AU761093B2

AU761093B2 - Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism

Info

Publication number: AU761093B2
Application number: AU10413/00A
Authority: AU
Inventors: Jorg Overhage; Horst Priefert; Jurgen Rabenhorst; Alexander Steinbuchel
Original assignee: Haarmann and Reimer GmbH
Current assignee: Symrise AG
Priority date: 1998-10-31
Filing date: 1999-10-20
Publication date: 2003-05-29
Anticipated expiration: 2019-10-20
Also published as: HUP0104772A2; AU1041300A; WO2000026355A3; PL348647A1; HUP0104772A3; EP1124947A2; KR20020022045A; SK5742001A3; WO2000026355A2; JP2003533166A; CN1325444A; BR9914930A; DE19850242A1; CA2348962A1; HK1041902A1; IL142272A0

Description

WO 00/26355 PCT/EP99/07952 -1- Constructing production strains for the preparation of substituted phenols by specifically inactivating genes of eugenol and ferulic acid catabolism The present invention relates to the construction of production strains and to a process for preparing substituted methoxyphenols, in particular vanillin.

DE-A 4 227 076 (process for preparing substituted methoxyphenols, and microorganism which is suitable for this purpose) describes the preparation of substituted methoxyphenols using a novel Pseudomonas sp.. The starting material in this context is eugenol and the products are ferulic acid, vanillic acid, coniferyl alcohol and coniferyl aldehyde.

An extensive review of the biotransformations which were possible using ferulic acid, which was written by Rosazza et al. (Biocatalytic transformation of ferulic acid: an abundant aromatic natural product; J. Ind. Microbiol. 15:457-471), also appeared in 1995.

The genes and enzymes for synthesizing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillic and vanillin acid from Pseudomonas sp. were described in EP-A 0 845 532.

The enzymes for converting trans-ferulic acid into trans-feruloyl-SCoA ester and subsequently into vanillin, and also the gene for cleaving the ester, were described by the Institute of Food Research, Norwich, GB, in WO 97/35999. In 1998, the content of the patent also appeared in the form of scientific publications (Gasson et al. 1998.

Metabolism of ferulic acid to vanillin. J. Biol. Chem. 273:4163-4170; Narbad and Gasson 1998. Metabolism of ferulic acid via vanillin using a novel CoA-dependent pathway in a newly isolated strain of Pseudomonas fluorescens. Microbiology 144:1397- 1405).

-2- DE-A 195 32 317 describes the use of Amycolatopsis sp. for obtaining vanillin from ferulic acid fermentatively in high yields.

The known processes suffer from the disadvantage that they either achieve only very low yields of vanillin or make use of relatively expensive starting compounds. While the last-mentioned process (DE-A 195 32 317) does achieve high yields, the use of Pseudomonas sp. HR 199 and Amycolatopsis sp. HR167 for biotransforming eugenol into vanillin requires a fermentation which is carried out in two steps, consequently leading to substantial expense and consumption of time.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.

The present invention constructs organisms which are able to convert the relatively inexpensive raw material eugenol into vanillin in a one-step process.

The invention is achieved by means of constructing production strains of unicellular or multicellular organisms, which strains are characterized in that enzymes of eugenol and/or ferulic acid catabolism are inactivated such that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid accumulate.

The production strain may be unicellular or multicellular. Accordingly, the invention 30 can relate to microorganisms, plants or animals. Furthermore, use can also be made of extracts which are obtained from the production strain. According to the invention, P:\WPDOCS\CRNPuniu\Spci'\7595580.spe.doc.24/3/03 -2apreference is given to using unicellular organisms. These latter organisms can be microorganisms or animal or plant cells. According to the invention, particular preference is given to using fungi and bacteria. The highest preference is given to bacterial species. Those bacteria which may in particular be used, after their eugenol and/or ferulic acid catabolism has/have been altered, are species of Rhodococcus, Pseudomonas und Escherichia.

In the simplest case, known, conventional microbiological methods can be used for isolating the organisms which may be employed in accordance with the invention.

S S o*D o*o o* Thus, the enzymic activity of the proteins involved in eugenol and/or ferulic acid catabolism can be altered by using enzyme inhibitors. Furthermore, the enzymic activity of the proteins involved in eugenol and/or ferulic acid catabolism can be altered by mutating the genes which encode these proteins. Such mutations can be generated in a random manner by means of classical methods, for example by using UV irradiation or mutation-inducing chemicals.

Recombinant DNA methods, such as deletions, insertions and/or nucleotide exchanges, are likewise suitable for isolating the novel organisms. Thus, the genes of the organisms can, for example, be inactivated using other DNA elements (2 elements). Suitable vectors can likewise be used for replacing the intact genes with gene structures which are altered and/or inactivated. In this context, the genes which are to be inactivated, and the DNA elements which are employed for the inactivation, can be obtained by means of classical cloning techniques or by means of polymerase chain reactions (PCR).

For example, in one possible embodiment of the invention, eugenol catabolism and ferulic acid catabolism can be altered by inserting Q elements, or introducing deletions, into appropriate genes. In this context, the abovementioned recombinant DNA methods can be used to inactivate the functions of the genes, which encode dehydrogenases, synthetases, hydratase-adolases, thiolases or demethylases, such that production of the relevant enzymes is blocked. Preferably, the genes are those which encode coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehdrogenases or vanillic acid demethylases. Very particular preference is given to genes which encode the amino acid sequences specified in EP-A 0845532 and/or nucleotide sequences which encode their allelic variations.

The invention accordingly also relates to gene structures for preparing transformed organisms and mutants.

-4- Preference is given to employing gene structures in which the nucleotide sequences encoding dehydrogenases, synthetases, hydratase-aldolases, thiolases or demethylases are inactivated for isolating the organisms and mutants. Particular preference is given to gene structures in which the nucleotide sequences encoding coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases are inactivated. Very particular preference is given to gene structures which exhibit the structures given in Figures la to Ir having the nucleotide sequences which are depicted in Figures 2a to 2r and/or nucleotide sequences encoding their allelic variations. In this context, particular preference is given to nucleotide sequences 1 to 18.

The invention also encompasses the part sequences of these gene structures as well as functional equivalents. Functional equivalents are to be understood as meaning those derivatives of the DNA in which individual nucleobases have been exchanged (wobble exchanges) without the function being altered. Amino acids may also be exchanged at the protein level without this resulting in an alteration in function.

One or more DNA sequences can be inserted upstream and/or downstream of the gene structures. By cloning the gene structures, it is possible to obtain plasmids or vectors which are suitable for the transformation and/or transfection of an organism and/or for conjugative transfer into an organism.

The invention furthermore relates to plasmids and/or vectors for preparing the organisms and mutants which are transformed in accordance with the invention.

These organisms and mutants consequently harbour the gene structures which have been described. The present invention accordingly also relates to organisms which harbour the said plasmids and/or vectors.

The nature of the plasmids and/or vectors depends on what they are being used for. In order, for example, to be able to replace the intact genes of eugenol and/or ferulic acid catabolism in pseudomonads with the genes which have been inactivated with omega elements, there is a need for vectors which, on the one hand, can be transferred into pseudomonads (conjugatively transferable plasmids) but which, on the other hand, cannot be replicated in these organisms and are consequently unstable in pseudomonads (so-called suicide plasmids). DNA segments which are transferred into pseudomonads with the aid of such a plasmid system can only be retained if they become integrated by homologous recombination into the genome of the bacterial cell.

The described gene structures, vectors and plasmids may be used for preparing different transformed organisms or mutants. The said gene structures can be used for replacing intact nucleic acid sequences with altered and/or inactivated gene structures. In the cells, which can be obtained by transformation or transfection or conjugation, the intact gene is replaced, by homologous recombination, with the altered and/or inactivated gene structure, as a consequence of which the resulting cells now only possess the altered and/or inactivated gene structure in their genome.

In this way, preferably genes can be altered and/or inactivated, in accordance with the invention, such that the relevant organisms are able to produce coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid.

Mutants of the strain Pseudomonas sp. HR199 (DSM 7063), which was described in detail in DE-A 4 227 076 and EP-A 0845532, are examples of production strains which have been constructed in this way in accordance with the invention, with the corresponding gene structures ensuing, inter alia, from Figures la to Ir, in combination with Figures 2a to 2r: 1. Pseudomonas sp. HR199calAQKm, which contains the Q2Km-inactivated calA gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. la; Fig. 2a).

-6- 2. Pseudomonas sp. HR199calAQGm, which contains the QGm-inactivated calA gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. lb; Fig. 2b).

3. Pseudomonas sp. HR199calAA, which contains the deletion-inactivated calA gene in place of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. 1c; Fig. 2c).

4. Pseudomonas sp. HR199calM Km, which contains the QKm-inactivated caiB gene in place of the intact caiB gene encoding coniferyl aldehyde, dehydrogenase (Fig. id; Fig. 2d) 5. Pseudomonas sp. HR199calBQ Gm, which contains the n2Gm-inactivated caiB gene in place of the intact caiB gene encoding coniferyl aldehyde, dehydrogenase (Fig. le; Fig. 2e).

6. Pseudomonas sp. HR199calBA, which contains the deletion -inactivated caiB gene in place of the intact caiB gene encoding coniferyl aldehyde, dehydrogenase (Fig.lf; Fig. 2f).

7. Pseudornonas sp. HR199fcsQ Km, which contains the 92Km-inactivated fcs gene in place of the intact fcs gene encoding feruloyl-CoA synthetase (Fig.lg; Fig. 2g).

8. Pseudomonas sp. HR199fcsf2Gm, which contains the 9 Gm-inactivated fes gene in place of the intact fcs gene encoding feruloyl-CoA synthetase (Fig.lh; Fig. 2h).

9. Pseutdornonas sp. HTRL99ftsA, which contains the deletion-inactivated fcs gene in place of the intact fcs gene encoding feruloyl-CoA synthetase (Fig.li; Fig. 2i).

10. Pseudomnonas sp. HR199echQ Km, which contains the 92Km-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase (Fig. lj; Fig. 2j).

11. Pseudonwnas sp. HR199eclzQGm, which contains the Q Gm-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase (Fig.lk; Fig. 2k).

-7- 12. Pseudomonas sp. HR199echA, which contains the deletion-inactivated ech gene in place of the intact ech gene encoding enoyl-CoA hydratase-aldolase (Fig.11; Fig. 21).

13. Pseudomonas sp. HR199aatQKm, which contains the QKm-inactivated aat gene in place of the intact aat gene ecnoding beta-ketothiolase (Fig. Im; Fig. 2m).

14. Pseudomonas sp. HR199aatQGm, which contains the QGm-inactivated aat gene in place of the intact aat gene encoding beta-ketothiolase (Fig.1n; Fig. 2n).

15. Pseudomonas sp. HR199aatA, which contains the deletion-inactivated aat gene in place of the intact aat gene encoding beta-ketothiolase (Fig. lo; 16. Pseudomonas sp. HR199vdh2Km, which contains the 92Km-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lp; Fig. 2p).

17. Pseudomonas sp. HR199vdhQGm, which contains the QGm-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.lq; Fig. 2q).

18. Pseudomonas sp. HR199vdhA, which contains the deletion-inactivated vdh gene in place of the intact vdh gene encoding vanillin dehydrogenase (Fig.1r; Fig. 2r).

19. Pseudomonas sp. HR199vdhBf2Km, which contains the QKm-inactivated vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase

II.

Pseudomonas sp. HR199vdhBQGm, which contains the 92Gm-inactivated vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase

II.

21. Pseudomonas sp. HR199vdhBA, which contains the deletion-inactivated vdhB gene in place of the intact vdhB gene encoding vanillin dehydrogenase II.

22. Pseudomonas sp. HR199adhQKm, which contains the g Km-inactivated adh gene in place of the intact adh gene encoding alcohol dehydrogenase.

23. Pseudomonas sp. HR199adh2Gm, which contains the 92Gm-inactivated adh gene in place of the intact adh gene encoding alcohol dehydrogenase.

24. Pseudomonas sp. HR199adhA which contains the deletion-inactivated adh gene in place of the intact adh gene encoding alcohol dehydrogenase.

25. Pseudomonas sp. HR199vanA2Km, which contains the QKm-inactivated vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic acid demethylase.

26. Pseudomonas sp. HR199vanAGm, which contains the 92Gm-inactivated vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic acid demethylase.

27. Pseudomonas sp. HR199vanAA, which contains the deletion-inactivated vanA gene in place of the intact vanA gene encoding the a-subunit of vanillic acid demethylase.

28. Pseudomonas sp. HR199vanB9Km, which contains the QKm-inactivated vanB gene in place of the intact vanB gene encoding the 3-subunit of vanillic acid demethylase.

29. Pseudomonas sp. HR199vanB92Gm, which contains the QGm-inactivated vanB gene in place of the intact vanB gene encoding the 3-subunit of vanillic acid demethylase.

30. Pseudomonas sp. HR199vanBA, which contains the deletion-inactivated vanB gene in place of the intact vanB gene encoding the 3-subunit of vanillic acid demethylase.

The invention additionally relates to a process for the biotechnological preparation of organic compounds. In particular, this process can be used to prepare alcohols, aldehydes and organic acids. The latter are preferably coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid.

The above-described organisms are employed in the novel process. The organisms which are very particularly preferred include bacteria, in particular the Pseudomonas species. Specifically, the abovementioned Pseudomonas species can preferably be employed for the following processes: I1. Pseudomonas sp. HRl99calAf Km, Pseudomonas sp. HR199calAQGm and Pseudomonas sp. fIR 99calAA for preparing coniferyl alcohol from eugenol.

2. Pseudomonas sp. HRl99calBL2Krn, Pseudomonas sp. IHRl99calBfQGm and Pseudomonas sp. HR199calBA for preparing coniferyl aldehyde from eugenol or coniferyl alcohol.

3. Pseudornonas sp. HR199fcs92Km, Pseudornonas sp. HR199fcsQ2Gm, Pseudomonas sp. HRl99fcsA, Pseudomonas sp. BR199echQ2Km, Pseudomonas sp. HR199echQ2Gm and Pseudomonas sp. HRl99echA for preparing ferulic acid from eugenol or coniferyl alcohol or coniferyl aldehyde.

4. Pseudomionas sp. HRl99vdhg2Km, Pseudomonas sp. HRl99vdhQGm, Pseudornonas sp. HR 199vdhA, Pseudomionas sp. HR 199vdlzQGmvdhBg Km, Pseudomonas sp. HR199vdh9 KmvdhB92Gm, Pseudomionas sp. HRl99vdhzA vdhB9 Gm and Pseudornonas sp. HRl99vdhiAvdhBf Km for preparing vanillin from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic acid.

Pseudomonas sp. HRl99vanA 2Km, Pseudornonas sp. HIR199vanAQ~m, Pseudoinonas sp. HR199vanAA, Pseudomonas sp. HR199vanBQ Km, Pseudomonas sp. HR199vanBgi Gm and Pseudomionas sp. HR199vanIBA for preparing vanillic acid from eugenol or coniferyl alcohol or coniferyl aldehyde or ferulic acid or vanillin.

Eugenol is the preferred substrate. However, it is also possible to add further substrates or even to replace the eugenol with another substrate.

Suitable nutrient media for the organisms which are employed in accordance with the invention are synthetic, semisynthetic or complex culture media. These media may comprise carbon-containing and nitrogen-containing compounds, inorganic salts, where appropriate trace elements, and vitamins.

Carbon-containing compounds which may be suitable are carbohydrates, hydrocarbons or organic standard chemicals. Examples of compounds which may preferably be used are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.

The sugar is preferably glucose. The organic acids which may preferably be employed are citric or acetic acid. Examples of the complex mixtures are malt extract, yeast extract, casein or casein hydrolysate.

Inorganic compounds are suitable nitrogen-containing substrates. Examples of these are nitrates and ammonium salts. Organic nitrogen sources can also be used. These sources include yeast extract, soya bean meal, casein, casein hydrolysate and corn steep liquor.

Examples of the inorganic salts which may be employed are sulphates, nitrates, chlorides, carbonates and phosphates. The metals which the said salts contain are preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.

The temperature for the culture is preferably in the range from 5 to 100°C. The range from 15 to 60 0 C is particularly preferred, with 22 to 37°C being most preferred.

The pH of the medium is preferably 2 to 12. The range from 4 to 8 is particularly preferred.

In principle, any bioreactor known to the skilled person can be employed for carrying out the novel process. Preferential consideration is given to any appliance which is 11 suitable for submerged processes. This means that vessels which do or do not possess a mechanical mixing device may be employed in accordance with the invention.

Examples of the latter are shaking apparatuses, and bubble column reactors or loop reactors. The former preferably include all the known appliances which are fitted with stirrers of any design.

The novel process can be carried out continuously or batchwise. The fermentation time required for achieving a maximum quantity of product depends on the specific nature of the organism employed. However, in principle, the fermentation times are between 2 and 200 hours.

The invention is explained in more detail below while referring to examples: Mutants of the eugenol-utilizing strain Pseudomonas sp. HR199 (DSM 7063) were generated in a targeted manner by specifically inactivating genes of eugenol catabolism by means of inserting omega elements or introducing deletions. The omega elements employed were DNA segments which encoded resistances to the antibiotics kanamycin (QKm) and gentamycin (QGm). These resistance genes were isolated from Tn5 and the plasmid pBBR1MCS-5 using standard methods. The genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which encode coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloyl-CoA synthetase, enoyl-CoA hydratase-aldolase, beta-ketothiolase, vanillin dehdrogenase, alcohol dehydrogenase, vanillin dehdrogenase 1 and vanillic acid demethylase, were isolated from genomic DNA of the strain Pseudomonas sp. HR199 using standard methods and cloned into pBluescript SK-. By means of digesting with suitable restriction endonucleases, DNA segments were removed from these genes (deletion) or substituted with Q elements (insertion), resulting in the respective gene being inactivated. The genes which had been mutated in this manner were recloned into conjugatively transferable vectors and subsequently introduced into the strain Pseudomonas sp. HR199. Suitable selection was used to obtain transconjugants which had replaced the respective functional wild-type gene with the newly -12introduced inactivated gene. The insertion and deletion mutants which were obtained in this way now only possessed the respective inactivated gene. This procedure was used to obtain both mutants possessing only one defective gene and multiple mutants, in which several genes had been inactivated in this manner. These mutants were employed for biotransforming a) eugenol into coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid; b) coniferyl alcohol into coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid; c) coniferyl aldehyde into ferulic acid, vanillin and/or vanillic acid; d) ferulic acid into vanillin and/or vanillic acid, and e) vanillin into vanillic acid.

-13- Materials and Methods Conditions for growing the bacteria.

Strains of Escherichia coli were propagated at 37°C in Luria-Bertani (LB) or M9 mineral medium Sambrook, E. F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Strains of Pseudomonas sp. were propagated at 0 C in Nutrient Broth (NB, wt/vol) or in mineral medium (MM) G.

Schlegel, et al. 1961. Arch. Mikrobiol. 38:209-222) or HR mineral medium (HR- MM) Rabenhorst, 1996. Appl. Microbiol. Biotechnol. 46:470-474.). Ferulic acid, vanillin, vanillic acid and protocatechuic acid were dissolved in dimethyl sulphoxide and added to the respective medium to give a final concentration of 0.1% (wt/vol).

Eugenol was either added directly to the medium to give a final concentration of 0.1% (vol/vol) or applied to filter paper (circular filter 595, Schleicher Schuell, Dassel, Germany) in the lids of MM agar plates. When transconjugants and mutants of Pseudomonas sp. were being propagated, tetracycline, kanamycin and gentamycin were employed in final concentrations of 25 Ig/ml, 100 pg/ml and 7.5 Ag/ml, respectively.

Qualitative and quantitative detection of metabolic intermediates in culture supernatants.

Culture supernatants were analysed by high pressure liquid chromatography (Knauer HPLC) either directly or after dilution with doubly distilled H20. The chromatography was carried out on Nucleosil 100 C18 (7 Am, 250 x 4 mm). 0.1% (vol/vol) formic acid and acetonitrile was used as the solvent. The course of the gradient employed for eluting the substances was as follows: 00:00 06:30 26% acetonitrile 06:30 08:00 100% acetonitrile 08:00 12:00 100% acetonitrile 12:00 13:00 26% acetonitrile 13:00 18:00 26% acetonitrile -14- Purification of vanillin dehydrogenase II.

The purification was carried out at 4 0

C.

Crude extract.

Pseudomonas sp. HR199 cells which had been propagated on eugenol were washed in 10 mM sodium phosphate buffer, pH 6.0, then resuspended in the same buffer and disrupted by being passed twice through a French press (Amicon, Silver Spring, Maryland, USA) at a pressure of 1000 psi. The cell homogenate was subjected to an ultracentrifugation (1 h, 100,000 x g, 4 0 resulting in the soluble fraction of crude extract being obtained as the supernatant.

Anion exchange chromatography on DEAE Sephacel.

The soluble fraction of the crude extract was dialysed overnight against 10 mM sodium phosphate buffer, pH 6.0. The dialysate was loaded onto a DEAE-Sephacel column (2.6 cm x 35 cm, bed volume[BV]: 186 ml) which had been equilibrated with 10 mM sodium phosphate buffer, pH 6.0, and which had a flow rate of 0.8 ml/min. The column was rinsed with two BV of 10 mM sodium phosphate buffer, pH 6.0. The vanillin dehydrogenase II (VDH II) was eluted with a linear salt gradient of from 0 to 400 mM NaCI in 10 mM sodium phosphate buffer, pH 6.0 (750 ml). 10 ml fractions were collected. Fractions having a high VDH II activity were combined to form the DEAE pool.

Determining the vanillin dehydrogenase activity.

The VDH activity was determined at 30 0 C using an optical enzymic test. The reaction mixture, whose volume was 1 ml, contained 0.1 mmol of potassium phosphate (pH 0.125 Itmol of vanillin, 0.5 Amol of NAD, 1.2 /mol of pyruvate (Na salt), lactate dehydrogenase (1 U; from pig heart) and enzyme solution. The oxidation of vanillin was monitored at X 340 nm (Evanillin 11.6 cm 2 /lmol). The enzyme activity was given in units with 1 U corresponding to the quantity of enzyme which converts 1 /mol of vanillin per minute. The protein concentrations in the samples were determined using the method of Lowry et al. H. Lowry, N. J.

Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).

Determining the coniferyl alcohol dehydrogenase activity.

The CADH activity was determined at 30 0 C using an optical enzymic test in accordance with Jaeger et al. L. Jaeger, Eggeling and H. Sahm. 1981. Current Microbiology. 6:333-336). The reaction mixture, whose volume was 1 ml, contained 0.2 mmol of tris/HCI (pH 0.4 Amol of coniferyl alcohol, 2 /mol of NAD, 0.1 mmol of semicarbazide and enzyme solution. The reduction of NAD was monitored at X 340 nm (E 6.3 cm 2 /Amol). The enzyme activity was given units with 1 U corresponding to the quantity of enzyme which converts 1 pmol of substrate per minute. The protein concentrations in the samples were determined by the method of Lowry et al. H. Lowry, N. J. Rosebrough, A. L. Farr and R.

J. Randall. 1951. J. Biol. Chem. 193:265-275).

Determining the coniferyl aldehyde dehydrogenase activity.

The CALDH activity was determined at 30 0 C using an optical enzymic test. The reaction mixture, whose volume was 1 ml, contained 0.1 mmol of tris/HCI (pH 8.8), 0.08 ttmol of coniferyl aldehyde, 2.7 imol of NAD and enzyme solution. The oxidation of coniferyl aldehyde to ferulic acid was monitored at X 400 nm (e 34 cm 2 /A/mol). The enzymic activity was given in units with 1 U corresponding to the quantity of enzyme which converts 1 pimol of substrate per minute. The protein concentrations in the samples were determined by the method of Lowry et al. H.

Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem.

193:265-275).

Determining the feruloyl-CoA synthetase (ferulic acid thiokinase) activity.

The FCS activity was determined at 30 0 C using an optical enzymic test which was a modification of that of Zenk et al. (Zenk et al. 1980. Anal. Biochem. 101:182-187).

The reaction mixture, whose volume was 1 ml, contained 0.09 mmol of potassium phosphate (pH 2.1 A/mol of MgCI2, 0.7 Amol of ferulic acid, 2 /mol of ATP, -16- 0.4 #jmol of coenzyme A and enzyme solution. The formation of the CoA ester from ferulic acid was monitored at X 345 nm (E 10 cm 2 The enzymic activity was given in units with 1 U corresponding to the quantity of enzyme which converts 1 Amol of substrate per minute. The protein concentrations in the samples were determined using the method of Lowry et al. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. J. Biol. Chem. 193:265-275).

Electrophoretic methods.

Protein-containing extracts were fractionated under native conditions in 7.4% (wt/vol) polyacrylamide gels using the method of Stegemann et al. (Stegemann et al.

1973. Z. Naturforsch. 28c:722-732) and under denaturing conditions in 11.5% (wt/vol) polyacrylamide gels using the method of Laemmli (Laemmli, U. K. 1970.

Nature (London) 227:680-685). Serva Blue R was used for non-specific protein staining. For specifically staining the coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase and vanillin dehydrogenase, the gels were rebuffered for min in 100 mM KP buffer (pH 7.0) and subequently incubated at 30 0 C in the same buffer to which 0.08% (wt/vol) NAD, 0.04% (wt/vol) p-nitro blue tetrazolium chloride, 0.003% (wt/vol) phenazine methosulphate and 1 mM of the respective substrate had been added until corresponding colour bands became visible.

Transfer of proteins from polyacrylamide gels to PVDF membranes.

Proteins were transferred from SDS-polyacrylamide gels to PVDF membranes (Waters-Millipore, Bedford, Mass., USA) using a Semidry Fastblot appliance (B32/33, Biometra, Gbttingen, Germany) in accordance with the manufacturer's instructions.

Determining N-terminal amino acid sequences.

N-terminal amino acid sequences were determined using a Protein Peptide Sequencer (Type 477 A, Applied Biosystems, Foster City, USA) and a PTH analyser in accordance with the manufacturer's instructions.

-17- Isolating and manipulating DNA Genomic DNA was isolated using the method of Marmur Marmur, 1961. J. Mol.

Biol. 3:208-218). Other plasmid DNA and/or DNA restriction fragments was/were isolated and analysed using standard methods E. Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratory Press, Cold Spring Habor, New York).

Transferring DNA.

Competent Escherichia coli cells were prepared and transformed using the method of Hanahan Hanahan, 1983. J. Mol. Biol. 166:557-580). Conjugative plasmid transfer between plasmid-harbouring Escherichia coli S17-1 strains (donor) and Pseudomonas sp.strains (recipient) was performed on NB agar plates in accordance with the method of Friedrich et al. Friedrich et al. 1981. J. Bacteriol. 147:198- 205), or by means of a "minicomplementation method" on MM agar plates containing 0.5% (wt/vol) gluconate as the C source and 25 pg of tetracycline/ml or 100 pg of kanamycin/ml. In this case, cells of the recipient were applied in one direction as an inoculation streak. After 5 min, cells of the donor strains were then applied as inoculation streaks, with these streaks crossing the recipient inoculation streak. After incubating at 30 0 C for 48 h, the transconjugants grew directly downstream of the crossing site whereas neither the donor strain nor the recipient strain was able to grow.

Hybridization experiments.

DNA restriction fragments were fractionated electrophoretically in a 0.8% (wt/vol) agarose gel in 50 mM tris- 50 mM boric acid- 1.25 mM EDTA buffer (pH 8.5) E.

Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual.

2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.).

The transfer of the denatured DNA out of the gel onto a positively charged nylon membrane (pore size: 0.45 Pall Filtrationstechnik, Dreieich, Germany), the subsequent hybridization with biotinylated or digoxigenin-labelled DNA probes, and the preparation of these DNA probes, were all performed using standard methods -18- E. Sambrook, F. Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

DNA sequencing.

Nucleotide sequences were determined "non-radioactively" in accordance with the Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74:5463-5467) dideoxy chain termination method using a "LI-COR" DNA Sequencer Model 4000L" (LI-COR Inc., Biotechnology Division, Lincoln, NE, USA) and using a "thermo sequenase fluorescent labelled primer cycle sequencing kit with 7-deaza-dGTP" (Amersham Life Science, Amersham International plc., Little Chalfont, Buckinghamshire, England), in each case in accordance with the manufacturer's instructions.

Synthetic oligonucleotides were used to carry out sequencing in accordance with the "primer-hopping strategy" of Strauss et al. C. Strauss et al. 1986. Anal. Biochem.

154:353-360).

Chemicals, biochemicals and enzymes.

Restriction enzymes, T4 DNA ligase, lambda DNA and enzymes and substrates for the optical enzymic tests were obtained from C.F. Boehringer Sihne (Mannheim, Germany) or from GIBCO/BRL (Eggenstein, Germany). [y- 3 2 P]ATP was from Amersham/Buchler (Braunschweig, Germany). Oligonucleotides were obtained from MWG-Biotech GmbH (Ebersberg, Germany). Type NA agarose was obtained from Pharmacia-LKB (Uppsala, Sweden). All other chemicals were from Haarmann Reimer (Holzminden, Germany), E. Merck AG (Darmstadt, Germany), Fluka Chemie (Buchs, Switzerland), Serva Feinbiochemica (Heidelberg, Germany) or Sigma Chemie (Deisenhofen, Germany).

-19- Examples Example 1 Constructing omega elements which mediate resistances to kanamycin (0Q Km) or gentamycin (2Gm).

For constructing the i2Km element, the 2099 bp BglI fragment of Transposons A. Auerswald, G. Ludwig and H. Schaller. 1981. Cold Spring Harb. Symp.

Quant. Biol. 45:107-113; E. Beck, G. Ludwig, E. A. Auerswald, B. Reiss and H.

Schaller. 1982. Genes 19:327-336; P. Mazodier, P. Cossart, E. Giraud and F. Gasser.

1985. Nucleic Acids Res. 13:195-205) was isolated on a preparative scale. The fragment was shortened down to approx. 990 bp by treating it with Bal 31 nuclease.

This fragment, which now only comprised the kanamycin resistance gene (encoding an aminoglycoside-3'-O-phosphotransferase), was then ligated to Sinal-cut pSKsym DNA (pBluescript SK- derivative which contains a symmetrically constructed multiple cloning site [Sail, Hind, EcoRI, Smal, EcoRI, HindmI, Sail]). It was possible to reisolate the QKm element from the resulting plasmid as a Smal fragment, an EcoRI fragment, a HindUl fragment or a Sail fragment.

For constructing the Q2Gm element, the 983 bp EaeI fragment of the plasmid E. Kovach, P. H. Elzer, D. S. Hill, G. T. Robertson, M. A. Farris, R. M. Roop and K. M. Peterson. 1995. Genes 166:175-176) was isolated on a preparative scale and then treated with mung bean nuclease (progressive digestion of single-stranded DNA molecule ends). This fragment, which now only comprised the gentamycin resistance gene (encoding a gentamycin-3-acetyltransferase), was then ligated to Smal-cleaved pSKsym DNA (see above). It was possible to reisolate the Q Gm element from the resulting plasmid as a SmaI fragment, an EcoRI fragment, a Hindlm fragment or a Sail fragment.

Example 2 Cloning the genes from Pseudomonas sp. HR199 (DSM7063) which were to be inactivated by inserting Q elements or by means of deletions.

The fcs, ech, vdh and aat genes were cloned separately proceeding from the E. coli S17-1 strains DSM 10439 and DSM 10440 and using the plasmids pE207 and pE5-1 (see EP-A 0845532). The given fragments were isolated on a preparative scale from these plasmids and treated as described below: For cloning the fcs gene, the 2350 bp Sall/EcoRI fragment from plasmid pE207 and the 3700 bp EcoRI/SalI fragment from plasmid pE5-1 were cloned together in pBluescript SK" such that the two fragments were joined together by way of the EcoRI ends. The 6050 bp Sall fragment was isolated on a preparative scale from the resulting hybrid plasmid and shortened down to approx. 2480 bp by being treated with Bal 31 nuclease. PstI linkers were subsequently ligated to the ends of the fragment and, after digestion with PstI, the fragment was cloned into pBluescript SK (pSKfcs). After transformation of E. coli XL1 blue, clones were obtained which expressed the fcs gene and exhibited an FCS activity of 0.2 U/mg of protein.

For cloning the ech gene, the 3800 bp Hindll/EcoRI fragment from plasmid pE207 was isolated on a preparative scale and shortened down to approx. 1470 bp by treating it with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the fragment and, after digestion with EcoRI, the fragment was cloned into pBluescript SK (pSKech).

For cloning the vdh gene, the 2350 bp SalIlEcoRI fragment from plasmid pE207 was isolated on a preparative scale. After cloning into pBluescript SK-, the fragment was truncated at one end by approx. 1530 bp using an exonuclease II/mung bean nuclease system. An EcoRI linker was then ligated to the end of the fragment and, after digestion with EcoRI, the fragment was cloned into pBluescript SK- (pSKvdh).

-21- Following transformation of E. coli XL1 blue, clones were obtained which expressed the VDH gene and exhibited a VDH activity of 0.01 U/mg of protein.

For cloning the aat gene, the 3700 bp EcoRIISalI fragment from plasmid pE5-1 was isolated on a preparative scale and shortened down to approx. 1590 bp by treating it with Bal 31 nuclease. EcoRI linkers were then ligated to the ends of the fragment and, after digestion with EcoRI, the fragment was cloned into pBluescript SK- (pSKaat).

Example 3 Inactivating the above-described genes by inserting elements or by deleting constituent regions of these genes.

Plasmid pSKfcs, which contained the fcs gene, was digested with BssHII, resulting in a 1290 bp fragment being excised from the fcs gene. Following religation, the deletion derivative of the fcs gene (fcsA) (see Figs. li and 2i) was obtained in cloned form in pBluescript SK- (pSKfcsA). In addition, after the fragment had been excised, the omega elements QKm and QGm were ligated in in its stead. This resulted in the G2-inactivated derivatives of the fcs gene (fcsQKm, see Figs. Ig and 2g) and (fcsQGm, see Fig. lh and 2h) being obtained in cloned form in pBluescript SK" (pSKfcsGKm and pSKfcsS2Gm). It was not possible to detect any FCS activity in crude extracts of the resulting E. coli clones, whose hybrid plasmids possessed an fcs gene which was inactivated by deletion or by 92 element insertion.

