U.S. patent application number 12/546450 was filed with the patent office on 2010-01-14 for p450rai-2 (p450 cytochrome 26b), encoding nucleic acid molecules and methods and uses thereof.
This patent application is currently assigned to CYTOCHROMA INC.. Invention is credited to Glenville Jones, Martin P. Petkovich, Heather Ramshaw, Jay A. White.
Application Number | 20100008902 12/546450 |
Document ID | / |
Family ID | 37103192 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100008902 |
Kind Code |
A1 |
White; Jay A. ; et
al. |
January 14, 2010 |
P450RAI-2 (P450 CYTOCHROME 26B), ENCODING NUCLEIC ACID MOLECULES
AND METHODS AND USES THEREOF
Abstract
The present invention provides a novel all-trans-RA inducible
all-trans-RA metabolizing cytochrome P450, P450RAI-2, that is
predominantly expressed in the brain, cerebellum in particular. It
is also expressed in normal and tumour lung tissue and in breast
cancer cells and may have a correlation with lung and breast
cancer. Human P450RAI-2 show 42% amino acid identity to human
P450RAI-1 and when transfected into COS-1 cells causes the rapid
conversion of all-trans-RA into more polar metabolites including
the inactive products 4-oxo-RA, 4-OH-RA and 18-OH-RA. P450RAI-2, as
with P450RAI-1, is also inducible in certain cultured cell lines
exposed to all-trans-RA. Methods for and uses of the new
polynucleotide, polypeptide, fragments thereof and inhibitors
thereof, include the treatment of dermatological disorders, cancer
and certain brain disorders.
Inventors: |
White; Jay A.; (Newmarket,
CA) ; Petkovich; Martin P.; (Kingston, CA) ;
Jones; Glenville; (Kingston, CA) ; Ramshaw;
Heather; (Napanee, CA) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
233 SOUTH WACKER DRIVE, 6300 SEARS TOWER
CHICAGO
IL
60606-6357
US
|
Assignee: |
CYTOCHROMA INC.
Markham
CA
|
Family ID: |
37103192 |
Appl. No.: |
12/546450 |
Filed: |
August 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11498250 |
Aug 3, 2006 |
7579329 |
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12546450 |
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10433485 |
Nov 13, 2003 |
7115558 |
|
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PCT/CA01/01805 |
Dec 17, 2001 |
|
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11498250 |
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PCT/CA00/01493 |
Dec 15, 2000 |
|
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10433485 |
|
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60171110 |
Dec 16, 1999 |
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60178314 |
Jan 27, 2000 |
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Current U.S.
Class: |
424/130.1 ;
435/325; 435/366; 435/6.1; 435/6.12; 435/6.13; 435/6.18; 435/7.21;
435/7.23 |
Current CPC
Class: |
C07K 16/30 20130101;
G01N 2800/52 20130101; A61P 35/00 20180101; A01K 2217/05 20130101;
Y10S 435/81 20130101; G01N 33/57423 20130101; C07K 16/40 20130101;
C12N 9/0077 20130101; G01N 33/573 20130101 |
Class at
Publication: |
424/130.1 ;
435/325; 435/366; 435/7.21; 435/7.23; 435/6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/06 20060101 C12N005/06; C12N 5/08 20060101
C12N005/08; G01N 33/53 20060101 G01N033/53; G01N 33/574 20060101
G01N033/574; C12Q 1/68 20060101 C12Q001/68; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method for inhibiting P450RAI-2 induced retinoic acid
hydroxylation in an organism or an in vitro system by administering
a polyclonal antibody to a cytochrome P450 retinoic acid
metabolizing peptide comprising the amino acid sequence of SEQ. ID.
No. 5 to the organism or the in vitro system.
2. The method of claim 1 wherein the organism is human.
3. The method of claim 1 wherein the organism is being treated for
a disease selected from the group consisting of cancer, actinic
keratosis, oral leukoplakia, a secondary tumor of the head and/or
neck, a non-small cell lung carcinoma, a basal cell carcinoma,
acute promyelocytic leukemia, skin cancer, and a premalignancy
associated actinic keratosis, acne, psoriasis and/or
ichthyosis.
4. The method of claim 3 wherein the disease is acute promyelocytic
leukemia.
5. The method of claim 3 wherein the disease is lung cancer.
6. The method of claim 5 wherein the lung cancer is non-small cell
lung cancer.
7. A method for the screening of the expression of P450RAI-2 in a
sample, comprising exposing the sample to a polyclonal antibody to
a cytochrome P450 retinoic acid metabolizing peptide comprising the
amino acid sequence of SEQ. ID. NO. 5 and assaying for the presence
P450RAI-2 and antibody interaction.
8. The method of claim 7 wherein the antibody is a labeled antibody
to enable detection of binding and non-binding to a P450RAI-2
substrate.
9. The method of claim 7 wherein the assay is an ELISA assay.
10. The method of claim 7 for diagnosing non-small lung cell
carcinoma in a patient.
11. The method of claim 8 for diagnosing non-small lung cell
carcinoma in a patient.
12. The method of claim 9 for diagnosing non-small lung cell
carcinoma in a patient.
13. A kit for determining the presence of a first nucleic acid
molecule of SEQ. ID NO. 4, the kit comprising a second nucleic acid
molecule capable of hybridizing with at least a portion of a first
nucleic acid molecule under high stringency conditions, in which
the second nucleic acid molecule is linked to a reporter system
wherein the reporter system produces a detectable response when a
predetermined amount of the first and second molecules are
hybridized together.
14. The kit of claim 13 wherein the second nucleic acid molecule is
at least 5 bases in length, or at least about 10 bases in length,
or at least about 15 bases in length, or at least about 20 bases in
length, or at least about 25 bases in length, or at least about 30
bases in length, or at least about 35 bases in length, or at least
about 40 bases in length, or at least about 45 bases in length, or
at least about 50 bases in length.
15. The kit of claim 13 for the diagnosis of non-small lung cell
carcinoma in a patient.
16. The kit of claim 14 for the diagnosis of non-small lung cell
carcinoma in a patient.
Description
PRIOR APPLICATIONS
[0001] This is a division of U.S. application Ser. No. 11/498,250
filed Aug. 3, 2006, now U.S. Pat. No. 7,115,558, which is a
continuation of U.S. application Ser. No. 10/433,485 filed Nov. 13,
2003, which is a national stage of International Application No.
PCT/US/CA01/01805 filed Dec. 17, 2001, which is a
continuation-in-part of PCT/CA00/01493 filed Dec. 15, 2000, which
claims priority to U.S. Provisional Patent Application No.
60/178,314 filed Jan. 27, 2000, and U.S. Provisional Patent
Application No. 60/171,110 filed Dec. 16, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel gene encoding a
polypeptide that is a member of the cytochrome P450 family. More
particularly the present invention relates to a polynucleotide
encoding the novel polypeptide, to an antisense polynucleotide
thereof and to fragments thereof. The invention further relates to
the novel cytochrome P450 as well as to vectors, host cells and
antibodies to the polypeptide and the recombinant methods for
producing the same. Uses and methods relating to any of the
foregoing are also included within the scope of the invention.
BACKGROUND OF THE INVENTION
Cytochrome P450s
[0003] The cytochromes P450 comprise a large gene superfamily that
encodes over 500 distinct heme-thiolate proteins that catalyze the
oxidation of drugs and numerous other compounds in the body [Nelson
et al., (1996); Guengerich (1991)]. Since there are at least 500
different cytochrome P450 enzymes, it is of considerable interest
in the pharmaceutical and other fields to identify which of these
enzymes are most important in the metabolism of individual
compounds. There are now numerous examples of adverse drug-drug
interactions and side effects that can now be understood in terms
of the cytochrome P450 enzymes.
[0004] P450 proteins are ubiquitous in living organisms, and have
been identified in bacteria, yeast, plants and animals [Nelson et
al (1996); and Nelson, (1999a)]. The P450 enzymes catalyze the
metabolism of a wide variety of drugs, xenobiotics carcinogens,
mutagens, and pesticides, and are responsible for the bioactivation
of numerous endogenous compounds including steroids,
prostaglandins, bile acids and fatty acids body [Nelson et al.,
(1996); Guengerich (1991); Nebert et al., (1989)].
[0005] Cytochrome P450 metabolism of xenobiotics can result in
detoxification of toxic compounds by their conjugation into
excretable forms or can result in activation of compounds into
metabolites that are toxic, mutagenic, or carcinogenic. Many
steroids are deactivated by cytochrome P450-catalyzed
oxidation.
Vitamin A and Retinoic Acid
[0006] Vitamin A metabolism gives rise to several active forms of
retinoic acid (RA) which are involved in regulating gene expression
during development, regeneration, and in the growth and
differentiation of epithelial tissues. [Maden, 1992; Chambon, 1995;
Mangelsdorf, 1995; Gudas, 1994; Lotan, 1995; Morriss-Kay, 1996] RA
has been linked to apoptosis, or programmed cell death in a number
of cell types; and to have anticarcinogenic and antitumoral
properties [Lotan, 1996].
[0007] Early studies of retinol deficiency indicated a correlation
between vitamin A depletion and a higher incidence of cancer and
increased susceptibility to chemical carcinogenesis [Chytil, 1984].
Several animal models have been used to demonstrate the
effectiveness of retinoids in suppressing carcinogenesis in a
variety of tissues including skin, mammary epithelia, oral cavity,
aerodigestive tract, liver, bladder and prostate [Moon, 1994].
These studies have led to the preventative use of retinoids to
treat premalignant lesions including actinic keratosis and oral
leukoplakia, as well as in the prevention of secondary tumors of
the head and neck and the recurrence of non-small cell lung
carcinomas, and basal cell carcinomas [Hong, 1994; Lippman, 1995].
RA itself has been found to be useful therapeutically, notably in
the treatment of cancers, including acute promyelocytic leukemia
(APL), tumors of the head and neck, and skin cancer, as well as in
the treatment of skin disorders such as the premalignancy
associated actinic keratoses, acne, psoriasis and ichthyosis. There
is evidence that the effectiveness of RA as an anti-tumor agent is
at least partially due to induction of cellular differentiation
and/or inhibition of proliferation [Lotan, 1996]. Studies over the
past several years indicate that a high proportion of patients with
acute promyelocytic leukemia (APL) achieve complete remission after
a short period of treatment with all-trans RA. Unfortunately, this
high rate of remission is in most cases brief. Following relapse,
patients are clinically resistant to further treatment with RA
[Warrell, 1994; Warrell, et al., 1994; Chomienne, 1996; Muindi,
1992]. The nature of this resistance is unknown. Interestingly,
leukemic cells taken from patients exhibiting clinical resistance
to RA have been shown to be sensitive to the differentiating action
of RA when grown in vitro [Muindi, 1992; Muindi, 1994]. This
suggests that pharmacokinetic mechanisms may account for the
acquired resistance to RA. This possibility is supported by studies
showing that peak plasma concentrations of RA were much higher in
patients after initial administration than in patients treated
following relapse. This decrease in peak plasma RA concentration
was accompanied by a 10-fold increase in urinary 4-oxo-retinoic
acid concentration. In addition, ketoconazole, a broad spectrum
inhibitor of cytochrome P450 function was shown to modulate RA
pharmacokinetics in vivo [Muindi, 1992; Muindi, 1994]. It is
therefore likely that RA increases the rate of its own metabolism
which in turn results in the inability to sustain effective
therapeutic doses of RA. Therapeutic administration of RA can
result in a variety of undesirable side effects and it is therefore
important to establish and maintain the minimal requisite doses of
RA in treatment. For example, RA treatments during pregnancy can
lead to severe teratogenic effects on the fetus. Adverse reactions
to RA treatment also include headache, nausea, chelitis, facial
dermatitis, conjunctivitis, and dryness of nasal mucosa. Prolonged
exposure to RA can cause major elevations in serum triglycerides
and can lead to severe abnormalities of liver function, including
hepatomegaly, cirrhosis and portal hypertension.
[0008] RA metabolism may also account for the lack of response of
certain tumors to RA treatment. For example, recent studies have
shown that cytochrome P450 inhibitors that block RA metabolism,
resulting in increased tissue levels of RA, may be useful
therapeutic agents in the treatment of prostate cancer [Wouters,
1992; De Coster, 1996]. Thus RA metabolizing cytochrome P450s may
be useful targets for the treatment of a number of different types
of cancer.
[0009] The classical view of vitamin A metabolism holds that all
trans-RA, the most active metabolite is derived from conversion of
retinol to retinaldehyde to RA through two oxidation steps and that
RA is further metabolized to the polar derivatives 4-OH RA and
4-oxo RA [Blaner, 1994; Napoli, 1995; Formelli, 1996; Napoli,
1996]. It is unknown whether the 4-oxo- and 4-OH-metabolites are
simply intermediates in the RA catabolic pathway or whether they
can also have specific activities which differ from those of
all-trans RA and 9-cis RA. Pijnappel et al. [Pijnappel, 1993] have
shown that, in Xenopus, 4-oxo-RA can efficiently modulate
positional specification in early embryos and exhibits a more
potent ability to regulate Hoxb-9 and Hoxb-4 gene expression than
all-trans RA. 4-oxo-RA has been found to bind to retinoic acid
receptor-.beta. (RAR-.beta.) with affinity comparable to all-trans
RA [Pijnappel, 1993] but poorly to RAR-.gamma. [Reddy, 1992],
suggesting that this metabolite exhibits some receptor selectivity.
4-oxo-RA also binds to cellular retinoic acid binding protein
(CRABP) but with an affinity slightly lower than that of all-trans
RA [Fiorella, 1993]. Takatsuka et al. [Takatsuka, 1996] have shown
that growth inhibitory effects of RA correlate with RA metabolic
activity but it is unknown whether there is a causal relationship
between production of RA metabolites and growth inhibition. The
asymmetric distribution of these metabolites in developing embryos
suggests that they may be preferentially sequestered or generated
by tissue specific isomerases [Creech Kraft, 1994]. The normal
balance of these metabolites is dependent upon rate of formation
from metabolic precursors, retinol and retinaldehyde [Leo, 1989],
and rate of catabolism. Little is presently known about the enzymes
involved in this metabolic scheme, in particular the catabolism of
RA.
[0010] The catabolism of RA is thought to be initiated by
hydroxylation either at the C4-, or C18-position of the
.beta.-ionone ring of RA [Napoli, 1996]. The C4-hydroxylation step
is mediated by cytochrome P450 activity, as judged by the ability
of broad spectrum P450 inhibitors such as ketoconazole and
liarazole to block 4-hydroxylation [Williams, 1987, Van Wauwe,
1988; Van Wauwe, 1990, Van Wauwe, 1992, Wouters, 1992]. In certain
tissues, including testis, skin and lung and in numerous cell
lines, such as NIH3T3 fibroblasts, HL 60 myelomonocytic leukemic
cells, F9 and P19 murine embryonal carcinoma cell lines and MCF7,
RA metabolism can be induced by RA pretreatment [Frolik, 1979,
Roberts, 1979a and b; Duell, 1992; Wouters, 1992]. Studies
involving targeted disruption of RAR genes in F9 cells suggest that
RAR-.alpha. and RAR-.gamma. isoforms may play a role in regulating
the enzymes responsible for this increased metabolism [Boylan,
1995].
[0011] The glucuronidation of RA is a significant metabolic step in
the inactivation of RA [Blaner, 1994; Formelli, 1996]. The
elimination of RA may require oxidation to 4-oxo, followed by
conjugation to form the 4-oxo all-trans RA glucuronide. This is
supported by studies in both primates and humans showing that the
4-oxo RA glucuronide is the only retinoid conjugate found in urine
[Muindi, 1992; Muindi, 1994]. The fact that following RA therapy,
4-oxo RA is not present or barely detectable in serum, suggests
that oxidation may be the rate limiting step in this process.
[0012] It has recently been shown that 4-oxoretinol (4-oxo-ROL) can
have greater biological activity than retinol. The 4-oxo-ROL is
inducible by RA in F9 and P19 mouse teratocarcinoma cells [Blumberg
et al., 1995; Achkar et al., 1996].
[0013] It is known that zebrafish fins regenerate through an RA
sensitive process which utilizes many gene regulatory pathways
involved in early vertebrate development [White, 1994; Akimenko,
1995a & b].
Cytochrome P450s and Retinoic Acid Metabolism
[0014] In 1979, Roberts et al., [Roberts (1979a] first postulated
that the catabolism of retinoic acid (RA) was mediated by a
cytochrome P450 enzyme. Several P450s have since been shown to
metabolize RA, including P450 proteins from human, zebrafish and
mouse. For example, human P450RAI, which is induced by RA,
metabolizes RA to more poplar derivatives including 4-hydroxy
retinoic acid (4-OHRA) and 4-oxo retinoic acid (4-oxo RA) [White et
al. (1996a)]. Since RA is useful as an antitumor agent, it is
desirable to maintain high tissue levels of RA. Thus, cytochrome
P450 inhibitors that block RA metabolism, resulting in increased
tissue levels of RA, may be useful therapeutic agents in the
treatment of cancers, such as prostate cancer [Wouters et al.,
(1992); and De Coster et al., (1996)].
[0015] International Patent Publication No. WO 97/49815, published
Dec. 31, 1997, describes a family retinoid metabolizing proteins,
CYP26A, including proteins from human, zebrafish and mouse and
their coding sequences. This earlier publication is incorporated
herein in its entirety. cDNAs encoding a cytochrome P450-dependent
enzyme (P450RAI) which is induced by RA have been cloned and
characterized from zebrafish and the protein metabolizes RA to more
polar derivatives including 4-hydroxy retinoic acid (4-OH RA) and
4-oxo retinoic acid (4-oxo RA) [White et al., 1996a]. The
identification of P450RAI gene is an important step in the
understanding of RA signaling but its presence has been known since
Roberts et al. (1979a) first postulated that the catabolism of RA
was mediated by a P450 enzyme [Frolik et al., 1979; Roberts et al.,
1979a]. More recently, the isolation of cDNAs which encode the
full-length human and mouse P450RAI orthologs whose expression,
like that of the fish cytochrome, is highly inducible by RA has
been achieved [Fujii et al., 1997; Ray et al., 1997]. Human and
mouse genomic P450RAI-1 sequences are identified herein as SEQ ID
NOS: 15 and 16. The mouse sequence encoding P450RAI-1 is identified
herein as SEQ ID NO: 17. Homologs have also been isolated from
human, mouse, chick and xenopus all exhibiting a high degree of
sequence conservation [Abu-Abed et al., 1998; Hollermann et al.,
1998; White et al., 1997]. There is extensive identity between the
human and fish P450RAI genes which overall is 68% at the amino acid
level (over 90% between mouse and human).
[0016] MCF7 cells have been shown to have RA inducible RA
metabolism [Butler and Fontana, 1992; Wouters et al., 1992]. The
expression of P450RAI in these cells is dependent on the continuous
presence of RA [White et al., 1997]. This suggests that P450RAI
regulation by RA forms an autoregulatory feedback loop that
functions to limit local concentrations of RA, such that when
normal physiological levels of RA are exceeded, induction of
P450RAI acts to normalize RA levels. The inducible expression of
P450RAI in mouse embryos also suggests that a similar
autoregulatory mechanism may limit exposure to RA sensitive tissues
during development [Iulianella et al., 1999].
Retinoic Acid, Cytochrome p450 and Embryonic Development
[0017] All-trans-RA is a critical regulator of gene expression
during embryonic development and in the maintenance of adult
epithelial tissues [Gudas, et al. (1994); Lotan, R. M. (1995);
Lotan, R. (1996); Morriss-Kay, G. M. (1996)]. The effects of
all-trans-RA are mediated by heterodimers of nuclear receptors for
retinoic acid (RARs) and retinoid-X-receptors, which are regulated
by the 9-cis isomer of RA. Three different subtypes exist for each
of these receptors (RAR.alpha., .beta. and .gamma.; RXR RAR
.alpha., .beta. and .gamma.) which individually are expressed in a
tissue specific manner but collectively can be found in essentially
all cell types, both during embryonic development and in the adult
[Chambon, P. (1995).]. The activity of RA in these tissues is
controlled, to a large extent, by enzymes involved in its synthesis
from retinaldehyde (ALDH-1 and RALDH-2) and its catabolism to 4-OH,
4-oxo and 18-OH products (P450RAI) [White J. A., et al. (1997);
Iulianella, A. et al. (1999); McCaffery P. et al., (1999)
Niederreither, K. et al. (1999) Swindell E., et al. (1999)].
[0018] The present inventor and others have shown that P450RAI-1
(CYP26A) from zebrafish, mouse, human, chick and xenopus which is
responsible for the metabolism of active all-trans-RA to inactive
polar metabolites including 4-OH-RA, 4-oxo-RA and 18-OH-RA [White
J., et al. (1997); Swindell E., et al. (1999); White, J. &
Petkovich, M. (1996); Abu-Abed, et al. (1998); Fujii, H. et al.
(1997); Ray, W. et al. (1997); Hollermann, T et al. (1998)].
P450RAI-1 expression can be induced by all-trans-RA pre-treatment
in multiple tissues, and cell types, and this expression is
concomitant with increased all-trans-RA catabolism. In MCF7 cells,
all all-trans-RA suggesting a feedback-loop mechanism is dependent
on the continued presence of all-trans-RA suggesting a
feedback-loop mechanism for the regulation of all-trans-RA levels
[White J., et al. (1997)]. Inducible expression of P450RAI-1 has
also been observed in vivo in zebrafish, chick, xenopus and mouse
embryos suggesting that this autoregulatory feedback-loop plays an
important role in balancing all-trans-RA levels in certain
developing tissues.
[0019] Studies from several groups show that tissues such as neural
folds in chick embryos [Swindell E., et al. (1999)], caudal
neuroepithelia [Iulianella, A et al. (1999); Fujii, H. et al.
(1997)] and developing retina [McCaffery P. et al. (1999)] from
mouse express P450RAI-1 constitutively thus forming a barrier to
all-trans-RA exposure. Comparison of the expression patterns of
RALDH-2 and P450RAI-1 in these models suggest that these enzymes
act together to form regions of RA synthesis and activity (where
RALDH-2 is expressed). RALDH-2 expressing tissues have been shown
to contain retinoid activity as measured by both retinoid
responsive reporter gene activity and direct measurement of RA
levels from tissue extracts; by similar analyses, P450RAI-1
expressing tissues do not [Iulianella, A et al. (1999); McCaffery
P. et al. (1999)]. In addition, over expression of P450RAI-1 in
xenopus embryos has been shown to abrogate the teratogenic effects
of exogenously applied RA, consistent with a catabolic role for its
enzyme [Hollermann, T et al. (1998)].
SUMMARY OF THE INVENTION
[0020] The present inventors have identified and characterized a
novel cytochrome P450 that can metabolize retinoic acid, preferably
all-trans retinoic acid, (hereinafter referred to as P450RAI-2) and
the nucleic acid sequence encoding therefor. In one embodiment the
novel cytochrome P450 metabolizes all-trans retinoic acid from into
more polar metabolites.
[0021] In one embodiment the P450RAI-2 is a mammalian P450RAI-2,
and preferably a murine (such as comprising amino acid sequence
depicted in SEQ. ID. NO. 11 or 17), rat (such as that comprising
SEQ. ID. NO. 27) or human P450RAI-2 (such as that of SEQ. ID. NO.
5). In an another embodiment the P450RAI-2 is a zebrafish P450RAI-2
(such as that comprising SEQ. ID. NO. 12). In another embodiment,
the invention provides functional homologs of the above-noted
sequences, or functional homologous fragments thereof.
[0022] Although, the P450RAI-2 and encoding nucleic acid sequence
of the invention can be isolated and characterized from a number of
different tissues such as spleen, kidney, skeletal muscle, brain,
liver, retina, heart or small intestine, it is preferably isolated
and characterized from brain cells, such as from the cerebellum,
cerebral cortex, medulla, occipital pole, frontal lobe and temporal
lobe, and most preferably from the cerebellum. In another
embodiment, P450RAI-2 is isolated from lung cells, preferably
tumour lung cells, such as human lung tissue or SKMES cells.
[0023] Accordingly, the present invention provides an isolated
nucleic acid molecule comprising a sequence encoding a P450RAI-2,
preferably a human P450RAI-2 (such as SEQ. ID. NO. 4 or 28 or
degenerate or corresponding RNA sequences (where T is U) thereof)
or a murine P450RAI-2 (such as comprising any one of SEQ. ID. NO.
17-26 or degenerate or corresponding RNA sequences (where T is U))
or fragments thereof, preferably fragments encoding functional
fragments of P450RAI-2.
[0024] In a preferred embodiment, an isolated nucleic acid molecule
is provided having: (a) a nucleic acid sequence as shown in SEQ ID
NOS: 4, 5, 28, 27, 7, 18, 26, 21, 22, 23, 24, or 25 where T can
also be U; (b) nucleic acid sequences complementary to (a); (c)
nucleic acid sequences which are homologous to (a) and (b); (d) a
nucleic acid molecule differing from any of the nucleic acid
molecules of (a) to (c) in codon sequences due to the degeneracy of
the genetic code; or (e) a fragment of (a) to (d) that is at least
15 bases, preferably 20 to 30 bases, and which will hybridize to
(a) to (c) under stringent hybridization conditions.
[0025] The present invention also includes the P450RAI-2
polypeptide itself. Accordingly, the invention provides a
polypeptide having an amino acid sequence of a P450RAI-2.
Preferably, the invention provides a polypeptide having either the
human (SEQ ID NO: 5) or a homologous mouse sequence or fragment
thereof. The invention also comprises peptides comprising fragments
of the amino acid sequence of SEQ ID NO: 5. Preferably the
fragments comprise amino acid sequences of SEQ ID NOS: 11 or 12 or
encoded by nucleic acid sequences SEQ ID NOS. 18, 26, or 21 to 25.
In another embodiment the fragments preferably comprise at least 14
amino acid residues and are most preferably antigenic or
immunogenic. In one embodiment the invention provides peptides
encoded by a nucleic acid sequence of SEQ ID NO: 4 or fragments
thereof and to an antisense nucleic acid molecule to all or part of
the nucleic acid molecule encoding P450RAI-2.
[0026] In another embodiment, homologs, analogs, and modified
proteins of the invention as described herein are encompassed
within the scope of this invention.
[0027] In one embodiment, the invention also provides a nucleic
acid molecule of the invention operationally linked to an
expression control sequence in a suitable expression vector. In
another embodiment, the expression vector comprising the nucleic
acid molecule of the invention is capable of being activated to
express the peptide which is encoded by the nucleic acid molecule
and is capable of being transformed or transfected into a suitable
host cell. Such transformed or transfected cells are also
encompassed with the scope of this invention.
[0028] The invention also provides a method of preparing a
P450RAI-2 protein of the invention utilizing a nucleic acid
molecule of the invention. In one embodiment, a method for
preparing a P450RAI-2 protein of the invention is provided
comprising: transforming a host cell with a recombinant expression
vector comprising a nucleic acid sequence of the invention; (b)
selecting transformed host cells from untransformed host cells; (c)
culturing a selected transformed host cell under conditions which
allow expression of the protein; and (d) isolating the protein.
[0029] The invention also encompasses an antibody specific for one
or more epitopes of a protein of the invention, such as a peptide
specific antibody or a polyclonal antibody, and more preferably a
monoclonal antibody. The invention also encompasses methods for
preparing the antibodies. Preferably the epitopes are selected from
the group consisting of SEQ ID NOS: 5, 11, 12, or those encoded by
nucelic acid sequences SEQ ID NOS; 4, 6, 8, 18, 21-25, 27 or 28 or
immunogenic fragments thereof.
[0030] The invention also includes a method for detecting a disease
or medical condition associated with P450RAI-2 expression or RA
metabolism in an animal. "A disease or medical condition associated
with P450RAI-2 expression" as used herein means any disease that
can be affected or characterized by the level of P450RAI-2
expression. This includes, without limitation, diseases affected
by, high, normal, reduced or non-existent expression of P450RAI-2
or expression of mutated P450RAI-2. A disease or medical condition
associated with P450RAI-2 expression includes diseases associated
with RA metabolism such as cell cycle regulation, particularly cell
growth and apoptosis, for instance cancer, dysplasia, autoimmune
disease, dermatological disorders and disabilities associated with
high order brain functions, such as learning and memory. The method
comprises assaying for the P450RAI-2 from a sample, such as a blood
sample, a biopsy, or other cellular or tissue sample, from an
animal susceptible of having such a disease. In one embodiment, the
method comprises contacting the sample with an antibody of the
invention that binds P450RAI-2, and measuring the amount of
antibody bound to P450RAI-2 in the sample, or unreacted antibody.
In another embodiment, the method involves detecting the presence
of a nucleic acid molecule having a sequence encoding a P450RAI-2,
comprising contacting the sample with a nucleotide probe which
hybridizes with the nucleic acid molecule, preferably mRNA or cDNA
to form a hybridization product under conditions which permit the
formation of the hybridization product, and assaying for the
hybridization product.
[0031] The invention further includes a kit for detecting a disease
or condition associated with P450RAI-2 expression in a sample
comprising an antibody of the invention, preferably a monoclonal
antibody. Preferably directions for its use is also provided. The
kit may also contain reagents that are required for binding of the
antibody to a P450RAI-2 protein in the sample.
[0032] The invention also provides a kit for detecting the presence
of a nucleic acid molecule having a sequence encoding a polypeptide
related to or analogous to a polypeptide of the invention,
comprising a nucleotide probe which hybridizes with the nucleic
acid molecule, reagents required for hybridization of the
nucleotide probe with the nucleic acid molecule, and directions for
its use.
[0033] The invention further provides a method of treating or
preventing a medical condition disease associated with P450RAI-2 or
RA expression comprising administering an effective amount of an
agent that activates, simulates or inhibits P450RAI-2 expression,
as the situation requires, to an animal in need thereof. In one
embodiment, P450RAI-2, a therapeutically active fragment thereof,
or an agent which activates or simulates P450RAI-2 expression is
administered to the animal in need thereof to treat cancer,
dysplasia, autoimmune disease or dermatological disorders or to
improve high order brain functions or dermatological disorders that
is related to the presence of too much RA or all-trans-RA. In
another embodiment the disease is associated with over expression
of P450RAI-2 or too little RA, or all-trans-RA or apoptosis, and
the method of treatment comprises administration of an effective
amount of an agent that inhibits P450RAI-2 expression such as an
antibody to P450RAI-2, a mutation thereof, or an antisense nucleic
acid molecule to all or part of the P450RAI-2 gene.
[0034] In another embodiment, the invention provides pharmaceutical
compositions comprising the nucleic acid molecules of proteins,
antibodies, vectors or cells of the invention and a
pharmaceutically acceptable carrier.
[0035] In another embodiment, the invention further provides a
method for identifying modulators of P450RAI-2 expression or
P450RAI-2 activity. In one embodiment, this involved incubating the
gene under conditions conducive to gene expression with a potential
modulator of gene expression and then assaying, for instance by
southern, northern or western blot for the presence of expression
of the gene and the effect of the modulator on such expression
(increased expression, inhibited expression or no effect on
expression). In another embodiment, the effect of the modulator on
such expression is determined by comparison to various controls run
without the potential modulator and/or with the potential modulator
but without the gene. Expression of P450RAI-2 can also be done by
radioactive or other labeling for such expression or by antibody
screening, such as using the P450RAI-2 polyclonal antibody
discloses herein.
[0036] In yet another embodiment, the invention further provides a
method of rational drug design.
[0037] In one embodiment the invention provides methods or uses of
the proteins and nucleic acid molecules of the invention, such as
in relation to medical treatment (where inhibitors of P450RAI-2,
such as antibodies or antisense molecules can be used to treat a
condition associated with too much RA metabolism (e.g., too little
RA), preferably all-trans RA metabolism. In another embodiment,
P450RAI-2 gene or protein or stimulators or functional fragments or
homologs thereof can be used in cases where there is too much RA or
all-trans-RA.
[0038] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only. Various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 is a schematic diagram showing the 6 exons of human
cytochrome p450RAI-2 on human clone having GenBank Accession No.
AC007002 (SEQ ID NO:3). The numbers above the schematic diagram
indicate the amino acid regions of the exons and the numbers below
the schematic refer to nucleotide positions on the human
sequence.
[0040] FIG. 2 Human P450RAI-2 cDNA and amino acid sequence (SEQ ID
NO: 28) comprising the coding sequence and the 3' untranslated
sequence. The full-length cDNA clone was isolated from human retina
cDNA. The deduced primary sequence using single-letter amino acid
codes, identifies a 512 amino acid protein which is shown directly
below the corresponding nucleotide sequence. Numbers on the right
refer to the nucleotide positions.
[0041] FIG. 3 Amino acid sequence comparison between human
P450RAI-2 (SEQ. ID. NO. 5) and human P450RAI-1 (amino acid sequence
shown in SEQ. ID. NO. 13). Predicted amino acid sequences were
aligned using Omiga software (Oxford Molecular, CA). Identical
amino acids are highlighted by the black boxes with white letters.
Conservatively substituted amino acids are indicated by the open
boxes. Gaps, indicated by the dashes, have been introduced in
several regions in order to optimize amino acid identity between
the two proteins. Overall the two protein sequences show 42%
identity at the amino acid level. Numbers on the right hand side
refer to the corresponding amino acid positions.
[0042] FIG. 4 Expression of P450RAI-2 in human tissues. RNA samples
from 76 normal human tissues were probed for expression of
P450RAI-2 transcripts using a commercially available dot blot (A).
Signals representing P450RAI-2 transcripts are observed in three
samples, a2, b2 and hl representing left and right cerebellum and
pons respectively. Sample a1 is whole brain and a11 is fetal brain
for comparison. Control hybridization with a human ubiquitin probe
(B) shows differences in RNA loading. Northern blot analyses for
expression of P450RAI-2 expression in human brain (D). 2 .mu.g poly
A+mRNA each from eight different human brain tissues was used to
analyze P450RAI-2 expression. Transcripts at approximately 5 Kb
corresponding to P450RAI-2 are indicated, with the highest levels
of expression seen in cerebellum.
[0043] FIG. 4C shows the location of the different human mRNAs on
the blot used in FIG. 4A.
[0044] FIG. 5 shows northern blot analysis of human cell lines
probed with an .alpha.-[.sup.32P]-dATP labeled probe having the
sequence identified as SEQ ID NO:4 SKMES; CALU-1 and MCF-7 (A).
Cells were treated with DMSO, all-trans retinoic acid, 9-cis
retinoic acid or 13-cis retinoic acid. Cells were exposed to
10.sup.-6 M final concentrations of each retinoid dissolved in
DMSO. Transcripts corresponding to P450RAI-2 are indicated by the
arrowhead. The blot was also probed with the human .beta.-actin
probe to control for RNA loading of the gel, shown in the middle
panel (B). The 18S and 28S portions of the mRNA as seen on the
ethidium bromide stained agarose gel are shown in the bottom panel
(C).
[0045] FIG. 6 is similar to FIG. 5 except that the cells,
HPK1A-RAS, were treated with either DMSO or all-trans retinoic
acid.
[0046] FIG. 7 All-trans-RA induction of P450RAI-2 expression.
Northern blot analysis (A) of cultured cells treated with
all-trans-RA or vehicle (DMSO). P450RAI-2 hybridizing transcripts
are identified. The positions of 28S and 18S ribosomal RNA are
indicated along with the ethidium bromide stained gel showing the
relative abundance of RNA in all samples. Multiple other cell lines
were tested using semiquantitative RT-PCR and southern blotting
(B). Transcripts for P450RAI-2 are regulated in a cell specific
manner. P450RAI-2 is constitutively expressed in SK-MES-1 and SW900
and possibly WTE; inducible in MCF-7, HPK1a-ras and HeLa; and
undetectable in SK-Luci-6, V79 or NB4. A timeline of induction of
mRNAs, for P450RAI-2 in HPK1a-ras cells (C). Within 2 hours of
addition of all-trans-RA maximal induction of transcripts for
P450RAI-2 is observed. GAPDH controls are shown below for each RNA
sample analyzed.
[0047] FIG. 8A shows the total aqueous soluble radioactivity
measured using aliquots of the aqueous soluble extracts from media
alone, as well as cells transfected with pcDNA3.1, pcDNA3.1-CYP26A
(human) (i.e., P450RAI) or pcDNA-P450RAI-2. Aqueous soluble
extracts were subjected to .beta.-scintillation counting.
[0048] FIG. 8B shows a summary of the peak integration analysis
performed after HPLC on the organic soluble radioactivity. Samples
were the same as in FIG. 8A. Cells transfected with either
pcDNA3.1-CYP26A (human) or pcDNA3.1-P450RAI-2 show a decrease in
all-trans retinoic acid substrate with a concomitant increase in
the production of 4-OH-retinoic acid, 4-oxo-retinoic acid and more
polar peaks.
[0049] FIG. 9 shows the same analysis as FIG. 8B except that cells
were treated with 1 .mu.M non-radioactive all-trans-retinoic
acid.
[0050] FIG. 10 shows a control HPLC trace from media alone in the
absence of the COS-1 cells. An all-trans-retinoic acid substrate
peak is observed at 20.808 minutes. Peaks at 1.409 and 1.874 are
contaminants not characteristic of retinoids. The top panel shows
photo diode array detection of the peaks. Retinoids have
characteristic UV maxima in the range of about 320 to about 380
nM.
[0051] FIG. 11 is similar to FIG. 10 except that the sample is from
COS-1 cells transfected with the pcDNA3.1 plasmid alone.
[0052] FIG. 12 is similar to FIG. 10 except that the sample is from
COS-1 cells transfected with pcDNA3.1-P450RAI. Multiple more polar
peaks characteristic of retinoids are generated in these cells.
Peaks which co-elute with standards for 4-OH-retinoic acid and
4-oxo-retinoic acid were observed at 8.191 and 9.661 minutes
respectively. Additional peaks which have not been characterized
are also evident.
[0053] FIG. 13 is similar to FIG. 12 except that the sample is from
COS-1 cells transfected with pcDNA3.1-P450RAI-2.
[0054] FIG. 14 shows HPLC analysis of all-trans-RA metabolism.
COS-1 cells were transiently transfected with pcDNA3.1-P450RAI-2
(A), pcDNA3.1-P450RAI-1 (B) or pcDNA3.1 alone (C) and exposed to
.mu.M all-trans-RA for 3 hours. Incubation with RA was followed by
total lipid extraction of the media and subsequent HPLC analysis
using a reverse-phase system. Expression of either P450RAI-2 or
P450RAI-1 in cells causes disappearance of all-trans-RA substrate
(compare C to A or B) in addition to the generation of more polar
metabolic products. Identities of the retinoids. labeled as
all-trans-RA, 4-OH-RA, 4-oxo-RA and 18-OH-RA (A and B) were
verified by co-elution with known standards and comparison of the
spectral properties of the peaks using photodiode array detection.
Multiple polar peaks are observed in P450RAI-2 expressing cells
exposed to all-trans-RA. Peaks labeled 1-4 (A) have spectral
properties characteristic of retinoids, specifically a UV maxima of
between 320 and 350 nm. The exact identity of these polar
metabolites remains to be established.
[0055] FIG. 15 shows metabolism of all-trans-RA. COS-1 cells
transiently transfected with pcDNA3.1-P450RAI-2, pcDNA3.1-P450RAI-1
or pcDNA3.1 alone and exposed to 100 nM [.sup.3H]all-trans-RA for 3
hours. Conversion of all-trans-RA to aqueous-soluble metabolites is
observed in P450RAI-2 or P450RAI-1 expressing cells compared to
pcDNA vector or media alone control samples (A). Using HPLC
analysis with subsequent .beta.-scintillation counting was used to
evaluate the lipid-soluble retinoids generated from P450RAI-2 or
P450RAI-1 expressing cells. Fractions from chromatography were
grouped into three regions, all-trans-RA substrate, 4-OH region and
polar region allowed quantification of the retinoid metabolites.
