Retinoid metabolizing protein

Amino acid sequences and corresponding nucelic acid sequence of retinoid metabolizing protein found in zebrafish and human are described.

BACKGROUND OF THE INVENTION 
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 adult epithelial 
tissues [Maden, 1992; Chambon, 1995; Mangelsdorf, 1995]. 
Retinoic acid 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. Unfortunately, a progressive resistance to 
RA has been observed in the treatment of APL [Muindi, 1992] and this has 
been attributed to increased RA metabolism [see Muindi, 1992; and Muindi, 
1994 for review]. 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. 
Many laboratory studies have involved metabolites of RA, particularly the 
activities of all-trans and 9-cis RA metabolites. The mechanism of 
conversion between all-trans RA and 9-cis RA in vivo is unclear; 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 [Lee, 1990], and rate of catabolism. 
RA catabolism is thought to proceed through the formation of polar 
intermediates, including 4-hydroxy-retinoic acid (4-OH-RA) and 
4-oxo-retinoic acid (4-oxo-RA) [Frolik, 1979]. 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 generation of 4-oxo-RA and 4-OH-RA metabolites is believed to be a 
cytochrome P450 dependent process. This is because of an observed 
effectiveness of general P450 inhibitors such as ketoconazole and 
liarozole in inhibiting the production of these metabolites from RA 
[Williams, 1987; Van Wauwe, 1992; Van Wauwe, 1988; Van Wauwe, 1990]. In 
certain tissues (testis, skin, lung) and cell lines (NIH 3T3, HL 60, F9, 
MCF-7) RA metabolism can be induced by RA pretreatment [Roberts, 1979a & 
b; Frolik, 1979; Duell, 1992; Wouters, 1992; Takatsuka, 1996]. Studies 
involving targetted 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]. 
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]. 
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]. 
As far as the inventors are aware, cytochrome P450s involved in the 
metabolism of RA in extrahepatic tissues remain uncharacterized at the 
molecular level. 
SUMMARY OF THE INVENTION 
The present inventors are the first to identify, clone and sequence a gene 
(cDNA) encoding a retinoic acid-inducible, retinoic acid-metabolizing 
protein, including a cDNA which is RA-inducible in humans. The protein has 
been found to be expressed in epithelia. 
A cDNA has been isolated from zebra fish and sequenced. A protein encoded 
by the cDNA has been expressed and shown to have the ability to 
hydroxylate retinoic acid at the 4 position of the .beta.-ionone ring of 
retinoic acid. The protein has been found to be inducible in epithelial 
cells exposed to retinoic acid. 
A human cDNA encoding a protein with similar functionality has also been 
isolated and sequenced. Homology between sequences from the two species, 
be they nucleic acids encoding the protein, or the amino acid sequences of 
the proteins, has been found to be relatively high and both proteins 
contain a heme-binding motif characteristic of the group of proteins known 
as cytochrome P450s. The overall homology between the amino acid sequences 
of these newly obtained proteins and known cytochrome P450s is less than 
30%. Notwithstanding this relatively low overall homology, a higher degree 
of homology has been observed in the heme binding region for certain other 
P450s. For example, homology between the approximately 20 amino acids 
defining respective heme binding regions of the new zebrafish protein and 
CYP4503A12 is about 50% and between the new zebrafish protein and hCYTFAOH 
is 65%. The homology between the heme binding region itself of a protein 
of the present invention and another P450 could well be 70%, 75%, 80%, 
85%, 90%, 95% or even 100%. 
A first aspect of the present invention is thus a purified protein having 
the ability to oxidize a retinoid, and having an amino acid sequence which 
is at least about 30% conserved in relation to the amino acid sequence 
identified as SEQ ID NO:2 or identified as SEQ ID NO:4, or a functionally 
equivalent homolog thereof. The amino acid sequence identified as SEQ ID 
NO:2 is of the protein, termed here "zP450RAI", obtained from zebrafish. 
The amino acid sequence of the human protein is identified as SEQ ID NO:4 
and the protein is referred to herein as "hP450RAI". 
Such a protein which is at least about 35% conserved in relation to the 
amino acid sequence identified as SEQ ID NO:2 or identified as SEQ ID 
NO:4, or a functionally equivalent homolog thereof, also forms part of the 
invention disclosed herein. Likewise, the degree of sequence conservation 
of a protein could be 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 
90%, 95% or of course 100% of either SEQ ID NO:2 or SEQ ID NO:4 or a 
functionally equivalent homolog thereof, variants being possible so long 
as the ability of the native protein to oxidize a retinoid is retained. 
Also within the scope of the invention is any such protein which has the 
ability to hydroxylate retinoic acid at the 4 position of the 
.beta.-ionone ring. Of course, conservatively substituted variants of 
proteins disclosed are within the scope of the present invention. 
A retinoid oxidized by a protein of the present invention may be a retinoic 
acid or a retinol and the protein may have the ability to oxidize the 
carbon occupying the 4-position of the .beta.-ionone ring of the retinoid. 
In particular, all-trans retinoids may be metabolized by proteins of the 
present invention. 
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, as is commonly known in the art, and 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. 
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. 
In another aspect, the present invention is an isolated nucleic acid 
molecule encoding a protein of the present invention. 
The present invention thus includes an isolated nucleic acid molecule 
encoding a protein having an amino acid sequence which is at least about 
30% conserved in relation to the amino acid sequence identified as SEQ ID 
NO:2 or identified as SEQ ID NO:4, or a functionally equivalent homolog 
thereof, for example, or a nucleic acid strand capable of hybridizing with 
the nucleic acid molecule under stringent hybridization conditions. Of 
course, the degree of conservation of the protein which the nucleic acid 
encodes can be higher, that is, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 
75%, 80%, 85%, 90%, 95% or more. 
Particularly, the invention is an isolated nucleic acid molecule encoding a 
protein having the ability to oxidize a retinoid at the carbon occupying 
the 4-position of the .beta.-ionone ring of the retinoid ring, and more 
particularly, having all-trans retinoic acid 4-hydroxylase activity. For 
the purposes of this invention, the term "isolated" refers to a nucleic 
acid that is substantially free of other cellular material or culture 
medium when produced by recombinant DNA techniques, or chemical precursors 
or other chemicals when produced by chemical synthesis. 
Cellular expression of preferred proteins of the present invention, 
preferred embodiments being described in more detail below, can for 
certain types of cells be induced by exposure of the cells to a retinoid, 
particularly, retinoic acid. A protein of the present invention, when 
described as being a "retinoic acid inducible protein", is a protein 
normally encoded by DNA of a cell and whose expression by that cell can be 
induced by exposure of the cell to retinoic acid. It will be appreciated 
that not every cell, even if it contains DNA encoding such a protein, 
possesses all the attributes necessary to express the protein on exposure 
to RA. It will be appreciated, however, that the DNA sequence encoding 
such a protein will occur in some proximity to a regulatory sequence which 
is necessary to cellular expression of the protein as it occurs in nature. 
That is, it is expected that RA induces expression of the gene through 
mediation of at least one regulatory element. It will be appreciated that, 
given the sequences described herein and modern genetic engineering 
techniques, a person skilled in the art would be capable of obtaining 
purified proteins of the present invention without the need for the 
regulatory sequence. In one respect, the present invention is thus a 
microbial cell containing and expressing heterologous DNA encoding a 
retinoic acid inducible protein having all-trans retinoic acid 
4-hydroxylase activity. 
The sequence of a nucleic acid molecule of the present invention can 
correspond to a part of a human genome or of a fish genome, or vary 
therefrom due to the degeneracy of the genetic code. More particularly, a 
nucleic acid molecule of the present invention can be a DNA molecule 
having the sequence identified as SEQ ID NO:3 (zP450RAI) or SEQ ID NO:5 
(hP450RAI), or the sequence can be one which varies from one of these 
sequences due to the degeneracy of the genetic code, or it can be a 
nucleic acid strand capable of hybridizing with at least one of these 
nucleic acid molecules under high or low stringency hybridization 
conditions. 
"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, 6x 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, N.Y. (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 100x Denhardt's 
solution, 2.5 ml deionized H.sub.2 O, 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.2 HPO.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 (&lt;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. 
Another aspect of this invention is isolated mRNA transcribed from DNA 
having a sequence encoding a protein of the present invention. 
In another aspect, the present invention is isolated DNA having a sequence 
according to a nucleotide sequence described above operatively linked in a 
recombinant cloning vector. In the context of this invention, the two-part 
term "operatively linked" means both that the regulatory sequence contains 
sufficient element(s) to allow expression of the nucleic acid in question 
and that the nucleic acid is linked to the regulatory sequence 
appropriately. For example, the nucleic acid of the invention is in the 
appropriate orientation and in phase with an initiation codon. The present 
invention thus includes a stably transfected cell line which expresses a 
protein having the ability to hydroxylate retinoic acid at the 4 position 
of the .beta.-ionone ring of retinoic acid. The invention includes a 
culture of cells transformed with a recombinant DNA molecule having a 
nucleic acid sequence which encodes a protein having the ability to 
hydroxylate retinoic acid at the 4 position of the .beta.-ionone ring of 
retinoic acid. 
Another aspect of the present invention is a host cell that has been 
engineered genetically to produce a protein of the invention described 
above, the cell having incorporated expressibly therein heterologous DNA 
encoding said protein. The cell may be selected such that production of 
the protein is inducible by exposing the cell to a retinoid, preferably, 
retinoic acid. The cell can be eukaryotic. 
The present invention also includes a process for producing an 
above-described protein of the invention. Such a process includes: 
preparing a DNA fragment containing a nucleotide sequence which encodes 
the protein; incorporating the DNA fragment into an expression vector to 
obtain a recombinant DNA molecule which contains the DNA fragment and is 
capable of undergoing replication; transforming a host cell with the 
recombinant DNA molecule to produce a transformant which can express the 
protein; culturing the transformant to produce the protein; and recovering 
the protein from resulting cultured mixture. 
