
in length.
Because two polynucleotides may each comprise (1) a sequence (i.e. only a
portion of the
complete polynucleotide sequence) that is similar between the two
polynucleotides, and (2)
a sequence that is divergent between the two polynucleotides, sequence
comparisons
between two (or more) polynucleotides are typically performed by comparing
sequences of
the two polynucleotides over a "comparison window" to identify and compare
local
regions of sequence similarity. A "comparison window" refers to a conceptual
segment of
typically 12 contiguous residues that is compared to a reference sequence. The
comparison
window may comprise additions or deletions (i.e. gaps) of about 20% or less as
compared
to the reference sequence (which does not comprise additions or deletions) for
optimal
alignment of the two sequences. Optimal alignment of sequences for aligning a
comparison
window may be conducted by computerized implementations of algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release
7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by
inspection
and the best alignment (i.e. resulting in the highest percentage homology over
the
comparison window) generated by any of the various methods selected. Reference
also
may be made to the BLAST family of programs as for example disclosed by
Altschul et al.
(Nucl. Acids Res. 25: 3389, 1997). A detailed discussion of sequence analysis
can be found
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in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John
Wiley &
Sons Inc, Chapter 15, 1994-1998). A range of other algorithms may be used to
compare
the nucleotide and amino acid sequences such as but not limited to PILEUP,
CLUSTALW,
SEQUENCHER or VectorNTI.
The terms "sequence similarity" and "sequence identity" as used herein refers
to the extent
that sequences are identical or functionally or structurally similar on a
nucleotide-by-
nucleotide basis over a window of comparison. Thus, a "percentage of sequence
identity",
for example, is calculated by comparing two optimally aligned sequences over
the window
of comparison, determining the number of positions at which the identical
nucleic acid
base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of
matched positions,
dividing the number of matched positions by the total number of positions in
the window
of comparison (i.e., the window size), and multiplying the result by 100 to
yield the
percentage of sequence identity. For the purposes of the present invention,
"sequence
identity" will be understood to mean the "match percentage" calculated by the
DNASIS
computer program (Version 2.5 for windows; available from Hitachi Software
engineering
Co., Ltd., South San Francisco, California, USA) using standard defaults as
used in the
reference manual accompanying the software. Similar comments apply in relation
to
sequence similarity.
Reference herein to a low stringency includes and encompasses from at least
about 0 to at
least about 15°/~ v/v formamide and from at least about 1 M to at least
about 2 M salt for
hybridization, and at least about 1 M to at least about 2 M salt for washing
conditions.
Generally, low stringency is at from about 25-30°C to about
42°C. The temperature may
be altered and higher temperatures used to replace formamide and/or to give
alternative
stringency conditions. Alternative stringency conditions may be applied where
necessary,
such as medium stringency, which includes and encompasses from at least about
16% v/v
to at least about 30% v/v formalnide and from at least about 0.5 M to at least
about 0.9 M
salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt
for washing
conditions, or high stringency, which includes and encompasses from at least
about 31%
v/v to at least about 50% v/v formarnide and from at least about 0.01 M to at
least about
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0.15 M salt for hybridization, and at least about 0.01 M to at least about
0.15 M salt for
washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)%
(Marmur
and Doty, J. Mol. Biol. 5: 109, 1962). However, the Tm of a duplex DNA
decreases by 1 °C
with every increase of 1% in the number of mismatch base pairs (Bormer and
Laskey, Eur.
J. Bioche~a. 46.~ 83, 1974). Fonnamide is optional in these hybridization
conditions.
Accordingly, particularly preferred levels of stringency are defined as
follows: low
stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate
stringency is 2 x SSC
buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C;
high stringency is 0.1 x
SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
The nucleotide sequence or amino acid sequence of the present invention may
correspond
to exactly the same sequence of the naturally occurring gene (or corresponding
cDNA) or
protein or other expression product or may carry one or more nucleotide or
amino acid
substitutions, additions and/or deletions. The nucleotide sequences set forth
in SEQ m
NO:1 (AGT-119), SEQ ID NO:2 (AGT-120) and SEQ ID N0:3 (AGT-121) or SEQ m
NO:S (AGT-122) or SEQ ID N0:6 (AGT-422) or SEQ ID N0:7 (AGT-123) or SEQ ID
NO:8 (AGT-504 genomic) or SEQ ID N0:9 (AGT-504 cDNA) correspond to novel genes
referred to in parenthesis. The corresponding expression products are AGT-119,
AGT-120,
AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504. Reference herein to AGT 119,
AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 includes, where
appropriate, reference to the genomic gene or cDNA as well as any naturally
occurring or
induced derivatives. For example, a genomic form of AGT-504 is represented as
SEQ )D
N0:8. Apart from the substitutions, deletions and/or additions to the
nucleotide sequence,
the present invention further encompasses mutants, fragments, parts and
portions of the
nucleotide sequence corresponding to AGT 119, AGT 120, AGT 121, AGT 122, AGT
422,
AGT 123 and AGT 504.
Another aspect of the present invention provides a nucleic acid molecule or
derivative,
homolog or analog thereof comprising a nucleotide sequence encoding, or a
nucleotide
sequence complementary to a sequence encoding an expression product wherein
said
nucleotide sequence is substantially as set forth in SEQ m NO:1 (AGT-119) or a
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derivative, homolog or mimetic thereof or having at least about 30% similarity
to all or
part of SEQ ID NO:1 or a nucleotide sequence capable of hybridizing to SEQ ID
NO:1 or
its complementary form under low stringency conditions.
Yet another aspect of the present invention provides a nucleic acid molecule
or derivative,
homolog or analog thereof comprising a nucleotide sequence encoding, or a
nucleotide
sequence complementary to a sequence encoding an expression product wherein
said
nucleotide sequence is substantially as set forth in SEQ >D N0:2 (AGT-120) or
a
derivative, homolog or mimetic thereof or having at least about 30% similarity
to all or
pan of SEQ m N0:2 or a nucleotide sequence capable of hybridizing to SEQ m
N0:2 or
its complementary form under low stringency conditions.
Still yet another aspect of the present invention provides a nucleic acid
molecule or
derivative, homolog or analog thereof comprising a nucleotide sequence
encoding, or a
nucleotide sequence complementary to a sequence encoding an expression product
wherein said nucleotide sequence is substantially as set forth in SEQ m NO:3
(AGT-121)
or a derivative, homolog or mimetic thereof or having at least about 30%
similarity to all
or part of SEQ )D N0:3 or a nucleotide sequence capable of hybridizing to SEQ
ID N0:3
or their complementary forms under low stringency conditions.
Even yet another aspect of the present invention provides a nucleic acid
molecule or
derivative, homolog or analog thereof comprising a nucleotide sequence
encoding, or a
nucleotide sequence complementary to a sequence encoding an expression product
wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:S
(AGT-122)
or a derivative, homolog or mimetic thereof or having at least about 30%
similarity to all
or part of SEQ ID NO:S or a nucleotide sequence capable of hybridizing to SEQ
ID NO:S
or its complementary form under low stringency conditions.
Even still another aspect of the present invention provides a nucleic acid
molecule or
derivative, homolog or analog thereof comprising a nucleotide sequence
encoding, or a
nucleotide sequence complementary to a sequence encoding an expression product
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wherein said nucleotide sequence is substantially as set forth in SEQ ID N0:6
(AGT-422)
or a derivative, homolog or mimetic thereof or having at least about 30%
similarity to all
or part of SEQ ID N0:6 or a nucleotide sequence capable of hybridizing to SEQ
ID N0:6
or its complementary form under low stringency conditions.
Another aspect of the present invention provides a nucleic acid molecule or
derivative,
homolog or analog thereof comprising a nucleotide sequence encoding, or a
nucleotide
sequence complementary to a sequence encoding an expression product wherein
said
nucleotide sequence is substantially as set forth in SEQ ID N0:7 (AGT-123) or
a
derivative, homolog or mimetic thereof or having at least about 30% similarity
to all or
part of SEQ ID N0:7 or a nucleotide sequence capable of hybridizing to SEQ ID
N0:7 or
its complementary form under low stringency conditions.
A further aspect of the present invention provides a nucleic acid molecule or
derivative,
homolog or analog thereof comprising a nucleotide sequence encoding, or a
nucleotide
sequence complementary to a sequence encoding an expression product wherein
said
nucleotide sequence is substantially as set forth in SEQ m N0:8 (AGT-504
genomic) or a
derivative, homolog or mimetic thereof or having at least about 30% similarity
to all or
part of SEQ ID NO:8 or a nucleotide sequence capable of hybridizing to SEQ ID
N0:8 or
its complementary form under low stringency conditions.
Yet another aspect of the present invention provides a nucleic acid molecule
or derivative,
homolog or analog thereof comprising a nucleotide sequence encoding, or a
nucleotide
sequence complementary to a sequence encoding an expression product wherein
said
nucleotide sequence is substantially as set forth in SEQ m N0:9 (AGT-504 cDNA)
or a
derivative, homolog or mimetic thereof or having at least about 30% similarity
to all or
part of SEQ ID N0:9 or a nucleotide sequence capable of hybridizing to SEQ ID
N0:9 or
its complementary form under low stringency conditions.
The expression pattern of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123
and AGT 504 has been determined, ihtef~ alia, to indicate an involvement in
the regulation
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of one or more of obesity, anorexia, weight maintenance, diabetes and/or
energy
metabolism. In addition to the differential expression of AGT 119, AGT 120,
AGT 121,
AGT 122, AGT 422, AGT 123 and AGT 504 in one or more of stomach, liver or
hypothalamus tissue of fed versus fasted or diabetic uersus non-diabetic
animals, these
genes may also be expressed in other tissues including but in no way limited
to brain,
muscle, adipose tissue, pancreas and gastrointestinal trait. The nucleic acid
molecule
encoding each of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or
AGT-504 is preferably a DNA such as a cDNA sequence or a genomic DNA. A
genomic
sequence may also comprise exons and introns. A genomic sequence may also
include a
promoter region or other regulatory regions.
A homolog is considered to be a gene from another aiumal species which has the
same or
greater than 30% similarity to one of AGT 119, AGT 120, AGT 121, AGT 122, AGT
422,
AGT 123 and AGT 504 and/or which has a similar function. The above-mentioned
genes
are exemplified herein from Psammom~s obesus stomach, liver or hypothalamus.
The
present invention extends, however, to the homologous gene, as determined by
nucleotide
sequence and/or function, from humans, primates, livestock animals (e.g. cows,
sheep,
pigs, horses, donkeys), laboratory test animals (e.g. mice, guinea pigs,
hamsters, rabbits),
companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents,
foxes, deer,
kangaroos).
The nucleic acids of the present invention and in particular AGT 119, AGT 120,
AGT 121,
AGT 122, AGT 422, AGT 123 and AGT 504 and their derivatives and homologs may
be in
isolated or purified form and/or may be ligated to a vector such as an
expression vector.
Expression may be in a eukaryotic cell line (e.g. mammalian, insect or yeast
cells) or in
prokaryote cells (e.g. E. coli) or in both. By "isolated" is meant a nucleic
acid molecule
having undergone at least one purification step and this is conveniently
defined, for
example, by a composition comprising at least about 10% subject nucleic acid
molecule,
preferably at least about 20%, more preferably at least about 30%, still more
preferably at
least about 40-50%, even still more preferably at least about 60-70%, yet even
still more
preferably 80-90% or greater of subject nucleic acid molecule relative to
other components
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as determined by molecular weight, encoding activity, nucleotide sequence,
base
composition or other convenient means. The nucleic acid molecule of the
present invention
may also be considered, in a preferred embodiment, to be biologically pure.
The nucleic
acid molecule may be ligated to an expression vector capable of expression in
a
prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal
cells, insect cells,
mammalian cells or plant cells). The nucleic acid molecule may be ligated or
fused or
otherwise associated with a nucleic acid molecule encoding another entity such
as, for
example, a signal peptide. It may also comprise additional nucleotide sequence
information
fused, linked or otherwise associated with it either at the 3' or 5' terminal
portions or at
both the 3' and 5' terminal portions. The nucleic acid molecule may also be
part of a
vector, such as an expression vector.
The derivatives of the nucleic acid molecule of the present invention include
oligonucleotides, PCR primers, antisense molecules, molecules suitable for use
in co-
suppression and fusion nucleic acid molecules. Ribozymes and DNAzymes are also
contemplated by the present invention directed to AGT 119, AGT 120, AGT 121,
AGT
122, AGT 422, AGT 123 and AGT 504 or their mRNAs. Derivatives and homologs of
AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 are
conveniently encompassed by those nucleotide sequences capable of hybridising
to one or
more of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:S or SEQ ~ NO:6
or SEQ ID N0:7 or SEQ ID N0:8 or SEQ ID N0:9 or their complementary forms
under
low stringency conditions.
Derivatives include fragments, parts, portions, mutants, variants and mimetics
from
natural, synthetic or recombinant sources including fusion nucleic acid
molecules.
Derivatives may be derived from insertion, deletion or substitution of
nucleotides.
Another aspect of the present invention provides an isolated expression
product or a
derivative, homolog, analog or mimetic thereof which is produced in larger or
lesser
amounts in one or more of stomach, liver or hypothalamus tissue in obese
animals
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compared to lean animals or in fed (including re-fed) compared to fasted
animals or in
animals under diabetic compared to non-diabetic conditions.
An expression product, as indicated above, may be RNA or protein. Insofar as
the product
is a protein, derivatives include amino acid insertional derivatives such as
amino and/or
carboxylic terminal fusions as well as intra-sequence insertions of single or
multiple amino
acids. Insertional amino acid sequence variants are those in which one or more
amino acid
residues are introduced into a predetermined site in a protein although random
insertion is
also possible with suitable screening of the resulting product. Deletional
variants are
characterized by the removal of one or more amino acids from the sequence.
Substitutional
amino acid variants are those in which at least one residue in the sequence
has been
removed and a different residue inserted in its place. An example of
substitutional amino
acid variants are conservative amino acid substitutions. Conservative amino
acid
substitutions typically include substitutions within the following groups:
glycine and
alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid;
asparagine and
glutamine; serine and threoiune; lysine and arginine; and phenylalanine and
tyrosine.
Additions to amino acid sequences include fusions with other peptides,
polypeptides or
proteins.
Chemical and functional equivalents of protein forms of the expression
products AGT-119,
AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 should be understood as
molecules exhibiting any one or more of the functional activities of these
molecules and
may be derived from any source such as being chemically synthesized or
identified via
screening processes such as natural product screening or screening of chemical
libraries.
The derivatives include fragments having particular epitopes or parts of the
entire protein
fused to peptides, polypeptides or other proteinaceous or non-proteinaceous
molecules.
Reference herein to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or
AGT-504 includes reference to isolated or purified naturally occurnng AGT-119,
AGT-
120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 as well as any derivatives,
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homologs, analogs and mimetics thereof. Derivatives include parts, fragments
and portions
of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 as well
as single and multiple amino acid substitutions, deletions a~id/or additions
to AGT-119,
AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 when the expression
products are proteins. A derivative of AGT-119, AGT-120, AGT-121, AGT-122, AGT-
422, AGT-123 or AGT-504 is conveniently encompassed by molecules encoded by a
nucleotide sequence capable of hybridizing to SEQ m NO:1 or SEQ m N0:2 or SEQ
m
N0:3 or SEQ )D NO:S or SEQ m N0:6 or SEQ m NO:7 or SEQ )D N0:8 or SEQ 117
N0:9 under low stringency conditions.
Other derivatives of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and
AGT-504 include chemical analogs. Analogs of AGT-119, AGT-120, AGT-121, AGT-
122, AGT-422, AGT-123 and AGT-504 contemplated herein include, but are not
limited
to, modifications to side chains, incorporation of unnatural amino acids
and/or their
derivatives during peptide, polypeptide or protein synthesis and the use of
crosslinkers and
other methods which impose confirmational constraints on the proteinaceous
molecule or
their analogs.
Examples of side chain modifications contemplated by the present invention
include
modifications of amino groups such as by reductive all~ylation by reaction
with an
aldehyde followed by reduction with NaBH4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups with cyanate;
trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulfonic acid
(TNBS);
acylation of amino groups with succinic anhydride and tetrahydrophthalic
anhydride; and
pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with
NaBH4.
The guanidine group of arginine residues may be modified by the formation of
heterocyclic condensation products with reagents such as 2,3-butanedione,
phenylglyoxal
and glyoxal.
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The carboxyl group may be modified by carbodiimide activation via O-
acylisourea
formation followed by subsequent derivitization, for example, to a
corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with
iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid;
formation of a
mixed disulphides with other thiol compounds; reaction with maleimide, malefic
anhydride
or other substituted maleimide; formation of mercurial derivatives using 4-
chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury
chloride, 2-
chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate
at alkaline
pH.
Tryptophan residues may be modified by, for example, oxidation with N-
bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl
bromide
or sulphenyl halides. Tyrosine residues on the other hand, may be altered by
nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished
by
alkylation with iodoacetic acid derivatives or N-carbethoxylation with
diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide
synthesis
include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-
amino-3-
hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine,
norvaline,
phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,
2-thienyl
alanine and/or D-isomers of amino acids. A list of unnatural amino acid,
contemplated
herein is shown in Table 3.
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TABLE 3
Codes fof~ szo~z-cohvesztiofaal amino acids
Non-conventional Code Non-conventional Code
amino acid amino acid
oc-aminobutyric Abu L-N-methylalanine Nmala
acid
a-amino-a,-methylbutyrateMgabu L-N-methylargiW ne Nmarg
aminocyclopropane- Cpro L-N-methylasparagine Nmasn
carboxylate L-N-methylaspartic acidNmasp
aminoisobutyric Aib L-N-methylcysteine Nmcys
acid
aminonorbornyl- Norb L-N-methylglutamine Nmgln
carboxylate L-N-methylglutamic acidNmglu
cyclohexylalanine Chexa L-Nmethylhistidine Nmhis
cyclopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaline Nmnva
D-glutamic acid Dglu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenylalanineNmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycineNmtbug
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D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr a-methyl-aminoisobutyrateMaib
D-valine Dval a-methyl-y-aminobutyrateMgabu
D-a-methylalanine Dmala a-methylcyclohexylalanineMchexa
D-a-methylarginine Dmarg a-methylcylcopentylalanineMcpen
D-a-methylasparagineDmasn a-methyl-a-napthylalanineManap
D-a-methylaspartate Dmasp a-methylpenicillasnine Mpen
D-a-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
10D-a-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-a-methylhistidine Dmhis N-(3-aminopropyl)glycineNorn
D-a-methylisoleucineDmile N-amino-a methylbutyrateNmaabu
D-a-methylleucine Dmleu a-napthylalanine Anap
D-a-methyllysine Dmlys N-benzylglycine Nphe
15D-a-methylmethioune Dmmet N-(2-carbamylethyl)glycineNgln
D-a-methylornithine Dmorn N-(carbamylmethyl)glycineNasn
D-a-methylphenylalanineDmphe N-(2-carboxyethyl)glycineNglu
D-a-methylproline Dmpro N-(carboxymethyl)glycineNasp
D-a-methylserine Dmser N-cyclobutylglycine Ncbut
20D-a-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-a-methyltryptophanDmtrp N-cyclohexylglycine Nchex
D-a-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-a-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala ~N-cyclooctylglycine Ncoct
25D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagineDnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycineNbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycineNbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycineNarg
30D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycineNthr
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D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucineDnmile N-(imidazolylethyl))glycineNhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycineNhtrp
D-N-methyllysine Dnmlys N-methyl-y-aminobutyrate Nmgabu
N-methylcyclohexylalanineNmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanineNmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnrnphe
N-methylaminoisobutyrateNmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycineNile D-N-methylserine Dnmser
10N-(2-methylpropyl)glycineNleu D-N-methylthreonine Dmnthr
D-N-methyltryptophanDnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycineNhtyr
15L-t-butylglycine Tbug N-(tluomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-a-methylalanine Mala
L-a-methylarginine Marg L-a-methylasparagine Masn
L-a-methylaspartate Masp L-a-methyl-t-butylglycineMtbug
20L-a-methylcysteine Mcys L-methylethylglycine Metg
L-a-methylglutamine Mgln L-a-methylglutamate Mglu
L-a-methylhistidine Mhis L-a-methylhomophenylalanineMhphe
L-a-methylisoleucineMile N-(2-methylthioethyl)glycineNmet
L-a-methylleucine Mleu L-a-methyllysine Mlys
25L-a-methylmethionineMmet L-a-methylnorleucine Mnle
L-a-methylnorvaline Mnva L-a-methylornithine Morn
L-a-methylphenylalanineMphe L-a-methylproline Mpro
L-a-methylserine Mser L-a-methylthreonine Mthr
L-a-methyltryptophanMtrp L-a-methyltyrosine Mtyr
30L-a-methylvaline Mval L-N-methylhomophenylalanineNmhphe
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N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe
carbamylmethyl)glycine carbamyhnethyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc
ethylamino)cyclopropane
Crosslinlcers can be used, for example, to stabilize 3I~ conformations, using
homo-
bifunctional crosslinkers such as the bifimctional imido esters having (CHZ)"
spacer groups
with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-
bifunctional
reagents which usually contain an amino-reactive moiety such as N-
hydroxysuccinimide
and another group specific-reactive moiety such as maleimido or dithio moiety
(SH) or
carbodiimide (COON). In addition, peptides can be conformationally constrained
by, for
example, incorporation of Ca and N ~methylamino acids, introduction of double
bonds
between Ca and Ca atoms of amino acids and the formation of cyclic peptides or
analogs
by introducing covalent bonds such as forming an amide bond between the N and
C
termini, between two side chains or between a side chain and the N or C
terminus.
All such modifications may also be useful in stabilizing the AGT-119, AGT-120,
AGT-
121, AGT-122, AGT-422, AGT-123 and AGT-504 molecule for use in iya vivo
administration protocols or for diagnostic purposes.
As stated above, the expression product may be a RNA or protein.
The term "protein" should be understood to encompass peptides, polypeptides
and
proteins. The protein may be glycosylated or unglycosylated and/or may contain
a range of
other molecules fused, lined, bound or otherwise associated to the protein
such as amino
acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
Reference
hereinafter to a "protein" includes a protein comprising a sequence of amino
acids as well
as a protein associated with other molecules such as amino acids, lipids,
carbohydrates or
other peptides, polypeptides or proteins.
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In a particularly preferred embodiment, the expression product is encoded by a
sequence of
nucleotides comprising SEQ ID NO:1 or a derivative, homolog or analog thereof
including
a nucleotide sequence having at least about 30% similarity to SEQ m NO:1 or a
nucleotide
sequence capable of hybridizing to SEQ ID NO:1 or its complementary form under
low
stringency conditions.
In another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ ID N0:2 or a derivative, homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ ID
N0:2 or a nucleotide sequence capable of hybridizing to SEQ ID N0:2 or its
complementary form under low stringency conditions.
In still another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ m NO:3 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ I~
N0:3 or a nucleotide sequence capable of hybridizing to SEQ m NO:3 or their
complementary form under low stringency conditions.
In yet another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ ID N0:5 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ m
NO:S or a nucleotide sequence capable of hybridizing to SEQ m NO:S or its
complementary form under low stringency conditions.
In another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ m NO:6 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ m
N0:6 or a nucleotide sequence capable of hybridizing to SEQ m N0:6 or its
complementary form under low stringency conditions.
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In a further particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ m N0:7 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ m
N0:7 or a nucleotide sequence capable of hybridizing to SEQ m NO:7 or its
complementary form under low stringency conditions.
In still another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ m N0:8 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ m
NO:8 or a nucleotide sequence capable of hybridizing to SEQ m NO:8 or its
complementary form under low stringency conditions.
In yet another particularly preferred embodiment, the expression product is
encoded by a
sequence of nucleotides comprising SEQ m N0:9 or a derivative homolog or
analog
thereof including a nucleotide sequence having at least about 30% similarity
to SEQ m
N0:9 or a nucleotide sequence capable of hybridizing to SEQ m NO:9 or its
complementary form under low stringency conditions.
Higher similarities are also contemplated by the present invention such as
greater than 40%
or 50% or 60% or 70% or 80% or 90% or 95% or 96% or 97% or 98% or 99% or
above.
Another aspect of the present invention is directed to an isolated expression
product
selected from the list consisting of:-
(i) an mRNA or protein encoded by a novel nucleic acid molecule which molecule
is
differentially expressed in one or more of stomach, liver or hypothalamus
tissue
from Psanafraonays obesus animals under fed or fasting conditions or animals
which
are diabetic or non-diabetic or a derivative, homolog, analog, chemical
equivalent
or mimetic thereof;
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(ii) an mRNA or protein encoded by a novel nucleic acid molecule which
molecule is
differentially expressed in one or more of stomach, liver or hypothalamus
tissue
from Psamnaomys obesus animals under fed or fasting conditions or animals
which
are diabetic or non-diabetic or a derivative, homolog, analog, chemical
equivalent
or mimetic thereof;
(iii) AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 or a
derivative, homolog, analog, chemical equivalent or mimetic thereof;
(iv) a protein encoded by a nucleotide sequence comprising SEQ ID NO:l or a
derivative, homolog or analog thereof or a sequence encoding an amino acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(vi) a protein encoded by a nucleotide sequence substantially comprising SEQ ~
N0:2
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(vii) a protein encoded by a nucleotide sequence substantially comprising SEQ
m NO:3
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to these sequences or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(viii) a protein comprising an amino acid sequence substantially as set forth
in SEQ m
N0:4 or a derivative, homolog or analog thereof or a sequence encoding an
amino
acid sequence having at least about 30% similarity to these sequences or a
derivative, homolog, analog, chemical equivalent or mimetic of said protein;
(ix) a protein encoded by a nucleotide sequence substantially comprising SEQ
)D NO:S
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
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sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(x) a protein encoded by a nucleotide sequence substantially comprising SEQ m
N0:6
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(xi) a protein encoded by a nucleotide sequence substantially comprising SEQ
ID N0:7
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(xii) a protein encoded by a nucleotide sequence substantially comprising SEQ
~ NO:~
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(xiii) a protein encoded by a nucleotide sequence substantially comprising SEQ
ID N0:9
or a derivative, homolog or analog thereof or a sequence encoding an amino
acid
sequence having at least about 30% similarity to this sequence or a
derivative,
homolog, analog, chemical equivalent or mimetic of said protein;
(xiv) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ ID NO:1 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions;
(xv) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ ID NO:2 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions;
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(xvi) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ m N0:3 or their complementary forms or a derivative,
homolog or analog thereof under low stringency conditions;
(xvii) a protein encoded by a nucleic acid molecule capable of hybridizing to
a nucleotide
sequence comprising SEQ m NO:S or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions;
(xviii) a protein encoded by a nucleic acid molecule capable of hybridizing to
a nucleotide
sequence comprising SEQ m N0:6 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions;
(xix) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ m N0:7 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions;
(xx) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ m N0:8 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions; and
(xxi) a protein encoded by a nucleic acid molecule capable of hybridizing to a
nucleotide
sequence comprising SEQ m NO:9 or its complementary form or a derivative,
homolog or analog thereof under low stringency conditions.
An example of an expression product is the amino acid sequence set forth in
SEQ III N0:4
(AGT-121 ).
The protein of the present invention is preferably in isolated form. By
"isolated" is meant a
protein having undergone at least one purification step and this is
conveniently defined, for
example, by a composition comprising at least about 10% subject protein,
preferably at
least about 20%, more preferably at least about 30%, still more preferably at
least about
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40-50%, even still more preferably at least about 60-70%, yet even still more
preferably
80-90% or greater of subject protein relative to other components as
detennined by
molecular weight, amino acid sequence or other convenient means. The protein
of the
present invention may also be considered, in a preferred embodiment, to be
biologically
pure.
Without limiting the theory or mode of action of the present invention, the
expression of
AGT-I19, AGT-120, AGT-121, AGT-122, AGT-422, AGT-I23 and/or AGT-504 is
thought to relate to regulation of body weight and glucose homeostasis.
Modulation of
expression of these genes is thought irztef- alia to regulate energy balance
via effects on
energy intal~e and also effects on carbohydrate/fat metabolism. The energy
intake effects
are likely to be mediated via the central nervous system but peripheral
effects on the
metabolism of both carbohydrate and fat are possible. The 'expression of these
genes may
also be regulated by fasting and feeding. Accordingly, regulating the
expression and/or
activity of these genes or their expression products provides a mechanism for
regulating
both body weight and energy metabolism, including carbohydrate and fat
metabolism.
The identification of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and
AGT 504 permits the generation of a range of therapeutic molecules capable of
modulating
expression of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504
or modulating the activity of AGT-I 19, AGT-120, AGT-121, AGT-122, AGT-422,
AGT-
123 and AGT-504. Modulators contemplated by the present invention include
agonists and
antagonists of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT
504
expression. Antagonists of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT
123
and AGT 504 expression include antisense molecules, ribozymes and co-
suppression
molecules (including any molecules which induce RNAi). Agonists include
molecules
which increase promoter activity or which interfere with negative regulatory
mechanisms.
Antagonists of AGT-I I9, AGT-120, AGT-I21, AGT-122, AGT-422, AGT-123 and AGT-
504 include antibodies and inhibitor peptide fragments. All such molecules may
first need
to be modified to enable such molecules to penetrate cell membranes.
Alternatively, viral
agents may be employed to introduce genetic elements to modulate expression of
AGT
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119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504. In so far as AGT
119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 act in
association
with other genes such as the ob gene which encodes leptin, the therapeutic
molecules may
target AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 and ob
genes or their translation products.
The present invention contemplates, therefore, a method for modulating
expression of
AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 in a mammal,
said method comprising contacting the AGT 119, AGT 120, AGT 121, AGT 122, AGT
422, AGT 123 and AGT 504 gene with an effective amount of a modulator of AGT
119,
AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 expression for a time
and under conditions sufficient to up-regulate or down-regulate or otherwise
modulate
expression ofAGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 andAGT 504.
