Note: Descriptions are shown in the official language in which they were submitted.
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GROWTH HORMONE-REGU7~ATABI~E BROWN ADIPOSE TISSUE
GENES AND PROTEINS AND USES THEREOF
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to the diagnosis of abnormal GH
activity or general pathological activity in brown adipose
tissue.
Description of the Background Art
Brown Adipose Tissue:
Brown adipose tissue (BAT) is known as a major site of
heat production or thermogenesis where it normally consumes fat
derived from white adipose tissues (WAT). Brown adipocytes
generally reside in capillary beds and are abundant in
cytochromes and proteins, particularly uncoupling protein-1
(UCP1). A BAT depot is located in the interscapular space in
rodents, but exists in the abdominal, neck, and upper back
areas in human neonates. In contrast to rodents, BAT gradually
disappears as children grow. Recent studies demonstrated that
brown adipocytes can also be found in rat and baboon WAT during
cold stress (Cousin et al. 1992 and Viguerie-Bascands et al.
1996) .
Growth Hormones:
The growth hormones are vertebrate proteins with about 191
amino acid residues, the number varying from species to
species. There are four cysteine residues, and two disulfide
bridges. The 3D-structure of porcine GH is known; it is
composed of four major antiparallel alpha-helices, at residues
7-34, 75-87, 106-127 and 152-183.
The 3D structure of the hGH:hGH receptor complex is also
known. Each molecule of hGH binds two molecules of the
receptor. hGH binds to two binding sites on hGH receptor.
Helix 4, the loop residues 54-74, and, to a lesser extent,
helix l, mediate binding to binding site 1. Helix 3 mediates
binding to binding site 2.
See generally Harvey, et al., Growth Hormone (CRC
Press:l995). GH is synthesized and secreted by the
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somatotrophic and somatomammotrophic cells of the lateral
anterior pituitary. The control of GH production and secretion
is complex, but is mainly under the influence of growth hormone
releasing hormone (GHRH) and somatostatin, which stimulate and
inhibit it, respectively. The shifting balance between these
regulatory agents is responsible for the pulsatile nature of
GH secretion, with normal human concentrations ranging from a
baseline value < 1 ug/L to peaks of 25-50 ~ag/L.
Glucocorticoids and thyroid hormones, and various
carbohydrates, amino acids, fatty acids and other biomolecules,
are also known to directly or indirectly regulate GH secretion.
Most GH is secreted at night, during deep sleep, but some
is secreted in response to exercise and other forms of physical
stress. About 500 ~tg/m2 body surface area are secreted by
women, and 350 by men. GH secretion rates are highest in
adolescents and lowest in the elderly. GH has a plasma half-
life of about 20-25 min. and is cleared at a rate of 100-150
ml/m2 body surface area.
Metabolic and Clinical Effects of Growth Hormone:
Chronic elevation of growth hormone levels in humans
usually results in either gigantism or acromegaly. GH, besides
affecting skeletal growth, can also influence other organ
systems, in particular, the liver and kidney. In the kidney,
it has been associated with glomerulosclerosis and nephropathy.
In the liver, it has been shown to cause an increase in liver
size, as a consequence of both hyperplasia and hepatocyte
hypertrophy. The hepatocellular lesions associated with high
GH levels progress with age. See Quaife, et al, Endocrinol.,
124: 49 (1989).
There is reason to believe that excessive GH activity in
the liver is deleterious to health. Mice that express GH
transgenes typically live to only about one year of age, while
the normal life expectancy for mice is 2-2.5 years. A major
cause of death in the GH transgenic mice has been liver
disease.
Growth hormone (GH) is an essential regulator of
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carbohydrate and lipid metabolism, participating in glucose
uptake and usage, accelerating fat expenditure, preventing
triglyceride accumulation, and facilitating lipid mobilization
in adipose tissues. Growth patterns and body compositions of
transgenic mice expressing GH analogs have been characterized
in our laboratory (Knapp et al. 1994). One transgenic mouse
line expresses a GH antagonist (GHA) and is dwarf. As these
mice age, they become obese.
Chronic depression of GH levels can also impair health.
Growth Hormone Antagonists:
In view of the foregoing, it has been suggested that if
a subject is suffering from excessive GH activity, it can be
useful to inhibit such activity by inhibiting the production,
release or action of GH, or facilitating the elimination of GH.
Among the agents useful for this purpose are those which
are competitive binding antagonists of GH. It was discovered
that certain mutants of GH are useful for this purpose.
Kopchick, USP 5,350,836.
In order to determine whether it is appropriate to
initiate or terminate use GH antagonists or other GH-inhibiting
drugs, it is important to be able to monitor GH activity.
Monitoring of GH Activity:
The most straightforward marker
of GH activity is the
serum level of GH per se. For humans, the mean GH
concentration (ug/L) in ood
bl is
preadolescent 4.6
early adolescent 4.8
late adolescent 13.8
adult 1.8
ISS (10y old) 3.5
GH deficient 1.4
IDDM (boys) 9.0
Obese (male) 0.66 (lower than controls)
Fasting 6.7 (higher than controls)
Hyperthyroid 1.9 (higher than controls)
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ISS - idiopathic short stature, IDDM - insulin dependent
diabetes mellitus
See Harvey (1995), supra.
While there is definitely a correlation between high
levels of GH in serum, and high levels of GH activity, it must
be recognized that both the total number of GH receptors, and
the distribution of those receptors among the various organs,
will vary from individual to individual. Hence, in determining
whether an individual is suffering from excessive GH activity,
and prone to develop adverse clinical sequelae, it is helpful
to identify a metabolite which is produced or released in
direct or indirect response to GH and, in particular, one which
is substantially liver-specific so that the specific threat to
liver function can be assessed.
Another marker of GH activity is insulin-like growth
factor-1 (IGF-1). IGF-1 is a 70 amino acid single chain
protein, with some structural similarity to proinsulin, which
is closely regulated by GH secretion. While the majority of
IGF-1 synthesis occurs in the liver, many other tissues,
including bone and skeletal muscle, also release IGF-1 in
response to GH. IGF-1 levels have been used by clinicians to
confirm suspected cases of acromegaly.
However, it would be desirable to have a marker, or
combination of markers, which was more liver specific than IGF
1, for use in monitoring and predicting the effect of chronic
elevation of GH levels on liver function.
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SUMMARY OF THE INVENTION
Applicants have identified certain genes whose expression
in brown adipose tissue is elevated or depressed as a result
of higher than normal GH levels.
5 By use of nucleic acid binding agents to bind messenger
RNA transcripts produced by the transcription of any of these
genes (or to bind the corresponding complementary DNAs
synthesized in vitro), or by use of a protein binding agent to
bind a protein encoded by any of these genes, it is possible
to assay the level of transcription of the gene in question,
or the level of expression and secretion of the corresponding
protein, and to correlate such level with the level of GH
activity in brown adipose tissue.
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BRIEF DESCRIPTION OF THE DRAWINGS
Fiqure 1 Analysis of Brown Adipose Tissue Growth in Transgenic
Mice. In upper panel (A), 10-week old male GHA mouse has
remarkably-enlarged size of iaT (p = 0.0131), iBAT (p = 3.5 X
10-6), and iWAT (p = 0.0155) comparing to their nontransgenic
littermates. This overgrowth has been observed at least at a
similar significant level for iAT (p = 2.0 X 10-~), iBAT (p =
5. 8 X 10-lE) , and iWAT (p = 4 . 4 X 10-5) if normalized by their
body weights. In lower panel (B), since the body size of
GHR/BP is about 51.80 of NT littermate, the size for those
adipose tissues are not significantly different from NT
littermate by gram. However, when normalized by its body
weight in percentage, the enlargement has been observed at a
significant level for iAT (p = 4.5 X 10-G), iBAT (p = 5.8 X 10-
1E) , and iWAT (p=1.0 X 10-') . In both dwarf mice, the size of
eWAT tends to be proportional to that of entire body by weight .
The significant difference for eWAT weight (p = 2.8 X 10-5 in
GHA group and p = 0.0013 in GHR/BPKO group) may be due to their
small body size. Although iBAT weight of bGH mouse is greater
than that of NT littermate (p = 0.0250), normalized iBAT weight
in percentage does not exhibit any significant difference for
iAT, iBAT, iWAT, and eWAT, suggesting that, in bGH mice, those
adipose tissues grow rather proportionally to the body weight
and that any impairment of GH signaling may result in an
overlarged size of interscapular adipose tissue which
constitutes of iBAT and iWAT.
Figure 2 Northern Blot of UCP1 in BAT from Different Transgenic
Mouse. Hybridizing total RNA prepared from various tissues of
10-week old male and female NT mice with 605-by probe, a
portion of UCP1 ORF sequence, the UCPl signals are only
observed in iBAT in male transgenic mice and NT littermates
after 2-hr exposure even prolonged exposure. The mRNA level
of UCP1 is enhanced in GHA and GHR/BPKO mice and is reduced in
bGH mice when comparing that in NT littermates. The ratio of
intensity volume of UCPl to ~3-actin demonstrates that these
changes are substantial.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
We have found that the BAT mass in the GHA mice (n=17) is
significantly greater (p =0.011) than that found in their non-
transgenic (NT) littermates (n=24) when normalized for body
weight. Hence, we proposed that genes in BAT may be up or
down-regulated by GH.
To examine this hypothesis, we employed a PCR-select cDNA
subtraction assay (Clontech) and constructed a forward
subtraction library by subtracting NT littermate BAT cDNAs from
GHA 3AT cDNAs and a reverse subtraction library by subtracting
GHA BAT cDNAs from NT BAT cDNAs . Positive clones were screened
by differential hybridization using probes made from the two
subtracted cDNA libraries. Partial cDNAs were isolated,
sequenced, and analyzed by BLAST searches. We found genes
encoding glucosephosphate isomerase, a-enolase, pyruvate
kinase, proteasome, ubiquitin, and heme oxygenase in the
forward subtraction library, indicating that these genes are
up-regulated in GHA BAT. We found genes encoding mitochondria
cytochrome b, mitochondria cytochrome C oxidase subunit I,
mitochondria NADH-ubiquinone oxidoreductase chain 4 and/or 6,
medium chain acyl-CoA dehydrogenase, adipocyte lipid binding
protein, and trans-Golgi network in the reverse subtraction
library, indicating that these genes are down-regulated in GHA
BAT. These results may partially explain why GHA mice become
obese.
The present invention relates to the use of these genes
and proteins as diagnostic markers in the analysis of brown
adipose tissue structure and function, in particular, its
differentiation, theriogenesis and pathologies.
It is now possible to determine the level of the mRNAs or
proteins corresponding to these genes, in normal adipose tissue
as compared to adipose tissue in a pathological state, and
thereby determine reference values of these mRNAs or proteins
which are indicative of a particular pathological state.
Known pathologic lesions in adipose tissues include:
white adipose tissues:
Aging; insulin resistance; hyperlipidemia; non-
insulin dependent diabetes mellitus; obesity;
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benign and malignant tumor
Brown adipose tissue and brown adipocyte;
Aging; insulin resistance; hyperlipidemia; non
insulin dependent diabetes mellitus; obesity;
benign and malignant tumor
Convertible adipose tissues and convertible
adipocytes:
Aging; insulin resistance; hyperlipidermia;
non-insulin dependent diabetes mellitus;
obesity; benign and malignant tumor.
The preferred screening assay for this purpose is an
antisense probe assay.
It additionally may be of advantage to ascertain the level
of the mRNAs and proteins in cells of the liver, kidney,
muscle, heart, spleen, intestine, brain, lung, testis, and
ovary, and correlate the level with a particular pathological
condition.
Definitions
Two proteins are connate if they are produced in different
species, but are sufficiently similar in structure and
biological activity to be considered the equivalent proteins
for those species. If the accepted scientific names for two
proteins are the same but for the species identification (e.g.,
human GH and shark GH), they should be considered cognate. If
not, the two proteins may still be considered cognate if they
have at least 50o amino acid sequence identity (when globally
aligned with a pam250 scoring matrix with a gap penalty of the
form q+r(k-1) where k is the length of the gap, q=-12 and r=-4;
percent identity=number of identities as percentage of length
of shorter sequence) and at least one biological activity in
common.
Two genes are cognate if they are expressed in different
species and encode cognate proteins.
Gene expression may be said to be specific to a particular
tissue if the average ratio of the specific mRNA to total mRNA
for the cells of that tissue is at least loo higher than the
average ratio is for the cells of some second tissue. Absolute
specificity is not required. Hence, a gene may be said to be
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expressed specifically in more than one tissue.
When the term "specific" is used in this specification,
absolute specificity is not intended, merely a detectable
difference.
Preferably the markers of the present invention are,
singly or in combination, more specific to the target tissue
than are serum GH or IGF-1 levels, or than GH mRNA or IGF-1
mRNA levels in the target tissue.
If this specifications calls for alignment of DNA
sequences, and one of the sequences is intended for the use as
a hybridization probe, the sequences are to be aligned using
a local alignment program with matches scored +5, mismatches
scored -4, the first null of a gap scored -12, and each
additional null of the same gap scored -2. Percentage identity
is the number of identities expressed as a percentage of the
length of the overlap, including internal gaps.
In Vitro Assays
The in vitro assays of the present invention may be
applied to any suitable analyte-containing sample, and may be
qualitative or quantitative in nature.
For the techniques to practice these assays, see, in
general, Ausubel, et al., Current Protocols in Molecular
Bioloay, and in particular chapters 2 ("Preparation and
Analysis of DNA"), 3 ("Enzymatic Manipulation of DNA and RNA"),
4 ("Preparation and Analysis of RNA"), 5 ("Construction of
Recombinant DNA libraries") 6 ("Screening of Recombinant DNA
Libraries"), 7 ("DNA Sequencing"), 10 ("Analysis of Proteins"),
11 ("Immunology"), 14 ("In situ hybridization and immune
histochemistry"), 15 ("The Polymerase Chain Reaction"), 19
("Informatics for Molecular Biologists"), and 20 ("Analysis of
Protein Interactions" ) . Also see, in general, Coligan, et al . ,
Current Protocols in Immunoloay, and in particular, chapters
2 ( "Induction of immune responses") , 8 ( "Isolation and Analysis
of Proteins"), 9 ("Peptides"), 10 ("Molecular Biology") and 17
("Engineering Immune Molecules and Receptors"). Also see
Coligan, et al., Current Protocols in Protein Science.
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The Assay Target (Analyte)
The assay target may be a positive or negative marker.
A positive marker is one for which a higher signal is
correlated with abnormally high growth hormone activity. A
5 negative marker is one for which a higher signal is correlated
with abnormally low growth hormone activity. Positive markers
are up-regulated in high GH mammals and down-regulated in low
GH mammals. Negative markers are up-regulated in high GH
mammals and down-regulated in low GH mammals.
10 A mammal which expresses a GH antagonist (GHA) is normally
considered a low GH level, because it expresses the endogenous
GH at presumably normal levels but the overall GH activity is
depressed as a result of the co-expression of the GHA.
Hence, genes which are up-regulated in GHA mice are
actually negative markers, while genes which are down
regulated in GHA mice are actually positive markers.
In one embodiment, the assay target is a messenger RNA
transcribed from a gene which, in brown adipose tissue, has
increased transcriptional activity if serum GH levels are
increased. This messenger RNA may be a full length transcript
of the gene, or merely a partial transcript. In the latter
case, it must be sufficiently long so that it is possible to
achieve specific binding, e.g., by nucleic acid hybridization.
For the purpose of conducting the assay, the messenger RNA is
extracted from brown adipose tissue by conventional means.
Alternatively, the assay target may be a complementary DNA
synthesized in vitro from the messenger RNA as previously
described.
For convenience, the term "gene" or "target sequence" will
be used to refer to both the messenger RNA or complementary DNA
corresponding to the induced gene, and to the coding gene
proper.
In another embodiment, the assay target is a protein
encoded by said gene and expressed at higher levels in response
to elevated GH levels. If the protein is secreted, the assay
may be performed on serum. If the protein is not secreted,
then cells of brown adipose tissue will be obtained from the
subject and lysed to expose the cytoplasmic contents.
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In either embodiment, one or more purification steps may
be employed prior to the practice of the assay in order to
enrich the sample for the assay target.
The proteins of particular interest are as follows:
Negative Markers:
glucosephosphate isomerase
neuroleukin
pyruvate kinase
heme oxygenase
ubiquitin/ribosomal fusion protein
a-enolase
proteasome 8 chain
Positive Markers
trans-Golgi network protein
medium chain acyl-CoA dehydrogenase
adipocyte lipid binding protein
cytochrome c oxidase
NADH-ubiquonone oxidoreductase
cytochrome b
The genes of particular interest are those encoding the
above proteins. These genes were identified, as described in
Example 1, on the basis of the identity or similarity of mouse
cDNAs obtained by subtractive hybridization methods to known
mouse genes or cDNAs . The mouse sequences are set forth in the
figures. However, if the assay is of a human subject, the
target gene or protein will of course be the cognate human gene
or protein. The sequence databank ID numbers for these
cognate human genes and proteins are given in Table A.
Certainly newly discovered DNAs are also of interest as
positive markers. These are identified below as clones
Ng-G119K2
Ng-G119K15
Ng-G119K36
Ng-G119K62
Ng-G119K42
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Ng-G119K58
Ng-G119K65
Ng-G119K66
The proteins encoded by the ORFs embedded in these DNAs
are also of interest.
Samples
The sample may be of any biological fluid or tissue which
is reasonably expected to contain the messenger RNA transcribed
from one of the above genes, or a protein expressed from one
of the above genes. The sample may be of brown adipose tissue
or interstitial fluid, or of a systemic fluid into which brown
adipose tissue proteins are secreted.
A non-invasive sample collection will involve the use of
urine samples from human subjects. Blood samples will also be
obtained in order to obtained plasma or serum from which
secreted proteins can be evaluated. Brown adipose tissue
aspirates can also be obtained to detect for the presence of
genes and proteins of interest . The most invasive method would
involve obtaining brown adipose tissue biopsies.
Analyte Binding Reagents (Molecules, ABM)
When the assay target is a nucleic acid, the preferred
binding reagent is a complementary nucleic acid. However, the
nucleic acid binding agent may also be a peptide or protein.
A peptide phage library may be screened for peptides which bind
the nucleic acid assay target . In a similar manner, a DNA
binding protein may be randomly mutagenized in the region of
its DNA recognition site, and the mutants screened for the
ability to specifically bind the target. Or the hypervariable
regions of antibodies may be mutagenized and the antibody
mutants displayed on phage.
When the assay target is a protein, the preferred binding
reagent is an antibody, or a specifically binding fragment of
an antibody. The antibody may be monoclonal or polyclonal.
It can be obtained by first immunizing a mammal with the
protein target, and recovering either polyclonal antiserum, or
immunocytes for later fusion to obtain hybridomas, or by
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constructing an antibody phage library and screening the
antibodies for binding to the target . The binding reagent may
also be a binding molecule other than an antibody, such as a
receptor fragment, an oligopeptide, or a nucleic acid. A
suitable oligopeptide or nucleic acid may be identified by
screening a suitable random library.
Binding and Reaction Assays
The assay may be a binding assay, in which one step
involves the binding of a diagnostic reagent to the analyte,
or a reaction assay, which involves the reaction of a reagent
with the analyte. The reagents used in a binding assay may be
classified as to the nature of their interaction with analyte:
(1) analyte analogues, or (2) analyte binding molecules (ABM).
They may be labeled or insolubilized.
In a reaction assay, the assay may look for a direct
reaction between the analyte and a reagent which is reactive
with the analyte, or if the analyte is an enzyme or enzyme
inhibitor, for a reaction catalyzed or inhibited by the
analyte. The reagent may be a reactant, a catalyst, or an
inhibitor for the reaction.
An assay may involve a cascade of steps in which the
product of one step acts as the target for the next step.
These steps may be binding steps, reaction steps, or a
combination thereof.
Signal Producing System (SPS)
In order to detect the presence, or measure the amount,
of an analyte, the assay must provide for a signal producing
system (SPS) in which there is a detectable difference in the
signal produced, depending on whether the analyte is present
or absent (or, in a quantitative assay, on the amount of the
analyte). The detectable signal may be one which is visually
detectable, or one detectable only with instruments. Possible
signals include production of colored or luminescent products,
alteration of the characteristics (including amplitude or
polarization) of absorption or emission of radiation by an
assay component or product, and precipitation or agglutination
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of a component or product. The term "signal" is intended to
include the discontinuance of an existing signal, or a change
in the rate of change of an observable parameter, rather than
a change in its absolute value. The signal may be monitored
manually or automatically.
