NZ253280A

NZ253280A - Recombinant truncated xylanases derived from anaerobic fungi

Info

Publication number: NZ253280A
Application number: NZ25328093A
Authority: NZ
Inventors: Geoffrey Peter Hazlewood; Harry John Gilbert
Original assignee: Univ Newcastle; Biotech & Biolog Scien Res
Priority date: 1992-06-17
Filing date: 1993-06-17
Publication date: 1997-09-22
Also published as: JPH08501444A; WO1993025693A1; BR9306580A; EP0652961A1; CA2138383A1; FI945928A0; FI945928A

Description

<div class="application article clearfix" id="description"> New Zealand No. 253280 International No. PCT/GB93/01283 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 17.06.1992; Complete Specification Filed: 17.06.1993 Classification:(6) C12N9/24; C12N15/56; A23K1/165; A21D8/04; D21C9/10 Publication date: 22 September 1997 Journal No.: 1420 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Recombinant xylanases Name, address and nationality of applicant(s) as in international application form: UNIVERSITY OF NEWCASTLE-UPON-TYNE, a United Kingdom University of 6 Kensington Terrace, Newcastle-Upon-Tyne NE1 7RU, United Kingdom; BIOTECHNOLOGY AND BIOLOGICAL DCIENCES RESEARCH COUNCIL, a United Kingdom body incorporated by Royal Charter of Central Office, Polaris House, North Star Avenue, Swindon SN2 1UH,United Kingdom New Zealand No. International No. 253280 PCT/GB93/01283 , A .. + NEW ZEALAND Substitution of Applicant Under Section 24 PATENTS ACT 1953 JttOTECHNOLOGY AND r^iv/ini ^PDLOGICAL SCIENCES COMPLETE SPECIFICATION RESEARCH COUNCIL; UNIVESITY OF NEW CASTLE-UPON-TYNE. Title of Invention: Recombinant xylanases Name, address and nationality of applicant(s) as in international application form: THEAetflCULTURAL AND FOOD RESEARCH COUNCIL, of Central Office, Polaris Htfuse, North Star Avenue, Swindon SN2 1 UH, United Kingdom; UNIVERSITY OF NEWCASTLE-UPON-TYNE, of 6 Kensington Terrace, Newcastle-upon-Tyne NE1 7RU, United Kingdom UK. Cow.pa^i&S. (FOLLOWED BY PAGE 1A) WO 93/25693 PCT/GB93/01283 -lA' RECOMBINANT XYLANASES This invention relates to recombinant xylanases derivable from an anaerobic fungus. 5 Xylan, a major component of plant hemicelluloses, consists of a polymer of 1,4-1 inked /3-D-xylopyranose units substituted with mainly acetyl, arabinosyl and glucuronosyl residues. Hardwood xylan is typically O-acetyl-4-O-methylglucuronoxylan with approximately ten percent of xylose units a-l,2-linked 10 to a 4-O-methylglucuronic acid side chain, and seventy percent of xylose residues acetylated at the C-2 or C-3 positions. Softwood xylans are commonly arabino-4-O-methyl-glucuronoxylans in which more than ten percent oi ->se u:uts are substituted with a-1,3-linked arabionfuranose residues. A repeno^- of microbial enzymes act co-operatively to convert xylan to its constituent simple sugars. These 15 include endo-/3-l,4-xylanases (EC 3.2.1.8), /3-xylosidase (EC 3.2.1.37) and a series of enzymes which cleave side-chain sugars (glycosidases) or remove acetyl groups from the xylan backbone (Dekker R.F.H., and Richards, G.N., Adv. Carbohydr. Chem. Biochem. 32: 277-352 (1976); Biely, Trends Biotechnol. 3: 286-290 (1985); Poutanen et al, "Accessory Enzymes Involved in the Hydrolysis 20 of Xylans" In: Enzymes in Biomass Conversion. ACS Symposium Series 460. pp426-436. Ed. G.F. Letham. (1991)). Xylanolytic micro-organisms generally express isoenzymic forms of xylanases which are encoded by multiple genes (Hazlewood et al, FEMS Microbiol. Lett. 51: 231-236 (1988); Gilbert et al, J. Gen. Microbiol. 134: 3239-3247 (1988); Clarke et al, FEMS Microbiol. Lett. 83: 25 305-310 (1991)). Some xylanases hydrolyse only xylan (Hall et al. Mol. Microbiol. 3: 1211-1219 (1989); Wong et al, Microbiol. Rev. 52: 305-317 (1988). Many microorganisms that hydrolyse xylan also degrade cellulose. In view of the similarity of the bond WO 93/25693 PCT/GB93/01283 -2- cleavcd (/3-1,4-glycosidic linkages), and the cross-specificity sometimes observed between ceilulases and xylanases, the phylogenetic relationships of these enzymes is an interesting question. Recently, sequence alignment and hydrophobic cluster analysis have been utilised to assign plant cell wall hydrolases to eight enzyme 5 families (Henrissat et al, Gene 81: 83-95 (1989); Gilkes et al, Microbiol. Rev. 55: 303-315 (1991)). Xylanases showed no convincing sequence identity with ceilulases suggesting that the two enzyme species evolved from distinct ancestral genes. 10 Many plant cell wall hydrolases consist of :no distinct domains; a catalytic domain (CD) linked by hydroxy amino acid/proline-rich linker sequences to a non-catalytic cellulose binding domain (CBD; Gilkes et al, Microbiol. Rev. 55: 303-315 (1991); Kelletr et al, Biochem. J. 272: 369-376 (1990); Gilben et al, Mol. Microbiol. 4: 9-767 (1990)). The precise role of the CBD is the subject of much debate; in 15 aerobic fungal ceilulases the CBD plays a critical role in the enzymes' hydrolysis of crystalline cellulose (Tomme et al, Eur. J. Biochem. 170: 575-581 (1988)). The role of this domain in prokaryotic ceilulases and xylanases is less certain (Ferreira et al, Biochem. J. 269: 261-264 (1990)). In addition to their modular structure, ceilulases often contain extended repeated sequences (Gilkes et al, 20 Microbiol. Rev. 55: 303-315 (1991)). The precise role of these tandem repeats is largely unresolved. Many cellulolytic and hemicellulolytic prokaiyotes reside in the rumen of cows and sheep. Recently, anaerobic rumen fungi have also been shown to degrade both 25 cellulose and xylan efficiently (Orpin and Letcher Curr. Microbiol. 3: 121-124 (1979); Lowe et al, Appl. Environ. Microbiol. 53: 1216-1223 (1987)) and similar fungi reside in the alimentary tracts of large herbivores (Orpin and Joblin, "Anaerobic fungi". In: The Rumen Microbial Ecosystem, P.N. Hobson (Ed), ppl29-150, Elsevier, London (1988)). The cellulase complex of the rumen fungus WO 93/25693 PCT/GB93/01283 -3- Neocailimastix frontalis has been characterised by Wood et al, Biochemistry and Genetics of Cellulose Degradation: FEMS Symp. 43: 31-52 (1988). The lower eukaryote synthesises a large multienzyme complex, of Mr 1-2 million, which rapidly hydroiyses crystalline cellulose. The complex contains substantial 5 endoglucanase, and some /3-glucosidase activity. The fungus also synthesises an Avicelase, presumably a cellobiohydrolase. Another rumen fungus, Neocallimastix patriciarum, produces extracellular enzymes which hydrolyse filter paper cellulose, Avicel'" (a trade mark for microcrystalline cellulose) and xylan (Williams and Orpin Can. J. Microbiol. 33: 418-426 (1987)). None of these enzymes has been 10 characterised. Limited information on Neocallimastix genes encoding plant cell wall hydrolases has been described (Reymond et al, FEMS Microbiol. Lett. 77: 107-112 (1991)). Xylans are found, in association with lignin, in the primary and secondary cell 15 walls of most plants. The association between xylan and lignin is the key to the commercial potential of xylanases in, among other things, paper pulp processing. Sandoz Products Ltd in the USA have already conducted practical trials using a crude fungal xylanase to replace, at least partially, the amount of chlorine and chlorine-derived compounds normally used to bleach the objectionable brown 2 0 lignin-derived residues in the treatment of wood pulp in the production of paper and other wood-derived products. The chlorine requirements of present day wood pulping plants are such that each plant may have its own chlorine dioxide production unit. 25 The advantages to the paper industry in avoiding the use of chlorine are clear: improvements in waste handling, operator safety and plant capital could be achieved if a suitable replacement for chlorine could be found. However, the paper industry is intensely competitive, and profit margins are slim, so any chlorine replacement must be capable of being produced reasonably economically WO 93/25693 PCT/GB93/01283 and must also, of course, be sufficiently effective to persuade pulp and paper manufacturers of the benefits of its use. The full length cDNA and protein sequence of a xylanase from NeocaUimastix pairiciarum were available from the EMBL databank in Heidelberg, Germany, as of 5 May 1992 under the accession number X65S26. The xylanase was designated XYLA and the corresponding gene xynA. It has now been found that modified xylanases derived from individual xylanases 10 from anaerobic fungi, such as the XYLA enzyme from N. pairiciarum, have properties which make them appropriate for industrial use, particularly in the manufacture of pulp and paper. It appears surprisingly that truncation can enhance the expression of the enzyme. 15 According to a first aspect of the present invention, there is provided a xylanase which has at least one catalytic domain which is substantially homologous with a xylanase of an anaerobic fungus and which is not a full length natural xylanase. Preferred catalytic domains are identical to catalytic domains of natural xylanases 2 0 from anaerobic fungi. However, for the purpose of the present invention, a first sequence is substantially homologous with a second sequence if, for example, it shares its biological activity and there is at least about 40% homology at the amino acid level; so a catalytic domain of a xylanase of this aspect of the invention has at least about 40% homology with a catalytic domain of a natural xylanase of an 25 anaerobic fungus. In general, it may be preferred for there to be at least 50%, 60%, 70%, 80% or 90% homology (in increasing order of preference) between the two amino acid sequences being compared. Homology may alternatively or additionally be assessed at the nucleic acid level. DNA encoding a first amino acid sequence may be substantially homologous with and hybridise to DNA (which SUBSTITUTE SHEET ISA/EP WO 93/25693 PCT/GB93/01283 -5- may be cDNA or genomic DNA) which encodes a second amino acid sequence or would so hybridise but for the degeneracy of the genetic code. Hybridisation conditions may be stringent, such as 65°C in a salt solution of approximately 0.9 molar. 5 Examples of anaerobic fungi, which may be alimentary tract (particularly rumen) fungi, include: Neocallimastix spp., such as N. pairiciarum, N. frontalis, N. hurleyensis and N. stanthorpensis; Sphaeromonas spp., such as S. communis; Caecomyces spp., such as C. equi', Piromyces spp., such as P. communis, P. equi, 10 P. dumbonica, P. lethargicus and P. mai; Ruminomyces spp., such as P. elegans; Anaeromyces spp., such as A. mucronatus and Orpinomyces spp., such as O. bovis and O. joyonii. Caecomyces equi, Piromyces equi, Piromyces dumbonica and Piromyces mai are 15 found in horses and not in the rumen of cattle like the other fungi listed above. Neocallimastix spp. are preferred, particularly N. pairiciarum. Xylanases in accordance with the invention may have a high specific activity. The specific activity may be significantly higher than that of bacterially derived 20 xylanases and may for example be at least 1000, 2000, 3000, 4000, 4500, 5000 or even 5500 U/mg protein, in increasing order of prrference. (A unit of xylanase activity is defined as the quantity of enzyme releasing 1 /xmole of product, measured as xylose equivalents, in 1 minute at 37°C.). More particularly, xylanases in accordance with this aspect of the invention may be significantly 25 better expressed than natural XYLA is expressed by N. pairiciarum: expression may be at least 10 fold improved or preferably at least 100 fold improved over the wild type enzyme. WO 93/25693 PCT/GB93/01283 -6- Xylanases in accordance with the invention may have the ability to degrade xylan at high efficiency. At least 0.1, and preferably at least 0.5 or even 0.75 g reducing sugar may be produced per g xylan substrate. 5 Xylanases in accordance with the invention may have no significant residual activity against cellulose, in contrast to many known xylanases. This property is particularly useful in the application of the invention to the pulp and paper industry, as the enzyme can remove xylan and dissociate lignin from plant fibre without damaging cellulose fibre. 10 Xylanases in accordance with the invention may have at least two catalytic domains. The arrangement of the catalytic domains may be as in a wild type xylanase enzyme, or they may be arranged in an artificial configuration to increase or otherwise improve the xylanolytic activity of the enzyme. 15 A particularly preferred xylanase as a source of catalytic domains for use in the invention, is that derived from Neocallimastix patriciarum and designated XYLA; it has the following properties: 20 (i) a specific activity of 5980 U/mg protein for the purified enzyme when prepared by the following protocol: Host cells (£. co/iXLl-Blue harbouring a plasmid expressing the enzyme) are harvested by centrifugation and resuspended in 50mM 25 Tris-HCl buffer, pH 8.0, and the cytoplasmic fraction prepared as described by Clarke et al, (FEMS Microbiol. Letts. 83 305-310 (1991)). Xylanase, precipitated by the addition of ammonium sulphate (0.39 g/ml), is redissolved in 10 mM Tris-HCl buffer, pH 8.0. After dialysis against three changes of the same buffer, the WO 93/25693 PCT/GB93/01283 -7- xyianase is substantially purified by anion-exchange chromatography on DEAE-Triacryl M essentially as described by Hall et al. (Mol. Microbiol. 3 1211-1219 (1989)). 5 (ii) the ability to degrade xylan at high efficiency, releasing 0.9g of reducing sugar per g of the substrate; (iii) no significant residual activity against cellulose (as determined by no detectable release of reducing sugar from carboxymethyl cellulose. 10 barley /3-glucan, laminarin or lichenan); and (iv) two catalytic domains. The structure of mature XYLA may be represented as follows (from the N-15 terminus to the C-tenninus); CAT1-LINK1-CAT2-LINK2-CTR1-CTR2 wherein: CAT1 represents a first catalytic domain, having the sequence: 20 RLTVGN GQTQHKGVADGYSYEIWLDNTGGSGSMTLGSGATFKAEWN ASVNRGNFLARRGLDFGSQK KATDYSYIGLDYTATYRQTG S ASGNSRLC VYGWFQNRGVQ GVPLVEYYHEDWVDWVPD A QGRMVTIDGAQYKIFQMDHT GPTINGGSETFKQYFSVRQQ 2 5 KRTSGHTTVSDHFKEWAKQG WGIGNLYEVAIv" \EGWQSSG IAD VTKLD VYTTQKGSNP AP; CAT2 represents a second catalytic domain having the sequence K FTVGNGQNQHKGVNDGFSYEIWLDNTGGNGSMTLGSGATF WO 93/25693 PCT/GB93/01283 KAEWNAAVNRGNFLARRGLDFGSQKKATDYDYIGLDYAAT YKQTASASGNSRLCVYGWFQ NRGLNGVPLVEYYIIEDWYD WVPDAQGKMVTIDGAQYKIF QMDHTGPnNGGSETFKQYF SVRQQKRTSGHTTVSDHFKE WAKQGWGIGNLYEVALNAEG 5 WQSSGVADVTLLDVYTTPKG SSPA; LINK1 represents a first linker having the sequence: TSTGTVPSSSAGGSTANGK; LINK2 represents a second linker having the sequence: TSAAPRTTTRTTTRTKSLPTNYNK; 10 CTR1 represents a first C-terminai repeat having the sequence: CSARITAQGYKCCSDPNCWYYTDEDGTWGVENNDWCGCG; and CTR2 represents a second C-terminai repeat having the sequence: VEQCSSKTTSQGYKCCSDPNCWFYTDDDGKWGVENNDWC 15 GCGF. All these partial sequences can be seen in SEQ ID NO: 1 and SEQ ID NO: 2. The structure of xylanases from other anaerobic fungi may be broadly similar, but 20 of course the precise sequences of the components will generally be different, unless the source organism is very closely related to N. pairiciarum. It may not be necessary for the entirety of the sequence of each region (particularly the catalytic domains) to be present for activity; in the present invention, although the entirety of a catalytic domain may be present, it is sufficient for the active portion 25 of the catalytic domain to be present (that is to say, the catalytic domain must be functionally present). The two catalytic domains can be seen to be very similar to each other but not identical. The difference between them gives an indication of the degree of WO 93/25693 PCT/GB93/01283 -9- homology to a natural sequence that is particularly preferred. The two C-terminai repeats can also be seen to be similar to each other (but less so than the two catalytic domains). The difference between them gives an indication of the degree of homology which is still highly preferred. The precise sequence of the two 5 linker sequences may not be particularly important; all that is necessary is that the spatial arrangement of the catalytic domain(s) is such as to enable them to function effectively (and preferably optimally). Preferred embodiments of the invention comprise a catalytic domain which is l o substantially homologous with at least one jf CAT1 and CAT2 and are missing at least pan of the amino acid sequence downstream (ie towards the C-terminus) of CAT2. At least pan of CTR2 may be missing; alternatively or (preferably) additionally, at least pan of CTR1 may be missing. 15 Particular embodiments of xylanases in accordance with the invention include those including (and preferably consisting essentially of) the following regions: A. CATl-IJNKl-CAT2-LINK2-CTRl(trunraff»d) - -NX3); B. CATl-UNKl-CAT2-UNK2(truncated) lct ■ X4); 20 C. LINKl(tnincated)-CAT2-LINK2(truncated) (eg pNX5); D. C ATI -LINKl(truncated) (eg pNX6); E. C ATI (truncated) (eg pNX7); F. LJNK1 (tmncated)-CAT2~LINK2-CTRl-CTR2 (eg pNX8); G. LINKl(tnincated)-CAT2 UNK2-CTR1 (truncated) (eg pNX9); 25 H. LINK1 (truncated)-C AT2(truncated) (eg pNXlO). (The plasmid designations in brackets refer to plasmids in the examples whose expression products are the xylanases shown.) Signal sequences may initially be present but will preferably be absent in the final molecule. Structures C, F, G and 3 0 H are preferred and structures C. G and H are particularly preferred. WO 93/25693 PCT/GB93/01283 -10- Eozymes in accordance with the invention may comprise a single CAT1 domain, a single CAT2 domain, or have two or more catalytic domains, each of which independently may be chosen from CAT1 and CATC. It may be that substantially only catalytic domains are present; and as indicated above it may be that not all 5 of the namrai catalytic domain sequences are essential for adequate activity. On the immature protein a signal peptide may be present; the sequence of the natural signal peptide is: MRTIKFFF AVAIATV AKAQWGGGG AS AGQ. 10 This sequence again is shown in SEQ ID "0:1 and SEQ ID NO:2. Xylanases in accordance with the invention may be prepared by any suitable means. While bulk fermentation of the source anaerobic fungus may be undertaken, and polypeptide synthesis by the techniques of organic chemistry may 15 be attempted, the method of preparation of choice will generally involve recombinant DNA technology. A xylanase as described above will therefore for preference be the expression product of heterologous xylanase-encoding DNA in a host cell. 20 According to a second aspect of the invention, there is provided an isolated or recombinant DNA molecule encoding a xylanase which has a catalytic domain substantially homologous with a xylanase of an anaerobic fungus, provided that the DNA molecule does not comprise a full length copy of natural mRNA encoding the xylanase. 25 cDNA (apparently comprising a full length copy of mRNA) encoding a xylanase of Neocallimastix frontalis has been described by Reymond et al, FEMS Microbiol. Lett. 77: 107-112 (1991), but no expression was reported. WO 93/25693 PCT/GB93/01283 -11- A1 though a full length copy of namrai mRNA is not present in DNA in accordance with this aspect of the invention, it should be understood that the invention is not limited to truncated cDNAs. It is contemplated that some or all of the introns (if any) naturally present in the corresponding wild type gene may be present. 5 However, at least some sequence that is present in the full length cDNA is absent in DNA in accordance with this aspect of the invention. It should also be understood that this aspect of the invention encompasses DNAs encoding full length xylanases; the absent portion of the DNA may be (and in some embodiments preferably is) in the 3' and/or 5' untranslated regions. Substantially 10 full length or truncated xylanases may therefore be produced from DNA in accordance with this aspea of the invention which (a) is substantially missing the 3' untranslated region, or (b) is substantially missing the 5' untranslated region or (c) is substantially missing both the 3' and 5' untranslated regions. 15 A full length cDNA encoding a xylanase of an anaerobic fungus (taking the xynA gene of N. pamciarum as the prototype) may have the following structure: 5 'urr-sig-cail -linkl -caf2-lirHQ.-ctr\-ctfl-3'uxr, wherein 20 5'uxr represents a 5' untranslated region; sig encodes a signal peptide; carl encodes a first catalytic domain; linkl encodes a first linker sequence; cat2 encodes a second catalytic domain; 25 linkl encodes a second linker sequence; crrl encodes a first C-terminal repeat; ctiQ. encodes a second C-terminal repeat: and 3'uir represents a 3' untranslated region. WO 93/25693 PCT/GB93/01283 -12- Genomic sequences may have one or more introns interspersed within the above structure. In the xynA gene encoding the XYLA enzyme of N. pairiciarum, the various DNA segments have the following sequences: 5 3'Utr: ttttattatatcaatctctaatttatttttttaggaaaaaaataaaaaaataaatataat aaatattagagagtaatatttaaaaacaaagaaatttaaaaacgtttatttagttatttt ttttactggttaaaaaaaaaataaaaaacaaaattaataaagatatttttgaaaaatatt gaattagaaaaaaa; 10 sig: atgagaactattaaattctttttcgcagtagctattgcaactgttg ctaaggcccaatggggtggaggtggtgcctctgctggtcaa ; 15 catl: agattaaccgtcggtaatg gtcaaacccaacataagggtgtagctgatggttacagttatgaaatctggttagataaca ccggtggtagtggttctatgactctcggtagtggtgcaaccttcaaggctgaatggaatg catctgttaaccgtggtaacttccttgcccgtcgtggtcttgacttcggttctcaaaaga 2 0 aggcaaccgattacagctacattggattggattatactgcaacttacagacaaactggta gcgcaagtggtaactcccgtctctgtgtatacggttggttccaaaaccgtggagttcaag gtgttccattggtagaatactacatcattgaagattgggttgactgggttccagatgcac aaggtagaatggtaaccattgatggagctcaatataagattttccaaatggatcacactg gtccaactatcaatggtggtagtgaaacctttaagcaatacttcagtgtccgtcaacaaa 25 agagaacttctggtcatattactgtctcagatcactttaaggaatgggccaaacaaggtt ggggtattggtaacctttatgaagttgctttgaacgccgaaggttggcaaagtagtggta tagctgatgtcaccaagttagatgtttacacaacccaaaaaggttctaatcctgcccct; liokl: 3 0 acctccactggtactgttccaagcagttctgctggtggaagtactgccaatggtaaa; cat2: aagt ttactgtcggtaatggacaaaaccaacataagggtgtcaacgatggtttcagttatgaaa 3 5 tctggttagataacactggtggtaacggttctatgactctcggtagtggtgcaactttca AGGCTGAATGGAATGCAGCTGTTAACCGTGGTAACTTCCTTGCCCGTCGTGGTCrTGACT tcggttctcaaaagaaggcaaccgattacgactacattggattagattatgctgctactt acaaacaaactgccagtgcaagtggt/actcccgtctctgtgtatacggatggttccaaa WO 93/25693 PCT/GB93/01283 -13- ACCGTGGACTTAATGGCGTTCCTTTAGTAGAATACTACATCATTGAAGATTGGGTTGACT GGGTTCCAGATGCACAAGGAAAAATGGTAACCATTGATGGAGCTCAATATAAGATTTTCC AAATGGAT CACACTG GT C CAACTAT CAAT GGT GGTAGTGAAAC CTTTAAG CAATACTT ca GTGTCCGTCAACAAAAGAGAACTTCTGGTCATATTACTGTCTCAGATCACTTTAAGGAAT 5 GGGCCAAACAAGGTTGGGGTATTGGTAACCTTTATGAAGTTGCTTTGAACGCCGAAGGTT GGCAAAGTAGTGGTGTTGCTGATGTCACCrTATTAGATGTTTACACAACTCCAAAGGGTT CTAGTCCAGCC; link2: 10 ACCTCTGCCGCTCCrCGTACTACTACCCGTACTACTACTCGTACCAAGTCTCTTCCAACC AATTACAATAAG; Ctrl: TGTTCTGCTAGAATTACTGCTCAAGGTTACAAGTGTTGTAGCGATCCAAATTGTGTTGTT 15 TACTACACTGATGAGGATGGTACCTGGGGTGTTGAAAACAACGACTGGTGTGGTTGTGGT ; ctr2 : GTTGAACAATGTTCTTCCAAGATCACTTCTCAAGGTTACAAGTGTTGTAGCGATCCAAAT 2 0 GGTTGTGGTTTC; and 5'utr: TAAGCAGTAAAATACTAATTAATAA AAAATTAAAGAATTATGAAAAATTTAAATTTAAAAATTTAAAAGAATTATGAAAAATTTA 2 5 AATTTAAAAATTTAAAAAAAACTAATTTAGTAAAAAATTAAAGAATTATTGAAAATTTTA AATGTAAAAATTTAAAAAATACAAATTTGTAAAAAAAAATGAAA.GAATTATGAAAAATTA AAATGTAAAAGTTTAAAAAATACAAATTTGTAAGAAAAATAAAjGAATTATAAAAAAAATA AAGAATTATGAAAAACCCAAATGTAAAGAAAAAAAAAAAAAAAAAAfiAAAAAAARAAA 3 0 (Note that the first three nucleotides of the 5 'utr segment constitute-a stop codon, which will generally be present.) The use of (less than the totality of) these-DNA segments, or sequences substantially homologous with them, is preferretTin this aspect of the invention. 3 5 Preferred embodiments correspond generally to the preferred embodiments:(if the xylanases per se in accordance with the first aspect of the invention, but with the WO 93/25693 P CT/GB93/01283 -14- added considerations that (a) it may be preferred for a DNA sequence encoding a peptide signal sequence to be present and/or (b) it may be preferred for one or both of the untranslated regions to be truncated or absent. Particular embodiments of this aspect of the invention include those including (and preferably consisting 5 essentially of, apart from vector-derived sequences) the following segments: a. 5 'iur-sig-caxl-linkl-cal2-link2-ctr\(truncated) (eg pNX3); b. 5 'iar'Sig-catl-linkl-cat2-link2(tnmcaied) (eg pNX4); c. Knifcl(truncated)-car2-/i/iA2(tiuncated) (eg pNX5); d. 5 'utr-sig-catl-linkl(tnmcated) (eg pNX6); e. 5 'idr-szg-car 1 (truncated) (eg pNX7); /• linkl(mxo&xed)-cat2-tink2-ctrl-ctrQ.-3 'utr (eg pNX8); 8- /mid(truncated)-car2-/zn/2-crrl(tnincated) (eg pNX9); h. /in/H(truncated)-azr2(truncated) (eg pNXlO). 15 (The plasmid designations in brackets refer to plasmids in the examples including the DNA sequences shown.) Structures c, /, g and h are preferred and structures c, g and h are particularly preferred. 20 Recombinant DNA in accordance with the invention may be in the form of a vector. The vector may for example be a plasmid, cosmid or phage. Vectors will frequently include one or more selectable markers to enable selection of cells transfected (or transformed: the terms are used interchangeably in this specification) with them and, preferably, to enable selection of cells harbouring 25 vectors incorporating heterologous DNA. Appropriate start and stop signals will generally be present. Additionally, if the vector is intended for expression, sufficient regulatory sequences to drive expression will be present. Vectors not including regulatory sequences are useful as cloning vectors; and. of course, expression vectors may also be useful as cloning vectors. WO 93/25693 PCT/GB93/01283 -15- Cloning vectors can be introduced into E. coli or another suitable host which facilitate their manipulation. According to another aspect of the invention, there is therefore provided a host cell transfected or transformed with DNA as described above. 5 DNA in accordance with the invention can be prepared by any convenient method involving coupling together successive nucleotides, and/or ligating oligo- and/or poly-micleotides, including in vitro processes, but recombinant DNA technology forms the method of choice. 10 Xylanase-encoding DNA may be cloned from a DNA library, which may be prepared from one of the above fungi. The library may be genomic, but a cDNA library may be easier to prepare and work with, particularly if steps are taken to enhance the likelihood of the presence of xylanase-encoding cDNA in the cDNA 15 library. Cultivation of a chosen fungus, such as N. pairiciarum, may proceed anaerobically in an appropriate culture medium containing rumen fluid; the sole or predominant carbon source may be xylan so as to promote xylanase expression and, hence, to 2 0 cause an increase in the amount of xylanase-encoding RNA. However, cultivation in the presence of xylan is not essential, and the carbon source may instead be a cellulose, such as the microcrystalline cellulose sold under the trade mark Avicel. After cultivation of the fungus, total RNA may be extracted in any suitable 25 manner. Fungal cells may be harvested by filtration and subsequently lysed in appropriate cell lysis buffer by mechanical disruption. A suitable RNA preserving compound, such as guanidinium thiocyanate, may also be added to the fungal cells to reduce or prevent RNase-mediated digestion. Total RNA may subsequently be isolated from the resulting homogenate by any suitable technique such as by WO 93/25693 PCT/GB93/01283 -16- ultracemrifugation through a CsCl2 cushion or as described in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989). 5 Another method for preparation of total fungal RNA in addition to that described above may be based on or adapted from the procedure described in Puissant and Houdebine Bio-Techniques 148-149 (1990). In this method, total fungal RNA can be isolated from the above homogenate by extraction with phenol/chloroform at pH 4 to remove DNA and associated protein. The resulting crude RNA was further 10 purified by washing with lithium chloride urea solution. A suitable further technique for fungal RNA extraction is that of Teeri et al. (Anal. Biochem. 164 60-67 (1987)). 15 Once total RNA has been extracted, by whichever method, poly-A+ mRNA may then be isolated from the total RNA, for example by affinity chromatography on a compound containing multiple thymidine or uracil residues, to which the poly-A tail of the mRNA can bind. Examples of suitable compounds include oligo-dT cellulose and poly-U Sephadex". Poly-A+ mRNA can, then be eluted by a 20 suitable buffer. A cDNA expression library may then be constructed using a standard technique based on conversion of the poly-A+ mRNA to cDNA by reverse transcriptase. While it is possible to construct a genomic library, a cDNA library is preferred 25 because it avoids any difficulties which may be caused by the presence of intxons in the fungal genomic DNA. The first strand of cDNA may be synthesised using reverse transcriptase and the second strand may be synthesised using any suitable DNA-directed DNA polymerase such as Escherichia coli DNA polymerase I (E. coli pol I). WO 93/25693 PCT/GB93/01283 -17- The cDNA may subsequently be fractionated to a suitable size and may be iigated to a suitable vector which is preferably a phage vector such as XZAP, XZAPII or Agt 11. Suitable kits for the purpose are available from Stratagene. Further or alternative guidance may be had from Reymond et al (FEMS Microbiol. Lett. 17 5 107-112 (1991)) which details the preparation of a cDNA library from N. frontalis. The resulting cDNA library may then be amplified after packaging in vitro, using any suitable host bacterial cell such as an appropriate strain of E. coli. The screening of xylanase positive recombinant clones may be carried out by any 10 suitable technique, which may be based on hydrolysis of xylan. In this procedure the clones may be grown on culture media incorporating xylan and hydrolysis may be detected by the presence of xylanase-positive plaques suitably assisted by a suitable colour indicator. Methods for selecting xylanase+ clones are described in the literature. Two examples are Clarice et al. (FEMS Microbiol. Lett. S3 305-310 15 (1991)) and Teather and Wood (Appl. Environ. Microbiol. 43 777-800 (1982)). Xylanase positive recombinant clones may then be purified (that is to say a plaque may be converted to a bacterial colony) by well established procedures. Suitable techniques can be found in Sambrook et al (1989) (loc. tit.), but it would be usual 20 simply to follow the manufacturer's instructions in whichever kit was being used and the cDNA insert in the clones may then be excised into a vector of choice, such as pBLUESCRlPT^*^ to name only one example. Other suitable plasmids can be used for subcloning; examples include the pUC plasmids and plasmids derived from them, as described in Sambrook et al. Molecular Cloning: A Laboratory 25 Manual, 2nd edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989). Expression vectors (particularly plasmids) in which the xylanase-encoding DNA is under the control of an appropriate promoter may also be formed by ligation and transformed and transfected into a suitable expression host. Examples of suitable expression vectors include the pUC series (which have WO 93/25693 PCT/C*?93/01283 -18- the lacZp promoter), the pMTL series (which also have the lacZp promoter and • pBluescript (which has both the lacZp promoter and the T7 promoter). The nature of the promoter is not in general believed to be particularly critical and 5 will depend on the expression host and the conditions under which expression is desired. As indicated above, a suitable example for a bacterial expression host such as E. coli is the lacZ promoter. Alternative promoters for bacterial hosts include the bacteriophage T7 promoter. 10 It may not be necessary to purify recoruoinant xylanases from their expression hosts. While E. coli as a host cell may be suitable for application of the xylanase of the invention in pulp manufacture, it will be appreciated that other host cells could be used such as gram positive bacteria inclusive of Bacillus subtilis, or lactic acid bacteria. Alternatively a eukaryotic expression host may be used; an example 15 would be yeast (such as Saccharomyces cerevisiae). Host cells expressing xylanases as described above and/or harbouring DNA sequences as described above (whether for expression or otherwise) themselves constitute a further aspect of the invention. Also included in the invention are 2 0 methods of preparing a host cell, in which xylanase-encoding DNA is transformed or transfected into a cell, and methods of producing a * xylanase, in which expression hosts are cultivated to express xylanase-encoding DNA. Depending on the nature of the host cell, it may be preferred for recombinant 2 5 DNA in accordance with the invention to include a signal sequence. Either a host-specific signal sequence may be included or, for expression in eukaryotes, the enzyme's own signal sequence may be used. A translational start site adapted for or preferred by the expression host may be provided; however, the protein's own translational start site may be adequate or even in some circumstances preferred. WO 93/25693 PCT/G B93/01283 -19- Recombinant xylanase enzyme from an expression host may then be characterised. Principal features that have been ascertained for certain embodiments of the invention are as follows: 5 (i) the cloned xylanase has a very high specific activity (S980 U/mg protein of the purified enzyme); this is in contrast to many cloned xylanases from bacteria which have been reported so far; (ii) the enzyme is able to degrade xylan at extraordinarily high efficiency, releasing 0.9g of reducing sugar per g of the substrate.; io (iii) the enzyme has no residue! activity again cellulose, while many other xylanases possess some cellulase activity; and (iv) the enzyme contains two catalytic domains, which may have potential for construction of a highly efficient xylanase-producing clone by further genetic manipulation of the xylanase cDNA. 15 The high specific activity of the full length cloned xylanase (hereinafter referred to as xylanase A) (5980 U/mg protein of the purified enzyme) is an intrinsic property of this fungal xylanase. However, the expression level of the present construct oixynA cDNA in pBluescript vector (pNXl) is relatively low in£. coli, 20 accounting for 0.3% of soluble protein synthesised by E. coli cells. Generally speaking, the expression of the cloned gene at the level of > 10% of total cellular E. coli protein is attainable. Truncated forms of xynA cDNA may be prepared by the use of restriction 25 enzymes. Some truncated forms, including that in the plasmid designated pNX5, produce several hundred-fold higher xylanase activity than pNXl. One explanation for this observation is that is a result of the utilisation of LacZ translation initiation sequence for the synthesis of the truncated xylanase A. Another explanation is that avoidance of AT-rich regions may result in higher expression levels; a theory is WO 93/25693 PCT/GB93/01283 -20- that Ac mRNA degrading activity of RNase E is the rate limiting step in protein synthesis, and that RNase E has a preference for AT-rich regions of mRNA. It is possible to further increase its expression level in E. coli by usiflg a stronger promoter, such as Bacteriophage T7 promoter. 5 Recombinant xylanase A (XYLA) purified from Escherichia coli harbouring xynA, had an Mr, of 53000 and hydrolysed oat spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of Mr 10 66192. The predicted primary structure of XYLA comprised of an N-terminal signal peptide followed by a 225 amino acid repeated sequence, which was separated from a tandem 40 residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full length enzyme. 15 Xylanases in accordance with the invention have a number of applications in the food, feed, and pulp and paper industries. The use of xylanases described herein in these industries is included within the scope of the invention. 20 Dealing first with the food industry, certain properties of dough and its resultant baked products are dependent on the pentosan and starch content of the flour used. These properties include the texture, volume and staling of bread. The use of xylanase could modify baked products to provide goods of potential commercial value. Among the properties that can be modified by xylanase treatment is the 25 specific volume of bread. The increase in specific volume is enhanced further when amylase is added in combination with xylanase. One of the factors contributing to this effect is the water-binding capacity of carbohydrates. The invention provides dough including a xylanase as described herein. WO 93/25693 PCT/GB93/01283 -21- ln the animal feed industry, the use of enzyme supplementation to improve feed for chicks was reported as early as 1957. More recent results suggest that, in certain grains such as wheat, and particularly rye, it is the pentosans in the endosperm that are mainly responsible for poor nutrient uptake and sticky 5 droppings from the chicks. Both problems appear to result from the high viscosity of the undigested oentosans. This hampers the diffusion of nutriems and binds water to malce excreta watery. The problems can be alleviated using xylanase preparations. Xylanase action can improve both the weight gain of chicks and their feed conversion efficiency. Jt app ~ at xyiu^»e supplementation could 10 be used to improve the nutritic- -alue i - so as to promote the use of this grain in chick feed. The effe: :ss of u. sztment may be dependent on the vi iety of rye. The invention . rovides the u f xylanase in chick feed and grain for these purposes. 15 In the pulp industry, dissolving pulps are purified celluloses used ftr iking viscose rayons, cellulose esters and cellulose ethers. They are r . from prehydrolysed kraft pulps or acid sulphate pulps. Their processing is characterised by the derivatisation of the cellulose at one stage, the derivative being soluble in common solvents and thus permitting the formation of fibres, films and plastics. 20 Impurities in the cellulose hamper derivatisation and thus lead to insolubles that block orifices in sprayers or form defects in the final product. Furthermore, certain xylan impurities can lead to colour, haze and thermal instability in acetate products. Xylanases may thus have a role to play in removing impurities, the use of xylanases described herein for this purpose is comprehended v ^ the 25 invention. The prebleaching of kraft pulp using cellulase-free xylanase has been identified as one of the biotechnologies most likely to be accepted in the pulp and paper industry in the near future, but only if suitable xylanases become available. The WO 93/25693 PCT/GB93/01283 -22- kraft (also known as alkaline or sulphate) process has become the predominant pulping technology in Canada because it produces strong wood fibres and because the chemicals used are recovered and recycled. Kraft pulps, particularly those derived from softwoods, are relatively difficult to bleach. A sequence of stages 5 using elemental chlorine and chlorine-containing compounds is traditionally required to bleach these pulps effectively to the desired full brightness of -90%. The bleaching process, particularly when using elemental chlorine, products chloro-organics that have traditionally been discharged from the bleach plant with the waste water. However, both public demand and legislated regulations are 10 presently pressurizing pulp mills to reduce or eliminate the emission of these pollutants. The pulp and paper industry is considering the implementation of various alternative technologies in order to reduce the environmental impact of its mills These options include xylanase prebleaching of kraft pulp. Xylanases in accordance with the present invention are particularly well suited to this purpose. 15 It is believed that the xylanases of the present invention are particularly applicable to the paper and pulp industry. While it is appreciated that the use of enzymes will never replace chemicals completely, there is pressure being exerted by those concerned with the environment to reduce the use of chemicals. There are also 20 practical reasons for reducing the use of chemicals in the paper and pulp industry. Pulping plants usually generate their own supplies of chlorine and chlorine dioxide on site, and this can limit capacity as well as being potentially hazardous. Treating the paper pulp (eg kraft pulp) to remove lignin involves the use of chlorine, 25 NaOH, H202 and chlorine dioxide. Sandoz in the USA have conducted practical trials using their CARTAZYME product, which is a fungal xylanase (crude), active at 30-55 °C, pH 3 to 5, and contains 2 xylanases, and have found that a 25-33% reduction in chlorine is possible using 1U xylanase/gm pulp. Also the product is brighter than when chemicals alone are used. Another advantage of the xylanase WO 93/25693 PCT/GB93/01283 -23- is that it is specific whereas chemicals can attack the cellulose at low lignin contents, leading to reduced fibre strength and other undesirable physical characteristics. It is therefore clear that xylanases could become more important in pulp bleaching and recombinant ones particularly so because of their specificity 5 and high yield. It is believed that lignin is bonded to hemicellulose, and if the hemicellulose (xylan) is depolymerised the lignin may be partially disassociated from cellulose and subsequently washed out. At present, however, some chemical treatment may still be necessary. The main points about xylanase of the present invention, with respect to commercial use, are (i) its very high specific activity and l o high level of expression would make it economical to produce on a large scale and (ii) its lack of cellulase activity make it particularly useful where it is necessary to remove xylan specifically as applied to the paper making and textile industry. It is also believed that the xylanase of the invention could find a valuable 15 application in the sugar industry and in relation to the treatment of bagasse or other products containing xylan for more efficient disposal. It was previously mentioned that the protein sequence of XYLA and the DNA sequence of xynA. were made available on 5 May 1992 on the EMBL database 20 under accession number X65526. This availability may not constitute effective prior art in the jurisdictions of all of the states designated in this application. For those jurisdictions where the EMBL database entry does not constitute effective prior art, notice is hereby given that the invention is and will be defined more broadly than as indicated above. In particular, the invention may then be seen to 25 reside in the following further aspects: a xylanase which has at least one catalytic domain which is substantially homologous with a xylanase of an anaerobic fungus; the xylanase may be a full length natural xylanase of an anaerobic fungus; and SUBSTITUTE SHEET ISA/EP WO 93/25693 PCT/GB93/01283 -24- an isolated or recombinant DNA molecule encoding a xylanase which has a catalytic domain substantially homologous with a xylanase of an anaerobic fungus, provided that if the DNA molecule is cDNA encoding a xylanase of Neocallimastix frontalis then the DNA molecule is operatively 5 coupled to a promoter; the DNA molecule may comprise a full length copy of natural mRNA encoding the xylanase. It will be apparent from the foregoing that the invention includes within its scope not only the recombinant xylanase described above but also xylanases derived from 10 other anaerobic fungi as described above ^'hich may be prepared by the methods described herein. The invention also includes within its scope any mutant derived from N. pairiciarum or strains derived from N. pairiciarum by selection or gene transfer. 15 The invention also includes within its scope (i) DNA sequences derived from pNXl, pNX4, pNX5, pNX6, pNX8, pNX9 and pNXlO and DNA sequences capable of hybridising thereto; 20 (ii) a DNA construct containing a DNA sequence as in (i) operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xylanase activity in a suitable expression host; (iii) a transformed microbial host capable of the expression or over-2 5 expression of a fungal xylanase containing an expression construct as in (ii); (iv) a polypeptide having xylanase activity produced by expression using a microbial host as in (iii); (v) amino acid sequence as shown in Figure 4 including WO 93/25693 PCT/GB93/01283 10 -25- componenis A, B, C and D and amino acid sequences derived from this xylanase; and (vi) plasmids described in Figure 1. The invention also includes within its scope a method of preparation of a xylanase from E. coli harbouring the recombinant plasmids as shown in Figure 1. Each preferred feature described above with reference to one aspect of the invention is equally preferred, mutatis mutandis, for each other aspect. The invention will now be illustrated by the following examples. The examples refer to the accompanying drawings, in which: FIGURE 1 is a restriction map of recombinant plasmids containing xynA. 15 The positions of the cleavage sites of EcoBl (R), Sstl (S), Seal (Sc), Hpal (Hp), Kpnl (K), Xhol (X), Smal (Sm), PvuTl (Pv), NaeI (Na), Nrul (Nr), Stul (St) and HindHl (H) are shown. Restriction sites of multiple cloning regions or vectors in parenthesis have been destroyed. Multiple cloning regions of vectors, designated by *, are derived from pSK(S), pMTL20(20) 20 and pMTL22(2) respectively. The solid line with an arrow shows the extent and orientation of the xynA open reading frame. Construction of the deletion mutants of xynA is detailed below. The phenotypes of E. coli strains harbouring the recombinant plasmids are shown. 25 FIGURES 2A and 2B show the purification of XYLA. SDS/PAGE of XYLA purified from cell-free extract E. coli XLl-Blue harbouring pNXl (A) or pNX5(B). Lane 1 contained XYLA purified by anion exchange chromatography, lane 2 contained cell-free extract from E. coli harbouring pNXl or pNX5 and lane 3 (B only) contained cell-free extract from E. coli WO 93/25693 PCT/GB93/01283 -26- comaining pBluescript SK. Gels depicted in A and B contained 10% (w/v) or 15 % (w/v) polyacrylamide, respectively. Protein sizes are shown in kD, deduced from the marker proteins which are high (Figure 2A) or low (Figure 2B) molecular weight markers from Sigma. FIGURE 3 shows the effect of purified XYLA on the specific viscosity of soluble xylan (0.5%) in PC buffer, pH 6.5 at 37°C. Specific viscosity (■) and reducing sugars (•) were measured as described below. 10 FIGURE 4 shows the primary structure o£.XYLA. The two homologous catalytic domains, designated A and B, together with the duplicated C-tenninal sequences (C and D) are boxed. FIGURE 5 shows the alignment of homologous regions of N. patririorum XYLA and prokaryote xylanases. The enzymes compared were as follows: B. pumilus xylanase A (XYLAB; Fukusaki et al, FEBS Lett. 171: 197-201 (1984)), B. circulans xylanase (XYLBC; Yang et al, Nucl. Acids Res. 16: 7178 (1988)) and C. acetoburylicum xylanase B (XYLBCA; Zappe et al., Nucl. Acids Res. 18 2179 (1990)). Residues which show identity or similarity in all primary sequences compared are boxed. The positions of the first and last residues of homologous regions, in their respective primary sequences, are shown. FIGURE 6 shows the structure of plasmid pNXl. 25 FIGURE 7 shows the cloning and characterisation of Neocallimastix pairiciarum xylanase A encoding cDNA. 15 20 WO 93/25693 PCT/GB93/01283 -27- EXAMPLE 1 - Prepararion of oNXl 1.1 Microbial strains, vectors and culture media The anaerobic fungus Neocallimastix pamciarum (type species) was isolated from 5 a sheep rumen by Orpin, C.G., and Munn, E.A., Trans. Br. Mycol. Soc. 86: 178-181 (1986). Host strains for cDNA cloning were E. coli PLK-F' and XLl-Blue. E. coli strain JM83 was used for characterisation of the xylanase+ cDNA clones. The vectors were XZAPII, pBLUESCRiPT51^ (Stratagene), pMTL20, pMTL22 and 10 pMTL22 "hambers et al, Gene 68: 139-149 (1988)). N. patrician** iture was maintained, in a medium containing 10% rumen fluid as describee up et al, J. Gen. Microbiol. 130: 27-37 (1984)). E. coli strains were grown in L-broth (Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989). The 15 recombinant phage were grown in E. coli strains using NZY medium according to Stratagene's instructions. 1.2 General recombinant DNA techniques Agarose-gel electrophoresis, transformation of E. coli and modification of DNA 2 0 using restriction enzymes and T4 DNA ligase were as described by Gilbert et al., J. Gen. Microbiol. 134 3239-3247 (1988). Large amounts of plasmid DNA was extracted from E. coli by Brij lysis' and subsequent CsCl d"-isity-gradient centrifugation (Clewell, D.B., and Helinski, D.R., Proc. Natl. Acua. Sci. USA 62: 1159-1166 (1969)). The rapid boiling method of Holmes, D.S., and C :igley, M., 25 Anal. Biochem. 114: 193-197 (1981) and the alkaline lysis method of Birnboim, H.L. and Doly, J., Nucl. Acids Res. 7: 1513-1523 (1979) were employed to isolate plasmid for rapid restriction analysis and nucleotide sequencing, respectively. Northern hybridisation was as described by Gilbert et al, J. Bacteriol. 161: 314-320 (1985). WO 93/25693 PCI7GB93/01283 -28- 1.3 Cultivation of rumen anaerobic fungus. N. patriciarum N. patriciarum was grown in a rumen fluid-containing medium (Kemp et al, J. Gen. Microbiol. 130: 27-37 (1984)) in the presence of 1% Avicel at 39°C and anaerobic conditions for 48hr (Alternative culture media, such as described by 5 Philips, M.W., and Gordon, G.L.R., Appl. Environ. Microbiol. 55: 1695-1702 (1989) and Lowe et al, J. Gen. Microbiol. 131: 2225-2229 (1985), can be used. 1.4 Total RNA isolation The frozen mycelia were ground to fine powder under liquid nitrogen with a 10 mortar and pestle. 5-10 vol of guanidiniuii; thiocyanate solution (4M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25mM sodium citrate, pH 7.0, ImM EDTA and 0.1M &■mercaptoethanol) was added to the frozen mycelial powder and the mixture was homogenised for 5 min with a mortar and pestle and for a further 2 min at full speed using a Polytron homogeniser. Total RNA was isolated from 15 the homogenate by ultracentrifuganon through a CsCl cushion (Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press (1989). (Alternative method for preparation of total fungal RNA, such as adaptation of the procedure described by Puissant, C., and Houdebine, L.M., Bio-Techniques 148-149 (1990), can be used). 20 1.5 Polv A+ mRNA purification Poly A+ mRNA was purified from the total RNA by Oligo (dT) cellulose chromatography (Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press 25 (1989). 1.6 Construction of a cDNA expression library of N. patriciarum The cDNA library was constructed, using Stratagene's XZAP cDNA synthesis kit, basically according to the manufacturer's instructions. WO 93/25693 PCT/GB93/01283 -29- The procedure is described briefly as follows: Poly A+ mRNA was converted to the first strand cDNA by reverse transcriptase, using Xhol linker - oligo (dT) primer and 5-methyi dCTP. Double-stranded cDNA was synthesised from the first-strand cDNA by the action of RNase H and DNA polymerase I. After 5 blunting cDNA ends, the cDNA a-as Iigated with EcoRl adaptor, phosphorylated and digested with Xhol to create cDNA with EcoKL site at 5' region and Xhol site at 3' region. The cDNA was size-fractionated by 1 % low-melting point agarose gel electro-phoresis and 1.2-8 Kb sizes of the cDNA were recovered by phenol extraction (Sambrook et al, Molecular Cloning. A Laboratory Manual, 2nd 10 edition. Cold Spring Harbor, New York. Cold Spring Harbor Laboratory Press (1989)). The size-fractionated cDNA was then Iigated to the EcoBUXhol digested XZAPII vector (other expression vectors can be used). The cDNA library was packaged in vitro and amplified using E. coli PLK-F as 15 plating cells. 1.7 Screening xvlanase-positive recombinant bacteriophage clones Recombinant phage were grown in E. coli XLl-Blue in 0.7 % top agar containing 0.1% xylan and lOmM isopropyl-jS-D-thiogalactopyranoside (IPTO, an inducer for 20 LacZ promoter controlled gene expression). After overnight incubation at 37°C, 0.5 % Congo red solution was added over the top agar. After incubation at RT for 15 min, the unbound dye was removed by washing with 1M NaCl. Xylanase-preducing phage plaques were surrounded by yellow haloes against a red background. 25 The xylanase-positive recombinant phage were purified to homogeneity by replating and rescreening the phage as above for 2-3 times. WO 93/25693 PCT/GB93/01283 -30- The cDNA inserts in xylanase-positive phage were excised into pBLUESCREPT SK* using VCS-M13 helper phage. 1.8 Xylanase and related-enzvme assays 5 The enzyme extracts from E. coli harbouring xylanase-positive recombinant plasmids were prepared as described by Kellett et al, Biochem. J. 272: 369-376 (1990). The enzymes were assayed for hydrolysis of xylan or other substrates at 37 °C in 10 50mM potassium phosphate /12mM citric add buffer, pH 6.5 and the reducing sugars released from xylan or other plant polysaccharides (carboxymethyl cellulose, barley /3-glucan, laminarin, lichenan) were measured as described by Kellett et al, Biochem. J. 272: 369-376 (1990) and Hazlewood et al, J. Gen. Microbiol. 136: 2089-2097 (1990). Assays for activities against artificial 15 substrates(methylumbelliferyl-|3-D-cellobiosidejnethylumbelliferyl-|3-D-glucoside, methylumbelliferyl-0-D-xyloside and p-nitrophenyl-/3-D-xyloside) were described by Hazlewood et al, J. Gen. Microbiol. 136: 2089-2097 (1990). 1.9 DNA sequencing 20 Plasmid DNA, denatured by alkali, was neutralised and further purified by spin dialysis (Murphy, G., and Kavanagh, T., Nucl. Acid Res. 16: 5198 (1988)). Sequencing of the resultant DNA was based on the protocol recommended by the manufacturer of the Sequenase DNA sequencing kit (USA, Cleveland, OH). Overlapping sequences were generated by cloning appropriate restriction fragments 25 into pMTL-based vectors. Sequences were compiled and ordered using the computer programs described by Staden, R., Nucl. Acids Res. 16: 3673-3694 (1980). The complete sequence of the cDNA contained in the plasmid designated pNXl was determined in both strands. The xylanase-encoding gene contained in the plasmid was designated xynA and the gene product, the xylanase enzyme itself, 3 0 was designated XYLA. WO 93/25693 PCT/GB93/01283 -31- EXAMPLE 2 - Construction of pNX4. a Deletion Mntant nf pNXl (xvnA\ pNXl was linearised by Xhol and the 3' region of xynA cDNA was removed by Bal-31 digestion (Hall, J., and Gilbert, H.J., Mol. Gen. Genet. 213: 112-117 5 (1988)). After blunt ending, the truncated cDNA was excised from pNXl by £coRI digestion and cloned into EcoSUSmaL digested pMTL22 vector. EXAMPLE 3 - Construction of pNX5. a Deletion Mutant nf pNXl (xvnA) 10 720bp ScdUNrul fragment was excised from pNX4 and cloned into pMTL20 vector. This resulted in a highly expressing clone, in which the enzyme expression levels were some hundreds higher than for pNXl. EXAMPLE 4 - Construction of pNX6. a Deletion Mutant of pNXI (xvnA) 15 pNX6 was constructed by cleaving pNXl with EcoBl/Scal and cloning the resulting fragment into EcoRl/Smal-cut pMTL22. EXAMPLE 5 - Construction of pNX8. a Deletion Mutant of pNXI (xvnA) 20 pNXl was digested with Seal and Xhol to obtain 1.3kb fragment which was cloned into pMTL20 so that the XynA sequence was in phase with the LacZ ATG contained in the vector. This resulted in a high expression clone in which the expression level was approximately fifteen times that of pNXl. 25 EXAMPLE 6 - Construction of pNX9. a Deletion Mutant of pNXI (xvnA) pNX8 was cut with Kpnl (1 site in vector poly linker) and the insert fragment, after electroelution was digested with Jfral (cuts in the PT linker region of the gene) to WO 93/25693 PCT/GB93/01283 -32- producc a -700bp fragment which was cloned into pMTL20 which had been cut with Kpnl and Stul. This resulted in a highly-expressing clone (much better than clone containing pNX8) with second catalytic domain in frame with vector LacZ N-tennmus. 5 EXAMPLE 7 - Construction of pNXIO. a Deletion Mutant of pNXI (xvnA) pNX8 was digested with Kpnl and the fragment (-850bp) was Iigated into Kpnl-cut pMTL20. This clone also expressed well but the protein expressed contains some 10 residues at the carboxy end, which wiien removed allow for the high level expression observed for pNX9. EXAMPLE 8 - Purification and amino acid sequencing of the N-terminus of xvlanase A 15 E. coli XL 1-Blue harbouring pNXl or pNX5 was cultured for 16 hours in LB broth containing ampicillin (lOOug/ml). Cells, harvested by centrifugation, were resuspended in 50mM Tris/HCl buffer, pH 8.0 and the cytoplasmic fraction prepared as described previously (Clarke et al, FEMS Microbiol. Lett. 83: 305-310 2 0 (1991)). Xylanase, precipitated by the addition of ammonium sulphate (0.39g/ml), was redissolved in lOmM Tris/HCl buffer, pH 8.0. After dialysing against 3 changes of the same buffer, the xylanase was substantially purified by anion exchange chromatography on DEAE-Trisacryl M essentially as described by Poole et al, Mol. Gen. Genet. 223: 217-223 (1990). 25 The xylanase (designated XYLA) purified from cell-free extract of E. coli XL1-Blue harbouring pNXl was fractionated by SDS/PAGE and electroblotted onto Problot" membrane (Applied Biosystems Inc). N-terminal sequence was determined by automated Edman sequencing using a 470 gas-phase sequenator WO 93/25693 PCT/GB93/01283 -33- equipped with a 120A on-line phenyithiohydantoin analyser (Applied Biosystems Inc: Hunkapillar et al, Methods Enzymol. 91: 399-413 (1983)). EXAMPLE 9 - .Summary of Isolation of xvnA 5 A cDNA library consisting of 106 clones was constructed using mRNA isolated from N. patriciarum cells grown with AviCEL as sole carbon source. Thirty one recombinant bacteriophages which hydrolysed xylan were identified after screening 5 x 104 clones from the library, and 16 strongly xylanase-positive phage were 10 isolated for further characterisation. Restiiction mapping and hybridisation data indicated that all the xylanase- positive recombinants contained cDNA sequences derived from the same mRNA species. A restriction map of the largest cDNA sequence encoding a functional xylanase, designated xynA, is shown in Figure 1. A nucleic acid probe consisting of 1.7kb of the 5' region of xynA, hybridised to 15 a single 2.5kb Neocallimastix RNA species. This suggests that the longest xynA cDNA isolated is almost full length. EXAMPLE 10 - Characterisation of xylanase A 20 The cDNA sequences encoding Neocallimastix xylanases were excised from XZAPII and rescued in E. coli XLl-Blue as recombinants of pBLUESCRlPT SK. Xylanase activity expressed by the recombinant strain harbouring the plasmid pNXl, which contained the longest form of xy/iA,was found predominantly in the cell-free extract, indicating that the enzyme was not efficiently secreted by E. coli. 25 The xylanase, designated xylanase A (XYLA), was purified to near homogeneity (>90% pure). Purified XYLA had a specific activity of 5980 U/mg protein, compared to the cell free extract value of 16 U/mg protein. This indicates that XYLA consists of 0.3% of soluble protein synthesised by E. coli cells harbouring pNXl. The purified enzyme bad an Mr of 53000 (Figure 2) and an N-tenninal WO 93/25693 PCT/GB93/01283 -34- sequence of IATVAKAQWGGGGAS. XYLA hydrolysed xylan but exhibited no activity against carboxymethyl cellulose, barley /3-glucan, laminarin, lichenan or the artificial substrates 4-methyl-umbellifery l-/3-D-xyloside and p-nitrophenyl-/3-D-xylopyranoside (Table 1). 5 TABLE 1 The enzyme activity of purified xylanase A from E. coli harbouring pNXl (xynA cDNA) plasmid. Substrate Activity1 Units/mg protein Barley /3-glucan 0 Carboxymethylcellulose 0 Xylan 5980 Xylobiose 0 p-Nitrophenyl /3-D-xylobiosidc (PNX) 0 Methyiumbelliferyi /S-D-cellobioside (MUC) 0 4-Methyiumbelliferyl 0-D-glucoside (MUG) 0 4-Methylumbellifcryl 0-D-xyloside (MUX) 0 2 0 1One unit of XYLA releases 1 /xmole of product per minute. The enzyme attacked soluble xylan in a manner typical of an endo-/3-l ,4-xylanase (EC 3.1.2.8), promoting a rapid decline in viscosity (Figure 3) and releasing 25 893mg of reducing sugar per g of substrate. Analysis of the hydrolysis products by HPLC revealed that XYLA liberated approximately equal amounts of xylobiose and xylose. No disaccharides containing arabinose, the major side-chain sugar of oat spelt xylan, were detected among the reaction products, suggesting that the WO 93/25693 PCT/GB93/01283 -35- enzyme does not hydrolyse glycosidic linkages involving xylose units linked to side chain sugars. EXAMPLE 11 - Nucleotide sequence 5 The 2.3kb Neocallimastix cDNA derived from pNXl was sequenced in both strands (Accession number X65S26 in EMBL/Genbank/DDBJ Nucleotide Sequence Data Libraries). Translation of the nucleotide sequence revealed a single open reading frame (ORF) of 1821 bp encoding a polypeptide of Mr66192. The 10 deduced primary structure of the encoded protein is shown in Figure 4. The N-tenninal IS residues of recombinant XYLA, purified from E. coli, exhibited a perfect match with amino acids 12 to 26 of the translated sequence. The assignment of the proposed translation initiation codon was based on the following observations: (i) there are not ATG sequences upstream of the ORF; (ii) 15 translational stop codons are in all 3 reading frames upstream of the pu:* *r*e translational start codon. Inspection of the nucleotide sequence in the vicini: a the putative ATG start codon did not reveal any alternative sequences which could act as translational start codon in E. coli. It is likely, therefore, that translational initiation of the xynA occurs at the same codon in the enteric bacterium and 20 anaerobic fungus. This is despite the fact that lower eukaryote mRNAs do not contain ribosome binding sequences which conform to the corresponding E. coli sequence. Presumably the sequence AGA, 7bp upstream of the ATG start codon, acts as weak ribosome binding sequence in the bacterium. Transcription initiation of xynA in E. coli is presumably at the vector's lacZp as subcloning of the xynA 25 cDNA, on a 2.3 kb EcoRI-Xhol restriction fragment, into pMTL22, generated a recombinant plasmid (pNX2) which did not direct a functional xylanase. The vector's lacZp is at the 3' of xynA in pNX2. Although XYLA is not secreted by E. coli, the deduced N-terminal region of the xylanase conforms to that of a signal peptide: comprising of an N-terminal hydrophilic basic region followed by a 3 0 sequence of 23 predominantly hydrophobic or neutral amino acids. WO 93/25693 PCT/GB93/01283 -36- The G + C content of the xynA. ORF was 43.4%, compared to 10.7% for the 5' and 3' non-coding regions (excluding the 3' polyA tail). The overall G + C content of Neocallimastix DNA is approximately 15% (Billon-Grand et al, FEMS Microbiol. Lett. 82: 267-270 (1991)), indicating that non-protein coding regions 5 of the genome are generally very A + T-rich. The bias in codon utilisation in xynA is evident from the absence of 14 of the 61 amino acid codons. There is a marked preference for T in the third position (-50% of all codons end in T) and an exclusion of G in the wobble position. Apart from ATG and TGG, which are the sole codons for Met and Trp respectively, only 3 codons contain G in the third 10 position; AAG, GAG and TTG. Inspection of the deduced primary structure of mature XYLA revealed several interesting features. Between residues 255-265 and 491-519 are regions rich in proline and hydroxy amino acids. Many ceilulases and xylanases consist of 15 multiple domains which are linked by sequences rich in proline/hydroxy amino acids (Gilkes et al, Microbiol. Rev. 55: 303-315 (1991)). The presence of 2 such "linker sequences" in XYLA suggests that the enzyme consists of at least 3 distinct domains. The Neocallimastix xylanase, in addition to comprising of linker regions, also contains a 225 amino acid repeated sequence at the N-terminus, and a C-20 terminal 40 residue reiterated domain (Figure 4). There is no obvious sequence conservation between the large and small repeated regions. The two N-terminal repeated sequences exhibited 91.6% and 95.6% identity and similarity, respectively. The 40 amino acid reiterated region displayed 82.9% and 95.1% identity and similarity, respectively. DNA encoding the two repeated regions also 25 showed sequence identity, with the 699 bp and 120 bp reiterated sequences exhibiting 92.7% and 90.8% identity, respectively. WO 93/25693 PCT/GB93/01283 -37- EXAMPLE 12 - Homology Studies Hydrophobic cluster analysis has shown that ceilulases and xylanases can be grouped into nine enzyme families. Proteins within a family are structurally 5 related and have probably evolved from a common ancestral gene (Henrissat et al, Gene 81: 83-95 (1989)). Comparison of XYLA with sequences in the SWISS-PROT database revealed homology between the fungal enzyme and Bacillus pumilis xylanase A (Fukusaki et al, FEBS Lett. 171: 197-201 (1984)), Bacillus circulans xylanase (Yang et al, Nucl. Acids Res. 16: 7178 (1988)), Clostridium 1 o acetobuiylicum xylanase B (Zappe et al, Nucl. Acids Res. 18: 2179 (1990)) and the N-terminal region of the multi-domainRuminococcus flavefaciens xylanase (Zhang & Flint, Mol. Microbiol. 6: 1013-1019 (1992)). The degree of homology between these enzymes and N. paxriciarum XYLA is shown in Figure 5. 15 It is interesting to note that only the large repeated sequence of XYLA exhibited homology with other hemiceilulases; the C-terminal reiterated region showed no identity with proteins in the database. This suggests that XYLA has a modular structure in which the N-terminal region constitutes the catalytic domain. 20 EXAMPLE 13 - Structure and function of XYLA To investigate the assertion that the N-terminal repeated sequence constituted the catalytic domain of XYLA, 5' and 3' regions of xynA were deleted, or subcloned into appropriate vectors, and the capacity of the resultant xynA derivatives to 2 5 express a functional xylanase was evaluated. A truncated form of xynA in which 291 bp of the 3' region encoding the 40 amino acid C-terminal repeat, had been deleted, still encoded a functional xylanase. The predicted Mr of the encoded enzyme was 53000. This is similar to the size of XYLA purified from E. coli harbouring pNXl. Thus, the recombinant xylanase synthesised from the full- WO 93/25693 PCT/ G B93/01283 -38- length gene by the emeric bacterium could also lack the C-terminal repeated sequeoce. Support for this view is provided by the fact that several multidomain ceilulases and xylanases are particularly sensitive to proteolytic cleavage within the linker sequences (Tomme et al, Eur. J. Biochem. 170: 575-581 (1988); Gilkes et 5 al, J. Biol. Chem. 263: 10401-10407 (1988), including a Pseudomonas xylanase, expressed by E. coli which was substantially cleaved within the serine-rich linker sequences (Hall et al, Mol. Microbiol. 3: 1211-1219 (1989)). A more substantial 3' deletion (pNX6), extending for 1011 bp did not affect the capacity of xynA to direct the synthesis of a functional xylanase. However, removal of 1324 bp from 10 the 3' region of xynA resulted in the synthesis of an inactive derivative of XYLA. These data suggest that the N-terminal 270 residues of the N. patriciarum xylanase folds imo a catalyticaily acdve enzyme. To determine whether both N-terminal reiterated sequences, fold into functional xylanases, the 720 bp Seal Nrul-restricdon fragment (Nrul cleaves in the multiple cloning region of pNX4) was 15 cloned into pMTL20 to generate pNX5, in which truncated xynA was in phase with the vectors lacZ' translation initiation codon (Figure 1). E. coli harbouring pNX5 expressed 15 times more XYLA compared to a clone harbouring full length xynA. This elevation in the expression of the fungal enzyme, is presumably a result of the utilisation of an E. coli translation initiation sequence in xynA encoded by 2 0 pNX5. XYLA purified from cell-free extract of (E. coli containing pNX5 had an My of 26000 (Figure 2B). These data confirm that the reiterated N-terminal 225 residues constitute distinct catalytic domains. Interestingly, a further increase in xylanase activity was achieved by deletion of a few amino residues from the C-terminus of the second catalytic domain to generate pNX9. 25 To investigate the substrate specificities of the N- and C-terminal catalytic domains, the capacity of the xylanases, encoded by pNX6 and pNX5, to cleave plant structural polysaccharides were assessed. The enzymes cleaved only xylan. releasing xylobiose and xylose in similar proportions to that of full-length XYLA. WO 93/25693 PCT/GB93/01283 -39- Thus, both catalytic domains displayed the same substrate specificities as full-length XYLA. Although many ceilulases and xylanases consist of multiple domains, celB from 5 Caldocellum saccharolyticum (Saul et al, Appl. Environ. Microbiol. 56:3117-3124 (1990)) is the only previous example of an enzyme containing 2 distinct catalytic domains. This enzyme consists of an N-terminal exoglucanase and a C-terminal endoglucanase which belong to different enzyme families. Thus, the gene encoding celB probably arose through the fusion of two discrete cellulase genes. 1 o This invention provides evidence that fungd xylanases can also consist of multiple catalytic domains. In contrast to the celB gene, xynA is clearly a result of tandem duplication of an ancestral gene. It is not apparent what selective advantage the gene duplication confers on the anaerobic fungus. Is it simply a mechanism for increasing the expression of XYLA catalytic domains? As this is the first 15 description of an anaerobic fungal xylanase, it is unclear whether multiple catalytic domains are a common feature of lower eukaryote hemicellulasss. WO 93/25693 PCT/GB93/01283 -40- SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Harry John GILBERT (B) STREET: 16 Kells Gardens, Low Fell, (C) CITY: Gateshead (D) STATE: Tyne and Wear (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): NE9 5XS (A) NAME: Geoffrey Peter HAZLEWOOD (B) STREET: 109A Duchess Drive (C) CITY: Newmarket (D) STATE: Suffolk (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): CBS 8AL (ii) TITLE OF INVENTION: Recombinant Xylanases (iii) NUMBER OF SEQUENCES: 18 (iv) COMPUTER READABLE FORM: 3.5" MS-DOS FLOPPY DISK CONTAINING ASCII FILE (93_01283.ASC) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER: WO PCT/GB93/01283 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2338 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Neocallimastix patriciarum (B) STRAIN: (type species) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 195..2018 (D) OTHER INFORMATION: /function= "Xylanolytic enzyme" /product= "XYLA" /standard_name= "Xylanase" (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 195..281 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 282..2018 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 282.-959 (D) OTHER INFORMATION: /label* CAT1 SUBSTITUTE SHEET WO 93/25693 PCT/GB93/01283 -41- /note= "1st catalytic domain" (ix) FEATURE: (A) NAME/KEY: miac feature (B) LOCATION: 10177.1691 (D) OTHER INFORMATION: /labels CAT2 /note- "2nd catalytic domain" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1764..1803 (D) OTHER INFORMATION: /labels CTR1 /notea "1st C-terminal repeat" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1884..2015 (D) OTHER INFORMATION: /labels CTR2 /note* "2nd C-terminal repeat" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1. .2338 (D) OTHER INFORMATION: /labels pNXlJLnsert (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..2338 (D) OTHER INFORMATION: /labels pNX2_ins».r: /notes "pNX2 insert is in reverse orientation to pNXl insert" (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1847 (D) OTHER INFORMATION: /labels pNX3_insert (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1725 (D) OTHER INFORMATION: /labels pNX4_insert (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1002..1725 (D) OTHER INFORMATION: /label- pNX5_iasert (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1001 (D) OTHER INFORMATION: /labels pNX6_insert (ix) FEATURE: (A) NAME/KEY: misc_feafcure (B) LOCATION: 1..690 (0) OTHER INFORMATION: /labels pNX7_inrer (ix) FEATURE: (A) NAME/KEY: misc feature (B) LOCATION: 10027.2338 (D) OTHER INFORMATION: /labels pNX8_insert (ix) FEATURE: (A) NAME/KEY: misc feature (B) LOCATION: 10027.1847 (D) OTHER INFORMATION: /labels DNX9_insert WO 93/25693 PCT/GB93/01283 A2- (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1002..1709 (D) OTHER INFORMATION: /labels pNX10_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTTTATTATA TCAATCTCTA ATTTATTTTT TTAGGAAAAA AATAAAAAAA TAAATATAAT 60 AAATATTAGA GAGTAATATT TAAAAACAAA GAAATTTAAA AACGTTTATT TAGTTATTTT 120 TTTTACTGGT TAAAAAAAAA ATAAAAAACA AAATTAATAA AGATATTTTT GAAAAATATT 180 GAATTAGAAA AAAA ATG AGA ACT ATT AAA TTC TTT TTC GCA GTA GCT ATT 230 Nec Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie -29 -25 -20 GCA ACT GTT GCT AAG GCC CAA TGG GGT GGA GGT GGT GCC TCT GCT GGT 278 Ala Thr Val Ala Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly -15 -10 -5 CAA AGA TTA ACC GTC GGT AAT GGT CAA ACC CAA CAT AAG GGT GTA GCT 326 Gin Arg Leu Thr Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala 15 10 15 GAT GGT TAC AGT TAT GAA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT 374 Asp Gly Tyr Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly 20 25 30 TCT ATG ACT CTC GGT AGT GGT GCA ACC TTC AAG GCT GAA TGG AAT GCA 422 Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala 35 40 45 TCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAC TTC GGT 470 Ser Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly 50 55 60 TCT CAA AAG AAG GCA ACC GAT TAC AGC TAC ATT GGA TTG GAT TAT ACT 518 Ser Gin Lys Lys Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr 65 70 75 GCA ACT TAC AGA CAA ACT GGT AGC GCA AGT GGT AAC TCC CGT CTC TGT 566 Ala Thr Tyr Arg Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys 80 85 90 95 GTA TAC GGT TGG TTC CAA AAC CGT GGA GTT CAA GGT GTT CCA TTG GTA 614 Val Tyr Gly Trp Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val 100 105 110 GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT CCA GAT GCA CAA 662 Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin 115 120 125 GGT AGA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG ATT TTC CAA ATG 710 Gly Arg Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met 130 135 140 GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA ACC TTT AAG CAA 758 Asp His Thr Gly Pro Thr. lie Asn Gly Gly Ser Glu Thr Phe Lys Gin 145 150 155 TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT CAT ATT ACT GTC 8 06 Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His He Thr Val 160 165 170 175 TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG GGT ATT GGT AAC 8 54 WO 93/25693 PCT/GB93/01283 -43- Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly He Gly Asn 180 185 190 CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA AGT AGT GGT ATA 902 Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie 195 200 205 GCT GAT GTC ACC AAG TTA GAT GTT TAC ACA ACC CAA AAA GGT TCT AAT 950 Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn 210 215 220 CCT GCC CCT ACC TCC ACT GGT ACT GTT CCA AGC AGT TCT GCT GGT GGA 998 Pro Ala Pro Thr Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly 225 230 235 AGT ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA 1046 Ser Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin 240 245 250 255 CAT AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC 1094 His Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn 260 265 270 ACT GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG 1142 Thr Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys 275 280 285 GCT GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT 1190 Ala Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg 290 295 300 GGT CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT 1238 Gly Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie 305 310 315 GGA TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT 1286 Gly Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly 320 325 330 335 AAC TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT 1334 Asn Ser Arg Leu Cvs Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn 340 345 350 GGC GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG 1382 Gly Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp 355 360 365 GTT CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT 1430 Val Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr 370 375 380 AAG ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT 1478 Lys lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser 385 390 395 GAA ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT 1526 Glu Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser 400 405 410 415 GGT CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT 1574 Gly His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly 420 425 430 TGG GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG 1622 Trp Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp 435 440 445 WO 93/25693 PCT/GB93/01283 -44- CAA AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT 1670 Gin Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr 450 455 460 CCA AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC 1718 Pro Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr 465 470 475 CGT ACT ACT ACT CGT ACC AAG TCT CTT CCA ACC AAT TAC AAT AAG TGT 1766 Arg Thr Thr Thr Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Lys Cys 480 485 490 495 TCT GCT AGA ATT ACT GCT CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT 1814 Ser Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn 500 505 510 TGT GTT GTT TAC TAC ACT GAT GAG GAT GGT ACC TGG GGT GTT GAA AAC 1862 Cys Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr Trp Gly Val Glu Asn 515 520 525 AAC GAC TGG TGT GGT TGT GGT GTT GAA CAA TGT TCT TCC AAG ATC ACT 1910 Asn Asp Trp Cys Gly Cys Gly Val Glu Gin Cys Ser Ser Lys lie Thr 530 535 540 TCT CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT TGC GTT GTT TTC TAC 1958 Ser Gin Gly Tyr Lys Cys Cys Ser Asd Pro Asn Cys Val Val Phe Tyr 545 550 555 ACT GAT GAC GAT GGT AAA TGG GGT GTT GAA AAC AAC GAC TGG TGT GGT 2006 Thr Asp Asp Asp Gly Lys Trp Gly Val Glu Asn Asn Asp Trp Cys Gly 560 555 570 575 TGT GGT TTC TAAGCAGTAA AATACTAATT AATAAAAAAT TAAAGAATTA 2055 Cys Gly Phe TGAAAAATTT AAATTTAAAA ATTTAAAAGA ATTATGAAAA ATTTAAATTT AAAAATTTJVA 2115 AAAAAACTAA TTTAGTAAAA AATTAAAGAA TTATTGAAAA TTTTAAATGT AAAAATTTAA 2175 AAAATACAAA TTTGTAAAAA AAAATGAAAG AATTATGAAA AATTAAAATG TAAAAGTTTA 2235 AAAAATACAA ATTTGTAAGA AAAATAAAGA ATTATAAAAA AAATAAAGAA TTATGAAAAA 2295 CCCAAATGTA AAGAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA 2338 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 607 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie Ala Thr Val Ala -29 -25 -20 -15 Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly Gin Arg Leu Thr -10 -5 1 Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala Asp Gly Tyr Ser 5 10 15 WO 93/25693 PCT/GB93/01283 -45- Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser MeC Thr Leu 20 25 30 35 Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala Ser Val Asn Arg 40 45 50 Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Gin Lys Lys 55 SO 65 Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr Ala Thr Tyr Arg 70 75 80 Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp 85 90 95 Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val Glu Tyr Tyr lie 100 105 110 115 lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin Gly Arg Met Val 120 125 130 Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Glu Met Asp His Thr Gly 135 140 145 Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin Tyr Phe Ser Val ISO 155 160 Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr Val Ser Asp His Phe 165 170 175 Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn Leu Tyr Glu Val 180 185 190 195 Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie Ala Asp Val Thr 200 205 210 Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn pro Ala Pro Thr 215 220 225 Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly Ser Thr Ala Asn 230 235 240 Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His Lys Gly Val 245 250 255 Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Asn 260 265 270 275 Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn 280 285 290 Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe 295 300 305 Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly Leu Asp Tyr 310 315 320 Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn Ser Arg Leu 325 330 335 Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly Val Pro Leu 340 345 350 355 Val Glu Tvr Tyr lie He Glu Asd Trp Val Asp Trp Val Pro Asp Ala 360 " 365 370 WO 93/25693 PCT/GB93/01283 -46- Gln Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin 375 3B0 385 Met Asp His Ttr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys 390 395 400 Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr 405 410 415 Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly 420 425 430 435 Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly 440 445 450 Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro Lys Gly Ser 455 460 465 Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg Thr Thr Thr 470 475 480 Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Lys Cys Ser Ala Arg lie 485 490 495 Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys Val V»i Tyr 500 505 510 515 Tyr Thr Asp Glu Asd Gly Thr Trp Gly Val Glu Asn Asn Asp Trp Cys 520 525 530 Gly Cys Gly Val Glu Gin Cys Ser Ser Lys lie Thr Ser Gin Gly Tyr 535 540 545 Lys Cys Cys Ser Asp Pro Asn Cys Val Val Phe Tyr Thr Asp Asp Asp 550 555 560 Gly Lys Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Cys Gly Phe 565 570 575 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1847 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 195..1847 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 195..281 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1847 (D) OTHER INFORMATION: /label* pNX3_insert (xx) SEQUENCE DESCRIPTION: SEQ ID NO: 3: WO 93/25693 PCT/GB93/01283 -47- ttttattata tcaatctcta atttattttt ttaggaaaa. .jitaaaaaaa taaatataat 60 aaatattaga gagtaatatt taaaaacaaa gaaatttaaa aacgtttatt tagttatttt 120 TTTTACTGGT TAAAAAAAAA ATAAAAAACA AAATTAATAA AGATATTTTT GAAAAATATT 180 GAATTAGAAA AAAA ATG AGA ACT ATT AAA TTC TTT TTC GCA GTA GCT ATT 230 Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie 15 10 GCA ACT GTT GCT AAG GCC CAA TGG GGT GGA GGT GGT GCC TCT GCT GGT 278 Ala Thr Val Ala Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly 15 20 25 CAA AGA TTA ACC GTC GGT AAT GGT CAA ACC CAA CAT AAG GGT GTA GCT 326 Gin Arg Leu Thr Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala 30 35 40 GAT GGT TAC AGT TAT GAA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT 374 Asp Gly Tyr Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly 45 50 5r 60 TCT ATG ACT CTC GGT AGT GGT GCA ACC TTC AAG GCT GAA TGG AAT GCA 422 Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala 65 70 75 TCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAC TTC GGT 470 Ser Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly 80 85 90 TCT CAA AAG AAG GCA ACC GAT TAC AGC TAC ATT GGA TTG GAT TAT ACT 518 Ser Gin Lys Lys Ala Thr Asp Tyr. Ser Tyr lie Gly Leu Asp Tyr Thr 95 100 105 GCA ACT TAC AGA CAA ACT GGT AGC GCA AGT GGT AAC TCC CGT CTC TGT 566 Ala Thr Tyr Arg Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg. Leu Cys 110 115 120 GTA -AC GGT TGG TTC CAA AAC CGT GGA GTT CAA GGT GTT CCA TTG GTA 614 Val Tyr Gly Trp Phe Gin Asn Arg Gly Val Gin Gly Val Pre Leu Val 125 130 135 140 GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT CCA GAT GCA CAA 662 Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin 145 150 155 GGT AGA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG ATT TTC CAA ATG 710 Gly Arg Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met 160 165 170 GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA ACC TTT AAG CAA 758 Asd His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin 175 180 185 TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT CAT ATT ACT GTC 806 Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His Xle Thr Val 190 195 200 TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG GGT ATT GGT AAC 854 Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn 205 210 215 220 CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA AGT AGT GGT ATA 902 Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie 225 230 235 WO 93/25693 PCT/GB93/01283 -48- GCT GAT GTC ACC AAG TTA GAT GTT TAC ACA ACC CAA AAA GGT TCT AAT 950 Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn 240 245 250 CCT GCC CCT ACC TCC ACT GGT ACT GTT CCA AGC AGT TCT GCT GGT GGA 998 Pro Ala Pro Thr Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly 255 260 265 AGT ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA 1046 Ser Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin 270 275 280 CAT AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC 1094 His Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn 285 290 295 300 ACT GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG 1142 Thr Gly Gly Asn Gly Ser Mee Thr Leu Gly Ser Gly Ala Thr Phe Lys 305 310 315 GCT GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT 1190 Ala Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg 320 325 330 GGT CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT 1238 Gly Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie 335 340 345 GGA TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT 1286 Gly Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly 350 355 360 AAC TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT 1334 Asn Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn 365 370 375 380 GGC GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG 1382 Gly Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp 385 390 395 GTT CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT 1430 Val Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr 400 405 410 AAG ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT 1478 Lys lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser 415 420 425 GAA ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT 1526 Glu Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser 430 435 440 GGT CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT 1574 Gly His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly 445 450 455 460 TGG GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG 1622 Trp Gly He Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp 465 470 475 CAA AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT 1670 Gin Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr 480 485 490 CCA AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC 1718 Pro Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr 495 500 505 WO 93/25693 PCT/GB93/01283 -49- CGT ACT ACT ACT CGT ACC AAG TCT CTT CCA ACC TAC AAT AAG TGT 1766 Arg Tfcr Thr Thr Arg Thr Lys Ser Leu Pro Thr : Tyr Asn Lys Cys 510 515 I ; TCT GCT AGA ATT ACT GCT CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT 1814 Ser Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn 525 530 535 540 TGT GTT GTT TAC TAC ACT GAT GAG GAT GGT ACC 1847 Cys Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr 545 550 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 551 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie Ala Thr Val Ala 15 10 15 Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly Gin Arg Leu Thr 20 25 30 Va l Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala Asp Gly Tvr Ser 35 40 45 Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu 50 55 60 Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala Ser Val Asn Arg 65 70 75 80 Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Gin Lys Lys 85 90 95 Ala Thr Asp Tyr Ser Tyr He Gly Leu Asp Tyr Thr Ala Thr Tyr Arg 100 105 110 Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp 115 120 125 Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val Glu Tyr Tyr lie 130 135 140 lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin Gly Arg Met Val 145 * 150 155 160 Thr lie Asd Gly Ala Gin Tyr Lys lie Phe Gin Met Asp His Thr Gly 165 170 175 Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin Tyr Phe Ser Val 180 185 190 Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr Val Ser Asp His Phe 195 200 205 Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn Leu Tyr Glu Val 210 215 220 WO 93/25693 PCT/GB93/01283 -50- Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie Ala Asp Val Thr 225 230 235 240 Lys Leu Asd Val Tyr Thr Thr Gin Lys Gly Ser Asn Pro Ala Pro Thr 245 250 255 Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly Ser Thr Ala Asn 260 265 270 Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His Lys Gly Val 275 280 285 Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Asn 230 295 300 Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn 305 310 315 320 Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe 325 330 335 Oly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly Leu Asp Tyr 340 345 350 Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn Ser Arg Leu 355 360 365 Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly Val Pro Leu 370 375 380 Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala 385 390 395 400 Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin 405 410 415 Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys 420 425 430 Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr 435 440 445 Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly 450 455 460 Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly 465 470 475 480 Val Ala Asd Val Thr Leu Leu Asp Val Tyr Thr Thr Pro Lys Gly Ser 485 490 495 Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg Thr Thr Thr 500 505 510 Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Lys Cys Ser Ala Arg lie 515 520 525 Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys Val Val Tyr 530 535 540 Tyr Thr Asd Glu Asp Gly Thr 545 550 (2) INFORMATION FOR SEQ ID NO; 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1725 base pairs WO 93/25693 PCT/GB93/01283 -51- (B) TYPE: nucleic acid (C) STRANDEDNESC: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 195..1724 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 195..281 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1725 (D) OTHER INFORMATION: /label- pNX4_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TTTTATTATA TCAATCTCTA ATTTATTTTT TTAGGAAAAA AATAAAAAAA TAAATATAAT 60 AAATATTAGA GAGTAATATT TAAAAACAAA GAAATTTAAA AACGTTTATT TAGTTATTTT 120 TTTTACTGGT TAAAAAAAAA ATAAAAAACA AAATTAATAA AGATATTTTT GAAAAATATT 180 GAATTAGAAA AAAA ATG AGA ACT ATT AAA TTC TTT TTC GCA GTA GCT ATT 230 Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie IS 10 GCA ACT GTT GCT AAG GCC CAA TGG GGT GGA GGT GGT GCC TCT GCT GGT 278 Ala Thr Val Ala Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly 15 20 25 CAA AGA TTA ACC GTC GGT AAT GGT CAA ACC CAA CAT AAG GGT GTA'GCT 32S Gin Arg Leu Thr Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala 30 35 40 GAT GGT TAC AGT TAT GAA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT 374 Asp Gly Tyr Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly 45 50 55 60 TCT ATG ACT CTC GGT AGT GGT GCA ACC TTC AAG GCT GAA TGG AAT GCA 422 Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala 65 70 75 TCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAC TTC GGT 470 Ser Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly 80 85 90 TCT CAA AAG AAG GCA ACC GAT TAC AGC TAC ATT GGA TTG GAT TAT ACT 518 Ser Gin Lys Lys Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr 95 100 105 GCA ACT TAC AGA CAA ACT GGT AGC GCA AGT GGT AAC TCC CGT CTC TGT 566 Ala Thr Tyr Arg Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys 110 115 120 GTA TAC GGT TGG TTC CAA AAC CGT GGA GTT CAA GGT GTT CCA TTG GTA 614 Val Tyr Gly Trp Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val 125 130 135 140 WO 93/25693 PCT/GB93/01283 -52- GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT CCA GAT GCA CAA 662 Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin 145 150 155 GGT AGA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG ATT TTC CAA ATG 710 Gly Arg Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met 160 165 170 GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA ACC TTT AAG CAA 758 Asd His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin 175 180 185 TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT CAT ATT ACT GTC 806 Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr Val 190 195 200 TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG GGT ATT GGT AAC 854 Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn 205 210 215 220 CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA AGT AGT GGT ATA 902 Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie 225 230 235 GCT GAT GTC ACC AAG TTA GAT GTT TAC ACA ACC CAA AAA GGT TCT AAT 950 Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn 240 245 250 CCT GCC CCT ACC TCC ACT GGT ACT GTT CCA AGC AGT TCT GCT GGT GGA 998 Pro Ala Pro Thr Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly 255 260 265 AGT ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA 1046 Ser Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin 270 275 280 CAT AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC 1094 His Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn 285 290 295 300 ACT GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG 1142 Thr Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys 305 310 315 GCT GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT 1190 Ala Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg 320 325 330 GGT CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT 1238 Gly Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie 335 340 345 GGA TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT 1286 Gly Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly 350 355 360 AAC TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT 1334 Asn Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn 365 370 375 380 GGC GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG 1382 Gly Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp 385 390 395 GTT CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT 1430 Val Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr 400 405 410 WO 93/25693 PCT/GB93/01283 -53- AAG ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT 1478 Lys lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser 415 420 425 GAA ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT 1526 Glu Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser 430 435 440 GGT CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT 1574 Gly His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly 445 450 455 460 TGG GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TOG 1622 Trp Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp 465 470 475 CAA AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT 1670 Gin Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr 480 485 490 CCA AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC 1718 Pro Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr 495 500 505 CGT ACT A 1725 Arg Thr 510 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 510 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie Ala Thr Val Ala 15 10 15 Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly Gin Arg Leu Thr 20 25 30 Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala Asp Gly Tyr Ser 35 40 45 Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu 50 55 60 Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala Ser Val Asn Arg 65 70 75 80 Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Gin Lys Lys 85 90 95 Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr Ala Thr Tyr Arg 100 105 110 Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp 115 120 125 Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val Glu Tyr Tyr lie 130 135 140 WO 93/25693 PCT/GB93/01283 -54- Ile Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin Gly Arg Met Val 145 150 155 160 Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met Asp His Thr Gly 165 170 175 Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin Tyr Phe Ser Val 180 185 190 Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr Val Ser Asp His Phe 195 200 205 Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn Leu Tyr Glu Val 210 215 220 Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie Ala Asp Val Thr 225 230 235 240 Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn Pro Ala Pro Thr 245 250 255 Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly Ser Thr Ala Asn 260 265 270 Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His Lys Gly Val 275 280 285 Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Asn 290 295 300 Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn 305 310 315 320 Ala Ala Val Asn Arg Gly Asn Phe Leu Ala krg Arg Gly Leu Asp Phe 325 330 335 Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly Leu Asp Tyr 340 345 350 Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn Ser Arg Leu 355 360 365 Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly Val Pro Leu 370 375 380 Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala 385 390 395 400 Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin 405 410 415 Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys 420 425 430 Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr 435 440 445 Val Ser Asd His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly 450 ~ 455 460 Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly 465 470 475 480 Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro Lys Gly Ser 485 490 495 WO 93/25693 PCT/GB93/01283 -55- Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg Thr 500 505 510 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 724 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..723 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..724 (D) OTHER INFORMATION: /label- p:JC5_insert (xi) SEQUENCE DESCRIPTICV- SEQ ID NO: 7: ACT GCC AAT GGT AAA AAG TTT ' T GTC GGT AAT GGA CAA AAC CAA CAT 48 Thr Ala Asn Gly Lys Lys Phe lor Val Gly Asn Gly Gin Asn Gin His 15 10 15 AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC ACT 96 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG GCT 144 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT ".32 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT GGA 240 Leu Asp Phe Gly Ser Gla Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT AAC 288 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT GGC 336 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT 384 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG 432 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA 480 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 WO 93/25693 PCT/GB93/01283 -56- ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT 528 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG 576 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp ISO 185 ISO GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA 624 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT CCA 672 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC CGT 720 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg 225 230 235 240 ACT A 724 Thr (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 241 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 15 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 Leu Asd Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 ' 70 75 80 Leu Asd Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 Pro Asd Ala Gin Gly Lys Met Val Thr He Asp Gly Ala Gin Tyr Lys 130 135 140 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 WO 93/25693 PCT/GB93/01283 Thr Phe Lys Gin Tyr Phe Ser Val 165 His lie Thr Val Ser Asp His Phe 180 Gly lie Gly Asn Leu Tyr Glu Val 195 200 Ser Ser Gly Val Ala Asp Val Thr 210 215 Lys Gly Ser Ser Pro Ala Thr Ser 225 230 Thr -57- Arg Gin Gin Lys Arg Thr Ser Gly 170 175 Lys Glu Trp Ala Lys Gin Gly Trp 185 190 Ala Leu Asn Ala Glu Gly Trp Gin 205 Leu Leu Asp Val Tyr Thr Thr Pro 220 Ala Ala Pro Arg Thr Thr Thr Arg 235 240 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1001 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 195..1001 (ix) FEATURE: (A) NAME/KEY: sig_peptide (B) LOCATION: 195..281 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1001 (D) OTHER INFORMATION: /label- pNX6_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TTTTATTATA TCAATCTCTA ATTTATTTTT TTAGGAAAAA AATAAAAAAA TAAATATAAT 60 AAATATTAGA GAGTAATATT TAAAAACAAA GAAATTTAAA AACGTTTATT TAGTTATTTT 120 TTTTACTGGT TAAAAAAAAA ATAAAAAACA AAATTAATAA AGATATTTTT GAAAAATATT 18 0 GAATTkGAAA AAAA ATG AGA ACT ATT AAA TTC TTT TTC GCA GTA GCT ATT 23 0 Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie 15 10 GCA ACT GTT GCT AAG GCC CAA TGG GGT GGA GGT GGT GCC TCT GCT GGT 278 Ala Thr Val Ala Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly 15 20 25 CAA AGA TTA ACC GTC GGT AAT GGT CAA ACC CAA CAT AAG GGT GTA GCT 326 Gin Arg Leu Thr Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala 30 35 40 GAT GGT TAC AGT TAT GAA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT 374 Asp Gly Tyr Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly 45 50 55 60 WO 93/25693 PCT/GB93/01283 -58- TCT ATG ACT CTC GGT AGT GGT GCA ACC TTC AAG GCT GAA TGG AAT GCA 422 Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala 65 70 75 TCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAC TTC GGT 470 Ser Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly 80 85 90 TCT CAA AAG AAG GCA ACC GAT TAC AGC TAC ATT GGA TTG GAT TAT ACT 518 Ser Gin Lys Lys Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr 95 100 105 GCA ACT TAC AGA CAA ACT GGT AGC GCA AGT GGT AAC TCC CGT CTC TGT 566 Ala Thr Tyr Arg Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys 110 115 120 GTA TAC GGT TGG TTC CAA AAC CGT GGA GTT CAA GGT GTT CCA TTG GTA 614 Val Tyr Gly Trp Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val 125 130 135 140 GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT CCA GAT GCA CAA 662 Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin 145 150 155 GGT AGA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG ATT TTC CAA ATG 710 Gly Arg Met Val Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met 160 165 170 GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA ACC TTT AAG CAA 758 Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin 175 180 185 TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT CAT ATT ACT GTC 806 Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly His He Thr Val 190 195 200 TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG GGT ATT GGT AAC 854 Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp Gly He Gly Asn 205 210 215 220 CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA AGT AGT GGT ATA 902 Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie 225 230 235 GCT GAT GTC ACC AAG TTA GAT GTT TAC ACA ACC CAA AAA GGT TCT AAT 950 Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn 240 245 250 CCT GCC CCT ACC TCC ACT GGT ACT GTT CCA AGC AGT TCT GCT GGT GGA 998 Pro Ala Pro Thr Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly 255 260 265 AGT 1001 Ser (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: WO 93/25693 PCT/GB93/01283 -59- Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie Ala Thr Val Ala 1 5 10 15 Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly Gin Arg Leu Thr 20 25 30 Val Gly Asa Gly Gin Thr Gin His Lys Gly Val Ala Asp Gly Tyr Ser 35 40 45 Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu 50 55 GO Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala Ser Val Asn Arg 65 70 75 80 Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Gin Lys Lys 85 90 95 Ala Thr Asd Tyr Ser Tyr lie Gly Leu Asp Tyr Thr Ala Thr Tyr Arg 100 105 110 Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp 115 120 125 Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val Glu Tyr Tyr lie 130 135 140 lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin Gly Arg Met Val 145 150 155 160 Thr lie Asp Gly Ala Gin Tyr Lys lie Phe Gin Met Asp His Thr Gly 165 170 175 Pro Thr lie Asn Gly Gly Ser Glu Thr Phe Lys Gin Tyr Phe Ser Val 180 185 190 Arg Gin Gin Lys Arg Thr Ser Gly His lie Thr Val Ser Asp His Phe 195 200 205 Lys Glu Trp Ala Lys Gin Gly Trp Gly lie Gly Asn Leu Tyr Glu Val 210 215 220 Ala Leu Asn Ala Glu Gly Trp Gin Ser Ser Gly lie Ala Asp Val Thr 225 230 235 240 Lys Leu Asp Val Tyr Thr Thr Gin Lys Gly Ser Asn Pro Ala Pro Thr 245 250 255 Ser Thr Gly Thr Val Pro Ser Ser Ser Ala Gly Gly Ser 260 265 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 690 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 195..689 (ix) FEATURE: WO 93/25693 PCT/GB93/01283 -60- (A) NAME/KEY: sig_peptide (B) LOCATION: 195..281 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..690 (D) OTHER INFORMATION: /label- pNX7_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTTATTATA TCAATCTCTA ATTTATTTTT TTAGGAAAAA AATAAAAAAA TAAATATAAT 60 AAATATTAGA GAGTAATATT TAAAAACAAA GAAATTTAAA AACGTTTATT TAGTTATTTT 120 TTTTACTGGT TAAAAAAAAA-ATAAAAAACA AAATTAATAA AGATATTTTT GAAAAATATT 180 GAATTAGAAA AAAA ATG AGA ACT ATT AAA TTC TTT TTC GCA GTA GCT ATT 230 Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie 15 10 GCA ACT GTT GCT AAG GCC CAA TGG GGT GGA GGT GGT GCC TCT GCT GGT 278 Ala Thr Val Ala Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly 15 20 25 CAA AGA TTA ACC GTC GGT AAT GGT CAA ACC CAA CAT AAG GGT GTA GCT 326 Gin Arg Leu Thr Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala 30 35 40 GAT GGT TAC AGT TAT GAA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT 374 Asp Gly Tyr Ser Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly 45 50 55 60 TCT ATG ACT CTC GGT AGT GGT GCA ACC TTC AAG GCT GAA TGG AAT GCA 422 Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala 65 70 75 TCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAC TTC GGT 470 Ser Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly 80 85 90 TCT CAA AAG AAG GCA ACC GAT TAC AGC TAC ATT GGA TTG GAT TAT ACT 518 Ser Gin Lys Lys Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr 95 100 105 GCA ACT TAC AGA CAA ACT GGT AGC GCA AGT GGT AAC TCC CGT CTC TGT 566 Ala Thr Tyr Arg Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys 110 115 120 GTA TAC GGT TGG TTC CAA AAC CGT GGA GTT CAA GGT GTT CCA TTG GTA 614 Val Tyr Gly Trp Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val 125 130 135 140 GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT CCA GAT GCA CAA 662 Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin 145 150 155 GGT AGA ATG GTA ACC ATT GAT GGA GCT C 6 90 Gly Arg Met Val Thr lie Asp Gly Ala 160 165 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 165 amino acids (B) TYPE: amino acid WO 93/25693 PCT/GB93/01283 -61- (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Arg Thr lie Lys Phe Phe Phe Ala Val Ala lie Ala Thr Val Ala 15 10 15 Lys Ala Gin Trp Gly Gly Gly Gly Ala Ser Ala Gly Gin Arg Leu Thr 20 25 30 Val Gly Asn Gly Gin Thr Gin His Lys Gly Val Ala Asp Gly Tyr Ser 35 40 45 Tyr Glu lie Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu 50 55 60 Gly Ser Gly Ala Thr Phe Lys Ala Glu Trp Asn Ala Ser Val Asn Arg 65 70 75 80 Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Gin Lys Lys 85 90 95 Ala Thr Asp Tyr Ser Tyr lie Gly Leu Asp Tyr Thr Ala Thr Tyr Arg 100 105 110 Gin Thr Gly Ser Ala Ser Gly Asn Ser Arg Leu Cys Val Tyr Gly Trp 115 120 125 Phe Gin Asn Arg Gly Val Gin Gly Val Pro Leu Val Glu Tyr Tyr lie 130 135 140 lie Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gin Gly Arg Met Val 145 150 155 160 Thr lie Asp Gly Ala 165 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1337 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1014 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..1337 (D) OTHER INFORMATION: /label- pNXe_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA CAT 4 8 Thr Ala Asn. Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 15 WO 93/25693 PCT/GB93/01283 -62- AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC ACT 96 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG GCT 144 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT 192 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT GGA 240 Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT AAC 288 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT GGC 336 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT 384 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG 432 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA 480 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 ISO 155 160 ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT 528 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG 576 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA 624 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT CCA 672 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC CGT 720 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg 225 230 235 240 ACT ACT ACT CGT ACC AAG TCT CTT CCA ACC AAT TAC AAT AAG TGT TCT 768 Thr Thr Thr Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Ly3 Cys Ser 245 250 255 GCT AGA ATT ACT GCT CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT TGT 816 Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys 260 265 270 GTT GTT TAC TAC ACT GAT GAG GAT GGT ACC TGG GGT GTT GAA AAC AAC 864 Val Val Tyr Tyr Thr Asp Glu Asd Gly Thr Trp Gly Val Glu Asn Asn 275 280 285 WO 93/25693 PCT/GB93/01283 -63- GAC TGG TGT GGT TGT GGT GTT GAA CAA TGT TCT TCC AAG ATC ACT TCT 912 Asp Trp Cys Gly Cys Gly Val Glu Gin Cys Ser Ser Lys lie Thr Ser 290 295 300 CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT TGC GTT GTT TTC TAC ACT 960 Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys Val Val Phe Tyr Thr 305 310 315 320 GAT GAC GAT GGT AAA TGG GGT GTT GAA AAC AAC GAC TGG TGT GGT TGT 1008 Asp Asp Asp Gly Lys Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Cys 325 330 335 GGT TTC TAAGCAGTAA AATACTAATT AATAAAAAAT TAAAGAATTA TGAAAAATTT 1064 Gly Phe AAATTTAAAA ATTTAAAAGA ATTATGAAAA ATTTAAATTT AAAAATTTAA AAAAAACTAA 1124 TTTAGTAAAA AATTAAAGAA TTATTGAAAA TTTTAAATGT AAAAATTTAA AAAATACAAA 1184 TTTGTAAAAA AAAATGAAAG AATTATGAAA AATTAAAATG TAAAAGTTTA AAAAATACAA 1244 ATTTGTAAGA AAAATAAAGA ATTATAAAAA AAATAAAGAA TTATGAAAAA CCCAAATGTA 1304 AAGAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA 1337 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 338 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 15 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 He Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 WO 93/25693 PCT/GR93/01283 -64- Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg 225 230 235 240 Thr Thr Thr Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Lys Cys Ser 245 250 255 Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys 260 265 270 Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr Trp Gly Val Glu Asn Asn 275 280 285 Asp Trp Cys Gly Cys Gly Val Glu Gin Cys Ser Ser Lys lie Thr Ser 290 295 300 Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys Val Val Phe Tyr Thr 305 310 315 320 Asp Asd Asp Gly Lys Trp Gly Val Glu Asn Asn Asp Trp Cys Gly Cys 325 330 335 Gly Phe (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 846 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..846 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..846 (D) OTHER INFORMATION: / label =. pNX9_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA CAT 48 Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 15 AAG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC ACT 96 Lvs Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 WO 93/25693 PCT/GB93/01283 -65- GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG GCT 144 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT 192 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT GGA 240 Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT AAC 288 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT GGC 336 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT 384 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG 432 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA 480 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT 528 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG 576 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA 624 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT CCA 672 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT ACT ACT ACC CGT 720 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg 225 230 235 240 ACT ACT ACT CGT ACC AAG TCT CTT CCA ACC AAT TAC AAT AAG TGT TCT 768 Thr Thr Thr Arg Thr Lys Ser Leu Pro Thr Asn Tvr Asn Lys Cys Ser 245 250 * 255 GCT AGA ATT ACT GCT CAA GGT TAC AAG TGT TGT AGC GAT CCA AAT TGT 816 Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys 260 265 270 GTT GTT TAC TAC ACT GAT GAG GAT GGT ACC 846 Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr 275 280 WO 93/25693 PCT/GB93/01283 -66- (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 282 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Thr Ala i\sn Gly Lys Lya Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 15 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg Thr Thr Thr Arg 225 230 235 240 Thr Thr Thr Arg Thr Lys Ser Leu Pro Thr Asn Tyr Asn Lys Cys Ser 245 250 255 Ala Arg lie Thr Ala Gin Gly Tyr Lys Cys Cys Ser Asp Pro Asn Cys 260 265 270 Val Val Tyr Tyr Thr Asp Glu Asp Gly Thr 275 280 WO 93/25693 PCT/GB93/01283 -67- (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 708 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..708 (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION: 1..708 (D) OTHER INFORMATION: /label* pNX10_insert (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1V : ACT GCC AAT GGT AAA AAG TTT ACT GTC GGT AAT GGA CAA AAC CAA CAT 48 Tnr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 15 10 IS AG GGT GTC AAC GAT GGT TTC AGT TAT GAA ATC TGG TTA GAT AAC ACT 96 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 GGT GGT AAC GGT TCT ATG ACT CTC GGT AGT GGT GCA ACT TTC AAG GCT 144 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 GAA TGG AAT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT 192 Glu Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 CTT GAC TTC GGT TCT CAA AAG AAG GCA ACC GAT TAC GAC TAC ATT GGA 240 Leu Asp Pne Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 TTA GAT TAT GCT GCT ACT TAC AAA CAA ACT GCC AGT GCA AGT GGT AAC 288 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 TCC CGT CTC TGT GTA TAC GGA TGG TTC CAA AAC CGT GGA CTT AAT GGC 336 Ser Arg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 GTT CCT TTA GTA GAA TAC TAC ATC ATT GAA GAT TGG GTT GAC TGG GTT 384 Val Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 CCA GAT GCA CAA GGA AAA ATG GTA ACC ATT GAT GGA GCT CAA TAT AAG 432 Pro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AGT GAA 480 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 ACC TTT AAG CAA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT 528 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 WO 93/25693 PCT/GB93/01283 -68- CAT ATT ACT GTC TCA GAT CAC TTT AAG GAA TGG GCC AAA CAA GGT TGG 576 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 GGT ATT GGT AAC CTT TAT GAA GTT GCT TTG AAC GCC GAA GGT TGG CAA 624 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 AGT AGT GGT GTT GCT GAT GTC ACC TTA TTA GAT GTT TAC ACA ACT CCA 672 Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 AAG GGT TCT AGT CCA GCC ACC TCT GCC GCT CCT CGT 708 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg 225 230 235 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 236 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Thr Ala Asn Gly Lys Lys Phe Thr Val Gly Asn Gly Gin Asn Gin His 1 5 10 15 Lys Gly Val Asn Asp Gly Phe Ser Tyr Glu lie Trp Leu Asp Asn Thr 20 25 30 Gly Gly Asn Gly Ser Met Thr Leu Gly Ser Gly Ala Thr Phe Lys Ala 35 40 45 Glu Tro Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly 50 55 60 Leu Asp Phe Gly Ser Gin Lys Lys Ala Thr Asp Tyr Asp Tyr lie Gly 65 70 75 80 Leu Asp Tyr Ala Ala Thr Tyr Lys Gin Thr Ala Ser Ala Ser Gly Asn 85 90 95 Ser .\rg Leu Cys Val Tyr Gly Trp Phe Gin Asn Arg Gly Leu Asn Gly 100 105 110 v,il Pro Leu Val Glu Tyr Tyr lie lie Glu Asp Trp Val Asp Trp Val 115 120 125 J?ro Asp Ala Gin Gly Lys Met Val Thr lie Asp Gly Ala Gin Tyr Lys 130 135 140 lie Phe Gin Met Asp His Thr Gly Pro Thr lie Asn Gly Gly Ser Glu 145 150 155 160 Thr Phe Lys Gin Tyr Phe Ser Val Arg Gin Gin Lys Arg Thr Ser Gly 165 170 175 His lie Thr Val Ser Asp His Phe Lys Glu Trp Ala Lys Gin Gly Trp 180 185 190 Gly lie Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gin 195 200 205 WO 93/25693 PCT/GB93/01283 -69- Ser Ser Gly Val Ala Asp Val Thr Leu Leu Asp Val Tyr Thr Thr Pro 210 215 220 Lys Gly Ser Ser Pro Ala Thr Ser Ala Ala Pro Arg 225 230 235 SUMMARY OF SEQUENCE LISTINGS SEQ ID NO: 1 pNXl DNA and coding region SEQ ID NO: 2 Protein sequence of SEQ ID NO: 1 SEQ ID NO: 3 pNX3 DNA and coding region SEQ ID NO: 4 Protein sequence of SEQ ID NO: 3 SEQ ID NO: 5 pNX4 DNA and coding region SEQ ID NO: 6 Protein sequence of SEQ ID NO: 5 SEQ ID NO: 7 pNXS DNA and coding region SEQ ID NO: 8 Protein sequence of SEQ ID NO: 7 SEQ ID NO: 9 pNX6 DNA and coding region SEQ ID NO: 10 Protein sequence of SEQ ID NO: 9 SEQ ID NO: 11 pNX7 DNA and coding region SEQ ID NO: 12 Protein sequence of SEQ ID NO: 11 SEQ ID NO: 13 pNX8 DNA and codina region SEQ ID NO: 14 Protein sequence of SEQ ID NO: 13 SEQ ID NO: 15 pNX9 DNA and coding region SEQ ID NO: 16 Protein seouence of SEQ ID NO: 15 SEQ ID NO: 17 pNXlO DNA and coding region SEQ ID NO: 18 Protein sequence of SEQ ID NO: 17 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> WO 93/25693 PCT/GB93/01283 2 5 3 2 8 0 -70-CLA1MS

1. A xylanase which has ax least one catalytic domain which is substantially homologous with a xylanase of an anaerobic fungus and which is not a full length

5 namrai xylanase.

2. A xylanase as claimed in claim 1, wherein the or each catalytic domain is identical to a catalytic domain of a namrai xylanase from an anaerobic fungus.

10 3. A xylanase as claimed in claim 1 or 2, wherein the anaerobic fungus is a rumen fungus.

4. A xylanase as claimed in claim 3, wherein the rumen fungus is of the genus Neocallimasrix.

IS

5. A xylanase as claimed in claim 4, wherein the fungus is Neocallimasrix patriciarum.

6. A xylanase as claimed in any one of claims 1 to 5, which is derived from 20 a xylanase having the structure (from the N-tenninus to the C-terminus):

CAT1-UNK1-CAT2-UNK2-CTR1-CTR2

wherein:

CAT1 represents a first catalytic domain.

25 CAT2 represents a second catalytic domain.

LINK1 represents a first linker,

UNK2 represents a second linker,

CTR1 represents a first C-terminal repeat, and CTR2 represents a second C-terminal repeat.

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7. A xylanase as claimed in claim 6, wherein CAT1 has a sequence which is identical or otherwise substantially homologou^ to the sequence:

RLTVGN

GQTQHKGVADGYSYEIWLDNTGGSGSMTLGSGATFKAEWN ASVNRGNFLARRGLDFGSQK KATDYSYIGLDYTATYRQTG SASGNSRLCVYGWFQNRGVQ GVPLVEYYQEDWVDWVPD A QGRMVTIDGAQYKIFQMDHT GPTINGGSETFKQYFSVRQQ KRTSGHTTVSDHFKEWAKQG WGIGNLYEVALNAEGWQSSG IADVTKLDVYTTQKGSNP AP.

8. A xylanase as claimed in claim 6 or 7, wherein CAT2 has a sequence /which is identical or otherwise substantially homologous^) the sequence:

K

FfVGNGQNQHKGVNDGFSYEIWLDNTGGNGSMTLGSGATF 15 KAEWNAAVNRGNFLARRGLDFGSQKKATDYDYIGLDYAAT

YKQTASASGNSRLCVYGWFQ NRGLNGVPLVEYYHEDWVD WVPDAQGKMVTIDGAQYKIF QMDHTGPTINGGSETFKQYF SVRQQKRTSGHITVSDHFKE WAKQGWGIGNLYEVALNAEG WQSSGVADVTLLDVYTTPKG SSPA.

20

9. A xylanase as claimed in claim 6, 7 or 8, wherein LINKl has a sequence which (is identical or otherwise substantially homologous) to the sequence:

TSTGTVPSSSAGGSTANGK.

25

10. A xylanase as claimed in any one of claims 6 to 9, wherein LINK2 has a sequence which is identical or otherwise substantially homologous/to the sequence: TSAAPRTTTRTTTRTKSLPTNYNK. '

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11. A xylanase as claimed in any one of claims 6 to 10, wherein CTR1 has a sequence ^hich is identical or otherwise substantially homologous^) the sequence: CSARUAQGYKCCSDPNCWYYTDEDGTWGVENNDWCGCG.

5 12. A xylanase as claimed in any one of claims 6 to 11, wherein CTR2 has a sequence (which is identical or otherwise substantially homologousjto the sequence: VEQCSSKITSQGYKCCSDPNC WFYTDDDGKWGVENNDWCGCGF.

13. A xylanase as claimed in any one of claims 6 to 12 comprising a catalytic 1 o domain ^vhich is substantially homologous 'with at least one of CAT1 and CAT2

and is missing at least part of the amino acid sequence jdownstream (iejtowards the C-terminus) of CAT2.

14. A xylanase as claimed in claim 13, wherein at least part of CTR2 is 15 missing.

15. A xylanase as claimed in claim 13 or 14, wherein at least part of CTR1 is missing.

20

16. A xylanase as claimed in any one of claims 6 to 15, which has the structure:

CAT 1-IJNK1-C AT2-IJNK2-CTR1 (truncated); CATl-lJNKl-CAT2-LINK2(tnincated);

LINKl (truncated)-CAT2-LINK2(truncated); 2 5 CAT1-LINK1 (truncated);

CATl(truncated);

LINKl(truncated)-C AT2-LINK2-CTR1-CTR2; LINKl(truncated)-CAT2-LINK2-CTRl(truncated); or LINK 1 (truncated)-C AT2(truncated).

30

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17. A xylanase as claimed in claim 15, which has the structure:

LINK l(truncated)-CAT2-LINK2( truncated).

18. An isolated or recombinant DNA molecule encoding a xylanase which has 5 a catalytic domain substantially^homologous with a xylanase of an aiiaerobic fungus, provided that the DNA molecule does not comprise a full length copy of natural mRNA encoding the xylanase.

19. A DNA molecule as claimed in claim 18, wherein the absent portion, or 10 one of the absent portions, of the DNA corresponds to the 3' and/or 5' untranslated region of the mRNA.

20. A DNA molecule as claimed in claim 18 or 19, which is derived from a DNA molecule having the following structure:

15

5 'utr-sig-catl-linkl-cafl-link2-ctrl-ctrJ2-3 'utr,

wherein

5 'utr represents a 5' untranslated region;

sig encodes a signal peptide;

20 cat 1 encodes a first catalytic domain;

linkl encodes a first linker sequence;

catl encodes a second catalytic domain;

linkl encodes a second linker sequence;

Ctrl encodes a first C-terminal repeat;

25 Ctrl encodes a second C-terminal repeat; and

3 'uxr represents a 3' untranslated region.

21. A DNA sequence as claimed in claim 20, wherein the 3'utr segment has a sequence which is identical to or otherwise substantially homologous with the

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following sequence:

TTTTATTATATCAATCTCTAATTTA1T1 I'ITi AGGAAAAAAATAAAAAAATAAATATAAT AAATATTAGAGAGTAATATTTAAAAACAAAGAAATTTAAAAACGTTTATTTAGTTATTTT TTTTACTGGTTAAAAAAAAAATAAAAAACAAAATTAATAAAGATATTTTTGAAAAATATT 5 GAATTAGAAAAAAA.

22. A DNA sequence as claimed in claim 20 or 21, wherein the sig segment has a sequence which is identical to or otherwise substantially homologous with the following sequence:

10 ATCAGAACTATTAAA 11C1111'l CGCAGTAGCTATTGCAAC llil'l G

CTAAGGCCCAATGGGGTGGAGGTGGTGCCTCTGCTGGTCAA.

23. A DNA sequence as claimed in claim 20, 21 or 22, wherein the carl segment has a sequence which is identical to or otherwise substantially homologous

15 with the following sequence:

AGATTAACCGTCGGTAATG

GTCAAACCCAACATAAGGGTGTAGCTGATGGTTACAGTTATGAAATCTGGTTAGATAACA CCGGTGGTAGTGGTTCTATGACTCTCGGTAGTGGTGCAACCTTCAAGGCTGAATGGAATG CA1L1L.11AACCGTGGTAAL1lOJl lUCCCURJUiUjlCl 1 GACITCGG 11C1 CAAAAGA 20 AGGCAACCGATTACAGCTACATTGGATTGGATTATACTGCAACTTACAGACAAACTGGTA

GTGTTCCATTGGTAGAATACTACATCATTGAAGATTGGGTTGACTGGGTTCCAGATGCAC AAGGTAGAATGGTAAC CATTGATGGAGCTCAATATAAGA I'i'l'IC CAAATGGATCACACTG GTCCAACTATCAATGGTGGTAGTGAAACCTTTAAGCAATACTTCAGTGTCCGTCAACAAA 25 AGAGAACTTCTGGTCATATTACTGTCTCAGATCACTTTAAGGAATGGGCCAAACAAGGTT

GGGGTATTGGTAACCTTTATGAAGTTGCTTTGAACGCCGAAGGTTGGCAAAGTAGTGGTA TAGCTGATGTCACCAAGTTAGA1G i'1'l'ACACAACCCAAAAAGG IHJIAATCCTGCCCCT.

24. A DNA sequence as claimed in any one of claims 20 to 23, wherein the 30 linkl segment has a sequence which is identical to or otherwise substantially homologous with the following sequence:

ACCTCCACTGGTACl G l'l CCAAGCAGl'lClGCl GG f GGAAGTACTGCCAATGGTAAA.

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25. A DNA sequence as claimed in any one of claims 20 to 24, wherein the cacti, segment has a sequence which is identical to or otherwise substantially homologous with the following sequence:

AAGT

5 TTACTGTCGGTAATGGACAAAACCAACATAAGGGTGTCAACGATGGTrTCAGTTATGAAA T(J 1 CKJ I' 1AGATAACACTGGTGGTAACGO I'l L'rATGACTCTCGGTAGTGGTGCAAL 1' 1 '1CA AGGCTGAATGGAATGCAGCTGTTAACCGTGGTAAC11CL' 11 (jCCQJ 1 CO 1OO1 (J 1TUACT TCGGTTCTCAAAAGAAGGCAACCGATTACGACTACATTGGATTAGATTATGCTGCTACTT

10 ACCGTGGACTTAATGGCGTTCCTTTAGTAGAATACTACATCATTGAAGATTGGGTTGACT

GGGTTCCAGATGCACAAGGAAAAATGGTAACCATTGATGGAGCTCAATATAAGATTTTCC AAATGGATCACACTGGTCCAACTATCAATGGTGGTAGTGAAACCTTTAAGCAATACTTCA GTGTCCGTCAACAAAAGAQAAC1111UO1 (JATATTACTGT CTCAQATCACTTTAAGGAAT GGGCCAAACAAGGTTGGGGTATTGGTAACCTTTATGAAGTTGCTTTOAACGCCGAAGGTT 15 GGCAAAGTAGTGGTGTTGCTGATGTCACCTTATTAGATGTTTACACAACTCCAAAGCGTT

CTAGTCCAGCC.

26. A DNA sequence as claimed in any one of claims 20 to 25, wherein the linkl segment has a sequence which is identical to or otherwise substantially

20 homologous with the following sequence:

ACCTCTGCCGCTCCTCGTACTACTACCCGTACTACTACTCGTACCAAG1C1LT1LCAACC AATTACAATAAG.

27. A DNA sequence as claimed in any one of claims 20 to 26, wherein the 25 ctri segment has a sequence which is identical to or otherwise substantially homologous with the following sequence:

TOl 1C1OCTAGAATTACTGCTCAAGGTTACAAO1O1101 AGCGATCCAAAl'l b 101lOl1 T ACTAC ACTGATGAGGATGGTACCrGGGO 101' 1UAAAACAACGAC1UO KjIOOIIOI OCT.

30

28. A DNA sequence as claimed in any one of claims 20 to 27, wherein the ctr2 segment has a sequence which is identical to or otherwise substantially homologous with the following sequence:

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(iTTGAACAAKi 1'ILTI LCAAGATCAL'i'I CICAAGGTTACAAUi li i'lUTAGCGATCCAAAT TGCCTTGTTTTCTACACTGATGACCATGGTAAATGGGGTCTTCAAAACAACGACTGGTGT GGTTGTGGTTTC.

5

29. A DNA sequence as claimed in any one of claims 20 to 28, wherein the 5'uxr segment has a sequence which is jrimriral to or otherwise substantially homologous with the following sequence:

TAAGCAGTAAAATACTAATTAATAA

AAAATTAAAGAATTATGAAAAATTTAAATTTAAAAATTTAAAAGAATTATGAAAAATTTA 10 AATTTAAAAATTTAAAAAAAACTAATTTAGTAAAAAATTAAAGAATTATTGAAAATTTTA

AATGTAAAAATTTAAAAAATACAAATrTGTAAAAAAAAATGAAACAATTATGAAAAATTA AAATGTAAAAGTTTAAAAAATACAAATTTGTAAGAAAAATAAAGAATTATAAAAAAAATA AAGAATTATGAAAAACCCAAATGTAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA.

IS

30. A DNA sequence as HamwH in any one of claims 18 to 29 encoding a xylanase as HamwH in any one of claims 1 to 17.

31. A DNA sequence as claimed in any one of claims 20 to 30 which comprises the following segments:

20

5 'uxr-sig-catl-linkl-cafl-linkl-arl (truncated);

5 W-nW/Kl -linH -rart-Iintr>(tnmmtrd)-

Z^l(tnmcated>-caz2-tirot2(trancatcd);

5 'utr-sig-catl-linkl(tiuiscaxed);

25 5 'utr-sig-catl (truncated);

linkl (tiuncated)-car2-#njfc2-crrl -ctf2-3 'uxr,

linkl (truncated)-cm2-tin*2-<3rl (truncated);

linkl (mincated)-azr2(truncated).

30

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32. A DNA molecule as claimed in any one of claims 18 to 31, which is in the form of a vector.

33. A DNA molecule as claimed in claim 32, wherein the vector is a plasmid.

5

34. A DNA molecule as claimed in claim 32 or 33, wherein the vector is an' • expression vector.

35. A DNA molecule which is; or~coxnprises the insert of, plasmid pNX3, 10 pNX4, pNX5, pNX6, pNX7, pNX8, pNX9 or pNXlO, as defined herein.

36. A DNA molecule which is, or comprises the insert of, plasmid pNX5, pNX9 or pNXlO, as defined herein.

37. A xylanase substantially as herein described with reference to any one of examples 1, 8 and 10.

38. An isolated or recombinant DNA molecule encoding a xylanase as claimed in claim 18 substantially as herein described with reference to the examples.

39. An isolated or recombinant DNA molecule encoding a xylanase substantially as herein described with reference to any one of figures 1, 2 and 4 to 6.

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15

40. A host ceil transfected or transformed with a DNA molecule as claimed in any one of claims 18 to 36, 38 and 39.

41 • The use of a xylanase as HafrrwH in any one of claims 1 to 17 and 37

in the modification of baked products. 20 - ' —

42. The use of a xylanase as claimed-in any one of claims 1 to 17 and 37 as an enzyme supplement for animal feed.

43. The use of a xylanase as claimed in any one of claims 1 to 17 and 37

25 as an impurity remover in pulp.

44> The use of a xylanase as Haimgri in any one of claims 1 to 17 and 37 in the prebleaching of kraft.pulp.

45. A xylanase which has at lease one catalytic domain which is substantially homologous with a xylanase of an anaerobic fungus and whi ch i s not a ful 1 length natural xylanase.

5

46. An isolated or recombinant DNA mohrnir a xylanase which has a catalytic doihain substantially homologous with a xylanase of an anaerobic fungus and which is not a full length natural xylanase, provided that if the DNA molecule is cDNA encoding a xylanase of Neocallimastix frontalis then the DNA molecule is operatively coupled to a promoter.

END OF CLAIMS

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