For example, a nucleic acid molecule encoding AGT 119, AGT 120, AGT 121, AGT
122,
AGT 422, AGT 123 and AGT 504 or a derivative or homolog thereof may be
introduced
into a cell to enhance the ability of that cell to produce AGT-119, AGT-120,
AGT-121,
AGT-122, AGT-422, AGT-123 and AGT-504, conversely, AGT 119, AGT 120, AGT 121,
AGT 122, AGT 422, AGT 123 and AGT 504 sense and/or antisense sequences such as
oligonucleotides may be introduced to decrease expression of the genes at the
level of
transcription, post-transcription or translation. Sense sequences preferably
encode hair pin
RNA molecules or double-stranded RNA molecules.
Another aspect of the present invention contemplates a method of modulating
activity of
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 in a
mammal, said method comprising administering to said mammal a modulating
effective
amount of a molecule for a time and under conditions sufficient to increase or
decrease
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 activity.
The molecule may be a proteinaceous molecule or a chemical entity and may also
be a
derivative of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-
504 or its ligand.
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Modulating levels of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and
AGT 504 expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123
and/or AGT-504 activity or function is important in the treatment of a range
of conditions
such as obesity, anorexia, energy imbalance, diabetes, metabolic syndrome,
dyslipidemia,
hypertension and insulin resistance. It may also be useful in the agricultural
industry to
assist in the generation of leaner animals, or where required, more obese
animals.
Accordingly, mammals contemplated by the present invention include but are not
limited
to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses,
donkeys),
laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits),
companion animals
(e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer). A
particularly
preferred host is a human, primate or livestock animal.
Accordingly, the present invention contemplates therapeutic and prophylactic
use of AGT-
119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 expression
products or AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504
genetic mutants and/or agonists or antagonists agents thereof.
The present invention contemplates, therefore, a method of modulating
expression of AGT
119, AGT 120 AGT 121, AGT 122, AGT 422, AGT 123 and/or AGT 504 in a mammal,
said method comprising contacting the AGT 119, AGT 120, AGT 121, AGT 122, AGT
422, AGT 123 and/or AGT 504 genes with an effective amount of an agent for a
time and
under conditions sufficient to up-regulate, down-regulate or otherwise module
expression
of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504.
Another aspect of the present invention contemplates a method of modulating
activity of
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 in a
subject, said method comprising achninistering to said subject a modulating
effective
amount of an agent for a time and under conditions sufficient to increase or
decrease AGT-
119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 activity or
function.
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Modulation of activity by the administration of an agent to a mammal can be
achieved by
one of several techniques, including, but in no way limited to, introducing
into a mammal a
proteinaceous or non-proteinaceous molecule which:
(i) modulates expression of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT
123 and/or AGT 504;
(ii) functions as an antagonist of AGT-119, AGT-120, AGT-121, AGT-122, AGT-
422,
AGT-123 and/or AGT-504; and/or
(iii) functions as an agonist of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422,
AGT-123 and/or AGT-504.
The molecules which may be administered to a mammal in accordance with the
present
invention may also be linked to a targeting means such as a monoclonal
antibody, which
provides specific delivery of these molecules to the target cells.
A further aspect of the present invention relates to the use of the invention
in relation to
mammalian disease conditions. For example, the present invention is
particularly useful in
a therapeutic or prophylactic treatment of obesity, anorexia, diabetes or
energy imbalance.
Accordingly, another aspect of the present invention relates to a method of
treating a
mammal suffering from a condition characterized by one or more symptoms of
obesity,
anorexia, diabetes and/or energy imbalance, said method comprising
administering to said
mammal an effective amount of an agent for a time and under conditions
sufficient to
modulate the expression of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT
123
and/or AGT 504 or sufficient to modulate the activity of AGT-119, AGT-120, AGT-
121,
AGT-122, AGT-422, AGT-123 and/or AGT-5 04.
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In another aspect, the present invention relates to a method of treating a
mammal suffering
from a disease condition characterized by one or more symptoms of obesity,
anorexia,
diabetes or energy imbalance, said method comprising administering to said
mammal an
effective amount of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123
and/or AGT-504 or AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and/or
AGT 504.
An agent includes proteinaceous or non-proteinaceous molecules such as
antibodies,
natural products, chemical entities or nucleic acid molecules (including
antisense
molecules, sense molecules, ribozymes, ds-RNA molecules or DNA-targeting
molecules).
An "effective amount" means an amount necessary at least partly to attain the
desired
immune response (e.g. against AGT-119, AGT-120, AGT-121, AGT-122, AGT-422,
AGT-123 or AGT-504) or to delay the onset or inhibit progression or halt
altogether the
onset or progression of a particular condition.
In accordance with these methods, AGT-119, AGT-120, AGT-121, AGT-122, AGT-422,
AGT-123 and/or AGT-504 or AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT
123 and/or AGT 504 or agents capable of modulating the expression or activity
of said
molecules may be co-administered with one or more other compounds or other
molecules.
By "co-administered" is meant simultaneous administration in the same
formulation or in
two different formulations via the same or different routes or sequential
administration by
the same or different routes. By "sequential" administration is meant a time
difference of
from seconds, minutes, hours or days between the administration of the two
types of
molecules. These molecules may be administered in any order.
In yet another aspect, the present invention relates to the use of an agent
capable of
modulating the expression of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT
123 and/or AGT 504 or a derivative, homolog or analog thereof in the
manufacture of a
medicament for the treatment of a condition characterized by obesity,
anorexia, weight
maintenance, diabetes and/or energy imbalance.
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In still yet another aspect, the present invention relates to the use of an
agent capable of
modulating the activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-
123
and/or AGT-504 or a derivative, homolog, analog, chemical equivalent or
mimetic thereof
in the manufacture of a medicament for the treatment of a condition
characterized by
obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
A further aspect of the present invention relates to the use of AGT 119, AGT
120, AGT
121, AGT 122, AGT 422, AGT 123 and/or AGT 504 or derivative, homolog or analog
thereof or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-
504 or derivative, homolog, analog, chemical equivalent or mimetic thereof in
the
manufacture of a medicament for the treatment of a condition characterized by
obesity,
anorexia, weight maintenance, diabetes and/or energy imbalance.
Still yet another aspect of the present invention relates to agents for use in
modulating the
expression of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and/or AGT
504 or a derivative, homolog or analog thereof.
A further aspect relates to agents for use in modulating AGT-119, AGT-120, AGT-
121,
AGT-122, AGT-422, AGT-123 and/or AGT-504 activity or a derivative, homolog,
analog,
chemical equivalent or mimetic thereof.
Still another aspect of the present invention relates to AGT 119, AGT 120, AGT
121, AGT
122, AGT 422, AGT 123 and/or AGT 504 or derivative, homolog or analog thereof
or
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or
derivative, homolog, analog, chemical equivalent or mimetic thereof for use in
treating a
condition characterized by one or more symptoms of obesity, anorexia, weight
maintenance, diabetes and/or energy imbalance.
In a related aspect of the present invention, the mammal undergoing treatment
may be a
human or an animal in need of therapeutic or prophylactic treatment.
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Accordingly, the present invention contemplates in one embodiment a
composition
comprising a modulator of AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123
and AGT 504 expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-
123 and AGT-504 activity and one or more pharmaceutically acceptable carriers
and/or
diluents. In another embodiment, the composition comprises AGT-119, AGT-120,
AGT-
12I, AGT-122, AGT-422, AGT-123 and AGT-504 or a derivative, homolog, analog or
mimetic thereof and one or more pharmaceutically acceptable Garners and/or
diluents. The
compositions may also comprise leptin or modulations of leptin activity or ob
expression.
For brevity, all such components of such a composition are referred to as
"active
components".
The compositions of active components in a form suitable for injectable use
include sterile
aqueous solutions (where water soluble) and sterile powders for the
extemporaneous
preparation of sterile injectable solutions. In all cases, the form must be
sterile and must be
fluid to the extent that easy syringability exists. It must be stable under
the conditions of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms such as bacteria and fungi.
The Garner can be a solvent or other medium containing, for example, water,
ethanol,
polyol (for example, glycerol, propylene glycol and liquid polyethylene
glycol, and the
like), suitable mixtures thereof, and vegetable oils.
The preventions of the action of microorganisms can be brought about by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic
acid, thirmerosal and the like. In many cases, it will be preferable to
include isotonic
agents, for example, sugars or sodium chloride. Prolonged absorption of the
injectable
compositions can be brought about by the use in the compositions of agents
delaying
absorption, for example, aluminum monostearate and gelatin.
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Sterile injectable solutions are prepared by incorporating the active
components in the
required amount in the appropriate solvent with optionally other ingredients,
as required,
followed by sterilization by, for example, filter sterilization, irradiation
or other convenient
means. In the case of sterile powders for the preparation of sterile
injectable solutions, the
preferred methods of preparation are vacuum drying and the freeze-drying
technique which
yield a powder of the active ingredient plus any additional desired ingredient
from
previously sterile-filtered solution thereof.
When AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504 or
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 are suitably
protected, they may be orally administered, for example, with an inert diluent
or with an
assimilable edible carrier, or it may be enclosed in hard or soft shell
gelatin capsule, or it
may be compressed into tablets, or it may be incorporated directly with the
food of the
diet. For oral therapeutic administration, the active compound may be
incorporated with
excipients and used in the form of ingestible tablets, buccal tablets,
troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such compositions and
preparations
should contain at least 1 % by weight of active compound. The percentage of
the
compositions and preparations may, of course, be varied and may conveniently
be between
about 5 to about 80% of the weight of the unit. The amount of active compound
in such
therapeutically useful compositions is such that a suitable dosage will be
obtained.
Preferred compositions or preparations according to the present invention are
prepared so
that an oral dosage unit form contains between about 0.1 ~g and 2000 mg of
active
compound.
The tablets, troches, pills, capsules and the like may also contain the
following: A binder
such as gum tragacanth, acacia, corn starch or gelatin; excipients such as
dicalcium
phosphate; a disintegrating agent such as corn starch, potato starch, alginic
acid and the
like; a lubricant such as magnesium stearate; and a sweetening agent such a
sucrose,
lactose or saccharin may be added or a flavouring agent such as peppermint,
oil of
wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it
may contain,
in addition to materials of the above type, a liquid Garner. Various other
materials may be
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present as coatings or to otherwise modify the physical form of the dosage
unit. For
instance, tablets, pills, or capsules may be coated with shellac, sugar or
both. A syrup or
elixir may contain the active compound, sucrose as a sweetening agent, methyl
and
propylparabens as preservatives, a dye and flavouring such as cherry or orange
flavour. Of
course, any material used in preparing any dosage unit form should be
pharmaceutically
pure and substantially non-toxic in the amounts employed. In addition, the
active
compound may be incorporated into sustained-release preparations and
formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active
substances is well known in the art. Except insofar as any conventional media
or agent is
incompatible with the active ingredient, use thereof in the therapeutic
compositions is
contemplated. Supplementary active ingredients can also be incorporated into
the
compositions.
It is especially advantageous to formulate parenteral compositions in dosage
unit form for
ease of administration and uniformity of dosage. Dosage unit form as used
herein refers to
physically discrete units suited as unitary dosages for the mammalian subjects
to be
treated; each unit containing a predetermined quantity of active material
calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical
carrier. The specification for the novel dosage unit forms of the invention
are dictated by
and directly dependent on (a) the unique characteristics of the active
material and the
particular therapeutic effect to be achieved, and (b) the limitations inherent
in the art of
compounding such an active material for the treatment of disease in living
subjects having
a diseased condition in which bodily health is impaired as herein disclosed in
detail.
The principal active component may be compounded for convenient and effective
administration in sufficient amounts with a suitable pharmaceutically
acceptable carrier in
dosage unit form. A unit dosage form can, for example, contain the principal
active
component in amounts ranging from 0.5 ~g to about 2000 mg. Expressed in
proportions,
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the active compound is generally present in from about 0.5 ~g to about 2000
mg/mI of
Garner. hi the case of compositions containing supplementary active
ingredients, the
dosages are determined by reference to the usual dose and manner of
administration of the
said ingredients.
In general terms, effective amounts of AGT 119, AGT 120, AGT 121, AGT 122, AGT
422,
AGT 123 and AGT 504 or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123
and AGT-504 will range from 0.01 ng/kglbody weight to above 10,000 mg/kg/body
weight. Alternative amounts range from 0.1 ng/kg/body weight to above 1000
mg/kg/body
weight. The active ingredients may be administered per minute, hour, day,
week, month or
year depending on the condition being treated. The route of administration may
vary and
includes intravenous, intraperitoneal, sub-cutaneous, intramuscular,
intranasal, via
suppository, via infusion, via drip, orally or via other convenient means.
The pharmaceutical composition may also comprise genetic molecules such as a
vector
capable of transfecting target cells where the vector carries a nucleic acid
molecule capable
of modulating AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123 and AGT 504
expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-
504 activity. The vector may, for example, be a viral vector.
Still another aspect of the present invention is directed to antibodies to AGT-
119, AGT-
120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and their derivatives and
homologs insofar as AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and
AGT-504 are proteins. Such antibodies may be monoclonal or polyclonal and may
be
selected from naturally occurnng antibodies to AGT-119, AGT-120. AGT-121, AGT-
122,
AGT-422, AGT-123 and AGT-504 or may be specifically raised to AGT-119, AGT-
120,
AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or derivatives or homologs
thereof. In the case of the latter, AGT-119, AGT-120, AGT-121, AGT-122, AGT-
422,
AGT-123 and AGT-504 or their derivatives or homologs may first need to be
associated
with a carrier molecule. The antibodies andlor recombinant AGT-119, AGT-120,
AGT-
121, AGT-122, AGT-422, AGT-123 and AGT-504 or their derivatives of the present
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invention are particularly useful as therapeutic or diagnostic agents. An
antibody "to" a
molecule includes an antibody specific for said molecule.
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and their
derivatives can be used to screen for naturally occurring antibodies to AGT-
119, AGT-
120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 which may occur in certain
autoimrnune diseases. Alternatively, specific antibodies can be used to screen
for AGT
119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504. Techniques for
such assays are well lrnown in the art and include, for example, sandwich
assays and
ELISA.
Antibodies to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-
504 of the present invention may be monoclonal or polyclonal and may be
selected from
naturally occurring antibodies to the AGT-119, AGT-120, AGT-121, AGT-122, AGT-
422,
AGT-123 and AGT-504 or may be specifically raised to the AGT-119, AGT-120 and
AGT-121or their derivatives. In the case of the latter, the AGT-119, AGT-120,
AGT-121,
AGT-122, AGT-422, AGT-123 and AGT-504 protein may need first to be associated
with
a Garner molecule. Alternatively, fragments of antibodies may be used such as
Fab
fragments. Furthermore, the present invention extends to recombinant and
synthetic
antibodies and to antibody hybrids. A "synthetic antibody" is considered
herein to include
fragments and hybrids of antibodies. The antibodies of this aspect of the
present invention
are particularly useful for immunotherapy and may also be used as a diagnostic
tool or as a
means for purifying AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and
AGT-504.
