MXPA98010433A - Protein that metabolizes retinol - Google Patents

Abstract

The present invention relates to amino acid sequences and corresponding nucleic acid sequence of protein that metabolizes retinoids found in human, mouse and zebrafish, as well as methods for using same.

Description

PROTEIN OUE METABOLIZA RETINOIDES DESCRIPTION OF THE INVENTION This application claims priority of U.S. Patent Application No. 08 / 724,466, filed October 1, 1996, and of U.S. Patent Application Serial No. 08 / 667,546, filed on June 21, 1996, the specifications of the applications that are incorporated herein for reference. The metabolism of Vitamin A gives rise to several active forms of retinoic acid (AR) which are involved in the regulation of gene expression during development, regeneration, and in the growth and differentiation of epithelial tissues [Maden, 1992; Chambón, 1995; Mangelsdorf, 1995; Gudas, 1994; Lotan, 1995; Morris-Kay, 1996]; apoptosis, or cell death programmed into a number of cell types, has been unit; and to have anticarcinogenic and antitumor properties. Recent studies of retinol deficiency indicate a correlation between vitamin A reduction and a higher incidence of cancer and increased susceptibility to chemical carcinogenesis [Chytil, 1984]. Several models have been used to demonstrate the efficacy of retinoids in the suppression of carcinogenesis in a variety of tissues including skin, breast epithelia, oral cavity, aerodigestive tract, liver, bladder and prostate [Moon, 1994]. These studies have led to the preventive use of retinoids to treat premalignant lesions including actinic keratosis and oral leucoplacia, as well as in the prevention of malignant tumors of the head and neck and the recurrence of small cell lung carcinomas, and basal cell carcinomas. , [Hong, 1994; Lippman, 1995]. It has been found that the same AR is 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 those of actinic keratoses associated with premalignancy, acne, psoriasis and ichthyosis. There is evidence that the efficacy of RA as an anti tumor agent is at least partially due to the induction of cell differentiation and / or inhibition of proliferation (Lotan, 1996. Studies in several past years indicate that a high proportion of patients with leukemia Acute promyelocytic (APL) achieve complete remission after a short period of treatment with all trans RA Unfortunately, this high remission rate is in most cases brief.After a relapse, patients are clinically resistant to additional treatment. with AR [arrell, 1994; arrell, et al., 1994; Chomienne, 1996; Muindi, 1992] The nature of this resistance is unknown Interestingly, it has been shown that leukemia cells taken from patients exhibiting clinical resistance AR are sensitive to the action of differentiation of AR when they grow in vitro (Muindi, 1992, Muindi, 1994) .This suggests that the pharmacokinetic mechanisms s can count for the acquired resistance to AR. This possibility is supported by studies that show that the maximum plasma concentrations of RA are much higher in patients after initial administration than in patients treated after relapse. This decrease in the maximum plasma AR concentration is accompanied by a ten-fold increase in urinary 4-oxo-retinoic acid concentration. In addition, it is shown that ketoconazole, a broad-spectrum inhibitor of cytochrome P450 function modulates the pharmacokinetics of RA in vivo [Muindi, 1992; Muindi, 1994]. Therefore it is likely that RA increases the proportion of its own metabolism which in turn results in the inability to sustain effective therapeutic doses of RA. The therapeutic administration of RA can result in a variety of undesirable side effects and it is therefore important to establish and maintain the minimum requirement doses of AR in the treatmentFor example, AR treatments during pregnancy can lead to severe teratogenic effects in the fetus. Adverse reactions for the treatment of RA also include headache, nausea, cheilitis, facial dermatitis, conjunctivitis, and dryness of the nasal mucosa. Prolonged exposure to RA can cause higher elevations in serum triglycerides and can lead to severe abnormalities of liver function, including hepatomegaly, cirrhosis, and portal hypertension. The metabolism of RA can also be taken into account for the lack of response of certain tumors for the treatment of RA. For example, recent studies have shown that cytochrome P450 inhibitors that block AR metabolism, resulting in increased levels of RA tissue, may be useful therapeutic agents in the treatment of prostate cancer [Wouters, 1992; De Coster, 1996]. In this way cytochromes P450 that metabolize AR can be useful targets for the treatment of a number of different types of cancer. The classic view of vitamin A metabolism maintains that all-trans RA, the most active metabolite is derived from the conversion of retinal to retinaldehyde to Ar through two oxidation stages and that the AR is further metabolized to the polar derivatives 4 -OH AR and 4 -oxo AR [Blaner, 1994; Napoli, 1995; Formelli, 1996; Napoli, 1996]. It is unknown if the metabolites 4 -oxo and 4 -OH are simply intermediaries in the catabolic pathway of RA or if they can also have specific activities which differ from that of all trans RA and AR 9-cis. Pijnappel et al. [Pijnappel, 1993] has shown that, in Xenopus, Ar 4 -oxo can efficiently modulate the positional specification in early embryos and exhibits a more potent capacity to regulate the expression of Hoxb-9 and Hoxb-4 genes than all trans RA. It has been found that AR 4 -oxo binds to the retinoic acid receptor β (RAR-β) with affinity comparable to all trans RA [Pijnappel, 1993] but deficiently to RAR-? [Reddy, 1992], suggesting that this metabolite exhibits some selectivity to the receptor. AR 4 -oxo also binds to the cellular retinoic acid binding protein (PEARC) but with slightly lower affinity than that of all trans RA [Fiorella, 1993]. Takatsuka et al. [Takatsuka, 1996] have shown that the inhibitory effects of RA growth correlate with the metabolic activity of RA but it is unknown if there is a causal relationship between the production of AR metabolites and growth inhibition. The asymmetric distribution of these metabolites in developing embryos suggests that they can be preferentially sequestered or generated by tissue-specific isomerases [Creeck Kraft, 1994]. The normal balance of these metabolites is dependent on the rate of formation of metabolic precursors, retinal and retinaldehyde [Leo, 1989], and a proportion of catabolism. Currently little is known about the enzymes involved in this metabolic scheme, particularly the catabolism of RA. It is thought that AR catabolism is initiated by hydroxylation either at the C4 position, or C18 of the ß-ionone ring of AR [Napoli, 1996]. The hydroxylation step of C4 is mediated by the activity of cytochrome P450, as judged by the ability of broad spectrum P450 inhibitors such as ketocoazole and liarazole to block 4-hydroxylation [Williams, 1987, Van Wauwe, 1988; Van Wauwe, 1990, Van Wauwe, 1992, Wouters, 1992]. In certain tissues, including testes, skin and lung and in numerous cell lines, such as NIH3T3 fibroblasts, myelomonocytic leukemia cells HL 60, mouse embryonal carcinoma cell lines F9 and P19 and MCF7, AR metabolism can be induced by pretreatment of the AR [Frolík, 1979, Roberts, 1979a and b; Duell, 1992; Wouters, 1992]. Studies involving the target alteration of RAR genes in F9 cells suggest that the RAR-a and RAR-? they may play a role in the regulation of enzymes responsible for this increased metabolism [Boylan, 1995]. The glucuronidation of RA is a significant metabolic step in the inactivation of RA [Blaner, 1994; Formelli, 1996]. The removal of the AR may require oxidation to 4 -oxo followed by conjugation to form the AR glucuronide all trans 4 -oxo. This is supported by studies in both primates and humans that show that AR-glucuronide 4 -oxo is the only retinoid conjugate found in urine [Muindi, 1992; Muindi, 1994]. The fact that by following RA therapy, RA 4 -oxo in serum is not present or barely detectable, suggests that oxidation may be the limiting stage of proportion in this process. It has recently been shown that 4-oxoretinol (4-oxo-ROL) may have higher biological activity than retinol. 4-Oxo-ROL is inducible by RA in mouse teratocarcinoma cells F9 and P19 [Blumberg et al., 1995; Achkar et al., 1996]. It is known that zebrafish fins are regenerated through the AR sensitive process which uses many regulatory pathways of genes involved in early vertebrate development [White, 1994; Akimenko, 1995a __ b]. As we are aware in the present, cytochromes P450 involved in the metabolism of AR in extrahepatic tissues remain uncharacterized at the molecular level. This is the first to identify, cloning and sequencing a gene (cDNA) that encodes a retinoic acid-inducible protein, which metabolizes retinoic acid, including a cDNA which is inducible by RA in humans. It has been found that the protein is expressed in epithelia. A zebra-fin cDNA has been isolated 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 β-ionone ring of retinoic acid. It has been found that the protein is inducible in epithelial cells exposed to retinoic acid. A human cDNA encoding a protein with similar functionality has also been isolated and sequenced. A mouse cDNA has also been isolated and sequenced. It has been found that the homology between the sequences of the three species, whether they are nucleic acids encoding the protein, or the amino acid sequences of the proteins, is relatively high and all three proteins contain a motif that binds a haem characteristic of the group of proteins. proteins known as cytochrome P450. The total homology between the amino acid sequences of these recently obtained proteins and known P450 cytochromes is less than 30%. Despite this relatively low total homology, a greater degree of homology has been observed in the heme binding region for certain other P450. For example, the homology between the approximately 20 amino acids that define respective heme linking regions of the new zebrafish protein and CYP4503A12 is approximately 50% and between the new zebrafish protein and hCYTFAOH is 65%. The homology between the same heme binding region of a protein of the present invention and another P450 can be either 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 identified as SEQ ID NO: 32, or a homology equivalent in functionality thereof. The amino acid sequence identified as SEQ ID NO: 2 is from the protein, named in the present "ZP450RAI", obtained from zebrafish fin. The amino acid sequence of the human protein is identified as SEQ ID NO: 4 and the protein is referred to herein as "hP450RAI". The mouse protein sequence is identified as SEQ ID NO: 32 and the protein is referred to herein as mP450RAI. Such a protein which is at least about 35% conserved in relation to the amino acid sequence identified as SEQ ID NO: 2 (zebrafish) or identified as SEQ ID NO: 4 (human) or identified as SEQ ID NO: 32 ( mouse), or a homology equivalent in functionality thereof, also forms part of the invention described herein. Similarly, the degree of sequence conservation of a protein can be 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or by 100% assumption of either SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 32 or equivalent homology in functionality thereof, variants that are possible while retaining the native protein's ability to oxidize a retinoid Also within the scope of the invention are any such proteins that have the ability to hydroxylate retinoic acid in the 4-position of the β-ionone ring. Of course, the conservatively substituted variants of proteins described 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 β-ionone ring of the retinoid. In particular, all-trans retinoids can be metabolized by proteins of the present invention. In the context of this specification, the term "conserved" describes similarity between sequences. The degree of preservation 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 positions compared by the same nucleotide or amino acid are occupied, as the case may be, the two sequences are retained in that position. The degree of preservation between two sequences is often expressed, as here, as a percentage representing the proportion of the number of coupling positions in the two sequences for 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 the development and differentiation in a wide variety of systems. For purposes of this description "retinoid" is also proposed to encompass an equivalent thereof having the same functional characteristics which can be produced, for example, by computational chemistry. 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 identified as SEQ ID NO: 32, or equivalent homology in functionality thereof, for example, or a nucleic acid strand capable of hybridizing to the nucleic acid molecule under the conditions of severe hybridization. Of course, the degree of preservation of the protein which encodes the nucleic acid 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 that encodes a protein that has the ability to oxidize a retinoid at the carbon occupying the 4-position of the β-ring of the retinoid ring, and more particularly, having all-trans retinoic acid activity 4-hydroxylase. For the purposes of this invention, the term "isolated" refers to a nucleic acid that is substantially free of other culture medium or cellular material when produced by recombinant DNA techniques, or chemical precursors or other chemicals when produced synthetically chemistry. The cellular expression of preferred proteins of the present invention can be induced, preferred embodiments which are described in greater detail below, for certain types of cells by exposure of the cells to 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 from a cell and whose expression by that cell can be induced by exposure of the cell to retinoic acid. It will be appreciated that no cell, even if it contains DNA encoding such a protein, possesses all the attributes necessary to express the protein on exposure to AR. The nucleotide sequences possessing the activity of the promoter associated with the proteins have been isolated and identified herein. The sequence of human nucleotides having the activity of the promoter is identified as SEQ ID NO: 33. The sequence of murine nucleotides having the activity of the promoter is identified as SEQ ID NO: 34. The nucleotide sequence of zebrafish having the activity of the promoter is identified as SEQ ID NO: 35. In fact, the invention includes genomic sequences for human (SEQ ID NO: 36 and 37) and mouse (SEQ ID NO: 38). In another aspect, the present invention is thus a microbial cell that contains and expresses heterologous DNA encoding a retinoic acid-inducible protein having the activity of all-trans 4-hydroxylase retinoic acid. The sequence of a nucleic acid molecule of the present invention may correspond to a portion of a human genome or a fish genome or a mouse 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 SEQ ID NO: 31 (mP450RAI) ), or the sequence may be one which varies from one of these sequences due to the degeneracy of the genetic code, or this may be a nucleic acid strand capable of hybridizing with at least one of these nucleic acid molecules under conditions of Hybridization of high or low rigor. "Rigid hybridization conditions" takes its common meaning for a person skilled in the art herein. Appropriate rigid conditions which promote nucleic acid hybridization, eg, 6x sodium chloride / sodium citrate (SSC) at about 45 ° 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 suitable first hybridization solution, mix together 24 ml formamide, 12 ml 20x SSC, 0.5 ml Tris HCl 2M pH 7.6, 0.5 ml 100 x Denhardt's solution, 2.5 ml of deionized HzO, 10 ml of 50% dextran sulfate, and 0.5 ml of 10% SDS. A second suitable hybridization solution can be 1% crystalline BSA (fraction V), 1 mM EDTA, 0.5 M Na2HP04 pH 7.2, 7% SDS. The salt concentration in the washing step can be selected from low stiffness of about 2x SSC at 50 ° C to a high rigidity of about 0.2x SSC at 50 ° C. both of these solutions may contain 0.1% SDS. In addition, the temperature in the washing stage can be increased from conditions of low stiffness to room temperature, about 22 ° C, under conditions of high rigidity, to about 65 ° C. The cited reference gives more detail, but the washing stiffness appropriate depends on the degree of homology and length of probe - the homology is 100%, a high temperature can be used (65 ° C to 75 ° C). If the homology is low, lower wash temperatures should be used. However, if the probe is very short (< 100 bp), lower temperatures should be used even with 100% homology. In general, you start by washing at low temperatures (37 ° C to 40 ° C), and the temperature increases by 3-5 ° C intervals until the baseline 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 operably linked in a recombinant cloning vector. In the context of this invention, the term "two operatively linked" means both that the regulatory sequence contains sufficient elements to allow expression of the nucleic acid in question and that the nucleic acid is bound to the regulatory sequence appropriately. For example, the nucleic acid of the invention is in the proper orientation and in the phase with a start codon. Thus, the present invention includes a stably transfected cell line which expresses a protein having the ability to hydroxylate the retinoic acid in the 4-position of the β-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 ß-ionone ring of retinoic acid. Another aspect of the present invention is a host cell that has been genetically engineered to produce a protein of the invention described above, the cell having expressively incorporated into the same heterologous DNA encoding the protein. The cell can be selected in such a way that the production of the protein is inducible by exposing the cell to retinoid, preferably retinoic acid. The cell can be eukaryotic. The present invention also includes a process for producing a protein of the invention described above. Such process includes: Preparing a DNA fragment that contains a nucleotide sequence that encodes the protein; Incorporate the DNA fragment into an expression vector to obtain a recombinant DNA molecule that contains the DNA fragment and is capable of replication; Transforming a host cell with the recombinant DNA molecule to produce a transformant that can express the protein; Cultivate the transformant to produce the protein; and Recover the protein from the resulting cultured mixture. The present invention includes an antibody for a protein of the invention. Here, the term "antibody" is proposed to include a Fab fragment and this may be a monoclonal antibody. The antibody can be originated specifically for the amino acid sequence identified as SEQ ID NO: 4, ie, hP450RAI. The present invention includes a purified protein for use in metabolizing retinoic acid in an organism or cell in need of such metabolization. Similarly, the invention includes a method for metabolizing retinoic acid in an organism or cell in need of metabolism of retinoic acid wherein the method includes administering a protein of the invention as described above. The invention includes a method for inhibiting the hydroxylation of retinoic acid in an organism in need of such inhibition, comprising introducing into the 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 may be in need of treatment against a cancerous disease or a disease selected from the group consisting of cancer, Actinic keratosis, oral leucoplacia, 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 an actinic keratosis associated with premalignancy, acne , psoriasis and / or ichthyosis. Such a method may include using at least one delivery vehicle or technique selected from the group of viral vectors, microinjection, electroporation, coprecipitation, liposomes, aerosol delivery and washing. The portion of the sequence can 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 can be found. The organism can be a human patient and the method can include treating the patient against a cancer disease. The invention also includes a method for inhibiting the hydroxylation of retinoic acid in an organism in need of such inhibition by administering to the organism an effective amount of an antibody, such antibodies as described above. An antibody particularly useful for the treatment of a human may be an antibody to the protein having the amino acid sequence identified as SEQ ID NO: 4, or a portion thereof. It may be advantageous to adapt such an antibody for administration to a human by "humanizing" the antibody, as 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 the DNA of the cell a DNA sequence encoding the endogenous protein at or near a site which is normally occupied by a DNA sequence encoding the protein; and exposing the cell to the retinoid to induce the 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: 32, or more likely to determine the presence of a protein (or peptide fragment) having an amino acid sequence identified as SEQ ID NO: 4, the human protein. The kit includes an antibody to the protein linked to a reporter system in which the reporter system produces a detectable response when a predetermined amount of the protein and the antibody bind to each other. In another aspect, the present invention is a kit for determining the presence of a nucleic acid encoding a protein of invention, or having the sequence identified as SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 31, or which varies in sequence due to the degeneracy of the genetic code, or a nucleic acid strand capable of hybridizing with at least the nucleic acid under rigid hybridization conditions. The kit includes a nucleic acid molecule capable of hybridizing with at least a portion of the nucleic acid or nucleic acid strand under rigid conditions in which the nucleic acid molecule is linked to a reporter system where the reporter system produces a response detectable when a predetermined amount of the nucleic acid or nucleic acid strand and nucleic acid molecule hybridizes to each other. The molecule can have 5 bases in length or more; between 5 and 50 bases in length, between 5 and 30 or 40 bases in length, or between 10 and 20 bases in length. Of course it may be possible to find a more adequate base length. The present invention includes an isolated DNA molecule having a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 33; (b) SEQ ID NO: 34; (c) SEQ ID NO: 35, and (d) A fragment of (a), (b) or (c), wherein the DNA molecule possesses promoter activity. In a more particular aspect, the invention includes a DNA molecule of one of the indicated sequences in which the DNA molecule includes the sequence TGAACT (N) XTGAACT, where x has a value of up to 5. This sequence is conserved in all three sequences identified as having promoter activity, particularly where x is 5. Even more particularly, the invention includes such a DNA molecule in which the sequence TCTGASSAAGKTAAC occurs downstream of the sequence TGAACT (N) XTGAACT. Even more particularly, the sequence includes the sequence AATT between the sequence TGAACT (N) x TGAACT and the sequence TCTGASSAAGKTAAC, AATT that has been found immediately downstream of the sequence TGAACT (N)? TGAACT. It has been observed that there may be up to six nucleotides between the sequence TGAACT (N) XTGAACT and the sequence TCTGASSAAGKTAAC. The CAATTAAAGA sequence upstream of the TGAACT (N) XTGAACT sequence can also be present. In a particular aspect, the sequence having the activity of the promoter includes the sequence CAATTAAAGATGAACTTTGGGTGAACTAATT and the sequence TATAA. Particularly, the TATAA sequence is current to the sequence TGAACT (N) XTGAACT, and more particularly, the sequence TATAA is downstream of the sequence TCTGASSAAGKTAAC and this may be spaced up to about 55 nucleotides downstream of the sequence TGAACT (N) XTGAACT. The invention includes a recombinant DNA having such a DNA sequence having promoter activity. The DNA Recombinant can also include one or more structural genes. Structural genes can code for cytochrome P450 proteins. The invention includes an expression plasmid that includes such recombinant DNA and an isolated cell that contains the recombinant DNA. The cell is usually eukaryotic. The invention includes a process for the production of recombinant protein that includes the steps of culturing a cell according to the invention containing recombinant DNA and recovering the protein produced. The proteins can be a cytochrome P450, but it is not very necessary. The protein can be a fusion protein. The invention further includes a method for screening drugs for their effect on activity of a retinoic acid-inducible protein, which comprises exposing a purified protein to the drug and determining the effect on activity. The activity may be hydroxylation of a retinoic acid, particularly all-trans retinoic acid. In particular, the activity may be hydroxylation of a retinoic acid, particularly all-trans retinoic acid, in the 4-position of the β-ionone ring thereof. The invention provides a method for screening drugs for their effect on the activity of any protein having P450RAI activity of the present invention, particularly determining the effect that a potential drug has on the oxidation of retinoic acid, specifically all-trans retinoic acid. In yet another aspect, the invention includes a method for examining drugs for their effect on the expression of a gene wherein the gene is inducible by a retinoid, including the step of exposing a recombinant DNA described above to a potential drug and determining the effect on it. the expression of genes. This may include exposing the recombinant DNA in the presence of a retinoid. The gene may be a reported gene which does not metabolize retinoic acid. The method may include determining the effect on transcription of the gene. The invention includes a method for examining a drug for its effect on the metabolism of a retinoid by a cytochrome P450 encoded by a nucleotide sequence of the invention which is incorporated into an expression system to be under the control of a nucleotide sequence having The activity of the native promoter for cytochrome P450, which comprises: Expose the system to the drug in the presence of retinoid to determine the effect of the drug on the metabolism of the retinoid. The retinoid can be retinoic acid and this can be all trans retinoic acid. The invention includes any drug identified according to such method as having the effect of modulating, preferably reducing, the activity of the protein or as having the effect of modulating, and preferably reducing, the expression of genes. The invention includes a method for inhibiting the metabolism of retinoic acid in an organism in need of such inhibition, comprising administering to the organism an effective amount of a drug identified according to a method of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the steps used to isolate genes that regulate retinoids using differential visualization of mRNA. Schematic representation of the stages involved in the isolation of genes regulated by retinoids using the differential visualization technique. The cloned products isolated in step 6 can then be used for sequencing, Northern blotting or examination of cDNA libraries. Pl, P2, and P3 correspond to mRNA fragments induced by AR. P4 represents a PCR product of an mRNA which is down-regulated. "Figure 2 (a) shows a polyacrylamide gel ofPCR amplified mRNA in duplicate obtained using retinoic-treated fish (lanes 1 and 2) and control fish treated with dimethylsulfoxide (DMSO) (lanes 3 and 4). The arrow indicates a band amplified by PCR present in samples treated with RA and is not observed in the controls.

Figure 2 (b) shows the nucleotide sequence (SEQ ID N0: 1) of the 337 base pair PCR product isolated from the band (Arrow) of Figure 2 (a). The arrows indicate the nucleotide sequences where the upstream and downstream promotion sites for differential visualization PCR amplification are located in the 3 'untranslated portion of ZP450RAI. Figure 2 (c) shows an amino acid sequence (SEQ ID NO: 2) corresponding to the cDNA (open reading structure of 492 amino acids). Framed residuals indicate the heme binding motif characteristic of cytochromes P450 Figure 2 (d) shows amino acid sequence comparisons between ZP450RAI and several other cytochromes P450 (SEQ ID NO: 6, 7, 8, 9, 10) in the area of the conserved heme binding motif found in the superfamily. The cysteine, designated 0 in the figure, which has been shown to be directly involved in the heme bond [Gotoh, 1989] is surrounded by several highly conserved amino acids. Figure 3 (a) shows the Northern blot analysis of MRNA obtained from a regenerated tissue of fish treated with AR in band 5, and controls (fish treated with DMSO) in band 4, using a ZP450RAI cDNA probe. The comparison to an RNA ladder (band 1) shows that the main ZP450RAI transcript is in the range of 1.4-2.4 kb.

Figure 3 (b) shows the location of ZP450RAI transcripts in regenerating caudal fin tissue 72 hours post amputation by total mounted in situ hybridization, (i) it is found that ZP450RAI transcripts are not detectable in regenerated treated with DMSO. The original plane of amputation is indicated by the white line with arrowhead; they are marked m (soft mesenchyme) and r (bony rays). (ii) in a sample obtained from a fish treated with RA, it is found that the ZP450RAI transcripts, indicated by the black arrowhead, are located in a band of cells that extend between the distal tip of the regenerated one. Lower levels of ZP450RAI expression are also evident in unregenerated tissue in the proximal base of the isolated fin, as indicated by the black line with arrow head. The amputation plane is indicated by the white line with the arrowhead as in Figure 3 (b) (i). (iii) A histological section taken through the plane is indicated by the line. (iv) a histological section of fins treated with RA after hybridization is shown. Localized expression of ZP450RAI is detected in a subset of epithelial cells (black arrowhead) which is at the distal tip of the regenerated one. The bottom membrane separating the dense blasteme and the epithelium rolled by the gray arrow head is indicated. Figure 4 shows elution profiles of lipid-soluble extracts obtained from treated media of COS-1 cells transfected with pSG5zP450RAI and control cells transfected with pSG5. Figures 4 (a) and 4 (b) are plots of cpm against fraction number for cells incubated with [11,12-3H] RA 575 pM for 4 hours and 24 hours, respectively, COS-1 cells pSG5-zP450RAI ( -) and control cells (-). The metabolism of [11,12-3H] RA to total aqueous soluble metabolites (% of total cpm) is measured using aliquots of the water soluble extract subjected to β-scintillation counting. See the boxes in Figures 4 (a) and (b). Figures 4 (c) and 4 (d) are absorbance versus retention time graphs for cells incubated with 1 μM AR for 4 and 24 hours, respectively. The peaks observed in the cell transfected with ZP450RAI are shaded in black. The chromatogram region has been expanded from 4 to 6 min. (see boxes of Figures 4 (c) and (d)). In cells transfected with ZP450RAI cDNA, the generation of peaks corresponding to AR 4 -oxo and AR 4-OH is observed. Figure 5 shows results obtained with human cell lines tested with a probe marked with - [32 P] -dATP having the sequence identified as SEQ ID NO: 11: HEK293; EL-E; HL-60; MCF10A; LC-T; SK-LC6; and MCF7. (+) indicates pretreatment with AR 10"6 M and (-) indicates no pretreatment with RA The blot with hGAPDH is also tested for control for RNA loading of the gel, shown in the lower panel Figure 6 is similar to the Figure 5 by the U937 and HepG2 cell lines Figure 7 is similar to Figure 5 by the NT2 cell line Figure 8 is similar to Figure 5 by the NB4 cell line (first two bands) and three cell lines derived from NB4 resistant to retinoic acid individually derived Figure 9 shows the amino acid sequence cAMP of murine (upper line, SEQ ID NO: 32) aligned with the amino acid sequence P450RAI human (middle line; SEQ ID NO: 4) and the amino acid sequence of zebrafish cDNA (lower line: SEQ ID NO: 2). Figure 10 (a) shows elution profiles of lipid soluble extracts obtained from treated media of COS-1 cells transfected with pSG5-hP450RAI and control cells transfected with pSG5. Graphs of cpm against fraction number are shown for cells incubated with [11,12- 3 H] RA per 24 hours of COS-1 cells pSG5-hP450RAI () and control cells (-). Figure 10 (b) shows measurement of aliquots of the aqueous soluble extract subjected to ß-scintillation counting taken to determine the metabolism of [11,12-3H] RA to total aqueous soluble metabolites. Figure 10 (c) shows absorbance plots against retention time for cells transfected with hP450RAI (-) and control cells (-) incubated with IμM AR for 24 hours. The box is the region around 10 minutes, expanded for clarity. Figure 11 (a) shows the production of 4-oxo-RA from COS-1 cells transfected with pSG5-hP450RAI and control cells transfected with pSG5. Figure 11 (b) shows the production of 4-OH-RA of COS-1 cells transfected with pSG5-hP450RAI and control cells transfected with pSG5. Figure 11 (c) shows the formation of aqueous soluble metabolites of COS-1 cells transfected with hP450f_AI and control cells transfected with pSG5. Figure 11 (d) shows unmetabolized AR of COS-1 cells transfected with pSG5-hP450RAI and control cells transfected with pSG5. Figure 12 (a) shows elution profiles of lipid soluble extracts obtained from cell media MCF10A exposed to AR and MCF10A control cells not exposed. The cpm graphs against the number of fraction for cells incubated with [11,12-3H] RA per 24 hours of MCF10A cells induced by AR (-) and control (-) are shown. Figure 12 (b) shows elution profiles of lipid-soluble extracts obtained from treated media of MCF7 cells "exposed to AR and MCF7 control cells not shown." Graphs of cpm against number of fraction are shown for cells incubated with [11, 12-3H] RA for 24 hours of MCF7 cells induced by AR (-) and control (-).

Figure 12 (c) shows the aqueous soluble metabolites measured using aliquots of the aqueous soluble extracts of the cell lines described in Figures 12 (a) and (b) subjected to β-scintillation counting. The first two are for MCF7 cells not exposed and MCF7 cells exposed to AR, respectively. The third and fourth bars are for MCF10A cells not exposed and MCF10A cells exposed to AR, respectively. Figure 13 (a) shows elution profiles of lipid soluble extracts obtained from microsomal preparations after incubation with radiolabelled AR for ninety minutes, as described in Example 7. Graphs of dpm against fraction number for HeLa P micromosomes (,) and microsomes HeLa RAÍ (,). Figure 13 (b) shows fractions of 5 to 15 of the Figure 13 (a) on a larger scale. Figure 14 (a) show SDS-PAGE of the fusion protein GST-hP450RAI. Lane 1 shows the whole cell lysate of E. coli expressing the plasmid containing the fusion protein, without induction. Lane 2 shows the whole cell lysate of E. coli expressing the plasmid containing the fusion protein after induction with 0.1 mM IPTG. Figure 14 (b) shows SDS-PAGE of the purified GST-hP450RAI fusion protein after induction with 0.1 mM IPTG, with molecular weight markers on the right side indicating the protein indicating the fusion protein to have a molecular weight of approximately 75 kDa. Figure 15 shows promoter sequences of human P450RAI, mouse and zebrafish. The quadr regions show highly conserved regions while the arrow indicates separate RARE consensus sequences. Figure 16 shows relative lucifera activity induced in cells containing a luciferase vector in which a portion of the putative promoter for mP450RAI is cloned. The activity of lucifera is measured in supernatants of cell extract transfected with 3 concentrations of expression vectors comprising cDNA encoding RXRa and RAR? (100 ng, 500 ng, and 1 μg) together with a reporter gene based on luciferase which already includes the sense or antisense promoter sequence, or no promoter sequence, which grows in the presence and absence of AR. Figure 17 shows inhibition of P450RAI mRNA in MCF7 cells by 4-hydroxy-phenylretinamine (4-HPR). Cells are treated for twelve hours with the indicated concentrations of all trans (atRA) and 4-HPR retinoic acid. Total RNA is extracted using TRIzol, and, after electrophoresis, s performed Northern blotting as described. The nitrocellulose is tested with radiolabeled P450RAI after GAPDH. Figure 18 shows expression of cytochrome P450RA in MCF7 cells by Northern blot analysis, over time, after administration of all trans retinoic acid. Figure 19 shows the expression of cytochrome P450RAI in MCF7 cells by northern blot analysis, over time, then administration of all trans retinoic acid, and in the presence of all trans retinoic acid and ketoconazal. Figure 20 shows cytochrome P450RAI expression in MCF7 cells by northern blot analysis, at t_j.en.po, then administration of all trans retinoic acid and then administration of Am580. Figure 1 projects the steps used to isolate genes regulated by retinoids using differential visualization of mRNA. The cloned products isolated in step 6 of Figure 1 are used to sequence and examine Danio rerio cDNA libraries (D.Rerio). Pl, P2 and P3 correspond to fragments of AR induced by mRNA. P4 is a PCR product of down regulated mRNA. Following are details of procedures followed in the determination of gene sequences described herein. Provisions Danio rerio D. Rerio is kept at 28.5 ° C in 40 liter tanks with 25-30 fish per tank in a cycle of 14 hours of light and 10 of darkness. The tap water is conditioned by the addition of 10 ml of Water Conditioning (Sera Aqutan) and 10 ml <; z \ e 250 g / 1 Aquarium Salt (Nutra Fin) for 20 liters. 2-3 liters of water are changed daily. The fins are amputated after anesthetizing the fish in a solution of ethyl-m-aminobenzoatometansulfonic acid 0.2% (ICN) in conditioned water. The treatment of retinoic acid is carried out by adding all trans RA, for a final concentration of 10"eM, directly in the tank water two days after the amputation, keeping both AR treated and control fish in the dark during the Differential mRNA visualization Differential mRNA visualization is performed essentially as described by Lian and Pardee (1992) with appropriate modifications as described herein.Regeneration tissues are harvested 3 days post amputation (24 hours post addition of AR) and freeze rapidly in liquid nitrogen Poly (A) + RNA is isolated using the Micro Fast-Track equipment.Inverse reverse transcription reactions are performed in duplicate in poly (A) + RNA isolated from both treated samples and not treated for each specific poly-T 3 'promoter used (5' -T12VN-3"). The symbol "V" represents A or C or G and not T or U. Various combinations of the 3 'poly-T promoters given in the first column of Table 1 and the upstream promoters given in the second column are used for amplification. of RCP. For each reaction, 0.1 μg of poly (A) + RNA is transcribed in reverse in a 20 μl reaction volume containing 300 U of Superscript Reverse Transcriptase (Gibco / BRL), IX buffer, 20 μM of dGTP, dATP, dCTP , and dTTP, 10 μM of dithiothrethitol (DTT) and 5 pmol of the 5'-T? 2VN-3 promoter. The reactions are mixed and incubated at 35 ° C for 60 minutes, followed by 5 minutes at 95 ° C. PCR amplification is performed on a Perkin Elmer Cetus PCR machine as follows: 1 μl of cDNA synthesis reaction, 5 U of Taq DNA polymerase (Gibco / BRL), IX of buffer for PCR, 2 μM each of dGTP, dATP, dCTP, and dTTP, 10 μCi a- [35 S] dATP (redivue, Amersham) 1.2 mM MgCl 2, 0.5 μM of upstream promoter and 0.5 μM of the 5'-T12VN-3 'promoter. The CPR conditions are as follows: 1 cycle, 94 ° C for 5 minutes; 40 cycles, 94 ° C for 30 seconds, 42 ° C for 1 minute, 72 ° C for 30 seconds, followed by a final extension of 5 minutes at 72 ° C. 4 μl of the PCR reactions are loaded on a denaturing polyacrylamide gel and the electrophoresis is performed at 60 watts, 45 ° C. Dry and expose the gel for 12 to 24 hours on a Kodak XAR film at room temperature. Table 1. Sequences of poly (T) oligonucleotides for differential visualization procedure Promoters 3 '-poly (T) 5'-degenerate promoters In Table 1, the sequences in the first column are identified as SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23, respectively. Sequences are identified in the second column as SEQ ID NO: 24, 25, 26, 27 and 28, respectively. Gel purification and amplification It is found that the bands demonstrating reproducible differential amplifications (see Figure 2a) are for the upstream promoter combination downstream of the 5'-TGCCAGTGGA-3 '-poly-T, 5' -TTT TTT promoter. TTT TTT AG-3 '(SEQ ID NO: 26 and 16, respectively). These gel bands are excised by overlapping the X-ray film and cutting the corresponding piece of dry gel and filter paper. The PCR product corresponding to the fragment of the protein described herein is isolated from the band in Figure 2 (a). The samples are placed in 100 μl of nuclease-free water, incubated for 10 minutes at room temperature, then boiled for 15 minutes. The supernatant is recovered after 15 minutes of centrifugation at 12,000 x g- In order to facilitate the cloning of the products CPR, several changes are made to the reactions. Promoters are used which include Eagl sites of restriction endonuclease in the re-amplification. Based on the results obtained in the differential display analysis, the 5'-TGCCAGTGGA-3 'promoter upstream is replaced by 5'-GTAGCGGCCGCTGCCAGTGGA-3' (SEQ ID NO: 29) and the poly T downstream promoter, '-TTT TTT TTT TTT AG-3', is replaced by 5'-GTAGCGGCCGCT12-3 '(SEQ ID NO: 30). In addition, the reaction volume is increased to 40 μl, the isotope and 20 opposite to 40 cycles are performed. 5 μl aliquots are removed from the PCR reactions and the products are visualized by electrophoresis on a 1% agarose gel followed by staining with ethidium bromide and UV illumination. Cloning of PCR products The reamplified products are purified by phenol / chloroform extraction followed by ethanol precipitation. The resulting DNA granule is resuspended in 17 μl of sterile water and digested at 37 ° C for 1 hour by the inclusion of 10 U Eagl (New England Biolabs), and lx NEB 3 buffer. The restriction endonuclease is heated Eagl inactivated by incubation at 65 ° C for 20 minutes. The pBluescript SK + vector is prepared by digestion with Eagl, followed by dephosphorylation using calf intestinal alkaline phosphatase (CAP, Promega). Restriction digestions are purified using the GeneClean II equipment (Bio 101) followed by electrophoresis on a 1% agarose gel. In a total ligation volume of 10 μl, 2 μl of digested PCR product, 1 μl of digested SK +, 1U of DNA T4 ligase (Gibco / BRL) and lx buffer were incubated at 16 ° C overnight. The bacterial strain of E. coli JM109 is transformed with 1 μl of the ligation product using a BioRad Gene Pulser, then it is plated on plates with LB + ampicillin and incubated overnight at 37 ° C. Colony selection Individual colonies are transferred in duplicate to fresh LB plates and grown overnight at 37 ° C. colonies are transferred to nitrocellulose membrane and denatured in a solution of 1.5 M NaCl, 0.5 M NaOH for 5 minutes, neutralized in 1.5 M NaCl, 0.5 M Tris HCl, pH 8.0 for 5 minutes, followed by 5 minutes of washes in 2x SSC. The membranes are then crosslinked by UV (Stratalinker UV Crosslinker, Stratagen). Prehybridization and hybridization are performed using Quickhyb (Stratagen), following the directions of the manufacturer. Each elevated colony is tested with the corresponding PCR product isolated during gel re-amplification and purification step. The probes labeled with a- [3 P] -dATP are generated using the Prime -It II (Stratagene) equipment. Subsequent to hybridization, the filters are washed twice for 20 minutes in 2X SSC, 0.1% SDS solution at room temperature and exposed to Kodak X-omat autoradiography film overnight at -70 ° C. the positive colonies of the plates are selected in duplicates, they are grown overnight in LB + Ampicline (100 μg / ml) and plasmid DNA is isolated using the Qiaprep Spin Plasmid (Qiagen). Clones are sequenced using the T7 sequencing kit (Pharmacia Biotech). Sequence comparisons are generated using the GeneWorks software package (Intelligenetics). Examination of a cDNA library D. Rerio. An embryonic cDNA library of 6-18 hours of randomly generated D. rerio constructed in UniZAP II is produced (Stratagene). 4.5 × 10 5 independent pfu * are examined using randomization, 336 bp PCR fragment labeled with α- [32 P] -dATP isolated by differential visualization of mRNA as a probe. The filters are prehybridized for 1-4 hours at 42 ° C in 50% formamide, 5X SSPE, 1 Denhardt's solution, 0.2 mg / ml denatured salmon sperm DNA. Hybridization is carried out at 42 ° C by adding denatured probe to the prehybridization solution. The filters are washed twice for 20 minutes in 2X SSC, 0.05% SDS at room temperature and exposed to Kodak XAR film overnight at -70 ° C. The positive plates are taken in 500 μl of SM buffer and subjected to additional cycles of reexamination until they are purified. Positive plaques are exposed to the in vivo excision protocol following the manufacturer's directions (Stratagene). Colonies containing pBluescript are plated on LB + amp plates and grown overnight at 37 ° C. Sequence data are generated using the T7 Sequencing equipment (Pharmacia) and analyzed using the Gene Works software package (Intelligenetics). Full assembled in situ hybridization Regenerates treated with RA and DMSO are isolated 72 hours post amputation (24 hours post AR / DMSO addition), washed in SAF and prepared for complete mounted in situ hybridization. Hybridizations are taken in situ as previously described [White, 1994]. Norther blot analysis Fish are allowed to regenerate their caudal fins for 72 hours. At 48 hours, 10"6 M of all trans RA in DMSO vehicle or DMSO alone is directly added to the tank water, mRNA is prepared using the Micro Fast-Track mRNA isolation kit (Invitrogen, CA), according to Manufacturer's directions Electrophoresis is performed at 3.0-5.0 μg of poly A + RNA, blot is performed and probed using a previously described method [White, 1994] with the full-length zP450RAI cDNA obtained as described below. Agarose gel stained with ethidium bromide shows that equivalent amounts of mRNA are used in the blotting experiments, see bands 2 and 3 of Figure 3 (a) HPLC Analysis Acid media of transfected cells incubated with 575 pM [11] 12-3] RA (Figures 4 (a) and 4 (B)) or 1 μM of AR (Figures 4 (c) and 4 (d)) by either 4 hours (Figures 4 (a) and 4 (c) ) or 24 hours (Figures 4 (b) and 4 (d)) with acetic acid 0. 1%. Lipid soluble metabolites are separated from the aqueous soluble metabolites using a total lipid extraction from the medium [Bligh, 1957]. The metabolism of [11,12-3H] RA to total aqueous soluble metabolites using aliquots of the aqueous soluble extract subjected to β-scintillation counting (see the boxes of Figures 4 (a) and 4 (b)). The lipid-soluble extracts are 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). The metabolites are separated by HPLC using a Zorbax-SIL column (3 μ, 8 x 0.62 cm) 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 The transcripts present in regenerating fin tissue are compared in the presence or absence of RA using the differential visualization PCR technique developed by Liang and Pardee [Liang, 1992] (Figure 2 (a). Use isolate and sequence one of the differential display products which exhibits a dependence on the presence of AR for its expression, indicated by the arrow in Figure 2 (a) .The sequence is identified as SEQ ID NO: 1 and is shown also coen Figure 2 (b) shows the amino acid sequence corresponding to the cDNA, named here, "zP450RAI", in Figure 2 (c) and identified as SEQ ID NO: 2. The BLAST search analyzes reveal sequence homology between ZP450RAI and multiple members of the cytochrome P450 superfamily.The alignments between ZP450RAI cDNA deduce amino acid sequence and that of other cytochrome P450 indicate that ZP450RAI exhibits less than 30% tot to the amino acid identity with members of previously defined subfamilies [Nelson, 1993]. ZP450RAI contains many structural motifs which are common to members of the cytochrome P450 family, including the heme binding domain located at the C-terminal portion of the protein, see Figure 2 (d). The P450RAI family has been designated "CYP26". EXAMPLE 2 Specific cellular induction of ZP450RAI by AR, all trans Northern blot analysis of mRNAs expressed in regenerated tissue isolated from control fish is performed (treated with dimethylsulfoxide) and treated with AR with a full-length zP450RAI cDNA probe. The ZP450RAI transcripts are not detectable in regenerated tissue of the control fish (Figure 3 (a), band 4) but are very notably present in isolated tissues of fish exposed to RA for 24 hours (Figure 3 (a), band 5). Full assembled in situ hybridization is used to determine the cellular location of zP450RAI expression in regenerating fin tissue. Figure 3 (b) shows regenerating fins of control and AR treated fish. Transcripts of ZP450RAI are not detectable in control fin tissue (Figure 3 (b) (i)). In the regenerating tissue of AR-treated fish, transcripts of ZP450RAI are found to be abundant in a layer of epithelial cells that extend between the distal edge of the injured epithelium as indicated by the black arrow head in Figure e3b) (ii) ). Some low-level staining is also observed in the inter-ray tissue as indicated by the black line with arrowhead in Figure 3 (b) (ii). A histological section of a fin treated with RA, taken along the line shown in Figure 3 (b) (iii), is shown in Figure 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. The complete assembled in situ hybridization thus illustrates the usefulness of the nucleic acid of the invention for the location of cytochrome P450RAI ARyst in the whole tissue. EXAMPLE 3 Metabolism of all trans RA by cells transfected by ZP450RAI Retinoic acid is studied as a ZP450RAI substance. The < cDNA is cloned P450RAI of full-length zebrafish in the eukaryotic expression vector pSG5 [Green, 1988]. COS-1 cells are temporarily transfected with either pSG5 or pSG5-zP450RAI and then incubated with either picomolar concentrations of all-trans [11,12-3H] AR or molar concentrations of non-radioactive all-trans RA. COS-1 cells are a kidney-like "fibroblast-like" cell line of African green monkey. The expression of ZP450RAI in COS-1 cells promotes the rapid transformation of AR into both lipid-soluble and aqueous metabolites. See Figures 4 (a) and 4 (b). Fractions of total lipid extracts from transfected cells are initially separated by normal phase HPLC on Zorbax-SIL. The comparison between the extracts of cells transfected with pSG5 and pSG5-zP450RAI indicate that ZP450RAI significantly increases the metabolism of RA. Incubation of cells transfected with ZP450RAI with 575 pMN of [11,12-3] all-trans RA for either 4 or 24 hours results in the accumulation of AR metabolites, one of which co-migrates in a column with synthetic standards 4- OH-RA and 18-OH-RA, and a slightly less polar second metabolite which co-migrates with the standard 4-oxo-RA (Figures 4 (a) and 4 (b)). The other process of chromatography of AR metabolites using other HPLC systems confirms the identity of these two metabolites such as 4-OH-RA and 4-oxo-RA (Table 2). It is possible that the aqueous soluble radioactivity represents glucuronides of AR metabolites or glucuronides of m. The rapid glucuronidation of 4 and 18-hydroxy-AR in extracts of mammalian cells by others has been reported [Wouters, 1992; Takatsuka, 1996]. Table 2 Chromatographic properties of metabolites of Metabolite Retention time (min.) Z - Sil Z-CNC Z-ODSc AR (est.) 2 .57 4.47 19 .92 4-oxo-RA (est.) 4 .79 11. -33 11 .73 4-OH-RA (est.) 5, .17 9.65 12 .65 18-OH-RA (est.) 5. .06 9.53 14, 3 Peak 1 (AR) 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 a HPLA conditions: Zorbax-SIL column eluted with 93.5 / 5/1 / 0.5 H / I / M / AA (1 ml / min) b HPLA conditions: Zorbax-CN column eluted with 93.5 / 5/1 / 0.5 H / I / M / AA (1 ml / min) cConditions of HPLA: Zorbax-ODS column eluted with a linear gradient of 20 min with solvent containing 10 mM sodium acetate which is in the range of 55.45 to 5.95 H20 / MeOH (2 ml / min). A similar pattern of ZP450RAI-dependent metabolism is also observed using a much higher concentration of AR (1 μM). COS-1 cells transfected with ZP450RAI incubated for 4 or 24 hours with 1 μM AR generate two close run peaks which are discernible in a 350 nm HPLC trace shown in Figures 4 (c) and 4 (d), but which are essentially undetectable in cells transfected with control pSG5 (See the boxes of Figures 4 (c) and 4 (d)). These peaks co-migrate with those of standards 4 -oxo-RA and 4-OH-RA, respectively. The spectrophotometric detection of diode array of the peaks generated by <; P450RAI shows that the spectral properties of the two metabolite peaks are coupled to the standard retinoids [in hexane-based solvents: 4-OH-RA,? Max = 350 nm; 4-oxo-RA,? Max = 355? M; in methanol-based solvents: 4-OH-RA,? max = 340 nm; 4-oxo-RA, In this way the invention includes a protein that metabolizes retinoic acid belonging to the family of cytochromes P450, designated CYP26, and generation of the protein in zebrafish caudal fin epithelium that is induced in response to the treatment with AR. While the metabolizing activity of AR has been previously detected in epithelial tissues of several species [Frolik, 1979; Roberts, 1979; Wouters, 1992; Duell, 1992], until now a real enzyme responsible for such activity has been unknown. ZP450RAI is upregulated by treatment with RA and apparently upregulation occurs in a specific group of cells in the wounded epithelium of caudal fins of regenerating zebrafish. It could be relevant to the regulation of the generation of this enzyme in vivo that the experiments with F9 cells where RAR has been selectively ablated indicate that RAR-a and RAR-? they may have a role in the regulation of AR metabolism [Boylan, 1995]. The expression of both RAR-a and RAR-? in caudal fin regenerant is consistent with the suggestion that may be involved in the regulation of expression of P450RAI by AR [White, 1994]. EXAMPLE 4 Cloning of human P450RAI The amino acid sequence corresponding to the zebrafish P450RAI DNA (zP450RAI) (SEQ ID NO: 2) is used to search a tag expression sequence (EST) database. A commercially available EST clone (SEQ ID NO: 11) having a high degree of homology with a C-terminal portion of ZP450RAI (from Glu 292 to Fen 410 of SEQ ID NO: 2) is purchased (Research Genetics, Huntsville, AL) . The clone is said to be from a human infant brain cDNA library (Bento Soares and M. Fatima Bonaldo) and apparently is similarly unpublished. The compared clone is sequenced using the T7 sequencing kit (Pharmacia) and the sequence data is generated using the sofWtare GeneWorks package (Intelligenetics). A cDNA library generated from an NT2 cell line treated with retinoic acid (Stratagene) is commercially available and this product is used for further studies. The cDNA library is tested with a nucleic acid having a sequence identified as SEQ ID NO: 11. Eleven positive hybridizing clones are isolated and purified according to the directions of the manufacturer. Sequence data for these clones is 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. EXAMPLE 5 Isolation of mouse P450RAI. It has been found in the present (results not shown) that the AR can induce transcripts of mRNA which cross-hybridize with a hP450RAI cDNA probe in either the F9 and P19 mouse cell lines that have 4-hydroxylase activity, such as it is described by Blumberg et al.

