AU4899300A - Nucleic acid molecule encoding a 11-cis retinol dehydrogenase - Google Patents

Description

1 "NUCLEIC ACID MOLECULE ENCODING A 11-CIS RETINOL DEHYDROGENASE" Field of the Invention This invention relates to a protein having ll-cis 15 retinol dehydrogenase activity, and which forms a complex with a specific portion of a membrane receptor for plasma retinol-binding protein (RBP) expressed, in retinal pigment epithelium (RPE), and more specifically a 32 kDa protein having ll-cis retinol dehydrogenase activity, which forms a complex with a 63 kDa RBP-binding membrane protein.

The invention also involves isolation of the 32 kDa protein (p32), as well as nucleic acid molecules coding for p 3 2 or complementary to coding sequences therefor, in addition to various applications of these materials.

Background of the Invention Retinoids (vitamin A-derivatives) have important physiological functions in a variety of biological processes. During embryonic growth and development, as well as during growth and differentiation of adult organisms, retinoids act as hormones and participate in the regulation of gene expression in a number of cell types.

See Lied et al. Trends Genet., 17:427-433 (1992). It is believed that these effects are mediated through two classes of nuclear ligand-controlled transcription factors, WO 97/19167 PCT/US96/18295 .2 the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), Benbrook et al., Nature, 333:669-672 (1988); Brand et al., Nature, 332:850-853 (1988); Giguere et al., Nature, 330:624-629 (1987); Mangelsdorf et al., Nature, 345:224-229 (1990); Mangelsdorf, et al. Genes Dev.

6: 329-344 (1992); Petkovich et al. Nature 330:440-450 (1987); and Zelent et al., Nature 339:714-717 (1989).

Apart from their role as hormones in cellular growth and differentiation, retinoids are also involved in the .0 visual process as the stereo isomer 11-cis retinaldehyde of retinaldehyde is the chromophore of the visual pigments.

See, e.g. Bridges, The Retinoids, Vol. 2, pp 125-176, Academic Press, Orlando, Florida, (1984).

Under normal physiological conditions most cells, both .5 ocular and non-ocular, obtain all-trans retinol as their major source of retinoids. Despite the many different metabolic events taking place in different tissues, it is known that a common extracellular transport machinery for retinol has evolved. Specifically, in plasma, retinol is !0 transported by plasma retinol binding protein (RBP). See Goodman et al., The Retinoids, Academic Press, Orlando Florida, Volume 2, pp. 41-88 (1984). The active derivatives of retinol, retinoic acid in non-ocular tissues and mostly 11-cis retinaldehyde for ocular tissues, are then generated by cellular conversion using specific mechanisms. To date, none of these mechanisms have been fully defined at the molecular level and several of the enzymes involved have only been identified by enzymatic activities. See Lion et al., Biochem. Biophys. Acta.

384:283-292 (1975); Zimmermann et al., Exp. Eye Res.

21:325-332 (1975); Zimmermann, Exp. Eye Res. 23:159-164 (1976) and Posch et al., Biochemistry 30:6224-6230 (1991).

Polarized retinal pigment epithelial cells (RPE) are unique with regard to retinoid uptake since all-trans retinol enters these cells via two different mechanisms.

Retinol accumulated from RBP is taken up through the basolateral plasma membrane, while all-trans retinol, WO 97/19167 PCT/US96/18295 3 presumably taken up from the interstitial retinol-binding protein (IRBP) following bleaching of the visual pigments, may enter through the apical plasma membrane. See Bok et al., Exp. Eye Res. 22:395-402 (1976); Alder et al., Biochem. Biophys. Res. Commun. 108:1601-1608 (1982); Lai et al., Nature 298:848-849 (1982); and Inu et al., Vision Res.

22:1457-1468 (1982).

The transfer of retinol from RBP to cells is not fully understood. In a number of cell types, includina RPF, specific membrane receptors for RBP have been identified, which is consistent with a receptor-mediated uptake mechanism for retinol. For example, isolated retinol binding protein receptors, nucleic acid molecule coding for these receptors and antibodies binding to the receptor have 15 been taught, in references relating to the first of the two mechanisms. See Bavik et al., J. Biol. Chem. 266:14978- 14985 (1991); Bavik, et al. J. Biol. Chem. 267:23035-23042 1992; Bavik et al., J. Biol. Chem. 267:20540-20546 (1993); and copending U.S. Application Serial No. 083,539 and International Publication WO 93/23538, all of which are incorporated by reference herein. See also Heller, J.

Biol. Chem. 250:3613-3619 (1975); and Bok et al., Exp. Eye Res. 22:395-402 (1976).

*Retinol uptake on the apical side of the RPE for the 25 regeneration of 11-cis retinaldehyde is less well characterized. Regardless of the origin of all-trans retinol, however, the synthesis and apical secretion of 11cis retinaldehyde seems to be the major pathway for accumulated retinol in the RPE. At present, it is not known whether similar mechanisms are used with regard to cellular retinol uptake through the basolateral and the apical plasma membranes. Available data do show that functional receptors for RBP are exclusively expressed on the basolateral. plasma membrane of RPE-cells. Bok et al., Exp. Eye Res. 22:395-402 (1976).

It is also known that pigment RPEs express a 63 kDa protein (p63). This molecular weight, and all others, is WO 97/19167 PCT/US96/18295 4 by the refernce to SDS-PAGE, unless stated otherwise. It has also been shown by chemical cross-linking that this protein may be part of an oligomeric protein complex which functions as a membrane receptor for plasma retinol-binding protein (RBP) in RPEs, or a component of the retinoid uptake machinery in RPE cells. See Bavik et al, J. Biol.

Chem. 266:14978-14875 (1991); Bavik et al., J. Biol, Chem.

267:23035-23042 (1992), and U.S. Application Serial No.

083,539 and PCT application W093/23538. The p63 protein has been isolated and the corresponding cDNA cloned. See Bavik et al., J. Biol. Chem. 267:20540-20546 (1993).

However, there is nothing in these references suggesting the existence of the protein which is a feature of this invention.

15 Summary of the Invention In accordance with this invention, RPE membrane associated proteins which have a molecular weight of about 32kd, as determined by SDS-PAGE, has now been discovered.

These proteins, referred to as "p32," form oligomeric 20 protein complexes with the previously characterized p 6 3 protein, a component of the membrane receptor for RBP.

Also disclosed are nucleic acid molecules which code for the p32 protein. Sequence analysis shows that the p 3 2 protein belongs to the family of short chain alcohol dehydrogenases, and exhibits 11-cis retinol dehydrogenase activity, the enzyme which catalyzes the stereospecific conversion of 11-cis-retinol into 11-cis retinaldehyde in the presence of cofactor NAD+.

As will be shown, p32 has many important uses. For example, owing to its membrane bound 11-cis-retinol dehydrogenase activity, which catalyzes the conversion of 11-cis-retinol to 11-cis-retinaldehyde, a major metabolic step in retinoid metabolism in RPE-cells, retinoid accumulation and metabolism which may lead to retinitis pigmentosa, may be directly or indirectly tied to the presence of p32, and/or its activation or inhibition. As WO 97/19167 PCT/US96/18295 p32 has also been found to be a member of the short chain alcohol dehydrogenase super family, many known alcohol dehydrogenase inhibitors (and activators) are available to develop activity assays, and thus diagnostic materials for retinol uptake, and ocular retinoid metabolism.

Also a part of this invention are nucleic acid molecules which encode mammalian forms of the proteins, such as the human, bovine, and murine forms. Also a part of the invention are probes, based upon the nucleotide sequences described herein.

These and other aspects of this invention are more fully discussed in the following Detailed Discussion with accompanying drawings.

Brief Description of the Drawings 15 FIG. 1A shows SDS-PAGE analysis of radiolabeled protein from RPE-membranes and immunoprecipatation with mAb A52 against p63.

FIG. 1B shows SDS-PAGE analysis of RPE-membrane proteins bound and eluted through an mAb A52 immunoaffinity 20 column, and the presence of p32 in the eluted faction from the immunoaffinity column.

FIG. 2A shows visualization in agarose gel electrophoresis of a 61 bp PCR-amplified fragment using oligonucleotide mixtures OM1 and OM3, both derived from peptide 321 deduced from partial amino acid sequence .i determination of trypsin digested p32.

FIG. 2B shows visualization of a 330 bp PCR-amplified fragment using oligonucleotide mixtures OM2 and OM3, derived from peptides p323 and p321, respectively, as deduced from partial amino acid sequence determination of trypsin digested p32.

FIG. 3 illustrates the nucleotide sequence of pX321 and the deduced amino acid sequence of p32, with the partial amino acid sequences determined from peptides isolated from trypsin digested p32.

WO 97/19167 PCT/US96/18295 6 FIG. 4 illustrates amino acid sequence alignments of p32 and some related proteins belonging to the family of short-chain alcohol dehydrogenases.

FIG. 5 illustrates analysis of the amino acid sequence of p32.

FIG. 6 illustrates membrane interaction of p32 synthesized in vitro.

FIG. 7 illustrates the restricted expression of transcripts corresponding to p32.

FIG. 8A illustrates expression of p32 in transfected cells for further enzymatic activity analysis of 11-cis retinol dehydrogenase activity.

FIG. 8B illustrates the expression of 11-cis retinol dehydrogenase activity in the presence of NAD+ as indicated 15 by the formation of 11-cis retinaldehyde.

FIG. 8C illustrates the lack of ll-cis-retinol dehydrogenase activity in the presence of cofactor NADP.

FIG. 8D illustrates control cells not expressing p32 which lack the ability to oxidize ll-cis-retinol into 11cis-retinaldehyde.

Figure 9 shows the structure of the human 11-cisretinol dehydrogenase gene.

*Detailed Discussion of Preferred Embodiments It is known that plasma retinol binding protein (RBP) can be chemically cross-linked to a high molecular weight complex of a 63 kDa protein (p63) receptor of retinal pigment epithelium membranes (RPE), forming an RBP-RBP receptor complex with elution properties of globular proteins of similar sizes having apparent molecular weights of approximately M, 150,000 and 450,000. See Bavik et al, J. Biol. Chem. 266:14978-14875 (1991), and Bavik et al., J.

Biol. Chem. 267:23035-23042 (1992). The protein responsible for binding of RBP, expression of which is restricted to RPE, has been identified as a 63 kDa protein (p63). Through the generation of a monoclonal antibody A52 (mAb A52) to the 63 kDa protein which binds the RBP-RBP WO 97/19167 PCT/US96/18295 7 receptor complex and p63, and immunoaffinity chromatographic analysis, a majority of p63 is eluted as a monomer, with a significant portion of the protein found in positions corresponding to higher molecular weight species.

This indicates that p63 exists in an oligomeric protein complex with other protein components. Bavik et al., J.

Biol. Chem. 266:14978-14985 (1991), and Bavik et al, J.

Biol. Chem. 267:23035-23042 (1992). Therefore, the following procedure was carried out to investigate the molecular characteristics of such oligomeric protein complexes, and whether p63 forms a complex with other proteins specific to RPE. The results show that a 32 kDa membrane associated protein (p32) indeed forms a complex with p63.

15 Example 1 Bovine RPE-cells were isolated and membrane fractions were prepared as described in Bavik et al, J. Biol. Chem.