For example, specific antibodies can be used to screen for AGT-119, AGT-120,
AGT-121,
AGT-122, AGT-422, AGT-123 and AGT-504 proteins. The latter would be important,
for
example, as a means for screening for levels of AGT-119, AGT-120, AGT-121, AGT-
122,
AGT-422, AGT-123 and AGT-504 in a cell extract or other biological fluid or
purifying
AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 made by
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recombinant means from culture supernatant fluid. Techniques for the assays
contemplated
herein are known in the art and include, for example, sandwich assays and
ELISA.
It is within the scope of this invention to include any second antibodies
(monoclonal,
polyclonal or fragments of antibodies) directed to the first mentioned
antibodies discussed
above. Both the first and second antibodies may be used in detection assays or
a first
antibody may be used with a commercially available anti-immunoglobulin
antibody. An
antibody as contemplated herein includes any antibody specific to any region
of AGT-119,
AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
Both polyclonal and monoclonal antibodies are obtainable by immunization with
the
enzyme or protein and either type is utilizable for immunoassays. The methods
of
obtaining both types of sera are well lcnown in the art. Polyclonal sera are
less preferred
but are relatively easily prepared by injection of a suitable laboratory
animal with an
effective amount of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and
AGT-504, or antigenic parts thereof, collecting serum from the animal, and
isolating
specific sera by any of the lffzown immunoadsorbent techniques. Although
antibodies
produced by this method are utilizable in virtually any type of immunoassay,
they axe
generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred
because of
the ability to produce them in large quantities and the homogeneity of the
product. The
preparation of hybridoma cell lines for monoclonal antibody production derived
by fusing
an immortal cell line and lymphocytes sensitized against the irnmunogenic
preparation can
be done by techniques which are well known to those who are skilled in the
art. (See, for
example, I~ouillard and Hoffinan, Basic Facts about Hybridomas, in Compendium
of
Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256:
495-499,
1975; Kohler and Milstein, European Journal oflmmunology 6: 511-519, 1976.)
Another aspect of the present invention contemplates a method for detecting
AGT-119,
AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or a derivative or
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homolog thereof in a biological sample from a subject, said method comprising
contacting
said biological sample with an antibody specific for AGT-119, AGT-120, AGT-
121, AGT
122, AGT-422, AGT-123 and AGT-504 or their antigenic derivatives or homologs
for a
time and under conditions sufficient for a complex to form, and then detecting
said
complex.
The presence of the complex is indicative of the presence of AGT-119, AGT-120,
AGT
121, AGT-122, AGT-422, AGT-123 and AGT-504. This assay may be quantitated or
semi-quantitated to determine a propensity to develop obesity or other
conditions or to
monitor a therapeutic regimen.
The presence of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and
AGT-504 may be accomplished in a number of ways such as by Western blotting
and
ELISA procedures. A wide range of immunoassay techniques are available as can
be seen
by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of
course,
include both single-site and two-site or "sandwich" assays of the non-
competitive types, as
well as in the traditional competitive binding assays. These assays also
include direct
binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays. A number
of
variations of the sandwich assay technique exist, and all are intended to be
encompassed
by the present invention. Briefly, in a typical forward assay, an unlabelled
antibody is
immobilized on a solid substrate and the sample to be tested brought into
contact with the
bound molecule. After a suitable period of incubation, for a period of time
sufficient to
allow formation of an antibody-AGT-119, AGT-120, AGT-121, AGT-122, AGT-422,
AGT-123 and AGT-504 complex, a second antibody specific to the AGT-119, AGT-
120,
AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504, labelled with a reporter
molecule capable of producing a detectable 'signal, is then added and
incubated, allowing
time sufficient for the formation of another complex of antibody-AGT-119, AGT-
120,
AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504-labelled antibody. Any
unreacted
material is washed away, and the presence of AGT-119, AGT-120, AGT-121, AGT-
122,
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AGT-422, AGT-123 and AGT-504 is determined by observation of a signal produced
by
the reporter molecule. The results may either be qualitative, by simple
observation of the
visible signal, or may be quantitated by comparing with a control sample
containng
known amounts of hapten. Variations on the forward assay include a
simultaneous assay,
in which both sample and labelled antibody are added simultaneously to the
bound
antibody. These techniques are well known to those skilled in the art,
including any minor
variations as will be readily apparent. In accordance with the present
invention, the sample
is one which might contain AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-
123 and AGT-504 including cell extract, tissue biopsy or possibly serum,
saliva, mucosal
secretions, lymph, tissue fluid and respiratory fluid. The sample is,
therefore, generally a
biological sample comprising biological fluid but also extends to fermentation
fluid and
supernatant fluid such as from a cell culture.
The solid surface is typically glass or a polymer, the most commonly used
polymers being
cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or
polypropylene. The
solid supports may be in the form of tubes, beads, discs or microplates, or
any other
surface suitable for conducting an immunoassay. The binding processes are well-
known in
the art and generally consist of cross-linlcing covalently binding or
physically adsorbing,
the polymer-antibody complex to the solid surface which is then washed in
preparation for
the test sample. An aliquot of the sample to be tested is then added to the
solid phase
complex and incubated for a period of time sufficient (e.g. 2-40 minutes or
overnight if
more convenient) and under suitable conditions (e.g. from room temperature to
about
37°C) to allow binding of any subunit present in the antibody.
Following the incubation
period, the antibody subunit solid phase is washed and dried and incubated
with a second
antibody specific for a portion of AGT-119, AGT-120, AGT-121, AGT-122, AGT-
422,
AGT-123 and AGT-504. The second antibody is linked to a reporter molecule
which is
used to indicate the binding of the second antibody to AGT-119, AGT-120, AGT-
121,
AGT-122, AGT-422, AGT-123 and AGT-504.
An alternative method involves immobilizing the target molecules in the
biological sample
and then exposing the immobilized target to specific antibody which may or may
not be
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labelled with a reporter molecule. Depending on the amount of target and the
strength of
the reporter molecule signal, a bound target may be detectable by direct
labelling with the
antibody. Alternatively, a second labelled antibody, specific to the first
antibody is exposed
to the target-first antibody complex to form a target-first antibody-second
antibody tertiary
complex. The complex is detected by the signal emitted by the reporter
molecule.
By "reporter molecule" as used in the present specification, is meant a
molecule which, by
its chemical nature, provides an analytically identifiable signal which allows
the detection
of antigen-bound antibody. Detection may be either qualitative or
quantitative. The most
commonly used reporter molecules in this type of assay are either enzymes,
fluorophores
or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent
molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second
antibody,
generally by means of glutaraldehyde or periodate. As will be readily
recognized, however,
a wide variety of different conjugation techniques exist, which are readily
available to the
skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose
oxidase,
13-galactosidase and alkaline phosphatase, amongst others. The substrates to
be used with
the specific enzymes are generally chosen for the production, upon hydrolysis
by the
corresponding enzyme, of a detectable colour change. Examples of suitable
enzymes
include alkaline phosphatase and peroxidase. It is also possible to employ
fluorogenic
substrates, which yield a fluorescent product rather than the chromogenic
substrates noted
above. In all cases, the enzyme-labelled antibody is added to the first
antibody hapten
complex, allowed to bind, and then the excess reagent is washed away. A
solution
containing the appropriate substrate is then added to the complex of antibody-
antigen-
antibody. The substrate will react with the enzyme linlced to the second
antibody, giving a
qualitative visual signal, which may be further quantitated, usually
spectrophotometrically,
to give an indication of the amount of hapten which was present in the sample.
A "reporter
molecule" also extends to use of cell agglutination or inhibition of
agglutination such as
red blood cells on latex beads, and the like.
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Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be
chemically coupled to antibodies without altering their binding capacity. When
activated
by illumination with light of a particular wavelength, the fluorochrome-
labelled antibody
absorbs the light energy, inducing a state to excitability in the molecule,
followed by
emission of the light at a characteristic colour visually detectable with a
light microscope.
As in the EIA, the fluorescent-labelled antibody is allowed to bind to the
first antibody-
hapten complex. After washing off the unbound reagent, the remaining tertiary
complex is
then exposed to the light of the appropriate wavelength. The fluorescence
observed
indicates the presence of the hapten of interest. linmunofluorescence and EIA
techniques
are both very well established in the art and are particularly preferred for
the present
method. However, other reporter molecules, such as radioisotope,
chemiluminescent or
bioluminescent molecules, may also be employed.
The present invention also contemplates genetic assays such as involving, for
example,
PCR analysis to detect AGT 119, AGT 120, AGT 121, AGT 122, AGT 422, AGT 123
and
AGT 504 or their derivatives.
Real-time PCR is also particularly useful for assaying for particular genetic
molecules.
The present invention is further described by the following non-limiting
Examples.
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EXAMPLE 1
Psasnsnonzys obesus
In the following examples, Psammomys obesus rats were used for differential
expression
studies under different conditions. The rats are divided into three groups,
based on
metabolic phenotype, as follows:-
Group A animals : lean
Group B animals : obese, non-diabetic
Group C animals . obese, diabetic.
EXAMPLE 2
Partial seqzzence of Psamnzosnys obesus AGT 119
AGT-119 was identified using differential display PCR of stomach cDNA from
fed, fasted
and re-fed Psan2monays obesus.
The partial nucleotide sequence is as follows:-
ZO AATGAAAGAATTGATTGATACGCAACCAAATTAGCCAGTGAGGTTAGNNNCNGGATTATCG
TGACCAGATAGGAGCCTTGGAAAATGACTAAGAAAAATGAAAA.ACAGCCTAAAATGTCATT
AGCCCAACAAGATGCGTTAAAACGCCTGGATCAAGTTAGAANGCAGAAAAGCGAAAGCC
[SEQ ID NO:1].
EXAMPLE 3
AGT 119 gene expression
Real-time PCR analysis of AGT-119 found dramatically lower levels of
expression in
fasted and re-fed animals when compared to fed animals (Figure 1). In most
fasted and re-
fed animals the levels of AGT-119 were undetectable. Fasting was for 16 hours,
fed
animals had ad libitum access to lab chow and re-fed animals were fasted for
16 hours then
allowed ad libituyra access to lab chow for 1 hour. These results were
confirmed with two
sets of primers targeting the gene of interest.
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EXAMPLE 4
AGT 119 sequence homology
The AGT-119 sequence does not show significant homology with anything on the
public
database (BLASTN version 2.2.1).
EXAMPLE 5
Partial seyueuce of Psammomys obesus AGT-120
AGT-120 was identified using differential display PCR of stomach cDNA from
fed, fasted
and re-fed Psarr2momys obesus.
The partial nucleotide sequence is as follows:-
GCTGATGGCGATGAATGAACACTGCGTTTGCTGGCCTTATCAATTCCCGCTTTTTCTTGGA
TGAAAAAGACTAACCATGATCGGGCCCAACGGCGGAAGTCGCTTTTGTCACCAGTAGTGAT
GGTGCCGGGATCAAGTGCCACGATTGAGCGTTTCAATTCACTGATTGCGATGCTTAATAAA
GATTCGCCGCACCCTCACAGTGTGTTAAAGATTAAAGTCATGAAAGATGGCAGTTTAAA
2O ATACAAGGGCAGCATTAATCGCGGTGATAATGAACCCTTTATTGTGATTGGTTTTGAAA
ATAATAAAGATGGCTATAGTAATATTAAGAAGCAAGCAAGCTGGCTAGATATTGCCTTTTA
TGAGATCTCGCNAACTTATAAATTTAACAACTTTAAGGCCTTTGGCCATTCAAATGGAG
GGCTGGTGTGGACATATTGGTTAGAGCATTATTATTCAGAGTATGAGTCAGAAA.TCA
AAATCAAGCGGTTGATGACTTTGGCTTCACCATTTAACTTTGACGAAGACAATCTGAAT
2S CACCGGACCCAGATGCTGGCTGACTTTATTAAATATCGGAAACGACTTCCAAAAACGCTC
AAAGTTTATTCACTGACTGGTGGCCAGACCTATGAATCTGACGGGATTGTTCCTGAAAA
TAGTGTAGCCGCAGCCAAGTATATTTTCCAAAATCAAGTGAAGAGCTTTATGGAAAT
TACGGTTACGGGTAAGCAGCTAATCACTCAGATTTACCGCAAAATGAACAAGTAGTGCTA
GTGATGAATCCACCACTCACTAAAGATAAT [SEQ ID N0:2].
EXAMPLE 6
AGT-120 gene expression
AGT-120 expression was significantly higher in the fed group (n=8) compared to
fasted
(n=12) and re-fed animals (n=8). There was no difference between fasted and re-
fed
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animals (Figure 2). No significant correlations were found between AGT-120
expression
and stomach weight, stomach content, glucose or insulin levels.
EXAMPLE 7
S AGT 120 gene homology
The AGT-120 sequence shows homology to a Lactobacillus gassey~i hypothetical
protein
(NZ AAABO1000011). A human homolog is yet to be identified.
EXAMPLE 8
Sequence of Psanafnonays obesus AGT 121
AGT-121 was identified using differential display PCR of hypothalamic cDNA
from
diabetic and non-diabetic Psammomys obesus.
1S
AGT-121 is a hypothalamic gene that was initially identified by differential
display. From
primary gene expression data in fed/fasted hypothalamus, it was of interest
because of the
large increase in its expression in Group B and C animals, as well as the
large disparity in
signal between the animals.
The nucleotide sequence is as follows:-
CAGACTCCTTGGAAATTAAGGAATGCAATTCTGCCACCATGATGGAAGGACTGAAAA.AACGT
ACAAGGAAGGCCTTTGGAATACGGAAGAAAGAAAAAGACACTGACTCTACAGGCTCACCAGA
2S TCGAGATGGAATGCAGCCCAGCCCACACGAGCTCCCCTACCATAGCAAAGCAGAGTGTGCCC
GAGAAGGAGGGAACAAAGCTTCGAAGAAAAGCAATGGGGCACCAAATGGATTTTATGCGGAA
ATTGATTGGGAAAGATATAACTCACCTGAGCTGGATGAAGAAGGTTACAGCATCAGACCTGA
GGAACCAGGCTCTACCAAAGGAAAGCACTTTTATTCTTCAAGTGAATCCGAAGAGGAGGAAG
AATCGCACAAGAAGTTCAATATCAAGATTAAACCCTTGCAGTCCAAGGACATCCTTAAGAAT
3O GCTGCAACAGTAGACGAGCTGAAGGCTTCCATAGGCAACATTGCACTTTCCCCTTCGCCTGT
GAGGAAAAGTCCGAGGCGCAGCCCGGGTGCAATTAAAAGGAACTTATCCAGTGAAGAAGTCG
CAAGACCCAGGCGTTCCACCCCAACTCCAGAACTTACAAGCAAGAAGCCTCTGGACGACACT
CTGGCCCTTGCTCCCCTCTTTGGCCCACCGTTAGAATCTGCTTTTGATGGACACAAGACGGA
AGTTCTTTTAGATCAGCCTGAGATATGGGGTTCAGGCCAACCAGTTAACCCAAGCATGGAGT
3S CACCAAAGCTAGCAAGACCTTTTCCCACTGGAACCCCTCCACCTCTGCCTCCAAAAACTGTA
CCAGCCACCCCGCCTCGGACAGGCTCCCCCTTAACAGTGGCGACAGGAAATGACCAGGCAGC
CACAGAGGCCAAAATTGAGAAACTACCATCCATCAGTGACCTGGACAGCATTTTTGGCCCCG
TGTTGTCCCCCAAGTCTGTTGCTGTTAATACTGAGGAGACGTGGGTCCATTTCTCTGATGCA
TCCCCGGAACATGTTACTCCAGAGTTGACTCCAAGGGAAAAGGTGGTGACCCCACCAGCTGC
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ATCAGACATCCCAGCTGACTCCCCAACTCCAGGCCCGCCTGGCCCCCCAGGCTCGGCAGGTC
CCCCAGGGCCTCCTGGTCCTCGCAATGTACCATCTCCGCTCAATTTAGAAGAAGTCCAGAAG
AAAGTCGCTGAGCAGACCTTCATTAAAGATGATTACTTAGAAACACTCTCATCTCCTAAAGA
GTGTGGGTTGGGACAGAGAGCAACTCCACCTCCCCCACCACCACCCACCTACAGGACTGTGG
S TTTCGTCCCCCGGACCTGGCTCGGGCAGTGGTACGGGGACCGCCAGTGGTGCATCGTCCCCT
GCTCGGCCAGCCACCCCCTTAGTTCCTTGCAGCTGCTCCACTCCGCCTCCACCTCCTCCCCG
GCCTCCATCCCGGCCAAAGCTACCTCCAGGAAAGCCTGGAGTTGGAGACGTGTCCAGACCTT
TTAGCCCACCCATACACTCCTCCAGCCCTCCTCCAATAGCACCCTTAGCCCGGGCTGAAAGC
ACTTCTTCAATATCATCAACCAATTCCCTGAGCGCAGCCACCACTCCCACAGTTGAGAATGA
ZO ACAGSCTTCCCTCGTTTGGTTTGACAGAGGAAAGTTTTATTTGACTTTTGAAGGTTCTTCCA
GGGGACCCAGTCCTCTAACTATGGGGGCCCAGGACACCCTCCCGGTTGCAGCAGCATTCACA
GAAACTGTCAATGCCTACTTCAAAGGAGCAGATCCAAGCAAATGCATTGTTAAGATCACGGG
AGAAATGGTGTTGTCCTTTCCTGCTGGCATCACCAGACACTTTGCCAACAACCCATCCCCAG
CTGCTCTGACTTTTCGAGTGATAAATTCCAGCAGGTTAGAGCACGTCCTGCCGAACCCCCAG
IS CTCCTCTGCTGCGATAACACACAAAATGATGCCAATACCAAGGAATTCTGGGTAAACATGCC
AAATTTGATGACCCACCTGAAGAAGGTCTCTGAACAAAAACCCCAGGCTACATATTACAATG
TGGACATGCTCAAGTATCAGGTGTCAGCCCAGGGCATTCAGTCCACACCTCTGAACTTGGCG
GTGAACTGGCGCTGTGAGCCTTCCAGCACTGACCTGCGCATAGATTATAAGTACAACACGGA
TGCCATGTCCACCGCAGTGGCCCTTAACAACGTGCAGTTCCTGGTCCCCATTGATGGAGGAG
2O TGACCAAGCTCCAGGCTGTCCTTCCTCCAGCAGTCTGGAATGCTGAACAACAAAGAATATTA
TGGAAGATTCCTGATATCTCCCAGAAGTCAGAAAATGGAGGCGTAGGTTCTTTACTGGCAAG
ATTTCAATTAGCCGAAGGCCCAAGCAAACCTTCCCCACTGGTCGTGCAGTTCACGAGTGAAG
GGAGCACTCTGTCTGGCTGCGACATTGAGCTTGTCGGAGCAGGGTACGGGTTTTCACTCATC
AAGAAGAGGTTTGCTGCAGGAAAATACTTGGCCGATAACTAATAAAATGTCATGCAAGGATT
~,S TTGAAGATCCATGTCCTGGAGAACTGTTGTCTGAGAGACATATTTTAATCTGGTTTGAGGAA
AACAAACCAACCGATGTCTGTACGTGGGCTCTGTCAGCTGGAAGGTCCCGGCTTTCAGCCGT
GATTTCCCACACCCAGTACAAGGAGGATCAGTTCTACAGTACTTACTTCTAGGTGTACTATT
GTTAATGGTTTTAAAATGTAATTATTGTATTTGTAAACTGTACCTTCATTCCAGTAAGGCAG
TTAGACACCTGAGTTTTAGCTTTTTTTTCCATTCCTGAAACGGATGTAATTTAAACTGCGGT
3O ATGTAAATTTAATAGTAGTACTGTCGAATGGCACAATGCTTACAGAGATACAGTGCATTTTG
TCAATATATAAAATTTAAATATAATGTTGATAGTTACCATAAAGGGGGTGCCACACATCAAG
AACCTTAAATGGAACCAGAAACAAGCAAGCAAACAAACAAACAAACAAACAAAACCTTACTT
TTCTTCACTCCTTATTACATTTTCCTCTAGAGCTAAAGAAACTTCTAGCTTCGGTTTAGTGG
GTTAAATTCAGAAACTATTTCAG TTCTGAAGTTACAGCATATTCAAAGA
3S GAAGCATTAATTACCACTTTTTTAAAAGCTTTTTTTTCAAACCGCAAATTTCATAAAAATGC
AAACTGTGTAAACAGGGCCTCTTATTTTTATAACTTGTGTAAAAAGGGAAAATCAATTCATA
TTTAAAGTTTAAGTAGTATTAAATTATATCCAAGAGTGAAGAGGATGTTGAAATCTTACCTG
ACCCCATGCCCCTTCTTTGCAGTTTAGCAAATGTTGAGATTGCTAAATCATCAGATTAAAGC
CAACTTGATTTTTAAAGTTTCAAGACTTTCTGAAGCTGAACTGGTTAAA.ACTTTTGCACAAT
4O TGCTTGGAACGGAGGGGGAGGGGCCTCTCTGGTCCAGCACAGGTACCTTGTTTCTTCCCTAC
TCACAAGAATCAAAACAATGAAAGTCAAGAACCACAGAGGGGGGAAATTAGTTCCCTGTTCA
GTCCAAAAGGAGAACTTTAAACTTATCATTTACGTCTTTGGGGAAGGAAGAAATAAGCTTTA
TAAGTGAAATCCTATTCACCTTGTTGTCCTATGAATGTTTTCGGGGTGACTTTAAGATTCAT
TGTATACATGTGCGAGTCTCTGCTATTCTTGGGGAGTTGAAAGCAGAGCCAGGCCAGTGGCC
4S TTGAAGTTCAGTAAATGCCACAGTTCTGGGGCAAAGGTAGGCATGAGGGTTCTGCCCCTCAG
CACAGGAATCAGAGCAGTGTCTTGTAAGGTCTAAAGATTAAGTCTTCCAGTAAGCCACAAGT
TATTTTGTAACAGAGTTGGGGAGTTTTGGCACTCGCTGCTGACTTTCATTTTGTATCCACTC
AAATGGAGTCTTCAACTCTTTTCAACTTTAGAATCAAATTAATTTTTTTTTTTTTTTTTTTT
TTTTTACACAAGGTTTACTCTGTGTAACTGTCCTGGATGTTCTGGAACTCTTTTTGTAGACC
SO AGGCTGGCCTCGAACTCAGAGAGATCCACCTGCCTGTGCTCCCCAAGTGCTGGGATTAAAGG
CGTGTGCCACCATGCCTGGCTTAGATTAAATTTTTTAAGTCTTACTTCACCAGTGAGATTGT
GATTGGCAGTTGTTTCGAGAGAGCTTTGTAGCTTAATCTATGTTCTCTTCAATCAATGCTTG
CTACCAAAAGAATGTCCAAAATGATCTATTTTTCCTGGGAACAATTCATCTATTTAAATAGG
CTCTTGCCTAGTTCCCCAAAGCAGCCTGTCTTTGAAGGTTTTTTTGAACAAAATAATTTTTT
SS CACAAAAAGTTTGGTTTTGAAATCAAAATAGAGAAATAAAATGTAAATTTTAAATCTAATGG
AACATGAGGAAATGAAAAAACTTAAGCCAATGGAGAGTAAAAGCAGAAAAAAATGAAACTTA
CCTAGAATGTGATTATATTATGTTTTTAAGTAGTCAATTCATGGAAAAATATTGAATATTAA
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CACAAAGCATATTAAAAATATGTAAATATTACTGTTTCTCATGTCTTTCTCTTTATATCTTA
TTTTATATAGTTTTAGAATGAATTGGTCATTAAATACAGTGTTTCTTTCCAAAGAATAATTT
TGTTGATATTGTAAAAATGTAATTAAAGATAGAGACTTGAATAGTCTCTAACATTATCCAAA
TGTTTCTAGGAACCAAATTCAAAGCTGTGAAGAAAGCTTGCAATCCCTGAATTGGCTTTTGT
S GAAATGGAATGACGGTGGGTAATCTCAAAATTCAGACTTGAATAGTCAGAGCTGAAGTGGGG
AATGGGTGGTTCCTTCTGGTTCAGAAAATAGGTCAAATAACAGCATTTGCTCGCATCAGGGA
TGGAGATGTTGGTGATGTTTGGTTTTACTCTCGCAGGCTTTCGTCTCCTGTTGAAGGTGTAT
CTGTAGCCCAGTGGGATAAGAGTTCATGTTCTGAGATGTGGTCCTAGACAAGGCAGGCAAGG
TTTCAGTCATCAATACCTATCAGGTCAGGTTCCCTTTTGTCTATACAAAATGGGTTAGCTCA
1O TAGCCAGATGGTTTGCAGGACAGTGAGCTAAATTAGGACAAGATTCTGGTTAGCCAAAGAGC
TGTTTCCTAAGCACTCTGATTTTTTTTTAAAGCTGATAGAAAGTGTAAATGTTCTATTTTGA
CGACATGGAAAGTATGTTTTCCTCTTCAAATAAATCCCTTATTTTTATGAAATTTTCAAAAA
TAAATTCTTGTTTAAAATAGTCTGAATGTTATCATAGTTGGAACTTGGCAATTACTAATTTG
AAATTCTATGAGATGTATCTCCAGCTAAAATGGCAATTCCCTGTATGCTATCTGGGGCTCAG
IS TTTACCTCTAAGGAAGACTGTCAGAGTGCAAATGGTTTTGAGTGACGGGAAAGTCAAAGGGC
AAATGTTTGTGCTTTTTTCTTTTTCTGTCTTATATACTTCTTCTTGGTCTCAGAATGCAAAG
TATCAGAGCCATAGTTACACACATTTCCACTTTTAACGCTTCTTTTGAAGGAAGCAGATCCA
CTTTTGCCCCGCCACTCATGCCTGCTGTGCAGACTCAGACGAGTCCCTGCCCTCTTCACGCC
TTTGGGGTGAGAGGGGAGCCATATGTAAGTAGTTTTCAAGCTTTTCTTAATGGGACTTTTCT
TTTTCTAATAAAATCATGCCTGGAATCCTGTAAAGATTGTTGCCTGGCTGTGAAGGGGCTTC
TCCAGATCCTGAAATATAGCATCACAATACGTAAATGACTCCCGATGGATCTCCCAGCTCTG
AAGACTTGCTCTTCTACTTCACATGTGTAGCCACGACGATCAGCTGGCACACAGTACAATTA
GCTGTGTAGTGAGTGCTCCCCAGCTATCAGTCATGAAACATATCACTTTGCTCAACCTGTTT
TTAAAAAAGCTCCAAAATGGTAAA.AATGCTTTTCAGTCTTTGTTTTCCCAATAATGGTATTG
~S AGGCCTAAGCTGATTAACTTCCCCCAAAGTGGTACCACAGCTGGTAACGACCCCAATGATCC
TGAAAA.AAATGGAATGAGTACCTTGCTGTTTCRTTTAGTTYATTTTGGGAAAATAATCCATT
TGAATGTCAAGATAAAAAGGCACCAGGAAAAGTCCTCATTGGAAGGATTAAAGATGAGCCTG
GTAAGATGTTAAGATGTAAGATGTTAAGATGTGTTACTGT GCTT [SEQ
ID NO:3].
The corresponding amino acid sequence is as follows:-
MMEGLKKRTRKAFGIRKKEKDTDSTGSPDRDGMQPSPHELPYHSKAECAREGGNKASKKSNG
APNGFYAEIDWERYNSPELDEEGYSIRPEEPGSTKGKHFYSSSESEEEEESHKKFNIKIKPL
3S QSKDILKNAATVDELKASIGNIALSPSPVRKSPRRSPGAIKRNLSSEEVARPRRSTPTPELT
SKKPLDDTLALAPLFGPPLESAFDGHKTEVLLDQPEIWGSGQPVNPSMESPKLARPFPTGTP
PPLPPKTVPATPPRTGSPLTVATGNDQAATEAKIEKLPSISDLDSIFGPVLSPKSVAVNTEE
TWVHFSDASPEHVTPELTPREKWTPPAASDIPADSPTPGPPGPPGSAGPPGPPGPRNVPSP
LNLEEVQKKVAEQTFIKDDYLETLSSPKECGLGQRATPPPPPPPTYRTVVSSPGPGSGSGTG
4O TASGASSPARPATPLVPCSCSTPPPPPPRPPSRPKLPPGKPGVGDVSRPFSPPIHSSSPPPI
APLARAESTSSISSTNSLSAATTPTVENEQASLVWFDRGKFYLTFEGSSRGPSPLTMGAQDT
LPVAAAFTETVNAYFKGADPSKCIVKITGEMVLSFPAGITRHFANNPSPAALTFRVINSSRL
EHVLPNPQLLCCDNTQNDANTKEFWVNMPNLMTHLKKVSEQKPQATYYNVDMLKYQVSAQGI
QSTPLNLAVNWRCEPSSTDLRIDYKYNTDAMSTAVALNNVQFLVPIDGGVTKLQAVLPPAVW
4S NAEQQRILWKIPDISQKSENGGVGSLLARFQLAEGPSKPSPLVVQFTSEGSTLSGCDIELVG
AGYGFSLIKKRFAAGKYLADN [SEQ ID N0:4].
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EXAMPLE 9
AGT 121 Taqtrzau tissue distribution
Tissue distribution of AGT-121 was investigated by Taqman PCR in multiple
tissues of P.
obesus (Figure 3). Highest levels were seen in the brain with very low levels
also evident
in the spleen.
EXAMPLE 10
AGT 121- Clouteclz MTN Izuman RNA blot
Tissue distribution of AGT-121 in human tissues was also examined by Northern
analysis
of a Clontech multiple tissue RNA blot. A specific band of approximately 6 kb
was seen in
the brain (Figure 4). AGT-121 is thought to be brain-specific.
EXAMPLE 11
AGT 121 alleles aze associated with obesity
The insertion and deletion alleles described in the original patent are
associated with
obesity. Eighty lean and obese individual Psafnnz~frzys obesus were genotyped
for the
presence of the deletion, or the insertion, or both. Diabetic animals were not
considered.
The genetoype is significantly associated with the obesity phenotype seen in
P. obesus.
The results are shown in Tables 4 and 5.
TABLE 4 Summary of results
Phenotype
Total
Lean Obese
Genotype Insertion 24 9 33
Heterozygote 17 15 32
Deletion 5 10 15
Total 46 34 ~0
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TABLE 5 Chi squared tests
Value df Asymp: Sig.
2_sided
Pearson Chi Square 6.967 2 .031
Likelihood Ratio 7.092 2 0.29
Linear-by-Linear Association6.879 1 .009
No. of valid cases 80
EXAMPLE 12
AGT 121 gene expressiotz ifz energy restricted lzypotlzalatzzus
Oligonuclotide primers were designed in the coding sequence of Psatnmomys
obesus
AGT-121. Expression of AGT-121 was analyzed in energy restricted hypothalamus.
Positive correlations were seen with body weight in control animals, change in
glucose in
all animals and subscapular fat mass in all animals (Figures 5-8).
EXAMPLE 13
AGT 121 sequezzce lzotzzology
The AGT-121: The ISR protein shows strong homology at both the nucleotide and
protein
level to human hypothetical protein DKFZp761D221 (DI~FZp761D221) [Accession:
NP_115667]. This protein is predicted to contain the pfam00928.5, Adap comp
sub
domain which is identified as adaptor complexes medium subunit family. This
family also
contains members which are cocatomer subunits. This gene has been localized to
human
chromosome 1p31.2.
EXAMPLE 14
Partial seqtteuce of Psatntzzotzzys obesus AGT 122
AGT-122 was identified using differential display PCR of liver cDNA from
diabetic and
non-diabetic Psammonzys obesus.
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The partial nucleotide sequence is as follows:-
TCGCGGATCCAGACGCTGCGTTTGCTGGCTTTGATGAAATTTTTAATTTTTCAATATCAGG
S AATGTTTAACTATGCCATGAATTTATGGTAGTCAAGGTTGGAAGGCAGGGGAGAGGACACA
GGGAGTAAAGGCACTTGCCCCCAAGCCTTACAACCTGAATTCCATCCCAGAGTGCCTAATG
GTTGAAGGACGGAACTGAATATCTCTAGCTGTCCTCTATCCTCCACAGATACACAGTGAAT
GCATCAACGTAAAAAATTACAGCTAGAAATAATGTCGTGCCATTCATTGTATTTTACATTN
GTNCATCTTNGNTTTTCCATANTAAAAATGTCTNAGACATACCACTTF~~;~AGCTT
[SEQ ID N0:5].
EXAMPLE 15
AGT 122 gene expression
There was a significant difference in AGT-122 gene expression in the fed state
between
Group A compared to Group B (p=0.001) and Group C (p=0.005) animals (Figure
9). In
the fasted state there were no significant differences between the groups.
Within the groups
of animals, significantly increased expression was seen only in the Group B
fasted animals
(p=0.009). When data from all groups were pooled, no siguficant differences
were seen
(Figure 10).
Expression of AGT-122 in grouped fasted animals showed no association with
body
weight, glucose, or insulin. Expression of AGT-122 in fed animals showed a
significant
negative correlation with body weight (p=0.005; Figure 11), glucose (p=0.003)
and insulin
(p=0.015).
EXAMPLE 16
AGT 122 sequence homology
The AGT-122 sequence shows significant homology with a number of regions on
different
mouse chromosomes. There was no homologous sequence on the public database
(BLASTN version 2.2.1). -_
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EXAMPLE 17
Partial seyuefzce of Psas~amomys obesus AGT 422
AGT-422 was identified by Suppression Subtractive Hybridization (SSH) [also
referred to
as Representational Difference Analysis (RDA)] of liver cDNA from diabetic and
non-
diabetic PsarnnZOmys obesus.
The partial nucleotide sequence is as follows:-
1O CCCTATCCGCTACCCCTGGGAGGGACACAAAAACACATTTTGTGTTTTTGAAAA.AACTGAG
GTCACCAGACTCTTGTATTGTCTTCTTGGACTTCTCTCAGGAACACTCAGGACTCTCCCCA
CACAACACCGTTCTTGAACCGTTCTAACAAATGTTTAAAGTGGTTTCCTTTGAACCACATT
AAATTTAGTTTAAGCAGTCACCAGTGGGCTAGCAGTTCTGGGTTGGGCAGCACATCTTGTA
CAAGCTCTTCCATCTGCCAGGATCACCACCTCTCTGACTTGCACATTTGTGGGTTCCCCAC
IS AGACGAATGGGATGAGTGAAAGAGTGAGTATGTTCTGTTGGGCCTTCAGTAACAGAAGACT
GATTCAGAAAGTAGCACACGTCACATTTTTCTGTAGGTTGGTTTGTTTAGTTTCATTTTTG
ATTTGTGGAACAAAA [SEQ ID N0:6].
EXAMPLE 18
20 AGT 422 ge~ze expression
AGT-422 was normally distributed. One way ANOVA with an LSD post IZOC test
found
gene expression tended to be higher in fed Group A animals than fed Group C
(p=0.060.
Gene expression was significantly greater in fed Group A animals than fasted
Group A
25 (p=0.002), fed Group B animals than fasted Group B p=0.014) and fed group C
animals
than fasted group C (p=0.039) [Figure 12J. An independent samples T test found
AGT-422
gene expression greater in fed animals than fasted animals when group data
were
combined (p<0.001; Figure 13).
30 EXAMPLE 19
AGT 422 sequence homology
The AGT-422 sequence shows sequence homology to Rattus nofvegicus clone CH230-
213K1. The full gene and open reading frame are yet to be identified and a
human
35 homolog is also as yet undetermined.
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EXAMPLE 20
Partial sequence of Psarrzrrzorrzys obesus AGT 123
AGT-123 was identified using differential display PCR of hypothalamic cDNA
from
diabetic and non-diabetic Psanzmomys obesus.
The partial nucleotide sequence is as follows:-
1O TGAGAGTCCATCTCAGCTTATTTCATTGAGATGTTTTGATTAAGAAGTATGACTAGATTAA
AAAATTCTATATAGCTTGGCATTGTTGATAGTTTATATATTTCACTGATTTCTGGTCCCTT
GAAAGTTACTTGGTGATCAACATAGTGTAGTGAAAGGATTGGGATGGACATTF~1AAAAAAA
AAGCTT [SEQ ID N0:7]
EXAMPLE 21
AGT 123 genre exp>"essiou
Hypothalamic AGT-123 expression tended to be increased with fasting in Group A
animals (p=0.06) and was significantly increased with fasting in Group B and
Group C
animals (p=0.02 and p=0.01, respectively) [Figure 14]. When all fed and fasted
animals
were combined, the fasted animals had significantly increased expression
compared to fed
animals (p=0.001) [Figure 14]. AGT-123 expression was not correlated with
glucose or
insulin concentrations. There was a tendency for a relationship between AGT-
123
expression and body weight, although this was not statistically significant
(p=0.0~).
EXAMPLE 22
AGT 123 sequence homology
The AGT-123 sequence matches three mouse expressed sequence tags
(gb~BG797393.1~BG797393 ic14h03.x1 I~aestner ngn3 wt Mus musculus;
gb~AI661150.1~AI661150 va01a02.x1 Soares mouse lymph node NbMLN;
dbj ~BB257743.1 ~BB257743 BB257743 RIKEN full-length enriched, 7 d) however,
the
corresponding gene has not yet been identified (BLASTN version 2.2.1).
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EXAMPLE 23
Paftial sequence of Psazumoszzys obesus AGT 504
S AGT-S04 was identified using Amplified Fragment Length Polymorphism (AFLP)
screening of genomic DNA from diabetic and non-diabetic Psanzmomys obesus. It
was
found to be expressed in the liver of diabetic and non-diabetic Psams~aomys
obesus.
The partial nucleotide sequences are as follow:-
Genomic DNA:-
TGGNTACTCTTGNAAGCACCTTGAAAGTTCAGCCTGCTAACTTACCTTTTTAGCTTTAGAT
TGCTTAGATATTCAAATGAGAGTGTGGTGCAAATCCTGATTACAAGGAGTTTGAGTTTGGA
IS GTGATTGGCAATGCACTTGTGAGGCTGTGTGCCTATGGTCCTAGGACTTAGGAGGCAGGGA
TAGAAGGACCAGGTGCTGAAAGACAGCCTTGGCTAGTTAGTGGACAGATACATAAATGTAC
TGCATGAGATTCTTTCAGAATAACAACCTCCTTTTAAAGAAGTTACTTCTGACATGGAATC
TGTTGCCTGCTTTTGGATCACTACCCCCTGGTGGGACACCTTTGCCAGACCATGGAGGAAG
AACTGTCTTGAT [SEQ ID N0:8].
cDNA:-
AACGTGAGCTTTTTGGAAGGCCCAGANAATTTAAGGAAAGTGTCCCGGCAAATCCTGATTA
CAAGGAGTTTGAGTTTGGAGTGATTGGCAATGCACTTGTGAGGCTGTGTGCCTATGGTCCT
2S AGGACTTAGGAGGCAGGGATAGAAGGACCAGGTGCTGAAAGACAGCCTTGGCTAGTTAGTG
GACAGATACATAAATGTACTGCATGAGATTCTTTCAGAATAACAACCTCCTTTTAAAGAAG
TTACTTCTGACATGGAATCTGTTGCCTGCTTTTGGATCACTACCCCCTGGTGGGACACCTT
TGCCAGACCATGGAGGAAGAACTGTCTTGATGGGANN [SEQ ID N0:9]
3 0 EXAMPLE 24
AGT 504 gene expressio~z
Hepatic AGT-S04 expression was normally distributed and ANOVA with LSD post
lZOc
analysis showed that Group C animals had significantly higher AGT-S04 gene
expression
3S compared to both Group A (p=0.014) and Group B (p=0.02) animals (Figure
1S).
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EXAMPLE 25
Amplified fragrrzeut length polymorphism (AFLP) technique
The AFLP technique is based on the amplification of subsets of genomic
restriction
fragments using polymerise chain reaction (PCR). DNA is cut with restriction
er~ymes
(EeoRI and MseI) and double-stranded adapters are ligated to the ends of the
DNA
fragments to generate template DNA for amplification. The sequences of
adapters and the
adj acent restriction site serve as primer binding sites for subsequent
amplification of the
restriction fragments. Selective nucleotides are included at the 3' ends of
the PCR primers,
which therefore prime DNA amplification only from a subset of the restriction
fragments.
This method will identify sequence differences in the restriction sites that
are associated
with the obesity/diabetes phenotype.
AFLP was performed using the GibcoBRL "AFLP Analysis System I" and "AFLP
Starter
Primer" Fits according to the manufacturer's instructions.
Six genomic DNA pools of each group of Psamm~mys obesus (n=15) were used in an
AFLP screen of 256 primer pairs, which equates to a genomic scan at a density
of
approximately 1.4 cM. The animals were divided by sex and then into three
groups
corresponding to lean (Group A), obese (Group B) and obese/diabetic (Group C).
The PCRs were performed in 20 ~1 under the following conditions: one cycle at
94°C for
s; 65°C for 30 s; and 72°C for 60 s, then for each consecutive
cycle the annealing
temperature was lowered by 0.7°C for 12 cycles. This gives a touchdowxn
phase of 13
25 cycles. This was followed by 23 cycles at 94°C for 30 s, 56°C
for 30 s and 72°C for 60 s.
Once amplified, the fragments were separated on a 6% (w/v) polyacrylamide gel
to
visualize the (typically) 50-150 bands. Those bands that were deemed to be
different
between the groups of animals were excised from the gel. Re-amplification of
the band of
30 interest was performed using the following PCR conditions: 94°C 2
min, 40 cycles of
(94°C 30 s; 54°C 30 s; 72°C 60 s); 72°C 5 min and
the following primers: F 5'-GTA GAC
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TGC GTA CCA ATT C-3' [SEQ )D NO:10] and R 5'-GAC GAT GAG TCC TGA GTA
A-3' [SEQ ID NO:11]. The amplified bands were sequenced with Applied
Biosystems Big
Dye sequencing lit.
EXAMPLE 26
Pfimers
Primer and probe sequences for amplification and analysis of each gene (shown
in the 5' to
3' direction).
SYBR Green analysis
AGT-119
Set 1
Forward: ggattatcgtgaccagataggagc [SEQ )D NO:12]
Reverse: acgcatcttgttgggctaatg [SEQ ~ N0:13]
Set 2
Forward: cgcaaccaaattagccagtg [SEQ m N0:14]
Reverse: gcatcttgttgggctaatgacat [SEQ JD NO:15]
AGT-120
Forward: acccagatgctggctgact [SEQ )D N0:16]
Reverse: ctggccaccagtcagtgaataa [SEQ JD NO:17]
AGT-121
Forward (insertion): aaaacatgagagaagccatactaattca [SEQ )D NO:1~]
Forward (deletion): cacaaaacatgagatactaattcataagtga [SEQ ID N0:19]
Reverse: tgaaaccaagatagcacaaacgaa [SEQ )D N0:20]
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AGT-122
Forward: catcccagagtgcctaatggtt [SEQ ID N0:21]
Reverse: acgttgatgcattcactgtgtatct [SEQ ID N0:22]
AGT-422
Forward: cagtcaccagtgggctagca [SEQ ID N0:23]
Reverse: cctggcagatggaagagctt [SEQ ID N0:24]
AGT-123
Forward: gcttggcattgttgatagtttatatatttc [SEQ ID N0:25]
ReVerSe: cactacactatgttgatcaccaagtaactt [SEQ ID N0:26]
AGT-504
Forward: gaatctgttgcctgcttttgg [SEQ ID N0:27]
Reverse: ctccatggtctggcaaaggt [SEQ ID N0:28]
Taqman analysis
(3-actin Forward: gcaaagacctgtatgccaacac [SEQ ID NO:29]
(3-actin Reverse: gccagagcagtgatctctttctg [SEQ ID NO:30]
Probe: FAM-tccggtccacaatgcctgggaacat-TAMRA [SEQ ID NO:31]
Cyclophilin Forward: cccaccgtgttcttcgaca [SEQ ID N0:32]
Cyclophilin Reverse: ccagtgctcagagcacgaaa - [SEQ ID NO:33]
Probe: FAM-cgcgtctccttcgagctgtttgc-TAMRA [SEQ ID N0:34]
Those spilled in the art will appreciate that the invention described herein
is susceptible to
variations and modifications other than those specifically described. It is to
be understood
that the invention includes all such variations and modifications. The
invention also
includes all of the steps, features, compositions and compounds referred to or
indicated in
this specification, individually or collectively, and any and all combinations
of any two or
more of said steps or features.
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Ausubel et al., "Current Protocols in Molecular Biology" John Wiley & Sons
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15, 1994-1998.
Barnett et al., Diabetologia 37: 671-676, 1994a.
Barnett et al., Int. J. ~besity 18: 789-794, 1994b.
Barnett et al., Diabete Nutz~. Metab. 8: 42-47, 1995.
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Collier et al., Ann. New Yo~kAcad. Sci. 827. 50-63, 1997a.
Collier et al., Exp Clin. Endoczrinol. Diabetes 105: 36-37, 1997b.