Plasmid pSKech, which contained the ech gene, was digested with NruI, resulting in a 53 bp fragment and a 430 bp fragment being excised from the ech gene. After religation, the deletion derivative of the ech gene (echA, see Fig. 11 and 21) was obtained in cloned form in pBluescript SK (pSKechA). In addition, after the fragments had been excised, the omega elements 9Km and QGm were ligated in in their stead. This resulted in the S-inactivated derivatives of the ech gene (echnKm -22and echQGm) being obtained in cloned form in pBluescript SK- (pSKechQKm and pSKech&2Gm).

Plasmid pSKvdh, which contained the vdh gene, was digested with BssHII, resulting in a 210 bp fragment being excised from the vdh gene. After religation, the deletion derivative of the vdh gene (vdhA, see Figs. lo and 20) was obtained in cloned form in pBluescript SK- (pSKvdhA). In addition, after the fragment had been excised, the omega elements 9SKm and DQGm were ligated in in its stead. This resulted in the SQinactivated derivatives of the vdh gene (vdhQKm and vdhSGm) being obtained in cloned form in pBluescript SK- (pSKvdhQKm, see Figs. Im and 2m) and (pSKvdhg2Gm, see Figs. In and 2n). It was not possible to detect any VDH activity in crude extracts of the resulting E. coli clones, whose hybrid plasmids possessed a vdh gene which was inactivated by deletion or by Q2 element insertion.

Plasmid pSKaat, which contained the aat gene, was digested with BssHII, resulting in a 59 bp fragment being excised from the aat gene. After religation, the deletion derivative of the aat gene (aatA, see Figs. Ir and 2r) was obtained in cloned form in pBluescript SK- (pSKaatA). In addition, after the fragment had been excised, the omega elements 2Km and Q2Gm were ligated in in its stead. This resulted in the 92inactivated derivatives of the aat gene (aatG2Km, see Figs. Ip and 2p) and (aatQGm, see Figs. lq and 2q) being obtained in cloned form in pBluescript SK (pSKaatQKm and pSKaatQGm).

-23- Example 4 Subcloning the Q element-inactivated genes into the conjugatively transferable "suicide plasmid" pSUP202.

In order to be able to replace the intact genes in Pseudomonas sp. HR199 with the ielement inactivated genes, there is a need for a vector which can, on the one hand, be transferred into pseudomonads (conjugatively transferable plasmids) but which, on the other hand, cannot replicate in these bacteria and is consequently unstable in pseudomonads ("suicide plasmid"). DNA segments which are transferred into pseudomonads using such a plasmid system can only be retained if they are integrated by means of homologous recombination (RecA-dependent recombination) into the genome of the bacterial cell. In the present case, the "suicide plasmid" pSUP202 (Simon et al. 1983. In: A. Ptihler. Molecular genetics of the bacteria-plant interaction. Springer Verlag, Berlin, Heidelberg, New York, pp. 98-106) was used.

Following digestion with PstI, the inactivated genes fcsQKm and fcsf2Gm were isolated from plasmids pSKfcsQKm and pSKfcs2Gm and ligated to PstI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPfcsgKm) contained the inactivated gene fcsQ2Km were obtained. The corresponding hybrid plasmid (pSUPfcsnGm) of the gentamycin-resistant transformants contained the inactivated genefcsGGm.

Following EcoRI digestion, the inactivated genes echQKm and echQGm were isolated from plasmids pSKechQKm and pSKechQGm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPechQKm) contained the inactivated gene echK2Km were obtained. The -24corresponding hybrid plasmid (pSUPechQGm) of the gentamycin-resistant transformants contained the inactivated gene echQGm.

Following EcoRI digestion, the inactivated genes vdhQKm and vdhSIGm were isolated from plasmids pSKvdhQKm and pSKvdhQ2Gm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPvdhiKm) contained the inactivated gene vdhQKm were obtained. The corresponding hybrid plasmid (pSUPvdhQ2Gm) of the gentamycin-resistant transformants contained the inactivated gene vdhSGm.

Following EcoRI digestion, the inactivated genes aatQKm and aatSGm were isolated from plasmids pSKaatQKm and pSKaatQGm and ligated to EcoRI-cleaved pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium which also contained kanamycin or gentamycin, respectively. Kanamycin-resistant transformants whose hybrid plasmid (pSUPaatQ2Km) contained the inactivated gene aatGKm were obtained. The corresponding hybrid plasmid (pSUPaatQGm) of the gentamycin-resistant transformants contained the inactivated gene aatQGm.

Example Subcloning the deletion-inactivated genes into the conjugatively transferable "suicide plasmid" PHE55, which possesses the "sacB selection system".

In order to be able to replace the intact genes in Pseudomonas sp. HR199 with the deletion-inactivated genes, there is a need for a vector which possesses the properties which have already been described in the case of pSUP202. Since no possibility (no antibiotic resistance) exists of selecting for successful replacement of the genes in Pseudomonas sp. HR199 in the case of deletion-inactivated genes, in contrast to the g element-inactivated genes, another selection system had to be used. In the "sacB selection system", the replacing, deletion-inactivated gene is cloned in a plasmid which possesses the sacB gene in addition to an antibiotic resistance gene. Following the conjugative transfer of this hybrid plasmid into a pseudomonad, the plasmid is integrated by means of homologous recombination at the site in the genome at which the intact gene is located (first crossover). This results in a "heterogenotic" strain which possesses both an intact gene and a deletion-inactivated gene, with these genes being separated from each other by the pHE55 DNA. These strains exhibit the resistance which is encoded by the vector and also possess an active sacB gene. The intention then is that the pHE55 DNA, together with the intact gene, should then be separated out of the genomic DNA by means of a second homologous recombination event (second crossover). This recombination event results in a strain which now only possesses the inactivated gene. In addition, the pHE55-coded antibiotic resistance and the sacB gene are both lost. If strains are streaked on sucrosecontaining media, the growth of strains which express the sacB gene is inhibited since the gene product converts sucrose into a polymer which is accumulated in the periplasm of the cells. The growth of cells which no longer carry the sacB gene as a result of the second recombination event having taken place is consequently not inhibited. In order to have a possibility of selecting phenotypically for the integration of the deletion-inactivated gene, this gene is not exchanged for an intact gene; instead, use is made of a strain in which the gene to be replaced is already "labelled" by the insertion of an 92 element. When successful replacement takes place, the resulting strain loses the antibiotic resistance which is encoded by the Q element.

Following digestion with Pstl, the inactivated gene fcsA was isolated from plasmid pSKfcsA and ligated to PstI-cleaved pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEfcsA) contained the inactivated genefcsA, were obtained.

Following digestion with EcoRI, the inactivated gene echA was isolated from plasmid pSKechA and treated with mung bean nuclease (generation of blunt ends).

-26- The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEechA) contained the inactivated gene echA, were obtained Following digestion with EcoRI, the inactivated gene vdhA was isolated from plasmid pSKvdhA and treated with mung bean nuclease. The fragment was ligated to BamHI-cleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEvdhA) contained the inactivated gene vdhA, were obtained.

Following digestion with EcoRI, the inactivated gene aatA was isolated from plasmid pSKaatA and treated with mung bean nuclease. The fragment was ligated to BamHIcleaved and mung bean nuclease-treated pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection took place on tetracycline-containing LB medium. Tetracycline-resistant transformants, whose hybrid plasmid (pHEaatA) contained the inactivated gene aatA, were obtained.

-27- Example 6 Generating mutants of the strain Pseudomonas sp. HR199 in which genes of eugenol catabolism have been specifically inactivated by inserting an Q-element.

The strain Pseudomonas sp. HR199 was employed as the recipient in conjugation experiments in which strains of E. coli S17-1 harbouring the hybrid plasmids of pSUP202 which are listed below were used as donors. The transconjugants were selected on gluconate-containing mineral medium which contained the antibiotic corresponding to the Q2 element. It was possible to distinguish between "homogenotic" (replacement of the intact gene with the SQ element insertioninactivated gene by means of a double crossover) and "heterogenotic" (integration of the hybrid plasmid into the genome by means of a single crossover) transconjugants on the basis of the pSUP202-encoded tetracycline resistance.

The mutants Pseudomonas sp. HR199 fcsgKm and Pseudomonas sp. HR199 fcsQGm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-1 (pSUPfcsQKm) and E. coli S17-1 (pSUPfcsQGm), respectively. The replacement of the intactfcs gene with the QKm-inactivated or 2Gm-inactivated gene (fcsG2Km and fcsSGm, respectively) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 echgKm and Pseudomonas sp. HR199 echQGm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-1 (pSUPechiKm) and E. coli S17-1 (pSUPechQGm), respectively. The replacement of the intact ech gene with the GKm-inactivated or QGm-inactivated gene (echQKm and echS2Gm, respectively) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 vdhQKm and Pseudomonas sp. HR199 vdhSGm were obtained after conjugating Pseudomonas sp. HR199 with E. coli S17-1 (pSUPvdh/iKm) and E. coli S17-1 (pSUPvdhQGm), respectively. The 28 replacement of the intact vdh gene with the Q Km-inactivated or QiGm-inactivated gene (vdhQKrn and vdhL2Gm, respectively) was verified by means of DNA sequencing.

The mutants Pseudomonas sp. HR199 aatQiKm and Pseudomonas sp. HR199 aatgi Gm were obtained after conjugating Pseudomonas sp. HR 199 with E. ccli S17-1 (pSLTPaatQiKm) and E. ccli S17-1 (pSUPaatQ2Gm), respectively. The replacement of the intact aat gene with the QKm-inactivated or L Gm-inactivated gene (aat9 Km and aatQiGm, respectively) was verified by means of DNA sequencing.

The mutant Pseudornonas sp. HR199 fcsgT KmvdhQ Gm was obtained after conjugating Pseudomonas sp. HR 199 fcsQ Km with E. ccli S 17-1 (pSU~vdh97 Gm).

The replacement of the intact vdh gene with the QiGm-inactivated gene (vdhK2Gm) was verified by means of DNA sequencing.

The mutant Pseudomionas sp. HIR199 vdhM Kmaat9QGm was obtained after conjugating Pseudomionas sp. H-R199 vdhzQKm with E. coli S 17-1 (pSUPaatQGm).

The replacement of the intact aat gene with the Q2Gm-inactivated gene (aatQ2Gm) was verified by means of DNA sequencing.) The mutant Pseudonionas sp. HR199 vdhQ KmechQ2Gm was obtained after conjugating Pseudomionas sp. HR 199 vdh 2Km with E. coli S 17-1 (pSUJPechn2Gm).

The replacement of the intact ech gene with the Q Gm-inactivated gene (echQ2Gm) was verified by means of DNA sequencing.

-29- Example 7 Generating of mutants of the strain Pseudomonas sp. HR199 in which genes of eugenol catabolism have been specifically inactivated by deleting a constituent region.

The strains Pseudomonas sp. HR199 fcsGQKm, Pseudomonas sp. HR199 echQKm, Pseudomonas sp. HR199 vdhL2Km and Pseudomonas sp. HR199 aatQKm were employed as recipients in conjugation experiments in which strains of E. coli S17-1 harbouring the hybrid plasmids of pHE55 which are listed below were used as donors. The "heterogenotic" transconjugants were selected on gluconate-containing mineral medium which also contained the antibiotic corresponding to the L element in addition to tetracycline (pHE55-encoded resistance). After streaking out on sucrose-containing mineral medium, transconjugants were obtained which had eliminated the vector DNA by means of a second recombination event (second crossover). By streaking out on mineral medium which was without antibiotic or which contained the antibiotic corresponding to the Q element, it was possible to identify the mutants in which the Q2 element-inactivated gene had been replaced with the deletion-inactivated gene (no antibiotic resistance).

The mutant Pseudomonas sp. HR199 fcsA was obtained after conjugating Pseudomonas sp. HR199fcsQ2Km with E. coli S17-1 (pHEfcsA). The replacement of the QKm inactivated gene (fcsQKm) with the deletion-inactivated gene (fcsA) was verified by means of DNA sequencing.

The mutant Pseudomonas sp. HR199 echA was obtained after conjugating Pseudomonas sp. HR199 echQKm with E. coli S17-1 (pHEechA). The replacement of the QKm-inactivated gene (ech2Km) with the deletion-inactivated gene (echA) was verified by means of DNA sequencing.

The mutant Pseudomonas sp. HR199 vdhA was obtained after conjugating Pseudomonas sp. HR199 vdhQKm with E. coli S17-1 (pHEvdhA). The replacement of the 2Km-inactivated gene (vdh&2Km) with the deletion-inactivated gene (vdhA) was verified by means of DNA sequencing.

The mutant Pseudomonas sp. HR199 aatA was obtained after conjugating Pseudomonas sp. HR199 aatSKm with E. coli S17-1 (pHEaatA). The replacement of the gKm-inactivated gene (aatQKm) with the deletion-inactivated gene (aatA) was verified by means of DNA sequencing.

Example 8 Biotransforming eugenol into vanillin using the mutant Pseudomonas sp. HR199 vdh2Km.

The strain Pseudomonas sp. HR199 vdhg2Km was propagated in 50 ml of HR-MM containing 6 mM eugenol up to an optical density of approx. OD600nm 0.6. After 17 h, it was possible to detect 2.9 mM vanillin, 1.4 mM ferulic acid and 0.4 mM vanillic acid in the culture supernatant.

Example 9 Biotransforming eugenol into ferulic acid using the mutant Pseudomonas sp.

HR199 vdhQ2GmaatQ2Km.

The strain Pseudomonas sp. HR199 vdhQGmaatQKm was propagated in 50 ml of HR-MM containing 6 mM eugenol up to an optical density of approx.OD600nm 0.6. After 18 h, it was possible to detect 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillic acid in the culture supernatant.

-31- Example Biotransforming eugenol into coniferyl alcohol using the mutant Pseudomonas sp. HR199 vdhQGmaatKm.

The strain Pseudomonas sp. HR199 vdhQGmaatQKm was propagated in 50 ml of HR-MM containing 6 mM eugenol up to an optical density of approx. OD600nm 0.4. After 15 h, it was possible to detect 1.7 mM coniferyl alcohol, 1.4 mM vanillin, 1.4 mM ferulic acid and 0.2 mM vanillic acid in the culture supernatant.

Example 11 Fermentatively producing natural vanillin from eugenol in a 10 1 fermenter using mutant Pseudomonas sp. HR 199 vdhgKm.

The production fermenter was inoculated with 100 ml of a 24-hour-old preliminary culture which had been propagated at 32 0 C on a shaking incubator (120 rpm) in a medium which was adjusted to pH 7.0 and which consisted of 12.5 g of glycerol/l, g of yeast extract/I and 0.37 g of acetic acid/I. The fermenter contained 9.9 1 of medium of the following composition: 1.5 g of yeast extract/i, 1.6 g of KH 2

PO

4 0.2 g of NaCI/1, 0.2 g of MgSO 4 The pH was adjusted to pH 7.0 with sodium hydroxide solution. After sterilization, 4 g of eugenol were added to the medium. The temperature was 32 0 C, the aeration was 3 Nl/min and the stirrer speed was 600 rpm.

The pH was maintained at pH 6.5 with sodium hydroxide solution.

At 4 hours after the inoculation, continuous addition of eugenol was begun such that 255 g of eugenol had been added to the culture when fermentation ended after hours. 40 g of yeast extract were also fed in during the fermentation. At the end of the fermentation, the concentration of eugenol was 0.2 g/l. The content of vanillin was 2.6 g/1. 3.4 g of ferulic acid/I were also present.

-32- The vanillin which is obtained in this way can be isolated by known physical methods such as chromatography, distillation and/or extraction and used for preparing natural flavourings.

Explanatory notes regarding the figures: FIG. la to Ir: Gene struktures for isolating organisms and mutants calA*: Part of the inactivated gene for coniferyl alcohol dehydrogenase calB*: Part of the inactivated gene for coniferyl aldehyde dehydrogenase fcs*: Part of the inactivated gene for feruloyl-CoA synthetase ech*: Part of the inactivated gene for enoyl-CoA hydratase-aldolase vdh*: Part of the inactivated gene for vanillin dehydrogenase aat*: Part of the inactivated gene for beta-ketothiolase While the restriction enzyme cleavage sites labelled were used for the construction, they are no longer functional in the resulting construct.

-33- FIG. 2a: Nucleotide sequence of the calAQKm gene structure FIG. 2b: Nucleotide sequence of the calAQ2Gm gene structure: FIG. 2c: Nucleotide sequence of the calAA gene structure FIG. 2d: Nucleotide sequence of the calBSKm gene structure FIG. 2e: Nucleotide sequence of the calBIGm gene structure FIG. 2f: Nucleotide sequence of the calBA gene structure FIG. 2g: Nucleotide sequence of the fcsQKm gene structure FIG. 2h: Nucleotide sequence of the fcsSgGm gene structure FIG. 2i: Nucleotide sequence of the fcsA gene structure FIG. 2j: Nucleotide sequence of the echQKm gene structure FIG. 2k: Nucleotide sequence of the echQGm gene structure FIG. 21: Nucleotide sequence of the echA gene structure FIG. 2m: Nucleotide sequence of the vdh2Km gene structure FIG. 2n: Nucleotide sequence of the vdhQGm gene structure FIG. 2o: Nucleotide sequence of the vdhA gene structure FIG. 2 p: Nucleotide sequence of the aatQKm gene structure FIG. 2q: Nucleotide sequence of the aatQGm gene structure FIG. 2r: Nucleotide sequence of the aatA gene structure EDITORIAL NOTE NO 52818/99 Sequence listing pages 1-75 is part of the description.

Claim pages are to follow.

CTGCAGCCAG

GGCTCCAATT

GCTAGGGAGA

CATTCTGCAT

AAGGTTGCTA

GCATGGAAAT

TGGAAGCACG

AAAGAGCATG

Met 1 Sequences GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGCGTTGGA CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT ATTCCTCCCC CGGTAGAGCC GTAACCGCGA CATTCAGCAC CAA CTG ACC AAC AAG AAA ATC GTC GTC ACC GGA Gin Leu Thr Asn Lys Lys Ile Val Val Thr Gly 5 10

GACGGCGCCT

GGAGAGTTCG

TGATTTTCTG

CACGGGATTG

TCAGCATGGT

TTTGGCGGAA

CGTAAAAAGG

GTG TCC TCC Val Ser Ser 120 180 240 300 360 420 472 GGT ATC GGT GCC GAA ACT GCC CGC GTT CTG CCC TCT CAC GGC CCC ACA Cly Ile Giy Ala Giu Thr Aia Arg Val Leu Arg Ser His Giy Aia Thr 25 GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC Val Ile Gly Val Asp Arg Asn Met Pro Ser Leu Thr Leu Asp Ala Phe 40 GTT CAC GCT GAC CTC AGC CAT CCT GAA GGC ATC GAT AAG GCC ATC GGG Val Gin Ala Asp Leu Ser His Pro Glu Giy Ile Asp Lys Aia Ile 55 60 62 ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTC GTAAGGTTGG GAAGCCCTGC AAAGTAAACT GGATCGCTTT CTTGCCGCCA ACGATCTCAT GGCGCAGGGG ATCAAGATCT CATCAAGAGA CAGCATCAGG ATCGTTTCGC ATC ATT GAA CAA CAT GGA TTC CAC Met Ile Giu Gin Asp Giy Leu His 1 CCA GGT TCT CCG GCC CCT TCG GTG GAG AGC CTA TTC GGC TAT CAC TGG Ala Giy Ser Pro Aia Aia Trp Vai Giu Arg Leu Phe Giy Tyr Asp Trp 15 CCA CAA CAG ACA ATC GGC TCC TCT CAT CCC CCC GTG TTC CGG CTG TCA Ala Gin Gin Thr Ile Cly Cys Ser Asp Aia Aia Val Phe Arg Leu Ser 30 35 GCG CAG GGG CCC CCG GTT CTT TTT GTC AAG ACC GAC CTC TCC GGT GCC Ala Gin Gly Arg Pro Val Leu Phe Vai Lys Thr Asp Leu Ser Cly Ala 50 CTC AAT CAA CTG CAG GAC GAG GCA GCG CGG CTA TCC TGG CTG GCC ACC Leu Asn Clu Leu Gin Asp Clu Ala Aia Arg Leu Ser Trp Leu Ala Thr 65 568 616 676 736 790 838 886 934 982 ACG GGC GTT Thr Gly Val CCT TGC GCA GCT Pro Cys Ala Ala

GTG

Val CTC GAC GTT GTC Leu Asp Val Val GAA GCG GGA Glu Ala Gly AGG GAC Arg Asp TGG CTG CTA TTG Trp Leu Leu Leu

GGC

Cly 95 GAA GTG CCC GGG Glu Val Pro Gly

CAG

Gin 100 GAT CTC CTG TCA Asp Leu Leu Ser

TCT

Ser 105 CAC CTT GCT CCT His Leu Ala Pro GAG AAA GTA TCC Glu Lys Val Ser ATG GCT GAT GCA Met Ala Asp Ala CGG CGG CTG CAT Arg Arg Leu His

ACG

Thr 125 CTT CAT CCG GCT Leu Asp Pro Ala

ACC

Thr 130 TGC CCA TTC GAC Cys Pro Phe Asp CAC CAA His Gin 135 GCG AAA CAT Ala Lys His GTC CAT CAG Val Asp Gin 155 ATC GAG CCA CCA Ile Glu Arg Ala

CGT

Arg 145 ACT CGG ATG GAA Thr Arg Met Glu CCC GGT CTT Ala Cly Leu 150 CCG CCA GCC Ala Pro Ala CAT CAT CTG GAC Asp Asp Leu Asp

CAA

Glu 160 GAG CAT CAG GGG Glu His Gin Cly

CTC

Leu 165 1030 1078 1126 1174 1222 1270 1318 1366 1414 1462 1510 GAA CTC Glu Leu 170 TTC CCC AGG CTC Phe Ala Arg Leu GCG CCC ATC CCC Ala Arg Met Pro GGC GAG CAT CTC Gly Glu Asp Leu GTC ACC CAT GGC Val Thr His Gly

CAT

Asp 190 CCC TCC TTC CCC Ala Cys Leu Pro ATC ATG GTG GAA Ile Met Val Glu

AAT

Asn 200 GGC CCC TTT TCT Gly Arg Phe Ser

GGA

Gly 205 TTC ATC CAC TGT Phe Ile Asp Cys

GGC

Cly 210 CGG CTC GGT CTG Arg Leu Cly Val GCG GAC Ala Asp 215 CGC TAT CAC Arg Tyr Gin GGC GGC GAA Cly Cly Glu 235 ATA GCG TTG GCT Ile Ala Leu Ala

ACC

Thr 225 CGT CAT ATT GCT Arg Asp Ile Ala CAA GAG CTT Glu Glu Leu 230 ATC CCC GCT Ile Ala Ala TGG CCT GAC CGC Trp Ala Asp Arg CTC GTC CTT TAC Leu Val Leu Tyr

GGT

Cly 245 CCC CAT Pro Asp 250 TCC CAC CCC ATC Ser Gin Arg Ile TTC TAT CCC CTT Phe Tyr Arg Leu CAC GAG TTC Asp Glu Phe TTC 1558 Phe 264 TGAGCGGGAC TCTGGGGTTC CAAATGACCG ACCAAGCGAC ATT GCA TTC ATC TGT CCT GAG GAG TCA CGT TGG Ile Ala Phe Met Cys Ala Glu Giu Ser Arg Trp 230 235 GCCCTG GCC GCG GTG Ala Ala Val 225 ATC AAC GGC ATA AAT Ile Asn Cly Ile Asn 240 1613 1661 -3- ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC Ile Pro Val Asp Gly Gly Leu Ala Ser Thr Tyr Val 245 250 255 GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG FIG. 2a: 1710 1770 1830 1890 1950 2010 2070 2130 2164 -4- CTGCAGCCAC GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCC' GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTC GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCT CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATT( AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAC CACGCGGGTT TCAGGATGG' GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGCCGGA.

TGGAAGCACG ATTCCTCGCC CCCTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGI AAAGAGCATG CAA CTG ACC AAC AAC AAA ATC GTC GTC ACC GGA GTG TCC Ti Met Gin Leu Thr Asn Lys Lys Ile Val Val Thr Gly Val Ser S 1 5 10 GGT ATC GGT GCC CAA ACT CCC CGC GTT CTC CGC TCT CAC GOC GCC ACA Gly Ile Gly Ala Giu Thr Ala Arg Val Leu Arg Ser His Gly Ala Thr 25 GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC Val Ilie Gly Val Asp Arg Asn Met Pro Ser Leu Thr Leu Asp Ala Phe 40 GTT CAG GCT GAC CTG AGC CAT CCT GAGGGGAGAG GCGGTTTGCG TATTGGGCGC Val Gin Ala Asp Leu Ser His Pro

T

G

3

G

er 120 180 240 300 360 420 472 520 568 622 682 742 802 862 922 982 1033 1081 1129 1177

ATGCATAAAA

GCATGATGAA

CCCATGGACG

CGGTTCGTAA.

CCGAACGCAG

TTGTACAGTC

TGATGTTATG

ACTGTTGTAA

CCTGAATCGC

CACACCGTGG

ACTGTA-ATGC

CGGTGGTAAC

TATGCCTCGG

GAGCAGCAAC

TTCATTAAGC ATTCTCCCGA CAGCGGCATC AGCACCTTGT AAACGGATGA AGGCACGAAC AAGTAGCGTA TGCGCTCACG GGCGCAGTGG CGGTTTTCAT GCATCCAAGC AGCAAGCGCG C ATG TTA CGC AGC AGC Met Leu Arg Ser Ser 1 5 CATGGAAGCC ATCACAAACG CCCCTTGCGT ATAATATTTG CCACTTGACA TAACCCTGTT CAACTGGTCC AGAACCTTGA GGCTTCTTA.T GACTCTTTTT TTACGCCCTG GGTCGATCTT AAC CAT GTT ACC CAC Asn Asp Val Thr Gin TCA ACT ATC CCC ATC Ser Ser Met Cly Ile AAA TCC ATG CCC CCT Lys Ser Met Arg Ala GTA CCC ACC TAC TCC Val Ala Thr Tyr Ser CAG CCC ACT CCC CCT AAA ACA AAG TTA CCT GC Gin Ciy Ser Arg Pro Lys Thr Lys Leu Ciy Cly 20 ATT CCC ACA TGT AGG CTC CCC CCT GAC CAA CTC Ile Arg Thr Cys Arg Leu Cly Pro Asp Gin Val 35 GCT CTT GAT CTT TTC GCT CCT GAG TTC GGA CAC Ala Leu Asp Leu Phe Gly Arg Glu Phe Giy Asp CAA CAT CAG CCG GAC TCC GAT Gin His Gin TTC ATC Phe Ile Pro Asp Ser Asp 65 GCG CTT GCT GCC Ala Leu Ala Ala 80 TAC CTC GGG Tyr Leu Gly TTC GAC CAA Phe Asp Gin Asn Leu Leu Arg Ser Lys AAC TTG CTC CGT AGT AAG

ACA

Thr

GAA

Glu GCG GTT GTT GGC Ala Val Val Gly CTC GCG GCT TAC Leu Ala Ala Tyr

GTT

Va1

CTC

Leu CTG CCC AGG TTT Leu Pro Arg Phe

GAG

Glu 100

GAG

Glu CAG CCG CGT AGT Gin Pro Arg Ser GAG ATC Glu Ile 105 TAT ATC TAT Tyr Ile Tyr GCC ACC GCG Ala Thr Ala 125 GCT TAT GTG Ala Tyr Val GCA GTC TCC Ala Val Ser

GGC

Gly 115

AAG

Lys CAC CGG AGG His Arg Arg ATC AAT CTC Ile Asn Leu CAT GAG GCC His Giu Ala

AAC

Asn 135

GAT

Asp CAG GGC ATT Gin Gly Ile 120 GCG CTT GGT Ala Leu Gly CCC GCA GTG Pro Ala Val ATC TAC GTG Ile Tyr Val GCA GAT TAC GGT Ala Asp Tyr Gly 140 GCT CTC Ala Leu TAT ACA AAG Tyr Thr Lys

TTG

Leu 160

GCC

Ala ATA CGG GAA Ile Arg Glu

GAA

Glu 165 ATG CAC TTT Met His Phe

GAT

Asp 170 1225 1273 1321 1369 1417 1465 1513 1567 1616 1665 1725 1785 1845 1905 1965 2025 2085 GAC CCA AGT Asp Pro Ser TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCT G ATT GCA TTC ATG TGT GCT GAG GAG TCA CGT TGG ATC AAC GGC ATA AAT Ile Ala Phe Met Cys Ala Giu Giu Ser Arg Trp Ile Asn Gly Ile Asn 228 235 240 ATT CCA GTC Ile Pro Va 245

GCCCTTTGCA

GAGGTAGCGG

GTTGTCCGTA

GCACTTTCAA

CAAACTCGAT

CTCAAGTATA

ACTGTCAATG

GAC GGA GGT TTG GCA TCG ACC TAC L Asp Gly Gly Leu Ala Ser Thr Tyr 250

CGCGCACTAT

GCGGAAAGGT

AACGAAAATA

AATAGCTACC

GCAGCTGGAT

GCCTTGCCTC

GTTATATCCG

ATCTCTATGC

GCAGAATGTC

AAAATAAAGA

CTGGCAGGCG

GTAGGTAGCT

TCGCCTGAAT

GATATTCAAA

AGCAGCTGAA

TAAATAATAA

GGAATGATAT

CCATTTATGC

CGGATTGGAC

GAGCAAGACT

GTCAGGGTGA

GTG TAA GTTCGTGGAC Va1 255 AGCAGCTTTG GTTTTGATCG AGGATTCTTG TGAAGCTTTA GAAAGCAAGT AGATCAGTCT AGCGCTGCCA ATGTCAGCTG GGTGCGTTGG GGACAACACC CAAGTCTGAC AAATGCGCCG TCGTAACTTT GACCGGGGGC TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 2119 FIG. 2b: -6-

CTGCAGCCAG

GGCTCCAATT

CCTAGCGAGA

CATTCTGCAT

AAGGTTGCTA

GCATGGAAAT

TGGAACCACG

AAAGACCATC

Met 1 GCCTGAAAAG GAGGGATTCA GTCAGGTCAT GAAGGGAGGG CCTCGATGGC GCCGCGATTG ACTGTCTTGG GCGCGGTCTT TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CAA CTG ACC AAC AAG AAA ATC GTC GTC ACC GGA Gin Leu Thr Asn Lys Lys Ile Val Val Thr Gly

GACGGCGCCT

GGAGAGTTCG

TGATTTTCTG

CACGGGATTG

TCAGGATGCT

TTTGGCGGAA

CGTAAAAAGG

GTG TCC TCC Vai Ser Ser 120 180 240 300 360 420 472 520 568 617 GCT ATC GGT CCC GAA ACT GCC CGC GTT CTG CGC Giy Ile Giy Aia Giu Thr Aia Arg Vai Leu Arg 25 GTG ATT GGC GTA GAT CGC AAC ATG CCC ACC CTC Vai Ilie Gly Vai Asp Arg Asn Met Pro Ser Leu 40 GTT CAG GCT GAC CTC AGC CAT CCT GAA GGC ATC Val Gin Aia Asp Leu Ser His Pro Ciu Giy Ile 55 58 ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC Ilie Pro Vai Asp Ciy Giy Leu Aia Ser Thr Tyr 245 250 CCCCTTTGCA CGCCCACTAT ATCTCTATCC AGCAGCTCAA CACGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA GTTGTCCGTA AACCAAAATA AAAATAAAGA GGAATGATAT GCACTTTCAA AATACCTACC CTGGCAGGCG CCATTTATGC CAAACTCGAT GCAGCTGGAT GTAGGTACCT CGGATTGGAC CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT ACTCTCAATG GTTATATCCC GATATTCAAA GTCAGCGTCA TTCCTATCCA ATCGTCTCGA TATTCTCGCT GCAG FIG. 2c: TCT CAC CCC CCC ACA Ser His Ciy Aia Thr ACT CTC CAT CCT TTC Thr Leu Asp Aia Phe CATC AAC CCC ATA AAT Asn Ciy Ile Asn 240 CTC TAA CTTCCTCCAC Val 255 ACCACCTTTC CTTTTCATCG ACGATTCTTC TCAACCTTTA CAAACCAACT AGATCACTCT ACCGCTCCCA ATCTCACCTC CCTGCCTTCC CCACAACACC CAACTCTCAC AAATCCGCCC TCCTAACTTT CACCCCCCCC 726 786 846 906 966 1026 1086 1120 -7-

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAACAT

CTCCAGCTCA

AGAATAACAA

TATCGCCCCG

TTTTCTTGC

GCTTCGCTTC

TAAGCATTCT

TTGAGGCCGA

AATTAAAATA

AGGGCAATTT

TTGACTCCTC

TTCTATCAGC

CATCCTTGTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGCAAACCGC

TTGGGCTATT

AGCAGGTCAG

GCCCCGCTTT

GCCTGAA.CCT

GCATCGAGAT

TGGTGGCTTT

CTTGCGCG

ATGGTTTCTT

GGCTGAGCAG

CG ATG AGC

CGAAAGTCAT

TCGTTGACAT

CCTGAGGTCA

GAACAGCCTG

TCGAAGCGAT

ATGTGAATTT

TTGCCTCTA.T

GGTCTTAGCC

AGGGCACAGG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

120 180 240 300 360 420 ATT CTT GGT TTG AAT Met Ser Ile Leu 1 GGT GCC CCC Gly Ala Pro GTC GGA GCT Val Gly Ala GAG CAG Giu Gin CAG GGG Gin Gly CTG CCC TCC GCT Leu Gly Ser Ala Gly Leu Asn GAT CCC ATC Asp Arg Met CTG CGT CTG Leu Arg Leu AAC AAC Lys Lys ACT AGG Ser Arg GCG CAC CTG GAG Ala His Leu Ciu CCT GCA AAC Pro Ala Asn 30

ATT

Ile

TTG

Leu

GAA

Glu CTC CAT CCT Leu Asp Arg

GCC

Ala 45

TCT

Ser CCA ATG CTT Ala Met Leu AAT CCT GAA Asn Arg Ciu

GCA

Ala ATT CCC GAC GCC Ile Ala Asp Ala

GTT

Val1 GCT GAC TTT Ala Asp Phe CCC AGC CGT Arg Ser Arg GAG CAA Ciu Gin ACA CTC CTT Thr Leu Leu CCC GAG CAC Arg Giu His TTT CCA GGG Phe Pro Gly CAC ATT GCT GC Asp Ile Ala Cly