266:14978-14875 (1991) incorporated by reference. RPEmembrane proteins were then solubilized in phosphatebuffered saline (PBS) (20 mM sodium phosphate, ph 7.2, containing 150 mM NaC) containing 1% 3-[(3-cholamidopropyl) -dimethylammonio] -1-propane sulfonic acid (CHAPS) at 1 mg of total membrane protein/mi of buffer. Remaining **material was removed by ultracentrifugation at 100,000 x g for 1 hour. Next, 500 ul aliquots of the solubilized membranes were subjected to gel filtration on a Sepharose 6 column equilibrated in PBS containing 1% CHAPS. The column was operated at a flow rate of 0.2 ml/min and 500 ul fractions collected. Proteins eluted in fractions corresponding to globular proteins of M, 150,000 to 400,000 were then radiolabelled with Na 5 I using the well-known Chloramin T procedure. Non-incorporated 5 I was removed by gel filtration on Sepahadex G-25 packed in a Pasteur pipette.

Aliquots of the radiolabelled proteins were then diluted in PBS containing 1% CHAPS and 1% bovine serum WO 97/19167 PCT/US96/18295 8 albumin and subsequently subjected to immunoprecipitation using the mAb A52 to p63 (5 ug per incubation) or using two polyclonal rabbit antisera to p63 (3 ul of serum per incubation) See Bavik et al., J. Biol. Chem. 267:23035- 23042 (1992). Non-specific immunoprecipitation was monitored in parallel incubations using an unrelated mAb and preimmune rabbit serum. Fifty ul of a 50% slurry of protein A-Sepharose was added to the incubations for minutes. The beads were subsequently carefully washed with PBS containing 1% CHAPS and the eluted material then prepared for SDS-PAGE analysis, was carried out according to Blobel et al. J. Cell. Bio. 67:835-851 (1975).

Referring now to FIG 1A, autoradiograms of the SDS- PAGE gels showed that both types of reagents reacted with 15 p63, whereas the unrelated mAb or preimmune rabbit serum did not precipitate p63. In all lanes containing immunoprecipitated p63 there was an enrichment of a M, 32,000 protein. Since both mAb A52 and the rabbit antisera to p63 are highly specific for p63 (See, for example, Bavik et al., J. Biol. Chem. 267:23035-23042 (1992)), it can be concluded that the M, 32,000 protein (p32) coprecipitated in the aforesaid analysis by binding to p 6 3. The analysis also identified a double band of M,50,000-52,000 which precipitated along with p32 and p63 (FIG. 1 d, e) o Example 2 Experiments were then carried out to identify p32.

Advantage was taken of the fact that p32 specifically interacts with p63 as shown supra. Thus, detergent solubilized RPE-membrane proteins were passed over an immunoaffinity column containing mAb A52. Referring now to FIG. 1B, lane b, following a washing procedure, bound proteins were eluted at high pH in a CHAPS-containing buffer, and SDS-PAGE analysis and Coomassie staining of the eluted fractions revealed p63 to be specifically retained and eluted from the immunoaffinity column. Further, a weekly stained band corresponding to p32 could be WO 97/19167 PCT/US96/18295 9 visualized in the eluate from the A52 column. As shown in FIG. 1B, a comparison of the total protein profile of solubilized RPE membranes and the eluted fraction from the A52 column show that the p32 protein is not efficiently retained therein. However, the appearance of p32 in the eluted fraction from the A52 column, but not in the eluted fraction from the column containing an unrelated Ig, indicate a specific interaction of p32 with p63. This result is consistent with previous immunoprecipitation data, and shows that p32 is complexed to p63 and is retained on the immunoaffinity column due to this complex formation.

Following identification of p32 as a component of a complex with p63 in RPE-membranes, the p32 protein itself 15 was isolated by SDS-PAGE of eluted fractions of solubilized RPE-membranes from the A52 immunoaffinity column, as set forth below in Example 3.

Example 3 RPE-membranes were solubilized in PBS containing 1% 20 CHAPS as set out above and then incubated with mAb A52 Ig •oi00. coupled to CNBr-activated Sepharose 4B beads (Pharmacia) in a Bio-Rad poly prep column (Bio-Rad) by end-over-end rotation at +4 0 C. Following a 2 hour incubation, the beads *oo* were allowed to settle and the column was quickly washed with 5 column volumes of PBS containing 1% CHAPS. Bound proteins were then eluted with 50 mM triethanolamine buffer (pH 11.2) containing 1% CHAPS. The pH of the eluate was quickly adjusted to 8.0 by the addition of 1 M Tris-HCl buffer containing 1% CHAPS. The eluted fractions were subjected to SDS-PAGE, and the separated proteins then visualized by Coomassie Blue staining. A band corresponding to p32 (SDS-PAGE, 32kDa), was found.

To determine the primary structure of p32 partial amino acid sequence analysis of the isolated protein was undertaken by first cutting out a portion of the aforesaid Coomassie stained band corresponding to approximately WO 97/19167 PCT/US96/18295 ug of the 32kDa protein, then lyophilizing the gel piece to dryness. The gel was rehydrated in buffer containing modified trypsin and incubated to generate various peptides for extraction and analysis. A preferred procedure is set forth below in Example 4.

Example 4 Coomassie stained bands containing the p32 protein from Example 2 were excised and treated according to Rosenfeld et al. Anal. Biochem. 15:173-179 (1992) with minor modifications. The gel pieces were washed twice for min at 30°C with 100 ul of 0.2 M ammonium bicarbonate buffer containing 50% acetonitrile and thereafter completely dried under a stream of nitrogen. The gel pieces were subsequently rehydrated with 5 ul of 0.2 M 15 ammonium bicarbonate buffer containing 0.02% Tween 20 and 0.5 ug of modified trypsin. Trypsin was added from a stock solution prepared in 1 mM HC1. Rehydration was continued by the addition of 5 ul portions of the 0.2 M ammonium g.e. bicarbonate buffer until the gel pieces rehydrated to their original sizes. The rehydrated gel pieces were then incubated overnight at 30 0 C. Protease activity was inhibited by the addition of trifluoroacetic acid (TFA) to a final concentration of The supernatant was recovered and combined with two extracts made with 150 ul of 0.1% TFA 25 in 60% acetonitrile. The organic phase was reduced and the digest was subjected to HPLC using a reverse phase mRPC C2/C18 SC 2.1/10 column operated in a SMART system. The sample was eluted with a gradient of acetonitrile in 0.065% TFA and fractions containing discrete peptides were collected using the automatic peak fractionation option.

Five of the identified peptides were selected for amino acid sequence analysis using an ABI 470A sequencer equipped with a model 120A PTH analyzer (applied Biosystems Inc.

Foster City, CA). The results are set forth below in Table 1.

WO 97/19167 PCT/US96/18295 11 TABLE 1 Amino acid sequences determinations of five peptides isolated from trypsin digested p32.

p321 L-V-E-A-V-L-A-E-V-L-P-K-P-A-Q-T-V-A (SEQ ID NO: 1)

(Y)

P322 Y-S-P-G-W-D-A-K (SEQ TI NO: 2) P323 T-P-V-T-N-L-E-T-L-E-D-T-L-Q-A (SEQ ID NO: 3) P324 D-V-A-P-F-G-V (SEQ ID NO: 4) 10 P325 L-H-T-T-L-L-D-V-T-D-P-Q-S-I (SEQ ID NO: The amino acid residues given within the parentheses i" are the residues deduced from the cDNA sequence in the same positions.

15 Protein SEQ ID NOS: 1-5 can be used alone or ligated to hapten by well known methods.

Next, to determine the complete primary structure of p32, four degenerative oligonucleotide mixtures, OM1-OM4, as set forth below in Table 2 were synthesized based on the 20 amino acid sequences of the p 3 21 and p323 sequenced peptides of Table 1. The procedure is as follows in Example Example Four degenerate oligonucleotide mixtures derived from peptides p321 and p323 were synthesized using well-known techniques. The two sense mixtures (OM1 and OM3) were derived from the N-terminal amino acids 1-5 of p321 and 2-6 of p323. The antisense mixtures (OM2 and OM4) were derived from amino acids 12-17 of p321 and 10-15 of p323. All nucleotide mixtures were synthesized with a 4 bp WO 97/19167 PCT/US96/18295 12 extension and an Eco RI-site for subsequent cloning of the PCR products. The sequences of the oligonucleotide mixtures are set out below in Table 2, and the Eco RI-site is underlined. Positions containing all four bases are marked N.

TABLE 2 OM1:ACGT GAA TTC TN GTN GA(A,G)GCN GT (SEQ ID NO: 6) OM2:ACGT GAA TTC AC NGT(T,C)TG NGC NGG(T,C)TT (SEO ID NO: 7) OM3:ACGT GAA TTC CCN GTN ACN AA(T,C) (C,T)T (SEQ ID NO: 8) OM4:ACGT GAA TTC GC(T,C)TG NA(A,G)NGT(A,G)TC(T,C)TC (SEQ ID NO: 9) Single stranded "complementary" cDNA from reverse transcribed RPE mRNA and four combinations of the above- 15 described degenerate nucleotide mixtures were employed in polymerase chain reactions (PCR) using a standard procedure. Following the amplification procedure, aliquots of the PCR reaction products were analyzed by agarose gel electrophoresis. The procedure is set out below in Example 20 6.

Example 6 To carry out the PCR amplifications, first strand cDNA was synthesized by standard procedures using avian myelostosis virus reverse transcriptase. Twenty ug of total RNA from isolated RPE-cells were used and the reaction was primed with oligo (dT) 15. Aliquots corresponding to 2 ug of total RNA was used in each subsequent PCR reaction. The PCR reactions were performed using a final concentration of 0.5 uM of the oligonucleotide mixtures in a 100 ul reaction. Taq polymerase was used. Following 30 cycles (2 minute at 1 minute at 55 0 C and 2 minute at 72 0 aliquots of the reactions were analyzed on 4% GTG agarose gel containing 5 ug/ml of ethidium bromide.

WO 97/19167 PCT/US96/18295 13 As shown in FIG. 2A, amplifications using the oligonucleotide mixtures OM1 and OM2, both derived from peptide p321, resulted in an amplified 61 bp fragment.

Amplifications using mixtures OM3-OM4 and OM1-OM4 failed to yield any products. Finally, as shown in FIG. 2B, amplification using OM3-0M2 resulted in an amplified 330 bp fragment.

Subsequent sequence analysis of the 61 bp and 330 bp fragments confirmed that cDNA sequences have been amplified which corresponded to the peptide sequences generated in the previous amino acid sequence analysis. Differences between the deduced amino acid sequences from amplified PCR fragments and the generated amino acid sequence of peptide p321 indicates the generation of specific probes suitable 15 for the isolation of full length cDNA clones encoding p32.

To isolate a full length cDNA clone, an RPE-specific lambda ZAP-II cDNA library was screened with the 330 bp fragment as the probe. Five independent lambda clones were isolated from approximately 200,000 clones, and subcloned 20 by in vivo excision. The cDNA clone pX321 contained the longest insert, (approximately 1.1 Kb), and was selected for use in further studies.

Both strands of pX321 were fully sequenced with the insert being 1104 bp long, excluding linkers used to 25 prepare the cDNA library. The procedure is set out below in Example 7.

Example 7 The amplified products using OM1-OM2 (61 bp) and OM3- OM2 (330 bp) were digested with EcoRl, gel purified and cloned into EcoRl-cut vector pBS. The 32 P-labelled 330 bp fragment was used to screen a RPE-specific XZAP II cDNA library as previously described by Bavik et al., J. Biol.

Chem. 267:20540-20546 (1993), incorporated by reference.

Five positive X clones were isolated and the inserts were subcloned in pBluescript by in vivo excision following the manufacturer's instructions. Clone pX321 contained an WO 97/19167 PCT/US96/18295 r r r insert of 1.1 Kb, and both strands were fully sequenced using Sequenase with T3, T7 or M13 universal primers or with internal primers.

