RECOMBINANT KYNURENINE-3-HYDROXYLASE ENZYME AND PROCESS FOR ITS PREPARATION

Abstract: Disclosed are isolated DNA sequences encoding kyn-3-OHase enzymes. Vectors and host cells containing the DNA sequences above, Xenopus laevis oocytes transiently expressing the rat kyn-3-OHase enzyme and E. coli cells stably producing human kyn-3-OHase enzyme, are also disclosed.

Description: RECOMBINANT KYNURENINE- 3-HYDROXYLASE ENZYME AND PROCESS FOR ITS PREPARATION BACKGROUND OF THE INVENTION The present invention concerns DNA sequences encoding for kynurenine- 3 -hydroxylase (kyn-3-OHase) . This enzyme is a flavin- containing monooxygenase which is localized in the outer mitochondrial membrane (Okamoto H., Yamamoto S., Nozaki M. and Hayaishi 0. 1967. Biochem. Biophys. Res. Commun. 26: 309-314); it catalyses the 3 hydroxylat ion of h kynurenine (L-kyn) , an intermediate in the oxidative metabolism of tryptophan (DeCastro F. T., Price J. M. and Brown R. R., 1956. J. Am. Chem. Soc . 78: 2900-2904). The kynurenine pathway (see the scheme below) is the major route of peripheral tryptophan metabolism in mammals: most of this metabolism takes place in the liver. The abbreviations used are the following: IDO, Indoleamine 2 , 3 -dioxygenase ; TDO, Tryptophan 2 , 3 - dioxygenase; HAD, 3-hydroxyanthranilate 3 , 4 -dioxygenase; QPRT, Quinolinate phosphoribosyltransferase ,- ATP, Adenosine 5'- triphosphate; NAD, Nicotinamide adenine dinucleotide ; NMN, Nicotinamide mononucleotide . Kynurenic Acid Xanthurenic Acid 3 -Hydrox kynurenine -Amino- 3-carboxymuconate Semlaldehyde Non-enzymatic Quinolinic Acid This pathway not only provides a route for total oxidation of tryptophan to acetyl-Co-A, but it is also responsible for the synthesis de novo of the nicotinamide nucleotide coenzymes NAD and NADP (Bendei D A and McCreanor G. M 1985 Bioche . Soc Trans 13-441 443) Most of the current interest in this pathway arises from the observations that two intermediate metabolites, kynuremc acid (KYNA) and quinolimc acid (QUIN) , seem to play a significant role in neurological diseases, the first acting as a neuroprotectant and the second as a neurotoxic agent Kyn-3 OHase is the first enzyme in the route of production ot QUIN The importance of QUIN as a neurotoxic agent was first evident from work by Lapm (1978 J Neural Trans 42 37-43) who demonstrated that the administration of QUIN to i ats caused convulsions This was not sufficient to classify QUIN as a neurotoxm, its action in the central nervous system was better clarified when electrophysiological studies revealed that it was an agonist at the excitatory ammo acid receptor sites normally activated by gluta ate and aspartate (Stone T and Perkins M N ,1981 Eur J Pharmacol 72 411-412) Moreover, using the mtrastriatal injection model, QUIN toxicity has been shown to be mediated through the N-methyl D-aspartate (NMDA) receptors (Beal M , Kowall N , Swartz K J , Ferrante R J and Martin J B 1989 J Neurosci . 8 3901-3908, Foster A C , Gill R and Woodruff G N 1988 J Neurosci 8 4745-4754) Consistent with the volvment of NMDA receptors were the studies that showed reversal of QUIN induced damage pathology by competitive NMDA antagonists (Foster A C , Vezzam A , French F D and Schwarcz R 1984.Neurosci Lett 48 273 278, Leeson P D , Baker R , Carl g R W , Curtis N R , Moore K W , Williams B J et al 1991 J Med Chem 34 1243 1252) It is becoming clear that some of the most important functions of the nervous system, such as synaptic plasticity and synapse formation, critically depend on the behavior of NMDA receptor channels and that neurological damages caused by a variety of pathological states can result from exaggerated activation of NMDA receptor channels (For a review see Mori H and M sh a M. 1995 Neuropharmacology 34 1219 1237) Excessive activation of these receptors may play an important role in the neuronal injury associated with several disease states, in luding hypoxia- ischemia (Simon R P , Swan J H , Griffiths 'I and Meldrum B A 1984 Science 226 850 852), hypoglycemia (Wieloch 1 198 r Science 230 681 683) and Hunt ingt on' s disease (Schwarc R , Whetsell W 0 Ji and Mangano R M 1983 Science 219 316 318 Koh J Y , Peters S and Choi D W 1986 Science 234 73 76) Assuming that QUIN is pathogenic for certain disorders, it is desirable to inhibit its formation To accomplish this goal, knowledge must be gained about the enzymes that make QUIN and the sites at which the pathway is controlled In theory, QUIN could be formed in the brain m several ways (see the Kynurenine pathway above) from tryptophan, as macrophages and in liver, oi from kynurenine or 3 -hydroxykynurenine which, having been formed peripherally, cross the blood-brain barrier to undergo final conversion to QUIN by brain kyn- -OHase , kynuren ase and 3 hydroxyanthranilate 3 , 4 -dioxygenase (in contrast peripherally formed 3 -hydroxyanthramlic acid enters the brain very poorly) The optimal target for the design of inhibitors should be the rate limiting step through the pathway Enzymatic studies m vi tro on all the currently known enzymes along the t o branches of the pathway have revealed that kyn-3-OHase, kynurenmase and qu olmate phosphorybosiltransferase (see the kynurenine pathway) could contribute to determine the cerebral QUIN levels, though it is still unclear which of these enzymes is most effective in the normal bram and under pathological conditions Studies on QUIN level, after administration to mice of different precursors (Erickson J B., Flanagan E M , Chang S Y , Salter M and Remhard J F Jr 1992. Soc Neurosci Abst 18 442 ) showed that bram and serum QUIN arise from qualitatively similar pathways However, bra QUIN appears to be controlled heavily at the kyn-3 OHase step As a consequence of these studies kyn 3- OHase can be considered an important enzyme target foi inhibition of QUIN biosynthesis Furthermore, being the first enzyme in the route of production of QUIN, the inhibition of kyn 3-OHase could lead to an accumulation of KYNA, the neuroprotectant metabolite of the pathway KYNA is an effective excitatory ammo acid receptor antagonist with a particularly high affinity to the glycme modulatory site (J Neurochem , 52, 1319-1328, 1989) As a naturally occurring brain metabolite (J Neurochem , 51, 177 180, 1988 and Bram Res , 454, 164-169, 1988), KYNA probably serves as a negative endogenous modulator of cerebral glutamatergic function (Ann N Y Acad Sci , 11, 290-296,1990); while applied directly into the bra , it exhibits anticonvulsant and neuroprotective properties (Neurosci Lett 48 273-278 1984) In confirmation of all these data kyn-3-OHase inhibitors have recently been successfully applied to demonstrate for the first time, in the rat bram m vivo, the functional interdependence of the two branches of the kynurenine pathway by shifting cerebral metabolism towards an enhanced production of KYNA (Neuroscience 61 237-244 1994, Soc Neurosci Abstr. 21, 436 3 1995) In particular, systemic administration of the new and potent kyn-3-OHase inhibitor (R, S) -3 , 4 -dichlorobenzoylalanine (FCE 28833) (see also Example 3b) causes a dose-dependent elevation in endogenous kynurenine and KYNA levels in rat brain tissue (Speciale et al Eur J of Pharmacology, vol 315, p.263-267, 1996) These chemicals clearly hold great promise as research tools and may also harbor therapeutic potential since a decrease in brain QUIN and a concomitant increase in brain KYNA could be clinically desirable . Moreover variants of the kyn-3-OHase enzyme could be present in different tissues and organs, and may constitute a possible target to develop more specific drugs. In this perspective it is fundamental to clone the gene encoding for kyn-3-OHase, so that the protein can be studied from a molecular point of view and the recombinant enzyme can be obtained in reasonable quantity for further studies. The purification of the enzyme and at least the partial sequence of the protein are the first steps to design degenerate oligonucleotides and to proceed with the cloning of the gene in a conventional hybridization way. In 1975 Nisimoto et al . described the isolation of kyn- 3 -OHase from the mitochondrial outer membrane of rat liver in a 5 steps purification procedure (Nisimoto Y., Takeuchi F. and Shibata Y. 1975. J. Biochem. 78: 573-581). They described the isolation of a single homogeneus protein with a molecular weight of 200,000 Da and an isoelectric point of pH 5.4. The purified enzyme had a specific activity of 140 nmol min g and an overall yield of 0.04%. In 1979, the same authors reported a 3 steps purification procedure with a better overall yield but a lower specific activity ( Nisimoto et al . J. of Chro atography 169: 357-364. 1979) . More recently any attempts to reproduce these results, also from our biochemistry laboratories, have been unsuccessful; this is probably due to an instability of the enzyme during purification that do not allow a further analysis of its amino acidic sequence, thus precluding the possibility to adopt a conventional cloning approach. Therefore, there is the need to set up an alternative cloning procedure and the invention aims to achieve this goal. According to the invention the problem is solved through the functional expression cloning Xenopus laevi s oocytes; this method is particularly useful to overcome the inconveniences deriving from the isolation of the protein by biochemical methods The functional expression cloning was first pioneered by No a et al (Noma Y , Sidcras P , Naito T , Bergstedt - Lmdquist S , Azuma C , Severmson E , lanabe T , K ashui T , Matsuda 1- , Yaoita Y and Honχo T 1986 Nature 319 640-646 ) and has been rapidly applied to the cloning of several plasma membrane proteins The strategy of functional expression cloning can be summarised as follow func ional expression, obtained with total mRNA from a given source, confirms the presence of mRNA coding for the piotein of interest The mRNA can then be fractionated according to size, the active fraction identified, and, aftei reverse transcription, used foi the generation of a cDNA library This library is then transcribed into polyadenylated cRNA, capped and expressed in the Xenopus oocytes Subsequent appearance of the function in the oocytes confirms the presence of a full length clone (or at least a consistent functional part of it) in the library Functional expression cloning has the advantage over classical strategies in that the danger of sequencing "false positive" clones, due to cross reactivity of antibodies or nucleotide probes, can be avoided Furthermore, most of the times full length clones will directly be obtained (Sigel E 1990 J Membrane Biol 117 201-221) Once cloned, the kyn- 3 -OHase cDNA obtained from a given source can be used to prepare probes for the screening of libraries derived from a different organisms I n this way, the expert in the art will recognize that, on the basis of the information provided herein, enzymes homologous to the ones specifically disclosed herein can be readily identified. SUMMARY OF THE INVENTION The present invention discloses the functional expression-cloning of rat kynurenine -hydroxylase and the molecula cloning of human kynurenine- 3 -hydroxylase . In a first aspect, the present invention provides an isolated single or double stranded polynuclcot ide , typically DNA, having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of: (a) the sense sequence of SEQ ID NO. 1 from about nucleotide position 45 to about nucleotide position 1481; the sense sequence of SEQ ID NO. 3 from about nucleotide position 34 to about nucleotide position 1494; (b) sequences complementary to the sequence of (a); (c) sequences that, on expression, encode polypeptides encoded by the sequence of (a); (d) analogous sequences that hybridize under stringent conditions to the sequences of (a) and (b) ; and (e) sequences homologous to the sequences of (a) that, on expression, encode polypeptides having kynurenine- 3-hydroxylase activity. A preferred embodiment is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. In another aspect, a DNA molecule of the present invention is contained in an expression vector. The expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide. In a preferred embodiment, the DNA molecule in the vector is the sequence of SEQ ID NO. 1 from about nucleotide position 45 to about nucleotide position 1481. In another preferred embodiment the DNA molecule in the vector is the sequence of SEQ ID NO. 3 from about nucleotide position 34 to about nucleotide position 1494. r lhe present invention still further provides for a host cell transformed with an expression vector of this invention. The host may be a prokaryotic or a eukaryotic cell. Example of a preferred prokaryotic host cell is E . coli , whereas, among the eukaryotic cells, preferred hosts are yeast, insect or mammalian cells Xenopus laevis oocytes transiently expressing the kyn-3-OHase enzyme are also part of the present invention In a still further aspect the invention provides an isolated and purified recombinant kyn-3 OHase which is coded foi by a nucleotide sequence selected from the group consisting of (a) the sense sequence of SEQ ID NO 1 from about nucleotide position 4b to about nucleotide position 1481, the sense sequence of SEQ ID NO ϊ from about nucleotide position 34 to about nucleotide position 1494, (b) a sequence complementary to the sequence of (a), (c) a sequence that, on expression, encodes a polypeptide encoded by the sequence of (a), (d) analogous sequences that hybridize under stringent conditions to the sequences of (a) and lb) , and (e) sequences homologous to the sequences of (a) that on expression, encode polypeptides having kynurenme-3 hydroxylase activity Furthermore, the invention provides a recombinant process for the transient expression of kyn-3-OHase, which process comprises extracting total mRNA from the tissues of a mammalian organism generating a cDNA expression library by reverse transcription of the said mRNA, obtaining RNA m vitro from the said cDNA library, injecting the RNA into Xenopus oocytes, measuring the enzymatic activity, dividing the said library into several pools, selecting the posi lve clone, linearizing the plasmid DNA extracted from the positive clone, synthesizing the cRNA from said DNA injecting the said cRNA into Xenopus laevi s oocytes thus obtaining expression of kyn-3-OHase enzyme BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Protein alignment between the deduced amino acid sequence of K30H clone and the deduced amino acid sequence of YBJ8_YEAST. Fig. 2. Northern blot hybridization using K30H cDNA as probe. Lane 1, heart; 2, brain; 3, spleen; 4, lung,- 5, livei ; 6, skeletal muscle; 7, kidney; 8, testis mRNA species of 2 kb from rat liver, kidney and testis strongly hybridize with the K30H probe; the signal obtained with testis is much less intense In all the three tissues a 4.3 kb transcript which hybridize with K30H probe is also present but the signal is less intense. Fig 3 In vitro translation of K30H cRNA. Lane 1, K30H clone; 2, negative control. Two translation products are present the upper band (-55,000 kDa) corresponds to a protein with the predicted molecular weight, the lower (-30,000 kDa) could be a partial translation product, due to a second ATG codon in frame, partially recognised as starting codon. Fig 4. Nucleotide alignment between the common portion of the sequences of the two human K30H clones obtained by screening of a human liver cDNA library. Fig 5. Protein alignment between the deduced amino acid sequences of rat and human K30H clones. DETAILED DESCRIPTION OF THE INVENTION The present invention provides isolated and purified polynucleotides that encode the enzyme kynurenine- 3 hydroxylase ( kyn- -OHase ) , vectors containing these polynucleotides, host cells transformed with these vectors, a process of making the kyn-3-OHase using the above polynucleotides and vectors, and isolated and purified recombinant kyn 3-OHase. I I For the purposes of the present invention as disclosed and claimed herein, the following is to be considered. The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence . The nucleotide sequences are presented by single strand only, in the 5' to 3' direction, from left, to right. Nucleotidcs and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letters code. In one aspect, the present invention provides isolated and purified polynucleotides that encode the enzyme kyn 3 -OHase from rat and human. A polynucleotide of the present invention is an isolated single or double stranded polynucleotide having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of: (a) the sense sequence of SEQ ID NO .1 from about nucleotide position 45 to about nucleotide position 1481; the sense sequence of SEQ ID NO. 3 from about nucleotide position 34 to about nucleotide position 1494; (b) sequences complementary to the sequences of (a) ,- (c) a sequence that, on expression, encodes a polypep ide encoded by the sequences of (a) ; (d) analogous sequences that hybridize under stringent conditions to the sequences of (a) and (b) ; and (e) sequences homologous to the sequences of (a) that, on expression, encode polypeptides having kynurenιne-3 -hydroxylase activity . A preferred polynucleotide is a DNA molecule. In another embodiment, the polynucleotide is an RNA molecule. The nucleotide sequence and deduced amino acid sequence of the rat kyn- 3 -OHase gene is set forth in SEQ ID NO. 1. The nucleotide sequences of human kyn-3 -OHase clones are set forth in SEQ ID NO. 3 and SEQ ID NO. 5 The nucleotide sequences of SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO 5 represent full length DNA clones of the sense strand of kyn- 3 -OHase genes and are intended to represent both the sense strand (shown on top) and its complementary strand The present invent ion also contemplates analogous DNA sequences which hybridize under stringent conditions to the DNA sequences set forth above Stringent hybridiza ion conditions are well known in the art and define a degree of sequence identity greater than about 80% preferably greater than 90% The term "analogous" refers to those nucleotide sequences that encode analogous polypeptides, analogous polypeptides being those which have only conservative differences and which retain the conventional characteristics and activities of kyn 3 OHase The present invention also contemplates naturally occur ling al elic variations and mutations of the DNA sequences set forth above so long as those variations and mutations code, on expi ession, for a kyn- 3 -OHase enzyme Moreover, the invention contemplates homologous DNA sequences that, on expression, encode polypeptides having kynurenine 3 hydroxylase activity As used herein, "homologous DNA sequences" are those sequences characterized by an ho ology, at the nucleotide level, greater than about 50%, preferably greater than about 60%, more preserably greater than about 70%, at least one functional domain of the encoded polypeptide, with respect to the corresponding region of the sequences disclosed in the present invention. Homologous DNA sequences include those sequences coding for isoforms of the kynurenine 3 hydroxylase enzyme encoded by the the DNA sequences set fort h above Such isoforms can be expressed m different tissues of the same organism as a result, e.g , of alternative splicing or because coded for by other genes. Homologous DNA sequences are also those sequences coding for a kyn-3-OHase of any species of origin. Preferably the DNA sequences code for enzymes of mammalian origin, more preferably the DNA sequences code for the human kyn- 3 -OHase enzyme As is well known the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptide as that encoded by the aforementioned kyn- -OHase genes. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode the polypeptides of SEQ ID NO 2, 4 and G Having identified the ammo acid residue sequence encoded by a kyn- -OHase gene, and with knowledge of all triplet codons foi each particular ammo acid residue, it is possible to describe all such encoding RNA and DNA sequences DNA and RNA molecules other than those specifically disclosed herein characterized simply by a change in a codon for a particular ammo acid, are within the scope of this invention. A table of ammo acids and their representative abbreviations, symbols and codons is set forth below in the following Table As is well known in the art, codons constitute triplet sequences of nucletotides in mRNA molecules and, as such, are characterized by the base uracil (U m place of base thymrdme (T) (which is present in DNA molecules) A simple change in a codon for the same ammo acid residue within a polynucleotide will not change the structure of the encoded polypept ide The kyn- 3 -OHase enzymes of the present invention include proteins homologous to, and having essentially the same biological properties as, the proteins coded tor by the nucleotide sequence herein disclosed. This definition is intended to encompass isoforms and natural allelic variants of kyn-3 -OHase sequences. With the knowledge of the sequence information disclosed in the present invention, the expert m the art can identify and obtain DNA sequences which encode the kyn- 3 -OHase enzyme from different sources (i e different tissues or different organisms) through a variety of means well known to him and disclosed by, for example, Maniatis et al , Molecular cloning a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) For example DNA which encodes the kyn-3 -OHase enzyme may be obtained by screening of mRNA, cDNA or genomic DNA with oligonucleotide probes generated from the kyn- 3 -OHase enzyme gene sequences information provided herein Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemilummescent group m accordance with known procedures and used in conventional hybridization assays, as described by, for example, Maniatis et al Moleculai cloning a laboratory manual, Second Edition, Cold Spring Harboi Press, Cold Spring Harboi, NY (1989) Kyn-3-OHase gene sequence may alternatively be recovered by use of the polymei ase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the kyn 3 OHase enzyme sequence provide herein See U S Pat Nos 4,683,195 to Mullis et al and U S Pat No 4,683,202 to Mullis The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particulai sample The method can be used to amplify eithei single- or double-stranded DNA The essence of the method involves the use of two oligonucleotides probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule The recombinant DNA molecules of the present invention can be produced through any of a variety of means well known to the experts in the art and disclosed by, tor example, Maniatis et al Molecular cloning. a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) Example 5 describes one of the possible procedures which can be followed for this purpose In order to replicate the kyn-3-OHase enzyme DNA sequence, this must be cloned in an appropriate vector A vector is a replicable DNA construct Vectors are used herein either to amplify DNA encoding the kyn-3 OHase enzyme and/or to express DNA which encodes the kyn-3-OHase enzyme An expression vector is a replicable DNA construct in which a DNA sequence encoding kyn-3-OHase enzyme is operably linked to suitable control sequences capable of effecting the expression of the kyn 3-OHase enzyme in a suitable host DNA regions are operably linked when they are functionally related to each other For example a promoter is operably linked to a coding sequence it it controls the transcription of the sequence Amplification vectors do not require expression contiol domains All that is needed is the ability to replicate m a host usually conferred by an origin of replication, and a selection gene to facilitate recognition of transf ormant s DNA sequences encoding kyn- -OHase enzyme may be recomb ed with vector DNA in accordance with conventional techniques including blunt-ended or staggered ended termini for ligation restriction enzyme digestion to provide appropi late termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesiderable joining, and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Maniatis et al Molecular cloning a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Haibor NY (1989) and are well known in the art Expression of the cloned sequence occurs when the expression vector is introduced into an appropriate host cell If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences, foi example E coli Similarly, if an eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence A yeast host may be employed, foi example S cerevis ae Alternatively, insect cells may be used, in which case a baculovnus vector system may be appropriate Another alternative host is a mammalian cell line, for example COS-1 cells The need for control sequences into the expression vector will vary depending upon the host selected and the transformation method chosen Generally, control sequences include a transcπptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, and sequences which control the termination of transcription and translation Vectors useful for practicing the present invention include plasmids, viruses (including phages), retroviruses , and mtegrable DNA fragments (i e fragments mtegrable into the host genome by homologous recombination) Tne vectors replicate and function independently of the host genome, or may, some instances, integrate into the genome itself Expression vectors should contain a promoter which is recognized by the host organism The promoter sequences of the piesent invention may be either prokaryotic, eukaryotic or vnal Example of suitable prokaryotic sequences include the P R and P L promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A D , Ed , Cold Spring Harbor Press, Cold Spring Harbor, NY (1973), Lambda II, Hendrix, R W , Ed , Cold Spring Harbor Press, Cold Spring Harbor, NY (1980) ), the trp, recA, heat shock, and lac/, promoters of E Coli and the SV40 early promoter (Benoist, C et al nature 290 304-310 (1981)) As far as the Shine Dalgarno sequence is concerned, preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno of the repl icase gene of the phage MS 2 and of the gene cll of bacteriophage lambda The Shme-Dalgarno sequence may be directly followed by the DNA encoding kyn-3 -OHase and result m the expression of the mature kyn-3-OHase protein Alternatively, the DNA encoding kyn-3-OHase may be preceded by a DNA sequence encoding a carrier peptide sequence In this case, a fusion protein is produced in which the N-termmus of kyn-3-OHase is fused to a carrier peptide, which may help to increase the protein expression levels and in racellular stability, and provide simple means of purification A preferred carrier peptide includes one or more of the IgG binding domains of protein A are easily purified to homogeneity by affinity chromatography e g on IgG-coupled Sepharose Alternatively, many vectors have the advantage of carrying a stretch of histidme residues that can be expressed at the N terminal or C-termmal end of the target protein Thus the protein of interest can be recovered by metal chelation chromatography (see the method described heiein below undei Example 5) A DNA sequence encoding a recognition site for a proteolytic enzyme such as enterokmase , factor X, pr ocollagenase or thrombine may immediately precede the sequence foi kyn-3 OHase to permit cleavage of the fusion protein to " • > obtain the mature kyn 3-OHase protein Moreover, a surtable expression vectoi includes an appropriate maiker which allows the screening of the transformed host cells The transformation of the selected host is carried out using any one of the various techniques well known to the expeit in the art 0 and described in Maniatis et al Molecular cloning- a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) One further embodiment of the invention is a prokaryotic host ce l transformed with the said expression vector and able to 5 produce, under appropriate culture conditions, the kyn 3 -OHase of the invention A peculiai example of expression m eukaryotic cells is represented by Xenopus laevi s oocytes This system has also been used as a strategy to clone the DNA sequence of the present 0 invention In this case the vector, which is not used to transform the cell, is linearized with an appropriate restriction enzyme and used for in vitro synthesis of cRNA, including capping, using RNA polymerase Techniques for such manipulation are well known the art, see for example Maniatis et al Molecular cloning a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) The cRNA is then injected into the oocytes according to the procedure described herein under Example 1 Ihese cells are capable of transiently expressing the functional enzyme and can be used to study the characteristics of variants of kyn- 3 -OHase and to test new compounds Moreover, cultures of cells derived from mul ticellulai organisms are a desiderable host fo recombinant kyn 3-OHase synthesis In principal, any eukaryotic cell culture is workable, whether from vertebrate or invertebrate culture, including insect and mammalian cells Propagation of such cells in cell culture has become a routine procedure See Tissue Culture, Academic Press Kruse and Patterson, eds (1973) Examples of useful host cell lines are HeLa cells, CHO and COS cell lines The transcript ronal and translational control sequences in expression vectors to be used in transforming vertebrate and invertebrate cells ai e often provided by viral sources, for example, commonly used promoters are derived from Adenovirus 2, polyo a and SV40 See, e g U S Pat No 4,599, 308 An origin of replication may be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vectoi is integrated into the host cell chromosome, the latter may be sufficient Rather than using vectors which contains viral origins of replication, one can transform mammalian cells by the method of cotransformation with a selectable marker and kyn-3 -OHase DNA An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidme kinase See U S Pat No 4,399,216 Cloned genes and vectors of the present invention are useful to transform cells which do not ord arly express kyn-3-OHase to thereafter express this enzyme. Such cells are useful as intermediates for making recombinant kyn-3-OHase preparations useful for drug screening. Furthermore, structural data deriving from the analysis of the deduced amino acid sequences of the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency. Sequence comparison of the two clones obtained by screening of the human liver cDNA library (SEQ ID NO. 3 and SEQ ID NO. 5) revealed 100% nucleotide homology in the coding region (Fig. ) . Notably, the cloned sequence in SEQ D NO. 5 shows a huge 3' untraslated region that could contain regulatory elements and need further investigations. Comparison between the deduced amino acid sequences of the rat Kyn30Hase (SEQ ID NO . 2) and the human Kyn30Hase (SEQ ID NOs . 2 and 6) showed 73% of identity (Fig. 5) . Comparison of the DNA sequences of the present invention with the sequences present in all the available data bases showed a significant homology with a portion of the Saccharo yces cerevisiae genomic DNA (YBJ8_YEAST) . This is a region containing a putative open reading frame of 1380 nucleotides corresponding to a hypothetical protein whose identity and function are so tar- unknown. Fig. 1 shows the protein alignment between rat Kyn30Hase and YBJ8_YEAST, the two deduced amino acid sequences show 35% of homology. Rat Kyn30Hase and therefore human Kyn30Hase are also homologous to p-hydroxybenzoate hydroxylase, a flavoprotein whose crystal structure has been resolved (Schreuder, H. A., et al . 1988, J. Mol . Biol . 199, 637-648). The homology between these proteins resulted to be highly significant in the regions containing the putative binding sites for FAD and NADP . Computer modelling could be therefore used to develop a putative tertiary structure of Kyn- 3 -OHase based on the available information on p hydroxybenzoate hydroxylase. This approach can help designing novel enzyme inhibitor based on the predicted structure of Kyn- 3- OHase. Variants of the kyn- 3 -OHase enzymes of the present invention (obtained as described above) could be present in different tissues and/or organs, and might represent potential new pharmacological targets to develop more specific drugs. Cloned genes of the present invention , and oligonucleotides derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated wi h certain disorders. Oligonucleotides derived from the kyn- 3 -OHase DNA sequences of the present invention are useful as diagnostic tools for probing kyn- 3 -OHase gene expression in various tissues. For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto. The present invention is explained in greater detail in the following examples. These examples are intended to be illustrative of the present invention, and should not be constructed as limiting thereof . EXAMPLE 1 Expression-cloning in Xenopus laeviβ oocytes a) Preparation of Xenopus laevis oocytes Ovarian tissue was dissected from adult female Xenopus laevis : a small incision was made in the skin and body wall in the posterior ventral side of the animal . The ovary consists of several lobes that were snipped off using scissors and placed in colture medium (ND solution: 5 mM hepes-NaOH, pH7.6 , 96 mM NaCl, 2 mM KC1, 1 mM MgCl 2 ) . The incision in the body wall was then sutured separately from the skm, which subsequently was sutured as well, and the animal was left to recover m shallow water (L D Smith et al 1991 Methods in Cell Biol 36 45-60) Each oocyte is closely surrounded by about 5,000 follicle cells, which greatly affect the composition and synthesis of ovarian material unless removed Individual oocytes of stages were obtained by gentle agitation in ND solution plus 1 2mg/ml collagenase for 60 90 min 'Ihey were then maintained at 18° C m ND solution enriched w th 1 8 mM CaCl 3 and 20 mg/1 gentamycm Oocytes of stages 5 and 6 were injected 24 hours later with 50 nl RNA or watei (N Dascal and I Lotan 1992 Methods in Mol Biol 13 205 225) b) mRNA isolation and injection The injected mRNA was poly (A) + RNA isolated from lat heart and ki dney Rat kidneys and hearts were removed and frozen in liquid nitrogen Total RNA was isolated with a guamdine thiocyanate extraction followed by CsCl 2 gradient centrifugation (Maniatis et al Molecular cloning a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harboi, NY (1989) mRNA was isolated by oligo dT chromatography (Pharmacia Biotech, Uppsala, Sweeden) Oocytes were injected with a Drummond Nanoject microdispenser To avoid accidental injection into the nucleus, the needle was inserted close to the equator or in the vegetal (light) half of the oocytes 1 3 days after injection the oocytes were assayed tor kyn 3 OHase c) Rat Kyn-3 -OHase activity expressed in X. laevis oocytes: biochemical assay. Kyn 3 -OHase activity was quantified by the conversion of L-kyn to 3 -OH Kyn Oocytes were sonicated m ice-cold 0 14 M KC1/20 mM potassium phosphate buffer (pH 7.0) to obtain a final protein concentration of 5-10 mg/ml The reaction mixture consisted of 100 ml of oocytes homogenates and 100 ml of 100 mM potassium phosphate buffer containing 4 mM MgCl 2 , 0 8 mM NADPH and different concentrations of L-kyn Aftei lh incubation at 37°C the reaction was terminated by adding 200 ml of 1M percloric acid (Saito K et al , J Neurochem 1993 60 180) 3 OH-Kyn was quantified by HPLC coupled to a coulometric detector ( + 0 2V) , (Heyes M P and Querry B J J Chrom 1994 428 340) d) Construction of a rat kidney cDNA expression library A directional cDNA library was constructed by using s ze selected rat kidney poly(A) * RNA that had been shown to induce expression of kyn-3-OHase activity in the oocytes The cDNA library was constructed by using a commercial kit (SupeiScrrpt plasmid system, pSPORIl vector, GIBCO/BRL) and precisely following the suppliers instructions The library contained about 5x10 colonies, initial pools for screening contained about 2000 colonies e) Library screening Plasmid DNA was isolated by standard procedures (alkaline ly i followed by a purification on Qiagen lesin, Kontron Zurich) Plasmids were linearized with Wot I and used for m vi tro synthesis and capping of cRNA using T7 RNA polymerase (Stratagene) and following the supplier's instructions Initially the library was divided into 25 pools of -2000 colonies, cRNA obtained transcribing the insert of each pool of colonies was injected in the oocytes and screened for the expressed kyn 3 OHase activity (See Example lc foi the methods) The positive pool identified for its kyn-3-OHase expressed activity (175 pmoles/h/mg protein- about 30 fold higher than that induced by the injection of total kidney poly (A) + RNA) was then split into several sub-pools and assayed with the same procedui e Following the same criterion, in the last lound of screening we had a single positive clone showing an activity of 3/47 pmoles/h/mg protein (about 400 fold higher than that induced by injection of total rat kidney poly (A) * RNA) EXA PLF 2 Characterization of the cDNA encoding rat kyn-3 -OHase a) DNA sequencing Both strands of the cDNA were sequenced Sequencing was earned out with universal and forward piimers and subsequently with a series of synthetic oligonucleotide primers ar-corαing to the dideoxy chain termination method (F Sangei et al '^roc Nat l Acad Sci USA 1977 74 5461 5467) using Sequenase (United States Biochemioals Corp , Cleveland OH) The sequenced cDNA, depicted in SEQ ID NO 1 , encodes a deduced protein of 478 ammo acid residues (the predicted ammo acid sequence is shown in SEQ ID NO 2) Using the protein algorithm described by Gavel and Von Heijne (Protein Engineering, 4, 33 3 1990) a potential mitochondr lal signal peptide is predicted in position 1 to 31 of the deduced protein b) Northern analysis A commercial premade Northern blot of poly (A)' RNA from different rat tissues was used (CLONTECH laboratories, USA) A full length cDNA probe of the clone was labeled by random priming (Amersha ) using [a 32 P] dCTP (DuPont/NEN) Blot was p ehybridized and hybridized in a buffer containing 50% (vol/vol) forma ide 5X standard saline citrate (SSC 750 mM NaCl , 75 mM sodium citrate) , IX Denhardt ' s solution, salmon sperm at 0 25 mg/ml , 1% SDS overnight at 42°C After hybridization, blot was washed three times in 2X SSC/0 1% SDS at 42°C (Fig. 2) c) In vi tro translation In vi tro translation of the rat Kyn-3-OHase cRNA was performed with a rabbit reticulocyte lysate system (Promega) following the supplier's instructions. Radiolabeled proteins synthesized in vitro using [3, 4, 5- H] L- leucine were resolved by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography (Fig. 5) (Maniatis et al . 1989. Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY) EXAMPLE 3 Biochemical characterization of rat kyn-3 -OHase expressed in Xenopus 1 . oocytes . Xenopus 1 . oocytes were first injected with 25 ng of poly (A) + RNA isolated from rat kidney. Three days after the injection, oocytes homogenate showed the production of 6.3 pmoles/h/mg protein of 3- OH-kyn after 1 hour incubation at 37°C in the presence of ImM L- kyn. The production of 3-OH-kyn in water- injected oocytes and in oocytes injected with poly (A) + RNA isolated from rat heart, a tissue in which the activity of the enzyme is known to be very low (E. Okuno and R. Kido "Kynurenine and Serotonin pathways" Ed. by Schwarcz et al . , Plenum Press, New York, 1991), was at the detection limit of the method and was similar to that observed in non- injected oocytes. a) cRNA dose-response in Xenopus 1 . oocytes Increasing doses (range 0.1-25 ng) of cRNA, obtained from the positive clone, were injected into oocytes which were then tested for kyn-3-OHase activity in the presence of lOO M of L- kyn . A linear dose-dependent effect was observed up to 5 ng cRNA/oocyte . At this dose the kyn-3 -OHase activity was 35.7 nmoles/h/mg protein. No further increase of the activity was measured when higher cRNA doses were injected b) Effect of a selective kyn-3 -OHase inhibitor FCE 28833A on the expressed enzyme activity. An inhibition experiment was performed using FCE 28833A, a well known Kyn-3-OHase mhibitoi (Molmaπ, A , M Cmi , M Marconi, H -Q Wu, R Schwarcz, A Bonsignori, R.A McArthur, M Varasi and C Speciale, 1995, Soc Neurosci Abstr 2], 436 3) Oocytes injected with the kynOH clone weie homogenized and incubated in the presence of 50 μM L kyn and different concentra ions of FCE 28833A In this experimental condition me IC 50 of the inhibitor was 0 29 μM , this is m agreement with the previous results obtained in rat brain mithocondria (Molinari, A , M. dm , M Marconi, H.-Q Wu, R Schwarcz, A Bonsignori, R A. McArthur, M Varasi and C Speciale, 1995, Soc Neurosci Abstr 21, 436.3) EXAMPLE 4 Molecular cloning of human Kyn-3 -OHase a) cDNA library screening About 1,000,000 recombinant phages of a λ ZapII human liver cDNA library (Stratagene) were plated on a lawn of E . col i XL1 Blue cells After an overnight growth at 37°C the recombinant phages were transferred in duplicate on nitrocellulose filters, their DNA was denatured, neutralized and baked under vacuum at 80°C for 2h. Prehybridization and hybridization were carried out under stringent conditions (6xSSC, 5xDenhardt, 0,1%SDS and Salmon sperm lOOμg/ml at 65°C) . The probe used was the Mlul-Hmdlll 668 bp fragment obtained from the rat Kyn-3 OHase cDNA (K30H) . The probe was gel-purified by Qiaex (Qiagen) and labeled with 3 P by Multipri e DNA labeling system (Amersham) The filters were washed at 65°C three times in 2xSSC, 0.1% SDS and once IxSSC, 0.05% SDS. Filters were exposed to Kodak X-AR film (Eastman Kodak Company, Rochester, N.Y., USA) with intensifying screen at -80°C. Positive phage plaques were isolated and screened again twice order to isolate single clones. b) Recombinant phage DNA extraction and Sequencing methods E . col i XL1 Blue cells were coinfected with about 10 phage particles corresponding to the positive clone selected and 1 μl of EX Assist helper phage (10 pfu/μl ; Stratagene) The mixture was incubated at 37° C for 15 mm and later incubated with 3 ml of LB medium for 3h Cells were spmned down and the supernatant was heated at 70°C for 15 mm. SORL cells at OD 600 -1 were mixed with the supernatant containing the phagemid pBluescript, incubated for 15 mm at 37°C and plated on LB-ampicillme (50 μg/ml) plates Single clones were incubated overnight in LB- ampicillme and DNA was extracted according to the instructions of Qiagen Plasmid Maxi Protocol Both strands of the cDNA were sequenced. Sequencing was carried out with universal and forward primers and subsequently with a series of synthetic oligonucleotide primers according to the dideoxy chain termination method (F. Sanger et al : Proc . Na l. Acad . Sci . USA 1977 74 5463-5467) using Sequenase (United States Biochemicals Corp , Cleveland, OH) . Eight positive clones were isolated and characterized by restriction analysis, to obtain the size of insert. All the positive clones were then sequenced on both strands seven clones had an insert of 1628 bp, depicted in SEQ ID NO. 3, while one had a bigger insert, of 5000 bp depicted SEQ ID NO 5 All the eight clones contained an identical (100% nucleotide level) open reading frame of 1458 bp encoding a deduced protein of 486 amino acid residues (the predicted amino acid sequence is shown in SEQ ID NO. 4 and 6) . The biggest clone had a huge 3' untraslated region (see Fig. 4) . Sequence comparison between the deduced amino acid sequences of the human K30H (SEQ ID NO. 4 and SEQ ID NO . 6) and the rat Kyn30Hase (SEQ ID NO. 2) showed 73% of identity (Fig. 5) . Also in this case, as for the rat protein, using the protein algorithm described by Gavel and Von Heijne (Protein Engineering, 4, 33-37, 1990) a potential mitochondria] signal peptide is predicted in position 1 to 31 of the deduced protein. EXAMPLE 5 Inducible expression in Escherichia col i a) Construction of the vector The expression plasmid encoding human Kyn30Hase was constructed as follows: a) PCR amplification was used to isolate the translated portion of the human Kyn30Hase cDNA sequence, to add the convenient restriction enzyme sites and to place the coding region in the proper reading frame. Two specific oligonucleotides were designed: the sense orientation oligonucleotide (5'- CAGGAATTCCATATGGACTCATCTGTC-3' ) (SEQ ID NO. 7) contains the Nde I restriction site that reconstitutes the ATG codon and therefore place the cDNA in frame; the antisense-orientation oligonucleotide ( ' -CGGGATCCCGCTATCACCTGCTAATGA- 3 ' ) (SEQ ID NO. 8) contains the BamH I restriction site and complements the sequence from the end of the coding region. In both the oligonucleotides the restriction site is flanked by a "spacer" at the 5' end to allow for efficient digestion. b) The Nde I/BamH I digested fragment, after being controlled by sequencing, was ligated into the vector pET-lla (Novagen, USA) cut with the same restriction enzymes and dephosphorylated . c) Initial cloning was done in DH5α strain to allow the examination of the construct sequence by restriction analysis. b) Transformation and induction of BL21 (DE3) pLysS strain After positive clones were identified, the plasmid was isolated (Easy-pure plasmid preps, Primm Labs, Italy) and used for transformation into BL21(DE3) pLysS according to the instructions of Novagen, USA. After the target plasmid was established in the expression host, expression of the human K30H DNA was induced by addition of IPTG to a growing culture . Following an induction protocol suggested by Novagen, a single colony or a few μl from a glycerol stock were grown into 2 ml LB medium containing the appropriate antibiotic at 37°C until the OD 60o reached 0.6-1. The culture was stored at 4°C overnight. The cells were then collected by centrifugation, resuspended in 2 ml fresh medium + antibiotics and used to inoculate 100 ml medium + antibiotics . The culture was incubated with shaking at 37°C until 0D 600 reached 0.4-1. Samples for the uninduced control were removed and IPTG was added to a final concentration of 0.1-1 mM . Induction time was 2-5 hours. Some induction experiments were performed at 30°C overnight. The cells were harvested by centrifugation at 3000 rpm for 10 min at 4°C, resuspended in 0.25 culture volume of cold 50 mM Tris-HCl pH 8.0, 2 mM EDTA and centrifuged as above. After removal of the supernatant, the cells were stored as frozen pellets at -70°C. c) Recombinant Kyn-3-OHase activity: biochemical assay. Kyn-3-OHase activity was quantified by the conversion of L-kyn to 3-OH-Kyn. E. coli pellets were resuspended in ice-cold 0.14 M KC1/20 mM potassium phosphate buffer (pH 7.0) to obtain a final protein concentration of 1-3 mg/ml and sonicated. The reaction mixture consisted of 100 μl of cells ho ogenates and 100 ml of 100 mM potassium phosphate buffer (pH 7.5) containing 4 mM MgCl 2 , 0.8 mM NADPH and increasing concentrations of L-kyn. After lh incubation at 37°C the reaction was terminated by adding 200 ml of 1M percloπc acid (Saito K. et al . , J Neurochem 1993 60.180) . 3-OH-Kyn was quantified by HPLC coupled to a coulometric detector ( + 0.2V), (Heyes M. P. and Querry B J J Chrom 1994 428 340) . EXAMPLE 6 Biochemical characterization of human recombinant kyn-3 -OHase expressed in E. coli Recombinant kyn-3-OHaεe activity measured in cells grown at 37°C until the OD 600 reached 0.6, induced for 2 hours at 37°C adding I mM IPTG was about 10 nmol/h mg protein, enzymatic activity of unmduced cells in the same conditions was less than 5% (homogenates incubated for 1 hour at 37°C with 500 μM L-kyn,- see Example 5c) . Not transformed cells did not show any kyn-3 -OHase activity . a) Kinetic analysis of human recombinant kyn-3 -OHase To further characterize human recombinant K30H activity, a kinetic study was performed by incubating E . col i extracts with increasing concentrations of L-kynurenine , ranging from 1 to 4,000 μM (Induction conditions: OD 600 =0.6, [iPTG]=imM, 2h at 37°c) . The kinetic parameters: K-, and V app (apparent maximal rate) measured in these conditions and calculated using the fitting curve analysis of Sigma Plot programme were 57 ± 2 μM and 10.5 ± 0.11 nmol/hour.mg of protein, respectively. The Km value is in the same order of magnitude of that measured in rat liver homogenate . b) Effect of a selective kyn-3-OHase inhibitor FCE 28833A on the recombinant enzyme activity. An inhibition experiment was performed using FCE 28833A, a well known Kyn-3-OHase inhibitor (Speciale C, Wu H.-Q., Cini M., Marconi M., Varasi M. and Schwarcz R. 1996, Eur . J. of Pharmacol. 315: 263-267) . E . col i extracts were incubated in the presence of 50 μM L-kyn and different concentrations of FCE 28833A. In this experimental condition the IC 50 of the inhibitor was 0.2 ± 0.06 μM , this is in agreement with the previous results obtained in rat brain mitochondria (Speciale C, Wu H.-Q., Cini M., Marconi M., Varasi M. and Schwarcz R. 1996, Eur. J. of Pharmacol. 315: 263-267). EXAMPLE 7 Recombinant expression of a fusion K30H protein with a cleavable His. Tag sequence for rapid affinity purification The plasmid pET-15b (Novagen, USA) was used. This vector has the advantage of carrying the His. Tag sequence, a stretch of 6 consecutive histidine residues that can be expressed at the N- terminal of the target protein. The His. Tag sequence binds to divalent cations immobilized on a metal chelation resin. After unbound proteins are washed away, the target protein can be recovered by elution with imidazole. The pET-15b construct expressing human K30H was constructed as follows: the insert contained in plasmid pET-lla (See Example 5) was excided by digestion with Nde I/BamH I and ligated into the vector pET-15b cut with the same restriction enzymes and dephosphorylated . Initial cloning was done in DH5α strain to allow the examination of the construct sequence by restriction analysis. After positive clones were identified, the plasmid was isolated (Easy-pure plasmid preps, Primm Labs, Italy) and used for transformation into BL21(DE3) pLysS according to the instructions of Novagen, USA. After the target plasmid was established in the expression host, expression of the human K30H DNA was induced by addition of IPTG to a growing culture. Induction conditions were the same used in Example 5. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Pharmacia & Upjohn S.p.A. (B) STREET: Via Robert Koch 1.2 (C) CITY: Milan (E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 20152 (G) TELEPHONE: +39-2-48381 (H) TELEFAX: +39-2-48385397 (ii) TITLE OF INVENTION: Recombinant kynurenine -3 -hydroxylase and process for its preparation (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1733 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA [ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 45..1481 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCACGCGTCC GAGCTCCTAC CTGAGCAGAG GTATTCTGGC AGCA ATG GCA TCG TCG 56 Met Ala Ser Ser 1 GAC ACT GAA GGA AAA AGA GTG GTT GTT ATC GGT GGT GGT TTG GTT GGA 104 Asp Thr Glu Gly Lys Arg Val Val Val He Gly Gly Gly Leu Val Gly 5 10 15 20 GCA TTG AAC GCG TGC TTT CTC GCA AAG AGG AAT TTC CAA GTT GAT GTG 152 Ala Leu Asn Ala Cys Phe Leu Ala Lys Arg Asn Phe Gin Val Asp Val 25 30 35 TAC GAA GCT AGG GAA GAT ATT CGA GTG GCT AAC TTT ATG CGT GGA AGA 200 Tyr Glu Ala Arg Glu Asp He Arg Val Ala Asn Phe Met Arg Gly Arg 40 45 50 AGC ATT AAT TTG GCC CTT TCT TAT AGA GGA CGG CAG GCC TTG AAG GCC 248 Ser He Asn Leu Ala Leu Ser Tyr Arg Gly Arg Gin Ala Leu Lys Ala 55 60 65 GTT GGT CTG GAA GAT CAG ATC GTG TCC AAA GGT GTG CCC ATG AAA GCC 296 Val Gly Leu Glu Asp Gin He Val Ser Lys Gly Val Pro Met Lys Ala 70 75 80 AGA ATG ATC CAC TCT CTC TCG GGA AAG AAG TCT GCA ATT CCC TAT GGG 344 Arg Met He His Ser Leu Ser Gly Lys Lys Ser Ala He Pro Tyr Gly 85 90 95 100 AAC AAG TCA CAG TAT ATC CTT TCA ATA AGC AGA GAA AAG TTA AAC AAG 392 Asn Lys Ser Gin Tyr He Leu Ser He Ser Arg Glu Lys Leu Asn Lys 105 110 115 GAT CTG CTG ACT GCC GTG GAG TCC TAC CCC AAT GCA AAG GTG CAC TTT 440 Asp Leu Leu Thr Ala Val Glu Ser Tyr Pro Asn Ala Lys Val His Phe 120 125 130 GGC CAC AAG CTG TCA AAA TGC TGT CCG GAG GAA GGG ATA CTC ACG ATG 488 Gly His Lys Leu Ser Lys Cys Cys Pro Glu Glu Gly He Leu Thr Met 135 140 145 CTT GGA CCC AAC AAA GTT CCC AGA GAC ATC ACG TGT GAC CTC ATT GTA 536 Leu Gly Pro Asn Lys Val Pro Arg Asp He Thr Cys Asp Leu He Val 150 155 160 GGA TGT GAT GGG GCC TAC TCA ACT GTC AGA GCT CAC CTC ATG AAG AAG 584 Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ala His Leu Met Lys Lys 165 170 175 180 CCC CGT TTT GAT TAC AGT CAG CAA TAT ATC CCT CAT GGC TAT ATG GAG 632 Pro Arg Phe Asp Tyr Ser Gin Gin Tyr He Pro His Gly Tyr Met Glu 185 190 195 CTG ACA ATT CCA CCT AAG AAC GGG GAG TAT GCC ATG GAA CCT AAC TGT 680 Leu Thr He Pro Pro Lys Asn Gly Glu Tyr Ala Met Glu Pro Asn Cys 200 205 210 CTT CAC ATT TGG CCT AGA AAT GCC TTT ATG ATG ATC GCC CTA CCG AAC 728 Leu His He Trp Pro Arg Asn Ala Phe Met Met He Ala Leu Pro Asn 215 220 225 ATG GAC AAA TCT TTC ACA TGC ACC TTG TTC ATG TCC TTT GAG GAG TTT 776 Met Asp Lys Ser Phe Thr Cys Thr Leu Phe Met Ser Phe Glu Glu Phe 230 235 240 GAA AAG CTT CCA ACG CAT AGT GAT GXG CTG GAC TTC TTC CAG AAG AAC 824 Glu Lys Leu Pro Thr His Ser Asp Val Leu Asp Phe Phe Gin Lys Asn 245 250 255 260 TTT CCA GAT GCC ATC CCT CTG ATG GGC GAG CAA GCC CTC ATG AGA GAT 872 Phe Pro Asp Ala He Pro Leu Met Gly Glu Gin Ala Leu Met Arg Asp 265 270 275 TTC TTT CTG TTG CCT GCC CAG CCC ATG ATA TCA GTA AAG TGC TCT CCC 920 Phe Phe Leu Leu Pro Ala Gin Pro Met He Ser Val Lys Cys Ser Pro 280 285 290 TTC CAC CTG AAG TCA CGC TGT GTG CTG ATG GGA GAT GCA GCT CAT GCC 968 Phe His Leu Lys Ser Arg Cys Val Leu Met Gly Asp Ala Ala His Ala 295 300 305 ATC GTC CCA TTT TTT GGG CAA GGA ATG AAT GCG GGC TTT GAA GAC TGC 1016 He Val Pro Phe Phe Gly Gin Gly Met Asn Ala Gly Phe Glu Asp Cys 310 315 320 TTG GTA TTT GAT GAG TTA ATG GAC AAA TTC AAT AAT GAT CTT AGT GTG 1064 Leu Val Phe Asp Glu Leu Met Asp Lys Phe Asn Asn Asp Leu Ser Val 325 330 335 340 TGC CTT CCT GAA TTC TCA AGA TTT AGG ATT CCT GAT GAC CAT GCA ATT 1112 Cys Leu Pro Glu Phe Ser Arg Phe Arg He Pro Asp Asp His Ala He 345 350 355 TCA GAC CTG TCT ATG TAC AAT TAC ATA GAG ATG CGA GCG CAT GTC AAC 1160 Ser Asp Leu Ser Met Tyr Asn Tyr He Glu Met Arg Ala His Val Asn 360 365 370 TCT AGG TGG TTC CTG TTT CAA AGG CTC CTG GAT AAA TTT CTT CAT GCA 1208 Ser Arg Trp Phe Leu Phe Gin Arg Leu Leu Asp Lys Phe Leu His Ala 375 380 385 CTA ATG CCA TCC ACT TTC ATC CCT CTC TAT ACC ATG GTC GCC TTC ACC 1256 Leu Met Pro Ser Thr Phe He Pro Leu Tyr Thr Met Val Ala Phe Thr 390 395 400 AGA ATA AGA TAC CAC GAG GCA GTG CTG CGC TGG CAT TGG CAA AAA AAG 1304 Arg He Arg Tyr His Glu Ala Val Leu Arg Trp His Trp Gin Lys Lys 405 410 415 420 GTG ATA AAC AGA GGA CTC TTT GTC CTT GGG TCC CTG GTA GCC ATT GGA 1352 Val He Asn Arg Gly Leu Phe Val Leu Gly Ser Leu Val Ala He Gly 425 430 435 AGT GCC TAC ATA CTC GTG CAC CAC CTG TCC CCG AGA CCT CTG GAA CTC 1400 Ser Ala Tyr He Leu Val His His Leu Ser Pro Arg Pro Leu Glu Leu 440 445 450 CTG AGA TCT GCC TGG ACG GGA ACC TCT GGC CAC TGG AAT AGG AGT GCA 1448 Leu Arg Ser Ala Trp Thr Gly Thr Ser Gly His Trp Asn Arg Ser Ala 455 460 465 GAC ATT TCT CCA CGA GTT CCA TCG AGT CAC TAG GACAAATGCC CC-AGTTCACT 1501 Asp He Ser Pro Arg Val Pro Trp Ser His * 470 475 ATCCATAGTG TCAACGTTCC GGGTAGCAAA TCCTTGATTC CTCTTCAATA TCAAGGGAGA 1561 AACTCATGTT CCCATTGCCG TCTTCAGTTC ACTATGGGAA AATCATTGTC AGCATATAAT 1621 TAAGTTCGGA GTGGAGGGCT GTTTTTACAG TGTCTCATTA TTTTGCATGC TTGGACTGGG 1681 TTCAATTTTT AAATTTAAAA ACACAATAAC CAAAAAAAAA AAAAAAAAAA AA 1733 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 479 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ala Ser Ser Asp Thr Glu Gly Lys Arg Val Val Val He Gly Gly 1 5 10 15 Gly Leu Val Gly Ala Leu Asn Ala Cys Phe Leu Ala Lys Arg Asn Phe 20 25 30 Gin Val Asp Val Tyr Glu Ala Arg Glu Asp He Arg Val Ala Asn Phe 35 40 45 Met Arg Gly Arg Ser He Asn Leu Ala Leu Ser Tyr Arg Gly Arg Gin 50 55 60 Ala Leu Lys Ala Val Gly Leu Glu Asp Gin He Val Ser Lys Gly Val 65 70 75 80 Pro Met Lys Ala Arg Met He His Ser Leu Ser Gly Lys Lys Ser Ala 85 90 95 He Pro Tyr Gly Asn Lys Ser Gin Tyr He Leu Ser He Ser Arg Glu 100 105 110 Lys Leu Asn Lys Asp Leu Leu Thr Ala Val Glu Ser Tyr Pro Asn Ala 115 120 125 Lys Val His Phe Gly His Lys Leu Ser Lys Cys Cys Pro Glu Glu Gly 130 135 140 He Leu Thr Met Leu Gly Pro Asn Lys Val Pro Arg Asp He Thr Cys 145 150 155 160 Asp Leu He Val Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ala His 165 170 175 Leu Met Lys Lys Pro Arg Phe Asp Tyr Ser Gin Gin Tyr He Pro His 180 185 190 Gly Tyr Met Glu Leu Thr He Pro Pro Lys Asn Gly Glu Tyr Ala Met 195 200 205 Glu Pro Asn Cys Leu His He Trp Pro Arg Asn Ala Phe Met Met He 210 215 220 Ala Leu Pro Asn Met Asp Lys Ser Phe Thr Cys Thr Leu Phe Met Ser 225 230 235 240 Phe Glu Glu Phe Glu Lys Leu Pro Thr His Ser Asp Val Leu Asp Phe 245 250 255 Phe Gin Lys Asn Phe Pro Asp Ala He Pro Leu Met Gly Glu Gin Ala 260 265 270 Leu Met Arg Asp Phe Phe Leu Leu Pro Ala Gin Pro Met He Ser Val 275 280 285 Lys Cys Ser Pro Phe His Leu Lys Ser Arg Cys Val Leu Met Gly Asp 290 295 300 Ala Ala His Ala He Val Pro Phe Phe Gly Gin Gly Met Asn Ala Gly 305 310 315 320 Phe Glu Asp Cys Leu Val Phe Asp Glu Leu Met Asp Lys Phe Asn Asn 325 330 335 Asp Leu Ser Val Cys Leu Pro Glu Phe Ser Arg Phe Arg He Pro Asp 340 345 350 Asp His Ala He Ser Asp Leu Ser Met Tyr Asn Tyr He Glu Met Arg 355 360 365 Ala His Val Asn Ser Arg Trp Phe Leu Phe Gin Arg Leu Leu Asp Lys 370 375 380 Phe Leu His Ala Leu Met Pro Ser Thr Phe He Pro Leu Tyr Thr Met 385 390 395 400 Val Ala Phe Thr Arg He Arg Tyr His Glu Ala Val Leu Arg Trp His 405 410 415 Trp Gin Lys Lys Val He Asn Arg Gly Leu Phe Val Leu Gly Ser Leu 420 425 430 Val Ala He Gly Ser Ala Tyr He Leu Val His His Leu Ser Pro Arg 435 440 445 Pro Leu Glu Leu Leu Arg Ser Ala Trp Thr Gly Thr Ser Gly His Trp 450 455 460 Asn Arg Ser Ala Asp He Ser Pro Arg Val Pro Trp Ser His * 465 470 475 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1628 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 34..1494 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GCACAGATAA TTGTGAAAAA TACTTCAGCA GTT ATC GAC TCA TCT GTC ATT CAA 54 Met Asp Ser Ser Val He Gin 480 485 AGG AAA AAA GTA GCT GTC ATT GGT GGT GGC TTG GTT GGC TCA TTA CAA 102 Arg Lys Lys Val Ala Val He Gly Gly Gly Leu Val Gly Ser Leu Gin 490 495 500 GCA TGC TTT CTT GCA AAG AGG AAT TTC CAG ATT GAT GTA TAT GAA GCT 150 Ala Cys Phe Leu Ala Lys Arg Asn Phe Gin He Asp Val Tyr Glu Ala 505 510 515 AGG GAA GAT ACT CGA GTG GCT ACC TTC ACA CGT GGA AGA AGC ATT AAC 198 Arg Glu Asp Thr Arg Val Ala Thr Phe Thr Arg Gly Arg Ser He Asn 520 525 530 TTA GCC CTT TCT CAT AGA GGA CGA CAA GCC TTG AAA GCT GTT GGC CTG 246 Leu Ala Leu Ser His Arg Gly Arg Gin Ala Leu Lys Ala Val Gly Leu 535 540 545 550 GAA GAT CAG ATT GTA TCC CAA GGT ATT CCC ATG AGA GCA AGA ATG ATC 294 Glu Asp Gin He Val Ser Gin Gly He Pro Met Arg Ala Arg Met He 555 560 565 CAC TCT CTT TCA GGA AAA AAG TCT GCA ATT CCC TAT GGG ACA AAG TCT 342 His Ser Leu Ser Gly Lys Lys Ser Ala He Pro Tyr Gly Thr Lys Ser 570 575 580 CAG TAT ATT CTT TCT GTA AGC AGA GAA AAT CTA AAC AAG GAT CTA TTG 390 Gln Tyr He Leu Ser Val Ser Arg Glu Asn Leu Asn Lys Asp Leu Leu 585 590 595 ACT GCT GCT GAG AAA TAC CCC AAT GTG AAA ATG CAC TTT AAC CAC AGG 438 Thr Ala Ala Glu Lys Tyr Pro Asn Val Lys Met His Phe Asn His Arg 600 605 610 CTG TTG AAA TGT AAT CCA GAG GAA GGA ATG ATC ACA GTG CTT GGA TCT 486 Leu Leu Lys Cys Asn Pro Glu Glu Gly Met He Thr Val Leu Gly Ser 615 620 625 630 GAC AAA GTT CCC AAA GAT GTC ACT TGT GAC CTC ATT GTA GGA TGT GAT 534 Asp Lys Val Pro Lys Asp Val Thr Cys Asp Leu He Val Gly Cys Asp 635 640 645 GGA GCC TAT TCA ACT GTC AGA TCT CAC CTG ATG AAG AAA CCT CGC TTT 5R? Gly Ala Tyr Ser Thr Val Arg Ser His Leu Met Lys Lys Pro Arg Phe 650 655 660 GAT TAC AGT CAG CAG TAC ATT CCT CAT GGG TAC ATG GAG TTG ACT ATT 630 Asp Tyr Ser Gin Gin Tyr He Pro His Gly Tyr Met Glu Leu Thr He 665 670 675 CCA CCT AAG AAC GGA GAT TAT GCC ATG GAA CCT AAT TAT CTG CAT ATT 678 Pro Pro Lys Asn Gly Asp Tyr Ala Met Glu Pro Asn Tyr Leu His He 680 685 690 TGG CCT AGA AAT ACC TTT ATG ATG ATT GCA CTT CCT AAC ATG AAC AAA 726 Trp Pro Arg Asn Thr Phe Met Met He Ala Leu Pro Asn Met Asn Lys 695 700 705 710 TCA TTC ACA TCT ACT TTG TTC ATG CCC TTT GAA GAG TTT GAA AAA CTT 774 Ser Phe Thr Cys Thr Leu Phe Met Pro Phe Glu Glu Phe Glu Lys Leu 715 720 725 CTA ACC AGT AAT GAT GTC GTA GAT TTC TTC CAG AAA TAC TTT CCG GAT 822 Leu Thr Ser Asn Asp Val Val Asp Phe Phe Gin Lys Tyr Phe Pro Asp 730 735 740 GCC ATC CCT CTA ATT GGA GAG AAA CTC CTA GTG CAA GAT TTC TTC CTG 870 Ala He Pro Leu He Gly Glu Lys Leu Leu Val Gin Asp Phe Phe Leu 745 750 755 TTG CCT GCC CAG CCC ATC ATA TCT GTA AAG TCC TCT TCA TTT CAC TTT 918 Leu Pro Ala Gin Pro Met He Ser Val Lys Cys Ser Ser Phe His Phe 760 765 770 AAA TCT CAC TGT GTA CTG CTG GGA GAT GCA GCT CAT GCT ATA GTG CCG 966 Lys Ser His Cys Val Leu Leu Gly Asp Ala Ala His Ala He Val Pro 775 780 785 790 TTT TTT GGG CAA GGA ATG AAT GCG GGC TTT GAA GAC TGC TTG GTA TTT 1014 Phe Phe Gly Gin Gly Met Asn Ala Gly Phe Glu Asp Cys Leu Val Phe 795 800 805 GAT GAG TTA ATG GAT AAA TTC AGT AAC GAC CTT AGT TTG TGT CTT CCT 1062 Asp Glu Leu Met Asp Lys Phe Ser Asn Asp Leu Ser Leu Cys Leu Pro 810 815 820 GTG TTC TCA AGA TTG AGA ATC CCA GAT GAT CAC GCG ATT TCA GAC CTA 1110 Val Phe Ser Arg Leu Arg He Pro Asp Asp His Ala He Ser Asp Leu 825 830 835 TCC ATG TAC AAT TAC ATA GAG ATG CGA GCA CAT GTC AAC TCA AGC TGG 1158 Ser Met Tyr Asn Tyr He Glu Met Arg Ala His Val Asn Ser Ser Trp 840 845 850 TTC ATT TTT CAG AAG AAC ATG GAG AGA TTT CTT CAT GCG ATT ATG CCA 1206 Phe He Phe Gin Lys Asn Met Glu Arg Phe Leu His Ala He Met Pro 855 860 865 870 TCG ACC TTT ATC CCT CTC TAT ACA ATG GTC ACT TTT TCC AGA ATA AGA 1254 Ser Thr Phe He Pro Leu Tyr Thr Met Val Thr Phe Ser Arg He Arg 875 880 885 TAC CAT GAG GCT GTC CAG CGT TCG CAT TGG CAA AAA AAG GTC ATA AAC 1302 Tyr His Glu Ala Val Gin Arg Trp His Trp Gin Lys Lys Val He Asn 890 895 900 AAA GGA CTC TTT TTC TTG GGA TCA CTG ATA GCC ATC AGC AGT ACC TAC 1350 Lys Gly Leu Phe Phe Leu Gly Ser Leu He Ala He Ser Ser Thr Tyr 905 910 915 CTA CTT ATA CAC TAC ATG TCA CCA CGA TCT TTC CTC TCC TTC AGA AGA 1398 Leu Leu He His Tyr Met Ser Pro Arg Ser Phe Leu Cys Leu Arg Arg 920 925 930 CCA TGG AAC TGG ATA GCT CAC TTC CGG AAT ACA ACA TGT TTC CCC GCA 1446 Pro Trp Asn Trp He Ala His Phe Arg Asn Thr Thr Cys Phe Pro Ala 935 940 945 950 AAG GCC GTG GAC TCC CTA GAA CAA ATT TCC AAT CTC ATT AGC AGG TCA 1494 Lys Ala Val Asp Ser Leu Glu Gin He Ser Asn Leu He Ser Arg * 955 960 965 TAGAAAGGTT TTCTGGTAGC AAATGCATGA TTTCTCTGTG ACCAAAATIA AGCATCAAAA 1554 AAATCTTTCC ATTGCCATAT TTCATTCACT AGTGGAAGAT AGTCTTCTGC TTATAATTAA 1614 ACTGAATGTA GAGT 1628 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Asp Ser Ser Val He Gin Arg Lys Lys Val Ala Val He Gly Gly 1 5 10 15 Gly Leu Val Gly Ser Leu Gin Ala Cys Phe Leu Ala Lys Arg Asn Phe 20 25 30 Gin He Asp Val Tyr Glu Ala Arg Glu Asp Thr Arg Val Ala Thr Phe 35 40 45 Thr Arg Gly Arg Ser He Asn Leu Ala Leu Ser His Arg Gly Arg Gin 50 55 60 Ala Leu Lys Ala Val Gly Leu Glu Asp Gin He Val Ser Gin Gly He 65 70 75 80 Pro Met Arg Ala Arg Met He His Ser Leu Ser Gly Lys Lys Ser Ala 85 90 95 He Pro Tyr Gly Thr Lys Ser Gin Tyr He Leu Ser Val Ser Arg Glu 100 105 110 Asn Leu Asn Lys Asp Leu Leu Thr Ala Ala Glu Lys Tyr Pro Asn Val 115 120 125 Lys Met His Phe Asn His Arg Leu Leu Lys Cys Asn Pro Glu Glu Gly 130 135 140 Met He Thr Val Leu Gly Ser Asp Lys Val Pro Lys Asp Val Thr Cys 145 150 155 160 Asp Leu He Val Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ser His 165 170 175 Leu Met Lys Lys Pro Arg Phe Asp Tyr Ser Gin Gin Tyr He Pro His 180 185 190 Gly Tyr Met Glu Leu Thr He Pro Pro Lys Asn Gly Asp Tyr Ala Met 195 200 205 Glu Pro Asn Tyr Leu His He Trp Pro Arg Asn Thr Phe Met Met He 210 215 220 Ala Leu Pro Asn Met Asn Lys Ser Phe Thr Cys Thr Leu Phe Met Pro 225 230 235 240 Phe Glu Glu Phe Glu Lys Leu Leu Thr Ser Asn Asp Val Val Asp Phe 245 250 255 Phe Gin Lys Tyr Phe Pro Asp Ala He Pro Leu He Gly Glu Lys Leu 260 265 270 Leu Val Gin Asp Phe Phe Leu Leu Pro Ala Gin Pro Met He Ser Val 275 280 285 Lys Cys Ser Ser Phe His Phe Lys Ser His Cys Val Leu Leu Gly Asp 290 295 300 Ala Ala His Ala He Val Pro Phe Phe Gly Gin Gly Met Asn Ala Gly 305 310 315 320 Phe Glu Asp Cys Leu Val Phe Asp Glu Leu Met Asp Lys Phe Ser Asn 325 330 335 Asp Leu Ser Leu Cys Leu Pro Val Phe Ser Arg Leu Arg He Pro Asp 340 345 350 Asp His Ala He Ser Asp Leu Ser Met Tyr Asn Tyr He Glu Met Arg 355 360 365 Ala His Val Asn Ser Ser Trp Phe He Phe Gin Lys Asn Met Glu Arg 370 375 380 Phe Leu His Ala He Met Pro Ser Thr Phe He Pro Leu Tyr Thr Met 385 390 395 400 Val Thr Phe Ser Arg He Arg Tyr His Glu Ala Val Gin Arg Trp His 405 410 415 Trp Gin Lys Lys Val He Asn Lys Gly Leu Phe Phe Leu Gly Ser Leu 420 425 430 He Ala He Ser Ser Thr Tyr Leu Leu He His Tyr Met Ser Pro Arg 435 440 445 Ser Phe Leu Cys Leu Arg Arg Pro Trp Asn Trp He Ala His Phe Arg 450 455 460 Asn Thr Thr Cys Phe Pro Ala Lys Ala Val Asp Ser Leu Glu Gin He 465 470 475 480 Ser Asn Leu He Ser Arg * 485 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5000 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 47..1507 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGCACGAGCA GAAGCAACAA TAATTGTGAA AAATACTTCA GCAGTT ATG GAC TCA 55 Met Asp Ser 490 TCT GTC ATT CAA AGG AAA AAA GTA GCT GTC ATT GGT GGT GGC TTG GTT 103 Ser Val He Gin Arg Lys Lys Val Ala Val He Gly Gly Gly Leu Val 495 500 505 GGC TCA TTA CAA GCA TGC TTT CTT GCA AAG AGG AAT TTC CAG ATT GAT 151 Gly Ser Leu Gin Ala Cys Phe Leu Ala Lys Arg Asn Phe Gin He Asp 510 515 520 GTA TAT GAA GCT AGG GAA GAT ACT CGA GTG GCT ACC TTC ACA CGT GGA 199 Val Tyr Glu Ala Arg Glu Asp Thr Arg Val Ala Thr Phe Thr Arg Gly 525 530 535 AGA AGC ATT AAC TTA GCC CTT TCT CAT AGA GGA CGA CAA GCC TTG AAA 247 Arg Ser He Asn Leu Ala Leu Ser His Arg Gly Arg Gin Ala Leu Lys 540 545 550 GCT GTT GGC CTG GAA GAT CAG ATT GTA TCC CAA GGT ATT CCC ATG AGA 295 Ala Val Gly Leu Glu Asp Gin He Val Ser Gin Gly He Pro Met Arg 555 560 565 570 GCA AGA ATC ATC CAC TCT CTT TCA GGA AAA AAG TCT GCA ATT CCC TAT 343 Ala Arg Met He His Ser Leu Ser Gly Lys Lys Ser Ala He Pro Tyr 575 580 585 GGG ACA AAG TCT CAG TAT ATT CTT TCT GTA AGC AGA GAA AAT CTA AAC 391 Gly Thr Lys Ser Gin Tyr He Leu Ser Val Ser Arg Glu Asn Leu Asn 590 595 600 AAG GAT CTA TTG ACT GCT GCT GAG AAA TAC CCC AAT GTC AAA ATG CAC 439 Lys Asp Leu Leu Thr Ala Ala Glu Lys Tyr Pro Asn Val Lys Met His 605 610 615 TTT AAC CAC AGG CTG TTC AAA TCT AAT CCA GAG GAA GGA ATC ATC ACA 487 Phe Asn His Arg Leu Leu Lys Cys Asn Pro Glu Glu Gly Met He Thr 620 625 630 GTG CTT GGA TCT GAC AAA GTT CCC AAA GAT GTC ACT TCT GAC CTC ATT 535 Val Leu Gly Ser Asp Lys Val Pro Lys Asp Val Thr Cyε Asp Leu He 635 640 645 650 GTA GGA TGT GAT GGA GCC TAT TCA ACT GTC AGA TCT CAC CTG ATC AAG 583 Val Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ser His Leu Met Lys 655 660 665 AAA CCT CGC TTT GAT TAC AGT CAG CAG TAC ATT CCT CAT GGG TAC ATC 631 Lys Pro Arg Phe Asp Tyr Ser Gin Gin Tyr He Pro His Gly Tyr Met 670 675 680 GAG TTG ACT ATT CCA CCT AAG AAC GGA GAT TAT GCC ATG GAA CCT AAT 679 Glu Leu Thr He Pro Pro Lys Asn Gly Asp Tyr Ala Met Glu Pro Asn 685 690 695 TAT CTC CAT ATT TGG CCT AGA AAT ACC TTT ATG ATG ATT GCA CTT CCT 727 Tyr Leu His He Trp Pro Arg Asn Thr Phe Met Met He Ala Leu Pro 700 705 710 AAC ATG AAC AAA TCA TTC ACA TCT ACT TTG TTC ATC CCC TTT GAA GAG 775 Asn Met Asn Lys Ser Phe Thr Cys Thr Leu Phe Met Pro Phe Glu Glu 715 720 725 730 TTT GAA AAA CTT CTA ACC AGT AAT GAT GTG GTA GAT TTC TTC CAG AAA 823 Phe Glu Lys Leu Leu Thr Ser Asn Asp Val Val Asp Phe Phe Gin Lys 735 740 745 TAC TTT CCG GAT GCC ATC CCT CTA ATT GGA GAG AAA CTC CTA GTG CAA 871 Tyr Phe Pro Asp Ala He Pro Leu He Gly Glu Lys Leu Leu Val Gin 750 755 760 GAT TTC TTC CTG TTG CCT GCC CAG CCC ATC ATA TCT GTA AAG TGC TCT 919 Asp Phe Phe Leu Leu Pro Ala Gin Pro Met He Ser Val Lys Cys Ser 765 770 775 TCA TTT CAC TTT AAA TCT CAC TGT GTA CTC CTG GGA GAT GCA GCT CAT 967 Ser Phe His Phe Lys Ser His Cys Val Leu Leu Gly Asp Ala Ala His 780 785 790 GCT ATA GTG CCG TTT TTT GGG CAA GGA ATC AAT GCG GGC TTT GAA GAC 1015 Ala He Val Pro Phe Phe Gly Gin Gly Met Asn Ala Gly Phe Glu Asp 795 800 805 810 TGC TTG GTA TTT GAT GAG TTA ATG GAT AAA TTC AGT AAC GAC CTT AGT 1063 Cys Leu Val Phe Asp Glu Leu Met Asp Lys Phe Ser Asn Asp Leu Ser 815 820 825 TTG TGT CTT CCT GTC TTC TCA AGA TTC AGA ATC CCA GAT GAT CAC GCG 1111 Leu Cys Leu Pro Val Phe Ser Arg Leu Arg He Pro Asp Asp His Ala 830 835 840 ATT TCA GAC CTA TCC ATG TAC AAT TAC ATA GAG ATC CGA GCA CAT GTC 1159 He Ser Asp Leu Ser Met Tyr Asn Tyr He Glu Met Arg Ala His Val 845 850 855 AAC TCA AGC TGG TTC ATT TTT CAG AAG AAC ATC GAG AGA TTT CTT CAT 1207 Asn Ser Ser Trp Phe He Phe Gin Lys Asn Met Glu Arg Phe Leu His 860 865 870 GCG ATT ATG CCA TCG ACC TTT ATC CCT CTC TAT ACA ATG GTC ACT TTT 1255 Ala He Met Pro Ser Thr Phe He Pro Leu Tyr Thr Met Val Thr Phe 875 880 885 890 TCC AGA ATA AGA TAC CAT GAG GCT GTC CAG CGT TCG CAT TCG CAA AAA 1303 Ser Arg He Arg Tyr His Glu Ala Val Gin Arg Trp His Trp Gin Lys 895 900 905 AAG GTG ATA AAC AAA GGA CTC TTT TTC TTG GGA TCA CTC ATA GCC ATC 1351 Lys Val He Asn Lys Gly Leu Phe Phe Leu Gly Ser Leu He Ala He 910 915 920 AGC AGT ACC TAC CTA CTT ATA CAC TAC ATC TCA CCA CGA TCT TTC CTC 1399 Ser Ser Thr Tyr Leu Leu He His Tyr Met Ser Pro Arg Ser Phe Leu 925 930 935 TCC TTG AGA AGA CCA TGG AAC TCG ATA GCT CAC TTC CGG AAT ACA ACA 1447 Cys Leu Arg Arg Pro Trp Asn Trp He Ala His Phe Arg Asn Thr Thr 940 945 950 TCT TTC CCC GCA AAG GCC GTC GAC TCC CTA GAA CAA ATT TCC AAT CTC 1495 Cys Phe Pro Ala Lys Ala Val Asp Ser Leu Glu Gin He Ser Asn Leu 955 960 965 970 ATT AGC AGG TCA TAGAAAGGTT TTGTGGTAGC AAATGCATGA TTTCTCTGTC 1547 He Ser Arg * ACCAAAATTA AGCATGAAAA AAATGTTTCC ATTGCCATAT TTCATTCACT AGTGGAAGAT 1607 AGTCTTCTGC TTATAATTAA ACTGAATCTA GAGTATCTCT GTATCTTAAT TGCAATTACT 1667 GGTTGGGGGG TGCATTTTAA AAGATGAAAC ATGCAGCTTC CCTACATTAC ACACACTCAG 1727 GTTGAGTCAT TCTAACTATA AAAGTGCAAT GACTAAGATC CTTCACTTCT CTGAAAGTAA 1787 GGCCCTAGAT GCCTCAGGGA AGACAGTAAT CATCCCTTTT CTTTAAAAGA CACAATAGGA 1847 CTCGCAACAG CATTGACTCA ACACCTAGGA CTAAAAATCA C.AACTTAACT AGCATCTTAA 1907 CTGCACTTTT CATTACGTGA ATGGAACTTA CCTAACCACA GGGCTCAGAC TTACTAGATA 1967 AAACCAGAAA TGGAAATAAG GAATTCAGGG GAGTTCCAGA GACTTACAAA ATGAACTCAT 2027 TTTATTTTCC CACCTTCAAA TATAAGTATT ATCATCTATC TCTTTATCGT CTATCTATCT 2087 ATCATCTATC TATCTATCTA TCATCTATCT ATCTATCTAT CTATCTATCT ATCTATCTAT 2147 CTATCTATCT ATCTCTATTT ATTTATGTAT TTAGAGATCA GGTCTCACTC TCTTGACCAG 2207 GCTGGAGTGC AGTCGTGAGA TCTGGGTTCA CTCCAACCTC TCCCTCCTGG GCTCAAGCAA 2267 TCCTCCCACT TCAGCCTCCC AAATAGCTGG GGCTACCATC GTATTTTTCA GTAGAGACCG 2327 GGTCTTGCCA TGCTGCCCAG GCCAGTCTCA AACTCCTGGC CTCATGTGAT CTGCCCACCT 2387 CAGCCTCCCA AAGTACAGGG ATTAGAGTTG TGAGCCACCG CTGCCAGCCC AGAGTTACCC 2447 TCTAAAGATA AGAAAAAGGC TATTAATATC ATACTAAGTC AAGGACAGGA AAGGGTTTTA 2507 TTCATAAATT AAATCTCTAC ATCTCCCAGA ATGGAAAGGA AACAAGGGGA GACAALT'ITT 2567 ATAGAAATAC AAAGCCATTA ( CTI ATTCAA TTTCAGACCC TCAGAAGCAA TTTACTAATT 2627 TATTCTTCGA CTACATACTG CAGCAGAACC AGCAATACAC TTCATTTTTA AAAGCACATT 2687 TAGTCAAATC TTTTCTTTGG TTCATCCTTC TTTAACAGGC TGCTGAGTCA CTCAGAAATC 2747 CTTCAAACAT GATTAATTAT GAAGATGAAA CACTAGAGTC ATATAAGAAA TAAAAATTGG 2807 GCAATAAAAT AAAATGATTC AGTCTTTCTT TTCTATATTC TCAATGAAAA CCTTCAGTTC 2867 TAATAATCCA TCTTCAGTTT GTAGGGAAAG AAAAAATAAT TTTTCCTTCT ACCCACTTTA 2927 ∞TTCCTTGG CTGGGGCCCC TATAACAAAA GACAGATTGA CAAGAGAAAA ACAAACATAA 2987 ATTTATTAGC GGGTATATGT AATATATATG TCGGAAATAC AGGGGAATGA GCAAATCTCA 3047 AAGAGCTCGC GTCTTAGAAC TCCCTGGCTT ATATAGCATC GACAAAGAAC AGTAAATTTT 3107 TAGAGAAACA ACAAAACAAA GAAAAAGAGC TTTGAGTCTC TAGGGGCAGC AATTTCGGGG 3167 AAGCAAATAT ATGGGAGTTT GCCTTGTAGA TTCCTCTGGT GGTGGTCTCC AGGCTGACAA 3227 GGATTCAAAG TTGTCTCTGA AACTCCTCTT TGTCATACTG CACATATAAA ACGTCTTTTG 3287 TTTCCAACAA GAGGATTTCT TTITCATTCT AGAATTATCT CCTT ATAAC TTGATCAGAT 3347 ATAGGACATG ACACTGAATA GAGTCCAACA GTACAAAAAA AATTCAGTAT GTTCTAGCTA 3407 CTTCACACAT GTGTACGCGA CAGTTATTTT TACAGTAAGG TATTTTCGAG AAAAATCCAT 3467 TACGTCTTTT GGAAAATAGA GTAATTTAAA AAATATATTT GAAATGAAAA TCTCCAACAC 3527 ATTAGAAGAT GATGATCTTA GATCCCCATC GTCTCCCACA AGTCGTTTTT TCATTATCTA 3587 AAGCACCCGT TGAATTAAAA GAATTTGTTT TTGTTCAACC TCITCCTGAG GCCCAAGAGC 3647 ATATGGGCAA TTCGGATTTC CTCCTGGACC ACAAGGTTCT GTTGATATTA CATAGAAACG 3707 GGTATTCCAG ACAL'iT lTA TGATCAAAGT CCAAAAGTCG C1ATCCAATTT AAGGCCCCAT 3767 CTTTCGTTGC CATTCTTCAT TCCTACAAAG GACGAACTTG C.ATTACATCA ACTTTGGACC 3827 CATTGGTTTT GTCGCTGTCG TCAACTGACA GT ATTCACC ACTGGTGATG ATAAAAATGA 3887 TGGAAGAAGA GTTGAAAGTC ALT1TTTTCT TTGGCCTCTC CCCATCTTTC TCTCACATCA 3947 CAATCGGTCT GAT TCCATT TCACTTCCAG CTGCTCGTAG GTCTTTAGCA GGCCTCTGGC 4007 ACCTCAGCAG TCGGAGGCAC AGAAGCTGCA AAAGGGATCT TCXGAAACTGG GCAGAGAAAA 4067 AATAAAGTGG AATATTAAGT AAAAGTTGGG CΑCTAATCTG GATTAACATT CGAGGAAATC 4127 AGTTGAGCTG ATTTAAGTTG TTTTTTGTTT GTTAGCAGGT GTGGATGTGG GGTTATGTGG 4187 TCATGCTCAG ATCTACCTAA ATCACCCCAG AC-CTTTATCT CTTTTATTCA TTCTAAATCT 4247 TATTAACCGG AATATCTAGG ACCATTTCAA TACCTTCTAA TCCTCCAAGC TTCAATCTCC 4307 ACACACT TC TATGAGGGCA GGTACAACTA TTAAGAGATT TTGAACATTA AGTTAGTCCA 4367 CAAATATTCA GTGGGCATCT ACTAGGTGAC AGCCACTCTG CTATAATTAG AGACTTTTTA 4427 CTATAAGCAT CAAAAACAGA TAAGGCTCTT CCTGGCAGAG TTTACAGCCT ∞TCTACTTG 4487 CTAATCTCTC TTTAATTAGG TGAAGAATTT TTTTTTTCTA TCGAAATTAC TAATCAGTTC 4547 GGGAAAAAAA TACTATAGCA GACAGCACTA ATCTiCATCAA CAAACATTCT TCTTCTCCGT 4607 GTCCTGGGTA C-AA ( _ΑTCGAA TAATATTTCT TGGCCTCCTT TCCX_CTTCTC CTCTCTGCTG 4667 TTCCTCTCTA CAAGAACCTG GGAGGCCAAC GCCTAAAGAT CATAATATCA CAATCGAAGG 4727 AACCTAGATT CCTAAATGAC TGCATAGGAC AGATCCCATC TCCTCCACCC AATACATTAT 4787 TAGACTGAAC TGTGACCTGA AATGAGCAAT AAACTCTGTA TTAATTCACT GAAATGTTGG 4847 GGTTGCTTGT TATAGTAGTC GGTCCATCAT GACCAGTAAA ACATAAATCA AAAGTTAATC 4907 TAATTGTTAT CCCATTATTT AGAGCGAAAT AAATCTTGAA TATATGGACT TTCTCAGATT 4967 AGGAAATACC AATTAAAAAT ATAATAAATA GCT 5000 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE O-ARACTERISTICS : (A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Asp Ser Ser Val He Gin Arg Lys Lys Val Ala Val He Gly Gly 1 5 10 15 Gly Leu Val Gly Ser Leu Gin Ala Cys Phe Leu Ala Lys Arg Asn Phe 20 25 30 Gin He Asp Val Tyr Glu Ala Arg Glu Asp Thr Arg Val Ala Thr Phe 35 40 45 Thr Arg Gly Arg Ser He Asn Leu Ala Leu Ser His Arg Gly Arg Gin 50 55 60 Ala Leu Lys Ala Val Gly Leu Glu Asp Gin He Val Ser Gin Gly He 65 70 75 80 Pro Met Arg Ala Arg Met He His Ser Leu Ser Gly Lys Lys Ser Ala 85 90 95 He Pro Tyr Gly Thr Lys Ser Gin Tyr He Leu Ser Val Ser Arg Glu 100 105 110 Asn Leu Asn Lys Asp Leu Leu Thr Ala Ala Glu Lys Tyr Pro Asn Val 115 120 125 Lys Met His Phe Asn His Arg Leu Leu Lys Cys Asn Pro Glu Glu Gly 130 135 140 Met He Thr Val Leu Gly Ser Asp Lys Val Pro Lys Asp Val Thr Cys 145 150 155 160 Asp Leu He Val Gly Cys Asp Gly Ala Tyr Ser Thr Val Arg Ser His 165 170 175 Leu Met Lys Lys Pro Arg Phe Asp Tyr Ser Gin Gin Tyr He Pro His 180 185 190 Gly Tyr Met Glu Leu Thr He Pro Pro Lys Asn Gly Asp Tyr Ala Met 195 200 205 Glu Pro Asn Tyr Leu His He Trp Pro Arg Asn Thr Phe Met Met He 210 215 220 Ala Leu Pro Asn Met Asn Lys Ser Phe Thr Cys Thr Leu Phe Met Pro 225 230 235 240 Phe Glu Glu Phe Glu Lys Leu Leu Thr Ser Asn Asp Val Val Asp Phe 245 250 255 Phe Gin Lys Tyr Phe Pro Asp Ala He Pro Leu He Gly Glu Lys Leu 260 265 270 Leu Val Gin Asp Phe Phe Leu Leu Pro Ala Gin Pro Met He Ser Val 275 280 285 Lys Cys Ser Ser Phe His Phe Lys Ser His Cys Val Leu Leu Gly Asp 290 295 300 Ala Ala His Ala He Val Pro Phe Phe Gly Gin Gly Met Asn Ala Gly 305 310 315 320 Phe Glu Asp Cys Leu Val Phe Asp Glu Leu Met Asp Lys Phe Ser Asn 325 330 335 Asp Leu Ser Leu Cys Leu Pro Val Phe Ser Arg Leu Arg He Pro Asp 340 345 350 Asp His Ala He Ser Asp Leu Ser Met Tyr Asn Tyr He Glu Met Arg 355 360 365 Ala His Val Asn Ser Ser Trp Phe He Phe Gin Lys Asn Met Glu Arg 370 375 380 Phe Leu His Ala He Met Pro Ser Thr Phe He Pro Leu Tyr Thr Met 385 390 395 400 Val Thr Phe Ser Arg He Arg Tyr His Glu Ala Val Gin Arg Trp His 405 410 415 Trp Gin Lys Lys Val He Asn Lys Gly Leu Phe Phe Leu Gly Ser Leu 420 425 430 He Ala He Ser Ser Thr Tyr Leu Leu He His Tyr Met Ser Pro Arg 435 440 445 Ser Phe Leu Cys Leu Arg Arg Pro Trp Asn Trp He Ala His Phe Arg 450 455 460 Asn Thr Thr Cys Phe Pro Ala Lys Ala Val Asp Ser Leu Glu Gin He 465 470 475 480 Ser Asn Leu He Ser Arg * 485 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CAGGAATTCC ATATGGACTC ATCTGTC 27 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CGGGATCCCG CTATCACCTG CTAATGA 27

Claims: CLAIMS 1. An isolated single or double stranded polynucleo ide having a nucleotide sequence which comprises a nucleotide sequence selected from the group consisting of (a) the sense sequence of SEQ ID NO .1 from about nucleotide position 45 to about nucleotide position 1481; the sense sequence of SEQ ID NO . 3 from about nucleotide position 34 to about nucleotide position 1494; (b) sequences complementary to the sequences of (a) ,- (c) a sequence that, on expression, encodes a polypeptide encoded by the sequences of (a) ,- (d) analogous sequences that hybridize under stringent conditions to the sequences of (a) and (b) ,- and (e) sequences homologous to the sequences of (a) that, on expression, encode polypeptides having ynurenine-3 hydroxylase activity. 2. The polynucleotide of claim 1 that is a DNA molecule. 3. The polynucleotide of claim 1 that is a RNA molecule. 4. The polynucleotide of claim 2 wherein the nucleotide sequence is the nucleotide sequence of (a) selected from the group consisting of (i) the sense sequence of SEQ ID NO. 1 from about nucleotide position 45 to about nucleotide position 1481; (ii) the sense sequence of SEQ ID NO. 3 from about nucleotide position 34 to about nucleotide position 1494. 5. A vector comprising a polynucleotide as defined in any one of claims 1 to 4. 6. A vector accoding to claim 5, which vector is a plasmid. 7. A vector according to claim 5, which vector is a virus. 8. A host cell transformed with a vector according to claim 5. 9. A transformed host cell according to claim 8, which cell is a bacterial cell. 10. A transformed host cell according to claim 8, which cell is a yeast cell. 11. A transformed host cell according to claim 8, which cell is an insect cell . 12. A transformed host cell according to claim 8, which cell is a mammalian cell. 13. A Xenopus laevis oocyte transiently expressing a kyn-3- OHase enzyme coded for by a polynucleotide according to any one of claims 1 to 4. 14. An isolated and purified kyn-3-OHase enzyme which is coded for by a polynucleotide according to any one of claims 1 to 4. 15. A recombinant process for the transient expression of kyn-3- OHase, which process comprises extracting total mRNA from the tissues of a mammalian organism; generating a cDNA expression library by reverse transcription of the said mRNA; obtaining RNA in vitro from the said cDNA library; injecting the RNA into Xenopus oocytes; measuring the enzymatic activity; dividing the said library into several pools,- selecting the positive clone,- linearizing the plasmid DNA extracted from the positive clone,- synthesizing the cRNA from said DNA; injecting the said cRNA into Xenopus laevis oocytes thus obtaining expression of kyn-3-OHase enzyme .

Inventors: BENATTI LUCA, CINI MASSIMO, COVINI NEVIE, MAGAGNIN SIMONA, SPECIALE CARMELA, BENATTI, LUCA, CINI, MASSIMO, COVINI, NEVIE, MAGAGNIN, SIMONA, SPECIALE, CARMELA
Applicants: BENATTI LUCA, CINI MASSIMO, COVINI NEVIE, MAGAGNIN SIMONA, PHARMACIA & UPJOHN SPA, SPECIALE CARMELA, BENATTI, LUCA, CINI, MASSIMO, COVINI, NEVIE, MAGAGNIN, SIMONA, PHARMACIA & UPJOHN S.P.A., SPECIALE, CARMELA
Application year: 1997
Main classification: C12N15
IPC codes: A01K67, A61K48, C07K14, C12N1, C12N5, C12N9, C12N15