The present invention includes an antibody to a protein of the invention. 
Here, the term "antibody" is intended to include a Fab fragment and it can 
be a monoclonal antibody. The antibody can be specifically to the amino 
acid sequence identified as SEQ ID NO:4, i.e., hP450RAI. 
The present invention includes a purified protein for use in metabolizing 
retinoic acid in an organism or cell in need of such metabolizing. 
Likewise, the invention includes a method for metabolizing retinoic acid 
in an organism or cell in need of retinoic acid metabolizing wherein the 
method includes administering a protein of the invention as described 
above. 
The invention includes a method for inhibiting retinoic acid hydroxylation 
in an organism in need of such inhibition, comprising introducing into 
cells of the organism an effective amount of an antisense RNA or 
oligonucleotide substantially complementary to at least a portion of the 
sequence identified as SEQ ID NO:5. The organism can be human and/or the 
organism can be in need of treatment against a cancerous disease. Such a 
method can include use of at least one delivery vehicle or technique 
selected from the set of viral vectors, microinjection, electroporation, 
coprecipitation, liposomes, aerosol delivery and lavage. The portion of 
the sequence may be 5 bases in length, between 5 and 50 bases in length, 5 
and 30 bases in length between 10 and 20 bases in length, or another 
suitable length may be found. The organism may be a human patient and the 
method can include treating the patient against a cancerous disease. 
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 above. 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:4, 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]. 
The invention includes a method for producing a desired protein, comprising 
providing a cell which can produce an endogenous protein in response to 
exposure to a retinoid; incorporating into DNA of the cell a DNA sequence 
encoding for the desired protein at or near a site which is normally 
occupied by a DNA sequence encoding for the endogenous protein; and 
exposing the cell to the retinoid so as to induce production of the 
desired protein. 
In another embodiment, the present invention is a kit for determining the 
presence of a protein having the ability to oxidize a retinoid, and having 
an amino acid sequence which is at least about 30% conserved in relation 
to the amino acid sequence identified as SEQ ID NO:2 or identified as SEQ 
ID NO:4, or more likely for determining the presence of a protein having 
an amino acid sequence identified as SEQ ID NO:4. The kit includes 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 another aspect, the present invention is a kit for determining the 
presence of a nucleic acid encoding a protein of invention, or a nucleic 
acid strand capable of hybridizing with the nucleic acid under stringent 
hybridization conditions, or having the sequence identified as SEQ ID NO:3 
or SEQ ID NO:5, or which varies from the sequence due to the degeneracy of 
the genetic code, or a nucleic acid strand capable or hybridizing with at 
least one said nucleic acid under stringent hybridization conditions. The 
kit includes a nucleic acid molecule capable of hybridizing with at least 
a portion of a said nucleic acid or nucleic acid strand under stringent 
conditions in which the nucleic acid molecule is linked to a reporter 
system wherein the reporter system produces a detectable response when a 
predetermined amount of the nucleic acid or nucleic acid strand and 
nucleic acid molecule are hybridized with each other. The molecule can be 
5 bases in length or longer; between 5 and 50 bases in length, 5 and 40 or 
30 bases in length, or between 10 and 20 bases in length. Of course it 
might be possible to find a more suitable base length.

DESCRIPTION OF PREFERRED EMBODIMENTS 
FIG. 1 outlines the steps used to isolate retinoid-regulated genes using 
differential display of mRNA. The cloned products isolated in step 6 of 
FIG. 1 were used for sequencing and screening of Danio rerio (D. rerio) 
cDNA libraries. P1, P2 and P3 correspond to fragments from RA induced 
mRNAs. P4 is a PCR product from a down-regulated mRNA. Details of 
procedures followed in determination of gene sequences described herein 
follow. 
Danio rerio Stocks 
D. rerio were kept at 28.5.degree. C. in 40 L tanks with 25-30 fish per 
tank on a 14 hour light-10 hour dark cycle. Tap water was conditioned by 
the addition of 10 ml of Water Conditioner (Sera Aqutan) and 10 ml of 250 
g/L Aquarium Salt (Nutra Fin) per 20 L. 2-3 L of water was changed daily. 
Amputation of fins was carried out following anaesthetization of the fish 
in a solution of 0.2% ethyl-m-aminobenzoate methanesulfonic acid (ICN) in 
conditioned water. Retinoic acid treatment was performed by adding 
all-trans RA, to a final concentration of 10.sup.-6 M, directly into the 
tank water two days following amputation. Both control- and RA-treated 
fish were kept in the dark during the experiments. 
Differential Display of mRNAs 
Differential mRNA display was performed essentially as described by Liang 
and Pardee (1992) with appropriate modifications as described herein. 
Regenerating tissues were collected 3 days post-amputation (24 hours 
post-RA addition) and quick frozen in liquid nitrogen. Poly (A).sup.+ RNA 
was isolated using the Micro Fast-Track kit. Duplicate independent reverse 
transcription reactions were performed on the isolated poly(A).sup.+ RNA 
from both the treated and untreated samples for each specific 3' poly-T 
primer used (5'-T.sub.12 VN-3'). The symbol "V" represents A or C or G and 
not Tor U. Several combinations of the 3' poly-T primers given in the 
first column of Table 1 and the upstream primers given in the second 
column were utilized for PCR amplification. For each reaction 0.1 .mu.g 
poly(A).sup.+ RNA was reverse transcribed in a 20 .mu.l reaction volume 
containing 300U Superscript Reverse Transcriptase (Gibco/BRL), 1.times. 
Buffer, 20 .mu.M each dGTP, dATP, dCTP and dTTP, 10 .mu.M dithiothreitol 
(DTT) and 5 pmol of 5'-T.sub.12 VN-3' primer. The reactions were mixed and 
incubated at 35.degree. C. for 60 minutes, followed by 5 minutes at 
95.degree. C. PCR amplification was performed in a Perkin Elmer Cetus PCR 
machine as follows: 1 .mu.l cDNA synthesis reaction, 5U Taq DNA polymerase 
(Gibco/BRL), 1.times. PCR Buffer, 2 .mu.M each DGTP, dATP, dCTP and dTTP, 
10 .mu.Ci .alpha.-[.sup.35 S]dATP (redivue, Amersham) 1.2 MM MgCl.sub.2, 
0.5 .mu.M upstream primer and 0.5 .mu.M of the corresponding 5'-T.sub.12 
VN-3' primer. PCR conditions were as follows: 1 cycle, 94.degree. C. for 5 
minutes; 40 cycles, 94.degree. C. for 30 seconds, 42.degree. C. for 1 
minute, 72.degree. C. for 30 seconds; followed by a final extension of 5 
minutes at 72.degree. C. 4 .mu.l of the PCR reactions were loaded o nto a 
6% non-denaturing polyacrylamide gel and electrophoresed at 60 watts, 
45.degree. C. The gel was dried and exposed for 12 to 24 hours on Kodak 
XAR film at room temperature. 
TABLE 1 
______________________________________ 
Sequences of the downstream Poly (T) 
oligonucleotides for the differential display 
procedure. 
3'-Poly(T) primers: 
5'-degenerate primers: 
______________________________________ 
5'-TTT.TTT.TTT.TTT.GG-3' 
5'-AAG.CGA.CCG.A-3' 
5'-TTT.TTT.TTT.TTT.GA-3'5'-TGT.TCG.CCA.G-3' 
5'-TTT.TTT.TTT.TTT.GT-3'5'-TGC.CAG.TGG.A-3' 
5'-TTT.TTT.TTT.TTT.GC-3'5'-GGC.TGC.AAA.C-3' 
5'-CCT.AGC.GTT.G-3' 
5'-TTT.TTT.TTT.TTT.AG-3' 
5'-TTT.TTT.TTT.TTT.AA-3' 
5'-TTT.TTT.TTT.TTT.AT-3' 
5'-TTT.TTT.TTT.TTT.AC-3' 
- 5'-TTT.TTT.TTT.TTT.CG-3' 
5'-TTT.TTT.TTT.TTT.CA-3' 
5'-TTT.TTT.TTT.TTT.CT-3' 
5'-TTT.TTT.TTT.TTT.CC-3' 
______________________________________ 
In Table 1, the sequences in the first column are identified as SEQ ID NOs: 
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, respectively. The 
sequences in the second column are identified as SEQ ID NOs: 24, 25, 26, 
27 and 28, respectively. 
Gel Purification and Reamplification 
Bands demonstrating reproducible differential amplifications (see FIG. 2a) 
were found for the upstream-downstream primer combination of 
5'-TGCCAGTGGA-3'-poly-T primer, 5'-TTT TTT TTT TTT AG-3' (SEQ ID NOs: 26 
and 16, respectively). These bands were excised from the gel by overlaying 
the X-ray film and cutting out the corresponding piece of dried gel and 
filter paper. The PCR product corresponding to a fragment of the protein 
described herein was isolated from the band in FIG. 2(a). Samples were 
placed in 100 .mu.l of nuclease free water, incubated for 10 minutes at 
room temperature, then boiled for 15 minutes. The supernatant was 
recovered following a 15 minute centrifugation at 12,000.times.g. 
In order to facilitate cloning of the PCR products, several changes were 
made to the reactions. Primers which included Eagl restriction 
endonuclease sites were used in the reamplification. Based on results 
obtained in the differential display analysis, the upstream 
5'-TGCCAGTGGA-3' primer was replaced by 5'-GTAGCGGCCGCTGCCAGTGGA-3' (SEQ 
ID NO: 29) and the downstream poly-T primer, 5'-TTT TTT TTT TTT AG-3', was 
replaced by 5'-GTAGCGGCCGCT.sub.12-3 ' (SEQ ID NO:30). In addition, the 
reaction volume was increased to 40 .mu.l, isotope was omitted and 20 as 
opposed to 40 cycles were performed. 5 .mu.l aliquots of the PCR reactions 
were removed and the products were visualized by electrophoresis in a 1% 
agarose gel followed by ethidium bromide staining and UV illumination. 
Cloning PCR Products 
The reamplified products were purified by phenol/chloroform extraction 
followed by ethanol precipitation. The resulting DNA pellet was 
resuspended in 17 .mu.l of sterile water and digested at 37.degree. C. for 
1 hour by the inclusion of 10U Eagl (New England Biolabs), and 1.times. 
NEB 3 buffer. Eagl restriction endonuclease was heat inactivated by 
incubation at 65.degree. C. for 20 minutes. pBluescript SK.sup.+ vector 
was prepared by digestion with Eagl, followed by dephosphorylation using 
calf intestinal alkaline phosphatase (CAP, Promega). Restriction digests 
were purified using the GeneClean II Kit (Bio 101) following 
electrophoresis in a 1% agarose gel. In a total ligation volume of 10 
.mu.l, 2 .mu.l of digested PCR product, 1 .mu.l digested SK.sup.+, 1U T4 
DNA ligase (Gibco/BRL) and 1.times. buffer were incubated at 16.degree. C. 
overnight. E. coli bacterial strain JM109 was transformed with 1 .mu.l of 
the ligation product using the BioRad Gene Pulser, then plated on 
LB+ampicillin plates and incubated overnight at 37.degree. C. 
Colony Selection 
Individual colonies were transferred in duplicate to fresh LB plates and 
grown overnight at 37.degree. C. Colonies were transferred to 
nitrocellulose membrane and denatured in a solution of 1.5M NaCl, 0.5M 
NaOH for 5 minutes, neutralized in 1.5M NaCl, 0.5M Tris-HCl, pH 8.0 for 5 
minutes, followed by two 5 minute washes in 2.times. SSC. Membranes were 
then UV cross-linked (Stratalinker UV Crosslinker, Stratagene). 
Prehybridization and hybridization were performed using Quickhyb 
(Stratagene) following the manufacturer's directions. Each colony lift was 
probed with the corresponding PCR product isolated during the gel 
reamplification and purification step. .alpha.-[.sup.32 P]-dATP labelled 
probes were generated using the Prime-It Kit II (Stratagene). Subsequent 
to hybridization, filters were washed twice for 20 minutes in 2.times. 
SSC, 0.1% SDS solution at room temperature and exposed to Kodak X-omat 
autoradiography film overnight at -70.degree. C. Positive colonies were 
selected from the duplicate plates, grown overnight in LB+ampicillin (100 
.mu.g/ml) and plasmid DNA isolated using the Qiaprep Spin Plasmid Kit 
(Qiagen). 
Clones were sequenced using the T7 Sequencing Kit (Pharmacia Biotech). 
Sequence comparisons were generated using the GeneWorks software package 
(Intelligenetics). 
Screening of a D. rerio cDNA Library 
A random primed D. rerio 6-18 hour embryo cDNA library constructed in 
Uni-ZAP II (Stratagene) was produced. 4.5.times.10.sup.5 independent pfu 
were screened using the random primed, .alpha.-[.sup.32 P]-dATP labelled 
337 bp PCR fragment isolated by mRNA differential display as a probe. 
Filters were prehybridized for 1-4 hours at 42.degree. C. in 50% 
formamide, 5.times. SSPE, 1.times. Denhardt's solution, 0.2 mg/ml 
denatured salmon sperm DNA. Hybridization was performed at 42.degree. C. 
by adding denatured probe to the prehybridization solution. Filters were 
washed two times for 20 minutes in 2.times. SSC, 0.05% SDS at room 
temperature and exposed to Kodak XAR film overnight at -70.degree. C. 
Positive plaques were picked into 500 .mu.l SM buffer and subjected to 
additional rounds of rescreening until purified. Positive plaques were 
exposed to the in vivo excision protocol following the manufacturer's 
directions (Stratagene). pBluescript containing colonies were plated onto 
LB+amp plates and grown overnight at 37.degree. C. Sequence data were 
generated using the T7 Sequencing Kit (Pharmacia) and analysed using the 
GeneWorks software package (Intelligenetics). 
Whole Mount in situ hybridization 
RA- and DMSO-treated regenerates were isolated 72 hours post-amputation (24 
hours post RA/DMSO addition), washed in PBS and prepared for whole mount 
in situ hybridization. In situ hybridizations were undertaken as 
previously described [White, 1994]. 
Northern Blot Analysis 
Fish were allowed to regenerate their caudal fins for 72 hours. At 48 hours 
10.sup.-6 M all-trans RA in DMSO vehicle or DMSO alone was added directly 
to the tank water. mRNA was prepared using the Micro Fast-Track mRNA 
isolation kit (Invitrogen, California) according to the manufacturer's 
directions. 3.0-5.0 .mu.g poly A.sup.+ RNA was electrophoresed, blotted 
and probed using a previously described method [White, 1994] with the full 
length zP450RAI cDNA according obtained as described below. Ethidium 
bromide stained agarose gel showed that equivalent amounts of mRNA were 
used in the blotting experiments. See lanes 2 and 3 of FIG. 3(a). 
HPLC Analysis 
Media from transfected cells incubated with 575 pM [11,12-.sup.3 H]RA 
(FIGS. 4(a) and 4(b)) or 1 .mu.M RA (FIGS. 4(c) and 4(d)) for either 4 hrs 
(FIGS. 4(a) and 4(c)) or 24 hrs (FIGS. 4(b) and 4(d)) were acidified with 
0.1% acetic acid. Lipid soluble metabolites were separated from aqueous 
soluble metabolites using a total lipid extraction of the medium [Bligh, 
1957]. Metabolism of [11,12-.sup.3 H]RA to total aqueous soluble 
metabolites was measured using aliquots of the aqueous soluble extract 
subjected to .beta.-scintillation counting (See the insets of FIGS. 4(a) 
and 4(b)). Lipid soluble extracts were evaporated to dryness under a 
stream of nitrogen and resuspended in 93.5/5/1/0.5 
hexane/isopropanol/methanol/acetic acid (H/I/M/AA). Metabolites were 
separated by HPLC using a Zorbax-SIL (3.mu., 8.times.0.62 cm) column 
eluted with a solvent system of 93.5/5/1/0.5 H/I/M/AA at a flow rate of 1 
ml/min. 
EXAMPLE 1 
Characterization of a Novel Cytochrome P450 
Transcripts present in fin tissue regenerating in the presence or absence 
of RA were compared using the differential display PCR technique developed 
by Liang and Pardee [Liang, 1992] (FIG. 2(a). One of the differential 
display products which exhibited a dependence on the presence of RA for 
its expression, indicated by the arrow in FIG. 2(a), was isolated and 
sequenced. The sequence is identified as SEQ ID NO:1 and is also shown in 
FIG. 2(b). The amino acid sequence corresponding to the cDNA, termed here, 
"zP450RAI", is shown in FIG. 2(c) and identified as SEQ ID NO:2. BLAST 
search analyses revealed sequence homology between zP450RAI and multiple 
members of the cytochrome P450 superfamily. Alignments between zP450RAI 
cDNA deduced amino acid sequence and those of other cytochrome P450s 
indicated that zP450RAI exhibited less than 30% overall amino acid 
identity with members of previously defined subfamilies [Nelson, 1993]. 
zP450RAI contains many of the structural motifs which are common to 
cytochrome P450 family members, including the heme-binding domain located 
in the C-terminal portion of the protein. See FIG. 2(d). 
EXAMPLE 2 
Cell Specific Induction of zP450RAI by All-trans RA 
Northern blot analysis of mRNAs expressed in regenerate tissue isolated 
from control (dimethyl sulfoxide-treated) and RA-treated fish was 
performed with a full-length zP450RAI cDNA probe. zP450RAI transcripts 
were not detectable in regenerate tissue from control fish (FIG. 3(a), 
lane 4) but were very noticeably present in tissues isolated from fish 
exposed to RA for 24 hours (FIG. 3(a), lane 5). 
Whole mount in situ hybridization was used to determine the cellular 
localization of zP450RAI expression in regenerating fin tissue. FIG. 3(b) 
shows regenerating fins from control and RA-treated fish. zP450RAI 
transcripts are not detectable in control fin tissue (FIG. 3(b)(i)). In 
regenerating tissue from RA-treated fish, zP450RAI transcripts were found 
to be abundant in a layer of epithelial cells extending across the distal 
edge of the wound epithelium as indicated by the black arrowhead in FIG. 
3(b)(ii). Some low level staining was also observed in inter-ray tissue as 
indicated by the black line with arrowhead in FIG. 3(b)(ii). A 
histological section of an RA-treated fin, taken along the line shown in 
FIG. 3(b)(iii), is shown in FIG. 3(b)(iv). The section indicates that 
cells expressing zP450RAI are located deep within the epithelial layer at 
the distal tip of the blastemal mesenchyme. 
EXAMPLE 3 
Metabolism of All-trans RA by zP450RAI Transfected Cells 
Retinoic acid as a substrate of zP450RAI was studied. The full-length 
zebrafish zP450RAI cDNA was cloned into the eukaryotic expression vector 
pSG5 [Green, 1988]. COS-1 cells were transiently transfected with either 
pSG5 or pSG5-zP450RAI and then incubated with either picomolar 
concentrations of [11,12-.sup.3 H]all-trans-RA or micromolar 
concentrations of non-radioactive all-trans-RA. COS-1 cells are an African 
green monkey kidney "fibroblast-like" cell line. zP450RAI expression in 
COS-1 cells promoted the rapid conversion of RA into both lipid- and 
aqueous-soluble metabolites. See FIGS. 4(a) and 4(b). Fractions of total 
lipid extracts of transfected cells were initially separated by 
normal-phase HPLC on Zorbax-SIL. Comparison between extracts from pSG5 and 
pSG5-zP450RAI-transfected cells indicated that zP450RAI significantly 
increased RA metabolism. Incubation of zP450RAI-transfected cells with 575 
pM [11,12-.sup.3 H]all-trans-RA for either 4 or 24 hours resulted in 
accumulation of RA metabolites, one of which co-migrated on a column with 
synthetic standards 4-OH-RA and 18-OH-RA, and a second slightly less polar 
metabolite which co-migrated with 4-oxo-RA standard (FIGS. 4(a) and 4(b)). 
Rechromatography of RA metabolites using other HPLC systems confirmed the 
identity of these two metabolites as 4-OH-RA and 4-oxo-RA (Table 2). It is 
possible that the aqueous-soluble radioactivity represents glucuronides of 
RA metabolites or glucuronides of RA itself. Rapid glucuronidation of 4- 
and 18-hydroxy-RA in mammalian cell extracts has been reported by others 
[Wouters, 1992; Takatsuka, 1996]. 
TABLE 2 
______________________________________ 
Chromatographic properties of RA metabolites. 
Retention Time (min) 
Metabolite Z-Sil.sup.a Z-CN.sup.b Z-ODS.sup.c 
______________________________________ 
RA (std) 2.57 4.47 19.92 
4-oxo-RA (std) 4.79 11.33 11.73 
4-OH-RA (std) 5.17 9.65 12.65 
18-OH-RA (std) 5.06 9.53 14.03 
Peak 1 (RA) 2.57 4.48 19.73 
Peak 2 (4-oxo-RA) 4.87 11.38 11.57 
Peak 3 (4-OH-RA) 5.16 9.68 12.68 
______________________________________ 
.sup.a HPLC conditions: ZorbaxSIL column eluted with 93.5/5/1/0.5 
H/I/M/A.A. (1 ml/min) 
.sup.b HPLC conditions: ZorbaxCN column eluted with 93.5/5/1/0.5 H/I/M/A. 
(1 ml/min) 
.sup.c HPLC conditions: ZorbaxODS column eluted with a 20 min linear 
gradient with solvent containing 10 mM ammonium acetate which ranged from 
55.45 to 5.95 H.sub.2 O/MeOH (2 ml/min). 
A similar pattern of zP450RAI-dependent metabolism was also observed using 
a much higher RA concentration (1 .mu.M). zP450RAI-transfected COS-1 cells 
incubated for 4 or 24 hours with 1 .mu.M RA generated two closely-running 
peaks which were discernible in a 350nm HPLC trace shown in FIGS. 4(c) and 
4(d), but which were essentially undetectable in control pSG5-transfected 
cells (See the insets of FIGS. 4(c) and 4(d)). These peaks co-migrated 
with those of 4-oxo-RA and 4-OH-RA standards, respectively. Diode array 
spectrophotometric detection of the zP450RAI-generated peaks showed that 
the spectral properties of the two metabolite peaks matched the standard 
retinoids [In hexane-based solvents: 4-OH-RA, .lambda..sub.max =350 nm; 
4-oxo-RA, .lambda..sub.max =355 nm; in methanol-based solvents: 4-OH-RA, 
.lambda..sub.max =340 nm; 4-oxo-RA, .lambda..sub.max =360 nm]. 
The invention thus includes a retinoic acid metabolizing protein belonging 
to the family of cytochrome P450s and generation of the protein in 
zebrafish caudal fin wound epithelium being induced in response to RA 
treatment. While RA metabolizing activity has previously been detected in 
epithelial tissues of several species [Frolik, 1979; Roberts, 1979; 
Wouters, 1992; Duell, 1992], an actual enzyme responsible for such 
activity has heretofore been unknown. 
zP450RAI is up-regulated by RA treatment and apparently this up-regulation 
occurs in a specific set of cells in the wound epithelium of regenerating 
zebrafish caudal fins. 
It might be of relevance to the regulation of the generation of this enzyme 
in vivo that experiments with F9 cells where RARs have been selectively 
ablated indicate that RAR-.alpha., and RAR-.gamma. might have a role in 
the regulation of RA metabolism [Boylan, 1995]. The expression of both 
RAR-.alpha. and RAR-.gamma. in the regenerating caudal fin is consistent 
with the possibility that they may be involved in the regulation of 
P450RAI expression by RA [White, 1994]. 
EXAMPLE 4 
Cloning of Human P450RAI 
The amino acid sequence corresponding to the DNA of zebrafish P450RAI 
(zP450RAI) (SEQ ID NO:2) was used to search an express sequence tag (EST) 
database. A commercially available EST clone (SEQ ID NO:11) having a high 
degree of homology with a C-terminal portion of the zP450RAI (from Glu 293 
to Phe 411 of SEQ ID NO:2) was purchased (Research Genetics, Huntsville, 
Ala.). The clone is reportedly from a human infant brain cDNA library 
(Bento Soares and M. Fatima Bonaldo) and is apparently otherwise 
unpublished. The purchased clone was sequenced using the T7 sequencing kit 
(Pharmacia) and sequence data was generated using the Geneworks Software 
Package (Intelligenetics). 
A cDNA library generated from an NT2 cell line treated with retinoic acid 
is commercially available (Stratagene, cat#939231) and this product was 
used for further studies. The cDNA library was probed with a nucleic acid 
having a sequence identified as SEQ ID NO:11. Eleven positively 
hybridizing clones were isolated and purified according to the 
manufacturer's directions. Sequence data for these clones were generated 
using the T7 Sequencing Kit (Pharmacia) and analyzed using the Geneworks 
package (Intelligenetics). The human DNA sequence is identified as SEQ ID 
NO:5 and the corresponding polypeptide as SEQ ID NO:4. FIG. 9 shows 
aligned portions of the amino acid sequence of the zebrafish protein (SEQ 
ID NO:2) with the amino acid sequence of the human protein (SEQ ID NO:4). 
EXAMPLE 5 
Transient Tranfection Analysis 
COS-1 cells were subcultured 20 hours prior to transfection which was 
carried out according to the standard DEAE-dextran method [Sambrook, 1989 
Maniatis, 1982]. Cells were transfected with pE-AR (adrenodoxin expression 
vector, 1 .mu.g/P100 plate) and pE-ADX (adrenodoxin reductase expression 
vector, 1 .mu.g/P100 plate) together with 3 .mu.g per plate of either pSG5 
(control) or hP450RAI-pSG5 (experimental). [11,12-.sup.3 H]all-trans 
retinoic acid (60,000 cpm per plate) was added 24 hours after 
transfection. Analyses were carried out as described in Example 3 and 
results obtained are shown in FIGS. 10 and 11(a) to 11(d). As indicated in 
the Figures, hP450RAI expression in COS-1 cells promoted conversion of RA 
into 4-OH-RA and 4-oxo-RA. Total amounts of 4-oxo-RA and 4-OH-RA produced 
in the transfected cells in comparison to amounts produced in the control 
cells are shown in FIGS. 11(a) and (b), respectively. Overall, greater 
amounts of aqueous soluble metabolites were produced in the transfected 
cells (FIG. 11 (c)) and greater amounts of unmetabolized RA were found in 
control cells (FIG. 11 (d)). 
The clone sequence (SEQ ID NO:11) was prepared as a .sup.32 [P]-dATP 
labeled probe to study the inducibility of hP450RAI by RA in several cell 
lines: HEK293; EL-E; HL-60; MCF10A; LC-T; SK-LC6; MCF7; U937; HepG2; NT2 
(See FIGS. 5 to 7). As can be seen, a variety of expression patterns were 
observed. The SK-LC6 human lung (epithelial) line appeared to 
constitutively express corresponding mRNA. There was apparently some 
increase in expression in the HEK293 (human embryonic kidney), LC-T (human 
lung epithelial), HepG2 (human liver, epithelial in morphology), NT2 
(pluripotent human embryonic carcinoma) and U937 (human monomyelocytes) 
cell lines in response to addition of RA. There was a large dependence on 
exposure to RA in the MCF7 (human breast carcinoma (epithelial)) cell 
line. Some cell lines showed no expression in the absence or presence of 
RA: EL-E; HL-60 and MCF10A. 
The .sup.32 [P]-dATP labeled probe was also used to study hP450RAI mRNA 
expression in a human acute promyelocytic leukemia cell line. Experiments 
were carried out using the NB4 cell line, isolated from a human acute 
promyelocytic leukemia patient, and three retinoic acid resistant cell 
lines were independently derived from NB4. Results are shown in FIG. 8. As 
can be seen, the normal cells expressed hP450RAI mRNA after treatment with 
10.sup.-6 M RA, while such expression appeared to be absent for the other 
cell lines both in the absence and presence of RA. 
Analysis of metabolites of MCF10A and MCF7 cell lines exposed to RA was 
carried out, MCF10A cells having displayed no expression of mRNA and 
latter having displayed a large dependence of mRNA expression on exposure 
to RA. The results are shown in FIGS. 12(a) to 12(c). Consistent with the 
results shown in FIG. 5, the results shown in FIG. 12(a) indicate there 
was little difference in the lipid soluble activity profiles of the MCF10A 
cell line exposed to RA and the control. The last two bars of FIG. 12 (c) 
indicate that total aqueous soluble metabolites were about the same for 
both the induced and control MCF10A cells. As indicated in FIG. 12(b), the 
MCF7 cell line exposed to RA had an elution profile which indicated 
significantly greater concentrations of 4-OH-RA and 4-oxo-RA than the same 
cell line not exposed to RA. FIG. 12(c) indicates that the amount of total 
aqueous soluble metabolites of the MCF7 cells exposed to RA was much 
greater than that for the control cells. Again, these results are 
consistent with those obtained in the blotting results shown in FIG. 5 for 
the MCF7 cell line. 
A 1.3 kb cDNA of hP450RAI was mapped using a P-1 derived artificial 
chromosome () library. Mapping of the cDNA and genomic clone was 
performed by fluorescence in situ hybridization [Lichter, 1990] to normal 
human lymphocyte chromosomes counterstained with propidium iodide and 
DAPI. Biotinylated probe was detected with avidin-fluorescein 
isothiocyanate (FITC). Images of metaphse preparations were captured by a 
thermoelectrically cooled charge coupled camera (Photometrics, Tucson, 
Ariz.). Separate images of DAPI banded chromosomes [Heng, 1993] and FITC 
targeted chromosomes were obtained. Hybridization signals were aequired 
and merged using image analysis software and pseudo colored blue (DAPI) 
and yellow (FTIC) [Boyle, 1992] and overlaid electronically. 
Positive hybridization signals were found to be localized to 10q23-24. The 
band assignment was determined by measuring the fractional chromosome 
length and by analyzing the banding pattern generated by the DAPI 
counterstained image. 
Genomic sequences can thus be sequenced. Oligonucleotides for use as 
primers are synthesized according to the DNA sequence of hP450RAI. These 
are then used to generate further primers corresponding to genomic DNA 
flanking hP450RAI and the complete sequence of the genomic locus 
determined. 
It is possible to compare the zP450RAI and hP450RAI sequences described 
above. Of the 492 amino acids of zP450RAI (SEQ ID NO:2), it is possible to 
align 334 amino acids with the 497 amino acids of hP450RAI (SEQ ID NO:4). 
See FIG. 9. On this basis, there is about 68% homology between the human 
and fish proteins. The degree of homology between the two amino acid 
sequences is slightly greater towards the C-terminus than in the 
N-terminus region. It also appears as though nucleic acid sequences 
encoding the conserved sequence Met-Lys-Arg-Gln-Lys (amino acid numbers 70 
to 74 of zP450RAI) can be used as a probe to obtain corresponding proteins 
from cDNA libraries of other species. 
It has also been found by the present inventors (results not shown) that RA 
can induce mRNA transcripts which cross hybridize with a P450RAI cDNA 
probe in either of the F9 and P19 mouse cell lines having 4-hydroxylase 
activity, as described by Blumberg et al. [Blumberg et al., 1995; Achkar 
et al., 1996]. 
As mentioned above, RA-induced expression of a protein by the cells 
described herein involves a regulatory sequence which is located upstream 
of the coding sequence of DNA that it controls. In the case of preferred 
embodiments described so far, the protein has been P450RAI, whether in 
cells of the zebrafish, human or other organism. Such a cell can be 
modified by incorporating DNA encoding a different protein into the region 
of the gene which encodes P450RAI. An approach very likely to succeed 
involves excision of the P450RAI DNA and replacement thereof with the 
different coding sequence. In this way, a cellular system for producing 
proteins that is inducible by exposure to a retinoid, preferably RA, is 
obtained. It may be that the regulatory sequence is directly responsive to 
the presence of RA, causing mRNA to be produced de novo with subsequent 
translation thereof into the protein. In such case it is possible to 
incorporate the regulatory DNA sequence operably linked to a 
protein-encoding sequence into a conventional genetically engineered 
protein-producing cell and induce the production of the desired protein by 
exposure of the cell to RA. 
RNA antisense sequences (nucleic acids or oligonucleotides) that inhibit 
cellular RA-induced P450RAI production can be used to inhibit metabolism 
of RA by P450RAI [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 hP450RAI can thus be used to design a 
series of oligonucleotides which together cover the 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. This can be done by exposing cells to the various 
oligonucletides and measuring subsequent changes in hP450 activity. 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. 
More directly, use of suitable antibodies that bind to the P450RAI protein 
so as to inhibit binding of RA would reduce RA metabolism by P450RAI. 
Other approaches involving inhibition of P450RAI action by might be more 
preferable. 
The present invention thus includes a method of screening drugs for their 
effect on activity of a retinoic acid inducible protein. The method 
includes exposing the protein to a prospective inhibitor 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, hP450RAI 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 hP450RAI 
over other P450s could be identified. 
Another system for screening for potential inhibitors of a P450RAI 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, 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 inducement 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 inducement which is indicated by the reporter protein. 
Exposure of the cells to RA in the presence of an agent that inhibits 
P450RAI 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 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 P4540RAI protein. 
There is the possibility that cellular retinoic acid-binding protein 
(CRABP) [Adamson, 1993] is involved in binding of a retinoid substrate to 
a P450RAI 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. 
It will of course be understood, without the intention of being limited 
thereby, that a variety of substitutions of amino acids is possible while 
preserving the structure responsible for retinoid metabolizing acitivity 
of the proteins disclosed herein. Conservative substitutions are described 
in the patent literature, as for example, in U.S. Pat. No. 5,2264,558. 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 of a variant protein with a naturally occuring protein, the 
greater the retention of metabolic activity. 
Also, an antibody can be linked to or conjugated with a reporter system 
which is set up to indicate positively binding of the protein to the 
antibody. Well known reporter systems include radioimmuno assays (RIAs) or 
immunoradiometric assays (IRMAs). Alternatively, an enzyme-linked 
immunosorbent assay (ELISA) would have in common with RIAs and IRMAs a 
relatively high degree of sensitivity, but would generally not rely upon 
the use of radioisotopes. A visually detectable substance may be produced 
or at least one detectable in a spectrophotometer. An assay relying upon 
fluroescence of a substance bound by the enzyme being assayed could be 
used. It will be appreciated that there are a number of reporter systems 
which may be used, according to the present invention, to detect the 
presence of a particular protein. With standardized sample collection and 
treatment, protein presence above a threshold amount in blood serum could 
well be determined. 
Such an antibody-linked reporter system could be used in a method for 
determining whether a fluid sample of a subject contains a deficient 
amount or an excessive amount of the protein. Given a normal threshold 
concentration of such a protein for a given type of subject, test kits 
could thus be developed. 
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 with a sequence coding for 
the C-terminal portion of E. coli .beta.-galactosidase to produce a fusion 
protein, for example. 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. 
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__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 30 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 337 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - TGCCAGTGGA CAATCTCCCT ACCAAATTCA CTAGTTATGT CCAGAAATTA GC - 
#CTAAACCG 60 
- - GAGCCTTTGT ACATATGTTT TTATTTTAGA TGAACTGTGA TGTATTGGAT AT - 
#TTTCTAAT 120 
- - TTGTTTATAT AAAGCAGATG TGTATATAAG TCTATGCGAA GAAGCGAAAA CG - 
#AGGGCACT 180 
- - ACTTTCTCAT GGATCACTGT AATGCTACAG AGTGTCTGTG ATGTATATTT AT - 
#AATGTAGT 240 
- - TGTGTCATAT AGCTTTTGTA CTGTATGCAA CTTATTTAAC TCGCTCTTTA TC - 
#TCATGGGT 300 
- - TTTATTTAAT AAAACATGTT CTTACAAAAA AAAAAAA - # 
- # 337 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 492 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - Met Gly Leu Tyr Thr Leu Met Val Thr Phe Le - #u Cys Thr Ile Val Leu 
1 5 - # 10 - # 15 
- - Pro Val Leu Leu Phe Leu Ala Ala Val Lys Le - #u Trp Glu Met Leu Met 
20 - # 25 - # 30 
- - Ile Arg Arg Val Asp Pro Asn Cys Arg Ser Pr - #o Leu Pro Pro Gly Thr 
35 - # 40 - # 45 
- - Met Gly Leu Pro Phe Ile Gly Glu Thr Leu Gl - #n Leu Ile Leu Gln Arg 
50 - # 55 - # 60 
- - Arg Lys Phe Leu Arg Met Lys Arg Gln Lys Ty - #r Gly Cys Ile Tyr Lys 
65 - #70 - #75 - #80 
- - Thr His Leu Phe Gly Asn Pro Thr Val Arg Va - #l Met Gly Ala Asp Asn 
85 - # 90 - # 95 
- - Val Arg Gln Ile Leu Leu Gly Glu His Lys Le - #u Val Ser Val Gln Trp 
100 - # 105 - # 110 
- - Pro Ala Ser Val Arg Thr Ile Leu Gly Ser As - #p Thr Leu Ser Asn Val 
115 - # 120 - # 125 
- - His Gly Val Gln His Lys Asn Lys Lys Lys Al - #a Ile Met Arg Ala Phe 
130 - # 135 - # 140 
- - Ser Arg Asp Ala Leu Glu His Tyr Ile Pro Va - #l Ile Gln Gln Glu Val 
145 1 - #50 1 - #55 1 - 
#60 
- - Lys Ser Ala Ile Gln Glu Trp Leu Gln Lys As - #p Ser Cys Val Leu 
Val 
165 - # 170 - # 175 
- - Tyr Pro Glu Met Lys Lys Leu Met Phe Arg Il - #e Ala Met Arg Ile Leu 
180 - # 185 - # 190 
- - Leu Gly Phe Glu Pro Glu Gln Ile Lys Thr As - #p Glu Gln Glu Leu Val 
195 - # 200 - # 205 
- - Glu Ala Phe Glu Glu Met Ile Lys Asn Leu Ph - #e Ser Leu Pro Ile Asp 
210 - # 215 - # 220 
- - Val Pro Phe Ser Gly Leu Tyr Arg Gly Leu Ar - #g Ala Arg Asn Phe Ile 
225 2 - #30 2 - #35 2 - 
#40 
- - His Ser Lys Ile Glu Glu Asn Ile Arg Lys Ly - #s Ile Gln Asp Asp 
Asp 
245 - # 250 - # 255 
- - Asn Glu Asn Glu Gln Lys Tyr Lys Asp Ala Le - #u Gln Leu Leu Ile Glu 
260 - # 265 - # 270 
- - Asn Ser Arg Arg Ser Asp Glu Pro Phe Ser Le - #u Gln Ala Met Lys Glu 
275 - # 280 - # 285 
- - Ala Ala Thr Glu Leu Leu Phe Gly Gly His Gl - #u Thr Thr Ala Ser Thr 
290 - # 295 - # 300 
- - Ala Thr Ser Leu Val Met Phe Leu Gly Leu As - #n Thr Glu Val Val Gln 
305 3 - #10 3 - #15 3 - 
#20 
- - Lys Val Arg Glu Glu Val Gln Glu Lys Val Gl - #u Met Gly Met Tyr 
Thr 
325 - # 330 - # 335 
- - Pro Gly Lys Gly Leu Ser Met Glu Leu Leu As - #p Gln Leu Lys Tyr Thr 
340 - # 345 - # 350 
- - Gly Cys Val Ile Lys Glu Thr Leu Arg Ile As - #n Pro Pro Val Pro Gly 
355 - # 360 - # 365 
- - Gly Phe Arg Val Ala Leu Lys Thr Phe Glu Le - #u Asn Gly Tyr Gln Ile 
370 - # 375 - # 380 
- - Pro Lys Gly Trp Asn Val Ile Tyr Ser Ile Cy - #s Asp Thr His Asp Val 
385 3 - #90 3 - #95 4 - 
#00 
- - Ala Asp Val Phe Pro Asn Lys Glu Glu Phe Gl - #n Pro Glu Arg Phe 
Met 
405 - # 410 - # 415 
- - Ser Lys Gly Leu Glu Asp Gly Ser Arg Phe As - #n Tyr Ile Pro Phe Gly 
420 - # 425 - # 430 
- - Gly Gly Ser Arg Met Cys Val Gly Lys Glu Ph - #e Ala Lys Val Leu Leu 
435 - # 440 - # 445 
- - Lys Ile Phe Leu Val Glu Leu Thr Gln His Cy - #s Asn Trp Ile Leu Ser 
450 - # 455 - # 460 
- - Asn Gly Pro Pro Thr Met Lys Thr Gly Pro Th - #r Ile Tyr Pro Val Asp 
465 4 - #70 4 - #75 4 - 
#80 
- - Asn Leu Pro Thr Lys Phe Thr Ser Tyr Val Ar - #g Asn 
485 - # 490 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1850 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - TGTCGCCGTT GCTGTCGGTT GCTGTCGGAC GCTGTCTCCT CTCCAGAAGC TT - 
#GTTTTTCG 60 
- - TTTTGGCGAT CAGTTGCGCG CTTCAAC ATG GGG CTG TAC ACC - #CTT ATG GTC 
ACC 114 
- # Met Gly Leu Ty - #r Thr Leu Met Val 
Thr 
- # 1 - # 5 
- - TTT CTC TGC ACC ATC GTG CTA CCC GTT TTA CT - #C TTT CTC GCC GCG 
GTG 162 
Phe Leu Cys Thr Ile Val Leu Pro Val Leu Le - #u Phe Leu Ala Ala Val 
10 - #15 - #20 - #25 
- - AAG TTG TGG GAG ATG TTA ATG ATC CGA CGA GT - #C GAT CCG AAC TGC AGA 
210 
Lys Leu Trp Glu Met Leu Met Ile Arg Arg Va - #l Asp Pro Asn Cys Arg 
30 - # 35 - # 40 
- - AGT CCT CTA CCG CCA GGT ACC ATG GGC TTG CC - #G TTC ATT GGA GAA ACG 
258 
Ser Pro Leu Pro Pro Gly Thr Met Gly Leu Pr - #o Phe Ile Gly Glu Thr 
45 - # 50 - # 55 
- - CTC CAG CTG ATC CTC CAG AGA AGG AAG TTT CT - #G CGC ATG AAA CGG CAG 
306 
Leu Gln Leu Ile Leu Gln Arg Arg Lys Phe Le - #u Arg Met Lys Arg Gln 
60 - # 65 - # 70 
- - AAA TAC GGG TGC ATC TAC AAG ACG CAC CTC TT - #C GGG AAC CCG ACT GTC 
354 
Lys Tyr Gly Cys Ile Tyr Lys Thr His Leu Ph - #e Gly Asn Pro Thr Val 
75 - # 80 - # 85 
- - AGG GTG ATG GGA GCT GAT AAT GTG AGG CAG AT - #T CTG CTG GGC GAA CAC 
402 
Arg Val Met Gly Ala Asp Asn Val Arg Gln Il - #e Leu Leu Gly Glu His 
90 - #95 - #100 - #105 
- - AAG CTG GTG TCT GTT CAG TGG CCA GCA TCA GT - #G AGA ACC ATC CTG GGC 
450 
Lys Leu Val Ser Val Gln Trp Pro Ala Ser Va - #l Arg Thr Ile Leu Gly 
110 - # 115 - # 120 
- - TCT GAC ACC CTC TCC AAT GTC CAT GGA GTT CA - #A CAC AAA AAC AAG AAA 
498 
Ser Asp Thr Leu Ser Asn Val His Gly Val Gl - #n His Lys Asn Lys Lys 
125 - # 130 - # 135 
- - AAG GCC ATT ATG AGG GCG TTC TCT CGA GAT GC - #T CTG GAG CAC TAC ATT 
546 
Lys Ala Ile Met Arg Ala Phe Ser Arg Asp Al - #a Leu Glu His Tyr Ile 
140 - # 145 - # 150 
- - CCC GTG ATC CAG CAG GAG GTG AAG AGC GCC AT - #A CAG GAA TGG CTG CAA 
594 
Pro Val Ile Gln Gln Glu Val Lys Ser Ala Il - #e Gln Glu Trp Leu Gln 
155 - # 160 - # 165 
- - AAA GAC TCC TGC GTG CTG GTT TAT CCA GAA AT - #G AAG AAA CTC ATG TTT 
642 
Lys Asp Ser Cys Val Leu Val Tyr Pro Glu Me - #t Lys Lys Leu Met Phe 
170 1 - #75 1 - #80 1 - 
#85 
- - CGG ATA GCT ATG AGA ATC CTG CTT GGT TTT GA - #A CCA GAG CAA ATA 
AAG 690 
Arg Ile Ala Met Arg Ile Leu Leu Gly Phe Gl - #u Pro Glu Gln Ile Lys 
190 - # 195 - # 200 
- - ACG GAC GAG CAA GAA CTG GTG GAA GCT TTT GA - #G GAA ATG ATC AAA AAC 
738 
Thr Asp Glu Gln Glu Leu Val Glu Ala Phe Gl - #u Glu Met Ile Lys Asn 
205 - # 210 - # 215 
- - TTG TTC TCC TTG CCA ATC GAC GTT CCT TTC AG - #T GGT CTG TAC AGG GGT 
786 
Leu Phe Ser Leu Pro Ile Asp Val Pro Phe Se - #r Gly Leu Tyr Arg Gly 
220 - # 225 - # 230 
- - TTG AGG GCA CGC AAT TTC ATT CAC TCC AAA AT - #T GAG GAA AAC ATC AGG 
834 
Leu Arg Ala Arg Asn Phe Ile His Ser Lys Il - #e Glu Glu Asn Ile Arg 
235 - # 240 - # 245 
- - AAG AAA ATT CAA GAT GAC GAC AAT GAA AAC GA - #A CAG AAA TAC AAA GAC 
882 
Lys Lys Ile Gln Asp Asp Asp Asn Glu Asn Gl - #u Gln Lys Tyr Lys Asp 
250 2 - #55 2 - #60 2 - 
#65 
- - GCC CTT CAG CTG TTG ATC GAG AAC AGC AGA AG - #A AGT GAC GAA CCT 
TTT 930 
Ala Leu Gln Leu Leu Ile Glu Asn Ser Arg Ar - #g Ser Asp Glu Pro Phe 
270 - # 275 - # 280 
- - AGT TTG CAG GCG ATG AAA GAA GCA GCT ACA GA - #G CTT CTA TTT GGA GGT 
978 
Ser Leu Gln Ala Met Lys Glu Ala Ala Thr Gl - #u Leu Leu Phe Gly Gly 
285 - # 290 - # 295 
- - CAT GAA ACC ACC GCC AGC ACT GCA ACC TCA CT - #T GTC ATG TTT CTG GGT 
1026 
His Glu Thr Thr Ala Ser Thr Ala Thr Ser Le - #u Val Met Phe Leu Gly 
300 - # 305 - # 310 
- - CTG AAC ACA GAA GTG GTG CAG AAG GTC AGA GA - #G GAG GTT CAG GAG AAG 
1074 
Leu Asn Thr Glu Val Val Gln Lys Val Arg Gl - #u Glu Val Gln Glu Lys 
315 - # 320 - # 325 
- - GTT GAA ATG GGC ATG TAT ACA CCT GGA AAG GG - #C TTG AGT ATG GAG CTG 
1122 
Val Glu Met Gly Met Tyr Thr Pro Gly Lys Gl - #y Leu Ser Met Glu Leu 
330 3 - #35 3 - #40 3 - 
#45 
- - TTG GAC CAG CTG AAG TAC ACT GGA TGT GTG AT - #T AAA GAG ACT CTT 
AGA 1170 
Leu Asp Gln Leu Lys Tyr Thr Gly Cys Val Il - #e Lys Glu Thr Leu Arg 
350 - # 355 - # 360 
- - ATC AAC CCT CCT GTT CCC GGA GGA TTC AGA GT - #C GCA CTC AAA ACC TTT 
1218 
Ile Asn Pro Pro Val Pro Gly Gly Phe Arg Va - #l Ala Leu Lys Thr Phe 
365 - # 370 - # 375 
- - GAA TTG AAT GGT TAC CAA ATT CCT AAA GGA TG - #G AAC GTC ATT TAC AGC 
1266 
Glu Leu Asn Gly Tyr Gln Ile Pro Lys Gly Tr - #p Asn Val Ile Tyr Ser 
380 - # 385 - # 390 
- - ATC TGT GAC ACG CAC GAT GTG GCC GAC GTC TT - #T CCA AAC AAA GAG GAG 
1314 
Ile Cys Asp Thr His Asp Val Ala Asp Val Ph - #e Pro Asn Lys Glu Glu 
395 - # 400 - # 405 
- - TTC CAG CCG GAG AGA TTC ATG AGC AAA GGT CT - #G GAG GAC GGG TCC AGG 
1362 
Phe Gln Pro Glu Arg Phe Met Ser Lys Gly Le - #u Glu Asp Gly Ser Arg 
410 4 - #15 4 - #20 4 - 
#25 
- - TTT AAC TAC ATC CCC TTC GGA GGA GGA TCC AG - #G ATG TGT GTG GGC 
AAA 1410 
Phe Asn Tyr Ile Pro Phe Gly Gly Gly Ser Ar - #g Met Cys Val Gly Lys 
430 - # 435 - # 440 
- - GAG TTC GCC AAA GTG TTA CTC AAG ATC TTT TT - #A GTT GAG TTA ACG CAG 
1458 
Glu Phe Ala Lys Val Leu Leu Lys Ile Phe Le - #u Val Glu Leu Thr Gln 
445 - # 450 - # 455 
- - CAT TGC AAT TGG ATT CTC TCA AAC GGA CCC CC - #G ACA ATG AAA ACA GGC 
1506 
His Cys Asn Trp Ile Leu Ser Asn Gly Pro Pr - #o Thr Met Lys Thr Gly 
460 - # 465 - # 470 
- - CCG ACT ATT TAC CCA GTG GAC AAT CTC CCT AC - #C AAA TTC ACT AGT TAT 
1554 
Pro Thr Ile Tyr Pro Val Asp Asn Leu Pro Th - #r Lys Phe Thr Ser Tyr 
475 - # 480 - # 485 
- - GTC AGA AAT TAGCCTAACC GGAGCTTTGT ACATATGTTT TTATTTTAG - #A 
1603 
Val Arg Asn 
490 
- - TGAACTGTGA TGTATTGGAT ATTTTCTATT TTGTTTATAT AAAGCAGATG TG - 
#TATATAAG 1663 
- - TCTATGCGAG GAAGCGAAAA CGAGGGCACT ACTTTCTCAT GGATCACTGT AA - 
#TGCTACAG 1723 
- - AGTGTCTGTG ATGTATATTT ATAATGTAGT TGTGTTATAT AGCTTTTGTA CT - 
#GTATGCAA 1783 
- - CTTATTTAAC TCGCTCTTTA TCTCATGGGT TTTATTTAAT AAAACATGTT CT - 
#TACAAAAA 1843 
- - AAAAAAA - # - # 
- # 1850 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 497 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - Met Gly Leu Pro Ala Leu Leu Ala Ser Ala Le - #u Cys Thr Phe Val 
Leu 
1 5 - # 10 - # 15 
- - Pro Leu Leu Leu Phe Leu Ala Ala Ile Lys Le - #u Trp Asp Leu Tyr Cys 
20 - # 25 - # 30 
- - Val Ser Gly Arg Asp Arg Ser Cys Ala Leu Pr - #o Leu Pro Pro Gly Thr 
35 - # 40 - # 45 
- - Met Gly Phe Pro Phe Phe Gly Glu Thr Leu Gl - #n Met Val Leu Gln Arg 
50 - # 55 - # 60 
- - Arg Lys Phe Leu Gln Met Lys Arg Arg Lys Ty - #r Gly Phe Ile Tyr Lys 
65 - #70 - #75 - #80 
- - Thr His Leu Phe Gly Arg Pro Thr Val Arg Va - #l Met Gly Ala Asp Asn 
85 - # 90 - # 95 
- - Val Arg Arg Ile Leu Leu Gly Asp Asp Arg Le - #u Val Ser Val His Trp 
100 - # 105 - # 110 
- - Pro Ala Ser Val Arg Thr Ile Leu Gly Ser Gl - #y Cys Leu Ser Asn Leu 
115 - # 120 - # 125 
- - His Asp Ser Ser His Lys Gln Arg Lys Lys Va - #l Ile Met Arg Ala Phe 
130 - # 135 - # 140 
- - Ser Arg Glu Ala Leu Glu Cys Tyr Val Pro Va - #l Ile Thr Glu Glu Val 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Ser Ser Leu Glu Gln Trp Leu Ser Cys Gl - #y Glu Arg Gly Leu 
Leu 
165 - # 170 - # 175 
- - Val Tyr Pro Glu Val Lys Arg Leu Met Phe Ar - #g Ile Ala Met Arg Ile 
180 - # 185 - # 190 
- - Leu Leu Gly Cys Glu Pro Gln Leu Ala Gly As - #p Gly Asp Ser Glu Gln 
195 - # 200 - # 205 
- - Gln Leu Val Glu Ala Phe Glu Glu Met Thr Ar - #g Asn Leu Phe Ser Leu 
210 - # 215 - # 220 
- - Pro Ile Asp Val Pro Phe Ser Gly Leu Tyr Ar - #g Gly Met Lys Ala Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - Asn Leu Ile His Ala Arg Ile Glu Gln Asn Il - #e Arg Ala Lys Ile 
Cys 
245 - # 250 - # 255 
- - Gly Leu Arg Ala Ser Glu Ala Gly Gln Gly Cy - #s Lys Asp Ala Leu Gln 
260 - # 265 - # 270 
- - Leu Leu Ile Glu His Ser Trp Glu Arg Gly Gl - #u Arg Leu Asp Met Gln 
275 - # 280 - # 285 
- - Ala Leu Lys Gln Ser Ser Thr Glu Leu Leu Ph - #e Gly Gly His Glu Thr 
290 - # 295 - # 300 
- - Thr Ala Ser Ala Ala Thr Ser Leu Ile Thr Ty - #r Leu Gly Leu Tyr Pro 
305 3 - #10 3 - #15 3 - 
#20 
- - His Val Leu Gln Lys Val Arg Glu Glu Leu Ly - #s Ser Lys Gly Leu 
Leu 
325 - # 330 - # 335 
- - Cys Lys Ser Asn Gln Asp Asn Lys Leu Asp Me - #t Glu Ile Leu Glu Gln 
340 - # 345 - # 350 
- - Leu Lys Tyr Ile Gly Cys Val Ile Lys Glu Th - #r Leu Arg Leu Asn Pro 
355 - # 360 - # 365 
- - Pro Val Pro Gly Gly Phe Arg Val Ala Leu Ly - #s Thr Phe Glu Leu Asn 
370 - # 375 - # 380 
- - Gly Tyr Gln Ile Pro Lys Gly Trp Asn Val Il - #e Tyr Ser Ile Cys Asp 
385 3 - #90 3 - #95 4 - 
#00 
- - Thr His Asp Val Ala Glu Ile Phe Thr Asn Ly - #s Glu Glu Phe Asn 
Pro 
405 - # 410 - # 415 
- - Asp Arg Phe Ser Ala Pro His Pro Glu Asp Al - #a Ser Arg Phe Ser Phe 
420 - # 425 - # 430 
- - Ile Pro Phe Gly Gly Gly Leu Arg Ser Cys Va - #l Gly Lys Glu Phe Ala 
435 - # 440 - # 445 
- - Lys Ile Leu Leu Lys Ile Phe Thr Val Glu Le - #u Ala Arg His Cys Asp 
450 - # 455 - # 460 
- - Trp Gln Leu Leu Asn Gly Pro Pro Thr Met Ly - #s Thr Ser Pro Thr Val 
465 4 - #70 4 - #75 4 - 
#80 
- - Tyr Pro Val Asp Asn Leu Pro Ala Arg Phe Th - #r His Phe His Gly 
Glu 
485 - # 490 - # 495 
- - Ile 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1494 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - ATG GGG CTC CCG GCG CTG CTG GCC AGT GCG CT - #C TGC ACC TTC GTG CTG 
48 
Met Gly Leu Pro Ala Leu Leu Ala Ser Ala Le - #u Cys Thr Phe Val Leu 
1 5 - # 10 - # 15 
- - CCG CTG CTG CTC TTC CTG GCT GCG ATC AAG CT - #C TGG GAC CTG TAC TGC 
96 
Pro Leu Leu Leu Phe Leu Ala Ala Ile Lys Le - #u Trp Asp Leu Tyr Cys 
20 - # 25 - # 30 
- - GTG AGC GGC CGC GAC CGC AGT TGT GCC CTC CC - #A TTG CCC CCC GGG ACT 
144 
Val Ser Gly Arg Asp Arg Ser Cys Ala Leu Pr - #o Leu Pro Pro Gly Thr 
35 - # 40 - # 45 
- - ATG GGC TTC CCC TTC TTT GGG GAA ACC TTG CA - #G ATG GTA CTG CAG CGG 
192 
Met Gly Phe Pro Phe Phe Gly Glu Thr Leu Gl - #n Met Val Leu Gln Arg 
50 - # 55 - # 60 
- - AGG AAG TTC CTG CAG ATG AAG CGC AGG AAA TA - #C GGC TTC ATC TAC AAG 
240 
Arg Lys Phe Leu Gln Met Lys Arg Arg Lys Ty - #r Gly Phe Ile Tyr Lys 
65 - #70 - #75 - #80 
- - ACG CAT CTG TTC GGG CGG CCC ACC GTA CGG GT - #G ATG GGC GCG GAC AAT 
288 
Thr His Leu Phe Gly Arg Pro Thr Val Arg Va - #l Met Gly Ala Asp Asn 
85 - # 90 - # 95 
- - GTG CGG CGC ATC TTG CTC GGA GAC GAC CGG CT - #G GTG TCG GTC CAC TGG 
336 
Val Arg Arg Ile Leu Leu Gly Asp Asp Arg Le - #u Val Ser Val His Trp 
100 - # 105 - # 110 
- - CCA GCG TCG GTG CGC ACC ATT CTG GGA TCT GG - #C TGC CTC TCT AAC CTG 
384 
Pro Ala Ser Val Arg Thr Ile Leu Gly Ser Gl - #y Cys Leu Ser Asn Leu 
115 - # 120 - # 125 
- - CAC GAC TCC TCG CAC AAG CAG CGC AAG AAG GT - #G ATT ATG CGG GCC TTC 
432 
His Asp Ser Ser His Lys Gln Arg Lys Lys Va - #l Ile Met Arg Ala Phe 
130 - # 135 - # 140 
- - AGC CGC GAG GCA CTC GAA TGC TAC GTG CCG GT - #G ATC ACC GAG GAA GTG 
480 
Ser Arg Glu Ala Leu Glu Cys Tyr Val Pro Va - #l Ile Thr Glu Glu Val 
145 1 - #50 1 - #55 1 - 
#60 
- - GGC AGC AGC CTG GAG CAG TGG CTG AGC TGC GG - #C GAG CGC GGC CTC 
CTG 528 
Gly Ser Ser Leu Glu Gln Trp Leu Ser Cys Gl - #y Glu Arg Gly Leu Leu 
165 - # 170 - # 175 
- - GTC TAC CCC GAG GTG AAG CGC CTC ATG TTC CG - #A ATC GCC ATG CGC ATC 
576 
Val Tyr Pro Glu Val Lys Arg Leu Met Phe Ar - #g Ile Ala Met Arg Ile 
180 - # 185 - # 190 
- - CTA CTG GGC TGC GAA CCC CAA CTG GCG GGC GA - #C GGG GAC TCC GAG CAG 
624 
Leu Leu Gly Cys Glu Pro Gln Leu Ala Gly As - #p Gly Asp Ser Glu Gln 
195 - # 200 - # 205 
- - CAG CTT GTG GAG GCC TTC GAG GAA ATG ACC CG - #C AAT CTC TTC TCG CTG 
672 
Gln Leu Val Glu Ala Phe Glu Glu Met Thr Ar - #g Asn Leu Phe Ser Leu 
210 - # 215 - # 220 
- - CCC ATC GAC GTG CCC TTC AGC GGG CTG TAC CG - #G GGC ATG AAG GCG CGG 
720 
Pro Ile Asp Val Pro Phe Ser Gly Leu Tyr Ar - #g Gly Met Lys Ala Arg 
225 2 - #30 2 - #35 2 - 
#40 
- - AAC CTC ATT CAC GCG CGC ATC GAG CAG AAC AT - #T CGC GCC AAG ATC 
TGC 768 
Asn Leu Ile His Ala Arg Ile Glu Gln Asn Il - #e Arg Ala Lys Ile Cys 
245 - # 250 - # 255 
- - GGG CTG CGG GCA TCC GAG GCG GGC CAG GGC TG - #C AAA GAC GCG CTG CAG 
816 
Gly Leu Arg Ala Ser Glu Ala Gly Gln Gly Cy - #s Lys Asp Ala Leu Gln 
260 - # 265 - # 270 
- - CTG TTG ATC GAG CAC TCG TGG GAG AGG GGA GA - #G CGG CTG GAC ATG CAG 
864 
Leu Leu Ile Glu His Ser Trp Glu Arg Gly Gl - #u Arg Leu Asp Met Gln 
275 - # 280 - # 285 
- - GCA CTA AAG CAA TCT TCA ACC GAA CTC CTC TT - #T GGA GGA CAC GAA ACC 
912 
Ala Leu Lys Gln Ser Ser Thr Glu Leu Leu Ph - #e Gly Gly His Glu Thr 
290 - # 295 - # 300 
- - ACG GCC AGT GCA GCC ACA TCT CTG ATC ACT TA - #C CTG GGG CTC TAC CCA 
960 
Thr Ala Ser Ala Ala Thr Ser Leu Ile Thr Ty - #r Leu Gly Leu Tyr Pro 
305 3 - #10 3 - #15 3 - 
#20 
- - CAT GTT CTC CAG AAA GTG CGA GAA GAG CTG AA - #G AGT AAG GGT TTA 
CTT 1008 
His Val Leu Gln Lys Val Arg Glu Glu Leu Ly - #s Ser Lys Gly Leu Leu 
325 - # 330 - # 335 
- - TGC AAG AGC AAT CAA GAC AAC AAG TTG GAC AT - #G GAA ATT TTG GAA CAA 
1056 
Cys Lys Ser Asn Gln Asp Asn Lys Leu Asp Me - #t Glu Ile Leu Glu Gln 
340 - # 345 - # 350 
- - CTT AAA TAC ATC GGG TGT GTT ATT AAG GAG AC - #C CTT CGA CTG AAT CCC 
1104 
Leu Lys Tyr Ile Gly Cys Val Ile Lys Glu Th - #r Leu Arg Leu Asn Pro 
355 - # 360 - # 365 
- - CCA GTT CCA GGA GGG TTT CGG GTT GCT CTG AA - #G ACT TTT GAA TTA AAT 
1152 
Pro Val Pro Gly Gly Phe Arg Val Ala Leu Ly - #s Thr Phe Glu Leu Asn 
370 - # 375 - # 380 
- - GGA TAC CAG ATT CCC AAG GGC TGG AAT GTT AT - #C TAC AGT ATC TGT GAT 
1200 
Gly Tyr Gln Ile Pro Lys Gly Trp Asn Val Il - #e Tyr Ser Ile Cys Asp 
385 3 - #90 3 - #95 4 - 
#00 
- - ACT CAT GAT GTG GCA GAG ATC TTC ACC AAC AA - #G GAA GAA TTT AAT 
CCT 1248 
Thr His Asp Val Ala Glu Ile Phe Thr Asn Ly - #s Glu Glu Phe Asn Pro 
405 - # 410 - # 415 
- - GAC CGA TTC AGT GCT CCT CAC CCA GAG GAT GC - #A TCC AGG TTC AGC TTC 
1296 
Asp Arg Phe Ser Ala Pro His Pro Glu Asp Al - #a Ser Arg Phe Ser Phe 
420 - # 425 - # 430 
- - ATT CCA TTT GGA GGA GGC CTT AGG AGC TGT GT - #A GGC AAA GAA TTT GCA 
1344 
Ile Pro Phe Gly Gly Gly Leu Arg Ser Cys Va - #l Gly Lys Glu Phe Ala 
435 - # 440 - # 445 
- - AAA ATT CTT CTC AAA ATA TTT ACA GTG GAG CT - #G GCC AGG CAT TGT GAC 
1392 
Lys Ile Leu Leu Lys Ile Phe Thr Val Glu Le - #u Ala Arg His Cys Asp 
450 - # 455 - # 460 
- - TGG CAG CTT CTA AAT GGA CCT CCT ACA ATG AA - #A ACC AGT CCC ACC GTG 
1440 
Trp Gln Leu Leu Asn Gly Pro Pro Thr Met Ly - #s Thr Ser Pro Thr Val 
465 4 - #70 4 - #75 4 - 
#80 
- - TAT CCT GTG GAC AAT CTC CCT GCA AGA TTC AC - #C CAT TTC CAT GGG 
GAA 1488 
Tyr Pro Val Asp Asn Leu Pro Ala Arg Phe Th - #r His Phe His Gly Glu 
485 - # 490 - # 495 
- - ATC TGA - # - # - 
# 1494 
Ile 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - Pro Phe Gly Gly Gly Pro Arg Leu Cys Pro Gl - #y Tyr Glu Leu Ala 
Arg 
1 5 - # 10 - # 15 
- - Val Ala Leu Ser 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - Pro Phe Ser Gly Gly Ala Arg Asn Cys Ile Gl - #y Lys Gln Phe Ala Met 
1 5 - # 10 - # 15 
- - Ser Glu Met Lys 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - Pro Phe Ser Gly Gly Ala Arg Asn Cys Ile Gl - #y Lys Gln Phe Ala Met 
1 5 - # 10 - # 15 
- - Asn Glu Leu Lys 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - Pro Phe Gly Thr Gly Pro Arg Asn Cys Ile Gl - #y Met Arg Phe Ala Ile 
1 5 - # 10 - # 15 
- - Met Asn Met Lys 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - Pro Phe Ser Gly Gly Ser Arg Asn Cys Ile Gl - #y Lys Gln Phe Ala Met 
1 5 - # 10 - # 15 
- - Asn Glu Leu Lys 
20 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 351 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - GAACTCCTCT TTGGAGGACA CGAAACCACG GCCAGTGCAG CCACATCTCT GA - 
#TCACTTAC 60 
- - CTGGGGCTCT ACCCACATGT TCTCCAGAAA GTGCGAGAAG AGCTGAAGAG TA - 
#AGGGTTTA 120 
- - CTTTGCAAGA GCAATCAAGA CAACAAGTTG GACATGGAAA TTTTGGAACA AC - 
#TTAAATAC 180 
- - ATCGGGTGTG TTATTAAGGA GACCCTTCGA CTGAATCCCC CAGTTCCAGG AG - 
#GGTTTCGG 240 
- - GTTGCTCTGA AGACTTTTGA ATTAAATGGA TACCAGATTC CCAAGGGCTG GA - 
#ATGTTATC 300 
- - TACAGTATCT GTGATACTCA TGATGTGGCA GAGATCTTCA CCAACAAGGA A - # 
351 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - TTTTTTTTTT TTGG - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
- - TTTTTTTTTT TTGA - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
- - TTTTTTTTTT TTGT - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
- - TTTTTTTTTT TTGC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
- - TTTTTTTTTT TTAG - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
- - TTTTTTTTTT TTAA - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
- - TTTTTTTTTT TTAT - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
- - TTTTTTTTTT TTAC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
- - TTTTTTTTTT TTCG - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
- - TTTTTTTTTT TTCA - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
- - TTTTTTTTTT TTCT - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
- - TTTTTTTTTT TTCC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO:24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
- - AAGCGACCGA - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
- - TGTTCGCCAG - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
- - TGCCAGTGGA - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
- - GGCTGCAAAC - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
- - CCTAGCGTTG - # - # 
- # 10 
- - - - (2) INFORMATION FOR SEQ ID NO:29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
- - GTAGCGGCCG CTGCCAGTGG A - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO:30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
- - GTAGCGGCCG CT - # - # 
- # 12 
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