The nucleotide sequence of pX321 and the predicted amino acid sequence of p32 are shown in FIG. 3 (SEQ ID NO: Nucleotides are numbered on the left and amino acid residues on the right. Amino acid 1 is initial methionine As shown in FIG. 3, the 1.1 kbp insert contains one long open reading frame encoding 318 amino acid residues with a calculated mass of 35,041D. The first methionine residue lies in a good context according to the Kozak rules for transcription initiation and is likely to be the initiation codon. See Kozak, Cell, 44:283-292 (1986).

This inference is strengthened by the fact that in vitro translation of synthetic mRNA transcribed from pX321 gives rise to a M, 32,000 protein (SDS-PAGE analysis), as set out below, but there is no stop codon in frame in the upstream bp 5'-untranslated region of the cDNA. As also shown in 20 FIG. 3, the 100 bp 3'-untranslated region ends with a putative polyA-tract, and a polyA-signal was identified in the upstream sequence (bp 1104-1110).

The deduced amino acid of pX321 and the amino acid sequences of the five generated tryptic peptides (Table 1) differ in only 3 positions out of the 62 residues available for a comparison. All 3 differences are found in the peptide p321 but the nucleotide sequence in this region of a second cDNA clone (pX324) is identical to that of pX321.

This indicates that the amino acid sequence determination of peptide p321 was probably incorrect although it cannot be excluded that the differences are due to the presence of different alleles of p32. These data demonstrate that pX321 contains the complete coding region of p32.

Again, referring to FIG. 3, a consensus sites for Nlinked glycosylation (amino acid residues N-I-T) could be found in the deduced amino acid sequence at position 160- 162.

WO 97/19167 PCTIUS96/18295 Additionally, it has been found that p32 shows sequence similarities to short-chain alcohol dehydrogenases. Referring now to FIG. 4 a search through the Swissprot protein data base revealed that p32 is structurally related to several previously sequenced proteins. It is most closely related to a mitochondrial matrix dehydrogenase, the D-f-hydroxybutyrate dehydrogenase (BDH) Churchill et al; Biochem. 31:3793-3799 (1992) and shows less but significant similarities to two other proteins, the 3-oxoacy[acyl carrier protein] reductase from E. coli (Rawlings et al., J. Biol. Chem. 267-5751-5754 (1992)) and the human estradiol 17 f-dehydrogenase (Peltoketo et al., FEBS Lett., 239:73-77 (1988) and Leu et al., Mol. Endocrinol. 3:1301-1309 (1989)). All the related 15 proteins fall into the protein super-family of shortalcohol dehydrogenases. This protein superfamily comprises approximately 50 different proteins (Persson et al, Eur. J.

Biochem, 200:537-593 (1991)). The overall sequence homology between p32 and BDH is around 39%. The level of 20 homology to the E. coli reductase and to the estradiol 17o-dehydrogenase is lower (31% and 33%, respectively).

Optimal multiple alignment identified several conserved regions shared by p32 and the most closely related proteins (boxed areas in FIG. The first region 25 involving residues 63-69 (using the numbering in FIG. 4) which displayed the conserved motif G-X-X-X-G-X-G is believed to be the binding site for cofactors NAD, NADP or its reduced forms. Another conserved region is found between residues 148-153 (consensus sequence L-V-N-N-A-G) but no functional characteristics have yet been attributed to that sequence motif. The sequence motif Y-X-X-X-K, thought to be the active site, is the most highly conserved motif in short-chain alcohol dehydrogenases and is present in p32 residues 175-179 See Persson et al., Eur. J.

Biochem, 200:537-593 (1991). These similarities demonstrate that p32 exhibits several features of a functional short-chain alcohol dehydrogenase.

PCT/US96/18295 WO 97/19167 16 As shown in FIG. 5, hydropathy analysis of the amino acid sequence of p32 reveals several hydrophobic stretches, indicating that p32 is a membrane-associated protein. The first 18 amino acids are hydrophobic, and this region has characteristics of a classical signal sequence. However, a consensus site for signal peptidase cleavage could not be identified. See Von Heijne, Nucl. Acid Res., 14:4683-4690 (1986). The amino acids between residues 130 to 150 are hydrophobic and there is a relatively long hydrophobic stretch near the C-terminus of the protein. Thus, p32 displays several hydrophobic regions which are potential membrane spanning segments. In light of the homology to the family of short-chain alcohol dehydrogenases as shown above, it is likely that the central hydrophobic region of p32 (residues 130-150) is not used as a membrane anchor.

Instead, both the N-terminal and the C-terminal regions are potential membrane anchoring domains.

To determine the mode of interaction of p32 with membranes, p32 was synthesized by in vitro translation using a reticulocyte lysate system with mRNA transcribed from linearized pX321. The procedure is set out in Example 8 below: 9* 25 25 Example 8 Expression of p32 by in vitro translation In vitro transcribed mRNA encoding p32 was synthesized from linearized PX321 using T7 RNA polymerase. In vitro translation reactions were carried out using nuclease treated rabbit reticulocyte lysate following the manufacturer's instructions. Fifty ng of mRNA was included in each reaction, with or without the addition of dog pancreatic microsomes. To isolate membrane inserted p32, the microsomes were collected by centrifugation at 12,000 x g for 10 min at 4°C. The microsomes were carefully resuspended in PBS and recentrifugated.

As shown in FIG. 6, translation in the presence of dog pancreatic microsomes showed that p32 becomes almost WO 97/19167 PCT/US96/18295 17 quantitatively membrane associated and migrates as a M, 32,000 species in SDS-PAGE. Translation in the absence of acceptor membranes similarly yields a M,32,000 protein.

These data indicate that the N-terminal hydrophobic sequence acts as a signal sequence but it is not removed by the signal peptidase, and such supports the previous observation that a consensus site for signal peptidase cleavage could not be identified in the deduced primary The tissue expression of p32 was analyzed by Northern blotting analyses using total RNA isolated from bovine RPE, liver, kidney, adrenal, lung, testis, brain and muscle.

The procedure is set out in Example 9.

Example 9 15 Northern blot analyses Twenty ug of total RNA isolated from a number of tissues was electrophoresed on a 1% agarose under denaturing conditions and transferred to a Hybond-N nylon filter. The filter was hybridized with 32P-labelled full length cDNA encoding p32 under stringent conditions. The details of the isolation of total RNA, hybridization conditions and washing procedure were identical to those previously described in Bavik, et al., J. Biol. Chem.

267:20540-20546 (1993).

25 Hybridization at high stringency with the 1.1 Kb insert of pX321 as the probe, revealed abundant expression of transcripts corresponding to p32 only in RPE but not at a detectable level in several other tissues. The size of the major transcript was 1.4 kb but other less abundant transcripts could be visualized after prolonged exposure of the filters both in RPE as well as in other tissues.

WO 97/19167 PCTIUS96/18295 18 Example Expression of p 3 2 in COS-cells and enzymatic analysis of the properties of recombinant p32.

p32 was first expressed in COS-cells using a eukaryotic expression vector, and then microsome fractions from transfected cells and control cells were subjected to immunoblotting analysis to verify the expression of p32 at the desired levels, as follows: Specifically, the EcoRI insert of p X 321 was cloned into the EcoRl-digested eucaryotic expression vector See Green et al., Nucl. Acid Res. 16:39 (1988). COS-cells were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 2mM glutamine and antibiotics. The cells were seeded into 60 mm petri dishes 15 (4 x 105 cells per dish) and transfected with 5 ug of plasmid per dish using DEAE dextran. Control cells were transfected with equal amounts of the parental vector alone. After treatment with 10% DMSO for 2 minutes, the cells were incubated for 72-96 hours, and then harvested by 20 scraping the dishes with a rubber policeman, and the cells thereafter collected by low speed centrifugation. The collected cell pellets were next resuspended in hypotonic buffer (10 mM Tris-HC1, pH 7.5 containing 1mM phenylmethylsufonyl fluoride), put on ice for 20 minutes, and 25 then homogenized using a Dounce homogenator. Unbroken cells and debris were removed by centrifugation (3000 x g)for 15 minutes. Microsomes were subsequently collected by ultra-centrifugation at 100,000 xg for 1 hour; membrane pellets were stored at -80 0 C until further analyzed.

Antisera to p32 were generated by injecting rabbits with p32 (amino acid residues 19-318) expressed as a fusion protein with GST. The bacterial expression vector pGEX 2T was used and induction and GST-fusion protein was induced and purified, as recommended by the supplier (Pharmacia).

Each rabbit received a subcutaneous injection of 75 ug of fusion protein emulsified in Freunds complete adjuvant.

The rabbits were boostered with 50 ug of fusion protein WO 97/19167 PCTIUS96/18295 19 emulsified in Freunds incomplete adjuvant every second week. Blood was collected every second week. The immune rabbit sera were passed over a column containing GST fusion-protein immobilized on CNBr activated Sepharose beads. Bound Ig was eluted with 0.1 M sodium citrate buffer (pH containing 0.5 M NaC1. To remove Ig to the GST portion of the fusion protein, the eluted Ig was similarly incubated with GST-coupled Sepharose beads and the unbound Ig fraction was used. For immunoblot analysis of the over expressed protein, the Ig was used at a concentration of 1 ug per ml. The details of the immunoblotting procedure are described in detail in Bavik et al., J. Biol. Chem., 267:23035-23042 (1992) the disclosure of which is herein incorporated by reference.

15 As shown in FIG. 8A, the above procedure resulted in expression of p32 in cells transfected with the recombinant expression vector, but not in control cells that were mock transfected.

Next, the enzymatic properties of p32 expressed in 20 COS-cells was assayed in a manner similar to that as described in the study of 11-cis-retinol dehydrogenase activity in microsomal fractions of RPE cells. See Saari Set al., Anal. Biochem 213:28-13226 (1993).

In particular, the enzymatic activity of p32 was 25 confirmed by incubating the microsomal fractions from the aforementioned transfected cells, and from control cells lacking p32, with varying combinations of the different stereo isomeric substrates, 11-cis-retinol or alltrans-retinol, in the presence of either cofactor NAD+ or

NADP.

To prepare the substrate, 11-cis retinol was synthesized from 11-cis-retinaldehyde using sodium borohydride, as set fourth in Heller et al., J. Biol. Chem.

248:6308-6316 (1973), and stored under argon at 80 0 C. HPLC analysis confirmed the quantitative reduction of 11-cisretinaldehyde to 11-cis-retinol, and all manipulations with the retinoids were done under subdued lighting conditions.

WO 97/19167 PCT/US96/18295 To assay for p32 activity in transfected cells, the final concentration of 11-cis-retinol and all-trans-retinol (obtained from Sigma Chemical Co.) in the incubations was reduced to 100 uM. Twenty ug of total membrane protein from COS-cells expressing p32 or from control cells were used in each incubation, and thereafter a 30 minute incubation at 37 0 C in the presence or absence of NAD+ or NADP followed. Reaction mixtures were then extracted with n-hexane, and organic phases removed and dried under argon.

The dried organic phases were then separately dissolved in ethanol and aliquots were analyzed on a normal phase silica HPLC column developed with n-hexane containing 4% dioxane at 1 ml per minute. See Saari et al. J. Biol. Chem.

257:13329-13333 (1982). Effluent was monitored at 330 nm.

15 Under these conditions, 11-cis-retinaldehyde and 11-cis retinol eluted at 7 minutes and 22.5 minutes, respectively, and all-trans-retinaldehyde and all-trans retinol eluted at 8 minutes and 23 minutes, respectively.

As indicated in FIG. 8B, the aforementioned

HPLC

20 analysis shows that fractions from transfected cells containing p32 expressed 11-cis-retinol dehydrogenase protein which was active in the presence of NAD+, as indicated by the formation of 11-cis-retinaldehyde.

A

second peak in the chromatogram is all-trans retinaldehyde; 25 however, control incubations with 11-cis retinaldehyde, in the absence of cellular membranes, show that, under the test procedure employed, a large amount of 11-cis retinaldehyde isomerizes to all-trans retinaldehyde. This indicates that the appearance of all-trans retinaldehyde is due to its generation during the incubation process used and extraction procedures, and is not an enzymatic reaction product. Further, incubations with all-trans retinol with cells containing p32 verify the stereo specificity of the enzyme, as no significant formation of all-trans retinaldehyde is detected.

As shown in FIG. 8C, p32 is not enzymatically active in the presence of cofactor NADP.

WO 97/19167 PCT/US96/18295 21 In FIG. 8D, assays of the control cells not expressing p32 show that these do not oxidize 11-cis-retinol into 11cis-retinaldehyde.

Therefore, the above shows conclusively that p32 is a stereo specific 11-cis-retinol dehydrogenase, which relies on NAD+ as its cofactor.

Example 11 F-ollowing the work described sura, using bovine materials, additional experiments were carried out to isolate and to clone a human sequence.

A cDNA library from human eye, in Xgtll was purchased from a commercial supplier Clontech). The bovine cDNA described supra, SEQ ID NO: 10, was used as a probe. The cDNA was 2 [P]dCTP labelled by random priming to 15 a high specific activity (about 109 cpm/pg of DNA).

The labelled bovine cDNA was then used to probe the human cDNA library. Conditions were as follows: S. hybridization in 6XSSC, 0.5% SDS, 5X Denhardt's solution, 25% formamide, at 68 0 C, with 100 Ag/ml of salmon sperm DNA, followed by four washes of 2XSSC, 0.5% SDS, at 65 0 C, for minutes each, and then a final wash at 2xSSC at 42 0 C for minutes.

When a positive cDNA was found, the insert the *I cDNA), was excised following the manufacturer's 25 instructions, and then subcloned into the commercially available vector pBluescript. The sequence of the insert was then determined, using well known methods. The sequence of 1128 nucleotides is set forth at SEQ ID NO: 14.

The corresponding amino acid sequence of 318 residues is se forth in SEQ ID Example 12 In further experiments, the information described in example 11, supra, was used to study and to analyze bovine neuroretina and murine 10 day embryos. Murine embryos were used because, apart from the general usefulness of the WO 97/19167 PCT/US96/18295 system for studying developmental biology, retinol dehydrogenases are extremely active in development. The model is extrapolatable to human development.

RNA (5-10ig), was isolated from bovine neuroretinas, or from murine 10 day embryos, using well known techniques.

The RNA was mixed in 41l of 5x AMVRT buffer, together with 4pl of dNTPs from 5mM stock solutions, 21l of oligo dT (18mer, at a final concentration of 10pM), together with 0.51 RNAse inhibitor, and 2ul of avian myelostosis virus .O reverse transcriptase (10U). The final volume was 20 pl.

This resulting mixture was incubated at 42 0 C for minutes, and then left on ice until used.

PCR was then carried out. In the PCR, two pl of cDNA were used. Primers were designed on the basis of the deduced amino acid sequence for bovine cDNA set forth supra. The following conserved amino acid sequences were noted: A) Cys Asp Ser Gly Phe Gly B) Pro Gly Trp Asp Ala C) Glu Ala Phe Ser Asp D) His Pro Arg Thr corresponds to amino acids 36-41 of SEQ ID NO:12.

is found at amino acids 283-287 of this sequence. "C" is found at positions 183-187, and at positions 276- 279. "Conserved" as used herein refers to conservation between the deduced sequence, the sequence for liver RDH shown by Chai, et al. J. Biol. Chem 270: 3900-3904 (1995), incorporated by reference in its entirety, and Simon, et al, J. Biol. Chem 270: 1107-1112 (1995), also incorporated by reference.

Degenerate oligomers were prepared. In a first set of PCR experiments, degenerate oligos based upon A and B were used as primers, i.e.: ACGTGAATTCTG YG AYTCNG G NWTY G G 3' (SEQ ID NO:16) WO 97/19167 PCT/US96/18295 23 AC G T GA AT T CTTN G C R T C CC A N CC 3' (SEQ ID NO:17) as forward and reverse primers respectively. The primers were mixed with the two 1p of cDNA discussed supra.

Conditions were 1 minute of denaturation at 94 0 C, 1 minute of annealing at 50 0 C, and two minutes at 72 0 C, for elongation. This constituted 1 cycle. Twenty-five cycles were carried out.

Following the first PCR. 5/L1 samples of PCR product were combined with primers based upon and i.e.: AC GT G A A T T C G AR GC NT T Y T C N G A 3' (SEQ ID NO:18) AC GT GAAT T C C G N G TN C K NG G R T G 3' (SEQ ID NO:19) 15 as forward and reverse primers, respectively. Conditions were exactly as used in the first set of experiments, except that the annealing temperature was 55 0

C.

Reaction products were analyzed on 1.5% agarose gels with ethidium bromide. The assumption was that the 20 amplification products should be about 300 base pairs in length. As such, any 300 base pair bands visualized confirmed that the PCR protocol generated products of appropriate size. The experiments were repeated, using 1% low melting point agarose gels, and the PCR products were eluted therefrom. The isolated products were reamplified, using the same protocols, and were cloned into plasmids (TA "cloning kit, Invitrogen). The plasmid DNA was prepared from transformants using standard protocols, and then analyzed by restriction digestion, using EcoRI. Any inserts of about 300 base pairs were analyzed further, using vector specific primers. The PCR products are presented in SEQ ID NOS: 20-23 with deduced amino acid sequences being presented as SEQ ID NOS: 24-27. SEQ ID NOS: 20 and 24 correspond to bovine sequences, while all others are murine sequences. In these nucleotide sequences, the first base is an artifact of the experiment, resulting from cleavage by a restriction WO 97/19167 PCT/US96/18295 24 endonuclease. Hence, one begins with the second nucleotide base in determining the deduced amino acid sequence.

Further, note that sequences corresponding to the degenerate oligos, which would normally be included at the 5' and 3' termini, are not included.

Example 13 The PCR product set forth in SEQ ID NO: 19 was then used in fuirther probing experimentR A murine 8.5 day cDNA library (in XgtO) was screened, using randomly labelled SEQ ID NO: 22, where the hybridization took place in 6XSSC, 0.5% SDS, 5x Denhardts's solution, 50% formamide at 42 0 C, with 100 pg/ml of salmon sperm DNA, followed by one wash at 2XSSC, 0.5% SDS at 50 0

C

for 30 minutes. A positive cDNA clone was identified, 15 subcloned into pBluescript, and sequenced. The nucleotide sequence, and deduced amino acid sequence, are set forth as SEQ ID NOS: 28 and 29.

Example 14 Following the work described, supra, the human cDNA 20 was used to probe a human genomic library. The probe was prepared by PCR and randomly labelled as described supra.

The primers used in the PCR were derived from the cDNA sequence of SEQ ID NO:11 and were as follows: Forward primer: G C T T C G G G C G C T G TA G T A-3' (SEQ ID and Reverse Primer: A A A A C A A T C T C T T G C T G G A A-3" (SEQ ID NO:31) PCR was carried out by denaturing at 95C, followed by annealing at 55 0 C, and elongation at 72 0 C. 2-5U of Taq polymerase and 0.2AM of each primer were used. Thirty cycles were carried out.

WO 97/19167 PCTIUS96/18295 The amplified fragment of 1056bp was isolated, following agarose gel electrophoresis analysis and cloned into vector PCR (commercially available from Invitrogen).

This probe was used to screen a human genomic library in XFXII vector, from Stratagene. The manufacturer's instructions were followed to screen approximately 1x10 6 plaque forming units. 106 cpm/ml of hybridization solution was used. Hybridization was carried out overnight with -xSSC, 0.5% SDS, 5 x Denhardt's solution, 50% formamide at 42 0 C, with 100g/ml of salmon sperm DNA, followed by one wash at 1 x SSC, 0.1% SDS at 50 0 C and a final wash at x SSC, 0.1% SDS at 65 0 C. Each wash was for 30 minutes.

Several positive plaques were isolated, rescreened, and XDNA prepared, using the glycerol step gradient method 15 described by Sambrook et al. Molecular Cloning, A Laboratory Manual (2d edition, 1989) incorporated by reference.

The isolated genomic clones were sequenced by analyzing fragments obtained following PCR reactions, using 20 100 mg of genomic X clones per reaction as templates. To carry out the PCR, different primers, derived from the cDNA sequence of SEQ ID NO:11 were used in two PCR reactions, numbered one and two Specifically, the primers used in PCR reaction 25 one were SEQ ID NO:30, supra (forward primer) and T C A G G C T G T C A GA GA A G G CC T-3' (SEQ ID NO:32) (reverse primer) The primers used in PCR reaction two were A C G A T T T C C A G C G G G T G C-3' (SEQ ID NO:33) (forward primer) and SEQ ID NO:31, supra PCR was carried out by denaturing at 95 0 C, followed by annealing at 55 0 C and elongation at 720C. 2-5U of Taq polymerase and 0.2AM of each primer was used. Thirty cycles were carried out. The amplified fragments from WO 97/19167 PCT/US96/18295 26 reactions one and two, were 2.2kb and 2.5kb respectively.

Each fragment was cloned into vector PCR commercially available from Invitrogen.

Sequence analysis of the cloned fragments using vector specific primers or internal primers, permitted identification of exon-intron boundaries.

The structure of the gene is presented in figure 9.

Example Studies.were carried out to identify the location of the gene on chromosomes.

To do this, high molecular weight DNA was isolated from human leuckoytes, Chinese hamster cells, murine liver cells, and from hamster/human and mouse/human somatic hybrid cell lines. These hybrid cell lines each retained one human chromosome, as well as rodent genomes.

The isolated DNA was digested with Hind III, fractionated on agarose gel via electrophoresis, and then transferred to a nylon filter. These were then probed, using the human cDNA described supra, labelled as described supra.

Slides of chromosomes were prepared from lymphocyte .cultures, using art recognized techniques. The ADNA from 3 genomic clones were mixed and labelled with biotinylated 16-dUTP, by Nick translation. A centromere specific probe 25 for human chromosome 12 centromere (obtained from the ATCC, under Accession Number D12Z1), was labelled with fluorored-dUTP, following the manufacturer's instructions. Preannealing of the probes, pretreatment of the slides, hybridization conditions, signal amplification, and detection, were in accordance with well known techniques.

Chromosomes were counter-stained with 4,6 diamino-2-phenyl indole (DAPI), and signal was visualized using a fluorescence microscope.

Analysis of all of these data indicated that the gene for human 11-cis RDH spans more than 4 kilobases, and is divided into 4 coding exons, which range from 165 to 342 WO 97/19167 PCT/US96/18295 27 base pairs in length. Further, an exon was found in the untranslated region, and the length of the last coding exon has not been established. Introns range in size from 250 base pairs to 1.9 kilobases. Figure 9 shows this schematically.

Study of exon/intron boundaries showed that all splice donor and acceptor sites follow the well known, canonical GT/AG rule. The Initiation codon and the conserved cofactor binding site are encoded by exon 2, while the active site, with invariant tyrosine residue, is encoded by exon 3. The gene for human 11-cis RDH maps to chromosome 12q13-14.

Example 16 The work set forth in example 12 was extended, using 15 the nucleic acid molecules set forth in SEQ ID NOS: 21 and 22.

A commercially available source of murine, multiple tissue Northern blots was used. Specifically, the nucleic 2. acid molecules set forth in SEQ ID NOS: 21 and 22 were labelled with 3 2 P, using standard methodologies. These labelled probes were then used to determine relative expression levels of transcript in murine tissue samples, using standard Northern blotting protocols. Blots were 2 hybridized, overnight, at 42 0 C, using 50% formamide, 6xSSPE buffer, 0.5% SDS, 2xDenhardt's solution, 100 ug/ml salmon sperm DNA, and 1x10 6 cpm/ml of labelled probe. Blots were washed at room temperature, twice, for 30 minutes per wash, using 2xSSC and 0.1% SDS, followed by two further washes, at 50 0 C, in 0.lxSSC containing 0.1% SDS. Blots were exposed at -70°C, overnight, using intensifying screens, and Kodak film. Relative expression levels, by visual inspection were as follows: WO 97/19167 PCT/US96/18295 28

PROBE

Tissue SEO ID NO: 21 SEQ ID NO: 22 Heart Brain Spleen Lung Liver Skeletal Muscle Kidney Testis Example 17 In view of the results of example 16, murine liver was used in the experiments which are described in this 15 example. A murine liver cDNA library was prepared in XZAP, using standard protocols. Probes, as described in example 16, supra, were used to screen the library. Specifically, manufacturer's instructions were followed for plating the library and preparing the filters. prehybridization was carried out at 42 0 C, in 50% formamide, 6xSSPE buffer, 2xDenhardt's solution, 100 ug/ml salmon sperm DNA. The filters were then hybridized, using 1x10 6 cpm/ml of hybridization-solution. Following overnight hybridization, filters were washed twice, in 2xSSC containing 0.1% SDS, 25 (30 minutes per wash, at 52C) followed by two, 20 minute washes in 0.1xSSC containing 0.1% SDS, at 52 0 C. Filters were then exposed, as described supra. Any positive clones were rescreened, twice, until all plaques on a plate were positive. Inserts from several, positive clones were subcloned into plasmid pBluescript using standard procedure by in vivo excision. Several of the resulting clone were sequenced.

When SEQ ID NO: 21 was used as a probe, three different cDNAs were identified. These are presented as SEQ ID NOS: 32, 33 AND 34, herein. When SEQ ID NO: 22 was used, SEQ ID NO: 35 was found. Amino acid sequences WO 97/19167 PCT/US96/18295 29 deduced therefrom are presented as SEQ ID NOS: 36-39, respectively.

Thus, as shown above, this invention provides a method for the isolation and characterization of a novel protein, p32, which associates with the p63 of RPE. The primary structure of p32 demonstrates that it has all the critical features of a functional short-chain alcohol dehydrogenase including a putative cofactor binding site and essential residues involved in thp cat-ayticr mPchanism, namely the almost invariant tyrosine containing sequence motif

Y-X-X-

X-K (Persson et al., Eur. J. Biochem. 200:537-543 (1991)).

The restricted tissue expression and the abundance of p32 in RPE indicates that this protein carries out a function Swhich is unique to the RPE. This possibility, and the fact 15 that it forms a complex with p63 which previously has been shown to be a component of the retinoid uptake machinery in RPE-cells (Bavik, et al., J. Biol. Chem. 267:23035-23042 (1992)), shows that the substrate for p32 is a retinoid.

A major metabolic step in retinoid metabolism in RPE- 20 cells is the conversion of ll-cis retinol to ll-cis retinaldehyde. Based on the results obtained hereinabove showing the restricted expression of p32 in the RPE, and :'the particular biochemical properties of this protein further investigation confirmed that p32 is in fact an 11cis-retinol dehydrogenase, the enzyme which catalyzes this I: reaction.

Thus, one aspect of the invention is the ability to produce recombinant il-cis-retinal dehydrogenase. The recombinant enzyme can be used to produce 1l-cisretinaldehyde in levels higher, and in purer form, than would be available using standard biochemical methodologies. Thus, the isolated nucleic acid molecules of the invention, which include SEQ ID NO: 10, as well as those nucleic acid molecules which hybridize to SEQ ID NO: 10 under stringent conditions can be used in this context.

By the term "stringent conditions" is meant hybridization in 6 X SSC, 0.5% SDS, 5 X Denhardt's solution at 68 0 C, with WO 97/19167 PCT/US96/18295 100 ug/ml of salmon sperm DNA, and a final wash with X SSC at 50 0 C. The term is also used herein to refer to any set of parameters which are at least as stringent as those set forth above. As is well known, equally stringent conditions can be created by changing one parameter to make it less stringent, with another parameter being changed to increase its stringency. Thus, any nucleic acid molecule which fulfills the hybridization criteria set forth herein will bh experted to code for p 3 2 or a p32 homologue. Th enzyme may be produced in an in vitro system, such as the one described above, or via transfecting or transforming eukaryotic or prokaryotic cell lines, such as CHO and COS cells or bacterial strains such as E. coli or the yeast strain S.cervisiae with the nucleic acid molecules of the 15 invention. In an especially preferred embodiment, the *nucleic acid molecules are contained within an expression vector, operably linked to a promoter. Complementary DNA, or "cDNA" is preferred, but genomic DNA and mRNA can also be used.

20 The identification of the p32, 11-cis retinol dehydrogenase, as a member of the short-chain alcohol dehydrogenase superfamily is important in view of the retinoid-metabolism which occurs in non-ocular tissues.

Studies show that generation of all-trans retinoic acid 25 from all-trans retinol is carried out in a two step process (Posch et al., Biochemistry 30:6224-6230 (1991)). First, retinol is oxidized to retinal by a membrane bound retinol dehydrogenase. In a second step, the retinal is oxidized to retinoic acid. Thus, the oxidation of retinol into retinal, occurring in non-ocular tissues, is similar to the reaction carried out during synthesis of 11-cis retinal from 11-cis retinol in the visual cycle. In light of these similarities, it can be proposed that formation of alltrans retinal from all-trans retinol is carried out by an enzyme which is structurally similar to the p 3 2 11-cis retinol dehydrogenase isolated by this invention. These findings are surprising in contrast to presently held views WO 97/19167 PCT/US96/18295 31 as it is generally believed that this metabolic step is carried out by members of the medium chain alcohol dehydrogenases. See Duester, Alcohol Clin. Exp. Res.

15:568-572 (1991); Yang et al., Alcohol Clin. Exp. Res.

17:496 (1993) and Zgombic-Knight et al., J. Biol. Chem.

269:6790-6795 (1994). Thus the identification and structural characterization of the p32 11-cis retinol dehydrogenase, provided by this invention also provides a previonsly 'ePxpfcted ave.niu for the isolq t i-orn and characterization of similar dehydrogenases involved in retinol metabolism is non-ocular tissues.

The p32 protein and nucleic acid encoding therefor and other aspects of this invention are also useful in many other important applications. For example, as it has been 15 shown that p32 is part of an oligomeric protein complex which functions as a membrane receptor for RBP in RPEcells, the nucleic acid sequence coding for p32 can be used in a phenotypic/genic diagnostic analysis to determine retinoid accumulation, which can lead to retinitis o 20 pigmentosa.

Additionally, as shown, p32 possesses 11-cis-retinol dehydrogenase activity, which catalyzes the conversion of 11-cis retinol to 11-cis-retinaldehyde, a major metabolic step in retinoid metabolism in RPE-cells carried out by a 25 membrane bound dehydrogenase. Thus, retinoid accumulation may be directly or indirectly tied to the presence of p32 and/or its activation or inhibition, for example, its complex formation with the RBP receptor p63.

In other applications the effect of potential retinoid drugs for treatment of various diseases on the ll-cisretinal dehydrogenase activity of p 3 2 may be assayed as such drugs may adversely effect the enzyme, and to thus determine which of the different drugs have limited or no adverse effect on enzyme activity.

Examples of such diseases include those of the eye and also skin disorders such as psoriasis and acne. Certain cancers such as T-cell leukemias may also be tested by WO 97/19167 PCT/US96/18295 32 retinoid drugs and hence be candidates for assaying p32 activity.

The various known functions of retinoids also suggests that various other retinoid linked pathological conditions may be diagnosed via assays for levels of the p63/p32 receptor complex associated with a particular retinol binding protein. Art recognized techniques may be used, such a immunoassays, and so forth, to determine whether p63/p32 receptor complex levels are too low or too high are at variance with a normal level.

Further, as p32 complexes with the p63 component of the retinoid uptake machinery in RPE cells, it may also be used in a therapeutic context, as it is well known that soluble receptors may be used to prevent binding of a 15 protein to its membrane-linked receptor. Thus, a subject characterized by enhanced levels of production of retinol binding protein may be treated via administering an amount of soluble receptor complex or antibody sufficient to inhibit binding of the retinol binding protein or other S. 20 related molecule to its target, namely, an inhibitor of p32's retinol dehydrogenase activity. Other aspects of the invention will be clear to the skilled artisan and need not S* be repeated here.

In yet another application, monoclonal and polyclonal 25 antibodies to p32 can be generated, which are useful, inter Salia, in monitoring the instance of pathological conditions characterized by aberrant levels of a receptor for retinol binding protein, by binding analysis of the antibody with body fluid or tissue samples. The generation of antibodies to p32 can be accomplished, for example, by using the procedure set out in Bavik et al., J. Biol. Chem.

268:20540-20546 (1993) for the generation of antibodies to p 6 3, including mAb A52.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features WO 97/19167 PCT/US96/18295 shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

000 0 0 00 0* a S 0*0* 0055

S*

.00.

S...o *0000S WO 97/19167 PCT/US96/18295 34 GENERAL INFORMATION: APPLICANTS: ERIKSSON ET AL.

(ii) TITLE OF INVENTION: ISOLATED NUCLEIC ACID MOLECULE WHICH CODES FOR A 32 KDA PROTEIN HAVING 11-CIS RETINOL DEHYDROGENASE ACTIVITY, AND WHICH ASSOCIATES WITH P63, A PORTION OF A RETINOL BINDING PROTEIN RECEPTOR (iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Felfe Lynch STREET: 805 Third Avenue CITY: New York City STATE: New York ZIP: 10022 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3.5 inch, 144 kb storage COMPUTER: IBM OPERATING SYSTEM: PC-DOS SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/562,114 FILING DATE: 22-November-1995 (vii) PRIOR APPLICATION DATA: oooo APPLICATION NUMBER: 08/375,962 S* FILING DATE: 20-January-1995 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/258,418 FILING DATE: 10-June-1994 (viii) ATTORNEY/AGENT INFORMATION: NAME: Hanson, Norman D REGISTRATION NUMBER: 30,946 REFERENCE/DOCKET NUMBER: LUD 5372.3-PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 688-9200 TELEFAX: (212) 838-3884 WO 97/19167 PCTUS96/18295 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 18 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Leu Val Glu Ala Val Leu Ala Glu Val Leu Pro Lys Pro Ala Gin Thr 5 10 Val Ala INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHAkACTEiRSTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Tyr Ser Pro Gly Trp Asp Ala Lys 5 S"INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 15 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Thr Pro Val Thr Asn Leu Glu Thr Leu Glu Asp Thr Leu Gin Ala 10 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: 1 LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Asp Val Ala Pro Phe Gly Val INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Leu His Thr Thr Leu Leu Asp Val Thr Asp Pro Gin Ser Ile WO 97/19167 PCT/US96/18295 36 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGTGAATTC TNGTNGARGC NGT 23 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: iiuceic cicid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: ACGTGAATTC ACNGTYTGNG CNGGYTT 27 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACGTGAATTC CCNGTNACNA AYYT 24 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACGTGAATTC GCYTGNARNG TRTCYTC 27 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1122 base pairs TYPE: nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: p32;ll-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID AGCTTTCCCC TGAGGAGGTC ACCTGGGCTC CAGCC ATG TGG CTG CCT CTG CAG 53 Met Trp Leu Pro Leu Leu TGC CTG CTG CTG GGT GTC TTG CTC TGG GCA GCA CTG TGG TTG CTC AGG 101 Leu Gly Val Leu Leu Trp Ala Ala Leu Trp Leu Leu Arg Asp Arg Gin 15 WO 97/19167 WO 9719167PCTIUS96/1 8295 GAO CGG CCA Cys Leu Pro GCC AGC GAT GC Ala Ser Asp Ala

TTT

Phe 30 ATC TTC ATC ACC Ile Phe Ile Thr

GGC

Gly TGT GAC TOG Cys Asp Ser 149 197 GGC TTT Gly Phe GGG CGG CTC CTT Gly Arg Leu Leu

GOT

Ala CTG AGG CTG GAC Leu Arg Leu Asp AGA GGC TTC CGA Arg Gly Phe Arg

GTA

Val CTG GCC AGO TGC Leu Ala Ser Cys ACA CCC TCG GGG GCG GAG GAO CTC CAG Thr Pro Ser Gly Ala Glu Asp Leu Gin 65

CGG

Arg GTC GCC TCC TCC Val Ala Ser Ser

CGC

Arg CTC CAC ACC ACC Leu His Thr Thr

CTG

tIeu so CTG GAT GTC ACA Leu Asp Val Thr GAT CCC Asp Pro CAG AGC ATC Gin Ser Ile GCA GGG OTT Ala Gly Leu 105

CGG

Arg CAG GOA GTC AAG Gin Ala Val Lys

TGG

Trp 95 GTG GAA ACG CAT Val Giu Thr His GTT GGG GAA Val Gly Glu 100 GCO ATC ATT Gly Ile Ile TTT GGT CTG GTG Phe Gly Leu Val

AAT

Asn 110 AAT GCT GOT GTG Asn Ala Gly Val

GOT

Ala 115 GGT COO Gly Pro 120 ACC CCA TGG CAG Thr Pro Trp Gin ACO CGG GAG GAO TTO CAG OGG GTG CTG AAT Thr Arg Glu Asp Phe Gin Arg Val Leu Asn 125 130

GTG

Val 135 AAO ACG OTG GGT Asn Thr Leu Gly 000 Pro 140 ATO GGG GTC ACC Ile Gly Val Thr

CTO

Leu 145 GCC CTG CTG COO Ala Leu Leu Pro

OTG

Leu 150 CTG OTG CAG GC Leu Leu Gin Ala

OGG

Arg 155 GGC CGA GTG ATC Gly Arg Val Ile

AAO

Asn 160 ATO ACC AGT GTC Ile Thr Ser Val OTT GGO Leu Gly 165 CGT OTO GOA Arg Leu Ala GAG CO TTC Glu Ala Phe 185

GCC

Ala 170 AAT GGA GCC GGO Asn Gly Gly Gly TGC GTO TCC AAG Cys Val Ser Lys_ TTT CGC CTG Phe Gly Leu 180 TTT GGG GTA Phe Gly Val 437 485 533 581 629 677 725 773 TOT GAO AGO OTG Ser Asp Ser Leu

AGG

Arg 190 CGA GAT GTG GOT Arg Asp Val Ala

CT

Pro 195 OGG GTO Arg Val 200 TOT ATO GTG GAA Ser Ile Val Glu

OCT

Pro 205 GGC TTC TTC OGA Gly Phe Phe Arg

ACC

Thr 210 COT GTG ACA AAO Pro Val Thr Asn

OTG

Leu 215 GAA ACT TTG GAG Glu Thr Leu Glu

GAO

Asp 220 ACC CTG CAG GC Thr Leu Gin Ala

TGO

Cys 225 TGG GCA COG OTG Trp Ala Arg Leu

COT

Pro 230 OCA G00 ACA CAG Pro Ala Thr Gin 000 Al a 235 OTO TAT GGG GAG Leu Tyr Gly Glii

GCO

Ala 240 TTC OTO ACC AAA Phe Leu Thr Lys TAO OTG Tyr Leu 245 AGA GTC CAG CAA CGT ATO ATG AAC ATG ATO TGT CAT COG GAO CTG CC Arg Val Gin Gin Arg Ile Met Asn Met Ile Cys Asp Pro Asp Leu Ala 250 255 260 AAG GTG AGO AGG TGO OTG GAG CAT GCC CTA ACT G00 CGT Lys Val Ser Arg Cys Leu Glu His Ala Leu Thr Ala Arg 265 270 275 CAC COO AGA His Pro Arg 869 WO 97/19167 WO 9719167PCT/US96/1 8295 ACC CGC TAC AGC CCA GGC TGG GAT GCC AAG CTG CTC TGG TTG CCA GCC Thr Arg Tyr Ser Pro Gly Tip Asp Ala Ly's Leu Leu Tip Leu Pro Ala 280 285 290 TCC TAC TTd CCA GCC AGG CTG GTG GAT GCT GTG CTC GCC TGG GTC CTT Ser Tyr Leu Pro Ala Arg Leu Val Asp Ala Val Leu Ala Tip Val Leu 295 300 305 310 CCC AAG CCT GCC CAG ACA GTC TAC TAA ATCCAGCCCT CCAGCAAAAG Pro Lys Pro Ala Gin Thr Val Tyr 315 ATGGTTGTTC AAGGCAAGGA CTCTGATTTA TTCTGTCCCC TACCCTGGTA CTGCCTGGTG TGTGGCATAA AACAGTCACT CAATAAATGT ATTATTCAAA ACAAAAAAAA 917 965 1012 1072 1122 INFORMATION FOR SEQ ID NO: 11: Wi SEQUENCE CHARACTERISTICS: LENGTH: 343 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Rat D-b-hydroxybutyrate (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: dehydrogenase (rBDH) Met Met Leu Ala Ala Arg Leu Ser Arg Lys Leu Ser Asp Phe Val Leu Arg Asn 145 Thr Ala Phe Gin 50 Ser Leu Arg Asn Ser 130 Al a Tyr Leu Ser Val Tyr Pro Ala Ala Asp Ala Gly Phe Gly Val Phe Ala Glu Leu Asp 100 Val Cys Asn 115 Gly Leu Lys Gly Ile Ser Lys Glu Val 165 Cys Asp Ser Phe Ala Ser 55 Phe Ser 70 Gly Cys Ser Leu Ser Glu Asp Pro 135 Thr Phe 150 Ala Giu Arg Glu 25 Ser Pro 40 Gly Lys Leu Ala Leu Leu Lys Ser 105 Giu Val 120 Glu Lys Gly Glu Val Asn Pro Leu Ser Asn Gly Thr Asp Thr Arg Ala Val Leu Lys His Leu 75 Lys Glu Gin 90 Asp Arg Leu Glu Lys Ala Gly Met Tip 140 Val Glu Phe 155 Leu Trp Gly 170 Gin Arg Arg Val His Gly Arg Val 125S Gly Thr Thr Leu His Thr Thr Ser Asp Thr 110 Glu Leu Ser Val Pro Thr Tyr Gly Lys Ala Ile Thr Val Met Arg 175 Gly Leu Thr Cys Gly Gly Gin Val Asn Giu 160 Thr Thr Lys Ser Phe Leu Pro Leu Leu 180 Arg 185 Arg Ala Lys Giy Arg Val Vai 190 WO 97/19167 WO 9719167PCTIUS96/I 8295 Asn Tyr Tyr 225 Asn Ile Tyr Cys Hius 305 Tyr Ile Ile Cys 210 Glu Phe Ala G1 y Asn 290 Ala Tyr Ser Ser 195 Ile Met Ile 275 Ser Leu Trp Asp Ser Thr His Ala Lys 260 Gly Thr Trp Lys Met Lys Pro Al a 245 Met Ser Al a Leu 325 Ile Leu Phe Leu 230 Thr Trp Thr Al a 310 Arg Tyr 340 Gly Gly 215 Gly Ser Asp Asp 295 Thr Met Ile Met Glu Lys Tyr Leu 265 1 y: Se r Tyr Val1 Ala Ala Val Ser 250 Pro Ser Thr Met 330 Asn Phe Ser 235 Pro Glu A 1- Val Arg 315 Thr Ala 205 Asp Val Arg Val1 e 285 Asn His Phe- Arg Cys Glu Ile Arg 270 Al a Pro Pro Ser Leu Pro Gin 255 Lys Thr Val Met Gly 335 Pro Arg Gly 240 Al a Asp Thr Asp 320 Al a INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids (B).TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Human estradiol 17-b dehydrogenase (hEDH) (xi) SEQUENCE DESCRIPTION:. SEQ ID NO:12: Ala Arg Thr Val Val Leu Ile Thr Gly Cys Ser Ser Gly Ile Gly Leu 10 His Leu Ala Val Arg Leu Ala Ser Asp Pro Ser Gin Ser Phe Lys Val 25 Tyr Ala Thr Leu Arg Asp Leu Lys Thr Gin Gly Arg Leu Trp Glu Ala 40 Ala Arg Ala Leu Ala Cys Pro Pro Gly Ser Leu Glu Thr Leu Gin Leu 55 Asp Val Arg Asp Ser Lys Ser Val. Ala Ala Ala Arg Glu Arg Val Thr 70 75 Glu Gly Arg Val Asp Val Leu Val Cys Asn Ala Gly Leu Gly Leu Leu 90 Gly Pro Leu Glu Ala Leu Gly Glu Asp Ala Val Ala Ser Val Leu Asp 100 105 110 WO 97/19167 WO 9719167PCT/US96/1 8295 Val Asn Val Val Gly Thr Val Arg Met Leu Gin Ala Phe Leu Pro Asp 115 Met Gly 145 Ala Gly Ilie Val1 225 Leu Giu Ser Ala Al a 305 Asp Lys 130 Leu Leu Val1 cilu His 210 Phe Thr Arg Asn Lys 290 Glu Pro Arg Met Glu His 195 Thr Arg Al a Phe Tyr 275 Al a Asp Pro Arg Gly Gly Leu 180 'zal Phe Glu Leu Leu 260 Val Glu Giu Ala Gly Ser Gly 135 Leu Pro Phe 150 Leu Cys Glu 165 Ser Leu Ile I cu C ly 53cr His Arg Phe 215 Ala Ala Gin 230 Arg Ala Pro 245 Pro Leu Leu Thr Ala Met Ala Gly Ala 295 Ala Gly Arg 310 Ala Pro Gin 325 120 Arg Asn Ser Glu 2 00 Tyr Asn Lys Arg His 280 Glu Val1 Asp Leu Cys 185 Gin Pro Pro Met 265 Arg Ala Leu Val1 Al a 170 Gly Tyr Glu Thr .250 Arg Glu Gly Val Tyr 155 Val1 Pro Leu Giu 235 Leu Leu Val1 Gly Gly 315 Thr 140 Cys Leu Val I i., Al a 220 Val1 Arg Asp Phe Gly 300 Gly Ala Leu His Acp 205 His Al a Tyr Asp Gly 285 Ala Ser Ser Leu Thr 190 Ser Giu Phe Pro 270 Asp Gly Val1 Lys Pro 175 Al a *Lys Val1 Thr 255 Ser Val1 Pro Gly Phe 160 Phe Phe Gin Phe 240 Thr Gly Pro Gly Ser Ala Val Asp Pro Glu Leu Gly 320 INFORMATION FOR SEQ ID NO: 13: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAMrE/KEY:E.coli 3-oxoacyl~acyl carrier protein) reductase

(FABG)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Asn Phe Glu Gly Lys Ile Ala Leu Val Thr Gly Ala Ser Arg Giy S 10 Ile Gly Arg Ala Ile Ala Glu Thr Leu Ala Ala Arg Gly Gly Lys Vai 25 Ilie Gly Thr Ala Thr Ser Glu Asn Gly Ala Gin Ala Ile Ser Asp Tyr 40 WO 97/19167 WO 9719167PCT/US96/1 8295 Leu Gly Ala Asn Gly Lys Gly Leu Met Leu Asn Thr Asp Pro Ala Ile Glu Ser Val Giu Lys Ile Arg Ala Giu Phe Gly Glu Asp Ile Leu Val Asn Ala Gly Ile Thr Arg Asp Asn Leu Leu Met Arg Met Lys Ser Val Phe 115 Asp 100 Giu Giu Trp Asn Asp 105 Ile Ile Giu Thr Asn Leu Ser 110 Met Lys Lys Arg Leu Ser Lys Ala 120 Val Met Arg Ala Ary Hizi 130 Gly Aig 119= lie 1 i 135 11eGly Sei Val Gly Thrx 114eL G1.y Asn 145 Gly Gly Gin Ala Asn 150 Tyr Ala Ala Ala Lys 155 Ala Gly Leu Ile Giy 160 Phe Ser Lys Ser Ala Arg Giu Val Ala 170 Ser Arg Gly Ile Thr Val 175 Asn Val Val Ser Asp Asp 195 Al a 180 Pro Gly Phe Ile Giu 185 Thr Asp Met Thr Arg Ala Leu 190 Ala Gly Arg Gin Arg Ala Gly Ile 200 Leu Ala Gin Val Pro 205 Leu Gly 210 Gly Ala Gin Glu Ile 215 Ala Asn Ala Val Phe Leu Ala Ser Asp 225 Glu Aia Ala Tyr Ile 230 Thr Gly Giu Thr Leu 235 His Val. Asn Gly Gly 240 Met Tyr Met Val INFORMATION FOR SEQ ID NO: 14: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1128 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: human li-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TAAGCTTCGG

ACCTGTAGGT

GCCTTACTCT

CGCCAGCAAT

GCCTTCTGGC

TGCCTGACCC

CCTCCACACC

CAGCCAAGTG

GTGAATAATG

CCGGGACGAT

GGGTCACCCT

ATCAACATCA

CTGTGTC TCC

ATGTAGCTCA

CGAACCCCTG

CTGGGCACGG

GCGCTGTAGT

CACTTGGGCT

GGGCAGTGCT

GCCTTTGTCT

ACTGCAGCTG

CCTCCGGGGC

ACCCTGTTGG

GGTGGAGATG

CTGGTGTGGC

TTCCAGCGGG

TGCCCTGCTG

CCAGCGTCCT

AAATTTGGCC

TTTTGGGATA

TGACCAACCT

CTGCCTCCTG

ACCTGCCAGC

CCAGCTATGT

GTGGTTGCTC

TCATCACCGG

GACCAGAGAG

CGAGGACCTG

ATATCACTGA

CACGTTAAGG

TGGTATCATC

TGCTGAATGT

CCTCTGCTGC

GGGTCGCCTG

TGGAGGCCTT

CGAGTCTCCA

GGAGAGTCTG

CCACACAGGC

TTTCGCCACA GGAGGCTGCC GGCTGCCTCT TCTGCTGGGT

AGGGACCGGC

CTGTGACTCA

GCTTCCGAGT

CAGCGGGTGG

TCCCCAGAGC

AAGCAGGGCT

GGACCCACAC

GAACACAATG

AGCAAGCCCG

GCAGCCAATG

CTCTGACAGC

TCGTGGAGCC

GAG.AAAACCC

CCACTATGGG

AGAGCCTGCC

GGCTTTGGGC

CCTGGCCAGC

CCTCCTCCCG

GTCCAGCAGG

TTTTGGTCTG

CATGGCTGAC

GGTCCCATCG

GGGCCGGGTG

GTGGGGGCTA

CTGAGGCGGG

TGGCTTCTTC

TGCAGGCCTG

GGGGCCTTCC

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 PCTIUS96/1 8295 WO 97/19167 TCACCAAGTA CCTGAAAATG CAACAGCGCA TCATGAACCT GATCTGTGAC CCGGACCTAA CCAAGGTGAG CCGATGCCTG GAGCATGCCC TGACTGC-TCG ACACCCCCGA ACCCGCTACA GCCCAGGTTG GGATGCCAAG CTGCTCTGGC TGCCTGCCTC CTACCTGCCA GCCAGCCTGG TGGATGCTGT GCTCACCTGG GTCCTTCCCA AGCCTGCCCA AGCAGTCTAC TGAATCCAGC CTTCCAGCAA GAGATTGTTT TTCAAGGACA AGGACTTTGA TTTATTTCTG CCCCCACCCT GGTACTGCCT GGTGCCTGCC ACAAAATA INFORMATION FOR SEQ, ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear iii I4C1,lC0= TYPE: pzotcirl (ix) FEATURE: NAME/KEY: human li-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 850 900 950 1000 1050 1100 1128 Met Trp Leu Pro Leu Leu Leu Leu Ile Asp Ala 65 Leu Glu Gly Phe Leu 145 Ile Val Val Arg Cys 225 Leu Thr Gin Glu Asp Met Val1 Gin 130 Ala Thr Ser Al a Thr 210 5 Arg Asp 20 Gly Cys 35 Arg Gly Asp. Leu Ile Thr 85 His Val 100 Aia Gly 115 Arg Val Leu Leu Arg Gin Asp Ser Phe Arg 55 Gin Arg 70 Asp Pro Lys Gilu Ile Ile Leu Asfl Pro Leu 150 Leu Gly 165 Gly Leu Gly Ile Thr Asn Ser Gly Val1 Val Gin Aia Gly Val 135 Leu Arg Gil Arc Gly Ala Leu 10 Leu Pro Ala 25 Phe Gly Arg Leu Ala Ser Ala Ser Ser 75 Ser Val Gin 90 Gly Leu Phe 105 Pro Thr Pro .120 Asn Thr Met Gin Gin Ala Leu Trp Ala Ser Asn Ala Leu Leu Ala Phe Leu Val1 Gin Val Leu Trp Cys Leu Thr Pro Ser Gly Arg Gin Gly Trp, Gly Arg 155 Asn Asp Val Leu His Ala Ala Leu Val Leu Thr 125 Pro Ile 140 Gly Arg Thr Lys Asn 110 Arg Gly Val Gly Arg 190 Gly Thr Trp Asn Asp Val1 Ile Tyr 175 Arg Phe Leu Val Ala Asp Thr Asn 160 Cys Asp Phe Ser Lys His 195 Pro Val Phe 180 Phe Val Leu Ala Val1 200 Al a Phe 185 Se r Al a 170 Ser Ile Gly Ser Glu Lys 220 205 Leu Giu Ser Leu Glu 215 Thr Leu Gin Ala Tyr Gly Giy Ala Trp Ala Arg Leu Pro Pro Ala Thr Gin Ala His 235 WO 97/19167 PCTIUS96/18295 Phe Leu Thr Lys Leu Lys Met Gin Gin 250 Arg Ile Met Asn Leu Ile 255 Cys Asp Pro Thr Ala Arg 275 Leu Leu Trp 290 Asp 260 Leu Thr Lys Val Ser 265 Arg Cys Leu His Pro Arg Thr Arg 280 Tyr Ser Pro Gly Glu His Ala Leu 270 Trp Asp Ala Lys 285 Leu Val Asp Ala Leu Pro Ala Ser 295 Tyr Leu Pro Ala Val 305 Leu Thr Trp Val Leu 310 Pro Lys Pro Ala Gin 315 Ala Val Tyr a a INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ACGTGAATTC TGYGAYTCNG GNWTYGG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ACGTGAATTC TTNGCRTCCC CANCC INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACGTGAATTC GARGCNTTYT CNGA WO 97/19167 PCT/US96/1 8295 44 INFORMATION FOR SEQ ID NO;19: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE:flucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ACGTGAATTC CGNGTNCKNG ORTG 24 INFORMATION FOR SEQ ID NO: ii SE-7ZC CHARACTEISTICS: LENGTH: 262 base pairs TYPE :nucleic. acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: CA) NAME/KEY: PCR. clone 194 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: *CTCTCTCAGA AGGGAGCTCT CCTACTTCGG AGTGAAGGTG GCTATGATTG AGCCTGGTT.A CTTTGTITACC AATATGACCC AAGATGAGGG TTTTATTGGA TACCTCCAGG CATTGTGGAA 120 CCGGGCCAGC CCAGAGCTGA AAGAACTCTA TGGAGAAAAC TTCCCTGCTG ACTTCTTGAA 180 GACATTGAGT TTACTGAAAC CACGGTGGAC TCAGAATCTG TCCTTGGTGA CCGACTGCAT 240 GGAGCACGCC CTGACTGCCT GC 262 *0.

INFORMATION FOR SEQ ID NO:21: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs TYPE:nucleic acid TOPOLOGY: linear MOLECULE TYPE: nucleic acid (ix) FEATURE: C, A) NAME/KEY: PCR clone 207 Cxi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CTCCCTCAGG AGGGGGCTCT CCTACTTTGG GGTGAAGGTG GCTATTATAG AGCCTGGCTT CTTCCTGACC GGTGTGACCA GTAGTGCCAG ATTATGCTCA AATACCCAGA TGCTGTGGGA 120 CCAGACCAGC TCAGAAATCA GGGAGATCTA TGGCGAGAAG TACCTGGCAT CCTATCTGAA 180 AAGGCTAAAC GAATTGGACA AGAGGTGCA.A CAAGGACCTG TCTTTGGTGA CTGACTGCAT 240 GGAGCATGCT CTGACTGCCTGC 262 WO 97/19167 PCTIUS96/1 8295 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 200 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22; CAGCCTGAGG CGGGACATGG CTCCGTTCGG AGTACAAGTC TCCATTGTGG AGCCTGGCTT CTTTCGAACC CCTGTGACCA ACCTGGAGAG TCTGGAGAGC ACCCTGAAGG CTTGTTGGGC 120 CCGGCTA6CCT COCCTATAC AGCCCCACTA CZCZAGGCTCTGT TACTCI AGTACAGCGC CGCATCATGA ACCTGATCTG TGACCCAGAA CTAACGAAGG TGACCAGCTG 240 CCTGGAGCAT GCCCTGACTG CTCGC 265 INFORMATION FOR SEQ ID NO:23: Wi SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid to 0(D) TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 215 (xi) SEQUENCE DESCRIPTION: SEQ ID 140:23: to..

CAGCCTGAGG CGAGATGTGG CTCCTTTTGG GGTACGGGTC TCTATCGTGG AACCTGGCTT "oo CTTCCGAACC CCTGTGACAA ACCTGGAAAC TTTGGAGGGC ACCCTGCAGG CCTGCTGGGC 120 ACGGCTGCC'r CCAGCCACAC AGGCCCTCTA TGGGGAGGCC TTCCTCACCA AATACCTGAG 180 o AGTGCAGCAA CGTATCATGA ACATGATCTG TGATCCGGAC CTGGCCAAGG TGAGCAGGTG 240 CCTGGAGCAT GCCCTAACTG CCCGT 265 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 87 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 194 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24 Ser Leu Arg Arg Glu Leu Ser Tyr Phe Gly Val Lys Val Ala Met Ile 10 Glu Pro Gly Tyr Phe Val Thr Asn Met Thr Gln Asp Glu Gly Phe Ile 25 Gly Tyr Leu Gin Ala Leu Trp Asn Arg Ala Ser Pro Glu Leu Lys Glu 40 WO 97/19167 PCTIUS96/1 8295 46 Leu Tyr Gly Glu Asn Phe Pro Ala Asp Phe Leu Lys Thr Leu Ser Leu 55 Leu Lys Pro Arg Trp Thr Gin Asn Leu Ser Leu Val Thr Asp Cys Met 70 75 Glu His Ala Leu Thr Ala Cys INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH:b7 amino acids TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAM~E/KEY: PCR clone 207 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2S: 00 Ser Leu Arg Arg Gly Leu Ser Tyr Phe Gly Val Lys Val Ala Ile Ile 10 Glu Pro Gly Phe Phe Leu Thr Gly Val Thr Ser Ser Ala Arg Leu Cys 25 'Ser Asn Thr Gin Met Leu Trp Asp Gin Thr Ser Ser Giu Ile Arg Glu 6%035 40 Ile Tyr Giy Giu Lys Tyr Leu Ala Ser Tyr Leu Lys Arg Leu Asn Glu 55 Leu Asp Lys Arg Cys Asn Lys Asp Leu Ser Leu Val Thr Asp Cys Met 70 75 s0 Giu His Ala Leu Thr Ala Cys INFORMATION FOR SEQ ID NO:26 SEQUENCE CHARACTERISTICS: LENGTH; 88 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 200 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26 Ser Leu Arg Arg Asp Met Ala Pro Phe Gly Val Gin Val Ser Ile Val 10 Giu Pro Gly Phe Phe Arg Thr Pro Val Thr Asn Leu Glu Ser Leu Glu 25 Ser Thr Leu Lys Ala Cys Trp Ala Arg Leu Pro Pro Ala Ile Gin Ala 40 His Tyr Gly Glu Ala Phe Leu Asp Thr His Leu Arg Vai Gin Arg Arg so 55 WO 97/19167 PCTIUS 96/18295 47 Ile Met Asn Leu Ile Cys Asp Pro Giu Leu Thr Lys Val Thr Ser Cys 70 75 Leu Glu His Ala Leu Thr Ala Arg INFORMATION FOR SEQ ID NO:27: SEQUENCE

CHARACTERISTICS:

LENGTH: 88 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NA~,i~Y: CTR clocfl 21.

(X)SEQUENCE DESCRIPTION: SEQ ID NO:27: 0 0.

Ser Leu Arg Arg Asp Val Ala Pro Phe Gay Val Arg Val 510 15 Glu Pro Gay Phe Phe Arg Thr Pro Val Thr Asn Leu Giu 25 30 Gly Thr Leu Gin Ala Cys Trp Ala Arg Leu Pro Pro Ala 40 45 Leu Tyr Gly Glu Ala Phe Leu Thr Lys Tyr Leu Arg Val so 55 60 Ile Met Asn Met Ile Cys Asp Pro Asp Leu Ala Lys Val 70 75 80 Ser Ile Val Thr Leu Glu Thr Gin Ala Gin Gln Arg Ser Arg Cys Leu Glu His Ala Leu Thr Ala Arg INFORMA~TION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 563 base pairs TYPE:nucieic acid TOPOLOGY: linear (ii) MOLECULTE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: clone ME207.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GAGGCGTTCT CGGACTCCCT CAGGAGGGGG CTCTCCTACT TTGGGGTGAA GGTGGCTATT ATAGAGCCTG GCTTCTTCCT GACCGGTGTG ACCAGTAGTG CCAGATTATG CTCAAATACC 120 CAGATGCTGT GGGACCAGAC CAGCTCAGAA ATCAGGGAGA TCTATGGCGA GAAGTACCTG 180 GCATCCTATC TGAAAAGGCT AAACGAATTG GACAAGAGGT GCAACAAGGA CCTGTCTTTG 240 GTGACTGACT GCATGGAGCA TGCTCTGACT GCCTGCCACC CTCGCACGCG ATACTCAGCT 300 GGCTGGGATG CTAAGCTCTT CTACCTCCCC TTGAGC'rACC TGCCTACCTT TCTTGTGGAT 360 GCCCTTCTCT ATTGGACTTC CCTGAAGCCT GAGAAAGCCC TCTGACGTGT TCACCTATGT 420 GCATACCTGG GGAGATGTAG GTAGAGTTTG AGAGAGAGAA TATTTAGGGG AAATTTG GAG 480 GGTTGAGGGA GGGAGTTTAT TACTCTGGGG TTCAGTCAAC ACACTTCATC TCATTAATTC 540 WO 97/19167 PCT/US96/18295 48 TCCTATGACA CTACTGAATA CTG 563 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 134 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: clone ME207 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Glu Ala Phe Ser Asp Ser Leu Arg Arg Gly Leu Ser Tyr Phe Gly Val 10 Lys Val Ala Ile Ile Glu Pro Gly Phe Phe Leu Thr Gly Val Thr Ser 25 Ser Ala Arg Leu Cys Ser Asn Thr Gin Met Leu Trp Asp Gin Thr Ser 40 Ser Glu Ile Arg Glu Ile Tyr Gly Glu Lys Tyr -Leu Ala Ser Tyr Leu 55 Lys Arg Leu Asn Glu Leu Asp Lys Arg Cys Asn Lys Asp Leu Ser Leu 65 70 75 Val Thr Asp Cys Met Glu His Ala Leu Thr Ala Cys His Pro Arg Thr 90 Arg Tyr Ser Ala Gly Trp Asp Ala Lys Leu Phe Tyr Leu Pro Leu Ser 100 105 110 Tyr Leu Pro Thr Phe Leu Val Asp Ala Leu Leu Tyr Trp Thr Ser Leu 115 120 125 Lys Pro Glu Lys Ala Leu 130 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID GCTTCGGGCG CTGTAGTA 18 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE:nucleic acid TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: Reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AAAACAATCT CTTGCTGGAA WO 97/19167 PCTIUS96/I 8295 49 INFORMATION FOR SEQ ID NO:32: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1613 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: CA7CCA-r CGjAG&= Ammm AAZT xmct =ICA Tm Gr GGA 7AM= M30*A CA2C7W G7 733=03MR GIJCIAX AA%9Mw774 C7C7GM= 7-CVAA MCfl9= 7G97 TC-'7AM~ T AX%332A' 7GXVAA C2~a-,t7 AZ"Vaom T~Aamimcc A3UP G7CIAAT-xGA Cr7Q.14 CTIA~TtT 2% *L-73 *G lk TAAA=AZC G712= =77C 7T.CA Go= AIA~AGT WO 97/19167 PCTJUS96/1 8295 INFORMATION FOR SEQ ID NO:33: Wi SEQUENCE CHARACTERISTICS: CA) LENGTH: 1384 base pairs TYPE:nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 7CrlrTTTZ cm.A ,S .S -Au C-ArG. 7VGGh= T=103 GA%ZATj 7=79 AA33CA Tr&Au Amm "MMAAo C7*,C *CrG CCXX= *h -5 7G2oa ar-c G3GG;6 2axAT3J AZT AGAIGT= G"mAr.-_ AkC C c -T AA= C7GMM ~Tmhm-T GGkVG CArnxT AM~.A =1032=~M MA~A~a- TZ;3GA ~=xv=,PCA 7~7CI~~AA LA oc2x~a1 WO 97/19167 PCTIUS96/1 8295 51 INFORMATION FOR SEQ ID NO:34: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1240 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic aciLd (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: t~C=CC 700T~V=T 1XA 7ATGI33C A72GZZAIG3 7GI T~7m7-ITZA aA~x3m A533% A".=nk TOACA-rAT, AWh AA G7929 GAAA GT7GA GAGGAZ W.G.lv AAAO GCTI C7 Tr r r! "I, G7GT C1G=33v= MA-7n= ZA =Txsh TG-t~b T=A 7a;=Za= 2CXkVAA CDAZ AM CASG CAT7IC7XVA 3~Az G~.CT 2 CAMAC CI1Zk1 -NV=M CAm7 GIG 7. CArA rCTrZT V7, A 7C 0CT~l 7*7 *XGAA WO 97/19167 PCT/US96/1 8295 52 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1146 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID 7=19=~ X-57T- TC%2- A=ZXA ASU:kxfTT Cr7G Ah k AWM ACr CCC== GhGATX= C..AOGTAZ'r C7 7AM= 33W C AAui2XC7 -A-1=3; =7VT 9*.CG= jC=,r ~3 2 X* GGThZ zcX=A= CGCx G~IAG=AA= C2GZ C! h *.CAM 9~7= C~~J4~ ;AAXAG WO 97/19167 PCTIUS96/1 8295 53 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 316 amino acids TYPE: protein TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: zssne. YCI G6ZA!'SD7LR RMLS~UUW sks~mrIy LLSVr?M~ Vmrxw 2TA= INFORMATION FOR SEQ ID NO:37: Wi SEQUENCE CHARACTERISTICS: LENGTH: 317 amino acids TYPE: protein TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: FGTU1.X: R.ZwL nvncr-i vxxuwF-Mt WVS-M-OS 22rY;ISK WJ.-r=-M7L 7STSSAP=.

ySA=,vr YLPIf2TF RFN"jMs= YV77GMSG Var;GLv IMISTPSG =NVL~ p WX9vt ymmrmm vj &NGMw s i==L WO 97/19167 WO 9719167PCT1US96/1 8295 54 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: protein TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: malm;11= LTGO-SSARL MS Iavo~ TSMCZWG !:SCM V4 DXSVL =L'~ew j lP 7 'yL. 'r L INFORMATION FOR SEQ, ID NO:39: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: protein TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: a. FVI? )LLVrAG YerG.~1cLL qWJYL 3rvGSFl Z'TfA==P mLsIVN GV1-,=V 3VLVA1Wf vJnvzn puAmSV3

Claims (9)

1. An isolated nucleic acid molecule whose nucleotide sequence consists of a nucleotide sequence which encodes a human protein having retinol dehydrogenase activity, the complementary sequence of which hybridizes, under stringent conditions, to at least one nucleic acid molecule whose nucleotide sequence consists of SEQ ID NO:14 or SEQ ID NO:28.

2. An isolated nucleic acid molecule according to claim 1, which encodes the amino acid sequence of SEQ ID

3. An isolated nucleic acid molecule according to claim 1, comprising cDNA. S 15 4. An isolated nucleic acid molecule according to claim 1, comprising genomic DNA. An isolated nucleic acid molecule according to claim 1, consisting of SEQ ID NO: 14.

6. An isolated nucleic acid molecule according to claim 1, which encodes the amino acid sequence of SEQ ID NO: 29.

7. An expression vector comprising the isolated nucleic acid molecule of claim 1 operably linked to a promoter.

8. A cell line or bacterial cell strain, transformed or transfected with the expression vector of claim 7.

9. An isolated nucleic acid molecule whose nucleotide sequence consists of a nucleotide sequence which encodes a human protein having retinol dehydrogenase activity, the complementary sequence of which hybridizes, under stringent conditions, to at least one nucleic acid molecule whose nucleotide sequence is selected from a group consisting of SEQ ID NOS:20-23 and 32-35.

10. An isolated nucleic acid molecule according to claim 9, the complementary sequence of which hybridizes, under stringent conditions, to at least one nucleic acid molecule whose nucleotide sequence is selected from a group consisting of SEQ ID NOS: 20, 21, 22 and 23.

11. An isolated nucleic acid molecule according to claim 9, the complementary sequence'of which hybridizes, under stringent conditions, to at least one nucleic acid molecule whose nucleotide sequence is selected from a group consisting of SEQ ID NOS: 32, 33, 34 and 15 12. An isolated protein comprising the amino acid sequence set forth in SEQ ID NOS: 36, 37, 38 OR 39. *DATED THIS FIRST DAY OF AUGUST 2000 *6 LUDWIG INSTITUTE FOR CANCER RESEARCH BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS