AU748675B2 - 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 11-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 p32 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 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 15 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 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, 25 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, including RPR, 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 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 p63 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 p32 protein belongs to the family of short chain alcohol dehydrogenases, and exhibits 11-cis retinol dehydrogenase S" 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 (k 0l 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 t* oligonucleotide mixtures OM1 and OM3, both derived from peptide 321 deduced from partial amino acid sequence 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 11-cis-retinol dehydrogenase activity in the presence of cofactor NADP.

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

Figure 9 shows the structure of the human 11-ciso* retinol 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 i: 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 n WO 97/19167 PCT/US96/18295 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 p 6 3.

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 NaCI), containing 1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonic acid (CHAPS) at 1 mg of total membrane protein/ml 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

C.

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'~I using the well-known Chloramin T procedure. Non-incorporated '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

I

a.

a

S

albumin and subsequently subjected to immunoprecipitation using the mAb A52 to p63 (5 ug per incubation) or using two polyclonal rabbit antisera to p 6 3 (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 .0 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 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 :0 et al., J. Biol. Chem. 267:23035-23042 (1992)), it can be concluded that the M, 32,000 protein (p 3 2 coprecipitated in the aforesaid analysis by binding to p63. The analysis also identified a double band of M,50,000-52,000 which precipitated along with p32 and p63 (FIG. 1 d, e).

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 a

S.

a WO 97/19167 PCT/US96/18295 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 coupled to CNBr-activated Sepharose 4B beads (Pharmacia) in a Bio-Rad poly prep column (Bio-Rad) by end-over-end rotation at +40C. Following a 2 hour incubation, the beads 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 0 C with 100 ul of 0.2 M ammonium bicarbonate buffer containing 50% acetonitrile and thereafter .o 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 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 2 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)

(D)

a

(Y)

P322 Y-S-P-G-W-D-A-K (SEQ TD 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) P325 L-H-T-T-L-L-D-V-T-D-P-Q-S-I (SEQ ID NO: a The amino acid residues given within the parentheses are the residues deduced from the cDNA sequence in the same positions.

o 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 S. 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 p321 and p 3 23 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 0 C, 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 e 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 3 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 14 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) 15 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) 25 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 PCT/US96/18295 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 17f-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) 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.

WO 97/19167 PCT/US96/18295 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 15 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 20 using a reticulocyte lysate system with mRNA transcribed from linearized pX321. The procedure is set out in Example 8 below: 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 40C. 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 sFPnnce 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.

*o*e 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 p32 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-HCl, 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 PCT/US96/18295 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 NaCl. 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 et al., Anal. Biochem 213:28-13226 (1993).

In particular, the enzymatic activity of p32 was 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°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 370C 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 Following the work described supra, 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 "[P]dCTP labelled by random priming to 15 a high specific activity (about 109 cpm/xg of DNA) The labelled bovine cDNA was then used to probe the human cDNA library. Conditions were as follows: hybridization in 6XSSC, 0.5% SDS, 5X Denhardt's solution, 25% formamide, at 68 0 C, with 100 fg/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°C for Sminutes.

When a positive cDNA was found, the insert the 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 0: 0..

0000 a 0 0000 0 0 0 *0 0 0:0..

0 system for studying developmental biology, retinol dehydrogenases are extremely active in development. The model is extrapolatable to human development.

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

The RNA was mixed in 4#1 of 5x AMVRT buffer, together with 4Al of dNTPs from 5mM stock solutions, 2pl of oligo dT (18mer, at a final concentration of 10pM), together with 0.5L1 RNAse inhibitor, and 2u1 of avian myelostosis virus .0 reverse transcriptase (10U). The final volume was 20 l.

This resulting mixture was incubated at 420C 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 .5 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 20 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.: ACGTGAATTCT.GYGA Y TCN GGNWTYGG 3' (SEQ ID NO:16) WO 97/19167 PCT/US96/18295 23 A C G T GA AT T CT TN G CR T C C CC A N CC 3' (SEQ ID NO:17) as forward and reverse primers respectively. The primers were mixed with the two 1i 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. 5I1L samples of PCR product were combined with primers based upon and i.e.: AC GTGAATTCGARGCNTTYTCNGA 3 (SEQ ID NO:18) AC GT GAATTCC G NGTNC 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 ulsed in further probing experiments.

A murine 8.5 day cDNA library (in XgtlO), 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 g/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 T A 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 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 were used. Thirty cycles were carried out.

WO 97/19167 PCT/US96/18295 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 SxSSC, 0.5% SDS, 5 x Denhardt's solution, 50% formamide at 42 0 C, with 100Jg/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 DNA 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 G A G A 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 550C and elongation at 72 0 C. 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 15 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 XDNA 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 acid molecules set forth in SEQ ID NOS: 21 and 22 were labelled with 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 hybridized, overnight, at 420C, using 50% formamide, 6xSSPE 25 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 500C, in O.1xSSC containing 0.1% SDS. Blots were exposed at -70 0 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 SEQ ID NO: 21 SEO 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 52 0 C) followed by two, 20 minute washes in 0.lxSSC 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 the catalytic mechanism, 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 which 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 l1-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 .reaction.

Thus, one aspect of the invention is the ability to produce recombinant 11-cis-retinal dehydrogenase. The recombinant enzyme can be used to produce ll-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 bp expected to code for p32 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 ll-cis retinal from ll-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 p32 11-cis retinol dehydrogenase isolated by this invention. These findings are surprising in contrast to presently held views WO 97/19167 PCT/US96/18295 4 *4 .4

S.

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 previously nexpPcted avnuen for the isol.tion a.nd 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 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 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 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 11-cisretinal dehydrogenase activity of p32 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 0* soluble receptors may be used to prevent binding of a 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 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 be repeated here.

In yet another application, monoclonal and polyclonal 25 antibodies to p32 can be generated, which are useful, inter alia, 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 p63, 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 33 shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

S. 60 S S

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5.55 OS 0 5.55 55 S. S 55 55 5 55 *650 0 *050 @045 55.5 @0 5 0

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5500 *055 5* S 05 @0 @505 6500 EDITORIAL NOTE THERE ARE NO NUMBERED PAGES BETWEEN 33 AND 55 BUT UNNUMBERED SEQUENCE LISTING PAGES.

GENERAL INFORMATION: APPLICANTS: Eriksson, Ulf; Simon, Andras; Romert, Anna (ii) TITLE OF INVENTION: ISOLATED NUCLEIC ACID MOLECULE WHICH CODES FOR A 32 KDA PROTEIN HAVING I-CIS RETINOL DEHYDROGENASE ACTIVITY, AND WHICH ASSOCIATES WITH P63, A PORTION OF A RETINOL BINDING PROTEIN RECEPTOR (iii) NUMBER OF SEQUENCES: 41 (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 e• (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: 08/729,594 FILING DATE: 11-October-1996 CLASSIFICATION: 435 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/562,114 FILING DATE: 22-Novenber-1995 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/375,962 FILING DATE: 20-January-1995 (vi) 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 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 688-9200 TELEFAX: (212) 838-3884 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 Leu Val Glu Ala Val Leu Ala Glu Val ID NO: 1: Leu Pro Lys Pro Ala Gln Thr Val Ala INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Tyr a..

a a a a a 4 (2) Ser Pro Gly Trp Asp Ala Lys 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: Pro Val Thr Asn Leu Glu Thr Leu Glu Asp Thr Leu Gln Ala 5 10 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: 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 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGTGAATTC TNGTNGARGC NGT INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: .o e o: *o .ACGTGAATTC ACNGTYTGNG CNGGYTT *o *o *oo** (2 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACGTGAATTC CCNGTNACNA AYYT INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACGTGAATTC GCYTGNARNG TRTCYTC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1122 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: p32;11-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID SA-CTTTCCCC TGAGGAGGTC ACCTGGGCTC CAGCC ATG TGG CTG CCT CTG CAG TC CTG CTG CTG eu Gly Val Leu 10 *"GAC CGG CCA GCC Cys Leu Pro Ala 25 :*tC TTT GGG CGG Phe Gly Arg 40 CTG GCC AGC *-al Leu Ala Ser 5 GTC GCC TCC TCC Val Ala Ser Ser CAG AGC ATC CGG Gln Ser Ile Arg GCA GGG CTT TTT Ala Gly Leu Phe 105 Met Trp Leu Pro Leu Leu GGT GTC TTG CTC TGG GCA GCA CTG TGG TTG CTC AGG Leu Trp Ala Ala Leu Trp Leu Leu Arg Asp Arg Gin 15 AGC GAT GCC TTT ATC TTC ATC ACC GGC TGT GAC TCG Ser Asp Ala Phe Ile Phe Ile Thr Gly Cys Asp Ser 30 CTC CTT GCT CTG AGG CTG GAC CAG AGA GGC TTC CGA Leu Leu Ala Leu Arg Leu Asp Gln Arg Gly Phe Arg 45 TGC CTG ACA CCC TCG GGG GCG GAG GAC CTC CAG CGG Cys Leu Thr Pro Ser Gly Ala Glu Asp Leu Gin Arg 60 65 53 101 149 197 245 293 341 389

CGC

Arg CTC CAC ACC ACC CTG CTG GAT GTC ACA GAT CCC Leu His Thr Thr Leu Leu Asp Val Thr Asp Pro 80 CAG GCA GTC AAG TGG GTG GAA ACG CAT GTT GGG GAA Gin Ala Val Lys Trp Val Glu Thr His Val Gly Glu 95 100 GGT CTG GTG AAT AAT GCT GGT GTG GCT GGC ATC ATT Gly Leu Val Asn Asn Ala Gly Val Ala Gly Ile Ile 110 115 GGT CCC ACC CCA Gly Pro Thr Pro 120 GTG AAC ACG CTG Val Asn Thr Leu 135 CTG CTG CAG GCC Leu Leu Gin Ala TGG CAG ACG CGG GAG Trp Gin Thr Arg Glu 125 GGT CCC ATC GGG GTC Gly Pro Ile Gly Val 140 CGG GGC CGA GTG ATC Arg Gly Arg Val Ile 155 AAT GGA GGG GGC TAC Asn Gly Gly Gly Tyr 175 GAC AGC CTG AGG CGA Asp Ser Leu Arg Arg 190 GTG GAA CCT GGC TTC Val Giu Pro Gly Phe 205 GAC TTC CAG CGG GTG CTG AAT Asp Phe Gin Arg Val Leu Asn 130 ACC CTC GCC CTG CTG CCC CTG Thr Leu Ala Leu Leu Pro Leu 145 150 AAC ATC ACC AGT GTC CTT GGC Asn Iae'Thr Ser Val Leu Gly 160 165 TGC GTC TCC AAG TTT GGC CTG Cys Val Ser Lys Phe Gly Leu 180 GAT GTG GCT CCT TTT GGG GTA Asp Val Ala Pro Phe Gly Val 195 437 485 533 CGT CTG Arg Leu GAG GCC Glu Ala GCA GCC Ala Ala 170 TTC TCT Phe Ser 185 TCT ATC Ser Ile 581 629 CGG GTC Arg Val 200 'CTG GAA 0-.;eu Glu *5 :-7CA GCC :P-o Ala AGA GTG Arg Val *Sa* *ara