Levels of all-trans-RA remaining after incubation were
substantially decreased in P450RAI-2 or P450RAI-1 transfected cells
compared to controls (B). Additionally, there is an increase in
radioactivity representing both the 4-OH and polar regions (B).
[0056] FIG. 16 A-E is competitive inhibition of P450RAI-mediated
all-trans-RA metabolism. COS 1 cells transiently transfected with
pcDNA3.1-P450RAI-2, pcDNA3.1-P450RAI-1 or pcDNA3.1 alone were used
to assess the ability of several retinoids to competitively inhibit
all-trans-RA metabolism. Cells were exposed to 2 nM
[.sup.3H]all-trans-RA with increasing concentrations of unlabelled
competitor and assayed for generation of aqueous soluble
radioactivity. Panels C and D show cells expressing P450RAI-2 with
panels A and B show cells expressing P450RAI-1. 9-cis-RA and
13-cis-RA (top panels 16A and 16C) are less effective than
all-trans-RA as competitors for either P450RAI-2 or P450RAI-1. FIG.
16E shows [.sup.3H]-RA-metabolism inhibition assay using microsomes
prepared from Hela-CYP26B (stable) cells. Microsomes (5 micrograms)
were incubated with [.sup.3H]-RA and increased concentration o
funlabeled all-trans-RA, 9-cis-RA, 13-cis-RA and ketoconazole.
Retinol and retinal were similarly tested (bottom panels 16B and
16D) and found to be less effective competitors. For comparison,
the nonspecific cytochrome P450 inhibitor ketoconazole is shown in
each panel.
[0057] FIG. 17 shows Northern blot (A) and RT-PCR (B) results for
total RNA samples from the various cell samples designated.
[0058] FIG. 18 shows expression of P450RAI-2 in 8.0 and 8.5 dpc
mouse embryos. (A) 8.0 dpc lateral view. No apparent staining. (B)
8.0 dpc, dorsal view. Staining at anterior end of neutral folds.
(C) 8.5 dpc, lateral view. Expression is evident, possibly in
presumptive rhombomeres 2, 5, and 6. (D) 8.5 dpc, dorsal view,
rhombomere expression of RAI2 is clearly evident. TL: Tail; YS Yolk
Sac; NF: Folds; pr: Presumptive Rhombomeres.
[0059] FIG. 19 shows expression of P450RAI2 in 9.0 and 10.5 dpc
mouse embryos. (A) 9.0 dpc, lateral view. Specific staining is
visible in the eye, and rhombomeres 5 and 6. Diffuse staining is
visible where the hind bud is beginning to form. (B) 9.0 dpc,
dorsal view. Rhombomeres 5 and 6 show RAI2 expression. (C) 10.5
dpc, lateral view. The otic vesicle and eye are stained. (D) 10.5
dpc, dorsal view. Specific staining is observed in both otic
vesicles as well as as the hind limb bud. HL: hind limb bud; OV:
Otic Vesicle.
[0060] FIG. 20 shows expression of P450RAI2 staining in 11.5 dpc
mouse embryo (A) 11.5 dpc, laterial view, staining is visible in
both the fore and hindlimb bud. (B) 11.5 dpc, ventral view.
Expression of RAI2 in both limb buds (C) Close up of forelimb bud,
showing a lack of expression in the apical ectodermal ridge FL:
Forelimb bud; HL: Hindlimb bud; AR: Apical Ectodermal Ridge.
[0061] FIG. 21 shows P450RAI2 staining in embryos treated with
Retinoic Acid (A) 8.5 dpc, lateral view, staining is observed in
rhombomere 5 and the tail mesoderm as indicated by the arrow. (B)
8.5 dpc, dorsal view clearly showing P450RAI2 expression in
rhombomere 5. (C) 9.5 dpc, lateral view, expression of P450RAI2 is
observed in rhombomeres 5 and 6, the developing hindlimb, somites
and posterior mesoderm. (D) 9.5 dpc, dorsal view, expression is
evident in rhombomeres 3, 5 and 6 and in trunk ectoderm as
indicated by the arrow. r: rhombomere; HL; Limb bud.
[0062] FIG. 22 shows P450RAI2 expression in 11.5 dpc embryos
treated with retinoic acid. (A) 11.5 dpc, lateral view, P450RAI2
expression is observed in both the developing hind and fore limb.
(B) 11.5 dpc, ventral view, as in embryos untreated with retinoic
acid, P450RAI2 expression is not observed in the aptical ectodermal
ridge.
[0063] FIG. 23 shows P450RAI2 (CYP26B), P450RAI-1 (CYP26A) and
P450RAI-3 (CYP26C) expression in normal (N) and tumour (T) lung
tissue. B-actin is a control.
[0064] FIG. 24 is an immunoblot indicating P450RAI-2 specificity of
the polyclonal antibody made against CYP26B (P450RAI-2) as compared
to other cytochromes (CYP26A, CYP4F11, and CYP4X1).
[0065] FIG. 25 shows the RT-PCR assay of CYP26A (P450RAI-1) and
CYP26B (P450RAI-2) expression in human retina and MCF-7 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0066] Retinoids, particularly all-trans retinoic acid
(all-trans-RA), are potent regulators of cell differentiation, cell
proliferation and apoptosis. As noted above, the role of
all-trans-RA during development and in the maintenance of adult
tissues has been well established. The control of all-trans-RA in
cells and tissues is regulated by the balance between its
biosynthesis and its catabolism to inactive metabolites. The
cytochrome P450 enzyme P450RAI-1 is partially responsible for this
inactivation of all-trans-RA.
[0067] The present inventors have now identified, cloned and
characterized a second related enzyme, here termed P450RAI-2, which
is also involved in the specific inactivation or metabolism of
all-trans-RA. A cDNA has been isolated from human retina and
sequenced. A protein encoded by the cDNA has been expressed and
shown to have the ability to metabolize all-trans-RA to more polar
metabolites. The metabolites so formed include products that have
been oxidized at the 4-position of the .beta.-ionone ring,
including the corresponding acid hydroxylated at the 4-position of
the .beta.-ionone ring. The mRNA has been found to be inducible in
multiple cell types including but not limited to human lung and
human skin upon the addition of all-trans retinoic acid. It has
also been shown to be expressed in human brain tissue and breast
and lung cancer cells.
[0068] The present inventors herein show that transiently
transfected P450RAI-2 can convert all-trans-RA to more polar
metabolites including 4-oxo, 4-OH, and 18-OH-retinoic acid.
Competition experiments with other retinoids suggest that
all-trans-RA is the preferred substrate, but not necessarily the
only retinoid substrate. The high level of expression of P450RAI-2
particularly in the cerebellum and pons of human adult brain,
suggests a unique role for this enzyme in the protection of
specific tissues from exposure to retinoids.
[0069] Homologs of the cloned sequence have also been identified.
Partial nucleotide sequences have been determined for the mouse,
rat and zebrafish P450RAI-2 coding sequence.
DEFINITIONS
[0070] The term "retinoids" as used herein means a group of
compounds which includes retinoic acid, vitamin A (retinol) and a
series of natural and synthetic derivatives that can exert profound
effects on development and differentiation in a wide variety of
systems. For purposes of this disclosure "retinoid" is also
intended to encompass an equivalent thereof having the same
functional characteristics which may be produced, for example, by
computational chemistry.
[0071] The following standard abbreviations for the amino acid
residues are used throughout the specification: A, Ala--alanine; C,
Cys--cysteine; D, Asp--aspartic acid; E, Glu--glutamic acid; F,
Phe--phenylalanine; G, Gly--glycine; H, His--histidine; I,
Ile--isoleucine; K, Lys--lysine; L, Leu--leucine; M,
Met--methionine; N, Asn--asparagine; P, Pro--proline; Q,
Gln--glutamine; R, Arg--arginine; S, Ser--serine; T,
Thr--threonine; V, Val--valine; W, Trp--tryptophan; Y,
Tyr--tyrosine; and p.Y., P.Tyr--phosphotyrosine.
I. Nucleic Acid Molecules of the Invention
[0072] The present invention provides an isolated nucleic aid
molecule comprising a sequence encoding a cytochrome P450RAI-2
polypeptide.
[0073] The term "isolated" refers to a nucleic acid substantially
free of cellular material or culture medium when produced by
recombinant DNA techniques, or chemical precursors, or other
chemicals when chemically synthesized.
[0074] The term "nucleic acid molecule" is intended to include
unmodified DNA or RNA or modified DNA or RNA. For example, the
nucleic acid molecules or polynucleotides of the invention can be
composed of single- and double stranded DNA, DNA that is a mixture
of single- and double-stranded regions, single- and double stranded
RNA, and RNA that is a mixture of single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically double stranded or a mixture of
single- and double stranded regions. In addition, the P450RAI-2
nucleic acid molecules can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. The nucleic acid
molecules of the invention may also contain one or more modified
bases or DNA or RNA backbones modified for stability or for other
reasons. "Modified" bases include, for example, tritiated bases and
unusual bases such as inosine. A variety of modifications can be
made to DNA and RNA; thus "nucleic acid molecule" embraces
chemically, enzymatically, or metabolically modified forms. The
term "polynucleotide" shall have a corresponding meaning.
[0075] One aspect of the present invention is thus an isolated
nucleic acid molecule comprising a nucleotide sequence selected
from the group consisting of:
[0076] (a) a nucleotide sequence as shown in SEQ ID NOS: 4 or 28,
but preferably SEQ ID NO: 4, wherein T can also be U;
[0077] (b) nucleotide sequences complementary to (a);
[0078] (c) nucleotide sequences which are homologous to (a) or
(b);
[0079] (d) a nucleotide sequence differing from any of the
nucleotide sequences of (a) to (c) in codon sequences due to the
degeneracy of the genetic code; or
[0080] (e) a fragment of (a) to (d) that is at least 15 bases,
preferably 20 to 30 bases, and which would hybridize to (a) to (d)
under stringent hybridization conditions. In another embodiment,
the fragment is at least 35, 40, 45, 50, 55, or 60 or more
nucleotides in length. In another embodiment the fragment is
capable of oxidizing a retinoid. In yet another embodiment it
preferably has at least about 55 percent homology with the protein
encoded by a nucleotide sequence of (a) or (b).
[0081] In another embodiment, the nucleotide sequence in (a) above
is a human P450RAI-2 sequence. In one embodiment, the nucleic acid
sequence can be used as a probe or a primer, such as for PCR, like
SEQ ID NOS: 9, 10, 29, 30, 33, 37 or 38, more preferably SEQ. ID.
Nos. 37 and/or 38. In yet another embodiment, the nucleotide
sequence of (a) above is selected from the group consisting of: a
mouse P450RAI-2 sequence, such as SEQ ID NO: 6, 18, 26, 21, 22, 23,
24 or 25; a rat P450RAI-2 sequence, SEQ ID NO: 27; or a zebrafish
P450RAI-2 sequence, SEQ ID NO: 8.
[0082] In one embodiment, the nucleic acid molecule of the
invention encodes a protein that is capable of oxidizing a
retinoid, preferably retinoic acid, preferably at the 4-position of
the .beta.-ionone ring. In another embodiment the nucleic acid
molecule of the invention encodes a protein having SEQ ID NO: 5. In
another embodiment, the nucleotide sequence inhibits P450RAI-2
expression, such as certain complimentary sequences to SEQ ID NO
4.
[0083] In one embodiment of the invention, the nucleic acid
molecule consists of any of the nucleotide sequences described
herein.
[0084] In all of the sequences referred to above, T can also be U.
As previously stated, the invention includes isolated DNA molecules
having such sequences of nucleotides, and RNA molecules having such
sequences. The invention thus includes isolated mRNA transcribed
from DNA having such a sequence. The invention further encompasses
nucleic acid molecules that differ from any of the nucleic acid
molecules of the invention in codon sequences due to the degeneracy
of the genetic code.
[0085] The invention also encompasses nucleic acid sequences or
molecules that are analogs of the nucleic acid sequences and
molecules described herein. The term "a nucleic acid sequence which
is an analog" means a nucleic acid sequence which has been modified
as compared to the sequences described herein, such as sequences of
(a), (b), (c), (d), or (e), above wherein the modification does not
alter the utility of the sequences described herein. The modified
sequence or analog may have improved properties over the sequence
shown in (a), (b), (c), (d) or (e). One example of a modification
to prepare an analog is to replace one of the naturally occurring
bases (i.e. adenine, guanine, cytosine or thymidine) of the
sequence shown in SEQ ID NO: 4, 6, 8-10, 18, or 21-28 with a
modified base such as such as xanthine, hypoxanthine,
2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo
uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza
thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,
8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl
adenine and other 8-substituted adenines, 8-halo guanines, 8 amino
guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine
and other 8-substituted guanines, other aza and deaza uracils,
thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl
uracil and 5-trifluoro cytosine.
[0086] Another example of a modification is to include modified
phosphorous or oxygen heteroatoms in the phosphate backbone, short
chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages in the nucleic
acid molecule shown in SEQ ID NOS: 4, 6, 8-10, 18, or 21-28. For
example, the nucleic acid sequences may contain phosphorothioates,
phosphotriesters, methyl phosphonates, and phosphorodithioates.
[0087] A further example of an analog of a nucleic acid molecule of
the invention is a peptide nucleic acid (PNA) wherein the
deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is
replaced with a polyamide backbone which is similar to that found
in peptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA
analogs have been shown to be resistant to degradation by enzymes
and to have extended lives in vivo and in vitro. PNAs also bind
stronger to a complimentary DNA sequence due to the lack of charge
repulsion between the PNA strand and the DNA strand. Other nucleic
acid analogs may contain nucleotides containing polymer backbones,
cyclic backbones, or acyclic backbones. For example, the
nucleotides may have morpholino backbone structures (U.S. Pat. No.
5,034,506). The analogs may also contain groups such as reporter
groups, a group for improving the pharmacokinetic or
pharmacodynamic properties of nucleic acid sequence.
[0088] It will be appreciated that the invention includes nucleic
acid molecules encoding truncations of proteins of the invention,
and analogs and homologs of proteins of the invention and
truncations thereof, as described below. It will further be
appreciated that variant forms of nucleic acid molecules of the
invention which arise by alternative splicing of an mRNA
corresponding to a cDNA of the invention are encompassed by the
invention. The invention further includes biologically active
fragments of the nucleic acid molecules of the invention. Such
fragments would include, but is not necessarily limited to any
nucleic acid molecules which are beneficial in the modulation,
including but not limited to simulation, inhibition or stimulation
of P450RAI-2 activity or P450RAI-2 expression, or in the
identification or production of such agents.
[0089] A nucleic acid molecule of the invention can encode a
protein (polypeptide) having a homology of at least about 50, at
least about 55, or more preferably at least about 58 percent with a
protein encoded by SEQ ID NO:4 or the full length anti-sense
sequence thereto. The level of homology, according to various
aspects of the invention is at least about 60 percent; at least
about 63 percent; at least about 65 percent; at least about 68
percent; at least about 70 percent; at least about 73 percent; at
least about 75 percent; at least about 78 percent; at least about
80 percent; at least about 83 percent; at least about 85 percent;
at least about 88 percent; at least about 90 percent; at least
about 93 percent; at least about 95 percent; or at least about 98
percent. Methods for aligning the sequences to be compared and
determining the level of homology between the sequences are
described in detail below.
[0090] In another aspect, the present invention includes a fragment
of the nucleotide sequence encoding P450RAI-2 (SEQ ID NO:4). Such a
fragment can find usefulness as a probe or depending on the
fragment may even have biological activity itself. The complement
of the probe can find utility in, for example, manufacture of the
probe or inhibition of any activity of the fragment, as the case
may be. In a particular use, the probe can be used to determine the
presence of an RNA molecule in a sample which might, or might not,
also include an RNA molecule encoding P450RAI-1. Such a probe would
generally be 20 nucleotides long or be at least 20 nucleotides
long. The probe could also be 25, 30, 35, 40, 45, 50, 55, 60 or
more nucleotides in length and the probe can include the full
length of the complement to the sequence to which it is intended to
bind. The sequence of the probe would also be sufficiently
distinguishable from any portion of the sequence encoding P450RAI-1
that it would not cross-hybridize to a significant extent to a
nucleotide sequence that encodes P450RAI-1, or portion thereof,
particularly to an RNA encoding P450RAI-1. Such a probe would thus
be sufficiently different from any sequence of contiguous
nucleotides selected from the nucleotide sequence encoding human
P450RAI-1 (SEQ ID NO:13) that there is no more than about 60%
homology between the two sequences when the two sequences are
directly aligned with each other. More preferably, the percent
homology would be less than about 55%, or less than about 50%, or
less than about 45%, or even less than about 40%. Certain probes of
the invention are selected so as span borders between introns of
the coding sequence as determined from the genomic sequence (SEQ ID
NO:3). A preferred P450RAI-2 specific probe or PCR primer are SEQ.
ID. NOs. 37 and/or 38.
[0091] The invention includes the method of determining the
presence of a nucleic acid molecule encoding P450RAI-2 in a sample
containing RNA isolated from a human cell, using such a probe.
[0092] In the context of this specification, the term "conserved"
describes similarity between sequences. The degree of conservation
between two sequences can be determined by optimally aligning the
sequences for comparison. Here, sequences were aligned using the
Omiga software program, Version 1.13. (Oxford Molecular Group,
Inc., Campbell, Calif.). The Omiga software uses the Clustal W
Alignment algorithms [Higgins et al., 1989; Higgins et al., 1991;
Thompson et al. 1994] Default settings used are as follows: Open
gap penalty 10.00; Extend gap penalty 0.05; Delay divergent
sequence 40 and Scoring matrix--Gonnet Series. Percent identity or
homology between two sequences is determined by comparing a
position in the first sequence with a corresponding position in the
second sequence. When the compared positions are occupied by the
same nucleotide or amino acid, as the case may be, the two
sequences are conserved at that position. The degree of
conservation between two sequences is often expressed, as it is
here, as a percentage representing the ratio of the number of
matching positions in the two sequences to the total number of
positions compared.
[0093] In one particular aspect, the present invention is a nucleic
acid molecule of any preceding claim which encodes a protein that
is a conservatively substituted variant of the protein encoded by
the nucleotide sequence of SEQ ID NO:4.
[0094] Further, it will be appreciated that the invention includes
nucleic acid molecules comprising nucleic acid sequences having
substantial sequence homology with the nucleic acid sequences as
shown in SEQ ID NO. 4, 6, 8-10, 18, or 21-28 and fragments thereof.
The term "sequences having substantial sequence homology" means
those nucleic acid sequences that have slight or inconsequential
sequence variations from these sequences, i.e., the sequences
function in substantially the same manner to produce functionally
equivalent proteins. The variations may be attributable to local
mutations or structural modifications.
[0095] Nucleic acid sequences having substantial homology include
nucleic acid sequences having at least 85%, preferably 90-95%
identity with the nucleic acid sequence as shown in SEQ ID NO: 4,
6, 8-10, 18, or 21-28. However, the invention is not to be
restricted by this homology, for instance, nucleic acid sequences
having at least a 50% homology with the sequence shown in 4, 6,
8-10, 18, or 21-28 are also encompassed within the scope of the
present invention.
[0096] "Stringent hybridization conditions" is a term known to a
person skilled in the art. Appropriate stringency conditions which
promote nucleic acid hybridization, for example, 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. are know. The
following examples are found in Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6: For 50 ml
of a first suitable hybridization solution, mix together 24 ml
formamide, 12 ml 20.times.SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml
100.times.Denhardt's solution, 2.5 ml deionized H.sub.2O, 10 ml 50%
dextran sulfate, and 0.5 ml 10% SDS. A second suitable
hybridization solution can be 1% crystalline BSA (fraction V), 1 mM
EDTA, 0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% SDS. The salt
concentration in the wash step can be selected from a low
stringency of about 2.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. Both of these
wash solutions may contain 0.1% SDS. In addition, the temperature
in the wash step can be increased from low stringency conditions at
room temperature, about 22.degree. C., to high stringency
conditions, at about 65.degree. C. The cited reference gives more
detail, but appropriate wash stringency depends on degree of
homology and length of probe. If homology is 100%, a high
temperature (65.degree. C. to 75.degree. C.) may be used. If
homology is low, lower wash temperatures must be used. However, if
the probe is very short (<100 bp), lower temperatures must be
used even with 100% homology. In general, one starts washing at low
temperatures (37.degree. C. to 40.degree. C.), and raises the
temperature by 3-5.degree. C. intervals until background is low
enough not to be a major factor in autoradiography.
[0097] Isolated nucleic acid molecules having sequences which
differ from the nucleic acid sequence shown in SEQ ID NO: 4, 6,
8-10, 18, or 21-28 due to degeneracy in the genetic code are also
within the scope of the invention. Such nucleic acids encode
functionally equivalent proteins but differ in sequence from the
above mentioned sequences due to degeneracy in the genetic
code.
[0098] An isolated nucleic acid molecule of the invention which
comprises DNA can be isolated by preparing a labelled nucleic acid
probe based on all or part of the nucleic acid sequences as shown
in SEQ ID NO: 4, 6, 8-10, 18, or 21-28 and using this labelled
nucleic acid probe to screen an appropriate DNA library (e.g. a
cDNA or genomic DNA library). For example, a genomic library
isolated can be used to isolate a DNA encoding a novel protein of
the invention by screening the library with the labelled probe
using standard techniques. Nucleic acids isolated by screening of a
cDNA or genomic DNA library can be sequenced by standard
techniques.
[0099] An isolated nucleic acid molecule of the invention which is
DNA can also be isolated by selectively amplifying a nucleic acid
encoding a novel protein of the invention using the polymerase
chain reaction (PCR) methods and cDNA or genomic DNA. It is
possible to design synthetic oligonucleotide primers from the
nucleic acid sequence as shown in SEQ ID NO: 4, 6, 8-10, 18, or
21-28 for use in PCR. A nucleic acid can be amplified from cDNA or
genomic DNA using these oligonucleotide primers and standard PCR
amplification techniques. The nucleic acid so amplified can be
cloned into an appropriate vector and characterized by DNA sequence
analysis. It will be appreciated that cDNA may be prepared from
mRNA, by isolating total cellular mRNA by a variety of techniques,
for example, by using the guanidinium-thiocyanate extraction
procedure of Chirgwin et al., Biochemistry, 18, 5294 5299 (1979).
cDNA is then synthesized from the mRNA using reverse transcriptase
(for example, Moloney MLV reverse transcriptase available from
Gibco/BRL, Bethesda, Md., or AMV reverse transcriptase available
from Seikagaku America, Inc., St. Petersburg, Fla.).
[0100] An isolated nucleic acid molecule of the invention which is
RNA can be isolated by cloning a cDNA encoding a novel protein of
the invention into an appropriate vector which allows for
transcription of the cDNA to produce an RNA molecule which encodes
a protein of the invention. For example, a cDNA can be cloned
downstream of a bacteriophage promoter, (e.g., a T7 promoter) in a
vector, cDNA can be transcribed in vitro with T7 polymerase, and
the resultant RNA can be isolated by standard techniques.
[0101] A nucleic acid molecule of the invention may also be
chemically synthesized using standard techniques. Various methods
of chemically synthesizing polydeoxynucleotides are known,
including solid-phase synthesis which, like peptide synthesis, has
been fully automated in commercially available DNA synthesizers
(See e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al.
U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and
4,373,071).
[0102] Determination of whether a particular nucleic acid molecule
encodes a novel protein of the invention may be accomplished by
expressing the cDNA in an appropriate host cell by standard
techniques, and testing the activity of the protein using the
methods as described herein. A cDNA having the activity of a novel
protein of the invention so isolated can be sequenced by standard
techniques, such as dideoxynucleotide chain termination or
Maxam-Gilbert chemical sequencing or by automated DNA sequencing,
to determine the nucleic acid sequence and the predicted amino acid
sequence of the encoded protein.
[0103] The initiation codon and untranslated sequences of nucleic
acid molecules of the invention may be determined using currently
available computer software designed for the purpose, such as
PC/Gene (IntelliGenetics Inc., Calif.). Regulatory elements can be
identified using conventional techniques. The function of the
elements can be confirmed by using these elements to express a
reporter gene which is operatively linked to the elements. These
constructs may be introduced into cultured cells using standard
procedures. In addition to identifying regulatory elements in DNA,
such constructs may also be used to identify proteins interacting
with the elements, using techniques known in the art.
[0104] The sequence of a nucleic acid molecule of the invention may
be inverted relative to its normal presentation for transcription
to produce an antisense nucleic acid molecule. The term "antisense"
nucleic acid molecule is a nucleotide sequence that is
complementary to its target. Preferably, an antisense sequence is
constructed by inverting a region preceding or targeting the
initiation codon or an unconserved region. In another embodiment
the antisense sequence targets all or part of the mRNA or cDNA of
P450RAI-2. In particular, the nucleic acid sequences contained in
the nucleic acid molecules of the invention or a fragment thereof
may be inverted relative to its normal presentation for
transcription to produce antisense nucleic acid molecules. In one
embodiment the antisense molecules can be used to inhibit P450RAI-2
expression and/or RA metabolism,
[0105] The antisense nucleic acid molecules of the invention or a
fragment thereof, may be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed with mRNA or the native
gene e.g. phosphorothioate derivatives and acridine substituted
nucleotides. The antisense sequences may be produced biologically
using an expression vector introduced into cells in the form of a
recombinant plasmid, phagemid or attenuated virus in which
antisense sequences are produced under the control of a high
efficiency regulatory region, the activity of which may be
determined by the cell type into which the vector is
introduced.
[0106] The invention also provides nucleic acids encoding fusion
proteins comprising a novel protein of the invention and a selected
protein, or a selectable marker protein (see below).
II. Polypeptides of the Invention
[0107] The invention further contemplates an isolated P450RAI-2
protein. In an embodiment of the invention, an isolated protein is
provided which has the human amino acid sequence as shown in SEQ ID
NO. 5 or a fragment, preferably biologically active fragment,
thereof. The present invention also encompasses peptides encoded by
the nucleic acid sequence of SEQ ID NO: 4 and all embodiments
therefor as described in reference to the peptides in SEQ ID NO. 5
described below.
[0108] Within the context of the present invention, a protein of
the invention may in one embodiment include various structural
forms of the primary protein which retain biological activity of
the cytochrome P450RAI-2. For example, a protein of the invention
may be in the form of acidic or basic salts or in neutral form. In
addition, individual amino acid residues may be modified by
oxidation or reduction. The biological activity of a active
cytochrome P450RAI-2 is the ability to oxidize a retinoid. Such
activity can be tested for as described herein.
[0109] In addition to the full length amino acid sequence (SEQ ID
NO: 5), the proteins of the present invention may also include
truncations of the proteins, and analogs, and homologs of the
proteins and truncations thereof as described herein. Truncated
proteins may comprise peptides of at least 10 and preferably at
least fourteen amino acid residues.
[0110] In one embodiment, the invention provides a peptide fragment
of human SEQ ID NO: 5. In another embodiment the invention provides
a peptide having an amino acid sequence of the partial mouse
P450RAI-2 sequence, SEQ ID NO: 11 or Zebrafish sequence (SEQ ID NO:
12). In another embodiment the invention provides a peptide encoded
by any of the nucleic acid molecules of the invention as described
above. In yet another embodiment the invention provides an
antigenic immunogenic fragment of the proteins of the
invention.
[0111] Analogs of the proteins having the amino acid sequences
shown in SEQ ID NO: 5 and/or truncations thereof as described
herein, may include, but are not limited to an amino acid sequence
containing one or more amino acid substitutions, insertions, and/or
deletions. Amino acid substitutions may be of a conserved or
non-conserved nature. Conserved amino acid substitutions involve
replacing one or more amino acids of the proteins of the invention
with amino acids of similar charge, size, and/or hydrophobicity
characteristics. When only conserved substitutions are made the
resulting analog should be functionally equivalent. Non-conserved
substitutions involve replacing one or more amino acids of the
amino acid sequence with one or more amino acids which possess
dissimilar charge, size, and/or hydrophobicity characteristics.
[0112] Without the intention of being limited thereby, in one
embodiment it is preferable that substitutions of amino acids are
made that preserve the structure responsible for retinoid
metabolizing activity of the proteins disclosed herein.
Conservative substitutions are described in the patent literature,
as for example, in U.S. Pat. No. 5,264,558. It is thus expected,
for example, that interchange among non-polar aliphatic neutral
amino acids, glycine, alanine, proline, valine and isoleucine,
would be possible. Likewise, substitutions among the polar
aliphatic neutral amino acids, serine, threonine, methionine,
asparagine and glutamine could possibly be made. Substitutions
among the charged acidic amino acids, aspartic acid and glutamic
acid, could probably be made, as could substitutions among the
charged basic amino acids, lysine and arginine. Substitutions among
the aromatic amino acids, including phenylalanine, histidine,
tryptophan and tyrosine would also likely be possible. These sorts
of substitutions and interchanges are well known to those skilled
in the art. Other substitutions might well be possible. Of course,
it would also be expected that the greater the percentage of
homology, i.e., sequence similarity, of a variant protein with a
naturally occurring protein, the greater the retention of metabolic
activity. Of course, as protein variants having the activity of
P450RAI-2 as described herein are intended to be within the scope
of this invention, so are nucleic acids encoding such variants.
[0113] One or more amino acid insertions may be introduced into the
amino acid sequences shown in SEQ ID NO: 5. Amino acid insertions
may consist of single amino acid residues or sequential amino acids
ranging from 2 to 15 amino acids in length. For example, amino acid
insertions may be used to destroy target sequences so that the
protein is no longer active. This procedure may be used in vivo to
inhibit the activity of a protein of the invention.
[0114] Deletions may consist of the removal of one or more amino
acids, or discrete portions from the amino acid sequence shown in
SEQ ID NO: 5. The deleted amino acids may or may not be contiguous.
The lower limit length of the resulting analog with a deletion
mutation is about 10 amino acids, preferably 100 amino acids.
[0115] Analogs of the proteins of the invention may be prepared by
introducing mutations in the nucleotide sequence encoding the
protein. Mutations in nucleotide sequences constructed for
expression of analogs of a protein of the invention must preserve
the reading frame of the coding sequences. Furthermore, the
mutations will preferably not create complementary regions that
could hybridize to produce secondary mRNA structures, such as loops
or hairpins, which could adversely affect translation of the
mRNA.
[0116] Mutations may be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked
by restriction sites enabling ligation to fragments of the native
sequence. Following ligation, the resulting reconstructed sequence
encodes an analog having the desired amino acid insertion,
substitution, or deletion.
[0117] Alternatively, oligonucleotide-directed site specific
mutagenesis procedures may be employed to provide an altered gene
having particular codons altered according to the substitution,
deletion, or insertion required. Deletion or truncation of a
protein of the invention may also be constructed by utilizing
convenient restriction endonuclease sites adjacent to the desired
deletion. Subsequent to restriction, overhangs may be filled in,
and the DNA religated. Exemplary methods of making the alterations
set forth above are disclosed by Sambrook et al (Molecular Cloning:
A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
1989).
[0118] Insertions, deletions or substitution mutations of P450RAI-2
can be used to generate dominant negative forms of P450RAI-2 that
can act as transdominant repressors of P450RAI-2 activity.
[0119] The proteins of the invention also include homologs of the
amino acid sequence shown in SEQ ID NO: 5 and/or truncations
thereof as described herein. Such homologs are proteins whose amino
acid sequences are encoded by nucleic acid sequences that hybridize
under stringent hybridization conditions (see discussion of
stringent hybridization conditions herein) with a probe used to
obtain a protein of the invention. Homologs of a protein of the
invention will have the same regions which are characteristic of
the protein.
[0120] A homologous protein includes a protein with an amino acid
sequence having at least 60%, preferably at least 76%, preferably
80-90% identity with the amino acid sequence as shown in SEQ ID NO:
5.
[0121] The invention also contemplates isoforms of the proteins of
the invention. An isoform contains the same number and kinds of
amino acids as a protein of the invention, but the isoform has a
different molecular structure. The isoforms contemplated by the
present invention are those having the same properties as a protein
of the invention as described herein.
[0122] The present invention also includes a protein of the
invention conjugated with a selected protein, or a selectable
marker protein (see below) to produce fusion proteins.
Additionally, immunogenic portions of a protein of the invention
are within the scope of the invention. Immunogenic portions of a
protein is that portion that if administered to a patient can
induce an immune response and preferably an antibody response.
[0123] A further advantage may be obtained through chimeric forms
of the protein, as known in the art. A DNA sequence encoding the
entire protein, or a portion of the protein, could thus be linked,
for example, with a sequence coding for the C-terminal portion of
E. coli .beta.-galactosidase to produce a fusion protein.
GST-P450RAI-2 fusion proteins are described in the above examples.
An expression system for human respiratory syncytial virus
glycoproteins F and G is described in U.S. Pat. No. 5,288,630
issued Feb. 22, 1994 and references cited therein, for example.
[0124] The proteins of the invention (including truncations,
analogs, etc.) may be prepared using recombinant DNA methods. These
proteins may be purified and/or isolated to various degrees using
techniques known in the art. Accordingly, nucleic acid molecules of
the present invention having a sequence which encodes a protein of
the invention may be incorporated according to procedures known in
the art into an appropriate expression vector which ensures good
expression of the protein. Possible expression vectors include but
are not limited to cosmids, plasmids, or modified viruses (e.g.,
replication defective retroviruses, adenoviruses and
adeno-associated viruses), so long as the vector is compatible with
the host cell used. The expression "vectors suitable for
transformation of a host cell", means that the expression vectors
contain a nucleic acid molecule of the invention and regulatory
sequences, selected on the basis of the host cells to be used for
expression, which are operatively linked to the nucleic acid
molecule. "Operatively linked" is intended to mean that the nucleic
acid is linked to regulatory sequences in a manner which allows
expression of the nucleic acid.
[0125] The invention therefore contemplates a recombinant
expression vector of the invention containing a nucleic acid
molecule of the invention, or a fragment thereof, and the necessary
regulatory sequences for the transcription and translation of the
inserted protein-sequence. Suitable regulatory sequences may be
derived from a variety of sources, including bacterial, fungal, or
viral genes (For example, see the regulatory sequences described in
Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990). Selection of appropriate
regulatory sequences is dependent on the host cell chosen, and may
be readily accomplished by one of ordinary skill in the art.
Examples of such regulatory sequences include: a transcriptional
promoter and enhancer or RNA polymerase binding sequence, a
ribosomal binding sequence, including a translation initiation
signal. Additionally, depending on the host cell chosen and the
vector employed, other sequences, such as an origin of replication,
additional DNA restriction sites, enhancers, and sequences
conferring inducibility of transcription may be incorporated into
the expression vector. It will also be appreciated that the
necessary regulatory sequences may be supplied by the native
protein and/or its flanking regions.
[0126] The invention further provides a recombinant expression
vector comprising a DNA nucleic acid molecule of the invention
cloned into the expression vector in an antisense orientation. That
is, the DNA molecule is operatively linked to a regulatory sequence
in a manner which allows for expression, by transcription of the
DNA molecule, of an RNA molecule which is antisense to a nucleotide
sequence of the invention preferably comprising the nucleotides as
shown in SEQ ID NO: 4 or fragments thereof. Regulatory sequences
operatively linked to the antisense nucleic acid can be chosen
which direct the continuous expression of the antisense RNA
molecule.
[0127] The recombinant expression vectors of the invention may also
contain a selectable marker gene that facilitates the selection of
host cells transformed or transfected with a recombinant molecule
of the invention. Examples of selectable marker genes are genes
encoding a protein which confers resistance to certain drugs, such
as G418 and hygromycin. Examples of other markers which can be used
are: green fluorescent protein (GFP), .beta.-galactosidase,
chloramphenicol acetyltransferase, or firefly luciferase.
Transcription of the selectable marker gene is monitored by changes
in the concentration of the selectable marker protein such as
.beta.-galactosidase, chloramphenicol acetyltransferase, or firefly
luciferase. If the selectable marker gene encodes a protein
conferring antibiotic resistance such as neomycin resistance
transformant cells can be selected with G418. Cells that have
incorporated the selectable marker gene will survive, while the
other cells die. This makes it possible to visualize and assay for
expression of recombinant expression vectors of the invention and
in particular to determine the effect of a mutation on expression
and phenotype. It will be appreciated that selectable markers can
be introduced on a separate vector from the nucleic acid of
interest.
[0128] The recombinant expression or cloning vectors of the
invention may also contain genes which encode a fusion moiety which
provides increased expression of the recombinant protein; increased
solubility of the recombinant protein; and aid in the purification
of a target recombinant protein by acting as a ligand in affinity
purification. For example, a proteolytic cleavage site may be added
to the target recombinant protein to allow separation of the
recombinant protein from the fusion moiety subsequent to
purification of the fusion protein.
[0129] Recombinant expression vectors can be introduced into host
cells to produce a transformed host cell. The term "transformed
host cell" is intended to include prokaryotic and eukaryotic cells
which have been transformed or transfected with a recombinant
expression vector of the invention. The terms "transformed with",
"transfected with", "transformation" and "transfection" are
intended to encompass introduction of nucleic acid (e.g. a vector)
into a cell by one of many possible techniques known in the art.
Prokaryotic cells can be transformed with nucleic acid by, for
example, electroporation or calcium chloride mediated
transformation. Nucleic acid can be introduced into mammalian cells
via conventional techniques such as calcium phosphate or calcium
chloride co precipitation, DEAE-dextran-mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory press (1989)), and other such laboratory
textbooks.
[0130] Suitable host cells include a wide variety of prokaryotic
and eukaryotic host cells. For example, the proteins of the
invention may be expressed in bacterial cells such as E. coli,
insect cells (using baculovirus), yeast cells or mammalian cells,
COS1 cells. Other suitable host cells can be found in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1991).
[0131] The invention includes a microbial cell that contains and is
capable of expressing a heterologous a nucleic acid molecule having
a nucleotide sequence as broadly encompassed by the invention. The
heterologous nucleic acid molecule can be DNA.
[0132] Isolated DNA of the invention can be contained in a
recombinant cloning vector.
[0133] The invention includes a stably transfected cell line which
expresses any one or more proteins as broadly defined by the
invention.
[0134] The invention includes a culture of cells transformed with a
recombinant DNA molecule having a nucleotide sequence as broadly
encompassed by the invention. Such a culture of cells can be
eukaryotic. They can be piscine--particularly zebrafish,
mammalian--particularly human, rat or mouse, for example.
[0135] The invention is also a process for producing any protein as
broadly defined by the invention. The process includes such steps
as: [0136] preparing a DNA fragment including a nucleotide sequence
which encodes said protein; [0137] incorporating the DNA fragment
into an expression vector to obtain a recombinant DNA molecule
which includes the DNA fragment and is capable of undergoing
replication; [0138] transforming a host cell with said recombinant
DNA molecule to produce a transformant which can express said
protein; [0139] culturing the transformant to produce said protein;
and [0140] recovering said protein from resulting cultured
mixture.
[0141] More particularly, the invention provides a method of
preparing a purified protein of the invention comprising
introducing into a host cell a recombinant nucleic acid encoding
the protein, allowing the protein to be expressed in the host cell
and isolating and purifying the protein. Preferably, the
recombinant nucleic acid is a recombinant expression vector.
Proteins can be isolated from a host cell expressing the protein
and purified according to standard procedures of the art, including
ammonium sulfate precipitation, column chromatography (e.g. ion
exchange, gel filtration, affinity chromatography, etc.),
electrophoresis, and ultimately, crystallization [see generally,
"Enzyme Purification and Related Techniques", Methods in
Enzymology, 22, 233-577 (1971)].
[0142] Alternatively, the protein or parts thereof can be prepared
by chemical synthesis using techniques well known in the chemistry
of proteins such as solid phase synthesis [Merrifield 1964] or
synthesis in homogeneous solution [Houbenwycl, 1987].
III. Applications
1. Diagnostic Applications
[0143] The above nucleic acid and peptide molecules of the
invention can be used to diagnose a disease affected by P450RAI-2
expression, such as a disease or medical condition associated with
or where RA, P450RAI-2 or inhibitors thereof treatment may be
indicated. Examples of such conditions have been outlined herein,
such as diseases associated with angiogenesis the regulation of the
cell cycle or apoptosis, such as cancer, dysplasia, various
autoimmune diseases and conditions related to cerebellum or pond
functions, such as memory or learning. Determination of peptide or
nucleic acid expression levels could assist not only in identifying
a medical condition but in determining the appropriate course of
treatment.
(i) Nucleic Acids
[0144] The above described nucleic acid molecules of the invention,
allow those skilled in the art to construct nucleotide probes for
use in the detection of nucleotide sequences homologous to
P450RAI-2 or a fragment thereof in a sample.
[0145] Accordingly, the present invention also relates to a method
of detecting the presence of nucleic acid molecules encoding a
P450RAI-2 in a sample comprising contacting the sample under
hybridization conditions with one or more nucleotide probes which
hybridize to the nucleic acid molecules and are labelled with a
detectable marker, and, determining the degree of hybridization
between the nucleic acid molecule in the sample and the nucleotide
probe(s).
[0146] A nucleotide probe may be labelled with a detectable marker
such as a radioactive label which provides for an adequate signal
and has sufficient half life such as 32P, 3H, 14C or the like.
Other detectable markers which may be used include antigens that
are recognized by a specific labelled antibody, fluorescent
compounds, enzymes, antibodies specific for a labelled antigen, and
chemiluminescent compounds. An appropriate label may be selected
having regard to the rate of hybridization and binding of the probe
to the nucleotide to be detected and the amount of nucleotide
available for hybridization.
[0147] Hybridization conditions which may be used in methods of the
invention are known in the art and are described for example in
Sambrook J, Fritch E F, Maniatis T. In: Molecular Cloning, A
Laboratory Manual, 1989. (Nolan C, Ed.), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. The hybridization
product may be assayed using techniques known in the art. The
nucleotide probe may be labelled with a detectable marker as
described herein and the hybridization product may be assayed by
detecting the detectable marker or the detectable change produced
by the detectable marker.
[0148] A nucleic acid molecule of the invention also permits the
identification and isolation, or synthesis of nucleotide sequences
which may be used as primers to amplify a nucleic acid molecule of
the invention, for example, in a polymerase chain reaction (PCR)
which is discussed in more detail below. The primers may be used to
amplify the genomic DNA or mRNA of other P450RAI-2 genes or coding
sequences. The PCR amplified sequences can be examined to determine
the relationship between the various P450RAI-2 genes.
[0149] The length and bases of primers for use in a PCR are
selected so that they will hybridize to different strands of the
desired sequence and at relative positions along the sequence such
that an extension product synthesized from one primer when it is
separated from its template can serve as a template for extension
of the other primer into a nucleic acid of defined length. Primers
which may be used in the invention are oligonucleotides, i.e.,
molecules containing two or more deoxyribonucleotides of the
nucleic acid molecule of the invention which occur naturally as in
a purified restriction endonuclease digest or are produced
synthetically using techniques known in the art such as for example
phosphotriester and phosphodiester methods (See Good et al. Nucl.
Acid Res 4:2157, 1977) or automated techniques (See for example,
Conolly, B. A. Nucleic Acids Res. 15:15(7): 3131, 1987). The
primers are capable of acting as a point of initiation of synthesis
when placed under conditions which permit the synthesis of a primer
extension product which is complementary to a DNA sequence of the
invention, i.e., in the presence of nucleotide substrates, an agent
for polymerization such as DNA polymerase and at suitable
temperature and pH. Preferably, the primers are sequences that do
not form secondary structures by base pairing with other copies of
the primer or sequences that form a hair pin configuration. The
primer preferably contains between about 7 and 25 nucleotides.
[0150] The primers may be labelled with detectable markers which
allow for detection of the amplified products. Suitable detectable
markers are radioactive markers such as P-32, S-35, I-125, and H-3,
luminescent markers such as chemiluminescent markers, preferably
luminol, and fluorescent markers, preferably dansyl chloride,
fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-1,3
diazole, enzyme markers such as horseradish peroxidase, alkaline
phosphatase, .beta.-galactosidase, acetylcholinesterase, or
biotin.
[0151] It will be appreciated that the primers may contain
non-complementary sequences provided that a sufficient amount of
the primer contains a sequence which is complementary to a nucleic
acid molecule of the invention or oligonucleotide fragment thereof,
which is to be amplified. Restriction site linkers may also be
incorporated into the primers allowing for digestion of the
amplified products with the appropriate restriction enzymes
facilitating cloning and sequencing of the amplified product.
[0152] In an embodiment of the invention a method of determining
the presence of a nucleic acid molecule having a sequence encoding
a protein of the invention is provided comprising treating the
sample with primers which are capable of amplifying the nucleic
acid molecule or a predetermined oligonucleotide fragment thereof
in a polymerase chain reaction to form amplified sequences, under
conditions which permit the formation of amplified sequences and,
assaying for amplified sequences.
[0153] Polymerase chain reaction as used herein refers to a process
for amplifying a target nucleic acid sequence as generally
described in Innis et al, Academic Press, 1990 in Mullis et al.,
U.S. Pat. No. 4,863,195 and Mullis, U.S. Pat. No. 4,683,202.
Conditions for amplifying a nucleic acid template are described in
M. A. Innis and D. H. Gelfand, PCR Protocols, A Guide to Methods
and Applications M. A. Innis, D. H. Gelfand, J. J. Sninsky and T.
J. White eds, pp 3-12, Academic Press 1989.
[0154] The amplified products can be isolated and distinguished
based on their respective sizes using techniques known in the art.
For example, after amplification, a DNA sample can be separated on
an agarose gel and visualized, after staining with ethidium
bromide, under ultra violet (uv) light. DNA may be amplified to a
desired level and a further extension reaction may be performed to
incorporate nucleotide derivatives having detectable markers such
as radioactive labelled or biotin labelled nucleoside
triphosphates. The primers may also be labelled with detectable
markers as discussed above. The detectable markers may be analyzed
by restriction enzyme digestion and electrophoretic separation or
other techniques known in the art.
[0155] Conditions which may be employed in the methods of the
invention using PCR are those which permit hybridization and
amplification reactions to proceed in the presence of DNA in a
sample and appropriate complementary hybridization primers.
Conditions suitable for a polymerase chain reaction are generally
known in the art. For example, see M. A. Innis and D. H. Gelfand,
PCR Protocols, A guide to Methods and Applications M. A. Innis, D.
H. Gelfand, J. J. Sninsky and T. J. White eds, pp 3-12, Academic
Press 1989. To amplify DNA template strands, preferably, the PCR
utilizes polymerase obtained from the thermophilic bacterium
Thermus aquatics (Taq polymerase, GeneAmp Kit, Perkin Elmer Cetus)
or other thermostable polymerase.
[0156] It will be appreciated that other techniques such as the
Ligase Chain Reaction (LCR) and NASBA may be used to amplify a
nucleic acid molecule of the invention (Barney in "PCR Methods and
Applications", August 1991, Vol. 1(1), page 5, and European
Published Application No. 0320308, published Jun. 14, 1989, and
U.S. Pat. No. 5,130,238 to Malek).
(ii) Antibodies
[0157] A P450RAI-2 protein of the invention or antigenic portion
thereof can be used to prepare antibodies specific for the proteins
of the invention, preferably to a protein having SEQ ID NO: 5 or
partial sequence SEQ ID NO: 11 or 12. Antibodies can be prepared
which bind a distinct epitope in an unconserved region of the
protein. An unconserved region of the protein is one which does not
have substantial sequence homology to other proteins.
Alternatively, a region from a well-characterized domain can be
used to prepare an antibody to a conserved region of a protein of
the invention. Antibodies having specificity for a protein of the
invention may also be raised from fusion proteins.
[0158] Conventional methods can be used to prepare the antibodies.
For example, by using a peptide of a protein of the invention,
polyclonal antisera or monoclonal antibodies can be made using
standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can
be immunized with an immunogenic form of the peptide which elicits
an antibody response in the mammal. Techniques for conferring
immunogenicity on a peptide include conjugation to carriers or
other techniques well known in the art. For example, the peptide
can be administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum. Standard ELISA or other immunoassay procedures can
be used with the immunogen as antigen to assess the levels of
antibodies. Following immunization, antisera can be obtained and,
if desired, polyclonal antibodies isolated from the sera.
[0159] To produce monoclonal antibodies, antibody producing cells
(lymphocytes) can be harvested from an immunized animal and fused
with myeloma cells by standard somatic cell fusion procedures thus
immortalizing these cells and yielding hybridoma cells. Such
techniques are well known in the art, (e.g., the hybridoma
technique originally developed by Kohler and Milstein (Nature 256,
495-497 (1975)) as well as other techniques such as the human
B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72
(1983)); the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy
(1985) Allen R. Bliss, Inc., pages 77-96); and screening of
combinatorial antibody libraries (Huse et al., Science 246, 1275
(1989)). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with the peptide and
the monoclonal antibodies can be isolated. Therefore, the invention
also contemplates hybridoma cells secreting monoclonal antibodies
with specificity for a protein of the invention.
[0160] The term "antibody" as used herein is intended to include
fragments thereof which also specifically react with a protein of
the invention, or peptide thereof. Antibodies can be fragmented
using conventional techniques and the fragments screened for
utility in the same manner as described above. For example, F(ab')2
fragments can be generated by treating antibody with pepsin. The
resulting F(ab')2 fragment can be treated to reduce disulfide
bridges to produce Fab' fragments.
[0161] Chimeric antibody derivatives, i.e., antibody molecules that
combine a non-human animal variable region and a human constant
region are also contemplated within the scope of the invention.
Chimeric antibody molecules can include, for example, the antigen
binding domain from an antibody of a mouse, rat, or other species,
with human constant regions. Conventional methods may be used to
make chimeric antibodies containing the immunoglobulin variable
region which recognizes a P450RAI-2 protein of the invention (See,
for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81, 6851
(1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S.
Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi
et al., European Patent Publication EP171496; European Patent
Publication 0173494, United Kingdom patent GB 2177096B).
[0162] Monoclonal or chimeric antibodies specifically reactive with
a protein of the invention as described herein can be further
humanized by producing human constant region chimeras, in which
parts of the variable regions, particularly the conserved framework
regions of the antigen-binding domain, are of human origin and only
the hypervariable regions are of non-human origin. Such
immunoglobulin molecules may be made by techniques known in the art
(e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312
(1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et
al., Meth. Enzymol., 92, 3-16 (1982); and PCT Publication
WO92/06193 or EP 0239400). Humanized antibodies can also be
commercially produced (Scotgen Limited, 2 Holly Road, Twickenham,
Middlesex, Great Britain.)
[0163] Specific antibodies, or antibody fragments reactive against
a protein of the invention may also be generated by screening
expression libraries encoding immunoglobulin genes, or portions
thereof, expressed in bacteria with peptides produced from nucleic
acid molecules of the present invention. For example, complete Fab
fragments, VH regions and FV regions can be expressed in bacteria
using phage expression libraries (See for example Ward et al.,
Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281
(1989); and McCafferty et al. Nature 348, 552-554 (1990)).
[0164] The antibodies may be labelled with a detectable marker
including various enzymes, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin; an example of a
luminescent material includes luminol; and examples of suitable
radioactive material include S-35, Cu-64, Ga-67, Zr-89, Ru-97,
Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186,
Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. The antibodies
may also be labelled or conjugated to one partner of a ligand
binding pair. Representative examples include avidin-biotin and
riboflavin-riboflavin binding protein. Methods for conjugating or
labelling the antibodies discussed above with the representative
labels set forth above may be readily accomplished using
conventional techniques.
[0165] Antibodies reactive against P450RAI-2 proteins of the
invention (e.g., enzyme conjugates or labelled derivatives) may be
used to detect a protein of the invention in various samples, for
example they may be used in any known immunoassays which rely on
the binding interaction between an antigenic determinant of a
protein of the invention and the antibodies. Examples of such
assays are radioimmunoassays, western immunoblotting, enzyme
immunoassays (e.g., ELISA), immunofluorescence,
immunoprecipitation, latex agglutination, hemagglutination, and
histochemical tests. Thus, the antibodies may be used to identify
or quantify the amount of a protein of the invention in a
sample.
[0166] A sample may be tested for the presence or absence of a
P450RAI-2 by contacting the sample with an antibody specific for an
epitope of a P450RAI-2 protein which antibody is capable of being
detected after it becomes bound to a P450RAI-2 protein in the
sample, and assaying for antibody bound to a P450RAI-2 protein in
the sample, or unreacted antibody.
[0167] In a method of the invention a predetermined amount of a
sample or concentrated sample is mixed with antibody or labelled
antibody. The amount of antibody used in the method is dependent
upon the labelling agent chosen. The resulting protein bound to
antibody or labelled antibody may be isolated by conventional
isolation techniques, for example, salting out, chromatography,
electrophoresis, gel filtration, fractionation, absorption,
polyacrylamide gel electrophoresis, agglutination, or combinations
thereof.
[0168] The sample or antibody may be insolubilized, for example,
the sample or antibody can be reacted using known methods with a
suitable carrier. Examples of suitable carriers are Sepharose or
agarose beads. When an insolubilized sample or antibody is used
protein bound to antibody or unreacted antibody is isolated by
washing. For example, when the sample is blotted onto a
nitrocellulose membrane, the antibody bound to a protein of the
invention is separated from the unreacted antibody by washing with
a buffer, for example, phosphate buffered saline (PBS) with bovine
serum albumin (BSA).
[0169] When labelled antibody is used, the presence of a P450RAI-2
can be determined by measuring the amount of labelled antibody
bound to a protein of the invention in the sample or of the
unreacted labelled antibody. The appropriate method of measuring
the labelled material is dependent upon the labelling agent.
[0170] When unlabelled antibody is used in a method of the
invention, the presence of a P450RAI-2 can be determined by
measuring the amount of antibody bound to the P450RAI-2 using
substances that interact specifically with the antibody to cause
agglutination or precipitation. In particular, labelled antibody
against an antibody specific for a protein of the invention, can be
added to the reaction mixture. The antibody against an antibody
specific for a protein of the invention can be prepared and
labelled by conventional procedures known in the art which have
been described herein. The antibody against an antibody specific
for a protein of the invention may be a species specific
anti-immunoglobulin antibody or monoclonal antibody, for example,
goat anti-rabbit antibody may be used to detect rabbit antibody
specific for a protein of the invention.
(iii) Methods of the Invention
[0171] The invention includes a protein of the invention for use in
metabolizing retinoic acid in an organism or cell in need of such
metabolizing. The invention also includes use of a protein of the
invention in the preparation of a medicament for use in
metabolizing retinoic acid in an organism. Preferably the protein
has sequence of SEQ ID NO: 5.
[0172] The invention includes a method for metabolizing retinoic
acid, particularly all-trans retinoic acid, in an organism or cell
comprising administering a protein as broadly defined by the
invention, most preferably of SEQ ID NO: 5.
[0173] The invention is also a method for inhibiting retinoic acid
hydroxylation in an organism in need of such inhibition, comprising
administering to the organism an effective amount of an antisense
nucleic acid or oligonucleotide substantially complementary to at
least a portion of the sequence identified as SEQ ID NO:4. In such
a method, the portion can be at least 5 bases in length, or at
least about 10 bases in length, or at least about 15 bases in
length, or at least about 20 bases in length, or at least about 25
bases in length, or at least about 30 bases in length, or at least
about 35 bases in length, or at least about 40 bases in length, or
at least about 45 bases in length, or at least about 50 bases in
length. Other inhibitors, of RA metabolism such as antibodies to
P450RAI-2, or inhibitors of P450RAI-2 activity could also be
used.
[0174] A preferred organism to be treated is human but could be any
organism with an RA metabolism or P450RAI-2 related condition.
[0175] The organism may be being treated for a medical condition or
disease wherein RA treatment may be indicated. In one embodiment
the disease or medical condition is selected from the group
consisting of cancer, angiogenesis, actinic keratosis, oral
leukoplakia, a secondary tumor of the head and/or neck, a non-small
cell lung carcinoma, a basal cell carcinoma, acute promyelocytic
leukemia, skin cancer, and a premalignancy associated actinic
keratosis, acne, psoriasis and/or ichthyosis, and particularly,
acute promyelocytic leukemia. In another embodiment the peptides
and nucleic and molecules of the invention may be used to treat
disorders of the brain such as memory loss or learning
deficiencies.
[0176] In one embodiment the invention includes a kit for
determining the presence of a protein as broadly described above,
or containing an amino acid sequence as identified by SEQ ID NO:5
(human P450RAI-2) SEQ ID NO:11 (mouse CYP26B) or SEQ ID NO:12
(zebrafish CYB26B), comprising an antibody to the protein linked to
a reporter system, wherein the reporter system produces a
detectable response when a predetermined amount of the protein and
the antibody are bound together. In a preferred aspect, the
antibody is specific for a protein that includes the amino acid
sequence identified as SEQ ID NO:5.
[0177] The invention also includes a kit for determining the
presence of a first said nucleic acid molecule as broadly defined
by the invention. The kit includes a second nucleic acid molecule
capable of hybridizing with at least a portion of a the first
nucleic acid molecule under the high stringency conditions of
paragraph (a), above, in which the second nucleic acid molecule is
linked to a reporter system wherein the reporter system produces a
detectable response when a predetermined amount of the first and
second molecules are hybridized together. The second nucleic acid
molecule can be at least 5 bases in length, or at least about 10
bases in length, or at least about 15 bases in length, or at least
about 20 bases in length, or at least about 25 bases in length, or
at least about 30 bases in length, or at least about 35 bases in
length, or at least about 40 bases in length, or at least about 45
bases in length, or at least about 50 bases in length.
[0178] The invention is also a method of screening drugs for their
modulating effect on activity of a protein as broadly defined by
the invention, which method includes exposing a purified said
protein to a said drug and determining the effect on the activity.
Usually, the modulating effect would be to inhibit the activity of
the protein in question. Typically, the activity of the protein is
oxidation, e.g., hydroxylation of a retinoic acid, particularly
all-trans retinoic acid. In a preferred embodiment, the activity is
oxidation of all-trans retinoic acid and the protein includes the
amino identified as SEQ ID NO:5.
[0179] The invention also includes a method of screening drugs for
their effect on expression of a gene, wherein the gene is an
inducible gene containing a nucleotide sequence as broadly defined
by the invention, by all-trans retinoic acid. The method includes
exposing a eukaryotic cell to a said drug and determining the
effect on gene expression. Preferably, the gene includes the
nucleotide sequence identified as SEQ ID NO:4.
[0180] The cell can be a mammalian cell, particularly, a human
cell.
[0181] The invention includes any drug identified according to a
method of the invention, particularly for a purpose related to its
related to its modulating effect on the activity of a protein of
the invention.
[0182] The invention includes a method for inhibiting retinoic acid
metabolism in an organism in need of such inhibition, or in cells
obtained from such an organism, comprising administering to the
organism an effective amount of a drug of the invention.
[0183] The invention includes a method of oxidizing a retinoid. The
method includes exposing the retinoid to a protein as broadly
defined by the invention, and particularly where the protein
includes the amino acid sequence identified as SEQ ID NO:5. The
retinoid can be a retinoic acid, particularly, all-trans retinoic
acid.
[0184] In another aspect, the invention is a method of screening a
drug for its activity on a protein. The method includes steps of:
[0185] (i) providing a cell line having heterologous DNA encoding a
functional protein as broadly defined by the invention incorporated
thereinto so as to be capable of expressing said protein; [0186]
(ii) exposing the cell line to the drug under conditions in which
said protein is expressed in an active form to expose the protein
to the drug; and [0187] (iii) determining the effect of the drug on
the activity of the protein.
[0188] The method can include exposing the cell line to a substrate
of the protein under conditions in which the protein is expressed
in an active form to expose the protein to the substrate.
[0189] In a particular aspect, the activity is oxidation.
[0190] Step (ii) of the method can include exposing the cell line
to the drug and the substrate simultaneously.
[0191] The substrate can be a retinoid, particularly, a retinoic
acid, more particularly, all-trans retinoic acid.
[0192] The oxidative activity can be oxidation of the .beta.-ionone
ring of the substrate, particularly, hydroxylation.
[0193] According to a particular aspect, the heterologous DNA
encodes the protein identified as SEQ ID NO:5.
[0194] The invention is also a method for screening an agent for
its effect on an activity of a first protein relative to its effect
on the activity of a second protein. This method includes steps of:
[0195] (a) providing a first protein, wherein the protein one
defined according this invention; [0196] (b) providing a second
protein, wherein the second protein is a cytochrome P450; [0197]
(c) exposing the first protein to the agent; [0198] (d) exposing
the second protein to the agent; and [0199] (e) determining the
effect of the agent on the activity of the first protein relative
to its effect on the activity of the second protein.
[0200] According to the method, the activity of each protein under
consideration can be the ability of the proteins to oxidize a
retinoid and it optionally limited to the ability to oxidize a
retinoid at the 4-position of the .beta.-ionone ring and/or to
hydroxylate a retinoid at the 4-position of the .beta.-ionone
ring.
[0201] The retinoid can be a retinoic acid, and it can simply be
all-trans retinoic acid.
[0202] The first protein can be a human protein and it can include
the sequence identified as SEQ ID NO:5.
[0203] The method can be such that the second protein is selected
from the group of proteins encoded by: (a) a nucleotide sequence
that hybridizes under high stringency conditions, wherein high
stringency conditions include a wash step of about 0.2.times.SSC at
50.degree. C., to the nucleotide sequence shown as SEQ ID NO:13 or
SEQ ID NO:14 or SEQ ID NO:17, and encodes a protein that oxidizes a
retinoid; and (b) a nucleotide sequence that hybridizes under high
stringency conditions, wherein high stringency conditions include a
wash step of about 0.2.times.SSC at 50.degree. C., to the
nucleotide sequence shown as SEQ ID NO:13 or SEQ ID NO:14, and
encodes a protein that hydroxylates retinoic acid at the 4 position
of the .beta.-ionone ring.
[0204] The second protein can be a human protein and it can include
a protein which includes the amino acid sequence encoded by the
nucleotide sequence identified as SEQ ID NO:13.
[0205] Step (c) of the method can include exposing the first
protein to a retinoid.
[0206] Step (d) of the method can include exposing the second
protein to a retinoid.
[0207] Each of steps (c) and (d) can include exposing the first
protein to a retinoid.
[0208] Step (c) can include exposing the first protein to said
retinoid at various concentrations and/or step (d) can include
exposing the second protein to said retinoid at various
concentrations.
[0209] In another broad aspect, the invention is a method for
screening an agent for its effect on an activity of a first protein
relative to its effect on the activity of a second protein where
the proteins are each expressed in a cell or group of cells. Such
method thus includes: [0210] (a) providing a group of first cells
having expressibly incorporated thereinto heterologous DNA encoding
a first protein wherein the protein is a protein of the invention
as defined herein; [0211] (b) providing a group of second cells
having expressibly incorporated thereinto heterologous DNA encoding
a second protein, where the second protein is a cytochrome P450;
[0212] (c) exposing the first cells to the agent under conditions
in which the first protein is expressed; [0213] (d) exposing the
second cells to the agent under conditions in which the second
protein is expressed; and [0214] (e) determining the effect of the
agent on the activity of the first protein relative to its effect
on the activity of the second protein.
[0215] Step (e) of the method can include monitoring the
disappearance of the agent in the presence the first cells and
monitoring the disappearance of the agent in the presence of the
second cells.
[0216] Step (e) can include monitoring the appearance of an
oxidized product or products formed from the agent on exposure to
the first cells and monitoring the appearance of the oxidized
product or products formed from the agent on exposure to the second
cells.
[0217] Step (c) can include exposing the first cells to a substrate
of the first protein in the presence of the agent and step (d) can
include exposing the second cells to the substrate in the presence
of the agent.
[0218] Step (e) can include monitoring the production of a reaction
product or products formed from the substrate on exposure to the
first protein in step (c) and, further, step (e) can include
monitoring the production of the reaction product or products
formed from the substrate on exposure to the second protein in step
(d).
[0219] Step (e) can include monitoring reduction in the amount of
substrate on exposure to the first protein in step (c) and step (e)
can include monitoring the reduction in the amount of the substrate
on exposure to the second protein in step (d).
[0220] The substrate can be a retinoid, which can be a retinoic
acid, which can be all-trans retinoic acid.
[0221] The observed activity of each protein can be its ability to
oxidize a retinoid, particularly, the ability to oxidize a retinoid
at the 4-position of the .beta.-ionone ring, or the ability to
hydroxylate a retinoid at the 4-position of the .beta.-ionone
ring.
[0222] The first protein can be a human protein and it can have the
sequence identified as SEQ ID NO:5.
[0223] The second protein can be selected from the group of
proteins encoded by: (a) a nucleotide sequence that hybridizes
under high stringency conditions, wherein high stringency
conditions include a wash step of about 0.2.times.SSC at 50.degree.
C., to the nucleotide sequence shown as SEQ ID NO:13 or SEQ ID
NO:14, and encodes a protein that oxidizes a retinoid; and (b) a
nucleotide sequence that hybridizes under high stringency
conditions, wherein high stringency conditions include a wash step
of about 0.2.times.SSC at 50.degree. C., to the nucleotide sequence
shown as SEQ ID NO:13 or SEQ ID NO:14, and encodes a protein that
hydroxylates retinoic acid at the 4 position of the .beta.-ionone
ring.
[0224] The second protein can be a human protein and it can include
the amino acid sequence encoded by the nucleotide sequence
identified as SEQ ID NO:13.
[0225] Step (c) of the method can include exposing the first cells
to a retinoid.
[0226] Step (d) can include exposing the second cells to a
retinoid.
[0227] Step (c) can include exposing the first cells to said
retinoid at various concentrations.
[0228] Step (d) can also include exposing the second cells to a
retinoid at various concentrations.
[0229] In another aspect, the present invention is a method of
inducing in an eukaryotic cell, production of RNA comprising the
nucleotide sequence identified as SEQ ID NO:4 The method includes
the steps of: [0230] exposing the cell to a retinoid; and [0231]
hybridizing the RNA with a probe comprising a nucleic acid molecule
comprising a nucleotide sequence which encodes a protein of the
present invention.
[0232] The cell can be a mammalian cell, particularly a human
cell.
[0233] The retinoid can be a retinoic acid, particularly, all-trans
retinoic acid.
[0234] The nucleic acid sequence can be a non-coding sequence
complementary to a coding sequence of a nucleic acid molecule
encoding the protein comprising the amino acid sequence identified
as SEQ ID NO: 5 and the probe is at least 10 nucleotide residues in
length, or the probe is at least 15 nucleotide residues in length,
or the probe is at least 20 nucleotide residues in length, or the
probe is at least 25 nucleotide residues in length, or the probe is
at least 30 nucleotide residues in length, or the probe is at least
35 nucleotide residues in length, or the probe is at least 40
nucleotide residues in length, or the probe is at least 45
nucleotide residues in length, or the probe is at least 50
nucleotide residues in length, or the probe is at least 55
nucleotide residues in length, or the probe is at least 56
nucleotide residues in length, or the probe is at least 60
nucleotide residues in length.
[0235] The invention includes a method of inducing expression in an
eukaryotic cell, a protein comprising the amino acid sequence
identified as SEQ ID NO: 5, the method comprising the steps of:
[0236] exposing the cell to a retinoid in an amount sufficient to
induce said expression; and [0237] isolating the protein from the
cell.
[0238] Isolating the protein can include exposing proteins produced
by the cell after said exposure step to an antibody which binds
specifically to the desired protein.
[0239] In the context of this invention, an antibody which
"specifically binds" (and grammatical equivalents) to a protein
refers to the phenomenon in which an antibody recognizes and binds
to a specific binding entity, e.g., protein, but substantially does
not recognize or bind to any other specific binding entity.
[0240] The cell can be a mammalian cell, particularly, a human
cell.
[0241] The retinoid can be a retinoic acid, particularly, all-trans
retinoic acid.
[0242] Human and mouse genomic P450RAI-1 sequences are identified
herein as SEQ ID NOs:15 and 16, respectively. The mouse sequence
encoding P450RAI-1 is identified herein as SEQ ID NO:17.
[0243] In another aspect, the present invention includes a fragment
of the nucleotide sequence encoding P450RAI-2 (SEQ ID NO:4). Such a
fragment can find usefulness as a probe. The complement of the
probe can find utility in, for example, manufacture of the probe.
In a particular use, the probe can be used to determine the
presence of an RNA molecule in a sample which might, or might not,
also include an RNA molecule encoding P450RAI-1. Such a probe would
generally be 20 nucleotides long or be at least 20 nucleotides
long. The probe could also be 25, 30, 35, 40, 45, 50, 55, 60 or
more nucleotides in length and the probe can include the full
length of the complement to the sequence to which it is intended to
bind. The sequence of the probe would also be sufficiently
distinguishable from any portion of the sequence encoding P450RAI-1
that it would not cross-hybridize to a significant extent to a
nucleotide sequence that encodes P450RAI-1, or portion thereof,
particularly to an RNA encoding P450RAI-1. Such a probe would thus
be sufficiently different from any sequence of contiguous
nucleotides selected from the nucleotide sequence encoding human
P450RAI-1 (SEQ ID NO:13) that there is no more than about 60%
homology between the two sequences when the two sequences are
directly aligned with each other. More preferably, the percent
homology would be less than about 55%, or less than about 50%, or
less than about 45%, or even less than about 40%. Certain probes of
the invention are selected so as span borders between introns of
the coding sequence as determined from the genomic sequence (SEQ ID
NO:3).
[0244] The invention includes the method determining the presence
of a nucleic molecule encoding P450RAI-2 in a sample containing RNA
isolated from human cell, using such a probe.
[0245] In the context of this specification, the term "conserved"
describes similarity between sequences. The degree of conservation
between two sequences can be determined by optimally aligning the
sequences for comparison. Here, sequences were aligned using the
Omiga software program, Version 1.13. (Oxford Molecular Group,
Inc., Campbell, Calif.). The Omiga software uses the Clustal W
Alignment algorithms [Higgins et al., 1989; Higgins et al., 1991;
Thompson et al. 1994] Default settings used are as follows: Open
gap penalty 10.00; Extend gap penalty 0.05; Delay divergent
sequence 40 and Scoring matrix--Gonnet Series. Percent identity or
homology between two sequences is determined by comparing a
position in the first sequence with a corresponding position in the
second sequence. When the compared positions are occupied by the
same nucleotide or amino acid, as the case may be, the two
sequences are conserved at that position. The degree of
conservation between two sequences is often expressed, as it is
here, as a percentage representing the ratio of the number of
matching positions in the two sequences to the total number of
positions compared.
[0246] The generic term "retinoids" means a group of compounds
which includes retinoic acid, vitamin A (retinol) and a series of
natural and synthetic derivatives that can exert profound effects
on development and differentiation in a wide variety of systems.
For purposes of this disclosure "retinoid" is also intended to
encompass an equivalent thereof having the same functional
characteristics which may be produced, for example, by
computational chemistry.
[0247] "Stringent hybridization conditions" takes on its common
meaning to a person skilled in the art here. Appropriate stringency
conditions which promote nucleic acid hybridization, for example,
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C. are known to those skilled in the art. The following examples
are found in Current Protocols in Molecular Biology, John Wiley
& Sons, NY (1989), 6.3.1-6.3.6: For 50 ml of a first suitable
hybridization solution, mix together 24 ml formamide, 12 ml
20.times.SSC, 0.5 ml 2 M Tris-HCl pH 7.6, 0.5 ml
100.times.Denhardt's solution, 2.5 ml deionized H.sub.2O, 10 ml 50%
dextran sulfate, and 0.5 ml 10% SDS. A second suitable
hybridization solution can be 1% crystalline BSA (fraction V), 1 mM
EDTA, 0.5 M Na.sub.2HPO.sub.4 pH 7.2, 7% SDS. The salt
concentration in the wash step can be selected from a low
stringency of about 2.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. Both of these
wash solutions may contain 0.1% SDS. In addition, the temperature
in the wash step can be increased from low stringency conditions at
room temperature, about 22.degree. C., to high stringency
conditions, at about 65.degree. C. The cited reference gives more
detail, but appropriate wash stringency depends on degree of
homology and length of probe. If homology is 100%, a high
temperature (65.degree. C. to 75.degree. C.) may be used. If
homology is low, lower wash temperatures must be used. However, if
the probe is very short (<100 bp), lower temperatures must be
used even with 100% homology. In general, one starts washing at low
temperatures (37.degree. C. to 40.degree. C.), and raises the
temperature by 3-5.degree. C. intervals until background is low
enough not to be a major factor in autoradiography.
[0248] The invention also includes a method of inhibiting retinoic
acid hydroxylation in an organism in need of such inhibition by
administering to the organism an effective amount of an antibody,
such antibodies being described herein. A particularly useful
antibody for the treatment of a human would be an antibody to the
protein having the amino acid sequence identified as SEQ ID NO:5,
or a portion thereof. It would be advantageous to adapt such an
antibody for administration to a human by "humanizing" the
antibody, as is understood by those skilled in the art [Hozumi,
1993].
(iv) Kits
[0249] Reagents suitable for conducting the above described
diagnostic and/or methods of the invention may be packaged into
convenient kits providing the necessary materials, packaged into
suitable containers. Such kits may include all the reagents
required to detect P450RAI-2 in a sample by means of the methods
described herein, and optionally suitable supports useful in
performing the methods of the invention.
[0250] In one embodiment of the invention the kit contains a
nucleotide probe which hybridizes with a nucleic acid molecule of
the invention, reagents required for hybridization of the
nucleotide probe with the nucleic acid molecule, and directions for
its use. In another embodiment of the invention the kit includes
antibodies of the invention and reagents required for binding of
the antibody to a P450RAI-2 protein in a sample. In still another
embodiment of the invention, the kit includes primers which are
capable of amplifying a nucleic acid molecule of the invention or a
predetermined oligonucleotide fragment thereof, all the reagents
required to produce the amplified nucleic acid molecule or
predetermined fragment thereof in the polymerase chain reaction,
and means for assaying the amplified sequences.
[0251] The methods and kits of the present invention have many
practical applications. For example, the methods and kits of the
present invention may be used to detect P450RAI-2 in any medical
sample suspected of containing or lacking P450RAI-2 and used to
diagnose diseases associated with P450RAI-2 or RA, expression or
metabolism or where RA, P450RAI-2 inhibitor or P450RAI-2 treatment
may be indicated. Examples of such diseases include cancer,
dysplasia, certain autoimmune diseases or dermatological disorders,
angiogenesis, conditions of high order brain functions or other
conditions as noted herein. Samples which may be tested include
bodily materials such as blood, urine, serum, tears, saliva, feces,
tissues and the like.
[0252] Before testing a sample in accordance with the methods
described herein, the sample may be concentrated using techniques
known in the art, such as centrifugation and filtration. For
hybridization and/or PCR-based methods described herein, nucleic
acids may be extracted from cell extracts of the test sample using
techniques known in the art.
2. Therapeutic Applications
Methods of Treatment/Pharmaceutical Compositions
[0253] P450RAI-2 may play a role in a number of diseases, such as
those associated with regulation of the cell cycle or apoptosis. In
particular P450RAI-2 may play a role in cancer or dysplasia by
activating apoptosis. In one embodiment, it is associated with lung
and/or breast cancer. In another embodiment, P450RI-2 may play role
in learning, memory and other brain functions, such as those
controlled by the cerebellum or pons. As such, the invention
comprises methods for modulating or simulating P450RAI-2 activity
or P450RAI-2 expression, preferably for treating or preventing a
P450RAI-2 or RA related condition. The invention further comprises
uses of the modulating or simulating agents disclosed herein for
the preparation of a medicament for treating or preventing a
condition associated with P450RAI-2 expression or activity. Such a
condition could be any condition that may benefit from the
treatment with a P450RAI-2 or a modulator thereof. In another
embodiment the invention provides a use of the modulating or
simulating agents for the treatment or prevention of a P450RAI-2
related condition. In another embodiment, the invention provides
for inhibitors of P450RAI-2 for the treatment of a related
condition. Such inhibitors could include antibodies to P450RAI-2 or
antisense molecules to P450RAI-s nucleic acid sequence or other
compounds such as ketoconazole.
[0254] Accordingly, the present invention provides a method of
treating or preventing a disease associated with P450RAI-2
expression or activity comprising administering an agent that
modulates or simulates P450RAI-2 expression or activity to an
animal in need thereof, such as in an animal with cancer (such as
lung or breast cancer), dysplasia, an autoimmune disease, a
dermatological disorder or a learning or memory or other brain
functions, such as those controlled by the cerebellum or pons. It
may also be used in the treatment of various embryonic development
disorders. In one embodiment, such modulators can be administered
to a pregnant women or to be pregnant women or delivered to the
fetus to avoid or correct certain developmental disorders.
[0255] In a one embodiment, such agents stimulate or simulate
P450RAI-2 activity. Examples of agents that activate or simulate
P450RAI-2 activity would include without limitations, P450RAI-2,
the gene encoding for P450RAI-2 with suitable promoters, such
promoters preferably being tissue specific promoters and
therapeutically effective fragments of the nucleic acid and amino
acid sequences of the invention.
[0256] Examples of agents that inhibit P450RAI-2 include antisense
nucleic acid molecules, antibodies and transdominant inhibitors, as
described herein.
[0257] Agents that inhibit, activate, or stimulate P450RAI-2 can be
formulated into pharmaceutical compositions with or without RA for
administration to subjects in a biologically compatible form
suitable for administration in vivo. As used herein "biologically
compatible form suitable for administration in vivo" means a form
of the substance to be administered in which therapeutic effects
outweigh any toxic effects. The substances may be administered to
animals in need thereof. Animals, as used herein refers to any
animal susceptible to a disease associated with P450RAI-2
expression preferably dogs, cats, mice, horses and humans.
[0258] Administration of an "effective amount" of pharmaceutical
compositions of the present invention is defined as an amount of
the pharmaceutical composition, at dosages and for periods of time
necessary to achieve the desired result. For example, a
therapeutically active amount of a substance may vary according to
factors such as disease state, age, sex, and weight of the
recipient, and the ability of the substance to elicit a desired
response in the recipient. Dosage regima may be adjusted to provide
an optimum Therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation.
[0259] An active substance may be administered in a convenient
manner such as by injection (subcutaneous, intravenous, topical,
intratumoral etc.), oral administration, inhalation, transdermal
application, or rectal administration. Depending on the route of
administration, the active substance may be coated in a material to
protect the compound from the action of enzymes, acids and other
natural conditions which may inactivate the compound.
[0260] The compositions described herein can be prepared by known
methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). On this basis, the compositions include,
albeit not exclusively, solutions of the substances in association
with one or more pharmaceutically acceptable vehicles or diluents,
and contained in buffered solutions with a suitable pH and
iso-osmotic with the physiological fluids.
[0261] Recombinant nucleic acid molecules comprising a sense, an
antisense sequence or oligonucleotide fragment thereof, may be
directly introduced into cells or tissues in vivo using delivery
vehicles known in the art such as retroviral vectors, adenoviral
vectors and DNA virus vectors. They may also be introduced into
cells in vivo using physical techniques known in the art such as
microinjection and electroporation or chemical methods such as
coprecipitation and incorporation of DNA into liposomes.
Recombinant molecules may also be delivered in the form of an
aerosol or by lavage.
[0262] The utility of the substances, antibodies, sense and
antisense nucleic acid molecules, and compositions of the invention
may be confirmed in animal experimental model systems. Suitable
animal model systems which can be used to determine P450RAI-2
activity may include, but is not limited or P450RAI-2 knock-out
transgenic animals.
3. Other Applications
Screening for P450RAI-2 Modulating Compounds
[0263] In another embodiment, the invention provides a method for
identifying a compound or molecule that modulates P450RAI-2 protein
activity or gene expression. "Modulate" as used herein can include
activation or increase of P450RAI-2 protein activity or gene
expression or suppression of P450RAI-2 protein activity or gene
expression. The method includes incubating components comprising
the compound and P450RAI-2 peptide or a recombinant cell expressing
P450RAI-2 peptide, under conditions sufficient to allow the
components to interact and determining the effect of the compound
on P450RAI-2 activity or expression. The effect of the compound on
P450RAI-2 activity can be measured by a number of assays and may
include measurements before and after incubation in the presence of
the compound. Compounds that affect P450RAI-2 activity or gene
expression include peptides, chemical compounds and biologic
agents. Assays include Northern blot analysis of P450RAI-2 mRNA
(for gene expression), Western blot analysis (for protein level)
and luciferase, apoptosis or growth suppression assays (for protein
activity).
[0264] In one embodiment, the above screening assays may be used
for detecting the compounds or molecules that bind to the P450RAI-2
protein or peptide, in isolating molecules that bind to the
P450RAI-2 gene, for measuring the amount of P450RAI-2 in a sample,
either peptide or RNA (mRNA), for identifying molecules that may
act as agonists or antagonists, and the like.
[0265] Incubating includes conditions which allow contact between
the test compound and P450RAI-2 peptide or with a recombinant cell
expressing P450RAI-2 peptide. Contacting includes in solution and
in solid phase, or in a cell. The test compound may optionally be a
combinatorial library for screening a plurality of compounds.
Compounds identified in the method of the invention can be further
evaluated, detected, cloned, sequenced and the like, either in
solution or after binding to a solid support by any method usually
applied to the detection of a specific DNA sequence such as PCR,
oligomer restriction, allele-specific oligonucleotide probe
analysis, and the like.
[0266] In another embodiment, modulators of P450RAI-2 activity can
be screened by incubating the potential modulator with P450RAI-2
and its substrate, such as all-trans-RA under conditions that
permit interaction between P450RAI-2 and the substrate and assaying
for substrate levels or levels of binding or other interaction
between P450RAI-2 and the substrate, or assaying for binding or
lack thereof between the potential modulator and/or P450RAI-2
and/or the substrate. Assaying for binding or activity levels can
be done by assays known in the art such as radiolabelling or
colourmetric assays, or ELISAs. Other ways of screening for
potential modulators of P450RAI-2 is to run the P450RAI-2 peptide
or nucleic acid molecule on a Southern, Northern or Western gel, as
the case may be and probing with a labeled (or radiolabelled)
potential modulator to check for binding. The potential modulator
can then be subsequently screened for modulating effects on RA
metabolism in the presence of P450RAI-2 and the all-trans-RA or
other suitable substrate. The gels can also be run in the reverse
by running the potential modulator on an appropriate gel and
screening for binding activity with P450RAI-2 and.or
all-trans-RA.
[0267] Competition assays can also be conducted to screen for
potential modulators of P450RAI-2 such as shown in FIG. 16.
Screening for P450RAI-2 or RA Related Disorders
[0268] Method for screening of P450RAI-2 protein activity and or
gene expression as described above, can also be used to screen for
P450RAI-2 related disorders. For instance, biological samples from
patients with a particular conditions, such as cancer or APL, or
other disorders outlined herein, such as dysplasia, lung or breast
cancer can be screened for P450RAI-2 protein activity and or gene
expression. P450RAI-2 gene can also be sequenced from patients with
a disorder to identify any mutations in the P450RAI-2 gene.
Correlation between P450RAI-2 activity and/or gene expression/and
or any mutations and the disorder can be determined by a number of
methods known in the art. For instance, P450RAI-2 activity and/or
gene expression in a biological sample, such as a tissue, blood or
other sample of a subject to be screened can be compared with that
from "healthy" individuals or healthy tissues. In one embodiment,
the level of P450RAI-2 activity and/or gene expression can be
compared with a cut off level for normal P450RAI-2 activity and/or
gene expression. In one embodiment, the cutoff level can be
determined by analysis of a database of levels from "healthy"
individuals.
Transgenic Animals and Methods of Making Same
[0269] Nucleic acids which encode proteins having biological
activity of P450RAI-2 can be used to generate either transgenic
animals or "knock out" animals which, in turn, are useful in the
development and screening of therapeutically useful reagents.
Preferably, non-human transgenic animals are encompassed within the
scope of this invention. A transgenic animal (e.g., a mouse) is an
animal having cells that contain a transgene, which transgene was
introduced into the animal or an ancestor of the animal at a
prenatal, e.g., an embryonic stage. A transgene is a DNA which is
integrated into the genome of a cell from which a transgenic animal
develops. In one embodiment, a P450RAI-2, preferably mouse or human
cDNA shown in SEQ ID NO: 4, or an appropriate sequence, can be used
to clone a murine P450RAI-2 gene in accordance with established
techniques and the genomic nucleic acid used to generate transgenic
animals that contain cells which express P450RAI-2 protein. Methods
for generating transgenic animals, particularly animals such as
mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, 5,616,491. In a
preferred embodiment, plasmids containing recombinant molecules of
the invention are microinjected into mouse embryos. In particular,
the plasmids are microinjected into the male pronuclei of
fertilized one-cell mouse eggs; the injected eggs are transferred
to pseudo-pregnant foster females; and, the eggs in the foster
females are allowed to develop to term. [Hogan, B. et al., (1986) A
Laboratory Manual, Cold Spring Harbor, N.Y., Cold Spring Harbor
Laboratory]. Alternatively, an embryonal stem cell line can be
transfected with an expression vector containing nucleic acid
encoding a protein having P450RAI-2 activity and cells containing
the nucleic acid can be used to form aggregation chimeras with
embryos from a suitable recipient mouse strain. The chimeric
embryos can then be implanted into a suitable pseudopregnant female
mouse of the appropriate strain and the embryo brought to term.
Progeny harbouring the transfected DNA in their germ cells can be
used to breed uniformly transgenic mice.
[0270] Typically, particular cells would be targeted for P450RAI-2
transgene incorporation by use of tissue specific enhancers
operatively linked to the P450RAI-2-encoding gene. For example,
promoters and/or enhancers which direct expression of a gene to
which they are operatively linked preferentially in cardiac muscle
cells can be used to create a transgenic animal which expresses a
P450RAI-2 protein. Examples of suitable promoters and enhancers
include those which regulate the expression of the genes for
cardiac myosin and cardiac actin. Transgenic animals that include a
copy of a P450RAI-2 transgene introduced into the germ line of the
animal at an embryonic stage can also be used to examine the effect
of increased P450RAI-2 expression in various tissues.
[0271] The pattern and extent of expression of a recombinant
molecule of the invention in a transgenic mouse is facilitated by
fusing a reporter gene to the recombinant molecule such that both
genes are co-transcribed to form a polycistronic mRNA. The reporter
gene can be introduced into the recombinant molecule using
conventional methods such as those described in Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual. Cold Spring Harbor
Laboratory press. Efficient expression of both cistrons of the
polycistronic mRNA encoding the protein of the invention and the
reporter protein can be achieved by inclusion of a known internal
translational initiation sequence such as that present in
poliovirus mRNA. The reporter gene should be under the control of
the regulatory sequence of the recombinant molecule of the
invention and the pattern and extent of expression of the gene
encoding a protein of the invention can accordingly be determined
by assaying for the phenotype of the reporter gene. Preferably the
reporter gene codes for a phenotype not displayed by the host cell
and the phenotype can be assayed quantitatively. Examples of
suitable reporter genes include lacZ (b-galactosidase), neo
(neomycin phosphotransferase), CAT (chloramphenicol
acetyltransferase) dhfr (dihydrofolate reductase), aphIV
(hygromycin phosphotransferase), lux (luciferase), uidA (b
glucuronidase). Preferably, the reporter gene is lacZ which codes
for b-galactosidase. b galactosidase can be assayed using the
lactose analogue X-gal(5-bromo-4-chloro-3-indolyl
b-D-galactopyranoside) which is broken down by b-galactosidase to a
product that is blue in color. (See for example Old R. W. &
Primrose S. B. Principles of Gene Manipulation. An Introduction to
Genetic Engineering, 4th ed. Oxford University Press at pages 63-66
for a discussion of procedures for screening for recombinants).
[0272] Additionally, the non-human homologues of genes encoding
proteins having P450RAI-2 activity can be used to construct a
P450RAI-2 "knock out" animal which has a defective or altered
P450RAI-2 gene. For example, a human P450RAI-2 cDNA, comprising the
nucleotide sequence shown in SEQ ID NO: 5, or a mouse P450RAI-2
cDNA appropriate sequence thereof, can be used to clone a murine
P450RAI-2 gene in accordance with established techniques. A portion
of the genomic P450RAI-2 DNA (e.g., an exon) can be deleted or
replaced with another gene, such as a gene encoding a selectable
marker which can be used to monitor integration. The altered
P450RAI-2 DNA can then be transfected into an embryonal stem cell
line. The altered P450RAI-2 DNA will homologously recombine with
the endogenous P450RAI-2 gene in certain cells and clones
containing the altered gene can be selected. Cells containing the
altered gene are injected into a blastocyst of an animal, such as a
mouse, to form aggregation chimeras as described for transgenic
animals. Chimeric embryos are implanted as described above.
Transmission of the altered gene into the germline of a resultant
animal can be confirmed using standard techniques and the animal
can be used to breed animals having an altered P450RAI-2 gene in
every cell. Accordingly, a knockout animal can be made which cannot
express a functional P450RAI-2 protein. Such a knockout animal can
be used, for example, to test the effectiveness of an agent in the
absence of a P450RAI-2 protein.
[0273] Although experimental animals used in the preferred
embodiment disclosed are mice, the invention should not be limited
thereto. It can be desirable to use other species such as rats,
hamsters and rabbits.
[0274] The transgenic animals of the invention can be used to
investigate the effect of P450RAI-2 expression and activity or lack
thereof and to test other compounds and molecules that can perhaps
be used to suppress or restore the P450RAI-2 or RA activity. The
transgenic animals of the invention can also be used to test
substances for the ability to prevent, slow or reverse apoptosis. A
transgenic animal can be treated with the substance in parallel
with an untreated control transgenic animal.
[0275] Cells from the transgenic animals of the invention can be
cultured using standard tissue culture techniques. In particular,
cells carrying the recombinant molecule of the invention can be
cultured and used to test substances for the ability to prevent,
slow or reverse apoptosis.
EXAMPLES
General Materials and Methods
Cell Culture
[0276] HPK1a-ras (ras-transformed human keratinocyte), COS-1
(African green monkey kidney and V79 cells (Chinese Hamster lung)
were maintained in DMEM supplemented with 10% FBS (5% for V79). WTE
(human non-small cell lung carcinoma and SW900 (human non-small
cell lung carcinoma) cells were maintained in RPMI supplemented
with 10% FBS. SK-Luci-6 (human non-small cell lung carcinoma) and
SK-MES-1 (human non-small cell lung carcinoma) cells in RPMI with
5% FBS. NB4 (human acute promyelocytic leukemia-serived) cells were
maintained in RPMI supplemented with 10% FBS and gentamicin (10
mg/ml) HeLa cells (human cervical carcinoma) were maintained in MEM
supplemented with 10% FBS. Previous cell lines were supplemented
with penicillin (50 units/ml) streptomycin (50 ug/ml) and fungizone
(0.1% final concentration). MCF-7 cells (human breast epithelial
adenocarcinoma) were grown in MEM supplemented with 10% FBS,
insulin (10 ng/ml), sodium pyruvate (0.5 mM), non-essential amino
acids (100 nM), L-glutamine (2 mM), penicillin (5 ug.ml),
streptomycin (5 ug/ml), fungizone (200 ng/ml), and gentamicin (10
mg/ml). All reagents were supplied by Life Technologies, NY. Cells
lines were maintained at 37.degree. C. in an atmosphere of 5%
CO.sub.2 and 95% air.
Transient Transfection of Cos-1 Cells
[0277] Exponentially growing cells were plated in triplicate into
6-well plates and transfected with 1 ug of pcDNA3.1-P450RAI-1 or
pcDNA3.1-P450RAI-2 using Fugene 6 transfection reagent with 3 ul
per sample as described by the manufacturer (Roche Molecular
Biochemicals, IN). Cells were maintained in media supplemented with
10% FBS during transfection.
RNA Preparation and Northern Blot Analysis
[0278] Total RNA was isolated from cultured cells using the
Oligotex Direct mRNA kit (Qiagen, CA) and electrophoresed on a
formaldehyde-agarose gel. Gels were photographed under ultraviolet
light and then blotted onto Hybond ECL nitrocellulose membrane
(Amersham Pharmacia Biotech, UK) and fixed to the membrane by
baking at 80.degree. C. for 2 hours under vacuum. Prehybridization
and hybridization steps were performed using ExpressHyb (Clontech,
CA) according to the manufacturer's directions. Full length
P450RAI-1 and P450RAI-2 cDNA were labeled with
.alpha.-[.sup.32P]dATP using Prime-A-Gene Labeling System (Promega,
WI). The blot was washed two times for 15 minutes in 2.times.SSC,
0.1% SDS at room temperature then for 15 minutes at 60.degree. C.
in 0.1.times.SSC, and 0.1% SDS and exposed at -70.degree. C. for 19
hours to Kodak X-Omat AR film (Eastman Kodak Company, NY).
Example 1
Determination cDNA Sequence Encoding P450RAI-2
[0279] The human expressed sequence tagged (EST) database at the
National Center for Biotechnology Information (NCBI), available
over the Internet at http://www.ncbi.nlm.nih.gov/BLAST/ was
searched using an amino acid sequence encoding a typical heme
binding motif found in all Cytochrome P450s. The database was
queried using the following sequence:
KKETFIPFGIGKRVCMGEQLAKMELFLMFV (SEQ ID NO1). The TBLASTN algorithm
of the Advanced BLAST program was used to search all 6 possible
reading frames for translation of all the human EST sequences
against the query sequence (SEQ ID NO:1). Parameters for all
searching were the defaults of Blosum 62 which use a gap existence
cost of 11, per residue cost of 1 and lambda ratio of 0.85. Subject
amino acid sequences which showed similarity to SEQ ID NO:1 were
retrieved from the GenBank database and their nucleotide sequences
used to search GenBank for nucleotide sequences showing similarity
to the EST nucleotide query sequence using the BLASTN
algorithm.
[0280] One of the subject sequences obtained from GenBank
(AA012833, identified here as SEQ ID NO:2, a 3.5 kb clone from the
Soares retina N2b4H2 library) showing similarity to the nucleotide
sequence encoding amino acid sequence identified as SEQ ID NO:1,
also showed similarity to a human genomic DNA sequence from GenBank
(Accession#, AC007002 (clone name NH0493L16), identified here as
SEQ ID NO:3). As described below, the present inventors determined
that this clone comprised within it the polynucleotide sequence
encoding the novel cytochrome P450 of the invention, P450RAI-2. A
BLASTN search of the EST database using SEQ ID NO:2 failed to
identify any more EST sequences showing similarity. In order to
check for protein sequences which may show similarity to the 6
possible reading frames for translation of SEQ ID NO:2, the BLASTX
program was run on the non-redundant GenBank database. Several
sequences with the highest degrees of amino acid similarity were
identified and included, human, mouse, Xenopus and zebrafish
P450RAI (CYP26A; protein encoded by SEQ ID NO:13 (human), protein
encoded by SEQ ID NO:14 (zebrafish)).
[0281] Using the amino acid sequence of human P450RAI-1 (SEQ ID NO.
4 of WO 97/49815) aligned to the 3 possible reading frame
translations of SEQ ID NO:3, a potential full-length amino acid
sequence for a novel CYP cDNA family member was assembled. The
intron/exon boundaries were deduced based on the loss of amino acid
similarity. The intron/exon boundaries of the novel cytochrome P450
is shown in FIG. 1. The amino acids within the respective exons are
identified above the schematic diagram and nucleotide positions in
relation to human sequence are provided below the diagram. However,
it would be appreciated that the positions of the exons noted in
FIG. 1 are approximate and may vary slightly from the actual
boundaries. The sequence has been termed P450RAI-2 based on its
sequence homology with CYP26A. An amino acid sequence comparison
between human P450RAI-2 and human p450RAI-1 is shown in FIG. 3.
Overall the two protein sequences show 42% identity at the amino
acid level and 52% at the nucleotide level over the region of the
predicted open reading frame. The overall similarity of the two
putative open reading frames is somewhat higher when conservatively
substituted amino acids are considered.
[0282] Human retina Marathon-Ready cDNA (Clontech, California) was
used as a template to amplify P450RAI-2 using the polymerase chain
reaction (PCR) according to the manufacturer's directions Using the
potential nucleotide sequence derived from the intron/exon mapping
exercise, two primers, (SEQ ID NO. 9 and SEQ ID NO. 10) one
upstream of the putative initial methionine and one downstream of
the putative stop codon, were synthesized and used to PCR amplify a
fragment of approximately 1600 base pairs representing the coding
region SEQ. ID NO. 4 See FIG. 2 [SEQ ID NO:28] which depicts the
original clone within the 1598 bp coding sequence and the 3
untranslated region which was cloned and sequenced. The 1598 bp
fragment including the coding sequence of the cDNA is indicated as
SEQ ID NO. 4 and appears to correspond to a full-length cDNA. SEQ
ID. 4 shows a single nucleotide change from "C" in the genomic
sequence SEQ ID NO. 3 to "T" at nucleotide 1401 of SEQ ID NO. 4
which, due to the degeneracy of the genetic code, does not change
the corresponding amino acid sequence, identified as SEQ ID NO.
5.
[0283] The cDNA 1600 bp PCR amplified product was gel purified
using the QIAEXII Gel Extraction kit (Qiagen, California) and
ligated into the pT-Adv vector using T4 DNA Ligase, heat shocked
into competent TOP10F' Escherichia coli, plated on Luria
Bertoni-kanamycin plates and incubated overnight at 37.degree. C.,
as per the manufactures instruction for the AdvanTAge PCR Cloning
Kit (Clontech, CA). White colonies were grown up in Luria
Bertoni-kanamycin medium, and DNA was prepared using the QIAprep
Spin Miniprep kit (Quiagen, CA). For transient expression studies
the P450RAI-2 cDNA was subcloned into the EcoR1 restriction
endonucleoase site of pcDNA3.1 (Invitrogen, CA).
Example 2
P450RAI-2 Tissue Expression
[0284] Given the presence of a full-length cDNA in a retinal cDNA
library, expression in human retinal tissues could be expected. In
order to find tissues in which P450RAI-2 is expressed, a
multi-tissue RNA dot blot containing samples from 76 different
normal human tissues a human poly A+ blot (Clontech, CA) was probed
using a labeled probe encoding the full length P450RAI-2
.alpha.-[.sup.32P]dATP-labeled probe for the corresponding cDNAs
were hybridized to blots using the conditions as described in the
manufacturers directions. A human brain multi-tissue northern blot
(Clontech, CA) was also hybridized with full-length
.alpha.-[.sup.32P]dATP-labelled P450RAI-2 probe according to the
manufacturer's directions. The blots were stripped and re-probed
with .alpha.-[32P]dATP-labelled ubiquitin and .beta.-actin
controls.
[0285] FIG. 4 illustrates the results. P450RAI-2 appears to be
expressed in a variety of tissues in differing levels (other data
not shown) and at higher levels in tissues including kidney, lung,
liver, spleen, fetal spleen, skeletal muscle, thymus, peripheral
blood leukocyte, lymph node, bone, stomach, placenta, duodenum and
pituitary gland. However, samples from human brain including pons
(FIG. 4A, sample hl) and left and right cerebellum (FIG. 4A,
samples a2 and b2 respectively) clearly show the highest levels of
expression. FIG. 4C depicts the tissue map of the 76 tissue
samples. In comparison, a similar blot probed with P450RAI-1 shows
low level expression in most of the tissues with the absence of a
distinct signal in any of the corresponding tissues from human
brain (data not shown). Blots shown in FIGS. 4A and 4B are
representative of multiple hybridization experiments. Two
independent blots were utilized and each blot was hybridized with
probes for P450RAI-2, P450RAI-1 and ubiquitin control (FIG. 4B in
order to verify the results.
[0286] A human Northern blot (FIG. 4D) (Clontech, CA) comprising
mRNAs from various brain tissues was also probed with P450RAI-2
[SEQ ID NO. 4] according to the manufacturers directions. A
hybridizing transcript of greater than 4.4 Kb was seen and is
consistent with the size predicted by the cDNA clone isolated from
human retina and from the predicted exons of the genomic clone that
was identified. Consistent with the dot blot analyses, considerably
higher levels of expression of transcripts for P450 RAI-2 are seen
in the cerebellum. Lower but detectable levels of expression are
observed in cerebral cortex, medulla, occipital pole, frontal lobe
and temporal lobe. Other data from a Northern blot comprising mRNAs
from multiple tissues indicated various levels of expression of
P450RAI-2 in brain, heart, skeletal muscle, spleen, kidney, liver
and small intestine. [Data not shown]
Example 3
Inducibility of P450RAI-2
[0287] The regulation of P450RAI-2 expression by various retinoids
was checked in three different human cell lines, SKMES, CALU-1 and
MCF-7. See FIG. 5A (P450RAI-2 probe), B (blot was stipped and
re-probed with .beta.-actin) and C is ethidium bromide stained gel
showing 18S and 28S RNAs. Tissue culture cells were incubated with
each retinoid for 12 hours, total RNA prepared and a northern blot
performed. The blot was hybridized with SEQ ID NO:4 using
ExpressHyb (Clontech, CA) according to the manufacturer's
directions at 65.degree. C. The blot was washed as follows:
2.times.SSPE & 0.1% SDS two washes of 5 minutes each;
1.times.SSPE & 0.1% SDS one wash of 15 minutes at 65.degree.
C.; 0.1.times.SSPE & 0.1.times.SDS one wash of 15 minutes at
65.degree. C. The blot was exposed to film for 66 hours. P450RAI-2
was found to be induced in MCF-7 cells in response to both
all-trans retinoic acid and 13-cis retinoic acid. Whether or not
9-cis retinoic acid induces expression of P450RAI-2 is not clear
from these results. It is known that aliquots of both 9-cis and
13-cis retinoic acid are frequently contaminated with all-trans
retinoic acid.
[0288] P450RAI-2 expression was found to be inducible in the human
keratinocyte cell line HPK1A-RAS. Cells in culture were incubated
with all-trans retinoic acid at a final concentration of 10.sup.-6
M for 12 hours. Total RNA was prepared and a northern blot
performed. The blot was hybridized at 65.degree. C. using
PerfectHyb (Sigma, Mo.) according to the manufacturer's directions.
The blot was washed as follows: 2.times.SSC & 0.1% SDS two
washes of 5 minutes each at room temperature; 1.times.SSC &
0.1% SDS one wash of 15 minutes at 65.degree. C.; 0.1.times.SSC
& 0.1% SDS three washes of 15 minutes each at 65.degree. C. The
blot was exposed to X-ray film overnight. The results are shown in
FIG. 6.
[0289] The potential induction of mRNAs for P450RAI-2 in response
to all-trans-RA was also evaluated.
[0290] Tissue culture cells were incubated with 1 .mu.M
all-trans-RA dissolved in dimethylsufoxide (DMSO) or DMSO alone for
12 hours, total RNA prepared and a northern blot analysis
performed. The blot was hybridized at 65.degree. C. using
ExpressHyb (Clontech, CA) according to the manufacturers
directions. The blot was washed as follows: 2.times.SSPE & 0.1%
SDS two washes of 5 minutes each; 1.times.SSPE & 0.1% SDS one
wash of 15 minutes at 65.degree. C.; O.1.times.SSPE &
0.1.times.SDS one wash of 15 minutes at 65.degree. C. The blot was
exposed to X-ray file for 66 hours.
[0291] RT-PCR was performed using 1 .mu.g total RNA and the
Advantage One-Step RT-PCR kit (Clontech, CA). The final primer
concentrations were 45 .mu.M for the P450RAI-2 primers and 11.25
.mu.M for the GAPDH-specific primers. The thermal cycling program
for cDNA synthesis and PCR amplification was 1 cycle at 50.degree.
C. for 1 hr, 1 cycle at 94.degree. C. for 5 min, followed by 30
cycles of 94.degree. C. for 30 sec, 65.degree. C. for 30 sec,
68.degree. C. for 1 min, and a final cycle for 68.degree. C. for 2
min. The P450RAI-2 upstream amplification primer was
5'-TCCCTGCCTGTCGACCTGCCCTTC-3' (SEQ ID NO: 29) and the downstream
primer was 5'-GACACTCCAGCCTTTGGGGATCTG-3' (SEQ ID NO: 30). The
upstream and downstream primers to detect human
glyceraldehyde-3-phosphate dehydrogenase mRNA were respectively,
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (SEQ ID NO:31) and
5'-CATGTGGGCCATGAGGTCCACCAC-3' (SEQ ID NO:32).
[0292] The RT-PCR products were electrophoresed on a 1.2% agarose
gel and blotted onto Hybond-N.sup.+ membrane (Amersham Pharmacia
Biotech, UK). Hybridization was performed at 42.degree. C. using
ExpressHyb hybridization buffer (Clontech Laboratories Inc., CA)
and probed with an interal P450RAI-2-specific oligonucleotide,
5'-GTGTGCCCTCGCAGGGGCAGCCGCCACTGTGC-3' (SEQ ID NO: 33) that had
been end-labeled using .gamma.[.sup.32P]ATP and T4 polynucleotide
kinase. The membrane was subsequently stripped and re-probed with
an internal-end labeled P450RAI-1-specific oligonucleotide,
5'-CGCCTCGGATGCCCGCAGCCC-GCAGATCTTGG-3' (SEQ ID NO: 34) The
membrane was again stripped and re-probed with an internal GAPDH
oligonucleotide. The final wash for all hydbridizations was
0.1.times.SSC/0.1%, 50.degree. C., 15 min, blots were exposed to
Kodak X-0mat AR film (Eastman Kodak Co., NY).
[0293] Several human cell lines in culture were tested for
expression and induction of expression of P450RAI-2 by treating
cells with 1 .mu.M all-trans-RA or DMSO for 12 hours followed by
Northern blot and RT-PCR analyses (FIG. 7A). Of the four cell lines
tested by Northern analysis, three (HPK1a-ras, HeLa and MCF-7) show
induction of P450RAI-2 transcripts in response to all-trans-RA with
MCF-7 showing the strongest induction.
[0294] Reverse transcription polymerase chain reaction (RT-PCR)
analyses of transcripts from both control and all-trans-RA treated
cultured cells demonstrate several important findings. As with
P450RAI-1, P450RAI-2 shows multiple distinct modes of regulation of
expression (FIG. 7B). NB4, V79, and SK-Luci-6 cells appear to have
little or no perceptible transcripts for P450RAI-2 in either
vehicle or all-trans-RA treated cells. Several cell lines,
including MCF-7, HeLa, HPK1a-ras and WT-E show evidence for
inducible expression of P450RAI-2 when cells are exposed to 1 .mu.M
all-trans-RA. Interestingly, SKMES-1 and SW900, shows constitutive
expression of P450RAI-2 in both treated and untreated samples.
[0295] Using RT-PCR we also evaluated a brief time course of
induction of PCR products corresponding to mRNA transcripts for
P450RAI-2 in HPK1a-ras cells. These results (FIG. 7C) indicate
that, at least in one cell line, transcripts for P450RAI-2 can be
induced by all-trans-RA within 2 hours post-addition of inducer
suggesting a direct transcriptional mechanism of induced.
Example 4
P450RAI-2 Homologs
[0296] The existence of homologs of P450RAI-2 in other species was
explored. SEQ ID NO. 4 and the BLASTN algorithm was used in a
search of the NCBI EST database. Initially, three homologs were
identified, one each from mouse, rat and zebrafish. The nucleotide
sequences of these clones, SEQ ID NO.6, (mouse) SEQ ID NO.7 (rat)
and SEQ ID NO.8 (zebrafish) show extensive similarity to SEQ ID
NO.5. The corresponding amino acid sequences of the mouse and
zebrafish are given as SEQ ID NOs.11 and 12, respectively.
[0297] The clones for mouse, zebrafish and rat are all partial
cDNAs and encoding portions corresponding to portions of the
full-length coding sequence of the human cDNA (SEQ ID NO. 4). The
zebrafish amino acid sequence, which shows homology to P450RAI-2,
contains 167 amino acid residues. Of the 167 amino acids, 115 are
identical to the human CYP26A sequence (i.e., when the amino acids
are aligned using the Omiga software program (Oxford Molecular,
Campbell, Calif.), version 1.13, with the default parameter
settings for protein alignments, 115 are the same), giving a
homology of about 68 percent. The rat sequence shows nucleotide
homology to the 3'-untranslated region of human P450RAI-2 so no
conclusion about the degree of homology to the coding region of
P450RAI-2 can be made. The mouse amino acid sequence shows an
absolute conservation with the human counterpart. Over a stretch of
92 amino acids beginning with the potential start methionine of the
human P450RAI-2, all 92 amino acids are conserved between the mouse
and human. At the nucleotide level there is 93.1 percent homology
between the mouse and human. This degree of homology is
exceptionally high, but this is a preliminary result in the sense
that the full-length mouse sequences have yet to be obtained. The
open reading frame of the mouse nucleotide sequence extends at
least 111 amino acids upstream of the putative initial methionine
in human P450RAI-2, raising a possible question as to the true
origin of the mouse sequence.
[0298] Further searching of the NCBI Mouse EST database available
over the internet at
http://www.ncbi.nlm.nih.gov/blast/blast.cgi?[form=1] using the
human P450RAI-2 sequence revealed three mouse EST clones,
AW488377.1 (SEQ ID NO:18) which corresponds to human cDNA
nucleotides 271-831, AW047279.1 (SEQ. ID. NO:19) which corresponds
to human cDNA nucleotides 1-276 and includes 5' UTR ATG is
underlined in bold, wherein the sequence should be read backwards
in antisense from this point, and EST BE864840.1 (SEQ. ID. NO:20)
which corresponds to human cDNA nucleotides 1-118 and includes
5'UTR; ATG is marked. In sequencing clone AW488377, the inventors
identified a novel sequence included therein, SEQ ID NO: 26. A
further rat EST, SEQ ID NO: 27 was also identified that corresponds
to human P450RAI-2 nucleotides 1-388.
[0299] The ESTs were used to search the HTGS database
http://www.ncbi.nl m.nih.gov/blast/blast.cgi.?[form=1] wherein
genomic mouse clone AC022779.3 was identified, potentially
comprising the complete mouse nucleotide coding sequence of
P450RAI-2. Using techniques similar to that used to identify the
intro/exon boundaries of human P450RAI-2, the mouse clone was
aligned with the human P450RAI-2 cDNA to detect similarities. Five
of Six putative mouse exon sequences were identified: SEQ ID NO: 21
(corresponds to exon 1, mouse sequence 185832-186036), SEQ ID NO:
22 (corresponds to exon 2, mouse sequence 189360-186590), SEQ ID
NO: 23 (corresponds to exon 3, mouse sequence 196630-196905, SEQ ID
NO: 24 (corresponds to exon 4, mouse sequence 197146-197303), and
SEQ ID NO: 25 (corresponds to exon 6, mouse sequence
90745-90366).
Example 5
Metabolism of all-Trans Retinoic Acid by P450RAI-2
[0300] Analysis of all-Trans-RA Metabolism by HPLC.
[0301] 48 hours post-transfection, cells were washed twice with
DMEM medium (without serum) and then incubated in 0.5 ml DMEM
medium containing 10% FBS and either 100 nM radiolabelled
all-trans-RA (0.1 .mu.Ci/ml[.sup.3H]-RA; 5nCi/nmol) or unlabelled 1
.mu.M all-trans-RA. After incubation for 3 hours at 37.degree. C.,
in a light protected environment, total lipids were extracted as
described previously by Bligh and Dyer [1957] as modified in White
and Petkovich [1996b]. The aqueous soluble retinoid metabolites
were quantified using .beta.-scintillation counting. The organic
soluble metabolites were dried under nitrogen gas, resuspended in
100 .mu.l acetonitrile:water:acetic acid in the ratio 50:50:0.5 and
analyzed by HPLC. HPLC was performed using a reverse phase column
(150.times.4.6 mm C18 Zorbax-SB, Hewlett Packard) at a flow rate of
1 ml/min. The Mobile phase contained 10 mM ammonium acetate and
consisted of an isocratic elution for 2 min with solvent A
(acetonitrile; water: acetic acid 50:50:0.5) followed by a linear
gradient over 18 min from solvent A to solvent B (acetonitrile:
water: acetic acid 90:10:0.04) and then an isocratic elution with
solvent B for an addition 5 min. Effluent from the HPLC column
flowed directly to a radioflow detector LB (EG&G Berthold). The
retinoids were detected at a wavelength of 351 nm and the
ultraviolet spectrum of each metabolite peak was determined using
photodiode array detection. Radioactivity as well as ultraviolet
spectrum data was analyzed using Millennium 32 software (Waters,
Mass.). Aqueous soluble radioactivity (FIGS. 14A and B) was
calculated by integration of selected regions of the chromatograms.
Three regions of the chromatograms were defined which represent:
(I) the substrate peak (all-trans-RA); (ii) peaks with retention
times between 8 and 12 minutes (4-OH region); and more polar peaks
with retention times between 2 and 6 minutes (polar region).
Example 5A
[0302] Retinoic acid as a substrate of P450RAI-2 was studied. The
full-length human P450RAI-2 cDNA was cloned into the eukaryotic
expression vector pcDNA 3.1 (Invitrogen, CA). Exponentially growing
COS-1 cells were plated in triplicate into 6 well plates and
transiently transfected with 1 ug of pcDNA 3.1, pcDNA3.1-P450RAI-1,
or pcDNA3.1-P450RAI-2 using Fugene 6 transfection reagent with 3 ul
per sample as described by the manufacturer (Roche Molecular
Biochemicals, IN) and then incubated with nanomolar concentrations
of [11,12-.sup.3H]all-trans retinoic acid or micromolar
concentrations of non-radioactive all-trans retinoic acid. COS-1
cells are an African green monkey kidney "fibroblast-like" cell
line. The cell line was maintained in DMEM supplemented with 10%
FBS at 37.degree. C. in an atmosphere of 5% CO.sub.2 and 95%
air.
[0303] P450RAI-2 expression in COS-1 cells promoted the rapid
conversion of RA into both lipid- and aqueous-soluble metabolites.
See FIGS. 8 to 13. Fractions of total lipid extracts of transfected
cells were initially separated by reverse-phase HPLC on Zorbax-SB
C18 column (Hewlett Packard). HPLC conditions used a linear
gradient of 50% acetonitrile, 49.9% H.sub.2O, 0.1% acetic acid to
90% acetonitrile, 9.9% H.sub.2O, 0.1% acetic acid with a flow rate
of 1 ml/minute. Radioactivity was detected using a Berthold
RadioFlow monitor. Using these conditions standard retinoids eluted
at the following times: all-trans retinoic acid--20.5 minutes;
4-OH-retinoic acid--8.1 minutes; 4-oxo-retinoic acid--9.6 minutes;
and 18-OH-retinoic acid--9.9 minutes. Comparison between extracts
from pcDNA 3.1 and pcDNA 3.1-P450RAI-2 cells indicated that
P450RAI-2 significantly increased all-trans retinoic acid
metabolism. Incubation of P450RAI-2 transfected cells with
micromolar concentrations of all-trans retinoic acid resulted in
the production of multiple more polar peaks, some of which
co-eluted with the standard retinoids. FIG. 8A shows an increase in
aqueous-soluble radioactivity in P450RAI-2 transfected cells
compared to media or pcDNA alone. FIG. 8B shows that there was an
increase in lipid-soluble metabolites of all-trans retinoic acid
when P450RAI-2 was transfected into the COS-1 cells. Metabolism of
micromolar concentrations of non-radioactive all-trans retinoic
acid was also evaluated. Transfected cells and controls were
exposed to 1 micromolar all-trans retinoic acid for 3 to 4 hours
and then analyzed for metabolism. Photo-diode array detection of
HPLC-separated peaks are shown in FIGS. 10 to 13. FIG. 10 shows a
background profile of media alone. FIG. 12 shows COS-1 cells
transfected with the pcDNA plasmid alone. FIG. 12 shows the
generation of more polar products when P450RAI (human) is
transfected. In comparison, in FIG. 13, P450RAI-2 also causes the
rapid metabolism of all-trans retinoic acid substrate to polar
metabolites, several of which have the same retention times as the
retinoid standards for 4-OH-retinoic acid and 4-oxo-retinoic acid.
Although similar, there appear to be some differences in the ratios
of individual metabolites in the profiles generated by P450RAI-2
compared to P450RAI.
Example 5B
[0304] COS-1 cells were transfected with either plasmid
(pcDNA3.1-P450RAI-2 or pcDNA3.1-P450RAI-1). The cells were then
incubated with, all-trans-RA substrate over a 3 hour incubation
period. The all-trans-RA substrate was extensively metabolized to
more polar aqueous soluble products (FIGS. 14 and 15). HPLC
analysis using photodiode array detection was performed on samples
prepared from transfected cells treated with 1 .mu.M unlabelled
all-trans-RA. FIGS. 14A, B and C show comparative chromatograms of
the lipid-soluble extracts from pcDNA3.1-P450RAI-2 (FIG. 14A),
pcDNA3.1-P450RAI-1 (FIG. 14B) and pcDNA3.1 transfected cells (FIG.
14C). In both pDNA3.1-P450RAI-2 and pcDNA3.1-P450RAI-1 transfected
cells the generation of multiple more polar peaks is observed.
There is also a significant decrease in all-trans-RA substrate when
compared to pcDNA3.1 controls (compare RA peaks in FIGS. 14A, B,
with C). Peaks labelled as 4-OH-retinoic acid, 4-oxo retinoic acid
and 18-OH-retinoic and co-elute with standards of and show
characteristics UV spectra of these metabolites. Additionally,
multiple unidentified peaks (labeled 1-4; FIG. 14A) which show
maxima characteristic of retinoids (data not shown) are generated
and appear to be qualitatively similar in both P450RAI-2 and
P450RAI-1 samples compared to controls.
[0305] To evaluate the efficiency of P450RAI-2 at more
physiological concentration of substrate, P450RAI-1 transfected
cells were exposed to 100 mM radiolabelled (spec. act.)
all-trans-retinoic acid (FIGS. 15A and B). In these cells,
34.+-.0.2% of the all-trans-RA substrate is converted to
aqueous-soluble products compared to 26.+-.1.5% in the P450RAI-1
transfected cells (FIG. 15A). Controls, including media alone or
pcDNA3.1 transfected cells, show 1.6.+-.0.1% (media alone) and
2.7.+-.0.2% (pcDNA3.1) conversion of substrate to aqueous
radioactivity.
[0306] The radioactivity remaining in the organic soluble fraction
in the transfected cells exposed to 100 nM [.sup.3H]
all-trans-retinoic acid (FIG. 15B) was also evaluated. HPLC
analysis identified many more polar metabolites in both the
P450RAI-2 and P450RAI-1 transfected cells compared to controls
(data not shown). In media from cells transfected with
pcDNA3.1-P450RAI-2 or pcDNA3.1-P450RAI-1 a high degree of
disappearance of substrate compared to controls was observed. As
well, there is a concomitant increase in the more polar
lipid-soluable retinoid metabolites which elute in both the 4-OH
and polar regions of the chromatograms. These results clearly
indicate that expression of either P450RAI-2 or P450RAI-1 causes
substantial metabolism of all-trans-RA to more polar metabolites
(FIG. 15B).
Example 6
Retinoid Substrate Specificity of P450RAI-2
[0307] Given the presence of two unique enzymes P450RAI-1 and
P450RAI-2, with the capacity to rapidly metabolize all-trans-RA the
specificity of these two enzymes were evaluated. Interestingly,
both P450RAI-1 and P450RAI-2 show approximately equal efficiencies
at metabolizing all-trans-RA (FIGS. 15 and 16 A-D). Competition
assays were also performed to evaluate the ability of five
retinoids, all-trans-RA, 9-cis-RA, 13-cis-RA, retinal and retinol
to compete out P450RAI-2 or P450RAI-1 mediated all-trans-RA
metabolism (FIG. 16). The non-specific cytochrome P450 inhibitor,
ketoconazole, was also tested.
[0308] COS-1 cells were transfected with either pcDNA3.1-P450RAI-1
or pcDNA3.1-P450RAI-2 in 6-well tissue culture plates as described
above. 48 hours post-transfection cells were harvested, pooled,
washed with DMEM medium and replated into duplicate 48-well plates
with 5.times.10.sup.5 cells per well. The cells were incubated in
0.2 ml DMEM medium containing 0.05 .mu.Ci/ml[.sup.3H]-RA (final
concentration 2 nM) in the present or absence of increasing
concentrations of the each unlabelled retinoids (all-trans-RA,
9-cis-RA, 13-cis-RA, retinol, retinal). Control cells were
incubated with increasing concentration of ketoconazole. After
incubation for 3 hours at 37.degree. C., the retinoids were
extracted using the Bligh and Dyer [1957] procedure and the aqueous
soluable RA-metabolites were counted in a scintillation counter as
described above.
[0309] These competition studies indicated that P450RAI-1 and, -2
exhibit comparable substrate specifics with all-trans-RA being the
preferred substrate for both enzymes having ID.sub.50 values of
approximately 3.0 .mu.M for P450RAI-2 and 2.5 .mu.M for P450RAI-1.
The other retinoids show varying abilities to compete out
metabolism of all-trans-RA by P450RAI-2 and P450RAI-1 in the
following rank order:
9-cis-RA>13-cis-RA>retinal.gtoreq.retinol (see Table 1 for
interpolated ID.sub.50 values). Using microsomes prepared from
stably-transfected P450RAI-1 cells we have also found the same
relative levels of competition suggesting that the differences in
ID.sub.50 values are not due differences in cellular uptake of the
retinoids (data not shown).
TABLE-US-00001 TABLE 1 INTERPOLATED ID.sub.50 VALUES P450RAI-1
P450RAI-2 All-trans-RA 2.5 3 9-cis-RA 32 25 13-cis-RA >75 55
Retinol >100 >100 Retinol >100 >100 Ketoconzole 16
12
FIG. 16E also illustrates a substrate competition in Mammalian
microsomes. [.sup.3H]-RA-metabolism inhibition assay was carried
out using microsomes prepared from Hela-CYP26B (stable) cells.
Briefly, microsomes (5 .mu.g) were incubated with [.sup.3H]-RA and
increased concentration of unlabeled all-trans-RA (ATRA), 9-cis-RA,
13-cis-RA, or ketoconazole. The reaction was started by adding
NADPH for 1 hour at 37.degree. C. and then total reinoids were
extracted using Bligh-Dyer procedure and water-soluble metabolites
counted. The results in FIG. 16E show that ATRA is the high
affinity substrate for CYP26B (P450RAI-2). 13-cis-RA did not
compete at lower concentrations. Ketoconazol is an inhibitor of
CYP26B.
Example 7
Expression of P450RAI-1 and P450RAI-2 in Human Cells
[0310] Total RNA aliquots (10 .mu.g) were electrophoresed on a 1.0%
formaldehyde-agarose gel and a northern blot performed. The gel was
photographed under ultraviolet light and then blotted on Hybond ECL
nitrocellulose membrane (Amersham Pharmacia Biotech, UK) and fixed
to the membrane by baking at 80.degree. C. for 2 hours under
vacuum. Prehybridization and hybridization steps were performed
using ExpressHyb (Clontech, CA) according to the manufacturer's
directions. Three individual cDNA fragments were labeled with
.alpha.-[.sup.32P]dATP using the Prime-A-Gene Labeling System
(Promega, WI). The probes were as follows; P450RAI-1 762-1217 bp of
SEQ ID NO:13, full length P450RAI-2 SEQ ID NO:4 and a human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR fragment. The
blot was hybridized with P450RAI-2, washed two times for 5 minutes
in 2.times.SSPE, 0.1% SDS at room temperature, then washed once at
65.degree. C. for 10 minutes in 1.times.SSPE, 0.1% SDS and a final
wash for 15 minutes at 65.degree. C. in 0.1.times.SSPE, 0.1% SDS.
The blot was exposed at -70.degree. C. for 48 hours to Kodak X-Omat
AR film (Eastman Kodak Company, NY). FIG. 17A shows P450RAI-2
expression was induced in the RAFT sample treated with Tazarotene.
P450RAI-2 appears to also be expressed in the Day 14 Psoriasis
Biopsy sample treated with Tazarotene. The blot was stripped by
washing two times for 30 minutes in boiling 0.5% SDS and exposed to
film overnight to ensure proper removal of probe. The above
protocol was repeated for P450RAI-1 and GAPDH. The P450RAI-1 and
GAPDH blots were exposed for 96 and 4 hours respectively. Two
separate hybridizations with a P450RAI-1 probe did not produce any
bands on the northern blot.
[0311] RT-PCR was performed using 1 ug of total RNA and the
Advantage One-Step RT-PCR kit (Clontech, CA). The final primer
concentrations were 45 .mu.M for the P450RAI-1 and P450RAI-2
primers and 11.25 .mu.M for the GAPDH-specific primers. Two
separate sets of reactions were performed using the primer sets,
P450RAI-1/GAPDH and P450RAI-2/GAPDH. The thermal cycling program
for cDNA synthesis and PCR amplification was 1 cycle at 50.degree.
C. for 1 hour, 1 cycle at 94.degree. C. for 5 minutes, followed by
30 cycles of 94.degree. C. for 30 seconds, 65.degree. C. for 30
seconds, 68.degree. C. for 1 minute and a final cycle of 68.degree.
C. for 2 minutes. The P450RAI-1, P450RAI-2 and GAPDH upstream and
downstream amplification primers sets were
5'-GCCTTCGAGGAAATGACCCG-3' (SEQ ID NO: 35) and
5'-CTGGATGCATCCTCTGGGTG-3' (SEQ ID NO: 36),
5'-GTCTACCAGCAGTTTGTGGAC-3' (SEQ ID NO: 37) and
5'-AGTCCAGGTAGCGCAGCCCACT-3' (SEQ ID NO: 38),
5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (SEQ ID NO: 39) and
5'-CATGTGGGCCATGAGGTCCACCAC-3' (SEQ ID NO: 40) respectively. Total
RNA isolated from MCF-7 cells treated with all-trans-RA was used as
a positive control. FIG. 17B also shows expression of P450RAI-2 in
the Tazarotene treated RAFT sample. Both the Psoriasis samples show
PCR products corresponding to P450RAI-2.
Example 8
Expression of P450RAI-2 in Mouse Embryos
[0312] Probe synthesis: All in situ hybridization experiments were
performed using an antisense copy of mouse EST AW488377.1 [SEQ ID
NO:18] labelled with digoxigenin. The EST was cloned into pT7T3D,
linearized with HindIII, and transcription was initiated from the
T7 promoter. RNA labeling reactions were performed using DIG RNA
labeling mix (Roche) as per instructed by the kit insert.
[0313] In Situ Hybridization: Mice were sacrificed by cervical
dislocation, and embryos were dissected in 1.times.PBS, before
being left to fix overnight in 4% paraformaldehyde at 4.degree. C.
The next day, embryos were washed twice with PBT (1.times.PBS, 0.1%
Tween-20) for 5 minutes at 4.degree. C. Embryos were washed 5
minutes each with 25, 50, 75% methanol in PBT, then twice with 100%
methanol and then stored at -20.degree. C. To rehydrate embryos,
they were taken through the previous methanol series in reverse and
washed twice with PBT. Then embryos were treated with 10 .mu.g/mL
proteinase K in PBT for the following times: 8.5 dpc (days post
coitum)--no treatment; 9.5 dpc--3 minutes; 10.5 dpc--4 minutes;
11.5 dpc--5 minutes. 3 washes of 2 mg/mL glycine in PBT were
performed for 5 minutes each. Embryos were refixed in 0.2%
glutaraldehyde/4% paraformaldehyde in PBT for 20 minutes, and then
washed twice with PBT for 5 min. 1 mL of prehybridization solution
(5.times.SSC, 1% SDS, 5 .mu.g/mL Yeast tRNA, .mu.g/mL heparin) was
added, and embryos were incubated at 70.degree. C. After 1 hour,
prehybridization solution was replaced with 1 mL of fresh solution
containing 1 .mu.g digoxigenin-labeled probe and left to hybridize
overnight at 70.degree. C. The next day, embryos were washed twice
with prehybridization solution for 30 minutes at 70.degree. C.
Embryos were washed for 20 minutes at 70.degree. C. with a 1:1 mix
of prehybridization solution and 1.times.MABT (DIG Wash and Block
Buffer Set, Roche). After 3 rinses with MABT, embryos were washed
twice for 30 minutes at 70.degree. C. with MABT. To preblock
embryos, 1.times.MABT/2% Blocking Reagent (DIG Wash and Block
Buffer Set, Roche) was added. After an hour, solution was removed
and replaced with 1.times. MABT/2% Blocking Reagent/20% sheep serum
and left for 1 hour. Next, 1.times. MABT/2% Blocking Reagent/20%
sheep serum/0.0005 Anti-Digoxigenin Antibody coupled to Alkaline
Phosphatase (Roche) was added at left overnight at 4.degree. C. The
next day, embryos were rinsed 3 times with 1.times.MABT with 2 mM
levamisole, and washed five times for 1 hour in 1.times. MABT/2 mM
levamisole. Two washes of 10 minutes each were performed in NTMT
(100 mM NaCl, 100 mM Tris-HCl, pH=9.5, 50 mM MgCl.sub.2, 2 mM
levamisole). 1 mL of fresh NTMT was added along with 3.5 .mu.L BCIP
and 4.5 .mu.L NBT (Gibco), and colour reaction was allowed to
proceed in the dark. When complete, embryos were rinse twice with
PBT, and refixed overnight at 4.degree. C. in 0.2%
glutaraldehyde/4% paraformaldehyde. The next day, embryos were
washed for 1 hour in 1:1 CMFeT:glycerol before transfer to 4:1
CMFeT:glycerol until ready to photograph.
[0314] FIG. 18 illustrates P450RAI-2 in 8.0 and 8.5 dpc mouse
embryos. (A) 8.0 dpc lateral view. No apparent staining. (B) 8.0
dpc, dorsal view. Staining at anterior end of neutral folds. (C)
8.5 dpc, lateral view. Expression is evident, possibly in
presumptive rhombomeres 2, 5, and 6. (D) 8.5 dpc, dorsal view,
rhombomere expression of RAI2 is clearly evident. TL: Tail; YS Yolk
Sac; NF: Folds; pr: Presumptive Rhombomeres.
[0315] FIG. 19 illustrates P450RAI2 in 9.0 and 10.5 dpc mouse
embryos. (A) 9.0 dpc, lateral view. Specific staining is visible in
the eye, and rhombomeres 5 and 6. Diffuse staining is visible where
the hind bud is beginning to form. (B) 9.0 dpc, dorsal view.
Rhombomeres 5 and 6 show RAI2 expression. (C) 10.5 dpc, lateral
view. The otic vesicle and eye are stained. (D) 10.5 dpc, dorsal
view. Specific staining is observed in both otic vesicles as well
as the hind limb bud. HL: hind limb bud; OV: Otic Vesicle.
[0316] FIG. 20 illustrates P450RAI2 staining in 11.5 dpc mouse
embryo (A) 11.5 dpc, laterial view, staining is visible in both the
fore and hindlimb bud. (B) 11.5 dpc, ventral view. Expression of
P450RAI2 in both limb buds (C) Close up of forelimb bud, showing a
lack of expression in the apical ectodermal ridge FL: Forelimb bud;
HL: Hindlimb bud; AR: Apical Ectodermal Ridge.
[0317] FIG. 21 illustrates P450RAI-2 Staining in Embryos Treated
with Retinoic Acid (A) 8.5 dpc, lateral view, staining is observed
in rhombomere 5 and the tail mesoderm as indicated by the arrow.
(B) 8.5 dpc, dorsal view clearly showing P450RAI2 expression in
rhombomere 5. (C) 9.5 dpc, lateral view, expression of P450RAI2 is
observed in rhombomeres 5 and 6, the developing hindlimb, somites
and posterior mesoderm. (D) 9.5 dpc, dorsal view, expression is
evident in rhombomeres 3, 5 and 6 and in trunk ectoderm as
indicated by the arrow. r: rhombomere; HL; Limb bud.
[0318] FIG. 22 illustrates P450RAI-2 expression in 11.5 dpc embryos
treated with retinoic acid. (A) 11.5 dpc, lateral view, P450RAI2
expression is observed in both the developing hind and fore limb.
(B) 11.5 dpc, ventral view, as in embryos untreated with retinoic
acid, P450RAI2 expression is not observed in the aptical ectodermal
ridge.
Example 9
Assays Using Cell Lines Expressing Different P450RAI's
[0319] P450RAI-1 and -2 expression plasmids can be used to generate
transfected cells. Plasmid vectors such as pcDNA3.1 (Invitrogen,
CA) are used to express cDNA of interest in the cells. Cells such
as COS-1 or Hela could be used as the host for this purpose. Cells
are then exposed to either low concentrations (picomolar) of a
radioactive agent (substrate) or higher concentrations (micromolar)
of non-radioactive agent (substrate) and the metabolic profile
determined (see Example 5 and international patent publication No.
WO 97/49815 in which all-trans retinoic acid is a substrate but
other retinoid or retinoid type compounds can be used). The time of
incubation of the cells can vary from 1 hour to 24 or 48 hours
depending on the amount of agent and levels of expression of the
protein of interest. The representative metabolite profiles are
determined using phase extraction and HPLC. In the case of
radioactive substrate .beta.-scintillation counting can also be
used if the metabolites produced segregate preferentially into one
phase, as in the case of P450RAI-1 and RAI-2 when looking at
all-trans retinoic acid metabolism. In comparative testing of
potential modulators of P450RAI-1 or P405RAI-2, the modulators
could be added to the cells at the same time as the substrate.
Specificity of the modulator is determined by examining the degree
to which its addition affects either the disappearance of substrate
or the production of metabolites.
Example 10
Preparation of Antibodies
Polyclonal Antibodies
[0320] A polyclonal antibody specific to P450RAI-2 was produced
using standard methodology outlined herein. Whole cell lysates of
Sf9 insect cells expressing his-tagges CYP26A CYP26B, CYP4511 and
CYP4X1 were loaded onto a Western gel and probed with primary
antibody, polyclonal antiserum 60350 used at a dilution of 1:300.
The secondary antibody was HRP conjugated donkey anti-rabbit Ig
used at 1:10,000. Results shown in FIG. 24 indicate that the
polyclonal antiserum 60350 produced by the present inventors is
specific for CYP26B (P450RAI-2) on the immunoblot.
Monoclonal Antibodies
[0321] Monoclonal antibodies (Mab's) specific for P450RAI-2 are
useful, for example, for diagnostic purposes such as for
determining P450RAI-2 protein levels in the identification of
normal and tumor tissues which metabolize RA. To produce these
antibodies, purified P450RAI-2 protein is prepared. The human
P450RAI-2 protein is produced in bacterial cells as a fusion
protein with glutathione-S-transferase using the vector pGEX2
(Pharmacia). This permits purification of the fusion protein by GSH
affinity chromatography. In another approach, P450RAI-2 is
expressed as a fusion protein with the bacterial maltose binding
domain. The fusion protein is thus recovered from bacterial
extracts by passing the extract over an amylose resin column
followed by elution of the fusion protein with maltose. For this
fusion construct, the vector pMalC2, commercially available from
New England Biolabs, is used. This vector has been used in the
past, for example, to overexpress nuclear receptor proteins which
were recovered in high yields for functional studies and the
production of receptor specific antisera [Ohno, 1993]. The
preparation of a second fusion protein is also useful in the
preliminary screening of MAb's.
[0322] The generation of hybridomas expressing monoclonal
antibodies recognizing P450RAI-2 protein is carried out as follows:
BALB/c mice are injected intraperitoneally with protein/adjuvant
three times at one-month intervals, followed by a final injection
into the tail vein shortly prior to cell fusion. Spleen cells are
harvested and fused with NS-1 myeloma cells (American Type Culture
Collection, Rockville, Md.) using polyethylene glycol 4000
according to standard protocols [Kennett, 1979; Mirski, 1989]. The
cell fusion process is carried out as described in more detail
below.
[0323] The fused cells are plated into 96-well plates with
peritoneal exudate cells and irradiated spleen cells from
BALB/Ccmice as feeder layers and selection with hypoxanthine,
aminopterin, and thymidine (HAT medium) is performed.
[0324] An ELISA assay is used as an initial screening procedure.
1-10 .mu.g of purified P450RAI-2 (cleaved from the fusion protein)
in PBS is used to coat individual wells, and 50-100 .mu.l per well
of hybridoma supernatants is incubated. Horseradish
peroxidase-conjugated anti-mouse antibodies are used for the
colorimetric assay.
[0325] As a secondary screening, cells which exhibit no detectable
expression of P450RAI-2 message and no detectable RA metabolizing
activity in the absence of retinoic acid, but in which P450RAI-2
exposure to RA induces expression of P450RAI-2, are used.
[0326] Positive hybridomas are cloned by limiting-dilution and
grown to large-scale for freezing and antibody production. Various
positive hybridomas are selected for usefulness in western blotting
and immunohistochemistry, as well as for cross reactivity with
P450RAI-2 proteins from different species.
[0327] The selected MAb's are useful for monitoring the levels of
expression of P450RAI-2 protein following RA treatment in cell
culture and in tissues. P450RAI-2 protein expression may be a
prognostic indicator for determining whether a particular tumor
will respond to RA treatment. There is also a wide intersubject
variability in baseline RA metabolism and there is evidence
suggesting that subjects with a high rate of RA metabolism have a
higher incidence of squamous or large cell cancers of the lung
[Rigas, 1996]. Once useful antibodies are characterized, these
antibodies are used to survey tumor tissue samples for P450RAI-2
expression.
Protocol for Production of Mouse Hybridomas
Fusion
[0328] Feeder cells (spleen and peritoneal exudate cells) are
plated. 24 to 48 hours before fusion, mouse myeloma cells are taken
off drug (8-azaguanine 20 .mu.g/ml) and counted to ensure that
there are at least 50.times.10.sup.6 cells. 2 g of PEG 4000 are
autoclaved in a glass tube for 15 minutes and maintained at
60.degree. C. for use or alternatively stored at room temperature
and remelted in a 60.degree. C. water bath when needed.
[0329] BALB/c mice are immunized as per desired schedule. The final
injection is given intravenously in the tail vein. Fusion of
immunized spleen cells is carried out 3 or 4 days after the
intravenous injection. Spleen from each animal is collected
separately; eye sera for ELISA if desired.
[0330] A single cell suspension is prepared using a Teflon pestle
and decanting connective tissue. The suspension is washed 1.times.
in serum-free medium. Each spleen has about 10.times.10.sup.6
cells. The myeloma cells are collected, counted and washed in
serum-free medium.
[0331] The cells are then fused. A small beaker of water, and
serum-free medium (37.degree. C.) are prepared and the PEG melted
at 50-60.degree. C. The immunized spleen cells and myeloma cells
are mixed in a 50 ml TC tube (recommended ratios vary from 1:1 to
2:1) and the cells are washed once with serum-free medium. The
supernatant is carefully discarded. 2.4 ml pre-warmed serum-free
medium is added immediately with pipette to the melted PEG and
mixed, maintaining the temperature at 37.degree. C. in beaker of
warm water. The PEG should be light pink. If it is yellow, another
aliquot should be used. 0.5-1.0 ml of PEG is added dropwise to the
cell pellet over 1 minute with gentle rotation of the tube or
gentle stirring to ensure mixing. The tip of the addition pipette
is placed directly over the cell pellet. The tube is swirled gently
in 37.degree. C. water bath for 90 seconds with the blunt end of a
3 ml pipette tip and 10 ml warm serum-free medium added slowly over
6-10 minutes while rotating tube gently to bring the volume up to
20-50 ml. The tube is maintained at 37.degree. C. for at least 20
minutes to obtain cell fusion and then the cells are washed
2.times. with serum-free medium. The cells are centrifuged and
gently resuspended in 100 ml of pre-warmed medium +10-20% FBS. 100
.mu.l/well aliquotted in 96-well plates. Assuming one spleen fused
with 100.times.10.sup.6 cells myeloma fusion partner, about 10
plates are needed. On the following day, 100 .mu.l medium is
removed and 100 .mu.l 2.times.HAT added. Feed with 1.times.HAT
medium for 1 to 3 weeks, then feed with HT medium (i.e., remove 1/2
HAT medium and replace with equal volume HT medium).
Preparation of Peritoneal Exudates and Spleen Feeder Cells
[0332] A sacrificed mouse is sprayed with 70% alcohol, skin is
nicked and torn apart, with care being taken not to cut the
peritoneum. The peritoneum is lifted with forceps and a needle is
introduced; 5 ml of serum-free medium is slowly injected. The
abdomen is massaged and the fluid is slowly sucked up, collected in
a sterile tube and kept on ice. The volume is brought up to 5 ml.
The spleen is obtained and placed in a sterile tube containing
serum-free medium. The spleen is gently mashed with a sterile
Teflon pestle. Clumps are allowed to settle and the cells are
decanted into a clean tube, care being taken to avoid including
connective tissue, in order to minimize fibroblast growth. The
sample is irradiated at 4500 R. Cells are washed once with
serum-free medium, placed into 96-well plates [one spleen/10 plates
(approximately 2-5.times.10.sup.5 cells/well) and peritoneal
exudate cell suspension (PECS) (<3.times.10.sup.3 cells/well) in
a total volume of 100 .mu.l/well] and incubated at 37.degree. C.
until ready to be used. They can also be stored in sterile tube
overnight at 4.degree. C.
Example 11
Expression of P450RAI-2 and P450RAI-1 in Normal and Tumour Lung
Cells
Materials and Methods
[0333] The RNA from human lung samples, both normal and tumour
(non-small cell cancer), were generously donated by Dr. Tsai from
the Ontario Cancer Institute, Toronto, Canada. The RNA was received
in 100% ethanol. The RNA was precipitated using standard protocols,
resuspended in DEPC-treated water and stored at -70.degree. C.
until use.
[0334] The RNA samples were analyzed spectrophotometrically
(260/280 ratios) (BeckmanDU 640 Spectrophotometer, USA), and the
RNA (1 .mu.g per sample) was run on an RNA formaldehyde gel with
sthidium bromide to visualize the 18S and 28S bands, using a
ChemiImager 4000 and Multi Image.TM. Light Cabinet (Alpha Innotech
Corp., USA).
[0335] RT-PCT was initially attempted using the Thermoscript RT
kit, followed by PCR according to standard lab conditions; however,
results were inconsistent and unsuccessful with this method. RT-PCR
was successfully conducted using the TIATANIUM.TM. Step RT-PCR Kit
(CLONETECH Laboratories, Inc., CA, USA) and a Mastercycler.TM.
gradient Thermo Cycler (Eppendorf Scientific, Inc. Westbury, N.Y.,
USA) Total RNA (0.5 .mu.g) was reverse transcribed at 50.degree. C.
for 1 hour and PCR amplification of entire cDNA conducted according
to the kit, in a total volume of 25 .mu.l. 45 picomoles of primer
pairs specific for human P450 CYP26A1 (5'-GGC TGC CTC TCT AAC CTG
CAC-3 (SEQ. ID. NO. 42); 5'GAT CAG AGA TGT GGC TGC TGC ACT G-3'
(SEQ. ID. NO. 43)) and human P450 CYP26B1 (SEQ. ID. NOs. 41 and 38,
although SEQ. ID. NO. 37 can be used also in place of SEQ. ID. NO.
41); human P450 CYP26C1 (5'-GCA AGG GAC CAG CTG CAT CGG CAC CTG-3
(SEQ. ID. NO. 44); 5'-CTC GTG CGT GTC CCG GAT GCT ATA C-3' (SEQ.
ID. NO. 45) and an alternative antisence primer containing the 8F
exon 5'GGA ATG GGC TAT GCC TCT GA-3' (SEQ. ID. NO. 46) and human
.beta.-actin (5'ATC TGG CAC CAC ACC TTC TAC AAT GAG CTG CG -3'
(SEQ. ID. NO. 47), 5'CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3'
(SEQ. ID. NO. 48) (Takizawa et al, Am. J. Respir. Crit. Care Med.
163, 1476-1483); were used to yield 550 bp, 450 bp, 555 bp, 850 bp
and 800 bp approximated sized cDNA products, respectively.
[0336] RNA from human breast cancer cell line, MCF-7, treated with
DMSO alone or retinoic acid in DMSo was used as a positive control,
in addition to the mouse liver RNA and mouse .beta.-actin primers
supplied with the kit. PCR was performed at 94.degree. C. for 5
min. followed by 35 cycles of 99.degree. C. for 30 sec., 65.degree.
C. for 30 sec., and 72.degree. C. for 1 min., with a final
72.degree. C. extension for 5 min. The amplified cDNA fragments
were separated on a 1% agarose gel containing ethidium bromide, and
visualized using a ChemiImager 4000 and Multi Image.TM. Light
Cabinet (Alpha Innotech Corp., USA).
[0337] The RT-PCR products (5 .mu.L/sample) were run on 1% agarose
gels for use in Southern blotting to confirm product. The gels were
soaked in ethidium bromide/1.times.TAE solution for 10 minutes
prior to transfer setup to allow visualization of DNA, Alkaline
southern blotting was conducted using a downward capillary action
setup with a sponge soaked in the alkaline 0.4 M NaOH transfer
solution and transfer run overnight. After disassembly, the gels
were visualized to ensure complete transfer occurred. The blots
were soaked in 2.times.SSC, and let to dry at 80.degree. C. for 2
hours; blots were wrapped and stored at 4.degree. C. until use.
[0338] Full length DNA probes for CYP26A1 AND CYP26B1 AND CYP26C1
were radioactively labeled using the Prime-A-Gene kit Stratagene).
Prehybridization and hybridization of the blots was done using the
QuickHyb.TM. Hybridization Solution exposed using the Storm
PhophoImager system (molecular Dynamics, USA); blots were also
exposed radiographically with X-OMAT film with similar results.
[0339] The results can be seen in FIG. 23. The results from the
same normal and tumour lung tissue from seven patients can be seen.
CYP26B1 (P450RAI-2) can be seen in both normal and tumour tissue.
However, the tumour tissue appears to have increased levels of CYP
26B1 expression compared to the normal tissue. The MCF-7 RNA
expressed Cyp26B1 in DMSO alone and increased levels in the reinoic
acid in DMSO treated cells, as was expected.
[0340] CYP26A1 was not or minimal expressed in normal lung tissue
but there was strong induction in tumour tissue. The MCF-7 RNA with
DMSo alone does not express P450 CYP26A1 while the MCF-7 RNA
treated with reinoic acid in DMSO had strongly induced expression
of CYP26A1, as expected.
[0341] There did not appear to be any expression of Cyp26C in
either normal or tumour lung tissue.
[0342] .beta.-actin was chosen as an internal control housekeeping
gene, since previous studies indicated that this was a more stable
control for the experimental system. As can be seen from the
figure, .beta.-actin was uniform for all samples, indicating that
the changes in CYP26 expression levels observed were true.
[0343] The southern blots (not shown) did indicate that that the
amplified products for CYP26A1 and CYP26B1 were indeed the specific
SYP26 targets.
[0344] Thus, these results indicate that P450RAI-1 and P450RAI-2
may be involved in lung cancer and inhibitors or antagonists of
these cytochromes may be beneficial in the treatment of lung
cancer. Further, screening tissue for P450RAI-1 and P450RAI-2
expression may assist in the diagnosis and potential early
detection of lung cancer or potential thereof.
Example 12
Expression of P450RAI-2 and P450RAI-2 in Retina and Breast Cancer
Cells
[0345] 1 .mu.g of total RNA was RT-PCR amplified (35 cycles) in the
presence of primers specific for human CYP26A, human 26B (SEQ. ID.
NOs. 37 or 38) or human GAPDH, respectively. PCR products were then
fractionated on 1.6% agarose gel and cisualized by ethidium
bromaide staining. Following products were expected: 0.55 kpb
CYP26A fragment, 0.45 kbp CYPSB fragment and 0.95 kBN GAPDH
fragment. One can see in FIG. 25 the expression of P450RAI-2
(CYP26B) in human retina and MCF-7 cells.
Example 13
Screening Potential Modulators of P450RAI-2
[0346] Antisense nucleic acids or oligonucleotides (RNA or
preferably DNA) that inhibit cellular RA-induced P450RAI-2
production can be used to inhibit metabolism of RA by P450RAI-2
[Monia, 1996]. Antisense oligonucleotides, typically 15 to 20 bases
long, bind to the sense mRNA or pre mRNA region coding for the
protein of interest, which can inhibit translation of the bound
mRNA to protein. The cDNA sequence encoding human P450RAI-2 can
thus be used to design a series of oligonucleotides which together
span a large portion, or even the entire cDNA sequence. These
oligonucleotides can be tested to determine which provides the
greatest inhibitory effect on the expression of the protein
[Stewart, 1996]. This can be done by exposing cells to the various
oligonucleotides and measuring subsequent changes in human
P450RAI-2 activity or by using antibodies to screen for inhibition
of P450RAI-2 synthesis. The most suitable mRNA target sites include
5'- and 3'-untranslated regions as well as the initiation codon.
Other regions might be found to be more or less effective.
Alternatively, an antisense nucleic acid or oligonucleotide may
bind to P450RAI-2 DNA coding or regulatory sequences.
[0347] Rather than reducing RA metabolism by inhibiting P450RAI-2
gene expression at the nucleic acid level, activity of the
P450RAI-2 protein may be directly inhibited by binding to an agent,
such as, for example, a suitable small molecule or a monoclonal
antibody.
[0348] The present invention thus includes a method of screening
drugs for their effect on activity (i.e., as a modulator,
preferably an inhibitor) of a retinoic acid inducible protein. The
method includes exposing the protein to a prospective inhibitor (or
modulating) drug and determining the effect on protein activity.
The measured activity might be hydroxylation of a retinoid,
particularly all-trans retinoic acid, or hydroxylation of a
retinoic acid, particularly all-trans retinoic acid, at the 4
position of the .beta.-ionone ring thereof. For screening drugs for
use in humans, P450RAI-2 itself is particularly useful for testing
the effectiveness of such drugs. Prospective drugs could also be
tested for inhibition of the activity of other P450 cytochromes,
which are desired not to be inhibited. In this way, drugs which
selectively inhibit P450RAI-2 over other P450s could be
identified.
[0349] Another system for screening for potential modulators,
preferably inhibitors, of a P450RAI-2 protein includes a stably
transfected cell line having incorporated therein DNA of a reporter
gene (e.g., .beta.-galactosidase, firefly luciferase, or the like)
and of the P450RAI-2, in which expression of both genes is
inducible by exposure of the cells to RA. Expression of the
reporter gene provides a measure of the induction of the expression
system and therefore provides an indication of the amount of RA
present. Exposure of the cells to RA leads to RA metabolism and,
with time, such metabolism leads to a decrease in the degree of
induction which is indicated by the reporter protein. Exposure of
the cells to RA in the presence of an agent that inhibits P450RAI-2
metabolism of RA results in decreased RA metabolism, whereas
exposure of the cells to RA in the presence of an agent that does
not inhibit P450RAI-2 metabolism of RA has no effect on RA
metabolism. A comparison of expression of the reporter gene in the
presence of RA alone and in the presence of both RA and a potential
inhibitory drug thus gives a measure of the effectiveness of the
drug in inhibiting metabolism of RA by the P450RAI-2 protein.
[0350] One system for screening for potential inhibitors of a
P450RAI-2 protein includes a cell line in which the endogenous
P450RAI-2 gene is not present or not functional or not expressed.
In this cell line, a cytochrome P450RAI-2 expression vector and an
RA-inducible reporter gene are incorporated such that exposure of
the cell line to RA results in metabolism of RA by the expressed
P450RAI-2 protein and a degree of induction of the reporter gene
based on remaining active RA. The addition of an inhibitor of
P450RAI-2 will decrease the rate of metabolism/degradation of RA
and therefore increase the activation/induction of the RA sensitive
reporter gene.
[0351] Another system for screening of potential inhibitors (or
other modulators) using the same cell line would be to add
radioactive substrate to the cells along with the potential
inhibitors (or modulators). Addition of the radioactive substrate,
which could be all-trans retinoic acid, causes the production of
radioactive metabolites. Using a phase extraction procedure, such
as that described herein, the amount of these metabolites can be
measured. The addition of an inhibitor will cause a decrease in the
generation of the radioactive metabolites, which can be measured
using .beta.-scintillation counting or HPLC.
[0352] The invention thus provides a system for screening potential
inhibitors of RA catabolism by a P450RAI-2 protein. The system
includes a transfected cell line having incorporated therein DNA of
a reporter gene, for example the luciferase gene exemplified above,
in which expression of the reporter gene is inducible by exposure
of the cells to RA. In this system, the P450RAI-2 gene is omitted,
that is the reporter gene is under the control of the native
promoter for the P450RAI-2 gene. Expression of the reporter gene
provides a measure of the induction of the expression system and
therefore provides an indication of the amount of mRNA produced in
response to exposure of the cells to RA. Exposure of the cells to
RA in the presence of an agent that inhibits induction of the
expression system indicates that the agent is a potential inhibitor
of RA catabolism, i.e., provides a measure of the effectiveness of
the agent as a drug in inhibiting the expression of P450RAI-2 gene
and thus metabolism of RA.
[0353] There is the possibility that cellular retinoic acid-binding
protein (CRABP) [Adamson, 1993] is involved in binding of a
retinoid substrate to a P450RAI-2 protein of the present invention.
The effect of the presence of CRABP, derivatives, synthetic
fragments or analogs thereof could thus be determined according to
screening methods of the present invention; effectiveness of such
agents in enhancing RA metabolism can also be determined.
[0354] The present invention allows the skilled artisan to prepare
bispecific antibodies and tetrameric antibody complexes. Bispecific
antibodies can be prepared by forming hybrid hybridomas [Staerz,
1986a & b].
[0355] The present invention includes three types of compounds
related to retinoids: those that inhibit enzymatic activity of
P450RAI-2, thereby inhibiting metabolism of RA; those retinoids
that evade metabolism by P450RAI-2; and those compounds that
repress induction of P450RAI-2 gene expression.
[0356] Compositions of the invention are administered to subjects
in a biologically compatible form suitable for pharmaceutical
administration in vivo. By "biologically compatible from suitable
for administration in vivo" is meant a form of the composition to
be administered in which any toxic effects are outweighed by the
therapeutic effects of the composition. The term "subject" is
intended to include living organisms in which a desired therapeutic
response can be elicited, e.g. mammals. Examples of subjects
include human, dogs, cats, mice, rats and transgenic species
thereof. Administration of a therapeutically active amount of the
therapeutic compositions of the present invention is defined as an
amount effective, at dosages and for periods of time necessary to
achieve the desired result. For example, a therapeutically active
amount of a compound that inhibits catabolism of RA by a P450RAI-2
protein may vary according to factors such as the disease state,
age, sex, and weight of the individual, as well as target tissue
and mode of delivery. Dosage regimes may be adjusted to provide the
optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation.
[0357] Compounds of the present invention, such as those that are
found to inhibit metabolism of RA by P450RAI-2 enzymes and that are
useful as anticancer agents and in the treatment, amelioration, or
prevention of skin disorders for which retinoic acid is useful, for
example, may be used topically. In this regard they may be included
in compositions for therapy in animals, including humans, for
premalignant epithelial cell lesions, as a prophylaxis against
tumor promotion in epithelial cells and treatment for dermatoses
such as ichthyoses, follicular disorders, benign epithelial
disorders, and other proliferative skin diseases, such as acne,
psoriasis, eczema, atopic dermatitis, nonspecific dermatitis and
the like.
[0358] Topical compositions are usually formulated with a
pharmaceutically acceptable carrier in liquid, semi-solid or solid
form. A pharmaceutically acceptable carrier is a material that is
nontoxic and generally inert and does not affect the functionality
of the active ingredients adversely. Such materials are well known
and include those materials sometimes referred to as diluents or
vehicles (excipients) in the pharmaceutical formulation art. The
carrier may be organic or inorganic in nature. Examples of
pharmaceutically acceptable carriers are water, gelatin, lactose,
starch, mineral oil, cocoa butter, dextrose, sucrose, sorbitol,
mannitol, gum, acacia, alginates, cellulose, talc, magnesium
stearate, polyoxyethylene sorbitan monolaurate, and other commonly
used pharmaceutical carriers. In addition to an active ingredient
and carrier, the formulation may contain minor amounts of additives
such as flavoring agents, coloring agents, thickening or gelling
agents, emulsifiers, wetting agents, buffers, stabilizers, and
preservatives such as antioxidants.
[0359] Certain compositions may be administered enterally. For oral
administration, suitable forms are, for example, tablets, pills,
syrups, suspensions, emulsions, solutions, powders and
granules.
[0360] As anti-tumor agents or as part of an anti-tumor
formulation, for example, compounds of the present invention can be
used in a similar manner to retinoids used for treating various
tumours, such as all-trans retinoic acid. The dose to be
administered, whether a single dose, multiple does or daily dose,
will of course vary with the particular compound employed because
of the varying potency of the active ingredient, the chosen route
of administration, the size of the recipient, the type of tumor,
and the nature of the patient's condition. The dosage to be
administered is not subject to definite bounds, but it will usually
be an effective amount, or the equivalent on a molar basis of the
pharmacologically active free form produced from a dosage
formulation upon the metabolic release of the active drug to
achieve its desired pharmacological and physiological effects. An
oncologist skilled in the art of cancer treatment will be able to
ascertain without undue experimentation, appropriate protocols for
the effective administration of the compounds of this present
invention.
[0361] Nucleic acids which encode proteins having biological
activity of a P450RAI-2 protein can be used to generate either
transgenic animals or "knock out" animals which, in turn, are
useful in the development and screening of therapeutically useful
reagents. A transgenic animal (e.g., a mouse) is an animal having
cells that contain a transgene, which transgene was introduced into
the animal or an ancestor of the animal at a prenatal, e.g., an
embryonic stage. A transgene is a DNA which is integrated into the
genome of a cell from which a transgenic animal develops. In one
embodiment, a human P450RAI-2 cDNA, comprising the nucleotide
sequence shown in SEQ ID NO:4, or an appropriate variant or
subsequence thereof, can be used to generate transgenic animals
that contain cells which express human P450RAI-2 protein. Methods
for generating transgenic animals, particularly animals such as
mice, have become conventional in the art are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009. In a preferred
embodiment, plasmids containing recombinant molecules of the
invention are microinjected into mouse embryos. In particular, the
plasmids are microinjected into the male pronuclei of fertilized
one-cell mouse eggs; the injected eggs are transferred to
pseudo-pregnant foster females; and, the eggs in the foster females
are allowed to develop to term. [Hogan, 1986]. Alternatively, an
embryonal stem cell line can be transfected with an expression
vector comprising nucleic acid encoding a protein having P450RAI-2
activity, and cells containing the nucleic acid can be used to form
aggregation chimeras with embryos from a suitable recipient mouse
strain. The chimeric embryos can then be implanted into a suitable
pseudopregnant female mouse of the appropriate strain and the
embryo brought to term. Progeny harboring the transfected DNA in
their germ cells can be used to breed uniformly transgenic
mice.
[0362] Typically, particular cells would be targeted for P450RAI-2
transgene incorporation by use of tissue specific enhancers
operatively linked to the P450RAI-2 encoding gene. For example,
promoters and/or enhancers which direct expression of a gene to
which they are operatively linked preferentially in cardiac muscle
cells can be used to create a transgenic animal which expresses a
P450RAI-2 protein preferentially in cardiac muscle tissue. Examples
of suitable promoters and enhancers include those which regulate
the expression of the genes for cardiac myosin and cardiac actin.
Transgenic animals that include a copy of an P450RAI-2 transgene
introduced into the germ line of the animal at an embryonic stage
can also be used to examine the effect of increased P450RAI-2
expression in various tissues.
[0363] The pattern and extent of expression of a recombinant
molecule of the invention in a transgenic mouse is facilitated by
fusing a reporter gene to the recombinant molecule such that both
genes are co-transcribed to form a polycistronic mRNA. The reporter
gene can be introduced into the recombinant molecule using
conventional methods such as those described in Sambrook et al.,
[Sambrook, 1989]. Efficient expression of both cistrons of the
polycistronic mRNA encoding the protein of the invention and the
reporter protein can be achieved by inclusion of a known internal
translational initiation sequence such as that present in
poliovirus mRNA. The reporter gene should be under the control of
the regulatory sequence of the recombinant molecule of the
invention and the pattern and extent of expression of the gene
encoding a protein of the invention can accordingly be determined
by assaying for the phenotype of the reporter gene. Preferably the
reporter gene codes for a phenotype not displayed by the host cell
and the phenotype can be assayed quantitatively. Examples of
suitable reporter genes include IacZ ($-galactosidase), neo
(neomycin phosphotransferase), CAT (chloramphenicol
acetyltransferase) dhfr (dihydrofolate reductase), aphIV
(hygromycin phosphotransferase), lux (luciferase), uidA
($-glucuronidase). Preferably, the reporter gene is lacZ which
codes for $-galactosidase. $-galactosidase can be assayed using the
lactose analogue X-gal
(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) which is broken
down by $-galactosidase to a product that is blue in color
[Old].
[0364] Although experimental animals used in the preferred
embodiment disclosed are mice, the invention should not be limited
thereto. It can be desirable to use other species such as, for
example, rats, hamsters, rabbits and sheep.
[0365] The transgenic animals of the invention can be used to
investigate the molecular basis of RA metabolism. The transgenic
animals of the invention can also be used to test substances for
the ability to prevent, slow or enhance RA metabolism. A transgenic
animal can be treated with the substance in parallel with an
untreated control transgenic animal.
[0366] Cells from the transgenic animals of the invention can be
cultured using standard tissue culture techniques. In particular,
cells carrying the recombinant molecule of the invention can be
cultured and used to test substances for the ability to prevent,
slow or enhance RA metabolism.
[0367] Additionally, the non-human homologs of genes encoding
proteins having P450RAI-2 activity can be used to construct a
"knock out" animal which has a defective or altered P450RAI-2 gene.
For example, with established techniques, a portion of murine
genomic P450RAI-2 DNA (e.g., an exon), can be deleted or replaced
with another gene, such as a gene encoding a selectable marker
which can be used to monitor integration. The altered P450RAI-2 DNA
can then be transfected into an embryonal stem cell line. The
altered P450RAI-2 DNA will homologously recombine with the
endogenous P450RAI-2 gene in certain cells and clones containing
the altered gene can be selected. Cells containing the altered gene
are injected into a blastocyst of an animal, such as a mouse, to
form aggregation chimeras as described for transgenic animals.
Chimeric embryos are implanted as described above. Transmission of
the altered gene into the germline of a resultant animal can be
confirmed using standard techniques and the animal can be used to
breed animals having an altered P450RAI-2 gene in every cell
[Lemoine, 1996]. Accordingly, a knockout animal can be made which
cannot express a functional P450RAI-2 protein. Such a knockout
animal can be used, for example, to test the effectiveness of an
agent in the absence of a P450RAI-2 protein.
[0368] The antisense nucleic acids and oligonucleotides of the
invention are useful for inhibiting expression of nucleic acids
(e.g. mRNAs) encoding proteins having P450RAI-2 activity. Since
proteins having P450RAI-2 activity are associated with metabolism
of agents which can act on the cell, e.g., RA, decreasing
expression of such proteins can increase sensitivity of the cell to
such agents. Antisense nucleic acids can be introduced into a drug
resistant cell in culture to inhibit P450RAI-2 expression. One or
more antisense nucleic acids, such as oligonucleotides, can be
added to cells in culture media, typically, for example, at 200
.mu.g/ml.
[0369] The antisense nucleic acids of the invention, or
oligonucleotides thereof, can thus be used in gene therapy to
correct or prevent retinoic acid or other retinoid resistance in a
subject. For example, antisense sequences can be used to render
retinoic acid or other retinoid resistant malignant cells sensitive
to chemotherapeutic agents. Administration of antisense nucleic
acids to a subject may be most effective when the antisense nucleic
acid is contained in a recombinant expression vector which allows
for continuous production of antisense RNA. Recombinant molecules
comprising an antisense nucleic acid or oligonucleotide thereof,
can be directly introduced into tissues, including lung tissue in
vivo, using delivery vehicles such as liposomes, retroviral
vectors, adenoviral vectors and DNA virus vectors. A delivery
vehicle can be chosen which can be targeted to a cell of interest
in the subject (e.g. a retinoid resistant tumor cell). Antisense
nucleic acids can also be introduced into isolated cells, such as
those of the haematopoietic system, ex vivo using viral vectors or
physical techniques such as microinjection and electroporation or
chemical methods such as coprecipitation and incorporation of DNA
into liposomes, and such cells can be returned to the donor.
Recombinant molecules can be delivered in the form of an aerosol or
by lavage.
[0370] Accordingly, the invention provides a method for inhibiting
retinoic acid or other retinoid resistance of a resistant cell by
introducing into the resistant cell a nucleic acid which is
antisense to a nucleic acid which encodes the protein identified as
SEQ ID NO:5.
[0371] The nucleic acids of the invention can further be used to
design ribozymes which are capable of cleaving a single-stranded
nucleic acid encoding a protein having P450RAI-2 activity, such as
an mRNA. A catalytic RNA (ribozyme) having ribonuclease activity
can be designed which has specificity for a P450RAI-2-encoding mRNA
based upon the sequence of a nucleic acid of the invention. For
example, a derivative of a Tetrahymena L-19IVS RNA can be
constructed in which the base sequence of the active site is
complementary to the base sequence to be cleaved in a
P450RAI-2-encoding mRNA. [Cech a and b]. Alternatively, a nucleic
acid of the invention could be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules [Bartel, 1993].
[0372] The isolated nucleic acids and antisense nucleic acids of
the invention can be used to construct recombinant expression
vectors as described previously. These recombinant expression
vectors are then useful for making transformant host cells
containing the recombinant expression vectors, for expressing
protein encoded by the nucleic acids of the invention, and for
isolating proteins of the invention as described previously. The
isolated nucleic acids and antisense nucleic acids of the invention
can also be used to construct transgenic and knockout animals as
described previously.
[0373] The isolated proteins of the invention are useful for making
antibodies reactive against proteins having P450RAI-2 activity, as
described previously. Alternatively, the antibodies of the
invention can be used to isolate a protein of the invention by
standard immunoaffinity techniques. Furthermore, the antibodies of
the invention, including bispecific antibodies are useful for
diagnostic purposes.
[0374] Molecules which bind to a protein comprising an amino acid
sequence shown in SEQ ID NO:5 can also be used in a method for
killing a cell which expresses the protein, wherein the cell takes
up the molecule. Preferably, the cell is a tumor cell. Destruction
of such cells can be accomplished by labeling the molecule with a
substance having toxic or therapeutic activity. The term "substance
having toxic or therapeutic activity" as used herein is intended to
include molecules whose action can destroy a cell, such as a
radioactive isotope, a toxin (e.g. diphtheria toxin or ricin), or a
chemotherapeutic drug, as well as cells whose action can destroy a
cell, such as a cytotoxic cell. The molecule binding to the
P450RAI-2 can be directly coupled to a substance having a toxic or
therapeutic activity or may be indirectly linked to the substance.
In one example, the toxicity of the molecule taken up by the cell
is activated by P450RAI-2 protein.
[0375] The invention also provides a diagnostic kit for identifying
tumor cells comprising a molecule which binds to a protein
comprising an amino acid sequence shown in SEQ ID NO:5, for
example, for incubation with a sample of tumor cells; means for
detecting the molecule bound to the protein, unreacted protein or
unbound molecule; means for determining the amount of protein in
the sample; and means for comparing the amount of protein in the
sample with a standard. Preferably, the molecule is a monoclonal
antibody. In some embodiments of the invention, the detectability
of the molecule which binds to P450RAI-2 is activated by said
binding (e.g., change in fluorescence spectrum, loss of
radioisotopic label). The diagnostic kit can also contain an
instruction manual for use of the kit.
[0376] The invention further provides a diagnostic kit for
identifying tumor cells comprising a nucleotide probe complementary
to the sequence, or an oligonucleotide fragment thereof, shown in
SEQ ID NO:4, for example, for hybridization with mRNA from a sample
of tumor cells; means for detecting the nucleotide probe bound to
mRNA in the sample with a standard. The diagnostic kit can also
contain an instruction manual for use of the kit.
DISCUSSION OF RESULTS
[0377] The present inventors have have identified a novel retinoic
acid metabolizing cytochrome P450. Consequently, at least two
genes, encoding all-trans-RA metabolizing enzymes P450RAI-1 [White,
J. A., et al. (1997), White, J., et al. (1998)] and P450RAI-2 are
expressed in humans. The predicted amino acid sequences of these
enzymes (P450RAI-1 and P450RAI-2) are 42% similar overall. Regions
corresponding to functional domains such as those for heme binding
and putative substrate binding exhibit the highest degrees of
similarity. P450RAI-1 and -2 are also conserved across species from
zebrafish to humans suggesting that these enzymes may be
functionally distinct [White, J. A., et al. (1997), Nelson, D.
(1999); White, J. A. et al. (1996)]. The genomic structures of
these two genes are quite distinct-, P450RAI-1 is comprised of 7
exons while P450RAI-2 has 6 and there is no clear evidence of
conserved intron/exon boundaries between the two genes [White, J.,
et al. (1998); Nelson, D. (1999)], These data support the
possibility that these genes diverged from a common ancestral gene
prior to the emergence of mammals.
[0378] P450RAI-2 can metabolize all-trans-RA with efficiency
comparable to that of P450RAI-1. HPLC analysis comparing these
enzymes reveals their striking similarities with respect to
conversion of all-trans-RA into 4-OH- and 4-oxo RA and
characteristic secondary products. Moreover, P450RAI-1 and
P450RAI-2 share surprisingly similar specificities for retinoids as
determined by our competition studies. The all-trans metabolite of
RA is clearly the preferred substrate for both enzymes with a
rank-order of: all-trans-RA>9 cis RA>13 cis. Other retinoids
such as retinol and retinaldehyde are very poor competitors
suggesting that they are unlikely to be natural substrates for
these enzymes under normal physiological conditions. This is in
contrast to a recent report suggesting that P450RAI (CYP26A) may be
involved in the activation step of retinol [Lane, M., et al.
(1999)]. These similarities would suggest that, at least with
respect to metabolic activity, these enzymes may be equivalent.
Although this is somewhat surprising given their differences in
sequence, there are many RA binding proteins--RARs, RXRs,
CRABPs--which are widely different in primary amino acid sequences.
It is possible that the differences in sequence reflect differences
in the abilities of these enzymes to interact with other proteins
such as CRABPs which have been proposed to modify all-transRA
metabolic activities in cells [Boylan, J. & Gudas, L. (1992);
Napoli, J. (1996)].
[0379] Although the enzymatic activities of P450RAI-1 and -2 may be
similar, it appears that their tissue specific expression is not.
Tissue dot blot and northern blot analyses indicate that in the
adult, P450RAI-2 is broadly expressed at low levels in most tissues
but is predominantly expressed in brain tissues, notably pons and
cerebellum. P450RAI-1, on the other hand, does not show appreciable
expression in any of the human brain tissues evaluated. During
development numerous studies have indicated that the role of
P450RAI-1 is to regulate local levels of all-trans-RA and restrict
certain tissues from all-trans-RA activity. Developing retina
exhibit an exquisite pattern of coordinated expression of
P450RAI-1, and the all-trans-RA synthesizing enzymes RALDH-2 and
ALDH-1 [McCafferty et al., (1999)]. Given that the EST
corresponding to P450RAI-2 (accession # AA012833) was derived from
an adult retinal library suggests that this enzyme may also play a
role in the balance of all-trans-RA in retinal tissue.
[0380] Expression of P450RAI-2 in adult human brain suggests that
all-trans-RA may play an important homeostatic role in brain
tissue. In this regard, it has recently been shown that
all-trans-RA signaling pathways may be important for memory and
learning since RARP/RXR (X knockout mice have impaired long-term
potentiation and limited ability to negotiate a water-maze [Chiang,
M. Y., (1998)]. If all trans-RA signaling pathways are involved in
maintenance of higher order brain function then the regulation and
function of enzymes like P450RAI-2 will be important regulators of
these pathways. The high level of expression of P450RAI-2 in adult
cerebellum suggests that it is protecting this tissue from exposure
to RA. Of note, developing cerebellum is highly sensitive to the
teratogenic effects of RA [Lammer, E. J., (1985); Lammer, E. &
Armstrong, D. (1992)]. Also, Yamamoto et al. [Yamamoto, M., et al.
(1998)], have reported evidence that RA may be synthesized from
retinol in the choroid plexus of developing cerebellum, and that RA
injected into the cerebellum is rapidly metabolized. These findings
support the notion of an important role for RA metabolism in
cerebellum during development which may also extend into adulthood.
It is likely that this metabolism is mediated by P450RAI-2.
[0381] Studies of several cell lines in culture indicate that
P450RAI-2 expression, similar to that of P450RAI-1, is regulated by
all-trans-RA. For example, the induction by all-trans-RA of
P450RAI-2 expression in the breast epithelial adenocarcinoma cell
line MCF-7 is comparable to that of P450RAI-1. Interestingly, the
transcriptional elements required for RA-induction of P450RAI-2 may
be different from those for induction of P450RAI-1-, inspection of
genomic sequence immediately upstream of the first exon of
P450RAI-2 has not revealed any elements previously demonstrated to
mediate a retinoid induction of transcription, whereas we have
identified a functional, conserved, canonical RA response element
(RARE) within the first 200 bp of the P450RAI-1 promoter (M.P.;
unpublished). Comparative studies of induction of these two genes
at the transcriptional level will help to discriminate possible
similarities in their regulation.
[0382] RA metabolism may be implicated in certain disease states
such as dermatological conditions angiogenesis, immunological
disorders, and cancer. The present work suggests that certain brain
functions may also depend on normal retinoid metabolism. There is
interest in inhibiting this activity to increase the cell
sensitivity to the differentiating or apoptotic affects of
all-trans-RA; recent clinical trials with all-trans-RA metabolism
inhibitors suggest that this may be a viable approach to treat
diseases which respond positively to retinoids. The identification
of a second P450RAI provides another potentially useful target for
rational drug design.
[0383] While the present invention has been described with
reference to what are presently considered to be the preferred
examples, it is to be understood that the invention is not limited
to the disclosed examples. To the contrary, the invention is
intended to cover various modifications and equivalent arrangements
included within the spirit and scope of the appended claims.
[0384] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
REFERENCES
[0385] Particulars of references cited above are given below. All
of the listed references are incorporated herein by reference.
[0386] Abu-Abed, S. S., Beckett, B. R., Chiba, H., Chithalen, J.
V., Jones, G., Metzger, D., Chambon, P., and Petkovich, M. (1998).
Mouse P450RAI (CYP26) expression and retinoic acid-inducible
retinoic acid metabolism in F9 cells are regulated by retinoic acid
receptor gamma and retinoid X receptor alpha. Journal of Biological
Chemistry 273, 2409-15. [0387] Achkar, C. C., Derguini, F.,
Blumberg, B., Langston, A., Arthur, A. L., Speck, J., Evans, R. M.,
Bolado, Jr., J. Nakanishi, K. and Buck, J. (1996) 4-Oxoreinol, a
new natural ligand and transactivator of the retinoic acid
receptors. Proc. Natl. Acad. Sci. USA 93, 4879-84. [0388] Adamson,
P. C., Boylan, J. F., Balis, F. M., Murphy, R. F., Godwin, K. A.,
Gudas, L. J. and Poplack, D. G. (1993). Time course of induction of
metabolism of all-trans retinoic acid and the up-regulation of
cellular retinoic acid-binding protein. Cancer Research 53,
472-476. [0389] Akimenko, M. A. and Ekker, M. (1995a). Anterior
duplication of the Sonic hedgehog expression pattern in the
pectoral fin buds of zebrafish treated with retinoic acid.
Developmental Biology 170, 243-7. [0390] Akimenko, M. A., Johnson,
S. L., Westerfield, M. and Ekker, M. (1995b). Differential
induction of four msx homeobox genes during fin development and
regeneration in zebrafish. Development 121, 347-57. [0391] Bartel,
D. and Szostak, J. W. (1993). Science 261, 1411-1418. [0392]
Blaner, W. (1994). Retinol and retinoic acid metabolism. In: The
Retinoids. (M. Sporn, Roberts, A. and Goodman, D. S., Editors)
Raven Press, Inc.: New York. [0393] Bligh, E. G. and Dyer, W. J.
(1957). A rapid method of total lipid extraction and purification.
Canadian Journal of Biochemistry 37, 911-917. [0394] Blumberg, B.,
Bolado, Jr., J., Derguini, F., Craig, A. G., Moreno, T. A.,
Chakravarti, D., Heyman, R. A., Buck, J. and Evans, R. M. (1996)
Novel retinoic acid receptor ligands in Xenopus embryos. Proc.
Natl. Acad. Sci. USA 93, 4873-78. [0395] Boss et al., U.S. Pat. No.
4,816,397. [0396] Boylan, J. & Gudas, L. (1992) J. Biol. Chem.
267, 21486-21491 [0397] Boylan, J. F., Lufkin, T., Achkar, C. C.,
Taneha, R., Chambon, P. and Gudas, L. J. (1995). Targeted
Disruption of Retinoic Acid Receptor a (RARa) and RARg Results in
Receptor-Specific Alterations in Retinoic Acid-Mediated
Differentiation and Retinoic Acid Metabolism. Mol. Cell. Biol. 15,
843-851. [0398] Butler, W. B., and Fontana, J. A. (1992). Responses
to retinoic acid of tamoxifen-sensitive and -resistant sublines of
human breast cancer cell line MCF-7. Cancer Research 52, 6164-7.
[0399] Cabilly et al., U.S. Pat. No. 4,816,567. [0400] Cech et al.,
(a) U.S. Pat. No. 4,987,071. [0401] Cech et al., (b) U.S. Pat. No.
5,116,742. [0402] Chambon, P. (1995). The molecular and genetic
dissection of the retinoid signaling pathway. [Review]. Recent
Progress in Hormone Research 50, 317-32. [0403] Chambon, P. (1996)
Faseb J. 10, 940-954 [0404] Chiang, M. Y., Misner, D., Kempermann,
G., Schikorski, T., Giguere, V., Sucov, H. M., Gage, F. H.,
Stevens, C. F. & Evans, R. M. (1998) Neuron 21, 1353-1361.
[0405] Chomienne, C., Fenaux and Degos, L. (1996). Retinoid
differentiation therapy in promyelocytic leukemia. FASEB J.
1025-1030. [0406] Chytil, F. (1984). Retinoic acid: Biochemistry,
toxicology, pharmacology, and therapeutic use. Pharmacol. Rev. 36,
93-99. [0407] Cole et al. (1985). Monoclonal Antibodies in Cancer
Therapy. Allen R. Bliss, Inc. [0408] Creech Kraft, J., Schuh, T.,
Juchau, M. R. and Kimelman, D. (1994). Temporal distribution,
localization and metabolism of all-trans retinol, didehydroretinol
and all-trans retinal during Xenopus development. Biochem. J. 301,
111-119. [0409] De Coster, R., Wouters, W. and Bruynseels, J.
(1996). P450-dependent enzymes as targets for prostate cancer
therapy. J. Ster. Biochem. Mol. Biol. 56, 133-43. [0410] Duell, E.
A., Astrom, A., Griffiths, C. E., Chambon, P. and Voorhees, J. J.
(1992). Human skin levels of retinoic acid and cytochrome
p-450-derived 4-hydroxyretinoic acid after topical application of
retinoic acid in vivo compared to concentrations required to
stimulate retinoic acid receptor-mediated transcription in vitro.
Journal of Clinical Investigation 90, 1269-74. [0411] Fiorella, P.
D., Giguere, V. and Napoli, J. L. (1993). Expression of Cellular
Retinoic Acid-binding Protein (Type II) in Escherichia coli. The
Journal of Biological Chemistry 268, 21545-21552. [0412] Formelli,
F., Barua, A. and Olson, J. (1996). Bioactivities of
N-(4-hydroxyphenyl) retinimide and retinoyl B-glucuronide. FASEB J.
10, 1014-1024. [0413] Frolik, C. A., Roberts, A. B., Tavela, T. E.,
Roller, P. P., Newton, D. L. and Sporn, M. B. (1979). Isolation and
identification of 4-hydroxy- and 4-oxoretinoic acid. In vitro
metabolites of all-trans retinoic acid in hamster trachea and
liver. Biochemistry 18, 2092-7. [0414] Fujii, H., Sato, T., Kaneko,
S., Gotoh, O., Fujii-Kuriyama, Y., Osawa, K., Kato, S., and Hamada,
H. (1997). Metabolic inactivation of retinoic acid by a novel P450
differentially expressed in developing mouse embryos. EMBO Journal
16, 4163-73. [0415] Gudas, L., Sporn, M. and Roberts, A. (1994).
Cellular biology and biochemistry of the retinoids. In: The
Retinoids. (M. Sporn, Roberts, A. and Goodman, D. S., Editors)
Raven Press, Inc.: New York. [0416] Guengerich, (1991) J. Biol.
Chem. 266:10019-10022 [0417] Higgins, D. G. and Sharp, P. M.
(1989). Fast and sensitive multiple sequence alignments on a
microcomputer. CABIOS 5, 151-153. [0418] Higgins, D. G., Bleasby,
A. J., and Fuchs, R. (1991). CLUSTAL V: improved software for
multiple sequence alignment. CABIOS 8, 189-191. [0419] Hogan, B. et
al., (1986). A Laboratory Manual, Cold Spring Harbor, N.Y., Cold
Spring Harbor Laboratory. [0420] Hollermann, T., Chen, Y., Grunz,
H., and Pieler, T. (1998). Regionalized metabolic activity
establishes boundaries of retinoic acid signalling. European
Molecular Biology Organization 17, 7361-7372. [0421] Hong, W.
(1994). Retinoids and human cancer. In: The Retinoids. (M. Sporn,
Roberts, A. and Goodman, D. S., Editors) Raven Press, Inc.: New
York. [0422] Houbenwcyl, (1987). Methods of Organic Chemistry, ed.
E. Wansch. Vol. 15 I and II. Thieme, Stuttgart. [0423] Hozumi, N
and Sandhu, J. S. (1993). Recombinant antibody technology, its
advent and advances. Cancer Invest. 11, 714-723. [0424] Huse et
al., (1989). Science 246, 1275-1281. [0425] Iulianella, A.,
Beckett, B., Petkovich, M., and Lohnes, D. (1999). A molecular
basis for retinoic acid-induced axial truncation. Developmental
Biology 205, 33-48. [0426] Jones, G., Ramshaw, H., Zhang, A., Cook,
R., Byford, V., White, J. & Petkovich, M (1999) Endocrinology
140, 3303-3310. [0427] Kennett, R. (1979). Cell fusion. Methods
Enzymol. 58, 345-359. [0428] Kohler and Milstein. (1975). Nature
256, 495-497. [0429] Kozbor et al. (1983). Immunol. Today 4, 72.
[0430] Lammer, E. j., Chen, D. T., Hoar, R. M., Agnish, N. D.,
Benke, P. J., Braun, J. T., curry, C. J., Fernhoff, P. M., Grix, A.
J., Lott, I. T. & et, a. l. (1985) N. Engl. J. Med. 313,
837-841. [0431] Lammer, E. & Armstrong, D. (1992) in Retinoids
in normal development and teratogenesis, ed. Morris-Kay, G. (Oxford
University Press, Oxford), pp. 281-295. [0432] Lane, M., Chen, A.,
Roman, S., Derguini, F. & Gudas, L. (1999) Proc. Natl. Acad.
Sci. USA 96, 13524-13529. [0433] Lemoine, N. R. and Cooper, D. N.
(1996). Gene Therapy, Human Molecular Genetics Series, BIOS
Scientific Publishers, Oxford, U.K. [0434] Leo et al. (1989).
Metabolism of retinol and retinoic acid by human liver cytochrome
P450IIC8. Arch. Biochem. Biophys. 269, 305-312. [0435] Lippman, S.
M., Heyman, R. A., Kurie, J. M., Benner, S. E. and Hong, W. K.
(1995). Retinoids and chemoprevention: clinical and basic studies.
J. Cellular Biochem. Supplement 22, 1-10. [0436] Lotan, R. M.
(1995). Squamous differentiation and retinoids. Cancer Treat. Res.
74, 43-72. [0437] Lotan, R. (1996). Retinoids in Cancer
Chemoprevention. Faseb J. 10, 1031-1039. [0438] Maden, M. and
Holder, N. (1992). Retinoic acid and development of the central
nervous system. [Review]. Bioessays 14, 431-8. [0439] Mangelsdorf,
D. J. and Evans, R. M. (1995). The RXR Heterodimers and Orphan
Receptors. Cell 83, 841-850. [0440] Merrifield, (1964]. J. Am.
Chem. Assoc. 85, 2149-2154. [0441] McCafferty et al., (1990).
Nature 348, 552-554. [0442] Mirski, S. and Cole, S. P. C. (1989).
Antigens associated with multidrug resistance in H69AR, a small
cell lung cancer cell line. Cancer Res. 49, 5719-5724. [0443]
Monia, B. P., Johnston, J. F., Geiger, T., Muller, M. and Fabbro,
D. (1996). Antitumor activity of a phosphorothioate antisense
oligodeoxynucleotide targeted against C-raf kinase. Nature Medicine
2, 668-75. [0444] Moon, R. C., Mehta, R. G. and Rao, K. V. N.
(1994). Retinoids and cancer in experimental animals. In: The
Retinoids. (M. Sporn, Roberts, A. and Goodman, D. S., Editors)
Raven Press, Inc.: New York. [0445] Morriss-Kay, G. M. (1996).
Embryonic development and pattern formation. FASEB J. 10, 961-968.
[0446] Morrison et al., (1985). Proc. Natl. Acad. Sci. USA 81,
6851. [0447] Muindi, J. R. F., Frankel, S. R., Huselton, C.,
DeGrazia, F., Garland, W., Young, C. W. and Warrell, R. P., Jr.
(1992). Clinical pharmacology of oral all-trans retinoic acid in
patients with acute promyelocytic leukemia. Cancer Research 52,
2138-2142. [0448] Muindi, J. R., Young, C. W. and Warrell, R. J.
(1994a). Clinical pharmacology of all-trans retinoic acid. Leukemia
8, 1807-1812. [0449] Muindi, J. R., Young, C. W. and Warrell, R. J.
(1994b). Clinical pharmacology of all-trans retinoic acid. Leukemia
8, s16-s21. [0450] Napoli, J. L., Boerman, M. H., Chai, X., Zhai,
Y. and Fiorella, P. D. (1995). Enzymes and binding proteins
affecting retinoic acid concentrations. J. Ster. Biochem. Mol.
Biol. 53, 497-502. [0451] Napoli, J. (1996). Retinoic acid
biosynthesis and metabolism. FASEB J. 10, 993-1001. [0452] Nebert
et al. (1989), DNA 8:1-13 [0453] Nelson, D. et al (1996),
Pharmacogenetics 6:1-42 [0454] Nelson, D. (1999a) Arch. Biochem.
Biophys 369:1-10 [0455] Nelson, D. (1999b) Arch. Biochem. Biophys.
371, 345-347. [0456] Niederreither, K., Subbarayan, V., Dolle, P.
& Chambon, P. (1999) Nature Genetics 21, 444-448. [0457] Old,
R. W. and Primrose, S. B., In: Principles of Gene Manipulation. An
Introduction to Genetic Engineering, 4th ed. Oxford University
Press. 63-66. [0458] Ohno, C. K. and Petkovich, M. (1993). FTZ-F1
beta, a novel member of the Drosophila nuclear receptor family.
Mechanisms of Development 40, 13-24. [0459] Pijnappel, W. W.,
Hendriks, H. F., Folkers, G. E., van den Brink, C., Dekker, E. J.,
Edelenbosch, C., van der Saag, P. and Durston, A. J. (1993). The
retinoid ligand 4-oxo-retinoic acid is a highly active modulator of
positional specification. Nature 366, 340-4. [0460] Ray, W. J.,
Bain, G., Yao, M., and Gottlieb, D. I. (1997). CYP26, a novel
mammalian cytochrome P450, is induced by retinoic acid and defines
a new family. Journal of Biological Chemistry 272, 18702-8. [0461]
Reddy, A. P., Chen, J., Zacharewski, T., Gronemeyer, H., Voorhees,
J. J. and Fisher, G. J. (1992). Characterization and purification
of human retinoic acid receptor-g1 overexpressed in the
baculovirus-insect cell system. Biochem. J. 287, 833-840. [0462]
Rigas, J., Miller, V., Zhang, Z. F., Klimstra, D., Tong, W., Kris,
M. and Warrell, R. (1996). Metabolic phenotypes of retinoic acid
and the risk of lung cancer. Cancer Res. 56, 2692-2696. [0463]
Roberts, A. B., Nichols, M. D., Newton, D. L. and Sporn, M. B.
(1979a). In vitro metabolism of retinoic acid in hamster intestine
and liver. Journal of Biological Chemistry 254, 6296-302. [0464]
Roberts, A. B., Frolik, C. A., Nichols, M. D. and Sporn, M. B.
(1979b). Retinoid-dependent induction of the in vivo and in vitro
metabolism of retinoic acid in tissues of the vitamin A-deficient
hamster. Journal of Biological Chemistry 254, 6303-9. [0465]
Sambrook, J., Fritsch E. F. and Maniatis, T. (1989). Molecular
Cloning: A Laboratory Manual. Cold Spring Harbor Lab Press, Cold
Spring Harbor, N.Y. [0466] Staerz & Bevan (1986a). Proc. Natl.
Acad. Sci. (USA) 83, 1453. [0467] Staerz & Bevan (1986b).
Immunology Today 7, 241. [0468] Stewart, A. J., Canitrot, Y.,
Baracchini, E., Dean, N. M., Deeley, R. G., and Cole, S. P. C.
(1996). Reduction of Expression of the multidrug resistance protein
(MRP) in human tumor cells by antisense phosphorothioate
oligonucleotides. Biochem. Pharamcol. 51, 461-469. [0469] Swindell,
E., Thaller, C., Sockanathan, S., Petkovich, M., Jessell, T. &
Eichele, G. (1999) Dev. Biol. 216, 282-296. [0470] Takatsuka, J.,
Takahashi, N. and De Luca, L. M. (1996). Retinoic Acid Metabolism
and Inhibition of Cell Proliferation: An Unexpected Liaison. Cancer
Research 56, 675-678. [0471] Takeda et al., (1985). Nature 314,
452. [0472] Tanaguchi et al., European Patent Publication EP171496.
[0473] Teng, et al. (1982) Meth. Enzymol. 92.3-16. [0474] Thompson,
J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W:
improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-specific gap
penalties and weight matrix choice. Nucleic Acids Research 22,
4673-4680. [0475] Van Wauwe, J. P., Coene, M.-C., Goossens, J., Van
Nijen, G., Cools, W. and Lauwers, W. (1988). Ketoconazole inhibits
the in vitro and in vivo metabolism of all-trans retinoic acid. The
Journal of Pharmacology and Experimental Therapeutics 245, 718-722.
[0476] Van Wauwe, J. P., Coene, M.-C., Goossens, J., Cools, W. and
Monbaliu, J. (1990). Effects of cytochrome P450 inhibitors on the
in vivo metabolism of all-trans-retinoic acid in rats. The Journal
of Pharmacology and Experimental Therapeutics 252, 365-369. [0477]
Van Wauwe, J., Van Nyen, G., Coene, M., Stoppie, P., Cools, W.,
Goossens, J., Borghgraef, P. and Janssen, P. A. J. (1992).
Liarazole, an Inhibitor of Retinoic Acid Metabolism, Exerts
Retinoid-Mimetic Effects in Vivo. The Journal of Pharmacology and
Experimental Therapeutics 261, 773-779. [0478] Ward et al., (1989).
Nature 341. 544-546. [0479] Warrell, R. J. (1994). Applications for
retinoids in cancer therapy. Seminars in Hematol. 31, 1-13. [0480]
Warrell, R. J., Maslak, P., Eardley, A., Heller, G., Miller, W. J.
and Frankel, S. R. (1994). Treatment of acute promyelocytic
leukemia with all-trans retinoic acid: an update of the New York
experience. Leukemia 8, 929-933. [0481] White, J. A., Boffa, M. B.,
Jones, B. and Petkovich, M. (1994). A zebrafish retinoic acid
receptor expressed in the regenerating caudal fin. Development 120,
1861-72. [0482] White, J. A., Guo, Y., Baetz, K., Beckett-Jones,
B., Bonasoro, J., Hsu, K., Dilworth, J., Jones, G., and Petkovich,
M. (1996a). Identification of the retinoic acid-inducible all trans
retinoic acid 4-hydroxylase. Journal of Biological Chemistry 271,
29922-29927. [0483] White, J.
& Petkovich, M. (1996b) Met. Mol. Biol. 89, 389-404. [0484]
White, J. A., Beckett-Jones, B., Guo, Y. D., Dilworth, F. J.,
Bonasoro, J., Jones, G., and Petkovich, M. (1997). cDNA cloning of
human retinoic acid-metabolizing enzyme (hP450RAI) identifies a
novel family of cytochromes P450. Journal of Biological Chemistry
272, 18538-41. [0485] White, J., Beckett, B., Scherer, S., Hebrick,
J. and Petkovick, M. (1998) Genomics 48, 270-272. [0486] Williams,
J. B. and Napoli, J. L. (1987). Inhibition of retinoic acid
metabolism by imidazole antimycotics in F9 embryonal carcinoma
cells. Biochemical Pharmacology 36, 1386-1388. [0487] Wouters, W.,
van, D. J., Dillen, A., Coene, M. C., Cools, W. and De, C. R.
(1992). Effects of liarazole, a new antitumoral compound, on
retinoic acid-induced inhibition of cell growth and on retinoic
acid metabolism in MCF-7 human breast cancer cells. Cancer Research
52, 2841-6.NCES [0488] Yamamoto, M., Drager, U., Ong, D. &
McCaffery, P. (1998) Eur. J. Biochem. 257, 344-350.
Sequence CWU 1
1
48130PRTArtificial Sequencetypical heme binding motif found in all
Cytochrome P450s 1Lys Lys Glu Thr Phe Ile Pro Phe Gly Ile Gly Lys
Arg Val Cys Met1 5 10 15Gly Glu Gln Leu Ala Lys Met Glu Leu Phe Leu
Met Phe Val 20 25 302194DNAArtificial Sequenceclone from soares
retinc N2b4 H2 library 2ggtgtccgga cctgcctggg caagcacctg gccaanctgt
tcctgaaggt gaaggcggtg 60gagctggcta gcaccagccg ctttnagctg gccacacgga
ccttcccccg catcaccttg 120gtccccgtcc tgcaccccgt ggatggcctc
agcgtcaagt tctttggcct ggactccaan 180cagaaagnga tatc
194311358DNAHomo sapiens 3gttcagagtg aggttggctg cctcatttaa
agccatgcat ggatttaagt gccagagtca 60tgtatttaac cattctgtgc ctcagtttcc
tcatctataa actgagtaat aacacatcac 120tggattaaaa gaaactatgt
atataaagct ttagaactgt gtctggcaca tagcttagtc 180tcagtaggat
ttagccttta actgactctc gtttgctgac acatgaggct ctggggctgg
240gagcagcgtc ctggggtacc ccgatctgcc aggcccagta gctgcagtcc
tagggatcat 300gactcacagg tactctgggg gaaccacttc agaacctctg
cctcccctcc caggctgggg 360tggctgggcc aatggaatga cctcagccag
gaggaatgag gggcgcggag tctgtaggca 420ggcccctgcc tacccctgga
gcatccgcag ccccggcttg cattcgctcc ctagcaggtg 480accacagcca
acaggacccc aggggtcacc ctggtgactc actgtagtct cttgatccag
540accttgttca agggacctcc cagcctcttc cttctgcagc ccactgctta
aaagttgatc 600ccagccccat tacccttgac ctatagctga gacctcaagc
tggcctctga ctaatgcatg 660accctgatct ttgactgacc tctgaccctg
gccgtggact ggctttgatt cagacagtcg 720cagttctggc ctctgctggg
ccctggccct ggctgcagat ggacccattg cacagacatg 780aaagacactc
agggggtttc tgggctgatc tcctctcagc accgcatctc cctcccttca
840gatctcctgg ctccaggctg aagtgctccg cctgccaggc agcctggctc
cctgaaggtg 900gggagtgcct gtctgccccc cttgctggga gaggcccaca
tggagaggag gcctcagcct 960tagagaagat acaggtggat tctgctggtg
ttatctcttt agtccacttc tcagtgatgc 1020ctcctaagga gcatccttcc
tccagcccct gctggctaca gctcccagat gaaagggaac 1080ccacaacccc
cgccaccact gttgccctgc cattagacac ttttccggga gcttcctaag
1140agcagcaaca aagcttccca gctggaccaa gtttctgtag gacagaacac
ggcaatcaga 1200ccgggagagc cccatggtca ggagttgtgt ctcagtcacc
catggtgggg cggggggggg 1260cccaaggagg ggctaagagc aacttgcgtg
ctgcagttga acacacttgg accctaacct 1320tggctccact atttactagc
tgtgtgtctt tgggccaatc tctgaacctc tctgaactca 1380gtttcctcac
ctgcaaaacg gggccagtgg cattacttac tgcagaatga tgaagatgga
1440atgggctaca ctctgggaag ccccaagcat gtctggcact tgtacttgct
cagtagatgt 1500cagtggtcag ggatagtctg tccctagcac atcaggggtt
ccacagggca gggcagctca 1560gtgaggctga gatctttacc cctgcagaag
cagggttttc tcactgaaga gggtcagaag 1620gcaatgtcta atctagcctc
ccctgcccgc ccttgctcct tgtagcttct cagggactct 1680gggcctgcag
ggctcagccc aacctctcca cagagctctc ctctgtcctt caccgtaaga
1740aagcctctcc tcctcagcct tacctattta tgtctacccg tccttcctgg
gttgcctgac 1800ccaacccacc ccctgcagga agcttctgag ggtgacgtct
gccagggcct ggctcttgaa 1860agctaagtca caaaagccag ggctgatcct
ccactgtgga tggccttgct gtgatccctg 1920gctgcctggt tctcctgaga
cagtgaggat cagggcccgg cctgccccgg caccatggac 1980cctagggtgg
ggccagggtg agacgtgttt cctgaacctg agccaccagg ccgggggcat
2040ctccattgca ctcattgccc aagcaggtac tcaaactgcc ctcagaaggg
tttggtttct 2100tttcctctct tttcagttaa ttaaatcaat taagtcattt
tgagtcattc ctgttggcca 2160cttggggctt tctccttagc gaggcctgat
tcattaaggc agcaggacag atgccaaggt 2220cagccatgac gtcatgatcc
ggggtcacac ttcctgtggg cggagaagag agccatacag 2280gcacccaggg
cgtgccccag gactgacctc tggggaggga cccagagagt aaggatatgg
2340gtccccgtgg ggtcacttgg ccaaagcaag tcatgaactt aggctgccac
ccatgttttc 2400agtcagcctc aagcattctt caagggtttc cttgacgcca
ggtgctgggg gaggatgaaa 2460agatggttca gtgcctcccg aagatgccct
ttttattagg ttgatttcaa aagtagtgtg 2520gccttttaat ggcagaaacc
acaattactt ttgcaccaac ttgtacctta gctgctagga 2580gcccagatag
aaagttaacc cttaatcaca cgatctgtgt tagcccctgt gtttggcgta
2640tttgagtctt tctttctcct cttgaatttt tatgtattat cattttttgt
tagcagcatt 2700atatgcatgt attctatggt aagctacttc atgtcctctt
tggaagtagg tagagtataa 2760atcataagtc aattaatggg gcctggagaa
actcatagac catcaagagg gactcatgca 2820aacatcctat acctttcccc
cttggatgga gaaaacctca agcgacaggg tgtgaaaacg 2880tgagtcttct
ggggcctgaa cacctggaaa caaattgcag ctgcaatctt cagttgagga
2940gaaagggagt aggggcctgg gctcaaacgg cagctctctg cggaaggaaa
ctggccgggg 3000tttaaggtct caagccctga ctgccggtcc cagctccacc
tgtctctggc agtgggaccc 3060tgggaagatt ctcttgcccc ttgggcctct
gtttatctaa gtgttaaacg gatttgatga 3120ggggcctgac cttggctctt
atggggctgc tttgaggctc ccttgagcca aagagtggat 3180ttagtgattt
ctctggtcag agctggtttg cccactaatc ttgctgctca aagaagaaac
3240gggagagttt gctcaggctg ggaaggccac ctactctcag cttctaggag
agaaatctat 3300tatataaatg actgtcccct gacccccgag agacccacta
cccatctgag gccccatgct 3360ctctggccac tgcctccctg ctggcctggg
ttcctccatg ccacactccc agcatccaag 3420ggtgaccgag catgtgagtc
agggctggct ccgacttcct tgaagcgggg tcctgctcct 3480tagcatgggg
tgccctccct aggctctggg caagaaaagc ctgccgggtg agctgagaga
3540agctttccgg gcacatctcc tgacctggaa aggggctcag tgggccgcga
cacacctgcc 3600tcctagtgat gctctgcaga tagcatcatg gtcacagtca
atatttattg agcacatgtt 3660aatccacaca ccagccccat gagtcaagta
ccattattaa cttaatttta caaataagcg 3720aactgaggca tgaagagatt
atgtcacaca gttaggaaat ggcagattac tagttctctt 3780ttccattaca
gtctactgat gcctattcac tcagtcacac tgtaagtatt gattgagcgc
3840atactgtgtg ccagatgcta aggatccttt aaagaacaag atagataaaa
agccctgccc 3900tcctggtgct aacattctag agggaggaga gagacaataa
acagcaaaca tagacgcatg 3960acagagtatg ttagaaggtc atatgaggtt
tggggaaaaa caaaggagag tcaagtggat 4020caggtctgct gccagggaag
ggtgttgcaa tggaagtgcg gaggccgagg agagcctggg 4080ttcagccaag
acttgaagga ggtcagggag ggagcgtgtg gctgcctggg agaaagacat
4140gcatgggccc tgagagagga gctgaacaca gcaaggggtg cttcggctgg
agaggaggga 4200gaagggcctg agaaccgaga ggtaatggag gacacagcat
gcagggccct gtcggccatt 4260gtaaggtctt ttgagttttt ttcccaaaac
cgagttggtt ttattgcaag aatagaacgg 4320tgcactgccc tcgaggcttt
gccttttact ctgaagcttc tgtggagttt gggccccaag 4380ggaccagacc
tgacttgagg tttaaaagga tccctctggc tgcagcaggg aatgctgatg
4440gtgggcggcg gggggggggg gggtggtggt ggcaaagcgt gggacagagt
gacccagagg 4500aggcttcctc gatggcccag aggaccacag gacctggacc
agtgaggaag aggtgaagca 4560agcatcagtg gaattctgga tctgctttga
aggtggcacc agagtatttg ctgctggatt 4620ggctgtgggg tagagagaaa
gagaggagtc caaattttgg gcctgaacaa ctggaaggat 4680ggagctgcct
tttactggga tacagggtga aggaggttct tttgtggcat tcggggatga
4740tcagagtttc atattgggca tgcagggttt gagctgccta gtgtgcattc
taggcagagt 4800tctagggaca gcagctggcc atgccagtgg gaggtatagg
gagacagagg tcacagctga 4860ggacctccct caggtcacac tcctgtcaga
gcaggatgga ctcttaggga ctgccgagga 4920agcacagcag caagtggtca
ggccacacag ctggcaaatg gcagggctgg ggctgcaacc 4980ccgggctgtt
cccaggtgct cctgtctcct gcagccactg cctcccatcc acacccacgt
5040caggaggagc ggctggggcc gactgagtcc accaccccct ttaccagagg
aggaaactag 5100gcctacgtga ttgcctgggc acacaccgag gacgagcagc
agagctggaa cccgggcctc 5160cctgacttgg ggttcagaga ttatgcatga
caagatagca ctcgtgaagg cgccccgaca 5220ggtctggaat gtgggagtga
gtgtggtggt gatgattatt gtgccccatt ggctggccag 5280gggcgcattt
tggcaggatt gccctcttac ccagtgaggg ctggggacca actcatgttc
5340ttcctgctgg gagaggccca ggcccctggg tgggggcccc acattcagtc
cctgccttcc 5400tgattcatcc cagctctctt ggaggcctga gtcacttttt
ttccaggtga gagctgcctg 5460tttggcaaat tcctggattt caggggaaga
aataggaact cctgcccagg gttggtctta 5520tgcacaggta aggcaggcag
caacccacgt ctgccccaaa tccctgccct ggcctggaat 5580tcactcactt
gtctatctat ccatggggcc agccatttct gagtaatgat ctccattctg
5640atattcagta ttttgcagtt tgcaaaacac tttcaaatac gggtagtcct
attttgtgga 5700tgagaagact gaggctcaga gaagggaaag gctgtttcaa
gagggcccag ctgggaaaca 5760gggagccagg gtgcagtccc aggcttgtct
gacacaggag ttcttccatt tacagtcact 5820actgtcccca catctggtcg
ccatttccaa ctctttgccc caattctctg agctagggaa 5880ggaagctgtg
accttcaaga agtctgattc cccttgcact gtgggtaaca ggagaaggag
5940gaggcatgga cactgaagaa cagtctcgag gacaactgca cagccaggtc
tggagagcgg 6000ccgtgggaga agccttgagt gggttgagtg cccagcagct
ccgtggaggc tatcacaaga 6060atatcagaga agcgaggtgg gtaggaggcg
ttctggacag ggaagcagcc agcagaatgg 6120gatgaactta cattcagggg
ccaggaacat aaggtatgtg tatgagtggg caggagggtc 6180aggttgcaga
agtttctgaa tgcctatgga atgttcgggg cagtactggt gaggggccca
6240gggctgctcc ctcttccccc aggaagcctt cctgcgctaa ctcctctggt
tcctcagccc 6300cggccggctc ctcttcccca ggcacagcct gtacttggag
acttcctggg tcatgtcctc 6360tctcccaggg aacagggcaa ggggccgctc
gtaggatacc taccttactg atagagatcg 6420tgggaagtta ggaagccaga
gtctttctca agaaggatca gggcttggag ggagggccaa 6480ccctacagac
aggcagactg tattcctcga ggtgtatgca gacgtctctt ctccaaagag
6540cccctgattg catgttacag tgcccagagt tccagcagag gtgattcaca
ctttcggaca 6600gggcacatcc cctcaccaca gatgaatttc cttgaaagga
tactgctgca cccatcactg 6660taaatggcac cagcaaatag tttcttccct
gggtaggggg ctgggggaag tgggagcaga 6720gaagagggtg accggagaac
ctcaagggaa ggtggggtag cagagacacc ttgggccact 6780tcatatccgt
gctataggaa gaccgtgtct atttggagcc tccacaataa tccatcctca
6840ctctttacca atgccctaac agctagttca gatttgcctt gtttcaatca
aattcctagt 6900gggtctcggg cttcttagta tagggggtgc aggggctgat
tcagggcacc atgaagaggg 6960tttttgaaaa ccagtttccc tcggcaggta
gaacctaatt cagagaaagg caatctgggc 7020atctgtcagg tagccagtca
gtagtgggca caaacggttc aagctggaaa taggatgcta 7080cctaactcag
gtttcactgt gaccacgtcg ctataactga caaaatgcaa ataagctcca
7140ctcccactga tgtagacaac gccctggagt agaattaaaa aaagaaaacg
ttgtgccaac 7200ctattttgat tgtgtacttc tgcaataagg atgcacatgt
tgcatggcac gattatatgc 7260cacgcctctc acctgggacc cagactcaaa
gctgcaaccg acccaaatcc tgcaaccaaa 7320taatacctgg tgcatacggc
tctgcttttt gagaaagggt gatataaagc ttagagtctt 7380tgacatgact
ctcagacaca gatccagttt aggaaaacaa agaccaagat cttgaggcag
7440gctgggcatg gtggctcacg cctgtactcc tagcactttg ggaggccaag
gcaggaggac 7500cccttgaatt taggagtttg agaccagcct ggacaacaca
gtgggatgct gtctctacaa 7560aaaataaaaa gttagccagg ggtggtggta
gccaggtgca cttgtagtcc tggctactca 7620ggaggctgag gtgggtggat
tgcttgaaac tgggaggtca aggctttgag ccatgattat 7680gccactgcac
tccagcctgg gcaacagagt gagaccctgt taaaaaacaa aacaaaacca
7740aaacaaaaac aaaaaaactt gaggtgttgg cagtgagtga ccacgaacca
gaataaaaca 7800atttttaaat atgctgtaaa taagccttag taatgatgtg
tatataagtt agaggatgtg 7860gctataagtg gccagttata aaaagtggga
agattataaa atgaagacaa tttcaaataa 7920ggaaaaatat attacattac
ttcaacttgt taattcctta ttctgaacat aaaaacatat 7980gtaatttata
ttgtttgaaa acctgtttct ttccatttct cctgaataaa agatagataa
8040gccaaaattt tagcggttta ttgagatcac caaattatta gccatcatgg
gttgaccagc 8100tgccttgttc ctccctgatg agaatggtgg gtgaaatcga
ggctgcttgc ggcgtgtgga 8160aaagccaagt ggaggcaact cagcttcaag
tctgtgctat ggcaacatat gtgcttctca 8220ttagtgattt acatcacaag
atctagcagc gggtctaata gtttttgtat aataatttct 8280gcattgggat
gagcgttttt gatatttcaa gatgtggatg agcgtttttg atatttcaag
8340atgtcttctc aactgtaatg atatgaaaat atctgatttc gattgctaac
aaagtcacag 8400gtacttttac atacttttgt ggtttgttgt ctccacacat
aactgaaaga aagttgagtt 8460aggcgttggt gaaaataatg gtgtaatttt
ctccgcattc aatccccaaa tcccctacat 8520tctctccttg gacgtccagt
gggttaatgg agcccaggtc aggtggtccc tgaagttccc 8580cacaatctct
gcccacagga cagacgcctg gagtgagagg ctctggggct gaactcccca
8640tggagggctc tgtctctctg acccactctt aagtgctcct ttcctccctc
tgaaatagac 8700aatgccctgt gtcccgcctc ctcacactct cccgtggagg
ctgctccttc atggctctcg 8760actctgtcca ctcctgccca cctgcccctc
tgagtcaggg ccccaggcct gggctgctgt 8820ggagcctccc gacagtctgc
tagctcctct ctggtggact cccattggtt ccccacatgg 8880taaccagtgg
gatgttaagt tgtagagttc atcatgtcac tctctgctta aagtccttca
8940atggttcccc agtaccctta gggtaggaac aagaacttga cttttgtccc
caaggccggc 9000ccacgctgcc tcccatctcc cactaggccc ctccgtagca
ccctggccac cttgcacttc 9060ctggctcatg ccatgtgcct ctctgccagg
cccttggcac tcaccgttcc ctctgtctgg 9120cccctaccct cgctctgccc
catagcttgt gaactcagct ctctcgcaga catcactttc 9180tccaggagat
gcccctgctc tctgtggagc ccccattcca cacctgctgc tctgctcaag
9240gctggtttgt tagaacatgc attttgtctt tcacattggg gatgtctgga
agccctttga 9300tgctacaaaa gggcatgggg tggggggcac caggaacatc
ccgctgcctg ggaggaccac 9360ccagcatgcg gacaactctg gatcaaggct
tgtgctgacg ttaggagatt ggagggcctg 9420aaaggcgatg ctaacacagt
gctggaaacc caggcccatg ggcctgtggt gtggggcgga 9480ggcaggctgc
cctccaggtg tggagcagag caagcactgt ctctgcgggc tctcctggca
9540ccatgaactt ttgggttaaa tcccctaatg ccattgcaac tttacttttc
tttgattaat 9600atcatttccc cactagacta taagttccaa ggaggcaaag
atggcattgg tggccagcat 9660ctagcatgtg cccgcctgca gtgggagttc
gaaagttgct attgaaggaa tgtccgagtt 9720ctctcttccc tattcacaga
aaagccctcc ggctccttct gtccctcttt gtttccccct 9780gccttttgag
agtctgcaca cccttggctg ggttttgtgg gtatttcccc attaactggc
9840taagtcatcc cttactcgtt tttcaggagg gctgctggtg actcagaccc
ggtgtggctg 9900ggcctggctc tgagggagct cctgagagcc tgagtagatg
ctcaacaaac atgtgccgag 9960ttgagcctca gccaagcttg gaggcagagg
gatggacagg atgacttggc tgcaggaggg 10020tgagtccttg gaggagggaa
aagacaggtc aggtgactga gaacagggtt cgattcccgg 10080tcaggtgact
ctggggaagg tgctggggtg tctatggagt ttcacacccc atgatctggg
10140ctgggtgctc cagataggat cctggcacac ccccggcggc ctgcccccac
ctgctcctgg 10200ggcaggggca acaggtgggg cccacttatc gccaaccagg
ccagcaggtg gagccagcct 10260ggccctgggg agatggcaat agcacaggca
ctcctcagtg gaaaatcacc acaccacacg 10320gcctcctcct gggaacggga
acagaaaggc ctggaagata tgggccagaa gtgtgtgggc 10380ctgtgcaaag
agctcaggct cccctcaaac tggggtaggc gggtgggcct tccctctatg
10440gtgccacatg tgggcagttc tcaggctggg tcggagtgag ccgagggtca
gtcctcagcc 10500ccatggatcc aggccctcca gggccttagg aagcatctgc
agtctccttt taggcccttg 10560agatttcaaa ttacgtctaa tcacaactcg
cagggctaga cctactccac ttgtgatccc 10620ataatgtctg ctggactccc
agcttgggtg tagcagtgtc cttggcaagg gccagcttga 10680gggccgaggg
cactgaacac ttttttattg gggcaatggc tgctggcccc tttacggtca
10740aggtgtgcag actgagaagt gtccatatca cttgctaatt gctttgctcg
aacatgaaga 10800cttggacata agaaatacgt gcacacactc agagcatact
ctgcacatgt tttcacacct 10860atgtccacac atgaagacat tttctcacat
aaccacagac aaaactgtgc cccagtgcac 10920acggatgcac agaggcctgc
gtggaaatcc atgcccatgc ctcggacacc tcacagtctc 10980ggtatgcagg
ctgagcacca caatcctgtc ctggagctgg ggagtcatgc ctctttcctt
11040ccagtccctt ttctatgcac ttacaaagca tacttgcatt tgaattattt
gtaccatttt 11100gtatctcatg tcatgaaaca ctctctgtaa acatcatttt
aaatcattgc ataatctacc 11160atcatatggg tggaccgtaa tttatttgac
caatccccta ttgttggaca tgtgagttgt 11220attccatttt tcacaacact
gtgtgtaaat ctttatccac ctgtctgatg atttccttgg 11280gctggcctcc
tagaagtcgc aatactgagc caaagggtaa gactgtttta aaggcctttg
11340aatcatgttg ctgaattc 1135841598DNAHomo sapiens 4atgctctttg
agggcttgga tctggtgtcg gcgctggcca ccctcgccgc gtgcctggtg 60tccgtgacgc
tgctgctggc cgtgtcgcag cagctgtggc agctgcgctg ggccgccact
120cgcgacaaga gctgcaagct gcccatcccc aagggatcca tgggcttccc
gctcatcgga 180gagaccggcc actggctgct gcagggttct ggcttccagt
cgtcgcggag ggagaagtat 240ggcaacgtgt tcaagacgca tttgttgggg
cggccgctga tacgcgtgac cggcgcggag 300aacgtgcgca agatcctcat
gggcgagcac cacctcgtga gcaccgagtg gcctcgcagc 360acccgcatgt
tgctgggccc caacacggtg tccaattcca ttggcgacat ccaccgcaac
420aagcgcaagg tcttctccaa gatcttcagc cacgaggccc tggagagtta
cctgcccaag 480atccagctgg tgatccagga cacactgcgc gcctggagca
gccaccccga ggccatcaac 540gtgtaccagg aggcgcagaa gctgaccttc
cgcatggcca tccgggtgct gctgggcttc 600agcatccctg aggaggacct
tgggcacctc tttgaggtct accagcagtt tgtggacaat 660gtcttctccc
tgcctgtcga cctgcccttc agtggctacc ggcggggcat tcaggctcgg
720cagatcctgc agaaggggct ggagaaggcc atccgggaga agctgcagtg
cacacagggc 780aaggactact tggacgccct ggacctcctc attgagagca
gcaaggagca cgggaaggag 840atgaccatgc aggagctgaa ggacgggacc
ctggagctga tctttgcggc ctatgccacc 900acggccagcg ccagcacctc
actcatcatg cagctgctga agcaccccac tgtgctggag 960aagctgcggg
atgagctgcg ggctcatggc atcctgcaca gtggcggctg cccctgcgag
1020ggcacactgc gcctggacac gctcagtggg ctgcgctacc tggactgcgt
catcaaggag 1080gtcatgcgcc tgttcacgcc catttccggc ggctaccgca
ctgtgctgca gaccttcgag 1140cttgatggtt tccagatccc caaaggctgg
agtgtcatgt atagcatccg ggacacccat 1200gacacagcgc ccgtgttcaa
agacgtgaac gtgttcgacc ccgatcgctt cagccaggcg 1260cggagcgagg
acaaggatgg ccgcttccat tacctcccgt tcggtggcgg tgtccggacc
1320tgcctgggca agcacctggc caagctgttc ctgaaggtgc tggcggtgga
gctggctagc 1380accagccgct ttgagctggc tacacggacc ttcccccgca
tcaccttggt ccccgtcctg 1440caccccgtgg atggcctcag cgtcaagttc
tttggcctgg actccaacca gaacgagatc 1500ctgccggaga cggaggccat
gctgagcgcc acagtctaac ccaagaccca cccgcctcag 1560cccagcccag
gcagcggggt ggtggttgtg ggaggtag 15985512PRTHomo sapiens 5Met Leu Phe
Glu Gly Leu Asp Leu Val Ser Ala Leu Ala Thr Leu Ala1 5 10 15Ala Cys
Leu Val Ser Val Thr Leu Leu Leu Ala Val Ser Gln Gln Leu 20 25 30Trp
Gln Leu Arg Trp Ala Ala Thr Arg Asp Lys Ser Cys Lys Leu Pro 35 40
45Ile Pro Lys Gly Ser Met Gly Phe Pro Leu Ile Gly Glu Thr Gly His
50 55 60Trp Leu Leu Gln Gly Ser Gly Phe Gln Ser Ser Arg Arg Glu Lys
Tyr65 70 75 80Gly Asn Val Phe Lys Thr His Leu Leu Gly Arg Pro Leu
Ile Arg Val 85 90 95Thr Gly Ala Glu Asn Val Arg Lys Ile Leu Met Gly
Glu His His Leu 100 105 110Val Ser Thr Glu Trp Pro Arg Ser Thr Arg
Met Leu Leu Gly Pro Asn 115 120 125Thr Val Ser Asn Ser Ile Gly Asp
Ile His Arg Asn Lys Arg Lys Val 130 135 140Phe Ser Lys Ile Phe Ser
His Glu Ala Leu Glu Ser Tyr Leu Pro Lys145 150 155 160Ile Gln Leu
Val Ile Gln Asp Thr Leu Arg Ala Trp Ser Ser His Pro 165 170 175Glu
Ala Ile Asn Val Tyr Gln Glu Ala Gln Lys Leu Thr Phe Arg Met 180 185
190Ala Ile Arg Val Leu Leu Gly Phe Ser Ile Pro Glu Glu Asp Leu Gly
195 200 205His Leu Phe Glu Val Tyr Gln Gln Phe Val Asp Asn Val Phe
Ser Leu 210 215 220Pro Val Asp Leu Pro Phe Ser Gly Tyr Arg Arg Gly
Ile Gln Ala Arg225
230 235 240Gln Ile Leu Gln Lys Gly Leu Glu Lys Ala Ile Arg Glu Lys
Leu Gln 245 250 255Cys Thr Gln Gly Lys Asp Tyr Leu Asp Ala Leu Asp
Leu Leu Ile Glu 260 265 270Ser Ser Lys Glu His Gly Lys Glu Met Thr
Met Gln Glu Leu Lys Asp 275 280 285Gly Thr Leu Glu Leu Ile Phe Ala
Ala Tyr Ala Thr Thr Ala Ser Ala 290 295 300Ser Thr Ser Leu Ile Met
Gln Leu Leu Lys His Pro Thr Val Leu Glu305 310 315 320Lys Leu Arg
Asp Glu Leu Arg Ala His Gly Ile Leu His Ser Gly Gly 325 330 335Cys
Pro Cys Glu Gly Thr Leu Arg Leu Asp Thr Leu Ser Gly Leu Arg 340 345
350Tyr Leu Asp Cys Val Ile Lys Glu Val Met Arg Leu Phe Thr Pro Ile
355 360 365Ser Gly Gly Tyr Arg Thr Val Leu Gln Thr Phe Glu Leu Asp
Gly Phe 370 375 380Gln Ile Pro Lys Gly Trp Ser Val Met Tyr Ser Ile
Arg Asp Thr His385 390 395 400Asp Thr Ala Pro Val Phe Lys Asp Val
Asn Val Phe Asp Pro Asp Arg 405 410 415Phe Ser Gln Ala Arg Ser Glu
Asp Lys Asp Gly Arg Phe His Tyr Leu 420 425 430Pro Phe Gly Gly Gly
Val Arg Thr Cys Leu Gly Lys His Leu Ala Lys 435 440 445Leu Phe Leu
Lys Val Leu Ala Val Glu Leu Ala Ser Thr Ser Arg Phe 450 455 460Glu
Leu Ala Thr Arg Thr Phe Pro Arg Ile Thr Leu Val Pro Val Leu465 470
475 480His Pro Val Asp Gly Leu Ser Val Lys Phe Phe Gly Leu Asp Ser
Asn 485 490 495Gln Asn Glu Ile Leu Pro Glu Thr Glu Ala Met Leu Ser
Ala Thr Val 500 505 5106616DNAMus musculus 6cggccgcccc agtaagtgtg
tcttgaaaac gttgccatac ttctcccggc gcgacgactg 60gaagccggaa ccctgtagca
accagtgacc agtctctccg atgagcggga atcccatgga 120gcccttgggg
atgggcagct tgcagctctt gtcgcgggtg gcagcccagc gcagctgcca
180cagctgctgc gacaccgcca gcagcagcgt cacggacacc aggcacgcgg
cgagggtggc 240cagcgccgac accaactcca agccttcaaa cagcatgttg
gcagccgctt gggggattgg 300ctgtgcggtc cgcgatgggg gaggggaggg
ccggaccaga gtgacggggg aggggaggct 360gcggcagggg gtcctggcac
ccctcggaga aacccaggag atggggccga ggggaagggg 420ctggaggctg
cgaggtgatc gaagagagag aggaaaaaga ttgcctgggg aggccttggc
480tccaaacact ttgcccagga ggaattattt tatagaagtg actcttccca
tcctccgcca 540tcaaagcccc aagcccagaa ttccttctgc cctacaacca
atacagtcta aagcaatctg 600aaaatggact tgccga 6167464DNArattus
7tttttttttt tttttgtgag tccccacagt ttattatgga agccaataaa agagcacatt
60gagtatttca caaaatgcag gacattcgtt gttgtaatat gcaacgtgga atattatata
120tacatcaaaa ccacacgaca gccaagacac acacacaagc accagctcat
accaagacga 180gacacacaag tgctttttca atattaaaac aactgtgata
aaagcatatt aatattttga 240aacatgttta caataaagaa caattcatat
tttactaaat aacaaatatt taacagcaaa 300aactttaaac taaatatcta
ttttgaatta taacaaaata gtacttataa tagtttataa 360agacggacac
aaaattataa catttatgaa aaaaaagttt tgtgtataaa ataacagaaa
420cctgtgcagg gtcaaaacgg ttgcccagaa tgcacacgcc agcg 4648503DNADanio
rerio 8ggttcttatg ggagagcaca gcctggtcac tgtggactgt ccccaaagca
ccagcactct 60tctggggcga aacagtttgg ccaactcaat aggagacatc catcgcaaaa
ggaggaagat 120ctttgctaaa gtttttagcc atgaggctct ggaaagctac
ctgcccaaga tccagcaggt 180cattcaggag accctacgtg tgtggagctc
caatcctgac cccatcaatg tgtatcgcga 240gtcccagcgg ctatccttca
acatggctgt acgcgtgctc ctgggtttcc gcatcccaga 300agaagagatg
cactgtttgt tcagcacctt ccaggagttt gtggagaacg tcttcagtct
360tcccatcgac ctgccattta gtggttacag aaagggtatt cgagcaagag
actcactcca 420aaaaagcata gagaaagcca tcagagagaa accactccac
acacagggga aagattacac 480tgatgctctt gatgtgcttc tag
503926DNAArtificial SequencePCR amplify fragment 9caacatgctc
tttgagggct tggatc 261027DNAArtificial SequencePCR amplify fragment
10ctacctccca caaccaccac cccgctg 2711203PRTMus musculus 11Arg Gln
Val His Phe Gln Ile Ala Leu Asp Cys Ile Gly Cys Arg Ala1 5 10 15Glu
Gly Ile Leu Gly Leu Gly Leu Trp Arg Arg Met Gly Arg Val Thr 20 25
30Ser Ile Lys Phe Leu Leu Gly Lys Val Phe Gly Ala Lys Ala Ser Pro
35 40 45Gly Asn Leu Phe Pro Leu Ser Leu Arg Ser Pro Arg Ser Leu Gln
Pro 50 55 60Leu Pro Leu Gly Pro Ile Ser Trp Val Ser Pro Arg Gly Ala
Arg Thr65 70 75 80Pro Cys Arg Ser Leu Pro Ser Pro Val Thr Leu Val
Arg Pro Ser Pro 85 90 95Pro Pro Ser Arg Thr Ala Gln Pro Ile Pro Gln
Ala Ala Ala Asn Met 100 105 110Leu Phe Glu Gly Leu Glu Leu Val Ser
Ala Leu Ala Thr Leu Ala Ala 115 120 125Cys Leu Val Ser Val Thr Leu
Leu Leu Ala Val Ser Gln Gln Leu Trp 130 135 140Gln Leu Arg Trp Ala
Ala Thr Arg Asp Lys Ser Cys Lys Leu Pro Ile145 150 155 160Pro Lys
Gly Ser Met Gly Phe Pro Leu Ile Gly Glu Thr Gly His Trp 165 170
175Leu Leu Gln Gly Ser Gly Phe Gln Ser Ser Arg Arg Glu Lys Tyr Gly
180 185 190Asn Val Phe Lys Thr His Leu Leu Gly Arg Pro 195
20012167PRTDanio rerio 12Val Leu Met Gly Glu His Ser Leu Val Thr
Val Asp Cys Pro Gln Ser1 5 10 15Thr Ser Thr Leu Leu Gly Arg Asn Ser
Leu Ala Asn Ser Ile Gly Asp 20 25 30Ile His Arg Lys Arg Arg Lys Ile
Phe Ala Lys Val Phe Ser His Glu 35 40 45Ala Leu Glu Ser Tyr Leu Pro
Lys Ile Gln Gln Val Ile Gln Glu Thr 50 55 60Leu Arg Val Trp Ser Ser
Asn Pro Asp Pro Ile Asn Val Tyr Arg Glu65 70 75 80Ser Gln Arg Leu
Ser Phe Asn Met Ala Val Arg Val Leu Leu Gly Phe 85 90 95Arg Ile Pro
Glu Glu Glu Met His Cys Leu Phe Ser Thr Phe Gln Glu 100 105 110Phe
Val Glu Asn Val Phe Ser Leu Pro Ile Asp Leu Pro Phe Ser Gly 115 120
125Tyr Arg Lys Gly Ile Arg Ala Arg Asp Ser Leu Gln Lys Ser Ile Glu
130 135 140Lys Ala Ile Arg Glu Lys Pro Leu His Thr Gln Gly Lys Asp
Tyr Thr145 150 155 160Asp Ala Leu Asp Val Leu Leu 165131494DNAHomo
sapiensCDS(1)..(1491) 13atg ggg ctc ccg gcg ctg ctg gcc agt gcg ctc
tgc acc ttc gtg ctg 48Met Gly Leu Pro Ala Leu Leu Ala Ser Ala Leu
Cys Thr Phe Val Leu1 5 10 15ccg ctg ctg ctc ttc ctg gct gcg atc aag
ctc tgg gac ctg tac tgc 96Pro Leu Leu Leu Phe Leu Ala Ala Ile Lys
Leu Trp Asp Leu Tyr Cys 20 25 30gtg agc ggc cgc gac cgc agt tgt gcc
ctc cca ttg ccc ccc ggg act 144Val Ser Gly Arg Asp Arg Ser Cys Ala
Leu Pro Leu Pro Pro Gly Thr 35 40 45atg ggc ttc ccc ttc ttt ggg gaa
acc ttg cag atg gta ctg cag cgg 192Met Gly Phe Pro Phe Phe Gly Glu
Thr Leu Gln Met Val Leu Gln Arg 50 55 60agg aag ttc ctg cag atg aag
cgc agg aaa tac ggc ttc atc tac aag 240Arg Lys Phe Leu Gln Met Lys
Arg Arg Lys Tyr Gly Phe Ile Tyr Lys65 70 75 80acg cat ctg ttc ggg
cgg ccc acc gta cgg gtg atg ggc gcg gac aat 288Thr His Leu Phe Gly
Arg Pro Thr Val Arg Val Met Gly Ala Asp Asn 85 90 95gtg cgg cgc atc
ttg ctc gga gac gac cgg ctg gtg tcg gtc cac tgg 336Val Arg Arg Ile
Leu Leu Gly Asp Asp Arg Leu Val Ser Val His Trp 100 105 110cca gcg
tcg gtg cgc acc att ctg gga tct ggc tgc ctc tct aac ctg 384Pro Ala
Ser Val Arg Thr Ile Leu Gly Ser Gly Cys Leu Ser Asn Leu 115 120
125cac gac tcc tcg cac aag cag cgc aag aag gtg att atg cgg gcc ttc
432His Asp Ser Ser His Lys Gln Arg Lys Lys Val Ile Met Arg Ala Phe
130 135 140agc cgc gag gca ctc gaa tgc tac gtg ccg gtg atc acc gag
gaa gtg 480Ser Arg Glu Ala Leu Glu Cys Tyr Val Pro Val Ile Thr Glu
Glu Val145 150 155 160ggc agc agc ctg gag cag tgg ctg agc tgc ggc
gag cgc ggc ctc ctg 528Gly Ser Ser Leu Glu Gln Trp Leu Ser Cys Gly
Glu Arg Gly Leu Leu 165 170 175gtc tac ccc gag gtg aag cgc ctc atg
ttc cga atc gcc atg cgc atc 576Val Tyr Pro Glu Val Lys Arg Leu Met
Phe Arg Ile Ala Met Arg Ile 180 185 190cta ctg ggc tgc gaa ccc caa
ctg gcg ggc gac ggg gac tcc gag cag 624Leu Leu Gly Cys Glu Pro Gln
Leu Ala Gly Asp Gly Asp Ser Glu Gln 195 200 205cag ctt gtg gag gcc
ttc gag gaa atg acc cgc aat ctc ttc tcg ctg 672Gln Leu Val Glu Ala
Phe Glu Glu Met Thr Arg Asn Leu Phe Ser Leu 210 215 220ccc atc gac
gtg ccc ttc agc ggg ctg tac cgg ggc atg aag gcg cgg 720Pro Ile Asp
Val Pro Phe Ser Gly Leu Tyr Arg Gly Met Lys Ala Arg225 230 235
240aac ctc att cac gcg cgc atc gag cag aac att cgc gcc aag atc tgc
768Asn Leu Ile His Ala Arg Ile Glu Gln Asn Ile Arg Ala Lys Ile Cys
245 250 255ggg ctg cgg gca tcc gag gcg ggc cag ggc tgc aaa gac gcg
ctg cag 816Gly Leu Arg Ala Ser Glu Ala Gly Gln Gly Cys Lys Asp Ala
Leu Gln 260 265 270ctg ttg atc gag cac tcg tgg gag agg gga gag cgg
ctg gac atg cag 864Leu Leu Ile Glu His Ser Trp Glu Arg Gly Glu Arg
Leu Asp Met Gln 275 280 285gca cta aag caa tct tca acc gaa ctc ctc
ttt gga gga cac gaa acc 912Ala Leu Lys Gln Ser Ser Thr Glu Leu Leu
Phe Gly Gly His Glu Thr 290 295 300acg gcc agt gca gcc aca tct ctg
atc act tac ctg ggg ctc tac cca 960Thr Ala Ser Ala Ala Thr Ser Leu
Ile Thr Tyr Leu Gly Leu Tyr Pro305 310 315 320cat gtt ctc cag aaa
gtg cga gaa gag ctg aag agt aag ggt tta ctt 1008His Val Leu Gln Lys
Val Arg Glu Glu Leu Lys Ser Lys Gly Leu Leu 325 330 335tgc aag agc
aat caa gac aac aag ttg gac atg gaa att ttg gaa caa 1056Cys Lys Ser
Asn Gln Asp Asn Lys Leu Asp Met Glu Ile Leu Glu Gln 340 345 350ctt
aaa tac atc ggg tgt gtt att aag gag acc ctt cga ctg aat ccc 1104Leu
Lys Tyr Ile Gly Cys Val Ile Lys Glu Thr Leu Arg Leu Asn Pro 355 360
365cca gtt cca gga ggg ttt cgg gtt gct ctg aag act ttt gaa tta aat
1152Pro Val Pro Gly Gly Phe Arg Val Ala Leu Lys Thr Phe Glu Leu Asn
370 375 380gga tac cag att ccc aag ggc tgg aat gtt atc tac agt atc
tgt gat 1200Gly Tyr Gln Ile Pro Lys Gly Trp Asn Val Ile Tyr Ser Ile
Cys Asp385 390 395 400act cat gat gtg gca gag atc ttc acc aac aag
gaa gaa ttt aat cct 1248Thr His Asp Val Ala Glu Ile Phe Thr Asn Lys
Glu Glu Phe Asn Pro 405 410 415gac cga ttc agt gct cct cac cca gag
gat gca tcc agg ttc agc ttc 1296Asp Arg Phe Ser Ala Pro His Pro Glu
Asp Ala Ser Arg Phe Ser Phe 420 425 430att cca ttt gga gga ggc ctt
agg agc tgt gta ggc aaa gaa ttt gca 1344Ile Pro Phe Gly Gly Gly Leu
Arg Ser Cys Val Gly Lys Glu Phe Ala 435 440 445aaa att ctt ctc aaa
ata ttt aca gtg gag ctg gcc agg cat tgt gac 1392Lys Ile Leu Leu Lys
Ile Phe Thr Val Glu Leu Ala Arg His Cys Asp 450 455 460tgg cag ctt
cta aat gga cct cct aca atg aaa acc agt ccc acc gtg 1440Trp Gln Leu
Leu Asn Gly Pro Pro Thr Met Lys Thr Ser Pro Thr Val465 470 475
480tat cct gtg gac aat ctc cct gca aga ttc acc cat ttc cat ggg gaa
1488Tyr Pro Val Asp Asn Leu Pro Ala Arg Phe Thr His Phe His Gly Glu
485 490 495atc tga 1494Ile141850DNADanio rerioCDS(88)..(1563)
14tgtcgccgtt gctgtcggtt gctgtcggac gctgtctcct ctccagaagc ttgtttttcg
60ttttggcgat cagttgcgcg cttcaac atg ggg ctg tac acc ctt atg gtc acc
114 Met Gly Leu Tyr Thr Leu Met Val Thr 1 5ttt ctc tgc acc atc gtg
cta ccc gtt tta ctc ttt ctc gcc gcg gtg 162Phe Leu Cys Thr Ile Val
Leu Pro Val Leu Leu Phe Leu Ala Ala Val10 15 20 25aag ttg tgg gag
atg tta atg atc cga cga gtc gat ccg aac tgc aga 210Lys Leu Trp Glu
Met Leu Met Ile Arg Arg Val Asp Pro Asn Cys Arg 30 35 40agt cct cta
ccg cca ggt acc atg ggc ttg ccg ttc att gga gaa acg 258Ser Pro Leu
Pro Pro Gly Thr Met Gly Leu Pro Phe Ile Gly Glu Thr 45 50 55ctc cag
ctg atc ctc cag aga agg aag ttt ctg cgc atg aaa cgg cag 306Leu Gln
Leu Ile Leu Gln Arg Arg Lys Phe Leu Arg Met Lys Arg Gln 60 65 70aaa
tac ggg tgc atc tac aag acg cac ctc ttc ggg aac ccg act gtc 354Lys
Tyr Gly Cys Ile Tyr Lys Thr His Leu Phe Gly Asn Pro Thr Val 75 80
85agg gtg atg gga gct gat aat gtg agg cag att ctg ctg ggc gaa cac
402Arg Val Met Gly Ala Asp Asn Val Arg Gln Ile Leu Leu Gly Glu
His90 95 100 105aag ctg gtg tct gtt cag tgg cca gca tca gtg aga acc
atc ctg ggc 450Lys Leu Val Ser Val Gln Trp Pro Ala Ser Val Arg Thr
Ile Leu Gly 110 115 120tct gac acc ctc tcc aat gtc cat gga gtt caa
cac aaa aac aag aaa 498Ser Asp Thr Leu Ser Asn Val His Gly Val Gln
His Lys Asn Lys Lys 125 130 135aag gcc att atg agg gcg ttc tct cga
gat gct ctg gag cac tac att 546Lys Ala Ile Met Arg Ala Phe Ser Arg
Asp Ala Leu Glu His Tyr Ile 140 145 150ccc gtg atc cag cag gag gtg
aag agc gcc ata cag gaa tgg ctg caa 594Pro Val Ile Gln Gln Glu Val
Lys Ser Ala Ile Gln Glu Trp Leu Gln 155 160 165aaa gac tcc tgc gtg
ctg gtt tat cca gaa atg aag aaa ctc atg ttt 642Lys Asp Ser Cys Val
Leu Val Tyr Pro Glu Met Lys Lys Leu Met Phe170 175 180 185cgg ata
gct atg aga atc ctg ctt ggt ttt gaa cca gag caa ata aag 690Arg Ile
Ala Met Arg Ile Leu Leu Gly Phe Glu Pro Glu Gln Ile Lys 190 195
200acg gac gag caa gaa ctg gtg gaa gct ttt gag gaa atg atc aaa aac
738Thr Asp Glu Gln Glu Leu Val Glu Ala Phe Glu Glu Met Ile Lys Asn
205 210 215ttg ttc tcc ttg cca atc gac gtt cct ttc agt ggt ctg tac
agg ggt 786Leu Phe Ser Leu Pro Ile Asp Val Pro Phe Ser Gly Leu Tyr
Arg Gly 220 225 230ttg agg gca cgc aat ttc att cac tcc aaa att gag
gaa aac atc agg 834Leu Arg Ala Arg Asn Phe Ile His Ser Lys Ile Glu
Glu Asn Ile Arg 235 240 245aag aaa att caa gat gac gac aat gaa aac
gaa cag aaa tac aaa gac 882Lys Lys Ile Gln Asp Asp Asp Asn Glu Asn
Glu Gln Lys Tyr Lys Asp250 255 260 265gcc ctt cag ctg ttg atc gag
aac agc aga aga agt gac gaa cct ttt 930Ala Leu Gln Leu Leu Ile Glu
Asn Ser Arg Arg Ser Asp Glu Pro Phe 270 275 280agt ttg cag gcg atg
aaa gaa gca gct aca gag ctt cta ttt gga ggt 978Ser Leu Gln Ala Met
Lys Glu Ala Ala Thr Glu Leu Leu Phe Gly Gly 285 290 295cat gaa acc
acc gcc agc act gca acc tca ctt gtc atg ttt ctg ggt 1026His Glu Thr
Thr Ala Ser Thr Ala Thr Ser Leu Val Met Phe Leu Gly 300 305 310ctg
aac aca gaa gtg gtg cag aag gtc aga gag gag gtt cag gag aag 1074Leu
Asn Thr Glu Val Val Gln Lys Val Arg Glu Glu Val Gln Glu Lys 315 320
325gtt gaa atg ggc atg tat aca cct gga aag ggc ttg agt atg gag ctg
1122Val Glu Met Gly Met Tyr Thr Pro Gly Lys Gly Leu Ser Met Glu
Leu330 335 340 345ttg gac cag ctg aag tac act gga tgt gtg att aaa
gag act ctt aga 1170Leu Asp Gln Leu Lys Tyr Thr Gly Cys Val Ile Lys
Glu Thr Leu Arg 350 355 360atc aac cct cct gtt ccc gga gga ttc aga
gtc gca ctc aaa acc ttt 1218Ile Asn Pro Pro Val Pro Gly Gly Phe Arg
Val Ala Leu Lys Thr Phe 365 370 375gaa ttg aat ggt tac caa att cct
aaa gga tgg aac gtc att tac agc 1266Glu Leu Asn Gly Tyr Gln Ile Pro
Lys Gly Trp Asn Val Ile Tyr Ser 380 385 390atc tgt gac acg cac gat
gtg gcc gac gtc ttt cca aac aaa gag gag 1314Ile Cys Asp Thr His Asp
Val Ala Asp Val Phe Pro Asn Lys Glu Glu 395 400 405ttc cag ccg gag
aga ttc atg agc aaa ggt ctg gag gac ggg tcc agg 1362Phe Gln Pro Glu
Arg
Phe Met Ser Lys Gly Leu Glu Asp Gly Ser Arg410 415 420 425ttt aac
tac atc ccc ttc gga gga gga tcc agg atg tgt gtg ggc aaa 1410Phe Asn
Tyr Ile Pro Phe Gly Gly Gly Ser Arg Met Cys Val Gly Lys 430 435
440gag ttc gcc aaa gtg tta ctc aag atc ttt tta gtt gag tta acg cag
1458Glu Phe Ala Lys Val Leu Leu Lys Ile Phe Leu Val Glu Leu Thr Gln
445 450 455cat tgc aat tgg att ctc tca aac gga ccc ccg aca atg aaa
aca ggc 1506His Cys Asn Trp Ile Leu Ser Asn Gly Pro Pro Thr Met Lys
Thr Gly 460 465 470ccg act att tac cca gtg gac aat ctc cct acc aaa
ttc act agt tat 1554Pro Thr Ile Tyr Pro Val Asp Asn Leu Pro Thr Lys
Phe Thr Ser Tyr 475 480 485gtc aga aat tagcctaacc ggagctttgt
acatatgttt ttattttaga 1603Val Arg Asn490tgaactgtga tgtattggat
attttctatt ttgtttatat aaagcagatg tgtatataag 1663tctatgcgag
gaagcgaaaa cgagggcact actttctcat ggatcactgt aatgctacag
1723agtgtctgtg atgtatattt ataatgtagt tgtgttatat agcttttgta
ctgtatgcaa 1783cttatttaac tcgctcttta tctcatgggt tttatttaat
aaaacatgtt cttacaaaaa 1843aaaaaaa 1850152677DNAHomo
sapiensmisc_feature(856)..(856)n is any nucleic acid 15gatcccagat
ctgcctattg cgcccgatgc cccgaggctc tctcttggac tctggccctg 60agttcttctg
cgcgatcctt cggagacgtc tggaggcctg ctttatgcat ctctcttgga
120cctcagtttc cccacacgtg ggaggaggca gctggacgat tcctgaaagg
actttccctt 180gcttcctcat cacgtggaag agagcccacc cggcacctgg
aaatggaaag ccagtgaagg 240ctgctttggg ccggggcakc gggtgggacc
gggcgggagg gattccaaag agaccgccgg 300gaaggctaga gcttggaatt
ccggctcctc ggagtcctgg ccctccccca ccgccgcctc 360ggagctcagc
acaccttgga tgggggaggc gggcagctcc tagccccgca ccccaggagg
420cgcgctcgga gggaagccgc caccgccgcc gcctctgcct cggcgcggaa
caaacggtta 480aagattttgg gccascgcct ccgcgggggg aggagccagg
ggccccaatc ccgcaattaa 540agatgaactt tgggtgaact aattgtctga
ccaaggtaac gtgggcagca acctgggccg 600cctataaagc ggcagcgccg
tggggtttga agcgctggcg gcggcggcag gtggcgcggg 660aggtcgcggc
gcgccatggg gctcccggcg ctgctggcca gtgcgctctg caccttcgtg
720ctgccgctgc tgctcttcct ggctgcgatc aagctctggg acctgtactg
cgtgagcggc 780cgcgaccgca gttgtgccct cccattgccc cccgggacta
tsggsttccc cttctttggg 840gaaaccttgc agatgntact ncaggtaagg
gagggtgggg cgggacaggc tgcttccccg 900gagcccggcg cggctctggg
cttctgctga agtcggggta ggcgcccccg ggaggcatgc 960tattgcggct
aggagcaggg ctggcgggag cgcggcgctc cccggmkymc sctcawgcsc
1020rcwwktmwcc tccgcctymc tcccamagcg garsaarwkc ykgmrgatga
agcgcaggaa 1080atacggcttc atctacaaga cgcatctgtt cgggcggccc
accgtacggg tgatgggcgc 1140ggacaatgtg cggcgcatct tgctcggaga
gcaccggctg gtgtcggtcc actggccagc 1200gtcggtgcgc accattctgg
gatctggctg cctctctaac ctgcacgact cctcgcacaa 1260gcagcgcaag
aaggtggggg caggaggcga cggctggaca gggaggggga ccccatttat
1320gagcggaatt ccggctgatg gatgctaggc gcgggctagc agcttgaggt
gggctaggac 1380cctctgccag ctccaggtta gctttcccag ctcggagagt
gccatgtgtc tggcaggact 1440gggggtgtct ggaaggggac ggcggtagac
gagaggggcg gatggaggct tttaacgctg 1500tcccctcctc gggactcagg
tgattatgcg ggccttcagc cgcgaggcac tcgaatgcta 1560cgtgccggtg
atcaccgagg aagtgggcag cagcctggag cagtggctga gctgcggcga
1620gcgcggcctc ctggtctacc ccgaggtgaa gcgcctcatg ttccgaatcg
ccatgcgcat 1680cctactgggc tgcgaacccc aactggcggg cgacggggac
tccgagcagc agcttgtgga 1740ggccttcgag gaaatgaccc gcaatctctt
ctcgctgccc atcgacgtgc ccttcagcgg 1800gctgtaccgg gtaagggcgg
caaacgggct gcggactagg ggcgcgggac ctgggcgtct 1860gctcaccgcc
gcgcgctctc tgcgctcagg gcatgaaggc gcggaacctc attcacgcgc
1920gcatcgagca gaacattcgc gccaagatct gcgggctgcg ggcatccgag
gcgggccagg 1980gctgcaaaga cgcgctgcag ctgttgatcg agcactcgtg
ggagagggga gagcggctgg 2040acatgcaggt gagtagcagc ttcagaccag
gcactgcgga gtttggtccc ctggctttcc 2100aaggcgctgt tcctggggcc
cccaaagcgc gcgcctgggg cccagctttc tggagtgggc 2160ggccggctca
gactacagct atggaatccc gaaggaaggc tgagacaccc ggtcaggaga
2220gctgcggaag gggctgcggm ggaaactggg agcatcccct agcctttamc
aggtttcaaa 2280gggaaagttg gaatttgcaa aaatgttaat aaagaacctt
gcgattttaa taaaactaag 2340actttaactc aggagtttcc ggtagrgcgg
ggtcgtactc gccttactgc tccagctgaa 2400ctaaagggac gttgcatttt
gtttaaagat attgctttcc ttgactttct gtcagcaaaa 2460catttagccc
ttctagtctt ccctccagaa ctctcagttc gattctgagt aatccttctg
2520tcaaaccgca ggcagacttg tgagaatgtg ggtctcactc tattcttagg
cactaaagca 2580atcttcaacc gaactcctct ttggaggaca cgaaaccacg
gccagtgcag ccacatctct 2640gatcacttac ctggggctct acccacatgt tctccag
267716683DNAHomo sapiens 16gatccaggtt gctgaaacat atctccatat
agggcagaac aattatcaaa agcataagaa 60ttgcagccac agcataggga agaaagagga
gtttttaaac cacaacaaaa gggagaaaga 120agagaatttt aacttacatt
taattcaaaa gtcttcagag caacccgaaa ccctcctgga 180actgggggat
tcagtcgaag ggtctcctta ataacacacc cgatgtatyt aagttgttcc
240aaaatttcca tgtccaactt gttgtcttga ttgctcttgc aaagtaaacc
ctaycaaaay 300agtcatacag aggtgaacag tyattttgtg ctccaattaa
aatcagccca gcagacgtaa 360acagggctta agtggagact aaacccaaag
ggccccatga tgggagagac tgggaggggg 420aaacagcagc taatggccat
ttgcctgccc aaatccacta tctatttaca atcccaggag 480aatgctgctc
accagttaga aggaccaagt ttctccccac gcccccccac cccacactca
540ccaccaccac ccacactaat cagctattca cactatgtat gcccttggac
acaccaattc 600aagaaaagtg gaacctatct gagaatctcc acggttcaca
aaaaggtgga ggaggggtag 660gaatacaagg tcaaaccctg ccc 683171725DNAMus
musculusCDS(25)..(1515) 17gcacgaggga ggctgaagcg tgcc atg ggg ctc
ccg gcg ctg ctg gcc agt 51 Met Gly Leu Pro Ala Leu Leu Ala Ser 1
5gcg ctc tgc acc ttc gtg ctg ccg ctg ctg ctc ttc ctg gcg gcg ctc
99Ala Leu Cys Thr Phe Val Leu Pro Leu Leu Leu Phe Leu Ala Ala Leu10
15 20 25aag ctc tgg gac ctg tac tgt gtg agc agc cgc gat cgc agc tgc
gcc 147Lys Leu Trp Asp Leu Tyr Cys Val Ser Ser Arg Asp Arg Ser Cys
Ala 30 35 40ctc ccc ttg ccc ccc ggt acc atg ggc ttc cca ttc ttt ggg
gaa aca 195Leu Pro Leu Pro Pro Gly Thr Met Gly Phe Pro Phe Phe Gly
Glu Thr 45 50 55ttg cag atg gtg ctt cag cgg agg aag ttt ctg cag atg
aag cgc agg 243Leu Gln Met Val Leu Gln Arg Arg Lys Phe Leu Gln Met
Lys Arg Arg 60 65 70aaa tac ggc ttc atc tac aag acg cat ctg ttt ggg
cgg ccc acg gtg 291Lys Tyr Gly Phe Ile Tyr Lys Thr His Leu Phe Gly
Arg Pro Thr Val 75 80 85cgg gtg atg ggc gcg gat aat gtg cgg cgc atc
ttg ctg gga gag cac 339Arg Val Met Gly Ala Asp Asn Val Arg Arg Ile
Leu Leu Gly Glu His90 95 100 105cgg ttg gtg tcg gtg cac tgg ccc gcg
tcg gtg cgc acc atc ctg ggc 387Arg Leu Val Ser Val His Trp Pro Ala
Ser Val Arg Thr Ile Leu Gly 110 115 120gct ggc tgc ctc tcc aac ctg
cac gat tcc tcg cac aag cag cga aag 435Ala Gly Cys Leu Ser Asn Leu
His Asp Ser Ser His Lys Gln Arg Lys 125 130 135aag gtg att atg cag
gcc ttc agc cgc gag gca ctc cag tgc tac gtg 483Lys Val Ile Met Gln
Ala Phe Ser Arg Glu Ala Leu Gln Cys Tyr Val 140 145 150ctc gtg atc
gct gag gaa gtc agc agt tgt ctg gag cag tgg cta agc 531Leu Val Ile
Ala Glu Glu Val Ser Ser Cys Leu Glu Gln Trp Leu Ser 155 160 165tgc
ggc gag cgc ggc ctc ctg gtc tac ccc gag gtg aag cgc ctc atg 579Cys
Gly Glu Arg Gly Leu Leu Val Tyr Pro Glu Val Lys Arg Leu Met170 175
180 185ttc cgc atc gcc atg cgc atc ctg ctg ggc tgc gag ccg ggt cca
gcg 627Phe Arg Ile Ala Met Arg Ile Leu Leu Gly Cys Glu Pro Gly Pro
Ala 190 195 200ggc ggc ggg gag gac gag caa cag ctc gtg gag gct ttc
gag gag atg 675Gly Gly Gly Glu Asp Glu Gln Gln Leu Val Glu Ala Phe
Glu Glu Met 205 210 215acc cgc aat ctc ttc tct ctt ccc att gac gtg
ccc ttt agc ggc ctg 723Thr Arg Asn Leu Phe Ser Leu Pro Ile Asp Val
Pro Phe Ser Gly Leu 220 225 230tac cgg ggc gtg aag gcg cgg aac ctt
ata cac gcg cgc atc gag gag 771Tyr Arg Gly Val Lys Ala Arg Asn Leu
Ile His Ala Arg Ile Glu Glu 235 240 245aac att cgc gcc aag atc cgc
cgg ctt cag gct aca gag ccg gat ggg 819Asn Ile Arg Ala Lys Ile Arg
Arg Leu Gln Ala Thr Glu Pro Asp Gly250 255 260 265ggt tgc aag gac
gcg ctg cag ctc ctg att gag cac tcg tgg gag agg 867Gly Cys Lys Asp
Ala Leu Gln Leu Leu Ile Glu His Ser Trp Glu Arg 270 275 280gga gag
agg ctg gat atg cag gca cta aaa caa tcg tca aca gag ctc 915Gly Glu
Arg Leu Asp Met Gln Ala Leu Lys Gln Ser Ser Thr Glu Leu 285 290
295ctc ttt ggt ggt cat gaa act aca gcc agt gct gcg aca tca ctg atc
963Leu Phe Gly Gly His Glu Thr Thr Ala Ser Ala Ala Thr Ser Leu Ile
300 305 310act tac cta gga ctc tac cca cat gtc ctc cag aaa gtt cga
gaa gag 1011Thr Tyr Leu Gly Leu Tyr Pro His Val Leu Gln Lys Val Arg
Glu Glu 315 320 325ata aag agc aag ggc tta ctt tgc aag agc aat caa
gac aac aag tta 1059Ile Lys Ser Lys Gly Leu Leu Cys Lys Ser Asn Gln
Asp Asn Lys Leu330 335 340 345gac atg gaa act ttg gaa cag ctt aaa
tac att ggg tgt gtc att aag 1107Asp Met Glu Thr Leu Glu Gln Leu Lys
Tyr Ile Gly Cys Val Ile Lys 350 355 360gag acc ctg cga ttg aat cct
ccg gtt cca gga ggg ttt cgg gtt gct 1155Glu Thr Leu Arg Leu Asn Pro
Pro Val Pro Gly Gly Phe Arg Val Ala 365 370 375ctg aag act ttt gag
ctg aat gga tac cag atc ccc aag ggc tgg aat 1203Leu Lys Thr Phe Glu
Leu Asn Gly Tyr Gln Ile Pro Lys Gly Trp Asn 380 385 390gtt att tac
agt atc tgt gac acc cac gat gtg gca gat atc ttc act 1251Val Ile Tyr
Ser Ile Cys Asp Thr His Asp Val Ala Asp Ile Phe Thr 395 400 405aac
aag gag gaa ttt aat ccc gac cgc ttt ata gtg cct cat cca gag 1299Asn
Lys Glu Glu Phe Asn Pro Asp Arg Phe Ile Val Pro His Pro Glu410 415
420 425gat gct tcc cgg ttc agc ttc att cca ttt gga gga ggc ctt cgg
agc 1347Asp Ala Ser Arg Phe Ser Phe Ile Pro Phe Gly Gly Gly Leu Arg
Ser 430 435 440tgt gta ggc aaa gag ttt gca aaa att ctt ctt aag ata
ttt aca gtg 1395Cys Val Gly Lys Glu Phe Ala Lys Ile Leu Leu Lys Ile
Phe Thr Val 445 450 455gag ctg gct agg cac tgt gat tgg cag ctt cta
aat gga cct cct aca 1443Glu Leu Ala Arg His Cys Asp Trp Gln Leu Leu
Asn Gly Pro Pro Thr 460 465 470atg aag aca agc ccc act gtg tac cct
gtg gac aat ctc cct gca aga 1491Met Lys Thr Ser Pro Thr Val Tyr Pro
Val Asp Asn Leu Pro Ala Arg 475 480 485ttc acc tac ttc cag gga gat
atc tgatagctat ttcaattctt ggacttattt 1545Phe Thr Tyr Phe Gln Gly
Asp Ile490 495gaagtgtata ttggtttttt ttaaaaatag tgtcatgttg
actttattta atttctaaat 1605gtatagtatg atatttatgt gtctctacta
cagtcccgtg gtctttaaat attaaaataa 1665tgaatttgta tgatttccca
ataaagtaaa attaaaaagt gaaaaaaaaa aaaaaaaaaa 172518690DNAMus
musculusmisc_feature(636)..(636)n is any nucleic acid 18gcggccgctg
attcgtgtga ccggtgcgga gaatgtgcgc aagatcctac tgggcgaaca 60ccagctagtg
agcaccgagt ggccgcggag cgcacgcgta ctgctgggtc ccaacacggt
120ggccaattcc attggcgaca tccaccgcaa caagcgcaag gtcttctcca
agatcttcag 180ccatgaggca cttgagagct acctgcccaa gatccaactg
gtgatccagg atacacttcg 240agcctggagc agccagcctg aggccatcaa
tgtatatcag gaggcccagc gacttacctt 300ccgaatggcc gtgcgtgtgc
tgctaggctt cagcatccct gaggaggacc tgggccacct 360ctttgaggta
taccagcagt ttgtggagaa tgtcttctct ctgccagtgg acctgccctt
420cagtggctac cggaggggca tccaagctcg gcagatcctt cagaagggcc
tagagaaggc 480tatccgtgag aagctgcagt gtacccaggg caaagactac
tcggacgccc tggacattct 540cattgagagc agcaaggaac atggcaagga
gatgaccatg caggagctga aggatggaac 600cctggagttg atcttcgcag
cctacgccac gacggncagc ctcgtgccga attcttggnc 660tcganggcca
aattccctat agngagtcgt 69019616DNAMus musculus 19cggccgcccc
agtaagtgtg tcttgaaaac gttgccatac ttctcccggc gcgacgactg 60gaagccggaa
ccctgtagca accagtgacc agtctctccg atgagcggga atcccatgga
120gcccttgggg atgggcagct tgcagctctt gtcgcgggtg gcagcccagc
gcagctgcca 180cagctgctgc gacaccgcca gcagcagcgt cacggacacc
aggcacgcgg cgagggtggc 240cagcgccgac accaactcca agccttcaaa
cagcatgttg gcagccgctt gggggattgg 300ctgtgcggtc cgcgatgggg
gaggggaggg ccggaccaga gtgacggggg aggggaggct 360gcggcagggg
gtcctggcac ccctcggaga aacccaggag atggggccga ggggaagggg
420ctggaggctg cgaggtgatc gaagagagag aggaaaaaga ttgcctgggg
aggccttggc 480tccaaacact ttgcccagga ggaattattt tatagaagtg
actcttccca tcctccgcca 540tcaaagcccc aagcccagaa ttccttctgc
cctacaacca atacagtcta aagcaatctg 600aaaatggact tgccga
61620477DNAMus musculus 20gcgccccttc caccttctat cggcaagtcc
attttcagat tgctttagac tgtattggtt 60gtagggcaga aggaattctg ggcttggggc
tttgatggcg gaggatggga agagtcactt 120ctataaaata attcctcctg
ggcaaagtgt ttggagccaa ggcctcccca ggcaatcttt 180ttcctctctc
tcttcgatca cctcgcagcc tccagcccct tcccctcggc cccatctcct
240gggtttctcc gaggggtgcc aggaccccct gccgcagcct cccctccccc
gtcactctgg 300tccggccctc ccctccccca tcgcggaccg cacagccaat
cccccaagcg gctgccaaca 360tgctgtttga aggcttggag ttggtgtcgg
cgctggccac cctcgccgcg tgcctggtgt 420ccgtgacgct gctgctggcg
gtgtcgcagc agctgtggca gctgcgctgg gctgcca 47721205DNAMus musculus
21atgctgtttg aaggcttgga gttggtgtcg gcgctggcca ccctcgccgc gtgcctggtg
60tccgtgacgc tgctgctggc ggtgtcgcag cagctgtggc agctgcgctg ggctgccacc
120cgcgacaaga gctgcaagct gcccatcccc aagggctcca tgggattccc
gctcatcgga 180gagactggtc actggttgct acagg 20522230DNAMus musculus
22agggttccgg cttccagtcg tcgcgccggg agaagtatgg caacgttttc aagacacact
60tactggggcg gccgctgatt cgtgtgaccg gtgcggagaa tgtgcgcaag atcctactgc
120gcgaacacca gctagtgagc accgagtggc cgcggagcgc acgcgtactg
ctgggtccca 180acacggtggc caattccatt ggcgacatcc accgcaacaa
gcgcaaggta 23023275DNAMus musculus 23aggtcttctc caagatcttc
agccatgagg cacttgagag ctacctgccc aagatccaac 60tggtgatcca ggatacactt
cgagcctgga gcagccagcc tgaggccatc aatgtatatc 120aggaggccca
gcgacttacc ttccgaatgg ccgtgcgtgt gctgctaggc ttcagcatcc
180ctgaggagga cctgggccac ctctttgagg tataccagca gtttgtggag
aatgtcttct 240ctctgccagt ggacctgccc ttcagtggct accgg 27524157DNAMus
musculus 24ggcatccaag ctcggcagat ccttcagaag ggcctagaga aggctatccg
tgagaagctg 60cagtgtaccc agggcaaaga ctactcggac gccctggaca ttctcattga
gagcagcaag 120gaacatggca aggagatgac catgcaggag ctgaagg
15725380DNAMus musculus 25ctcaacatgg cctctgtctc cggcagaatc
tcattctggt tggagtccag accaaagaac 60ttgacactga ggccatccac ggggtgcaag
acggggacca gagtgatgcg agggaaggtc 120cgggtggcca gctcgaagcg
gctagtgctg gccagctcca ctgccagcac cttcaggaac 180agcttggcca
agtgcttgcc caggcaggtc cgcacgccac cgccaaacgg gaggtaatgg
240aagcggccat ccttatcctc actgcgtgcc tgactgaagc ggtccgggtc
aaacacattc 300acgtccttga acacgggcgc tgtgtcgtga gtgtctcgga
tgctatacat gacactccag 360cccttgggga tctggaaacc 3802683DNAMus
musculusmisc_feature(83)..(83)n is any nucleic acid 26aaggaacatg
gcaaggagat gaccatgcag gagctgaagg atggaaccct ggagttgatc 60ttcgcagcct
acgccacgac ggn 8327399DNArattus 27cggccgccaa catgctgttt gagggcttgg
agttggtgtc ggcgctggcc accctcgccg 60cgtgcctggt gtccgtgacg ctgcttctgg
cggtgtcgca gcagctgtgg cagctgcgct 120gggctgccac ccgcgacaag
agctgcaagc tgcctatccc caagggctcc atgggattcc 180cgctcatcgg
agagactggt cactggttgc tacagggttc cggcttccag tcgtcgcgcc
240gcgagaagta tggcaacgtt ttcaagacac acttactggg gcggccgttg
atccgtgtga 300ccggtgcgga gaacgtgcgc aagatcctac tgggcgaaca
ccagctagtg agcacggagt 360gggcgcggag cgcacgcgtg ctgctgggac ccaacacgg
399284445DNAHomo sapiensCDS(1)..(1536) 28atg ctc ttt gag ggc ttg
gat ctg gtg tcg gcg ctg gcc acc ctc gcc 48Met Leu Phe Glu Gly Leu
Asp Leu Val Ser Ala Leu Ala Thr Leu Ala1 5 10 15gcg tgc ctg gtg tcc
gtg acg ctg ctg ctg gcc gtg tcg cag cag ctg 96Ala Cys Leu Val Ser
Val Thr Leu Leu Leu Ala Val Ser Gln Gln Leu 20 25 30tgg cag ctg cgc
tgg gcc gcc act cgc gac aag agc tgc aag ctg ccc 144Trp Gln Leu Arg
Trp Ala Ala Thr Arg Asp Lys Ser Cys Lys Leu Pro 35 40 45atc ccc aag
gga tcc atg ggc ttc ccg ctc atc gga gag acc ggc cac 192Ile Pro Lys
Gly Ser Met Gly Phe Pro Leu Ile Gly Glu Thr Gly His 50 55 60tgg ctg
ctg cag ggt tct ggc ttc cag tcg tcg cgg agg gag aag tat 240Trp Leu
Leu Gln Gly Ser Gly Phe Gln Ser Ser Arg Arg Glu Lys Tyr65 70 75
80ggc aac gtg ttc aag acg cat ttg ttg ggg cgg ccg ctg ata cgc gtg
288Gly Asn Val Phe Lys Thr His Leu Leu Gly Arg Pro Leu Ile Arg Val
85 90 95acc ggc gcg gag aac gtg cgc aag atc ctc atg ggc gag cac cac
ctc 336Thr Gly Ala Glu Asn Val Arg Lys Ile Leu Met Gly Glu His His
Leu 100 105 110gtg agc acc gag tgg cct cgc agc acc cgc atg ttg ctg
ggc ccc aac 384Val Ser Thr Glu Trp Pro Arg Ser Thr Arg Met Leu Leu
Gly
Pro Asn 115 120 125acg gtg tcc aat tcc att ggc gac atc cac cgc aac
aag cgc aag gtc 432Thr Val Ser Asn Ser Ile Gly Asp Ile His Arg Asn
Lys Arg Lys Val 130 135 140ttc tcc aag atc ttc agc cac gag gcc ctg
gag agt tac ctg ccc aag 480Phe Ser Lys Ile Phe Ser His Glu Ala Leu
Glu Ser Tyr Leu Pro Lys145 150 155 160atc cag ctg gtg atc cag gac
aca ctg cgc gcc tgg agc agc cac ccc 528Ile Gln Leu Val Ile Gln Asp
Thr Leu Arg Ala Trp Ser Ser His Pro 165 170 175gag gcc atc aac gtg
tac cag gag gcg cag aag ctg acc ttc cgc atg 576Glu Ala Ile Asn Val
Tyr Gln Glu Ala Gln Lys Leu Thr Phe Arg Met 180 185 190gcc atc cgg
gtg ctg ctg ggc ttc agc atc cct gag gag gac ctt ggg 624Ala Ile Arg
Val Leu Leu Gly Phe Ser Ile Pro Glu Glu Asp Leu Gly 195 200 205cac
ctc ttt gag gtc tac cag cag ttt gtg gac aat gtc ttc tcc ctg 672His
Leu Phe Glu Val Tyr Gln Gln Phe Val Asp Asn Val Phe Ser Leu 210 215
220cct gtc gac ctg ccc ttc agt ggc tac cgg cgg ggc att cag gct cgg
720Pro Val Asp Leu Pro Phe Ser Gly Tyr Arg Arg Gly Ile Gln Ala
Arg225 230 235 240cag atc ctg cag aag ggg ctg gag aag gcc atc cgg
gag aag ctg cag 768Gln Ile Leu Gln Lys Gly Leu Glu Lys Ala Ile Arg
Glu Lys Leu Gln 245 250 255tgc aca cag ggc aag gac tac ttg gac gcc
ctg gac ctc ctc att gag 816Cys Thr Gln Gly Lys Asp Tyr Leu Asp Ala
Leu Asp Leu Leu Ile Glu 260 265 270agc agc aag gag cac ggg aag gag
atg acc atg cag gag ctg aag gac 864Ser Ser Lys Glu His Gly Lys Glu
Met Thr Met Gln Glu Leu Lys Asp 275 280 285ggg acc ctg gag ctg atc
ttt gcg gcc tat gcc acc acg gcc agc gcc 912Gly Thr Leu Glu Leu Ile
Phe Ala Ala Tyr Ala Thr Thr Ala Ser Ala 290 295 300agc acc tca ctc
atc atg cag ctg ctg aag cac ccc act gtg ctg gag 960Ser Thr Ser Leu
Ile Met Gln Leu Leu Lys His Pro Thr Val Leu Glu305 310 315 320aag
ctg cgg gat gag ctg cgg gct cat ggc atc ctg cac agt ggc ggc 1008Lys
Leu Arg Asp Glu Leu Arg Ala His Gly Ile Leu His Ser Gly Gly 325 330
335tgc ccc tgc gag ggc aca ctg cgc ctg gac acg ctc agt ggg ctg cgc
1056Cys Pro Cys Glu Gly Thr Leu Arg Leu Asp Thr Leu Ser Gly Leu Arg
340 345 350tac ctg gac tgc gtc atc aag gag gtc atg cgc ctg ttc acg
ccc att 1104Tyr Leu Asp Cys Val Ile Lys Glu Val Met Arg Leu Phe Thr
Pro Ile 355 360 365tcc ggc ggc tac cgc act gtg ctg cag acc ttc gag
ctt gat ggt ttc 1152Ser Gly Gly Tyr Arg Thr Val Leu Gln Thr Phe Glu
Leu Asp Gly Phe 370 375 380cag atc ccc aaa ggc tgg agt gtc atg tat
agc atc cgg gac acc cat 1200Gln Ile Pro Lys Gly Trp Ser Val Met Tyr
Ser Ile Arg Asp Thr His385 390 395 400gac aca gcg ccc gtg ttc aaa
gac gtg aac gtg ttc gac ccc gat cgc 1248Asp Thr Ala Pro Val Phe Lys
Asp Val Asn Val Phe Asp Pro Asp Arg 405 410 415ttc agc cag gcg cgg
agc gag gac aag gat ggc cgc ttc cat tac ctc 1296Phe Ser Gln Ala Arg
Ser Glu Asp Lys Asp Gly Arg Phe His Tyr Leu 420 425 430ccg ttc ggt
ggc ggt gtc cgg acc tgc ctg ggc aag cac ctg gcc aag 1344Pro Phe Gly
Gly Gly Val Arg Thr Cys Leu Gly Lys His Leu Ala Lys 435 440 445ctg
ttc ctg aag gtg ctg gcg gtg gag ctg gct agc acc agc cgc ttt 1392Leu
Phe Leu Lys Val Leu Ala Val Glu Leu Ala Ser Thr Ser Arg Phe 450 455
460gag ctg gct aca cgg acc ttc ccc cgc atc acc ttg gtc ccc gtc ctg
1440Glu Leu Ala Thr Arg Thr Phe Pro Arg Ile Thr Leu Val Pro Val
Leu465 470 475 480cac ccc gtg gat ggc ctc agc gtc aag ttc ttt ggc
ctg gac tcc aac 1488His Pro Val Asp Gly Leu Ser Val Lys Phe Phe Gly
Leu Asp Ser Asn 485 490 495cag aac gag atc ctg ccg gag acg gag gcc
atg ctg agc gcc aca gtc 1536Gln Asn Glu Ile Leu Pro Glu Thr Glu Ala
Met Leu Ser Ala Thr Val 500 505 510taacccaaga cccacccgcc tcagcccagc
ccaggcagcg gggtggtggt tgtgggaggt 1596agaaacctgt gtgtgggagg
gggccggaac ggggagggcg agtggccccc atacttgccc 1656tcccttgctc
cccctccctg gcaaacccta cccaaagcca gtgggcccca ttcctagggc
1716tgggctcccc ttctggctcc agcttccctc cagccactcc ccatttacca
tcagctcagc 1776ccctgggaag ggcgtggcag gggctctgca tgcccgtgac
agtgttaggt gtcagcgcgt 1836gctacagtgt ttttgtgatg ttctgaactg
ctcccttccc tccgttcctt tcggaccctt 1896ttagctgggg ttgggggacg
ggaagagccg tgcccccttg ggcgcactct tcagcgtctc 1956ctcctcctgc
gcccccactg cgtctgccca ggaacagcat cctgggtagc agaacaggag
2016tcaaccttgg cggggcgggg gctgcgtcca acctggagat tgcccttccc
tatgccacgg 2076ttcccaccct ccctcaccag tttggacaat ttgaaattac
ctattgctgc tacttgttct 2136gtcctctgac cttggggcaa aggagcccca
ggccctgtct ccccagcatc ctccctggtg 2196gccctgggca ggtgcactga
cacccccacc ttcccatccc ctgctgaacc aggccctgtt 2256acacacagcc
gcctaaggcc cgcggctcat gtgctgcccg cccccatatt tattcactga
2316tagagaatct tggggatgct ggggtctgga gtgaacatct cctccccttc
atgccctagc 2376ctgtgttcta gctgtcctgg cgagacttct gtgagtgaag
aggaaggggt ctctggtcaa 2436acccagcccc cagggcctag ggttgaaagc
cttccccggc tccgggcatt atttgggttt 2496aatctcggag cctcactcct
ggactgaagt ccggtgcctc tgccttatcc ctggtggaga 2556tggaatgtgg
cccattgcct cctccctctc ctgtcaaaaa ccctgatcag gtagatttgg
2616aggcggccac gatttcctgt ttggcccctg ttcaccccag tgcactggcc
ctgactccag 2676gcgtgagtat ggggaaggat acgggttctt ctgacgggga
gcaagggcct ccgtcttccc 2736ttccttaact ctcccccttt gccctccgcc
ctgaaaaagg tgtccttgaa gtcccttcca 2796cctctatgcc actgtctgct
tagcccagct caggggtggg gaagaggcga aagcgtgggg 2856gaggtgagcg
cagcggcagt tctgcctcgg agctgattgc agggccctgt gtggtctccg
2916gacagctgcg ggaaggctgc cgcagctgaa gctgaagagg cggctacgtg
cggtttgtca 2976gggggattgg gttgaaaact ggccagtcgg gatgactggg
tgaaagagga gtagctcctg 3036ccactggcgt tttgagtgtt ggcaatttgg
gatgcctcct ggggaaggtt tccgggcgtt 3096tggtgagtct ctagattttt
ccttgctttc tgtgtttatt ggtttttgat gttgtaaaag 3156caatgaatcc
cctttacaag aaaatcgaaa acacagaaga atgaaggaca tgccagtccc
3216cgatcgctgc tgtgagcacc tcagtggctc cctcagacca gatcccgtag
gcagccccac 3276agaccgaccc tgaccccact cacagccacc ctgaagatag
actataggaa cgggcccata 3336ccacacagac tgctctccaa tccctgagtc
tcagatgttt catttatttc ctacttttcc 3396actactaaaa aacagtgtgg
aatagacatt attggcaaaa ttgctcatcc ctaatcctga 3456aaaacaggcc
agaatgggta aagacttgtc aaagcttgca acatagctac atggtgcacc
3516cggacctgta ccccctcccc ccaacacaaa accagtgtct gggaggttca
ttttccttta 3576aactgatcca gctggccctg aaccaattgt ttttgactga
gtatctagga gagcagtaag 3636tggaacttca gacaagccca ctgggtctgg
tccaggtgag gggcaggggg catggggctg 3696ggaggtctca ggggccttcc
ctgggggtgg ccagcctggt agggggcaga gaaggaaaag 3756ctgagggggg
tccctgtgag ggaggaaaga aggatcattt gccccgctgg gtctcaaagg
3816cagtgagaag agagctgaag aaagctctgg ctggctgaca ggatccctgt
gttgtaattg 3876gtccctcctt tcagctctct agtgagatgc ccgtgtctgt
gcgtgtgcgt gtgtgtttca 3936tacagctagc attagatggg tgatgtttct
tacttatcat ccctaactat tgcaacttga 3996ccttaaaaag acaaaacccc
acaaaactct tcctgccacg ggcttgcaga ttgaagcact 4056ttcgatgttg
ggcgctggcg tttgtgttct gggcaccacc gtgaccctgc ccagatggct
4116ataatattat tttatacaca aacctttttt ttcataaatg ttataatttt
gtgtctgtct 4176ttataaacta ttataagtac tatttttgtt ataattcaaa
atagatattt agtataaagt 4236ttttgctgtt aaatatttgt tatttagtaa
aatatgaatt ttgctctatt gtaaacatgg 4296ttcaaaatat taatatgttt
ttatcacagt cgttttaata ttgaaaaagc acttgtgtgt 4356tttgttttga
tatgaaactg gtaccgtgtg agtgtttttg ctgtcgtggt tttaatctgt
4416atataatatt ccatgttgca tattaaaaa 44452924DNAArtificial
Sequenceprimers and probes 29tccctgcctg tcgacctgcc cttc
243024DNAArtificial Sequenceprimers and probes 30gacactccag
cctttgggga tctg 243126DNAArtificial Sequenceprimers and probes
31tgaaggtcgg agtcaacgga tttggt 263224DNAArtificial Sequenceprimers
and probes 32catgtgggcc atgaggtcca ccac 243332DNAArtificial
Sequenceprimers and probes 33gtgtgccctc gcaggggcag ccgccactgt gc
323432DNAArtificial Sequenceprimers and probes 34cgcctcggat
gcccgcagcc cgcagatctt gg 323520DNAArtificial Sequenceprimers and
probes 35gccttcgagg aaatgacccg 203620DNAArtificial Sequenceprimers
and probes 36ctggatgcat cctctgggtg 203721DNAArtificial
Sequenceprimers and probes 37gtctaccagc agtttgtgga c
213822DNAArtificial Sequenceprimers and probes 38agtccaggta
gcgcagccca ct 223926DNAArtificial Sequenceprimers and probes
39tgaaggtcgg agtcaacgga tttggt 264024DNAArtificial Sequenceprimers
and probes 40catgtgggcc atgaggtcca ccac 244121DNAArtificial
Sequenceprimers and probes 41gtctaccagc agtttctcga c
214221DNAArtificial Sequenceprimer 42ggctgcctct ctaacctgca c
214325DNAArtificial Sequenceprimer 43gatcagagat gtggctgctg cactg
254427DNAArtificial Sequenceprimer 44gcaagggacc agctgcatcg gcacctg
274525DNAArtificial Sequenceprimer 45ctcgtgcgtg tcccggatgc tatac
254620DNAArtificial Sequenceprimer 46ggaatgggct atgcctctga
204732DNAArtificial Sequenceprimer 47atctggcacc acaccttcta
caatgagctg cg 324832DNAArtificial Sequenceprimer 48cgtcatactc
ctgcttgctg atccacatct gc 32
* * * * *
References