TCG

Ser

GAG

Giu GCA AGC CTC Ala Ser Leu GCC AAA TGG Ala Lys Trp CCC CAA CAT Pro Ciu His

CAC

His 100

CTG

Leu AAG CAT AGC Lys Asp Ser AAC CCG ATG Lys Aia Met GGT GTC GTT Gly Val Val CC GAG CCA Ala Glu Ala 105 GCG CTC Cly Val

CGC

Arg 110

AAC

Asn GAC TTT CAG Giu Phe Gin ATT ACT CCC Ile Ser Pro 120

CTG

Leu

TCC

Trp 125

CCA

Ala TTC CCT ATC Phe Pro Ile

CTA

Val1 130

CC

Ala CCC TTT GC Ala Phe Gly 809 857 905 CCC CCC ATA Ala Cly Ile

TTC

Phe 140

CCC

Arg CCA CGT AAT Ala Gly Asn

CC

Arg 145

CTT

Leu ATG CTC AAC Met Leu Lys CCC TCC Pro Ser 150 CCT CGT Ala Arg GAG CTT ACC Giu Leu Thr

CCC

Pro 155 ACT TCT CC Thr Ser Ala CC GAC CTA Ala Glu Leu

ATT

Ile 165 TAC TTC GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC Tyr Phe Asp Glu Thr Giu Leu Thr Thr Val Leu Gly Asp 170 175 180 GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC Gly Ala Leu Phe Ser Ala Gin Pro Phe Asp His Leu Ile 185 190 195 GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG Gly Thr Ala Val Ala Lys His lie Met Arg Ala Ala Ala 200 205 210 GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG Val Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Val 220 225 CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG Arg Ser Ala Asp Met Ala Asp Val Ala Gin Arg Val Leu 235 240 ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CCG GAC TAT Thr Phe Asn Ala Gly Gin Ile Cys Leu Ala Pro Asp Tyr 250 255 260 CCG GAA GGGACAGCAA GCGAACCGGA ATTGCCAGCT GGGGCGCCCT Pro Glu 265 TGGGAAGCCC TGCAAAGTAA ACTGGATGGC TTTCTTGCCG CCAAGGA GGGATCAAGA TCTGATCAAG AGACAGGATG AGGATCGTTT CGC ATG GCT GAA GTC Ala Glu Val TTC ACC GGC Phe Thr Gly GAT AAC CTA Asp Asn Leu 215 ATC GTT TCC Ile Val Ser 230 ACG GTG AAA Thr Vai Lys 245 GTG CTG CTG Val Leu Leu

CTGGTAAGGT

TCT GATGGCGCAG ATT GAA CAA 1001 1049 1097 1145 1193 1241 1297 1357 1412 1460 1508 1556 1604 1652

GAT

Asp

GGC

Gly

TTC

Phe

CTG

Leu

TGG

Trp

GGA

Gly

TAT

Tyr

CGG

Arg

TCC

Ser

CTG

Leu

TTG

Leu

GAG

Asp

CTG

Leu

GGT

Gly

GCC

Ala

CAC

His

TGG

Trp

TCA

Ser

GCC

Ala

ACG

Thr

GCA

Ala

GCA

Ala

GCG

Ala

CTG

Leu

ACG

Thr

GGT

Gly 10

CAA

Gin

CAG

Gin

AAT

Asn

GGC

Gly

TCT

Ser

CAG

Gin

GGG

Gly

GAA

Glu

OTT

Va1 75

CCC

Pro

ACA

Thr

CGC

Arg

CTG

Leu 60

CCT

Pro

GCC

Ala

ATC

Ile

CCC

Pro 45

CAG

Gin

TGC

Gys

GCT

Ala

GGC

Gly 30

GTT

Val

GAG

Asp

OCA

Ala

TGG

Trp 15

TGC

Cys

GTT

Leu

GAG

Glu

OCT

Ala Met Ile Glu Gin 1 GTG GAG AGG CTA TTC Val Glu Arg Leu Phe TCT OAT GCC GCC GTG Ser Asp Ala Ala Val TTT GTC AAG ACC GAC Phe Val Lys Thr Asp OCA GCG CGG CTA TCG Ala Ala Arg Leu Ser GTG CTC GAC GTT GTC Val Leu Asp Val Val -9-

ACT

Thr GAA GCG GGA AGG Glu Ala Gly Arg TGG CTG CTA TTG Trp Leu Leu Leu

GGC

Gly GAA GTG CCG GGG Glu Val Pro Gly GAT CTC CTG TCA Asp Leu Leu Ser

TCT

Ser 105 CAC CTT GCT CCT His Leu Ala Pro

GCC

Ala 110 GAG AAA GTA TCC Glu Lys Val Ser ATC ATG Ile Met 115 GCT GAT GCA Ala Asp Ala TTC GAC CAC Phe Asp His 135 CGG CGG CTG CAT Arg Arg Leu His

ACG

Thr 125 CTT GAT CCG GCT Leu Asp Pro Ala ACC TGC CCA Thr Cys Pro 130 ACT CGG ATG Thr Arg Met CAA GCG AAA CAT Gin Ala Lys His ATC GAG CGA GCA Ile Glu Arg Ala

CGT

Arg 145 GAA GCC Glu Ala 150 GGT CTT GTC GAT Gly Leu Val Asp

CAG

Gln 155 GAT GAT CTG GAC Asp Asp Leu Asp

GAA

Glu 160 GAG CAT CAG GGG Glu His Gin Gly GCG CCA GCC GAA Ala Pro Ala Glu

CTG

Leu 170 TTC GCC AGG CTC Phe Ala Arg Leu

AAG

Lys 175 GCG CGC ATG CCC Ala Arg Met Pro

GAC

Asp 180 GGC GAG GAT CTC Gly Glu Asp Leu GTG ACC CAT GGC Val Thr His Gly

GAT

Asp 190 GCC TGC TTG CCG Ala Cys Leu Pro AAT ATC Asn Ile 195 1700 1748 1796 1844 1892 1940 1988 2036 2084 2132 2180 2235 2283 2331 ATG GTG GAA Met Val Glu GGT GTG GCG Gly Val Ala 215

AAT

Asn 200 GGC CGC TTT TCT Gly Arg Phe Ser TTC ATC GAC TGT Phe Ile Asp Cys GGC CGG CTG Gly Arg Leu 210 CGT GAT ATT Arg Asp Ile GAC CGC TAT CAG Asp Arg Tyr Gin

GAC

Asp 220 ATA GCG TTG GCT Ile Ala Leu Ala

ACC

Thr 225 GCT GAA Ala Glu 230 GAG CTT GGC GGC Glu Leu Gly Gly

GAA

Glu 235 TGG GCT GAC CGC Trp Ala Asp Arg

TTC

Phe 240 CTC GTG CTT TAC Leu Val Leu Tyr

GGT

Gly 245 ATC GCC GCT CCC Ile Ala Ala Pro TCG CAG CGC ATC Ser Gin Arg Ile TTC TAT CGC CTT Phe Tyr Arg Leu GAC GAG TTC Asp Glu Phe CGC CAT GCC His Ala 444 445 TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC Phe 264 AAG CCT GTT CTC GTG CAA AGT CCT GTG GGT GAG TCG AAC Lys Pro Val Leu Val Gln Ser Pro Val Gly Glu Ser Asn 450 455 TTG GCG Leu Ala 460 ATG CGC GCA CCC Met Arg Ala Pro TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT Tyr Gly Glu Ala Ile His Gly Leu Leu Ser 465 470 GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA Val Leu Leu Ser Thr Glu Cys 475 480 481 GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGC TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTT ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCT CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAG CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGC ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACG TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATG

T

c

T

2385 2445 2505 2565 2625 2685 2745 2805 2822

CCAACCTTGA

FIG. 2d:

GGAATTC

I1I

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAAGAT

CTCCAGCTCA

AGAATAACAA

TATCGCCCGG

TTTTCTTGGC

GCTTCGCTTC

TAAGCATTCT

TTGAGCCCA

AATTAAAATA

AGGGCAATTT

TTGACTCCTC

TTCTATCAGC

CATGCTTCTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGGAAACCGC

TTGGGCTATT

AGGAGGTCAG

GGGCCGCTTT CGAP GCCTGAACCT TCGI GCATCGAGAT GCTG TGGTGGCTTT GAAC CTTGGCGGCG TCG ATGGTTTCTT ATGI CGCTGAGCAG TTGC CG ATG AGC ATT Met Ser Ile

AGTCAT

TGACAT

;ACGTCA

AGCCTG

LAGCGAT

~GAATTT

CTCTAT

GGTGTTAGCC

AGGGCAGACG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

120 180 240 300 360 420 473 CTT CCT TTG AAT Leu Gly Leu Asn GCT CCC CCG Gly Ala Pro AAG AAG, GCG Lys Lys Ala GTC GGA GCT GAG Val Gly Ala Clu CAG CTG Gin Leu 15 GGG CCT Gly Pro GGC TCG GCT Gly Ser Ala

CTT

Leu

GAG

Glu GAT CGC ATG Asp Arg Met CTG CGT CTG Leu Arg Leu CAC CTG GAG Hius Leu Clu GCA AAC Ala Asn AGT AGG Ser Arg

TTG

Leu

GAA

Giu CTG CAT CGT Leu Asp Arg

CCG

Ala ATT GCA ATG CTT Ile Ala Met Leu AAT CGT GAA Asn Arg Giu

ATT

Ile CCC GAC CC Ala Asp Ala TCT GCT GAC TTT Ser Ala Asp Phe AAT CGC AGC CGT Asn Arg Ser Arg GAG CAA Ciu Gin 665 ACA CTC CTT Thr Leu Leu CCC GAG CAC Arg Ciu His TTT CCA CCC Phe Pro Cly

TGC

Cys ATT CCT GC Ile Ala Cly CTC CCA ACC CTC Val Ala Ser Leu CTC CCC AAA TCC Val Ala Lys Trp

ATC

Met 95

CTT

Val1 GAG CCC CAA CAT Clu Pro Ciu His

CAC

His 100

CTC

Leu AAC CAT AC Lys Asp Ser AAC CC ATC Lys Aia Met CCT CTC CTT Gly Val Val CC GAG CCA Ala Clu Ala 105 CCC CTC Cly Val

CC

Arg 110 GAG TTT CAC Giu Phe Gin

CCC

Pro ATT ACT CCC Ile Ser Pro 120

CTC

Leu TCC AAC TTC Trp Asn Phe 125 CCT ATC Pro Ile CTC CCC TTT CCC Leu Ala Phe Cly

CCC

Pro 135 857 905 CCC GC Ala Cly ATA TTC Ile Phe 140 CCC CCC Pro Arg 155 CCA CCA CCT AAT CCC CCC ATC CTC AAC Ala Ala Cly Asn Arg Ala Met Leu Lys 145 ACT TCT CCC CTC CTT CC GAG CTA ATT Thr Ser Ala Leu Leu Ala Ciu Leu Ile 160 165 CCC TCC Pro Ser 150 CCT CCT Ala Arg GAG CTT ACC Ciu Leu Thr -12- TAC TTC GAT Tyr Phe Asp 170 GAA ACT GAG CTG Giu Thr Glu Leu

ACT

Thr 175

CCT

Pro ACA GTG CTG GGC Thr Val Leu Gly

GAC

Asp 180

ATC

Ile GCT GAA GTC Ala Giu Val TTC ACC GGC Phe Thr Gly GGT GCG Gly Ala 185 GGC ACT Giv Thr CTG TTC AGT GCT Leu Phe Ser Ala

GAG

Gin 190

CAG

His TTG GAT CAT Phe Asp His

GTG

Leu 195

GG

Ala GGC GTG GCG Ala Val Ala

AAG

Lys 205

GAA

Glu ATG ATG GGT Ile Met Arg

GCG

Ala 210

TG

Ser GCG GAT AAC Ala Asp Asn

CTA

Leu 215 CCC GTT ACC Pro Val Thr TTG GGT GGG Leu Gly Gly

AAA

Lys 225

CAA

Gin CGG GTG ATG Pro Val Ile CGC AGT GGA Arg Ser Ala ACC TTG AAT Thr Phe Asn 250

GAT

Asp 235

GCC

Ala GGG GAC GTT Ala Asp Val

GCA

Ala 240

CTG

Leu GGG GTG TTG Arg Val Leu

AG

Thr 245

GTG

Val GTT TC Val Ser 230 GTG AAA Val Lys CTG GGG Leu 262 GGG CAA ATC Gly Gin Ile

TGT

Cys 255 GCA GCG GAG Ala Pro Asp

TAT

Tyr 260

GAGAGGCGGT

GCCGACATGG

CTTGTCGCCT

CGAACCCAGT

TCACGCAAGT

TTGATGGCTT

GCGCGTTACG

TTGCGTATTG

AAGCCATGAC

TGCGTATAAT

TGACATAAGC

GGTCGAGAAC

GTTATGAGTG

CCGTGGGTG

GGCGATGGA

AAACGGGATG

ATTTGCCGAT

GTGTTGGGTT

CTTGACCGAA

TTTTTTTGTA

ATGTTTGATG

TAAAAACTGT

ATGAACCTGA

GGACGCAGAC

CGTAAACTGT

CGGAGCGGTG

GAGTGTATGG

TTATGGAGCA

TGTAATTCAT

ATCGCCAGG

GGTGGAAACG

AATGCAAGTA

GTAACGGCGC

GTGGGGATC

GCAACG ATG Met 1

TAAGCATTCT

GCATGAGGAC

GATGAAGGCA

GCGTATGCGC

AGTGGCGGTT

CAAGCAGCAA

TTA CGC Leu Arg 1001 1049 1097 1145 1193 1241 1301 1361 1421 1481 1541 1601 1656 1704 1752 1800 1848 1896 1944 AGC AGC Ser Ser AAC GAT GTT AG Asn Asp Val Thr

GAG

Gin 10 GAG GGG ACT CG Gin Gly Ser Arg

GGT

Pro AAA ACA AAG TTA Lys Thr Lys Leu

GT

Gly

GAA

Gin GGG TGA AGT ATG Oly Ser Ser Met ATG ATT GG ACA Ile Ile Arg Thr AGG GTG GG GGT Arg Leu Gly Pro

GAG

Asp GTG AAA TGG Val Lys Ser

ATG

Met OCT GGT GTT Ala Ala Leu

GAT

Asp 45

CG

Gin GTT TTC GGT GGT Leu Phe Gly Arg GAG TTG Giu Phe OGA GAG GTA Oly Asp Val GGG AAG TTG Gly Asn Leu AGG TAG TGG GAA Thr Tyr Ser Gin CCG GAG TGG Pro Asp Ser GAT TAG GTG Asp Tyr Leu 0CC TTG GAG Ala Phe Asp GGT AGT AAG Arg Ser Lys ATG GGG GTT Ile Ala Leu

GGT

Ala CAA GAA GGG GTT GTT GGG GGT GTG GGG GGT TAG GTT GTG CCC AGG TTT 13- Gin Giu Ala Val Val Gly Ala 90 Leu Ala Ala Tyr Va1

CTC

Leu Leu Pro Arg Phe GAG CAG CCG Glu Gin Pro 100 GAG CAC CGG Glu His Arg CAT GAG GCC His Giu Ala TAC GGT GAC Tyr Gly Asp 150 GAA GAA GTG Glu Giu Val 165 CGT AGT Arg Ser AGG CAG Arg Gin 120 AAC GCG Asn Ala GAG ATC Glu Ile TAT ATC TAT Tyr Ile Tyr GCA GTC TCC Ala Val Ser 105

GGC

Gly

GGC

Gly 115 ATT GCC ACC Ile Ala Thr

GCG

Ala 125

GTG

Val ATC AAT CTC Ile Asn Leu CTC AAG Leu Lys 130 CTT GGT GCT Leu Giy Ala

TAT

Tyr 140

CTC

Leu ATC TAC GTG Ile Tyr Val CCC GCA GTG Pro Ala Val TAT ACA AAG Tyr Thr Lys

TTG

Leu 160

GCC

Ala CAA GCA GAT Gin Ala Asp 145 GGC ATA CGG Gly Ile Arg ACC TAA CAA Thr 177 ATG CAC TTT GAT Met His Phe Asp 170 GAC CCA AGT Asp Pro Ser

ACC

Thr 175 TTCGTTCAAG CCGAGATCGG CTTCCCTG CAA AGT CCT GTG GGT GAG TCG AAC Gin Ser Pro Val Gly Giu Ser Asn 451 455 1992 2040 2088 2136 2184 2236 2284 2338 2398 2458 2518 2578 2638 2698 2758 2775 TTG GCG Leu Ala 460 GTC CTC Val Leu 475

GGAGCATG

TCGAGCCT

ATCTTCGC

CCGATGTC

CCAGACAC

ATTTCGTC

TGTACGTC

CCAACCT9 FIG. 2e: ATG CGC GCA Met Arg Ala CTT TCA ACG Leu Ser Thr ;CG CTTCTGGGC 'GC ACAGCTGAJ CA TTCATGAC( CG ATTCCACG( ;CA ACAAATGA( AA TGTGACGA TT GGTGGCGA( ?GA GGAATTC Cc P2 C TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT o0 Tyr Gly Giu Ala Ile His Gly Leu Leu Ser 465 470 GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA Glu Cys 480 481 C CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGC' 'G AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTT( ;G AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCT' C GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAG( 3T AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGC.

rG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGI ,C AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATG'

T

r

A

T

14

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAAGAT

CTCCAGCTCA

TATCGCCCGG

TTTTCTTGGC

GCTTCGCTTC

TAAGCATTCT

TTGAGGCCGA

AATTAAAATA

AGGGCAATTT

TTCTATCAGC

CATGCTTGTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGGAAACCGC

TTGGGCTATT

GGGCCGCTTT

GCCTGAACCT

GCATCGAGAT

TGGTGGCTTT

CTTGGCGGCG

ATGGTTTCTT

GGCTGAGCAG

CGAAAGTCAT

TCGTTGACAT

GCTGAGGTCA

GAACAGCCTG

TCGAAGCGAT

ATGTGAATTT

TTGCCTCTAT

GGTGTTAGCC

AGGGCAGAGG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

120 180 240 300 360 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT Met Ser Ile CTT GGT TTG AAT Leu Gly Leu Asn GGT GCC CCG Gly Ala Pro AAG AAG GCG Lys Lys Ala GTC GGA GCT GAG Val Gly Ala Glu CAG CTG Gin Leu 15 GGG CCT Gly Pro GGC TCG GCT Gly Ser Ala

CTT

Leu GAT CGC ATG Asp Arg Met CAC CTG GAG His Leu Glu GCA AAC Ala Asn AGT AGG Ser Arg

TTG

Leu

GAA

Glu GAG CTG CGT CTG Glu Leu Arg Leu CTG GAT CGT Leu Asp Arg

ATT

Ile

GCG

Ala 45

TCT

Ser GCA ATG CTT Ala Met Leu

CTG

Leu

AAT

Asn AAT CGT GAA Asn Arg Giu GCC GAC GCG Ala Asp Ala

GTT

Val1

GAC

Asp GCT GAC TTT Ala Asp Phe CGC AGC CGT Arg Ser Arg 665 713 ACA CTG CTT Thr Leu Leu CGC GAG CAC Arg Giu His TTT CCA GGG Phe Pro Gly ATT GCT GGC Ile Ala Gly GTG GCA AGC CTG Val Ala Ser Leu GTG GCC AAA TGG Val Ala Lys Trp

ATG

Met 95

GTT

Val1 GAG CCC GAA CAT Giu Pro Glu His

CAC

His 100

CTG

Leu AAG GAT AGC Lys Asp Ser AAG GCG ATG Lys Ala Met GGT GTC GTT Gly Val Val GCG GAG GCA Ala Giu Ala GAG TTT CAG Giu Phe Gin 105 GOG GTC Gly Val

CCG

Pro 115

CTG

Leu ATT AGT CCC Ile Ser Pro 120

CTG

Leu

TGG

Trp 125

GCA

Ala TTC CCT ATC Phe Pro Ilie GCC TTT GGG Ala Phe Gly

CCG

Pro 135 GCC GGC ATA Ala Gly Ile

TTC

Phe 140

CGG

Arg GCA GGT AAT Ala Gly Asn

CGC

Arg 145

CTT

Leu GCC ATG CTC AAG Ala Met Leu Lys CCG TCC Pro Ser 150 GCT CGT Ala Arg GAG CTT ACC Giu Leu Thr ACT TCT GCC Thr Ser Ala

CTG

Leu 160 GCG GAG CTA Ala Giu Leu 15 TAC TTC GAT Tyr Phe Asp 170 GGT GCG CTG Gly Ala Leu GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT GAA GTC Glu Thr Glu Leu Thr Thr Val Leu Gly Asp Ala Giu Val 175 180 TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC Phe Ser Ala Gin Pro Phe Asp His Leu Ile Phe Thr Gly 185 GGC ACT Glv Thr 195

GCG

Ala GCC GTG GCC Ala Val Ala ATC ATG CGT Ile Met Arg GCG GAT AAC Ala Asp Asn 200

GTG

Va1

CTA

Leu 215 CCC GTT ACC Pro Val Thr

CTG

Leu 220

ATG

Met TTG GGT GGC Leu Gly Gly

AAA

Lys 225 CGC AGT GCA Arg Ser Ala ACC TTC AAT Thr Phe Asn 250 TTG GCG ATG Leu Ala Met 460 GCG GAC GTT Ala Asp Val GCA CAA Ala Gin 240 TCG CCG GTG ATC GTT TCC Ser Pro Val Ile Val Ser 230 CGG GTG TTG ACG GTG AAA Arg Val Leu Thr Val Lys 245 CC GTG GGT GAG TCG AAC Val Gly Glu Ser Asn 454 455 ATC CAC GGA CTG CTC TCT Ile His Gly Leu Leu Ser 470 GGG CAA ATC Gly Gin Ile TGT CTG GCA Cys Leu Ala 255 257 GGA GAA GCG Gly Glu Ala 1001 1049 1097 1145 1193 1240 1288 1342 1402 1462 1522 1582 1642 1702 1762 1779 CGC GCA CCC Arg Ala Pro

TAC

Tyr 465 CTC CTT TCA ACG Leu Leu Ser Thr GAG TGT Glu Cys 480 481 TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA

GGAGCATGCG

TCGAGCCTGC

ATCTTCGCCA

CCGATGTCCG

CCAGACAGCA

ATTTCGTCAA

TGTACGTCTT

CCAACCTTGA

FIG. 2f:

CTTCTGGGCC

ACAGCTGATG

TTCATGACGG

ATTCCACGCC

ACAAATGAGT

TGTGACGATG

GGTGGCGAGC

GGAATTC

CGTTTCTTGA

AGCACCCTGG

AACTCCGTTC

GCCTGACGCT

AGGCTCTTGG

GATTCGATTG

AAGGAAGAAC

GTATTCATTG

AAGGCGCGCT

CCCAGTACCG

AAGCGGGGGC

ATGCCGCGGC

CCCGTGCTGC

TCATTTCCCG

GATAGTCACG

GTACGCGGAC

CGATGACTAT

GGGGGCGCCC

GGCTGAGATT

CGGCGTCTCA

GTTAGTGGCT

CGTGGTAGCT

GACTGGGTTC

TTTGCCTCTT

GCATCCCAGC

GGTAACGGCA

AAAAAAACGC

CGAGACATGT

-16-

CTGCAGCCGA

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

GCATCGATTG

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

AGCACTTTAC

AAATCGATCT

AACTTGATAA

CTCGAAGAGG

TCTGCTCAGC

GACGCTGGAG

CCAGCTGCGC

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

TGGCTGACCA TTCAGAATGG CGGGCATCAT GCCCGCGGCG GTTCTCCGGT CTTGGTGGAT GAACGCCGAG TCCACATTGC CAGTGTGTCG ATTGGTCATC G ATG CGT TCT CTC GAG Met Arg Ser Leu Giu 120 180 240 300 356 GC CTT CTT CCC Ala Leu Leu Pro

TTC

Phe CCG GGT CGA ATT Pro Gly Arg Ile

CTT

Leu 15

GTT

Val1 GAG CGT CTC GAG Glu Arg Leu Giu CAT TGG His Trp, GCT AAG ACC Ala Lys Thr COG GAA TGG Cly Glu Trp GCC ATC GCA Ala Ile Ala CCA GAA CAA ACC Pro Glu Gin Thr

TGC

Cys

GCG

Ala GCT GCC AGG Ala Ala Arg CGT CGT ATC AGC Arg Arg Ile Ser GAA ATG TTC Glu Met Phe

CAC

His

GCA

Ala GCG GCA AAT Ala Ala Asn AAC GTC CGC Asn Val Arg GAG CGT CCG Glu Arg Pro CAG AGC TTG Gin Ser Leu TAC GGA CTA Tyr Gly Leu CTG CTT Leu Leu

TCG

Ser

CTT

Leu ATC GTC TCT Ile Val Ser

GGA

Gly

GGC

Gly GAC CTG GAA Asp Leu Clu

CAT

His

CCG

Pro CAG CTG GCA Gin Leu Ala 596 644 OCT ATO TAT Ala Met Tyr

GCG

Ala

CAA

Gin ATT CCC TAT Ile Pro Tyr

TGC

Cys 95

CTG

Leu GTC TCT CCT Val Ser Pro TCA CTO CTG Ser Leu Leu CTG CAA CCG Leu Gin Pro 120

TCG

Ser 105

GGA

Gly GAT TTG GCG Asp Leu Ala

AAG

Lys 110

GCC

Ala COT CAC ATC Arg His Ile

GTA

Val1 115

TTC

Phe GCT TAT Ala Tyr 100 GGT CTT Gly Leu CAG GGG Gin 132 CTG GTC TTT Leu Val Phe

GCT

Ala 125 GAT GCA GCA Asp Ala Ala

CCT

Pro 130 ACAGCAAGCG AACCGGAATT GCCAGCTGGO OCOCCCTCTG AAAGTAAACT GCATGGCTTT CTTOCCCCCA AOOATCTGAT

GTAAGGTTGG

GGCGCAGGGO

GAAGCCCTGC

ATCAAGATCT

GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATG ATT OAA CAA GAT GGA TTG CAC Met Ile Glu Gin Asp Gly Leu His 1 GCA GOT TCT CCG GCC GCT TOO GTG GAG AGG CTA TTC 0CC TAT CAC TGG Ala Oly Ser Pro Ala Ala Trp Val Clu Arg Leu Phe Cly Tyr Asp Trp 15

GCA

Ala CAA CAG ACA ATC Gin Gin Thr Ile

GGC

Gly TGC TCT GAT GCC Cys Ser Asp Ala GTG TTC CGG CTG Val Phe Arg Leu GCG CAG GGG CGC Ala Gin Gly Arg GTT CTT TTT GTC Val Leu Phe Val ACC GAC CTG TCC Thr Asp Leu Ser GGT GCC Gly Ala CTG AAT GAA Leu Asn Glu ACG GGC GTT Thr Gly Val

CTG

Leu CAG GAC GAG GCA GCG CGG CTA TCG TGG Gin Asp Glu Ala Ala Arg Leu Ser Trp 65 CTG GCC ACG Leu Ala Thr GAA GCG GGA Glu Ala Gly CCT TGC GCA GCT Pro Cys Ala Ala

GTG

Val CTC GAC GTT GTC Leu Asp Val Val AGG GAC Arg Asp TGG CTG CTA TTG Trp Leu Leu Leu GAA GTG CCG GGG Glu Val Pro Gly

CAG

Gin 100 GAT CTC CTG TCA Asp Leu Leu Ser

TCT

Ser 105 CAC CTT GCT CCT His Leu Ala Pro GAG AAA GTA TCC Glu Lys Val Ser ATG GCT GAT GCA Met Ala Asp Ala

ATG

Met 120 CGG CG CCTG CAT Arg Arg Leu His

ACG

Thr 125 CTT GAT CCG GCT Leu Asp Pro Ala

ACC

Thr 130 TGC CCA TTC GAC Cys Pro Phe Asp CAC CAA His Gln 135 1010 1058 1106 1154 1202 1250 1298 1346 1394 1442 1490 1538 1586 1634 GCG AAA CAT Ala Lys His GTC GAT CAG Val Asp Gin 155 ATC GAG CGA GCA Ile Glu Arg Ala ACT CGG ATG GAA Thr Arg Met Glu GCC GGT CTT Ala Gly Leu 150 GCG CCA GCC Ala Pro Ala GAT GAT CTG GAC Asp Asp Leu Asp GAG CAT CAG GGG Glu His Gin Gly GAA CTG Glu Leu 170 TTC GCC AGG CTC Phe Ala Arg Leu

AAG

Lys 175 GCG CGC ATG CCC Ala Arg Met Pro

GAC

Asp 180 GGC GAG GAT CTC Gly Glu Asp Leu

GTC

Val 185 GTG ACC CAT GGC Val Thr His Gly

GAT

Asp 190 GCC TGC TTG CCG Ala Cys Leu Pro

AAT

Asn 195 ATC ATG GTG GAA Ile Met Val Glu GGC CGC TTT TCT Gly Arg Phe Ser TTC ATC GAC TGT Phe Ile Asp Cys

GGC

Gly 210 CGG CTG GGT GTG Arg Leu Gly Val GCG GAC Ala Asp 215 CGC TAT CAG Arg Tyr Gin GGC GGC GAA Gly Gly Glu 235

GAC

Asp 220 ATA GCG TTG GCT ACC CGT GAT ATT GCT Ile Ala Leu Ala Thr Arg Asp Ile Ala 225 GAA GAG CTT Glu Glu Leu 230 ATC GCC GCT Ile Ala Ala TGG GCT GAC CGC Trp Ala Asp Arg

TTC

Phe 240 CTC GTG CTT TAC Leu Val Leu Tyr

GGT

Gly 245 -18- CCC GAT TCG GAG CCC ATC GCC TTC TAT CGC CTT CTT GAG GAG TTC TTC Pro Asp Ser Gin Arg Ile Ala Phe Tyr Arg Leu Leu Asp Clu Phe Phe 250 255 260 264 TGACCGCGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC CCCCCT GTT TTG CAA Val Leu Gin 563 565 TGG CGG TCG GCG AAA GTT GAT GCG CTG TAT CGT GGT GAA GAT CAA TCC Trp Arg Ser Ala Lys Val Asp Ala Leu Tyr Arg Gly Glu Asp Gin Ser 570 575 580 ATG CTG CCT CAC GAG CC ACA CTC TGA CTTCGTCAGG CCCGGCTTAC Met Leu Arg Asp Glu Ala Thr Leu 585 589 TCGGCGTTTT CCCACACTGC CTTGGTTGCC CCAGTGCGCA CCCCCTGGAT TG GGTGCCCTCT CCCTGCTCTC CCCTATCCAC TTAGGGGTAA AGGTCCCTCG CCG ATCCGTCCGT CGCTTGAACC ACAAATGGTC GATACCGTAC TCGCAGCCTC TA CCAAGCTTTG ATCCTTACCT GCTCCCGCGC CACATTCGCT TGTACACCGG TG TCGGTTCCGG CCTTGGCGGT GCAGCCCATT TCCGGCACAG CCTTCGAACT C GCCGGCGAGC AGATTTCCCA ACCCGCTGAT CACGTCCTGT CTGTCCCGGG CT FIG. 2g: 1682 1737 1785 1832 1892 1952 2012 2072 2132 2188 AiTTGC CCC

ACTTCTG

TGCCTCAA

TTCCCAAG

TTCGGCAG

GCAG

19

CTGCAGCCGA

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

GCATCGATTG

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

AGCACTTTAC

AAATCGATCT

AACTTGATAA

CTCGAAGAGG

TCTGCTCAGC

GACGCTGGAG

CCAGCTGCGC

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

TGGCTGACCA TTCAGAATGG CGGGCATCAT GCCCGCGGCG GTTCTCCGGT CTTGGTGGAT GAACGCCGAG TCCACATTGC CAGTGTGTCG ATTGGTCATC G ATG CGT TCT CTC GAG Met Arg Ser Leu Giu GCG CTT CTT CCC Ala Leu Leu Pro

TTC

Phe CCG GGT CGA Pro Gly Arg GCT AAG ACC Ala Lys Thr GGG GAA TGG Gly Glu Trp CCC ATC GCA Ala Ilie Ala CCA GAA CAA ACC Pro Giu Gin Thr ATT CTT Ile Leu 15 TGC GTT Cys Val GCG GAA Ala Ciu GAG CGT CTC GAG Giu Arg Leu Giu CAT TGG His Trp 404 452 GCT GCC AGG Ala Ala Arg CCT ATC AGC Arg Ile Ser ATG TTC Met Phe

CAC

His

GCA

Ala GCG GCA AAT Ala Ala Asn AAC GTC CGC Asn Val Arg GAG CGT CCG Giu Arg Pro CAG AGC TTG Gin Ser Leu CTC CTT Leu Leu

CTT

Leu 60

AAT

Asn TAC GCA CTA Tyr Cly Leu

TCG

Ser ATC GTC TCT Ile Val Ser GAC CTG GAA Asp Leu Giu CTT CAG CTG GCA Leu Gin Leu Ala

CCC

Cly GCT ATG TAT Ala Met Tyr

C

Ala

CAA

Gin ATT CCC TAT Ile Pro Tyr GTG TCT CCT Val Ser Pro TCA CTG CTG Ser Leu Leu CTG CAA CCG Leu Gin Pro 120

TCG

Ser 105

GGA

Gly GAT TTG GCG Asp Leu Ala

AAG

Lys 110

GCC

Ala CCT CAC ATC Arg His Ile CCT TAT Ala Tyr 100 GGT CTT Cly Leu CAG CCC Gin 132 CTC GTC TTT Leu Val Phe

GCT

Ala 125 CAT CCA CCA Asp Ala Ala

CCT

Pro 130

CACAGCCCT

CCCACATGC

CTTGTCCCCT

CGAACCCACT

TCACCCAACT

TTCATCGCTT

TTCCTATTC

AACCCATCAC

TGCGTATAAT

TGACATAAC

GCTCCAGAAC

CTTATCACTC

GGCGCATCCA

AAACCCCATC

ATTTGCCCAT

CTGTTCGCTT

CTTGACCCAA

TTTTTTTCTA

TAAAAACTCT

ATCAACCTCA

GGACGCACAC

CCTAAACTCT

CGCACCCTC

CAGTCTATC

TCTAATTCAT

ATCCCCAGCC

CGTCGAAACC

AATCCAAGTA

GTAACGCCGC

CTCGCCCATC

TAAGCATTCT

CCATCACCAC

CATGAAGGCA

CCTATGCGC

AGTGCCTT

CAAGCAGCAA

800 860 920 980 1040 1100 GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA GCAACG ATG TTA CGC Met Leu Arg 1 AGC AGC AAC GAT GTT ACG Ser Ser Asn Asp Val Thr CAG GGC AGT CGC Gin Gly Ser Arg

CCT

Pro AAA ACA AAG TTA Lys Thr Lys Leu

GGT

Gly GGC TCA AGT ATG Gly Ser Ser Met

GGC

Gly ATC ATT CGC ACA Ile Ile Arg Thr AGG CTC GGC CCT Arg Leu Gly Pro CAA GTC AAA TCC Gin Val Lys Ser CGG GCT GCT CTT GAT CTT TTC GGT CGT Arg Ala Ala Leu Asp Leu Phe Gly Arg GAG TTC Glu Phe GGA GAC GTA Gly Asp Val GGG AAC TTG Gly Asn Leu ACC TAC TCC CAA Thr Tyr Ser Gin CAG CCG GAC TCC Gin Pro Asp Ser GAT TAC CTC Asp Tyr Leu GCC TTC GAC Ala Phe Asp CTC CGT AGT AAG Leu Arg Ser Lys TTC ATC GCG CTT Phe Ile Ala Leu

GCT

Ala CAA GAA Gin Glu GCG GTT GTT GGC Ala Val Val Gly

GCT

Ala 90 CTC GCG GCT TAC Leu Ala Ala Tyr

GTT

Val CTG CCC AGG TTT Leu Pro Arg Phe 1155 1203 1251 1299 1347 1395 1443 1491 1539 1587 1635 1683 1735 1783

GAG

Glu 100 CAG CCG CGT AGT Gin Pro Arg Ser ATC TAT ATC TAT Ile Tyr Ile Tyr

GAT

Asp 110 CTC GCA GTC TCC Leu Ala Val Ser GAG CAC CGG AGG Glu His Arg Arg

CAG

Gin 120 GGC ATT GCC ACC Gly Ile Ala Thr CTC ATC AAT CTC Leu Ile Asn Leu CTC AAG Leu Lys 130 CAT GAG GCC His Glu Ala TAC GGT GAC Tyr Gly Asp 150

AAC

Asn 135 GCG CTT GGT GCT Ala Leu Gly Ala

TAT

Tyr 140 GTG ATC TAC GTG Val Ile Tyr Val CAA GCA GAT Gin Ala Asp 145 GGC ATA CGG Gly Ile Arg GAT CCC GCA GTG Asp Pro Ala Val

GCT

Ala 155 CTC TAT ACA AAG Leu Tyr Thr Lys

TTG

Leu 160 GAA GAA Glu Glu 165 GTG ATG CAC TTT Val Met His Phe ATC GAC CCA AGT Ile Asp Pro Ser ACC GCC ACC TAA CAA Thr Ala Thr 175 177 TTCGTTCAAG CCGAGATCGG CTTCCCCT GTT TTG CAA TGG CGG TCG GCG AAA Val Leu Gin Trp Arg Ser Ala Lys 563 565 570 GTT GAT GCG CTG TAT CGT GGT GAA GAT CAA TCC ATG CTG CGT GAC GAG Val Asp Ala Leu Tyr Arg Gly Glu Asp Gin Ser Met Leu Arg Asp Glu 575 580 585 -21- GCC ACA CTG TCA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC Ala Thr Leu 589 GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA AGGCGCTGAT CACGTGCTGT GTGTCGCGGG CTGCAG FIG. 2h: 1835 1895 1955 2015 2075 2135 2171 -22- CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

AAATCGATCT

AACTTGATAA

CTCGAAGAGG

TCTGCTCAGC

GACGCTGGAG

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

CGGGCATCAT GCCCGCGGCG GTTCTCCGGT CTTGGTGGAT GAACGCCGAG TCCACATTGC CAGTGTGTCG ATTGGTCATC G ATG CGT TCT CTC GAG Met Arg Ser Leu Glu GCG CTT CTT CCC Ala Leu Leu Pro

TTC

Phe

CCA

Pro CCG GGT CGA ATT Pro Gly Arg Ile GAG CGT CTC GAG Glu Arg Leu Glu CAT TGG His Trp GCT AAG ACC Ala Lys Thr GGG GAA TGG Gly Glu Trp GCC ATC GCA Ala Ile Ala GAA CAA ACC Glu Gin Thr

TGC

Cys

GCG

Ala GCT GCC AGG Ala Ala Arg CGT ATC AGC Arg Ile Ser GAA ATG TTC Glu Met Phe

CAC

His

GCA

TCG

Ser

CTT

Leu ATC GTC TCT Ile Val Ser

GGA

Gly

GGC

Gly GAC CTG GAA Asp Leu Glu

CAT

His

CCG

Pro CAG CTG GCA Gin Leu Ala 596 644 GCT ATG TAT Ala Met Tyr ATT CCC TAT Ile Pro Tyr

TGC

Cys

CTG

Leu GTG TCT CCT Val Ser Pro TCA CTG CTG Ser Leu Leu CTG CAA CCG Leu Gin Pro 120 GCT GTT TTG Ala Val Leu

TCG

Ser 105

GGA

Gly

CAA

Gin 565

TCC

Ser GAT TTG GCG Asp Leu Ala CGT CAC ATC Arg His Ile CTG GTC TTT Leu Val Phe TGG CGG TCG Trp Arg Ser ATG CTG CGT Met Leu Arg

GCT

Ala 125

GCG

Ala

GAC

Asp 585 GAT GCA GCA Asp Ala Ala

CCT

Pro 130

CTG

Leu

GTA

Va1 115

TTC

Phe

TAT

Tyr 575 GCT TAT Ala Tyr 100 GGT CTT Gly Leu CAG CGC Gin Arg 133 CGT GGT Arg Gly 562

GAA

Glu AAA GTT GAT GCG Lys Val Asp Ala 570 GAG GCC ACA CTG Glu Ala Thr Leu 589 GAT CAA Asp Gin 580 TGA GTTGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC GTTGGTTGCG TGATTGCGGG GGTGCCCTGT CGCTGGTGTC GCCTATCGAC GCAGTGCGCA CCCCCTGGAT TTAGGGGTAA AGGTCGCTCG 23 CGAAGTTCTG ATGCGTGCGT CGCTTGAACC ACAAATGGTC GATAGCGTAC TCGCAGGCTC 1017 TATGGCTCAA GCAAGCTTTG ATGCTTACCT GCTCCCGCGG CACATTGGCT TGTACAGCGG 1077 TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT GCAGCGCATT TGCGGCACAG GCTTCGAACT 1137 GCTTCGGCAG GCCGGCGAGC AGATTTCCCA AGGCGCTGAT CACGTGCTGT GTGTCGCGGG 1197 CTGCAG 1203 FIG. 2i: -24- GAATTCCCCT GGCGACGAAA GGGCGGCAGG GCTTGGGTTA ATCGTTAACC GTTTGAAATT GGGTACGCCT TTGCGTGCGC TTTGATCTGC AATTGAGAGA ACTATAGGTT CGCAGTAGCT GGTGCACG ATG AAT AGC TAG GAT GGG Met Asn Ser Tyr Asp Gly 1 5 CCGCATGGCC ACGGCTGGGC GGTAACTGAT GCTTGCCAAA TTTCGGGAG AGAATCATGG GCTTCCGTGG CTTGAATCAG AAAAATAGTT TTTGCTCACC CACCAAATCG ACAGGACTGG GGT TGG TCT ACC GTT GAT GTG AAG Arg Trp Ser Thr Val Asp Val Lys

GTT

Va1

AAG

Asn GAA GAA GGT ATG Glu Giu Gly Ile

GCT

Ala 20 TGG GTG AGG CTG Trp Val Thr Leu GCA ATG AGC Ala Met Ser

GCA

Pro

GAG

Asp ACT GTC AAT GGA Thr Leu Asn Arg AAC CGC CCG GAG AAG GGC Asn Arg Pro Giu Lys Arg 25 ATG GTG GAG GTT GTG GAG Met Val Glu Val Leu Glu GTT GTT GTG AGT GGT GCA Leu Val Leu Thr Gly Ala 120 180 240 290 338 386 434 482 531 591 651 703 GTG GTG GAG GAG Val Leu Glu Gin GGG GAA TCC TGG Gly Glu Ser Trp ACC GAT GGT GGG Thr Asp Ala Gly GCA GAT GCT Ala Asp Ala

CGC

Arg 55 ACC GGG GGG ATG Thr Ala Gly Met 70 CCC GAA ATT GTG Pro Glu Ile Leu 85 GAC CTG AAG Asp Leu Lys GAA GAG AAG Gin Glu Lys AAGCGAACCG GAATTGCGAG GTGGGGCGCC CTCTGGTAAG AAACTGGATG GGTTTGTTGC CGCCAAGGAT GTGATGGCGC AGAGAGAGGA TGAGGATGGT TTCGC ATG ATT GAA CAA Met Ile Giu Gin 1 GGT TGT CCG GCC GGT TGG GTG GAG AGG GTA TTG Gly Ser Pro Ala Ala Trp Val Giu Arg Leu Phe 15 20 CAA GAG ACA ATC GGG TGG TGT GAT GCC GCC GTG Gin Gin Thr Ile Gly Gys Ser Asp Ala Ala Val 35 GAG GGG GGC CCG GTT CTT TTT GTG AAG ACC GAG Gin Gly Arg Pro Vai Leu Phe Val Lys Thr Asp 50 AAT GAA CTG GAG GAG GAG GCA GGG GGG GTA TCG Asn Giu Leu Gin Asp Glu Ala Ala Arg Leu Ser 65 GAG TAT TTC CGC GAG Glu Tyr Phe Arg Glu ATT CGT GGGGGAGAGC Ile Arg 90 91 GTTGGGAAGC CCTGCAAAGT AGGGGATGAA GATCTGATCA GAT GGA TTG CAC GCA Asp Gly Leu His Ala GGC TAT GAG TGG GCA Gly Tyr Asp Trp Ala TTC CGG CTG TGA GCG Phe Arg Leu Ser Ala GTG TCC GGT GCG CTG Leu Ser Gly Ala Leu TGG GTG GCC ACG ACG Trp Leu Ala Thr Thr 799 GGC GTT Gly Val CCT TGC GCA GCT Pro Cys Ala Ala

GTG

Val 80 CTC GAC GTT GTC Leu Asp Val Val

ACT

Thr GAA GCG GGA AGG Glu Ala Gly Arg

GAC

Asp TGG CTG CTA TTG Trp Leu Leu Leu GAA GTG CCG GGG Glu Val Pro Gly GAT CTC CTG TCA Asp Leu Leu Ser CAC CTT GCT CCT His Leu Ala Pro

GCC

Ala 110 GAG AAA GTA TCC Glu Lys Val Ser ATG GCT GAT GCA Met Ala Asp Ala ATG CGG Met Arg 120 CGG CTG CAT Arg Leu His AAA CAT CGC Lys His Arg 140

ACG

Thr 125 CTT GAT CCG GCT Leu Asp Pro Ala

ACC

Thr 130 TGC CCA TTC GAC Cys Pro Phe Asp CAC CAA GCG His Gin Ala 135 GGT CTT GTC Gly Leu Val ATC GAG CGA GCA Ile Glu Arg Ala ACT CGG ATG GAA Thr Arg Met Glu

GCC

Ala 150 GAT CAG Asp Gin 155 GAT GAT CTG GAC Asp Asp Leu Asp GAG CAT CAG GGG Glu His Gin Gly

CTC

Leu 165 GCG CCA GCC GAA Ala Pro Ala Glu

CTG

Leu 170 TTC GCC AGG CTC Phe Ala Arg Leu

AAG

Lys 175 GCG CGC ATG CCC Ala Arg Met Pro GGC GAG GAT CTC Gly Glu Asp Leu GTG ACC CAT GGC Val Thr His Gly

GAT

Asp 190 GCC TGC TTG CCG Ala Cys Leu Pro

AAT

Asn 195 ATC ATG GTG GAA Ile Met Val Glu AAT GGC Asn Gly 200 1039 1087 1135 1183 1231 1279 1327 1375 1423 1471 1525 1573 CGC TTT TCT Arg Phe Ser TAT CAG GAC Tyr Gin Asp 220

GGA

Gly 205 TTC ATC GAC TGT Phe Ile Asp Cys

GGC

Gly 210 CGG CTG GGT GTG Arg Leu Gly Val ATA GCG TTG GCT Ile Ala Leu Ala CGT GAT ATT GCT Arg Asp Ile Ala

GAA

Glu 230 GCG GAC CGC Ala Asp Arg 215 GAG CTT GGC Glu Leu Gly GCC GCT CCC Ala Ala Pro GGC GAA Gly Glu 235 TGG GCT GAC CGC Trp Ala Asp Arg CTC GTG CTT TAC Leu Val Leu Tyr GGT ATC Gly Ile 245

GAT

Asp 250 TCG CAG CGC ATC GCC TTC TAT CGC CTT Ser Gin Arg Ile Ala Phe Tyr Arg Leu GAC GAG TTC Asp Glu Phe TTC TGA Phe 264 GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CC GAG CAG GGC ATG Glu Gin Gly Met 255 GGC TTG CAG ACC TAC Gly Leu Gin Thr Tyr 270 AAG CAG Lys Gin 260 TTC CTT GAC GAG Phe Leu Asp Glu AAA AGC ATC AAG CCG Lys Ser Ile Lys Pro 265 26 AAG CGC TGA TAAATGCGCC GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA TAATGACAAT 1632 Lys Arg 275 276 AATGAGGAGT GCCCAATGTT TCACGTGCCC CTGCTTATTG GTGGTAAGCC TTGTTCAGCA 1692

TCTGATGAGC

GCTGCTGCCA

GAATGGGCGG

CTAGAGGACC

TGGTATGGGT

FIG. 2j:

GCACCTTCGA

GTTTGGAAGA

CGCTTGCTCC

GTTCTTCCGA

TTAACGTTTA

GCGTCGTAGC

TGCGGACGCC

GAGCGAACGC

GTTCACCGCC

CCTGGCGGCG

CCGCTGACCG

GCAGTGGCCG

CGTGCCCGAC

GCAGCGAGTG

GGCATGTTGC

GAGAAGTGGT ATCGCGCGTC CTGCACAGGC TGCGTTTCCT TGCTGCGAGC GGCGGATCTT AAACTGGCGC AGCGGGAAAC

GGGGAATTC

1752 1812 1872 1932 1981 27 GAATTCCCCT GGCGACGAAA GGGCGGCAGG GCTTGCGTTA ATCGTTAACC GTTTGAAATT GGGTACGCCT TTCCGTGCGC TTTGATCTGC AATTGACAGA ACTATAGGTT CGCAGTAGCT GGTGCACG ATG AAT AGC TAC GAT GGC Met Asn Ser Tyr Asp Gly 1 5 CCGCATGGCC ACGGCTGGGC GGTAACTGAT CCTTGCCAAA TTTCGGCGAG AGAATCATGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT TTTGCTCACC CACCAAATCC ACAGCACTGG CGT TGG TCT ACC GTT GAT GTG AAG Arg Trp Ser Thr Val Asp Val Lys 120 180 240 290 338

GTT

Val

AAC

Asn GAA GAA GGT ATC Glu Glu Gly Ile TGG GTC ACG CTG Trp, Val Thr Leu GCA ATG AGC Ala Met Ser

CCA

Pro

CAC

Asp ACT CTC AAT Thr Leu Asn GCA CAT GCT Ala Asp Ala GTG CTG GAG CAC Val Leu Giu Gin CGC CAA TCC TGC Cly Giu Ser Trp CGA GAG Arg Ciu 40 CGC CTC Arg Val 55 GAC CTG Asp Leu

AAC

Asn 25

ATG

Met

CTT

Leu CGC CCC GAG AAG CGC Arg Pro Giu Lys Arg CTC GAG CTT CTC GAG Val Ciu Val Leu Giu GTT CTC ACT GGT GCA Val Leu Thr Gly Ala ACC CC CCC ATC Thr Ala Cly Met 70 AAG GAG TAT Lys Ciu Tyr TTC CCC GAG Phe Arg Ciu ACC CAT GC' Thr Asp Al~

GCGGTTTGCC

CATGGAAGCC

CCCCTTCT

CCACTTCACA

CAACTGGTCC

CCCTTCTTAT

TTACCCCTG

P CCC CCC GAA ATT CTG CAA GAG AAC i Cly Pro Glu Ile Leu Gin Ciu Lys

TATTCGCC

ATCACAAACG

ATAATATTTG

TAAGCCTGTT

AGAACCTTGA

CACTCTTTTT

GGTCGATCTT

ATGCATAAAA

GCATGATGAA

CCCATCCACC

CGGTTCGTAA.

CCGAACGCAG

TTCTACACTC

TCATGTTATG

ACTGTTGTAA

CCTGAATCGC

CACACCCTCC

ACTGTAATGC

CGGTGGTAAC

TATCCCTCG

GAGCAGCAAC

ATT CCT CCCCCCACAC Ile Arg 90 91 TTCATTAAGC ATTCTGCCCA CACCCATC ACCACCTTCT AAACGGATGA AGGCACGA.AC AAGTAGCGTA TCCTCACC GCCCACTCC CCCTTTTCAT GCATCCAAGC AGCAAGCGCG C ATG TTA CCC ACC AC Met Leu Arg Ser Ser 1 ACA AAC TTA CCT GC Thr Lys Leu Gly Cly CCC CCT CAC CAA CTC Cly Pro Asp Gin Val 531 591 651 711 771 831 891 947 995 1043 AAC CAT CTT ACC CAC CAC CCC ACT CCC CCT AAA Asn Asp Val Thr Gin Gin Cly Ser Arg Pro Lys 15 TCA ACT ATC CCC ATC ATT CCC ACA TCT ACC CTC Ser Ser Met Cly Ilie Ile Arg Thr Cys Arg Leu 30 -28- AAA TCG ATG CGG GCT GCT CTT GAT GTT TTG Lys Ser Met Arg Ala Ala Leu Asp

CAG

Gin Leu Phe GGT CGT GAG TTG GGA GAG Gly Arg Glu Phe Gly Asp GTA GCC Val Ala TTG GTC Leu Leu TAC TCC CAA Tyr Ser Gin GGG GAC TC Pro Asp Ser

CAT

Asp

CC

Ala CTG GGC AAC Leu Gly Asn GGT AGT AAG Arg Ser Lys

AGA

Thr 75 TTC ATC CG CTT Phe Ile Ala Leu

GCG

Ala

GCT

Ala 80

CTG

Leu TTC GAG GAA Phe Asp Gin

CAA

Giu GTT GTT GGC Val Val Gly CTC GGG GGT TAG Leu Ala Ala Tyr GGG AGC TTT Pro Arg Phe GAG GAG Glu Gin 100 CCC GT AGT Pro Arg Ser CG ACC GAG Arg Arg Gin 120 GCC AACGCC Ala Asn Aia

GAG

Ciu 105

GGG

Gly TAT ATC TAT Tyr Ile Tyr

GAT

Asp 110

GTG

Leu CA GTG TGG Ala Val Ser ATT CG AGG Ile Ala Thr

GG

Ala 125

CTG

Val1 ATG AAT GTG Tie Asn Leu

GTG

Leu 130

CA

Ala GGG GAG GAG Gly Glu His 115 AAG GAT GAG Lys His Giu GAT TACGGCT Asp Tyr Cly 1091 1139 1187 1235 1283 1331 1379 1427 1476 GTT CCT GGT Leu Cly Ala 135 CAC CAT Asp Asp 150

TAT

Tyr 140

GTG

Leu ATG TACGCTC Ile Tyr Val CCC CA CTC Pro Ala Val TAT ACA AAC Tyr Thr Lys

CAA

Gin 145

GC

Cly

ACC

Thr 177 ATA CCC CAA CAA Ile Arg Giu Clu 165 TAA CAATTGTTC CTC ATGCGAG TTT Val Met His Phe

CAT

Asp 170 GC GGA ACT Asp Pro Ser

ACC

Thr 175 AACCGACAT CGGTTGGGCC GAC CCC ATC AAGCGAG TTG GTT GC GAG 1526 Clu Gin Cly Met Lys Gin Phe Leu Asp Giu 255 260 AAA AC ATG AAC CCC CCC TTGCGAG ACC TAG AAC Lys Ser Ile Lys Pro Cly Leu Gin Thr Tyr Lys 265 270 275 GGGCCCGTCG CTGCGGGGG GGGGTTGGAA TAATGAGAAT TGACGTGGGG GTGGTTATTC CTCCTA.AC TTCTTCGCA CGCGTAC GCGCTGAGGG CAGAACTCCT ATCGCGCGC TCGCG GACTGCGC GTGGAGCCG CTCCCTCGTTTGGT CACGAACGGCCGGCGC TCGCGAC GCGATGTT CCC TCA TAAATCGCG Arg 276 AATCACGACT GGGGAATGTT TTGATCAC G CAGGTTGGA CGTGCA CTTTCGAACA GAATCCCGCGG GCGTTCTGG GTACAAGGC CTTTTCGA 1575 1635 1695 1755 1815 1875 -29 GTTCACCGCC GCAGCGAGTG AAACTGGCGC AGCGGGAAAC TGGTATGGGT TTAACGTTTA 1935 CCTGGCGGCG GGCATGTTGC GGGGAATTC 1964 FIG. 2k: 30 GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG GGTGCACG ATG AAT AGC TAC GAT GGC CGT TGG TCT ACC GTT GAT GTG AAG Met Asn Ser Tyr Asp Gly Arg Trp Ser Thr Val Asp Val Lys

GTT

Val1

AAC

Asn GAA GAA GGT ATC Giu Glu Gly Ile

GCT

Ala 20

ACT

Thr TGG GTC ACG Trp Val Thr CTC AAT CGA Leu Asn Arg CTG AAC Leu Asn GAG ATG Giu Met CGC CCG GAG AAG Arg Pro Giu Lys GCA ATG AGC Ala Met Ser GTC GAG GTT Vai Glu Val

CTG

Leu GTG CTG GAG Val Leu Giu GGC GAA TCC Gly Glu Ser ACC GAT GCT Thr Asp Ala

CAG

Gin

TGG

Trp

GGC

Gly GCA GAT GCT Ala Asp Ala GTG CTT GTT CTG Val Leu Val Leu CTG AAG GAG TAT Leu Lys Giu Tyr ACC GCG GGC Thr Ala Giy CCC GAA ATT Pro Giu Ile 85 TTC CTT GAC Phe Leu Asp

ATG

Met 70 ACT GGT GCA Thr Gly Ala TTC CGC GAG Phe Arg Giu CGC GAG CAG Arg Glu Gin 92 255 GGC TTG CAG Gly Leu Gin 270 482 530 CTG CAA GAG AAG Leu Gin Giu Lys ATT CGT Ile Arg 90 GGC ATG AAG CAG Gly Met Lys Gin 260 ACC TAC AAG CGC Thr Tyr Lys Arg 275 276 GAG AAA Giu Lys 265 AGC ATC AAG CCG Ser Ile Lys Pro TGA TAAATGCGCC GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA

TAATGACAAT

TTGTTCAGCA

ATCGCGCGTC

TGCGTTTCCT

GGCGGATCTT

AGCGGGAAAC

FIG. 21:

AATGAGGAGT

TCTGATGAGC

GCTGCTGCCA

GAATGGGCGG

CTAGAGGACC

TGGTATGGGT

GCCCAATGTT

GCACCTTCGA

GTTTGGAAGA

CGCTTGCTCC

GTTCTTCCGA

TTAACGTTTA

TCACGTGCCC

GCGTCGTAGC

TGCGGACGCC

GAGCGAACGC

GTTCACCGCC

CCTGGCGGCG

CTGCTTATTG

CCGCTGACCG

GCAGTGGCCG

CGTGCCCGAC

GCAGCGAGTG

GGCATGTTGC

GTGGTAAGCC

GAGAAGTGGT

CTGCACAGGC

TGCTGCGAGC

AAACTGGCGC

GGGGAATTC

-31 GAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATO TTT CAC GTG CCC CTC CTT Met Phe His Val Pro Leu Leu

ATT

Ile

CCT

Arg

TTG

Leu

GAA

Giu

GCG

Ala

AGT

Ser

CC

Ala

GGC

Gly 120

CGA

Arg

GTA

Val1

ACC

Thr

ATT

Ile

GGT

Gly

AGC

Ser

GAA

Glu

TGG

Trp

GCG

Ala

GAA

Glu

GCG

Ala 105

GAT

Asp

CAC

Gin

ATC

Ile

GTG

Val

GGT

Gly 185

GT

Gly

CCC

Pro

CAT

Asp

C

Ala

CAT

Asp

ACT

Thr

GC

Cly

GTC

Val1

CCA

Pro

CTT

Leu

CTC

Val 170

CAC

Gin

AAC

Lys

CTC

Leu

CC

Ala

C

Ala

CTT

Leu

GC

Cly

ATG

Met

ATT

Ile

TGT

Cys

GC

Cly 155

TTC

Leu

CTC

Val1 CCT TCT Pro Cys ACC GCA Thr Gly GAC GCC Asp Ala 45 CTT CCT Leu Ala CTA GAG Leu Glu CCA CC Ala Ala TTC CCC Leu Arg CCC TCC Pro Ser 125 CCC GTC Cly Val 140 CTA CCC Val Arg AAA AC Lys Ser TTG CAT Leu His

TCA

Ser

CAA

Clu 30

CCA

Ala

CCC

Pro

CAC

Asp

GCA

Cly

CAA

Clu 110

AAT

Asn

CTG

Val

GCT

Ala

TCT

Ser

CAT

Asp 190 CCA TCT Ala Ser 15 CTC CTA Val Val GT CC Val Ala ACC CAA Ser Clu CCT TCT Arg Ser 80 AAC TCC Asn Trp 95 CCC CC Ala Ala CTG CCC Val Pro CTC GCT Leu Cly CTT CC Val Ala 160 GAG CTG Glu Leu 175 CCT GGT Ala Cly

CAT

Asp

TCC

Ser

GCT

Ala

CC

Arg 65

TCC

Ser

TAT

Tyr

CC

Al a

GGT

Gly

ATT

Ile 145

ATG

Met

ACT

Ser

CTC

Leu GAG CCC ACC TTC GAG CCT Clu

CC

Arg

CCA

Ala 50

CCT

Arg

GAG

Clu

CCC

Cly

ATC

Met

AC

Ser 130

CC

Ala

CCC

Pro

CCC

Pro

CCC

Gly Arg Thr CTC CCT Val Ala CAC CCT Gin Ala CCC CGA Ala Arg TTC ACC Phe Thr TTT AAC Phe Asn 100 ACC ACA Thr Thr 115 TTT GCC Phe Ala CCT TGG Pro Trp TTC CCA Leu Ala TTT ACC Phe Thr 180 CAT GC Asp Gly 195 Phe

GCT

Ala

CC

Ala

CTC

Leu

CC

Ala

GTT

Val

CAC

Gin

ATC

Met

AAT

Asn

TC

Cys 165

CAT

His

CTC

Val1 Clu

CC

Ala

TTT

Phe

CTG

Leu

GCA

Ala

TAC

Tyr

ATT

Ile

CC

Ala

CCT

Ala 150

GC

Gly

CC

Arg

GTC

Val1 Arg

ACT

Ser

CCT

Pro

CGA

Arg

CC

Ala

CTG

Leu

CAC

Gin

GTT

Val 135

CCC

Pro

AAT

Asn

CTG

Leu

AAT

Asn 535 CTC ATC AGC AAT GCC CCC CAA CAC CCT CCT CC GTC CTC GAG CGA CTC Ile Ser Asn Ala Pro 205 Gin Asp Ala Pro Val Val Glu Arg Leu 215 -32- ATT GCA AAT CCT Ile Ala Asn Pro OTA CGT CGA Val Arg Arg GTO AAC Val Asn 225 TTC ACC GOT TCG Phe Thr Gly Ser ACC CAC Thr His 230 GTT GGA CGG Val Gly Arg GTG CTG GAA Val Leu Glu 250

ATC

Ile 235 ATT GOT GAG CTO Ile Gly Glu Leu

TCT

Ser 240 GCG CGT CAT CTG Ala Arg His Leu AAO CCT OCT Lys Pro Ala 245 GAC OAT 0CC Asp Asp Ala TTA GOT GOT AAO Leu Gly Gly Lys

OCT

Ala 255 CCG TTC TTG GTC Pro Phe Leu Val

TTG

Leu 260 GAC CTC Asp Leu 265 OAT GCG GCG Asp Ala Ala OTC GAA Val Glu 270 GCG GCG GCC TTT Ala Ala Ala Phe

GGT

Gly 275 0CC TAC TTC AAT Ala Tyr Phe Asn

CAG

Gin 280 GGT CAA ATC TGC Gly Gin Ile Cys

ATG

Met 285 TCC ACT GAG CGT Ser Thr Giu Arg ATT GTG ACA OCA Ile Val Thr Ala

OTC

Va1 295 OCA GAC 0CC TTT Ala Asp Ala Phe

OTT

Va1 300 GAA AAG CTG GCG Glu Lys Leu Ala

AGO

Arg 305 AAG GTC 0CC ACA CTG CGT Lys Val Ala Thr Leu Arg 310 OCT GOC OAT Ala Gly Asp 0CC AAT OCA Ala Asn Ala 330 AAT OAT CCG CAA Asn Asp Pro Gin

TCG

Ser 320 OTC TTO GGT Val Leu Gly TC0 TTG ATT OAT Ser Leu Ile Asp 325 OAT GCG CTC GGG Asp Ala Leu 340 342 GGT CAA COC ATC Gly Gin Arg Ile OTT CTG GTC OAT Val Leu Val Asp OACAGCAAOC OAACCGOAAT TGCCAGCTGG GGCOCCCTCT OGTAAGGTTG OGAAOCCCTO CAAAGTAAAC TGGATGGCTT TCTTOCCGCC AAGGATCTOA TGGCGCAGOG OATCAAGATC TGATCAAGAG ACAGGATOAG GATCOTTTCG C ATO ATT OAA CAA OAT OGA TTG Met Ile Glu Gin Asp Gly Leu 1015 1063 1123 1183 1235 1283 1331 1379 1427 CAC OCA GGT His Ala Oly TCT CCG GCC OCT Ser Pro Ala Ala

TGG

Trp 15 GTG GAG AGO CTA TTC GOC TAT GAC Val Glu Arg Leu Phe Gly Tyr Asp TGG GCA Trp Ala CAA CAG ACA ATC Gin Gin Thr Ile TGC TCT OAT 0CC Cys Ser Asp Ala GTG TTC COG CTG Val Phe Arg Leu TCA GCG CAG GGG COC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT Ser Ala Gin Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly 45 50 0CC CTO AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TOG CTG 0CC Ala Leu Asn Olu Leu Gin Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala 65 -33- ACG ACG GGC Thr Thr Gly GGA AGG GAC Gly Arg Asp CCT TGC GCA GCT Pro Cys Ala Ala

GTG

Val CTC GAC GTT GTC Leu Asp Val Val ACT GAA GCG Thr Glu Ala GAT CTC CTG Asp Leu Leu TGG CTG CTA TTG Trp Leu Leu Leu GAA GTG CCG GGG Glu Val Pro Gly

CAG

Gin 100 TCA TCT Ser Ser 105 CAC CTT GCT CCT His Leu Ala Pro

GCC

Ala 110 GAG AAA GTA TCC Glu Lys Val Ser

ATC

Ile 115 ATG GCT GAT GCA Met Ala Asp Ala

ATG

Met 120 CGG CGG CTG CAT Arg Arg Leu His CTT GAT CCG GCT Leu Asp Pro Ala

ACC

Thr 130 TGC CCA TTC GAC Cys Pro Phe Asp CAA GCG AAA CAT Gin Ala Lys His

CGC

Arg 140 ATC GAG CGA GCA Ile Glu Arg Ala ACT CGG ATG GAA Thr Arg Met Glu GCC GGT Ala Gly 150 CTT GTC GAT Leu Val Asp GCC GAA CTG Ala Glu Leu 170

CAG

Gin 155 GAT GAT CTG GAC Asp Asp Leu Asp GAG CAT CAG GGG Glu His Gin Gly CTC GCG CCA Leu Ala Pro 165 GGC GAG GAT Gly Glu Asp TTC GCC AGG CTC Phe Ala Arg Leu

AAG

Lys 175 GCG CGC ATG CCC Ala Arg Met Pro

GAC

Asp 180 1475 1523 1571 1619 1667 1715 1763 1811 1859 1907 1955 2003 2057 2105 CTC GTC Leu Val 185 GTG ACC CAT GGC Val Thr His Gly GCC TGC TTG CCG Ala Cys Leu Pro

AAT

Asn 195 ATC ATG GTG GAA Ile Met Val Glu

AAT

Asn 200 GGC CGC TTT TCT Gly Arg Phe Ser TTC ATC GAC TGT Phe Ile Asp Cys CGG CTG GGT GTG Arg Leu Gly Val

GCG

Ala 215 GAC CGC TAT CAG Asp Arg Tyr Gin

GAC

Asp 220 ATA GCG TTG GCT Ile Ala Leu Ala

ACC

Thr 225 CGT GAT ATT GCT Arg Asp Ile Ala GAA GAG Glu Glu 230 CTT GGC GGC Leu Gly Gly GCT CCC GAT Ala Pro Asp 250 TGG GCT GAC CGC Trp Ala Asp Arg

TTC

Phe 240 CTC GTG CTT TAC Leu Val Leu Tyr TCG CAG CGC ATC Ser Gin Arg Ile TTC TAT CGC CTT Phe Tyr Arg Leu

CTT

Leu 260 GGT ATC GCC Gly Ile Ala 245 GAC GAG TTC Asp Glu Phe CG GCC CAG Ala Gin 421 GTG CAT GAC Val His Asp TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC Phe 264 CGC GTC GAT TCG GGC ATT TGC CAT ATC AAT GGA CCG ACT Arg Val Asp Ser Gly Ile Cys His Ile Asn Gly Pro Thr 425 430 435 -34- GAG GCT CAG ATG CCA TTC GGT GGG GTG AAG TCC AGC GGC TAC GGC AGC Giu Ala Gin Met Pro Phe Gly Gly Val Lys Ser Ser Gly Tyr Gly Ser 440 445 450 TTC GGC AGT CGA GCA TCG ATT GAG CAC TTT ACC CAG CTG CGC TGG CTG Phe Gly Ser Arg Ala Ser Ile Giu His Phe Thr Gin Leu Arg Trp Leu 455 460 465 470 ACC ATT CAG AAT GGC CCG CGG CAC TAT CCA ATC TAA ATCGATCTTC Thr Ile Gin Asn Gly Pro Arg His Tyr Pro Ile 475 480 481 2153 2201 2247 2307 2367 2427 2487 2539

GGGCGCCGCG

ACAGAGCTGT

AGTACAGTGA

CGCTACCGCA

ATTGTCCGGA

FIG. 2m:

GGCATCATGC

TCTCCGGTCT

ACGCCGAGTC

GTGTGTCGAT

TGCGTTCTCT

CCGCGGCGCT

TGGTGGATCA

CACATTGCAA

TGGTCATCCT

CGAGGCGCTT

CGCCTCATTT

AGGCCAGTCG

CCGCAGGCAT

CCGGTTGAGG

CTTCCCTTCC

CAATCTCTAA

CGGAGAGTCT

CATCATGCTC

TTACGCAAGA

CGGGTGGAAT

CTTGATAAAA

CGAAGAGGAG

TGCTCAGCCA

CGCTGGAGGT

TC

GAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATG TTT CAC GTG CCC CTG CTT Met Phe His Val Pro Leu Leu ATT GGT GGT Ile Gly Gly AAG CCT TGT TCA Lys Pro Cys Ser TCT GAT GAG CGC Ser Asp Glu Arg

ACC

Thr TTC GAG CGT Phe Glu Arg CGT AGC Arg Ser CCG CTG ACC GGA Pro Leu Thr Gly GTG GTA TCG CGC Val Val Ser Arg GCT GCT CCC AGT Ala Ala Ala Ser

TTG

Leu GAA GAT GCG GAC GCC GCA GTG GCC GCT Glu Asp Ala Asp Ala Ala Val Ala Ala CAG GCT GCG TTT Gin Ala Ala Phe 199 247 CAA TGG GCG GCG Glu Trp Ala Ala GCT CCG AGC GAA Ala Pro Ser Glu

CGC

Arg CGT GCC CGA CTG Arg Ala Arg Leu CTG CGA Leu Arg GCG GCG GAT Ala Ala Asp AGT GAA ACT Ser Glu Thr CTA GAG GAC CGT Leu Glu Asp Arg TCC GAG TTC ACC Ser Giu Phe Thr GCC GCA GCG Ala Ala Ala GTT TAC CTG Val Tyr Leu GGC GCA GCG GGA Gly Ala Ala Gly

AAC

Asn TGG TAT GCG TTT Trp Tyr Gly Phe GCC GCG Ala Ala 105 GGC ATG TTG CGG Gly Met Leu Arg

GAA

Glu 110 GCC GCC GCC ATG Ala Ala Ala Met

ACC

Thr 115 ACA CAG ATT CAG Thr Gin Ile Gin GAT GTC ATT CCG Asp Val Ile Pro AAT GTG CCC GGT Asn Val Pro Cly TTT GCC ATG GCG Phe Ala Met Ala

GTT

Va1 135 CGA CAG CCA TGT Arg Gin Pro Cys

GGC

Gly 140 GTG GTG CTC GGT Val Val Leu Cly GCG CCT TGG AAT Ala Pro Trp Asn CCT CCG Ala Pro 150 GTA ATC CTT Val Ile Leu ACC GTG GTG Thr Val Val 170

GGC

Cly 155 GTA CGG CCT GTT Val Arg Ala Val

GCG

Ala 160 ATG CCG TTG CCA Met Pro Leu Ala TGC CGC AAT Cys Gly Asn 165 CAT CGC CTG His Arg Leu TTG AAA AGC TCT Leu Lys Ser Ser CTG ACT CCC TTT Leu Ser Pro Phe

ACC

Thr 180 ATT GGT Ile Cly 185 CAG GTG TTG CAT Cln Val Leu His CCT GGT CTG GGG Ala Cly Leu Cly

GAT

Asp 195 GGC GTG GTG AAT Cly Val Val Asn ATC ACC AAT GCC Ile Ser Asn Ala

CCC

Pro 205 CAA CAC GCT CCT Gin Asp Ala Pro

CCG

Ala 210 CTG CTG GAG CGA Val Val Glu Arg 679 -36- ATT GCA AAT CCT Ile Ala Asn Pro GTA COT CGA GTG Val Arg Arg Val

AAC

Asn 225

GCG

Ala TTC ACC GOT TCG Phe Thr Gly Ser ACC CAC Thr His 230 727 775 GTT OGA CGG Val Oly Arg GTG CTC GAA Val Leu Clu 250 CAC CTC OAT Asp Leu Asp

ATC

Ile 235

TTA

Leu GGT GAG CTG Gly Giu Leu

TCT

Ser 240

CCC

Pro CGT CAT CTO Arg His Leu GGT GOT AAC Gly Oly Lys

OCT

Ala 255

GCG

Ala TTC TTG GTC Phe Leu Val

TTO

Leu 260 0CC Ala AAO CCT OCT Lys Pro Ala 245 GAC OAT 0CC Asp Asp Ala TAC TTC AAT Tyr Phe Asn GCG GCG GTC Ala Ala Val

OAA

Glu 270 GCG 0CC TTT Ala Ala Phe

GGT

Oly 275 265 CAG GGT Gin Oly CAA ATC TGC Gin Ile Cys 280

OCA

Ala

ATG

Met 285

GAA

Glu TCC ACT GAO CGT Ser Thr Giu Arg ATT CTG ACA GCA Ile Val Thr Ala

GTC

Val 295 GAC CCC TTT Asp Ala Phe

GTT

Val 300

AAT

Asn AAG CTG GCG Lys Leu Ala

AGO

Arg 305

GTC

Val GTC CCC ACA Val Ala Thr CTC CGT Leu Arg 310 ATT OAT Ile Asp OCT GGC OAT Ala Cly Asp GCC AAT OCA Ala Asn Ala 330 CAT CCC CAA Asp Pro Gin

TCC

Ser 320 TTG GGT TCC Leu Cly Ser GGT CAA CCC ATC Gly Gin Arg Ile CTGGGGAGAG GCGGTTTGCG TATTGGGCGC

ATGCATAAAA

GCATCATCAA

CCCATGGACG

CGGTTCGTAA

CCGAACGCAG

TTCTACACTC

TGATCTTATG

ACTGTTGTAA

CCTGAATCGC

CACACCGTGG

ACTGTAATGC

CGGTCGTAAC

TATCCCTCGG

GACCAGCAAC

TTCATTAACC ATTCTGCCGA CAGCGGCATC ACCACCTTGT AAACCGATCA ACGCACGAAC AAGTACCGTA TGCGCTCACC GGCGCAGTGG CGGTTTTCAT GCATCCAACC AGCAAGCGCC C ATO TTA CCC ACC AGC Met Leu Arg Ser Ser CATGCAACCC ATCACAAACC CGCCTTGCGT ATAATATTTC CCACTTCACA TAACCCTCTT CAACTCGTCC ACAACCTTGA GGCTTGTTAT CACTGTTTTT TTACGCCGTG GGTCCATOTT AAC CAT OTT ACO CAG Asn Asp Val Thr Gin TCA ACT ATG GGC ATC Ser Ser Met Cly Ile AAA TCC ATC CGG GCT Lys Ser Met Arg Ala 871 919 967 1015 1069 1129 1189 1249 1309 1369 1429 1480 1528 1576 CAG GGC ACT CCC CCT AAA ACA AAC TTA GGT GGC Cmn Gly Ser Arg Pro Lys Thr Lys Leu Cly Cly 20 ATT CCC ACA TOT AGO CTC GGC CCT CAC CAA CTC Ile Arg Thr Cys Arg Leu Cly Pro Asp Cmn Val 35 -37- GCT CTT GAT CTT TTC GGT CGT GAG TTC GGA GAC GTA GCC ACC TAC TCC 1624 Ala Leu Asp Leu Phe Gly Arg Glu Phe Gly Asp Val Ala Thr Tyr Ser 50 CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG 1672 Gin His Gin Pro Asp Ser Asp Tyr Leu Gly Asn Leu Leu Arg Ser Lys 65 ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT 1720 Thr Phe Ile Ala Leu Ala Ala Phe Asp Gin Glu Ala Val Val Gly Ala 80 85 CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC 1768 Leu Ala Ala Tyr Val Leu Pro Arg Phe Glu Gin Pro Arg Ser Glu Ile 100 105 TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT 1816 Tyr Ile Tyr Asp Leu Ala Val Ser Gly Glu His Arg Arg Gin Gly Ile 110 115 120 GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT 1864 Ala Thr Ala Leu Ile Asn Leu Leu Lys His Glu Ala Asn Ala Leu Gly 125 130 135 GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG 1912 Ala Tyr Val Ile Tyr Val Gin Ala Asp Tyr Gly Asp Asp Pro Ala Val 140 145 150 GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT 1960 Ala Leu Tyr Thr Lys Leu Gly Ile Arg Glu Glu Val Met His Phe Asp 155 160 165 170 ATC GAC CCA AGT ACC GCC ACC TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCA 2014 Ile Asp Pro Ser Thr Ala Thr 175 177 A TTG GCC CAG CGC GTC GAT TCG GGC ATT TGC CAT ATC AAT GGA CCG ACT 2063 Leu Ala Gin Arg Val Asp Ser Gly Ile Cys His Ile Asn Gly Pro Thr 420 425 430 435 GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT GGG GTG AAG TCC AGC GGC 2111 Val His Asp Glu Ala Gin Met Pro Phe Gly Gly Val Lys Ser Ser Gly 440 445 450 TAC GGC AGC TTC GGC AGT CGA GCA TCG ATT GAG CAC TTT ACC CAG CTG 2159 Tyr Gly Ser Phe Gly Ser Arg Ala Ser Ile Glu His Phe Thr Gin Leu 455 460 465 CGC TGG CTG ACC ATT CAG AAT GGC CCG CGG CAC TAT CCA ATC TAA 2204 Arg Trp Leu Thr Ile Gin Asn Gly Pro Arg His Tyr Pro Ile 470 475 480 481 ATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT CGCCTCATTT CAATCTCTAA 2264 CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA AGGCCAGTCG CGGAGAGTCT 2324 -38- CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA CCGCAGGCAT CATCATGCTC 2384 TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT CCGGTTGAGG TTACGCAAGA 2444 CGCTGGAGGT ATTGTCCGGA TCCGTTCTCT CGAGGCGCTT CTTCCCTTCC CGGGTGGAAT 2504 TC 2506 FIG. 2n: -39- GAATTCCAAT AATGACAATA ATGAGGAGTG CCCA ATG TTT CAC GTG CCC CTG CTT Met Phe His Val Pro Leu Leu ATT GGT GGT Ile Gly Gly AAG CCT TGT TCA Lys Pro Cys Ser TCT GAT GAG CGC Ser Asp Glu Arg

ACC

Thr TTC GAG CGT Phe Clu Arg CGT AGC Arg Ser CCG CTG ACC GGA Pro Leu Thr Gly GTG OTA TCG CGC Val Val Ser Arg GCT GCT GCC AGT Ala Ala Ala Ser

TTG

Leu GAA GAT CCG GAC Glu Asp Ala Asp

GCC

Ala 45 GCA GTC GCC GCT Ala Val Ala Ala

GCA

Ala CAG OCT GCG TTT Gin Ala Ala Phe 199 247 GAA TGG GCG GCG Glu Trp Ala Ala GCT CCG AGC GAA Ala Pro Ser Glu

CGC

Arg CGT GCC CGA CTG Arg Ala Arg Leu CTG CGA Leu Arg GCG GCG GAT Ala Ala Asp AGT CAA ACT Ser Glu Thr

CTT

Leu CTA GAG GAC CGT Leu Glu Asp Arg TCC GAG TTC ACC Ser Glu Phe Thr CCC GCA GCG Ala Ala Ala GTT TAC CTG Val Tyr Leu GGC GCA GCG GGA Cly Ala Ala Cly

AAC

Asn TGG TAT GGG TTT Trp Tyr Cly Phe GCG GCG Ala Ala 105 GGC ATC TTC CGC Cly Met Leu Arg

CAA

Clu 110 CCC GCG GCC ATG Ala Ala Ala Met

ACC

Thr 115 ACA CAC ATT CAC Thr Gin Ile Gin

GGC

Cly 120 CAT GTC ATT CCC Asp Val Ile Pro AAT GTC CCC GGT Asn Val Pro Cly TTT CCC ATG GCG Phe Ala Met Ala

GTT

Va1 135 CGA CAC CCA TGT Arg Gin Pro Cys

GGC

Gly 140 GTG GTC CTC CGT Val Val Leu Cly GCG CCT TGG AAT Ala Pro Trp Asn CCT CCC Ala Pro 150 CTA ATC CTT Val Ile Leu ACC GTG GTC Thr Val Val 170

GGC

Cly 155 GTA CGG GCT GTT Val Arg Ala Val

GCG

Ala 160 ATG CCC TTC GCA Met Pro Leu Ala TGC GGC AAT Cys Gly Asn 165 CAT CCC CTC His Arg Leu TTC AAA ACC TCT Leu Lys Ser Ser

GAG

Clu 175 CTG ACT CCC TTT Leu Ser Pro Phe

ACC

Thr 180 ATT GGT Ile Cly 185 CAG GTG TTC CAT Gin Val Leu His CCT CGT CTG GGG Ala Cly Leu Cly GGC GTG GTG AAT Cly Val Val Asn

CTC

Va1 200 ATC ACC AAT GCC CCC CAA CAC CCT CCT GCG GTG GTG GAG CGA Ile Ser Asn Ala Pro Gin Asp Ala Pro Ala Val Val Clu Arg CTG 679 Leu 215 ATT GCA AAT CCT GCG GTA CGT CGA GTG AAC TTC ACC GGT TCG ACC CAC 727 Ile Ala Asn Pro Ala Val Arg Arg Val Asn Phe Thr Gly Ser Thr His 220 225 230 GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775 Val Gly Arg Ile Ile Gly Glu Leu Ser Ala Arg His Leu Lys Pro Ala 235 240 245 GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823 Val Leu Glu Leu Gly Gly Lys Ala Pro Phe Leu Val Leu Asp Asp Ala 250 255 260 GAC CTC GAT GCG GCG GTC GAA GCG GCG GCC TTT GGT GCC TAC TTC AAT 871 Asp Leu Asp Ala Ala Val Glu Ala Ala Ala Phe Gly Ala Tyr Phe Asn 265 270 275 CAG GGT CAA ATC TGC ATG TCC ACT GAG CGT CTG ATT GTG ACA GCA GTC 919 Gin Gly Gin Ile Cys Met Ser Thr Glu Arg Leu Ile Val Thr Ala Val 280 285 290 295 GCA GAC GCC TTT GTT GAA AAG CTG GCG AGG AAG GTC GCC ACA CTG CGT 967 Ala Asp Ala Phe Val Glu Lys Leu Ala Arg Lys Val Ala Thr Leu Arg 300 305 310 GCT GGC GAT CCT AAT GAT CCG CAA TCG GTC TTG GGT TCG TTG ATT GAT 1015 Ala Gly Asp Pro Asn Asp Pro Gin Ser Val Leu Gly Ser Leu Ile Asp 315 320 325 GCC AAT GCA GGT CAA CGC ATC CAG GTT CTG GTC GAT GAT GCG CTC GCA 1063 Ala Asn Ala Gly Gin Arg Ile Gin Val Leu Val Asp Asp Ala Leu Ala 330 335 340 AAA GGC GCG CAATGGAA TTG GCC CAG CGC GTC GAT TCG GGC ATT TGC CAT 1113 Lys Gly Ala Leu Ala Gin Arg Val Asp Ser Gly Ile Cys His 345 346 420 425 430 ATC AAT GGA CCG ACT GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT GGG 1161 Ile Asn Gly Pro Thr Val His Asp Glu Ala Gin Met Pro Phe Gly Gly 435 440 445 GTG AAG TCC AGC GGC TAC GGC AGC TTC GGC AGT CGA GCA TCG ATT GAG 1209 Val Lys Ser Ser Gly Tyr Gly Ser Phe Gly Ser Arg Ala Ser Ile Glu 450 455 460 CAC TTT ACC CAG CTG CGC TGG CTG ACC ATT CAG AAT GGC CCG CGG CAC 1257 His Phe Thr Gin Leu Arg Trp Leu Thr Ile Gin Asn Gly Pro Arg His 465 470 475 TAT CCA ATC TAA ATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT 1309 Tyr Pro Ile 480 481 CGCCTCATTT CAATCTCTAA CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA 1369 AGGCCAGTCG CGGAGAGTCT CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA 1429 -41- CCGCAGGCAT CATCATGCTC TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT 1489 CCGGTTGAGG TTACGCAAGA CGCTGGAGGT ATTGTCCGGA TCCGTTCTCT CGAGGCGCTT 1549 CTTCCCTTCC CGGGTGGAAT TC 1571 FIG. 2o: -42- GAATTCCGCG GTCGGCGAAA GTTGATGCOC TGTATCGTGG TGAAGATCAA TCCATGCTGC GTGACGAGGC CACACT GTO AGT TGG .TCA GGG GGG GCT TAC TCG GCG TTT TCC Met Ser Trp Ser Gly Gly Ala Tyr Ser Ala Phe Ser 1 5 GAC ACT GCG TTG GTT GCG GCA Asp Thr Ala Leu Val Ala Ala

GTG

Va1 CGC ACC CCC TGO Arg Thr Pro Trp

ATT

Ile GAT TGC GGG Asp Cys Gly 160 208 GGT GCC Gly Ala CTG TCO CTG GTO Leu Ser Leu Val CCT ATC GAC TTA Pro Ile Asp Leu

GGG

Gly GTA AAG OTC OCT Val Lys Val Ala

CGC

Arg GAA OTT CTO ATO Olu Val Leu Met

CGT

Arg 50 GCG TCO CTT OAA Ala Ser Leu Glu CAA ATG GTC OAT Gin Met Val Asp

AGC

Ser OTA CTC OCA GGC Val Leu Ala Oly ATG GCT CAA OCA Met Ala Gin Ala

AGC

Ser TTT OAT OCT TAC Phe Asp Ala Tyr CTO CTC Leu Leu CCG CGG CAC Pro Arg His TTG GGG GTG Leu Oly Vai GGC TTO TAC AGC Oly Leu Tyr Ser OTT CCC AAO TCO Val Pro Lys Ser OTT CCG GCC Val Pro Ala CTT CGG CAG Leu Arg Gin CAG COC ATT TGC Gin Arg Ile Cys

GGC

Oly 100 ACA GOC TTC OAA Thr Oly Phe Olu

CTO

Leu 105 GCC GGC Ala Gly 110 GAG CAG ATT TCC CAA OGC OCT OAT CAC Glu Gin Ile Ser Gin Oly Ala Asp His 115

GTG

Va1 120 CTO TOT OTC GCG Leu Cys Val Ala GAG TCC ATO TCG Glu Ser Met Ser AAC CCC ATC GCG Asn Pro Ile Ala

TCG

Ser 135 TAT ACA CAC CGG Tyr Thr His Arg GGG TTC COC CTC Gly Phe Arg Leu GCG CCC OTT GAG Ala Pro Val Glu AAG GAT TTT TTG Lys Asp Phe Leu TGG GAO Trp Glu 155 OCA TTO TTT OAT CCT OCT CCA OGA CTC GAC ATO ATC OCT Ala Leu Phe Asp Pro Ala Pro Gly Leu Asp Met Ile Ala ACC OCA OAA Thr Ala Glu 170 AAC CTG GGGACAOCAA GCGAACCGGA ATTOCCAOCT GGGGCGCCCT CTGGTAAGGT Asn Leu 174 TGGGAAOCCC TGCAAAGTAA ACTOGATGGC TTTCTTGCCO CCAAGGATCT GATGGCGCAC GGGATCAAGA TCTOATCAAO AGACAGOATO AGGATCOTTT COC ATO ATT GAA CAA Met Ile Giu Gin 1 -43-

GAT

Asp GGA TTG CAC GCA Gly Leu His Ala TCT CCG GCC GCT Ser Pro Ala Ala GTG GAG AGG CTA Val Glu Arg Leu GGC TAT GAC TGG Gly Tyr Asp Trp

GCA

Ala CAA CAG ACA ATC Gin Gin Thr Ile

GGC

Gly 30 TGC TCT GAT GCC Cys Ser Asp Ala GCC GTG Ala Val TTC CGG CTG Phe Arg Leu CTG TCC GGT Leu Ser Gly

TCA

Ser GCG CAG GGG CGC Ala Gin Gly Arg

CCG

Pro 45 GTT CTT TTT GTC Val Leu Phe Val AAG ACC GAC Lys Thr Asp CGG CTA TCG Arg Leu Ser GCC CTG AAT GAA Ala Leu Asn Glu CAG GAC GAG GCA Gin Asp Glu Ala

GCG

Ala TGG CTG Trp Leu GCC ACG ACG GGC Ala Thr Thr Gly CCT TGC GCA GCT Pro Cys Ala Ala CTC GAC GTT GTC Leu Asp Val Val

ACT

Thr GAA GCG GGA AGG Glu Ala Gly Arg TGG CTG CTA TTG Trp Leu Leu Leu

GGC

Gly 95 GAA GTG CCG GGG Glu Val Pro Gly

CAG

Gin 100 GAT CTC CTG TCA Asp Leu Leu Ser

TCT

Ser 105 CAC CTT GCT CCT His Leu Ala Pro

GCC

Ala 110 GAG AAA GTA TCC Glu Lys Val Ser ATC ATG Ile Met 115 GCT GAT GCA Ala Asp Ala TTC GAC CAC Phe Asp His 135

ATG

Met 120 CGG CGG CTG CAT Arg Arg Leu His

ACG

Thr 125 CTT GAT CCG GCT Leu Asp Pro Ala ACC TGC CCA Thr Cys Pro 130 ACT CGG ATG Thr Arg Met 859 907 955 1003 1051 1099 1147 1195 1243 1291 1339 1387 1435 CAA GCG AAA CAT Gin Ala Lys His ATC GAG CGA GCA Ile Glu Arg Ala

CGT

Arg 145 GAA GCC Glu Ala 150 GGT CTT GTC GAT Gly Leu Val Asp GAT GAT CTG GAC Asp Asp Leu Asp GAG CAT CAG GGG Glu His Gin Gly GCG CCA GCC GAA Ala Pro Ala Glu

CTG

Leu 170 TTC GCC AGG CTC Phe Ala Arg Leu

AAG

Lys 175 GCG CGC ATG CCC Ala Arg Met Pro

GAC

Asp 180 GGC GAG GAT CTC Gly Glu Asp Leu

GTC

Val 185 GTG ACC CAT GGC Val Thr His Gly

GAT

Asp 190 GCC TGC TTG CCG Ala Cys Leu Pro AAT ATC Asn Ile 195 ATG GTG GAA Met Val Glu GGT GTG GCG Gly Val Ala 215

AAT

Asn 200 GGC CGC TTT TCT Gly Arg Phe Ser

GGA

Gly 205 TTC ATC GAC TGT Phe Ile Asp Cys GGC CGG CTG Gly Arg Leu 210 CGT GAT ATT Arg Asp Ile GAC CGC TAT CAG Asp Arg Tyr Gln GAC ATA GCG TTG GCT ACC Asp Ile Ala Leu Ala Thr 220 225 -44- GCT GAA Ala Glu 230 GAG CTT GGC GGC Glu Leu Gly Gly TGG GCT GAC Trp Ala Asp CGC TTC CTC GTG CTT TAC Arg Phe Leu Val Leu Tyr 240 GCC TTC TAT CGC CTT CTT Ala Phe Tyr Arg Leu Leu 255 260

GGT

Gly 245 ATC GCC GCT CCC Ile Ala Ala Pro

GAT

Asp 250 TCG CAG CGC ATC Ser Gin Arg Ile GAC GAG TTC TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC Asp Glu Phe Phe 264 CA TTG AGG GCG CAA GAG GAG AAA TGG ATT GAC CAA GAG ATC GTG GCT Leu Arg Ala Gin Glu Glu Lys Trp Ile Asp Gin Glu Ile Val Ala GTT ACG GAT Val Thr Asp GAA CTG CCT Glu Leu Pro 230

GAA

Glu 215 CAG TTC GAT TTA Gin Phe Asp Leu

GAG

Glu 220 GGC TAC AAC AGT Gly Tyr Asn Ser CGA GCA ATT Arg Ala Ile 225 ATC CGC GGC Ile Arg Gly CGG AAG GCA AAA Arg Lys Ala Lys TTG ATC GTG ACA Leu Ile Val Thr

GTC

Val 240 CTA GCA Leu Ala 245 GTC TTT GAA GCC Val Phe Glu Ala

CTT

Leu 250 TCC CGA TTG AAG Ser Arg Leu Lys GTT CAT TCT GGC Val His Ser Gly 1483 1531 1586 1633 1681 1729 1777 1825 1873 1921 1969 2017 2065 2113

GGG

Gly 260 GTG CAG ACT GCG Val Gin Thr Ala

GGC

Gly 265 AAC AGC TGT GCC Asn Ser Cys Ala

GTA

Val 270 GTG GAC GGC GCC Val Asp Gly Ala

GCG

Ala 275 GCG GCT TTG GTG Ala Ala Leu Val

GCT

Ala 280 CGA GAG TCG TCT Arg Glu Ser Ser

GCG

Ala 285 ACA CAG CCG GTC Thr Gin Pro Val TTG GCT Leu Ala 290 AGG ATA CTG Arg Ile Leu CTC GGC CCT Leu Gly Pro 310

GCT

Ala 295 ACC TCC GTA GTC Thr Ser Val Val ATC GAG CCC GAG Ile Glu Pro Glu CAT ATG GGG His Met Gly 305 AGT GAT CTT Ser Asp Leu GCG CCC GCG ATT Ala Pro Ala Ile CTG CTG CTT GCG Leu Leu Leu Ala

CGT

Arg 320 AGT TTG Ser Leu 325 AGG GAT ATC GAC Arg Asp Ile Asp

CTC

Leu 330 TTT GAG ATA AAC Phe Glu Ile Asn

GAG

Glu 335 GCG CAG GCC GCC Ala Gin Ala Ala

CAA

Gin 340 GTT CTA GCG GTA Val Leu Ala Val CAT GAA TTG GGT His Glu Leu Gly GAG CAC TCA AAA Glu His Ser Lys AAT ATT TGG GGC Asn Ile Trp Gly

GGG

Gly 360 GCC ATT GCA CTT Ala Ile Ala Leu GGA CAC Gly His 365 CCG CTT GCC Pro Leu Ala GCG ACC Ala Thr 370 GGA TTG CGT CTC TGC ATG ACC CTC GCT CAC CAA Gly Leu Arg Leu Cys Met Thr Leu Ala His Gin 375 380 TTT CGA TAT GGA ATT CCC TCG GCA TGC ATT GGT Phe Arg Tyr Gly Ile Ala Ser Ala Cys Ile Gly 390 395 GCG GTT CTT TTA GAG AAT CCC CAC TTC GGT TCG Ala Val Leu Leu Giu Asn Pro His Phe Gly Ser 405 410 TCG ATG ATT AAC AGA GTT GAC CAC TAT CCA CTG Ser Met Ile Asn Arg Val Asp His Tyr Pro Leu 420 425 430 CTTTGTTGCT TTGAGGTGGC GCACGAAGGA GGGCTCGAAA GAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAACTTGCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TTCTACAGTG TACATACCTT GTCAGGGTTG GTGGGAATTC FIG. 2p: TTG CAA GCT AAT AAC Leu Gin Ala Asn Asn 385 GGG GGA CAG GGG ATG Gly Giy Gin Gly Met 400 TCC TCT GCA CGA AGT Ser Ser Ala Arg Ser 415 AGC TAA CGGGCATCTC Ser 431 ATCTCTGCTA AAAACAAGAA AAAGTTTAGG AGTCCAGGCT TGATTGTTAC CGGCACGGGT 2161 2209 2257 2306 2366 2426 2486 2526 -46 GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT TAC TCG GCG TTT TCC Met Ser Trp, Ser Gly Gly Ala Tyr Ser Ala Phe Ser

GAC

Asp

GGT

Cly

CGC

Arg

GTA

Val1

CCG

Pro

TTG

Leu

GCC

Ala

GCA

Ala 125

GGG

Gly

GCA

ACT GCG Thr Ala GCC CTG Ala Leu GAA GTT Clu Val CTC GCA Leu Ala CGG CAC Arg His GGG GTC Gly Val GGC GAG Gly Clu 110 GAG TCC Clu Ser TTC CC Phe Arg TTC TTT

TTG

Leu

TCG

Ser

CTC

Leu

GGC

Gly

ATT

Ile

CAG

Gin

CAG

Gln

ATG

Met

CTC

Leu

CAT

GTT GCG GCA CTC CCC ACC CCC TCC ATT CAT TCC CCC Val

CTC

Leu

ATG

Met

TCT

Ser

GGC

Gly

CGC

Arg

ATT

I le

TCC

Ser

CCT

Gly 145

CCT

Ala

GTG

Val

CGT

Arg 50

ATG

Met

TTG

Leu

ATT

Ile

TCC

Ser

CCT

Arg 130

GCG

Ala

GCT

Ala

TCG

Ser 35

GCG

Ala

GCT

Ala

TAC

Tyr

TC

Cys

CAA

Gin 115

AAC

Asn

CCC

Pro

CCA

Val1 20

CCT

Pro

TCC

Ser

CAA

Gin

AGC

Ser

GC

Gly 100

GC

Gly

CCC

Pro

GTT

Val

GGA

Arg

ATC

Ile

CTT

Leu

CCA

Ala

CGT

Gly 85

ACA

Thr

GCT

Al a

ATC

Ile

GAG

Glu

CTC

Thr

GAC

Asp

GAA

Glu

AGC

Ser 70

GTT

Val1

GGC

Gly

GAT

Asp

GCG

Ala

TTC

Phe 150

GAC

Pro

TTA

Leu

CCA

Pro 55

TTT

Phe

CCC

Pro

TTC

Phe

CAC

His

TCG

Ser 135

AAG

Lys

ATG

Trp Ile GGG GTA Gly Val CAA ATG Gin Met GAT GCT Asp Ala AAG TCG Lys Ser GAA CTG Glu Leu 105 GTG CTG Val Leu 120 TAT ACA Tyr Thr CAT TTT Asp Phe ATC GCT Ile Ala Asp

AAG

Lys

GTC

Val

TAC

Tyr

GTT

Val1

CTT

Leu

TGT

Cys

CAC

His

TTG

Leu

ACC

Cys Gly GTC CCT Val Ala CAT AGC Asp Ser CTG CTC Leu Leu CCG CC Pro Ala CGG CAG Arg Gin GTC GCG Val Ala CGC GGC Arg Gly 140 TGG GAG Trp Ciu 155 CCA CAA 160 208 256 304 352 544 Ala Leu Phe Asp Pro Ala Pro Cly Leu Asp Met Thr Ala Ciu 170 AAC CTG GGGGAGAGGC GGTTTGCGTA TTGGGCGCAT CCATAAAAAC TCTTCTAATT Asn Leu 174 CATTAACCAT TCTGCCCACA TCCAAGCCAT CACAAACCCC ATCATCAACC TCAATCCCCA GCGGCATCAC CACCTTGTCG CCTTGCCTAT AATATTTCCC CATGGACGCA CACCCTCCAA ACCCATGAAC CCACCAACCC AGTTGACATA AGCCTGTTCC CTTCCTAAAC TCTAATCCAA GTAGCGTATG CGCTCACGCA ACTGGTCCAG AACCTTCACC CAACCCAC CTCCTAACCC -47- CGCAGTGGCG GTTTTCATGG CTTGTTATGA CTGTTTTTTT GTACAGTCTA TGCCTCGGGC ATCCAAGC AGCAAGCGCG TTACGCCGTC GGTCGATGTTTG ATGTTATGGA GCAGCAACG ATG TTA CGC AGC Met Leu Arg Ser 1

AGC

Ser 5 AAC GAT GTT ACG CAG CAG GGC AGT CGC Asn Asp Val Thr Gin Gin Gly Ser Arg 10 CCT AAA Pro Lys ACA AAG TTA Thr Lys Leu GGC CCT GAC Cly Pro Asp GGC TCA ACT ATG Cly Ser Ser Met ATC ATT CCC ACA Ile Ile Arg Thr TGT AGC CTC Cys Arg Leu CTT TTC GGT Leu Phe Cly CAA CTC AAA TCC Gin Vai Lys Ser

ATC

Met 40 CGG GCT CCT CTT Arg Aia Aia Leu

CAT

Asp CGT GAG Arg Glu TTC GCA GAC CTA Phe Gly Asp Vai

GCC

Ala 55 ACC TAC TCC CAA Thr Tyr Ser Gin

CAT

His CAC CCC GAC TCC Gin Pro Asp Ser

CAT

Asp TAC CTC GGG AAC Tyr Leu Cly Asn CTC CCT ACT AAC Leu Arg Ser Lys TTC ATC GCG CTT Phe Ile Ala Leu GCC TTC CAC CAA Ala Phe Asp Gin GCG GTT CTT CGC Ala Val Val Gly CTC GCG GCT TAC Leu Aia Ala Tyr GTT CTG Vai Leu 948 1007 1055 1103 1151 1199 1247 1295 1343 1391 1439 1487 1535 1589 1637 CCC AGG TTT Pro Arg Phe CTC TCC GGC Val Ser Cly 115

GAG

Glu 100 CAC CCC CCT ACT GAG ATC TAT ATC TAT Gin Pro Arg Ser Clu Ile Tyr Ile Tyr 105 CAT CTC CCA Asp Leu Ala 110 CTC ATC AAT Leu Ile Asn GAG CAC CGG AGG Glu His Arg Arg

CAC

Gin 120 GGC ATT CCC ACC Cly Ile Aia Thr

GCG

Ala 125 CTC CTC Leu Leu 130 AAC CAT GAG GCC Lys His Glu Ala GCG CTT GGT CCT Ala Leu Cly Ala

TAT

Tyr 140 GTG ATC TAC GTG Val Ile Tyr Val

CAA

Gin 145 CCA CAT TAC GGT Ala Asp Tyr Gly CAT CCC CCA GTG GCT CTC TAT ACA AAG Asp Pro Aia Vai Ala Leu Tyr Thr Lys 155

TTG

Leu 160 GGC ATA CGG GAA Cly Ile Arg Glu

CAA

Glu 165 GTG ATC CAC TTT Val Met His Phe

CAT

Asp 170 ATC CAC CCA AGT Ile Asp Pro Ser ACC GCC Thr Ala 175 ACC TAA CAATTCCTTC AAGCCGAGAT CGGCTTCCCA TTC AGG GCG CAA GAG GAG Thr Leu Arg Aia Gin Glu Clu 177 197 200 AAA TGG ATT CAC CAA GAG ATC CTG CCT CTT ACG GAT Lys Trp Ile Asp Gin Glu Ile Val Aia Vai Thr Asp 205 210 GAA CAC TTC CAT Glu Gin Phe Asp 215 -48

TTA

Leu

TTG

Leu 235

TCC

Ser

AGC

Ser

TCG

Ser

GTC

Val1

CGC

Arg 315

TTT

Phe

GAA

Giu

GCA

Ala

CTC

Leu

GCA

Ala 395

CAC

His

GGC

Gly

ATC

Ile

TTG

Leu

GCC

Ala

GCG

Ala 285

ATC

Ile

CTG

Leu

ATA

Ile

GGT

Gly

GGA

Gly 365

CAC

His

ATT

Ile

GGT

Gly

TAC

Tyr

GTG

Val

AAG

Lys

GTA

Val1 270

ACA

Thr

GAG

Glu

CTT

Leu

AAC

Asn

ATT

Ile 350

CAC

His

CAA

Gin

GGT

Gly

TCG

Ser

AAC

Asn

ACA

Thr

CCT

Pro 255

GTG

Val

CAG

Gin

CCC

Pro

GCG

Ala

GAG

Giu 335

GAG

Giu

CCG

Pro

TTG

Leu

GGG

Gly

TCC

Ser 415

AGT

Ser

GTC

Val1 240

GTT

Val1

GAC

Asp

CCG

Pro

GAG

Giu

CGT

Arg 320

GCG

Ala

CAC

His

CTT

Leu

CAA

Gin

GGA

Gly 400

TCT

Ser

CGA

Arg 225

ATC

Ile

CAT

His

GGC

Gly

GTC

Val

CAT

His 305

AGT

Ser

CAG

Gin

TCA

Ser

CC

Ala

GCT

Ala 385

CAG

Gin

GCA

Ala

CGC

Arg

TCT

Ser

GCC

Ala

TTG

Leu 290

ATG

Met

GAT

Asp

CC

Ala

AAA

Lys

C

Ala 370

AAT

Asn

GGG

Gly

CGA

ATT

Ile

GGC

Gly

GGC

Gly

C

Ala 275

GCT

Ala

GGG

Gly

CTT

Leu

CC

Ala

CTT

Leu 355

ACC

Thr

AAC

Asn

ATC

Met

AGT

GAA

Giu

CTA

Leu

CCC

Cly 260

CCG

Ala

AGG

Arg

CTC

Leu

ACT

Ser

CAA

Gin 340

AAT

Asn

GGA

Gly

TTT

Phe

GCG

Ala

TCG

CCT

Pro 230

GTC

Val1

CAG

Gin

TTG

Leu

CTG

Leu

CCT

Pro 310

AGG

Arg

CTA

Leu

TCG

Trp

CGT

Arg

TAT

Tyr 390

CTT

Leu

ATT

CGG

Arg

TTT

Phe

ACT

Thr

GTG

Val1

GCT

Ala 295

GCG

Ala

GAT

Asp

CCG

Ala

GCC

Cly

CTC

Leu 375

CGA

Cly

TTA

Leu

AAC

GCA

Ala

GCC

Ala

GGC

Gly 265

CGA

Arg

TCC

Ser

GCC

Ala

GAC

Asp

CAC

Gin 345

CC

Ala

ATG

Met

CC

Ala

AAT

Asn

GTT

AAA

Lys

CTT

Leu 250

AAC

Asn

GAG

Giu

GTA

Val

ATT

Ile

CTC

Leu 330

CAT

His

ATT

Ile

ACC

Thr

TCG

Ser

CCC

Pro 410

GAC

Asp 1685 1733 1781 1829 1877 1925 1973 2021 2069 2117 2165 2213 2261 2309 Ala Arg Ser Ser Met Ile Asn Arg Val CAC TAT CCA CTG AGC TAA CCCCCATCTC CTTTCTTCCT TTGAGGTGGC His Tyr Pro Leu Ser 430 431 -49 GCACGAAGGA GGGCTCGAAA ATCTCTGCTA AAAACAAGAA GAAGGAACAG GGAACATGAT 2369 TAGTTTCGCT CGTATGGCAG AAAGTTTAGG AGTCCAGGCT AAACTTGCCC TTGCCTTCGC 2429 ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT TTCTACAGTG TACATACCTT 2489 GTCAGGGTTG GTGGGAATTC 2509 FIG. 2q: GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT TAC TCG GCG TTT TCC 112 Met Ser Trp Ser Gly Gly Ala Tyr Ser Ala Phe Ser 1 5 GAC ACT GCG TTG GTT GCG GCA GTG CGC ACC CCC TGG ATT GAT TGC GGG 160 Asp Thr Ala Leu Val Ala Ala Val Arg Thr Pro Trp Ile Asp Cys Gly 20 GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208 Gly Ala Leu Ser Leu Val Ser Pro Ile Asp Leu Gly Val Lys Val Ala 35 CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC 256 Arg Glu Val Leu Met Arg Ala Ser Leu Glu Pro Gln Met Val Asp Ser 50 55 GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304 Val Leu Ala Gly Ser Met Ala Gin Ala Ser Phe Asp Ala Tyr Leu Leu 70 CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352 Pro Arg His Ile Gly Leu Tyr Ser Gly Val Pro Lys Ser Val Pro Ala 85 TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400 Leu Gly Val Gin Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg Gin 100 105 GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448 Ala Gly Glu Gin Ile Ser Gln Gly Ala Asp His Val Leu Cys Val Ala 110 115 120 GCA GAG TCC ATG TCG CGT AAC CCC ATC GCG TCG TAT ACA CAC CGG GGC 496 Ala Glu Ser Met Ser Arg Asn Pro Ile Ala Ser Tyr Thr His Arg Gly 125 130 135 140 GGG TTC CGC CTC GGT GCG CCC GTT GAG TTC AAG GAT TTT TTG TGG GAG 544 Gly Phe Arg Leu Gly Ala Pro Val Glu Phe Lys Asp Phe Leu Trp Glu 145 150 155 GCA TTG TTT GAT CCT GCT CCA GGA CTC GAC ATG ATC GCT ACC GCA GAA 592 Ala Leu Phe Asp Pro Ala Pro Gly Leu Asp Met Ile Ala Thr Ala Glu 160 165 170 AAC CTG GCG CGC A TTG AGG GCG CAA GAG GAG AAA TGG ATT GAC CAA GAG 641 Asn Leu Ala Arg Leu Arg Ala Gin Glu Glu Lys Trp Ile Asp Gin Glu 175 176 197 200 205 ATC GTG GCT GTT ACG GAT GAA CAG TTC GAT TTA GAG GGC TAC AAC AGT 689 Ile Val Ala Val Thr Asp Glu Gin Phe Asp Leu Glu Gly Tyr Asn Ser 210 215 220 CGA GCA ATT GAA CTG CCT CGG AAG GCA AAA TTG TTG ATC GTG ACA GTC 737 Arg Ala Ile Glu Leu Pro Arg Lys Ala Lys Leu Leu Ile Val Thr Val 225 230 235 240 -51- ATC CGC GGC CTA Ile Arg Gly Leu

GCA

Ala 245 GTC TTT GAA GCC Val Phe Glu Ala

CTT

Leu 250 TCC CGA TTG AAG Ser Arg Leu Lys CCT GTT Pro Val 255 CAT TCT GGC His Ser Gly GGC CCC GCG Gly Ala Ala 275

GGG

Gly 260 GTG CAG ACT GCG Val Gin Thr Ala

GGC

Gly 265 AAC AGC TGT GCC Asn Ser Cys Ala GTA GTG GAC Val Val Asp 270 ACA CAG CCG Thr Gin Pro GCG GCT TTG GTG Ala Ala Leu Val CGA GAG TCG TCT Arg Glu Ser Ser

GCG

Ala 285 GTC TTG Val Leu 290 GCT AGG ATA CTG Ala Arg Ile Leu ACC TCC GTA GTC GGG ATC GAG CCC GAG Thr Ser Val Val Gly Ile Giu Pro Glu ATG GGG CTC GGC Met Gly Leu Gly

CCT

Pro 310 GCG CCC GCG ATT CGC CTG CTG CTT GCG Ala Pro Ala Ile Arg Leu Leu Leu Ala 315

CGT

Arg 320 AGT GAT CTT AGT Ser Asp Leu Ser

TTG

Leu 325 AGG GAT ATC GAC Arg Asp Ile Asp

CTC

Leu 330 TTT GAG ATA AAC Phe Giu Ile Asn GAG GCG Glu Ala 335 CAG CCC GCC Gin Ala Ala TCA AAA CTT Ser Lys Leu 355

CAA

Gin 340 GTT CTA GCG GTA Val Leu Aia Val CAT GAA TTG GGT His Glu Leu Gly ATT GAG CAC Ile Giu His 350 CAC CCG CTT His Pro Leu AAT ATT TGG GGC Asn Ile Trp Gly

GGG

Gly 360 CCC ATT GCA CTT Ala Ile Ala Leu

GGA

Gly 365 CCC GCG Ala Ala 370 ACC GGA TTG CGT Thr Gly Leu Arg

CTC

Leu 375 TGC ATG ACC CTC Cys Met Thr Leu

GCT

Ala 380 CAC CAA TTG CAA His Gin Leu Gin

GCT

Ala 385 AAT AAC TTT CGA Asn Asn Phe Arg GGA ATT CCC TCG Gly Ile Ala Ser TGC ATT GGT GGG Cys Ile Gly Gly

GGA

Gly 400 1025 1073 1121 1169 1217 1265 1313 1373 1433 1493 1543 CAG GGG ATG GCG Gin Cly Met Ala CTT TTA GAG AAT Leu Leu Giu Asn

CCC

Pro 410 CAC TTC GGT TCG His Phe Gly Ser TCC TCT Ser Ser 415 GCA CGA AGT Ala Arg Ser

TCG

Ser 420 ATG ATT AAC AGA Met Ile Asn Arg

GTT

Va1 425 GAC CAC TAT CCA Asp His Tyr Pro CTG AGC TAA Leu Ser 430 431 CGGGCATCTC CTTTGTTGCT TTGAGGTGGC CCACGAGGA GGGCTCCAAA ATCTCTGCTA AAAACAACAA CAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAACTTTAGG AGTCCAGGCT AAACTTCCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT TTCTACAGTG TACATACCTT GTCAGGGTTG CTGGCAATTC FIG. 2r: 52 Sequence 1

CTGCAGCCAG

GGCTCCAATT

GCTAGGGAGA

CATTCTGCAT

AAGGTTGCTA

GCATGGAAAT

TGGAAGCACG

AAAGAGCATG

TGCCGAAACT

CATGCCGAGC

CGATAAGGCC

GGTTGGGA-AG

CAGGGGATCA

TGGATTGCAC

ACAACAGACA

GGTTCTTTTT

GCGGCTATCG

TGAAGCGGGA

TCACCTTGCT

GCTTGATCCG

TACTCGGATG

CGCGCCAGCC

CGTGACCCAT

ATTCATCGAC

CCGTGATATT

TATCGCCGCT

AGCGGGACTC

TCATGTGTGC

TGGCATCGAC

AGCAGCTGAA

TAAATAATAA

GGAATGATAT

CCATTTATGC

CGGATTGGAC

GAGCAAGACT

GTCAGGGTGA

GCAG

GGCTGAAAAG

GCTCGATGGC

TAAATTTGCT

CATGAAATTC

GGAGAGTGCA

GGCATGAGCT

ATTCCTCGCC

CAACTGACCA

GCCCGCGTTC

CTGACTCTGG

ATCGGGACAG

CCCTGCAAAG

AGATCTGATC

GCAGGTTCTC

ATCGGCTGCT

GTCAAGACCG

TGGCTGGCCA

AGGGACTGGC

CCTGCCGAGA

GCTACCTGCC

GAAGCCGGTC

GA.ACTGTTCG

GGCGATGCCT

TGTGGCCGGC

GCTGAAGAGC

CCCGATTCGC

TGGGGTTCGA

TGAGGAGTCA

CTACGTGTAA

AGCAGCTTTG

AGGATTCTTG

GAAAGCAAGT

AGCGCTGCCA

GGTGCGTTGG

CAAGTCTGAC

TCGTAACTTT

GAGGGATTCA

GCCGCGATTG

GGCCATGGTG

ATGAAATCAT

TTGCTCGTAA

TTGCTGGATA

CGGTAGAGCG

ACAAGAAAAT

TGCGCTCTCA

ATGCTTTCGT

CAAGCGAACC

TAAACTGGAT

AAGAGACAGG

CGGCCGCTTG

CTGATGCCGC

ACCTGTCCGG

CGACGGGCGT

TGCTATTGGG

AAGTATCCAT

CATTCGACCA

TTGTCGATCA

CCAGGCTCAA

GCTTGCCGAA

TGGGTGTGGC

TTGGCGGCGA

AGCGCATCGC

AATGACCGAC

CGTTGGATCA

GTTCGTGGAC

GTTTTGATCG

TGAAGCTTTA

AGATCAGTCT

ATGTCAGCTG

GGACAACACC

AAATGCGCCG

GACCGGGGGC

GTGAGGTCAT

AGTGTCTTGG

GCGGCCCCTG

CACTTTTCGG

GCCCAGGAAG

TGATTAGAGA

GTAACCGCGA

CGTCGTCACC

CGGCGCCACA

TCAGGCTGAC

GGAATTGCCA

GGCTTTCTTG

ATGAGGATCG

GGTGGAGAGG

CGTGTTCCGG

TGCCCTCAAT

TCCTTGCGCA

CGAAGTGCCG

CATGGCTGAT

CCAAGCGAAA

GGATGATCTG

GGCGCGCATG

TATCATGGTG

GGACCGCTAT

ATGGGCTGAC

CTTCTATCGC

CAAGCGACGC

ACGGCATAAA

GCCCTTTGCA

GAGGTAGCGG

GTTGTCCGTA

GCACTTTCAA

CAAACTCGAT

CTCAAGTATA

ACTGTCAATG

TTGGTATCCA

GAAGGGAGGG

GCGCGCTCTT

ATGGGTTGGA

GGGGTGGGTG

CACGCGGGTT

CATTAACTAT

CATTCAGGAC

GGAGTGTCCT

GTGATTGGCG

CTGAGCCATC

GCTGGGGCGC

CCGCCAAGGA

TTTCGCATGA

CTATTCGGCT

CTGTCAGCGC

GAACTGCAGG

GCTGTGCTCG

GGGCAGGATC

GCAATGCGGC

CATCGCATCG

GACGAAGAC

CCCGACGGCG

GAAAATGGCC

CAGGACATAG

CGCTTCCTCG

CTTCTTGACG

CCTGGCCGCG

TATTCCAGTG

CGCGCACTAT

GCGGAAAGGT

AACGAAkAATA

AATAGCTACC

GCAGCTGGAT

GCCTTGCCTC

GTTATATCCG

ATCGTCTCGA

GACGGCGCCT

GGAGAGTTCG

TGATTTTCTG

CACGGGATTG

TCAGGATGGT

TTTGGCGGAA

CGTAAAAAGG

CCGGTATCGG

TAGATCGCAA

CTGAAGGCAT

CCTCTGGTAA

TCTGATGGCG

TTGAACAAGA

ATGACTGGGC

AGGGGCGCCC

ACGAGGCAGC

ACGTTGTCAC

TCCTGTCATC

GGCTGCATAC

AGCGAGCACG

ATCAGGGGCT

ACGATCTCGT

GCTTTTCTGG

CGTTGGCTAC

TGCTTTACG

AGTTCTTCTG

GTGATTGCAT

GACGGAGGTT

ATCTCTATGC

GCAGAATGTC

AAAATAAAGA

CTGGCAGGCG

GTAGGTAGCT

TCGCCTGAAT

GATATTCAA

TATTCTGGCT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2164 53

CTGCAGCCAG

GGCTCCAATT

GCTAGGGAGA

CATTCTGCAT

AAGGTTGCTA

GCATGGAAAT

TGGAAGCACG

AAAGAGCATG

TGCCGAAACT

CATGCCGAGC

AGGCGGTTTG

GACATGGAAG

GTCGCCTTGC

ACCCAGTTGA

CGCAACTGGT

ATGGCTTGTT

CGTTACGCCG

CGATGTTACG

CATTCGCACA

TTTCGGTCGT

CCTCGGGAAC

GGTTGTTGGC

CTATATCTAT

CATCAATCTC

AGATTACGGT

GATGCACTTT

CTTCCCTGAT

CAGTGGACGG

ACTATATCTC

AAGGTGCAGA

AAATAAAAAT

CTACCCTGGC

TGGATGTAGG

GCCTCTCGCC

ATCCGGATAT

CTCGATATTC

GGCTGAAAAG

GCTCGATGGC

TAAATTTGCT

CATGAAATTC

GGAGAGTGCA

GGCATGAGCT

ATTCCTCGCC

CAACTGACCA

GCCCGCGTTC

CTGACTCTGG

CGTATTGGGC

CCATCACAAA

GTATAATATT

CATAAGCCTG

CCAGAACCTT

ATGACTGTTT

TGGGTCGATG

CAGCAGGGCA

TGTAGGCTCG

GAGTTCGGAG

TTGCTCCGTA

GCTCTCGCGG

GATCTCGCAG

CTCAAGCATG

GACGATCCCG

GATATCGACC

TGCATTCATG

AGGTTTGGCA

TATGCAGCAG

ATGTCTAAAT

AAAGAGGAAT

AGGCGCCATT

TAGCTCGGAT

TGAATGAGCA

TCAAAGTCAG

TGGCTGCAG

GAGGGATTCA

GCCGCGATTG

GGCCATGGTG

ATGAAATCAT

TTGCTCGTAA

TTGCTGGATA

CGGTAGAGCG

ACAAGAAAAT

TGCGCTCTCA

ATGCTTTCGT

GCATGCATAA

CGGCATGATG

TGCCCATGGA

TTCGGTTCGT

GACCGAACGC

TTTTGTACAG

TTTGATGTTA

GTCGCCCTAA

GCCCTGACCA

ACGTAGCCAC

GTAAGACATT

CTTACGTTCT

TCTCCGGCGA

AGGCCAACGC

CAGTGGCTCT

CAAGTACCGC

TGTGCTGAGG

TCGACCTACG

CTGAAAGCAG

AATAAAGGAT

GATATCAAAG

TATGCAGCGC

TGGACGGTGC

AGACTCAAGT

GGTGATCGTA

Sequence 2

GTGAGGTCAT

AGTGTCTTGG

GCGGCCCCTG

CACTTTTCGG

GCCCAGGAAG

TGATTAGAGA

GTAACCGCGA

CGTCGTCACC

CGGCGCCACA

TCAGGCTGAC

AAACTGTTGT

AACCTGAATC

CGCACACCGT

AAACTGTAAT

AGCGGTGGTA

TCTATGCCTC

TGGAGCAGCA

AACAAAGTTA

ACTCAAATCC

CTACTCCCAA

CATCGCGCTT

GCCCAGGTTT

GCACCGGAGG

GCTTGGTGCT

CTATACAAAG

CACCTALACAA

AGTCACGTTG

TGTAAGTTCG

CTTTGGTTTT

TCTTGTGAAG

CAAGTAGATC

TGCCAATGTC

GTTGGGGACA

CTGACAAATG

ACTTTGACCG

GAAGGGAGGG

GCGCGGTCTT

ATGGGTTGGA

GGGGTGGGTG

CACGCGGGTT

CATTAACTAT

CATTCAGGAC

GGAGTGTCCT

GTGATTGGCG

CTGAGCCATC

AATTCATTAA

GCCAGCGGCA

GGAAACGGAT

GCAAGTAGCG

ACGGCGCAGT

GGGCATCCAA

ACGATGTTAC

GGTGGCTCAA

ATGCGGGCTG

CATCAGCCGG

GCTGCCTTCG

GAGCAGCCGC

CAGGGCATTG

TATGTGATCT

TTGGGCATAC

TTCGTTCAAG

GATCAACGGC

TGGACGCCCT

GATCGGAGGT

CTTTAGTTGT

AGTCTGCACT

AGCTGCAAAC

ACACCCTCAA

CGCCGACTGT

GGGGCTTGGT

GACGGCGCCT

GGAGAGTTCG

TGATTTTCTG

CACGGGATTG

TCAGGATGGT

TTTGGCGGAA

CGTAAAAAGG

CCGGTATCGG

TAGATCGCAA

CTGAGGGGAG

GCATTCTGCC

TCAGCACCTT

GAAGGCACGA

TATGCGCTCA

GGCGGTTTTC

GCAGCAAGCG

GCAGCAGCAA

GTATGGGCAT

CTCTTGATCT

ACTCCGATTA

ACCAAGAAGC

GTAGTGAGAT

CCACCGCGCT

ACGTGCAAGC

GGGAAGAAGT

CCGAGATCGG

ATAAATATTC

TTGCACGCGC

AGCGGGCGGA

CCGTAAACGA

TTCAAAATAG

TCGATGCAGC

GTATAGCCTT

CAATGGTTAT

ATCCAATCGT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2119 -54- Sequence 3

CTGCAGCCAG

GGCTCCAATT

GCTAGGGAGA

CATTCTGCAT

AAGGTTGCTA

GCATGGAAAT

TGGAAGCACG

AAAGAGCATG

TGCCGAAACT

CATGCCGAGC

CGATCAACGG

GTGGACGCCC

TGATCGGAGG

GCTTTAGTTG

CAGTCTGCAC

CAGCTGCAAA

AACACCCTCA

GCGCCGACTG

GGGGGCTTGG

GGCTGAAAAG

GCTCGATGGC

TAAATTTGCT

CATGAAATTC

GGAGAGTCCA

GGCATGAGCT

ATTCCTCGCC

CAACTGACCA

GCCCGCGTTC

CTGACTCTGG

CATAAATATT

TTTGCACGCG

TAGCGGGCGG

TCCGTAAACG

TTTCAAAATA

CTCGATGCAG

AGTATAGCCT

TCAATGGTTA

TATCCAATCG

GAGGGATTCA

GCCGCGATTG

GGCCATGGTG

ATGAAATCAT

TTGCTCGTAA

TTGCTGGATA

CGGTAGAGCG

ACAAGAAAAT

TGCGCTCTCA

ATGCTTTCGT

CCAGTGGACG

CACTATATCT

AAAGGTGCAG

AAAATAAAAA

GCTACCCTGG

CTGGATGTAG

TGCCTCTCGC

TATCCGGATA

TCTCGATATT

GTGAGGTCAT

AGTGTCTTGG

GCGGCCCCTG

CACTTTTCGG

GCCCAGGAAG

TGATTAGAGA

GTAACCGCGA

CGTCGTCACC

CGGCGCCACA

TCAGGCTGAC

GAGGTTTGGC

CTATGCAGCA

AATGTCTAA.A

TAAAGAGGAA

CAGGCGCCAT

GTAGCTCGGA

CTGAATGAGC

TTCAAAGTCA

CTGGCTGCAG

GAAGGGAGGG

GCGCGGTCTT

ATGGGTTGGA

GGGGTGGGTG

CACGCGGGTT

CATTAACTAT

CATTCAGGAC

GGAGTGTCCT

GTGATTGGCG

CTGAGCCATC

ATCGACCTAC

GCTGAAAGCA

TAATAAAGGA

TGATATGAAA

TTATGCAGCG

TTGGACGGTG

AAGACTCAAG

GGGTGATCGT

GACGGCGCCT

GGAGAGTTCG

TGATTTTCTG

CACGGGATTG

TCAGGATGGT

TTTGGCGGAA

CGTAAAAAGG

CCGGTATCGG

TAGATCGCAA

CTGAAGGCAT

GTGTAAGTTC

GCTTTGGTTT

TTCTTGTGAA

GCAAGTAGAT

CTGCCAATGT

CGTTGGGGAC

TCTGACAAAT

AACTTTGACC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1120 55 Sequence 4

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAAGAT

CTCCAGCTCA

AGAATAACAA

CGGTCGGAGC

AGGGGCCTGC

TGGAAAATCG

AGCAAACACT

ACGTGGCCAA

GCGTTGAGTT

TACTGGCCTT

CGTCCGAGCT

ATGAAACTGA

AGCCTTTCGA

CCGCGGCGGA

TTTCCCGCAG

ATGCCGGGCA

AACCGGALATT

GGATGGCTTT

CAGGATGAGG

CTTGGGTGGA

CCGCCGTGTT

CCGGTGCCCT

GCGTTCCTTG

TGGGCGAAGT

CCATCATGGC

ACCACCAAGC

ATCAGGATGA

TCAAGGCGCG

CGAATATCAT

TGGCGGACCG

GCGAATGGGC

TATCGCCCGG

TTTTCTTGGC

GCTTCGCTTC

TAAGCATTCT

TTGAGGCCGA

AATTAAAATA

AGGGCAATTT

TTGACTCCTC

TGAGCAGCTG

AAACTTGGAG

TGAAGCAATT

GCTTTGCGAC

ATGGATGGAG

TCAGCCGCTG

TGGGCCGCTG

TACCCCGCGG

GCTGACTACA

TCATCTGATC

TALACCTAGTG

TGCAGATATG

ALATCTGTCTG

GCCAGCTGGG

CTTGCCGCCA

ATCGTTTCGC

GAGGCTATTC

CCGGCTGTCA

GAATGAACTG

CGCAGCTGTG

GCCGGGGCAG

TGATGCAATG

GAAACATCC

TCTGGACGAA

CATGCCCGAC

GGTGGAAAAT

CTATCAGGAC

TGACCGCTTC

TTCTATCAGC

CATGCTTGTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGGAAACCGC

TTGGGCTATT

AGGAGGTCAG

GGCTCGGCTC

CTGCGTCTGA

GCCGACGCGG

ATTGCTGGCT

CCCGAACATC

GGTGTCGTTG

GCCGGCATAT

ACTTCTGCCC

GTGCTGGGCG

TTCACCGGCG

CCCGTTACCC

GCGGACGTTG

GCACCGGACT

GCGCCCTCTG

AGGATCTCAT

ATGATTGAAC

GGCTATGACT

GCGCAGGGGC

CAGGACGAGG

CTCGACGTTG

GATCTCCTGT

CGGCGGCTGC

ATCGAGCGAG

GAGCATCAGG

GGCGAGGATC

GGCCGCTTTT

ATAGCGTTGG

CTCGTGCTTT

GGGCCGCTTT

GCCTGAACCT

GCATCGAGAT

TGGTGGCTTT

CTTGGCGGCG

ATGGTTTCTT

GGCTGAGCAG

CGATGAGCAT

TTGATCGCAT

GTAGGCTGGA

TTTCTGCTGA

CGGTGGCAAG

ACAAGGCGAT

GGGTCATTAG

TCGCAGCAGG

TGCTTGCGGA

ACGCTGAAGT

CCACTGCCGT

TGGAATTGCG

CACAACGGGT

ATGTGCTGCT

GTAAGGTTGG

GGCGCAGGGG

AAGATGGATT

GGGCACAACA

GCCCGGTTCT

CAGCGCGGCT

TCACTGAAGC

CATCTCACCT

ATACGCTTGA

CACGTACTCG

GGCTCGCGCC

TCGTCGTGAC

CTGGATTCAT

CTACCCGTGA

ACGGTATCGC

CGAAAGTCAT

TCGTTGACAT

GCTGAGGTCA

GALACAGCCTG

TCGAAGCGAT

ATGTGAATTT

TTGCCTCTAT

TCTTGGTTTG

GAAGAAGGCG

TCGTGCGATT

CTTTGGCAAT

CCTGAAGGAT

GTTTCCAGGG

TCCCTGGAAC

TAATCGCGCC

GCTAATTGCT

CGGTGCGCTG

GGCCAAGCAC

TGGCAAATCG

GTTGACGGTG

GCCGGAAGGG

GAAGCCCTGC

ATCAAGATCT

GCACGCAGGT

GACAATCGGC

TTTTGTCAAG

ATCGTGGCTG

GGGAAGGGAC

TGCTCCTGCC

TCCGGCTACC

GATGGAAGCC

AGCCGAACTG

CCATGGCGAT

CCACTGTGGC

TATTGCTGAA

CGCTCCCGAT

GGTGTTAGCC

AGGGCAGAGG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

AATGGTGCCC

CACCTGGAGC

GCAATGCTTC

CGCAGCCGTG

AGCCGCGAGC

GCGGAGGCAC

TTCCCTATCG

ATGCTCAAGC

CGTTACTTCG

TTCAGTGCTC

ATCATGCGTG

CCGGTGATCC

AAAACCTTCA

ACAGCAAGCG

A.AAGTAAACT

GATCAAGAGA

TCTCCGGCCG

TGCTCTGATG

ACCGACCTGT

GCCACGACGG

TGGCTGCTAT

GAGAAAGTAT

TGCCCATTCG

GGTCTTGTCG

TTCGCCAGGC

GCCTGCTTGC

CGGCTGGGTG

CAGCTTGGCG

TCGCAGCGCA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 56

TCCCCTTCTA

CGACCAAGCG

AACTTGGCGA

TCAACGGAGT

GGGCCCGTTT

TGATGAGCAC

GACGGAACTC

ACGCCGCCTG

TGAGTACGCT

CGATGGATTC

CGAGCAAGGA

TC

TCGCCTTCTT

ACGCCCGCCA

TGCGCGCACC

GTTAGAACCG

CTTGAGTATT

CCTGGAAGGC

CGTTCCCCAG

ACGCTAAGCG

CTTGGATGCC

GATTGCCCGT

AGAACTCATT

GACGAGTTCT

TGCCAAGCCT

CTACGGAGAA

TTGGTAGTGG

CATTGGATAG

GCGCTGTACG

TACCGCGATG

GGGGCGGGGG

GCGGCGGCTG

GCTGCCGGCG

TCCCGGTTAG

TCTGAGCGGG

GTTCTCGTGC

GCGATCCACG

TTTTGGACGG

TCACGCGTGG

CGGACGACTG

ACTATTTTGC

CGCCCGCATC

AGATTGGTAA

TCTCAAAAAA

TGGCTCGAGA

ACTCTGGGGT

AAAGTCCTGT

GACTGCTCTC

GCCCAGGAGC

TAGCTTCGAG

GGTTCATCTT

CTCTTCCGAT

CCAGCCCAGA

CGGCAATTTC

AACGCTCTAC

CATGTCCAAC

TCGAAATGAC

GGGTGAGTCG

TGTCCTCCTT

ATGCGCTTCT

CCTGCACAGC

CGCCATTCAT

GTCCGATTCC

CAGCAACAAA

GTCAATGTGA

GTCTTGGTGG

CTTGAGGAAT

2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2822 57 Sequence

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAAGAT

CTCCAGCTCA

AGAATAACAA

CGGTCGGAGC

AGGGGCCTGC

TGGAAAATCG

AGCAAACACT

ACCTGGCCAA

GCGTTGAGTT

TACTGGCCTT

CGTCCGACCT

ATGAAACTGA

AGCCTTTCGA

CCGCGGCGGA

TTTCCCGCAG

ATGCCGGGCA

GGGCGCATGC

CAAACGGCAT

TATTTGCCCA

CCTGTTCGGT

CCTTGACCGA

GTTTTTTTGT

GATGTTTGAT

GGCAGTCGCC

CTCGGCCCTG

GGAGACGTAG

CGTAGTAAGA

GCGGCTTACG

GCAGTCTCCG

CATGAGGCCA

CCCGCAGTGG

TATCGCCCGG

TTTTCTTGGC

GCTTCGCTTC

TAAGCATTCT

TTGACGCCGA

AATTAAAATA

AGGCCAATTT

TTGACTCCTC

TGAGCAGCTG

AAACTTGGAG

TGAAGCAATT

GCTTTGCGAC

ATGGATGGAG

TCAGCCGCTG

TGGGCCGCTG

TACCCCGCGG

GCTGACTACA

TCATCTGATC

TAACCTAGTG

TGCAGATATG

AATCTGTCTG

ATAAAAACTG

GATGAACCTG

TGGACGCACA

TCGTAAACTG

ACGCAGCGGT

ACAGTCTATG

GTTATGGAGC

CTAAAACAAA

ACCAAGTCAA

CCACCTACTC

CATTCATCGC

TTCTGCCCAG

GCGAGCACCG

ACGCGCTTGG

CTCTCTATAC

TTCTATCAGC

CATGCTTGTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGGAAACCGC

TTGGGCTATT

AGGAGGTCAG

GGCTCGGCTC

CTGCGTCTGA

GCCGACGCGG

ATTGCTGGCT

CCCGAACATC

GGTGTCGTTG

GCCGGCATAT

ACTTCTGCCC

GTGCTGGGCG

TTCACCGGCG

CCCGTTACCC

GCGGACGTTG

GCACCGGACT

TTGTAATTCA

AATCGCCAGC

CCGTGGAAAC

TAATGCAAGT

GGTAACGGCG

CCTCGGGCAT

AGCAACGATG

GTTAGCTGGC

ATCCATGCGG

CCAACATCAG

GCTTGCTGCC

GTTTGAGCAG

GAGGCAGGGC

TGCTTATGTG

AAAGTTGGGC

GGGCCGCTTT

GCCTGAACCT

GCATCGAGAT

TGGTGGCTTT

CTTGGCGGCG

ATGGTTTCTT

GGCTGAGCAG

CGATGAGCAT

TTGATCGCAT

GTAGGCTGGA

TTTCTGCTGA

CGGTGGCAAG

ACAAGGCGAT

GGGTCATTAG

TCGCAGCAGG

TGCTTGCGGA

ACGCTGAAGT

GCACTGCCGT

TGGAATTGGG

CACAACGGGT

ATGTGCTGGG

TTAAGCATTC

GGCATCAGCA

GGATGAAGGC

AGCGTATGCG

CAGTGGCGGT

CCAAGCAGCA

TTACGCAGCA

TCAAGTATGG

GCTGCTCTTG

CCGGACTCCG

TTCGACCAAG

CCGCGTAGTG

ATTGCCACCG

ATCTACGTGC

ATACGGGAAG

CGAAAGTCAT

TCGTTGACAT

GCTGAGGTCA

GAACAGCCTG

TCGAAGCGAT

ATGTGAATTT

TTGCCTCTAT

TCTTGGTTTG

GAAGAAGGCG

TCGTGCGATT

CTTTGGCAAT

CCTGAAGGAT

GTTTCCAGGG

TCCCTGGAAC

TAATCGCGCC

GCTAATTGCT

CGGTGCGCTG

GGCCAAGCAC

TGGCAAATCG

GTTGACGGTG

GGAGAGGCGG

TGCCGACATG

CCTTGTCGCC

ACGAACCCAG

CTCACGCAAC

TTTCATGGCT

AGCGCGTTAC

GCAACGATGT

GCATCATTCG

ATCTTTTCGG

ATTACCTCGG

AAGCGGTTGT

AGATCTATAT

CGCTCATCAA

AAGCAGATTA

AAGTGATGCA

GGTGTTAGCC

AGGGCAGAGG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

AATGGTGCCC

CACCTGGAGC

GCAATGCTTC

CGCAGCCGTG

AGCCGCGAGC

GCGGAGGCAC

TTCCCTATCG

ATGCTCAAGC

CGTTACTTCG

TTCAGTGCTC

ATCATGCGTG

CCGGTGATCG

AAAACCTTCA

TTTGCGTATT

GAAGCCATCA

TTGCGTATAA

TTGACATAAG

TCGTCCAGAA

TGTTATGACT

GCCGTGGGTC

TACGCAGCAG

CACATGTAGG

TCGTGAGTTC

GAACTTGCTC

TGGCGCTCTC

CTATGATCTC

TCTCCTCAAG

CGGTGACGAT

CTTTGATATC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 58

GACCCAAGTA

TGTGGGTGAG

CTCTGTCCTC

AGCATGCGCT

GAGCCTGCAC

CTTCGCCATT

GATGTCCGAT

AGACAGCAAC

TTCGTCAATG

TACGTCTTGG

AACCTTGAGG

CCGCCACCTA

TCGAACTTGG

CTTTCAACGG

TCTGGGCCCG

AGCTGATGAG

CATGACGGAA

TCCACGCCGC

AAATGAGTAG

TGACGATGGA

TGGCGAGCAA

AATTC

ACAATTCGTT

CGATGCGCGC

AGTGTTAGAA

TTTCTTGAGT

CACCCTGGAA

CTCCGTTCCC

CTGACGCTAA

GCTCTTGGAT

TTCGATTGCC

GGAAGAACTC

CAAGCCGAGA

ACCCTACGGA

CCGTTGGTAG

ATTCATTGGA

GGCGCGCTGT

CAGTACCGCG

GCGGGGGCGG

GCCGCGGCGG

CGTGCTGCCG

ATTTCCCGGT

TCGGCTTCCC

GAAGCGATCC

TGGTTTTGGA

TAGTCACGCG

ACGCGGACGA

ATGACTATTT

GGGCGCCCGC

CTGAGATTGG

GCGTCTCAAA

TAGTGGCTCG

TGCAAAGTCC

ACGGACTGCT

CGGGCCCAGG

TGGTAGCTTC

CTGGGTTCAT

TGCCTCTTCC

ATCCCAGCCC

TAACGGCAAT

AAAAACGCTG

AGACATGTCC

2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2775 59 Sequence 6

GAATTCCGCG

GGTAGGGTCT

TGCGTTTGCC

TTAACTCGCG

GTCTCGCCCT

CGATTAAGAT

CTCCAGCTCA

AGAATAACAA

CGGTCGGAGC

AGGGGCCTGC

TGGAAAATCG

AGCAAACACT

ACGTGGCCAA

GCGTTGAGTT

TACTGGCCTT

CGTCCGAGCT

ATGAAACTGA

AGCCTTTCGA

CCGCGGCGGA

TTTCCCGCAG

ATGCCGGGCA

CGGAGAAGCG

GTAGTGGTTT

TGGATAGTCA

CTGTACGCGG

CGCGATGACT

GCGGGGGCGC

GCGGCTGAGA

GCCGGCGTCT

CGGTTAGTGG

TATCGCCCGG

TTTTCTTGGC

GCTTCGCTTC

TAAGCATTCT

TTGAGGCCGA

AATTAAAATA

AGGGCAATTT

TTGACTCCTC

TGAGCAGCTG

AAACTTGGAG

TGAAGCAATT

GCTTTGCGAC

ATGGATGGAG

TCAGCCGCTG

TGGGCCGCTG

TACCCCGCGG

GCTGACTACA

TCATCTGATC

TAACCTAGTG

TGCAGATATG

AATCTGTCTG

ATCCACGGAC

TGGACGGGCC

CGCGTGGTAG

ACGACTGGGT

ATTTTGCCTC

CCGCATCCCA

TTGGTAACGG

CAAAAAAAAC

CTCGAGACAT

TTCTATCAGC

CATGCTTGTT

GCGATGAACC

GTCATTTTTT

TTCTTGGGCG

AGGAAACCGC

TTGGGCTATT

AGGAGGTCAG

GGCTCGGCTC

CTGCGTCTGA

GCCGACGCGG

ATTGCTGGCT

CCCGAACATC

GGTGTCGTTG

GCCGGCATAT

ACTTCTGCCC

GTGCTGGGCG

TTCACCGGCG

CCCGTTACCC

GCGGACGTTG

GCACCGTGGG

TGCTCTCTGT

CAGGAGCATG

CTTCGAGCCT

TCATCTTCGC

TTCCGATGTC

GCCCAGACAG

CALATTTCGTC

GCTGTACGTC

GTCCAACCTT

GGGCCGCTTT

GCCTGAACCT

GCATCGAGAT

TCGTGGCTTT

CTTGGCGGCG

ATGGTTTCTT

GGCTGAGCAC

CGATGAGCAT

TTGATCGCAT

GTAGGCTGGA

TTTCTGCTGA

CGGTGGCAAG

ACAAGGCGAT

GGGTCATTAG

TCGCAGCAGG

TGCTTGCGGA

ACGCTGAAGT

GCACTGCCGT

TGGAATTGGG

CACAACGGGT

TGAGTCGAAC

CCTCCTTTCA

CGCTTCTGGG

GCACAGCTGA

CATTCATGAC

CGATTCCACG

CAACAAATGA

AATGTGACGA

TTGGTGGCGA

GAGGAATTC

CGAAAGTCAT

TCGTTGACAT

GCTGAGGTCA

GAACAGCCTG

TCGAAGCGAT

ATGTGAATTT

TTGCCTCTAT

TCTTGGTTTG

GAAGA.AGGCG

TCGTGCGATT

CTTTGGCAAT

CCTGA-AGGAT

GTTTCCAGGG

TCCCTGGAAC

TAATCGCGCC

GCTAATTGCT

CGGTCCGCTG

GGCCA.AGCAC

TGGCAAATCG

GTTGACGGTG

TTGGCGATGC

ACGGAGTGTT

CCCGTTTCTT

TGAGCACCCT

GGAACTCCGT

CCGCCTGACG

GTAGGCTCTT

TGGATTCGAT

GCAAGGAAGA

GGTGTTAGCC

AGGGCAGAGG

GGATTTTTCC

ATGAAAGGTG

GCTCCACTAC

GTCTGGCATA

ATGGTTATTC

AATGGTGCCC

CACCTGGAGC

GCAATGCTTC

CGCAGCCGTG

AGCCGCGAGC

GCGGAGGCAC

TTCCCTATCG

ATGCTCA.AGC

CGTTACTTCG

TTCAGTGCTC

ATCATGCGTG

CCGGTGATCG

AAAACCTTCA

GCGCACCCTA

AGAACCGTTG

GAGTATTCAT

CGAAGGCGCG

TCCCCAGTAC

CTAAGCGGGG

GGATGCCGCG

TGCCCGTCCT

ACTCATTTCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 60 Sequence 7

CTGCAGCCGA

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

TTCTTCCCTT

AACAAACCTG

AAATGTTCCA

AGCGTCCGCT

CTATGTATGC

ATTTGGCGAA

ATGCAGCACC

GTAAGGTTGG

GGCGCAGGG

AAGATGGATT

GGGCACAACA

GCCCGGTTCT

CAGCGCGGCT

TCACTGAAGC

CATCTCACCT

ATACGCTTGA

CACGTACTCG

GGCTCGCGCC

TCGTCGTGAC

CTGGATTCAT

CTACCCGTGA

ACGGTATCGC

TCTGAGCGGG

CGGTCGGCGA

GCCACACTGT

CGGCAGTCCG

ACTTAGGGGT

TCGATAGCGT

GGCACATTGG

TTTGCGGCAC

ATCACGTGCT

GCATCGATTG

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

CCCGGGTCGA

CGTTGCTGCC

CAACGTCCGC

GCTTATCGTC

GGGCATTCCC

GCTGCGTCAC

TTTCCAGGGG

GAAGCCCTGC

ATCAAGATCT

GCACGCAGGT

GACAATCGGC

TTTTGTCAAG

ATCGTGGCTG

GGGAAGGGAC

TGCTCCTGCC

TCCGGCTACC

GATGGAAGCC

AGCCGAACTG

CCATGGCGAT

CGACTGTGGC

TATTGCTGAA

CGCTCCCGAT

ACTCTGGGGT

AAGTTGATGC

GAGTTGGTCA

CACCCCCTGG

AAAGGTCGCT

ACTCGCAGGC

CTTGTACAC

AGGCTTCGAA

GTGTGTCGCG

AGCACTTTAC

AAATCGATCT

AACTTGATAA

CTCGAAGAGG

TCTCCTCAGC

GACGCTGGAG

ATTCTTGAGC

AGGGCGGCAA

GCCATCGCAC

TCTGGAAATG

TATTGCCCGG

ATCGTAGGTC

ACACCAAGCG

AAAGTAAACT

GATCAAGAGA

TCTCCGGCCG

TGCTCTGATG

ACCGACCTGT

GCCACGACGG

TGGCTGCTAT

GAGAA.AGTAT

TGCCCATTCG

GGTCTTGTCG

TTCGCCAGGC

GCCTGCTTGC

CGGCTGGGTG

GAGCTTGGCG

TCGCAGCGCA

TCGAAATGAC

GCTGTATCGT

GGGGGGGCTT

ATTGATTGCG

CGCGAAGTTC

TCTATGGCTC

GGTGTTCCCA

CTGCTTCGGC

GGCTGCAG

CCAGCTGCGC

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

GTCTCGAGCA

ATGGGGAATG

AGAGCTTGCT

ACCTGGAACA

TGTCTCCTGC

TTCTGCAACC

AACCGGAATT

GGATGGCTTT

CAGGATGAGG

CTTGGGTGGA

CCGCCGTGTT

CCGGTGCCCT.

GCGTTCCTTG

TGGGCGAAGT

CCATCATCGC

ACCACCAAGC

ATCAGGATGA

TCAAGGCGCG

CGAATATCAT

TGGCGGACCG

GCCAATGGGC

TCGCCTTCTA

CGACCAAGCG

GGTGAAGATC

ACTCGGCGTT

GGGGTGCCCT

TGATGCGTGC

AAGCAACCTT

AGTCGGTTCC

AGGCCGGCGA

TGGCTGACCA

CGGGCATCAT

GTTCTCCGGT

GAACGCCGAG

CAGTGTCTCG

GATGCCTTCT

TTGGGCTAAG

GCGTCGTATC

TCCTTACGGA

TCTTCAGCTG

TTATTCACTG

GGGACTGGTC

GCCAGCTGGG

CTTGCCGCCA

ATCGTTTCGC

GAGGCTATTC

CCGGCTGTCA

GAATGAACTG

CGCAGCTGTG

GCCGGGGCAC

TGATGCAATG

CPAACATCGC

TCTGGACGALA

CATGCCCGAC

GGTGCAAA.AT

CTATCAGGAC

TGACCGCTTC

TCGCCTTCTT

ACGCCCCTGT

AATCCATGCT

TTCCGACACT

GTCGCTGCTG

GTCGCTTGAA

TGATGCTTAC

GGCCTTGGGG

GCAGATTTCC

TTCAGAATGG

GCCCGCGGCG

CTTGGTGGAT

TCCACATTGC

ATTGGTCATC

CTCGAGGCGC

ACCCGTCCAG

AGCTACGCGG

CTATCGGCAG

GCATTTGGGG

CTGTCGCAAG

TTTGCTGCCG

GCGCCCTCTG

AGGATCTGAT

ATGATTGAAC

GGCTATGACT

GCCCAGGGGC

CACGACGAGG

CTCGACGTTG

GATCTCCTGT

CGGCGGCTGC

ATCGAGCGAG

GAGCATCAGG

GGCGAGGATC

GGCCGCTTTT

ATAGCGTTGG

CTCGTGCTTT

GACGAGTTCT

TTTGCAATGG

GCGTGACGAG

GCGTTGGTTG

TCGCCTATCG

CCACAAATGG

CTGCTCCCGC

GTGCAGCGCA

CAAGGCGCTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2188 -61- Sequence 8

CTGCAGCCGA

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

TTCTTCCCTT

AACAAACCTG

AAATGTTCCA

AGCGTCCGCT

CTATGTATGC

ATTTGGCGAA

ATGCAGCACC

TGTAATTCAT

ATCGCCAGCG

CGTGGAAACG

AATGCAAGTA

GTAACGGCGC

CTCGGGCATC

GCAACGATGT

TTAGGTGCCT

TCCATGCGGG

CAACATCAGC

CTTGCTGCCT

TTTGAGCAGC

AGGCAGGGCA

GCTTATGTGA

AAGTTGGGCA

CAATTCGTTC

TGCGCTGTAT

TCAGGGGGGG

TGGATTGATT

GCTCGCGAAG

GGCTCTATGG

AGCGGTGTTC

GAACTGCTTC

GCGGGCTGCA

GCATCGATTG

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

CCCGGGTCGA

CGTTGCTGCC

CAACGTCCGC

GCTTATCGTC

GGGCATTCCC

GCTGCGTCAC

TTTCCAGGGG

TAAGCATTCT

GCATCAGCAC

GATGAAGGCA

GCGTATGCGC

AGTGGCGGTT

CAAGCAGCAA

TACGCAGCAG

CAAGTATGGG

CTGCTCTTCA

CGGACTCCGA

TCGACCAAGA

CGCGTAGTGA

TTGCCACCGC

TCTACGTGCA

TACGGGAAGA

AAGCCGAGAT

CGTGGTGAAG

CTTACTCGGC

GCGGGGGTGC

TTCTGATGCG

CTCAAGCAAG

CCAAGTCGGT

GGCAGGCCGG

G

AGCACTTTAC

AAATCGATCT

AACTTGATAA

CTCGAAGAGG

TCTGCTCAGC

GACGCTGGAG

ATTCTTGAGC

AGGGCGGCAA

GCCATCGCAC

TCTGGAAATG

TATTGCCCGG

ATCGTAGGTC

GAGAGGCGGT

GCCGACATGG

CTTGTCGCCT

CGAACCCAGT

TCACGCAACT

TTCATGGCTT

GCGCGTTACG

CAACGATGTT

CATCATTCGC

TCTTTTCGGT

TTACCTCGGG

AGCGGTTGTT

GATCTATATC

GCTCATCAAT

AGCAGATTAC

AGTGATGCAC

CGGCTTCCCC

ATCAATCCAT

GTTTTCCGAC

CCTGTCGCTG

TGCGTCGCTT

CTTTGATGCT

TCCGGCCTTG

CGAGCAGATT

CCAGCTGCGC

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

GTCTCGAGCA

ATGGGGAATG

AGAGCTTGCT

ACCTGGAACA

TGTCTCCTGC

TTCTGCAACC

TTGCGTATTG

AAGCCATCAC

TGCGTATAAT

TGACATAAGC

GGTCCAGAAC

GTTATGACTG

CCGTGGGTCG

ACGCAGCAGG

ACATGTAGGC

CGTGAGTTCG

AACTTGCTCC

GGCGCTCTCG

TATGATCTCG

CTCCTCAAGC

GGTGACGATC

TTTGATATCG

TGTTTTGCAA

GCTGCGTGAC

ACTGCGTTGG

GTGTCGCCTA

GAACCACAAA

TACCTGCTCC

GGGGTGCAGC

TCCCA-AGGCG

TGGCTGACCA

CGGGCATCAT

GTTCTCCGGT

GAACGCCGAG

CAGTGTGTCG

GATGCGTTCT

TTGGGCTAAG

GCGTCGTATC

TCCTTACGGA

TCTTCAGCTG

TTATTCACTG

GGGACTGGTC

GGCGCATGCA

AAACGGCATG

ATTTGCCCAT

CTGTTCGGTT

CTTGACCGAA

TTTTTTTGTA

ATGTTTGATG

GCAGTCGCCC

TCGGCCCTGA

CAGACGTAGC

GTAGTAAGAC

CGGCTTACGT

CAGTCTCCGG

ATGAGGCCAA

CCGCAGTGGC

ACCCAAGTAC

TGGCGGTCGG

GAGGCCACAC

TTGCGGCAGT

TCGACTTAGG

TGGTCGATAG

CGCGGCACAT

GCATTTGCGG

CTGATCACGT

TTCAGAATGG

GCCCGCGGCG

CTTGGTGGAT

TCCACATTGC

ATTGGTCATC

CTCGAGGCGC

ACCCGTCCAG

AGCTACGCGG

CTATCGGCAG

GCATTTGGGG

CTGTCGCAAG

TTTGCTGCCG

TAAAAACTGT

ATGAACCTGA

GGACGCACAC

CGTAAACTGT

CGCAGCGGTG

CAGTCTATGC

TTATGGAGCA

TAAAACAAAG

CCAAGTCAAA

CACCTACTCC

ATTCATCGCG

TCTGCCCAGG

CGAGCACCGG

CGCGCTTGCT

TCTCTATACA

CGCCACCTAA

CGAAAGTTGA

TGTGAGTTGG

GCGCACCCCC

GGTAAAGGTC

CGTACTCGCA

TGGCTTGTAC

CACAGGCTTC

GCTGTGTGTC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2171 -62 Sequzence 9

CTGCAGCCGA

CCCGCGGCAC

CTCGCCTCAT

CAAGGCCAGT

AACCGCAGGC

CTCCGGTTGA

TTCTTCCCTT

AACAAACCTG

AAATGTTCCA

AGCGTCCGCT

CTATGTATGC

ATTTGGCGAA

ATGCAGCACC

ATCGTGGTGA

GGCTTACTCG

TTGCGGGGGT

AGTTCTGATG

GGCTCAAGCA

TCCCAAGTCG

TCGGCAGGCC

CAG

GCATCGATTG

TATCCAATCT

TTCAATCTCT

CGCGGAGAGT

ATCATCATGC

GGTTACGCAA

CCCGGGTCGA

CGTTGCTGCC

CAACGTCCGC

GCTTATCGTC

GGGCATTCCC

GCTGCGTCAC

TTTCCAGCGC

AGATCAATCC

GCGTTTTCCG

GCCCTGTCGC

CGTGCGTCGC

AGCTTTGATG

GTTCCGGCCT

GGCGAGCAGA

AGCACTTTAC

AAATCGATCT

AACTTGATAA

CTCGAAGAGC

TCTGCTCAGC

GACGCTGGAG

ATTCTTGAGC

AGGGCGGCAA

GCCATCGCAC

TCTCGAAATG

TATTGCCCGG

ATCGTAGGTC

GCTGTTTTGC

ATGCTGCGTG

ACACTGCGTT

TGGTGTCGCC

TTGAACCACA

CTTACCTGCT

TGGGGGTGCA

TTTCCCAAGG

CCAGCTGCGC

TCGGGCGCCG

AAACAGAGCT

AGAGTACAGT

CACGCTACCG

GTATTGTCCG

GTCTCGAGCA

ATGGGGAATG

AGAGCTTGCT

ACCTGGAACA

TGTCTCCTGC

TTCTGCAACC

AATGGCGGTC

ACGAGGCCAC

GGTTCCGGCA

TATCGACTTA

AATGGTCGAT

CCCGCGGCAC

GCGCATTTGC

CGCTGATCAC

TGGCTGACCA

CGGGCATCAT

GTTCTCCGGT

GAACGCCGAG

CAGTGTGTCG

GATGCGTTCT

TTGGGCTAAG

GCGTCCTATC

TCCTTACGGA

TCTTCAGCTG

TTATTCACTG

GGGACTGGTC

GGCGAAAGTT

ACTGTGAGTT

GTGCGCACCC

GGGGTAAAGG

AGCGTACTCG

ATTGGCTTGT

GGCACAGGCT

GTGCTGTGTG

TTCAGAATGG

GCCCGCGGCG

CTTGGTGGAT

TCCACATTGC

ATTGGTCATC

CTCGAGGCGC

ACCCGTCCAG

AGCTACGCGG

CTATCGGCAG

GCATTTGGGG

CTGTCGCAAG

TTTGCTGCCG

GATGCGCTGT

GGTCAGGGGG

CCTGGATTGA

TCGCTCGCGA

CAGGCTCTAT

ACAGCGGTGT

TCGAACTGCT

TCGCGGGCTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1203 63 Sequence

GAATTCCCCT

GCTTGCGTTA

GGGTACGCCT

AATTGACAGA

GGTGCACGAT

GTATCGCTTG

ATCGAGAGAT

TTCTGACTGG

AGACCGATGC

GGAATTGCCA

GGCTTTCTTG

ATGAGGATCG

GGTGGAGAGG

CGTGTTCCGG

TGCCCTGAAT

TCCTTGCGCA

CGAAGTGCCG

CATGGCTGAT

CCAAGCGAAA

GGATGATCTG

GGCGCGCATG

TATCATGGTG

GGACCGCTAT

ATGGGCTGAC

CTTCTATCGC

CAACCGACGC

CTTGCAGACC

AATAATGACA

CCTTGTTCAG

GTATCGCGCG

GCTGCGTTTC

GCGGCGGATC

GCAGCGGGAA

C

GGCGACGAAA

ATCGTTAACC

TTCCGTGCGC

ACTATAGGTT

GAATAGCTAC

GGTCACGCTG

GGTCGAGGTT

TGCAGGCGAA

TGGCCCCGAA

GCTGGGGCGC

CCGCCAAGGA

TTTCGCATGA

CTATTCGGCT

CTGTCAGCGC

GAACTGCAGG

GCTGTGCTCG

GGGCAGGATC

GCAATGCGGC

CATCGCATCG

GACGAAGAGC

CCCGACGGCG

GAAAATGGCC

CAGGACATAG

CGCTTCCTCG

CTTCTTGACG

CCCGAGCAGG

TACAAGCGCT

ATAATGAGGA

CATCTGATGA

TCGCTGCTGC

CTGAATGGGC

TTCTAGAGGA

ACTGGTATGG

GGGCGGCAGG

GTTTGAAATT

TTTGATCTGC

CGCAGTAGCT

GATGGCCGTT

AACCGCCCGG

CTGGAGGTGC

TCCTGGACCG

ATTCTGCAAG

CCTCTGGTAA

TCTGATGGCG

TTGAACAAGA

ATGACTGGGC

AGGGGCGCCC

ACGAGGCAGC

ACCTTGTCAC

TCCTGTCATC

GGCTGCATAC

AGCGAGCACG

ATCAGGGGCT

AGGATCTCCT

GCTTTTCTGG

CGTTGGCTAC

TGCTTTACGG

AGTTCTTCTG

GCATGAAGCA

GATAAATGCG

GTGCCCAATG

GCGCACCTTC

CAGTTTGGAA

GGCGCTTGCT

CCGTTCTTCC

GTTTAACGTT

CCGCATGGCC

CCTTGCCAAA

GCTTCCGTGC

TTTGCTCACC

GGTCTACCGT

AGAAGCGCAA

TGGAGCAGGA

CGGGCATGGA

AGAAGATTCG

GGTTGGGAAG

CAGGGGATCA

TGGATTGCAC

ACAACAGACA

GGTTCTTTTT

GCGGCTATCG

TGAAGCGGGA

TCACCTTGCT

GCTTGATCCG

TACTCGGATG

CGCGCCAGCC

CGTGACCCAT

ATTCATCGAC

CCGTGATATT

TATCGCCGCT

AGCGGGACTC

GTTCCTTGAC

CCGGGGCCCT

TTTCACGTGC

GAGCGTCGTA

GATCCGGACG

CCGAGCGAAC

GAGTTCACCG

TACCTGGCGG

ACGGCTGGGC

TTTCGGCGAG

CTTGAATCAG

CACCAAATCC

TGATGTGAAG

CGCAATGAGC

CGCAGATGCT

CCTGAAGGAG

TCGGGGACAG

CCCTGCAAAG

AGATCTGATC

GCAGGTTCTC

ATCGGCTGCT

GTCAAGACCG

TGGCTGGCCA

AGGGACTGGC

CCTGCCGAGA

GCTACCTGCC

GAAGCCGGTC

GAACTCTTCG

GGCGATGCCT

TGTGGCCGGC

GCTGAAGAGC

CCCGATTCGC

TGGGGTTCGA

GAGAAAAGCA

CGCTCCGCCC

CCCTGCTTAT

GCCCGCTGAC

CCGCAGTGGC

GCCGTGCCCG

CCGCAGCGAG

CGGGCATGTT

GGTAACTGAT

AGAATCATGC

AAAAATAGTT

ACAGCACTGG

GTTGAAGAAG

CCAACTCTCA

CGCGTGCTTG

TATTTCCGCG

CAAGCGAACC

TAAACTGGAT

AAGAGACAGG

CGGCCGCTTG

CTGATGCCGC

ACCTGTCCGG

CGACGGGCGT

TGCTATTGGG

AAGTATCCAT

CATTCGACCA

TTGTCGATCA

CCAGGCTCAA

GCTTGCCGAA

TGGGTGTGGC

TTGGCGGCGA

AGCGCATCGC

AATGACCGAC

TCAAGCCGGG

CCGGCCTTCC

TGGTGGTAAG

CGGAGAAGTG

CGCTGCACAG

ACTGCTGCGA

TGAAACTGGC

GCGGGGAATT

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 1981 64 Sequence 11

GAATTCCCCT

GCTTGCGTTA

GGGTACGCCT

AATTGACAGA

GGTGCACGAT

GTATCGCTTG

ATCGAGAGAT

TTCTGACTGG

AGACCGATGC

GTATTGGGCG

CATCACAAAC

TATAATATTT

ATAAGCCTGT

CAGAACCTTG

TGACTGTTTT

GGGTCGATGT

AGCAGGGCAG

GTAGGCTCGG

AGTTCGGAGA

TGCTCCGTAG

CTCTCGCGGC

ATCTCGCAGT

TCA-AGCATGA

ACGATCCCGC

ATATCGACCC

AGGGCATGAA

GCTGATAAAT

GGAGTGCCCA

TGAGCGCACC

TGCCAGTTTG

GGCGGCGCTT

GGACCGTTCT

TGGGTTTAAC

GGCGACGAAA

ATCGTTAACC

TTCCGTGCGC

ACTATAGGTT

GAATAGCTAC

GGTCACGCTG

GGTCGAGGTT

TGCAGGCGAA

TGGCCCCGAA

CATGCATAAA

GGCATGATGA

GCCCATGGAC

TCGGTTCGTA

ACCGAACGCA

TTTGTACAGT

TTGATGTTAT

TCGCCCTAAA

CCCTGACCAA

CGTAGCCACC

TAAGACATTC

TTACGTTCTG

CTCCGGCGAG

GGCCAACGCG

AGTGGCTCTC

AAGTACCGCC

GCAGTTCCTT

GCGCCGGGGC

ATGTTTCACG

TTCGAGCGTC

GAAGATGCGG

GCTCCGAGCG

TCCGAGTTCA

GTTTACCTGG

GGGCGGCAGG

GTTTGAAATT

TTTGATCTGC

CGCAGTAGCT

GATGGCCGTT

AACCGCCCGG

CTGGAGGTGC

TCCTGGACCG

ATTCTGCAAG

AACTGTTGTA

ACCTGAATCG

GCACACCGTG

AACTGTAATG

GCGGTGGTAA

CTATGCCTCG

GGAGCAGCAA

ACAAAGTTAG

GTCAAATCCA

TACTCCCAAC

ATCGCGCTTG

CCCAGGTTTG

CACCGGAGGC

CTTGGTGCTT

TATACAAAGT

ACCTAACAAT

GACGAGAAAA

CCTCGCTGCG

TGCCCCTGCT

GTAGCCCGCT

ACGCCGCAGT

AACGCCGTGC

CCGCCGCAGC

CGGCGGGCAT

CC GCATGGC C

CCTTGCCAAA

GCTTCCGTGC

TTTGCTCACC

GGTCTACCGT

AGAAGCGCAA

TGGAGCAGGA

CGGGCATGGA

AGAAGATTCG

ATTCATTAAG

CCAGCGGCAT

GAAACGGATG

CAAGTAGCGT

CGGCGCAGTG

GGCATCCAAG

CGATGTTACG

GTGGCTCAAG

TGCGGGCTGC

ATCAGCCGCA

CTGCCTTCGA

AGCAGCCGCC

AGCGCATTGC

ATGTGATCTA

TGGCCATACG

TCGTTCAAGC

GCATCAAGCC

CCCCCGGCCT

TATTGGTGGT

GACCGGAGAA

GGCCGCTGCA

CCGACTGCTG

GACTGAAACT

GTTGCGGGGA

ACGGCTGGGC

TTTCGGCGAG

CTTGAATCAG

CACCAAATCC

TGATGTGAAG

CGCAATGAGC

CGCAGATGCT

CCTGAAGGAG

TCGGGGGAGA

CATTCTGCCG

CAGCACCTTG

AAGGCACGAA

ATGCGCTCAC

GCGGTTTTCA

CAGCAAGCC

CAGCAGCAAC

TATGGGCATC

TCTTGATCTT

CTCCGATTAC

CCAAGAAGCG

TAGTGAGATC

CACCGCGCTC

CGTGCAAGCA

GGAAGAAGTG

CGAGATCGGC

GGGCTTGCAG

TCCAATAATG

AAGCCTTGTT

GTGGTATCGC

CAGGCTGCGT

CGAGCGGCGG

GGCGCAGCGG

ATTC

GGTAACTGAT

AGAATCATGC

AAAAATAGTT

ACAGCACTGG

GTTGAAGAAG

CCAACTCTCA

CGCGTGCTTG

TATTTCCGCG

GGCGGTTTGC

ACATGGAAGC

TCGCCTTGCG

CCCAGTTGAC

GCAACTGGTC

TGGCTTGTTA

GTTACGCCGT

GATGTTACGC

ATTCGCACAT

TTCGGTCGTG

CTCGGCAACT

GTTGTTGGCG

TATATCTATG

ATCAATCTCC

GATTACGGTG

ATGCACTTTG

TTCCCCGAGC

ACCTACAAGC

ACAATAATGA

CAGCATCTGA

GCGTCGCTGC

TTCCTGAATG

ATCTTCTAGA

GAAACTGGTA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1964 65 Sequence 12

GAATTCCCCT

GCTTGCGTTA

GGGTACGCCT

AATTGACAGA

GGTGCACGAT

GTATCGCTTG

ATCGAGAGAT

TTCTGACTGG

AGACCGATGC

AGTTCCTTGA

GCCGGGGCCC

GTTTCACGTG

CGAGCGTCGT

AGATGCGGAC

TCCGAGCGAA

CGAGTTCACC

TTACCTGGCG

GGCGACGAAA

ATCGTTAACC

TTCCGTGCGC

ACTATAGGTT

GAATAGCTAC

GGTCACGCTG

GGTCGAGGTT

TGCAGGCGAA

TGCCCCCGAA

CGAGAAAAGC

TCGCTGCGCC

CCCCTGCTTA

AGCCCGCTGA

GCCGCAGTGG

CGCCGTGCCC

GCCGCAGCGA

GCGGGCATGT

GGGCGGCAGG

GTTTGAAATT

TTTGATCTGC

CGCAGTAGCT

GATGGCCGTT

AACCGCCCGG

CTGGAGGTGC

TCCTGGACCG

ATTCTGCAAG

ATCAAGCCGG

CCCGGCCTTC

TTGGTGGTAA

CCGGAGAAGT

CCGCTGCACA

GACTGCTGCG

GTGAAACTGG

TGCGGGGAAT

CCGCATGGCC

CCTTGCCAAA

GCTTCCGTGC

TTTGCTCACC

GGTCTACCGT

AGAAGCGCAA

TGGAGCAGGA

CGGGCATGGA

AGAAGATTCG

GCTTGCAGAC

CAATAATGAC

GCCTTGTTCA

GGTATCGCGC

GGCTGCGTTT

AGCGGCGGAT

CGCAGCGGGA

TC

ACGGCTGGGC

TTTCGGCGAG

CTTGAATCAG

CACCAAATCC

TGATGTGAAG

CGCAATGAGC

CGCAGATGCT

CCTGAAGGAG

TCGCGAGCAG

CTACAAGCGC

AATAATGAGG

GCATCTGATG

GTCGCTGCTG

CCTGAATGGG

CTTCTAGAGG

AACTGGTATG

GGTAACTGAT

AGAATCATGC

AAAAATAGTT

ACAGCACTGG

GTTGAAGAAG

CCAACTCTCA

CGCGTGCTTG

TATTTCCGCG

GGCATGAAGC

TGATAAATGC

AGTGCCCAAT

AGCGCACCTT

CCAGTTTGGA

CGGCGCTTGC

ACCGTTCTTC

GGTTTAACGT

-66 Sequence 13 GAATTCCAAT AATGACAATA

TGGTAAGCCT

AGAAGTGGTA

TGCACAGGCT

GCTGCGAGCG

AACTGGCGCA

GGAAGCCGCG

TAGCTTTGCC

TGCTCCGGTA

GGTGTTGAAA

TGATGCTGGT

TGCGGTGGTG

GACCCACGTT

GGAATTAGGT

CGAAGCGGCG

TCTGATTGTG

ACTGCGTGCT

TGCAGGTCAA

AATTGCCAGC

CTTTCTTGCC

GAGGATCGTT

TGGAGAGGCT

TGTTCCGGCT

CCCTGAATGA

CTTGCGCAGC

AAGTGCCGGG

TGGCTGATGC

AAGCGAA.ACA

ATGATCTGGA

CGCGCATGCC

TCATGGTGGA

ACCGCTATCA

GGGCTGACCG

TCTATCGCCT

AGCGACGCCC

ATGACGAGGC

TGTTCAGCAT

TCGCGCGTCG

GCGTTTCCTG

GCGGATCTTC

GCGGGAAACT

GCCATGACCA

ATGGCGGTTC

ATCCTTGGCG

AGCTCTGAGC

CTGGGGGATG

GAGCGACTGA

GGACGGATCA

GGTAAGGCTC

GCCTTTGGTG

ACAGCAGTCG

GGCGATCCTA

CGCATCCAGG

TGGGGCGCCC

GCCAAGGATC

TCGCATGATT

ATTCGGCTAT

GTCAGCGCAG

ACTGCAGGAC

TGTGCTCGAC

GCAGCATCTC

AATGCGGCGG

TCGCATCGAG

CGAAGAGCAT

CGACGGCGAG

AAATGGCCGC

GGACATAGCG

CTTCCTCGTG

TCTTGACGAG

GGCCCAGCGC

TCAGATGCCA

ATGAGGAGTG

CTGATGAGCG

CTGCTGCCAG

AATGGGCGGC

TAGAGGACCG

GGTATGGGTT

CACAGATTCA

GACAGCCATG

TACGGGCTGT

TGAGTCCCTT

GCGTGGTGAA

TTGCAAATCC

TTGGTGAGCT

CGTTCTTGGT

CCTACTTCAA

CAGACGCCTT

ATGATCCGCA

TTCTGGTCGA

TCTGGTAAGG

TGATGGCGCA

GAACAAGATG

GACTGGGCAC

GGGCGCCCGG

GACGCAGCGC

GTTGTCACTG

CTGTCATCTC

CTGCATACGC

CGAGCACGTA

CAGGGGCTCG

GATCTCGTCG

TTTTCTGGAT

TTGGCTACCC

CTTTACGGTA

TTCTTCTGAG

GTCGATTCGG

TTCGGTGGGG

CCCAATGTTT

CACCTTCGAG

TTTGGAAGAT

GCTTGCTCCG

TTCTTCCGAG

TAACGTTTAC

GGGCGATGTC

TGGCGTCGTG

TGCGATCCCG

TACCCATCGC

TGTCATCAGC

TGCGGTACGT

GTCTGCGCGT

CTTGGACGAT

TCAGGGTCAA

TGTTGAAAAG

ATCGGTCTTG

TGATGCGCTC

TTGGGAAGCC

GGGGATCAAG

GATTGCACGC

AACAGACAAT

TTCTTTTTGT

GGCTATCGTG

AAGCGGGAAG

ACCTTGCTCC

TTGATCCGGC

CTCGGATGGA

CGCCAGCCGA

TGACCCATGG

TCATCGACTG

GTGATATTGC

TCGCCGCTCC

CGGGACTCTG

GCATTTGCCA

TGAAGTC CAG

CACGTGCCCC

CGTCGTAGCC

GCGGACGCCG

AGCGAACGCC

TTCACCGCCG

CTGGCGGCGG

ATTCCGTCCA

CTCGGTATTG

TTGGCATGCG

CTGATTGGTC

AATGCCCCGC

CGAGTGAACT

CATCTGAAGC

GCCGACCTCG

ATCTGCATGT

CTGGCGAGGA

GGTTCCTTGA

GGGGACAGCA

CTGCAAAGTA

ATCTGATCAA

AGGTTCTCCG

CGGCTGCTCT

CAAGACCGAC

GCTGGCCACG

GGACTGGCTG

TGCCGAGAAA

TACCTGCCCA

AGCCGGTCTT

ACTGTTCGCC

CGATGCCTGC

TGGCCGGCTG

TGAAGAGCTT

CGATTCGCAG

GCGTTCGAAA

TATCAATGGA

CGGCTACGC

TGCTTATTGG

CGCTGACCGG

CAGTGGCCGC

GTGCCCGACT

CAGCGAGTGA

GCATGTTGCG

ATGTGCCCGG

CGCCTTGGAA

GCAATACCGT

AGGTGTTGCA

AAGACGCTCC

TCACCGGTTC

CTGCTGTGCT

ATGCGGCGGT

CCACTGAGCG

AGGTCGCCAC

TTGATGCCAA

AGCGAACCGG

AACTGGATGG

GAGACAGGAT

GCCGCTTGGG

GATCCGCCG

CTGTCCGGTG

ACGGGCGTTC

CTATTGGGCG

GTATCCATCA

TTCGACCACC

GTCGATCAGG

AGGCTCAAGG

TTGCCGAATA

GGTGTGGCGC

GGCGGCGAAT

CGCATCGCCT

TGACCGACCA

CCGACTGTGC

AGCTTCGGCA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 -67

GTCGAGCATC

GGCACTATCC

CTCATTTCAA

CCAGTCGCGG

CAGGCATCAT

GTTGAGGTTA

CCCTTCCCGG

GATTGAGCAC

AATCTAAATC

TCTCTAACTT

AGAGTCTCGA

CATGCTCTGC

CGCAAGACGC

GTGGAATTC

TTTACCCAC

GATCTTCGGG

GATAAAAACA

AGAGGAGAGT

TCAGCCACGC

TGGAGGTATT

TGCGCTGGCT

CGCCGCGGGC

GAGCTGTTCT

ACAGTGAACG

TACCGCAGTG

GTCCGGATGC

GACCATTCAG

ATCATGCCCG

CCGGTCTTGG

CCGAGTCCAC

TGTCGATTGG

GTTCTCTCGA

AATGGCCCGC

CGGCGCTCGC

TGGATCAAGG

ATTGCAACCG

TCATCCTCCG

GGCGCTTCTT

2220 2280 2340 2400 2460 2520 2539 -68- Sequence 14

GAATTCCAAT

TGGTAAGCCT

AGAAGTGGTA

TGCACAGGCT

GCTGCGAGCG

AACTGGCGCA

GGAAGCCGCG

TAGCTTTGCC

TGCTCCGGTA

GGTGTTGAAA

TGATGCTGGT

TGCGGTGGTG

GACCCACGTT

GGAATTAGGT

CGAAGCGGCG

TCTGATTGTG

ACTGCGTGCT

TGCAGGTCAA

CTGTTGTAAT

CTGAATCGCC

ACACCGTGGA

CTGTAATGCA

GGTGGTAACG

ATGCCTCGGG

AGCAGCAACG

AAAGTTAGGT

CAAATCCATG

CTCCCAACAT

CGCGCTTGCT

CAGGTTTGAG

CCGGAGGCAG

TGGTGCTTAT

TACAAAGTTG

CTAACAATTC

TTTGCCATAT

AGTCCAGCGG

AATGACAATA

TGTTCAGCAT

TCGCGCGTCG

GCGTTTCCTG

GCGGATCTTC

GCGGGAAACT

GCCATGACCA

ATGGCGGTTC

ATCCTTGGCG

AGCTCTGAGC

CTGGGGGATG

GAGCGACTGA

GGACGGATCA

GGTAAGGCTC

GCCTTTGGTG

ACAGCAGTCG

GGCGATCCTA

CGCATCCAGG

TCATTAAGCA

AGCGGCATCA

AACGGATGAA

AGTAGCGTAT

GCGCAGTGGC

CATCCALAGCA

ATGTTACGCA

GGCTCAAGTA

CGGGCTGCTC

CAGCCGGACT

GCCTTCGACC

CAGCCGCGTA

GGCATTGCCA

GTGATCTACG

GGCATACGGG

GTTCAAGCCG

CAATGGACCC

CTACGGCAGC

ATGAGGAGTG

CTGATGAGCG

CTGCTGCCAG

AATGGGCGGC

TAGAGGACCG

GGTATGGGTT

CACAGATTCA

GACAGCCATG

TACGGGCTGT

TGAGTCCCTT

GCGTGGTGAA

TTGCAAATCC

TTGGTGAGCT

CGTTCTTGGT

CCTACTTCAA

CAGACGCCTT

ATGATCCGCA

TGGGGAGAGG

TTCTGCCGAC

GCACCTTGTC

GGCACGAACC

CCGCTCACGC

GGTTTTCATG

GCALAGCGCGT

GCAGCAACGA

TGGGCATCAT

TTGATCTTTT

CCGATTACCT

AAGAAGCGGT

GTGAGATCTA

CCGCGCTCAT

TGCAAGCAGA

AAGAAGTGAT

AGATCGGCTT

ACTGTGCATG

TTCGGCAGTC

CCCAATGTTT

CACCTTCGAG

TTTGGAAGAT

GCTTGCTCCG

TTCTTCCGAG

TAACGTTTAC

GGGCGATGTC

TGGCGTGGTG

TGCGATGCCG

TACCCATCGC

TGTCATCAGC

TGCGGTACGT

GTCTGCGCGT

CTTGGACGAT

TCAGGGTCAA

TGTTGAAAAG

ATCGGTCTTG

CGGTTTGCGT

ATGGAAGCCA

GCCTTGCGTA

CAGTTGACAT

AACTGGTCCA

GCTTGTTATG

TACGCCGTGG

TGTTACGCAG

TCGCACATGT

CGGTCGTGAG

CGGAACTTG

TGTTGGCGCT

TATCTATGAT

CAATCTCCTC

TTACGGTGAC

GCACTTTGAT

CCCAATTGGC

ACGAGGCTCA

GAGCATCGAT

CACGTGCCCC

CGTCGTAGCC

GCGGACGCCG

AGCGAACGCC

TTCACCGCCG

CTGGCGGCGG

ATTCCGTCCA

CTCGGTATTG

TTGGCATGCG

CTGATTGGTC

AATGCCCCGC

CGAGTGAACT

CATCTGAAGC

GCCGACCTCG

ATCTGCATGT

CTGGCGAGGA

GGTTCGTTGA

ATTGGGCGCA

TCACAAACGG

TAATATTTGC

AAGCCTGTTC

GAACCTTGAC

ACTGTTTTTT

GTCCATGTTT

CAGGGCAGTC

AGGCTCGGCC

TTCGGAGACG

CTCCGTAGTA

CTCGCGGCTT

CTCGCAGTCT

AAGCATGAGG

GATCCCGCAG

ATCGACCCALA

CCAGCGCGTC

GATGCCATTC

TGAGCACTTT

TGCTTATTGG

CGCTGACCGG

CAGTGGCCGC

GTGCCCGACT

CAGCGAGTGA

GCATGTTGCG

ATGTGCCCGG

CGCCTTGGAA

GCAATACCGT

AGGTGTTGCA

AAGACGCTCC

TCACCGGTTC

CTGCTGTGCT

ATGCGGCGGT

CCACTGAGCG

AGGTCGCCAC

TTGATGCCAA

TGCATAAAAA

CATGATGAAC

CCATGGACGC

GGTTCGTAAA

CGAACGCAGC

TCTACAGTCT

GATGTTATGG

GCCCTAAAAC

CTGACCAAGT

TAGCCACCTA

AGACATTCAT

ACGTTCTGCC

CCGGCGAGCA

CCAACGCGCT

TCGCTCTCTA

GTACCGCCAC

GATTCGGGCA

GGTGGGGTGA

ACCCAGCTGC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 -69

GCTGGCTGAC

CGCGGGCATC

CTGTTCTCCG

GTGAACGCCG

CGCAGTGTGT

CGGATGCGTT

CATTCAGAAT

ATGCCCGCGG

GTCTTGGTGG

AGTCCACATT

CGATTGGTCA

CTCTCGAGGC

GGCCCGCGGC

CGCTCGCCTC

ATCAAGGCCA

GCAACCGCAG

TCCTCCGGTT

GCTTCTTCCC

ACTATCCAAT

ATTTCAATCT

GTCGCGGAGA

GCATCATCAT

GAGGTTACGC

TTCCCGGGTG

CTAAATCGAT

CTAACTTGAT

GTCTCGAAGA

GCTCTGCTCA

AAGACGCTGG

GAATTC

CTTCGGGCGC

AAAAACAGAG

GGAGAGTACA

GCCACGCTAC

AGGTATTGTC

2220 2280 2340 2400 2460 2506 70 Sequence

GAATTCCAAT

TGGTAAGCCT

AGAAGTGGTA

TCCACAGGCT

GCTGCGAGCG

AACTGGCGCA

GGAAGCCGCG

TAGCTTTGCC

TGCTCCGGTA

GGTGTTGAAA

TGATGCTGGT

TGCGGTGGTG

GACCCACGTT

GGAATTAGGT

CGAAGCGGCG

TCTGATTGTG

ACTGCGTGCT

TGCAGGTCAA

TTGGCCCAGC

GCTCAGATGC

TCGATTGAGC

CCAATCTAAA

AATCTCTAAC

GGAGAGTCTC

ATCATGCTCT

TACGCAAGAC

GGGTGGAATT

AATGACAATA

TGTTCAGCAT

TCGCGCGTCG

GCGTTTCCTG

GCGGATCTTC

GCGGGAAACT

GCCATGACCA

ATGGCGGTTC

ATCCTTGGCG

AGCTCTGAGC

CTGGGGGATG

GAGCGACTGA

GGACGGATCA

GGTAAGGCTC

GCCTTTGGTG

ACAGCAGTCG

GGCGATCCTA

CGCATCCAGG

GCGTCGATTC

CATTCGGTGG

ACTTTACCCA

TCGATCTTCG

TTGATAAAAA

GAAGAGGAGA

GCTCACCCAC

GCTGGAGGTA

C

ATGAGGAGTG

CTGATGAGCG

CTGCTGCCAG

AATGGGCGGC

TAGAGGACCG

GGTATGGGTT

CACAGATTCA

GACAGCCATG

TACGGGCTGT

TGAGTCCCTT

GCGTGGTGAA

TTGCAAATCC

TTGGTGAGCT

CGTTCTTGGT

CCTACTTCAA

CAGACGCCTT

ATGATCCGCA

TTCTGGTCGA

GGGCATTTGC

GGTGAAGTCC

GCTGCGCTGG

GCCGCCGCGG

CAGAGCTGTT

GTACAGTGAA

GCTACCGCAG

TTGTCCGGAT

CCCAATGTTT

CACCTTCGAG

TTTGGAACAT

GCTTGCTCCG

TTCTTCCGAG

TAACGTTTAC

GGGCGATGTC

TGGCGTGGTG

TGCGATGCCG

TACCCATCGC

TGTCATCAGC

TGCGGTACGT

GTCTGCGCGT

CTTGGACGAT

TCAGGGTCAA

TGTTGAAAAG

ATCGGTCTTG

TGATGCGCTC

CATATCAATG

AGCGGCTACG

CTGACCATTC

GCATCATGCC

CTCCGGTCTT

CGCCGAGTCC

TGTGTCGATT

GCGTTCTCTC

CACGTGCCCC

CGTCGTAGCC

GCGGACGCCG

AGCGAACGCC

TTCACCGCCG

CTGGCGGCGG

ATTCCGTCCA

CTCGGTATTG

TTGGCATGCG

CTGATTGGTC

AATGCCCCGC

CGAGTGAACT

CATCTGAAGC

GCCGACCTCG

ATCTGCATGT

CTGGCGAGGA

GGTTCGTTGA

GCAAAAGGCG

GACCGACTGT

GCAGCTTCGG

AGAATGGCCC

CGCGGCGCTC

GGTGGATCAA

ACATTGCA-AC

GGTCATCCTC

GAGGCGCTTC

TGCTTATTGG

CGCTGACCGG

CACTGGCCGC

GTGCCCGACT

CAGCGAGTGA

GCATGTTGCG

ATGTGCCCGG

CGCCTTGGAA

GCAATACCGT

AGGTGTTGCA

AAGACGCTCC

TCACCGGTTC

CTGCTGTGCT

ATGCGGCGGT

CCACTGAGCG

AGGTCGCCAC

TTGATGCCAA

CGCAATGGAA

GCATGACGAG

CAGTCGAGCA

GCGGCACTAT

GCCTCATTTC

GGCCACTCGC

CGCAGGCATC

CGGTTGAGCT

TTCCCTTCCC

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1571 -71- Sequence 16

GAATTCCGCG

GTGACGAGGC

GTTGGTTGCG

GCCTATCGAC

ACAAATGGTC

GCTCCCGCGG

GCAGCGCATT

AGGCGCTGAT

GTATACACAC

GGAGGCATTG

GACAGCAAGC

CAAAGTAAAC

TGATCAAGAC

TTCTCCGGCC

CTGCTCTGAT

GACCGACCTG

GGCCACGACG

CTGGCTGCTA

CGAGAAAGTA

CTGCCCATTC

CGGTCTTGTC

GTTCGCCAGG

TGCCTGCTTG

CCGGCTGGGT

AGAGCTTGGC

TTCGCAGCC

TTCGAAATGA

AGAGATCGTG

TCAACTGCCT

TGAAGCCCTT

CTGTGCCGTA

GCCGGTCTTG

GCTCGGCCCT

TATCGACCTC

ATTGGGTATT

GCTTGCCGCG

GTCGGCGAAA

CACACTGTGA

GCAGTGCGCA

TTAGGGGTAA

GATAGCGTAC

CACATTGGCT

TGCGGCACAG

CACGTGCTGT

CGGGGCGGGT

TTTGATCCTG

GAACCGGAAT

TGGATGGCTT

ACAGGATGAG

CCTTGGGTGG

GCCGCCGTGT

TCCGGTGCCC

GGCGTTCCTT

TTGGGCGAAG

TCCATCATGG

GACCACCAAG

GATCAGGATG

CTCAAGGCGC

CCGAATATCA

GTGGCGGACC

GGCGAATGGG

ATCGCCTTCT

CCGACCAAGC

GCTGTTACGG

CGGAAGGCAA

TCCCGATTGA

GTGGACGGCG

GCTAGGATAC

GCGCCCGCGA

TTTGAGATAA

GAGCACTCAA

ACCGGATTGC

GTTGATGCGC

GTTGGTCAGG

CCCCCTGGAT

AGGTCGCTCG

TCGCAGGCTC

TGTACAGCGG

GCTTCGAACT

GTGTCGCGGC

TCCGCCTCGG

CTCCAGGACT

TGCCAGCTGG

TCTTGCCGCC

GATCGTTTCG

AGACGCTATT

TCCGGCTGTC

TGAATGAACT

GCGCAGCTGT

TGCCGGGGCA

CTGATGCAAT

CGAAACATCG

ATCTGGACGA

GCATGCCCGA

TGGTGGAAAA

GCTATCAGGA

CTGACCGCTT

ATCGCCTTCT

GACGCCCATT

ATGAACAGTT

AATTGTTGAT

AGCCTGTTCA

CCGCGGCGGC

TGGCTACCTC

TTCGCCTGCT

ACGAGGCGCA

AACTTAATAT

GTCTCTGCAT

TGTATCGTGG

GGGGGCTTAC

TGATTGCGGG

CGAAGTTCTG

TATGGCTCAA

TGTTCCCAAG

GCTTCGGCAG

AGAGTCCATG

TGCGCCCGTT

CGACATGATC

GGCGCCCTCT

AAGGATCTGA

CATGATTGAA

CGGCTATGAC

AGCGCAGGGG

GCAGGACGAG

GCTCGACGTT

GGATCTCCTG

GCGGCGGCTG

CATCGAGCGA

AGAGCATCAG

CGGCGAGCAT

TGGCCGCTTT

CATAGCGTTG

CCTCGTGCTT

TGACGAGTTC

GAGGGCGCAA

CGATTTAGAG

CGTGACAGTC

TTCTGGCGGG

TTTGGTGCCT

CGTAGTCGGG

GCTTGCGCGT

GGCCGCCCAA

TTGGGCGGG

GACCCTCGCT

TGAAGATCAA

TCGGCGTTTT

GGTGCCCTGT

ATGCGTGCGT

GCAAGCTTTG

TCGGTTCCGG

GCCCGCGAGC

TCGCGTAACC

GAGTTCAAGG

GCTACCGCAG

GGTAAGGTTG

TGGCGCAGGC

CAAGATGGAT

TGGGCACAAC

CGCCCGGTTC

GCAGCGCGGC

GTCACTGAAG

TCATCTCACC

CATACGCTTG

GCACGTACTC

GGGCTCGCGC

CTCGTCGTGA

TCTGGATTCA

GCTACCCGTG

TACGGTATCG

TTCTGAGCCG

GAGGAGAAAT

GGCTACAACA

ATCCGCGGCC

GTGCAGACTG

CGAGAGTCGT

ATCGAGCCCG

AGTGATCTTA

GTTCTAGCGG

GCCATTGCAC

CACCAATTGC

TCCATGCTC

CCGACACTGC

CGCTGGTGTC

CGCTTGAACC

ATGCTTACCT

CCTTGGGGGT

AGATTTCCCA

CCATCGCGTC

ATTTTTTGTG

AAAACCTGGG

GGAAGCCCTG

GATCAAGATC

TGCACGCAGG

AGACAATCGG

TTTTTGTCAA

TATCGTGGCT

CGGGAAGGGA

TTGCTCCTGC

ATCCGGCTAC

GGATGGAAGC

CAGCCGAACT

CCCATGGCGA

TCGACTGTGG

ATATTGCTGA

CCGCTCCCGA

GACTCTGGGG

GGATTGACCA

GTCGAGCAAT

TAGCAGTCTT

CGGGCAACAG

CTGCGACACA

AGCATATGGG

GTTTGAGGGA

TACAGCATGA

TTGGACACCC

AAGCTAATAA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 72

CTTTCGATAT

AGAGAATCCC

CTATCCACTG

TCGAAAATCT

TGGCAGAAAG

TCGGGCTGAT

GAATTC

GGAATTGCCT CGGCATGCAT CACTTCGGTT CGTCCTCTGC AGCTAACGGG CATCTCCTTT CTGCTAAAAA CAAGAAGAAG TTTAGGAGTC CAGGCTAAAC TGTTACCGGC ACGGGTTTCT

TGGTGGGGGA

ACGAAGTTCG

GTTGCTTTGA

GAACAGGGAA

TTGCCCTTGC

ACAGTGTACA

CAGGGGATGG

ATGATTAACA

GGTGGCGCAC

CATGATTAGT

CTTCGCACTC

TACCTTGTCA

CGGTTCTTTT

GAGTTGACCA

GAAGGAGGGC

TTCGCTCGTA

GTATTATGTG

GGGTTGGTGG

2220 2280 2340 2400 2460 2520 2526 73 Sequence 17

GAATTCCGCG

GTGACGAGGC

GTTGGTTGCG

GCCTATCGAC

ACAAATGGTC

GCTCCCGCGG

GCAGCGCATT

AGGCGCTGAT

CTATACACAC

GGAGGCATTG

GGAGAGGCGG

TGCCGACATG

CCTTGTCGCC

ACGAACCCAG

CTCACGCAAC

TTTCATGGCT

AGCGCGTTAC

GCAACGATGT

GCATCATTCG

ATCTTTTCG

ATTACCTCG

AAGCGGTTGT

AGATCTATAT

CGCTCATCAA

AAGCAGATTA

AAGTGATGCA

TCGGCTTCCC

CGGATGAACA

CAAAATTGTT

TGAAGCCTGT

GCGCCGCGGC

TACTGGCTAC

CGATTCGCCT

TAAACGAGC

CAAAACTTAA

TGCGTCTCTG

GTCGGCGAAA

CACACTGTGA

GCAGTGCGCA

TTAGGGGTAA

GATAGCGTAC

CACATTGGCT

TGCGGCACAG

CACGTGCTGT

CGGGGCGGGT

TTTGATCCTG

TTTGCGTATT

GAAGCCATCA

TTGCGTATAA

TTGACATAAG

TGGTCCAGAA

TGTTATGACT

GCCGTGGGTC

TACGCAGCAG

CACATGTAGG

TCGTGAGTTC

GAACTTGCTC

TGGCGCTCTC

CTATGATCTC

TCTCCTCAAG

CGGTGACGAT

CTTTGATATC

ATTGAGGGCG

GTTCGATTTA

GATCGTGACA

TCATTCTGC

GGCTTTGGTG

CTCCGTAGTC

GCTGCTTGCG

GCAGCCCC

TATTTGGGGC

CATGACCCTC

GTTGATCCC

GTTGGTCAGG

CCCCCTGGAT

AGGTCGCTCG

TCGCAGGCTC

TGTACAGCGG

GCTTCGAACT

GTGTCGCGGC

TCCGCCTCGG

CTCCAGGACT

GGGCGCATGC

CAAACGGCAT

TATTTGCCCA

CCTGTTCGGT

CCTTGACCGA

GTTTTTTTGT

GATGTTTGAT

GGCAGTCCC

CTCGGCCCTG

GGAGACGTAG

CGTAGTAAGA

GCGGCTTACG

GCAGTCTCCG

CATGAGGCCA

CCCGCAGTGG

GACCCAAGTA

CAAGAGGAGA

GAGGGCTACA

GTCATCCGCG

GGGGTGCAGA

GCTCGAGAGT

GGGATCGAGC

CGTAGTGATC

CAAGTTCTAG

GGGGCCATTG

GCTCACCAAT

TGTATCGTGG

GGGGGCTTAC

TGATTGCCGGG

CGAAGTTCTG

TATGGCTCAA

TGTTCCCAAG

GCTTCGGCAG

AGAGTCCATG

TGCGCCCGTT

CGACATGATC

ATAAAAACTG

GATGAACCTG

TGGACGCACA

TCGTAAACTG

ACGCAGCGGT

ACAGTCTATG

GTTATGGAGC

CTAAAACAAA

ACCAAGTCAA

CCACCTACTC

CATTCATCGC

TTCTGCCCAG

GCGAGCACCG

ACGCGCTTGG

CTCTCTATAC

CCCCCAC CTA

AATGGATTGA

ACAGTCGAGC

GCCTAGCACT

CTCCCGGCAA

CGTCTGCGAC

CCGAGCATAT

TTAGTTTGAG

CGGTACAGCA

CACTTGGACA

TGCAAGCTAA

TGAAGATCAA

TCGGCGTTTT

GGTGCCCTGT

ATGCGTGCGT

GCAAGCTTTG

TCGGTTCCCG

GCCGGCGAGC

TCGCGTAACC

GAGTTCAAGG

GCTACCGCAG

TTGTAATTCA

AATCGCCAGC

CCGTGGAAAC

TAATGCAAGT

GGTAACGGCG

CCTCGGGCAT

AGCAACGATG

GTTAGGTGGC

ATCCATGCGG

CCAACATCAG

GCTTGCTGCC

GTTTGAGCAG

GAGGCAGGGC

TGCTTATGTG

AAAGTTGGGC

ACAATTCGTT

CCAAGAGATC

AATTGAACTG

CTTTGAAGCC

CAGCTGTGCC

ACAGCCGGTC

GGGGCTCGGC

GGATATCGAC

TGAATTGGGT

CCCGCTTGCC

TAACTTTCGA

TCCATGCTGC

CCGACACTC

CGCTGGTGTC

CGCTTGAACC

ATGCTTACCT

CCTTGGGGGT

AGATTTCCCA

CCATCGCGTC

ATTTTTTGTG

AAAACCTGGG

TTAAGCATTC

GGCATCAGCA

GGATGAAGGC

AGCGTATGCG

CAGTGGCGGT

CCAAGCAGCA

TTACGCAGCA

TCAAGTATGG

GCTGCTCTTG

CCGGACTCCG

TTCGACCAAG

CCGCGTAGTG

ATTCCACCG

ATCTACGTGC

ATACGGGAAG

CAAGCCGAGA

GTGGCTGTTA

CCTCGGAAGG

CTTTCCCGAT

GTAGTGGACG

TTGGCTAGGA

CCTGCGCCCG

CTCTTTGAGA

ATTGAGCACT

GCGACCGGAT

TATGGAATTG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 -74- CCTCGGCATG CATTGGTGG GTTCGTCCTC TGCACGAAGT GGGCATCTCC TTTGTTGCTT AAACAAGAAG AAGGAACAGG GTCCAGGCTA AACTTGCCCT GGCACGGGTT TCTACAGTGT

GGACAGGGGA

TCGATGATTA

TGAGGTGGCG

GAACATGATT

TGCCTTCGCA

ACATACCTTG

TGGCGGTTCT

ACAGAGTTGA

CACGAAGGAG

AGTTTCGCTC

CTCGTATTAT

TCAGGGTTGG

TTTAGAGAAT

CCACTATCCA

GGCTCGAAAA

GTATGGCAGA

GTGTCGGGCT

TGGGAATTC

CCCCACTTCG

CTGAGCTAAC

TCTCTGCTAA

AAGTTTAGGA

GATTGTTACC

2220 2280 2340 2400 2460 2509 75 Sequence 18

GAATTCCGCG

GTGACGAGGC

GTTGGTTGCG

GCCTATCGAC

ACAAATGGTC

GCTCCCGCGG

GCAGCGCATT

AGGCGCTGAT

GTATACACAC

GGAGGCATTG

GCGCATTGAG

AACAGTTCGA

TGTTGATCGT

CTGTTCATTC

CGGCGGCTTT

CTACCTCCGT

GCCTGCTGCT

AGGCGCAGGC

TTAATATTTG

TCTGCATGAC

CATGCATTGG

CCTCTGCACG

CTCCTTTGTT

GAAGAAGGAA

GCTAAACTTG

GGTTTCTACA

GTCGGCGAAA

CACACTGTGA

GCAGTGCGCA

TTAGGGGTAA

GATAGCGTAC

CACATTGGCT

TGCGGCACAG

CACGTGCTGT

CGGGGCGGGT

TTTGATCCTG

GGCGCAAGAG

TTTAGAGGC

GACAGTCATC

TGGCGGGGTG

GGTGGCTCGA

AGTCGGGATC

TGCGCGTAGT

CGCCCAAGTT

GGGCGGGGCC

CCTCGCTCAC

TGCGGGACAG

AAGTTCGATG

GCTTTGAGGT

CAGGGAACAT

CCCTTGCCTT

GTGTACATAC

GTTGATGCGC

GTTGGTCAGG

CCCCCTGGAT

AGGTCGCTCG

TCGCAGGCTC

TGTACAGCGG

GCTTCGAACT

GTGTCGCGGC

TCCGCCTCGG

CTCCAGGACT

GAGAAATGGA

TACAACAGTC

CGCGGCCTAG

CAGACTGCGG

GAGTCGTCTG

GAGCCCGAGC

GATCTTAGTT

CTAGCGGTAC

ATTGCACTTG

CAATTGCAAG

GGGATGGCGG

ATTAACAGAG

GGCGCACGAA

GATTAGTTTC

CGCACTCGTA

CTTGTCAGGG

TGTATCGTGG

GGGGGCTTAC

TGATTGCGGG

CGAAGTTCTG

TATGGCTCAA

TGTTCCCAAG

GCTTCGGCAG

AGAGTCCATG

TGCGCCCGTT

CGACATGATC

TTGACCAAGA

GAGCAATTGA

CAGTCTTTGA

GCAACAGCTG

CGACACAGCC

ATATGGGGCT

TGAGGGATAT

AGCATGAATT

GACACCCGCT

CTAATAACTT

TTCTTTTAGA

TTGACCACTA

GGAGGGCTCG

GCTCGTATG

TTATGTGTCG

TTGGTGGGAA

TGAAGATCAA

TCGGCGTTTT

GGTGCCCTGT

ATGCGTGCGT

GCAAGCTTTG

TCGGTTCCGG

GCCGGCGAGC

TCGCGTAACC

GAGTTCAAGG

GCTACCGCAG

GATCGTGGCT

ACTGCCTCGG

AGCCCTTTCC

TGCCGTAGTG

GGTCTTGGCT

CGGCCCTGCG

CGACCTCTTT

GGGTATTGAG

TGCCGCGACC

TCGATATGGA

GAATCCCCAC

TCCACTGAGC

AAAATCTCTG

CAGAAAGTTT

GGCTGATTGT

TTC

TCCATGCTGC

CCGACACTGC

CGCTGGTGTC

CGCTTGAACC

ATGCTTACCT

CCTTGGGGGT

AGATTTCCCA

CCATCGCGTC

ATTTTTTGTG

AAAACCTGGC

GTTACGGATG

AAGGCAAAAT

CGATTGAAGC

GACGGCGCCG

AGGATACTGG

CCCGCGATTC

GAGATAAACG

CACTCAAAAC

GGATTGCGTC

ATTCCCTCGG

TTCGGTTCGT

TAACGGGCAT

CTAAAAACAA

AGGAGTCCAG

TACCGGCACG

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1543

Claims

1. Transformed and/or mutagenized unicellular or multicellular organism, wherein an enzyme of eugenol and/or ferulic acid catabolism is inactivated by inserting an omega element or introducing a deletion into a corresponding gene, such that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid accumulate.

2. Organism according to claim 1, characterized in that a gene encoding any of the enzymes coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases is inactivated.

3. Organism according to one of claims 1 or 2, characterized in that it is unicellular, preferably a microorganism or a plant or animal cell.

4. Organism according to one of claims 1 to 3, characterized in that it is a bacterium, preferably a Pseudomonas species. 20

5. Gene structure for preparing transformed organisms and mutants, comprising a nucleotide sequence encoding any of the enzymes coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, vanillin dehydrogenases or vanillic acid demethylases, said nucleotide sequence being inactivated.

6. Gene structure, comprising a nucleotide sequence encoding for any of the enzymes coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, .i feruloyl-CoA synthetases, enoyl-CoA hydratase-aldolases, beta-ketothiolases, P:\WPDOCS\CRN\Punia\Speci\7595580.claims.doc-24/3/03 vanillin dehydrogenases or vanillic acid demethylases, said nucleotide sequence being inactivated by inserting an omega element or introducing a deletion.

7. Gene structure having any of the structures given in Figures la to Ir.

8. Gene structure having any of the sequences given in Sequence 1 to Sequence 18. 0* S S S S

9. Vector, comprising at least one gene structure according to one of claims 5 to 8.

10. Organism according to one of claims 1 to 4, characterized in that it harbours a vector according to claim 9.

11. Organism according to one of claims 1 to 4, characterized in that it contains a gene structure according to one of claims 5 to 8, which is integrated into the genome instead of the respective intact gene.

12. Process for the biotechnological preparation of organic compounds, in particular alcohols, aldehydes and organic acids, characterized in that an organism according to one of claims 1 to 4, 10 or 11 is employed.

13. Use of an organism according to one of claims 1 to 4, 10 or 11 for preparing coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillic acid.

14. Use of a gene structure according to one of claims 5 to 8 or a vector according 25 to claim 9 for preparing transformed and/or mutagenized organisms.

S .00. 6600 a S S 5S *S S *5 S 50 S S P:\WPDOCS\CRN\Punia\Spmd\759580.cimsdoc243/03 36 Transformed organism according to any one of claims 1 to 4, substantially as described herein with reference to the examples.

16. A gene structure according to claim 5 or 6, substantially as described herein with reference to the examples.

17. A vector according to claim 9 substantially as described herein with reference to the examples.

18. Process according to claims 12 substantially as described herein with reference to the examples.

19. Use according to claim 13 or 14 substantially as described herein with reference to the examples. DATED this 24th day of March, 2003 HAARMANN REIMER GMBH 0000 By its Patent Attorneys S

20 DAVIES COLLISON CAVE *oo 0*OS *0,0 *ft