GTG

Val a ACT TTG GAG Thr Leu Glu GAC ACC CTG CAG Asp Thr Leu Gin 220 CTC TAT GGG GAG Leu Tyr Giy Glu ACA CAG Thr Gin CAG CAA Gin Gin 250 AGC AGG Ser Arg 265

GCC

Ala 235 TTC CGA ACC Phe Arg Thr 210 GCC TGC TGG Ala Cys Trp 225 GCC TTC CTC Ala Phe Leu 240 ATC TGT GAT Ile Cys Asp CTA ACT GCC Leu Thr Ala AAG CTG CTC Lys Leu Leu 290 CGT ATC ATG AAC ATG Arg Ile Met Asn Met 255 TGC CTG GAG CAT GCC Cys Leu Giu His Ala 270 CCA GGC TGG GAT GCC Pro Gly Trp Asp Ala 285 CCT GTG ACA AAC Pro Val Thr Asn GCA CGG CTG CCT Ala Arg Leu Pro 230 ACC AAA TAC CTG Thr Lys Tyr Leu 245 CCG GAC CTG GCC Pro Asp Leu Ala 260 CGT CAC CCC AGA Arg His Pro Arg 275 TGG TTG CCA GCC Trp Leu Pro Ala GCC TGG GTC CTT Ala Trp Val Leu 310 677 725 773 821 869 917 .=CQC CGC TAC AGC 4 ir Arg Tyr Ser 9: 280 TCC TAC TTG CCA Ser Tyr Leu Pro 295 CCC AAG CCT GCC Pro Lys Pro Ala GCC AGG CTG GTG GAT GCT GTG CTC Ala Arg Leu Val Asp Ala Vai Leu 300 305 965 CAG ACA GTC TAC TAA ATCCAGCCCT CCAGCAAAAG Gln Thr Val Tyr 315 1012 ATGGTTGTTC AAGGCAAGGA CTCTGATTTA TTCTGTCCCC TACCCTGGTA CTGCCTGGTG 1072 TGTGGCATAA AACAGTCACT CAATAAATGT ATTATTCAAA ACAAAAAAAA 1122 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 344 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NANE/KEY: Rat D-b-hydroxybutyrate (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: dehydrogenase (rBDH) Met Met Leu Ala Ala ArgLeu Ser Arg Lys Leu Ser asp &**P'he a *g :-s Th.

sn Tyr0 Tyr Ala Phe Gin Ser Leu Arg Asn Ser 130 Ala Tyr Lys Ilie Cys 210 Glu Leu Tyr Ala Gly Val Giu Val 115 Gly Gly Lys Ser Ser 195 Ile Met Ser Val Cys Pro Ala Ser Asp Ala Ala Phe Gly Phe Phe Ala Gly Leu Asp Ser 100 Cys Asn Ser Leu Lys Asp Ile Ser Thr 150 Giu Val Ala 165 Phe Leu Pro 180 Ser Met Leu Thr Lys Phe His Pro Leu Asp Phe Ser 55 Ser Cys Leu Glu Pro 135 Phe Glu Leu Gly Gly 215 Gly Arg Giu 25 Ser Pro 40 Gly Lys Leu Ala Leu Leu Lys Ser 105 Glu Val 120 Glu Lys Gly Glu Val Asn Leu Arg 185 Arg Met 200 Pro f0 Asn Asp Ala Lys Lys Asp Glu Gly Val Leu 170 Arg Ala Leu Ser Gin Leu Gly Thr Arg His Thr Arg Arg Thr Val Leu Val Thr His Leu His Ser 75 Giu Gln Gly Asp Arg Leu Arg Thr 110 Lys Ala Val Glu 125 Met Trp Gly Leu 140 Glu Phe Thr Ser 155 Trp Gly Thr Val Ala Lys Gly Arg 190 Asn Pro Ala Arg 205 Phe Ser Asp Cys 220 Ser Val Val Giu Pro Thr Tyr Gly Lys Ala Ile Thr Val Met Arg 175 Val Ser Leu Pro Gly Leu Thr Cys Gly Gly Gin Val Asn Giu 160 Thr Val Pro Arg Gly Val Glu Ala Val Lys Val 225 Asn Ile Tyr Cys His 305 Tyr Ile '0 t 0 0O

O

Ala s

S

His

*SSS

*Ty* r Ala Asp Glu ?he Ile Ala Ala Lys.Lys 260 Gly Lys Lys 275 Asn Ser Gly 290 Ala Leu Thr Tyr Trp Trp Ser Asp Lys 340 230 Ala Thr Ser Leu Tyr 245 Met Trp Asp Glu Leu 265 Tyr Phe Asp Glu Lys 280 Ser Thr"Asp Thr Ser 295 Ala Ala Thr Pro Tyr 310 Leu Arg Met Gin Val 325 Ile Tyr Ile His 235 Ser Pro 250 Pro Glu Ile Ala Ser Val Thr Arg 315 Met Thr 330 Glu Arg Ile Val Val Arg 270 Lys Met Glu 285 Ile Asn Ala 300 Tyr His Pro His Phe Pro 240 Gin Ala 255 Lys Asp Thr Tyr Val Thr Met Asp 320 Gly Ala 335 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids 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: Arg Thr Val Val Leu Ile Thr Gly Cys Ser Ser Gly Ile Gly Leu 5 10 Leu Ala Val Arg Leu Ala Ser Asp Pro Ser Gin Ser Phe Lys Val 25 Ala Thr Leu Arg Asp Leu Lys Thr Gin Gly Arg Leu Trp Glu Ala 35 40 Arg Ala Leu Ala Cys Pro Pro Gly Ser Leu Glu Thr Leu Gin Leu 55 Val Arg Asp Ser Lys Ser Val Ala Ala Ala Arg Glu Arg Val Thr 75 Gly Arg Val Asp Val Leu Val Cys Asn Ala Gly Leu Gly Leu Leu 90 Gly Pro Leu Glu Ala Leu Gly Glu 100 Asp 105 Ala Val Ala Ser Val Leu Asp 110 Val Asn Met Lys 130 Gly Leu 145 Ala Leu Gly Val Met Giu Ile His 210 Val Phe .::4eu Thr ::61u Arg :.:ser Asn Lys 290 :TCla Glu :..305 :A sp Pro VJal 1.15 .krg Met Giu His L~ys 195 Thr Arg Ala Phe Tyr 275 Ala Asp Prc Val Arg Gly Gly Leu 180 Val Phe Glu Leu Leu 260 Val *Giu Giu Ala Gly Gly Leu Leu 165 Ser Leu His Ala Arg 245 Pro Thr Ala Ala Ala 325 Thr Val Ser Gly 135 Pro Phe 150 Cys Glu Leu Ile Gly Ser Arg Phe 215 Ala Gin 230 Ala Pro Leu Leu Ala Met Gly Ala 295 Gly Arg 310 Pro Gin krg 1.20 krg ~sn Ser Glu Pro 200 Tyr Asn Lys Ar9 His 280 Git Met Val Asp Leu Cys 185 Glu Gin Pro Pro Met 265 Arg Ala Leu Leu Val Al L 170 Gly Glu Tyr Glu Thr 250 Arg Giu Gly Gin.

Val Tyr 155 Val Pro Val Leu Giu 235 Leu Leu Val Gly Gly 315 Al a Thr 140 Cys Leu Val Leu Ala 220 Val Arg Asp Phe Gly 300 Phe 125 Gly Al a Leu His Asp 205 His Ala Tyr Asp Gly 285 Ala Leu Ser Ser Leu Thr 190 Arg Ser Glu Phe Pro 270 Asp Gly Pro Val Lys Pro 175 Ala Thr L~ys Val Thr 255 Ser Val Pro Asp Gly Phe 160 Phe Phe Asp Gin Phe 240 Thr Gly Pro Gly .Gly 320 Ser Ala Val Asp Pro Glu Leu INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/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 Met Asn Phe Glu Gly Lys Ile Ala Leu Val Thr Gly lie lie Leu Ser Ar .Ash SLe o "Asn Ser Leu Gly Gly Gly Ile Ile Met Val His 130 Gly Ser Val Asp Gly 210 Arg Ala Thr Ala Ala Asn Glu Ser Leu Val Lys Asp 100 Phe Arg 115 Gly Arg Gly Gin Lys Ser Val Ala 180 Asp Gin 195 Gly Ala Ile Thr Gly Val Asn Glu Leu Ile Ala Leu 165 Pro Arg Gin Ala Glu Ser Glu Lys Gly 55 Leu Glu 70 Asn Ala Glu Trp Ser Lys Ile Thr 135 Asn Tyr 150 Ala Arg Gly Phe Ala Gly Glu Ile 215 Thr Asn 40 Leu Lys Gly Asn Ala 120 Ile Ala Glu Ile Ile 200 Ala 10 Leu Ala 25 Gly Ala Met Leu Ile Arg Ile Thr 90 Asp Ile 105 Val Met Gly Ser Ala Ala Val Ala 170 Glu Thr 185 Leu Ala Asn Ala Ala Arg Gin Ala Asn Val Ala Glu 75 Arg Asp Ile Glu Arg Ala Val Val 140 Lys Ala 155 Ser Arg Asp Met Gin Val Val Ala 220 Ala Gly Ile Thr Phe Asn Thr Met 125 Gly Gly Gly Thr Pro 205 Phe Ser Arg Gly Lys Ser Asp Asp Pro Gly Glu Leu Leu Asn Leu 110 Met Lys Thr Met Leu Ile Ile Thr 175 Arg Ala 190 Ala Gly Leu Ala Gly Val Tyr Ala Val Met Ser Lys Gly Gly 160 Val Leu Arg Ser Asp Glu Ala Ala Tyr Ile Thr Gly Glu Thr Leu His Val Asn Gly Gly 225 230 235 240 Met Tyr Met Val INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 1128 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: human il-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TAAGCTTCGG GCGCTGTAGT ACCTGCCAGC TTTCGCCACA GGAGGCTGCC ACCTGTAGGT CACTTGGGCT CCAGCTATGT GGCTGCCTCT TCTGCTGGGT GCCTTACTCT GGGCAGTGCT 120 GTGGTTGCTC AGGGACCGGC AGAGCCTGCC CGCCAGCAAT GCCTTTGTCT TCATCACCGG 180 CTGTGACTCA GGCTTTGGGC GCCTTCTGGC ACTGCAGCTG GACCAGAGAG GCTTCCGAGT 240 CCTGGCCAGC TGCCTGACCC CCTCCGGGGC CGAGGACCTG CAGCGGGTGG CCTCCTCCCG 300 C('E'CCACACC ACCCTGTTGG ATATCACTGA TCCCCAGAGC GTCCAGCAGG CAGCCAAGTG 360 TGGAGATG CACGTTAAGG AAGCAGGGCT TTTTGGTCTG GTGAATAATG CTGGTGTGGC 420 .:'GsTATCATC GGACCCACAC CATGGCTGAC CCGGGACGAT TTCCAGCGGG TGCTGAATGT 480 tAACACAATG GGTCCCATCG GGGTCACCCT TGCCCTGCTG CCTCTGCTGC AGCAAGCCCG 540 GGGCCGGGTG ATCAACATCA CCAGCGTCCT GGGTCGCCTG GCAGCCAATG GTGGGGGCTA 600 :ITGTGTCTCC AAATTTGGCC TGGAGGCCTT CTCTGACAGC CTGAGGCGGG ATGTAGCTCA 660 .1TTGGGATA CGAGTCTCCA TCGTGGAGCC TGGCTTCTTC CGAACCCCTG TGACCAACCT 720