[Blumberg et al., 1995; Achkar et al., 1996]. The p19 teratocarcinoma cDNA library treated with retinoic acid in Unizap XR lambda (Stratagene) is examined using 32 P-labeled full length human P450RAI as a probe. The nitrocellulose filters are hybridized overnight at 37 ° C in a buffer containing 50% formamide, 1x Denhardt, 5x SSPE, 0.1% SDS, and 100 μg / ml salmon sperm DNA by boiling, and washed in 2x SSC, 0.1% SDS for 2x 15 minutes at room temperature followed by 20 minutes at 37 ° C. From 600,000 plate-forming units, 72 positive ones are isolated. Of these, 18 are purified, as described above, and one having the 1.7 kb full-length cDNA insert in full length homolog to the full-length human clone previously isolated is found. The murine DNA sequence is identified as SEQ ID NO: 31 and the corresponding polypeptide as SEQ ID NO: 32. Figure 9 shows aligned portions of the amino acid sequence of the mouse protein (SEQ ID NO: 32) with those of the human protein (SEQ ID NO: 4) and the zebrafish protein (SEQ ID NO: 2). EXAMPLE 6 Temporal transfection analysis COS-I cells are subcultured 20 hours before transfection which is performed according to the DEAE standard dextran method [Sambrook, 1989; Maniatis, 1982]. Cells are transfected with pE-AR (adrenodoxin expression vector, 1 μg / PlOO plate) and pE-ADX (adrenodoxin reductase expression vector, 1 μg / PlOO plate) together with 3 μg per plate of either pSG5 ( control) or hP450RAI-pSG5 (experimental). [11, 12 -3H] All trans retinoic acid is added (600,000 cpm per plate) 24 hours after transfection. The analysis is performed as described in Example 3 and results obtained in Figures 10 and 11 (a) through 11 (d) are shown. As indicated in the Figures, hP450RAI expression in COS-1 cells promotes the transformation of AR into 4-OH-RA and 4-oxo-RA. Total amounts of 4-oxo-RA and 4-OH-RA produced in the transfected cells are shown in comparison to amounts produced in the control cells in Figures 11 (a) and (b), respectively. Fully, larger amounts of aqueous soluble metabolites are produced in the transfected cells (Figure 11 (c)) and larger amounts of unmetabolized RA are found in the control cells (Figure 11 (D)). The sequence of clones (SEQ ID NO: 11) is prepared as a probe labeled with 32 [P] -dATP to study the inducibility of hP450RAI by AR in several cell lines: HEK293; EL-E; HL-60; MCF10A; LC-T; SK-LC6; MCF7; U937; HepG2; NT2 (See Figures 5 to 7). As can be seen, a variety of expression patterns are observed. The human lung (epithelial) SK-LC6 line appears to expressly constitute the corresponding mRNA. There is apparently some increase in expression in cell lines HEK293 (human embryonic kidney), LC-T (human lung epithelial), HepG2, (liver, epithelial in morphology), NT2 n (human pluripotent embryonic carcinoma) and U937 (human monomyelocytes) ) in response to the addition of AR. There is a high dependence on exposure to AR in the cell line MCF7 (human breast carcinoma (epithelial)). Some of the cell lines show no expression in the absence or presence of RA; EL-E; HL-60 and MCF10A. The 32 [P] -dATP-labeled probe is also used to study expression of hP450RAI mRNA in a human acute promyelocytic leukemia cell line. Experiments are performed using the NB4 cell line, isolated from a patient of human acute promyelocytic leukemia and three retinoic acid-resistant cell lines independently derived from NB4 are independently derived. The results are shown in Figure 8. As can be seen, normal cells express hP450RAI mRNA after treatment with 10"6M AR, whereas such expression seems to be absent for the other cell lines both in the absence and presence of RA. performs the analysis of metabolites of MCF10A and MCF7 cell lines exposed to AR, MCF10A cells that have not exhibited mRNA expression and the last one that has exhibited a high dependency on mRNA expression not exposed to AR. The results are shown in Figures 12 (a) to 12 (c). Consistent with the results shown in Figure 5, the results shown in Figure 12 (a) indicate. The results shown in Figure 12 (a) indicate little difference in the profiles of lipid-soluble activity of the MCF10A cell line exposed to AR and the control. The last two bars of Figure 12 (c) indicate that the total aqueous soluble metabolites are approximately the same for both the MCF10A induced and control cells. As indicated in Figure 12 (b), the MCF7 cell line exposed to AR has an elution profile which indicates significantly higher concentrations of 4-OH-RA and 4-oxo-RA than the same cell line not exposed to AR . Figure 12 (c) indicates that the amount of the total aqueous soluble metabolites of the MCF7 cells exposed to AR is much greater than that of the control cells. Again, these results are consistent with those obtained in the Northern blot analysis shown in Figure 5 for the MCF7 cell line. EXAMPLE 7 Generation of a Stable Cell Line using human P450RAI: HeLa cells expressing the sense construct For expression in HeLa cells, the human cytochrome P450RAI cDNA is inserted into the Xhol-Notl sites of the vector multiple cloning site on the basis of to the Epstein-Barr virus pCEBV7 [Wilson, 1995]. Stable transfection is carried out via the calcium phosphate method [Sambrook, 1989]. Before the day of transfection, the HeLa cells are sown in 3.0 x 10 cells per 100 mm of plaque. Approximately 12 μg of DNA is transfected per plate and triplicate plates are used for transfection. Selection using hygromycin B begins three days after transfection and is continued for approximately three weeks until the development of foci in the plaques. The concentration of hygromycin B (100 μg / ml) is chosen by selecting cells with high construction expression. A death curve is determined before selection, which shows that 50 μg / ml of hygromycin is sufficient to kill 50% of the cells in the body. •days. Confirmation of selected HeLa cells expressing the sense construct is determined by Northern blot analysis and probed with full-length hP450RAI cDNA (data not shown). Microsomes are prepared from HeLa cells transfected with pCEBV7 alone (Hela P) or Hela cells expressing P450RAI (HeLa RAI) and exposed to radiolabeled AR for ninety minutes. The results are shown in Figures 13 (a) and 13 (b), in which it can be observed that when microsomes prepared from these cells are incubated with RA, only that of HeLa RAÍ show any transformation of retinoic acid to retinoic acid 4 - hydroxy or 4-oxo retinoic acid. EXAMPLE 8 Expression of a fusion protein containing human P450RAI The hP450RAI cDNA is inserted into the fusion vector of the GST gene, pGEX3X using the Sma 1 site. The generated construct lacks 177 bp of the N-terminal portion of the hP450RAI cDNA, which encodes a hydrophobic region characteristic of a signal sequence. E. coli cells (JM109) are used for vector expression and construction. After examining for positive colonies expressing the construct in the proper orientation, expression of the fusion protein is performed according to the manual provided by Pharmacia. The GST-hP450RAJ fusion protein is generated via electroporation of E. coli cells (JM109) with the construct, followed by the induction of gene expression. The expression of the fusion protein is under the control of the tac promoter which is inducible by the lactose analog, isopropyl beta-thiogalactosidase (IPTG). The cultures in LB-ampicillin broth are grown overnight. The next day, approximately 1: 100 dilution factor of the culture is transferred overnight to a fresh tube containing LB-ampicillin broth. The culture is allowed to grow by three hours and is induced with IPTG at a final concentration of 0.1 mM for an additional 2.5 hours. After induction, the culture is centrifuged at 7,700x g for 10 minutes at 4 ° C to pellet the cells. The pellet is resuspended in a ratio of 50 μl culture / ml of Ix SAF cooled in ice. The cells are lysed with a sonicator at 30 second intervals while immersing in ice water. Triton X-100 is added to. the cells used at a final concentration of 1% and incubate the mixture for 30 minutes at 4 ° C to allow solubilization. The suspension is subjected to centrifugation at 12,000 x g for 10 minutes at 4 ° C. The binding of the GST portion of the fusion protein to glutathione beads is used to purify the protein. 2 ml of a 50% suspension of reduced glutaione beads equilibrated with IX SAF per 100 ml of sonicated mixture of the above is used. After the addition of the beads, the material is gently stirred for 30 minutes at room temperature to allow binding of the fusion protein to the beads. The suspension is centrifuged at 500 x g for 5 minutes to separate glutathione beads with bound fusion protein from other cellular components. The glutathione beads are washed three times with cold SAF to remove unbound material specifically. The fusion protein is eluted from glutathione beads using an elution buffer containing reduced glutathione. Approximately 1.0 ml of the glutathione elution buffer is used per 100 ml of sonicated suspension. The bound glutathione beads are allowed to mix at room temperature for 10 minutes to elute the fusion protein. Centrifugation of the beads eluted at 500 x g for 5 minutes is performed. Both the elution and centrifugation is performed three times. Figures 14 (a) and (b) show the SDS-PAGE results of the GST-hP450RAI fusion protein. It is generally possible to perform some functional tests with the GST protein covalently bound to the protein of interest, but elimination is preferred for the production of antibodies. The GST-hP450RAI fusion protein contains a site recognized by factor Xa. Treatment of the fusion protein eluted with factor Xa produces both the GST protein and hP450RAI protein. Alternatively, it is possible to cleave the fusion protein before its elution from the beads, but inadequate cleavage can occur. Approximately 10 μg Factor Xa / g fusion protein is used. The material is mixed gently and incubated for approximately 2-16 hours at room temperature. The completion of the digestion by electrophoresis is checked at several time points. To purify the protein of interest from the GST protein, suspension linkage of cleaved fusion protein with glutathione beads is performed, followed by centrifugation. The protein of interest remains in the supernatant. The GST fusion protein can be tested immunologically or biochemically. Biochemically, the treatment of the GST fusion protein with the substrate GST, l-chloro-2,4-dinitrobenzene (CDNB), indicates the amount of the fusion protein axis. The GST protein catalyzes the conjugation of CDNB with glutathione, resulting in a product of CDNB-glutathione with a strong molar absorptivity at 340 nm. Immunologically, a Western blot of the fusion protein can be tested with an anti-GST antibody which can be easily detected using a secondary antibody such as a goat anti-alkaline phosphatase conjugate IgG, and the standard colorimetric reaction. EXAMPLE 9 Mapping of human P450RAI A 1.3 kb cDNA of hP450RAI is mapped using an artificial chromosome library derived from P-1 (CAP). The cDNA and genomic CAP were mapped by fluorescence in situ hybridization [Lichter, 1990] to human lymphocyte chromosomes counterstained with propido iodide and DAPI. The biostained probe with avidin-fluorescein isothiocinanto (FITC) is detected. Images of metaphase preparations are captured by thermoelectrically cooled charge coupled chamber (Photometrics, Tucson, Arizona). Separate images of chromosomes banded by DAPI are obtained [Heng, 1993] and chromosomes with FITC target. Hybridization signals are acquired and emerge using pseudo-colorful image analysis software (DAPI) and pseudo-yellow coloring software (FTIC) [Boyle, 1992] and electronically overlap. It is found that the positive hybridization signals are located at 10q23-24. The band assignment is determined by measuring the length of the fractional chromosome and analyzing the band pattern generated by the image against dyeing with DAPI. EXAMPLE 10 Transcription regulation P450RAI cloning of P450RAI promoters Cloning of the zebrafish P450RAI promoter. An adult zebrafish genomic library (1.0 x 10e pfu) is examined with the full-length cDNA corresponding to ZP450RAI and positive plaques purified by examination. secondary and tertiary education. Lambda DNA corresponding to positive plates is prepared, and the inserts of these clones are cleaved by restriction enzyme digestion with Notl and sub-alluded to the SK + plasmid (Stratagene). The genomic alleles are analyzed by restriction enzyme digestion using enzymes from the SK + polylinker, followed by Southern blotting using an oligonucleotide (5'-GTAGCACGGATGGTG-3 ') which hybridizes to the nucleotide sequence encoding the N-terminus of the ZP450RAI cDNA. to identify fragments of the genomic clones that encode the N-terminal region. The 772 base pair PstI fragment which hybridizes with the oligonucleotide probe is purified, ligated into the SK + vector and sequenced using the Core Facility for Protein / DNA Chemistry from Queen's University, Kingston, Canada. The sequence analysis identifies this clone as containing the putative initial methionine followed by 129 base pairs of the coding sequence, plus 651 nucleotides upstream (5 '). Within this 772 base pair fragment, a 402 base pair HindIII fragment is found to contain the retinoic acid response element (RARE). This fragment is subcloned into the pGL3B luciferase vector, while forward and reverse orientations, (Promega) for temporal transfection analysis. Cloning of the human P450RAI promoter. The full-length hP450RAI cDNA is used as a probe to identify clones of CAP (artificial chromosome Pl) which contain the hP450RAI gene. The hP450RAI cDNA is sent to the Canadian Genome Analysis and Technology Program at the Hospital for Sick Children in Toronto, Ontario, Canada to examine the CAP libraries. Five CAP clones are obtained from this examination, which is verified to contain the hP450RAI gene by restriction enzyme digestion and Southern blotting using the full-length hP450RAI cDNA as a probe. It is found that one of these clones, 245C7, hybridizes to an N-terminal probe of hP450RAI. The probe used has a Notl fragment of approximately 130 bp generated from hP450RAI cDNA. The digestion and Southern blotting of clone 245C7 identifies a Ba Hl fragment of 3.5 kb which hybridizes with the Notl fragment. This fragment is subcloned into the SK + plasmid and the sequence data are generated in Core Facility for Protein / DNA Chemistry at Queen's University, Kingston, Canada. The comparison of the generated sequence data with the c hP450RAI DNA identifies this 3.5 kb clone as containing the potential initial methionine and approximately 675 bp upstream (5 '). Cloning of the mouse P450RAI promoter. A mouse P450RAI genomic DNA clone approximately 17 kb long is isolated from an SV129? DASH library and subcloned in SK. DNA prepared from this plasmid is digested with several restriction endonucleases, an electrophoresis is performed on an agarose gel, and Southern blotting is performed on nitrocellulose. The resulting blot is hybridized with a 230 base pair probe labeled with 32 P from the N-terminal region of a mouse P450RAI cDNA clone. It is found that a Sacl fragment of approximately 520 base pairs in length hybridize strongly to the probe. This fragment is subcloned in SK cleaved with Sacl. The sequence analysis reveals the presence of RARE type DR5 in the proximal promoter. The flanking of this RARE are two BssHII sites 193 separate base pairs. This fragment of BssHII of 193 base pairs is subcloned to isolate clones with BssHII fragments in both forward and reverse orientations. Within the fragment of 193 base pairs is also the TATA box, 45 base pairs downstream of RARE. Also included in the BssHII fragment of 193 base pairs are 83 base pairs of DNA upstream of RARE DR5. The existence of retinoic acid response elements (RARE), which bind retinoic acid (RAR) receptors and retinoid x RXR receptors, is known. In this way, the three P450RAI promoters of different species described above are examined for the presence of RARE, which indicates that the regulation of P450RAI expression is at the level of transcription. RAREs bind RARs in the heterodimeric form and typically include a direct repeat of the consensus motif 5'- (A / G) G (T / G) TCA-3 '. Sequences repeated by 1, 2 or 5 nucleotides can be separated. Figure 5 shows the promoter sequences for P450RAI genes from zebrafish, human and mouse. There is a highly conserved motif, indicated by the arrows, which corresponds to a consensus retinoic acid response element known as DR5, which consists of a direct repeat of an AGTTCA motif separated by 5 nucleotides. This motif in the P450RAI promoters extends to the TGAACT sequence (the complement of AGTTCA). There is also a highly conserved region that stretches on either the DR5 side, the entire region that is in box. The degree of conservation of this region of the promoter among the three species suggests that this region is important for controlling the response of RA and that RAR / RXR are acting together or competing with other binding factors to this region whose identities are currently unknown. A portion of the conserved region corresponds to the sequence 5'-CAATTAAA-3 'which corresponds very closely to a hemodominium binding motif, suggesting that the frame proteins may facilitate or compete with RAR / RXR heterodimers in the regulation of expression P450RAI EXAMPLE 11 Transfection with the mouse P450RAI promoter A fragment of 195 base pairs of genomic DNA containing a portion of the putative promoter for mP450RAI in the luciferase vector, pGL3B (Promega) is cloned. For analysis of promoter activity, HepG2 cells are transfected in 6-well boxes with 2 μg of BssHI-pGL3B sense or antisense constructs, described in Example 190, using 5 μl of lipofectAMINE reagent (Gibco, BRL). On the first day, 48 hours before transfection, the cells are re-plated in 6-well plates, with 2.5-3 ml of Minimum Essential Medium (MEM) (Gibco, BRL) + 10 fetal calf serum. % (Gibco, BRL). On the third day, the cells are generally approximately 50% confluent (HepG2). Before starting transfection, the medium is replaced with fresh medium and the cells are allowed to grow while preparing the DNA / lipofectamine mixture. Individual wells of a 48-well plate are mixed with 1-5 μg of DNA in 100 μl of optiMEM (Gibco, BRL) and 5 μl of lipofectAMINE reagent in 100 μl optiMEM, by adding DNA / optiMEM to lipofectAMINE / optiMEM with mixing soft. This is allowed to settle for 15 minutes to 1 hour at room temperature. 800 μl of optiMEM are added to each well to obtain a final volume of 1 ml. These cells are washed twice in lx SAF and once with optiMEM. 1 ml of DNA / lipofectamine / optiMEM mixture is added to the cells and incubated for 20 hours at 37 ° C. The effects of retinoic acid on promoter activity are analyzed by cotransfection with various amounts (100 ng to 1 μg) of expression vectors including cDNA sequences encoding the retinoic acid receptor of gamma zebrafish (RAR-?) And receptor x alpha zebrafish retinoid (RXR-a). Comparisons are made between the cells transfected with the control vector pGL3B, those with the construction with sense and those with the anti-sense construct incubating a group of the cells transfected with DMSO and the other group with AR 10"GM in DMSO. On the fourth day, the medium is removed and replaced with fresh medium (SAF + 10%) .The AR or vehicle is added as required to the wells and mixed gently by moving the plate.It is added to a plate of 6 wells. AR 10"3 M in DMSO. The control wells receive 3 μl of MSO. The plates are covered in metallic foil and incubated for 24 hours at 37 ° C. On the fifth day, the cells were harvested by removing the medium and washing twice with 1CX SAF. Then, on ice, add 100 μl of lysis buffer (1% Triton X-10Q, 25 mM glycylglycine, pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT (added just before use), and the cells are scraped from the bottom of the plate and transferred to 1.5 ml of microcentrifuge tube, rotated for 65 minutes at 12,000 x g, and the supernatant recovered. To assay the luciferase activity, 20 μl of supernatant of lysed, granulated cells is transferred to a fresh tube. 80 μl of the luciferase assay buffer is added and a reading in millivolts is immediately taken in a luminometer. The luciferase assay buffer has 20 M tricine, 1.07 mM (MgC03) 4Mg (OH) 2 * 5H20, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 0.27 mM Coenzyme A, 0.47 mM Luciferin, 0.53 mM ATP. The results are shown in Figure 16, in which it can be seen that the increased luciferase activity is observed in the presence of AR, RXRa and RARa, for both orientations of the promoter sequence, although the improvement seems to be greater for the construction with sense. EXAMPLE 12 Inhibition of P450RAI induction by 4-hydroxyphenylretinamide It has recently been reported that 4-hydroxyphenylretinamide (4-HPR) inhibits the catabolism of AR induced by AR by NB4 cells [Taimi, 1997]. It is suggested that 4-HPR can inhibit the cytochrome P450 enzymes responsible for AR oxidation to competitively inhibit AR catabolism. However, any such enzymes are not identified, no explanation of the 4-HPR inhibition nature of AR catabolism is provided, and no evidence of metabolism by 4-HPR is observed. Experiments are thus carried out to determine the effect of 4-HPR on the induction of P450RAI. The Figure 17 illustrates the ability of the synthetic retinoid, 4-HPR to inhibit the induction of P450RAI by AR. MCF7 cells are grown in culture in minimal essential medium (MEM) (Gibco) supplemented with 10% fetal calf serum, insulin (0.01 mg / ml), MEM non-essential amino acids (as directed by the manufacturer-Gibco ), sodium pyruvate (500 nM), L-glutamine (2 mM) gentamicin (10 μg / ml), penicillin (5 μg / ml), streptomycin (5 μg / ml), and fungizone (200 ng / ml). MCF7 cells cultured in parallel for 12 hours are treated with: 10 μM 4 HPR; 1 μM 4 HPR; 1 μM AR; and 1 μM AR and 1 μM 4-HPR. At the end of the 12 hour treatment, total RNA is extracted from the cells using TRIzol reagent (as indicated by the manufacturer Gico). The P450RAI message is analyzed in the total RNA preparations by northern blot hybridization. The blotting is retested with a probe corresponding to GAPDH cDNA to control an equivalent load of RNA in each band of the blotting. The results indicate that the 4 -HPR treatment does not induce the P450RAI message. As expected, the AR treatment of MCF7 cells results in a marked induction of P450RAI message after 12 hours of incubation. However, when the cells are treated with AR in the presence of 10 μM 4-HPR, there is a remarkable suppression of induction by P450RAI. EXAMPLE 13 Expression P450RAI in the presence of retinoic acid Figure 18 shows an expression course of cytochrome p450 RAI after treatment of MCF7 breast epithelial adenocarcinoma cells with 1 μM all trans retinoic acid. In this northern blot analysis, the total RNA is extracted at the indicated time points and transferred to nitrocellulose after electrophoresis in 1% agarose, 0.66 M formaldehyde gel. The nitrocellulose is then tested with human cytochrome p450RAI cDNA or GAPDH cDNA. to control the equivalence of loading mRNA. This shows that after 3 hours of incubation with retinoic acid, MCF7 cells express high levels of p450RAI message and after 12 hours of exposure, the message decreases rapidly, possibly indicating that the metabolic activity of induced p450RAI protein is reducing the concentration of active retinoic acid in the surrounding medium. This strongly suggests that the induction of P450RAI in MCF7 cells forms a self-regulatory negative feedback loop. EXAMPLE 14 Expression P450RAI in the presence of retinoic acid and ketoconazole Figure 19 shows a time course of expression of P450RAI mRNA in MCF7 cells similar to that described in Figure 18, except that the effect of cytochrome P450 inhibitor ketoconazole is examined. broad spectrum in the expression of P450RAI. In the absence of ketoconazole, bands 1 to 5 in Figure 19 are shown a time course similar to that shown in Figure 18. In cells that are exposed to 1 μM ketoconazole (replaced every 12 hours after initial treatment) , the message of cytochrome P450RAI is detectable at high levels at 24 and 48 hours point of time indicating that the decomposition of retinoic acid by the cytochrome P450 inhibitor can be inhibited and that the metabolism of P450RAI may be responsible for the deep fall in the P450RAI message in the absence of ketoconazole. The example illustrates a method for identifying P450RAI inhibitors. EXAMPLE 15 Expression P450RAI in the presence of Am580 Figure 20 shows a time course of expression of P450RAI mRNA in MCF7 cells similar to those described in Figure 18, except that a comparison is made between the retinobenzoic acid derivative Am580 and retinoic acid all trans. The retinoic acid shows a typical P450RAI message induction time course, bands 1 to 4, Figure 20. Am580 induces the P450RAI message at levels comparable to those observed after treatment with retinoic acid. Notably, while the message of P450RAI decreases drastically between 24 and 48 hours, for the cells treated with retinoic acid, the levels of P450RAI in cells treated with Am580 remain high at this point in time. This indicates that Am580 synthetic retinoid is recovered for metabolism in these cells and illustrates the utility of identifying such compounds for therapeutic use. For example, resistance to treatment with retinoic acid observed in acute promyelocytic leukemia due to increased retinoic acid metabolism [Warrell, 1994] can be avoided by treatment with a retinoid resistant to metabolism. The P450RAI protein can be a useful agent to screen for these types of compounds. EXAMPLE 16 Monoclonal Antibodies for P450RAI Monoclonal Antibodies (Mab's) specific for P450RAI are useful, for example, for diagnostic purposes such as to determine p450RAI protein levels in the identification of normal and tumor tissues which metabolize AR. To produce these antibodies, purified P450RAI protein is prepared. The human P450RAI protein is produced in bacterial cells as a fusion protein with glutathione S transferase using the vector pGEX2 (Pharmacia). This allows the purification of the fusion protein by affinity chromatography GSH. In another method, P450RAI is expressed as a fusion protein with the bacterial maltose binding domain. In this way the fusion protein of the bacterial extracts is recovered by passing the extract on a column of amylose resin followed by elution of the maltose fusion protein. For this fusion construct, the vector pMaIC2, commercially available from New England Biolabs, is used. This vector has been used in the past, for example, for over expressing protein nucelar receptors which are recovered in high yields by functional studies and the production of specific antiserum to receptor [Ohno, 1993]. The preparation of a second fusion protein is also useful in the preliminary examination of Mab's. The generation of hybridomas expressing monoclonal antibodies is performed recognizing the P450RAI protein as follows: BALB / c mice are injected intraperitoneally with protein / adjuvant three times at one month intervals, followed by final injection into the tail vein quickly prior to administration. cellular 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 performed as described in more detail below. The fused cells are brought to 96-well plate with peritoneal exudate cells and irradiated BALB / Cc spleen cells as feeder layers and selection is made with hypoxanthine, aminopterin, and thymidine (HAT medium). An ELISA assay is used as an initial screening procedure. 1-10 μg of purified P450RAI (cleaved from the fusion protein) in SAF is used to cover individual wells, and 50-100 μl per well of hybridoma supernatants is incubated. Anti-mouse antibodies conjugated with horseradish peroxidase are used for the colorimetric assay. As a secondary examination, MCF-7 cells from breast epithelial carcinoma are used as a source of P450RAI determinant. As indicated above, untreated MCF-7 cells do not exhibit detectable expression of P450RAI message and no detectable AR metabolising activity, while there is rapid and strong induction of both AR treated cells. In this way the untreated MCF-7 cells serve as a negative antecedent control. Aliquots of MCF-7 cells are taken in 96-well plates one day before the assay. The cells are fixed and permeabilized with methanol. Hybridoma supernatants are added and anti-mouse antibodies conjugated to fluorescein isothiocyanate (FITC) are used to examine using a standard fluorescence microscope. Positive hybridomas are cloned by limiting dilution and grown on a large scale by freezing and antibody production. Several positive hybridomas are selected for use in western blotting and immunochemistry, as well as cross-reactivity with P450RAI proteins of different species. The selected Mabs are useful for monitoring the expression levels of P450RAI protein after treatment with RA in cell culture and tissues. The expression of the P450RAI protein may be a prognostic indicator to determine if a particular tumor will respond to treatment with RA. There is also a wide intersubject variability in the metabolism of RA-based and there is evidence suggesting that subjects with a high rate of AR 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 for samples of surviving tumor tissue for expression of P450RAI. Protocol for the production of mouse hybridomas Fusion. The feeder cells (spleen cells and peritoneal exudate) are brought to plates. 24 to 48 hours before the fusion, the mouse myeloma cells are removed from the drug (8-azaguanine 20 μg / (ml) and counted to ensure that there are at least 50 × 10 5 cells. of PEG 4000 in a glass tube for 15 minutes and kept at 60 ° C for use or alternatively stored at room temperature and re-melted in a 60 ° C water bath when necessary. The final injection into the vein of the tail is given intravenously The fusion of spleen cells immunized 3 or 4 days after the intravenous injection is performed separately The spleen of each animal is collected separately. of eyes by ELISA if desired.A single cell suspension is prepared using a Teflon pistil and decanting connective tissue.Ix suspension is washed in serum free medium.Each spleen has approximately 10 x 10e cells.The myeloma cells are harvested, they are counted and washed in serum-free medium. The cells are then fused. Prepare a small beaker of water, and serum-free medium (37 ° C) and melt PEG at 50-60 ° C. Immunized spleen cells and myeloma cells are mixed in a 50-TC tube (the recommended ratios vary from 1: 1 to 2: 1) and the cells are washed once with serum-free medium. The supernatant is carefully discharged. Immediately add 2.4 ml of pre-warmed serum free medium with pipette to the melted PEG and mix, maintaining the temperature at 37 ° C in a hot water beaker. The PEG should be light pink. If it is yellow, another aliquot should be used. 0.5-1.0 ml of PEG are added in drops to the cell granule in 1 minute with soft tube rotation or gentle agitation to ensure mixing. The tip of the addition pipette is placed directly in the cell pellet. Gently rotate the tube in a 37 ° C water bath for 90 seconds with the blunt end of a 3 ml pipette tip and 10 ml of serum free medium added slowly in 6-10 minutes while gently rotating the tube to bring the volume up to 20-50 ml. The tube is maintained at 37 ° C for at least 20 minutes to obtain cell fusion and then the cells are washed 2x with serum-free medium. The cells are centrifuged and gently resuspended in 100 ml of pre-heated medium + 10-20% SAF. 100 μl / well in aliquot in 96-well plates. Assuming a spleen fused with 100 x 105 myeloma fusion standard cells, approximately 10 plaques are necessary. On the next day, 100 μl of medium and 100 ul of added 2x HAT are removed. Feed with medium lx HAT for 1 to 3 weeks, then feed HT medium (ie, eliminate fear 1/2 H T and replace with equal volume of HT medium). Preparation of peritoneal exudates and spleen feeder cells. It is sprinkled mouse sacrificed with 70% alcohol, the skin is stripped and separated, it is taken with care not to cut the peritoneum. The peritoneum is elevated with forceps and a needle is inserted; 5 ral of serum free medium is injected slowly. The abdomen is massaged and the fluid is slowly sucked, collected in a sterile tube and kept on ice. The volume is brought to 5 ml. The spleen is obtained and placed in a sterile tube containing serum-free medium. The spleen is gently crushed with a sterile Teflon pistil. They are allowed to sediment the clots and decant in clean tube, care is 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 in 96-well plates [one spleen / 10 plates (approximately 2-5 x 10 5 cells / well) and cell suspension of peritoneal exudate (PECS) (<3 x 105 cells / well) in a total volume of 100 μl is incubated at 37 ° C until ready for use. Discussion It is possible to compare the ZP450RAI, mP450RAI 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 497 amino acids of hP450RAI (SEQ ID N0: 4). See Figure 9.

On this basis, there is approximately 68% homology between human proteins and fish. The degree of homology between the two amino acid sequences is slightly higher towards the C-terminus than at the N-terminal region. It also seems that the nucleic acid sequences encoding the conserved Met-Lis-Arg-Gln-Lis sequence can be used. (amino acid numbers 70 to 74 of ZP450RAI) as a probe to obtain corresponding proteins from cDNA libraries of other species. The amino acid identity between the human P450RAI sequences of mouse is greater than 93%. As shown, the AR-induced expression of a protein by the cells described herein involves a regulatory sequence which is located upstream of the DNA coding sequence it controls. As it happens in nature, the protein is P450RAI, whether in the cells of the zebrafish, human, mouse or other organism. Such a cell can be modified by incorporating DNA encoding a different protein in the region of the gene which encodes P450RAI, as exemplified above with DNA encoding luciferase. One aspect of the present invention is thus a regulatory DNA sequence, responsive to the presence of AR, operably linked to a sequence encoding the protein and incorporated into a cell that produces genetically engineered engineered protein. The production of the desired protein can be induced by exposure of the cell to AR. Antisense or oligonucleotide nucleic acids (RNA or preferably DNA) that inhibit production of P450RAI induced by cellular AR can be used to inhibit the metabolism of AR by P450RAI [Monia, 1996]. Antisense oligonucleotides, typically 15 to 20 bases in length, linked to sense mRNA or pre-mRNA region encoding the protein of interest, which may or may not inhibit the translation of protein-bound mRNA. The cDNA sequence encoding hP450RAI can be used in this way to design a series of oligonucleotides which expand 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 hP450RAI activity or using antibodies to examine inhibition of P450RAI synthesis. The most suitable mRNA target sites include 5 'and 3' untranslated regions as well as the start codon. Other regions can be found to be more or less effective. Alternatively, an antisense or oligonucleotide nucleic acid can be linked to P450RAI DNA or regulatory sequences. Unlike the metabolism of reducing AR by inhibiting the expression of P450RAI genes at the level of nucleic acid, activity of the P450RAI protein can be inhibited directly by binding to an agent such as, for example, a suitable small molecule or a monoclonal antibody. Thus, the present invention includes a method for screening drugs for their effect on retinoic acid-inducible protein activity. The method includes exposing the protein to a prospective inhibitory drug and determining the effect on protein activity. The measured activity may be hydroxylation of a retinoid, particularly all-trans retinoic acid, or hydroxylation of a retinoic acid, particularly all-trans retinoic acid, in the 4-position of the β-ionone ring thereof. To examine drugs for use in humans, the same hP450RAI is particularly useful for testing the efficacy of such drugs.

Prospective drugs can also be tested for inhibiting the activity of other cytochromes P450, which are desired to be inhibited. In this form, drugs which selectively inhibit hP450RAI other than P450 can be identified. Another system for screening potential inhibitors of a P450RAI protein includes a stably transfected cell line that is incorporated into the same DNA of a reporter gene (e.g., β-galactosidase, firefly luciferase, or the like) and P450RAI, in which the expression of both genes is inducible by exposure of the cells to AR. The expression of the reporter gene provides a measure of the induction of the expression system and therefore provides an indication of the amount of the AR present. Exposure of the cells to AR leads to AR metabolism and, with time, such metabolism leads to a decrease in the degree of induction which is indicated by the reporter protein. The exposure of the cells to AR in the presence of an agent that inhibits the metabolism of P450RAI from RA results in decreased AR metabolism, whereas exposure of the cells to AR in the presence of an agent that does not inhibit the metabolism of P450RAI of AR has no effect on AR metabolism. In this way a comparison of expression of the reporter gene in the presence of AR alone and in the presence of both AR and potential inhibitory drug gives a measure of the drug's efficacy in the metabolism of AR inhibition by the P450RAI protein. A system for screening potential inhibitors of a P450RAI protein includes a cell line in which the endogenous P450RAI gene is not present or does not work or is not expressed. In this cell line, a cytochrome P450RAI expression vector and an AR-inducible reporter gene are incorporated in such a way that exposure of the cell line to AR results in the metabolism of AR by the expressed P450RAI protein and a degree of induction of the reporter gene based on the remaining active AR. The addition of a P450RAI inhibitor will decrease the rate of AR metabolism / degradation and therefore increase the activation / induction of the AR responsive reporter gene. Given the high degree of conservation of the promoter regions of the P450RAI promoter regions of mouse, human and zebrafish, it is likely that AR regulates the expression of <; P450RAI through a transcriptional mechanism involving the RARE conserved in the promoters of the three species. This is supported by studies which show the rapid and AR-dependent expression of P450RAI in a number of cell lines. Since the P450RAI message is very strongly induced a reporter gene can be a useful indicator of AR activity in vivo as well as in vitro. In this way, the P450RAI promoter linked to a reporter gene provides a tool to examine retinoids or other compounds which have the ability to block or inhibit P450RAI induction. For example, the P450RAI reporter gene can be stably or temporarily introduced into a cell line in such a way that when the cells are exposed to a certain level of retinoid or other agent, the concentration will be reflected in the activity of the reporter gene. Such transfection assays can be performed in a manner similar to those described by Petkovich et al., For example [Petkovich, 1987; Ohno, 1994]. In this way the invention provides a system for screening for potential AR catabolism inhibitors by a P450RAI protein. The system includes a transfected cell line that incorporates in the DNA DNA of a reporter gene, for example the luciferase gene exemplified above, the P450RAI gene is omitted, ie the reporter gene is under the control of the native promoter of the P450RAI gene. The 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 the exposure of the cells to AR. The exposure of the cells to AR in the presence of an agent that inhibits induction of the expression system indicates that the agent is a potential AR catabolism inhibitor, ie, it provides a measure of the agent's efficacy as a drug in inhibiting the expression of the P450RAI gene and in this way the metabolism of AR. There is a possibility that cellular retinoic acid binding protein (PEARL) [Adarason, 1993]} is involved in the binding of a retinoid substrate to a P450RAI protein of the present invention. The effect of the presence of PEARL, derivatives, synthetic fragments or analogs thereof can be determined according to examination methods 4e of the present invention, the effectiveness of such agents in increasing AR metabolism can also be determined. It will of course be understood, without the intention to be limiting therefore, that a variety of amino acid substitutions are possible while preserving the structure responsible for retinoid metabolizing activity of the proteins described herein. Conservative substitutions are described in the patent literature, such as, for example, in U.S. Patent No. 5,264,558. In this way, it is expected, for example, that it may be possible to exchange neutral non-polar aliphatic amino acids, glycine, alanine, proline, valine and isoleucine. Similarly, substitutions between neutral polar aliphatic amino acids, serine, threonine, methionine, asparagine and glutamine may possibly be made. Probably substitutions can be made between the charged acidic amino acids, aspartic acid and glutamic acid, as substitutions can be made between the charged basic amino acids, lysine and arginine. Substitutions between aromatic amino acids, including phenylalanine, histidine, tryptophan and tyrosine, may also be possible. These types of substitutions and exchanges are well known to those skilled in the art. Other substitutions may be possible. Of course, it could also be expected that the higher the percentage of homology, ie, sequence similarity, of a variant protein corj a naturally occurring protein, the greater the retention of metabolic activity. Of course, as proposed by protein variants having the activity of P450RAI as described herein to be within the scope of this invention, are therefore nucleic acids encoding such variants. An additional advantage can be obtained through the chimeric forms of the protein, as is known in the art. In this way, an ADJST sequence encoding the entire protein, or a portion of the protein, for example, can be linked with a sequence encoding the C-terminal portion of E. coli β-galactosidase to produce a fusion protein. . The GST-CYPRAI fusion proteins are described in the previous examples. An expression system for human respiratory syncytial virus F and G glycoproteins is described and in U.S. Pat. ,288,630 filed on February 22, 1994 and references cited therein, for example. A recombinant expression vector of the invention can be a plasmid, as described above. The recombinant expression vector of the invention may also be a virus, or a portion thereof, which allows the expression of a nucleic acid introduced into the viral nucleic acid. For example, retroviruses defective in replication, adenoviruses and adeno-associated viruses can be used. The invention provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a form which allows expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to the nucleotide sequence of SEQ ID NO: 3 , SEQ ID NO: 5, SEQ ID NO: 31. Regulatory sequences operably linked to the antisense nucleic acid can be chosen which directs the continuous expression of the antisense RNA molecule in a variety of cell types, for example a viral promoter and / or enhancer, or other regulatory sequences, which direct specific expression of the tissue or cell type of antisense RNA.

The recombinant expression vectors of the invention can be used to make a transforming host cell that includes the recombinant expression vector. The term "transformant host cell" is proposed 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 proposed to encompass the introduction of nucleic acid (for example 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. The nucleic acid can be introduced into mammalian cells via conventional techniques such as coprecipitation with calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, electroporation or microinjection. Suitable methods for transforming and transfecting host cells are known [Sambrook, 1989]. The number of host cells transformed with a recombinant expression vector of the invention by techniques such as those described above will depend on the type of recombinant expression vector used and the type of transformation technique used. Plasmid vectors introduced into mammalian cells are integrated into the DNA of the host cell at only a low frequency. In order to identify these members, a gene containing a selectable marker (e.g., antibiotic resistance) is generally introduced into the host cells together with the gene of interest. Preferred selectable markers include those which confer resistance to certain drugs, such as G418 and hygromycin. Selectable markers can be introduced into a plasmid separated from the nucleic acid of interest or, preferably, introduced into the same plasmid. Transformed host cells can be identified with one or more recombinant expression vectors containing a nucleic acid of the invention and a gene for a selectable marker by selecting cells using the selectable marker. For example, if the selectable marker encodes a gene that confers resistance to neomycin (such as pRc / CMV), the transforming cells can be selected with G418. The cells that have the selectable marker gene will survive, while the other cells will die. Certain nucleic acids of the invention encode proteins which "have a cytochrome P450RAI biological activity". The biological activity of a cytochrome P450RAI is the ability to oxidize a retinoid. Such activity can be tested as described above.

The invention provides purified proteins having biological activity of P450RAI. The terms "isolated" and "purified" each refer to a protein substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. In certain preferred embodiments, the protein having P450RAI biological activity comprises an amino acid sequence identified as SEQ ID NO: 2 or SEQ ID NO: 4 or SEQ ID NO: 32. Alternatively, preferred proteins encoded by a protein are encompassed by the invention. nucleic acid comprising the nucleotide sequence identified as SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 31, as defined above. Additionally, proteins having biological activity of P450RAI that are encoded by nucleic acids that hybridize under rigid conditions, as discussed above, to a nucleic acid comprising a nucleotide sequence identified as SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 31. P450RAI of the invention can be obtained by expression in a suitable host cell using techniques known in the art. Suitable host cells include prokaryotic or eukaryotic organisms or lines, for example, yeast, E. coli, insect cells and COS 1 cells. The recombinant expression vectors of the invention, described above, can be used to express a protein that has P450RAI activity in a host cell in order to isolate the protein. The invention provides a method for 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. The proteins can be isolated from a host cell that expresses the protein and purified according to standard procedures of the art, including precipitation with ammonium sulfate, column chromatography (e.g., ion exchange, gel filtration, affinity chromatography). , etc.), electrophoresis, and finally, crystallization [see generally, "Eμzyme Purification and Related Techniques", Methods in Enzymology, 22, 233-577 (1971)]. 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 homogeneous solution synthesis [Houbenwcyl, 1987]. The protein of the invention, or portions thereof, can be used to prepare antibodies specific for the proteins. Antibodies can be prepared which bind to a distinctive epitope in a non-conserved region of a particular protein. A non-conserved region of the protein is one which has no substantial sequence homology to other proteins, for example other members of the P450 family of cytochromes. Conventional methods can be used to prepare the antibodies. For example, using a peptide from a P450RAI protein, polyclonal antiserum or monoclonal antibodies can be prepared using standard methods. As demonstrated in Example 16, a mammal (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which produces 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. The progress of immunization can be monitored by detection of antibody titres in plasma or serum. Standard ELISA or other immunoassay can be used to evaluate antibody levels. After immunization, antiserum can be obtained and, if desired, polyclonal antibodies isolated from the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) from an immunized animal can be harvested and fused to myeloma cells by standard somatic cell fusion procedures, thereby immortalizing these cells and producing hybridoma cells. Such techniques are well known in the art. For example, the hybridoma technique originally developed by Kohler and Milstein [Kohler, 1975] as well as other techniques such as the human B-cell hybridoma technique [Kozbor, 1983], the EBV hybridoma technique to produce human monoclonal antibodies [Colé , 1985], and examine combinatorial antibody libraries [Huse, 1989]. Hybridoma cells can be re-examined immunochemically for production of antibodies specifically reactive with the peptide, and isolated monoclonal antibodies. The term "antibody" is proposed as used herein to include fragments thereof which are also specifically reactive with a protein having the biological activity of P450RAI, or a peptide fragment thereof. The antibodies can be fragmented using conventional techniques and the fragments examined for utility in the same manner as described above for whole antibodies. 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 the disulfide bridges to produce Fab' fragments. It is also known in the art to prepare chimeric antibody molecules with human constant regions. See, for example, Morrison et al., Takeda et al., Cabilly et al., Boss et al., Tanaguchi et al., Teng et al. [Morrison, 1985; Takeda, 1985; Cabily; Boss; Tanaguchi; Teng, 1982]; European Patent Publication 0173494, UK Patent GB 2177096B, PCT Publication WO92 / 06193 and PE 0239400. It is expected that such chimeric antibodies may be less immunogenic in a human subject than the corresponding non-chimeric antibody. Another method for generating specific antibodies, or antibody fragments, reagents against protein having the biological activity of P450RAI, or a peptide fragment thereof, is to examine expression libraries that encode 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 display libraries. See for example Ward et al., Huse et al., And McCafferty et al. [Ward. 1989; Huse, 1989; McCaffferty, 1990]. Examining such libraries with, for example, a P450 RAI peptide can identify immunoglobulin fragments reactive with P450RAI. Alternatively, the SCID-hu mouse developed by Genpharm can be used to produce antibodies or fragments thereof. Polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect proteins of the invention, portions thereof or closely related isoforms in various biological materials, for example they can be used in ELISA, radioimmunoassay or histochemical tests. In this way, the antibodies can be used to quantify the amount of a P450RAI protein of the invention, portions thereof or closely related isoforms in a sample in order to determine the role of P50RAI proteins in particular particular cellular events or states. pathological Using methods described hereinabove, polyclonal, monoclonal or chimeric monoclonal antibodies can be increased for non-conserved regions of P450RAI and are used to distinguish a particular P450RAI from other proteins. Polyclonal or monoclonal antibodies can be coupled to a detectable substance or reporter system. The term "coupled" is used to mean that the detectable substance is physically bound to the antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansyl chloride and phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include 125 I, 131 I, 35 S and 3 H. In a preferred embodiment, the reporter system allows quantification of the amount of protein (antigen) present. Such antibody-linked reporter system can be used in a method to determine whether a fluid sample or tissue from a subject contains a deficient or excessive amount of the protein. Given a normal threshold concentration of such a protein for a given type of subject, test kits can be developed in this way. 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]. The present invention includes three types of compounds related to retinoids; those that inhibit the enzymatic activity of P450RAI, by which the metabolism of AR is inhibited; those retinoids that evacuate microorganisms by P450RAI, for example Am580, shown above; and those compounds that suppress the induction of expression of P450RAI genes, for example 4 -HPR, as shown above. The compositions of the invention are administered to subjects in a biologically compatible form by pharmaceutical administration in vivo. By "biologically compatible form by in vivo administration" is meant a form of the composition to be administered in which any toxic effects are heavier by the therapeutic effects of the composition. The term "subject" is proposed to include living organisms in which a therapeutic response can be produced, for example mammals. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. The administration of a therapeutically active amount of the therapeutic compositions of the present invention is defined as an effective amount, in doses or for periods of time necessary to achieve the desired result. For example, a therapeutically active amount of a compound that inhibits AR catabolism by a P450RAI 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 regimens can be adjusted to provide the optimal 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. The compounds of the present invention, such as those found to inhibit the metabolism of AR by P450RAI enzymes and which are useful as anticancer agents and in the treatment, amelioration, or prevention of skin disorders for the patients, may be used topically. which is useful retinoic acid, for example. In this aspect they can be included in compositions for animal therapy, including humans, for premalignant epithelial cell lesions, such as prophylaxis against tumor cell promotion in epithelial cells and treatment for dermatoses such as ichthyosis, follicular disorders, benign epithelial disorders, and other diseases. of proliferative skin, such as acne, psoriasis, eczema, atopic dermatosis, non-specific dermatitis and the like. Topical compositions are usually formulated with a pharmaceutically acceptable carrier in liquid, semisolid or solid form. A pharmaceutically acceptable carrier is a material that is non-toxic and generally inert and does not adversely affect the functionality of the active ingredients. Such materials are well known and include those materials sometimes referred to as diluents or carriers (excipients) in the pharmaceutical formulating art. The carrier can be organic or inorganic in nature. Examples of pharmaceutically acceptable carriers are water, gelatin, lactose, starch, mineral oil, cocoa fat, dextrose, sucrose, sorbitol, mannitol, gum, acacia, alginates, cellulose, talc, magnesium stearate, polyoxyethylene sorbitan monolaurate and other pharmaceutical carriers. commonly used In addition to an active ingredient and carrier, the formulation may contain minor amounts of additive such as flavoring agents, coloring agents, thickening or gelling agents, emulsifiers, wetting agents, buffers, stabilizers, and preservatives such as antioxidants. Certain enteric compositions can be administered. For oral administration, suitable forms are, for example, tablets, pills, syrups, suspensions, emulsions, solutions, powders and granules. They can be used as anti-tumor agents or as part of an anti-tumor formulation, for example, composed of the present invention in a retinoid-like manner used to treat various tumors, such as all-trans retinoic acid. The dose to be administered, whether it is a single dosi, multiple dose or daily dose will, of course, vary with the particular compound employed due to the variant potency of the active ingredient, the route of administration chosen, the size of the container, the type of tumor, and the nature of the patient's condition. The dose to be administered is not subject to definitive theories ^ but usually it will be an effective amount, or the equivalent on a molar basis of the pharmacologically active libr form produced from a dosi formulation after the metabolic release of the active drug to achieve its effects pharmacological and physiological. An oncologist skilled in the art of cancer treatment will be able to determine without prolonged experimentation, appropriate protocols for the effective administration of the compounds of this present invention. Nucleic acids encoding proteins having biologically active activity of a P450RAI protein can be used to generate either transgenic or "nullified" animals which, in turn, are useful in the development and examination of therapeutically useful reagents. A transgenic anima (e.g., a mouse) is an animal having cells that contain a transgene, transgene that is introduced into the animal or an animal ancestor in a prenatal, e.g., an embryonic stage. A transgene is a DNA that is integrated into the genome of a cell from which a transgenic animal developed. In one embodiment, human P450RAI DNA, comprising the nucleotide sequence shown in SEQ ID NO: 5, or an appropriate variant thereof variant can be used to generate transgenic animals which contain cells expressing P450 protein. Methods for generating transgenic animals, particularly animals such as mice, have become conventional, and are described, for example, in U.S. Patent Nos. 4,735.86 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 male pronucleus of fertilized cell mouse eggs; the injected eggs are transferred to pseudo-pregnant surrogate females; and, eggs are allowed to develop on the surrogate females [Hogan, 1986]. Alternatively, an embryonic germ cell line can be transfected with an expression vector comprising nucleic acid encoding a protein having P450RAI activity, and cells containing the nucleic acid can be used to form aggregation chimeras with embryos from a mouse strain proper receiver. The chimeric embryos can then be implanted in an appropriate pseudo-pregnant female mouse of the appropriate strain and the embryo is brought to term. The progeny that harvested the transfected DNA in their germ cells can be used to uniformly reproduce transgenic mice. Typically, particular cells can be targeted for transgene P450RAI incorporation by use of tissue-specific enhancers operably linked to P450RAI that encode genes. For example, promoters and / or enhancers can be used which direct expression of a gene to which they are operatively linked preferentially in cardiac muscle cells to create a transgenic animal which expresses a P450RAI 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 can also be used to be used transgenic animals that include a copy of a transgene P450RAI introduced into the germ line of the animal in an embryonic stage to examine the effect of increased P450RAI expression in various tissues. The pattern and degree of expression of a recombinant molecule of the invention in a transgenic mouse is facilitated by fusing a reporter gene for the recombinant molecule such that both genes are co-transcribed to form polycistronic mRNA. The reporter gene may be introduced into the recombinant molecule using conventional methods such as those described in Sambrook et al., [Sambrook, 1989] '. The efficient expression of both cistron polycistronic mRNAs encoding the protein of the invention can be achieved by including a known internal translation start sequence such as that present in poliovirus mRNA. The reporter gene must 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 therefore be determined by assaying the phenotype of the reporter gene. Preferably the reporter gene encodes a phenotype not exhibited by the host cell and the phenotype can be assayed quantitatively. Examples of suitable reporter genes include lac Z (β-galactosidase), neo (neomycin phosphotransferase), CAT (chloramphenicol acetyltransferase) dhr (dihydrofolate reductase), aphlV (hygromycin phosphotransferase), lux (luciferase), uidA (β-glucuronidase). Preferably, the reporter gene is lacZ which encodes β-galactosidase. Β-galactosidase can be assayed using the lactose analogue X-gal (5-bromo-4-chloro-3-indolyl-bD-galactopyranoside) which is decomposed by β-galactosidase to a product that is blue in color [ old] . Although the experimental animals used in the preferred embodiment described are mice, the invention should not be limited thereto. It may be desirable to use other species such as, for example, rats, hamsters, rabbits and sheep. The transgenic animals of the invention can be used to investigate the molecular basis of AR metabolism. The transgenic animals of the invention can also be used to test substances for the ability to prevent, delay or increase the metabolism of RA. A transgenic animal can be treated with the substance in parallel with an untreated control transgenic animal. The cells of transgenic animals can be cultured using standard tissue culture techniques. E particular, the cells carrying the recombinant molecule of the invention can be cultured and used to test substances for the ability to prevent, delay or increase the metabolism of RA. Additionally, non-human homologs of genes encoding proteins having P450RAI activity can be used to construct a "nullified" animal which has a defective or altered P450RAI gene. For example, with established techniques, a portion of murine genomic P450RAI DNA (eg, 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 DNA can be transfected into an embryonic germ cell line. The altered P450RAI DNA will recombine in an homologous manner with the endogenous P450RAI gene in certain cells and clones containing the altered gene can be selected. The 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. The transmission of the altered gene in the germline of a resulting animal can be confirmed using standard techniques and the animal can be used to reproduce animals that have an altered P450RAI gene in each cell [Lemoine, 1996]. Accordingly, a nullified animal can be made which can not express a functional P450RAI protein. Such a nullified animal can be used, for example, to test the efficacy of an agent in the absence of a P450RAI protein. The antisense and oligonucleotide nucleic acids of the invention are useful for displaying expression of nucleic acids (e.g., mRNA) encoding proteins having P450RAI activity. Since proteins having P450RAI activity are associated with metabolism of agents which can act in the cell, for example, AR, decreasing the expression of such proteins can increase the sensitivity of the cell to such agents. Antisense nucleic acids may be introudic in a drug-resistant cell in culture to inhibit the expression of P450RAI. One or more antisense nucleic acids, such as oligonucleotides can be added to cells in culture media, typically, for example, at 200 μg / ml. In this way, the antisense nucleic acids of the invention, or oligonucleotides in gene therapy can be used to correct or prevent resistance to retinoic acid or other retinoid in a subject. For example, antisense sequences can be used to bring the malignant cells resistant to retinoic acid or another retinoid to be sensitive to chemotherapeutic agents. The administration of antisense nucleic acids to a subject can be more effective when the antisense nucleic acid is contained in a recombinant expression vector which allows the continuous production of antisense RNA. Recombinant molecules comprising an antisense nucleic acid or oligonucleotides thereof can be introduced directly 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 (eg, a retinoid-resistant tumor cell). Antisense nucleic acids can also be introduced into isolated cells, such as those from the hematopoietic system, ex vivo using viral vectors or physical techniques such as microinjection and electroporation methods or chemicals such as coprecipitation and incorporation of DNA 'into liposomes, and such cells can be returned to the donor. Recombinant molecules can be supplied in the form of an aerosol or by washing. Accordingly, the invention provides a method for inhibiting resistance to retinoic acid or another retinoid 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: 2, SEQ ID NO: 32, or particularly, in the case of human cells SEQ ID NO: 4. In addition, the nucleic acids of the invention can be used to design ribozymes which are capable of cleaving a nucleic acid from a chain encoding a protein having P450RAI activity, such as mRNA. A catalytic RNA (ribozyme) having ribonuclease activity which has specificity for mRNA encoding P450RAI based on the sequence of a nucleic acid of the invention can be designed. For example, an AR L-19IVS derivative of Tetrahymena may be constructed in which the base sequence of the active site is complementary to the base sequence to be excised in an mRNA encoding P450RAI [Cech a and b]. Alternatively, a nucleic acid of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a group of AR molecules [Bartel, 1993]. The antisense nucleic acid-isolated nucleic acids of the invention can be used to construct recombinant expression vectors as previously described. These recombinant expression vectors are then useful for making transforming 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 previously described. The isolated nucleic acids and antisense nucleic acids of the invention can also be used to construct transgenic and nullified animals as previously described. The isolated proteins of the invention are useful for making antibodies reactive against proteins having P450RAI activity, as previously described. Alternatively, the antibodies of the invention can be used to isolate a protein of the invention by standard immunoaffinity techniques. Additionally, antibodies of the invention, including bispecific antibodies, are useful for diagnostic purposes. Molecules which bind a protein comprising an amino acid sequence shown in SEQ ID NO: can also be used in a method for killing a cell expressing the protein, wherein the cell takes the molecule. Preferably, the cell is a tumor cell. The destruction of such cells can be done 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. toxin diphtheria or ricin), or a chemotherapeutic drug. , as well as cells whose action can destroy a cell, such as a cytotoxic cell. The P450RAI binding molecule can be directly coupled to a substance that has toxic or therapeutic activity or can be directly linked to the substance.

In one example, the toxicity of the molecule captured by the cell is activity by the P450RAI protein. 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: 4, 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 to determine the amount of protein in the sample; and means to compare 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 is activated which binds to P450RAI via the linkage (eg, change in the fluorescence spectrum, loss of isotopic radio label). The diagnostic equipment may also contain an instruction manual for the use of the equipment. Furthermore the invention 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: 3, 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 equipment may also contain an instruction manual for the use of the equipment. Those skilled in the art will know, or will be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are proposed to be encompassed by the following claims. REFERENCES Individual references are given below. All references listed herein are incorporated for reference. 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. Achkar, C.C., Derguini, F., Blumberg, B., Langston, A., Arthr, A. L., Speck, J., Evans, R.M., Bolado, Jr., J. Nakanishi, K. And Buck, J. (1996) 4-0xoreinol, a new natural ligand and transactivator of the retinoic acid receptors. Proc. Nati Acad. Sci. USA 93, 4879-84. Akimenko, M.A. and Ekker, M. (1995a). Previous duplication of th Sonic hedgehog expression pattern in th pectoral fin bus of zebrafish treated with retinoic acid. Developmental Biology 170, 243-7. Akimenko, M.A. , Johnson, S.L. Westeerfield, M. and Ekker, M (1995b). Differenctial induction of flour msx homeobox genes during fin development and regeneraation in zebrafish. Development 121, 347-57. Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. And Yabusaki, Y. (1994). Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli. European Journal of Biochemistry 224, 335-43. Bartel, D. And Szostak, J.W. (1993). Science 261, 1411-1418. Blaneer, W. (1994). Retinol and retinoic acid metabolism. In: The Retinoids. (M. Sporn, Roberts, A. And Goodman, D.S., Editors) Raven Press, Inc .; New York Bligh, E.G. and Dyer, W. J. (1957). Arapid method of total lipid extraction and purification. Canadian Journal of Biochemistry 37, 911-917. 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. Nati Acad. Sci. USA 93, 4873-78. Boss et al., United States Patent No. 4,816,397. Boylan J.F., Lufkin, T., Achkar, C.C., Taneha, R., Chambon, P. and Gudas, L.J. (nineteen ninety five) . 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. Boyle, A.L. et al. (1992). Genomics 12, 106-15. Cabilly et al. U.S. Patent No. 4,816,567. Cech et al., (A) U.S. Patent No. 4,987,071.

Cech et al., (B) United States Patent no. 5,116,742.

Chambón, P. (1995). The molecular and genetic dissection of retinoid signaling pathway. [Review] Recent Progress in Hormone Research 50, 317-32. Chen, K.S. and DeLuca, H.F. (nineteen ninety five). Cloning of the human 1 alpha, 25-dihydroxyvitamin D-3 24-hydroxylase gene promoter and identification of two vitamin D-responsive elements. Biochimica et Biophysica Acta 1263, 1-9 Chomienne, C., Fenaux and Degos, L. (1996). Retinoid differentiation therapy in promyelocytic leukemia. FASEB J. 1025-1030. Chytil, F. (1984). Retinoic acid: Biochemistry, toxicology, pharmacology, and therapeutic use. Pharmacol. Rev. 36,93-99.

Cole et al. (1985). Monoclanal Anitibodies in Cancer Therapy. Alien R.Bliss, Inc. Costaridis, P., Horton, C., Zeitlinger, J., Holdor, N. and Maden, M. (1996) Endogenous Retinoids in the Zebrafish Embryo and Adult. Developmental Dynamics 205, 41-51. Creech Kraft, J., Schuh, T., Juchau, M.R. and Kimelman, D. (1994). Temporal distribution, ocalization and metabolism of all-trans retinol, didehdroretinol and all-trans retinal during Xenopus development. Biochem. J. 301, 111-119. De Coster, R., Wouters, W.and Bruynseels,. /nineteen ninety six) . P450-dependent enzymes as targets for prostate cancer therapy. J. Ster. Biochem.Mol. Biol.56, 133 -43. Duell, E .A. , Astrom, A., Griffiths, CE., Chambón, P. and Voorhees, J. J. (1992). Human skin levéis of retinoic acid and cytochrome p-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo. Journal of Clinical Investigation 90 ,. 1269-74. Duell, E.A., Astrom, A., Kang, S., Griffithes, C.E.M. and Voorhees, J. (1994). All-trans, 9-cis and 13-cis retinoic acid each induce cytochrome P450 4 -retinoic acid hydroxylase which causes all-trans but not 9-cis or 13-cis retinoic acid to self-metabolize. Society for Investigation Dermatology Abstracts 102, 641. Fiorella P.D., Giguere, V. AND nAPOLI, j.L. (1993). Expression of Cellular Retinoic Acid-binding Protein (Type 11) in Escherichia coli. The Journal of Biological Chemistry 268, 21545-21552. Formelli, F., Barua, A.and Olson, J. (1996) .Biactivities of N- (4-hydroxyphenyl) retinimide and retinoyl B-glucuronide. FASEB J. 10, 1014-1024.

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 vito metabolites of all-trans retinoic acid in hamster trachea and liver. Biochemistry 18, 2092-7. Gotoh, 0.and Fuj ii-Kuriyama, Y. (1989). Evolution, structure, and gene regulation of cytochrome P-450. Green, S., Issemann, I. and Sheer, E. (1988). Aversatile in vivo and invitro eukarytic expression vector for protein engineering. Nucleic Acids Research 16, 369-370 Gudas L., Sporn, M. and Roberts, A. (1994). Cellular biology and biochemistry of the retionoids. In: The Retinoids. (M. SPORN, Roberts, A. and Goodman, D.S., Editors) Raven Press, Inc,: New York. Heng, H.and Tsui, L-C. (1993). Chromosome 102, 325-32. Hogan, B. et al., (1986). A Laboratory Manual, Cold Spring Harbor , N.Y., Cold Spring Harbor Laboratory. Hong, W. (1994). Retinoids and human cancer. In: The Retinoids.

(M. Sporn, Roberts, A. and Goodman, D.S., Editors) Raven Press, Inc.: New York. Houbenwcyl, (1987). Methods of Organic Chemistry, by E. Wansch.

Vol. 15 1 and 11. Thieme, Stuttgart. Hozumi, Nand Sandhu, J.S. (1993). Recombinant antibody technology, its advent and advances. Cancer invest. 11,714-723. Huse et al., (1989). Scince 246, 1275-1281 Jones, B.B., Ohno, CV.K., Allendy, G., Boffa, M., Levin, A.A. , Grippo, J.F. and Petkovich. M. (1995). New Retinoid X Receiver Subtypes in Zebra Fish (Danio rerio) Differentially Modulate Transcription and Do Not Bind 9-cis Retinoic Acid Mol. Cell Biol. 15 5226-5234. Kennett, R. (1979). Cell fusion. Methods Enzymol. 58, 345-359.

Kohler and Milstein. (1975). Nature 256, 495-497. Kozbor et al. (1983). Immunol. Today 4,72. 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., Loptt, I.T. et al. (1985) Retinoic acid ambryopathy. New England Journal of Medicine 313, 837-41. Lansdorp, United States Patent 4,868, 109. Lemoine, N.R. and Cooper, D.N. (nineteen ninety six) . Genen Therapy, Human Molecular Genetics Series, BIOS Scientific Publishers, Oxford, U.K. Leo et al. (1989). Metabolism of retinol and retinoic acid and human liver cytochrome P45IIC8. Arch.Biochem.Biophyys. 269, 305-312. Liang, P.et al. (1990). High Resolution Mapping of Chromosome 11 d and in situ hybridization by cosmid clones. Science 247, 64 -; 9.

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. Supplemet 22,1-10. Lotan R.M. (nineteen ninety five) . Squamous differentiation and retinoids.

Cancer Treaut. Res. 74, 43-72. Lotan, R. (1996). Retinoids in Cancer Chemoprevention. Fased J.10, 1031-1039. Maden, M.and Holder, N. (1992). Retinoic acid and development of the central nervous system. (Review). Bioessays 14 431-8.

Makin, G., Lohnes, D., Byford, V., Ray, R. and Jones, G. (1989).

Target cell metabolism of 1, 25 -dihydroxyvitamin D3 to calcitroic acid. Evidence for a pathway in kidney and bone involving 24-oxidation. Biochemical journal 262, 173-80. Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York Mangelsdorf, D. and Evans, R.M. (nineteen ninety five) . The RXR Heterodimers and Orphan Receptors. CELL 83, 841-850. Merrifield, (1964). J.am. Chem. Assoc. 85, 2149-2154. McCafferty et al., (1990). Nature 348, 552-554. Mirki, A. and Cole, S. P. C. (1989) Antigens associated with multidrug resistance in H69AR, to amall cell lung cancer cell line. Cancer Res. 49, 51719-5724. 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. Moon, R. C, Mehta, R.G. and Rao, K. V.N. (1994). Retinoids and cancer in experimental animáis. In: The Retinoids. (M. Sporn, Roberts, A. and Goodman, D.S., Editors) Raven Press, Inc.,: New York Morriss-Kay, G. (1993). Retinoic acid and craniofacial development: molecules and morphogenesis. (Review). Bioessays 15, 9-15. Morriss-Kay, G.M. (1996) Embryonic development and oattern formation. FASEB J. 10, 961-968. Morrison et al., (1985). Proc, Nati. Acad. Sci. USA 81, 6861.

Muindi, J.R., F., Frankel, S.R., Huselton, C, De Grazia, F., Garland, W., Young, C. W. and Warell, R.P., Jr., (1992) Clinical pharmacology of oral all-trans retinoic acid in patients with promyelocytic leukemia. Cancer Research 52, 2138-2142. Muindi, J.R., Young, C. W. and Warell, R.J. (994a), Clinical pharmacology of all-trans retinoic acid, Leukemia 8, 1807-1812.

Muindi, J. R., Young, C.W. and Warell, R. J.! 994b), Clinical pharmacology of all-trans retinoic acid, Leukemia 8, sl6-s21.

Napoli, J.L., Boerman, M.H., Chai, X., Zhai, Y. and Fiorella, P.S. (nineteen ninety five). Enzymes and binding proteins affecting retinoic acid concentrations, J. Ster. Biochem. Mol. Biol. 53, 497-502.

Napoli, J. (1996). Retinoic acid biosynthesis and metabolism.

FASEB J. 10, 993-1001. Nelson, D.R., Kamataki, T., Waxman, D.J., Guengerich, F.P., Estabrook, R. W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsalus, Y.C., Gotoh, O., Okuda, K. and Nebert, D.W. (1993).

The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial ñames of enzymes, and nomenclature. DNA _ Cell Biology 12, 1-51. Listen, R. W. and Primrose, s.B., In: Principles of Gene Manipilation. An introdiction to Genetic Engineering. 4 th of. Oxford University Press. 63-66. 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. Ohno, C.K., Ueda, H. and Petkovich, M. (1994). The Drosophila nuclear receptors FTZ-F1 alpha and FTZ-F1 beta compete as monomers for binding to a site in the fushi taraze gene Mol. Cell Biol. 14, 3166-75. Ohyama, Y., Ozone, K., Uchida, M., Shinki, T., Kato, S., Suda, T., Yamamoto, O., Noshiro, M. and Kato, Y. (1994). Identification of a vitamin D-responsive element in the 5-flanking region of the rat 25-hydroxyvitamin D3 24 -hydroxylase gene. Journal of Biological Chemistry 269, 10545-50. Petkovich, M., Brand, N.J., Krust, A. and Chambón, P. (1987). A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 330, 444-50. 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. Reddy, A. P., Chen, J., Zacharewski, T., Gronemeyer, H., Voorhees, J. J. and Fisher, G. J. (1992). Characterization and purification of humen retinoic acid receptor-gl overexpressed in the baculoviruis-insect cell system. Biochem. J. 287, 833-840. 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. Roberts, A. B., Nichols. M. D., Newton, D. L. ans Sporn, M. B. (1979a). In vitro metabolism of retinoic acid in hamster intestine and liver. Journal of Biological Chemistry 254, 6296-302. Roberts, A.B., Frolik, C.A., Nichols. M. D., and Sporn, M. B. (1979b). Retinoic-dependent induction of the in vivo and in vitro metabolism of retonoic acid in tissues of the vitamin A-deficient hamster. Journal of Biological Chemistry 254, 6303-9. Sambrook, J., Fritsch E. F .. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Lab Press. Cold Spring Harbor, New York. Staerz __ Bevan (1986a). Proc. Nati Acad. Sci. (USA) 83, 1453 Staerz & Bevan (1986b). Immunology Today 7. 241. Stewart, A.J., Canitrot, Y., Baracchini, E., Dean, N.M., Deeley, R.G., and Cole, S.P.C. (nineteen ninety six). Reduction of Expression of the multidrug resitance protein (MRP) in human tumor cells by antisense phophorothioate oligonocleotides. Biochem. Pharamcol. 51, 461-469. Taime, M and Breitman, T.R. (1997). N-4 hydroxyphenylretinamide enhances retinoic acid-induced differentiation and retinoxylation of proteins in the human promyelocytic leukemia cell line. NB4, by a mechanism that may involve inhibition of retinoic acid catabolism, Biochemical and Biophysical Research Communications 232, 432-435. Takatsuka, J., Takahashi, N. and De Luca, L.M. (nineteen ninety six). Retinoic Acid Metabolism and Inhibition of Cell Proliferation: An Unexpected Liaison. Cancer Research 56, 675-678. Takeda et al., (1985). Nature 314,452. Tanaguchi et al European Patent Publication EP171496. Teng, et al., (1982) Meth. Enzymol. 92. 3-16. Thaller, C.and Eichele, G. (1990). Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature 345, 815-9. Van Wauwe, JP., 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 in rats. The Journal of Pharmacology and Experimental Therapeutics 245, 365-369. Van Wauwe, J.P., Coene, M.-C, Goossens, J., Cools, W.and Molbaliu, J. (1990). Effecvts 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. 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. Ward et al., (1989). Nature 341. 544-546. Warrell, R.J. (1994). Applications for retinoids in cacer therapy. Seminars in Hematol .31, 1-13. Warrell, R. , 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. White, J.A. , Boffa, M.B., Jones, B. and Petkovich, M. (1994).

Azebrafish retinoic acid receptor expressed in the regenerating end. Development 120.1861-72. William, J.B. and Napoli, J.L. (1987). Inhibition of retinoic acid metabolism by imidazole antimycotics in F9 ambrional carcinoma cells. Biochemical Pharmacology 36, 1386-1388. Windhorst, D.B. (1982). The use of isotretinoin in disorders of keratinization. Journal of the American Academy of Dermatology 6, 708-9.

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-ionduced inhibition of cell growth and on retinoic acid metabolism in MCF-7 human breast cancer cells. Cancer Research 52.2841-6. Zierold, C., Darwish, H.M. and DeLuca, H.F. (nineteen ninety five). Two vitamin D response elements fuction in the mouse 1, 25-dihydroxyvitamin D24 -hydroxylase promoter. Journal of Biological Chemistry 270, 1675-8. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANTS: (A) NAME: QUEEN'S UNIVERSITY AT KINGSTON (B) STREET: (C) CITY: Kingston (D) PROVINCE: Ontario (E) COUNTRY: CA (F) POSTAL CODE (ZIP): K7L 1J7 (A) NAME: PETKOVICH, P. Martin (B) STREET: 875 Everitt Avenue (C) CITY: Kingston (D) PROVINCE: Ontario (E) COUNTRY: CA (F) ZIP CODE: K7M 4R1 (TO NAME) : WHITE, Jay A. (B STREET: 913 Purcell Crescent (C CITY: Kingston (D PROVINCE: Ontario (E COUNTRY: CA (F ZIP CODE): K7P 1C1 (A NAME: BECKETT, Barbara R. (B STREET: 112 Westmorland road (C CITY: Kingston (D PROVINCE: Ontario (E COUNTRY: CA (F ZIP CODE): K7M 1J7 (A) NAME: JONES, Glenville (B) STREET: 66 Inverness Crescent (C) CITY: Kingston (D) PROVINCE: Ontario (E) COUNTRY: CA (F) ZIP CODE: K7M 6N7 (ii) TITLE THE INVENTION: PROTEIN THAT METABOLIZES RETINOIDS (iii) NUMBER OF SEQUENCES: 38 (iv) COMPUTER LEADABLE FORM: (A) TYPE MEDIUM: Diskette, 3 INCH, 1.4 Mb storage (B) COMPUTER: COMPAQ, IBM PC compatible (C) OPERATING SYSTEM: MS-DOS 5.1 (D) SOFTWARE: WORD PERFECT (v) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (vi) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 667,546; US 08 / 724,466 (B) DATE OF SUBMISSION: JUNE 21, 1996; 01-OCT-1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 337 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: TGCCAGTGGA CAATCTCCCT ACCAAATTCA CTAGTTATGT CCAGAAATTA GCCTAAACCG 60 GAGCCTTTGT ACATATTTTT TTATTTTAG? TGAACTGTGA TGTATTGGAT ATTTTCTAAT 120 TTGTTTATAT AAAGCAGATG TGT? TATAAG TCTATGCGAA GAAGCGAAAA CGAGGGCACT 180 ACTTTCTCAT GGATCACTGT AATGCTACAG AGTGTCTGTG ATGTATATTT ATAATGTAGT 240 TGTGTCATAT AGCTTTTGTA CTGTATGCAA CTTATTTAAC TCGCTCTTTA TCTCATGGGT 300 TTTATTTAAT AAAACATGTT CTTACAAAAA AAAAAAA 337 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 492 amino acids (B) TYPE: nucleic amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Gli Leu Tir Tre Leu Met Val Tre Fen Leu Cis Tre lie Val Leu 1 5 10 15 Pro Val Leu Leu Fen Leu Ala Ala Val Lis Leu Trp Glu Met Leu Met 20 25 30 lie Arg Arg Val Asp Pro Asn Cis Arg Ser Pro Leu Pro Pro Gli Tre 40"45 Met Gli Leu Pro Fen lie Gli Glu Tre Leu Gln Leu lie Leu Gl.n Arg 50 55 60 Arg Lis Fen Leu Arg Met Lis Arg Gln Lis Tir Gli Cis lie Tir Lis 65 70 75 80 Tre His Leu Fen Gli Asn Pro Tre Val Arg Val Met Gli Wing Asp Asn 85 90 95 Val Arg Gln lie Leu Leu Gli Glu His Lis Leu Val Ser Val Gln Trp 100 105 110 Pro Wing Ser Val Arg Tre lie Leu Gli Ser Asp Tre Leu Ser Asn Val 115 120 125 His Gli Val Gln His Lis Asn Lis Lis Lis Ala lie Met Arg Ala Fen 130 135 140 Ser Arg Asp Ala Leu Glu His Tir lie Pro Val lie Gln Gln Glu Val 145 150 155 160 Lis Ser Ala lie Gln Glu Trp Leu Gln Lis Asp Ser Cis Val Leu Val 165 170 175 Tir Pro Glu Met Lis Lis Leu Met Fen Arg lie Ala Met Arg lie Leu 180 185 190 Leu Gli Fen Glu Pro Glu Gln lie Lis Tre Asp Glu Gln Glu Leu Val 195 200 205 Glu Ala Fen Glu Glu Met lie Lis Asn Leu Fen Ser Leu Pro lie Asp 210 215 220 Val Pro Fen Ser Gli Leu Tir Arg Gli Leu Arg Ala Arg Asn Fen lie 225 230 235 240 His Ser Lis lie Glu Glu Asn lie Arg Lis Lis lie Gln Asp Asp Asp 245 250 255 Asn Glu Asn Glu Gln Lis Tir Lis Asp Ala Leu Gln Leu Leu lie Glu 260 265 270 Asn Ser Arg Arg Ser Asp Glu Pro Fen Ser Leu Gln Ala Met Lis Glu 275 280 285 Ala Ala Tre Glu Leu Leu Fen Gli Gli His Glu Tre Tre Ala Ser Tre 290 295 300 Ala Tre Ser Leu Val Met Fen Leu Gli Leu Asn Tre Glu Val Val Gln 305 310 315 320 Lis Val Arg Glu Glu Val Gln Glu Lis Val Glu Met Gli Met Tir Tre 325 330 335 Pro Gli Lis Gli Leu Ser Met Glu Leu Leu Asp Gln Leu Lis Tir Tre 340 345 350 Gli Cis Val lie Lis Glu Tre Leu Arg lie Asn Pro Pro Val Pro Gli 355 360 365 Gli Fen Arg Val Ala Leu Lis Tre Fen Glu Leu Asn Gli Tir Gln lie 370 375 380 Pro Lis Gli Trp Asn Val lie Tir Lie lie Cis Asp Tre His Asp Val 385 390 395 400 Wing Asp Val Fen Pro Asn Lis Glu Glu Fen Gln Pro Glu Arg Fen Met 405 410 415 Ser Lis Gli Leu Glu Asp Gli Ser Arg Fen Asn Tir lie Pro Fen Gli 420 425 430 Gli Gli Ser Arg Met Cis Val Gli Lis Glu Fen Wing Lis Val Leu Leu 435 440 445 Lis Lie Fen Leu Val Glu Leu Tre Gln His Cis Asn Trp Lie Leu Ser 450 455 460 Asn Gli Pro Pro Tre Met Lis Tre Gli Pro Tre Lie Tir Pro Val Asp 465 470 475 480 Asn Leu Pro Tre Lis Fen Tre Ser Tir Val Arg Asn 485 490 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1850 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: TGTCGCCGTT GCTGTCGGTT GCTGTCGGAC GCTGTCTCCT CTCCAGAAGC TTGTTTTTCG 60 TTTTGGCGAT CAGTTGCGCG CTTCAAC ATG GGG CTG TAC ACC CTT ATG GTC ACC 114 Met Gli Leu Tir Tre leu met val tre 1 5 TTT CTC TGC ACC ATC GTG CTA CCC GTT TTA CTC TTT CTC GCC GCG GTG 162 Fen leu cis tre ile ile leu pro val leu leu fen leu ala ala val 10 15 20 25 AAG TTG TGG GAG ATG TTA ATG ATC CGA CGA GTC GAT CCG AAC TGC AGA 210 Lis leu trp glu met leu met ile arg arg val asp pro asn cis arg 30 35 40 AGT CCT CTA CCG CCA GGT ACC ATG GGC TTG CCG TTC ATT GGA GAA ACG 258 Ser pro leu pro gli tre met gli leu pro fen ile gli glu tre 45 50 55 CTC CAG CTG ATC CTC CAG AGA AGG AAG TTT CTG CGC ATG AAA CGG CAG 306 Leu gln leu ile leu gln arg arg lis fen leu arg met ls arg gln 60 65 70 AAA TAC GGG TGC ATC TAC AAG ACG CAC CTC TTC GGG AAC CCG ACT GTC 354 LIS TROLL GI CIS ILL TIRE LIS HIS LEGAL F GIL ASN PRO TRE VAL 75 80 85 AGG GTG ATG GGA GCT GAT AAT GTG AGG CAG ATT CTG CTG GGC GAA CAC 402 Arg val met gli ala asp asn val arg gln ile leu leu gli glu his 90 95 100 AAG CTG GTG TCT GTT CAG TGG CCA GCA TCA GTG AGA ACC ATC CTG GGC 450 Lis leu val be val gln trp pro wing be val arg tre ile leu gli 110 115 120 TCT GAC ACC CTC TCC AAT GTC CAT GGA GTT CAA CAC AAA AAC AAA AAA 498 Be asp tre leu be asn val his gli val gln his lis asn lis lis 125 130 135 AAG GCC ATT ATG AGG GCG TTC TCT CGA GAT GCT CTG GAG CAC TAC ATT 546 The wing wing is arg wing be arg asp ala leu glu his tir ile 140 145 150 CCC GTG ATC CAG CAG GAG GTG AAG AGC GCC ATA CAG GAA TGG CTG CAA 594 Pro val ile gln gln glu val lys be wing ile gln glu trp leu gln 155 160 165 AAA GAC TCC TGC GTG CTG GTT TAT CCA GAA ATG AAG AAA CTC ATG TTT 642 Lis asp is cis val leu val tir pro glu met lis lis leu met fen 170 175 180 CGG ATA GCT ATG AGA ATC CTG CTT GGT TTT GAA CCA GAG CAA ATA AAG 690 Arg ile ile arg ile leu leu gli fen glu glu glu ile ile 190 195 200 ACG GAC GAG CAA GAA CTG GTG GAA GCT TTT GAG GAA ATG ATC AAA AAC 738 Tre asp glu glu glu leu val gl ala fen glu glu met ile lis asn 205 210 215 TTG TTC TCC TTG CCA ATC GAC GTT CCT TTC AGT GGT CTG TAC AGG GGT 786 Leu fen be leu pro ile asp val pro fen se gli leu tir arg gli 220 225 230 TTG AGG GCA CGC AAT TTC ATT CAC TCC AAA ATT GAG GAA AAC ATC AGG 834 Leu arg ala arg asn fen ile his ser ile ile glu glu sn ile arg 235 240 245 AAG AAA ATT CAA GAT GAC GAC AAT GAA AAC GAA CAG AAA TAC AAA GAC 882 Lis lis ile gln asp asp asp asn glu asn glu gln lis tir lis asp 250 255 260 265 GCC CTT CAG CTG TTG ATC GAG AAC AGC AGA AGA AGA GAC GAA CCT TTT 930 Ala leu gln leu leu ile glu asn be arg arg be asp glu glu 270 275 280 AGT TTG CAG GCG ATG AAA GAA GAC GCT ACA GAG CTT CTA TTT GGA GGT 978 Be leu gln wing metal wing wing wing glu leu leu fen gli gli 285 290 295 CAT GAA ACC ACC GCC AGC ACT GCA ACC TCA CTT GTC ATG TTT CTG GGT 1026 His glu tre tre to be tre tre tre leu val met fen leu gli 300 305 310 CTG AAC ACA GAA GTG GTG CAG AAG GTC AGA GAG GAG GTT CAG GAG AAG 1074 Leu asn tre val val valn lis val val glu glu val gln glu lis 315 320 325 GTT GAA ATG GGC ATG TAT ACA CCT GGA AAG GGC TTG AGT ATG GAG CTG 1122 Val glu met gli met tir tre pro gli lis gli leu ser met glu leu 330 335 340 345 TTG GAC CAG CTG AAG TAC ACT GGA TGT GTG ATT AAA GAG ACT CTT AGA 1170 Leu asp gln leu lis tir tre gli cis val ile lisus glu tre leu arg 350 355 360 ATC AAC CCT CCT GTT CCC GGA GGA TTC AGA GTC GTC CTC AAA ACC TTT 1218 lie asn pro pro val pro gli gli fen arg val wing leu lis tre fen 365 370 375 GAA TTG AAT GGT TAC CAA ATT CCT AAA GGA TGG AAC GTC ATT TAC AGC 1266 Glu leu asn gli tir gln ile pro lis gli trp asn val tir be 380 385 390 ATC TGT GAC ACG CAC GAT GTG GCC GAC GTC TTT CCA AAC AAA GAG GAG 1314 lie cis asp asp his asp val asp wing fen fen pro asn lis glu glu 395 400 405 TTC CAG CCG GAG AGA TTC ATG AGC AAA GGT CTG GAG GAC GGG TCC AGG 1362 Fen gln pro glu arg fen met be lis gli leu glu asp gli be arg 410 415 420 425 TTT AAC TAC ATC CCC TTC GGA GGA GGA TCC AGG ATG TGT GTG GGC AAA 1410 Fen asn tir pro gli gli gli gli be arg met cis val gli lis 430 435 440 GAG TTC GCC AAA GTG TTA CTC AAG ATC TTT TTA GTT GAG TTA ACG CAG 1458 Glu fen ala lis val leu leu lis ile fen leu val glu leu tre gln 445 450 455 CAT TGC AAT TGG ATT CTC TCA AAC GGA CCC CCG ACA ATG AAA ACA GGC 1506 His cis asn trp ile leu be asn gli pro pro tre met lis tre gli 460 465 470 CCG ACT ATT TAC CCA GTG GAC AAT CTC CCT ACC AAA TTC ACT AGT TAT 4-554 Pro tre ile pro val asp asn leu pro tre lis fen tre tir 475 480 485 GTC AGA AAT TAGCCTAACC GGAGCTTTGT ACATATGTTT TTATTTTAGA 1603 Val arg asn 490 TGAACTGTGA TGTATTGGAT ATTTTCTATT TTGTTTATAT AAAGCAGATG TGTATATAAG 16S3 TCTATGCGAG GAAGCGAAAA CGAGGGCACT ACTTTCTCAT GGATCACTGT AATGCT? CAG ^ .723 AGTGTCTGTG ATGTATATTT ATAATGTAGT TGTGTTATAT AGCTTTTGTA CTGTATGCAA 1783 CTTATTTAAC TCGCTCTTTA TCTCATGGGT TTTATTTAAT AAAACATGTT CTTACAAAAA 1843 AAAAAAA 1850 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 497 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Gli Leu Pro Ala Leu Leu Ala Be Ala Leu Cis Tre Fen Val Leu 1 5 10 15 Pro Leu Leu Leu Fen Leu Ala Wing Ele Leu Trp Asp Leu Tir Met 20 25 30 Val Ser Gli Arg Asp Arg Ser Cis Wing Leu Pro Leu Pro Pro Gli Tre 35 40 45 Met Gli Fen Pro Fen Fen Gli Glu Tre Leu Gln Met Val Leu Gln Arg 50 55 60 Arg Lis Fen Leu Gln Met Lis Arg Arg Lis Tir Gli Fen lie Tir Lis 65 70 75 80 Tre His Leu Fen Gli Arg Pro Tre Val Arg Val Met Gli Ala Asp Asn 85 90 95, Val Arg Arg lie Leu Leu Gli Asp Asp Arg Leu Val Ser Val His Trp 100 105 110 Pro Wing Ser Val Arg Tre lie Leu Gli Ser Gli Cis Leu Ser Asn Leu 115 120 125 His Asp Ser Ser His Lis Gln Arg Lis Lis Val lie Met Arg Wing Fen 130 135 140 Ser Arg Glu Wing Leu Glu Cis Tir Val Pro Val lie Tre Gln Glu Val 145 150 155 160 Gli Ser Ser Leu Glu Gln Trp Leu Ser Cis Gli Glu Arg Gli Leu Leu 165 170 175 Val Tir Pro Glu Val Lis Arg Leu Met Fen Arg lie Wing Met Arg lie 180 185 190 Leu Leu Gli Cis Glu Pro Gln Leu Wing Gli Asp Gli Asp Ser Glu Gln 195 200 205 Gln Leu Val Glu Ala Fen Glu Glu Met Tre Arg Asn Leu Fen Ser Leu 210 215 220 Pro lie Asp Val Pro Fen Ser Gli Leu Tir Arg Gli Met Lis Arg 225 230 235 240 Asn Leu lie His wing Arg lie Glu Gln Asn lie Arg Ala Lis lie Cis 245 250 255 Gli Leu Arg Wing Ser Glu Wing Gli Gln Gli Cis Lis Asp Wing Leu Gln 260 265 270 Leu Leu lie Glu His Ser Trp Glu Arg Gli Glu Arg Leu Asp Met Gln 275 280 285 Wing Leu Lis Gln Ser Ser Tre Glu Leu Leu Fen Gli Gli His Glu Tre 290 295 300 Tre Ala Ala Ala Ala Tre Ser Leu lie Tre Tir Leu Gli Leu Tir Pro 305 310 315 320 His Val Leu Gln Lis Val Arg Glu Glu Leu Lis Ser Lis Gli Leu Leu 325 330 _ 335 Cis Lis Ser Asn Gln Asp Asn Lis Leu Asp Met Glu lie Leu Glu Gln 340 345. 350 Leu Lis Tir lie Gli Cis Val lie Lis Glu Tre Leu Arg Leu Asn Pro 355 360 365 Pro Pro Pro Gli Gli Fen Arg Val Ala Leu Lis Tre Fen Glu Leu Asn 370 375 380 Gli Tir Gln He Pro Lis Gli Trp Asn Val He Tir Ser He Cis Asp 385 390 395 400 Tre His Asp Val Ala Glu He Fen Tre Asn Lis Glu Glu Fen Asn Pro 405 410 415 Asp Arg Fen Be Pro Wing Pro Pro Glu Asp Wing Ser Arg Fen Ser Fen 420 425 430 He Pro Fen Gli Gli Gli Leu Arg Ser Cis Val Gli Lis Glu Fen Wing 435 440 445 Lis He Leu Leu Lis He Fen Tre Val Val Glu Leu Ala Arg His Cis Asp 450 455 460 Trp Gln Leu Leu Asn Gli Pro Pro Tre Met Lis Tre Ser Pro Tre Val 465 470 475 480 Tir Pro Val Asp Asn Leu Pro Wing Arg Fen Tre His Fen His Gli Gli 485 490 495 He (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1494 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATG GGG CTC CCG GCG CTG CTG GCC AGT GCG CTC TGC-ACC TTC GT CTG 8 Met Gli Leu Pro Ala Leu Leu Ala Ser Ala Leu Cis Tre Fen Val Leu 1 5 10 15 CCG CTG CTG CTC TTC CTG GCT GCG ATC AAG CTC TGG GAC CTG TAC TGC 96 Pro Leu Leu Leu Fen Leu Ala wing Lis Leu Trp Asp Leu Tir Cis 20 25 30 GTG AGC GGC CGC GAC CGC AGT TGT GCC CTC CCA TTG CCC CCC GGG ACT 144 Val Ser Gli Arg Asp Arg Ser Cis Ala Leu Pro Leu Pro Pro Gli Tre 35 40 45 ATG GGC TTC CCC TTC TTT GGG GAA ACC TTG CAG ATG GTA CTG CAG CGG 192 Met Gli Fen Pro Fen Fen Gli Glu Tre Leu Gln Met Val Leu Gln Arg 50 55 60 AGG AAG TTC CTG CAG ATG AAG CGC AGG AAA TAC GGC TTC ATC TAC AAG 240 Arg Lis Fen Leu Gln Met Lis Arg Arg Lis Tir Gli Fen He Tir Lis 65 70 75 80 ACG CAT CTG TTC GGG CGG CCC ACC GTA CGG GTG ATG GGC GCG GAC AAT 288 Tre His Leu Fen Gli Arg Pro Tre Val Arg Val Met Gli Wing Asp Asn 85 90 95 GTG CGG CGC ATC TTG CTC GGA GAC GAC CGG CTG GTG TCG GTC CAC TGG 336 Val Arg Arg He Leu Leu Gli Asp Asp Arg Leu Val Ser Val His Trp 100 105 110 cea gcg teg gtg cgc acc att ctg gga tet ggc tge ctc tet aac ctg 384 Pro Ala Ser Val Arg Tre He Leu Gli Ser Gli CIS Leu Ser Asn Leu 115 120 125 CAC GAC TCC TCG CAC AAG CAG CGC AAG AAG GTG ATT ATG CGG GCC TCC 432 His Asp Ser Ser His Lis Gln Arg Lis Lis Val He Met Arg Ala Fen 130 135 140 AGC CGC GAG GCA CTC GAA TGC TAC GTG CCG GTG ATC ACC GAG GAA GTG 480 Ser Arg Glu Ala Leu Glu Cis Tir Val Pro Val He Tre Glu Glu Val 145 150 155 160 GGC AGC AGC CTG GAG CAG TGG CTG AGC TGC GGC GAG CGC GGC CTC CTG 538 Gli Ser Ser Leu Glu Gln Trp Leu Ser Cis Gli Glu Arg Gli Leu Leu 165 170 175 GTC TAC CCC GAG GTG AAG CGC CTC ATG TTC CGA ATC GCC ATG CGC ATC 576 Val Tir Pro Glu Val Lis Arg Leu Met Fen Arg lie Wing Met Arg lie 180 185 190 CTA CTG GGC TGC GAA CCC CAA CTG GCG GGC GAC GGG GCC TCC GAG CAG 624 Leu Leu Gli Cis Glu Pro Gln Leu Wing Gli Asp Gli Asp Ser Glu Gln 195 200 205 CAG CTT GTG GAG GCC TTC GAG GAA ATG ACC CGC AAT CTC TTC TCG CTG 672 Gln Leu Val Glu Ala Fen Glu Glu Met Tre Arg Asn Leu Fen Ser Leu 210 215 220 CCC ATC GAC GTG CCC TTC AGC GGG CTG TAC CGG GGC ATG AAG GCG CGG 720 Pro He Asp Val Pro Fen Ser Gli Leu Tir Arg Gli Met Lis Ala Arg 225 230 235 240 AAC CTC ATT CAC GCG CGC ATC GAG CAG AAC ATT CGC GCC AAG ATC TGC 768 Asn Leu He His Wing Arg He Glu Gln Asn lie Arg Wing Lis He Cis 245 250 255 GGG CTG CGG GCA TCC GAG GCG GGC CAG GGC TGC AAA GAC GCG CTG CAG 826 Gli Leu Arg Wing Ser Glu Wing Gli Gln Gli Cis Lis Asp Wing Leu Gln 260 265 270 CTG TTG ATC GAG CAC TCG TGG GAG AGG GGA GG CGG CTG GAC ATG CAG 864 Leu Leu He Glu His Ser Trp Glu Arg Gli Glu Arg Leu Asp Met Gln 275 280 285 GCA CTA AAG CAA TCT TCA ACC GAA CTC TTT GGA GGA CAC GAA ACC 912 Wing Leu Lis Gln Ser Ser Tre Glu Leu Leu Fen Gli Gli His Glu Tre 290 295 300 ACG GCC AGT GCC GCC ACA TCT CTG ATC ACT TAC CTG GGG CTC TAC CCA 960 Tre Wing Wing Ala Wing Tre Ser Leu He Tre Tre Leu Gli Leu Tir Pro 305 310 315 320 CAT GTT CTC CAG AAA GTG CGA GAA GAG CTG AAG AGT AAG GGT TTA CTT 1008 His Val Leu Gln Lis Val Arg Glu Glu Leu Lis Ser Lis Gli Leu Leu 325 330 335 TGC AAG AGC AAT CAA GAC AAC AAG TTG GAC ATG GAA ATT TTG GAA CAA 1056 CIS Lis Ser Asn Gln Asp Asn Lis Leu Asp Met Glu He Leu Glu Gln 340 345 350 CTT AAA TAC ATC GGG TGT GTT ATT AAG GAG ACC CTT CGA CTG AAT CCC 1104 Leu Lis Tir He Gli Cis Val He Lis Glu Tre Leu Arg Leu Asn Pro 355 360 365 CCA GTT CCA GGA GGG TTT CGG GTT GCT CTG AAG ACT TTT GAA TTA AAT 1152 Pro Val Pro Gli Gli Fen Arg Val Ala Leu Lis Tre Fen Glu Leu Asn 370 375 380 GGA TAC CAG ATT CCC AAG GGC TGG AAT GTT ATC TAC AGT ATC TGT GAT 1200 Gli Tir Gln He Pro Lis Gli Trp Asn Val He Tir Ser He Cis Asp 385 390 395 400 ACT CAT GAT GTG GCA GAG ATC TTC ACC AAC AAG GAA GAA TTT AAT CCT 1248 Tre His Asp Val Wing Glu Lie Fen Tre Asn Lis Glu Glu Fen Asn Pro 405 410 415 GAC CGA TTC AGT GCT CCT CAC CCA GAG GAT GCC TCC AGG TTC AGC TTC 1296 Asp Arg Fen Be Pro Wing Pro Glu Asp Wing Ser Arg Fen Ser Fen 420 425 430 ATT CCA TTT GGA GGA GGC CTT AGG TGT GTA GGC AAA GAA TTT GCA 1344 He Pro Fen Gli Gli Gli Leu Arg Ser Cis Val Gli Lis Glu Fen Wing 435 440 445 AAA ATT CTT CTC AAA ATA TTT ACA GTG GAG CTG GCC AGG CAT TGT GAC 1392 Lis He Leu Leu Lis He Fen Tre Valu Glu Leu Ala Arg His Cis Asp 450 455 460 TGG CAG CTT CTA AAT GGA CCT CCT ACA ATG AAA ACC AGT CCC ACC GTG 1440 Trp Gln Leu Leu Asn Gli Pro Pro Tre Met Lis Tre Ser Pro Tre Val 465 470 475 480 TAT CCT GTG GAC AAT CTC CCT GCA AGA TTC ACC CAT TTC CAT GGG GAA 1488 Tir Pro Val Asp Asn Leu Pro Wing Arg Fen Tre His Fen His Gli Gli 485 490 495 ATC TGA 1494 He (2) INFORMATION FOR SEQ ID NO: 6 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Pro Fen Gli Gli Gli Pro Arg Leu Cis Pro Gli Tir 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 (D) TOPOLOGY : linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Pro Fen Ser Gli Gli Wing Arg Asn Cis He Gli Lis Gln Fen Wing Met 1 5 10 15 Lis 20 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Pro Fen Ser Gli Gli Wing Arg Asn Cis He Gli Lis Gln Fen Wing Arg 1 5 10 15 Asn Glu Leu Lis 20 (.2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Pro Fen Gli Tre Gli Pro Arg Asn Cis He Gli Met Arg Fen Ala He 1 5 10 15 Met Asn Met Lis 20 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Pro Fen Ser Gli Gli Ser Arg Asn Cis He Gli Lis Gln Fen Ala Met 1 5 10 15 Asn Glu Leu Lis 20 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 351 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: GAACTCCTCT TTGGAGGACA CGAAACCACG GCCAGTGCAG CCACATCTCT GATCACTTAC 60 CTGGGGCTCT ACCCACATGT TCTCCAGAAA GTGCGAGAAG AGCTGAAGAG TAAGGGTTTA 120 CTTTGCAAGA GCAATCAAGA CAACAAGTTG GACATGGAAA TTTTGGAACA ACTTAAATAC 180 ATCGGGTGTG TTATTAAGGA GACCCTTCGA CTGAATCCCC CAGTTCCAGG AGGGTTTCGG 240 GTTGCTCTGA AGACTTTTGA ATTAAATGGA TACCAGATTC CCAAGGGCTG GAATGTTATC 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 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 12: TTTTTTTTTT TT GG 14 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (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 (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 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : fifteen TTTTTTTTTT TTGC 14 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (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 (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 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 18 TTTTTTTTTTT TTAT 14 (2) INFORMATION FOR SEQ ID NO: 19 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base pairs (B) TYPE: nucleic acid (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) CHAIN: one (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: 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 (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 (D) TOPOLOGY: linear i) 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 (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 (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 (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 (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 (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 (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 (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 (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: GTAGCGGCCG CT 12 (2) INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1725 base pairs (B) TYPE: nucleic acid (C) CHAIN: one (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: GCACGAGGGA GGCTGAAGCG TGCC ATG GGG CTG CCG GCG CTG CTG GCC AGT 51 Met Gli Leu pro wing leu leu wing 2 5 GCG CTC TGC ACC TTC GTG CTG CCG CTG CTG CTC TTC CTG GCG GCG CTC 99 Wing leu cis tre fen leu pro leu leu leu wing leu wing wing leu 10 15 20 25 AAG TTG TGG GAC CTG TAC TGT GTG AGC AGC CGC GAT CGC AGC TGC GCC 147 Lis leu trp asp leu tir cis val be be arg asp arg be cis wing 30 35 40 CTC CCC -TTG CCC CCC GGT ACC ATG GGC TTC CCA TTC TTG GGG GAA ACA 195 Leu pro leu pro pro gli tre met gli fen pro fen fen gli glu tre 45 50 55 TTG CAG ATG GTG CTT CAG CGG AGG AAG TTT CTG CAG ATG AAG CGC AGG 243 Leu gln leu val leu gln arg arg lis fen leu gln met lis arg gln 60 65 70 AAA TAC GGG TTC ATC TAC AAG ACG CAC CTC TTC GGG CGG CCG ACT GTC 291 Lys tir gli fen ile il lis tre his leu fen gli arg pro tre val 75 80 85 AGG GTG ATG GGA GCT GAT AAT GTG AGG CGC ATT CTG CTG GGC GAA CAC 339 Arg val met gli wing asp asn val arg arg ile leu leu gli glu his 90 95 100 CGG CTG GTG TCT GTT CAC TGG CCA GCA TCA GTG AGA ACC ATC CTG GGC 387 arg leu val ser val his trp pro wing be val arg tre ile leu gli 110 115 120 GCT GGC TGC CTC TCC AAT CTG CAC GAT TCC TCG CAC AAG CAG CGA AAG 435 Wing gli cis leu be asn leu his asp be his lys gln arg lis 125 130 135 AAG GTG ATT ATG CAG GCG TTC TCT CGA GAG GCT CTG CAG TGC TAC GTG 483 Lys val ile gln wing fen be arg glu wing leu gln cis tir val 140 145 150 CTC GTG ATC GCT GAG GAA GTC AGC AGT TGT CTG GAG CAG TGG CTA AGC 531 leu val wing glu glu val val ser ser cis leu glu gln trp leu be 155 160 165 TGC GGC GAG CGC GGC CTC CTG GTC TAC CCC GAG GTG AAG CGC CTC ATG 579 Cis gli glu arg gli leu leu val tir by glu val lis arg leu met 170 175 180 TTC CGC ATC GCC ATG CGC ATC CTG CTG GGC TGC GAG CCG GGT CCA GCG 627 Feng arg ile W arg ile leu leu gli cis glu pro gli pro ala 190 195 200 GGC GGC GGG GAG GAC GAG CAA CAG CTC GTG GAG GCT TTC GAG GAG ATG 675 Gli gli gli glu asp glu gln gln leu val glu glu fen fen glu glu metallic 205 210 215 ACC CGC AAT CTC TTC TCT CTT CCC ATT GAC GTG CCC TTT AGC GGC CTG 723 Tre arg asn leu fen ser leu pro ile asp val pro fen ser gli leu 220 225 230 TAC CGG GGC GTG AG GCG CGG AAC CTT ATA CAC GCG CGC ATC GAG GAG 771 Tir arg gli val lis ala arg asn leu ile his ala arg ile glu glu 235 240 245 AAC ATT CGC GCC AAG ATC CGC CGG CTT CAG GCT ACA GAG CCG GAT GGG 819 Asn ile arg ala lis arg arg leu gln ala tre glu asp asp gli 250 255 260 265 GGT TGC AAG GAC GCG CTG CAG CTC CTG ATT GAG CAC TCG TGG GAG AGG 867 Gli cis lis asp ala leu gln leu leu ile glu his ser trp glu arg 270 275 280 GGA GAG AGG CTG GAT ATG CAG GCA CTA AAA CAA TCG TCA ACA GAG CTC 915 Gli glu arg leu asp met gln wing leu lys gln be tre glu leu 285 290 295 CTC TTT GGT GGT CAT GAA ACT ACA GCC AGT GCT GCG ACA TCA CTG ATC 963 Leu fen gli gli his glu tre tre ala ala tre tre leu ile 300 305 310 ACT TAC CTA GGA CTC TAC CCA CAT GTC CTC CAG AAA GTT CGA GAA GAG 1011 Tre tir leu gli leu tir pro his val leu gln lis val arg glu glu 315 320 325 ATA AAG AGC AAG GGC TTA CTT TGC AAG AGC AAT CAA GAC AAC AAG TTA 1059 I have to be lis gli leu leu cis lis be asn gln asp asn lis leu 330 335 340 345 GAC ATG GAA ACT TTG GAA CAG CTT AAA TAC ATT GGG TGT GTC ATT AAG 1107 Asp met glu tre leu glu g ln leu lis tir ile gli cis val ile ile 350 355 360 GAG ACC CTG CGA TTG AAT CCT CCG GTT CCA GGA GGG TTT CGG GTT GCT 1155 Glu tre leu arg leu asn pro for val pro gli gli fen arg val ala 365 370 375 CTG AAG ACT TTT GAG CTG AAT GGA TAC CAG ATC CCC AAG GGC TGG AAT 1203 Leu lis tre fen leu asn gli tir gln ile pro lis gli trp asn 380 385 390 GTT ATT TAC AGT ATC TGT GAC ACC CAC GAT GTG GCA GAT ATC TTC ACT 1251 Val ile tir be ile cis asp tre asp asp asp fen fen 395 400 405 AAC AAG GAG GAA TTT AAT CCC GAC CGC TTT ATA GTG CCT CAT CCA GAG 1299 Asn lis glu glu fen asn pro asp arg fen ile val pro-glu 410 415 420 425 GAT GCT TCC CGG TTC AGC TTC ATT CCA TTT GGA GGC GGC CTG CGG AGC 1347 Asp wing be arg fen be fen gli gli gli leu arg be 430 435 440 TGT GTA GGC AAA GAG TTT GCA AAA ATT CTT CTT AAG ATA TTT ACA GTG 1395 Cis val gli lis glu fen ala lis ile leu leu lis ile fen tre val 445 450 455 GAG CTG GCT AGG CAC TGT GAT TGG CAG CTT CTA AAT GGA CCT CCT ACA 1443 Glu leu wing arg his cis asp trp gln leu leu asn gli pro pro tre 460 465 470 ATG AAG ACA AGC CCC ACT GTG TAC CCT GTG GAC AAT CTC CCT GCA AGA 1491 Met lis tre pro pro val val pro val asp asn leu pro ala arg 475 480 485 TTC ACC TAC TTC CAG GGA GAT ATC TGATAGCTAT TTCAATTCTT 1535 Fen tre tir fen gln gli asp 490 495 GGACTTATTT GAAGTGTATA TTGGTTTTTT TTAAAAATAG TGTCATGTTG ACTTTATTTA 1595 ATTTCTAAAT GTATAGTATG ATATTTATGT GTCTCTACTA CAGTCCCGTG GTCTTTAAAT 1655 ATTAAAATAA TGAATTTGTA TGATTTCCCA ATAAAGTAAA ATTAAAAAGT GAAAAAAAAA 1715 AAAAAAAAAA 1725 (2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 497 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Met Gli Leu Pro Ala Leu Leu Ala Be Ala Leu Cis Tre Fen Val Leu 1 5 10 15 Pro Leu Leu Leu Fen Leu Ala Ala Leu Lis Leu Trp Asp Leu Tir Met 20 25 30 Val Ser Ser Arg Asp Arg Ser Cis Ala Leu Pro Leu Pro Pro Gli Tre 35 40 45 Met Gli Fen Pro Fen Fen Gli Glu Tre Leu Gln Met Val Leu Gln Arg 50 55 60 Arg Lis Fen Leu Gln Met Lis Arg Arg Lis Tir Gli Fen He Tir Lis 65 70 75 80 Tre His Leu Fen Gli Arg Pro Tre Val Arg Val Met Gli Wing Asp Asn 85 90 95 Val Arg Arg He Leu Leu Gli Glu His Arg Leu Val Ser Val His Trp 100 105 110 Pro Wing Ser Val Arg Tre He Leu Gli Wing Gli Cis Leu Ser Asn Leu 115 120 125 His Asp Ser Ser His Lis Gln Arg Lis Lis Val He Met Arg Ala Fen 130 135 140 Ser Arg Glu Ala Leu Gln Cis Tir Val Leu Val He Ala Glu Glu Val 145 150 155 160 Ser Ser Cis Leu Glu Gln Trp Leu Ser Cis Gli Glu Arg Gli Leu Leu 165 170 175 Val Tir Pro Glu Val Lis Arg Leu Met Fen Arg He Ala Met Arg He 180 185 190 Leu Leu Gli Cis Glu Pro Gli Pro Wing Gli Gli Gli Glu Asp Glu Gln 195 200 205 Gln Leu Val Glu Wing Fen Glu Glu Met Tre Arg Asn Leu Fen Ser Leu 210 215 220 Pro He Asp Val Pro Fen Ser Gli Leu Tir Arg Gli Val Lis Ala Arg 225 230 235 240 Asn Leu He His Wing Arg He Glu Gln Asn He Arg Wing Lis He Arg 245 250 255 Arg Leu Gln Ala Tre Glu Pro Asp Gli Gli Cis Lis Asp Ala Leu Gln 260 265 270 Leu Leu He Glu His Ser Trp Glu Arg Gli Glu Arg Leu Asp Met Gln 275 280 285 Ala Leu Lis Gln Ser Ser Tre Glu Leu Leu Fen Gli Gli His Glu Tre 290 295 300 Tre Wing Wing Ala Wing Tre Ser Leu He Tre Tre Leu Gli Leu Tir Pro 305 310 315 320 His Val Leu Gln Lis Val Arg Glu Glu He Lis Ser Lis Gli Leu Leu 325 330 335 Cis Lis Ser Asn Gln Asp Asn Lis Leu Asp Met Glu Tre Leu Glu Gln 340 345 350 Leu Lis Tir He Gli Cis Val He Lis Glu Tre Leu Arg Leu Asn Pro 355 360 365 Pro Val Pro Gli Gli Fen Arg Val Ala Leu Lis Tre Fen Glu Leu Asn 370 375 380 Gli Tir Gln He Pro Lis Gli Trp Asn Val He Tir Ser He Cis Asp 385 390 395 400 Tre-His Asp Val Wing Asp He Fen Tre Asn Lis Glu Glu Fen Asn Pro 405 410 415 Asp Arg Fen He Val Pro His Pro Glu Asp Wing Ser Arg Fen Ser Fen 420 425 430 He Pro Fen Gli Gli Gli Leu Arg Ser Cis Val Gli Lis Glu Fen Ala 435 440 445 Lis He Leu Leu Lis He Fen Tre Val Glu Leu Ala Arg His Cis Asp 450 455 460 Trp Gln Leu Leu Asn Gli Pro Pro Tre Met Lis Tre Ser Pro Tre Val 465 470 475 480 Tir Pro Val Asp Asn Leu Pro Wing Arg Fen Tre Tir Fen Gln GH Asp 485 490 495 He (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 273 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: CGCACCCCAG GAGGCGCGCT CGGAGGGAAG CCGCCACCGC CGCCGCCTCT GCCTCGGCGC 60 GGAACAAACG GTTAAAGATT TTGGGCCASC GCCTCCGCGG GGGGAGGAGC CAGGGGCCCC 120 AATCCCGCAA TTAAAGATGA ACTTTGGGTG AACTAATTGT CTGACCAAGG TAA.CGTGGGC 180 AGCAACCTGG GCCGCCTATA AAGCGGCAGC GCCGTGGGGT TTGAAGCGCT GGCGGCGGCG 240 GCAGGTGGCG CGGGAGGTCG CGGCGCGCCA TGG 273 (2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 274 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT. 34: CGCACCCCCA GGAGGCGCGC TCAGAGGGAA GCCGCCAGTG CGCCGCCTCT GCCTCGGCGC 60 GGAACAAACG GTTAAAGATT TTTTTGGGCA GCGCCTCGAG GGGGGAGGAG CCAGGGGCCC 120 GATCCGCAAT TAAAGATGAA CTTTGGGTGA ACTAATTTGT CTGACCAAGG TAACGTGGGC 180 AGTAACCTGG GCGGCCTTAT AAAGAGGGCG CGCGGCGGGG TTCGGAGCTA GGGAGGCGGC 240 GGCAGGTGGC GCGGGAGGCT GAAGCGTGCC ATGG 274 (2) INFORMATION FOR SEQ ID NO: 35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 319 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xi i) SEQUENCE DESCRITION: SEQ ID NO. 35: TCGGGGG? AT TAACACCTTT TCAAAGTGAA ATCTCAGGAT TGTCTGCCTT CTACAGGAGG 60 TGGTATTAAA ATGCGCCTAT AACAAATGGT TGAGAGTTTG GAGCCGCTTC TGCCCTGTGG 120 GCGGGGCGAG ATGACACCAC AATTAAAGAT GAACTTTGGG TGAACTAATT TATCTGAGGA 180 AGTTAACAGG AGGAGACCTG CGCGCAATGG ATATATAAGG GCGCGCAGGC GAGGACGCCC 240 TCAGTTTGTG CGTAAAGACG CGTCTCCTCT CCAGAAGCTT GTTTTTCGTT TTGGCGATCA 300 GTTGCGCGCT TCAACATGG 319 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xiü) DESCRIPTION OF SEQUENCE: SEQ ID NO. 36: GATCCCAGATCTGCCTATTGCGCCCGATGCCCCGAGGCTCTCTCTTGGAC TCTGGCCCTGAGTTCTTCTGCGCGATCCTTCGGAGACGTCTGGAGGCCTG CTTTATGCATCTCTCTTGGACCTCAGTTTCCCCACACGTGGGAGGAGGCA GCTGGACGATTCCTGAAAGGACTTTCCCTTGCTTCCTCATCACGTGGAAG AGAGCCCACCCGGCACCTGGAAATGGAAAGCCAGTGAAGGCTGCTTTGGG CCGGGGCAKCGGGTGGGACCGGGCGGGAGGGATTCCAAAGAGACCGCCGG GAAGGCTAGAGCTTGGAATTCCGGCTCCTCGGAGTCCTGGCCCTCCCCCA CCGCCGCCTCGGAGCTCAGCACACCTTGGATGGGGGAGGCGGGCAGCTCC TAGCCCCGCACCCCAGGAGGCGCGCTCGGAGGGAAGCCGCCACCGCCGCC GCCTCTGCCTCGGCGCGGAACAAACGGTTAAAGATTTTGGGCCASCGCCT CCGCGGGGGGAGGAGCCAGGGGCCCCAATCCCGCAATTAAAGATGAACTT TGGGTGAACTAATTGTCTGACCAAGGTAACGTGGGCAGCAACCTGGGCCG CCTATAAAGCGGCAGCGCCGTGGGGTTTGAAGCsCTGGCGGCGGCGGCAG GTGGCGCGGGAGGTCGCGGCGCGCCATGGGGCTCCCGGCGCTGCTGGCCA GTGCGCTCTGCACCTTCGTGCTGCCGCTGCTGCTCTTCCTGGCTGCGATC AAGCTCTGGGACCTGTACTGCGTGAGCGGCCGCGACCGCAGTTGTGCCCT CCCATTGCCCCCCGGGACTATSGGSTTCCCCTTCTTTGGGGAAACCTTGC AGATGNTACTNCAGGTAAGGGAGGGTGGGGCGGGACAGGCTGCTTCCCCG GAGCCCGGCGCGGCTCTGGGCTTCTGCTGAAGTCGGGGTAGGCGCCCCCG GGAGGCATGCTATTGCGGCTAGGAGCAGGGCTGGCGGGAGCGCGGCGCTC CCCGGMKYMCSCTCA GCSCRC WKTMWCCTCCGCCTYMCTCCCAMAGCG GARSAAWKCyKGMRGATGAAGCGCAGGAAATACGGCTTCATCTACAAGA CGCATCTGTTCGGGCGGCCCACCGTACGGGTGATGGGCGCGGACAATGTG CGGCGCATCTTGCTCGGAGAGCACCGGCTGGTGTCGGTCCACTGGCCAGC GTCGGTGCGCACCATTCTGGGATCTGGCTGCCTCTCTAACCTGCACGACT CCTCGCACAAGCAGCGCAAGAAGGTGGGGGCAGGAGGCGACGGCTGGACA GGGAGGGGGACCCCATTTATGAGCGGAATTCCGGCTGATGGATGCTAGGC GCGGGCTAGCAGCTTGAGGTGGGCTAGGACCCTCTGCCAGCTCCAGGTTA GCTTTCCCAGCTCGGAGAGTGCCATGTGTCTGGCAGGACTGGGGGTGTCT GGAAGGGGACGGCGGTAGACGAGAGGGGCGGATGGAGGCTTTTAACGCTG TCCCCTCCTCGGGACTCAGGTGATTATGCGGGCCTTCAGCCGCGAGGCAC TCGAATGCTACGTGCCGGTGATCACCGAGGAAGTGGGCAGCAGCCTGGAG CAGTGGCTGAGCTGCGGCGAGCGCGGCCTCCTGGTCTACCCCGAGGTGAA GCGCCTCATGTTCCGAATCGCCATGCGCATCCTACTGGGCTGCGAACCCC AACTGGCGGGCGACGGGGACTCCGAGCAGCAGCTTGTGGAGGCCTTCGAG GAAATGACCCGCAATCTCTTCTCGCTGCCCATCGACGTGCCCTTCAGCGG GCTGTACCGGGTAAGGGCGGCAAACGGGCTGCGGACTAGGGGCGCGGGAC CTGGGCGTCTGCTCACCGCCGCGCGCTCTCTGCGCTCAGGGCATGAAGGC GCGGAACCTCATTCACGCGCGCATCGAGCAGAACATTCGCGCCAAGATCT GCGGGCTGCGGGCATCCGAGGCGGGCCAGGGCTGCAAAGACGCGCTGCAG CTGTTGATCGAGCACTCGTGGGAGAGGGGAGAGCGGCTGGACATGCAGGT GAGTAGCAGCTTCAGACCAGGCACTGCGGAGTTTGGTCCCCTGGCTTTCC AAGGCGCTGTTCCTGGGGCCCCCAAAGCGCGCGCCTGGGGCCCAGCTTTC TGGAGTGGGCGGCCGGCTCAGACTACAGCTATGGAATCCCGAAGGAAGGC TGAGACACCCGGTCAGGAGAGCTGCGGAAGGGGCTGCGGMGGAAACTGGG AGCATCCCCTAGCCTTTAMCAGGTTTCAAAGGGAAAGTTGGAATTTGCAA AAATGTTAATAAAGAACCTTGCGATTTTAATAAAACTAAGACTTTAACTC AGGAGTTTCCGGTAGRGCGGGGTCGTACTCGCCTTACTGCTCCAGCTGAA CTAAAGGGACGTTGCATTTTGTTTAAAGATATTGCTTTCCTTGACTTTCT GTCAGCAAAACATTTAGCCCTTCTAGTCTTCCCTCCAGAACTCTCAGTTC GATTCTGAGTAATCCTTCTGTCAAACCGCAGGCAGACTTGTGAGAATGTG GGTCTCACTCTATTCTTAGGCACTAAAGCAATCTTCAACCGAACTCCTCT TTGGAGG? CACGAAACCACGGCCAGTGCAGCCACATCTCTGATCACTTAC CTGGGGCTCTACCCACATGTTCTCCAG (2) INFORMATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xiv) DESCRIPTION OF SEQUENCE: SEQ ID DO NOT . 37 GATCCAGGTTGCTGAAACATATCTCCATATAGGGCAGAACAATTATCAAA AGCATAAGAATTGCAGCCACAGCATAGGGAAGAAAGAGGAGTTTTTAAAC CACAACAAAAGGGAGAAAGAAGAGAATTTTAACTTACATTTAATTCAAAA GTCTTCAGAGCAACCCGAAACCCTCCTGGAACTGGGGGATTCAGTCGAAG GGTCTCCTTAATAACACACCCGATGTATYTAAGTTGTTCCAAAATTTCCA TGTCCAACTTGTTGTCTTGATTGCTCTTGCAAAGTAAACCCTAYCAAAAY AGTCATACAGAGGTGAACAGTYATTTTGTGCTCCAATTAAAATCAGCCCA GCAGACGTAAACAGGGCTTAAGTGGAGACTAAACCCAAAGGGCCCCATGA TGGGAGAGACTGGGAGGGGGAAACAGCAGCTAATGGCCATTTGCCTGCCC AAATCCACTATCTATTTACAATCCCAGGAGAATGCTGCTCACCAGTTAGA AGGACCAAGTTTCTCCCCACGCCCCCCCACCCCACACTCACCACCACCAC CCACACTAATCAGCTATTCACACTATGTATGCCCTTGGACACACCAATTC AAGAAAAGTGGAACCTATCTGAGAATCTCCACGGTTCACAAAAAGGTGGA GGAGGGGTAGGAATACAAGGTCAAACCCTGCCC (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF SEQUENCE: (A) LENGTH: base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (xv) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 38: TCGCGAGGAGCGACCACGGCTTGAAGAGGGGTAGACGAGACCAGATGCTC CCCGGCGCCCCCTCATGCGGGTTGCGGTCTCTCTCCTCCACCTCCCTCTC AGCGGAGGAAGTTTCTGCAGATGAAGCGCAGGAAATACGGCTTCATCTAC AAGACGCATCTGTTTGGGCGGCCCACGGTGCGGGTGATGGGCGCGGATAA TGTGCGGCGCATCTTGCTGGGAGAGCACCGGTTGGTGTCGGTGCACTGGC CCGCGTCGGTGCGCACCATCCTGGGCGCTGGCTGCCTCTCCAACCTGCAC GATTCCTCGCACAAGCAGCGAAAGAAGGTGAGGGTGAGCTGGCAACTCCT TGGCTGGCAGGGAGACCTCATCCTATGGCTTGGTTCAGGCAAAATAGAAT GCGGGGCGAGGGCTAGTCCTATGTGGTGGGGACCAGGACCCTCTCTATCT GAGATCCACTTTAGCTTTTCTGCTAGCACGTGGGTTAGTCCTGGGGGGGA CTGAAATTCTTGAAAGGGTACTCGGAAAGGCGAAGGGGGGGGGGCTGAGG GAAAGTAGAGGATTGTAACACTCTCTGCTCCTGGGGGGTGCTCAGGTGAT TATGCAGGCCTTCAGCCGCGAGGCACTCCAGTGCTACGTGCCCGTGATCG CTGAGGAAGTCAGCAGTTGTCTGGAGCAGTGGCTAAGCTGCGGCGAGCGC GGCCTCCTGGTCTACCCCGAGGTGAAGCGCCTCATGTTCCGCATCGCCAT GCGCATCCTGCTGGGCTGCGAGCCGGGTCCAGCGGGCGGCGGGGAGGACG AGCAGCAGCTCGTGG? GGCTTTCGAGGAGATGACCCGCAATCTCTTCTCT CTTCCCATTGACGTGCCCTTTAGCGGCCTGTACCGGGTAAGGGCGGTTTG CGGAGTCGGAGTAGGGGAACGCAAGCTCGGGCATCCGCTCACCGCCACGC TCTCTCCGCGCTCAGGGCGTGAAGGCGCGGAACCTTATACACGCGCGCAT CGAGGAGAACATTCGCGCCAAGATCCGCCGGCTTCAGGCTACAGAGCCGG ATGGGGGTTGCAAGGACGCGCTGCAGCTCCTGATTGAGCACTCGTGGGAG AGGGGAGAGAGGCTGGATATGCAGGTGAGAAGCAATTTCAAAAGGTGCCA AGGGCCGGGGAGTGCCTCTGACTTTCCAGACACACTTTCTGGGGTCTCCA AAGCCCTGTCAAGGCCCCAGCTACTTCCAAGTGGGCGGCGATGCTAGGTC TAGAGCTTTTCAACCTGTGGGTCGTGACCCCTTCACGGAGCCAAACAACC CTTTCAGAAGGGTCGCCTAAGAGCATCTGCATATCCGATATTTACATCAA GAAACATAACAGTAGCAAAATTACCGTTATGAAGTAGCAACAAAGATAAT TTTATCGTTGGGGGTCACCACAACACGAGGAACCGTATTAAAGGGTGGCA TTGGTCTAGAGAGCTGTGGAAGGGGGTGGCTGAGCAATGGGGAAGATCCC AAAGTTCAAAGGGCAAGGCTCATCTACAAAGGTTAAAGCGGAAGAGCAGG ATTAAGGGAGTTTTGCGTTTTTGTTTGTGGTCTTTGACTTTCTATGAACA AAACGGATTTTACCCTTGAAGTCTTCCGTGCAATATTCTCAGGTCAGGTC TTTGTAACAGTGCTATAAACTGCACTCAGATCTGTATAAACTTCCGTTTT TATCCTTAGGCACTAAAACAATCGTCAACAGAGCTCCTCTTTGGTGGTCA TGAAACTACAGCCAGTGCTGCGACGTCACTGATCACTTACCTAGGACTCT ACCCACATGTCCTCCAGAAAGTTCGAGAAGAGATAAAsAGCAAGGTAGGA TGATTCTAGAGGTTCCCCATTTGCCTAGGACATTCCTCTATTAACCACCA CCACCACCCCCACTGTATATAAGTTTGCTCGATACACCCAGTACTATGAC AGTGAAGATCTGAGAGCTAGGTGGGACTGTGGGGGAGAGACTCCACCTCG TGAATTTAAAAAGGCAGTTGTTTGTACTGGGCTCTCTCTTGGGCAGAATT TGACCCTCTCCTCCTCCTCCTCCTCCTCCTCCTCTTCCTCCTCCACCACC ACCACCATCACCACCTTTTATAGAGCAAGGTTCTCCTTTCCCTGACCAAG AACATGAATAATGTGATTAGAGCCAATAGCTGATCAGGGTCGCAGTGTTG GTGAGGGCTCAGGGTATG? CCCTTTATATACCTGATAAGCAACATTGTCT GGATAATGGGTTTAGGCTGAGGAAGTGTGGAAAGGAAGGCCATCAGGCCA TCAGCTCTTTCCCTTTTATCCTCTCCCATCCAGACGCCTTCAGGTTTAGT TAACAGGTGAGTCCTGCTGGGCTGACTTTTTTTTTGGAGTGCCCAGGGAT CCATCACTCACTTTTTTATCTGTTTCCATAGGGCTTACTTTGCAAGAGCA ATCAAGACAACAAGTTAGACATGGAAACTTTGGCACAGCTTAAATACACT GGGTGTGTCATTAAGGAGACCCTGCGATTGAATCCTCCGGTTCCAGGAGG GTTTCGGGTTGCTCTGAAGACTTTTGAGCTGAATGTGAGTGCACCTCCTG TCCCCCACCCCCAGCCCTCGTCCACGTCCACTCTGCTATGCTGTTGAGCA TCAGCTGCCCAGAGCAGTGGCTCACTGCCCTTGACAGTGTCCTGCCTCCT ATGGTACTGGGAACCAATTTGCTCTCCTCTCTTAATGCCATCCATGCTAG TAATGACTTTTTGTTGTTGCAAGCTCAGGGCCGGGATTGTCAATTCTT? G GATTTTTTTTTTTTTTTAAACAGGGATACCAGATCCCCAAGGGCTGGAAT GTTATTTACAGTATCTGTGACACCCACGATGTGGCAGATATCTTCACTAA CAAGGAGGAATTTAATCCCGACCGCTTTATAGTGCCTCATCCAGAGGATG CTTCCCGGTTCAGCTTCATTCCATTTGGAGGAGGCCTTCGGAGCTGTGTA GGCAAAGAGTTTGCAAAAATTCTTCTTAAGATATTTACAGTGGAGCTGGC TAGGCACTGTGATTGGCAGCTTCTAAATGGACCTCCTACAATGAAGACAA GCCCCACTGTGTACCCTGTGGACAATCTCCCTGCAAGATTTACCCACTTC CAGGGAGATATCTGATAGCTATTTCAATTCTTGGACTTATTTGAAGTGTA TATTGTTTTTTTTAAAATAGTGTCATGTTGACTTTATTTAATTTCTAAAT GTATAGTATGATATTTATGTGTCTCTACTACAGTCCCGTGGTCTTAAATA TTAAAATAATGAATTTGTATGATTTCCCAATAAAGTAAAATTAAAAAGTG CTTCTCTTGCTTTTTAAGATTCTTGTTGGCAAGCTGCCCATGGTGGTACA TTGCTGTAATACTAGGACTTGGAAGGTGGAGGCAAGAAGAGCAGGCATTC AAGGCTAGCCTGGGCTACAGAAATCCTGTCTTAAACAAACACTACAACAA AAAGTCCTGTTAGGGAATCTGACTGGCTCAGTGTTTGTACTTTGTGTATT TAAAATGATTTAGAGTGAAACCATAGGTCTCTCCCCCATGTCAGAAAATA TATATTATTATGTGTATGCTGATCCAAAGTATCTTTGTAACTTTTTCTAA GGTCATTGAGACTTCATATTTTGAAATTGTATGGAGGCTAGTTATATTAC ATTATTT? TTTATTTATTTATTTACATTTTTATGGTGCTGGGGATTGGAT CGAAGGCTTCACACCTCTAGGGCAAGCCCTTTGTCATTAAGGCGCTGCCT CTCCCTTTCAGCCCAACGTTAATTCTAGATTCTTTTTCTTTGGTGCTTTT GGGAGGTAAACCTGGGATGCTGCAGTTATTTGGTGGTGGTCGTTGGTTTT ACTCTAGAGAGAAGGCAACTTTGGGAAGGCAACACTGCTGCTGGTGAGTC GGGAAGCATCATCCCAGAGCAACGGGGTCAGCATAGCTAACATTTTAAAT CAGCATAATGAATCCCTGTCATATGGAGGAGGCAGAACTCCTCTTTGAAG TTGATATTTTAGATAAGACAGAGCCAGCCCCTCTGGTTATGGACAGTTCT TACCCAAAATGAAACAGAGAAGAAAACCACTGGTGTGTCACCTTT.CCTTA GAAGTGCTTCAGGA

Claims (79)

1. CLAIMS 1. A purified protein characterized by oxidizing 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 identified as SEQ ID NO: 32.
2. 2. The protein according to claim 1, characterized in that it 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 identified as SEQ ID NO: 32.
3. 3. The protein according to claim 1, characterized in that it is at least about 50% conserved in relation to the amino acid sequence identified as SEQ ID NO: 2 or identified as SEQ ID NO: 4 or identified as SEQ ID NO: 32.
4. 4. A protein which is a conservatively substituted variant of the protein according to claim 1, characterized in that 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 identified as SEQ ID NO: 32 and which oxidizes a retinoid.
5. 5. The protein according to claim 1, characterized in that the retinoid is a retinol and / or a retinoic acid.
6. 6. The protein according to claim 1, characterized in that the protein oxidizes the carbon in the 4-position of the β-ionone ring. Of the retinoid.
7. 7. The protein according to claim 6, characterized in that the retinoid is an all-trans retinoid.
8. 8. A purified protein characterized by having the ability to hydroxylate retinoic acid in the 4-position of the ß-ionone ring of retinoic acid, and having a sequence of amino acid which is at least approximately 30% conserved in relation to the sequence of amino acid identified as SEQ ID NO: 2 or as SEQ ID NO: 4 or identified as SEQ ID NO: 32.
9. 9. A purified protein characterized by oxidizing a retinoid at the carbon in the 4-position of the ß-ionone ring of the retinoid .
10. 10. The protein according to claim 9, characterized in that the retinoid is an all-trans retinoid.
11. 11. The protein according to claim 10, characterized in that the retinoid is a retinoic acid.
12. 12. An isolated nucleic acid molecule characterized in that it encodes a protein according to claim 1, or a nucleic acid strand which hybridizes to the nucleic acid molecule under rigid hybridization conditions the complementary coding strand of which it encodes a rotein which is at least 30% conserved in relation to the amino acid sequence identified as SEQ ID NO: 2 or identified as SEQ ID NO: 4 or identified as SEQ ID NO: 32 and which oxidizes a retinoid.
13. 13. An isolated nucleic acid molecule characterized in that it encodes a protein according to claim 8.
14. 14. An isolated nucleic acid molecule characterized in that it comprises the sequence identified as SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO. : 31, or which varies in sequence due to the degeneracy of the genetic code, Q a nucleic acid chain which hybridizes with at least one nucleic acid molecule under rigid hybridization conditions the complementary coding strand of which it encodes a protein which is at least about 30% conserved relative to the amino acid sequence identified as SEQ ID NO: 2 or identified as SEQ ID NO: 4 or identified as SEQ ID NO: 32 and which oxidizes a retinoid.
15. 15. A nucleic acid molecule according to claim 14, characterized in that the sequence of the nucleic acid molecule corresponds to a part of a human genome or a genome of fish or a mouse genome, or varies thereof. to degeneration of the genetic code.
16. 16. Isolated mRNA transcribed from DNA characterized in that it has a sequence which corresponds to a nucleic acid molecule according to claim 14 and which encodes a protein 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 identified as SEQ ID NO: 32 and which oxidizes a retinoid.
17. 17. A microbial cell characterized in that it contains and expresses heterologous DNA encoding a retinoid-inducible protein.
18. 18. A microbial cell according to claim 17 characterized in that the protein oxidizes a retinoid.
19. 19. A microbial cell characterized in that it contains and expresses heterologous DNA encoding a retinoid-inducible protein having all-trans 4-hydroxylase retinoic acid activity.
20. 20. The cell in accordance with the claim 17 characterized because the retinoid is retinoic acid.
21. 21. A microbial cell characterized in that it contains and expresses heterologous DNA comprising a nucleic acid strand according to claim 12.
22. 22. Isolated DNA characterized in that it has a sequence according to claim 14 in a recombinant cloning vector.
23. 23. A stably transfected cell line characterized in that it expresses a protein according to claim 1.
24. 24. A culture of cells transformed with a recombinant DNA molecule characterized in that it has a DNA sequence according to claim 14.
25. 25. A cell host characterized in that it has been engineered to produce a protein according to claim 1, the cell having expressively incorporated in the same heterologous DNA encoding the protein.
26. 26. The cell according to claim 25 characterized in that the production of the protein is inducible by exposing the cell to retinoic acid.
27. 27. The cell according to claim 25 characterized in that the cell is eukaryotic.
28. 28. A process for producing the protein according to claim 1, characterized in that it comprises: Preparing a DNA fragment that includes a nucleotide sequence which encodes the protein; Incorporate the DNA fragment into an expression vector to obtain a recombinant DNA molecule which includes the DNA fragment and is capable of replication; Transforming a host cell with the recombinant DNA molecule to produce a transformant which can express the protein; Cultivate the transformant to produce the protein; and Recovering the protein from the resulting cultured mixture
29. 29. An antibody to a protein according to claim 1.
30. 30. An antibody according to claim 29 characterized in that the antibody is a monoclonal antibody.
31. 31. An antibody to a protein characterized in that it has a sequence included in the sequence identified as SEQ ID NO: 4.
32. 32. A protein according to claim 1 for use in the metabolism of retinoic acid in an organism or cell in need of such metabolization.
33. 33. A method for metabolizing retinoic acid in an organism or cell in need of retinoic acid metabolism characterized in that it comprises administering a protein according to claim 1.
34. 34. A method for inhibiting hydroxylation of retinoic acid in an organism in need of such a inhibition, characterized in that it comprises 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: 3 or SEQ ID NO: 5 or SEQ ID NO: 31 where the portion has at least 5 bases in length.
35. 35. The method according to claim 34 characterized in that the organism is a human.
36. 36. The method of compliance with the claim Characterized in that the organism is being treated for a disease selected from the group consisting of cancer, actinic keratosis, oral leucoplacia, 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 actinic keratosis associated with premalignancy, acne, psoriasis and / or ichthyosis.
37. 37. The method according to claim 36 characterized in that the disease is acute promyelocytic leukemia.
38. 38. A method for producing a desired protein, characterized in that it comprises: Providing a cell which can produce an endogenous protein in response to exposure to a retinoid; Incorporate into DNA of the cell a DNA sequence that encodes the desired protein at or near a site which is normally occupied by a DNA sequence that encodes the endogenous protein; and exposing the cell to the retinoid to induce the production of the desired protein.
39. 39. A device for determining the presence of a protein according to claim 1 or having an amino acid sequence identified as SEQ ID N0: 4, characterized in that it comprises an antibody for the protein linked to a reporter system, wherein the system The reporter produces a detectable response when a predetermined amount of the protein and the antibody bind to each other.
40. 40. A device for determining the presence of a first nucleic acid molecule according to claim 12, the device characterized in that it comprises a second nucleic acid molecule at least approximately 5 bases in length which hybridizes with at least a portion of a first nucleic acid molecule under rigid 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 ones hybridize to each other molecule.
41. 41. An isolated DNA molecule characterized in that it has a nucleotide sequence selected from the group consisting of: (a) SEQ ID NO: 33 (b) SEQ ID NO: 34 (c) SEQ ID NO: 35. (d) A fragment of (a), (b), or (c), Where the DNA molecule possesses promoter activity.
42. 42. A DNA molecule according to claim 41 characterized in that the DNA molecule includes the sequence TGAACT (N) XTGAACT, wherein x has a value of up to
43. 43. A DNA molecule according to claim 42 characterized because x is 5.
44. 44. A DNA molecule according to claim 42 characterized in that the DNA molecule further includes the sequence TCTGASSAAGKTAAC downstream of the sequence TGAACT (N) XTGAACT.
45. 45. A DNA molecule according to claim 44 characterized in that it further includes the AAT sequence between the sequence TGAACT (N) XTGAACT and the sequence TCTGASSAAGKTAAC.
46. 46. A DNA molecule according to claim 44 characterized in that there are up to six nucleotides between the sequence TGAACT (N) XTGAACT, and the sequence TCTGASSAAGKTAAC.
47. 47. A DNA molecule according to claim 42, characterized in that it also comprises the sequence CAATTAAAGA upstream of the sequence TGAACT (N) xTGAACT.
48. 48. A DNA molecule according to claim 41 characterized in that the DNA molecule includes the sequence CAATTAAAGATGAACTTTGGGTGAACTAATT.
49. 49. A DNA molecule according to claim 41, 42, 43, 44, 45, 46, 47, or 48 characterized in that the DNA molecule includes the TATAA sequence.
50. 50. A DNA molecule according to claim 42, 43 or 47, characterized in that the DNA molecule includes the sequence TATAA downstream of the sequence TGAACT (N) XTGAACT.
51. 51. A DNA molecule according to claim 44 or 45, characterized in that the DNA molecule includes the sequence TATAA downstream of the sequence TCTGASSAAGKTAAC.
52. 52. A DNA molecule according to claim 42 characterized in that the DNA molecule includes the TATAA sequence located downstream of the TGAACT (N) XTGAACT sequence and separated up to about 55 nucleotides thereof.
53. 53. A recombinant DNA characterized in that it comprises the isolated DNA molecule according to claim 41.
54. 54. A recombinant DNA according to claim 53, characterized in that it also comprises one or more structural genes.
55. 55. A recombinant DNA according to claim 54, characterized in that the one or more structural genes encode the cytochrome P450 protein.
56. 56. An expression plasmid characterized in that it comprises the recombinant DNA according to claim 53.
57. 57. An isolated cell characterized in that it contains the recombinant DNA according to claim 5,
58. 58. An isolated cell according to claim 57, characterized in that the cell is eukaryotic.
59. 59. A process for the production of protein, characterized in that it comprises culturing a cell according to claim 57 and recovering the proteins produced.
60. 60. A process according to claim 59 characterized in that the protein comprises a cytochrome P450.
61. 61. A process according to claim 59 characterized in that the protein comprises a fusion protein.
62. 62. A method for screening drugs for their effect on the activity of a retinoic acid-inducible protein characterized in that it comprises exposing the purified protein to a drug and determining the effect on the activity.
63. 63. The method according to claim 62 characterized in that the activity is hydroxylation of a retinoic acid, particularly all trans retinoic acid.
64. 64. A method for examining drugs for their effect on the activity of a protein according to claim 1, characterized in that it comprises exposing the purified protein to a drug and determining the effect on the activity.
65. 65. The method according to claim 64, characterized in that the method includes determining the effect that the drug has on the oxidation of the retinoic acid.
66. 66 The method according to claim 65 characterized in that the retinoid is all trans retinoic acid.
67. 67. A method for examining drugs for their effect on the expression of a gene wherein the gene is inducible by a retinoid, characterized in that it comprises exposing a recombinant DN according to claim 54 to the drug and determining the effect on the expression of the gene .
68. 68. The method according to claim 67, characterized in that it includes exposing the recombinant DNA in the presence of a retinoid.
69. 69 The method according to claim 67 characterized in that the structural gene is a reporter gene which does not metabolize retinoic acid.
70. 70. A method for examining drugs for its effect on the expression of a gene wherein the gene is inducible by a retinoid, characterized in that it comprises exposing a recombinant DNA according to claim 53 to the drug and determining the effect on the expression of the gene wherein determining the effect on gene expression includes determining the effect on transcription of the gene.
71. 71. A method for examining a drug for its effect on the metabolism of a retinoid by a cytochrome P450 encoded by a nucleotide sequence according to claim 12 which is incorporated into an expression system to be under control of a sequence of nucleotide having native promoter activity by cytochrome P450, characterized in that it comprises: Exposing the system to the drug in the presence of the retinoid to determine the effect of the drug on the metabolism of the retinoid.
72. 72. The method according to claim 71 characterized in that the retinoid is retinoic acid.
73. 73. The method according to claim 72 characterized in that the retinoic acid is all trans retinoic acid.
74. 74. A drug identified according to the method of claim 62, 63, 64, 65 or 66 as having the effect of modulating the activity of the protein.
75. 75. A drug identified according to a method of claim 67, 68, 69, 70, 72 or 72 as having the effect of modulating the expression of the gene.
76. 76. A drug identified according to a method of claim 73 as having the effect of modulating the expression of the gene.
77. 77. A method for inhibiting the metabolism of retinoic acid in an organism in need of such inhibition, characterized in that it comprises administering to the organism an effective amount of a drug of claim 74.
78. 78. A method for inhibiting the metabolism of retinoic acid in a organism in need of such inhibition, characterized in that it comprises administering to the organism an effective amount of a drug of claim 75.
79. 79. A method for inhibiting the metabolism of retinoic acid in an organism in need of such inhibition characterized in that it comprises administering to the organism an effective amount of a retinoic acid drug. claim 76.