In a reaction assay, the signal is often a product of the
reaction. In a binding assay, it is normally provided by a
label borne by a labeled reagent.
Labels
The component of the signal producing system which is most
intimately associated with the diagnostic reagent is called the
"label". A label may be, e.g., a radioisotope, a fluorophore,
an enzyme, a co-enzyme, an enzyme substrate, an electron-dense
compound, an agglutinable particle.
The radioactive isotope can be detected by such means as
the use of a gamma counter or a scintillation counter or by
autoradiography. Isotopes which are particularly useful for
the purpose of the present invention are 'H, 3=p, i-JI, 1'~I, 3'S,
'4C, and, preferably, 1=SI.
The label may also be a fluorophore. When the
fluorescently labeled reagent is exposed to light of the proper
wave length, its presence can then be detected due to
fluorescence. Among the most commonly used fluorescent
labelling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
Alternatively, fluorescence-emitting metals such as 1-SEu,
or others of the lanthanide series, may be incorporated into
a diagnostic reagent using such metal chelating groups as
diethylenetriaminepentaacetic acid (DTPA) of ethylenediamine-
tetraacetic acid (EDTA).
The label may also be a chemiluminescent compound. The
presence of the chemiluminescently labeled reagent is then
determined by detecting the presence of luminescence that
arises during the course of a chemical reaction. Examples of
particularly useful chemiluminescent labeling compounds are
luminol, isolumino, theromatic acridinium ester, imidazole,
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acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used for
labeling. Bioluminescence is a type of chemiluminescence found
in biological systems in which a catalytic protein increases
5 the efficiency of the chemiluminescent reaction. The presence
of a bioluminescent protein is determined by detecting the
presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
10 Enzyme labels, such as horseradish peroxidase and alkaline
phosphatase, are preferred. When an enzyme label is used, the
signal producing system must also include a substrate for the
enzyme. If the enzymatic reaction product is not itself
detectable, the SPS will include one or more additional
15 reactants so that a detectable product appears.
An enzyme analyte may act as its own label if an enzyme
inhibitor is used as a diagnostic reagent.
Conjugation Methods
A label may be conjugated, directly or indirectly (e. g.,
through a labeled anti-ABM antibody), covalently (e. g., with
SPDP) or noncovalently, to the ABM, to produce a diagnostic
reagent.
Similarly, the ABM may be conjugated to a solid phase
support to form a solid phase ("capture") diagnostic reagent.
Suitable supports include glass, polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural
and modified celluloses, polyacrylamides, agaroses, and
magnetite. The nature of the carrier can be either soluble to
some extent or insoluble for the purposes of the present
invention.
The support material may have virtually any possible
structural configuration so long as the coupled molecule is
capable of binding to its target. Thus the support
configuration may be spherical, as in a bead, or cylindrical,
as in the inside surface of a test tube, or the external
surface of a rod. Alternatively, the surface may be flat such
as a sheet, test strip, etc.
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Binding Assay Formats
Binding assays may be divided into two basic types,
heterogeneous and homogeneous. In heterogeneous assays, the
interaction between the affinity molecule and the analyte does
not affect the label, hence, to determine the amount or
presence of analyte, bound label must be separated from free
label. In homogeneous assays, the interaction does affect the
activity of the label, and therefore analyte levels can be
deduced without the need for a separation step.
In one embodiment, the ABM is insolubilized by coupling
it to a macromolecular support, and analyte in the sample is
allowed to compete with a known quantity of a labeled or
specifically labelable analyte analogue. The "analyte
analogue" is a molecule capable of competing with analyte for
binding to the ABM, and the term is intended to include analyte
itself. It may be labeled already, or it may be labeled
subsequently by specifically binding the label to a moiety
differentiating the analyte analogue from analyte. The solid
and liquid phases are separated, and the labeled analyte
analogue in one phase is quantified. The higher the level of
analyte analogue in the solid phase, i.e., sticking to the
ABM, the lower the level of analyte in the sample.
In a "sandwich assay", both an insolubilized ABM, and a
labeled ABM are employed. The analyte is captured by the
insolubilized ABM and is tagged by the labeled ABM, forming a
ternary complex. The reagents may be added to the sample in
either order, or simultaneously. The ABMs may be the same or
different. The amount of labeled ABM in the ternary complex
is directly proportional to the amount of analyte in the
sample.
The two embodiments described above are both heterogeneous
assays. However, homogeneous assays are conceivable. The key
is that the label be affected by whether or not the complex is
formed.
Detection of Genes of Interest
For the detection of genes in the sample, PCR can be done
using primers specific for the genes of interest. This would
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amplify the genes of interest. Primers may be designed to
anneal to any site within the open reading frames of the genes
of interest. Resolution of the fragments by electrophoresis on
agarose gel may be used to determine the presence of the genes .
PCR product may be quantitated by densitometry in order to
estimate the concentration of the genes in the samples.
Detection of genes of interest may also be done by
Northern blot analysis on liver biopsies. Tissue sample from
patients may be obtained and the total RNA extracted using
RNAStat 60. The total RNA sample may then be resolved on
denaturing gel by electrophoresis and then transferred onto a
nylon membrane. After transfer of RNA onto the membrane, the
membrane may then be used in hybridization with a suitable
probe, which may be a synthetic probe directed against a gene
already known to be a marker, or which may be a cDNA probe
prepared directly from subtractive hybridization, wherein the
fragment encoding the gene of interest, that is enriched in GH-
overproducing subjects, will be labeled, preferably either
radioactively with 3=P or non-radioactively with DIG
(Digoxigenin). A negative control, such as one composed of RNA
sample from brown adipose tissue of normal subjects, may be
resolved side by side with the patients' sample, to determine
quantitatively whether there is a significant increase in the
level of gene expression. Elevation of the messenger RNA
transcript from this gene would imply that brown adipose tissue
damage might have occurred.
The DNA sequences of the present invention may be used
either as hybridization probes per se, or as primers for PCR.
In a hybridization assay, a nucleic acid reagent may be
used either as a probe, or as a primer. For probe use, only
one reagent is needed, and it may hybridize to all or just a
part of the target nucleic acid. Optionally, more than one
probe may be used to increase specificity. For the primer-
based assay, two primers are needed. These hybridize the non-
overlapping, separated segments of the target sequence. One
primer hybridizes to the plus strand, and the other to the
minus strand. By PCR techniques, the target nucleic acid
region starting at one primer binding site and ending at the
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18
other primer binding site, along both strands, is amplified,
including the intervening segment to which the primers do not
hybridize. In a primer-based assay, the primer thus will not
correspond to the entire target, but rather each primer will
correspond to one end of the target sequence.
In probe-based assays, hybridizations may be carried out
on filters or in solutions. Typical filters are
nitrocellulose, nylon, and chemically-activated papers. The
probe may be double stranded or single stranded, however, the
double stranded nucleic acid will be denatured for binding.
To be successful, a hybridization assay, whether primer-
or probe-based, must be sufficiently sensitive and specific to
be diagnostically useful.
For probe-based assays, sensitivity is affected by the
amount and specific activity of the probe, the amount of the
target nucleic acid, the detectability of the label, the rate
of hybridization, and the duration of the hybridization. The
hybridization rate is maximized at a Ti (incubation
temperature) of 20-25°C. below Tm for DNA:DNA hybrids and 10
15°C. below Tm for DNA:RNA hybrids. It is also maximized by
an ionic strength of about 1.5M Na'. The rate is directly
proportional to duplex length and inversely proportional to the
degree of mismatching.
For primer-based PCR assays, sensitivity is not usually
a major issue because of the extreme amplification of the
signal.
For probe-based assays, specificity is a function of the
difference in stability between the desired hybrid and
"background" hybrids. Hybrid stability is a function of duplex
length, base composition, ionic strength, mismatching, and
destabilizing agents (if any).
The Tm of a perfect hybrid may be estimated.
for DNA:DNA hybrids, as
Tm = 81.5°C + 16.6 (log M) + 0.41 (oGC) -
0.61 (o form) - 500/L
and for DNA:RNA hybrids, as
Tm = 79.8°C + 18.5 (log M) + 0.58 (oGC) -
11.8 (oGC)2 - 0.56(o form) - 820/L
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where
M, molarity of monovalent can ons, 0.01-0.4 M NaCl,
oGC, percentage of G and C nucleotides in DNA, 300-750,
o form, percentage formamide in hybridization solution,
and
L, length hybrid in base pairs.
Tm is reduced by 0.5-1.5°C for each to mismatching.
Tm may also be estimated by the method of Tinoco et al.,
developed originally for the determination of the stability of
a proposed secondary structure of an RNA. Tm may also be
determined experimentally.
Filter hybridization is typically carried out at 68°C.,
and at high ionic strength (e.g., 5 - 6 x SSC), which is
nonstringent, and followed by one or more washes of increasing
stringency, the last was being of the ultimately desired
stringency. The equations for Tm can be used to estimate the
appropriate Ti for the final wash, or the Tm of the perfect
duplex can be determined experimentally and Ti then adjusted
accordingly.
While a mouse cDNA was used to probe a mouse liver cDNA
library, and could be used to probe nonmurine liver cDNA
libraries, it would be expected that there would be some
sequence divergence between cognate mouse and nonmouse DNAs,
possibly as much as 25-500.
Hence, when the human DNA cognate to the original mouse
cDNA is known, it is better to use that DNA, or a fragment
thereof, to probe a human liver cDNA library. The practitioner
may use the complete genomic DNA or cDNA sequence of the human
gene as a probe, or, for the sake of greater specificity or
synthetic convenience, a partial sequence.
It is also noted that while some of the mouse clones were
identical to subsequences of a databank mouse DNA, others
diverged slightly (up to 5a). This divergence could be
artifactual (sequencing error) or real (allelic variation).
Hybridization conditions should be chosen so as to permit
allelic variations, but avoid hybridizing to other genes. In
general, stringent conditions are considered to be a Ti of 5°C.
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below the Tm of a perfect duplex, and a 1% divergence
corresponds to a 0.5-1.5°C. reduction in Tm. Typically, the
mouse clones were 95-100% identical to database mouse
sequences. Hence, use of a Ti of 5-15°C. below, more
5 preferably 5-10°C. below, the Tm of the double stranded form
of the probe is recommended.
If the sequences of the major allelic variants are known,
one may use a mixed probe, and optionally increase the
stringency.
10 If there is no known human gene cognate to the mouse (or
rat) gene homologous to the clone, then the mouse (or rat)
gene, or other known nonhuman cognate gene, may be used as a
probe. In this case, more moderate stringency hybridization
conditions should be used. The nonhuman gene may be modified
15 to obey a more human set of codon preferences.
Alternatively, the mouse (or rat) gene may be used once
as a probe to isolate the human gene, and the human gene then
used for diagnostic work. If a partial human cDNA is obtained,
it may be used to isolate a larger human cDNA, and the process
20 repeated as needed until the complete human cDNA is obtained.
For cross-species hybridization, the Ti should be reduced
further, by about 0.5-1.5°C, e.g., 1°C, for each expected to
divergence in sequence. The degree of divergence may be
estimated from the known divergence of the most closely related
pairs of known genes from the two species.
If the desired degree of mismatching results in a wash
temperature less than 45°C., it is desirable to increase the
salt concentration so a higher temperature can be used.
Doubling the SSC concentration results in about a 17°C.
increase in Tm, so washes at 45°C in 0.1 x SSC and 62°C in 0.2
x SSC are equivalent (1 x SSC = 0.15 M NaCl, 0.015M trisodium
citrate, pH 7.0).
The person skilled in the art can readily determine
suitable combinations of temperature and salt concentration to
achieve these degrees of stringency.
The hybridization conditions set forth in the examples may
be used as a starting point, and then made more or less
stringent as the situation merits.
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Examples of successful cross-species-hybridization
experiments include Braun, et al., EMBO J., 8:701-9 (1989)
(mouse v. human), Imamura, et al., Biochemistry, 30:5406-11
(1991) (human v. rat), Oro, et al, Nature, 336:493-6 (1988)
(human v. Drosophila), Higuti, et al., Biochem. Biophys. Res.
Comm., 178:1014-20 (1991) (rat v. human), Jeung, et al., FEBS
Lett., 307:224-8 (1992) (rat, bovine v. human), Iwata, et°al.,
Biochem. Biophys. Res. Comm., 182:348-54 (1992) (human v.
mouse), Libert, et al., Biochem. Biophys. Res. Comm., 187:919-
926 (1992) (dog v. human), Wang, et al., Mamm. Genome, 4:382-7
(1993) (human v. mouse), Jakubiczka, et al., Genomics, 17:732-5
(1993) (human v. bovine), Nahmias, et al., EMBO J., 10:3721-7
(1991) (human v. mouse), Potier, et al., J. DNA Sequencing and
Mapping, 2:211-218 (1992) (rat v. human), Chan, et al., Somatic
Cell Molec. Genet., 15:555-62 (1989) (human v. mouse), Hsieh,
et al., Id., 579-590 (1989) (human, mouse v. bovine), Sumimoto,
et al., Biochem. Biophys. Res. Comm., 165:902-6 (1989) (human
v . mouse ) , Boutin, et al . , Molec . Endocrinol . , 3 : 1455-61 ( 198 9 )
(rat v. human), He, et al., Biochem. Biophys. Res. Comm.,
171:697-704 (1990) (human, rat v. dog, guinea pig, frog,
mouse), Galizzi, et al., Int. Immunol., 2:669-675 (1990) (mouse
v. human). See also Gould, et al., Proc. Nat. Acad. Sci. USA,
86:1934-8 (1989).
In general, for cross-species hybridization, Ti = 25-35°C.
below Tm. Wash temperatures and ionic strengths may be
adjusted empirically until background is low enough.
For primer-based PCR assays, the specificity is most
dependent on reagent purity.
The final considerations are the length and binding site
of the probe. In general, for probe-based assays, the probe
is preferably at least 15, more preferably at least 20, still
more preferably at least 50, and most preferably at least 100
bases (or base pairs) long. Preferably, if the probe is not
complementary to the entire gene, it targets a region low in
allelic variation.
In general, for primer-based PCR assays, the primer is
preferably at least 18-30 bases in length. Longer primers do
no harm, shorter primers may sacrifice specificity. The
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22
distance between the primers may be as long as 10 kb, but is
preferably less than 3kb, and of course should taken into
account the length of the target sequence (which is likely to
be shorter for mRNA or cDNA than for genomic DNA) . Preferably,
primers have similar GC content, minimal secondary structure,
and low complementarily to each other, particularly in the 3'
region. Also, their targets are preferably relatively
invariant from allete to allete.
For theoretical analysis of probe design considerations,
see Lathe, et al., J. Mol. Biol., 183:1-12 (1985).
Detection of Proteins of Interest
ELISA can be done on blood plasma or serum from patients
using antibodies specific to the protein of interest. Samples
will be incubated with primary antibodies on plates. This
primary antibody is specific to the protein of interest.
Another method that can be conducted will involve the use
of chemical or enzymatic reactions in which the protein of
interest will act as a substrate (or, if the protein is an
enzyme, as a catalyst) to cause a reaction that lead to the
production of colored solution or emission of fluorescence.
Spectrometric analysis can be done in order to determine the
concentration of the proteins in the sample.
Western blot analysis can also be done on the
plasmalserum, tissue aspirate, tissue biopsies or urine
samples. This would involve resolving the proteins on an
electrophoretic gel, such as an SDS PAGE gel, and transferring
the resolved proteins onto a nitrocellulose or other suitable
membrane. The proteins are incubated with a target binding
molecule, such as an antibody.
This binding reagent may be labeled or not. If it is
unlabeled, then one would also employ a secondary, labeled
molecule which binds to the binding reagent. One approach
involves avidinating one molecule and biotinylating the other.
Another is for the secondary molecule to be a secondary
antibody which binds the original binding reagent.
To improve detection of the specific protein,
immunoprecipitation can be conducted. This typically will
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23
involve addition of a monoclonal antibody against the protein
of interest to samples, then allowing the Ig-protein complex
to precipitate after the addition of an affinity bead (ie
antihuman Ig sepharose bead). The immunoprecipitates will
undergo several washings prior to transfer onto a
nitrocellulose membrane. The Western blot analysis can be
perform using another antibody against the primary antibody
used.
Interpretation of Assay Results
The assay may be used to predict the clinical state of the
brown adipose tissue if the level of GH activity remains
unchanged.
A scheme for the diagnostic interpretation of the level
of the target in question is determined in a conventional
manner by monitoring the level of GH, the level of the target,
and the brown adipose tissue condition in a suitable number of
patients, and correlating the level of the target at an earlier
time point with the simultaneous or subsequent brown adipose
tissue state.
This correlation is then used to predict the future
clinical state of the brown adipose tissue in new patients with
high GH levels.
The diagnosis may be based on a single marker, or upon a
combination of markers, which may include, besides the markers
mentioned above, the level of GH or of IGF-1. A suitable
combination may be identified by any suitable technique, such
as multiple regression, factor analysis, or a neural network
using the scaled levels of the markers as inputs and the
current or subsequent brown adipose tissue state as an output.
In vivo Diagnostic Uses
Radio-labelled ABM which are not rapidly degraded in blood
may be administered to the human or animal subject.
Administration is typically by injection, e.g., intravenous or
arterial or other means of administration in a quantity
sufficient to permit subsequent dynamic and/or static imaging
using suitable radio-detecting devices. The dosage is the
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24
smallest amount capable of providing a diagnostically effective
image, and may be determined by means conventional in the art,
using known radio-imaging agents as a guide.
Typically, the imaging is carried out on the whole body
of the subject, or on that portion of the body or organ
relevant to the condition or disease under study. The amount
of radio-labelled ABM accumulated at a given point in time in
relevant target organs can then be quantified.
A particularly suitable radio-detecting device is a
scintillation camera, such as a gamma camera. A scintillation
camera is a stationary device that can be used to image
distribution of radio-labelled ABM. The detection device in
the camera senses the radioactive decay, the distribution of
which can be recorded. Data produced by the imaging system can
be digitized. The digitized information can be analyzed over
time discontinuously or continuously. The digitized data can
be processed to produce images, called frames, of the pattern
of uptake of the radio-labelled ABM in the target organ at a
discrete point in time. In most continuous (dynamic) studies,
quantitative data is obtained by observing changes in
distributions of radioactive decay in target organs over time.
In other words, a time-activity analysis of the data will
illustrate uptake through clearance of the radio-labelled
binding protein by the target organs with time.
Various factors should be taken into consideration in
selecting an appropriate radioisotope. The radioisotope must
be selected with a view to obtaining good quality resolution
upon imaging, should be safe for diagnostic use in humans and
animals, and should preferably have a short physical half-life
so as to decrease the amount of radiation received by the body.
The radioisotope used should preferably be pharmacologically
inert, and, in the quantities administered, should not have any
substantial physiological effect.
The ABM may be radio-labelled with different isotopes of
iodine, for example lz3l, lzsl, or 1311 (see for example, U.S.
Patent 4,609,725). The extent of radio-labeling must, however
be monitored, since it will affect the calculations made based
on the imaging results (i.e. a diiodinated ABM will result in
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twice the radiation count of a similar monoiodinated ABM over
the same time frame).
In applications to human subjects, it may be desirable to
use radioisotopes other than 1=SI for labelling in order to
5 decrease the total dosimetry exposure of the human body and to
optimize the detectability of the labelled molecule (though
this radioisotope can be used if circumstances require) . Ready
availability for clinical use is also a factor. Accordingly,
for human applications, preferred radio-labels are for example,
10 95mTc~ 6,Ga~ 6eGa~ 90~,~ ~mln~ m3mln~ m3I~ lesRe~ '-b"Re or =mAt.
The radio-labelled ABM may be prepared by various methods .
These include radio-halogenation by the chloramine - T method
or the lactoperoxidase method and subsequent purification by
HPLC (high pressure liquid chromatography), for example as
15 described by J. Gutkowska et al in "Endocrinology and
Metabolism Clinics of America: (1987) 16 (1):183. Other known
method of radio-labelling can be used, such as IODOBEADSTM.
There are a number of different methods of delivering the
radio-labelled ABM to the end-user. It may be administered by
20 any means that enables the active agent to reach the agent's
site of action in the body of a mammal. Because proteins are
subject to bering digested when administered orally, parenteral
administration, i.e., intravenous subcutaneous, intramuscular,
would ordinarily be used to optimize absorption of an ABM, such
25 as an antibody, which is a protein.
Other Uses
The markers in question may also be used to determine if
the subject is suffering from or prone to develop a disorder
associated with insufficient GH activity in the brown adipose
tissue.
Presumably, in that event the positive markers will be at
abnormally low levels, and the negative markers are abnormally
low levels.
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Examples
BLASTN searches were performed with the default parameters
match +1, mismatch -3, gap q=-5 r=-2, penalty q+rk for gap
length k. For BLASTP, BLOSUM62 matrix with q=-l, r=-1, lambda
ratio=0.85.
Examgle 1
Brown Adipose Tissue (BAT) Total RNA Preparation
70-day old male G119K growth hormone antagonist (GHA) mice
and their non-transgenic (NT) littermates were sacrificed by
neck dislocation. Their interscapular brown adipose tissues
(BAT) were immediately dissected, weighed, placed in 10 volumes
of cold RNA STAT-60TH solution (TEL-TEST ~~B", Friendswood,
Texas), and carefully homogenized on ice. Total RNAs were
prepared by following the manufacture's protocol. Total RNA
pellets were usually stored in 75o ethanol at ~80°C for not
more than 6 months.
SMARTz'''' PCR cDNA Synthesis
Fresh BAT total RNAs from GHA and NT mice were prepared
with RNA STAT-6OT"' kit (TEL-TEST ~~B", Friendswood, Texas ) and
further purified with QIAEX° II Rneasy Mini Kit (Qiagen,
Chatsworth, California). Purified total RNAs were quantified
by their spectrum ratio of A260/A280 and their band intensity
ratio of 18S/28S on to formaldehyde-Agarose gel. 1 ~zg of each
purified total RNA (as starting material) was then applied to
first-strand cDNA synthesis with SMARTTM PCR cDNA Synthesis Kit
(CLONTECH, Palo Alto, California). Major components, cDNA
synthesis (CDS) primer (AAGCAGTGGTAACAACGCAGAGTACT~3o~N_1N),
SMART II oligonucleotide (AAGCAGTGGTAACAACGCAGAGTACGCGGG), and
MMLV reverse transcriptase (Gibco BRL, Palo Alto, California),
were included in these reactions. The second-strand cDNAs were
synthesized in the presence of Advantage KlenTaq Polymerase Mix
and PCR primer (AAGCAGTGGTAACAACGCAGAGT). The double-stranded
(ds) cDNAs were then simultaneously amplified under the Kit-
recommended PCR program (95°C for 1 minute and 1521 cycles of
95°C for 15 seconds, 65°C for 30 seconds, and 68°C for 6
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27
minutes). 20 for GHA and 18 for NT were determined as optimal
number of PCR cycles by electrophoresing 5 ~l of each PCR
product on a 1.2o Agarose/EtBr gel so that all BAT SMARTTM PCR
cDNAs were equally synthesized for following subtraction.
PCR-Selects cDNA Subtraction
BAT SMARTTM PCR cDNAs were sized with Column
chromatography and cleaved with Rsa I by following the protocol
provided with PCR-SelectTM cDNA Subtraction Kit (CLONTECH, Palo
Alto, California) . The digested cDNAs were purified with QIAEX
II Agarose Gel Extraction Kit (Qiagen, Chatsworth, California),
microfiltrated and precipitated with this Subtraction Kit.
Final concentrations of 300 ng/~.zl were made to both GHA and NT
mouse Rsa I-restricted BAT SMART'' PCR cDNAs.
Standard adapter legations were performed in the presence
of either adaptor 1
(CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT) or adaptor 2R
(CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT) under the
direction of PCR-SelectT"~ cDNA Subtraction Kit so that GHA-
adaptor 1, GHA-adaptor 2R, NT-adaptor l, and NT-adaptor 2R were
respectively prepared to serve as experimental tester cDNAs
while unligated Rsa I-restricted cDNAs were used as
experimental driver cDNAs.
The GHA-adaptor 1 and GHA-adaptor 2R experimental tester
cDNAs were first cross-hybridized with Rsa I-restricted NT
experimental driver cDNAs respectively; while the NT-adaptor
1 and NT-adaptor 2R tester cDNAs were done with Rsa I-
restricted GHA driver cDNAs respectively. After 8-hour air-
incubation at 68°C, the second hybridization was followed by
simply mixing the first cross-hybridization products and
incubating at 68°C overnight: GHA-adaptor 1 tester/NT driver
and GHA-adaptor 2R/NT driver, or NT-adaptor 1 tester/GHA driver
and NT-adaptor 2R/GHA driver.
The final hybridized cDNAs were primarily amplified in the
presence of PCR primer 1 (CTAATACGACTCACTATAGGGC) and Advantage
KlenTaq Polymerase Mix under the Kit-recommended PCR program
(94°C for 25 seconds and 27-32 cycles of 94°C for 10 seconds,
66°C for 30 seconds, and 72°C for 1.5 minutes); then those PCR
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cDNA products were secondarily amplified again in the presence
of Nested PCR primer 1 (TCGAGCGGCCGCCCGGGCAGGT) and Nested PCR
primer 2R (AGCGTGGTCGCGGCCGAGGT) for additional 9-15 cycles
(94°C for 10 seconds, 68°C for 30 seconds, and 72°C for
1.5
minutes) followed by 72°C for 5 minutes. The number of primary
PCR cycles were 27 for GHA and 29 for NT; while that of
secondary PCR cycles were 9 for both GHA and NT . These optimal
number of PCR cycles were determined by electrophoresing 8 ~.Zl
of each PCR product on a 2o Agarose/EtBr gel to minimize the
non-specific BAT SMARTTM PCR-Select subtracted cDNA products.
Analyses of doub~_e-stranded cDNA synthesis products, Rsa
I digestion, adaptor ligation, and subtraction efficiency were
performed according to the recommendation of PCR-Select'"' cDNA
Subtraction Kit. End products from each manipulation were
visualized on 1.2-2o Agarose/EtBr gel before proceeding to do
the next step.
Subtraction Zibrary Construction
Fresh secondary PCR amplification products after PCR
Select cDNA subtraction were ligated to 3.9 kb PCRT"-' II vector
with a standard method provided by TA Cloning Kit (Invitrogen,
Carlsbad, California). After 16-hour incubation at 14°C, these
products were used to transform library efficiency DHSaTM
competent cells (Life Technologies, Palo Alto, California) onto
LB-ampicillin plates by using a recommended small-scale
protocol. a-complementation of the ~3-galactosidase gene
within this vector was employed to produce blue/white screening
of colonies on bacterial plates containing X-gal. 160 white
colonies were isolated from each subtraction library: forward
subtraction library (GHA subtracting NT) and reverse
subtraction library (NT subtracting GHA); total 320 colonies
were maintained.
PCR-Select Differential Screening
The adaptor sequences of the secondary PCR amplification
products after PCR-Select cDNA subtraction were removed by
restricting with Rsa I digestion; and digested products were
purified with QIAEX II Agarose Gel Extraction Kit (Qiagen,
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Chatsworth, California) and precipitated with NH9AC and ethanol
at ~20°C overnight. The down-stream products were then used
to random-prime PCR Dig-labeled probes by incubating at 37°C
for 5 hours with DIG High Prime DNA Labeling Kit (Boehringer
Mamheim, Indianapolis, Indiana). The concentration of both
forward and reverse subtraction library probes were estimated
with the series dilution of Dig-labeled marker.
cDNA arrays were at mean time made with the PCR-Select
Differential Screening Kit (CLONTECH, Palo Alto, California)
under the provided instruction. All PCR inserts were examined
with Nested Primer 1 and Nested Primer 2R on 2o Agarose/EtBr
gels before the cDNA dot blot duplicate were prepared onto the
positively-charged nylon membrane (Boehringer Mamheim,
Indianapolis, Indiana) from either the forward or the reverse
subtraction library for further library screening.
The cDNA dot blot duplicate from forward subtraction
library were pre-hybridized with DIG Easy Hyb solution
(Boehringer Mamheim, Indianapolis, Indiana) at 50°C for 1 hour
and hybridized respectively with random-primed Dig-labeled
probe prepared from forward subtraction library and reverse
subtraction at same temperature for 14 hours. The same
protocol was simultaneously applied to the duplicate from
reverse subtraction library. Washing procedures and detection
of Dig-labeled nucleic acids were standardized under the
GeniusT~' System User's Guide for membrane hybridization
(Boehringer Mamheim, Indianapolis, Indiana). By observing the
differential signals present in dot blot duplicates, 26
positive clones were screened from forward subtraction library
and 14 from reverse subtraction library. Plasmids with
positive inserts were prepared with Plasmid Midi Kit (Qiagen,
Chatsworth, California). These inserts were sequenced with
dGTP mix by using Thermo Sequenase 33P radiolabeled terminator
cycle sequencing Kit (Amersham, Cleveland, Ohio). All
sequences were applied to BLAST search
(http://www.ncbi.nlm.nih.qov/BLAST) so that they could be
determined whether a known sequence was identified at both
nucleic acid and amino acid levels by its alignment to the DNA
and protein databases.
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Among the 26 clones from the forward subtraction library,
25 were homologous to known genes, as follows:
Gene Number
glucosoephosphate
5 isomerase and
neuroleukin 1
pyruvate kinase 1
heme oxygenase 1
ubiquitin/ribosomal fusion
10 protein 1
alpha-enolase 2
proteasome theta chain 2
G119K BGH mutant 17
One clone was considered irrelevant.
15 Among 14 clones from the reverse subtraction library, ten
were homologous to known genes, as follows:
Gene Number
trans-Golgi network
protein 1
20 medium chain acyl-
CoA dehydrogenase 1
adipocyte lipid binding
protein 2
mitochondrial
25 cytochrome c oxidasel
NADFl-ubiquonone
oxidoreductase 2
cytochrome b 3
There were also four novel sequences: Ng-G119K2, Ng
30 G119K15, Ng-G119K36 and Ng-G119K62. Two of these were further
studied in Ex. 2.
Example 2
Brown Adipose Tissue (BAT) Total RNA Preparation
70-day old male G119K growth hormone antagonist (GHA) mice
and their non-transgenic (NT) littermates were sacrificed by
neck dislocation. Their interscapular brown adipose tissues
(BAT) were immediately dissected, weighed, placed in 10 volumes
of cold RNA STAT-6OTM solution (TEL-TEST "B", Friendswood,
Texas), and carefully homogenized on ice. Total RNAs were
prepared by following the manufacturers protocol. Total RNA
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31
pellets were usually stored in 75o ethanol at -80°C for not
more than 6 months.
SMARTT''s PCR cDNA Synthesis
Fresh BAT total RNAs from GHA and NT mice were prepared
with RNA STAT-60TM kit (TEL-TEST "B", Friendswood, Texas) and
further purified with QIAEX° II Rneasy Mini Kit (Qiagen,
Chatsworth, California). Purified total RNAs were quantified
by their spectrum ratio of A260/A280 and their band intensity
ratio of 18S/28S on to formaldehyde-Agarose gel. 1 ~g of each
purified total RNA (as starting material) was then applied to
first-strand cDNA synthesis with SMARTTM PCR cDNA Synthesis Kit
(CLONTECH, Palo Alto, California). Major components, cDNA
synthesis (CDS) primer (AAGCAGTGGTAACAACGCAGAGTACT~3~;N_=N),
SMART II oligonucleotide (AAGCAGTGGTAACAACGCAGAGTACGCGGG), and
MMLV reverse transcriptase (Gibco BRL, Palo Alto, California),
were included in these reactions. The second-strand cDNAs were
synthesized in the presence of Advantage KlenTaq Polymerase Mix
and PCR primer (AAGCAGTGGTAACAACGCAGAGT). The double-stranded
(ds) cDNAs were then simultaneously amplified under the Kit-
recommended PCR program (95°C for 1 minute and 15-21 cycles of
95°C for 15 seconds, 65°C for 30 seconds, and 68°C for 6
minutes). 20 for GHA and 18 for NT were determined as optimal
number of PCR cycles by electrophoresing 5 u1 of each PCR
product on a 1.2o Agarose/EtBr gel so that all BAT SMARTT~'" PCR
cDNAs were equally synthesized for following subtraction.
PCR-SelectT''' cDNA Subtraction
BAT SMARTTM PCR cDNAs were sized with Column
chromatography and cleaved with Rsa I by following the protocol
provided with PCR-SelectTM cDNA Subtraction Kit (CLONTECH, Palo
Alto, California). The digested cDNAs were purified with QIAEX
II Agarose Gel Extraction Kit (Qiagen, Chatsworth, California),
microfiltrated and precipitated with this Subtraction Kit.
Final concentrations of 300 ng/ul were made to both GHA and NT
mouse Rsa I-restricted BAT SMARTTM PCR cDNAs.
Standard adapter ligations were performed in the presence
of either adaptor 1
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(CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT) or adaptor 2R
(CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT) under the
direction of PCR-SelectTr' cDNA Subtraction Kit so that GHA-
adaptor 1, GHA-adaptor 2R, NT-adaptor 1, and NT-adaptor 2R were
respectively prepared to serve as experimental tester cDNAs
while unligated Rsa I-restricted cDNAs were used as
experimental driver cDNAs.
The GHA-adaptor 1 and GHA-adaptor 2R experimental tester
cDNAs were first cross-hybridized with Rsa I-restricted NT
experimental driver cDNAs respectively; while the NT-adaptor
1 and NT-adaptor 2R tester cDNAs were done with Rsa I-
restricted GHA driver cDNAs respectively. After 8-hour air-
incubation at 68°C, the second hybridization was followed by
simply mixing the first cross-hybridization products and
incubating at 68°C overnight: GHA-adaptor 1 tester/NT driver
and GHA-adaptor 2R/NT driver, or NT-adaptor 1 tester/GHA driver
and NT-adaptor 2R/GHA driver.
The final hybridized cDNAs were primarily amplified in the
presence of PCR primer 1 (CTAATACGACTCACTATAGGGC) and Advantage
KlenTaq Polymerase Mix under the Kit-recommended PCR program
(94°C for 25 seconds and 2732 cycles of 94°C for 10 seconds,
66°C for 30 seconds, and 72°C for 1.5 minutes); then those PCR
cDNA products were secondarily amplified again in the presence
of Nested PCR primer 1 (TCGAGCGGCCGCCCGGGCAGGT) and Nested PCR
primer 2R (AGCGTGGTCGCGGCCGAGGT) for additional 915 cycles
(94°C for 10 seconds, 68°C for 30 seconds, and 72°C for
1.5
minutes ) followed by 72 °C for 5 minutes . The number of primary
PCR cycles were 27 for GHA and 29 for NT; while that of
secondary PCR cycles were 9 for both GHA and NT. These optimal
number of PCR cycles were determined by electrophoresing 8 u1
of each PCR product on a 2o Agarose/EtBr gel to minimize the
non-specific BAT SMARTTM PCR-Select subtracted cDNA products.
Analyses of double-stranded cDNA synthesis products, Rsa
I digestion, adaptor ligation, and subtraction efficiency were
performed according to the recommendation of PCR-SelectTM cDNA
Subtraction Kit. End products from each manipulation were
visualized on 1.2~2o Agarose/EtBr gel before proceeding to do
the next step.
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Subtraction Library Construction
Fresh secondary PCR amplification products after PCR-
Select cDNA subtraction were ligated to 3.9 kb PCRT"' II vector
with a standard method provided by TA Cloning Kit (Invitrogen,
Carlsbad, California). After 16-hour incubation at 14°C, these
products were used to transform library efficiency DHSaT""
competent cells (Life Technologies, Palo Alto, California) onto
LB-ampicillin plates by using a recommended small-scale
protocol. a-complementation of the (3-galactosidase gene
within this vector was employed to produce blue/white screening
of colonies on bacterial plates containing X-gal. 160 white
colonies were isolated from each subtraction library: forward
subtraction library (GHA subtracting NT) and reverse
subtraction library (NT subtracting GHA); total 320 colonies
were maintained.
PCR-Select Differential Screening
The adaptor sequences of the secondary PCR amplification
products after PCR-Select cDNA subtraction were removed by
restricting with Rsa I digestion; and digested products were
purified with QIAEX II Agarose Gel Extraction Kit (Qiagen,
Chatsworth, California) and precipitated with NHAC and ethanol
at D20°C overnight. The down-stream products were then used
to random-prime PCR Dig-labeled probes by incubating at 37°C
for 5 hours with DIG High Prime DNA Labeling Kit (Boehringer
Mamheim, Indianapolis, Indiana). The concentration of both
forward and reverse subtraction library probes were estimated
with the series dilution of Dig-labeled marker.
cDNA arrays were at mean time made with the PCR-Select
Differential Screening Kit (CLONTECH, Palo Alto, California)
under the provided instruction. All PCR inserts were examined
with Nested Primer 1 and Nested Primer 2R on 2o Agarose/EtBr
gels before the cDNA dot blot duplicate were prepared onto the
positively-charged nylon membrane (Boehringer Mamheim,
Indianapolis, Indiana) from either the forward or the reverse
subtraction library for further library screening.
The cDNA dot blot duplicate from forward subtraction
library were pre-hybridized with DIG Easy Hyb solution
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(Boehringer Mamheim, Indianapolis, Indiana) at 50°C for 1 hour
and hybridized respectively with random-primed Dig-labeled
probe prepared from forward subtraction library and reverse
subtraction at same temperature for 14 hours. The same
protocol was simultaneously applied to the duplicate from
reverse subtraction library. Washing procedures and detection
of Dig-labeled nucleic acids were standardized under the
GeniusTM System UserOs Guide for membrane hybridization
(Boehringer Mamheim, Indianapolis, Indiana). By observing the
differential signals present in dot blot duplicates, 26
positive clones were screened from forward subtraction library
and 14 from reverse subtraction library. Plasmids with
positive inserts were prepared with Plasmid Midi Kit (Qiagen,
Chatsworth, California). These inserts were sequenced with
dGTP mix by using Thermo Sequenase 1'P radiolabeled terminator
cycle sequencing Kit (Amersham, Cleveland, Ohio). All
sequences were applied to BLAST search
(http://www.ncbi.nlm.nih.gov/BLAST) so that they could be
determined whether a known sequence was identified at both
nucleic acid and amino acid levels by its alignment to the DNA
and protein databases. Based on these Internet searches, two
novel partial cDNA sequences from the reverse subtraction
library, Ng-G119K36 and Ng-G119K62, have been chosen for
further study.
SMARTS PCR cDNA Library Construction
NT BAT SMARTTM PCR cDNAs were synthesized from fresh
purified NT BAT total RNAs with CDS primer and SMART II
oligonucleotide at PCR cycle 20 by using SMARTTM PCR cDNA
Library Construction Kit (CLONTECH, Palo Alto, California).
The integrity of this SMART cDNA was examined with 5 pairs of
primers: 540 by mouse (3-actin, 606 by mouse uncoupling protein
1 (UCP1), 521 by mouse GHR, 452 by mouse glycerol-3-phosphate
dehydrogenase (G3PDH), and approximately 250 by hypoxanthine
phosphoribosyltransferase (HPRT). The PCR program was 1 cycle
of 94°C for 2 minutes, 55 cycles of 94°C for 30 seconds,
60°C
for 30 seconds, and 6872°C for 1~2 minutes, 1 cycle of 6872°C
for 7 minutes, and held at 4°C.
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The polished SMART ds cDNA were ligated with 5' overhang
EcoR I adaptor (13-mer: 5'-OH-AATTCGGCACGAG-3'; 9-mer: 3'-
GCCGTGCTC-Pi-5') by using the ZAP-cDNAO Gigapack0 III Gold
Cloning Kit (STRATAGEM, La Jolla, California), purified by
5 organic reagent extraction, precipitated with NaAC and ethanol,
and physphorylated in the presence of T4 polynucleotide kinase
and ATP, then size-fractionated with column provided in this
ZAP-cDNAO Gigapack0 III Gold Cloning Kit. Fractional drops
from number 12 through 18 were collected together and further
10 precipitated with cold ethanol at ~720°C overnight. 25100 ng
of this SMART cDNA were then ligated to 1 ug of 41 kb ?~ZAPII
predigested vector at 12°C overnight and packaged with
Gigapack0 III Gold packaging extract at 22°C for 1.5 hours.
Following the instruction of ZAP-cDNAO Gigapack0 III Gold
15 Cloning Kit, 200 u1 of 0.5 ODE~,~ fresh XL-1 Blue MRF- strain
was mixed with 0.1-1 ~l of packaged SMART cDNAs in 2-3 ml of
NZY top agar at 4855°C, then immediately inoculated onto a
fresh NZY agar plate (100-mm), and incubated at 37°C for 8
hours . The titer ranged from 1 . 5-1 . 8 X lOc plaque forming unit
20 (pfu) every 1 ug vector arm. 600 u1 of 0.5 OD6o~ fresh XL-1
Blue MRF- strain was mixed with a twentieth aliquot of packaged
SMART cDNA in 6.5 ml of NZY top agar at 4 855°C, then
immediately inoculated onto a fresh NZY agar plate (150-mm),
and incubated at 37 °C for 8 hours . The titer was approximately
25 4.05 X 10~ pfu per plate (150-mm). The amplified library was
made by pooling all samples together with SM dilution buffer
and stored in 70 (v/v) dimethylsulfoxide (DMSO) solution at
~780°C. The titer was similarly determined on the NZY plate
(100-mm): 4.30 X 105 pfu/ul amplified library. A mini-PCR
30 reactions were set up as 3.25 u1 dH~O, 0.5 u1 lOX PCR buffer,
0.05 u1 10 uM T, primer and 0.05 u1 10 uM M13R(-48) primer, 0.1
u1 10 mM dNTPs, 0.05 u1 5 unit/ml Taq DNA polymerase (Promega,
Madison, Wisconsin), and 1.0 u1 DNA template-containing
released particle from every isolated plaque with the
35 parameters : 1 cycle of 95C for 2 minutes, 50 cycles of 94 °C for
30 seconds, 55°C for 30 seconds, and 72°C for 5 minutes, 1
cycle of 72°C for 7 minutes, and 4°C to hold. All total 5 u1
reaction volume per reaction was loaded to to Agarose/EtBr gel
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to determine the recombinational rate.
SMARTT's PCR cDNA Library Screening
600 u1 of 0.5 ODE~o fresh XL-1 Blue MRF- strain was mixed
with approximately 2 u1 of amplified library in 6.5 ml of NZY
top agar at 4 855°C, then immediately inoculated onto a fresh
NZY agar plate ( 150-mm) , and incubated at 37 °C for 8 hours .
Duplicates of pre-shrinked (sterilization cycle for 10 minutes)
nylon membranes (132 mm ~) (Boehringer Mamheim, Indianapolis,
Indiana) were placed to pre-cooled plaques on NZY agar plates
for 2 minutes to make the first duplicate and for 4 minutes to
make the second duplicate, orientated with insoluble ink by
needling at its edge through the NZY agar, then blotted onto
the filter papers. These plaque lifts were immobilized when
autoclaving at sterilization cycle for 2 minutes.
Two entire inserts of positive clone Ng-G119K36 and Ng-
G119K62 from reverse subtraction library were used to prepare
the probes in the presence of Nested Primer 1 and Nested Primer
2R with the PCR DIG Probe Synthesis Kit (Boehringer Mamheim,
Indianapolis, Indiana). The concentration of both Ng-G119K36
and Ng-G119K62 probes were estimated with the series dilution
of Dig-labeled marker.
The plaque lift duplicates were pre-hybridized with DIG
Easy Hyb solution (Boehringer Mamheim, Indianapolis, Indiana)
at 42°C for 1 hour and hybridized respectively with PCR Dig-
labeled probe prepared from either clone Ng-G119K36 or clone
Ng-G119K62 from reverse subtraction library for about 12 hours .
Washing procedures and detection of Dig-labeled nucleic acids
were standardized under the GeniusTM System User's Guide for
membrane hybridization (Boehringer Mamheim, Indianapolis,
Indiana). Plaques showing signals in plaque lift duplicates
were isolated and prepared for further multiple screening on
NZY agar plate (100-mm) until plaques were purified.
Following the protocol provided with ZAP-cDNA~ Gigapack~
III Gold Cloning Kit (STRATAGEM, La Jolla, California), SOLR
strain was prepared to excise the pBluescript phagemid out from
each purified plaque screened from full-length NT BAT SMART
cDNA library. Colonies were isolated from LB-ampicillin
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plates, prepared with Plasmid Maxi Kit (Qiagen, Chatsworth,
California), and maintained for further studies.
Inserts within purified plasmids were sequenced with
either dITP mix or dGTP mix by using Thermo Sequenase 33P
radiolabeled terminator cycle sequencing Kit (Amersham,
Cleveland, Ohio). Downstream sequence determined from each
reaction was used to design the primer for next run sequencing
reaction until all sequences from a single insert can be
overlapped together to deduce a completed full-length cDNA
sequence. All full-length sequences were applied to BLAST
search (http://www.ncbi.nlm.nih.gov/BLAST) so that they could
be determined whether a known sequence was called at both
nucleic acid and amino acid levels by its alignment to the DNA
and protein databases. An automated DNA sequencing approach
was also employed in this project as well. Half reactions
followed by isopropanol precipitation were chosed by using ABI
PRISM° BigDyeTr'' Terminator Cycle Sequencing Ready Reaction Kit
(Perkin-Elmer, Foster City, California). All samples were
arranged to the ABI PRISM 310 Genetic Analyzer. Sequences done
with automated fashion were edited with the ABI PRISM EditView
1Ø1.sea software which is downloaded from the website
(http://www2.perkin-
elmer.com/ab/techsupp/softlib/SeqAnal/installs/mac/EditView
1Øl.sea.hqx).
In general, two novel cDNAs sequences were identified.
Clone 42 was screened with a Ng-G119K36 PCR Dig-labeled probe.
Clone 42 codes a 2478-by mRNA with an open reading frame
encoding 346-amino acid sequence. Clone 42 has two isoforms,
2.4-kb & 1.2-kb, which seem down-regulated in GHA mice. Clone
42 is widely expressed in most tissues; there are significant
levels of both isoforms in BAT, a pronounced level of the long
isoform in brain and a striking level of the short isoform in
testis. Predicted secondary structure seems a helix-like
polypeptide with a 18-amino acid signal peptide and relatively
low hydrophobicity value. Predicted tertiary structure
contains 7-8 hydrophobic regions through the sequence. The
folding model seems similar to bacteriorhodopsin which is a
important protein for proton conductance in archaebacterial.
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A BLAST search (Fig. 2 (e) ) demonstrates that Clone 42 is highly
homologous to Clone 25077 mRNA from human female fetal brain
tissue and PTD 010 mRNA from human pituitary tumor at both mRNA
level and amino acid level.
Three other clones, Clone 58 (1380 bp), Clone 65 (2437
bp), and Clone 66 (1613 bp) were screened with the same Ng-
G119K62 PCR Dig-labeled probe.
Clone 58, 65, & 66 seem like triple alternative splice
forms which are 1379-bp, 2436=bp, 1612-by mRNAs respectively.
All these isoforms may encode a possible open reading frame
containing 86 amino acids. The predicted open reading frame
has a homology of two human genomic DNA sequences from clone
41562 on chromosome 22 which contains synapsin IIa exon 1, EST
and GSS. All these isoforms do not match up with any sequence
in public protein database. The expression of long isoform
tend to be BAT-specific.
Conclusions
Body weight and subcutaneous adipose tissue seems
accumulated with age in GHA mice. An impairment of GH-induced
signaling leads to abnormal growth of interscapular adipose
tissues. GH-induced signaling has also been observed to down
regulate uncoupling protein-1 at transcriptional level.
In interscapular brown adipose tissue, GH may down
regulate genes coding glycolytic enzymes, ubiquitin/proteasome
degradation machinery, and heme oxygenase; GH may up-regulate
genes coding adipocyte lipid binding protein, trans-Golgi
network protein (TGN38), medium chain acyl-CoA dehydrogenase,
and mitochondrial innermembrane proteins for electron
respiration chain. These GH-regulatable genes may be used as
potential molecular markers to help explain obesity in GHA
mice.
Clone 42 codes a 2478-by mRNA with an open reading frame
encoding 346-amino acid sequence. Two isoforms, 1.2-kb & 2.4-
kb, are up-regulatable by GH and are widely expressed in most
tissues with significant levels in BAT, pronounced level of
long isoform in brain, and striking level of the short isoform
in testis. It seems a helix-like polypeptide with a 18-amino
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39
acid signal peptide and relatively low hydrophobicity value.
Clone 42 is highly homologous to Clone 25077 mRNA from human
female fetal brain tissue and PTD O10 mRNA from human pituitary
tumor at both mRNA level and amino acid level.
Clone 58, 65, & 66 seem like triple alternative splice
forms which are 1379-bp, 2436-bp, 1612-by mRNAs respectively.
They may encode a possible open reading frame containing 86
amino acids, with a homology of two human genomic DNA sequences
from clone 41562 on chromosome 22 containing synapsin IIIa exon
1, EST and GSS. The expression of clone 65 tend to be BAT-
specific.
Example 3
A. Use of Mouse BAT genes in Assay of Human BAT
Brown adipose tissues are obtained from the human subject
in a conventional, medically acceptable manner. Total RNA is
then extracted using mL RNAStat60 per gram of tissue.
To 15-20 ug of brown adipose tissue RNA isolates, 1X MOPS,
formaldehyde, formamide and ethidium bromide will be added,
heat denatured at 60 °C then loaded on a formamide containing
denaturing to agarose gel. The RNA will then be resolved by
electrophoresis at 50V for about 2-2~h. After electrophoresis,
the gel will be washed twice briefly with deionized water; then
once with 0.05N NaOH, with O.1M Tris at pH 7.5, and with lOX
SSC at washing times of at least 30 min in each case.
The resolved RNA after electrophoresis will be transferred
onto a nylon membrane by upward gradient adsorption using lOX
SSC as transfer buffer. The RNA on the membrane will be UV
crosslinked at 120 mJ, after which the RNA blots will be ready
for hybridization.
B. Northern Blot Hybridization involving Non-radioactive DIG-
labeled probe
Northern blot hybridization using digoxigenin (DIG)-
labeled probe will be conducted to determine whether the genes
of interest are present in brown adipose tissue RNA blots . The
probes to be used for hybridization will be prepared from pCR2
clones, which contain as inserts the fragments isolated by
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subtractive hybridization of brown adipose tissue genes from
GHA mice versus WT mice.
1. Preparation of DIG-labeled probe
The DIG-labelled probe preparation will require PCR
5 amplification of the inserts in pCR2 clones using Taq
polymerase as polymerization enzyme and pCR 2.1A and pCR 2B as
primers. The conditions for PCR amplification will be 95°C for
2 min. ; 55 cycles at three temperature conditions of 95°C forl5
sec., 58°C for 20 sec., and 72 °C for 45 sec.; then 72°C
for 7
10 min. The amplified double-stranded cDNA fragment will undergo
a second PCR amplification using a single primer, pCR 2.1A, in
the presence of DIG labeled dNTPs to produce a single stranded
DIG-labeled PCR product which will serve as the probe for RNA
blot hybridization. The concentrations of the DIG labeled
15 probe will be determined by comparing the signals produced by
the probe to that of control DIG-labeled DNA upon exposure to
radiographic film.
2. RNA Blot Hybridization
The concentration of DIG-labeled probe to be used for
20 hybridization will be 50ng/mL of DIG Easy Hyb solution
(Boehringer-Mannheim). Prior to hybridization, the RNA blots
will be prehybridized in DIG Easy Hyb solution at 42 °C for 30
60 min. Following prehybridization, the RNA blots will undergo
hybridization using the probes prepared form the different pCR
25 2 clones. Hybridization will be done at 42 °C for at least 8
hours.
Posthybridization washings of the membrane will then be
performed at room temperature for 5min using a solution of 2X
SSC and 0.1o SDS; and twice at 60 °C for 15 min. using a
30 solution of 0.5X SSC and O.loSDS. The RNA blots will then be
incubated with DIG antibody, which is conjugated to alkaline
phosphatase. This antibody recognizes the DIG labeled hybrids
in the RNA blot. CSPD (Boehringer-Mannheim), which is a
chemiluminescent substrate for alkaline phosphatase, will be
35 use to achieve detection of the RNA of interest in the blot.
The presence of bands that is specific to the brown adipose
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41
tissue genes of interest could be diagnostic of brown adipose
tissue damage.
C. Northern Blot Hybridization involving 32P-labeled probe
1. Preparation of 32P-labeled probe
The 3=P-labeled probe will be prepared by first isolating
the cDNA fragments that were inserted into the pCR 2 vector by
performing EcoRI restriction enzyme digestion. The fragments
will be purified though a Qiaex~ agarose gel extraction column
(Qiagen). A 25ng of the purified fragment will serve as a
template for the production of single-stranded '=P-labeled
probe using Random Primed DNA Labeling kit (Boehringer-
Mannheim). The unincorporated dNTPs will be separated from the
radiolabeled fragments using STE Select D G-25 column. The
purified radiolabeld probe will then be quantified t,o determine
the activity of the probe per ug of the DNA template. A good
labeling of the template would have a specific activity range
of l0a-lOq cpm/ug of the template DNA.
2. RNA Blot hybridization
Prior to hybridization, prehybridization of the RNA blots
will be performed by incubating the membrane in
prehybridization solution made up of 50o formamide, to SDS, 1M
NaCl, and loo Dextran sulfate for 1 hour at 42 °C.
Hybridization of the RNA blot with the J'-P-labeled probes
prepared will follow after prehybridization. This will be
conducted at 42 °C for at least 8 hours. Washing of the blots
will be conducted once with 2X SSC at room temp for 5 min. and
then with 2X SSC, O.lo SDS at 56 °C which could last for about
5 minutes to an hour depending on the intensity of the
radiactive signal. Radiographic exposure of the blots will
determine whether the genes of interest are present.
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References
Cousin, B, Cinti, S, Morroni, M, Raimbault, S, Ricquier,
D, and Penicaud, L. 1992. Occurrence of brown adipocytes in rat
white adipose tissue: molecular and morphological
characterization. Journal of Cell Science 103, 931-42.
Knapp, JR, Chen, WY, Turner, ND, Byers, FM, and Kopchick,
JJ. 1994. Growth patterns and body composition of transgenic
mice expressing mutated bovine somatotropin genes. Journal of
Animal Science 72, 2812-9.
Viguerie-Bascands, N, Bousquet-Melou, A, Galitzky, J,
Larrouy, D, Ricquier, D, Berlan, M, and Casteilla, L. 1996.
Journal of Clinical Endocrinology and Metabolism 81, 368-75.
All patents or publications cited anywhere in this
specification are hereby incorporated by reference in their
entirety.
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Table A
Human Genes regulated by Growth hormone (GH) and its
antagonist in Brown Adipose Tissue
Gene
DNA
ID
Protein
ID
GHA
NT
GH
1 neuroleukin N/A N/A + 1
gucosephosphate NM000175 NP000166 + 1
isomerase
2 a-enolase X84907 NP001419 + 1
3 pyruvate kinase NM002659 A33983/564635 + 1
4 proteasome 0 chain NM002795 NP002786 + 1
5 heme oxygenase D21243 NP002125/P30519+ 1
6 Ubiquitin/ NM00333 NP003324 + 1
ribosomal fusion
protein
7 trans-Golgi network N/A N/A + 1
38
8 adipocyte lipid NP001442/J NP001433 + l
binding protein 02874
9 medium chain acyl-CoAU07159 N/A + l
dehydrogenase
10 NADH-ubiquonone V00711 P03905/CAA24035 + t
oxidoreductase
11 cytochrome b V00711 AAC28269-88 + i
12 cytochrome c oxidaseV00711 BAA07292 + ?
13 Ng-G119K2 (Novel) + l
14 Ng-G119K15 (Novel) + T
15 Ng-G119K36 (Novel) + 1
2 16 Ng-G119K62 (Novel _ _.
0 ) ~
GHA: present in GH antagonist mouse cDNA substraction library
(forward)
NT: present in nontransgenic mouse cDNA substraction library
(reverse)
GH: presumed regulatory effect of GH on gene expression
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Table B Identification of Homologous Mouse/Rat Genes and
Proteins
Genes DNA Accession Protein Clone
# Accession #s
#
alucosephosphate U89408 & M14220 P06745 44
isomerase & neuroleukin
a-enolase X52379 P17182 27, 141
~;rruvate kinase X97047 CAA65761 12
proteasome theta chainD21800 (rat) P40112 (rat) 19, 59
heme oxyaenase AF029874 2984774 or 128
3169816
Ubiauitin/ribosomal AF118902 511248 30
fusion protein
traps-Golgi network D50031, D50032 68
38
adipocyte lipid bindingK02109 or M13385 lALB or 99, 23
protein P04117
medium chain acyl-CoA U07159 128
dehydrogenase
NADH-ubiquonone V00711 P03925 19
oxidoreductase
cytochrome b V00711 CAA24092 or 18
CAB09443
cytochrome c oxidase V00711 P00397 45
Underlined genes were more strongly expressed in GHA mice, and hence are
down-regulated by GH (negative markers). The remaining genes were more
strongly expressed in normal mice than in GHA mice, and hence are up-
regulated by GH (positive markers).
2 5 DNA and protein #s are for mouse unless otherwise stated.
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Table C
Clone Related Mouse SeguenceIdentities
49 neuroleukin 359/365 (98%)
glucosephosphate 265/275 (960)
isomerase
27 alpha-enolase 520/527 (98%)
359/363 (98%)
5 141 pyruvate kinase 291/303 (96%)
19 & 59 proteasome theta 255/268 (95%)
128 heme oxygenase 311/317 (980)
30 ubiquitin 280/284 (980)
68 trans-golgi network 48/49 (970)
protein 308/329 (93)
10 99 adipocyte lipid 278/286 (970)
binding 211/224 (940)
protein 63/65 (960)
123 same 355/364 (97%)
211/224 (940)
103/105 (980)
127 medium chain acyl-CoA540/542 (99%)
dehydrogenase
19 NADH-ubiquonone 722/726 (990)
oxidoreductase
160 same 130/131 (99%)
15 18 cytochrome b 596/598 (990)
45 cytochrome c 323/334 (960)
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Table D Summary of genes regulated by Growth Hormone (GH) and
GH antagonist (GHA) in Brown Adipose Tissue (BAT)
Message present
in PCR-Select
mouse cDNA subtraction
libraries
Identified Gene GHA(25) NT(14)
f IVR f IVR
Bovine Growth Hormone 14 21.2-
a-enolase/Neuroleukin 5 4.7-257.0
Glucosphosphate Isomerase 1 3.9
Pyruvate Kinase 1 8.4
Ubiquitin/ribosomal 1 11.2
Fusion Protein
Proteasome 8 Chain 2 35. 478.7
Heme Oxygenase 1 31.3
Trans-Golgi Network 1 2.4
Protein (TGN38)
Adipocyte Lipid Binding 2 3.6-11.5
Protein
Medium Chain Scyl-CoA 1 31.1
Dehydrogenase
NADH-ubiquonone 2 7.1-12.3
Oxidoreductase
Cytochrome c Oxidase 2 1.9-5.2
Cytochrome b 2 1.8-2.3
Novel partial cDNAs I4 35.2-
Table D. Summary of genes regulated by Growth Hormone (GH) &
GH antagonist (GHA) in Brown Adipose Tissue (BAT). In the
table, ".f" indicates number of positive clones, whereas "IVR"
is abbreviated from Intensity Volume Ratio estimated from
library screening Dot Blots. "GHA" represents the forward
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47
subtraction library ( subtracting NT mouse BAT cDNA from GHA' s ) .
17 out of 26 sequences were found to be bGH G119K EST. 1 out
of 26 was found to be a contaminant. "NT" represents the
reverse subtraction library (subtracting GHA mouse BAT cDNA
from NT's). 4 out of 14 were determined as novel ESTs after
applying BLAST searches.
Results: Genes encoding glucosephosphate isomearse, a-
enolase, pyruvate kinase, proteasome 8 chain, ubiquitin, and
heme oxygenase were found in the forward subtraction library,
indicating that these genes are up-regulated in GHA mouse BAT.
Genes encoding mitochondria cytochrome b, mitochondria
cytochrome C oxidase subunit I, mitochondria NADH-ubiquinone
oxidoreductase chain 4 and/or 6, medium chain aryl-CoA
dehydrogenase, adipocyte lipid binding protein, and trans-Golgi
network protein (TGN38) were found in the reverse subtraction
library, indicating that these genes are down-regulated in GHA
mouse BAT. All these GH-regulated genes may be used as
potential molecular markers to help explain obesity in GHA
mice.
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Table 1 shows certain of the nucleotide sequences identified
by subtractive hybridization against the mouse brown adipose
tissue library. These are the sequences which appeared to be
identical, or nearly identical, to a databank sequence.
Presumably, the corresponding human genes are similarly
regulated. The sequence names, sequence lengths, and the names
of the most closely related databank sequences, are set forth
below:
(A) G119K-Ng 44 (361 bp)(glucosephosphate isomerase;
neuroleukin)
(B) G119K-Ng27 (550 bp) and G119K-Ng 141 (363 bp)(alpha-
enolase)
(C) G119K-Ngl2 (300 bp)(pyruvate kinase)
(D) G119K-Ngl9 & 59 (299 bp)(proteasome theta chain)
(E) G119K-Ng128 (336 bp)(heme oxygenase)
(F) G119K-Ng30 (303 bp)(ubiquitin)
(G) G119K-Ng68 (345 bp)(trans-Golgi network protein)
(H) G119K-Ng99 (313 bp) and G119K-Ng123 (374 bp)(adipocyte
lipid binding protein)
(I) G119K-Ng127 (542 bp)(medium chain aryl-CoA dehydrogenase)
(J) G119K-Ngl9 (725 bp) and 160 (131 bp)(NADH-ubiquinone
oxireductase)
(K) G119K-Ng45 (343 bp)(cytochrome c oxidase)
For each sequence, the complete clone sequence, and the
highest scoring BLAST alignment, are given.
Table 2 (a) shows the sequences of Ng-G119K42, and (as
boldfaced subsequence) Ng-G119K36. (B) shows ORF.
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The ORF of clone 42 spanning from nucleotide 112 through
1152 is believed to encode a 346 amino acid long polypeptide,
which possesses a predicted cleavage site most likely between
residue Q18 and P19 based on the output of Signal V1.1 World
Wide Web Server (htt~://www.cbs.dlu.dlk/services/SiqnalP/).
The clone 42 protein has a theoretical Molecular Weight
of 37.2751 Kda, theoretical Isoelectric Point of 9.82, 97.05
in aliphatic index, and 0.382 in grand average of
h y d r o p a t h i c i t y , b y P r o t P a r a m t o 0 1
(http://www.expasy.ch/tools/protparam.html).
The secondary structure prediction for clone 42 suggests
an all-helices protein which contains 38.44° for a-helix,
22.54° for extended strand, 12.72° for (3-turn, and 26.300 for
random coil, by the SOPM method (htt~://pbil.ibcp.fr/eai-
bin/npsa sopm.html) [Geourjon, C. & Deleage, G., SOPM: a self
optimised method for protein secondary structure prediction,
Protein Engineering (1994) 7, 157-164]. By the SOPMA method
( h t t p . / / p b i 1 . i b c p . f r / c g i -
bin/npsa automat.pl?page=npsa sopma.html), we get 51.730 for
a-helix, 16.180 for extended strand, 5.490 for (3-turn, and
26.59° for random coil.
The polypeptide encoded by clone 42 may have 6-8
transmembrane (TM) regions, based on the output from "DAS" -
Transmembrane Prediction server (http://www.biokemi.su.se-
server/DAS/). Depended on the criteria applied for prediction,
several other free public servers suggest 6 TM, see TMHMM (v.
0.1) program (http://www.cbs.dtu.dk/services/TMHMM-1.0/), 7 TM
by TopPred 2 program (http://www.biokemi.su.se/
server/to~pred2/toppredServer. cai) , or 8 TM, see Tmpred program
(http://www.ch.embnet.orc~/software/TMPRED form.html).
The Tmpred program makes a prediction of membrane-spanning
regions and their orientation. The algorithm is based on the
statistical analysis of Tmbase, a database of naturally
occurring transmembrane proteins. The prediction is made using
a combination of several weight-matrices for scoring.
No suitable target has been found in searching sequences
of known 3D structures from the SWISS-MODEL Protein Modelling
Server (http:/www.expasy.ch/swissmod/SWISS-MODEL.hlml).
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However, the protein folding encoded by clone 42 is suggested
as being similar to a membrane and cell surface protein and
peptide, such as bacteriorhodopsin ( 0 . 4 65 of SAWTED E-value and
0.364 of PSSM E-value) at low significant level, based on the
5 Program 3D-PSSM (http://www.bmm.icnel.uk/-3dpssm/) which has
a capacity to recognize protein fold using 1D and 3D sequence
profiles coupled with secondary structure information
(Foldfil).
Potential type O-glycosylation sites S186, T163, and T269
10 are predicted from NetOGlyc 2.0 Prediction Server
(http://www.cbs.dlu.dk/services/NclOGlycl) which produces
neural network predictions of mucin type GalNAc O-glycosylation
sites in mammalian proteins. Potential phosphorylations sites
at 571, S127, S182, T50, T54, T59, T306, T322, Y46, and Y183
15 are predicted from the NetPhos WWW server
(http://www.cbs.dlu.dk/services/NetPhos/) which produces neural
network predictions for serine, threomine and tyrosine
phosphorylation sites in eukaryotic proteins.
Results: Predicted secondary structure of clone 42 seems
20 a helix-like polypeptide with a 18-amino acid signal peptide
and relatively low hydrophobicity value. Predicted tertiary
structure of clone 42 is a protein with a 6-8 hydrophobic
regions through the sequence, leading to a folding model
similar to bacteriorhodopsin, which is a important protein for
25 proton conductance in archaebacteria.
Applying the advanced version of Blast search with a
controlled expect value below 0.0001, 12 bits have been found
matchable to the sequence of clone 42 in positive sense strand
at amino acid level. Among them, three sequences from Genebank
30 database share a protein homogy at a very significant level as
listed in this figure: C25077 gene product from female human
infant brain with an expect value 1.0 x10-1'°, PTDO10 gene
product from human pituitary tumor with an expect value 1.0 x
10-19', and CG1287 gene product from drosophila melanogaster
35 with an expect value 4.0 x 10-'3. The C25077 gene product from
female human infant brain possesses a same size of deduced
polypeptide as DERP2. Clone 42-encoded polypeptide shares 860
(2980aa out of 346-aa) identities and 900 (313-as out of 346-
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aa) positives to C25077 gene product, 740 (256-as out of 346-
aa) identities and 770 (268-as out of 346-aa) positives to
PTDO10 gene product, and 400 (139-as out of 346-aa) identities
and 540 (188-as out of 346-aa) positives to CG1287 gene
product.
Results: Since the PTDO10 is missing the C-terminal
portion of sequence which remains intact in C25077 and Clone
42, it suggests that the C-terminal region may be important for
normal biological function.
At the nucleic acid level, applying the advanced version
of BLAST search with a ccntrolled expect value below 0.0001,
many genes have been found matchable beyond the nucleotide
position 1794 in positive sense stand of clone 42. They span
approximately 100-bp. However, a few of matched sequences
exhibited similarity from 5' end through most part of clone 42
with a zero expect value, such as C25077 gene (345-aa) of
female human infant brain, PTDO10 gene product from human
pituitary tumor, and human dermal papilla derived protein 2
gene (DERP2). DERP2 is identical to C25077. Along with their
matched portion, clone 42 shares 90o homology with both
sequences, 946-by out of 1049-by for C25077 or 945-by out of
1049-by for PTDO10.
Table 3 shows the sequences of (a) Ng-G119K58, (b) Ng-G119K65,
(c) Ng-G119K66, and the alignment of EST NG-G119K62 with each
of (a) - (c) . 3 (d) shows the triple alignment of (a) , (b) and
(c). 3(e) shows ORF of greatest interest.
Clones 58, 65, and 66 are 1379-bp, 2437-bp, and 1613-by
long mRNA respectively and have 3 ORFs, 7 ORFs, and 3 ORFs
respectively. 5 out of total 9 OFRs are derived from positive
strand of cDNAs: 1 specific to clone 65, 2 specific to clone
65 and 66, and 2 specific to clone 58, 65 and 66. The sequence
of Reverse 62 completely matches all these three clones at 3'
end which is upstream of the multiple polyadenylational tail
signals (AAUAAA). Additional polyadenylational signal sequence
is unusually seen in 5' end of clone 65, but whole context
suggests it may not functional.
Clone 58, 65, and 66 seem like triple alternative splice
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forms which are 1379-bp, 2436-bp, 1612-by mRNAs respectively,
which support the prediction by sequence analyses of these
clones. Because multiple bands were observed under prolonged
exposure, other unknown spliced forms may exist in this gene
family. Messages from ORF region encoded multiple sequences
and clone 65 itself seem negatively regulated by GH at
transcriptional level in mouse BAT.
Applying the advanced version of Blast search with a
controlled expect value below 0.000.1, many genes have been
found matchable beyond the nucleotide position 1203 for clone
58, 1856 for clone 65, and 1437 for clone 66 in antisense
strand for these cDNAs. However, in positive sense strand,
only human DNA sequence from clone 41562 on chromosome has been
matched to clone 58 with an expect value 3.0 x 10-J'', to clone
65 with an expect value 6.0 x10-J°, and to clone 66 with an
expect value 4.0 X 10-5° as indicated in A and B. These
alignment searches suggest that all these three isoforms share
a common open reading frame which encodes 86 amino acids.
Table 4 shows the sequences of (a) Ng-G119K2, first strand and
second strand and (b) Ng-G119K15 first strand and second
strand.
Applying the advanced version of BLAST search with a
controlled expect value below 0.0001, the polypeptide sequence
of clone 58 shares 490 (42-as out of 86-aa) identities, 770
(66-as out of 86-aa) positives with CG6115 gene product of
Drosophila melanogaster with an expect value 1.0 X 10-16 for
clone 58 and 2.0 X 10-16 for both clone 65 and 66, though there
is no similarity at nucleic acid sequence level. This output
further supports the prediction of ORF. Additionally, DNA
sequence homology only occurs between human and mice, not
between mice and fruit fly, suggesting a evolutionary role
within these species.
Table 5 shows the sequence of Ng62D'4-2-1-4 cDNA.
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Table 1
mouse ctlucosephosphate isomerase & neuroleukin
partial (+) strand cDNA Open Reading Frame
G119K-Ng 44 sequence (361 bp):
CTCTTTATAATCGCCTCCAAGACCTTCACCACCCAGGAGACCATCACCAATGCAGAGACA
GCAAAGGATGGTTTCTCGAAGCGGCCAAGATCCATCTGCAGTTGCAAAGCACTTTGTCGC
CCTGTCTACGAACACGGCCAAAGTGAAAGAGTTTGGAATTGACCCTCAAAACATGTTCGA
GTTCTGGGATTGGGTAGGTGGCCTATTCGCTGTGGTCAGCCATTGGACTTTCCATTGCTC
TGTATGTAGGTTTTGACCACTTCGAGCAGCTGCTGTCCGGGGCTCACTGGATGGACCTGC
ACTTCCTCAAGACGCCCCTGGAGAAGAATGCCCCCGTCCTGCTGGCTCTACTGGGCATCT
G
Sequence 1 1c11G119k-Ng 94 Length 361 from:l to = 361
Sequence 2 gi 200064 Mouse neuroleukin mRNA, complete cds.
Length 1985 from: 1 to +1985
NOTE: The statistics (bitscore and expect value) is calculated based on
the size of nr database
Score = 639 bits (332), Expect = 0.0
Identities = 359/365 (98o), Positives = 359/365 (980), Gaps = 4/365
(1°)
Aligned query 1-361 to subject 665-1029.
2 0 Sequence 1 G119K-Ng 44 Length 361 from:l to = 361
Sequence 2 gi 3642648 Mus musculus strain BALB/c glucosc-6-phosphate
isomerase mRNA, partial cds.
Length 477 from:l to = 477
Score = 462 bits (240), Expect = e-128
2 5 Identities = 265/275 (96o), Positives = 265/275 (960), Gaps = 2/275 (0o)
Aligned query 89-361 to subject 1-275.
2. mouse a-enolase
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partial (+) strand cDNA Open Reading Frame
G119K-Ng 27 sequence (550 bp)
ACGCGGAAGCAGTGGTAACAACGCAGAGTACACCGCAAAAGGTCTCTTCCG
AGCTGCGGTGCCCAGCGGTGCGTCCACTGGCATCTACGAGGCCTAGAACTC
CGAGACAATGATAAGACCCGCTTCATGGGGGAAGGGTGTCTCACAGGCTGTT
GAGCACATCAATAAAACTATTGCGCCTGCTCTGGTTAGCAAGAAAGTGAATG
TTGTGGAGCAAGAGAAGATTGACAAGCTGATGATCGAGATGGACGGCACAGA
GAATAAATCTAAATTTGGTGCAAATGCCATCCTGGGAGTGTCCCTGGCTGTC
TGCAAAGCTGGTGCCGTGGAAAAGGGGTGCCCCTTTACCGCCACATTGCTGA
CTTGGCCGGCAACCCTGAAGTCATCCTGCCTGTCCCGGCTTTCAATGTGATC
AACGGTGGTTCTCATGCTGGCAACAAAGCTGGCCATGCAAAGAGTTCATGAT
CCTGCCTGTGGGGCATCCAGCTCCGGGAAGCCATGCGCATTGGAGCAGAGGT
TTACCACAACCTGAAGAACGTGATCAAGGAGA
Sequence 1 G119K-Ng 27 Length 550 from:l to = 550
Sequence 2 gi 55490 Mouse mRNA for alpha-enolase (2-phospho-D-
glycerate hydrolase) (EC 4.2.1.11)
Length 1720 from:l to = 1720
Score = 906 bits (471), Expect = 0.0
Identities = 520/527 (98°), Positives = 520/527 (98°), Gaps
= 7/527 (1s)
2 0 Aligned query 28-550 to subject 167-690.
G119K-Ng 141 sequence (363 bp)
ACAAGTCCTTCGTCCAGAACTACCCAGTGGTGTCCATCGAAGATCCCTTTGA
CCAGGACGACTGGGGCGCCTGGCAGAAGTTCACGGCTAGTGCGGGCATCCAG
GTGGTGGGCGATGACCTCACAGTGACCAACCCTAAGCGGATTGCCAAGGCTG
CGAGCGAGAAGTCCTGCAACTGCCTCTTGCTCAAAGTGAACCAGATCGGCTC
TGTGACCGAATCCCTGCAGGCGTGTAAGCTGGCCCAATCCAATGGCTGGGGT
GTCATGGTGTCCCACCGATCTGGGGAAACTGAGGACACTTTCATCGCAGACC
TGGTGGTGGGGCTCTGCACTGGGCAGATCAAGACTGGTGCCCCTTGCCGAT
Sequence 1 G1119K-Ng 141 Length 363 from:l to = 363
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Sequence 2 gi 55490 Mouse mRNA for alpha-enolase (2-phospho-D-
glycerate hydrolase) (EC 4.2.1.11)
Length 1720 from:l to = 1720
Score = 675 bits (351), Expect = 0.0
5 Identities = 359/363 (980), Positives = 359/363 (980)
Aligned query 1-360 to subject 934-1293.
3. mouse pyruvate kinase
partial (+) strand cDNA Open Reading Frame
G119K-Ng 12 sequence (300 bp):
10 CATGCAGAGACCATCAAGAATGTCCGTGAAGCCACAGAAAGCTTTGCATCTG
ATCCCATTCTCTACCGTCCTGTTGTGGTGGCTCTGGATACAAAGGGACCTGA
GATCCGGACTGGACTCATCAAGGGCAGCGGCACCGCTGAGGTGGAGCTGAAG
AAGGGAGCCACTCTGAAGATCACCCTGGACAAGCTTACATGGAGAAGTGTGA
CGAGAACATCCTGTGGCTGGACTACAGACATCTGCAAGGTGTGAGTGGCAGC
15 AAGATCTACGTGGACGATGGCTCATCTCACTGCAGTGAAG
Sequence 1 G119K-Ng 12 Length 300 from:l to = 300
Sequence 2 gi 1405932 M.musculus mRNA from M2-type pyruvate kinase
Length 2134 from:l to = 2134
Score = 465 (242), Expect = e-129
2 0 Identities = 291/303 (960), Positives = 291/303 (960), Gaps = 8/303 (2%)
Aligned query 1-295 to subject 256-558.
4. mouse proteasome 8 chain
partial (+) strand cDNA Open Reading Frame
G119K-Ng 19 & 59 sequence (299 bp):
25 GCGGGGACTCCAGCGCAATCATGTCTATTATGTCCTATAATGGAGGGGCCGT
CATGGCATGAAGGGAAAGAACTGTGTGGCCATCGCTGCAGACAGACGTTTCG
GGATCCAGGCCCAGATGGTGACCACGGACTTCCAGAAGATCTTTCCCATGGG
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TGACAGGCTCTACATAGGCCTGGCCGCCTGGCCACTGACGTCCAGACAGTTG
CCCAGCGTCTCAAGTTCCGACTGAACTTGTATGAGCTGAAGAAGGTCGACAG
ATCAGCCTTACACCTACTGAGACTGGTGGCACTCTGTAT
Sequence 1 G119K-Ng 19 & 59 Length 299 from:l to = 299
Sequence 2 gi 958730 Rat mRNA for proteasome subunit RC10-II, complete
cds.
Length 828 from:l to = 828
Score = 402 bits (209), Expect = e-110
Identities = 255/268 (95%), Positives = 255/268 (95°), Gaps =
4/268 (lo)
1 0 Aligned query 11-274 to subject 68-335.
5. mouse heme oxycrenase
partial (+) strand cDNA Open Reading Frame
G119K-Ng 128 sequence (336 bp):
TCCAGTTGTCAAGACTTCTTGAAGGAAACATTAAGAAGGAGCTATTTAAGAT
GGCACCACTGCACTTTACTTCACATACTCAGCCCTTGAGAGGAATGGACCGC
AACAAGGACACCCAGCCTTCGCCCCCTTATATTTCCCCACGGAGCTACACCG
GAAGGCAGCACTGATCAAGGACATGAAGTATTTCTTTGGTGAAAACTGGGAG
GAGCAGGTGAAGTGCTCTGAGGCTGCCCAGAAGTATGTGGATCGGATTCACT
ATGTAGGGCAAAATGAGCCAGAGCTGCTGGTGGCCCATGCTTATACTCGTTA
CATGGGGGACTTTCAGGGGGTTAG
Sequence 1 G119K-Ng 128 Length 336 from:l to = 336
Sequence 2 gi 2984773 Length 1255 from:l to = 1255
Score = 527 bits (274), Expect = e-148
Identities = 311/317 (980), Positives = 311/317 (980), Gaps = 5/317 (1%)
2 5 Aligned query 22-303 to subject 1-284.
6. mouse ubiquitin
partial (+) strand cDNA Open Reading Frame
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G119R-Ng 30 sequence (303 bp):
GGGCTTTCTCTTCAACGAGGCGGCCGAGCGGCAGACGCCAACATGCAGATCT
TCGTGAAGACCTGACGGGCAAGACCATCACTCTTGAGGTCGAGCCCAGTGAC
ACCATCGAGAATGTCAAGGCCAAGATCCAAGACAAGGAAGGCATCCCACCTG
ACCAGCAGAGGCTGATATTCGCGGGCAAACAGCTGGAGGATGGCCGCACCCT
GTCCGACTACAACATCCAGAAAGAGTCCACCTTCGACCTGGTGCTGCGTCTG
CGCGGTGGCATCATTGAGCCATCCTTCGTCAGCTTGCCCAGAA
Sequence 1 G119K-Ng 30 Length 303 from:l to = 303
Sequence 2 gi 4262554 Length 500 from:l to = 500
1 0 Score = 509 bits (262), Expect = e-141
Identities = 280/284 (98~), Positives = 280/284 (980), Gaps = 2/284 (0o)
Aligned query 22-303 to subject 1-284.
7. mouse Trans-Golg~i Network protein (TF GN38)
partial (+) strand cDNA
Ng-G119K 68 sequence (345 bp):
TAGCATAAAAGGGACTCGAGGTTTCTGAAAGTAAAATCACTGTTTGATGGGA
TTTTTTAAAAAAATGATCATTGAACAAGTGTGTTCTTGCATACATTCACCCC
AATAAGGGCTTCCTGGAAAGGGACAGGTTCATGCTTTGTGGAAGAAAACACA
TAGGAGGGATTTAGTATGCAGGAAAGAGGTTTTCTACAAATTGAGTTTTGCT
TTTATTGCCCGCAGTAGATAGATATTTAGAAACTAACTGCATTCTTCACACT
CCTCCTTGCTGTTTAAGATGTGCAGGATAGGAAATCTTCCTATCCTGTCATA
TCTGGTCATGAACTGTAGAACTAATAGTCCTGA
Sequence 1 Ng-G119K 68 Length 345 from:l to = 345
Sequence 2 gi 949828 Mouse mRNA for TGN38A, complete cds.
Length 1673 from:l to = 1673
Score = 89.1 bits (46), Expect = 3e-16
Identities = 48/49 (970), Positives = 48/49 (97s)
Aligned query 1-49 to subject 1625-1673.
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Sequence 1 Ng-G119K 68 Length 345 from:l to = 345
Sequence 2 gi 949830 Mouse mRNA for TGN38B, complete cds.
Length 2265 from:l to = 2265
Score = 487 bits (253), Expect = e-136
Identities = 308/329 (930), Positives = 308/329 (930), Gaps = 6/329 (lo)
Aligned query 1-324 to subject 1656-1983.
8. mouse adipocyte lipid bindincLprotein
partial (+) strand cDNA Open Reading Frames
Ng-G119K 99 sequence (313 bp):
1o GCAGAAGTGGGATGGAAAGTCGACCACAATAAAGAGAAAACGAGATGGTGAC
AAGCTGGTGGTGGAATGTGTTATGAAAGGCGTGACTTCCACAAGAGTTTATG
AAAGGGCATGAGCCAAAGGAAGAGGCCTGGATGGAAATTTGCATCAAACACT
ACAATAGTCAGTCGGATTTATTGTTTTTTTTAAAGATATGATTTTCCACTAA
TAAGCAAGCAATTAATTTTTTCTGAAGATGCATTTTATTGGATATGGTTATG
TTGATTAAATAAAACCTTTTTAGACTCAAA.AAA GG
T
Sequence 1 Ng-G119K 99 Length 313 from:l to = 313
Sequence 2 gi 198716 Mouse 3T3-Ll lipid binding protein mRNA, complete
cds.
2 0 Length 614 from:l to = 614
Score = 519 bits (270), Expect = e-146
Identities = 278/286 (970), Positives = 278/286 (970)
Aligned query 1-286 to subject 328-613.
Sequence 1 G119K-Ng 99 Length 313 from:l to = 313
2 5 Sequence 2 gi 198718 Mouse adipocyte lipid binding protein gene,
complete cds.
Length 5212 from:l to = 5212
Score = 333 bits (173), Expect = 9e-90
Identities = 211/224 (940), Positives = 211/224 (94%), Gaps = 4/224 (lo)
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Aligned query 64-286 to subject 4490-4710.
Score = 114 bits (59), Expect = 8e-24
Identities = 63/65 (960), Positives = 63/65 (960)
Aligned query 1-65 to subject 3760-3824.
Ng-G119K 123 sequence (374 bp):
GAATTCGATGAAATCACCGCAGACGACAGGAAGGTGAAGAGCATCATAACCC
TAGATGGCGGGGCCCTGGTGCAGGTGCAGAAGTGGGATGGAAAGTCGACCAC
AATAAAGAGAAAACGAGATGGTGACAAGCTGGTGGTGGAATGTGTTATGAAA
GGCGTGACTTCCACAAGAGTTTATGAAAGGGCATGAGCCAAAGGAAGAGGCC
l0 TGGATGGAAATTTGCATCAAACACTACAATAGTCAGTCGGATTTATTGTTTT
TTTTTAAAGATATGATTTTCCACTAATAAGCAAGCAATTAATTTTTTCTGAA
GATGCATTTTATTGGATATGGTTATGTTGATTAAATAAAACCTTTTTAGACT
CAAAAAAAAA
Sequence 1 Ng-G119K 123 Length 379 from:l to = 374
Sequence 2 gi 198716 Mouse 3T3-Ll lipid binding protein mRNA, complete
cds.
Length 614 from:l to = 619
Score = 654 bits (340), Expect = 0.0
Identities = 355/364 (97°s), Positives = 355/364 (97°),
Gaps = 1/364 (0~)
2 0 Aligned query 1-364 to subject 251-613.
Sequence 1 Ng-G119K 123 Length 374 from:l to = 379
Sequence 2 gi 198718 Mouse adipocytc lipid protein gene, complete cds.
Length 5212 from:l to = 5212
Score = 344 bits (179), Expect = 4e-93
2 5 Identities = 211/224 (940), Positives = 211/224 (94o), Gaps = 3/224 (1%)
Aligned query 141-364 to subject 4490-4710.
Score = 191 bits (99), Expect = 7e-47
Identities = 103/105 (98a), Positives = 103/105 (98%)
Aligned query 38-142 to subject 3720-3824.
3 0 Score = 75.7 bits (39), Expect = 4e-12
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Identities = 39/39 (100x), Positives = 39/39 (1000)
Aligned query 1-39 to subject 3097-3135.
9. mouse medium chain acyl-CoA dehydrogenase
partial (+) strand cDNA
s Ng-G119K 127 sequence (542 bp):
GAGGCTGATCATAGCTCGTGAGCACATTGAAAAGTATAAAAATTAACAGGA
ATTACTATTGAACGATGCATCACCCTCGTGTAACTAAGCTCCAAGCACTGT
TGCTGCTTCAGGGGAAAAGGGCTTTACTGTCTTCCCAAGGAAATGAGATCAA
AGACGAGTTTGGATCTGTGCAGCGGATTCCCATGGCGGAGGAACCTGTCTTC
10 AGCTCTATGGTGACCCTTTCTAGATAGGTTTGGCTTTTGGACAATGATTGGT
CCTTAGCCCCGAATTGTGTTAGTTTGCTCTTTGATCACTTAAAATGGAAAAA
CACCCTGGACTTTTAATGTTCATTCAAGTGACAGGAAAGGCGGCTTGTCAAG
GAAGAACTCATGATTCTAACATAAACACTGAAAATTTGTGGTAGATTGGACA
CGTCAGACTGTGACATAGCAGCATTTCTGTGCTGAACTGTTAATTTTATAAT
15 TTTGATTATATTTGCTTTGTTTTGCACAAAAGAGTAAAAAGTTTATATTCAC
ATTCTCCCATTATAAAACTAAAAC
Sequence 1 Ng-G119K 127 Length 542 from:l to = 542
Sequence 2 gi 463908 Mus musculus medium-chain acyl-CoA dehydrogenase
mRNA, complete cds.
2 0 Length 1846 from:l to = 1846
Score = 1021 bits (531), Expect = 0.0
Identities = 540/542 (99%), Positives = 540/542 (990), Gaps = 1/542 (0%)
Aligned query 1-542 to subject 1228-1768.
10. mouse NADH-ubiquonone oxidoreductase
25 partial (+) strand cDNA Open Reading Frame from
mitochondria genome
Ng-G119K 19 sequence (725 bp): chain 4
GATCCGTTCGTAGTTGGAGTTTGCTAGGCAGAATAGGAGTGATGATGTGAG
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GCCATGTGCGATTATTAGTATTGTTGCTCCTATGAAGCTTCATGGAGTTTG
GATTATGATTGATGCAATAACAAGTGCTATGTGGCTAACTGAGGAGTAGGC
GATTAGTGATTTTAAATCTGTTTGGCGTACAGAGATTGAGCTAGTTATAAT
TATTCCTCATAGGGAGAGAAGGATGAAGGGGTATGCTATATATTTTGTTAG
TGGGTCTAGAATAATGGAGATGCGAATTATTCCGTAACTACCTAATTTTAG
AAGAATAGCTGCTAGAATTATTGACCCAGCAATTGGAGCTTCAACATGGGCT
TTTGGTAGTCATAGGTGAACTCCATATAATGGTATTTTAATAAGAAATGCTA
TTATGCATGCCAACCATAGTAAGTTGTTAGATCATGAAGCGTCTAAGGTGTG
TGTTGTGAATGATAAAATTATGAGGTTTAGGGTTCCTACATGGTTTTGGATT
AAGATGAGGGCAATTAGCAGTGGAATAGAACCGATTAGGGTATAAAATAGGA
AATAAATCCCTGCGTTTAGGCGTTCAGTTTGGTTCCTCATCGGGTAATAATA
ATAAGTGTTGGGATTAAGGTTGCTTCAAATAAAATATAAAATATAATTAGTT
CAGTTGCTGAAAAGGTTATGATTAGGAGAATTTGTAAGCTGATTAGTATTGA
GAT
Sequence 1 Ng-G119K 19 Length 725 from:l to = 725
Sequence 2 gi 13838 Mus musculus mitochondrial genome
Length 16295 from:l to = 16295
Score = 1363 bits (709), Expect = 0.0
Identities = 722/726 (990), Positives = 722/726 (990), Gaps = 1/726 (0o)
2 0 Aligned query 1-725 to subject 11171-10446.
Ng-G119R 160 sequence (131 bp): chain 6
ATACTCAATTAATCTCGAGTAATCTCGATAATAATAAAAGATACCCGCAAAC
AAAGATCACCCAGCTACTACCATCATTCAAGTAGCACAACTATATATTGCCG
CTACCCCAATCCCTCCTTCCAACATAA
2 5 Sequence 1 Ng-G119K 160 Length 131 from:l to = 131
Sequence 2 gi 13838 Mus musculus mitochondrial genome.
Length 16295 from:l to = 16295
Score = 237 bits (123), Expect = 3e-61
Identities = 130/131 (990), Positives = 130/131 (990), Gaps = 1/131 (0o)
3 0 Aligned query 1-131 to subject 13538-13667.
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11. mouse cytochrome b
partial (+) strand cDNA Open Reading Frame from
mitochondria genome
Ng-G119K 18 sequence ( 5 9 9 by )
GAGTCATAGCCACAGCATTTATAGGCTACGTCCTTCCATGAGGACAAATAT
CATTCTGAGGTGCCACAGTTATTACAAACCTCCTATCAGCCATCCCATATA
TTGGAACAACCCTAGTCGAATGAATTTCAGGGGGCTTCTCAGTAGACAAAG
CCACCTTGACCCGATTCTTCGCTTTCCACTTCATCTTACCATTTATTATCGC
GGCCCTAGCAATCGTTCACCTCCTCTTCCTCCACGAAACAGGATCAAACAAC
l0 CCAACAGGATTAAACTCAGATGCAGATAAAATTCCATTTCACCCCTACTATA
CAATCAAAGATATCCTAGGTATCCTAATCATATTCTTAATTCTCATAACCCT
AGTATTATTTTTCCCAGACATACTAGGAGACCCAGACAACTACATACCAGCT
AATCCACTAAACACCCCACCCCATATTAAACCCGAATGATATTTCCTATTTG
CATACGCCATTCTACGCTCAATCCCCAATAAACTAGGAGGTGTCCTAGCCTT
AATCTTATCTATCCTAATTTTAGTCCTAATACCTTTCCTTCATACCTCAAAG
CAACGAAGCCTAATATTCCGCCCAATCACA
Sequence 1 Ng-G119K 18 Length 599 from:l to = 599
Sequence 2 gi 13838 Mus musculus mitochondrial genome.
Length 16295 from:l to = 16295
2 0 Score = 1138 bits (592), Expect = 0.0
Identities = 596/598 (990), Positives = 596/598 (990)
Aligned query 2-599 to subject 14504-15101.
12. mouse cytochrome c oxidase
partial (+) strand cDNA Open Reading Frame from
mitochondria genome
Ng-G119K 45 sequence (343 bp):
TCCAGCTATACTATGAGCCTTAGGCTTATTTCTATTACAGTTGGTGGTCTAA
CCGGAATGTTTATCCAACTCATCCCTTGACATCGTGCTTCACGATAACATAC
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TATGTAGTAGCCCATTTCCACTATGTTCTATCAATGGGAGCAGTGTTTGCTA
TCATAGCAGGATTTGTTCACTGATTCCCATTATTTTCAGGCTTCACCCTAGA
TGACACATGAGCAAAAGCCCACTTCGCCATCATATTCGTAGGAGTAAACATA
ACATTCTTCCCTCAACATTTCCTGGGCTTTCAGGAATACCACGACGCTACTC
AGACTACCCAGATGC AAAAAAA
Sequence 1 lcllseq 1 Length 343 from:l to = 343
Sequence 2 gi 13838 Mus musculus mitochondrial genome.
Length 16295 from:l to = 16295
Score = 531 bits (276), Expect = e-149
Identities = 323/334 (960), Positives = 323/339 (96s), Gaps = 8/334 (20)
Aligned query 1-327 to subject 6332-6664.
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Table 2A
mouse Brown Adipose Tissue Reverse (NT-GAA) 36 cDNA
(EST) ; 463 BP. (in bold)
mouse Brown Adipose Tissue Clone 42 cDNA (full-length);
2475 BP.
Start Codon:
112 (underlined)
Stop Codon : 1152
Open Reading
Frame: 396-as
GAGAGGGAGG TCGCACACTC TGAGTTTCGG TGACCCGGAA GGAGCCCCGT
GGTAGAGGTG ACCGGAGCTG AGCATTTCAG ATCTGCTTAG TAAACCGGTG
TATCGCCCAC CATGTTGGCT GCAAGGCTTG TGTGTCTCCG GACACTACCT
TCCAGGGTTT TCCAGCCCAC TTTCATCACC AAGGCCTCTC CACTTGTGAA
GAATTCCATC ACAAAGAACC AATGGCTCGT AACACCCAGC AGGGAATATG
CTACCAAGAC AAGAATTAGG ACTCACCGTG GGAAAACTGG ACAAGAACTG
AAAGAGGCAG CCAAGGAACC ATCAATGGAA AAAATCTTTA AAATCGATCA
AATGGGAAGG TGGTTTGTTG CTGGAGGAGC AGCTGTTGGT CTTGGAGCGC
TCTGCTACTA TGGCTTGGGA ATGTCTAATG AGATTGGAGC TATCGAAAAG
GCTGTAATTT GGCCTCAGTA TGTAAAGGAT AGAATTCATT CTACTTACAT
GTACTTAGCA GGAAGTATTG GTTTAACAGC TTTGTCTGCC TTGGCAGTAG
CCAGAACACC TGCTCTCATG AACTTCATGA TGACAGGCTC TTGGGTGACA
ATTGGTGCGA CCTTTGCAGC CATGATTGGA GCTGGAATGC TTGTACACTC
AATATCATAT GAGCAGAGCC CAGGCCCAAA GCATCTGGCT TGGATGCTGC
ATTCTGGTGT GATGGGTGCA GTTGTGGCTC CTCTGACGAT CTTAGGGGGG
CCTCTTCTCC TGAGAGCCGC ATGGTACACC GCTGGTATTG TGGGAGGCCT
CTCTACTGTG GCCATGTGTG CGCCTAGTGA GAAGTTTCTG AACATGGGAG
CACCCCTGGG AGTGGGCCTG GGTCTTGTCT TTGCGTCTTC TCTGGGGTCT
ATGTTTCTTC CCCCTACCTC TGTGGCTGGT GCCACTCTGT ACTCAGTGGC
AATGTATGGT GGATTAGTTC TTTTCAGCAT GTTCCTTCTG TATGATACTC
AGAAAGTAAT CAAACGTGCA GAAATAACAC CCATGTATGG AGCTCAAAAG
TATGATCCCA TCAATTCGAT GTTGACAATC TACATGGATA CATTAAATAT
ATTTATGCGA GTTGCAACTA TGCTAGCAAC TGGAAGCAAC AGAAAGAAAT
GAAGTAACCG CTTGTGATGT CTCCGCTCAC TGATGTCTTG CTTGTTTAAT
AGGAGCAGAT AGTCATTACA GTTTGCATCA GCAGAATTCC TTGAGGTTTA
GAAGATAGCC TGTCACCATG TTTAAAATGT GCAGTAATGC GACCCTTCAG
GCATGCCTTT TCTTTTAGAA AATAAATGCA ATAGATGTCT TCCAAATATA
TTTTCATCTC TTATGCTTTC ATACTTTAAA ACTGCTTTGA TGAATGTGTG
AACAAATATA TTTTAGAAGA TTTCAAGTAT TGTTTTATGT ATTGGATAAG
TAAAATTTAG CAAATTTGCG TGTCTTCATA TTGTGGAAGC CTGCAGAATA
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TTTCAGTGGC ATCATGAGTG ACAAGTTTTT TGTATAGAGGTCAGAGAGAT
AAAAGGCACC TGCAGTCAGT TTGAATGCCC AGGACAACACTGATTGTGGT
GAGCCAGTGA AAGACATCAG AGATGTGGAA CAAGGGACCACCAAATGTGG
GGTTAACAAA GACACGGATG TTTCTTCTGT GCTCTTAATGTCCTTGAGGT
5 TGACTGCTCA TTGTCAGGAC AGTCCAGAGT GTTAACCATACAGAGAATCT
CTGCTGGAAT TATGTCTGTG TTTTACTATG AAGTCTTTAGAACAAGCAGG
TTGGTGGTGG CGCACACCTT TAGTCCCATC ATCTGGGAGGCAGAGGCAAG
CAGATCTCTA AATTCAAGGC CAGCCAGGTC TACAAAGTGAGTTCCAAGCC
AGACAAGGAC CTGTCTCTAA TACAAGCAAA CAAACAACAACAAACACTAC
10 CGCTATGCTC GGTATGATGT ACTACTCCAA AGCTCAAGACTCCTTTGCTG
TCAGATGTGT GGTGTATATG CAGTTGGACA GGATTTAGGTTTTGGTTTTT
GGTTTTGTTT TATTTTGATA TTTTTCTCAG TGTCTAATTGAAAGCATGCT
TGCTTTCTCA TCACAGCTTT GACAGCTGTC AGAAAAGCCTCTTTGTGGCT
TATGCTAAGA TTAGGATTGG TTTTTCTTCT AAAACTGTTGGCTTCCTCCG
15 TTCCCTCTCA GCTTAAGCAT GAACAAAGCA AATTTAGTTGACCTTGGGAA
GTATTTGAAT GAAAACTGGA ATGGGGAGGT GCTCAGCTTCCTTGTGACAT
AAGATTTTAA TACAGATCAC TTGTTTGTGG TGAGGGGTTCTTCATTGAAG
TCTGTATGTA TTTGCAAAAT AACTATTTTT GAGAAGTATTTATTACAGTA
ATCCATAAGT AATTCTTTTA ATCACTTTAA AGTACACTGAATGCTAATTT
20 CTGAAATAAA AGTTTCAGCT AAGTG
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Table 2B
Open Reading Frame Sequence (346-aa) of Clone 42:
Codes (~-~l~x, Str.jn_i, no prediction)
MLAARLVCLRTLPSRVFQPTF'ITKASPLVKNSITKNQWLVTPSREYATKT~;IRTH
RGKTGQELKEAALEPSMEKIFKIDL~MGRWFVAGGAAVGLGALCYYGLGMSNEIGA
IEKAVIWPQYVKDRIHST YMYLAGSIGLTAL,SALAVARTPALMNFMMTGSWVTIG
ATFAAMIGAGMLVHSISYEQSPGPKHL:'~WMLHSGVMGAWAPLTILGGPLLLR.~A
WY TAGIVGGLS TVP.MCAPSEKF LNMGAPLGVGLGLVFASSLGSMFLPPTSVAGAT
LYSVAMYGGLVLFSMFLLYDT~;KVIKRAEITPMYGAQKYDPINSMLTIYMDTLNI
FMRVATMLATGSNRKK
//
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Table 3A.
mouse Brown Tissue Reverse
Adipose (NT-GHA)
62
cDNA (EST) (in bold)
; 735 BP.
mouse Brown Adipose lone 58
Tissue cDNA
C
(full-length);
1379 BP.
Start Codon: 151 (underlined )
Stop Codon: (underlined )
411
Open Reading
Frame:
86-as
GACTTCCGGC AGACGGTCGG AGCATTTACG GCCGTGGTGC CGCAAAGGCC
TGGAGTGAGG CGGTCTGAGC AAGCTGTCGT CTGGACCCCA GACCTGCTGG
TGGTGAAGTA TATCATGTAT AAAAGTGGAT CAATTCCATG TTAAGTGAAA
ATGGCCAATT CGTTACGAGG AGAAGTACTG ACTCTTTATA AAAATCTGCT
GTATCTTGGA CGGGACTATC CAAAAGGAGC AGACTATTTT AAAAGGCGTT
TGAAGAACGT TTTCC'rTAAAAACAAGGATG TGGAGGACCC AGAGAAGATC
AAAGAACTTA TCGCACGAGG AGAATTTGTA ATGAAGGAGC TAGAGGCCTT
GTACTTCCTT AGGAAATACA GAGCTATGAA GCAACGTTAC TATTCAGATA
CCAAAGTCTG ACCAATCATT GCACCAGTCG AGCTGACAAC CAGTGCTGGC
TGTTTGCCTG TACCAACTAT TAAAAAATAA TTCAGTTTAA AAGGGTGAGA
TACATGGTTT TTAAAAAAAT GAGTTGCCCT ACTGTACTGA AATAGGTTTC
AACCTTATTG ATACTGAGAG CTTTGCCCAT AATCCTTTTA TTACTGAAAT
AGTAACTTTA GTACCTTTCA TGATAATATA ATTTTGAAAG AAAATACACT
TAATTTTTAA ACATGTTATA GCCAATTTTC TTAAGTCTAT TTCTTCATTT
ACTGATGAGA TTGTCACTAT CGAATGGTGT CTGACAGGCT TGCCCTTTAG
CTTCTAGAGT GTCTTTGTCC TTGTTTTTTG TTGTTTTGTT AGCCCATCTA
GTATACTAAA GTGCATATTC AAGGCTCTCT ACAGACACCT CAAAATGATT
TAAATGCAGT TATCAAAATA AGACATGTGA AGGTGACCTC TATCTTGAGA
AGCTCAGTGG GTGACTAGCA TTGTGTAGCT ATTATTCCCA TTATTCTTTG
TGCTGCTGGC CTGCCTTAAG TTCTGAACCA CTTCAAGTAG CTTTCATGAG
GAGTTGTAAT GTTCCTCTAT TTCTGCCATT AAAGCTGGTA TATTTTCTGT
CGACCTGTAA CCGAGTCCAT GTGGCAGTGG ACCTAACCCA GGCAGGACTG
TAAGTTTAAG CAAAAATGTT TATGTAATGT TTTTAGCAAC GTTATAAATA
ACATTTCTAA CTTAAAAGCT GCAAATAGTG TTGCTTATAG GATTCTGTAT
CAGGCTGGAG AGATGGCTCA GTGGTTAAGA GCACTGACTG CTCTTCCAGA
GGTCCTGAAT TTAATTCCCA GCAACCATAT GGTGGCTTAC AACCATCTGT
AATGGGATCT GATGTCCACT TCTGGTGTGT CTGAACACAG ACAGTGTACT
CATAGAATAA ATAAATAAAC GAATAAATC
//
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Table 3B
mouse Brown Adipose Tissue Reverse (NT-GHA) 62
cDNA (EST) ; 735 BP. (in bold)
mouse Brown Adipose Tissue Clone 65 cDNA
full-length); 2436 BP.
Start Codon: 804 (underlined)
Stop Codon: 1064 (underlined)
Open Reading Frame: 86-as
GAGACGGTCG GAGCATTTAC GGCCGTGGTG CCGCAAAGCG CTGGAGTGAG
10GCGGTCTGAG CAAGCTGTCG TCTGGACCCC AGACCTGCTG GTGGTGAACT
AAAGCACCGA GTCAAAAGCA TGGTCAGCAG CATGGATGCT GTCTGCTCTG
CCTCCCGTGG AACCTTTCCA AGTGCTCCCT TTGCCCGCTG CCTCTTACTC
TGCATTCTCC TTAAGGACCA ACCTTCTTGA TCTTGATCGA ACAACCCAAT
TTATCTTAGT TTTAAAATTT CCTCCAAGAA TACTCTTCTA GATTTGGGCT
15CTTAGTTTCT TCCAAATAAT CAAGCCAAGC CTTGAGAGCA GGGCAGACAG
CTTTACTTTT GGTAAGGAAA GCAGGCTTAG AAAAGTGGTG TTACCCAGTG
CCTCAATAAA ACAGCTCAGT ACAAATAACC ATTTGGGGGG ATAAGAAGTC
TTAATGGCAA AGCACTTGCA CAAACAAGAG GGTCCTGTAG ACCTGCAAGT
TTGTAATCCC AGTGTACATA CAGGGGGGTG AGAGGTAGGA GAATCCCTAA
20ATGAAGGAAG GGCCAGCTGT TTGCAGCAAC AACTAAGACC CGTGGAAAGG
ACTGACAGCT GAGGTCATCA GCTCCAAATG CACACTGGCA AGTACAAGTC
TGTACACAAG AATGAAAAGC CAGCTCACCA GCTCCATGGG AAGATCTCTG
GTTCTTTAAG ATTTACAATG CAGTTATTTG CAAAAAAAAG AAAATCTTCC
TTTTCTTTAG GTATATCATG TATAAAAGTG GATCAATTCC ATGTTAAGTG
25AAAATGGCCA ATTCGTTACG AGGAGAAGTA CTGACTCTTT ATAAAAATCT
GCTGTATCTT GGACGGGACT ATCCAAAAGG AGCAGACTAT TTTAAAAGGC
GTTTGAAGAA CGTTTTCCTT AAAAACAAGG ATGTGGAGGA CCCAGAGAAG
ATCAAAGAAC TTATCGCACG AGGAGAATTT GTAATGAAGG AGCTAGAGGC
CTTGTACTTC CTTAGGAAAT ACAGAGCTAT GAAGCAACGT TACTATTCAG
30ATACCAAAGT CTGACCAATC ATTGCACCAG TCGAGCTGAC AACCAGTGCT
GGCTGTTTGC CTGTACCAAC TATTAAAAAA TAATTCAGTT TAAAAGGGTG
AGATACATGG TTTTTAAAAA AATGAGTTGC CCTACTGTAC TGAAATAGGT
TTCAACCTTA TTGATACTGA GAGCTTTGCC CATAATCCTT TTATTACTGA
AATAGTAACT TTAGTACCTT TCATGATAAT ATAATTTTGA AAGAAAATAC
35ACTTAATTTT TAAACATGTT ATAGCCAATT TTCTTAAGTC TATTTCTTCA
TTTACTGATG AGATTGTCAC TATCGAATGG TGTCTGACAG GCTTGCCCTT
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TAGCTTCTAG AGTGTCTTTG TCCTTGTTTT TTGTTGTTTT GTTAGCCCAT
CTAGTATACT AAAGTGCATA TTCAAGGCTC TCTACAGACA CCTCAAAATG
ATTTAAATGC AGTTATCAAA ATAAGACATG TGAAGGTGAC CTCTATCTTG
AGAAGCTCAG TGGGTGACTA GCATTGTGTA GCTATTATTC CCATTATTCT
TTGTGCTGCT GGCCTGCCTT AAGTTCTGAA CCACTTCAAG TAGCTTTCAT
GAGGAGTTGT AATGTTCCTC TATTTCTGCC ATTAAAGCTG GTATATTTTC
TGTCGACCTG TAACCGAGTC CATGTGGCAG TGGACCTAAC CCAGGCAGGA
CTGTAAGTTT AAGCAAAAAT GTTTATGTAA TGTTTTTAGC AACGTTATAA
ATAACATTTC TAACTTAAAA GCTGCAAATA GTGTTGCTTA TAGGATTCTG
TATCAGGCTG GAGAGATGGC TCAGTGGTTA AGAGCACTGA CTGCTCTTCC
AGAGGTCCTG AATTTAATTC CCAGCAACCA TATGGTGGCT TACAACCATC
TGTAATGGGA TCTGATGTCC ACTTCTGGTG TGTCTGAACA CAGACAGTGT
ACTCATAGAA TAAATAAATA AACGAATAAA TCTTAAAGTC TTAAAGGAGT
CTTTATCAAC TACCAAGCAG ACATTTCCAC CAAGAAATAC CTATAGCCAG
GATGGGGATG AGGCTCAGTG TTAAGTACTT GCCTAAGGAA CACGTGAGGC
TCCAAAATTG AGCCTTAACC ACAATTAAAA CTACATAATT ACACACTTCA
TAGTCACCAT AACTATTTTT ATTACATTAC AATGATTAGG AGCAGTACGG
TTCATGACAA AAATATTACA AATTTCAGAT CACTTCACAG CACGTACTCC
TATAAACATT TAAAAGTTAA TTTTAATTAA GAGTGGTCAC TTTTAAATTT
AATGTTTGAT ATGACCAACA TTCCCTAGGT CAGCGCAACC AAAGGATGGA
AAACAACTGG ATCACACTGC ATATGTCCCA TAACAA//
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Table 3C.
mouse Brown Reverse
Adipose (NT-GHA)
Tissue 62
cDNA (EST)
; 735 BP.
(in bold)
5 mouse Brown Adipose ssue Clone 66 cDNA
Ti
(full- length); 12 BP.
16
Start Codon: 385 (underlined)
Stop C odon: 645 (underlined)
Open R eading Frame:
86-as
10 GGCCGTGGTG CCGCAAAGCG CTGGAGTGAG GCGGTCTGAG CAAGCTGTCG
TCTGGACCCC AGACCTGCTG GTGGTGAACT AAAGCACCGA GTCAAAAGCA
TGGTCAGCAG CATGGATGCT GTCTGCTCTG CCTCCCGTGG AACCTTTCCA
AGTGCTCCCT TTGCCCGCTG CCTCTTACTC TGCATTCTCC TTAAGGACCA
ACCTTCTTGA TCTTGATCGA ACAACCCAAT TTATCTTAGT TTTAAAATTT
15 CCTCCAAGAA TACTCTTCTA GATTTGGACT CTTAGTTTCT TCCAAATAAT
CAAGCCAAGC CTTGAGAGCA GGGCAGACAG CTTTACTTTT GGTATATCAT
GTATAAAAGT GGATCAATTC CATGTTAAGT GAAAATGGCC AATTCGTTAC
GAGGAGAAGT ACTGACTCTT TATAAAAATC TGCTGTATCT TGGACGGGAC
TATCCAAAAG GAGCAGACTA TTTTAAAAGG CGTTTGAAGA ACGTTTTCCT
20 TAAAAACAAG GATGTGGAGG ACCCAGAGAA GATCAAAGAA CTTATCGCAC
GAGGAGAATT TGTAATGAAG GAGCTAGAGG CCTTGTACTT CCTTAGGAAA
TACAGAGCTA TGAAGCAACG TTACTATTCA GATACCAAAG TCTGACCAAT
CATTGCACCA GTCGAGCTGA CAACCAGTGC TGGCTGTTTG CCTGTACCAA
CTATTAAAAA ATAATTCAGT TTAAAAGGGT GAGATACATG GTTTTTAAAA
25 AAATGAGTTG CCCTACTGTA CTGAAATAGG TTTCAACCTT ATTGATACTG
AGAGCTTTGC CCATAATCCT TTTATTACTG AAATAGTAAC TTTAGTACCT
TTCATGATAA TATAATTTTG AAAGAAAATA CACTTAATTT TTAAACATGT
TATAGCCAAT TTTCTTAAGT CTATTTCTTC ATTTACTGAT GAGATTGTCA
CTATCGAATG GTGTCTGACA GGCTTGCCCT TTAGCTTCTA GAGTGTCTTT
30 GTCCTTGTTT TTTGTTGTTT TGTTAGCCCA TCTAGTATAC TAAAGTGCAT
ATTCAAGGCT CTCTACAGAC ACCTCAAAAT GATTTAAATG CAGTTATCAA
AATAAGACAT GTGAAGGTGA CCTCTATCTT GAGAAGCTCA GTGGGTGACT
AGCATTGTGT AGCTATTATT CCCATTATTC TTTGTGCTGC TGGCCTGCCT
TAAGTTCTGA ACCACTTCAA GTAGCTTTCA TGAGGAGTTG TAATGTTCCT
35 CTATTTCTGC CATTAAAGCT GGTATATTTT CTGTCGACCT GTAACCGAGT
CCATGTGGCA GTGGACCTAA CCCAGGCAGG ACTGTAAGTT TAAGCAAAAA
TGTTTATGTA ATGTTTTTAG CAACGTTATA AATAACATTT CTAACTTAAA
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AGCTGCAAAT AGTGTTGCTT ATAGGATTCT GTATCAGGCT GGAGAGATGG
CTCAGTGGTT AAGAGCACTG ACTGCTCTTC CAGAGGTCCT GAATTTAATT
CCCAGCAACC ATATGGTGGC TTACAACCAT CTGTAATGGG ATCTGATGTC
CACTTCTGGT GTGTCTGAAC ACAGACAGTG TACTCATAGA ATAAATAAAT
AAACGAATAA AT//
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Table 3D
Alignment of Clones 66, 58 and 65
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 GAGACGGTCG GAGCATTTAC GGCCGTGGTG CCGCAAAGCG CTGGAGTGAG 50
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 GCGGTCTGAG CAAGCTGTCG TCTGGACCCC AGACCTGCTG GTGGTGAACT 100
C66 __________ __________ __________ __________ __________
C58
C65 AAAGCACCGA GTCAAAAGCA TGGTCAGCAG CATGGATGCT GTCTGCTCTG 150
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 CCTCCCGTGG AACCTTTCCA AGTGCTCCCT TTGCCCGCTG CCTCTTACTC 200
1 5 C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 TGCATTCTCC TTAAGGACCA ACCTTCTTGA TCTTGATCGA ACAACCCAAT 250
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
2 O C65 TTATCTTAGT TTTAAAATTT CCTCCAAGAA TACTCTTCTA GATTTGGGCT 300
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 CTTAGTTTCT TCCAAATAAT CAAGCCAAGC CTTGAGAGCA GGGCAGACAG 350
C66 __________ __________ __________ __________ __________
25 C58
C65 CTTTACTTTT GGTAAGGAAA GCAGGCTTAG AAAAGTGGTG TTACCCAGTG 400
C66 __________ __________ __________ __________ __________
C58 __________ __________ __________ __________ __________
C65 CCTCAATAAA ACAGCTCAGT ACAAATAACC ATTTGGGGGG ATAAGAAGTC 450
3 O C66 -----GGCCG TGGTGCCGCA -AAGCGCTGG AGTGAGGCGG TCTGAGCAAG 44
C58 __________ __________ __________ __________ __________
C65 TTAATGGCAA AGCACTTGCA CAAACAAGAG GGTCCTGTAG ACC-TGCAAG 499
C66 -CTGTCGT-C TGGACCCCAG ACCTGCTGGT G-G-TGAACT AAAGCACCGA 90
C58 __________ __________ __________ __________ __________
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C65 TTTGTAATCC CAGTGTACAT ACAGGGGGGT GAGAGGTAGG AGAATCCCTA 549
C66 GTCAAAAGCA TGGTCAGCAG CATGGATGCT GTCTGCTCTG CCTCCCGTGG 140
C58 __________ __________ __________ __________ __________
C65 AATGAAGGAA GGGCCAGCTG TTTGCA-GC- AACAACTAAG --ACCCGTGG 595
C66 AACCTTTCCA AGTGCTCCCT TTGCCCGCTG CCTCTTACTC TGCATTCTCC 190
C58 __________ __________ __________ __________ __________
C65 AA-AGGACTG ACAGCTGAGG TCATCAGCT- CC----A-AA TGCACACT-G 637
C66 TTAAGGACCA ACCTTCTTGA TCTTGATCGA ACAACCCAAT TTATC-TTAG 239
C58 ---------- ---------- ---------- ---'----GA CTTCCGGCAG 12
C65 GCAAGTA-CA AGTCTGTACA -CAAGAATGA A-AAGCCAGC TCACCAGC-- 682
C66 TT--TT--AA AATTTCCTCC AAGAATACTC TTCTAGATTT GGACTCTTAG 285
C58 ACGGTCGGAG CATTTACGGC -CGTGGTGCC GCAAAGGCCT GGAGT-GAGG 60
C65 TCCATGGGAA GATCT----- -C-TGGT-TC TTTAAGATTT ACAAT-GCAG 723
C66 TTTCTTCCAA ATAATCAAGC CAAGCCTTGA GAGCAGGGCA GA-CAGCTTT 334
C58 CG--GTC--- -TGAGCAAG- CTGTCGTCTG GACC---CCA GACCTGCTGG 100
C65 TT--ATT--- -T--GCAAA- AAAAAG--AA AATC---TT- --CCT-TT-- 753
C66 ACTTTTGGTA TATCATGTAT AAAAGTGGAT CAATTCCATG TTAAGTGAAA 384
C58 TGGTGAAGTA TATCATGTAT AAAAGTGGAT CAATTCCATG TTAAGTGAAA 150
C65 TCTTTAGGTA TATCATGTAT AAAAGTGGAT CAATTCCATG TTAAGTGAAA 803
2 O C66 ATGGCCAATT CGTTACGAGG AGAAGTACTG ACTCTTTATA AAAATCTGCT 434
C58 ATGGCCAATT CGTTACGAGG AGAAGTACTG ACTCTTTATA AAAATCTGCT 200
C65 ATGGCCAATT CGTTACGAGG AGAAGTACTG ACTCTTTATA AAAATCTGCT 853
C66 GTATCTTGGA CGGGACTATC CAAAAGGAGC AGACTATTTT AAAAGGCGTT 484
C58 GTATCTTGGA CGGGACTATC CAAAAGGAGC AGACTATTTT AAAAGGCGTT 250
2 5 C65 GTATCTTGGA CGGGACTATC CAAAAGGAGC AGACTATTTT AAAAGGCGTT 903
C66 TGAAGAACGT TTTCCTTAAA AACAAGGATG TGGAGGACCC AGAGAAGATC 534
C58 TGAAGAACGT TTTCCTTAAA AACAAGGATG TGGAGGACCC AGAGAAGATC 300
C65 TGAAGAACGT TTTCCTTAAA AACAAGGATG TGGAGGACCC AGAGAAGATC 953
C66 AAAGAACTTA TCGCACGAGG AGAATTTGTA ATGAAGGAGC TAGAGGCCTT 584
3 O C58 AAAGAACTTA TCGCACGAGG AGAATTTGTA ATGAAGGAGC TAGAGGCCTT 350
C65 AAAGAACTTA TCGCACGAGG AGAATTTGTA ATGAAGGAGC TAGAGGCCTT 1003
C66 GTACTTCCTT AGGAAATACA GAGCTATGAA GCAACGTTAC TATTCAGATA 634
C58 GTACTTCCTT AGGAAATACA GAGCTATGAA GCAACGTTAC TATTCAGATA 400
C65 GTACTTCCTT AGGAAATACA GAGCTATGAA GCAACGTTAC TATTCAGATA 1053
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C66 CCAAAGTCTG ACCAATCATT GCACCAGTCG AGCTGACAAC CAGTGCTGGC 684
C58 CCAAAGTCTG ACCAATCATT GCACCAGTCG AGCTGACAAC CAGTGCTGGC 450
C65 CCAAAGTCTG ACCAATCATT GCACCAGTCG AGCTGACAAC CAGTGCTGGC 1103
C66 TGTTTGCCTG TACCAACTAT TAAAAAATAA TTCAGTTTAA AAGGGTGAGA 734
C58 TGTTTGCCTG TACCAACTAT TAAAAAATAA TTCAGTTTAA AAGGGTGAGA 500
C65 TGTTTGCCTG TACCAACTAT TAAAAAATAA TTCAGTTTAA AAGGGTGAGA 1153
C66 TACATGGTTT TTAAAA.AAAT GAGTTGCCCT ACTGTACTGA AATAGGTTTC 784
C58 TACATGGTTT TTAAAAAAAT GAGTTGCCCT ACTGTACTGA AATAGGTTTC 550
C65 TACATGGTTT TTAAAAAAAT GAGTTGCCCT ACTGTACTGA AATAGGTTTC 1203
1 O C66 AACCTTATTG ATACTGAGAG CTTTGCCCAT AATCCTTTTA TTACTGAAAT 834
C58 AACCTTATTG ATACTGAGAG CTTTGCCCAT AATCCTTTTA TTACTGAAAT 600
C65 AACCTTATTG ATACTGAGAG CTTTGCCCAT AATCCTTTTA TTACTGAAAT 1253
C66 AGTAACTTTA GTACCTTTCA TGATAATATA ATTTTGAAAG AAAATACACT 884
C58 AGTAACTTTA GTACCTTTCA TGATAATATA ATTTTGAAAG AAAATACACT 650
C65 AGTAACTTTA GTACCTTTCA TGATAATATA ATTTTGAAAG AAAATACACT 1303
C66 TAATTTTTAA ACATGTTATA GCCAATTTTC TTAAGTCTAT TTCTTCATTT 934
C58 TAATTTTTAA ACATGTTATA GCCAATTTTC TTAAGTCTAT TTCTTCATTT 700
C65 TAATTTTTAA ACATGTTATA GCCAATTTTC TTAAGTCTAT TTCTTCATTT 1353
C66 ACTGATGAGA TTGTCACTAT CGAATGGTGT CTGACAGGCT TGCCCTTTAG 984
C58 ACTGATGAGA TTGTCACTAT CGAATGGTGT CTGACAGGCT TGCCCTTTAG 750
C65 ACTGATGAGA TTGTCACTAT CGAATGGTGT CTGACAGGCT TGCCCTTTAG 1403
C66 CTTCTAGAGT GTCTTTGTCC TTGTTTTTTG TTGTTTTGTT AGCCCATCTA 1034
C58 CTTCTAGAGT GTCTTTGTCC TTGTTTTTTG TTGTTTTGTT AGCCCATCTA 800
C65 CTTCTAGAGT GTCTTTGTCC TTGTTTTTTG TTGTTTTGTT AGCCCATCTA 1453
2 5 C66 GTATACTAAA GTGCATATTC AAGGCTCTCT ACAGACACCT CAAAATGATT 1084
C58 GTATACTAAA GTGCATATTC AAGGCTCTCT ACAGACACCT CAAAATGATT 850
C65 GTATACTAAA GTGCATATTC AAGGCTCTCT ACAGACACCT CAAAATGATT 1503
C66 TAAATGCAGT TATCAAAATA AGACATGTGA AGGTGACCTC TATCTTGAGA 1134
C58 TAAATGCAGT TATCAAAATA AGACATGTGA AGGTGACCTC TATCTTGAGA 900
3 O C65 TAAATGCAGT TATCAAAATA AGACATGTGA AGGTGACCTC TATCTTGAGA 1553
C66 AGCTCAGTGG GTGACTAGCA TTGTGTAGCT ATTATTCCCA TTATTCTTTG 1184
C58 AGCTCAGTGG GTGACTAGCA TTGTGTAGCT ATTATTCCCA TTATTCTTTG 950
C65 AGCTCAGTGG GTGACTAGCA TTGTGTAGCT ATTATTCCCA TTATTCTTTG 1603
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C66 TGCTGCTGGC CTGCCTTAAG TTCTGAACCA CTTCAAGTAG CTTTCATGAG 1234
C58 TGCTGCTGGC CTGCCTTAAG TTCTGAACCA CTTCAAGTAG CTTTCATGAG 1000
C65 TGCTGCTGGC CTGCCTTAAG TTCTGAACCA CTTCAAGTAG CTTTCATGAG 1653
C66 GAGTTGTAAT GTTCCTCTAT TTCTGCCATT AAAGCTGGTA TATTTTCTGT 1284
5 C58 GAGTTGTAAT GTTCCTCTAT TTCTGCCATT AAAGCTGGTA TATTTTCTGT 1050
C65 GAGTTGTAAT GTTCCTCTAT TTCTGCCATT AAAGCTGGTA TATTTTCTGT 1703
C66 CGACCTGTAA CCGAGTCCAT GTGGCAGTGG ACCTAACCCA GGCAGGACTG 1334
C58 CGACCTGTAA CCGAGTCCAT GTGGCAGTGG ACCTAACCCA GGCAGGACTG 1100
C65 CGACCTGTAA CCGAGTCCAT GTGGCAGTGG ACCTAACCCA GGCAGGACTG 1753
10 C66 TAAGTTTAAG CAAAAATGTT TATGTAATGT TTTTAGCAAC GTTATAAATA 1384
C58 TAAGTTTAAG CAAAAATGTT TATGTAATGT TTTTAGCAAC GTTATAAATA 1150
C65 TAAGTTTAAG CAAAAATGTT TATGTAATGT TTTTAGCAAC GTTATAAATA 1803
C66 ACATTTCTAA CTTAAAAGCT GCAAATAGTG TTGCTTATAG GATTCTGTAT 1434
C58 ACATTTCTAA CTTAAAAGCT GCAAATAGTG TTGCTTATAG GATTCTGTAT 1200
15 C65 ACATTTCTAA CTTAAAAGCT GCAAATAGTG TTGCTTATAG GATTCTGTAT 1853
C66 CAGGCTGGAG AGATGGCTCA GTGGTTAAGA GCACTGACTG CTCTTCCAGA 1484
C58 CAGGCTGGAG AGATGGCTCA GTGGTTAAGA GCACTGACTG CTCTTCCAGA 1250
C65 CAGGCTGGAG AGATGGCTCA GTGGTTAAGA GCACTGACTG CTCTTCCAGA 1903
C66 GGTCCTGAAT TTAATTCCCA GCAACCATAT GGTGGCTTAC AACCATCTGT 1534
2 O C58 GGTCCTGAAT TTAATTCCCA GCAACCATAT GGTGGCTTAC AACCATCTGT 1300
C65 GGTCCTGAAT TTAATTCCCA GCAACCATAT GGTGGCTTAC AACCATCTGT 1953
C66 AATGGGATCT GATGTCCACT TCTGGTGTGT CTGAACACAG ACAGTGTACT 1584
C58 AATGGGATCT GATGTCCACT TCTGGTGTGT CTGAACACAG ACAGTGTACT 1350
C65 AATGGGATCT GATGTCCACT TCTGGTGTGT CTGAACACAG ACAGTGTACT 2003
2 5 C66 CATAGAATAA ATAAATAAAC GAATAAAT-- ---------- ---------- 1612
C58 CATAGAATAA ATAAATAAAC GAATAAATC- ---------- ---------- 1379
C65 CATAGAATAA ATAAATAAAC GAATAAATCT TAAAGTCTTA AAGGAGTCTT 2053
C66 __________ __________ __________ __________ __________ 1612
C58 __________ __________ __________ __________ __________ 1379
3 O C65 TATCAACTAC CAAGCAGACA TTTCCACCAA GAAATACCTA TAGCCAGGAT 2103
C66 __________ __________ __________ __________ __________ 1612
C58 __________ __________ __________ __________ __________ 1379
C65 GGGGATGAGG CTCAGTGTTA AGTACTTGCC TAAGGAACAC GTGAGGCTCC 2153
C66 __________ __________ __________ __________ __________ 1612
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C58 __________ __________ __________ __________ __________ 1379
C65 AAAATTGAGC CTTAACCACA ATTAAAACTA CATAATTACA CACTTCATAG 2203
C66 __________ __________ __________ __________ __________ 1612
C58 __________ __________ __________ __________ __________ 1379
C65 TCACCATAAC TATTTTTATT ACATTACAAT GATTAGGAGC AGTACGGTTC 2253
C66 __________ __________ __________ __________ __________ 1612
C58 __________ __________ __________ __________ __________ 1379
C65 ATGACAAAAA TATTACAAAT TTCAGATCAC TTCACAGCAC GTACTCCTAT 2303
C66 __________ __________ __________ __________ __________ 1612
1 0 C58 __________ __________ __________ __________ __________ 1379
C65 AAACATTTAA AAGTTAATTT TAATTAAGAG TGGTCACTTT TAAATTTAAT 2353
C66 __________ __________ __________ __________ __________ 1612
C58 __________ __________ __________ __________ __________ 1379
C65 GTTTGATATG ACCAACATTC CCTAGGTCAG CGCAACCAAA GGATGGAAAA 2403
C66 __________ __________ __________ ___ 1612
C58 __________ __________ __________ ___ 1379
C65 CAACTGGATC ACACTGCATA TGTCCCATAA CAA 2436
Position of probe 62 shown by underlining.
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Table 3E
Open Reading Frame Sequence (86-aa) for clones 66, 58, 65:
MANSLRGEVLTLYKNLLYLGRDYPKGADYFKRRLKNVFLKNKDVEDPEKIKELIARGEFV
MKELEALYFLRKYRAMKQRYYSDTKV
Table 3F
Polypeptide Sequence Alignments:
Sequence 1: ORF of Clone 58, 65, & 66
Sequence 2 : CG6I 15 gene encoding a 85-as polypeptide in Drosophila
melanogaster
(GI: 7298358)
in~e~°edi~le a~°g indicates the identities (letter) and
similarities (+)
MANSLRGEVLTLYKNLLYLGRDYP--KGAD
M -~ LR ~~~~LYK-P~L '.:LGH.-~YPG
M-SQLRSKVISLYKHLQYLGREYPGLNGPQ
YFFCRRLKNVFLKNKDVEDPEKIKELIARGE
F-~-~~-~ ~- ~f°~ ~KD ~DP~-KI L.+T3+G
KFRKQIHDAFMNHKDEQDPKKIVALLAQGR
FVMICELEALYFLRKYRAMKQRY-YSDTKV
-~-~ KE+ Y L-~KYR+-~-KQRY Y+D
YLAKEVEALYSLKKYRSVKQRYSYND
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Table 4. Unknown Estimated Sequence Tags (EST) Identified in
Reverse Subtraction Library (NT-GHA)
Table 4a.
mouse Brown Adipose Tissue Reverse (NT-GHA) 2 cDNA
(EST); 556 BP.
1St strand:
ACATTTCAAG AGATGGAGAA ACATTTAGGT CCAGTAAATT TCTTGGTAAA
TGCAGCCGGT ATCAACAGAG ACAGTCTTCT AGTAAGAACA AAGACTGAAG
ACATGATCTC TCAGCTGCAC ACTAACCTCC TGGGCTCCAT GCTGACCTGT
AAAGCTGCCA TGGAGACAAT GATTCAGCAG GGAGGGTCTA TTGTTAATGT
GGGAAGTATT ATTGGTTTGA AAGGCAACGT TGGCCAGTCT GCATACAGTG
CCACCAAAGG AGGACTCGTT GGGTTTTCAC GCTCGCTTGC TAAAGAGGTT
GCACGGAAGA AAAATCAGAG TGAATGTGGT GGCACCAGGA TTTATTCGCA
CGGATATGAC AAGACACTTG AAAGAAGAAC ACTTCAAGAA AAACATTCCT
CTTGGGAGGT TTGGAGAAAC TCCTTGAGGT AGCACATGCC GTTGTGTTTC
TTTTAGAGTC ACCATACATC ACAGGCCATG TTCTTACCGT GGATGGAGGA
TTGCAGCTCA CCGTCTAATT AGAGATGATG TTACTGTGAT GCGCTTTGGG
TCAAGT
2nd strand:
ACTTGACCCA AAGCGCATCA CAGTAACATC ATCTCTAATT AGACGGTGAG
CTGCAATCCT CCATCCACGG TAAGAACATG GCCTGTGATG TATGGTGACT
CTAAAAGAAA CACAACGGCA TGTGCTACCT CAAGGAGTTT CTCCAAACCT
CCCAAGAGGA ATGTTTTTCT TGAAGTGTTC TTCTTTCAAG TGTCTTGTCA
TATCCGTGCG AATAAATCCT GGTGCCACCA CATTCACTCT GATTTTTCTT
CCGTGCAACC TCTTTAGCAA GCGAGCGTGA AAACCCAACG AGTCCTCCTT
TGGTGGCACT GTATGCAGAC TGGCCAACGT TGCCTTTCAA ACCAATAATA
CTTCCCACAT TAACAATAGA CCCTCCCTGC TGAATCATTG TCTCCATGGC
AGCTTTACAG GTCAGCATGG AGCCCAGGAG GTTAGTGTGC AGCTGAGAGA
TCATGTCTTC AGTCTTTGTT CTTACTAGAA GACTGTCTCT GTTGATACCG
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GCTGCATTTA CCAAGAAATT TACTGGACCT AAATGTTTCT CCATCTCTTG
AAATGT
Table 4B. mouse Brown Adipose Tissue Reverse (NT-GHA) 15
eDNA (EST); 681 BP.
1st strand:
ACCCATTAGC CAAACAGAAC TCCTGAATAT ATCTTGAAAG CCTTTCTTGT
ATTGTTTCTT CATCTGTAGG TTTGAACACA GCAGGAGATT TTATCATGGC
CTCCACCTGA TCCACCTCTA TTTCCCAGTC CCTAGCTAAT CTCTGCAAAG
ATGTTTCATC CACTCCAAAC ACAGTGCGGT AGAATTTCAT GCTTTTCTTC
AGAGTCTCCA AATCACTGTC CAAGAGAAAG GTCAGAGAAG GGATGATATT
CACTAGGTCA GCAGCAAATC CTTCCAGCCA AATCCTCTGC TTCAGAAATT
GCCGCTTCTT TTCAATGACT GAATCTGTGA TATTGGGTAA GGAGACCATA
AAATTGTGTC TCTTGTAGAT AGGGAGGTCA CTTATCAGCT TGTCCATCAG
GACGGGGAAG TCATAGTGAC AAACATTTTT GTTAGAGAGC AGGAAGATTG
GTGGCTCAGC AATGCCATTC TCCCTAAAGG TGTTCACACA GTTAAGGCGG
ATGTCCTGCA GGACCTTTTC TTTGTCAAAG GTTTGAGGTT TGCCATCTGC
TTCATTTGTT ATGTCAGAGT CCACCTTGGT TCTCACGAAG TAGAATTCCT
TCTTCATCAT GCTGATTGCT TTGGCAATGT CTATATCATT TTTCTTGAAG
CGTGTGGCCG AAATAATAAT //
GAAGAAATCG
T
2na strand:
ACGATTTCTT CATTATTATT TCGGCCACAC GCTTCAAGAA AAATGATATA
GACATTGCCA AAGCAATCAG CATGATGAAG AAGGAATTCT ACTTCGTGAG
AACCAAGGTG GACTCTGACA TAACAAATGA AGCAGATGGC AAACCTCAAA
CCTTTGACAA AGAAAAGGTC CTGCAGGACA TCCGCCTTAA CTGTGTGAAC
ACCTTTAGGG AGAATGGCAT TGCTGAGCCA CCAATCTTCC TGCTCTCTAA
CAAAAATGTT TGTCACTATG ACTTCCCCGT CCTGATGGAC AAGCTGATAA
GTGACCTCCC TATCTACAAG AGACACAATT TTATGGTCTC CTTACCCAAT
ATCACAGATT CAGTCATTGA AAAGAAGCGG CAATTTCTGA AGCAGAGGAT
TTGGCTGGAA GGATTTGCTG CTGACCTAGT GAATATCATC CCTTCTCTGA
CCTTTCTCTT GGACAGTGAT TTGGAGACTC TGAAGAAAAG CATGAAATTC
TACCGCACTG TGTTTGGAGT GGATGAAACA TCTTTGCAGA GATTAGCTAG
GGACTGGGAA ATAGAGGTGG ATCAGGTGGA GGCCATGATA AAATCTCCTG
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CTGTGTTCAA ACCTACAGAT GAAGAAACAA TACAAGAAAG GCTTTCAAGA
TATATTCAGG AGTTCTGTTT GGCTAATGGG T//
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Table 5. Unknown Full Length cDNA Sequence Identified in
Brown Adipose Tissue Full Length cDNA Library.
mouse Brown Adipose Tissue NG62D'4-2-1-4 cDNA
(Full-Length); 2280 BP.
Start Codon: 163
Stop Codon: 2241
Open Reading Frame: 692-as
GACAGTGGGA GAGGCAAAAT GGCCGCGGGA GTGGCGGCGA GTGGATCGCT
TCCCACAGCG GGCATTATAA TTGATTAGGT TTCTGATATC AAGATATCTT
CCTAAGAAGT AAATTAACAA GCCTCACGTT TCTGTGCAAA CACTGAGGAG
CCAGTTGGCA CCATGAAGGT CTTCTGTGGC CGTGCCAATC CTACCACGGG
ATCCCTGGAG TGGCTGGAGG AGGATGAACA CTATGATTAC CACCAGGAGA
TTGCCAGGTC ATCCTATGCC GACATGCTAC ATGACAAAGA CAGAAATATA
AAATACTACC AGGGTATCCG GGCAGCTGTG AGCAGGGTGA AAGACAGAGG
ACAGAAGGCC TTGGTTCTTG ACATTGGCAC TGGCACAGGC CTCTTGTCAA
TGATGGCAGT TACTGCAGGG GCTGACTTCT GCTATGCTAT CGAGGTTTTT
AAGCCTATGG CTGAGGCTGC TGTGAAGATT GTGGAGAGGA ATGGCTTCAG
TGATAAGATT AAAGTCATTA ACAAGCACTC CACTGAGGTG ACAGTCGGAC
CAGATGGTGA CTTGCCGTGT CGTGCTAACA TTCTGATCAC GGAGCTGTTT
GACACAGAGC TGATTGGGGA GGGAGCGCTG CCCTCTTATG AGCATGCACA
CAAGCATCTT GTCCAGGAAG ACTGCGAGGC AGTGCCACAC AGGGCAACTG
TCTATGCCCA GCTGGTGGAG TCCCGAAGGA TGTGGTCCTG GAACAAGCTG
TTTCCCGTCC GTGTCCGGAC GAGTCTAGGC GAGCAGGTCA TCGTCCCCCC
CTCAGAATTG GAGAGGTGTC CTGGTGCGCC TTCAGTCTGT GACATTCAGC
TGAACCAGGT GTCGCCTGCT GACTTCACTG TCCTCAGTGA TGTGCTGCCA
ATGTTCAGCG TGGACTTCAG CAAGCAAGTC AGCAGCTCGG CAGCGTGCCA
TAGCAGGCAG TTTGTACCTT TGGCGTCTGG CCAAGCACAG GTGGTTCTGT
CCTGGTGGGA CATTGAAATG GACCCTGAGG GCAAGATCAA GTGCACCATG
GCACCCTTTT GGGCACAGAC AGATCCGCAG GAGCTTCAGT GGCGGGACCA
CTGGATGCAG TGTGTGTACT TCCTGCCGCA GGAGGAGCCT GTTGTGCAGG
GCTCACCCCG GTGCCTGGTA GCCCACCATG ATGACTACTG TGTGTGGTAC
AGCCTTCAGA GAACCAGCCC TGATGAGAAC GACAGCGCCT ACCAAGTGCG
ACCTGTGTGT GACTGTCAGG CTCACTTGCT CTGGAACCGG CCTCGGTTTG
GAGAAATCAA TGATCAGGAC AGAACTGATC ACTATGCCCA GGCCCTGAGG
ACTGTGCTGC TGCCAGGTAG CGTCTGCCTT TGTGTGAGTG ATGGCAGTCT
CCTCTCCATG CTGGCCCATC ACCTCGGAGC GGAGCAGGTG TTTACAGTTG
AGAGTTCAGT AGCTTCCTAT AGACTGATGA AAAGGATCTT CAAGGTTAAC
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CACTTGGAAG ATAAAATCAG TGTCATCAAT AAACGGCCTG AGTTGCTGAC
AGCTGCAGAC CTGGAGGGCA AGAAGGTCTC CCTCCTCCTG GGTGAACCCT
TTTTCACCAC CAGCCTGCTG CCATGGCACA ACCTGTACTT CTGGTATGTC
CGTACCTCTG TGGACCAGCA CCTAGCACCT GGAGCTGTGG TGATGCCTCA
GGCTGCCTCA CTGCATGCCG TGATTGTGGA GTTCAGGGAC CTGTGGCGGA
TCCGGAGTCC TTGCGGTGAC TGCGAAGGTT TTGATGTGCA CATCATGGAT
GATATGATCA AGCACTCCCT GGATTTCCGA GAGAGCAGAG AGGCAGAGCC
ACACCCACTG TGGGAATACC CCTGCAGAAG CCTCTCCAAG CCTCAAGAGA
TCCTGACTTT TGATTTCCAG CAGCCCATCC CCCAACAGCC TATGCAATCC
AAGGGCACAA TGGAGCTGAC AAGACCCGGG AAGAGCCATG GGGCTGTCCT
GTGGATGGAG TATCAGCTCA CTCCAGACAG CACGATCAGC ACTGGCCTCA
TAAACCCTGC AGAAGACAAG GGGGACTGCT GCTGGAACCC CCACTGCAAG
CAAGCTGTGT ACTTCCTCAG CACCACGCTG GATCTCAGAG TGCCTCTGAA
TGGCCCTCGG TCAGTCAGCT ATGTTGTGGA GTTTCACCCC CTCACTGGAG
ACATCACCAT GGAGTTTAGG CTTGCAGACA CCTTGAGCTG ATCTCTTATT
GAGAAATAAA ATGGCCAGCA CTGCAGAC
GG
ORF Sequence:
MKVFCGRANPTTGSLEWLEEDEHYDYHQEIARSSYADMLHDKDRNIKYYQGIRAAVSRVK
DRGQKALVLDIGTGTGLLSMMAVTAGADFCYAIEVFKPMAEAAVKIVERNGFSDKIKVIN
KHSTEVTVGPDGDLPCRANILITELFDTELIGEGALPSYEHAHKHLVQEDCEAVPHRATV
YAQLVESRRMWSWNKLFPVRVRTSLGEQVIVPPSELERCPGAPSVCDIQLNQVSPADFTV
LSDVLPMFSVDFSKQVSSSAACHSRQFVPLASGQAQVVLSWWDIEMDPEGKIKCTMAPFW
AQTDPQELQWRDHWMQCVYFLPQEEPWQGSPRCLVAHHDDYCVWYSLQRTSPDENDSAY
QVRPVCDCQAHLLWNRPRFGEINDQDRTDHYAQALRTVLLPGSVCLCVSDGSLLSMLAHH
LGAEQVFTVESSVASYRLMKRIFKVNHLEDKISVINKRPELLTAADLEGKKVSLLLGEPF
FTTSLLPWHNLYFWYVRTSVDQHLAPGAVVMPQAASLHAVIVEFRDLWRIRSPCGDCEGF
DVHIMDDMIKHSLDFRESREAEPHPLWEYPCRSLSKPQEILTFDFQQPIPQQPMQSKGTM
ELTRPGKSHGAVLWMEYQLTPDSTISTGLINPAEDKGDCCWNPHCKQAVYFLSTTLDLRV
PLNGPRSVSYVVEFHPLTGDITMEFRLADTLS