:*U

6 GAGTCTG GAGAANACCC TGCAGGCCTG CTGGGCACGG CTGCCTCCTG CCACACAGGC 780 LC1CTATGGG GGGGCCTTCC TCACCAAGTA CCTGAAAATG CAACAGCGCA TCATGAACCT 840 *GA'TCTGTGAC CCGGACCTAA CCAAGGTGAG CCGATGCCTG GAGCATGCCC TGACTGCTCG 920 ACACCCCCGA ACCCGCTACA GCCCAGGTTG GGATGCCAAG CTGCTCTGGC TGCCTGCCTC 980 CTACCTGCCA GCCAGCCTGG TGGATGCTGT GCTCACCTGG GTCCTTCCCA AGCCTGCCCA 1020 AGCAGTCTAC TGAATCCAGC CTTCCAGCAA GAGATTGTTT TTCAAGGACA AGGACTTTGA 1080 TTTATTTCTG CCCCCACCCT GGTACTGCCT GGTGCCTGCC ACAAAATA 1128 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: human 11-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Leu Ile Asp Ala 65 *..Leu ly Plie Val Val a Trp Leu Leu Arg Thr Gly Gin Arg Glu Asp Asp Ile Met His Val Ala 115 Gin Arg 130 Ala Leu Thr Ser Ser Lys Ala His 195 Pro Asp Cys Gly Leu Thr Val 100 Gly Val Leu Val Phe 180 Phe Leu Arg Asp Phe Gin Asp 85 Lys Ile Leu Pro Leu 165 Gly Gly 6- Leu Leu Gin Ser Ser Gly Arg Val 55 Arg Val 70 Pro Gin Glu Ala Ile Gly Asn Val 135 Leu Leu 150 Gly Arg Leu Glu Gly Leu Phe 40 Leu Ala Ser Gly Pro 120 Asn Gin Leu Ala Ala Pro 25 Gly Ala Ser Val Leu 105 Thr Thr Gin Ala Phe 185 Ser Leu Leu 10 Ala Ser Arg Leu Ser Cys Ser Arg 75 Gin Gin 90 Phe Gly Pro Trp Met Gly Ala Arg 155 Ala Asn 170 Ser Asp Ile Val Trp Asn Leu Leu Leu Ala Leu Leu Pro 140 Gly Gly Ser Glu Ala Val Leu Trp Ala Phe Val Phe Ala Leu Gin Leu Thr Pro Ser Gly His Thr Thr Leu Ala Lys Trp Val Val Asn Asn Ala 110 Thr Arg Asp Asp 125 Ile Gly Val Thr Arg Val Ile Asn 160 Gly Gly Tyr Cys 175 Leu Arg Arg Asp 190 Pro Gly Phe Phe 205 Ile Arg Val 200 Arg Thr Pro Val Thr Asn Leu Glu Ser Leu Glu Lys Thr Leu Gin Ala 210 215 220 Cys Trp Ala Arg Leu Pro Pro Ala Thr Gin Ala His Tyr Gly Gly Ala 225 230 235 240 Phe Leu Thr Lys Tyr Leu Lys Met Gin Gin Arg Ile Met Asn Leu Ile 245 250 255 Cys Asp Pro Asp Leu Thr Lys Val Ser Arg Cys Leu Glu His Ala Leu 260 265 270 Thr Ala Arg His Pro Arg Thr Arg Tyr Ser Pro Gly Trp Asp Ala Lys 275 280 285 Leu Leu Trp Leu Pro Ala'Ser Tyr Leu Pro Ala Ser Leu Val Asp Ala 290 295 300 Val Leu Thr Trp Val Leu Pro Lys Pro Ala Gin Ala Val Tyr 305 310 315 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: .:*PTGAATTC TGYGAYTCNG GNWTYGG 27 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE:nucleic acid STRANDEDNESS: single 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 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACGTGAATTC GARGCNTTYT CNGA INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: *.*ACGTGAATTC CGNGTNCKNG GRTG 2) INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 194 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: r r r :'*CTCTCTCAGA AGGGAGCTCT CCTACTTCGG AGTGAAGGTG GCTATGATTG AGCCTGGTTA ***.CTTTGTTACC AATATGACCC AAGATGAGGG TTTTATTGGA TACCTCCAGG CATTGTGGAA CCGGGCCAGC CCAGAGCTGA AAGAACTCTA TGGAGAAAAC TTCCCTGCTG ACTTCTTGAA GACATTGAGT TTACTGAAAC CACGGTGGAC TCAGAATCTG TCCTTGGTGA CCGACTGCAT 120 180 240 GGAGCACGCC CTGACTGCCT GC 262 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid.

(ix) FEATURE: NAME/KEY: PCR clone 207 (xi) 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 AGAGGTGCAA CAAGGACCTG TCTTTGGTGA CTGACTGCAT 240 GGAGCATGCT CTGACTGCCT GC 262 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid STRANDEDNESS: double 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 :"TPGGCTACCT CCAGCTATAC AGGCCCACTA CGGGGAGGCC TTCCTCGATA CTCATCTTCG 180 :.AGTACAGCGC CGCATCATGA ACCTGATCTG TGACCCAGAA CTAACGAAGG TGACCAGCTG 240 ***CCTGGAGCAT GCCCTGACTG CTCGC 265 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 215 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CAGCCTGAGG CGAGATGTGG CTCCTTTTGG GGTACGGGTC TCTATCGTGG AACCTGGCTT CTTCCGAACC CCTGTGACAA ACCTGGAAAC TTTGGAGGGC ACCCTGCAGG CCTGCTGGGC 120 ACGGCTGCCT CCAGCCACAC AGGCCCTCTA TGGGGAGGCC TTCCTCACCA AATACCTGAG 180 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 S (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 194 S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24 Ser Leu Arg Arg Glu Leu Ser Tyr Phe Gly Val Lys Val Ala Met Ile 10 :.Gu Pro Gly Tyr Phe Val Thr Asn Met Thr Gin Asp Glu Gly Phe Ile 20 25 :'ly Tyr Leu Gin Ala Leu Trp Asn Arg Ala Ser Pro Glu Leu Lys Glu 35 40 .T Tyr Gly Glu Asn Phe Pro Ala Asp Phe Leu Lys Thr Leu Ser Leu 50 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 SEQUENCE CHARACTERISTICS: LENGTH:87 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 207 (xi) SEQUENCE DESCRIPTION: SEQ ID 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 Glu Ile Arg Glu 40 Ile Tyr Gly Glu Lys Tyr Leu Ala Ser Tyr Leu Lys 55 Arg Leu Asn Glu Leu Asp Lys Arg Cys Asn Lys Asp Leu Ser Leu Val Thr Asp Cys Met 70 75 *Glu His Ala Leu Thr Ala Cys INFORMATION FOR SEQ ID NO:26: S SEQUENCE CHARACTERISTICS: LENGTH: 88 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: S(A) NAME/KEY: PCR clone 200 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: .*So .'Se.1 "Gluu oa, Leu Arg Arg Asp Met Ala Pro Phe Gly Val Gin Val 5 10 Ser Ile Val Pro Gly Phe Phe Arg Thr Pro Val Thr Asn Leu Glu Ser 20 25 Leu Glu Ser Thr Leu Lys Ala Cys Trp Ala Arg Leu Pro Pro Ala 40 His Tyr Gly Glu Ala Phe Leu Asp Thr His Leu Arg Val 55 Ile Met Asn Leu Ile Cys Asp Pro Glu Leu Thr Lys Val 70 75 Ile Gin Ala Gin Arg Arg Thr Ser Cys 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: NAME/KEY: PCR clone 215 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Ser Leu Arg Arg Asp Val Ala Pro Phe Gly Val Arg Val 10 Glu Pro Gly Phe Phe Arg Thr Pro Val Thr Asn Leu Glu 25 Gly Thr Leu Gin Ala Cys Trp Ala Arg Leu Pro Pro Ala 40 Leu Tyr Gly Glu Ala Phe Leu Thr Lys Tyr Leu Arg Val 50 55 Met Asn Met Ile Cys Asp Pro Asp Leu Ala Lys Val 70 75 Glu His Ala Leu Thr Ala Arg :*85 Ser Ile Val Thr Leu Glu Thr Gin Ala Gin Gin Arg Ser Arg Cys INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 563 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE 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 TTGAGCTACC TGCCTACCTT TCTTGTGGAT 360 GCCCTTCTCT ATTGGACTTC CCTGAAGCCT GAGAAAGCCC TCTGACGTGT TCACCTATGT 420 GCATACCTGG GGAGATGTAG GTAGAGTTTG AGAGAGAGAA TATTTAGGGG AAATTTGGAG 480 GGTTGAGGGA GGGAGTTTAT TACTCTGGGG TTCAGTCAAC ACACTTCATC TCATTAATTC 540 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 5 10 :L Val Ala Ile Ile Glu Pro Gly Phe Phe Leu Thr Gly Val 20 25 Sb Ala Arg Leu Cys Ser Asn Thr Gin Met Leu Trp Asp Gln 35 40 'er Glu Ile Arg Glu Ile Tyr Gly Glu Lys Tyr Leu Ala Ser 55 LS Arg Leu Asn Glu Leu Asp Lys Arg Cys Asn Lys Asp Leu 70 75 "V I Thr Asp Cys Met Glu His Ala Leu Thr Ala Cys His Pro 90 Tyr Ser Ala Gly Trp Asp Ala Lys Leu Phe Tyr Leu Pro 100 105 110 Tyr Leu Pro Thr Phe Leu Val Asp Ala Leu Leu Tyr Trp Thr 115 120 125 Lys Pro Glu Lys Ala Leu 130 Gly Val Thr Ser Thr Ser Tyr Leu Ser Leu Arg Thr Leu Ser Ser Leu t GENERAL INFORMATION: APPLICANTS: Eriksson, Ulf; Simon, Andras; Romert, Anna (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: 41 (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: 08/729,594 FILING DATE: 1l-October-1996 CLASSIFICATION: 435 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/562,114 FILING DATE: 22-November-1995 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/375,962 FILING DATE: 20-January-1995 (vi) 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 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 688-9200 TELEFAX: (212) 838-3884

L

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 Gln Thr 10 Val Ala INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: yr Ser Pro Gly Trp Asp Ala Lys 5 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: r'hr Pro Val Thr Asn Leu Glu Thr Leu Glu Asp Thr Leu Gin Ala 5 10 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: 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 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear S (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: :..;kGTGAATTC TNGTNGARGC NGT INFORMATION FOR SEQ ID NO: 7: 9090 9 0 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: *:ACGTGAATTC ACNGTYTGNG CNGGYTT INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ACGTGAATTC CCNGTNACNA AYYT INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ACGTGAATTC GCYTGNARNG TRTCYTC INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1122 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: p32;ll-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID .OCTTTCCCC TGAGGAGGTC ACCTGGGCTC CAGCC ATG TGG CTG CCT CTG CAG Met Trp Leu Pro Leu Leu TGC CTG CTG CTG GGT GTC TTG CTC TGG GCA .**Leu Gly Val Leu Leu Trp Ala Ala Leu Trp 10 15 CGG CCA GCC AGC GAT GCC TTT ATC TTC Leu Pro Ala Ser Asp Ala Phe Ile Phe 30 .GC TTT GGG CGG CTC CTT GCT CTG AGG CTG *"91y Phe Gly Arg Leu Leu Ala Leu Arg Leu 40 45 GTA CTG GCC AGC TGC CTG ACA CCC TCG GGG Val Leu Ala Ser Cys Leu Thr Pro Ser Gly 60 GTC GCC TCC TCC CGC CTC CAC ACC ACC CTG Val Ala Ser Ser Arg Leu His Thr Thr Leu 80 CAG AGC ATC CGG CAG GCA GTC AAG TGG GTG Gln Ser Ile Arg Gln Ala Val Lys Trp Val 95 GCA CTG TGG TTG CTC AGG Leu Leu Arg Asp Arg Gln ATC ACC GGC TGT GAC TCG Ile Thr Gly Cys Asp Ser GAC CAG AGA GGC TTC CGA Asp Gln Arg Gly Phe Arg GCG GAG GAC CTC CAG CGG Ala Glu Asp Leu Gln Arg 65 CTG GAT GTC ACA GAT CCC Leu Asp Val Thr Asp Pro GAA ACG CAT GTT GGG GAA Glu Thr His Val Gly Glu 100 53 101 149 197 245 293 341 389 GCA GGG CTT TTT GGT CTG GTG AAT AAT GCT GGT GTG GCT GGC ATC ATT Ala Gly Leu Phe Gly Leu Val Asn Asn Ala Gly Val Ala Gly Ile Ile 105 110 115 GGT CCC ACC CCA TGG CAG ACG CGG GAG GAC TTC CAG Gly Pro Thr Pro Trp Gin Thr Arg Giu Asp Phe Gin 120 125 130 GTG AAC ACG CTG GGT CCC ATC GGG GTC ACC CTC GCC Val Asn Thr Leu Gly Pro Ile Gly Vai Thr Leu Ala 135 140 145 CGG GTG CTG AAT Arg Val Leu Asn' CTG CTG CCC CTG Leu Leu Pro Leu 150 AGT GTC CTT GGC Ser Val Leu Gly 165 437 485 CTG CTG Leu Leu CGT CTG Arg Leu CAG GCC CGG GGC CGA GTG ATC AAC ATC ACC Gin Ala Arg Gly Arg Val 1le Asn Ile'Thr 155 160 GCA GCC AAT GGA GGG GGC TAC TGC GTC TCC Ala Ala Asn Gly Gly Gly Tyr Cys Val Ser 170 175 AAG TTT GGC Lys Phe Gly 180

CTG

Leu 533 581 629 677

GAG

Glu 'Arg

CT'G

.eu 2*15 GCC TTC Ala Phe 185 TCT GAC AGC CTG AGG CGA GAT GTG GCT Ser Asp Ser Leu Arg Arg Asp Val Ala 190 CCT TTT GGG GTA Pro Phe Gly Val 195 CCT GTG ACA AAC Pro Val Thr Asn

GTC

Val 200 TCT ATC GTG GAA CCT GGC TTC TTC CGA ACC Ser Ile Val Giu Pro Gly Phe Phe Arg Thr 205 210

O(;CA

Pto A7AG .J#sk GAA ACT Glu Thr GCC ACA Ala Thr GTG CAG Val Gin GTG AGC Val Ser 265 TTG GAG GAC ACC CTG CAG GCC TGC TGG Leu Glu Asp Thr Leu Gin Ala Cys Trp 220 225 CAG GCC CTC TAT GGG GAG GCC TTC CTC Gin Ala Leu Tyr Gly Glu Ala Phe Leu 235 240 CAA CGT ATC ATG AAC ATG ATC TGT GAT Gin Arg Ile Met Asn Met Ile Cys Asp 250 255 AGG TGC CTG GAG CAT GCC CTA ACT GCC Arg Cys Leu Giu His Ala Leu Thr Ala 270 GCA CGG CTG Ala Arg Leu

CCT

Pro 230 725 ACC AAA TAC CTG Thr Lys Tyr Leu 245 CCG GAC CTG GCC Pro Asp Leu Ala 260 CGT CAC CCC AGA Arg His Pro Arg 275 TGG TTG CCA GCC Trp Leu Pro Ala GCC TGG GTC CTT Ala Trp Val Leu 310 773 821 869 ACC CGC Thr Arg 280 TAC AGC CCA GGC TGG GAT GCC AAG CTG CTC Tyr Ser Pro Gly Trp Asp Ala Lys Leu Leu 285 290 917 965

TCC

Ser 295 TAC TTG Tyr Leu CCA GCC AGG CTG GTG GAT GCT GTG CTC Pro Ala Arg Leu Val Asp Ala Val Leu 300 305 CCC AAG CCT Pro Lys Pro GCC GAG ACA GTC TAC TAA ATCCAGCCCT CCAGCAAAAG Ala Gin Thr Val Tyr 315 1012 ATGGTTGTTC AAGGCAAGGA CTCTGATTTA TTCTGTCCCC TACCCTGGTA CTGCCTGGTG 1072 TGTGGCATAA AACAGTCACT CAATAAATGT ATTATTCAAA ACAAAAAAAA 1122 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 344 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 Pro Leu Lys Ala Leu Phe Gin S 50 Ser **Phe Leu Arg :lTeu Asn .:Arg Ser 130 Asn Ala 145 Thr Tyr Thr Lys Asn Ile Tyr Cys 210 Leu Tyr Ala Gly Val Glu Val 115 Gly Gly Lys Ser Ser 195 Ile Ser Pro Asp Phe Phe Leu 100 Cys Leu Ile Glu Phe 180 Ser Thr Val Ala Ala Gly Ala Asp Asn Lys Ser Val 165 Leu Met Lys Cys Ser Ala Phe Gly Ser Ser Asp Thr 150 Ala Pro Leu Phe Asp Arg Phe Ser 40 Ser Gly 55 Ser Leu Cys Leu Leu Lys Glu Glu 120 Pro Glu 135 Phe Gly Glu Val Leu Leu Gly Arg 200 Gly Val Glu Asn 25 Pro Asp Lys Ala Ala Lys Leu Lys 90 Ser Asp 105 Val Glu Lys Gly Glu Val Asn Leu 170 Arg Arg 185 Met Ala Glu Ala Gly Thr Val His 75 Glu Arg Lys Met Glu 155 Trp Ala Asn Phe Ser Gin Thr Arg Arg Arg Leu Val Leu His Gin Gly Leu Arg Ala Val 125 Trp Gly 140 Phe Thr Gly Thr Lys Gly Pro Ala 205 Ser Asp 220 Leu Pro Gly His Thr Leu Thr Tyr Thr Thr Gly Cys Ser Lys Gly Asp Ala Gly Thr Ile Gin 110 Glu Thr Val Leu Val Asn Ser Met Glu 160 Val Arg Thr 175 Arg Val Val 190 Arg Ser Pro Cys Leu Arg 215 Tyr Glu Met His Pro Leu Gly Val Lys Val Ser Val Val Glu Pro Gly 225 Asn Ile Tyr Cys His 305 S..Tyr :ele e 2) a Ala G* *u o 5 G u His Tyr Ala Asp Glu 230 ?he lie Ala Ala Lys.Lys 260 Gly Lys Lys 275 Asn Ser Gly 290 Ala Leu Thr Tyr Trp Trp Ser Asp Lys 340 Ala Thr Ser 245 Met Trp Asp Tyr Phe Asp Ser Thr Asp 295 Ala Ala Thr 310 Leu Arg Met 325 Ile Tyr Ile Leu Glu Glu 280 Thr Pro Gin His Tyr Leu 265 Lys Ser Tyr Val Ser 250 Pro Ile Ser Thr Met 330 235 Pro Glu Ala Val Arg 315 Thr Glu Val Lys Ile 300 Tyr His Arg Val Met 285 Asn His Phe Ile Arg 270 Glu Ala Pro Pro Gin 255 Lys Thr Val Met Gly 335 240 Ala Asp Tyr Thr Asp 320 Ala INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 327 amino acids 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: Arg Thr Val Val Leu Ile Thr Gly Cys Ser Ser Gly Ile Gly Leu 5 10 Leu Ala Val Arg Leu Ala Ser Asp Pro Ser Gin Ser Phe Lys Val 25 Ala Thr Leu Arg Asp Leu Lys Thr Gin Gly Arg Leu Trp Glu Ala 40 Arg Ala Leu Ala Cys Pro Pro Gly Ser Leu Glu Thr Leu Gin Leu 55 Val Arg Asp Ser Lys Ser Val Ala Ala Ala Arg Glu Arg Val Thr 70 75 Gly Arg Val Asp Val Leu Val Cys Asn Ala Gly Leu Gly Leu Leu 90 Gly Pro Leu Glu Ala Leu Gly 100 Glu Asp Ala Val Ala Ser Val Leu Asp 105 Val Met Gly 145 Ala Gly Met .:225 Leu 41 Ala Asn Val.

115 Lys Arg 130 Leu Met Leu Glu Val His Glu Lys 195 His Thr 210 Phe Arg Thr Ala Arg Phe Asn Tyr 275 Lys Ala 290 LGlu Asp Val Arg Gly Gly Leu 180 Val Phe Glu Leu Leu 260 Val Glu Gly Thr Va1 Arg 120 Gly Ser Gly Arg 135 Leu Pro Phe Asn 150 Leu Cys Glu Ser 165 Ser Leu Ile Glu Leu Gly Ser Pro 200 His Arg Phe Tyr 215 Ala Ala Gln Asn 230 Arg Ala Pro Lys 245 Pro Leu Leu Arg Thr Ala Met His 280 Ala Gly Ala Glu Met Val Asp Leu Cys 185 Glu Gln Pro Pro Met 265 Arg Ala Leu Leu Val Ala 170 Gly Glu Tyr Glu Thr 250 Arg Glu Gly Gln.

Val Tyr 155 Val Pro Val Leu Glu 235 Leu Leu Val Gly Gly 315 Ala Thr 140 Cys Leu Val Leu Ala 220 Val Arg Asp Phe Gly 300 Phe 125 Gly Al a Leu His Asp 205 His Ala Tyr Asp Gly 285 Ala 110 Leu Ser Ser Leu Thr 190 Arg Ser Glu Phe Pro 270 Asp Gly Pro Val Lys Pro 175 Ala Thr Lys Val.

Thr 255 Ser Val Pro Asp Gly Phe 160 Phe Phe Asp Gln Phe 240 Thr Gly Pro Gly 295 Asp Pro Pro Ala Ala Gly Arg Ser Ala Val 310 Ala Pro Gln 325 Asp Pro Glu Leu Gly 320 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 244 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY:E.coli 3-oxoacyl[acyl carrier protein] reductase

(FABG)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Ile I 2Set Asp A.e Asn 145 Phe Ser Leu Nrg Asn Ser Asn Gly Gly Gly Ile Ile Met Val His 130 Gly Ser Val Asp Gly 210 Phe Glu Gly Lys Ile Ala Arg Ala Ile Ala Glu Thr Thr Ala Thr Ser Glu Asn 35 40 Ala Glu Leu Lys Phe 115 Gly Gly Lys Val Asp 195 Gly Asn Gly Lys Gly Leu 55 Ser Val Leu Glu Lys Val Asn Asn Ala Gly 85 Asp Glu Glu Trp Asn 100 Arg Leu Ser Lys Ala 120 Arg Ile Ile Thr Ile 135 Gin Ala Asn Tyr Ala 150 Ser Leu Ala Arg Glu 165 Ala Pro Gly Phe Ile 180 Gin Arg Ala Gly Ile 200 Ala Gin Glu Ile Ala 215 Leu Val Thr Gly 10 Leu Ala Ala Arg 25 Gly Ala Gin Ala Met Leu Asn Val Ile Arg Ala Glu 75 Ile Thr Arg Asp 90 Asp Ile Ile Glu 105 Val Met Arg Ala Gly Ser Val Val 140 Ala Ala Lys Ala 155 Val Ala Ser Arg 170 Glu Thr Asp Met 185 Ala Gly Ile Thr Phe Asn Thr Met 125 Gly Gly Gly Thr Pro 205 Phe Ser Arg Gly Lys Ser Asp Asp Pro Gly Glu Leu Leu Asn Leu 110 Met Lys Thr Met Leu Ile Ile Thr 175 Arg Ala 190 Ala Gly Leu Ala Gly Val Tyr Ala Val Met Ser Lys Gly Gly 160 Val Leu Arg Ser Leu Ala Gin Val Asn Ala Val Ala 220 Asp Glu Ala Ala Tyr Ile Thr Gly Glu Thr Leu His Val Asn Gly Gly 225 230 235 240 Met Tyr Met Val INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 1128 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: human il-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

TAAGCTTCGG

CA12TTGGGCT

:..T'GGTTGCTC

4CT1GTGACTCA

CTGGCCAGC

CCTCCACACC

G'MUGGAGATG

3TATCATC 4ZdZACACAATG C6GCCGGGTG

:.G*GTGTCTCC

TTTTGGGATA

GGAGAGTCTG

CCACTATGGG

GATCTGTGAC

ACACCCCCGA

CTACCTGCCA

AGCAGTCTAC

GCGCTGTAGT

CCAGCTATGT

AGGGACCGGC

GGCTTTGGGC

TGCCTGACCC

ACCCTGTTGG

CACGTTAAGG

GGACCCACAC

GGTCCCATCG

ATCAACATCA

AAATTTGGCC

CGAGTCTCCA

GAGAAAA CCC

GGGGCCTTCC

CCGGACCTAA

ACCCGCTACA

GCCAGCCTGG

TGAATCCAGC

ACCTGCCAGC

GGCTGCCTCT

AGAGCCTGCC

GCCTTCTGGC

CCTCCGGGGC

ATATCACTGA

AAGCAGGGCT

CATGGCTGAC

GGGTCACCCT

CCAGCGTCCT

TGGAGGCCTT

TCGTGGAGCC

TGCAGGCCTG

TCACCAAGTA

CCAAGGTGAG

GCCCAGGTTG

TGGATGCTGT

CTTCCAGCAA

TTTCGCCACA

TCTGCTGGGT

CGCCAGCAAT

ACTGCAGCTG

CGAGGACCTG

TCCCCAGAGC

TTTTGGTCTG

CCGGGACGAT

TGCCCTGCTG

GGGTCGCCTG

CTCTGACAGC

TGGCTTCTTC

CTGGGCACGG

CCTGAAAATG

CCGATGCCTG

GGATGCCAAG

GCTCACCTGG

GAGATTGTTT

GGAGGCTGCC

GCCTTACTCT

GCCTTTGTCT

GACCAGAGAG

CAGCGGGTGG

GTCCAGCAGG

GTGAATAATG

TTCCAGCGGG

CCTCTGCTGC

GCAGCCAATG

CTGAGGCGGG

CGAACCCCTG

CTGCCTCCTG

CAACAGCGCA

GAGCATGCCC

CTGCTCTGGC

GTCCTTCCCA

TTCAAGGACA

ACCTGTAGGT

GGGCAGTGCT

TCATCACCGG

GCTTCCGAGT

CCTCCTCCCG

CAGCCAAGTG

CTGGTGTGGC

TGCTGAATGT

AGCAAGCCCG

GTGGGGGCTA

ATGTAGCTCA

TGACCAACCT

CCACACAGGC

TCATGAACCT

TGACTGCTCG

TGCCTGCCTC

AGCCTGCCCA

AGGACTTTGA

120 180 240 300 '360 420 480 540 600 660 720 780 840 920 980 1020 1080 1128 TTTATTTCTG CCCCCACCCT GGTACTGCCT GGTGCCTGCC ACAAAATA INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: human 11-cis retinol dehydrogenase (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Leu le SLe Leu Ile Valu Val he Leu 145 Ile Val Val Trp Leu Pro L( Leu Arg Asp A: Thr Gly Cys A! Gin Arg Gly P] 50 Glu Asp Leu G Asp Ile Thr A 8 Met His Val L 100 Val Ala Gly I 115 Gin Arg Val L 130 Ala Leu Leu P Thr Ser Val L 1 Ser Lys Phe G 180 Ala His Phe G 195 eu Leu rg sp he In sp 5 ys le eu ro eu 65 ly ly Gin Ser Arg Arg Pro Glu Ile Asn Leu 150 Gly Leu Ile Leu Gly Ala Ser Leu Pro 25 Gly Phe Gly 40 Val Leu Ala 55 Val Ala Ser Gin Ser Val Ala Gly Leu 105 Gly Pro Thr 120 Val Asn Thr 135 Leu Gin Gin Arg Leu Ala Glu Ala Phe 185 Arg Val Ser 200 Leu Leu 10 Ala Ser Arg Leu Ser Cys Ser Arg 75 Gin Gin 90 Phe Gly Pro Trp Met Gly Ala Arg 155 Ala Asn 170 Ser Asp Ile Val Trp Ala Val Asn Ala Phe Leu Ala Leu Leu Thr Pro Leu His Thr Ala Ala Lys Leu Val Asn 110 Leu Thr Arg 125 Pro Ile Gly 140 Gly Arg Val Gly Gly Gly Ser Leu Arg 190 Glu Pro Gly 205 Leu Trp Val Phe Gin Leu Ser Gly Thr Leu Trp Val Asn Ala Asp Asp Val Thr Ile Asn 160 Tyr Cys 175 Arg Asp Phe Phe Arg Thr Pro Val Thr Asn Leu Glu Ser Leu Glu Lys Thr Leu Gin Ala 210 215 220 Cys Trp Ala Arg Leu Pro Pro Ala Thr Gin Ala His 225 230 235 Phe Leu Thr Lys Tyr Leu Lys Met Gin Gin Arg Ile 245 250 Tyr Gly Gly Ala 240 Met Asn Leu Ile 255 Cys Asp Pro Asp Leu Thr Lys Val Ser Arg 260 265 Thr Ala Arg His Pro Arg Thr Arg Tyr Ser 275 280 Cys Leu Glu His Ala Leu 270 Asp Ala Lys Pro Gly Trp 285 Leu Leu Trp Leu Pro Ala Ser Tyr Leu Pro Ala Ser 290 295 300 Leu Val Asp Ala Val Tyr Val Leu 305 Thr Trp Val Leu Pro Lys Pro Ala Gin Ala 310 315 a INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: a AGTGAATTC TGYGAYTCNG GNWTYGG a.

•2 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE:nucleic acid STRANDEDNESS: single 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 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: ACGTGAATTC GARGCNTTYT CNGA 24 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: .*'ACGTGAATTC CGNGTNCKNG GRTG 24 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs TYPE:nucleic acid o STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 194 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTCTCTCAGA AGGGAGCTCT CCTACTTCGG AGTGAAGGTG GCTATGATTG AGCCTGGTTA CTTTGTTACC AATATGACCC AAGATGAGGG TTTTATTGGA TACCTCCAGG CATTGTGGAA 120 CCGGGCCAGC CCAGAGCTGA AAGAACTCTA TGGAGAAAAC TTCCCTGCTG ACTTCTTGAA 180 GACATTGAGT TTACTGAAAC CACGGTGGAC TCAGAATCTG TCCTTGGTGA CCGACTGCAT 240 GGAGCACGCC CTGACTGCCT GC 262 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 262 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid.