De Looper, M and Bhatia I~, Austf~alia's Health Ti~ends 2001. Australian
Institute of
Health and Welfare (AIHW) Cat. No. PHE 24. Canberra: AIHW, 2001.
Douillard and Hoffinan, Basic Facts about Hybridomas, in Conzpendiunz of
Immunology
Vol. II, ed. by Schwartz, 1981.
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-69-
Kohler and Milstein, Nature 256. 495-499, 1975.
Kohler and Milstein, Europea~z Jouf°raal of Immunology 6: 511-519,
1976.
Koopmans, Experimental studies on the control of food intake. In: Handbook of
Obesity,
Eds. GA Bray, C Bouchard, WPT Jasnes pp 273-312, 1998.
Kopehnan et al., Iyat. J. Obesity I8: 188-191, 1994.
Kopehnan, Natuf°e 404: 635-643, 2000.
Marmur and Doty, J. Mol. Biol. 5: 109, 1962.
Mokdad et al., JAMA. 282(16): 1519-1522, 1999.
Mokdad, Diabetes CaYe 24(2): 412, 2001.
Must et al., JAMA. 282(16): 1523-1529, 1999.
National Health and Medical Research Council, Acting on Australia's weight: A
strategy
for the prevention of overweight and obesity. Canberra: National Health and
Medical
Research Council, 1996.
Ravussin, Metabolisfra 44(Suppl 3): 12-14, 1995.
Russek, M., A hypothesis on the participation of hepatic glucoreceptors in the
control of
food intake. Natuf°e 200: 176, 1963.
Shafrir and Gutman, J. Basic Clif2. Physiol. Pharm. 4: 83-99, 1993.
Stellar, Psyclaol. Rev. 61: 5-22, 1954.
CA 02463578 2004-04-13
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Walder et al., Obesity Res. S: 193-200, 1997a.
Wolf and Colditz, Obes. Res. 6: 97-106, 1998.
Zimmet, Diabetes Cage 15(2): 232-247, 1992.
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-1-
SEQUENCE LISTING
<110> Autogen Research Pty Ltd (all other states other than US)
International Diabetes Institute (all other states other than US)
Deakin University (all other states other than US)
Collier, Greg (US only)
~immet, Paul (US only) Z
Walder, Ken (US only)
McMillan, Janine (US only)
de Silva, Andrea (US only) M
Windmill, Kelly (US only)
<120> A gene and uses therefor
<130> 2574822/EJH
<140> International
<141> 2002-10-16
<150> US 60/330,149
<151> 2001-10-16
<160> 34
<170> PatentIn version 3.1
<210> 1
<211> 181
<212> DNA
<213> Psammomys obesus
<220>
<221> misc_feature
<222> (48) . (50)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (52) . (52)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (164) . . (164)
<223> n = any nucleotide
<400> 1
aatgaaagaa ttgattgata cgcaaccaaa ttagccagtg aggttagnnn cnggattatc 60
gtgaccagat aggagccttg gaaaatgact aagaaaaatg aaaaacagcc taaaatgtca 120
ttagcccaac aagatgcgtt aaaacgcctg gatcaagtta gaangcagaa aagcgaaagc 180
c 181
CA 02463578 2004-04-13
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-2-
<210> 2
<211> 814
<212> DNA
<213> Psammomys obesus
<220>
<221> misc_feature
<222> (374)..(374)
<223> n = any nucleotide
<400>
2
gctgatggcgatgaatgaacactgcgtttgctggccttatcaattcccgctttttcttgg60
atgaaaaagactaaccatgatcgggcccaacggcggaagtcgcttttgtcaccagtagtg120
atggtgccgggatcaagtgccacgattgagcgtttcaattcactgattgcgatgcttaat180
aaagattcgccgcaccctcacagtgtgttaaagattaaagtcatgaaagatggcagttta240
aaatacaagggcagcattaatcgcggtgataatgaaccctttattgtgattggttttgaa300
aataataaagatggctatagtaatattaagaagcaagcaagctggctagatattgccttt360
tatgagatctcgcnaacttataaatttaacaactttaaggcctttggccattcaaatgga420
gggctggtgtggacatattggttagagcattattattcagagtatgagtcagaaatcaaa480
atcaagcggttgatgactttggcttcaccatttaactttgacgaagacaatctgaatcac540
cggacccagatgctggctgactttattaaatatcggaaacgacttccaaaaacgctcaaa600
gtttattcactgactggtggccagacctatgaatctgacgggattgttcctgaaaatagt660
gtagccgcagccaagtatattttccaaaatcaagtgaagagctttatggaaattacggtt720
acgggtaagcagctaatcactcagatttaccgcaaaatgaacaagtagtgctagtgatga780
atccaccactcactaaagataataaaaaaaaaaa 814
<210> 3
<211> 6317
<212> DNA
<213> Psammomys obesus
<400> 3
cagactcctt ggaaattaag gaatgcaatt ctgccaccat gatggaagga ctgaaaaaac 60
gtacaaggaa ggcctttgga atacggaaga aagaaaaaga cactgactct acaggctcac 120
cagatcgaga tggaatgcag cccagcccac acgagctccc ctaccatagc aaagcagagt 180
gtgcccgaga aggagggaac aaagcttcga agaaaagcaa tggggcacca aatggatttt 240
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atgcggaaattgattgggaaagatataactcacctgagctggatgaagaaggttacagca300
tcagacctgaggaaccaggctctaccaaaggaaagcacttttattcttcaagtgaatccg360
aagaggaggaagaatcgcacaagaagttcaatatcaagattaaacccttgcagtccaagg420
acatccttaagaatgctgcaacagtagacgagctgaaggcttccataggcaacattgcac480
tttccccttcgcctgtgaggaaaagtccgaggcgcagcccgggtgcaattaaaaggaact540
tatccagtgaagaagtcgcaagacccaggcgttccaccccaactccagaacttacaagca600
agaagcctctggacgacactctggcccttgctcccctctttggcccaccgttagaatctg660
cttttgatggacacaagacggaagttcttttagatcagcctgagatatggggttcaggcc720
aaccagttaacccaagcatggagtcaccaaagctagcaagaccttttcccactggaaccc780
ctccacctctgcctccaaaaactgtaccagCCaCCCCgCCtcggacaggctcccccttaa840
cagtggcgacaggaaatgaccaggcagccacagaggccaaaattgagaaactaccateca900
tcagtgacctggacagcatttttggccccgtgttgtcccccaagtctgttgctgttaata960
ctgaggagacgtgggtccatttctctgatgcatccccggaacatgttactccagagttga1020
ctccaagggaaaaggtggtgaccccaccagctgcatcagacatcccagctgactccccaa1080
ctccaggcccgcctggccccccaggctcggcaggtcccccagggcctcctggtcctcgca1140
atgtaccatctccgctcaatttagaagaagtccagaagaaagtcgctgagcagaccttca1200
ttaaagatgattacttagaaacactctcatctcctaaagagtgtgggttgggacagagag1260
caactccacctcccccaccaccacccacctacaggactgtggtttcgtcccccggacctg1320
gctcgggcagtggtacggggaccgccagtggtgcatcgtcccctgctcggccagccaccc1380
CCttagttCCttgcagctgctCCaCt CtCCdCCtCCtCCCCggCCtCCatGCCggC1440
CCgC
caaagctacctccaggaaagcctggagttggagacgtgtccagaccttttagcccaccca1500
tacactcctccagccctcctccaatagcacccttagcccgggctgaaagcacttcttcaa1560
tatcatcaaccaattccctgagcgcagccaccactcccacagttgagaatgaacagsctt1620
ccctcgtttggtttgacagaggaaagttttatttgacttttgaaggttcttccaggggac1680
ccagtcctctaactatgggggcccaggacaccctcccggttgcagcagcattcacagaaa1740
ctgtcaatgcctacttcaaaggagcagatccaagcaaatgcattgttaagatcacgggag1800
aaatggtgttgtcctttcctgctggcatcaccagacactttgccaacaacccatccccag1860
ctgctctgacttttcgagtgataaattccagcaggttagagcacgtcctgccgaaccccc1920
agctcctctgctgcgataacacacaaaatgatgccaataccaaggaattctgggtaaaca1980
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tgccaaatttgatgacccacctgaagaaggtctctgaacaaaaaccccaggctacatatt2040
acaatgtggacatgctcaagtatcaggtgtcagcccagggcattcagtccacacctctga2100
acttggcggtgaactggcgctgtgagccttccagcactgacctgcgcatagattataagt2160
acaacacggatgccatgtccaccgcagtggcccttaacaacgtgcagttcctggtcccca2220
ttgatggaggagtgaccaagctccaggctgtccttcctccagcagtctggaatgctgaac2280
aacaaagaatattatggaagattcctgatatctcccagaagtcagaaaatggaggcgtag2340
gttctttactggcaagatttcaattagccgaaggcccaagcaaaccttccccactggtcg2400
tgcagttcacgagtgaagggagcactctgtctggctgcgacattgagcttgtcggagcag2460
ggtacgggttttcactcatcaagaagaggtttgctgcaggaaaatacttggccgataact2520
aataaaatgtcatgcaaggattttgaagatccatgtcctggagaactgttgtctgagaga2580
catattttaatctggtttgaggaaaacaaaccaaccgatgtctgtacgtgggctctgtca2640
gctggaaggtcccggctttcagccgtgatttcccacacccagtacaaggaggatcagttc2700
tacagtacttacttctaggtgtactattgttaatggttttaaaatgtaattattgtattt2760
gtaaactgtaccttcattccagtaaggcagttagacacctgagttttagcttttttttcc2820
attcctgaaacggatgtaatttaaactgcggtatgtaaatttaatagtagtactgtcgaa2880
tggcacaatgcttacagagatacagtgcattttgtcaatatataaaatttaaatataatg2940
ttgatagttaccataaagggggtgccacacatcaagaaccttaaatggaaccagaaacaa3000
gcaagcaaacaaacaaacaaacaaacaaaaccttacttttcttcactccttattacattt3060
tcctctagagctaaagaaacttctagcttcggtttagtgggttaaattcagaaactattt3120
cagaaaaaaaaaaaaattctgaagttacagcatattcaaagagaagcattaattaccact3180
tttttaaaagcttttttttcaaaccgcaaatttcataaaaatgcaaactgtgtaaacagg3240
gcctcttatttttataacttgtgtaaaaagggaaaatcaattcatatttaaagtttaagt3300
agtattaaattatatccaagagtgaagaggatgttgaaatcttacctgaccccatgcccc3360
ttctttgcagtttagcaaatgttgagattgctaaatcatcagattaaagccaacttgatt3420
tttaaagtttcaagactttctgaagctgaactggttaaaacttttgcacaattgcttgga3480
acggagggggaggggcctctctggtccagcacaggtaccttgtttcttccctactcacaa3540
gaatcaaaacaatgaaagtcaagaaccacagaggggggaaattagttccctgttcagtcc3600
aaaaggagaactttaaacttatcatttacgtctttggggaaggaagaaataagctttata3660
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agtgaaatcctattcaccttgttgtcctatgaatgttttcggggtgactttaagattcat3720
tgtatacatgtgcgagtctctgctattcttggggagttgaaagcagagccaggccagtgg3780
ccttgaagttcagtaaatgccacagttctggggcaaaggtaggcatgagggttctgcccc3840
tcagcacaggaatcagagcagtgtcttgtaaggtctaaagattaagtcttccagtaagcc3900
acaagttattttgtaacagagttggggagttttggcactcgctgctgactttcattttgt3960
atccactcaaatggagtcttcaactcttttcaactttagaatcaaattaatttttttttt4020
tttttttttttttttacacaaggtttactctgtgtaactgtcctggatgttctggaactc4080
tttttgtagaccaggctggcctcgaactcagagagatccacctgcctgtgctccccaagt4140
gctgggattaaaggcgtgtgccaccatgcctggcttagattaaattttttaagtcttact4200
tcaccagtgagattgtgattggcagttgtttcgagagagctttgtagcttaatctatgtt4260
ctcttcaatcaatgcttgctaccaaaagaatgtccaaaatgatctatttttcctgggaac4320
aattcatctatttaaataggctcttgcctagttccccaaagcagcctgtctttgaaggtt4380
tttttgaacaaaataattttttcacaaaaagtttggttttgaaatcaaaatagagaaata4440
aaatgtaaattttaaatctaatggaacatgaggaaatgaaaaaacttaagccaatggaga4500
gtaaaagcagaaaaaaatgaaacttacctagaatgtgattatattatgtttttaagtagt4560
caattcatggaaaaatattgaatattaacacaaagcatattaaaaatatgtaaatattac4620
tgtttctcatgtctttctctttatatcttattttatatagttttagaatgaattggtcat4680
taaatacagtgtttctttccaaagaataattttgttgatattgtaaaaatgtaattaaag4740
atagagacttgaatagtctctaacattatccaaatgtttctaggaaccaaattcaaagct,4800
gtgaagaaagcttgcaatccctgaattggcttttgtgaaatggaatgacggtgggtaatc4860
tcaaaattcagacttgaatagtcagagctgaagtggggaatgggtggttccttctggttc4920
agaaaataggtcaaataacagcatttgctcgcatcagggatggagatgttggtgatgttt4980
ggttttactctcgcaggctttcgtctcctgttgaaggtgtatctgtagcccagtgggata5040
agagttcatgttctgagatgtggtcctagacaaggcaggcaaggtttcagtcatcaatac5100
ctatcaggtcaggttcccttttgtctatacaaaatgggttagctcatagccagatggttt5160
gcaggacagtgagctaaattaggacaagattctggttagccaaagagctgtttcctaagc5220
actctgatttttttttaaagctgatagaaagtgtaaatgttctattttgacgacatggaa5280
agtatgttttcctcttcaaataaatcccttatttttatgaaattttcaaaaataaattct5340
tgtttaaaatagtctgaatgttatcatagttggaacttggcaattactaatttgaaattc5400
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tatgagatgtatctccagctaaaatggcaattccctgtatgctatctggggctcagttta5460
cctctaaggaagactgtcagagtgcaaatggttttgagtgacgggaaagtcaaagggcaa5520
atgtttgtgcttttttctttttctgtcttatatacttcttcttggtctcagaatgcaaag5580
tatcagagccatagttacacacatttccacttttaacgcttcttttgaaggaagcagatc5640
C2.CttttgCCCCgCCdCtCatgCCtgCtgtgcagactcagacgagtccctgCCCt CttCa5700
cgcctttggggtgagaggggagccatatgtaagtagttttcaagcttttcttaatgggac5760
ttttctttttctaataaaatcatgcctggaatcctgtaaagattgttgcctggctgtgaa5820
ggggcttctccagatcctgaaatatagcatcacaatacgtaaatgactcccgatggatct5880
cccagctctgaagacttgctcttctacttcacatgtgtagccacgacgatcagctggcac5940
acagtacaattagctgtgtagtgagtgctccccagctatcagtcatgaaacatatcactt6000
tgctcaacctgtttttaaaaaagctccaaaatggtaaaaatgcttttcagtctttgtttt6060
cccaataatggtattgaggcctaagctgattaacttcccccaaagtggtaccacagctgg6120
taacgaccccaatgatcctgaaaaaaatggaatgagtaccttgctgtttcrtttagttya6180
ttttgggaaaataatccatttgaatgtcaagataaaaaggcaccaggaaaagtcctcatt6240
ggaaggattaaagatgagcctggtaagatgttaagatgtaagatgttaagatgtgttact6300
gtaaaaaaaaaaagctt 6317
<210>
4
<211>
827
<212>
PRT
<213>
Psammomys
obesus
<400> 4
Met Met Glu G1y Leu Lys Lys Arg Thr Arg Lys Ala Phe Gly Ile Arg
1 5 10 15
Lys Lys Glu Lys Asp Thr Asp Ser Thr Gly Ser Pro Asp Arg Asp Gly
20 25 30
Met Gln Pro Ser Pro His Glu Leu Pro Tyr His Ser Lys Ala Glu Cys
35 40 45
Ala Arg Glu Gly Gly Asn Lys Ala Ser Lys Lys Ser Asn Gly Ala Pro
50 55 60
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Asn Gly Phe Tyr Ala Glu Ile Asp Trp Glu Arg Tyr Asn Ser Pro Glu
65 70 75 80
Leu Asp Glu Glu Gly Tyr Ser Ile Arg Pro Glu Glu Pro Gly Ser Thr
85 90 95
Lys Gly Lys His Phe Tyr Ser Ser Ser Glu Ser Glu Glu Glu Glu Glu
100 105 110
Ser His Lys Lys Phe Asn Ile Lys Ile Lys Pro Leu Gln Ser Lys Asp
115 120 125
Ile Leu Lys Asn Ala Ala Thr Val Asp Glu Leu Lys Ala Ser Ile Gly
130 135 140
Asn Ile Ala Leu Ser Pro Ser Pro Val Arg Lys Ser Pro Arg Arg Ser
145 150 155 160
Pro Gly Ala Ile Lys Arg Asn Leu Ser Ser Glu Glu Val Ala Arg Pro
165 170 175
Arg Arg Ser Thr Pro Thr Pro Glu Leu Thr Ser Lys Lys Pro Leu Asp
180 185 190
Asp Thr Leu Ala Leu Ala Pro Leu Phe Gly Pro Pro Leu Glu Ser Ala
195 200 205
Phe Asp Gly His Lys Thr Glu Val Leu Leu Asp Gln Pro Glu Ile Trp
210 215 220
Gly Ser Gly Gln Pro Val Asn Pro Ser Met Glu Ser Pro Lys Leu Ala
225 230 235 240
Arg Pro Phe Pro Thr Gly Thr Pro Pro Pro Leu Pro Pro Lys Thr Val
245 250 255
Pro Ala Thr Pro Pro Arg Thr Gly Ser Pro Leu Thr Val Ala Thr Gly
260 265 270
Asn Asp Gln Ala Ala Thr Glu Ala Lys Ile Glu Lys Leu Pro Ser Ile
275 280 285
Ser Asp Leu Asp Ser I1e Phe Gly Pro Val Leu Ser Pro Lys Ser Val
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_g_
290 295 300
Ala Val Asn Thr Glu Glu Thr Trp Val His Phe Ser Asp Ala Ser Pro
305 310 315 320
Glu His Val Thr Pro Glu Leu Thr Pro Arg Glu Lys Val Val Thr Pro
325 330 335
Pro Ala Ala Ser Asp Ile Pro Ala Asp Ser Pro Thr Pro Gly Pro Pro
340 345 350
Gly Pro Pro Gly Ser Ala Gly Pro Pro Gly Pro Pro Gly Pro Arg Asn
355 360 365
Val Pro Ser Pro Leu Asn Leu Glu Glu Val Gln Lys Lys Val Ala Glu
370 375 380
Gln Thr Phe Ile Lys Asp Asp Tyr Leu Glu Thr Leu Ser Ser Pro Lys
385 390 395 400
Glu Cys Gly Leu Gly Gln Arg Ala Thr Pro Pro Pro Pro Pro Pro Pro
405 410 415
Thr Tyr Arg Thr Val Val Ser Ser Pro Gly Pro Gly Ser Gly Ser Gly
420 425 430
Thr Gly Thr Ala Ser Gly Ala Ser Ser Pro Ala Arg Pro Ala Thr Pro
435 440 445
Leu Val Pro Cys Ser Cys Ser Thr Pro Pro Pro Pro Pro Pro Arg Pro
450 455 460
Pro Ser Arg Pro Lys Leu Pro Pro Gly Lys Pro Gly Val Gly Asp Val
465 470 475 480
Ser Arg Pro Phe Ser Pro Pro Ile His Ser Ser Ser Pro Pro Pro Ile
485 490 495
Ala Pro Leu Ala Arg Ala Glu Ser Thr Ser Ser Ile Ser Ser Thr Asn
500 505 510
Ser Leu Ser Ala Ala Thr Thr Pro Thr Val Glu Asn Glu Gln Ala Ser
515 520 525
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Leu Val Trp Phe Asp Arg Gly Lys Phe Tyr Leu Thr Phe Glu Gly Ser
530 535 540
Ser Arg Gly Pro Ser Pro Leu Thr Met Gly Ala Gln Asp Thr Leu Pro
545 550 555 560
Val Ala Ala Ala Phe Thr Glu Thr Val Asn Ala Tyr Phe Lys Gly Ala
565 570 575
Asp Pro Ser Lys Cys Ile Val Lys Ile Thr Gly Glu Met Val Leu Ser
580 585 590
Phe Pro Ala Gly Ile Thr Arg His Phe Ala Asn Asn Pro Ser Pro Ala
595 600 605
Ala Leu Thr Phe Arg Val Ile Asn Ser Ser Arg Leu Glu His Val Leu
610 615 620
Pro Asn Pro Gln Leu Leu Cys Cys Asp Asn Thr Gln Asn Asp Ala Asn
625 630 635 640
Thr Lys Glu Phe Trp Val Asn Met Pro Asn Leu Met Thr His Leu Lys
645 650 655
Lys Val Ser Glu Gln Lys Pro Gln Ala Thr Tyr Tyr Asn Val Asp Met
660 665 670
Leu Lys Tyr Gln Val Ser Ala Gln Gly Ile Gln Ser Thr Pro Leu Asn
675 680 685
Leu Ala Val Asn Trp Arg Cys Glu Pro Ser Ser Thr Asp Leu Arg Ile
690 695 700
Asp Tyr Lys Tyr Asn Thr Asp Ala Met Ser Thr Ala Val Ala Leu Asn
705 710 715 720
Asn Val Gln Phe Leu Val Pro Ile Asp Gly Gly Val Thr Lys Leu Gln
725 730 735
Ala Val Leu Pro Pro Ala Val Trp Asn Ala Glu Gln Gln Arg Ile Leu
740 745 750
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Trp Lys Ile Pro Asp Ile Ser Gln Lys Ser Glu Asn Gly Gly Val Gly
755 760 765
Ser Leu Leu Ala Arg Phe Gln Leu Ala Glu Gly Pro Ser Lys Pro Ser
770 775 780
Pro Leu Val Val Gln Phe Thr Ser Glu Gly Ser Thr Leu Ser Gly Cys
785 790 795 800
Asp Ile Glu Leu Val Gly Ala Gly Tyr Gly Phe Ser Leu Ile Lys Lys
805 810 815
Arg Phe Ala Ala Gly Lys Tyr Leu Ala Asp Asn
820 825
<210> 5
<211> 366
<212> DNA
<213> Psammomys obesus
<220>
<221> misc_feature
<222> (305) . . (305)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (308) . . (308)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (315) . . (315)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (317) . . (317)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (327) . . (327)
<223> n = any nucleotide
<220>
CA 02463578 2004-04-13
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-11-
<221> misc_feature
<222> (339) . . (339)
<223> n = any nucleotide
<400>
tcgcggatccagacgctgcgtttgctggctttgatgaaatttttaatttttcaatatcag 60
gaatgtttaactatgccatgaatttatggtagtcaaggttggaaggcaggggagaggaca 120
cagggagtaaaggcacttgcccccaagccttacaacctgaattccatcccagagtgccta 180
atggttgaaggacggaactgaatatctctagctgtcctctatcctccacagatacacagt 240
gaatgcatcaacgtaaaaaattacagctagaaataatgtcgtgccattcattgtatttta 300
cattngtncatcttngnttttccatantaaaaatgtctnagacataccacttaaaaaaaa 360
aagctt 366
<2l0> 6
<211> 442
<212> DNA
<213> Psammomys obesus
<400>
6
ccctatccgctacccctgggagggacacaaaaacacattttgtgtttttgaaaaaactga 60
ggtcaccagactcttgtattgtcttcttggacttctctcaggaacactcaggactctccc 120
cacacaacaccgttcttgaaccgttctaacaaatgtttaaagtggtttcctttgaaccac 180
attaaatttagtttaagcagtcaccagtgggctagcagttctgggttgggcagcacatct 240
tgtacaagCtCttCCatCtgccaggatcacCdCCtCtCtgaCttgcaCatttgtgggttc 300
cccacagacgaatgggatgagtgaaagagtgagtatgttctgttgggccttcagtaacag 360
aagactgattcagaaagtagcacacgtcacatttttctgtaggttggtttgtttagtttc 420
atttttgatt tgtggaacaa as 442
<210> 7
<211> 189
<212> DNA
<213> Psammomys obesus
<400> 7
tgagagtcca tctcagctta tttcattgag atgttttgat taagaagtat gactagatta 60
aaaaattcta tatagcttgg cattgttgat agtttatata tttcactgat ttctggtccc 120
ttgaaagtta cttggtgatc aacatagtgt agtgaaagga ttgggatgga cattaaaaaa 180
aaaaagctt 1gg
CA 02463578 2004-04-13
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-12-
<210> 8
<211> 378
<212> DNA
<213> Psammomys obesus
<220>
<221> misc_feature
<222> (4) . (4)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (13) .(13)
<223> n = any nucleotide
<400>
8
tggntactcttgnaagcaccttgaaagttcagcctgctaacttacctttttagctttaga60
ttgcttagatattcaaatgagagtgtggtgcaaatcctgattacaaggagtttgagtttg120
gagtgattggcaatgcacttgtgaggctgtgtgcctatggtcctaggacttaggaggcag180
ggatagaaggaccaggtgctgaaagacagccttggctagttagtggacagatacataaat240
gtactgcatgagattctttcagaataacaacctccttttaaagaagttacttctgacatg300
gaatctgttg cctgcttttg gatcactacc ccctggtggg acacctttgc cagaccatgg 360
aggaagaact gtcttgat 378
<210> 9
<211> 342
<212> DNA
<213> Psammomys obesus
<220>
<221> misc_feature
<222> (27) . (27)
<223> n = any nucleotide
<220>
<221> misc_feature
<222> (341)..(342)
<223> n = any nucleotide
<400> 9
aacgtgagct ttttggaagg cccaganaat ttaaggaaag tgtcccggca aatcctgatt 60
acaaggagtt tgagtttgga gtgattggca atgcacttgt gaggctgtgt gcctatggtc 120
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-13-
ctaggactta ggaggcaggg atagaaggac caggtgctga aagacagcct tggctagtta 180
gtggacagat acataaatgt actgcatgag attctttcag aataacaacc tccttttaaa 240
gaagttactt ctgacatgga atctgttgcc tgcttttgga tcactacccc ctggtgggac 300
acctttgcca gaccatggag gaagaactgt cttgatggga nn 342
<210> 10
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 10
gtagactgcg taccaattc 19
<210> 11
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> primer
<400> 11
gacgatgagt cctgagtaa 19
<210> 12
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> AGT-119 (set 1) forward primer
<400> 12
ggattatcgt gaccagatag gagc 24
<210> 13
<211> 21
<212> DNA
<213> artificial sequence
<220>
<223> AGT-119 (set 1) reverse primer
<400> 13
acgcatcttg ttgggctaat g 21
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<210> 14
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> AGT-119 (set 2) forward primer
<400> 14
cgcaaccaaa ttagccagtg 20
<210 > 15
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> AGT-119 (set 2) reverse primer
<400> 15
gcatcttgtt gggctaatga cat 23
<210> 16
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> AGT-120 forward primer
<400> 16
acccagatgc tggctgact 19
<210> 17
<211> 22
<212 > DNA
<213> artificial sequence
<220>
<223> AGT-120 reverse primer
<400> 17
ctggccacca gtcagtgaat as 22
<210> 18
<211> 28
<212> DNA
<213> artificial sequence
<220>
<223> AGT-121 forward (insertion) primer
<400> 18
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-15-
aaaacatgag agaagccata ctaattca 28
<210> 19
<211> 31
<212> DNA
<213> artificial sequence
<220>
<223> AGT-121 reverse (deletion) primer
<400> 19
cacaaaacat gagatactaa ttcataagtg a 31
<210> 20
<211> 24
<212> DNA
<213> artificial sequence
<220>
<223> AGT-121 reverse primer
<400> 20
tgaaaccaag atagcacaaa cgaa 24
<210> 21
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> AGT-122 forward primer
<400> 21
catcccagag tgcctaatgg tt 22
<210> 22
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> AGT-122 reverse primer
<400> 22
acgttgatgc attcactgtg tatct 25
<210> 23
<211> 20
<212 > DNA
<213> artificial sequence
<220>
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-16-
<223> AGT-422 forward primer
<400> 23
cagtcaccag tgggctagca 20
<210> 24
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> AGT-422 reverse primer
<400> 24
cctggcagat ggaagagctt 20
<210> 25
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> AGT-123 forward primer
<400> 25
gcttggcatt gttgatagtt tatatatttc 30
<210> 26
<211> 30
<212> DNA
<213> artificial sequence
<220>
<223> AGT-123 reverse primer
<400> 26
cactacacta tgttgatcac caagtaactt 30
<210> 27
<211> 21
<2l2> DNA
<213> artificial sequence
<220>
<223> AGT-504 forward primer
<400> 27
gaatctgttg cctgcttttg g 21
<210> 28
<211> 20
<212> DNA
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-17-
<213> artificial sequence
<220>
<223> AGT-504 reverse primer
<400> 28
ctccatggtc tggcaaaggt 20
<210> 29
<211> 22
<212> DNA
<213> artificial sequence
<220>
<223> Beta-actin forward primer
<400> 29
gcaaagacct gtatgccaac ac 22
<210> 30
<2l1> 23
<212> DNA
<213> artificial sequence
<220>
<223> Beta-actin reverse primer
<400> 30
gccagagcag tgatctcttt ctg 23
<210> 31
<211> 25
<212> DNA
<213> artificial sequence
<220>
<223> Beta-actin probe
<400> 31
tccggtccac aatgcctggg aacat 25
<210> 32
<211> 19
<212> DNA
<213> artificial sequence
<220>
<223> Cyclophilin forward primer
<400> 32
cccaccgtgt tcttcgaca 19
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-18-
<210> 33
<211> 20
<212> DNA
<213> artificial sequence
<220>
<223> Cyclophilin reverse primer
<400> 33
ccagtgctca gagcacgaaa 20
<210> 34
<211> 23
<212> DNA
<213> artificial sequence
<220>
<223> Cyclophilin probe
<400> 34
cgcgtctcct tcgagctgtt tgc 23
