Note: Descriptions are shown in the official language in which they were submitted.
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GROWTH HORMONE-REGULATABT~E LIVER 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 the liver.
Description of the Background Art
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 1, mediate binding to binding site 1. Helix 3
mediates binding to binding site 2.
See generally Harvey, et al., Growth Hormone (CRC
Press: 1995).
GH is synthesized and secreted by the 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 ~g/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
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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
S 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. (Diabetic glumerosclerosis and nephropathy has
been attributed to a GH effect.) 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); Sharp, et al., Lab. Anim. Sci., 45:607-612 (1995).
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.
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 the third alpha helix of
GH are useful for this purpose. Kopchick, USP 5,350,836.
In order to determine whether it is appropriate to
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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 blood 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)
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
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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-l, for use in monitoring and predicting the effect of
chronic elevation of GH levels on liver function. It is
known that mice transgenic for IGF-1 do not develop the same
abnormalities as mice transgenic for GH, in particular, they
do not develop similar liver and kidney abnormalities. See
Quafe, supra, and Yang, et al., Lab. Invest., 68:62-70
(1993) .
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SUN~lARY OF THE INVENTION
Applicants have identified certain genes whose
expression in liver cells is elevated as a result of higher
than normal GH levels. In contrast, Applicants were unable
5 to identify similarly GH-regulated genes in kidney cells.
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 the liver.
In addition, transgenic mammals, especially mice, rats
and rabbits, which overproduce these proteins may be useful
as animal models of liver pathologies.
Finally, agents which inhibit expression of these
proteins (i.e., antisense nucleic acids) or the binding of
these proteins to their receptors (by binding either the
protein or the receptor) may be useful therapeutically in
inhibiting the development of liver pathologies associated
with the expression of that protein.
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
We have determined some of the differences in the
patterns of gene expression between transgenic mice with a
giant phenotype and nontransgenic mice with a normal
phenotype. This is indicative of the effect of
overproduction of GH on the expression of other genes. GH-
mediated liver pathology is presumably the result of such
expression.
Results of BLAST sequence similarity analyses
identified several genes in the GH TG subtraction library
suggesting that they are differentially expressed in GH TG
mouse liver. These include interferon-cx/~3 receptor
(IFNRcx(3) , corticosteroid binding globulin (CBG) , cx-
fetoprotein, cytochrome P450, fetuin (Ahsg), 3-(3-
hydroxysteroid (3(3HSD), paraoxonase-3 (PON-3), rab8
interacting protein and coagulation factor V. We have also
identified two previously unknown genes affected by GH,
cDNAs 5 and 45. This differential expression has been
confirmed, in the case of IFNRa(3 and CBG, by using the
differentially expressed cDNAs as probes. 3-(3-hydrosteroid
dehydrogenase is down-regulated, the others are up-
regulated.
Assays for expression of these genes may be useful in
the diagnosis of liver pathologies. Such diagnosis is not
limited to the diagnosis of liver pathologies associated
with giantism or acromegaly, or with diabetes, as other
causative agents may act directly or indirectly upon the
same genes.
Liver pathologies include:
1. Liver cirrhosis (hepatic disease of various
etiology) such as
-Alcoholic liver disease
-portal hypertension
2. Liver tumor: .
-Benign: adenomas and focal nodular
hyperplasia
-Malignant: primary carcinomas and
metastatic tumors
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3. Infections of the liver: viral hepatitis
and liver abscesses of whatever origin
4. Hepatic failure or deterioration of the
liver function due to some chronic
progressive disorder or acute injuries
or massive necrosis
5. Drug related liver injury due to
hepatotoxicity of therapeutic agents
Reference to the pathologies is: Cotran, R.S., Kumar, V.,
Robbins, S.L. 1989. Robbin's Pathologic Basis of Disease (qth
ed.) W.B. Saunders Co.; Philadelphia, PA; pp. 911-980.
By preliminary screening assays using nucleic acids,
antibodies, or other binding agents, carried out an mRNA,
cDNA or protein samples from cells of various livers with
known pathologic lesions, we may determine whether the level
of expression of each of the genes mentioned above is
correlated with the presence (or degree of severity) of a
particular liver lesion.
Also, we may make transgenic mammals (e. g. mice) that
overexpress the cDNA in a liver-specific manner (using a
liver-specific promoter like the albumin or PEPCK promoter),
and determine if these transgenic mammals develop liver
histopathologies, or other signs of aging (GH transgenic
mice die prematurely of liver and kidney disease).
Conversely, transgenic mammals in which expression of
these genes is knocked out can be examined to determine if
they provide any protection to the liver against any of the
agents known to cause liver pathology, e.g., viral infection
(esp. hepatitis), alcoholism, hepatoxic drugs, tumors, etc.
if so, then an agent interfering with the expression or
activity of the gene product would have therapeutic value.
The proteins of interest include both secreted and
intracellular proteins.
Secreted proteins can potentially disrupt normal
signaling mechanisms through ligand/receptor interaction.
They can also be used as indicators of a pathophysiological
state. Also, they may be "peptide hormones". Thus, they
could have diagnostic or therapeutic value. Depending upon
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the scenario, recombinant agonists or antagonists may emerge
from these molecules.
Intracellular proteins, on the other hand, could
regulate the intrinsic biological functions of certain
cells. These proteins could be potential drug targets in
that one may design molecules to activate or inhibit them.
1. a- fetoprotein- Closely related to serum albumin
but is found primarily during fetal development, during
which elevated levels can be indicative of neural tube
defects. Elevated levels have been reported in
patients with alcoholic liver disease and
hepatocellular calcinoma (HCC). [Scand J Gastroenterol
2000 Mar;35(3):333-6] This is a secreted protein.
2. Fetuin (AHSG)- A 52 kDa glycoprotein that has
been reported to be an inhibitor of the insulin
receptor tyrosine kinase. [Kalabay L, Horm Metab Res
1998 Jan;30(1):1-6] AHSG has also been reported to
inhibit protease activities and to act as a regulator
of calcium metabolism and osteogenesis. [Banine F, et
al. Eur J Biochem 2000 Feb;267(4):1214-22] This
protein may be important in GH's diabetogenic activity.
Elimination or down regulation of this activity may
allow cells to become more sensitive to the action of
insulin. Thus, inhibitors of this action could be used
as "insulin sensitizers".
3. 3-~3-Hydroxysteroid Dehydroaenase (3-~f3-HSD) -
Isomerase and Dehydrogenase that plays an important
role in all aspects of steroid production. It is
present in many different isoforms which indicates
multiple functionality. It acts in the liver as a key
enzyme in the cholesterol biosynthetic pathway and as a
transporter of bile acids [Marscall HU, et al.
Hepatology 2000 Apr;31(4):990-6] It has also been
reported that GH administration to cultured cells
stimulated the activity of 3-(3-HSD. [Gregoraszczuk EL,
et al. Anim Reprod Sci 2000 Feb 28;58(1-2):113-25]
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Since the activity goes up in the livers of these GH
animals and since it has been shown to be involved in
cholesterol synthesis, it could be used as a target for
the down regulation of cholesterol production.
4. Rab8 interacting protein- Rab proteins are small
GTP binding proteins involved in vesicular transport
during endocytosis and exocytosis. They are distant
relatives of the ras family of oncogenes, but are not
oncogenic themselves. RabBip shows similarity to the
GC kinase, a serine/threonine kinase that has recently
been identified in stress activated human lymphoid
tissue. It is thought that RabBip may have a role in
modulation of secretion in response to stress stimuli.
[Ren M, et al. Proc Natl Acad Sci USA 1996 May
14; 93 (10) :5151-5]
5. Paraoxonase 3 (PON3)- Although little is known
about PONS, the PON family of gene products are active
in cholesterol biosynthesis. PON1 is an enzyme found
in serum which is associated with high density
lipoprotein (HDL) and is thought to protect low density
lipoprotein (LDL) from peroxidation. Decreased
activity of PON enzymes is found in sufferers of
chronic renal failure. [Dantoine TF, J Am Soc Nephrol
1998 Nov;9(11):2082-8] There has been recent
speculation as to the merit of potential testing for
genetic variation in the PON gene family or whether the
gene products might be good candidates for therapeutic
interventions. [Hegele RA, Ann Med 1999 Jun;31(3):217-
24 ]
6. S-2-hydroxy acid oxidase (Glycolate oxidase)-
This gene was just recently cloned in mice. [Kohler SA,
J Biol Chem 1999 Jan 22;274(4):2401-7] It is a
peroxisomal protein that is involved in the oxidation
of hydroxy acids such as L-lactate. Any method to
reduce lactic acid in a diabetic individual would be
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beneficial.
7 . Interferon a/~'~ receptor (IFNa~3R) - Interferons are
antiviral, antiproliferative, immune responsive
cytokines. Recombinant forms have been in use for the
5 treatment of various malignancies. Serum levels of
soluble IFNa(3R have been found to be elevated in
patients with chronic hepatitis C. [Mizukoshi E, et al.
Hepatology 1999 Nov;30(5):1325-31] It is thought that
resistance to IFN therapy in patients with chronic
10 hepatitis C may be due to low levels of hepatic IFNa(3R.
[Yatsuhashi H, J Hepatol 1999 Jun;30(6):995-1003].
Thus any method by which this IFN "binding protein"
would be increased could be beneficial. Since the
soluble version of this has been found, and it is
secreted, it could be used as a diagnostic marker.
8. Growth Hormone Receptor (GHR)- All physiological
attributes of growth hormone are mediated via signaling
though binding with the GHR. Low levels of GHR have
been indicated in cirrhotic liver. [Shen XY, J Clin
Endocrinol Metab 1998 Ju1;83(7):2532-8]
9. Cytochrome P450- The cytochromes are an extensive
family of Heme containing electron transport molecules
found in liver microsomes. They convert a wide range
of substrates to forms that are more easily excreted by
the cell, some of which may be carcinogenic. The
cytochromes are also involved in steroid and
prostaglandin biosynthesis.
10. Proteosome subunit Z- A component of the
multicatalytic Proteinase complex found in the
eukaryotic cytosol and nucleus that is responsible for
ubiquitin dependent protein degradation. It has
recently been reported that GHR internalization
requires proteosome action and active ubiquitin
conjugation system. [van Kerkhof P, J Biol Chem 2000
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Jan 21;275(3):1575-80]. Any substance that could
control ubiquitination could be of value.
11. Corticosteroid Binding Globulin (CBG)- The major
function of CBG is to regulate the bioavailability of
plasma cortisol by restriction it's exit from the
capillaries. [Alexander SL, J Endocrinol 1998
Jun;157(3):425-32] CBG is regulated by many factors,
including stress, steroid sex hormones, and GH (when
dosed continuously). [Jansson JO, J Endocrinol 1989
Sep;122(3):725-32]
12. Coagulation Factor V- Coagulation factors are a
group of protease enzymes and cofactors involved in
clotting. Their activation is triggered by tissue
injury and phospholipoprotein release, which ultimately
leads to the production of thrombin. Again, any
substance that could up or down regulated blood
clotting could be of value.
Definitions
Two proteins are cognate 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
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particular tissue if the average ratio of the specific mRNA
to total mRNA for the cells of that tissue is at least l00
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 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").
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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.
The Assay Target (Marker) (Analyte)
In one embodiment, the assay target is a messenger RNA
transcribed from a gene which, in liver cells, 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 liver cells 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 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 liver cells will be obtained from the subject
and lysed to expose the cytoplasmic contents.
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:
alpha-fetoprotein
fetuin
3-~i-hydroxysteroid
rab8-interacting protein
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paraoxonase-3
interferon cx/(3 receptor
proteasome z-subunit
corticosteroid binding globulin
growth hormone receptor
cytochrome P450IIIA
cytochrome P450
coagulation factor V
S-2 hydroxyacid oxidase
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 (or, in the case of the S-2
hydroxyacid oxidase, to a known rat gene). The mouse
sequencens were transferred onto a nylon membrane. After
transfer of RNA onto the membrane, the membrane may then be
used in a hybridization reaction 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-radiactively with DIG
(Digoxigenin). A negative control, such as one composed of
RNA sample from liver of normal subjects, may be resolved
side by side with the patients' sample, Detection of this
gene or protein could therefore indicate the presence of
liver problem.
Certainly newly discovered DNAs are also of interest.
These are identified below as clones 5 and 45. The
proteins encoded by the ORFs embedded in these DNAs are also
of interest.
Samp1 es
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
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expressed from one of the above genes. The sample may be of
liver tissue or interstitial fluid, or of a systemic fluid
into which liver proteins are secreted.
A non-invasive sample collection will involve the use
5 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. Liver aspirates
can also be obtained to detect for the presence of genes and
proteins of interest. The most invasive method would
10 involve obtaining liver 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
15 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 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,
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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 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.
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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 3H, 3zp, lzsl,
1311, 3sS, 19C, and, preferably, lzsl.
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
lzsEu, 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, 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 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.
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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 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.
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
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19
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
performed using primers specific for the genes of interest.
This would 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.
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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
5 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
10 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-radiactively with DIG (Digoxigenin). A
15 negative control, such as one composed of RNA sample from
liver 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
20 this gene would imply that liver 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 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
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21
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)' - 0.56(o form) - 820/L
where
M, molarity of monovalent cations, 0.01-0.4 M NaCl,
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and
oGC, percentage of G and C nucleotides in DNA, 300-750,
o form, percentage formamide in hybridization solution,
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 25-50o.
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. This divergence (up to 50) 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
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23
be a Tm of 5°C. below the Tm of a perfect duplex, and a to
divergence corresponds to a 1-1.5°C. reduction in Tm.
Hence, use of a Tm of 5-15°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.
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 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 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 1o 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 this degree of stringency.
The hybridization conditions set forth in the Examples
may be used as a starting point, and then made more or less
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24
stringent as the situation merits.
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.,
Ma mm. 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
(1989) (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
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preferably at least 18-30 bases in length. Longer primers
do no harm, shorter primers may sacrifice specificity. The
distance between the primers may be as long as 10 kb, but is
preferably less than 3kb, and of course should taken into
5 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.
10 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
15 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
20 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
25 determine the concentration of the proteins in the sample.
Western blot analysis can also be done on the
plasma/serum, liver aspirate, Liver 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
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secondary antibody which binds the original binding reagent.
To improve detection of the specific protein,
immunoprecipitation can be conducted. This typically will
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 liver 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 liver condition in a suitable number
of patients, and correlating the level of the target at an
earlier time point with the simultaneous or subsequent liver
tissue state.
This correlation is then used to predict the future
clinical state of the liver 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 liver 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
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27
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 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.
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The ABM may be radio-labelled with different isotopes
of iodine, for example 1z31, lzsl, or 13=I (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 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 lzsl for labelling in order
to 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, 99mTc, 6'Ga, 6aGa, 9°y, 111In,
Ilsmln ~ iz3l ~ le6Re ~ ieeRe or 2liAt .
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 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 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 being digested when
administered orally, parenteral administration, i.e.,
intravenous subcutaneous, intramuscular, would ordinarily be
used to optimize absorption of an ABM, such as an antibody,
which is a protein.
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EXAMPLES
BLASTIN and BLASTP searches were performed with the
default parameters match +l, mismatch -3, gap q=-5 r=-2,
penalty q+rk for gap length k. For BLASTP, BLOSUM62 matrix
with q=-l, r=-l, lambda ratio=0.85.
Preliminary results indicate that proteosome z-subunit,
GH receptor, rab8 interacting protein, alpha-fetoprotein,
fetuin are elevated in livers of GH TM when compared to NT,
whereas, 3-beta-HSD is decreased. IFNR, CBG, clone 45 and
clone 5 are expressed in GH TM and not in NT littermates.
Example 1
Introduction: GH-inducible Liver Genes for Diaanosis of GH
Action on Liver Patholoay
Human growth hormone (hGH) upon binding to its receptor
induces expression of a number of genes. These growth
hormone (GH)-inducible genes can be identified in transgenic
mice (TM) expressing bovine GH (bGH). These mice are twice
as big as wild type (WT) mice and are also reported to show
some form of liver pathology in their later stages of life.
Our work aimed to create a library of liver GH inducible-
genes in liver and to identify genes that are associated
with the progression of liver disease that may eventually be
use to diagnose pathologic liver in humans as observed on
patients with acromegaly, liver cirrhosis, and viral
infections causing hepatitis.
Production of differentially expressed cDNAs from GH TM by_
Subtractive Hybridization
The method employed to determine the GH-inducible genes
in bGH TM involves subtractive hybridization using
Clontech's PCR-Select cDNA Subtraction kit. This method
requires that mRNAs be isolated first and then converted
into cDNAs. The mRNAs from liver of 60 days old bGH TM and
WT mice were isolated by passing through oligo-dT columns
(Invitrogen's Fastract 2.0) total RNAs prepared by RNAStat
60. Conversion of mRNAs to cDNAs involves the use of AMV
reverse transcriptase (Clontech). The primer used for the
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first strand cDNA synthesis is 5° TTTTGTACAAGCTT 3' (SEQ ID
N0:6) which binds to polyA tail of the mRNA. This primer
introduces a unique restriction site Rsa 1 downstream of
polyA tail. The second strand cDNA synthesis involves the
5 use of an enzyme cocktail composed of RNase H, DNA
polymerase and lipase enzymes.
Once the double-stranded cDNAs from bGH TM (tester) and
WT (driver) were prepared, these two cDNA populations were
subjected to Rsal digestion to produce shorter, blunt ended
10 fragments. The tester was divided into two halves and each
half was then ligated with different adaptors, adaptor 1 and
adaptor 2R. These two adaptors have stretches of identical
sequences (in bold characters) which serve as sites for
binding of PCR primer 1 during the PCR amplification:
15 Adaptor 1:5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3'
(SEQ ID NO:1)
3'-GGCCCGTCCA-5'
(SEQ ID N0:2)
Adaptor 2R:5'-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT-3'
20 (SEQ ID N0:3)
3'-GCCGGCTCCA-5'
(SEQ ID N0:4)
Since only one end of the adaptors is phosphorylated,
ligation of adaptors to tester cDNAs can occur only at the
25 5' ends of the cDNAs.
Isolation of differentially expressed genes from GH TM
(tester) is achieved by performing two hybridization steps.
The first hybridization step involved mixing each of the
adapter ligated testers with excess of drivers. This
30 resulted in annealing of identical ss cDNA fragments common
to both the tester and driver. Differentially expressed
sequences from GH TM that did not form hybrids with the
driver sequences underwent a second hybridization step.
This step involved mixing two reaction products from the
first hybridization in the presence of more drive cDNA.
This resulted in the formation of new hybrids between
adaptor ligated ss cDNAs from GH TM. After fill in of the
ends of these new hybrids using 50X Advantage cDNA
polymerase mix (Clontech), primer sites for PCR primer 1(5'-
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CTAATACGACTCACTATAGGGC-3', bases 1-22 of SEQ ID NO: l) were
generated.
Subtraction is achieved by preventing the tester-driver
hybrid sequences from being amplified during PCR
amplification while hybrids between testers with adaptor 1
and adaptor 2R can. Thus, those cDNA fragments that undergo
PCR amplification correspond to differentially expressed GH
TM.
Two steps of PCR amplification were conducted to enrich
the pool of differentially expressed cDNA from GH TM. PCR
primer 1 was used in the first PCR amplification at 94°C for
25 sec followed by 27 cycles at three different temperatures
of 94°C for 10 sec, 66°C for 30 sec, and 72°C for 1.5
min.
After the first PCR amplification step which resulted to
exponential amplification of differentially expressed
sequences from GH TM, nested PCR primer 1(5'-
TCGAGCGGCCGCCCGGGCAGGT-3', bases 23-44 of SEQ ID NO:1) and
nested PCR primer 2R(5'-AGCGTGGTCGCGGCCGAGGT-3', bases 23-42
of SEQ ID N0:3) were added to the first PCR amplified
reaction mixture. Then the second PCR amplification step
was conducted at 10-12 cycles of amplification at 94°C for
10 sec, 68°C for 30 sec and 72°C for 1.5 min. to further
enrich the differentially expressed sequence from GH TM.
The integrity of the products from each manipulation was
determined by gel electrophoresis of an aliquot of the
reaction mixtures. The differentially expressed sequences
obtained by subtractive hybridization were subcloned
directly into PCR II cloning vector.
Subcloninct and Seauencina of Differentiallv Expressed
Subtraction Products
The pool of partial cDNA fragments was ligated into a
pCRO 2.1 expression vector using the TA Cloning~ Kit from
Invitrogen~. The ligation mixture was subsequently
transformed into Library Efficiency DHSaTM Competent Cells
from Life Technologies. Ampicillin resistant colonies were
propagated and plasmid DNA was extracted and purified using
an alkaline lysis miniprep protocol (Birnhoim, H.C. 1983).
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The purified plasmid DNAs containing different partial
cDNA fragments were then sequenced using S labeled dNTPs and
the T7 SequenaseTT~ version 2.0 DNA polymerase from Amersham
Life Science Products. The sequencing primer, 5'
TACTCAAGCTATGCATCAAG 3' (SEQ ID N0:5), hybridized to the
pCR~ 2.1 expression vector in the multiple cloning site ~60
bases 5' of the partial cDNA insert. The sequence data was
analyzed and matched against known sequences using BLAST
(Basic Local Alignment Search Tools), available through the
National Centers for Biotechnology Information (NCBI)
Internet database. Our search results indicated that out of
56 sequences analyzed, 13 were identifiable as perfectly or
almost perfectly identical to subsequences of known genes on
the database. These GH-inducible genes in the liver of GH
TM are mouse a-fetoprotein, fetuin, 3-(3-Hydroxysteroid,
rab8-interacting protein, paraoxonase-3, interferon oc/(3
receptor (IFNR a~i), proteasome z-subunit, corticosteroid
binding globulin (CBG), growth hormone receptor, cytochrome
P450IIIA, cytochrome P450, and coagulation factor V, and rat
S-2-hydroxyacid oxidase. It follows that the cognate human
genes may be used as probes for observing GH-regulated
expression of those genes in the liver, which genes are
presumed to be regulated in a similar manner.
Northern Analysis of RNA Extracted from Wildtype and bGH
Transaenic Mice
Total RNA was extracted and purified from the livers of
both bGH trangenic and nontransgenic littermates. 60 day
old mice were euthanized and dissected to obtain the tissues
we needed. Tissues were then homogenized in lml RNA STAT-
60TM Total RNA/mRNA Isolation Reagent per 100mg of tissue.
The RNA was quantitated by spectrophotometry (O. D. 260/280)
and electrophoresed on agarose-formaldehyde gels and
transferred onto Boehringer-Mannheim nylon membranes.
probes were generated using an EcoRI which cleaves out the
partial cDNA insert from the plasmid DNAs. The fragments
were purified using the Qiaex~ II Agarose Gel Extraction Kit
from Qiagen~. The purified fragments were then labeled
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using the Random Primed DNA Labeling Kit from Boehringer
Mannheim. The membrane bound RNA was then hybridized with
cx~=P labeled DNA probes specific for the aforementioned
partial cDNA sequences (see previous page). Preliminary
results indicate that IFNR a(3 and CBG mRNA are expressed in
livers of GH TM and not in NT littermates.
Additional information on preparation of DIG-labeled
probe for Northern blot analysis.
Non-radioactive DIG-labeled probe for Northern blot was
constructed by amplification of the target sequence in the
first PCR step followed incorporation of digoxigenin-11-UTP
or DIG-UTP (Roche) on the antisense strand during the second
PCR. In the construction of probe for used in the
confirmation of differential gene expression in GH
transgenic mice versus non-transgenic mice, fragments from
subtractive hybridization that were subcloned into pCR2.l
cloning vector were PCR amplified using primers pCR 2.1A (5'
ATTACGCCAAGCTTGGTACCG 3') and pCR IIB (5' CCCTCTAGATGCATGCTC
3'). Incorporation of DIG-UTP is accomplished using primer
pCR 2.1A or pCRIIB in the second PCR step. pBluescript
plasmid with the 'full-length' cDNA 45 and cDNA 5 probes,
respectively. T3 (5' AATTAACCCTCACTAAAGGG 3') and mKS (5'
CCTCGAGGTCGACGGTATC 3') primers were used for the first PCR
amplification step and mKS primer for the second DIG-UTP
incorporation step.
Example 2
A cDNA library was constructed from the liver of growth
hormone (GH) trangenic mice. The cDNA that was used in the
construction of the cDNA library was prepared from mRNAs,
which was obtained from total RNA isolated from the liver of
GH transgenic mice. The cDNA prepared was then used to
produce the lambda zap (Stratagene) cDNA library. The titer
of the amplified library was 109 pfu/ml and the recombination
efficiency determined to be 75%. Screening of the cDNA
library for novel genes was done by probe hybridization of
the nitrocellulose plaque lifts. The probe used in the
screening was prepared by PCR amplification of gene
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fragment, which previously was identified by subtractive
hybridization as differentially expressed in GH transgenic
mice and not in wild type mice. After screening of
approximately 2.5 X 105 plaques, five plaques that hybridized
with the probe were purified and then the pBluescript
plasmids, which contain the cDNA inserts, were excised out
of the lambda zap vector utilizing helper phages following
the manufacturer's protocol. The cDNA sequence of the
insert was determined by "walking" through the sequence
starting with T3 and KS primers complementary to sequences
in the plasmid vector.
Clone 5
The sequencing of cDNA for one of the positive clones
that hybridize with probe 5 is completed (Table 2A).
Using GeneRunner software program, the translational
reading frames were determined. The DNA sequence (SEQ ID
N0:7) of Clone 5 has several ORFs; the longest,
corresponding to 1548 bases, encodes a protein of 515 amino
acid residues (SEQ ID N0:8). All ORFs are set forth in
Table 3B.
Using BLAST (Basic Local Alignment Search Tool)
programs, which are designed to compare DNA and protein
sequences available in the database, the DNA and the
corresponding protein sequences were found to be novel.
Protein motif search utilizing PROSITE database
indicate that the protein corresponding to the longest open
reading frame in cDNA sequence of Clone 5 possess the
following motifs: N-glycosylation, protein kinase C
phosphorylation, casein kinase II phosphorylation, and
amidation sites. The protein appears to have a signal
peptide but no transmembrane region found. Thus, this
protein encoded by the longest open reading from in Clone 5
could be cytoplasmic in location.
Clone 45
The sequencing of cDNA (SEQ ID N0:9) for one of the
positive clones that hybridize with probe 45 is complete
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(Table 3A).
Using GeneRunner software program, the translational
reading frames were determined. The DNA sequence of Clone
has several ORFs; the longest, corresponding to 1029
5 bases (SEQ ID N0:9) which encodes a protein of 342 amino
acids (SEQ ID N0:10). All ORFs are set forth in Table 3B.
Using BLAST (Basic Local Alignment Search Tool)
programs, which are designed to compare DNA and protein
sequences available in the database, the DNA and the
10 corresponding protein sequences were found to be novel.
Protein motif search utilizing PROSITE database
indicates that the protein corresponding to the longest open
reading frame in cDNA sequence of Clone 45 possess the
following motifs: N-glycosylation, protein kinase C
15 phosphorylation, casein kinase II phosphorylation, and
amidation sites, as well as a Myc-type helix-loop-helix
dimerization domain. The protein appears to have signal
peptide at the N-terminal and transmembrane region close to
the N-terminal. This could indicate that the protein
20 encoded by longest open reading frame in Clone 45 is
membrane bound and/or secreted.
Significance of the protein motifs found in novel cDNA
sequences isolated from the livers of GH transgenic mice:
N-glycosylation: post-translational modification of
25 proteins involving attachment of carbohydrate residues.
This modification is seen in secreted and membrane proteins.
Glycosylation is associated with lengthening biological life
of a protein by decreasing its rate of clearance from the
serum. For membrane bound proteins, carbohydrates are
30 usually involve in interaction with other cells or
molecules, such as immunoglobulins, cell surface receptors,
and proteases.
Phosphorylation sites: site of attachment of phosphate
group. Reversible phosphorylation-dephosphorylation of
35 protein is associated with regulation of activity of the
protein. Some proteins are activated when phosphorylated
and inactivated when unphosphorylated, or vice versa.
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N-myristoylation: (usually at the N-terminus) this
protein modification involves addition of myristoyl group
which is believed to cause some of the attached proteins to
be loosely associated with membranes. Some myristoylated
proteins are not associated with membranes to any
significant extent. Some of myristoylated proteins such as
protein kinases and phosphatases have important roles in
modulating cellular metabolism.
Amidation: is usually seen on carboxy-terminus of
peptide hormones. Enzymes involve in amidation reaction are
usually found in secretory granules.
Myc-type, helix-loop-helix (HhH) dimerization domain:
HLH dimerization domains are usually present in proteins
that interact with DNA. Myc proteins are involve in growth
regulation.
References:
Creighton, T.E. 1993. Proteins: Structures and
Molecular Properties, 2nd ed. W.H. Freeman and Co., NY, pp.
78-99.
Lewin, B. 1994. Gene V. Oxford University Press, NY,
pp. 899-902.
Example 3
Assav for using mouse DNAs presence of genes from liver of
human patients
Total RNA preparation human liver
Total RNA will be extracted from liver biopsy using 10
mL RNAStat60 per gram of liver tissue. To 15-20 ug of liver
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
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transferred onto a nylon membrane by upward gradient
adsorption using 10X 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 liver RNA blots. The
probes to be used for hybridization will be prepared from
pCR2 clones, which contain as inserts the fragments isolated
by subtractive hybridization of liver genes from GH mice
versus WT mice. The sequence homology of the fragments to
that of the human genes range from about 74o to 940, which
were obtained using the default parameters of Blast 2.0
sequence alignment version blastn 2Ø8.
1. Preparation of DIG-labeled probe
The DIG-labelled probe preparation will require PCR
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 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 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
hybridization will be 50ng/mL of DIG Easy Hyb solution
(Boehringer-Mannheim). Prior to hybridization, the RNA
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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 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 O.lo SDS; and twice at 60 °C for 15 min. using a
solution of 0.5X SSC and 0.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 use to achieve detection of
the RNA of interest in the blot. The presence of bands that
is specific to the liver genes of interest could be
diagnostic of liver damage.
Northern Blot Hybridization involving 32P-labeled probe
1. Preparation of 32P-labeled probe
The 32P-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 QiaexR agarose gel
extraction column (Qiagen). A 25ng of the purified fragment
will serve as a template for the production of single-
stranded 32P-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 to 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 10~-109 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
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prehybridization solution made up of 50o formamide, 1o SDS,
1M NaCl, and 10o Dextran sulfate for 1 hour at 42 °C.
Hybridization of the RNA blot with the 3zP-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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Table A
Human Genes (counterpart of Mouse Genes) regulated by Grorath
Hormone in Diver Tissue
Genes Nucleotide Protein Accession
Accession No. Number
5 Human alpha-fetoprotein NM 001134 NP 001125
V01514 CAA24758
Human Fetuin (A2HS) WOHU
M16961 AAA51683
P02765
S04467
Human 3-beta-hydroxysteroidM27137 AAA36015 dehydrogenase
Type 1
Human Rab8 Interacting NM_003618 NP_003609
10 prote in-like 1
Human Paraoxonase-3 L48516 AAC41996
Human IFN alpha/Beta ReceptorA32391 CAA02098
Human GHR AAA52555 M28458
Human Cytochrome P450 IIIANM 000777 NM 000768
X12387 CAA30944
M18907 AAA35745
J04449 AAA35747
NM 000765 NP 000756
M14096 AAA35744
NM 000776 NM 000767
15 Human proteasome z-subunitD38048 BAA07238
Human Corticosteroid bindingNM 001756 NP_001747
Globulin
Human Coagulation Factor M16967 AAA52424
V
NM 000130 NP 000121
2 0 Rat S-2-hydroxyacid oxidase X67156 CAA47629
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Table B: Result of Blast Search
Clone Closest Match Identities
2 Mouse alpha fetoprotein 77/78
M16111
6 Mouse fetuin
AJ002146 70/70
596534 78/78
7 mouse 3-beta hydroxysteroid
dehydogenase
M77015 78/78
13 mouse rab8-interacting
protein
U50595 66/66
14 mouse paraoxonase-3
L76193 64/64
21 rat S2 hydroxyacid
oxidase
X67156 58/65
26 mouse interferon a/(3
receptor
M89641 78/79
U06244 78/79
29 mouse low MW GH
receptor
M31680 59/61
M33324 59/61
20 mouse cytochrome
P450 IIIA
X60452 62/64
36 same 69/78
39 mouse cytochrome
P450 III A
D26137 75/81
37 mouse proteazome
Z subunit
D83585 77/78
34 corticosteroid-binding
protein
X70533 46/46
35 same
X70533 37/37
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52 same
X70533 121/123
49 same
X70533 46/46
56 mouse coagulation factor 104/106
V
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Table 1 shows the sequence of each clone, and its BLASTN
alignment to the known mouse (or rat) gene as found in a
sequence databank, to which it appears to be most closely
related. The known genes are as follows:
(A) mouse alpha-fetoprotein (WLAC #2)
(B) mouse fetuin (WLAC #6)
(C) mouse 3-(3-hydroxysteroid (WLAC #7)
(D) mouse rab8 interacting protein (WLAC #13)
(E) mouse paraoxonase-3 (WLAC #14)
(F) rat S-2-hydroxyacid oxidase (WLAC #21)
(G) mouse interferon ec/(3 receptor (WLAC #26)
(H) mouse growth hormone receptor (WLAC #29)
(I) mouse cytochrome P450IIIA (WLAC #20, #36)
(J) mouse cytochrome P450 (WLAC #39)
(K) mouse proteasome z-subunit (WLAC#37)
(L) mouse corticosteroid binding globulin ((WLAC #3, 34, 35,
52)
(M) mouse coagulation factor V (WLAC #56)
Table 2 (A) full-length single stranded nucleotide sequence
of clone 5 and (B) ORFs 1-16 corresponding to clone 5.
Table 3 (A) full-length single stranded nucleotide sequence
of clone 45 (B) ORFs 1-9 corresponding to clone 45.
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Table 1
WLAC #2 -fetoprotein
WLAC #2 SEQUENCE
TCCTAGGCTTCTTGCAGCCTCCACGAGAGTTGGGGTTGACACCTGAGGTGCTTTCTGGGTGTAGCGAA
CTAGAATGGCATTTTGGAATCCATATTCTCCACCGCCCTCC
Sequence 1 1c11secL1 WLAC #2 Length 109 from:l to = 109
Sequence 2 gi191764 Mouse alpha-fctoprotein mRNA, partial cds.
Length 1254 from: l to = 1254
NOTE: The statistics (bitscore and expect value) is calculated based on the
size of nr
database
Score = 187 bits (97), Expect 2e-46
Identities = 99/100 (99%), Positives = 99/100 (99%)
Aligned query 1-100 to subject 1071-972.
WLAC #3, 34, 35, 52 Corticosteroid Binding Globulin
WLAC #3 SEQUENCE
CATTGGTGGGAGCCAGGTCTCGGTGAGAACTTGAATCCTCATCAGTGACAGCC
TGGGTGGTCCAGAGGCCACTGGTGCAGAGCCAGAAGAGACAGGTATACAGGG
CGAGCGACATTGTTTTGG
2 0 Sequence 1 1 c 11 seq-1 WLAC#3 Length 124 from: l to = 124
Sequence 2 gi 298114 M. musculus mRNA for corticosteroid-binding globulin
Length 1462 from:l to = 1462
Score = 217 bits (113), Expect = 1 e-55
Identities = 122/124 (98%), Positives = 122/224 (98%), Gaps = 1/124 (0%)
Aligned query 1-124 to subject 169-47.
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WLAC #34 SEQUENCE
GGCAGCAGGCAGCACATTCCCTTCATCCAGTTGCAGCATGGCCTTGCTGGCGC
TACCGCCCTCCGCACCACGCCCTAAGCCGAATTCTGCCATACTATCCATCACA
CTGG
5 Sequence 1 1 c 11 seq-1 WLAC #34 Length 110 from: l to = 110
Sequence 2 gi 298114 M. musculus mRNA for corticosteroid-binding globulin
Length 1462 from: l to = 1462
Score = 89.1 bits (46), Expect = 8e-17
Identities = 46/46 (100%), Positives = 46/46 (100%)
10 Aligned query 1-46 to subject 116-1071.
WLAC #35 SEQUENCE
GCAACTGGATGAAGGGAATGTGCTGCCTGCTGCCACCAATGGAAATCCTGTAC
CGCCCTCCGCACCACGCCCTAAGCCCGAATTCTGCAGTCTAT
Sequence 1 lcl lseq_1 WLAC #35 Length 95 from: 1 to = 95
15 Sequence 2 gi 298114 M. musculus mRNA for corticosteroid-binding globulin
Length 1462 from:l to = 1462
Score = 85.3 bits (44), Expect = 9e-16
Identities = 50/53 (94%), Positives = 50/53 (94%)
Aligned query 1-53 to subject 1083-1135.
2 0 WLAC #52 SEQUENCE
GCCTGACTGGACCATCATGGGCACCTTCACTGTGCTTGTCTCATTCACATAGAA
GTCCTCCTCTCAGTATTTTCTGGGCTGAAGGGAAGTTTCCATATTCCTTTGAGG
AAGAGTAGTTGAT
Sequence 1 1 c 11 seq 1 WLAC #52 Length 121 from: l to = 121
2 5 Sequence 2 gi 298114 M. musculus mRNA for corticosteroid-binding globulin
Length 1462 from:l to = 1462
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Score = 206 bits (107), Expect = 4e-52
Identities = 121/123 (98%), Positives = 121/123 (98%), Gaps = 2/123 (1%)
Aligned query 1-121 to subject 747-625.
WLAC #6 Fetuin (AHSG~
WLAC #6 SEQUENCE
GGGAGAGGCACATTTTGAGCCCGGGAAATCTCCACCACTTTGGGGTAGGTTCC
ATTATTCTGTGTGTTGAAGCAGCCAGGGCAGTGTTGAC
Sequence 1 lcl lseq 1 WLAC #6 Length 91 from:l to = 91
Sequence 2 gi 2546994 Mus musculus fe- (Ahsg) gene, complete cds.
Length 8946 from: l to = 8496
Score = 117 bits (61 ), Expect = 1 e-25
Identities = 78/84 (92%), Positives = 78/84 (92%), Gaps = 1/84 (1%)
Aligned query 9-91 to subject 4638-4555.
WLAC #7 3--Hydroxysteroid
WLAC #7 SEQUENCE
GGGTCAGTGACTGGCAAGGCTTTGGTGACTTGATTAAGGCACTAAATTGGCCT
CTGTGTCAAAAGAAGGCAACAGCACCTGTGTTGTGCTTTTATCCTTACTG
Sequence 1 1 c 11 seq-1 WLAC #7 Length 103 from: l to = 103
Sequence 2 gi 194006 Mouse 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4
2 0 isomerase mRNA sequence
Length 1533 from:l to = 1533
Score = 198 bits (103), Expect = 7e-50
Identities = 103/103 9100%), Positives = 103/103 (100%)
Aligned query 1-103 to subject 1325-1223.
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WLAC #13 rab8 Interacting Protein
WLAC # 13 SEQUENCE
GGAAAGATCCAACTGATAACCCCGGGGCACACAGCAACCTCTACATCCTCAC
GGGTCACCAGAGCAGCTACTGAGCTATCTCCCCGATGACGCCAAGCCCTCGGC
CTC
Sequence 1 1 c 11 seq-1 WLAC # 13 Length 108 from: l to = 108
Sequence 2 gi1330327 Mus musculus RabB-interacting protein mRNA, complete
cds.
Length 2466 from:l to 2466
Score = 127 bits (66), Expect = 2e-28
Identities = 66/66 (100%), Positives = 66/66 (100%)
Aligned query 9-74 to subject 2401-2466.
WLAC #14 Paraoxonase-3
WLAC #14 SEQUENCE
GGCATAGAACTGCTCTGGCCCAAGAACCACAATGTCATTCACACTCTTGAGAA
GTTCATGTTTTGAGATTTTCAGGTGGATGAGAGAGAGCGTTGTTGTTCTTCAAA
Sequence 1 lcl lseq_1 WLAC #14 Length 107 from: 1 to = 107
Sequence 2 gi 1333639 Mus musculus paraoxonase-3(pon3- mRNA, complete cds.
Length 1121 from: l to = 1121
2 0 Score = 162 bits (84), Expect = 7c-39
Identities = 101/107 (94%), Positives = 101/107 (94%), Gaps = 2/107 (1%)
Aligned query 1-107 to subject 537-433.
WLAC #20, #36 Cytochrome P450IIIA
WLAC #20 SEQUENCE
GGAGCATGAGTTTCCCTCAAGGAGTTCTGCTGAGTTCTTCAGAAAGGCAGTGT
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CTAAGAACATCAGATATG
Sequence 1 1 c 11 seq_1 WLAC #20 Length 71 from: l to = 71
Sequence 2 gi 50534 M. musculus mRNA for cytochrome P-450IIIA
Length 1690 from:l to =1690
Score = 112 bits (58), Expect = 4e-24
Identities = 62/64 (96%), Positives = 62/64 (96%)
Aligned query 1-64 to subject 1581-1644.
WLAC #36 SEQUENCE
AAAGGATCACAAAAGTCAACTATTAAAATCCCTTTGGCTTTCTCCACAAAGGG
ATCCTCTAAACTTGTTGAGGGAATCCACATTCACTCCAAA
Sequence 1 1 c 11 secLl WLAC #36 Length 93 from: 1 to = 93
Sequence 2 gi 50534 M. musculus mRNA for cytochrome P-450111A
Length 1690 from: l to = 1690
Score = 94.9 bits (49), Expect = 1e-18
Identities = 80/93 (86%), Positives = 80/93 (86%), Gaps = 1/93 (1%)
Aligned query 1-93 to subject 730-639.
WLAC #21 S-2-Hydroxy acid oxidase
WLAC #21 SEQUENCE
AACCCAAGTTCCTACAGCATCTTTGCAGCTGTTGATCTCACTCTTTCGTTCTAT
TGGAGAAACTACCGGCCCAGCAATGTCTTTG
Sequence 1 1 c 11 seq-1 WLAC #21 Length 85 from: l to = 85
Sequence 2 gi 311832 R. norvegicus mRNA for (s)-2-hydroxy acid oxidase
Length 1648 from: l to = 1648
Score = 79.5 bits (41 ). Expect = 4e-14
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Identities = 75/87 (86%), Positives = 75/87 (86%), Gaps = 2/87 (2%)
Aligned query 1-85 to subject 125-211.
WLAC #26 Interferon a/~3 Receptor
WLAC #26 SEQUENCE
GGCCACACTGAGATCTTAAACAACGCCAGCTCCTCCAGTTAGTGTCCCTTTCTC
CATGGTTCAGTGACTTCTGGTCAGAAG
Sequence 1 lcl lseq-1 WLAC #26 Length 82 from:l to = 82
Sequence 2 gi 194111 Mus musculus interferon alpha/beta receptor (IFNAR)
mRNA, complete cds.
to Length 3894 from:l to = 3894
Score = 144 bits (75), Expect = 8e-34
Identities = 82/83 (98%), Positives = 82/83 (98%), Gaps = 1/83 (1%)
Aligned query 1-82 to subject 2222-2140.
WLAC #29 GHR
WLAC #29 SEQUENCE
TTGCTGGACCCGGGGGTCGTTTCACTGTTGACCGAAATAGTGCAACCTGATCC
ACCCATTGGCCTAACTGGACTTTACTAAA
Sequence 1 1 c 11 seq_1 WLAC #29 Length 82 from: l to = 82
Sequence 2 gi 193508 Mouse high molecular weight growth hormone
2 0 receptor/binding protein, complete cds.
Length 2288 from: l to = 2288
Score = 94.9 bits (49), Expect = 9e-19
Identities = 63/65 (96%), Positives = 63/65 (96%), Gaps = 2/65 (3%)
Aligned query 19-82 to subject 541-604.
2 5 WLAC #37 Proteasome z-subunit
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WLAC #37 SEQUENCE
TGTCCTCACCGAGAAAGTTACCCCTCTGGAGATTGAGGTGCTAGAAGAGACTG
TTCAGACAATGGATACTTCGTAATGGTG
Sequence 1 1 c 11 seq-1 WLAC #37 Length 81 from: l to = 81
5 Sequence 2 gi 1632754 Mouse mRNA proteasome Z subunit, complete cds.
Length 969 from: l to = 969
Score = 142 bits (74), Expect = 3e-33
Identities = 74/74 (100%), Positives = 74/74 (100%)
Aligned query 1-74 to subject 763-836.
l0 WLAC #56 Coagulation Factor V
WLAC #56 SEQUENCE
TGTGGCTTCTGAAAAGGGTAGTTATGAAATAATAGCAGCAAATGGCGAAGAC
ACAGATGTGGATAAGCTGACCAACAGTACCTCAAAATCAGAATATCACAGTA
CCGCCCTCCGCACCACGCCCCTAAGCCCGAATTCTCGAGAT
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Table 2A
Full-length nucleotide sequence of Clone 5
(5' -~ 3' direction of the + strand)
AAGACCCGCCATGTCTCTGCTGGCTACTGTACTGCTGCTCTGGGGG
TTCACTCTGGGCCCAGGAAATACTCTAATGCTCGATTCTGGCAGTG
AACCTAAACTATGGGCAGAGCCTCAGTCCCTGCTGGAACCCTGGG
CAAACCTGACCCTGGTGTGTGCAGTTGATTTGCCGACTAAGGTCTT
CGAGCTGATCCAGAACGGGTGGTTCCTGAGTCAAGTCCGACTTGA
GACACAGGTGCTGTCATACCGCTTTTCCCTGGGGGCCATTACAAGT
to AACAACAGTGGCATCTACCGCTGCAGATGTGGCGTGGAACCCCCT
GTTGACATTCACCTGCCAGCACTGAACAAGTGGACCATGCTAAGC
AATGCTGTGGAGGTGACAGGGAAAGAGCCCTTGCCTCGGCCCTTG
GCTCATGCTGATCCAGTCGACTGGATCACACCTGGTGGCCTGCCTG
TATACGTGATGTGCCAGGTTGCAATGCGGGGTGTGACCTACCTGCT
15 GAGGCAGGAAGGAGTGGATGGCGTCCAGAAACCTGATGTCCAGC
ACAAGGGAACAGCTGGCTTTTTAATCTACAAGCCTGGCAACTACA
GCTGCAGCTACCTAACCCATGCAGCAGGTGAACCCTCTGAGCCCA
GTGATATTGTGACCATCAAGATGTATGCCTCACAGGCTCCACCCAC
TCTGTGTTTGATGGGAAACTACCTAATGATCTACCCCCAGAAGACA
2o TATGAGACCCTTGCCTGCAAAGCTCCTCGGAATGCAGCTGAATTCC
AACTCAGGCAAGGAGGGAAGGTGCTGAAAATTCATGGGTTTAGCC
CCACCAGAGATGCTATCCTGTACTATGTGAACTTGAAGGAACTGG
ATAACCCAGGTCCTTTCACCTGCCGCTACCGGATGCACAAATACAT
GCACGTTTGGTCAGAGGACAGCAAGCCCGTAGAGTTAATGTGGAG
25 TGATGAGACTCTACAAGCTCCGGTACTTACTGCAGAGCCATCGAG
TAGGGACCTTGAGCCTGGTTCAACGGTGCAGCTTCGATGTACTGCA
CCCGTATCCGGCCTGCGCTTTGGCCTGCAACGCCAGGGCAAACCG
GAATTAGTTGTGGTGCAAATGCTGAATTCGTCTGGGACCGAAGCA
GTCTTTGAGCTGCACAATATCTCAACAATAGACTCTGGAAACTACA
3o GCTGTATCTACATGGAACAGGCACCGCCATTCTCAGGATCTTCTTC
CAGTGAGCCCGTGGAGCTGCGGGTGAATGGGCCACCACCCAAGCC
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AAGGCTGGAAGCTCTGTGGAAAAGCACAGTACATCTGGGTCAGGA
AGCCATCTTTCGATGCCACGGCCATGTGCCTAGGGTCAGCATGGA
GCTGGTACGTGAGGGCTTTAAAACACCCTTCGCGGTGGCCTCCAC
AAGAAGCACCTCAGCTTACCTGAAGCTGCTCTTCGTCGGTCCCCAA
CATGCAGGCAACTACAGCTGCCGCTATACGGCCCTGCCGCCCTTC
ACATTTGAATCAGGGATCAGCGACCCTGTGGAGGTTATAGTAGAA
GGTTAGGCTCTCCTGAGCTGTGTTTGAGGTTTTGGGTTCTTAATAT
TTCCAGAGCTGTACACTGGCTAATTGCTTCACCAAGGTCAGTGTGG
AAAGGCCCTGTGGCAACTTGCTGAGTCAATGAAGCCATTTCTTTGT
z o CTAGGCCGCTAATGTGGCTGCAGACACA,~~AAAAAGTGTTCTTGGG
AAGGGTTCAAGACAGGTATAATACCCATTCTTCTCAATGTAAGAT
AACTTCATTTTCTCTGGACTTAATAAAGGTCAAGTAAAAACCCGTT
TAAAAAAAAAAA,AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAA GCTTGA
CAAAA
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Table 2B
Open Reading Frames found in Clone 5
MSLLATVLLLWGFTLGPGNTLMLDSGSEPKLWAEPQSLLEPWANLTL
VCAVDLPTKVFELIQNGWFLSQVRLETQVLSYRFSLGAITSNNSGIYR
CRCGVEPPVDIHLPALNKWTMLSNAVEVTGKEPLPRPLAHADPVDWI
TPGGLPVYVMCQVAMRGVTYLLRQEGVDGVQKPDVQHKGTAGFLI
YKPGNYSCSYLTHAAGEPSEPSDIVTIKMYASQAPPTLCLMGNYLMIY
PQKTYETLACKAPRNAAEFQLRQGGKVLKIHGFSPTRDAIL~'YVNLK
ELDNPGPFTCRYRMHKI'NIFIVWSEDSKPVELMWSDETLQAPVLTAEP
zo SSRDLEPGSTVQLRCTAPVSGLRFGLQRQGKPELVVVQMLNSSGTEA
VFELHNISTIDSGNYSCIYMEQAPPFSGSSSSEPVELRVNGPPPKPRLEA
LWKSTVHLGQEAIFRCHGHVPRVSMELVREGFKTPFAVASTRSTSAY
LKLLFVGPQHAGNYSCRYTALPPFTFESGISDPVEVIVEG@
(SEQ ID N0:8)
MWLQTQKKCSWEGFKTGIIPILLNVR#
MYCTRIRPALWPATPGQTGISCGANAEFVWDRSSL&
MQATTAAAIRPCRPSHLNQGSATLWRL@
MRLYKLRYLLQSHRVGTLSLVQRCSFDVLHPYPACALACNARANRN
@
2 o MGHHPSQGWKLCGKAQYIWVRKPSFDATAMCLGSAWSWYVRALK
HPSRWPPQEAPQLT&
MKLSYIEKNGYYTCLEPFPRTLFLCLQPH@
MSSGGRSLGSFPSNTEWVEPVRHTS&
MGIIPVLNPSQEHFFCVCSHISGLDKEMASLTQQVATGPFHTDLGEAIS
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QCTALEILRTQNLKHSSGEPNLLL#
MAVPVPCRYSCSFQSLLLRYCAAQRLLRSQTNSAFAPQLIPVCPGVAG
QSAGRIRVQYIEAAPLNQAQGPYSMALQ#
MNFQHLPSLPELEFSCIPRSFAGKGLICLLGVDH@
MAVASKDGFLTQMYCAFPQSFQPWLGWWPIHPQLHGLTGRRS&
KTRHVSAGYCTAALGVHSGPRKYSNARFWQ&
FCQAFFFFFFFFFFFFFFFFFFFFFFFFFFKRVFT&
FVKLFFFFFFFFFFFFFFFFFFFFFFFFFLNGFLLDLY#
LSSFFFFFFFFFFFFFFFFFFFFFFFFFF#
to
Note: First 12 sequences are ORF starting from met to stop codon, the next
four sequences were also identified as ORFs from the beginning of the
sequence to the stop codon. ORF analysis conducted using GeneRunner
version 3.05 software by Hastings Software, Inc.
@ = TAG, & = TGA, # = TAA
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Table 3A
Full-length nucleotide sequence of Clone 45
(5' -~ 3' direction of the + strand)
5 GCTGAACTGAAGACCCGCCATGTCTCTGCTGGCTACTGTACTG
CTGCTCTGGGGTTTCACTCTGGACCCAGCAACGGATGCAGCCA
CCTGTACATTCAAGGATGCCATAAAAAACAATTCCTTGCCCAG
GCCCTGGATTTTGCCTTATCCTGTGCCTTGGATCATACCTGGC
CTGATCACGTCCGTGTTGTGCCTGGGGAGAGTGAAAGGGGCA
to GCCTTCCTGCTGAGGCGGGAAGGAGATGATGACTTCCTAGAG
GTAGCTGAAAATACCACTGTTTTCGGGGATGAAACTCAGGCAG
GATACAGGGAACAAGCCATGTTTCGAGTCTATCAACCGGGCAA
CTACAGCTGCAGCTATCAAACTCACGGAGAATGTACCTCCTCT
ACGCCCAGTAGGATTGTGACCATCAAGAAGTTTGCCAAACCAC
15 CGCCACCCCTGCTGACCTCCTCAGAAAGTTCCACAGTGGAGCC
ACCCCACATGGCCCGTATGACCCTTCTCTGTTCCACTTTTCTG
AACGACGTTGAATTTCAGCTGAGGCAGGGAAAGCGTGAGATG
AAGGTCCTTATGTTCAGCACCAGCCCAGAGCAAGTCAACTTCT
ATCTGAAATTGTCAGACATGGGTGACCAGAGCCCCTTCACCTG
2o CCGCTACCGTCTAAGCAACATGACAGCTTGGTCGGAAGACAGT
GAGCCCGTAGAGCTAATGTGGAGCGACGAAAGACTACCAGCA
CCAGTGTTGACTGCAGAGCCATCGACGAATCAGAGCTTTGAGC
CGGGTTCGACGGTGCAGTTTCGATGTACCGCACCCAAGGCTG
GCCTACGCTTTGAGTCTGGCCTGCGCTTTGGCCTGCATACCGA
25 AGACTTGTATGAGCGCAGCCTGATCCAGATACTGAAGTCTTCT
GGTCATGAAACTGTATTCCAGCTGCAAAACCTCTCAGCCGCAG
ATTCTGCCAGCTACAGCTGCATCTATACTGAACTGAAACCACC
CTTCTCTGGATCTGCTCCCAGCAACCTTGTGCCTCTGATGGTG
GACGGATCCTACGAGTACTGAACTCCTATAGTAAACTGGAGCT
3o GCATTTTGTGGGTCCCGAACATACAGGAAACTATACCTGCCGT
TATACCTCCTGGCAGCCTGAGCCCGTCCACTCAGAGCCCAGCA
ACTCCGTGGAGCTCCTAGTGGAAGGTATGGCAGTGGTTGGGT
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TTTGCCTCTTGATCTTTGTTGGACTATGCATTCAGTTAATTGTG
TGATCTAGCCTGGTATTCAAAGGCCCCGTGGCAGTTTGCTGAG
TCAAGTCACCTACTACTTTGTCTGGGAAACTGAAGTAGCTGCA
GACACAGGACCAAACATTGTTCTTGGAAAGAGCAGAAGACAG
ACGGGCAGAACTCCTATTCTTCCTGCTGCAAGATGTATTTCCC
TCAAACTCCCTCCACTTAATAAAGATCAAAAAAAAAAAAA
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Table 3B
Open Reading Frames for Clone 45
MSLLATVLLLWGFTLDPATDAATCTFKDAIKNNSLPRPWILPYPV
PWIIPGLITSVLCLGRVKGAAFLLRREGDDDFLEVAENTTVFGDE
TQAGYREQAMFRVYQPGNYSCSYQTHGECTSSTPSRIVTIKKFAK
PPPPLLTSSESSTVEPPHMARMTLLCSTFLNDVEFQLRQGKREMK
VLMFSTSPEQVNFYLKLSDMGDQSPFTCRYRLSNMTAWSEDSEP
VELMWSDERLPAPVLTAEPSTNQSFEPGSTVQFRCTAPKAGLRFE
SGLRFGLHTEDLYERSLIQILKSSGHETVFQLQNLSAADSASYSCI
1 o YTELKPPFSGSAPSNLVPLMVDGSYEY&
(SEQ ID NO:10)
MKLRQDTGNKPCFESINRATTAAAIKLTENVPPLRPVGL&
MTRRLQYLDQAALIQVFGMQAKAQARLKA@
MWGGSTVELSEEVSRGGGGLANFLMVTILLGVEEVHSP&
MALQSTLVLVVFRRSTLALRAHCLPTKLSCCLDGSGR&
MHSPTKIKRQNPTTAIPSTRSSTELLGSEWTGSGCQEV#
AELKTRHVSAGYCTAALGFHSGPSNGCSHLYIQGCHKKQFLAQA
LDFALSCALDHTWPDHVRVVPGESERGSLPAEAGRR&
FFFLIFIKWREFEGNTSCSRKNRSSARLSSALSKNNVWSCVCSYFS
2 o FPDKVVGDLTQQTATGPLNTRLDHTIN&
MASLNVQVAASVAGSRVKPQSSSTVASRDMAGLQFS
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Note: First six sequences are ORFs starting from met to stop codon, the
next two sequences were also identified as ORFs from the beginning of
the sequence to the stop codon and the last ORF is the sequence starting
from met to the end of the sequence. ORF analysis conducted using
GeneRunner version 3.05 software by Hastings Software, Inc.