(ix) FEATURE: NAME/KEY: PCR clone 207 (xi) 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 AGAGGTGCAA CAAGGACCTG TCTTTGGTGA CTGACTGCAT 240 GGAGCATGCT CTGACTGCCT GC 262 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid.

STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 200 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: .AGCCTGAGG CGGGACATGG CTCCGTTCGG AGTACAAGTC TCCATTGTGG AGCCTGGCTT GTTTCGAACC CCTGTGACCA ACCTGGAGAG TCTGGAGAGC ACCCTGAAGG CTTGTTGGGC 120 CCGGCTACCT CCAGCTATAC AGGCCCACTA CGGGGAGGCC TTCCTCGATA CTCATCTTCG 180 AGTACAGCGC CGCATCATGA ACCTGATCTG TGACCCAGAA CTAACGAAGG TGACCAGCTG 240 CCTGGAGCAT GCCCTGACTG CTCGC 265 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 265 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: PCR clone 215 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CAGCCTGAGG CGAGATGTGG CTCCTTTTGG GGTACGGGTC TCTATCGTGG AACCTGGCTT CTTCCGAACC CCTGTGACAA ACCTGGAAAC TTTGGAGGGC ACCCTGCAGG CCTGCTGGGC 120 ACGGCTGCCT CCAGCCACAC AGGCCCTCTA TGGGGAGGCC TTCCTCACCA AATACCTGAG 180 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

S..

S

S

.5 0SSS TOPOLOGY: linear S (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 194 S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24 ,Ser Leu Arg Arg Glu Leu Ser Tyr Phe Gly Val Lys 5 10 Glu Pro Gly Tyr Phe Val Thr Asn Met Thr Gin Asp 25 Gly Tyr Leu Gin Ala Leu Trp Asn Arg Ala Ser Pro 40 Leu Tyr Gly Glu Asn Phe Pro Ala Asp Phe Leu Lys 55 Leu Lys Pro Arg Trp Thr Gln Asn Leu Ser Leu Val 70 75 Glu His Ala Leu Thr Ala Cys Val Ala Met Ile Glu Gly Phe Ile Glu Leu Lys Glu Thr Leu Ser Leu Thr Asp Cys Met INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH:87 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: PCR clone 207 (xi) SEQUENCE DESCRIPTION: SEQ ID 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 25 Ser Asn Thr Gln Met Leu Trp Asp Gln Thr Ser Ser Glu Ile 40 Leu Cys Arg Glu I,!e u 0000 **ft @090 0000 00 0 00 0 Tyr Gly Glu Lys Tyr Leu Ala Ser Tyr Leu Lys 50 55 Asp Lys Arg Cys Asn Lys Asp Leu Ser Leu Val 70 75 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: Arg Leu Asn Glu Thr Asp Cys Met Ser Leu Arg Arg Asp Met Ala Pro Phe Gly Val Gin Val 10 Ser Ile Val Glu Pro Gly Phe Phe Arg Thr Pro Val Thr Asn Leu Glu Ser 25 Leu Glu Ser Thr Leu Lys Ala Cys Trp Ala Arg Leu Pro Pro Ala 40 His Tyr Gly Glu Ala Phe Leu Asp Thr His Leu Arg Val 55 Ile Gin Ala Gin Arg Arg Ile Met Asn Leu Ile Cys Asp Pro Glu 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: NAME/KEY: PCR clone 215 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Ser Leu Arg Arg Asp Val Ala Pro Phe Gly Val Arg Val 10 Glu Pro Gly Phe Phe Arg Thr Pro Val Thr Asn Leu Glu 25 'ty Thr Leu Gln Ala Cys Trp Ala Arg Leu Pro Pro Ala 35 40 X4u Tyr Gly Glu Ala Phe Leu Thr Lys Tyr Leu Arg Val 55 j1e Met Asn Met Ile Cys Asp Pro Asp Leu Ala Lys Val 70 75 Leu Glu His Ala Leu Thr Ala Arg .r o Ser Ile Val Thr Leu Glu Thr Gln Ala Gln Gln Arg Ser Arg Cys :2)

GAGGC

ATAGA

CAGAT

GCATC

GCATC

INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 563 base pairs TYPE:nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: clone ME207.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 'GTTCT CGGACTCCCT CAGGAGGGGG CTCTCCTACT TTGGGGTGAA GGTGGCTATT GCCTG GCTTCTTCCT GACCGGTGTG ACCAGTAGTG CCAGATTATG CTCAAATACC 120 GCTGT GGGACCAGAC CAGCTCAGAA ATCAGGGAGA TCTATGGCGA GAAGTACCTG 180 CTATC TGAAAAGGCT AAACGAATTG GACAAGAGGT GCAACAAGGA CCTGTCTTTG 240 GTGACTGACT GCATGGAGCA TGCTCTGACT GCCTGCCACC CTCGCACGCG ATACTCAGCT 300 GGCTGGGATG CTAAGCTCTT CTACCTCCCC TTGAGCTACC TGCCTACCTT TCTTGTGGAT 360 GCCCTTCTCT ATTGGACTTC CCTGAAGCCT GAGAAAGCCC TCTGACGTGT TCACCTATGT 420 GCATACCTGG GGAGATGTAG GTAGAGTTTG AGAGAGAGAA TATTTAGGGG AAATTTGGAG 480 GGTTGAGGGA GGGAGTTTAT TACTCTGGGG TTCAGTCAAC ACACTTCATC TCATTAATTC 540 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: :Giu Ala Phe Ser Asp Ser Leu Arg Arg Gly Leu Ser Tyr Phe 10 Lnro Val Ala Ile Ile Glu Pro Gly Phe Phe Leu Thr Gly Val 25 .g';Ala Arg Leu Cys Ser Asn Thr Gin Met Leu Trp Asp Gin 40 ~rGiu Ile Arg Glu Ile Tyr Gly Glu Lys Tyr Leu Ala Ser 55 Is- Arg Leu Asn Glu Leu Asp Lys Arg Cys Asn Lys Asp Leu 75 *Va;7 Thr Asp Cys Met Glu His Ala Leu Thr Ala Cys His Pro 90 Arg Tyr Ser Ala Gly Trp Asp Ala Lys Leu Phe Tyr Leu Pro 100 105 110 Tyr Leu Pro Thr Phe Leu Val Asp Ala Leu Leu Tyr Trp Thr 115 120 125 Lys Pro Giu Lys Ala Leu 130 Gly Val Thr Ser Thr Ser Tyr Leu Ser Leu Arg Thr Leu Ser Ser Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: forward primer (xi) SEQUENCE DESCRIPTION: SEQ ID GCTTCGGGCG CTGTAGTA INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE:nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: nucleic acid (ix) FEATURE: NAME/KEY: Reverse primer (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: .AAAkACAATCT CTTGCTGGAA :~(SZ INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 1613 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: r :t!AjCCATACT

M.EGTAAGCA

:AAACCATGTG

*CTGGCTTTGG

C4;PGTCTGAC

-C!ATGATCCT

*.krIGTGTTGG

CTATCAATGA

GCATGATCGA

TCAACATCTC

AGTATGGTGT

AGGTGGCTAT

CACACAGCAT

AGAATTTTCT

ACCTGTCTGT

GATACTCAGC

GGTCAGAGGA

GCGGCCAGGC

GCTCTACCTG

GGTNGTGAGC

GAACCTTCTG

GGAGAAGGGA

GGATGTCACC

GAACAGAGGA

GTGGCTGAAA

GGTGACTCTG

CAGCTCCATG

AGAGGCCTTC

CATAGAGCCT

AGAGAAGCTG

GGACTCCTAT

GGTAACTGAC

TGGCTGGGAT

ACATGATAGA

TCATTTTGAC

GTTGCACTGG

CATCTCCAAG

GCCAGACAAC

GCTGAGCAGC

AAGACAGAGA

CTCTGGGGCC

AAAGAGGACT

AGCATGCTGC

GGTCGAGTGT

TCAGACTCCC

GGCGGGTTCA

TGGGACCAGA

ATCAAAGCAA

TGCATGGAGC

GCCAAGCTCT

AACCTGACAT

ACAGAATATC

TGGGCCTGTG

ACAAGTATGT

TGGACAGGAG

TGAGGAACAA

GTATTGTGGC

TGGTCAACAA

TTGCAAATAT

CCTTAGTGAG

CTTTGTGTGG

TCAGGAGGGA

GGACTAATGT

CATCCTCGGA

TACAGTCATT

ACGCTCTGAC

TCTACCTACC

TCTCAGTGCC

TCTCTCTGCT

GACGCTCCTG

CTTCATCACG

AGGCATGAGG

GACATCTGAC

AGCCACTCAG

TGCTGGCATC

ACTGGATGTG

GAAGGCGAGG

TGGTGGTTAC

GATCTCCTAT

CTCCAACTAC

GGTCAAGGAG

GACAGACACA

TGCCTGTCAC

CTTGAGCTAC

TATACCTTCC

TGACTTCTAC

CGCTTCTTCA

GGCTGTGACT

GTGCTAGCTG

AGGCTGGAGA

TGGGTGAAGG

TGTGTCTTTG

AACCTGTTGG

GGCCGTGTGT

TGCATCTCCA

TTTGGGGTGA

GAGAGGCTAT

GTCTATGACA

TGCTCAGATG

CCTCGCACAA

ATGCCCACCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020

TCCTGGTAGA

GCACGTGTGT

TGAGACTCAT

AGAGGCAGTA

CTTAGCTTCT

TAAACACCTA

CATGGAAGAT

GCC CC CACC C

AATCTTGCT.A

TTGGATTCCT

TGCCATGTTG

GCAGACTTGT

TAAAACAATT

A.AGTTCTGCC

TGGGGCTTCA

GGATGAGCTT

CGAAAGAATC

GCACTCCACA

AGATAAACAA

TTCATTTCAG

TACTGGAGCT

GGCGGGTGGA

CAGCTTCCAT

AAGGGGGAGT

TCTTGGCCTA

TGCCTCTNNC

CACTTCACAA

TCCTA.ATCGG

CAACAACAAT

AATAAAAGTT

CTGTAAAGCC

GGGAGATAAT

ACTACTCAGA

GATACAAAGG

TTGTGAGA.AT

CTTCCTC.ATG

TCACCTTTTT

CTTTAGGAGG

TTTTATTTGT

GAAAAGATAA

TGCCCAAGCC

GGCACAGGGC

ACCAGTAAAG

GGCTGGCAAT

CCACAGGACT

ATGTCCATGG

CCATGGTGTC

TGGTTTTGCT

CTCAAAACCA

AAAAAAAAAA

CTGTGAATCT

ATGTGGTTCT

TTCAGGGGAA

ATCCTGTGAA

GCAGAGATTG

GCCTGGCCAT

AGGAGGGAGG

GGTGGGATAG

TGGTTTTTCT

AA

1080 1140 1200 1260 1320 1380 1440 1500 1560 1613 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 1384 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

CTTTTTTTTT

TGTGGCTCTA

GGCAGGTGGT

I*eff;GGACCCT .TrzACGGAGAA *.VfCtTGGATGT

!*:%GGAACAG

*eC.AACGAGTG GtTCGAGGT 'A2GTCTCCAG *.4rGTGTAGA

TGGCTATTAT

.gAATACCCA

AGTACCTGGC

.T4TCTGGGGT 6-TCAGCTGG :*IriTGGATGC

:CACCTATGTG

M.AtTAGGAGA

=TAATTCTC

TGCCAAGGG

A*tATGAAGG

TGGTCCTCCT

AATT

TTTTTTTTTG

CCTGGTGGCA

GAGCCATCTC

GCTGGCCAGA

GGGAGCCGAG

CACCAAGACA

AGGACTCTGG

GATGAAAAAG

GACTCTGAGC

TGTCATGGGT

GGCCTTCTCA

AGAGCCTGGC

GATGCTGTGG

ATCCTATCTG

GACTGACTGC

CTGGGATGCT

CCTTCTCTAC

CATACCTGGG

GTTGGGGGGG

CTATGACACT

ATTCAGTTAC

TCCACACAGA

CTGCAGGAAA

ACACAGAGTA

CTGGTGGGCC

CAAGACAAAT

CAGCTGGACA

GAGCTGAGGA

GAGAGTATTG

GGCCTGGTCA

CAGGACTTTG

ATGCTGCCTT

CGAGTGTCTC

GACTCC CT CA

TTCTTCCTGA

GACCAGACCA

AAAAGGCTAA

ATGGAGCATG

AAGCTCTTCT

TGGACTTCCC

GAGATGTAGG

GATTTTATTA

ACTCAAGACT

AAAAGAGCTG

TGAGGAGGCT

TGTACATGCC

TCTCTCTCTC

TGTGGACGCT

ATGTCTTCAT

GGAGAGGCAT

ACAAGACATC

TGACAGCCAC

ACAACGCTGG

CACATGTACT

TAGTGAGGAA

TCTTTGGTGG

GGAGGGAGCT

CCGGTGTGAC

GCTCAGAAAT

ACAAATTGGA

CTCTGACTGC

ACCTCCCCTT

TGAGGCCTGA

TAGAGTTTGA

CTCTGGGGTT

GATGATGACC

GCTGATGCCC

GGGACAAGTT

CTGGCTAGTT

TGCTTGACTT

TCTGCGCTTC

CACGGGCTGT

GAGGGTGCTG

TGACAGGCTG

TCAGTGGGTG

CATCTCCACC

GGATGTGAAC

GGCGAGGGGT

TGGTTACTGC

CCGCTACTTT

CAGTAGTGCC

CAGGGAGATC

CAAGAGGTGC

C TGTCAC CC T

GAGCTACCTG

AAAAGCCCTC

GAGAGAGAAT

CAGTCAATAC

AAAGAAATAG

AGATTATGAG

TGTGCCAAAG

TAAACCCCTA

CTACAAGCCA

TTCAGGGTGA

GACTCTGGCT

GCTGCATGTC

GAGACAGTGA

AAGGAGCATG

CCCTCGGGTC

CTGTTGGGCA

CGTGTGGTCA

ATCTCTAAGT

GGGGTGAAGG

AGATTATGCT

TATGGCGAGA

AACAAGGACC

CGTACCCGAT

CCCACCTTTC

TGAAAGTATT

ATTTAGGGGA

ACTTCATCTT

GCAAAGAATT

CATCATGGCT

CACTCTCCTG

TGCAAGATGG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1384 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 1240 base pairs TYPE: nucleic acid STRJANDEDNESS: double TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:

CTTCTCTCTC

GCTACTGGGC

TCAAGACAAG

ACAGCTGGAC

GGAGCTGAGG

AGAGAATATT

GGGCCTGGTC

ACAGGACTTT

CATGCTGCCC

CAGAGTGTCA

CTCAGACTCC

TGGCTTCTTC

GTGGGACCAG

TCTGAAAAAC

CTGCATGGAG

TGCTAAGCTC

ATACTGGACT

*GGGTCAATGG

:eATCCCACAT .4TGGGACCA

.AACGACCT

INFOR

(i)

TCTCTCTCTC

CTGTGGATGC

TATGTCTTCA

AGGAGAGGCA

AGAAAGACAT

GTGGCAGCCA

AACAACGCTG

GCAAGTGTAC

TTAGTGAGGA

CTTGGTGGTA

CTTAGGAGGG

CTGACTGGTA

ACCAGCTCAG

CTAAACGAAT

CACGCTCTGA

TTCTTCACCC

TCTCCAAAAC

AAGTGCTGTG

GTGCTGTGCA

CAGGAATCTG

CCCCTGTCTC

TCTCTCCTCT

TCCTGCGCTT

TCACAGGCTG

TGAC*.kGTGTT

CAGAAAGGCT

CTCAGTGGGT

GCATCTCCGT

TGGATGTGAA

AGGCGAGGGG

GTGGTGGTTA

ACGTCCGCTA

TGGCCAGTAG

AAATGCGGGA

TGGACCAGAG

CTGCCTGTCA

CCTTGAGCTA

CTGACAAAGC

TGAGGGTGCA

TAGGTGCCTG

CTATGCTTAA

TCTACAAACC

TTTTAGAGAG

TGGCTCTGGC

GGCTGCATGT

GGAGACAGTG

GAAGGAGCGT

CCCCTCGGGT

CCTGTTGGGC

CCGTGTGGTC

CTGCATCTCC

CTTTGGGGTG

TGCCAGATTA

GATCTATGGA

GTGCAACAAG

CC CTCGCACG

CCTGCCCACC

CCTGTGAAAG,

GANNCTCAGG

GCTTTTTCTT

ATGATCATTA

ATGTGGCTCT

AGGCAAGTGG

TTTGGGAACC

CGGAAGGAGG

ATCCTGGATG

GTTGGGAACA

CCCAACGAGT

TTGATCGAGG

AACGTCTCCA

AAGTATGGTA

AAGGTGGCT.A

TGCTCAAATA

GAGAAATACC

GACCTGTCTG

AGATACTCAG

TTTCTTGTGG

CTAGATCCTT

AGGAGGAAGA

CCTCTGTATA

CCATTGTTGG

ACATGGTGGC

TGGACCATCT

TGCTGGCCAG

AGGGAGCCGA

TCACCAAGAC

GAGGACTCTG

GGATGAAAAA

TGACTCTGAG

GCATCTTGGG

TAGAGGCCTT

TTATAGAGCC

TCCAGATGCT

TGGCATCCTA

TGGTGACTGA

CTGGCTGGGA

ATGCTCTTCT

TTGTGTTGTT

TTCCTGTTCT

ACATGGGGGA

TGAAAGAAAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1240 GGGTTAAAAA AAAAAA 9**a 9*9* (xi) 4ATION FOR SEQ ID EQUENCE CHARACTERISTICS: LENGTH: 1146 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear 3EQUENCE DESCRIPTION: SEQ ID

,IGCTTGAGAG

hQ,6TCTGCTT

C=GCCGGCC

QtTGGCACTG -V(!;iAGCAGAA

CACTGATCCC

TGGACTTTTT

GCTAACACAG

CACCCTTGCC

TGTCTTGGGC

GGCCTTCTCT

GGAGCCTGGC

GGCTTGTTGG

TACTTATCTT

CTTTCCCCAG AGGCTGCCCT CTGGGTGCCT TGCTGTGGGC AGTGATGCTT TCATCTTCAT CAACTTGACC AGAAGGGCTT GACCTGCAGC AGATGGCCTC CAGAATGTCC AGCAAGTTGC GGTCTGGTG.A ATAACGCTGG GATGATTTCC AGAGAGTACT

CTGCTGCCCC'TGCTACAGCA

CGCATAGCAG CCAATGGCGG GACAGCCTGA GGCGGGACAT TTCTTTCGAA CCCCTGTGAC GCCCGGCTAC CTCCAGCTAT CGAGTACAGC GCCGCATCAT

CAGCAGGGCA

AGTGCTGTGG

CACTGGCTGT

CCAAGTCCTG

CTCCCGCCTC

CAAGTGGGTG

CGTAGCTGGT

GAGTGTGAAC

GGCCAGGGGT

GGGCTACTGT

GGCTCCGTTC

CAACCTGGAG

ACAGGCCCAC

TCTCATCCCA TCATGTGGCT

TTGCTCAGAG

GACTCTGGCT

GCCGGCTGCC

CACACAACAC

AAGACACGTG

ATCATCGGGC

ACACTGGGGC

CGGGTGGTCA

GTCTCCAAGT

GGAGTACAAG

AGTCTGGAGA

TACGGGGAAG

ACCGGCAGAG

TTGGGCGCCT

TGACCCCCTC

TACTGGATAT

TTGGAGAAAC

CCACACCATG

CCATCGGTGT

ACATCACCAG

TTGGCCTGGA

TCTCCATTGT

GCACCCTGAA

CCTTCCTCGA

AACTAACGAA

120 180 240 300 360 420 480 540 600 660 720 780 840 GAACCTGATC TGTGACCCAG GGTGACCAGC TGCCTGGAGC ATGCCCTGAC TGCTCGCCAC CCCCGAACAC GCTACAGCCC AGGCTGGGAT GCCAAGCTGC TCTGGCTGCC TGCCTCCTAC CTTCCAGCCA GGGTGGTGGA TGCTGTGCTC ACCTGGATCC TTCCCCGGCC CGCCCAGTCA GTCTCCTGAT TCCAGCTTTA CAGCAAGAGG CTGATTTTGA AAAGCAAGGC ATCTATTTCT GTGTCTACCC AGTGCTGCCT GGTTTCTGAT ACCAATTAGG CTCTCAATAA ATATGTATTG CTTTAAAAAA AAAAAA

AAAAAG

900 960 1020 1080 1140 114.6 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS:- LENGTH: 31-$ amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: Met Trp Leu Tyr Leu Val Ala Leu Val Gly Leu Trp Thr Leu Leu Arg 10 Phe Phe Arg Glu Arg Gin Val Val Ser His Leu Gin Asp Lys Tyr Val 25 Phe Ilie Thr Gly Cys Asp Ser Gly Phe Gly Asn Leu Leu Ala 40 Arg Gin Glu Lys Leu

*J

sec.9 -Me 9 Asp Arg Arg Gly Met Arg Val Leu Ala Ala Cys Leu Thr 55 Ala Glu Gin Leu Asp Val Leu Arg Asn Lys Thr Ser Asp Arg Leu Giu Thr Val 70 75 Thr Lys Thr Giu Ser Ile Val Ala Ala Thr Gin Trp 90 Lys Glu Arg Val Gly Asn Arg Gly Leu Trp Gly Leu Val 100 105 110 Gly Ile Cys Val Phe Ala Ile Asn Giu Trp Leu Lys Lys 115 120 125 Ala Asn Ile Leu Asp Val Asn Leu Leu Gly Met Ile Glu 130 135 140 Asn Asn Giu Asp Val Thr .Leu Ser Met Leu Ie Ser Ser Ser Pro Leu Val Arg Lys Ala Arg Giy Arg Val Phe Asn 150 155 160 Met Gly Arg Vai Ser Leu Cys Gly Gly Gly Tyr Cys 165 170 175 Ilie Ser Lys Tyr Gly Val Glu Ala Phe Ser Asp Ser Leu Arg 180 185 190 Ile Ser Tyr Phe Gly Val Lys Val Ala Ile Ile Giu Pro Gly 195 200 205 Arg Giu Gly Phe Arg Thr Asn 210 Leu Trp Asp 225 Phe Leu Asp Ser Asp Asp Ala Cys His 275 Phe Tyr Leu 290 Leu Tyr Trp 305 Val Gin Ser Leu 260 Pro Pro Ser Ser Asn Thr Ser 230 Tyr Ile 245 Ser Val Arg Thr Leu Ser Ser Val 310 Tyr 215 Ser Lys Val Itrg Tyr 295 Lays Glu Arg Glu Val Ala Ile Thr Asp 265 Tyr Ser 280 Met Pro Pro Ala Leu Ser Lys Giu 235 Gin Ser 25.0 Cys Met Ai1 Gly Thr Phe Gin Ala 315 His 220 Val Leu Giu Trp Leu 300 Leu Ser Ile Tyr Asp Thr Asp His Ala 270 Asp Ala 285 Val Asp Giu Lys Thr 255 Leu Lys Ala Lys Asn 240 Cys Thr Leu Met (2) S0 0e 0 *so& 0 0 0Ile al* AlaO INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 317 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Trp Leu Tyr Leu Vai Ala Leu Val Gly Leu Trp 10 Phe Arg Val Arg Gin Val Val Ser His Leu Gin 20 25 Ile Thr Gly Cys Asp Ser Gly Phe Gly Thr Leu 35 40 Asp Arg Arg Giy Met Arg Val Leu Ala Ala Cys 55 Ala Giu Giu Leu Arg Asn Lys Thr Ser Asp Arg 75 Leu Asp Val Thr Lys Thr Giu Ser Ile Val Thr 90 Lys Giu His Vai Gly Asn Arg Gly Leu Trp Gly 100 105 Gly Ile Ser Thr Pro Ser Gly Pro Asn Giu Trp, 115 120 Thr Asp Leu Leu Leu Ala Leu Met 125 Leu Lys Ala Thr Giu Thr Val 110 Lys Leu Tyr Arg Glu Thr Gin Asn Lys Arg Val Gin Lys Vai Trp Asn Gin Asp Phe Ala His Val Leu 130 Asp Val 135 Asn Leu Leu Thr 145 Asn Cys Giu Phe Met 225 Lys *Qys Le.

Le Leu Ser Val Ser Ile Ser Leu Arg 195 Leu Thr 210 Leu Trp, Tyr Leu Asn Lys Ala Cys 275 Phe Tyr 290 Leu Tyr Met Ser Lys 180 Tyr Gly Asp Ala Asp 260 His Leu Trp Leu Val 165 Tyr Phe Val Gin Ser 245 Leu Pro Pro Thr Pro 150 Met Gly Giy Thr Thr 230 Tyr Ser Arg Leu Ser 310 Leu Val Gly Arg Val Giu Val Lys 200 Ser Ser 215 Ser Ser Leu Lys Gly Val Thr Arg 280 Ser Tyr 295 Arg Val Ala 185 Val Ala Giu Arg Thr 265 Tyr Leu Lys Ser 170 Phe Ai~a Arg Ile Leu 250 Asp Ser Pro Ala 155 Leu Ser Ile Leu Arg 235 Asn Cys Ala Thr Lys 315 Gly 140 Arg Phe Asp Ile Cys 220 Giu Lys Met Gly Phe 300 Gly Arg Gly Gly Ser Leu 190 Giu Pro 205 Ser Asn Ile Tyr Leu Asp Giu His 270 Trp Asp 285 Leu Val Val Gly 175 Arg Gly Thr Gly Lys 255 Ala Ala Asp Val 160 Tyr Arg Phe Gin Giu 240 Arg Leu Lys Ala Met Ile Glu Val Leu Arg Pro Glu Ala Leu INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Trp Leu Tyr Met Val Ala Leu Leu Gly Leu Trp Met Leu Leu Arg 10 Phe Arg Giu Arg Gin Val Val Asp His Leu Gin Asp Lys Tyr Val 25 Ilie Thr Gly Cys Gly Ser Gly Phe Gly Asn Leu Leu Ala Arg Gin 40 Leu Asp Arg Arg Gly Met Arg 55 Val Leu Ala Ala Cys Arg Lys Giu Giu Gly Ala Ilie Leu Val Lys Ala Gly Asp Phe 130 Thr Leu 145 Asn Val *Tyr Cys .:7rg Asp *:Phe Phe *5S0 0.0 210 Qln Met iu Lys ~ieu Thr Lys Leu 290 Glu Asp Glu Ile 115 Ala Ser Ser Ile Val 195 Leu Leu Tyr Asn Ala 275 Phe Giu Val Arg 100 Ser Ser Met Ser Ser 180 Arg Thr Trp, Leu Lys 260 Cys Phe Leu Thr Val Val Val Leu Ile 165 Lys Tyr Gly Asp Ala 245 Asp His Thr Arg.

70 Lys Gly Pro Leu Pro 150 Leu Tyr Phe Met Gln 230 Ser Leu Pro Pro Arg Thr Asn Ser Asp 135 Leu Gly Gly Gly Ala 215 Thr Tyr Ser Arg Leu 295 Lys Glu.

Arg Gly 120 Val Val Arg Ile Val 200 Ser Ser Leu Val Thr 280 Ser Thr Asn Gly 105 Pro Asn Arg Val Glu 185 Lys Ser Ser Lys Val 265 Arg Tyr Ser Glu Arg 75 Ile Val Ala 90 Leu Trp Gly Asn Giu Trp Leu Leu Gly 140 Lys Ala Arg 155 Ser Leu Gly 170 Ala Phe Ser Val Ala Ile Ala Arg Leu 220 Giu Met Arg 235 Asn Leu Asn 250 Thr Asp Cys Tyr Ser Ala Leu Pro Thr 300 Leu Ala Leu Met 125 Leu Gly Gly Asp Ile 205 Cys Giu Glu Met Gly 285 Phe Giu Thr Thr Gin Val Asn 110 Lys Lys Ile Giu Arg Val Ser Giy 175 Ser Leu 190 Giu Pro Ser Asn Ile Tyr Leu Asp 255 Giu His 270 Trp Asp Leu Val Val Trp Asn Gin Val Val 160 Gly Arg Giy Ile Giy 240 Gin Aia Ala Asp Ala Leu Leu Tyr Trp Thr Ser Pro Lys Pro Asp 315 Lys Ala Leu 305 310 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: Met Trp Let' Pro Leu Let' Leu Gly

C

I

Let' Ile Asp Ala Let' y GiyPh Zeu ::::145 Me9t 9 225 Leu Thr Gin Giu Asp Thr Vai Gin 130 Ala Thr Ser Ala Thr 210 Trp Arg Gly Lys Asp Ile Arg Ala 115 Arg Let' Ser Lys Prc 195 Prc Ala Asp.

Cys Gly Let' Thr Val 100 Gly Val Let' Val Phe 180 Phe Val ~Arg Arg Asp Phe Gin Asp Gly Ile Let' Pro Let' 165 Gly Gly Thr Leu Gin Ser Gin Gin 70 Pro Git' Ile Ser Leu 150 Gly Leu Val Asn Prc 230 Ser Gly Val 55 Met Gin Thr Gly Val 1~35 Let' Arg Giu Gin Leu 215 Prc Leu Phe 40 Leu' Ala Asn Gly Pro 120 Asn Gin Ile Ala .Val 200 Git' Ala 3EQ kl a Pro 25 G1y Al a Ser Val Let' 105 Thr Thr Gin Ala Phe 185 Sex Sei IlE ID NO: 39: Let' Let' T 10 Ala Ser A Arg Let' L Gly Cys L Ser Arg I 75 Gin Gin N 90 Phe Gly I Pro Trp, I Let' Gly *Ala Arg 155 *Ala Asn 170 Ser Asp Ilie Val -Let' Gt' SGin Ala 235 *sp et' set set Tai let iet Pro L4 0 3ily ,iy Ser Glu Ser 220 His Ala Phe Ala Leu Thr Pro His Thr Ala Lys Vai Asn 110 Thr Gin 125 Ile Gly Arg Val Gly Gly Let' Arg 190 Pro Gly 205 Thr Let' Tyr Gly Ile Gin Ser Thr Trp Asn Asp Val Val Tyr 175 Arg Phe Lys Glu rp, Ala Val Let' Trp Phe.

Let' Gly Let' Val Ala Asp Thr Asn 160 Cys Asp Phe Ala Ala 240 Phe Let' Asp Thr Tyr Let' Arg Val Gin Arg Arg Ile Met Asn Let' Ile 245 250

,Z!D!D

Cys Asp Pro Glu Leu Thr Lys Val Thr Ser Cys Leu Glu His Ala Leu 260 265 270 Thr Ala Arg His Pro Arg Thr Arg Tyr Ser Pro Gly Trp 275 280 285 Leu Leu Trp Leu Pro Ala Ser Tyr Leu Pro Ala Arg Val 290 295 300 Val Leu Thr Trp Ile Leu Pro Arg Pro Ala Gin Ser Val 305 310 315 Asp Ala Lys Val Asp Ala Ser (2) INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CTCAGGCTGT CAGAGAAGGC CT a INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: a a GACGATTTCC AGCGGGTGC

Claims (12)

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. 15

4. An isolated nucleic acid molecule according to claim 1, comprising genomic DNA.

5. 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 LUDWIG INSTITUTE FOR CANCER RESEARCH *B BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS