SK5742001A3 - Construction of production strains for producing substituted phenols by specifically inactivating genes of the eugenol and ferulic acid catabolism - Google Patents

Abstract

The invention relates to a transformed and/or mutagenated unicellular or multicellular organism which is characterized in that enzymes of the eugenol and/or ferulic acid catabolism are deactivated in such a manner that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and/or vanillinic acid are accumulated.

Description

The present invention relates to the construction of production strains and to a process for the preparation of substituted methoxyphenols, in particular vanillin.

BACKGROUND OF THE INVENTION

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

In 1995, Rosazza et al. (Biocatalytic transformation of ferulic acid: an abundant aromatic natural product; J. Ind. Microbiol. 15: 457471) an extensive review of biotransformations feasible on ferulic acid.

In EP-A 0 845 532, genes and enzymes from Pseudomonas sp. for the synthesis of coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid.

The Institute of Food Research, Norwich, UK, described vWO

97/35999 enzymes for converting trans-ferulic acid to trans-feruloylSCoA ester and successively vanillin, as well as a gene for hydrolyzing said ester. In year

In 1998, the contents of the patent were published in the form of scientific publications (Gasson et al. 1998. Metabolism of ferulic acid to vanillin. Metabolism of ferulic acid

31700 h · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· 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).

DE-A 195 32 317 describes the use of Amycolatopsis sp. in fermentative recovery of vanillin from ferulic acid in high yields.

The known processes have the disadvantage that they either achieve only very low yields of vanillin or use expensive starting compounds. While the latter process (DE-A 195 32 317) achieves high yields, the use of Pseudomonas sp. HR199 and Amycolatopsis sp. HR167 for the biotransformation of eugenol to vanillin requires fermentation which takes place in two steps, resulting in substantial cost and time-consuming costs.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to construct organisms which are capable of converting the relatively cheap raw material eugenol into vanillin in a one-step process.

This objective is achieved by constructing production strains of unicellular or multicellular organisms, which strains are characterized in that the enzymes involved in the catabolism of eugenol and / or ferulic acid are inactivated, so that the coniferyl alcohol, coniferyl aldehyde, or ferulic acid / ferulic acid intermediates accumulate. vanillic.

The production strain may be single-cell or multicellular. Therefore, the invention may relate to microorganisms, plants, or animals. In addition, extracts obtained from production strains may also be used. by

31700 h • The invention is preferred the use of unicellular organisms, which may be microorganisms or plant or animal cells. According to the invention, the use of filamentous fungi and bacteria is particularly preferred. Most preferred is the use of bacteria. Among the bacteria which can be used primarily after altering their catabolism of eugenol and / or ferulic acid, they are species of the genera Rhodococcus, Pseudomonas and Escherichia.

In the simplest case, known, commonly used microbiological methods can be used to isolate the organisms useful in the invention. In this case, the enzymatic activity of the proteins involved in catabolic conversions of eugenol and / or ferulic acid can be altered by the use of enzyme inhibitors. furthermore, the enzymatic activity of the proteins involved in the catabolism of eugenol and / or ferulic acid can be altered by mutating the genes encoding these proteins. These mutations can be generated randomly by classical methods, for example using ultraviolet radiation or mutants.

Recombinant DNA methods such as deletions, insertions and / or nucleotide exchanges are also useful for isolating new organisms. Thus, for example, the genes of organisms can be inactivated using other DNA elements (Ω elements). Appropriate vectors can also be used to replace intact genes with gene structures that are altered or inactivated. In this case, the genes to be inactivated and the DNA elements that are used for inactivation can be obtained by classical cloning techniques or by chain polymerase reactions (PCR).

31700 h

For example, according to one embodiment of the invention, the catabolism of eugenol and the catabolism of ferulic acid may be altered by insertion

Ω elements into the respective genes or deletions carried out on these genes. In this case, to inactivate the functions of genes that code for dehydrogenases, synthetases, hydratase-aldolase, thiolases, or demethylases, they can be inactivated. Using the previously mentioned recombinant DNA methods, the production of key enzymes is then blocked. Preferably, they are genes that code for coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetases, enoyl-CoA-hydratase aldolase, beta-ketothiolase, vanillin dehydrogenase, or vanilla acid demethylase. Particularly preferred are genes that encode the amino acid sequences specified in EP-A 0845532 and / or nucleotide sequences that encode their allelic variations.

Accordingly, the present invention also relates to gene structures for the preparation of transformed organisms and mutants.

Preferably, gene structures are used in which nucleotide sequences encoding dehydrogenases, synthetases, hydratase-aldolase, thiolases, or demethylases are inactivated to isolate these organisms and mutants. Particularly preferred are gene structures in which the nucleotide sequences encoding coniferyl alcohol dehydrogenases, coniferyl aldehyde dehydrogenases, feruloyl-CoA synthetase, enoyl-CoA-hydratasealdolase, beta-thiolase, vanillin dehydrogenase, or vanilla acid demethylase are inactivated. Particularly preferred are gene structures having the structure shown in Figures 1a to 1r and having the nucleotide sequences described in Figures 2a to 2r and / or nucleotide sequences encoding allelic variants thereof. In this regard, nucleotide sequences of 1 to 18 are particularly preferred.

The invention also encompasses portions of the sequences of said gene structures as well as functional equivalents thereof. Functional equivalents are those DNA derivatives in which individual nucleobases have been exchanged (wobble exchanges) without changing the function. Also, amino acids can be exchanged at the protein level without altering function.

31700 h ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ··

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

The invention furthermore relates to plasmids and / or vectors for the preparation of organisms and mutants which are transformed in accordance with the invention. These organisms and mutants subsequently harbor the gene structures described above. The invention therefore also relates to organisms harboring said plasmids and / or vectors.

The nature of the plasmids and / or vectors depends on the purpose for which they are to be used. For example, in order to replace the intact eugenol and / or ferulic acid catabolism genes in pseudomonads with genes that have been inactivated by omega elementary vectors are needed which, on the one hand, can be transferred to pseudomonads (conjugate transferable plasmids) but these organisms are replicated and are therefore unstable in pseudomonads (so-called suicide plasmids). DNA segments transferred to pseudomonads using such a plasmid system are only maintained in the bacterial cell genome if they are integrated into it by homologous recombination.

The described gene structures, vectors and plasmids can be used to prepare various transformed organisms or mutants. Said gene structures may be used to replace intact nucleic acid sequences with altered and / or inactivated gene structures. In cells obtainable by transformation or transfection or conjugation, the intact gene is replaced by an altered and / or inactivated gene structure by homologous recombination, as a result of which the resulting cells contain only altered and / or inactivated gene structure in their genome. In this way, genes can be altered and / or inactivated in accordance with the invention so that the relevant organisms are capable of producing

31700 h ·· ·· ·· ·· ··· · · · ··· · e ····· · e ··· · · ······ « Coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid.

Mutants of Pseudomonas sp. HR199 (DSM 7063), which has been 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 according to the invention, with respective gene structures resulting inter alia from Figures 1a to 1r. in combination with Figures 2a to 2r.

1. Pseudomonas sp. HR199ca / QQKm, which contains the QKm-inactivated calA gene instead of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. 1a; Fig. 2a).

2. Pseudomonas sp. HR199ca / AQGm, which contains the QGm-inactivated calA gene instead of the intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. 1b; Fig. 2b).

3. Pseudomonas sp. HR199ca / AA, which contains a deletion inactivated calA gene instead of an intact calA gene encoding coniferyl alcohol dehydrogenase (Fig. 1c; Fig. 2c).

4. Pseudomonas sp. HR199ca / SQKm, which contains the QKm-inactivated calB gene instead of the intact calB gene encoding coniferylaide dehydrogenase (Fig. 1d; Fig. 2d).

5. Pseudomonas sp. HR199ca / fQGm, which contains the QGm-inactivated calB gene instead of the intact calB gene encoding coniferylaide dehydrogenase (Fig. 1e; Fig. 2e).

6. Pseudomonas sp. HR199ca / SA, which contains a deletion-inactivated calB gene instead of an intact calB gene encoding coniferylaide dehydrogenase (Fig. 1f; Fig. 2f).

7. Pseudomonas sp. HR199fcsQKm, which contains the QKm-inactivated fcs gene instead of the intact fcs gene encoding feruloyl-CoA synthetase (Fig. 1g; Fig. 2g).

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· · · · · · · ·  · · • · • · • • • • • · · • • · • • · · · • • • • e • • • · • • ··· · · · • · · · · · • ·

8. Pseudomonas sp. HR199fcsOGm, which contains the QGm-inactivated fcs gene instead of the intact fcs gene encoding feruloyl-CoA synthetase (Fig. 1h; Fig. 2h).

9. Pseudomonas sp. HR199fcsA, which contains a deletion-inactivated fcs gene instead of an intact fcs gene encoding coniferyl alcohol dehydrogenase (Fig. 1i; Fig. 2i).

10. Pseudomonas sp. HR199echQKm, which contains the QKm-inactivated ech gene instead of the intact ech gene encoding enoyl-CoAhydratase-aldolase (Fig. 1j; Fig. 2j).

11. Pseudomonas sp. HR199echQGm, which contains the QGm-inactivated ech gene instead of the intact ech gene encoding enoyl-CoAhydratase-aldolase (Fig. 1k; Fig. 2k).

12. Pseudomonas sp. HR199ec / 7A, which contains a deletion-inactivated ech gene instead of an intact ech gene encoding enoyl-CoAhydratase-aldolase (Fig. 11; Fig. 21).

13. Pseudomonas sp. HR199a and qKm, which contains the QKm-inactivated aat gene instead of the intact aat gene encoding beta-ketothiolase (Fig. 1m; Fig. 2m).

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

15. Pseudomonas sp. HR199aaA, which contains a deletion inactivated aat gene instead of an intact aat gene encoding beta-ketothiolase (Fig. 1o; Fig. 2o).

16. Pseudomonas sp. HR199vdhQKm, which contains the QKm-inactivated vdh gene instead of the intact vdh gene encoding vanillin dehydrogenase (Fig. 1p; Fig. 2p).

17. Pseudomonas sp. HR199vd / 7QGm, which contains the QGm-inactivated vdh gene instead of the intact vdh gene encoding vanillin dehydrogenase (Fig. 1p; Fig. 2p).

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9999

99 ··· · · · ··· · · · ···

18. Pseudomonas sp. HR199vdbA, which contains a deletion-inactivated vdh gene instead of an intact vdh gene encoding vanillin dehydrogenase (Fig. 1 r; Fig. 2r).

19. Pseudomonas sp. HR199vBQKm, which contains the QKm-inactivated vdhB gene instead of the intact vdhB gene encoding vanillin dehydrogenase II.

20. Pseudomonas sp. HR199vdhBQGm, which contains a QGm-inactivated vdhB gene instead of an intact vdhB gene encoding vanillin dehydrogenase II.

21. Pseudomonas sp. HR199vd /? BA, which contains a deletion-inactivated vdhB gene instead of an intact vdhB gene encoding vanillin dehydrogenase II.

22. Pseudomonas sp. HR199addQKm, which contains a QKm-inactivated adh gene instead of an intact adh gene encoding an alcohol dehydrogenase.

23. Pseudomonas sp. HR199ad / 7QGm, which contains a QGm-inactivated adh gene instead of an intact adh gene encoding an alcohol dehydrogenase.

24. Pseudomonas sp. HR199addA, which contains a deletion-inactivated adh gene instead of an intact adh gene encoding an alcohol dehydrogenase.

25. Pseudomonas sp. HR199vanAQKm, which contains the QKm-inactivated vanA gene instead of the intact vanA gene encoding the vanilla acid demethylase α-subunit

26. Pseudomonas sp. HR199vanAQGm, which contains the QGm-inactivated vanA gene instead of the intact vanA gene encoding the vanilla acid demethylase α-subunit

27. Pseudomonas sp. HR199vanA, which contains a deletion-inactivated vanA gene instead of an intact vanA gene encoding the vanilla acid demethylase α-subunit

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28. Pseudomonas sp. HR199ViOKm which contains the OKm-inactivated vanB gene instead of the intact vanB gene encoding the vanilla acid demethylase β-subunit

29. Pseudomonas sp. HR199vanfiQGm, which contains the QGm-inactivated vanB gene instead of the intact vanB gene encoding the vanilla acid demethylase β-subunit

30. Pseudomonas sp. HR199vanSA, which contains a deletion inactivated vanB gene instead of an intact vanB gene encoding the vanilla acid demethylase β-subunit

The invention also relates to a process for the biotechnological preparation of organic substances. In particular, the process can be used to prepare alcohols, aldehydes and organic acids, preferably coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, and vanilla acid.

The above described organisms were used in this new process. Particularly preferred organisms include bacteria, especially Pseudomonas species. More specifically, the aforementioned species of the genus Pseudomonas may preferably be used in the following processes:

1. Pseudomonas sp. HR199ca / AQKm, Pseudomonas sp. HR199ca / AQGm and Pseudomonas sp. HR199ca / AA for the preparation of coniferyl alcohol from eugenol

2. Pseudomonas sp. HR199ca / BQKm, Pseudomonas sp. HR199ca / BQGm and Pseudomonas sp. HR199ca / SA for the preparation of coniferyl aldehyde from eugenol or coniferyl alcohol

3. Pseudomonas sp. HR199fcsQKm, Pseudomonas sp. HR199fcsQGm, Pseudomonas sp. HR199fcsA, Pseudomonas sp. HR199echQKm, Pseudomonas sp. HR199echQGm and Pseudomonas sp. HR199echA na

31700 h • Preparation of feruic acid zeugenol or coniferyl alcohol or coniferyl aldehyde.

4. Pseudomonas sp. HR199vdhQKm, Pseudomonas sp. HR199vdhQGm.

Pseudomonas sp. HR199vdrA, Pseudomonas sp. HR199vdrqQGmvdhBQKm, Pseudomonas sp.

HR199vd / 7QKmvd / 7µGm, Pseudomonas sp. HR199vdrvAvdrvBQGm and Pseudomonas sp. HR199vd / 7AvdriBQKm for the preparation of vanillin from eugenol or coniferyl alcohol or coniferyl aldehyde or feruic acid

5. Pseudomonas sp. HR199vanADKm, Pseudomonas sp. HR199vanAQGm, Pseudomonas sp. HR199vanAA, Pseudomonas sp. HR199Vanfin, Pseudomonas sp. HR199vanSDGm and Pseudomonas sp. HR199VanfiA for the preparation of vanillic acid zeugenol or coniferyl alcohol, or coniferyl aldehyde or feruic acid or vanillin

The preferred substrate is eugenol. However, it is also possible to add other substrates or even replace eugenol with another substrate.

Suitable nutrient media for the organisms used according to the invention are synthetic, semi-synthetic or complex culture media. These media may contain carbon and nitrogen substances, inorganic salts where trace elements are needed, and vitamins.

Suitable carbonaceous substances may be carbohydrates, hydrocarbons or common organic compounds. Examples of preferred substances are sugars, alcohols or sugar alcohols, organic acids or complex mixtures.

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The sugar is preferably glucose. The organic acids used may preferably be citric acid and acetic acid. Examples of complex mixtures are malt extract, yeast extract, casein or casein hydrolyzate.

Inorganic compounds are suitable nitrogenous substrates. Examples are nitrates and ammonium salts. Organic nitrogen sources can also be used. These include yeast extract, soybean meal, casein, casein hydrolyzate and corn steep liquor.

Examples of inorganic salts that can be used are sulfates, nitrates, chlorides, carbonates and phosphates. The metals containing these salts are preferably sodium, potassium, magnesium, manganese, calcium, zinc and iron.

The culture temperature is preferably in the range of 5 to 100 ° C. In particular, a range of 15 to 60 ° C is preferred, a range of 22 to 37 ° C is most preferred.

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

In principle, any fermenter with which the skilled person can work can be used for this new process. All devices suitable for submerged processes are preferred. That is, according to the invention, vessels equipped with or without a mechanical agitator can be used. Examples of vessels without agitator are shaker, bubble column or recirculation reactors. Apparatus with a mixing device preferably includes all known apparatuses equipped with stirrers of any possible type.

This new process can be carried out continuously or batchwise.

Fermentation time required to reach the maximum amount of product

31700 h • ··· · · depends on the specific nature of the organism used. In principle, however, the fermentation times are between 2 and 200 hours.

The subject matter of the invention is explained in more detail with reference to the examples as follows:

Mutants of Pseudomonas sp. HR199 (DSM 7063) utilizing eugenol was specifically generated by specific inactivation of eugenol catabolism genes by insertion of omega elements or deletions. The omega elementary used were DNA segments that encode antibiotic resistance of kanamycin (ΩΚπί) and gentamycin (Gm). These resistance genes were isolated using standard methods of zTn5 and plasmid pBB1MCS-5. The genes calA, calB, fcs, ech, aat, vdh, adh, vdhB, vanA and vanB, which code for coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloyl-CoA synthetase, enoyl-CoA-hydratase-aldolase dehydrogenase, beta-vanotheolase, demethylase of vanilic acid were isolated by standard methods of genomic DNA of Pseudomonas sp. HR199 and cloned into pBluescript SK '. By digesting with the appropriate restriction endonucleases, the DNA segments were removed from these genes (deletion) or replaced by Ω elemental (insertion), thereby inactivating the genes of interest. The genes mutated in this way were then recloned into conjugally transferable vectors and sequentially introduced into the Pseudomonas sp. HR199. By appropriate selection, transconjugants were obtained in which the respective functional genes of the parent strain were replaced with established inactivated genes. The insertion and deletion mutants obtained in this way contained only the respective inactivated genes. This procedure was used both to obtain mutants having only one defective gene as well as to obtain multiple mutants having several genes inactivated in this way. These mutants were used for biotransformation:

(a) eugenol to coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid;

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9 9 e ··· e · e ···············

b) coniferyl alcohol to coniferyl aldehyde, ferulic acid, vanillin and / or vanillaic acid;

c) coniferyl aldehyde to ferulic acid, vanillin and / or vanillaic acid;

d) ferulic acid to vanillin and / or vanillic acid;

e) vanillin to vanillic acid.

Materials and methods

Conditions for the cultivation of bacteria.

Escherichia coli strains were propagated at 37 ° C in mineral medium Luria-Bertani (LB) or M9 (J. Sambrook, EF Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. Molecular cloning: laboratory manual. 2nd Edition. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Pseudomonas sp. were propagated at 30 ° C in Nutrient Medium (SM, 0.8%, w / v) or in Mineral Medium (MM) (HG Schlegel, et al. 1961. Árch. Microbiol. 38: 209-222), or in HR mineral medium (HR-MM) (J. Rabenhorst, 1996. Appl. Microbiol. Biotechnol. 46: 470-474). Ferulic acid, vanillin, vanilic acid and protocatechic acid were dissolved in dimethylsulfoxide and added to the appropriate medium in an amount such that the final concentration was 0.1% (w / v). Eugenol was added directly to the medium to a final concentration of 0.1% (v / w) or loaded onto filter paper (595 round filter, Schleicher & Schueil, Dassel, Germany) in MM agar plate lids. In promoting the transconjugants and mutants of Pseudomonas sp. tetracycline at a final concentration of 25 pg / ml, kanamycin at a final concentration of 100 gg / ml, and gentamycin at a final concentration of 7.5 pg / ml were used.

Qualitative and quantitative detection of metabolic intermediates in cultures.

Culture medium supernatants were analyzed by high performance liquid chromatography (Knauer HPLC) either directly or after dilution with

31700 h Double distilled H2O. Chromatography was performed on a Nucleosil 100 C18 column (7 gm, 250 x 4 mm). A mixture of 0.1% (v / v) formic acid and acetonitrile was used as solvent. The gradient of the gradient elution used 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

Purification of vanillin dehydrogenase II.

Purification was performed at 4 ° C.

Coarse extract

Pseudomonas sp. HR199 propagated on eugenol was washed in 10 mM sodium phosphate buffer, pH 6.0, resuspended in the same buffer and broken by double pass through a French press (Amicon, Silver Spring, Maryland, USA) at 1000 psi pressure. The cell homogenate was subjected to centrifugation (1h, 100,000 x g, 4 ° C) to obtain a soluble fraction of the crude extract as a supernatant.

Anion exchange chromatography on DEAE Sephacel.

The soluble crude extract fraction was dialyzed overnight against 10 mM sodium phosphate buffer, pH 6.0. The dialysate was applied to a DEAE -ephacel column (2.6 cm x 35 cm, column volume 186 ml) stabilized in 10 mM sodium phosphate buffer, pH 6.0, at a flow rate of 0.8 ml / min. The column was washed with two column volumes of 10 mM phosphate buffer, pH 6.0. Vanillin dehydrogenase II (VDH II) was eluted with a linear salt gradient from 0 to 400 mM NaCl in 10 mM sodium phosphate buffer, pH 6.0 (750 mL), collecting fractions of

31700 h ·· ·· ·· ·· ·· ········ 9 9 9 9 Volume 10 ml. High VDH II fractions were pooled into a common DEAE fraction.

Determination of vanillin dehydrogenase activity

VDH activity was determined at 30 ° C using an optical enzyme assay. The 1 ml reaction mixture contained 0.1 mmol sodium phosphate (pH 7.1), 0.125 μιτιοΙ vanillin, 0.5 μιτιοΙ NAD, 1.2 μιτιοΙ sodium pyruvate, lactate dehydrogenase (1U, from pig heart), and enzyme solution . Vanillin oxidation was monitored at a wavelength of λ = 340 nm (e _va niiin = 11.6 ατι ² / μΐτ) οΙ). Enzyme activity was expressed in units (U), with 1 U corresponding to the amount of enzyme that converts 1 μπιοΙ vanillin per minute. Protein concentrations in the samples were determined by the method of Lowry et al. (OH Lowry, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of coniferyl alcohol dehydrogenase activity.

Coniferyl alcohol dehydrogenase activity was determined at 30 ° C by the optical enzyme assay of Jaeg et al. (EL Jaeger, Eggeling and H. Sahm. 1981. Current Microbiology. 6: 333-336). The 1 ml reaction mixture contained 0.2 mmol tris / HCl (pH 9.0), 0.4 μmτιοΙ coniferyl alcohol, 2 μΓηοΙ NAD, 0.1 mmol semicarbazide and enzyme solution. NAD reduction was monitored at λ = 340 nm (ε = 6.3 ατι ² / μηιοΙ). Enzyme activity was expressed in units (U), with 1 U corresponding to the amount of enzyme that converts 1 μιτιοΙ of substrate per minute. Protein concentrations in the samples were determined by the method of Lowry et al. (OH Lowry, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Determination of coniferyl aldehyde dehydrogenase activity.

Coniferyl aldehyde dehydrogenase activity was determined at 30 ° C by an optical enzyme assay. The 1 ml reaction mixture contained 0.1 mmol tris / HCl (pH 8.8), 0.08 μιτιοΙ coniferyl aldehyde, 2.7 μητο! NAD and solution

31700 h · ································· ·· ·· ·· enzyme. The oxidation of coniferyl aldehyde to ferulic acid was monitored at λ = 400 nm (ε = 34 cm ² / pmol). Enzyme activity was expressed in units (U), with 1 U corresponding to the amount of enzyme that converts 1 μηηοΙ of substrate per minute. Protein concentrations in the samples were determined by the method of Lowry et al. (OH Lowry, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

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

The activity of feruloyl-CoA synthetase was determined at 30 ° C by modification of the optical enzyme assay of Zen et al. (Zenk et al. 1980. Anal. Biochem. 101: 182-187). The 1 ml reaction mixture contained 0.09 mmol of potassium phosphate (pH 7.0), 2.1 pmol MgCl 2, 0.7 µl ferulic acid, 2 µmol ATP, 0.4 µmol of coenzyme A and the enzyme solution. CoA ester formation from ferulic acid was monitored at λ = 345 nm (ε = 10 cm ² / pmol). Enzyme activity was expressed in units (U), with 1 U corresponding to the amount of enzyme that converts 1 μιτιοΙ of substrate per minute. Protein concentrations in the samples were determined by the method of Lowry et al. (OH Lowry, NJ Rosebrough, AL Farr and RJ Randall. 1951. J. Biol. Chem. 193: 265-275).

Electrophoretic methods

Protein-containing extracts were fractionated under native conditions in 7.4% (w / v) polyacrylamide gels by the method of Stegermann et al. (Stegermann et al. 1973. Z. Naturforsch. 28c: 722-732) and under denaturing conditions in 11.5% (w / v) polyacrylamide gels by the method of Laemmli (Laemmli, UK 1970. Nature (London) 227: 680-685). Non-specific protein staining was performed with Serva Blue R. For specific staining of coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase and vanillin dehydrogenase, gels were gassed for 20 min. buffered in 100 mM potassium phosphate buffer (pH 7.0) and incubated successively at 30 ° C in the same buffer to which 0.08% (w / v) NAD was added,

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9 · · · ·· ·

0.04% (w / v) p-nitro blue tetrazolium chloride, 0.003% (w / v) phenazine metosulfate and 1 mM of the respective substrate until the color bands become 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 (B32 / 33, Biometra, Gottingen, Germany) according to the manufacturer's instructions.

Determination of N-terminal amino acid sequences.

N-terminal amino acids were determined using a Protein Peptide Sequencer (Type 477 A, Applied Biosystems, Foster City, USA) and a PTH analyzer according to the manufacturer's instructions.

DNA isolation and processing

Genomic DNA was isolated by the method of Marmur (J. Marmur, 1961. J. Mol. Biol. 3: 208-218). Other plasmid DNA and / or DNA restriction fragments were isolated and analyzed by standard methods (JE Sambrook, F Fritsch and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2nd Edition., Cold Spring Harbor Laboratories Press, Cold Spring Harbor, New York).

DNA transfers.

Competent Escherichia coli cells were prepared and transformed by the Hanahan method (D. Hanahan, J. Mol. Biol. 166: 557-580). Conjugative transfer of plasmids between Escherichia coli S17-1 strains harboring plasmids (donor) and Pseudomonas sp. (recipient) was performed on nutrient agar plates according to the method of Friedrich et al. (B. Friedrich et al. 1981. J. Bacteriol. 147: 198-205), or by the "mini-complementation method", on mineral medium agar plates containing 0.5% (w / v) gluconate as a carbon source and 25 pg tetracycline / ml or 100 μ9 kanamycin / ml. In this case, the cells

31700 h ························· They were applied to the recipient by dashing in one direction as the inoculation line. After five minutes, donor strains were applied as inoculation lines, crossing the recipient inoculation line. After a 48 hour incubation at 30 ° C, transconsultants grew directly at the crossing points of the inoculation line, while neither the donor strain nor the recipient strain were able to grow.

Hybridization experiments

The restriction DNA fragments were electrophoretically fractionated in a 0.8% (w / v) agarose gel in 50 mM tris-50 mM boric acid-1.25 mM EDTA buffer (pH 8.25) (JE Sambrook, F. Fritch) , and T. Maniatis, 1989, Molecular Cloning: a Laboratory Manual Molecular Cloning: Laboratory Manual 2 ^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Transfer of denatured DNA from the gel to a positively charged nylon membrane (pore size: 0.45 mm, Pall Filtrationtechnik, Dreieich, Germany), sequential hybridization with biotinylated or digoxigenin labeled DNA samples and preparation of these DNA samples were performed by standard methods (Sambrook NP, F) Fritch, and T. Maniatis 1989, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

DNA sequencing

Nucleotide sequences were determined "non-radioactive" by the dideoxy end method of Sanger et al. (Sanger et al. 1977. Proc. Natl. Acad. Sci USA 74: 5463-5467) using the "LI-COR" DNA Sequencer Model 4000L (LI-COR Inc., Biotechnology Division, Lincoln NE, USA) and using "Thermosequenase cyclic sequencing kits with a fluorescently labeled 7deaza-dGTP primer" (Amersham Life Science, Amersham International plc., Little Chalfont, Buckinghamshire, England), each according to the manufacturer's instructions.

For sequencing by a "jumping primer strategy" according to Strauss et al.

(E. C. Strauss et al. 1986. Anal. Biochem. 154: 353-360) synthetic oligonucleotides were used.

31700 h ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · · · · · · · · · ·

Chemicals, biochemicals and enzymes.

Restriction enzymes, DNA ligand T4, lambda DNA, and enzymes and substrates for optical enzyme assays were obtained from CF Boehringer & Sohne (Mannheim, Germany), or from GIBCO / BRL (Eggenstei, Germany). [γ- ³² Ρ] ΑΤΡ was from Amersham / Buchler (Braunschweig, Germany). Oligonucleotides were from MWG-Biotech GmbH (Ebersberg, Germany). Agarose type NA was 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).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Construction of omega elements that mediate resistance to kanamycin (Ω Km) or gentamycin (QGm).

The 2099 bp Sg / I fragment from Transposons Tn5 was isolated on a preparative scale for the construction of the element ΩΚιτι (EA Auierswald, G. Ludwig and H. Schuller. 1981. Cold Sprin Harb. Symp. Quant. Biol. 45: 107-113; E. Beck, G. Ludwig, EA Auerswald, B. Reiss, and H. Schaller, 1982. Genes 19: 327-336, P. Mazodier, P. Cossart, E. Giraud, and F. Gasser, 1985. Nucleic Acids Res. 195-205). This fragment was truncated to approximately 990 bp using nuclease Bal 31. This fragment, which now contained only the kanamycin resistance gene (encoding aminoglycoside-3'-O-phosphotranferase), was then ligated into a SmaI section of pSKsym DNA (a pBluescript SK derivative containing symmetrically) constructed multiple cloning site [Sa / I, H / ndIII, EcoRI, HindIII, Sa / I]). It was possible to reisolate výsledιτι from the resulting plasmid

31700 h

· · · · · · · · • · · · • · · · · · · • · · · · · · _«e • · · · · · · · · · · ···· element as a SmaI fragment, an EcoRI fragment, a HindIII fragment or an Sa / I fragment.

For the construction of the QGm element, a 983 bp Eael fragment was isolated from plasmid pBR1MCS-5 (ME Kovach, PH Elzer, DS Hill, GT Robertson, MA Farris, RM Roop and KM Peterson. 1995. Genes 166: 175-176). ) and digested with nuclease from the bean (progressive hydrolysis of the ends of the single-stranded DNA molecules). This fragment, which now contained only the gentamycin resistance gene (encoding gentamycin-3-acetyltransferase), was then ligated into SmaI digested pSKsym DNA (see above). From the plasmid thus prepared, it was possible to reisolate the QGm element as a SmaI fragment, an EcoRI fragment, a HindIII fragment or an Sa / I fragment.

Example 2

Cloning of Pseudomonas sp. HR199 (DSM7063) to inactivate them by inserting Ω elements or by deletion.

Each of the fcs, ech, vdh and aat genes were separately cloned from E. coli S17-1 strains DSM 10439 and DSM 10440 using plasmids pE207 and pE5-1 (see EP-A 0845532). From these plasmids, the fragments were isolated on a preparative scale and processed as follows:

For cloning of the fcs gene, the 2350 bp Sa / l / EcoRI fragments from plasmid pE207 and the 3700 bp EcoRI / Sa / l from plasmid pE5-1 were cloned together in pBluescript SK 'by joining the two fragments with their EcoRI ends. From the resulting hybrid plasmid, a 6050 bp Sa / I fragment was isolated on a preparative scale and truncated to approximately 2480 bp by Bal nuclease

31. Psti linkers were sequentially ligated to the ends of the fragment, and after digestion with Psfi, the fragment was cloned into pBluescript SK '(pSKfcs). After transformation of E. coli XL1 blue, clones were obtained with fcs genes that exhibited FCS activity of 0.2U / mg protein.

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For cloning the ech gene, a 3800 bp H / ndlll / EcoRI fragment was isolated from plasmid pE207 on a preparative scale and truncated to approximately 1470 bp by nuclease Bal 31. The EcoRI linkers were then ligated to the ends of the fragment and cloned into pBluescript SK '. (pSKech).

For cloning of the vdh gene, a 2350 bp Sa / l / EcoRI fragment was isolated from plasmid pE207 on a preparative scale. After cloning into pBluescript SK ', the fragment was truncated at one end by about 1530 bp with the exonuclease III / bean nuclease system. The EcoRI linker was ligated to the end of the fragment, and after digestion with EcoRI, the fragment was cloned into pBluescript SK '(pSKvdh). Transformation of E. coli XL1 blue resulted in clones with VDH genes that showed VDH activity of 0.01 U / mg protein.

For the cloning of the aat gene, a 3700 bp EcoRI / Sa / 1z fragment of plasmid pE5-1 was isolated on a preparative scale and truncated to ca. 1590 bp by nuclease Bal 31. The fragment was then chained with EcoRI linkers and after hydrolysis with EcoRI the fragment was cloned into pBluescript SK (pSKaar).

Example 3

Inactivation of the genes described above by inserting Ω elements or cutting out regions with the basic components from these genes.

The plasmid pSKrcs containing the fcs gene was digested with BssHII to excise the 1200 bp fragment from the fcs gene. Subsequent religions yielded the deletion derivative of the fcs gene (fcsN) (see Figures 1i and 2i) in cloned form in pBluescript SK '(pSKfcsA). In addition, the omega elements QKm and OGm were ligated instead after excision of said fragment. Thus, Ω-inactivated derivatives of the fcs gene (fcsQKm, see Figures 1g and 2g) and (fcsQGm, see Figures 1h and 2h) were generated in cloned form in pBluescript SK '(pSKfcsOKm and pSKfcsOGm). In the extracts of the E. coli clones thus obtained,

31700 h ··

9 9 9 9 9 9 9 9 9 9 9 9 hybrid plasmids had fcs genes inactivated by deletion or insertion of Ω elements, no FCS activity was detected.

The plasmid pSKech, which carried the ech gene, was hydrolyzed with, / ruler, thereby cleaving two fragments from the ech gene, 53 bp and 430 bp. After religation, the ech gene deletion derivative (echa, see Figures 11 and 21) was obtained in cloned form in pBluescriptSK '(pSKec / ιΔ). In addition, the omega elements fragmentγπ and QGm were ligated into the gene after cleavage of the fragments. Thus, inactivated ech gene derivatives (echQKm and echQGm) were obtained in cloned form in pBluescript SK '(pSKec ^ Km and pSKec ^ Gm).

The plasmid pSKvd / 7 containing the vdh gene was hydrolyzed with fissHII to cleave a 210 bp fragment from the vdh gene. After religation, the deletion derivative of the vdh gene (vdhA, see Figs. 1o and 2o) was obtained in cloned form in pBluescript SK- (pSKvc / ΛΔ). Moreover, after cleavage of the fragment, omega elements γγπ and ΩΘιτι were ligated into the gene instead. Thus, Ω-inactivated derivatives of the vdh gene (vdhQKm and vdMMGm) were obtained in cloned form in pBluescript SK '(pSKvd ^ Km, see Figures 1m and 2m) and (pSKvddGm, see Figures 1n and 2n). No VDH activity was detected in the crude extracts of the thus obtained E. coli clones whose hybrid plasmids contained the vdh gene inactivated by deletion or insertion of Ω elements.

Plasmid pSKaaf, which contained the aat gene, was hydrolyzed with SssHII, leaving a 59 bp fragment from the aat gene. After religation, the aat gene derivative (aat & see Figs. 1r and 2r) was obtained in cloned form in pBluescript SK '(pskaaàA). In addition, after cleavage of the fragment, omega elements ΩΚγπ and Ωβιτι were ligated into the gene instead. Thus, Ω-inactivated derivatives of the aat gene (aaft1Km, see Figures 1p and 2p) and (aaaQGm, see Figures 1q and 2q) were obtained in cloned form in pBluescript SK '(pSKaatoKm and pSKaaffiGm).

31700 h

Example 4

Subcloning of Ω-element inactivated genes into the conjugally transferable "suicide plasmid" pSUP202.

In order to replace the intact genes in Pseudomonas sp. HR199 genes inactivated by the element-element, a vector is required which, on the one hand, can be transferred to pseudomonads (conjugally transferable plasmids), but which on the other hand cannot replicate in these bacteria and is therefore unstable in the pseudomonads ("suicide plasmid") . DNA segments that are transferred into such pseudomonads by such a plasmid system are only retained therein if they are integrated into the genome of the bacterial cell by homologous recombination (Rec A-dependent recombination). In our case, the "suicide plasmid" pSUP202 (Simon et al. 1983. In: A. Puhler. Molecular genetics of the bacterial-plant interaction. Springer Verlag, Berlin, New York, pp. 98-106) was used.

After digestion with PsIII, the inactivated genes (οδΩΚπι and / οβΩΘιτι) were isolated from plasmids pSKfcsf1Km and pSKfcsQGm and ligated into DNA from pSUP202 digested with sPsl1. The ligation mixtures were transferred to E. coli S17-1. Selection was performed on LB medium containing tetracycline as well as either kanamycin or gentamycin. Thus, kanamycin resistant transformants were obtained whose hybrid plasmid (pSUPft ^ Km) contained the inactivated fcsQKm gene. The respective hybrid plasmid (pSUPft.RTM.) Of gentamicin-resistant transformants contained an inactivated .beta.Gm gene.

After digestion with EcoRI, inactivated ec / iQKm and echQGm genes were isolated from the plasmids pSKecbQKm and pSKec1Gm and ligated into EcoRI digested pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline as well as either kanamycin or gentamycin. Thus, kanamycin resistant transformants were obtained whose hybrid plasmid (pSUPec ^ Km) contained inactivated

31700 h echQKm gene. The respective hybrid plasmid (pSUPecbQGm) of gentamicin-resistant transformants contained the inactivated echQGm gene.

After digestion with EcoRI, inactivated genes in c / qKm and vdhQGm were isolated from plasmids pSKvhQKm and pSKvdQQm and ligated into EcoRI digested pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline as well as either kanamycin or gentamycin. Thus, kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUPvdQQm) contained an inactivated gene. The respective hybrid plasmid (pSUPvdhQGm) of gentamicin resistant transformants contained the inactivated vdhQGm gene.

After digestion with EcoRI, inactivated genes aaqKm and aaqQm were isolated from the plasmids pSKaaqKm and pSKaaqQm and ligated into EcoRI digested pSUP202 DNA. The ligation mixtures were transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline as well as either kanamycin or gentamycin. Thus, kanamycin-resistant transformants were obtained whose hybrid plasmid (pSUPaaQKm) contained the inactivated aaQKm gene. The respective hybrid plasmid (pSUPaa / QGm) of gentamicin-resistant transformants contained the inactivated aafQGm gene.

Example 5

Subcloning of deletion-inactivated genes into a conjugally transferable " suicide plasmid " pHE55 that contains a " sacB selection system ".

In order to replace the intact genes in Pseudomonas sp. HR199 by deletion-inactivated genes, a vector is required which has the properties already described for pSUP202. Since, unlike in-inactivated genes, in the case of deletion-inactivated genes, there is no possibility of selection (no antibiotic resistance) for successful gene replacement

31700 h * in Pseudomonas sp. HR199, another system had to be used. In the "sacB selection system", a replacement, deletion-inactivated gene is cloned into a plasmid containing the sacB gene along with an antibiotic resistance gene. In conjugative transfer of this hybrid plasmid to pseudomonads, the plasmid is inserted by homologous recombination at the site of the genome where the intact gene (first crossover) is located. This results in a "heterogenotic" strain that contains both an intact and a deletion inactivated gene, separated from the pHE55 DNA. These strains exhibit resistance encoded by the vector while having an active sacB gene. The intention then is to remove the pHE55 DNA genome even with the intact gene by means of a second homologous recombination (second crossover). Such recombination results in a strain having only an inactivated gene, and both the pHE55 encoded and the sacB gene have been removed from the genome of that strain. When strains are applied to a medium containing sucrose, the growth of strains exhibiting the sacB gene is inhibited since the genetic product converts sucrose into a polymer that is stored in the periplasm of cells. The growth of those cells that no longer carry the sacB gene due to said second recombination is not inhibited. In order to distinguish phenotypically the deletion of the deletion-inactivated gene, this gene is not confused with the intact gene; instead, the strain in which the gene to be replaced is indicated by the insertion of the Ω element is used. If the substitution is successful, the resulting strain loses antibiotic resistance encoded in the Ω element.

After digestion with Pst I, the inactivated fcsA gene was isolated from plasmid pSKfcsA and ligated into Pst I digested pHE55 DNA. The ligation mixture was transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline. Thus, tetracycline resistant transformants were obtained whose hybrid plasmid (pHEfcsA) contained the inactivated fcsA gene.

31700 h · · · · · · · ·

After digestion with EcoRI, the inactivated echa gene was isolated from plasmid pSKechA and hydrolyzed with bean nuclease (blunt end formation). The fragment was ligated into SamHI digested pHE55 DNA and treated with bean nuclease. The ligation mixture was transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline. This resulted in tetracycline resistant transformants whose plasmid (pHEechA) contained the inactivated echo gene.

After digestion with EcoRI, the inactivated vdhk gene was isolated from plasmid pSKvdhA and processed by bean nuclease. The fragment was ligated into SamHI digested pHE55 DNA and treated with bean nuclease. The ligation mixture was transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline. This resulted in tetracycline resistant transformants whose plasmid (pHEvdhA) contained the inactivated vdhk gene.

After digestion with EcoRI, the inactivated aa? A gene was isolated from plasmid pSKaa? A and processed with bean nuclease. The fragment was ligated into SamHI digested pHE55 DNA and treated with bean nuclease. The ligation mixture was transformed into E. coli S17-1. Selection was performed on LB medium containing tetracycline. Thus, tetracycline resistant transformants were obtained whose plasmid (pHEaa? A) contained the inactivated aa? A gene.

Example 6

Generation of strains of Pseudomonas sp. HR199 in which eugenol catabolism genes were specifically inactivated by Ω-element insertion.

Pseudomonas sp. HR199 was used as a recipient in conjugation experiments in which E. coli S17-1 strains harboring the above hybrid plasmids from pSUP202 were used as donors. The transconjugants were selected on a mineral medium containing gluconate and an antibiotic belonging to the appropriate Ω element. Based on tetracycline resistance

31700 h · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The encoded pSUP202 was able to distinguish between "homogeneous" (replacement of the intact gene by a gene inactivated by pomocou element insertion by a double crossover) and "heterogenotic" (integration of a hybrid plasmid into the genome with a single crossover) transconjugants.

Mutants of Pseudomonas sp. HR199 fcsQKm and Pseudomonas sp. HR199 ΖόδΩΘηη were obtained by conjugating Pseudomonas sp. HR199 with either E. Coli S17-1 (pSUPft? Gm) or with E. Coli S17-1 (pSUPft? Gm). Replacement of the intact fcs gene by genes inactivated with ΩΚγπ and OGm (/ όδΩΚιτι or ft ^Gm) was confirmed by DNA sequencing.

Mutants of Pseudomonas sp. HR199 echQKm and Pseudomonas sp. HR199 echQGm were obtained by conjugating Pseudomonas sp. HR199 with either E. Coli S17-1 (pSUPecretKm) or E. coli 17-1 (pSUPechQGm). Replacement of the intact gene by ech genes inactivated with ΩΚγπ and ΩΟηη (ecriQKm or echQGm) was confirmed by DNA sequencing.

Mutants of Pseudomonas sp. HR199 vdhQKm and Pseudomonas sp. HR199 vdhQGm were obtained after conjugation of Pseudomonas sp. HR199 with either E. coli S171 (pSUPvd ^ Km) or E. coli 17-1 (pSUPvcWGm). Replacement of the intact gene with vdh genes inactivated with ΩΚγπ and Ωθιη (vdhQKm or vdhQGm) was confirmed by DNA sequencing.

Mutants of Pseudomonas sp. HR199 and QKm and Pseudomonas sp. HR199 and αK G Gm were obtained by conjugating Pseudomonas sp. HR199 with either E. coli S17-1 (pSUPaaK7Gm) or E. coli 17-1 (pSUPaaK7Gm). Replacement of the intact gene with the aat genes inactivated with ΩΚγπ (aaíQKm) and ΩΟγπ (aaK1Gm) was confirmed by DNA sequencing.

Mutant Pseudomonas sp. HR199 ft ^ KmvW2Gm was obtained by conjugating Pseudomonas sp. HR199fcsQKm with E. coli S17-1

31700 h (pSUPvdhQGm). Replacement of the intact gene with the vdh gene inactivated with QGm (vddQGm) was confirmed by DNA sequencing.

Mutant Pseudomonas sp. HR199 vddQKmaa / QGm was obtained by conjugating Pseudomonas sp. HR199 in QKm with E. coli S17-1 (pSUPαarQGm). Replacement of the intact gene with the aat gene inactivated with QGm (aa / QGm) was confirmed by DNA sequencing.

Mutant Pseudomonas sp. HR199 was obtained by conjugating Pseudomonas sp. HR199vd / 7QKm with E. coli S17-1 (pSUPecdQGm). Replacement of the intact ech gene with QGm inactivated gene (ec6QGm) was confirmed by DNA sequencing.

Example 7

Generation of Pseudomonas sp. HR199 in which eugenol catabolism genes have been specifically inactivated by excision of the constituent regions from these genes.

Pseudomonas sp. HR199 fcsQKm, Pseudomonas sp. HR199 ecdQKm, Pseudomonas sp. HR199 vddQKm, Pseudomonas sp. HR199 and QFKm, were used as recipients in conjugation experiments in which E. coli S17-1 strains harboring the above hybrid plasmids from pHE55 were used as donors. The "heterogeneous" transconjugants were selected on a mineral medium containing gluconate and an antibiotic belonging to the appropriate Q element as well as tetracycline (resistance encoded by pHE55). After loading onto sucrose containing mineral broth, transconjugants were obtained in which the vector DNA was removed by a second recombination (second crossover). By applying to an antibiotic-free mineral soil or containing an antibiotic corresponding to the respective Q element, it was possible to identify mutants in which the Q element inactivated gene was replaced by a deletion-inactivated gene (without antibiotic resistance).

31700 h · · · ··· · · · · · ···

Conjugation of Pseudomonas sp. HR199 fcsQKm with E. coli S17-1 (pHEfcsA), a mutant of Pseudomonas sp. HR199 fcsA. Replacement of the gene inactivated with QKm (fcsQKm) by the deletion inactivated gene (fcsA) was confirmed by DNA sequencing.

Conjugation of Pseudomonas sp. HR199 echQKm with E. coli S17-1 (pHEecnA), a mutant of Pseudomonas sp. HR199 echk. Replacement of the gene inactivated with QKm (ec / QQKm) by the deletion inactivated gene (echA) was confirmed by DNA sequencing.

Conjugation of Pseudomonas sp. HR199 in E. coli S17-1 (pHEvdA) gave a mutant of Pseudomonas sp. HR199 vdhh. Replacement of the gene inactivated with QKm (vdhQKm) by the deletion-inactivated gene (vddA) was confirmed by DNA sequencing.

Conjugation of Pseudomonas sp. HR199 and QKm with E. coli S17-1 (pHEaaŕA), a mutant of Pseudomonas sp. HR199 aaàA. Replacement of the gene inactivated with QKm (aa? QKm) by the deletion inactivated gene (aa? A) was confirmed by DNA sequencing.

Example 8

Biotransformation of eugenol to vanillin by mutant Pseudomonas sp. HR199 vdríQKm.

Pseudomonas sp. HR199 d / µKm was propagated in 50 ml HRMM containing 6 mM eugenol until an optical density of approximately OD600nm = 0.6 was reached. After 17 h, 2.9 mM vanillin, 1.4 mM feruic acid and 0.4 mM vanillic acid could be determined in the culture supernatant.

31700 h • B

B ··· • B B B B

B ··· ·· ·· ·· ··

Example 9

Biotransformation of eugenol to ferulic acid by mutant Pseudomonas sp. HR199 vdhQGmaaťQKm.

Pseudomonas sp. HR199 in GmA and QKm was propagated in 50 ml HR-MM containing 6 mM eugenol until an optical density of approximately OD600nm = 0.6 was reached. After 18 h it was possible to determine 1.9 mM vanillin, 2.4 mM ferulic acid and 0.6 mM vanillic acid in the culture supernatant.

Example 10

Biotransformation of eugenol to coniferyl alcohol by Pseudomonas sp. HR199 vdftQGmaaťQKm.

Pseudomonas sp. HR199 in qGmaa / QKm was propagated in 50 ml HR-MM containing 6 mM eugenol until an optical density of approximately OD600nm = 0.4 was reached. After 15 h, it was possible to determine 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

Fermentation production of natural vanillin from eugenol in a 10 L fermentor with Pseudomonas sp. HR199 vdbQKm.

The production fermenter was inoculated with 100 ml of a 24 hour old culture propagated at 32 ° C in a shaker incubator (120 rpm) in a medium adjusted to pH 7.0 consisting of 12.5 g glycerol (1.10 g yeast extract) and 0.37 g of acetic acid / l. The fermenter contained 9.9 L of the following composition: 1.5 g yeast extract / L, 1.6 g KH ₂ PO ₄ / L, 0.2 g NaCl / L, 0.2 g MgSO ₄ / L. The pH was adjusted to pH 7.0 with sodium hydroxide solution. After sterilization, 4g of eugenol was added to the medium. The temperature was 32 ° C,

31700 h * 3 * aeration 3 Nl / min and stirrer speed 600 rpm. The pH was maintained at pH 6.5 with sodium hydroxide solution.

After 4 hours of inoculation, continuous dosing of eugenol was initiated such that when fermentation was complete after 65 hours, the total addition was 255 g of eugenol. 40 g of yeast extract was also added during the fermentation. At the end of the fermentation, the eugenol concentration was 0.2 g / l. The vanillin content was 2.6 g / l, and ferulic acid 3.4 g / l was also present.

The vanillin obtained in this way can be isolated by known physical methods such as chromatography, distillation and / or extraction, and can be used to prepare natural flavorings.

Description of the drawings

FIG. 1 to 1 years:

Gene structures for isolation of organisms and mutants of calA * \ Part of the inactivated coniferyl alcohol dehydrogenase gene calB *: Part of the inactivated coniferyl aldehyde dehydrogenase gene fcs *: Part of the inactivated gene for feruloyl-CoA-synthase-echo * co for inactivated gol *: *: Part of the inactivated vanillin dehydrogenase gene aaf: Part of the inactivated beta-ketothiolase gene

While the restriction enzyme hydrolysis points labeled in the resulting products were used for construction, they were non-functional.

FIG. 2a: Nucleotide sequence for the structure of the ca / ΑΩΚηΊ gene

FIG. 2b: Nucleotide sequence for the structure of the ca / AQGm gene

FIG. 2c: Nucleotide sequence for the structure of the ca / ΑΔ gene

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FIG. 1d: Nucleotide sequence for the ca / 8QKm gene structure

FIG. 1e: Nucleotide sequence for the structure of the ca / BOGm gene

FIG. 1f: Nucleotide sequence for the ca / štrukt gene structure

FIG. 1g: Nucleotide sequence for fcsOKm gene structure

FIG. 1h: Nucleotide sequence for fcsOGm gene structure

FIG. 11 i: Nucleotide sequence for fcsA gene structure

FIG. 1j: Nucleotide sequence for ecftQKm gene structure

FIG. 2k: Nucleotide sequence for ecftQGm gene structure

FIG. 2I: Nucleotide sequence for ech < > gene structure

FIG. 2m: Nucleotide sequence for gene structure

FIG. 2n: Nucleotide sequence for gene structure in hOGm

FIG. 2o: Nucleotide sequence for vdhb gene structure

FIG. 2p: Nucleotide sequence for aa / ΩΚπι gene structure

FIG. 2q: Nucleotide sequence for aaOGOG gene structure

FIG. 2r: Nucleotide sequence for aa? A gene structure

31700 h • · · ·

JJ · · · · · · · φ ·

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Claims (16)

1. A transformed and / or mutagenized unicellular or multicellular organism, characterized in that the enzymes for the catabolism of eugenol and / or ferulic acid are inactivated so that the intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanilla acid accumulate. An organism according to claim 1, characterized in that the catabolism of eugenol and / or ferulic acid is altered by the introduction of Ω elements or by deletion on the respective genes. Organism according to claim 1 or 2, characterized in that the one or more genes encoding the coniferyl alcohol dehydrogenase (s), coniferyl aldehyde dehydrogenase (s), feruloyl-CoA synthetase (s), enoyl-CoAhydratase (s) aldolase, beta-ketothiolases, vanillin dehydrogenases or demethylase / and / or inactivated. The organism according to one of claims 1 to 3, characterized in that it is a single-cell, preferably a microorganism or a plant or animal cell. Organism according to one of Claims 1 to 4, characterized in that it is a bacterium, preferably of the genus Pseudomonas. 6. Structures of genes in which the nucleotide sequences encoding the coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydrogenase, feruloylCoA synthetase, enoyl-CoA-hydratase-aldolase, beta-ketothiolase, vanillin dehydrogenase, or the demethylase or vanemic acid enzymes or demethylases, or vanillaic acid or methylene enzymes. 31700 h ································· ·· ·· ·· · 7. Gene structures having the sequences shown in Figures 1a to 1r. 8. Gene structures having the sequences shown in Figures 2a to 2r. Vectors comprising at least one gene structure according to one of claims 6 to 8. Transformed organism according to one of claims 1 to 5, characterized in that it houses at least one vector according to claim 9. An organism according to any one of claims 1 to 5, characterized in that it comprises at least one gene structure according to one of claims 6 to 8 integrated into the genome instead of the respective intact gene. 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 5 or 10 to 11 is used. Method for the preparation of the organisms as claimed in one of claims 1 to 5, characterized in that the catabolism of eugenol and / or ferulic acid is achieved by known microbiological culture methods. Method for the preparation of an organism according to any one of claims 1 to 5 or 10 to 11, characterized in that the change in the catabolism of eugenol and / or ferulic acid and / or the inactivation of the respective genes is achieved by recombinant DNA methods. 31700 h ··· ··················· ·· ·· Use of organisms according to one of claims 1 to 5 or 10 to 11 for the preparation of coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and / or vanillic acid. Use of the gene structures according to one of claims 6 to 8 or of the vector according to claim 9 for the preparation of transformed and / or mutagenized organisms. 31700 h ·· ··· · · · · β ·· ·· ········· · · -Ι- sequences CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG Met 1 CAA CTG ACC AAC AAA ATC GTC GTC ACC GGA Gin Leu Thr 5 10 GTG TCC TCC Val Ser 15 472 GGT Gly ATC GGT GCC GAA ACT GCC CGC Thr Ala Arg GTG CTG CGC TCT CAC GGC GCC Ala 30 ACA Thr 520 Ile Gly Ala Glu 20 wall Leu Arg Ser His Gly GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC 568 wall Ile Gly wall Asp Arg same time Met for Ser Leu Thr Leu Asp Ala Phe 35 40 45 GTT CAG GCT GAC CTG AGC CAT CCT GAA GGC ATC GAT AAG GCC ATC GGG 616 wall gin Ala Asp Leu Ser His for Glu Gly Ile Asp Lys Ala Ile 55 60 62 ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC 676 AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT 736 GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATG ATT GAA CAA Met Ile Glu Aso Gly Leu His 1 5 GCA GGT Ala Gly TCT Ser CCG GCC GCT TGG GTG 15 GAG AGG CTA TTC GGC TAT GAC TGG 838 for Ala Glu Arg Leu Phe 20 Gly Tyr Asp Trp 10 GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA 886 Ala gin gin Thr Ile Gly Cys Ser Asp Ala Ala wall Phe Arg Leu Ser 25 30 35 40 GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC 934 Ala gin Gly Arg for wall Leu Phe wall Lys Thr Asp Leu Ser Gly Ala 45 50 55 CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG 982 Leu same time Glu Leu gin Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr 60 65 70 -2 ·························· • · · · · · · · · ACG Thr GGC Gly GTT CCT TGC Cys GCA Ala GCT GTG CTC 1030 wall 75 for Ala Val Leu Asp wall wall Thr 85 Glu Ala Gly AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA 1078 Arg Asp Trp Leu Leu Leu Gly Glu wall for Gly gin Asp Leu Leu Ser 90 95 100 TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG 1126 Ser His Leu Ala for Ala Glu Lys wall Ser Ile Met Ala Asp Ala Met 105 110 115 120 CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA 1174 Arg Arg Leu His Thr Leu Asp for Ala Thr Cys for Phe Asp His. gin 125 130 135 GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT 1222 Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu 140 145 150 GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC 1270 wall Asp gin Asp Asp Leu Asp Glu Glu His gin Gly Leu Ala for Ala 155 160 165 GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC 1318 Glu Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp Gly Glu Asp Leu 170 175 180 GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT 1366 wall wall Thr His Gly Asp Ala Cys Leu for same time Ile Met wall Glu same time 185 190 195 200 GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC 1414 Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly wall Ala Asp 205 210 215 CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT 1462 Arg Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu 220 225 230 GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT 1510 Gly Gly Glu Trp Ala Asp Arg Phe Leu wall Leu Tyr Gly Ile Ala Ala 235 240 245 CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC 1558 for Asp Ser gin Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 250 255 260 264 TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCTG GCC GCG GTG 1613 Ala Ala Val ATC AAC GGC ATA AAT 1661 white Ala Phe Met Cys Ala Glu Ser Arg Trp white Asn Gly white Asn 230 235 240 ····························· ·· ·· ·· -3ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC 1710 White Pro Val Gly Gly Gly Leu Ala Ser Thr Tyr Val 245 250 255 GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 1770 GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 1830 GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 1890 GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 1950 CAAACTCGAT GCAGCTGGAT GTAGGTAGCT GGATTGGAC GGTGCGTTGG GGACAACACC 2010 CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 2070 ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 2130 TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 2164 FIG. 2a: -4 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG CAACTG ACC AAC AAG AAA ATC GTC GTC ACC GGA GTG «TCC 472 Met Gin Leu Thr Asn Lys Lys Clay Val Val Thr Gly Val Ser Ser 15 10 15 GGT ATC GGT GCC ACA 520 Gly Ala Glu Ala Glu Thr Ala Arg Val Leu Arg Ser Gly Ala Thr 20 25 30 GTG ATT GGC GTA GAT GCT TTC 568 Val ile Gly Val Asp Arg Asn Met Pro Leu Thr Leu Asp Ala Phe 34 40 45 GTT CAG GCT GAC CTG AGC CAT CCT GAGGGGAGAG GCGGTTTGCG TATTGGGCGC 622 Val Gin Ala, Asp Leu Ser His Pro 50 55 ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA CATGGAAGCC ATCACAAACG 682 GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT CGCCTTGCGT ATAATATTTG 742 CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC CCAGTTGACA TAAGCCTGTT 802 CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC AGAACCTTGA 862 CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT GACTGTTTTT 922 TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTT 982 TGATGTTATG GAGCAGCAAC G ATG TTA GC AGC AGC AAC GAT GTT ACG CAG 1033 Met Leu Arg Ser Asn Asp Val Thr Gin 15 10 CAG GGC AGT CCT AAA ACA AAG TAG GGT GGC TCA AGT ATG GGC ATC 1081 Gin Gly Ser Arg For Lys Thr Lys Leu Gly Ser Gly Ser Ser Gly White 15 20 25 ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT 1129 White Arg Thr Cys 30 GCT GTC GTC GTC GTC GTC GTC GTC GTC GTC TTC TCC 1177 Ala Leu Asp Phu Gly Arg Glu Phe Gly Val Val Thr Tyr Ser 45 50 55 ·· • · · · ····· -5 ·· · · · ··· · · · · · CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC 1225 Gin His Gin For Asp Ser Asp 65 Tyr Leu Gly same time Leu 70 Leu Arg Ser Lys ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT 1273 Thr Phe White Ala Leu Ala Ala Phe Asp gin Glu Ala Val Val Gly Ala 75 80 85 90 CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC 1321 Leu Ala Tyr Val Leu for Arg Phe Glu gin for Arg Ser Glu ile 95 100 105 ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT 1369 Tyr ile Tyr Asp Leu Ala wall 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 1417 Ala Thr Ala Leu ile same time 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 1465 Ala Tyr Val ile Tyr wall 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 1,513 Ala Leu Tyr Thr Lys Leu Gly Ile Arg Glu Glu wall Met His Phe Asp 155 160 165 170 ATC GAC CCA AGT ACC GCC ACC TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCT 1567 Asp Pro Ser Thr Ala Thr 175 177 G ATT GCA TTC ATG TGT GCT GAG GAG TCA CGT TGG ATC AAC GGC ATA AAT 1616 white Ala Phe Met Cys Ala Glu Glu Ser Arg Trp white Asn Gly white Asn (+420) 228 230 235 240 ATT CCA GTG GAC GGA GGT TTG GCA TCG ACC TAC GTG TAA GTTCGTGGAC 1665 White Pro Val Gly Gly Gly Leu Ala Ser Thr Tyr Val 245 250 255 GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 1725 GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 1785 GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 1845 GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 1905 CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC 1965 CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 2025 ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 2085 TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 2119 FIG. 2b: • • • • • (• · ···· B ·· ·· CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG CAA CTG ACC AAC AAA ATC GTC GTC ACC. GGA GTG TCC 472 Met Gin Leu Thr Asn Lys Lys Clay Val Val Thr Gly Val Ser Ser 15 10 15 GGT ATC GGT GCC GAA ACT GCC CGC GTT CTG CGC TCT CAC GCC GCC ACA 520 Gly Ile Gly Ala Glu 20 Thr Ala Arg wall Leu 25 Arg Ser His Gly Ala Thr 30 GTG ATT GGC GTA GAT CGC AAC ATG CCG AGC CTG ACT CTG GAT GCT TTC 568 wall Ile Gly wall 35 Asp Arg same time Met for 40 Ser Leu Thr Leu Asp Ala Phe 45 GTT wall CAG gin GCT Ala 50 GAC Asp CTG Leu AGC Ser CAT His CCT for 55 GAA Glu GGC Gly ATC Ile 58 GATC AAC Asn Gly White Asn 240 617 ATT Ile CCA for GTG wall GAC Asp GGA Gly GGT Gly TTG Leu GCA Ala TCG Ser ACC Thr TAC Tyr GTG wall TAA GTTCGTGGAC 666 245 250 255 GCCCTTTGCA CGCGCACTAT ATCTCTATGC AGCAGCTGAA AGCAGCTTTG GTTTTGATCG 726 GAGGTAGCGG GCGGAAAGGT GCAGAATGTC TAAATAATAA AGGATTCTTG TGAAGCTTTA 786 GTTGTCCGTA AACGAAAATA AAAATAAAGA GGAATGATAT GAAAGCAAGT AGATCAGTCT 846 GCACTTTCAA AATAGCTACC CTGGCAGGCG CCATTTATGC AGCGCTGCCA ATGTCAGCTG 906 CAAACTCGAT GCAGCTGGAT GTAGGTAGCT CGGATTGGAC GGTGCGTTGG GGACAACACC 966 CTCAAGTATA GCCTTGCCTC TCGCCTGAAT GAGCAAGACT CAAGTCTGAC AAATGCGCCG 1026 ACTGTCAATG GTTATATCCG GATATTCAAA GTCAGGGTGA TCGTAACTTT GACCGGGGGC 1086 TTGGTATCCA ATCGTCTCGA TATTCTGGCT GCAG 1120 FIG. 2c: • · ·· -7 ·· ·· · a · a · a · a aaa · ··· a a a a a a · a a a a ···· ·· aa aa GAATTCCGCG GGTAGGGTCT TGCGTTTGCC TATCGCCCGG TTTTCTTGGC GCTTCGCTTC TTCTATCAGC CATGCTTGTT GCGATGAACC GGGCCGCTTT GCCTGAACCT GCATCGAGAT CGAAAGTCAT TCGTTGACAT GCTGAGGTCA GGTGTTAGCC AGGGCAGAGG GGATTTTTCC 120 180 TTAACTCGCG TAAGCATTCT GTCATTTTTT GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CGATTAAGAT AATTAAAATA AGGAAACCGC CTCCAGCTCA AGGGCAATTT TTGGGCTATT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT CTT GGT TTG AAT Met 1 Ser Ile Leu Gly 5 Leu same time GGT GCC CCG GTC GGA GCT GAG CAG CTG GGC TCG GCT CTT GAT CGC ATG Gly Ala for 10 wall Gly Ala Glu gin 15 Leu Gly Ser Ala Leu 20 Asp Arg Met AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG Lys Lys 25 Ala His Leu Glu gin 30 Gly for Ala same time Leu 35 Glu Leu Arg Leu AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA Ser 40 Arg Leu Asp Arg Ala 45 Ile Ala Met Leu Leu 50 Glu same time Arg Glu Ala 55 ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA Ile Ala Asp Ala wall 60 Ser Ala Asp Phe Gly 65 same time Arg Ser Arg Glu 70 gin ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC Thr Leu Leu Cys 75 Asp Ile Ala Gly Ser 80 wall Ala Ser Leu Lys 85 Asp Ser CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG Arg Glu His 90 wall Ala Lys Trp Met 95 Glu for Glu His His 100 Lys Ala Met TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT Phe for 105 Gly Ala Glu Ala Arg 110 wall Glu Phe gin for 115 Leu Gly wall wall GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG Gly 120 wall Ile Ser for Trp 125 same time Phe for Ile wall 130 Leu Ala Phe Gly for 135 CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC Leu Ala Gly Ile Phe 140 Ala Ala Gly same time Arg 145 Ala Met Leu Lys for 150 Ser GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT Glu Leu Thr for 155 Arg Thr Ser Ala Leu 160 Leu Ala Glu Leu Ile 165 Ala Arg 473 521 569 617 665 713 761 809 857 905 953 • · ··· -8 · ··· · · · · · · · · · · · · TAT TTC GAT Tyr Phe Asp GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT GAA GTC 1001 Glu Thr Glu Leu Thr Thr Val Leu Asp 180 Ala Glu wall 170 GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC 1049 Gly Ala Leu Phe Ser Ala gin for Phe Asp His Leu Ile Phe Thr Gly 185 190 195 GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT AAC CTA 1097 Gly Thr Ala wall Ala Lys His Ile Met Arg Ala Ala Ala Asp same time Leu 200 205 210 215 GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC GTT TCC 1145 wall for wall Thr Leu Glu Leu Gly Gly Lys Ser for wall Ile wall Ser 220 225 230 - CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG GTG AAA 1193 Arg Ser Ala Asp Met Ala Asp wall Ala gin Arg wall Leu Thr wall Lys 235 240 245 ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CCG GAC TAT GTG CTG CTG 1241 Thr Phe same time Ala Gly gin Ile Cys Leu Ala for Asp Tyr wall Leu Leu 250 255 260 CCG GAA GGGACAGCAA GCGAACCGGA ATTGCCAGCT GGGGCGCCCT Pro Glu 265 TGGGAAGCCC TGCAAAGTAA ACTGGATGGC TTTCTTGCCG CCAAGGATCT GATGGCGCAG 1357 GGGATCAAGA TCTGATCAAG AGACAGGATG AGGATCGTTT Met Ile Glu GAT Asp 5 GGA TTG GCA Gly Leu His Ala GGT TCT. CCG GCC GCT 1460 Gly Ser for Ala Ala Trp 15 wall Glu Arg Leu Phe 20 GGC TAT GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG 1508 Gly Tyr Asp Trp Ala gin gin Thr Ile Gly Cys Ser Asp Ala Ala wall 25 30 35 TTC CGG CTG TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC 1556 Phe Arg Leu Ser Ala gin Gly Arg for wall Leu Phe wall Lys Thr Asp 40 45 50 CTG TCC GGT GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG 1604 Leu Ser Gly Ala Leu same time Glu Leu gin Asp Glu Ala Ala Arg Leu Ser 55 60 65 TGG CTG GCC ACG ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC 1652 Trp Leu Ala Thr Thr Gly wall for Cys Ala Ala wall Leu Asp wall wall 70 75 80 • • • -9 ··· ·· »·· · · · · · ACT Thr 85 GAA GCG GGA AGG GAC TGG CTG CTA TTG GGC GAA 1700 Glu Ala Gly Arg Asp 90 Trp Leu Leu Leu Gly Glu wall For Gly Gin 100 GAT CTC CTG TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG 1748 Asp Leu Leu Ser Ser His Leu Ala for Ala Glu Lys wall Ser Ile Met 105 110 115 GCT GAT GCA ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA 1796 Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp for Ala Thr Cys for 120 125 130 TTC GAC CAC CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG 1844 Phe Asp HLS gin Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met 135 140 145 GAA GCC GGT CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG 1892 Glu Ala Gly Leu wall Asp gin Asp Asp Leu Asp Glu Glu His gin Gly 150 155 160 CTC GCG CCA GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC 1940 Leu Ala for Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp 165 170 175 180 GGC GAG GAT CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC 1988 Gly Glu Asp Leu wall wall Thr His Gly Asp Ala Cys Leu for same time Ile 185 190 195 ATG GTG GAA AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG 2036 Met wall Glu same time Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu 200 205 210 GGT GTG GCG GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT 2084 Gly wall Ala Asp Arg Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile 215 220 225 GCT GAA GAG CTT GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC 2132 Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu wall Leu Tyr 230 235 240 GGT ATC GCC GCT CCC GAT TCG CAG Gly Ala Ala Ala For Asp Ser Gin Arg Ala Ala Phe Tyr Arg Leu Leu 245 250 255 260 GAC GAG TTC TTC GGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 2235 Asp Glu Phe Phe 264 CGC CAT GCC AAG CCT GTT CTC GTG AAC 2283 His 444 Ala Lys Pro Val Leu Val Gin Ser Pro Val Gly Glu Ser Asn TTG GCG ATG CGC GCA CCC TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT 2331 Leu Ala Met Arg Ala for Tyr Gly Glu Ala Ile His Gly Leu Leu Ser 460 465 470 ·· • · - 10GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 2385 Val Leu Le Thr Glu Cys 475 480 481 GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 2445 TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 2505 ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 2565 CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 2625 CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 2685 ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 2745 TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 2805 CCAACCTTGA GGAATTC 2822 FIG. 2d: ································ GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 CGATTAAGAT AATTAAAATA AGGAAACCGC atggtttctt ATGTGAATTT GTCTGGCATA 360 CTCCAGCTCA AGGGCAATTT TTGGGCTATT GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC Met Ser ATT CTT GGT TTG AAT White Leu Gly Leu Asn 473 1 5 GGT GCC CCG GTC GGA Gly GCT GAG CAG CTG GGC TCG 521 Gly Ala Pro wall Ala Glu Gin Leu Gly Ser Ala Leu Asp Arg Met 10 15 20 AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG 569 Lys Lys Ala His Leu Glu gin Gly for Ala same time Leu Glu Leu Arg Leu 25 30 35 AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA 617 Ser Arg Leu Asp Arg Ala Ile Ala Met Leu Leu Glu same time Arg Glu Ala 40 45 50 55 ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA 665 Ile Ala Asp Ala wall Ser Ala Asp Phe Gly same time Arg Ser Arg Glu gin 60 65 70 ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC 713 Thr Leu Leu Cys Asp Ile Ala Gly Ser wall Ala Ser Leu Lys Asp Ser 75 80 85 CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG 761 Arg Glu His wall Ala Lys Trp Met Glu for Glu His His Lys Ala Met 90 95 100 TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT 809 Phe for Gly Ala Glu Ala Arg wall Glu Phe gin for Leu Gly wall wall 105 110 115 GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG 857 Gly wall Ile Ser for Trp same time Phe for Ile wall Leu Ala Phe Gly for 120 125 130 135 CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC 905 Leu Ala Gly Ile Phe Ala Ala Gly same time Arg Ala Met Leu Lys for Ser 140 145 150 GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT 953 Glu Leu Thr for Arg Thr Ser Ala Leu Leu Ala Glu Leu Ile Ala Arg 155 160 -12 · · 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12 TAC TTC GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT Tyr Phe Asp 170 Glu Thr Glu Leu Thr 175 Thr wall Leu Gly Asp 180 Ala GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC Gly Ala 185 Leu Phe Ser Ala gin 190 for Phe Asp His Leu 195 Ile Phe GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT Gly 200 Thr Ala wall Ala Lys 205 His Ile Met Arg Ala 210 Ala Ala Asp GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC wall for wall Thr Leu 220 Glu Leu Gly Gly Lys 225 Ser for wall Ile CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG Arg Ser Ala Asp 235 Met Ala Asp wall Ala 240 gin Arg wall Leu Thr 245 ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CCG GAC TAT GTG Thr Phe same time 250 Ala Gly gin Ile Cys 255 Leu Ala for Asp Tyr 260 wall GTC Glu Glu Val ACC GGC Thr Gly AAC CTA Asn Leu 215 GTT TCC Val Ser 230 GTG AAA Val Lys CTG GGG Leu 262 1001 1049 1097 1145 1193 1241 GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT TGTAATTCAT TAAGCATTCT 1301 GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA ATCGCCAGCG GCATCAGCAC 1361 CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC CGTGGAAACG GATGAAGGCA 1421 CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT AATGCAAGTA GCGTATGCGC 1481 TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG GTAACGGCGC AGTGGCGGTT 1541 TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC CTCGGGCATC CAAGCAGCAA 1601 GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGC GCAACG ATG TTA CGC Met Leu Arg 1656 AGC AGC AAC GAT GTT ACG CAG CAG 1704 Ser Ser Asn Asp Val 4 Thr Gin Gin Gly Ser Arg Pro Lys Thr Lys Leu GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC 1752 Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly for Asp 20 25 30 35 CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT CGT GAG TTC 1800 gin wall Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly Arg Glu Phe 40 45 50 GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC 1848 Gly Asp wall Ala Thr Tyr Ser gin His gin for Asp Ser Asp Tyr Leu 55 60 65 GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC 1896 Gly same time Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala Ala Phe Asp 70 75 80 CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT 1944 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · · · · ····· Gin Glu Ala wall wall Gly Ala Leu Ala Ala Tyr Val Leu Pro Arg Phe 85 90 95 GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC 1992 Glu gin for Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala wall Ser Gly 100 105 110 115 GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG 2040 Glu His Arg Arg gin Gly Ile Ala Thr Ala Leu Ile same time Leu Leu Lys 120 125 130 CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT 2088 His Glu Ala same time Ala Leu Gly Ala Tyr wall Ile Tyr wall gin Ala Asp 135 140 145 TAC GGT GAC Gath CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA 'CGG 2136 Tyr Gly Asp Asp for Ala wall Ala Leu Tyr Thr Lys Leu Gly Ile Arg 150 155 160 GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC ACC TAA CAA 2184 Glu Glu wall Met His Phe Asp Ile Asp for Ser Thr Ala Thr 165 170 175 177 TTCGTTCAAG CCGAGATCGG CTTCCCTG CAA AGT CCT GTG Gin Ser Pro Val Gly Ser Asn 451 455 TTG GCG CCC TAC GGA GAA GCG CTC TCT 2284 Leu Ala Met 460 Arg Ala For Tyr 465 Gly Glu Ala Ile His Gly Leu Leu Ser 470 GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 2338 Val Leu Ser Thr Glu Cys 475 480 481 GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 2398 TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 2458 ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 2518 CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 2578 CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 2638 ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 2698 TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 2758 CCAACCTTGA GGAATTC 2775 FIG. 2e: ························································· - 14GAATTCCGCG TATCGCCCGG TTCTATCAGC GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GGTAGGGTCT TTTTCTTGGC CATGCTTGTT GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 TGCGTTTGCC GCTTCGCTTC GCGATGAACC GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TTAACTCGCG TAAGCATTCT GTCATTTTTT TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CGATTAAGAT AATTAAAATA AGGAAACCGC CTCCAGCTCA AGGGCAATTT TTGGGCTATT CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 AGAATAACAA TTGACTCCTC AGGAGGTCAG CG ATG AGC ATT CTT GGT TTG AAT Met 1 Ser Ile Leu Gly 5 Leu same time GGT GCC CCG GTC GGA GCT GAG CAG CTG GGC TCG GCT CTT GAT CGC ATG Gly Ala for 10 wall Gly Ala Glu gin 15 Leu Gly Ser Ala Leu 20 Asp Arg Met AAG AAG GCG CAC CTG GAG CAG GGG CCT GCA AAC TTG GAG CTG CGT CTG Lys Lys 25 Ala His Leu Glu gin 30 Gly for Ala same time Leu 35 Glu Leu Arg Leu AGT AGG CTG GAT CGT GCG ATT GCA ATG CTT CTG GAA AAT CGT GAA GCA Ser 40 Arg Leu Asp Arg Ala 45 Ile Ala Met Leu Leu 50 Glu same time Arg Glu Ala 55 ATT GCC GAC GCG GTT TCT GCT GAC TTT GGC AAT CGC AGC CGT GAG CAA Ile Ala Asp Ala wall 60 Ser Ala Asp Phe Gly 65 same time Arg Ser Arg Glu 70 gin ACA CTG CTT TGC GAC ATT GCT GGC TCG GTG GCA AGC CTG AAG GAT AGC Thr Leu Leu Cys 75 Asp Ile Ala Gly Ser 80 wall Ala Ser Leu Lys 85 Asp Ser CGC GAG CAC GTG GCC AAA TGG ATG GAG CCC GAA CAT CAC AAG GCG ATG Arg Glu His 90 wall Ala Lys Trp Met 95 Glu for Glu His His 100 Lys Ala Met TTT CCA GGG GCG GAG GCA CGC GTT GAG TTT CAG CCG CTG GGT GTC GTT Phe for 105 Gly Ala Glu Ala Arg 110 wall Glu Phe gin for 115 Leu Gly wall wall GGG GTC ATT AGT CCC TGG AAC TTC CCT ATC GTA CTG GCC TTT GGG CCG Gly 120 wall Ile Ser for Trp 125 same time Phe for Ile wall 130 Leu Ala Phe Gly for 135 CTG GCC GGC ATA TTC GCA GCA GGT AAT CGC GCC ATG CTC AAG CCG TCC Leu Ala Gly Ile Phe 140 Ala Ala Gly same time Arg 145 Ala Met Leu Lys for 150 Ser GAG CTT ACC CCG CGG ACT TCT GCC CTG CTT GCG GAG CTA ATT GCT CGT Glu Leu Thr for 155 Arg Thr Ser Ala Leu 160 Leu Ala Glu Leu Ile 165 Ala Arg 473 521 569 617 665 713 761 809 857 905 953 -15 · Φ · 15 15 15 15 15 15 15 15 15 · · · · · · · · · · · · GAT GAA ACT GAG CTG ACT ACA GTG CTG GGC GAC GCT 1001 Tyr Phe Asp 170 Glu Thr Glu Leu Thr 175 Thr wall Leu Gly Asp Ala Glu Val 180 GGT GCG CTG TTC AGT GCT CAG CCT TTC GAT CAT CTG ATC TTC ACC GGC 1049 Gly Ala Leu Phe Ser Ala Gin for Phe Asp His Leu Phe Thr Gly 185 190 195 GGC ACT GCC GTG GCC AAG CAC ATC ATG CGT GCC GCG GCG GAT AAC CTA 1097 Gly Thr Ala wall Ala Lys His Ile Met Arg Ala Ala Ala Asp Asn Leu 200 205 210 215 GTG CCC GTT ACC CTG GAA TTG GGT GGC AAA TCG CCG GTG ATC 1145 Val Pro Val Thr Leu Glu Leu Gly Gly Lys Ser Pro Val ile Val Ser 220 225 230 CGC AGT GCA GAT ATG GCG GAC GTT GCA CAA CGG GTG TTG ACG GTG AAA 1193 Arg Ser Ala Asp Met Ala Asp wall Ala gin Arg Val Leu Thr Val Lys 235 240 245 ACC TTC AAT GCC GGG CAA ATC TGT CTG GCA CC GTG GGT GAG TCG AAC 1240 Thr Phe Asn Ala Gly Gin ile Cys Leu Ala Val Gly Glu Ser Asn 250 255 257 454 455 TTG GCG ATG CGC GCA CCC TAC GGA GAA GCG ATC CAC GGA CTG CTC TCT 1288 Leu Ala Met Arg Ala Pro Tyr Gly Glu Ala ile His Gly Leu Leu Ser 460 465 470 GTC CTC CTT TCA ACG GAG TGT TAG AACCGTTGGT AGTGGTTTTG GACGGGCCCA 1342 Val Leu Ser Thr Glu Cys 475 480 481 GGAGCATGCG CTTCTGGGCC CGTTTCTTGA GTATTCATTG GATAGTCACG CGTGGTAGCT 1402 TCGAGCCTGC ACAGCTGATG AGCACCCTGG AAGGCGCGCT GTACGCGGAC GACTGGGTTC 1462 ATCTTCGCCA TTCATGACGG AACTCCGTTC CCCAGTACCG CGATGACTAT TTTGCCTCTT 1522 CCGATGTCCG ATTCCACGCC GCCTGACGCT AAGCGGGGGC GGGGGCGCCC GCATCCCAGC 1582 CCAGACAGCA ACAAATGAGT AGGCTCTTGG ATGCCGCGGC GGCTGAGATT GGTAACGGCA 1642 ATTTCGTCAA TGTGACGATG GATTCGATTG CCCGTGCTGC CGGCGTCTCA AAAAAAACGC 1702 TGTACGTCTT GGTGGCGAGC AAGGAAGAAC TCATTTCCCG GTTAGTGGCT CGAGACATGT 1762 CCAACCTTGA GGAATTC 1779 FIG. 2f: ··· ··· ······· -16 · ··· · · · · · · · CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG ATG CGT TCT CTC GAG 356 Met Arg Ser Leu Glu GCG Ala CTT CTT CCG TTC CCG GGT CGA ATT CTT GAG CGT CTC GAG CAT TGG 404 Leu Leu For Phe 10 For Gly Arg Ile Leu 15 Glu Arg Leu Glu His Trp GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452 Ala Lys Thr Arg for Glu gin Thr Cys wall Ala Ala Arg Ala Ala same time 25 30 35 GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500 Gly Glu Trp Arg Arg Ile Ser Tyr Ala Glu Met Phe His same time wall Arg 40 45 50 GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548 Ala Ile Ala gin Ser Leu Leu for Tyr Gly Leu Ser Ala Glu Arg for 55 60 65 CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596 Leu Leu Ile wall Ser Gly same time Asp Leu Glu His Leu gin Leu Ala Phe 70 75 80 85 GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644 Gly Ala Met Tyr Ala Gly Ile for Tyr Cys for wall Ser for Ala Tyr 90 95 100 TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692 Ser Leu Leu Ser gin Asp Leu Ala Lys Leu Arg His Ile wall Gly Leu 105 110 115 CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG GGG 740 Leu gin for Gly Leu wall Phe Ala Ala Asp Ala Ala for Phe gin 120 125 130 132 ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC 800 AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT 860 GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATG ATT GAA CAA Methyl Glu Gin Asp Gly Leu His 1 5 GCA GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG 962 Ala Gly Ser Pro Ala Ala Trp Val Glu Arg Leu Phe 10 15 20 BB BB • B -17BB BB B • BBB B • BB • B B · B B B · BB B BBB • BB B B B B GCA Ala 25 CAA CAG ACA ATC GGC Thr Gly 30 TGC Cys TCT GAT GCC GCC GTG TTC CGG CTG TCA 1010 gin gin Ser Asp Ala Ala 35 wall Phe Arg Leu Ser 40 GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC 1058 Ala gin Gly Arg for wall Leu Phe wall Lys Thr Asp Leu Ser Gly Ala 45 50 55 CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC ACG 1106 Leu same time Glu Leu gin Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr 60 65 70 ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC ACT GAA GCG GGA 1154 Thr Gly wall for Cys Ala Ala wall Leu Asp wall wall Thr Glu Ala Gly 75 80 85 AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA 1202 Arg Asp Trp Leu Leu Leu Gly Glu wall for Gly gin Asp Leu Leu Ser 90 95 100 TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG 1250 Ser His Leu Ala for Ala Glu Lys wall Ser Ile Met Ala Asp Ala Met 105 110 115 120 CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA 1298 Arg Arg Leu His Thr Leu Asp for Ala Thr Cys for Phe Asp His gin 125 130 135 GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT 1346 Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu 140 145 150 GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC 1394 wall Asp gin Asp Asp Leu Asp Glu Glu His gin Gly Leu Ala for Ala 155 160 165 GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC 1442 Glu Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp Gly Glu Asp Leu 170 175 180 GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT 1490 wall wall Thr His Gly Asp Ala Cys Leu for same time Ile Met wall Glu same time 185 190 195 200 GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC 1538 Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly wall Ala Asp 205 210 215 CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT 1586 Arg Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu 220 225 230 GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT 1634 Gly Gly Glu Trp Ala Asp Arg Phe Leu wall Leu Tyr Gly Ile Ala Ala 235 240 245 ·· · · · · -18 · · · • 18 18 18 18 18 18 18 18 18 18 18 18 18 18 CCC GAT TCG CAG CGC ATC GCC TTC TTC For Asp Ser Gin Arg Ala Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 250 255 260 264 TGAGCGGGAC TCTGGGGTTC GAAATGACCG ACCAAGCGAC GCCCCT GTT TTG CAA 1737 Val Leu Gin 565 565 TGG CGG TCG GCG AAA GTT GAT GCG CTG TAT CGT GGT GAA GAT CAA TCC 1785 Trp Arg Ser Ala Lys wall Asp Ala Leu Tyr Arg Gly Glu Asp gin Ser 570 575 580 ATG CTG CGT GAC GAG GCC ACA CTG TGA GTTGGTCAGG GGGGGCTTAC 1832 Met Leu Arg Asp Glu Ala Thr Leu 585 589 TCGGCGTTTT CCGACACTGC GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG 1892 GGTGCCCTGT CGCTGGTGTC GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG 1952 ATGCGTGCGT CGCTTGAACC ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA 2012 GCAAGCTTTG ATGCTTACCT GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG 2072 TCGGTTCCGG CCTTGGGGGT GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG 2132 GCCGGCGAGC AGATTTCCCA AGGCGCTGAT CACGTGCTGT GTGTCGCGGG CTGCAG 2188 FIG. 2G ··························· -19 · · · 19 19 19: 19 19 19 19 19 19 i 19 i CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG ATG CGT TCT CTC GAG 356 Met Arg Ser Leu Glu GCG CTT CTT CCC TTC CCG GGT CGA ATT CTT GAG CAT His 20 TGG Trp 404 Ala Leu Leu for Phe 10 for Gly Arg ile Leu 15 Glu Arg Leu Glu GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452 Ala Lys Thr Arg for Glu gin Thr Cys wall Ala Ala Arg Ala Ala same time 25 30 35 GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500 Gly Glu Trp Arg Arg Ile Ser Tyr Ala Glu Met Phe His same time wall Arg 40 45 50 GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548 Ala Ile Ala gin Ser Leu Leu for Tyr Gly Leu Ser Ala Glu Arg for 55 60 65 CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596 Leu Leu Ile wall Ser Gly same time Asp Leu Glu His Leu gin Leu Ala Phe 70 75 80 85 GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644 Gly Ala Met Tyr Ala Gly Ile for Tyr Cys for wall Ser for Ala Tyr 90 95 100 TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692 Ser Leu Leu Ser gin Asp Leu Ala Lys Leu Arg His Ile wall Gly Leu 105 110 115 CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG GGG 740 Leu gin for Gly Leu wall Phe Ala Ala Asp Ala Ala for Phe gin 120 125 130 132 GAGAGGCGGT TTGCGTATTG GGCGCATGCA TAAAAACTGT TGTAATTCAT TAAGCATTCT 800 GCCGACATGG AAGCCATCAC AAACGGCATG ATGAACCTGA ATCGCCAGCG GCATCAGCAC 860 CTTGTCGCCT TGCGTATAAT ATTTGCCCAT GGACGCACAC CGTGGAAACG GATGAAGGCA 920 CGAACCCAGT TGACATAAGC CTGTTCGGTT CGTAAACTGT AATGCAAGTA GCGTATGCGC 980 TCACGCAACT GGTCCAGAAC CTTGACCGAA CGCAGCGGTG GTAACGGCGC AGTGGCGGTT 1040 TTCATGGCTT GTTATGACTG TTTTTTTGTA CAGTCTATGC CTCGGGCATC CAAGCAGCAA 1100 ·· • · -20GCGCGTTACG CCGTGGGTCG ATGTTTGATG TTATGGAGCA GCAACG ATG TTA CGC 1155 Met Leu Arg AGC Ser AGC Ser 5 AAC GAT GTT ACG CAG CAG 1203 same time Asp Val Thr Gin gin Gly Ser Arg Pro Lys Lys Leu Thr GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC 1251 Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly for Asp 20 25 30 35 CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT CGT GAG TTC 1299 gin wall Lys Ser Met Arg Ala. Ala Leu Asp Leu Phe Gly Arg Glu Phe 40 45 50 GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC 1347 Gly Asp wall Ala Thr Tyr Ser gin His gin for Asp Ser Asp Tyr Leu 55 60 65 GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC 1395 Gly same time Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala Ala Phe Asp 70 75 80 CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT 1443 gin Glu Ala wall wall Gly Ala Leu Ala Ala Tyr wall Leu for Arg Phe 85 90 95 GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC 1491 Glu gin for Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala wall Ser Gly 100 105 110 115 GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG 1539 Glu His Arg Arg gin Gly Ile Ala Thr Ala Leu Ile same time Leu Leu Lys 120 125 130 CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT 1587 His Glu Ala same time Ala Leu Gly Ala Tyr wall Ile Tyr wall gin Ala Asp 135 140 145 TAC GGT GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA CGG 1635 Tyr Gly Asp Asp for Ala wall Ala Leu Tyr Thr Lys Leu Gly Ile Arg 150 155 160 GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC ACC TAA CAA 1683 Glu Glu wall Met His Phe Asp Ile Asp for Ser Thr Ala Thr 165 170 175 177 TTCGTTCAAG CCGAGATCGG CTTCCCCT GTT TTG CAA TCG'GCG AAA 1735 Val Leu Gin Trp Arg Ser Ala Lys 563,565,570 GTT GAT GCG CTG TAT CGT GGT GAA GAT Val Asp Ala Leu Tyr Arg Gly Glu Asp Gin Ser Met Leu Arg Asp Glu 575 580 585 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· -21GCC ACA CTG TGA GTGGGTCAGG GGGGGCTTAC TCGGCGTTTT CCGACACTGC 1835 Ala Thr Leu 589 GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 1895 GCCTATCGAC TTAGGGGTAA AGGTCGCTCG CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 1955 ACAAATGGTC GATAGCGTAC TCGCAGGCTC TATGGCTCAA GCAAGCTTTG ATGCTTACCT 2015 GCTCCCGCGG CACATTGGCT TGTACAGCGG TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 2075 GCAGCGCATT TGCGGCACAG GCTTCGAACT GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 2135 AGGCGCTGAT CACGTGCTGT GTGTCGCGGG CTGCAG 2171 FIG. 2h: ·· • · -22 ········································ ·· II _e CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG ATG CGT TCT CTC GAG 356 Met Arg Ser Leu Glu GCG Ala CTT Leu CTT CCC TTC CCG GGT CGA ATT CTT GAG 404 Leu for Phe 10 Pro Gly Arg Ile Leu Glu Arg Leu Glu His Trp 20 GCT AAG ACC CGT CCA GAA CAA ACC TGC GTT GCT GCC AGG GCG GCA AAT 452 Ala Lys Thr Arg 25 for Glu Gin Thr Cys 30 Val Ala Ala Arg Ala Ala Asn 35 GGG GAA TGG CGT CGT ATC AGC TAC GCG GAA ATG TTC CAC AAC GTC CGC 500 Gly Glu Trp 40 Arg Arg íle Ser Tyr 45 Ala Glu Met Phe His 50 Asn Val Arg GCC ATC GCA CAG AGC TTG CTT CCT TAC GGA CTA TCG GCA GAG CGT CCG 548 Ala Ile 55 Ala gin Ser Leu Leu for Tyr Gly Leu Ser 65 Ala Glu Arg Pro CTG CTT ATC GTC TCT GGA AAT GAC CTG GAA CAT CTT CAG CTG GCA TTT 596 Leu 70 Leu Ile wall Ser Gly Asn Asp Leu Glu His 80 Leu gin Leu Ala Phe85 GGG GCT ATG TAT GCG GGC ATT CCC TAT TGC CCG GTG TCT CCT GCT TAT 644 Gly Ala Met Tyr Ala 90 Gly ile for Tyr Cys Pro 95 wall Ser For Ala Tyr 100 TCA CTG CTG TCG CAA GAT TTG GCG AAG CTG CGT CAC ATC GTA GGT CTT 692 Ser Leu Leu Ser 105 gin Asp Leu Ala Lys 110 Leu Arg His Ile Le Gly Leu 115 CTG CAA CCG GGA CTG GTC TTT GCT GCC GAT GCA GCA CCT TTC CAG CGC 740 Leu gin for 120 Gly Leu Val Phe Ala 125 Ala Asp Ala Ala for 130 Phe Gin Arg 133 GCT GTT TTG CAA TGG CGG TCG GCG AAA GTT GAT GCG CTG TAT CGT GGT 788 Ala 562 wall Leu gin 565 Trp Arg Ser Ala Lys 570 Val Asp Ala Leu Tyr Arg Gly 575 GAA Glu GAT Asp CAA gin TCC Ser ATG Met CGT Leu Arg GAC Asp GAG Glu GCC ACA Ala Thr CTG Leu TGA GTTGGTCAGG 837 580 585 589 GGGGGCTTAC TCGGCGTTTT CCGACACTGC GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT 897 TGATTGCGGG GGTGCCCTGT CGCTGGTGTC GCCTATCGAC TTAGGGGTAA AGGTCGCTCG 957 ··· · · 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 CCGCATGGCC ACGGCTGGGC GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC GGTAACTGAT AGAATCATGC AAAAATAGTT ACAGCACTGG 120 180 240 GGTGCACG ATG AAT AGC TAC GAT GGC CGT Met Asn Ser Thr Asp Gly Arg Trp Thr Val Th Val Lys 1 5 10 GTT GAA GAA GGT ATC GCT TGG GTC CGC CCG GAG Val Glu Glu Gly white Ala Trp Val Thr Leu Asn Arg Pro Glu Lys Arg 30 AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT GG 386 Asn Ala Met Ser Pro Thr Leu Asn Arg Glu Met Val Glu Val Glu 45 GTG CTG GAG CAG GAC GCA GAT GCT GTT CTG ACT GGT GCA 434 Val Leu Glu Gin Asp 50 Ala Asp Ala Arg Val Leu Val Leu Thr Gly Ala 60 GGC GAA TCC TGG ACC GCG GGC ATG GAG TAT TTC CGC GAG 482 Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys 71 Glu Tyr Phe Arg Glu 75 ACC GAT Thr Asp 80 GCT GGC CCC Ala Gly Pro GAA ATT CTG CAA GAG AAG Glu White Leu Gin Glu Lys 85 ATT CGT CGGGGACAGC Arg 90 91 AAGCGAACCG GAATTGCCAG CTGGGGCGCC CTCTGGTAAG GTTGGGAAGC CCTGCAAAGT 591 AAACTGGATG GCTTTCTTGC CGCCAAGGAT CTGATGGCGC AGGGGATCAA GATCTGATCA 651 AGAGACAGGA TGAGGATCGT TTCGC ATG ATT 1 GAT GGA TTG CAC GCA 703 Asp Gly Leu His Ala 5 GGT TCT CCG GCC GCT TGG GTG GAG AGG CTA TTC GGC TAT GAC TGG GCA 751 Gly Ser For Al Ala 20 Trp Val Glu Arg Leu Phe Gly Tyr Asp Trp Ala 25 CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG TCA GCG 799 Gin Gin Thr ile Gly 30 Cys Ser Asp Ala Ala Val 34 Phe Arg Leu Ser Ala 40 CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT GCC CTG 847 Gin Gly Arg Pro Val Leu Phe Val Lys Thr Asp Leu Ser Gly Ala Leu 55 AAT GAA CTG CAG GAC GAG GCA GCG TGG CTG GCC ACG ACG 895 Asn Glu Leu Gin Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala Thr Thr 60 65 70 GGC GTT CCT TGC GCA GCT GTC GG GTC GTC GGA 943 Gly Val for Cys Ala Ala wall 80 Leu Asp Val Thr Glu Ala Gly Arg GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG TCA TCT 991 Asp Trp Leu Leu Leu Gly Glu wall for Gly gin Asp Leu Leu Ser Ser 90 95 100 105 CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA ATG CGG 1039 His Leu Ala for Ala Glu Lys wall Ser Ile Met Ala Asp Ala Met Arg 110 115 120 CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC CAA GCG 1087 Arg Leu His Thr Leu Asp for Ala Thr Cys for Phe Asp His gin Ala 125 130 135 AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT CTT GTC 1135 Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly Leu wall 140 145 150 GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA GCC GAA 1183 Asp gin Asp Asp Leu Asp Glu Glu His gin Gly Leu Ala for Ala Glu 155 160 165 CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT CTC GTC 1231 Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp Gly Glu Asp Leu wall 170 175 180 185 GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA AAT GGC 1279 wall Thr His Gly Asp Ala Cys Leu for same time Ile Met wall Glu same time Gly 190 195 200 CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG GAC CGC 1327 Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly wall Ala Asp Arg 205 210 215 TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG CTT GGC 1375 Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu Leu Gly 220 225 230 GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC GCT CCC 1423 Gly Glu Trp Ala Asp Arg Phe Leu wall Leu Tyr Gly Ile Ala Ala for 235 240 245 GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC TTC TGA 1471 Asp Ser gin Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe Phe 250 255 260 264 GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC CC GAG CAG GGC ATG 1525 Glu Gin Gly Met 255 AAG CAG TTC CTT GAC GAG AAA AGC ATC Lys Gin Thr Tyr 260 265 270 ···················· • · · ·· ·· a -26AAG CGC TGA TAAATGCGCC GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA TAATGACAAT 1632 AATGAGGAGT GCCCAATGTT TCACGTGCCC CTGCTTATTG GTGGTAAGCC TTGTTCAGCA 1692 TCTGATGAGC GCACCTTCGA GCGTCGTAGC CCGCTGACCG GAGAAGTGGT ATCGCGCGTC 1752 GCTGCTGCCA GTTTGGAAGA TGCGGACGCC GCAGTGGCCG CTGCACAGGC TGCGTTTCCT 1812 GAATGGGCGG CGCTTGCTCC GAGCGAACGC CGTGCCCGAC TGCTGCGAGC GGCGGATCTT 1872 CTAGAGGACC GTTCTTCCGA GTTCACCGCC GCAGCGAGTG AAACTGGCGC AGCGGGAAAC 1932 TGGTATGGGT TTAACGTTTA CCTGGČGGCG GGCATGTTGC GGGGAATTC 1981 FIG. 2 j: · · · -27GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTGCACG ATG AAT AGC TAC GAT GGC CGT Met Asn Ser Thr Asp Gly Arg Trp Thr Val Th Val Lys 15 10 GTT wall 15 GAA GAA GGT ATC Glu Glu Gly White GCT TGG GTC ACG CTG AAC CGC 338 Ala Trp wall Thr Leu same time 25 Arg for Glu Lys Arg 30 AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT CTG GAG 386 same time Ala Met Ser for Thr Leu same time Arg Glu Met wall Glu wall Leu Glu 35 40 45 GTG CTG GAG CAG GAC GCA GAT GCT CGC GTG CTT GTT CTG ACT GGT GCA 434 wall Leu Glu gin Asp Ala Asp Ala Arg wall Leu wall Leu Thr Gly Ala 50 55 60 GGC GAA TCC TGG ACC GCG GGC ATG GAC CTG AAG GAG TAT TTC CGC GAG 482 Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu 65 70 75 ACC GAT GCT GGC CCC GAA ATT CTG CAA GAG AAG ATT CGT CGGGGGAGAG 531 Thr Asp Ala Gly for Glu Ile Leu gin Glu Lys Ile Arg 80 85 90 91 GCGGTTTGCG TATTGGGCGC ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA 591 CATGGAAGCC ATCACAAACG GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT 651 CGCCTTGCGT ATAATATTTG CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC 711 CCAGTTGACA TAAGCCTGTT CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG 771 CAACTGGTCC AGAACCTTGA CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT 831 GGCTTGTTAT GACTGTTTTT TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG 891 TTACGCCGTG GGTCGATGTT TGATGTTATG GAGCAGCAACG ATG TTA CGC AGC AGC 947 Met Leu Arg Ser AAC same time GAT Asp GTT wall ACG CAG Thr Gin 10 CAG gin GGC AGT CGC CCT 1 AAA ACA AAG TTA 5 GGT GGC 995 Gly Ser Arg for 15 Lys Thr Lys Leu Gly 20 Gly TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC 1043 Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu Gly for Asp gin wall 25 30 35 ·· ·· • · • · φ ΦΦ · -28φφφφ ·· • Φ ·· φ φ φ 9 9 φ Φ ΦΦ AAA Lys TCC ATG CGG GCT GCT Ala CTT GAT GTC GTC GTC GTC GTC GTC GTC 1091 Ser Met 40 Arg Ala Leu Asp 45 Leu Phe Gly Arg Glu Phe. Gly Asp GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC 1139 wall Ala 55 Thr Tyr Ser gin His 60 gin for Asp Ser Asp Tyr Leu Gly Asn TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA 1187 Leu 70 Leu Arg Ser Lys Thr 75 Phe Ile Ala Leu Ala 80 Ala Phe Asp Gin Glu 85 GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG 1235 Ala wall wall Gly Ala Leu Ala Ala Tyr wall 95 Leu For Arg Phe Glu 100 CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC 1283 for Arg Ser Glues 105 Tyr Ile Tyr Asp 110 Leu Ala Val Ser Gly Glu His 115 CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG 1331 Arg Arg gin 120 Gly ile Ala Thr Ala 125 Leu Ile same time Leu Leu Lys His Glu GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT 1379 Ala same time 135 Ala Leu Gly Ala Tyr 140 wall Ile Tyr wall Gin Ala Asp Tyr Gly GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA 1427 Asp 150 Asp for Ala Val Ala 155 Leu Tyr Thr Lys Leu 160 Gly ile Arg Glu Glu 165 GTG wall ATG Met CAC His TTT GAT Phe Asp 170 ATC Ile GAC Asp CCA for AGT Ser ACC Thr 175 GCC Ala ACC TAA Thr 177 CAATTCGTTC 1476 AAGCCGAGAT CGGCTTCCCC GAG CAG GGC ATG Glu Gin Gly Met Lys Gin Phe Leu Asp Glu 255 260 AAA AGC ATC AAG CCG GGC TTG CAG ACC TAC AAG CGC TGA TAAATGCGCC 1575 Lys Serie Lys Pro Gly Leu Gin Thr Tyr Lys Arg 265 270 275 276 GGGGCCCTCG CTGCGCCCCC GGCCTTCCAA TAATGACAAT AATGAGGAGT GCCCAATGTT 1635 TCACGTGCCC CTGCTTATTG GTGGTAAGCC TTGTTCAGCA TCTGATGAGC GCACCTTCGA 1695 GCGTCGTAGC CCGCTGACCG GAGAAGTGGT ATCGCGCGTC GCTGCTGCCA GTTTGGAAGA 1755 TGCGGACGCC GCAGTGGCCG CTGCACAGGC TGCGTTTCCT GAATGGGCGG CGCTTGCTCC 1815 GAGCGAACGC CGTGCCCGAC TGCTGCGAGC GGCGGATCTT CTAGAGGACC GTTCTTCCGA 1875 -29GTTCACCGCC GCAGCGAGTG AAACTGGCGC AGCGGGAAAC TGGTATGGGT TTAACGTTTA 1935 CCTGGCGGCG GGCATGTTGC GGGGAATTC 1964 FIG. 2k: ······················· 9 9 -30 • · · · · · · · ··· 999 i: ·· GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTGCACG ATG AAT AGC TAC GAT GGC CGT Met Asn Ser Thr Asp Gly Arg Trp Thr Val Th Val Lys 15 10 GTT GAA Val Glu 15 GAA Glu GGT ATC GCT TGG GTC ACG CTG AAC CGC 338 Gly Ile Ala 20 Trp Val Thr Leu same time 25 Arg Pro Glu Lys Arg 30 AAC GCA ATG AGC CCA ACT CTC AAT CGA GAG ATG GTC GAG GTT CTG GAG 386 same time Ala Met Ser for Thr Leu same time Arg Glu Met wall Glu wall Leu Glu 35 40 45 GTG CTG GAG CAG GAC GCA GAT GCT CGC GTG CTT GTT CTG ACT GGT GCA 434 wall Leu Glu gin Asp Ala Asp Ala Arg wall Leu wall Leu Thr Gly Ala 50 55 60 GGC GAA TCC TGG ACC GCG GGC ATG GAC CTG AAG GAG TAT TTC CGC GAG 482 Gly Glu Ser Trp Thr Ala Gly Met Asp Leu Lys Glu Tyr Phe Arg Glu 65 70 75 ACC GAT GCT GGC CCC GAA ATT CTG CAA GAG AAG ATT CGT CGC GAG CAG 530 Thr Asp Ala Gly for Glu Ile Leu gin Glu Lys Ile Arg Arg Glu gin 80 85 90 92 255 GGC ATG AAG CAG TTC CTT GAC GAG AAA AGC ATC AAG CCG GGC TTG CAG 578 Gly Met Lys gin Phe Leu Asp Glu Lys Ser Ile Lys for Gly Leu gin 260 265 270 ACC TAC AAG TGA TAAATGCGCC GGGGCCCTCG GGCCTTCCAA 633 Thr Tyr Lys Arg 275 276 TAATGACAAT AATGAGGAGT GCCCAATGTT TCACGTGCCC CTGCTTATTG GTGGTAAGCC 693 TTGTTCAGCA TCTGATGAGC GCACCTTCGA GCGTCGTAGC CCGCTGACCG GAGAAGTGGT 753 ATCGCGCGTC GCTGCTGCCA GTTTGGAAGA TGCGGACGCC GCAGTGGCCG CTGCACAGGC 813 TGCGTTTCCT GAATGGGCGG CGCTTGCTCC GAGCGAACGC CGTGCCCGAC TGCTGCGAGC 873 GGCGGATCTT CTAGAGGACC GTTCTTCCGA GTTCACCGCC GCAGCGAGTG AAACTGGCGC 933 AGCGGGAAAC TGGTATGGGT TTAACGTTTA CCTGGCGGCG GGCATGTTGC GGGGAATTC 992 FIG. 21: -31GAATTCCAAT AATGACAATA ATGAGGAGTG CCG ATT TTG CAC GTG CCC CTG CTT 55 Met Phe His Val For Leu Leu 1 5 ·· ·· ·· ·· ATT GGT GGT AAG White Gly Gly Lys CCT TGT TCA GCA TCT GAT GAG GC ACC TTC GAG CGT 103 for Cys Ser Ala 15 Ser Asp Glu Arg Thr 20 Phe Glu Arg 10 CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151 Arg Ser for Leu Thr Gly Glu wall wall Ser Arg wall Ala Ala Ala Ser 25 30 35 TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199 Leu Glu Asp Ala Asp Ala Ala wall Ala Ala Ala gin Ala Ala Phe for 40 45 50 55 GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247 Glu Trp Ala Ala Leu Ala for Ser Glu Arg Arg Ala Arg Leu Leu Arg 60 65 70 GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295 Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala 75 80 85 AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343 Ser Glu Thr Gly Ala Ala Gly same time Trp Tyr Gly Phe same time wall Tyr Leu 90 95 100 GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391 Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr gin Ile gin 105 110 115 GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439 Gly Asp wall Ile for Ser same time wall for Gly Ser Phe Ala Met Ala wall 120 125 130 135 CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487 Arg gin for Cys Gly wall wall Leu Gly Ile Ala for Trp same time Ala for 140 145 150 GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535 wall Ile Leu Gly wall Arg Ala wall Ala Met for Leu Ala Cys Gly same time 155 160 165 ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583 Thr wall wall Leu Lys Ser Ser Glu Leu Ser for Phe Thr His Arg Leu 170 175 180 ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631 Ile Gly gin wall Leu His Asp Ala Gly Leu Gly Asp Gly wall wall same time 185 190 195 GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679 wall Ile Ser same time Ala for gin Asp Ala for Ala wall wall Glu Arg Leu 200 205 210 215 ·· a .2. -32 · · 1 1 1 t a 1 1 1 1 1 1 1 1 1 1 1 a a a a a a a a a a a a a ATT Ile GCA AAT CCT GCG Pro Ala 220 GTA wall CGT CGA GTG AAC TTC ACC 727 Ala same time Arg Arg wall Asn Phe Thr Gly Ser Thr His 230 GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775 wall Gly Arg Ile Ile Gly Glu Leu Ser Ala Arg His Leu Lys for Ala 235 240 245 GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823 wall Leu Glu Leu Gly Gly Lys Ala for Phe Leu wall 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 wall Glu Ala Ala Ala Phe Gly Ala Tyr Phe same time 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 wall Thr Ala wall 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 wall Glu Lys Leu Ala Arg Lys wall 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 for same time Asp for gin Ser wall Leu Gly Ser Leu Ile Asp 315 320 325 GCC AAT GCA GGT CAA CGC ATC CAG GTT CTG GTC GAT GAT GCG CTC GGG 1063 Ala same time Ala Gly gin Arg Ile gin wall Leu wall Asp Asp Ala Leu 330 335 340 342 GACAGCAAGC GAACCGGAAT TGCCAGCTGG CAAAGTAAAC TGGATGGCTT TCTTGCCGCC TGATCAAGAG ACAGGATGAG GATCGTTTCG GGCGCCCTCT GGTAAGGTTG GGAAGCCCTG 1123 AAGGATCTGA TGGCGCAGGG GATCAAGATC 1183 C ATG ATT GAA GAT GGA TTG 1235 Met Bad Glu Gin Asp Gly Leu 1 5 CAC His GCA GGT TCT CCG GCC Ala 10 GCT TGG GTG GAG AGG CTA TTC GGC 1283 Ala Trp 15 wall Glu Arg Leu Phe 20 Gly Tyr Asp TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG TTC CGG CTG 1331 Trp Ala gin gin Thr Ile Gly Cys Ser Asp Ala Ala wall Phe Arg Leu 25 30 35 TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC CTG TCC GGT 1379 Ser Ala gin Gly Arg for wall Leu Phe wall Lys Thr Asp Leu Ser Gly 40 45 50 55 GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG TGG CTG GCC 1427 Ala Leu same time Glu Leu gin Asp Glu Ala Ala Arg Leu Ser Trp Leu Ala 60 65 70 -33 ····························· ·· · · · · · · ACG ACG GGC GTT CCT TGC Cys GCA GCT GTG CTC 1475 Thr Thr Gly wall 75 for Ala Ala wall 80 Leu Asp Val Val Thr Glu Ala GGA AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG GAT CTC CTG 1523 Gly Arg Asp Trp Leu Leu Leu Gly Glu wall for Gly gin Asp Leu Leu 90 95 100 TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG GCT GAT GCA 1571 Ser Ser His Leu Ala for Ala Glu Lys wall Ser Ile Met Ala Asp Ala 105 110 115 ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA TTC GAC CAC 1619 Met Arg Arg Leu His Thr Leu Asp for Ala Thr Cys for Phe Asp His 120 125 130 135 CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG GAA GCC GGT 1667 gin Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met Glu Ala Gly 140 145 150 CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG CTC GCG CCA 1715 Leu wall Asp gin Asp Asp Leu Asp Glu Glu His gin Gly Leu Ala for 155 160 165 GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC GGC GAG GAT 1763 Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp Gly Glu Asp 170 175 180 CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC ATG GTG GAA 1811 Leu wall wall Thr His Gly Asp Ala Cys Leu for same time Ile Met wall Glu 185 190 195 AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG GGT GTG GCG 1859 same time Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu Gly wall Ala 200 205 210 215 GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT GCT GAA GAG 1907 Asp Arg Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile Ala Glu Glu 220 225 230 CTT GGC GGC GAA TGG GCT GAC CGC TTC CTC GTG CTT TAC GGT ATC GCC 1955 Leu Gly Gly Glu Trp Ala Asp Arg Phe Leu wall Leu Tyr Gly Ile Ala 235 240 245 GCT CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT GAC GAG TTC 2003 Ala for Asp Ser gin Arg Ile Ala Phe Tyr Arg Leu Leu Asp Glu Phe 250 255 260 TTC TGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC Phe Gin 264 421 CGC GTC GAT TCG GGC ATT TGC CAT Arg Val Asp Ser Gly Ile Cys His White Asn Gly Pro Thr Val His Asp 425 430 435 ·· -34 ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · GAG GCT Clu Glu Ala Gin ATG Met CCA TTC Pro Phe Gly Gly Gly Gly 445 GTG wall AAG TCC AGC GGC TAC GGC AGC 2153 Lys Ser Ser 450 Gly Tyr Gly Ser 440 TTC GGC AGT CGA GCA TCG ATT GAG CAC TTT ACC CAG CTG CGC TGG CTG 2201 Phe Gly Ser Arg Ala Ser Ile Glu 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 2247 Thr Ile gin same time Gly for Arg His Tyr for Ile 475 480 481 GGGCGCCGCG GGCATCATGC CCGCGGCGCT CGCCTCATTT CAATCTCTAA CTTGATAAAA 2307 ACAGAGCTGT -TCTCCGGTCT TGGTGGATCA AGGCCAGTCG CGGAGAGTCT CGAAGAGGAG 2367 AGTACAGTGA ACGCCGAGTC CACATTGCAA CCGCAGGCAT CATCATGCTC TGCTCAGCCA 2427 CGCTACCGCA GTGTGTCGAT TGGTCATCCT CCGGTTGAGG TTACGCAAGA CGCTGGAGGT 2487 ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT CTTCCCTTCC CGGGTGGAAT TC 2539 FIG. 2 m: ·· ·· ·· • · -35GAATTCCAAT AATGACAATA ATGAGGAGTG CCG ATT TTG CAC GTG CCC CTG CTT 55 Met Phe His Val For Leu Leu 1 5 ································· ATT GGT GGT AAG CCT TGT TCA 103 Ile Gly Gly 10 Lys Pro Cys Ser Ala Ser Glu Arg Thr Phe Glu Arg 15 20 CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151 Arg Ser for Leu Thr Gly Glu wall wall Ser Arg wall Ala Ala Ala Ser 25 30 35 TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199 Leu Glu Asp Ala Asp Ala Ala wall Ala Ala Ala gin Ala Ala Phe for 40 45 50 55 - GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247 Glu Trp Ala Ala Leu Ala for Ser Glu Arg Arg Ala Arg Leu Leu Arg 60 65 70 GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295 Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala 75 80 85 AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343 Ser Glu Thr Gly Ala Ala Gly same time Trp Tyr Gly Phe same time wall Tyr Leu 90 95 100 GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391 Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr gin Ile gin 105 110 115 GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439 Gly Asp wall Ile for Ser same time wall for Gly Ser Phe Ala Met Ala wall 120 125 130 135 CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487 Arg gin for Cys Gly wall wall Leu Gly Ile Ala for Trp same time Ala for 140 145 150 GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535 wall Ile Leu Gly wall Arg Ala wall Ala Met for Leu Ala Cys Gly same time 155 160 165 ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583 Thr wall wall Leu Lys Ser Ser Glu Leu Ser for Phe Thr His Arg Leu 170 175 180 ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631 Ile Gly gin wall Leu His Asp Ala Gly Leu Gly Asp Gly wall wall same time 185 190 195 GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679 wall Ile Ser same time Ala for gin Asp Ala for Ala wall wall Glu Arg Leu 200 205 210 215 ·· · · · · · · · · · · · · -36 ·· ·························· · · · · · · · ATT Ile GCA AAT CCT GCG Pro Ala 220 GTA wall CGT CGA GTG AAC TTC ACC 727 Ala same time Arg Arg wall Asn Phe Thr Gly Ser Thr His 230 GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775 wall 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 wall Leu Glu Leu Gly Gly Lys Ala for Phe Leu wall 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 wall 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 Cys Met Ser Thr Glu Arg Leu Ile wall 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 wall 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 for gin Ser Val Leu Gly Ser Leu Asp 315 320 325 GCC AAT GCA GGT CAA CGC ATC CAG GTGGGGAGAG GCGGTTTGCG TATTGGGCGC 1069 Ala same time Ala Gly Gin Arg Ile gin 330 335 ATGCATAAAA ACTGTTGTAA TTCATTAAGC ATTCTGCCGA CATGGAAGCC ATCACAAACG 1129 GCATGATGAA CCTGAATCGC CAGCGGCATC AGCACCTTGT CGCCTTGCGT ATAATATTTG 1189 CCCATGGACG CACACCGTGG AAACGGATGA AGGCACGAAC CCAGTTGACA TAAGCCTGTT 1249 CGGTTCGTAA ACTGTAATGC AAGTAGCGTA TGCGCTCACG CAACTGGTCC AGAACCTTGA 1309 CCGAACGCAG CGGTGGTAAC GGCGCAGTGG CGGTTTTCAT GGCTTGTTAT GACTGTTTTT 1369 TTGTACAGTC TATGCCTCGG GCATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTT 1429 TGATGTTATG GAGCAGCAAC G ATG Met TTA CGC AGC Ser AGC Ser 5 AAC GAT GTT ACG CAG 1480 Leu Arg same time Asp Val Thr Gin 10 1 CAG GGC AGT CCT AAA ACA AAG TTA GGT GGC TCA AGT ATG GGC ATC 1528 gin Gly Ser Arg Lys Thr 15 Lys Leu Gly 20 Gly Ser Ser Met Gly ile 25 ATT CGC ACA TGT AGG CTC GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT 1576 Ile Arg Thr Cys Arg Leu Gly for Asp 35 gin wall Lys Ser Met Arg Ala -37 ·· ·· · · · · 9 9 9 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 9 9 9 9 9 99 99 99 GCT CTT GGT Gly CGT Arg GAG TTC GGA GAC GTA GCC ACC TAC TCC Tyr Ser 1624 Ala Leu Asp Leu Phe Glu Phe Gly Asp wall Ala 55 Thr CAA CAT CAG CCG GAC TCC GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG 1672 gin His 60 Gin Pro Asp Ser Asp 65 Tyr Leu Gly Asn Leu 70 Leu Arg Ser Lys ACA TTC ATC GCG CTT GCT GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT 1720 Thr 75 Phe Ala Ala Leu Ala 80 Ala Phe Asp Gin Glu Ala wall wall Gly Ala 90 CTC GCG GCT TAC GTT CTG CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC 1768 Leu Ala Ala Tyr wall 95 Leu for Arg Phe Glu Gin 100 for Arg Ser Glu ile 105 TAT ATC TAT GAT CTC GCA GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT 1816 Tyr Ile Tyr Asp 110 Leu Ala wall Ser Gly Glu His Arg Arg gin 120 Gly ile GCC ACC GCG CTC ATC AAT CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT 1864 Ala Thr Ala Leu Ile same time Leu Leu Lys His Glu Ala same time 135 Ala Leu Gly GCT TAT GTG ATC TAC GTG CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG 1912 Ala Tyr 140 Val ile Tyr wall gin 145 Ala Asp Tyr Gly Asp 150 Asp for Ala Val GCT CTC TAT ACA AAG TTG GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT 1960 Ala 155 Leu Tyr Thr Lys Leu 160 Gly Arg Glu Glu wall Met His Phe Asp 170 ATC Ile GAC Asp CCA AGT Pro Ser ACC Thr GCC Ala ACC Thr TAA CAATTCGTTC AAGCCGAGAT CGGCTTCCCA 2014 175 177 A TTG GCC CAG CGC GTC GAT TCG GGC ATT Leu Ala Gin Arg Val Asp Ser Gly White Cys His White Asn Gly Pro Thr 420 425 430 435 GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT 2111 wall His Asp Glu Ala 440 gin Met for Phe Gly Gly 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 same time Gly for Arg His Tyr for Ile 470 475 480 481 ATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT CGCCTCATTT CAATCTCTAA 2264 CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA AGGCCAGTCG CGGAGAGTCT 2324 ·························· CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA CCGCAGGCAT CATCATGCTC 2384 TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT CCGGTTGAGG TTACGCAAGA 2444 CGCTGGAGGT ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT CTTCCCTTCC CGGGTGGAAT 2504 TC 2506 FIG. 2n: ·· ·· -39GAATTCCAAT AATGACAATA ATGAGGAGTG CCG ATT TTG CAC GTG CCC CTG CTT 55 Met Phe His Val For Leu Leu 1 5 ATT GGT White Gly GGT Gly 10 AAG CCT TGT TCA GCA TCT GAT GAG 103 Lys for Cys Ser Ala Ser Asp Glu Arg Thr 20 Phe Glu Arg CGT AGC CCG CTG ACC GGA GAA GTG GTA TCG CGC GTC GCT GCT GCC AGT 151 Arg Ser for Leu Thr Gly Glu wall wall Ser Arg wall Ala Ala Ala Ser 25 30 35 TTG GAA GAT GCG GAC GCC GCA GTG GCC GCT GCA CAG GCT GCG TTT CCT 199 Leu Glu Asp Ala Asp Ala Ala wall Ala Ala Ala gin Ala Ala Phe for 40 45 50 55 GAA TGG GCG GCG CTT GCT CCG AGC GAA CGC CGT GCC CGA CTG CTG CGA 247 Glu Trp Ala Ala Leu Ala for Ser Glu Arg Arg Ala Arg Leu Leu Arg 60 65 70 GCG GCG GAT CTT CTA GAG GAC CGT TCT TCC GAG TTC ACC GCC GCA GCG 295 Ala Ala Asp Leu Leu Glu Asp Arg Ser Ser Glu Phe Thr Ala Ala Ala 75 80 85 AGT GAA ACT GGC GCA GCG GGA AAC TGG TAT GGG TTT AAC GTT TAC CTG 343 Ser Glu Thr Gly Ala Ala Gly same time Trp Tyr Gly Phe same time wall Tyr Leu 90 95 100 GCG GCG GGC ATG TTG CGG GAA GCC GCG GCC ATG ACC ACA CAG ATT CAG 391 Ala Ala Gly Met Leu Arg Glu Ala Ala Ala Met Thr Thr gin Ile gin 105 110 115 GGC GAT GTC ATT CCG TCC AAT GTG CCC GGT AGC TTT GCC ATG GCG GTT 439 Gly Asp wall Ile for Ser same time wall for Gly Ser Phe Ala Met Ala wall 120 125 130 135 CGA CAG CCA TGT GGC GTG GTG CTC GGT ATT GCG CCT TGG AAT GCT CCG 487 Arg gin for Cys Gly wall wall Leu Gly Ile Ala for Trp same time Ala for 140 145 150 GTA ATC CTT GGC GTA CGG GCT GTT GCG ATG CCG TTG GCA TGC GGC AAT 535 wall Ile Leu Gly wall Arg Ala wall Ala Met for Leu Ala Cys Gly same time 155 160 165 ACC GTG GTG TTG AAA AGC TCT GAG CTG AGT CCC TTT ACC CAT CGC CTG 583 Thr wall wall Leu Lys Ser Ser Glu Leu Ser for Phe Thr His Arg Leu 170 175 180 ATT GGT CAG GTG TTG CAT GAT GCT GGT CTG GGG GAT GGC GTG GTG AAT 631 Ile Gly gin wall Leu His Asp Ala Gly Leu Gly Asp Gly wall wall same time 185 190 195 GTC ATC AGC AAT GCC CCG CAA GAC GCT CCT GCG GTG GTG GAG CGA CTG 679 wall Ile Ser same time Ala for gin Asp Ala for Ala wall wall Glu Arg Leu 200 205 210 215 · · -40 · 40 · 40 40 40 40 40 • · · · · · ATT GCA AAT CCT GCG GTA CGT CGA GTG 727 Ile Ala Asn Pro Ala wall Arg Arg wall Asn Phe Thr Gly Ser Thr His 230 GTT GGA CGG ATC ATT GGT GAG CTG TCT GCG CGT CAT CTG AAG CCT GCT 775 wall Gly Arg tions 235 Gly Glu Leu Ser 240 Ala Arg His Leu Lys Pro Ala GTG CTG GAA TTA GGT GGT AAG GCT CCG TTC TTG GTC TTG GAC GAT GCC 823 wall Leu Glu Leu Gly 250 Gly Lys Ala 255 for Phe Leu Val Leu Asp 260 Asp Ala GAC CTC GAT GCG GTC GAA GCG GCG GCC TTT GCC GCC TAC TTC AAT 871 Asp Leu 265 Asp Ala Ala wall Glu 270 Ala Ala Ala Phe Gly Ala Tyr Phe Asn CAG GGT CAA ATC TGC ATG TCC ACT GAG CGT CTG ATT GTG ACA GCA GTC 919 gin 280 Gly Gin White Cys Met 285 Ser Thr Glu Arg Leu Val Thr Ala Val 295 GCA GAC GCC TTT GTT GAA AAG CTG GCG AGG AAG GTC GCC ACA CTG CGT 967 Ala Asp Ala Phe Val 300 Glu Lys Leu Ala Arg Lys Val Ala Thr Leu Arg 310 GCT GGC GAT CCT AAT GAT CCG CAA TCG GTC TTG GGT TCG TTG ATT GAT 1015 Ala Gly Asp Pro Asn 315 Asp for gin Ser 320 Val Leu Gly Ser Leu 325 Asp GCC AAT GCA GGT CAA CGC ATC CAG GTT CTG GTC GAT GAT GCG CTC GCA 1063 Ala same time Ala Gly Gin Arg Ile gin 335 wall Leu Val Asp Asp Ala 340 Leu Ala AAA Lys GGC Gly 345 GCG CAATGGAA TTG GCC CAG CGC GTC GAT TCG GGC ATT TGC CAT Ala Leu 1113 ATC AAT GGA CCG ACT GTG CAT GAC GAG GCT CAG ATG CCA TTC GGT GGG 1161 Ile same time Gly Pro Thr 434 wall His Asp Glu Ala Gin Met Pro Phe Gly Gly 445 GTG AAG TCC AGC GGC TAC GGC AGC TTC GGC AGT CGA GCA TCG ATT GAG 1209 wall Lys Ser Ser Gly 450 Tyr Gly Ser Phe 455 Gly Ser Arg Ala Ser 460 ile Glu CAC TTT ACC CAG CTG CGC TGG CTG ACC ATT CAG AAT GGC CCG CGG CAC 1257 His Phe Thr Gin Leu 466 Arg Trp Leu 470 Thr ile Gin Asn Gly Pro 474 Arg His TAT Tyr CCA for 480 ATC TAA ATCGATCTTC GGGCGCCGCG GGCATCATGC CCGCGGCGCT 481 1309 CGCCTCATTT CAATCTCTAA CTTGATAAAA ACAGAGCTGT TCTCCGGTCT TGGTGGATCA 1369 AGGCCAGTCG CGGAGAGTCT CGAAGAGGAG AGTACAGTGA ACGCCGAGTC CACATTGCAA 1429 · t 29 29 29 29 29 29 1429 • ··· • · · · · · · CCGCAGGCAT CATCATGCTC TGCTCAGCCA CGCTACCGCA GTGTGTCGAT TGGTCATCCT 1489 CCGGTTGAGG TTACGCAAGA CGCTGGAGGT ATTGTCCGGA TGCGTTCTCT CGAGGCGCTT 1549 CTTCCCTTCC CGGGTGGAAT TC 1571 FIG. 2o: ·· · · ·· ·· -42 · e e 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT Met Ser Trp Ser 15 10 GAC Asp ACT GCG TTG GTT GCG GCA GTG 160 Thr Ala 15 Leu wall Ala Ala wall 20 Arg Thr for Trp Ile 25 Asp Cys Gly GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208 Gly Ala 30 Leu Ser Leu wall Ser 35 for Ile Asp Leu Gly 40 wall Lys wall Ala CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC 256 Arg 45 Glu wall Leu Met Arg 50 Ala Ser Leu Glu for 55 gin Met wall Asp Ser 60 GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304 wall Leu Ala Gly Ser 65 Met Ala gin Ala Ser 70 Phe Asp Ala Tyr Leu 75 Leu CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352 for Arg His Ile 80 Gly Leu Tyr Ser Gly 85 wall for Lys Ser wall 90 for Ala TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400 Leu Gly wall 95 gin Arg Ile Cys Gly 100 Thr Gly Phe Glu Leu 105 Leu Arg gin GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448 Ala Gly 110 Glu gin Ile Ser gin 115 Gly Ala Asp His wall 120 Leu Cys wall Ala GCA GAG TCC ATG TCG CGT AAC CCC ATC GCG TCG TAT ACA CAC CGG GGC 496 Ala 125 Glu Ser Met Ser Arg 130 same time for Ile Ala Ser 135 Tyr Thr His Arg Gly 140 GGG TTC CGC CTC GGT GCG CCC GTT GAG TTC AAG GAT TTT TTG TGG GAG 544 Gly Phe Arg Leu Gly 145 Ala for wall Glu Phe 150 Lys Asp Phe Leu Trp 155 Glu GCA TTG TTT GAT CCT GCT CCA GGA CTC GAC ATG ATC GCT ACC GCA GAA 592 Ala Leu Phe Asp 160 for Ala for Gly Leu 165 Asp Met Ile Ala Thr 170 Ala Glu AAC CTG GGGACAGCAA GCGAACCGGA ATTGCCAGCT GGGGCGCCCT CTGGTAAGGT 648 Asn Leu 174 TGGGAAGCCC TGCAAAGTAA ACTGGATGGC TTTCTTGCCG CCAAGGATCT GATGGCGCAG 708 GGGATCAAGA TCTGATCAAG AGACAGGATG AGGATCGTTT Met Giles Gin · G · • · · · · · · · · · • · • · • · GAT GGA Asp Gly 5 TTG Leu CAC GCA GGT TCT. CCG GCC GCT 811 His Ala Gly 10 Ser Pro Ala Ala Trp Val Leu Phe 15 20 GGC TAT GAC TGG GCA CAA CAG ACA ATC GGC TGC TCT GAT GCC GCC GTG 859 Gly Tyr Asp Trp Ala gin gin Thr Ile Gly Cys Ser Asp Ala Ala wall 25 30 35 TTC CGG CTG TCA GCG CAG GGG CGC CCG GTT CTT TTT GTC AAG ACC GAC 907 Phe Arg Leu Ser Ala gin Gly Arg for wall Leu Phe wall Lys Thr Asp 40 45 50 CTG TCC GGT GCC CTG AAT GAA CTG CAG GAC GAG GCA GCG CGG CTA TCG 955 Leu Ser Gly Ala Leu same time Glu Leu gin Asp Glu Ala Ala Arg Leu Ser 55 60 65 TGG CTG GCC ACG ACG GGC GTT CCT TGC GCA GCT GTG CTC GAC GTT GTC 1003 Trp Leu Ala Thr Thr Gly wall for Cys Ala Ala wall Leu Asp wall wall 70 75 80 ACT GAA GCG GGA AGG GAC TGG CTG CTA TTG GGC GAA GTG CCG GGG CAG 1051 Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu wall for Gly gin 85 90 95 100 GAT CTC CTG TCA TCT CAC CTT GCT CCT GCC GAG AAA GTA TCC ATC ATG 1099 Asp Leu Leu Ser Ser His Leu Ala for Ala Glu Lys wall Ser Ile Met 105 110 115 GCT GAT GCA ATG CGG CGG CTG CAT ACG CTT GAT CCG GCT ACC TGC CCA 1147 Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp for Ala Thr Cys for 120 125 130 TTC GAC CAC CAA GCG AAA CAT CGC ATC GAG CGA GCA CGT ACT CGG ATG 1195 Phe Asp His gin Ala Lys His Arg Ile Glu Arg Ala Arg Thr Arg Met 135 140 145 GAA GCC GGT CTT GTC GAT CAG GAT GAT CTG GAC GAA GAG CAT CAG GGG 1243 Glu Ala Gly Leu wall Asp gin Asp Asp Leu Asp Glu Glu His gin Gly 150 155 160 CTC GCG CCA GCC GAA CTG TTC GCC AGG CTC AAG GCG CGC ATG CCC GAC 1291 Leu Ala for Ala Glu Leu Phe Ala Arg Leu Lys Ala Arg Met for Asp 165 170 175 180 GGC GAG GAT CTC GTC GTG ACC CAT GGC GAT GCC TGC TTG CCG AAT ATC 1339 Gly Glu Asp Leu wall wall Thr His Gly Asp Ala Cys Leu for same time Ile 185 190 195 ATG GTG GAA AAT GGC CGC TTT TCT GGA TTC ATC GAC TGT GGC CGG CTG 1387 Met wall Glu same time Gly Arg Phe Ser Gly Phe Ile Asp Cys Gly Arg Leu 200 205 210 GGT GTG GCG GAC CGC TAT CAG GAC ATA GCG TTG GCT ACC CGT GAT ATT 1435 Gly wall Ala Asp Arg Tyr gin Asp Ile Ala Leu Ala Thr Arg Asp Ile 215 220 225 • · ·········· GCT GAA GAG GGC GAA TGG GCT GTC Ala Glu Glu 230 Leu Gly Gly Glu 235 Trp Ala Asp Arg Phe Leu Val Leu 240 Tyr GGT ATC GCC GCT CCC GAT TCG CAG CGC ATC GCC TTC TAT CGC CTT CTT Gly Ile Ala Ala for Asp Ser gin Arg Ile Ala Phe Tyr Arg Leu Leu 245 250 255 260 GAC GAG TTC TTC GGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 1586 Asp Glu Phe Phe 264 CA TTG AGG GCG CAA GAG GC AAA TGG ATT GAC Leu Arg Ala Gin Glu Lys Trp White Asp Gin Glu White Val Ala 197 200 -205 210 GTT ACG GAT GAA CAG TTC Gin Phe GAT GGC TAC AAC 1681 wall Thr Asp Glu 215 Asp Leu Glu 220 Gly Tyr Asn. Ser Arg Ala 225 Ile GAA CTG CCT CGG AAG GCA AAA TTG TTG ATC GTG ACA GTC ATC CGC GGC 1729 Glu Leu for Arg Lys Ala Lys Leu Leu Ile wall Thr wall Ile Arg Gly 230 235 240 CTA GCA GTC TTT GAA GCC CTT TCC CGA TTG AAG CCT GTT CAT TCT GGC 1777 Leu Ala wall Phe Glu Ala Leu Ser Arg Leu Lys for wall His Ser Gly 245 250 255 GGG GTG CAG ACT GCG GGC AAC AGC TGT GCC GTA GTG GAC GGC GCC GCG 1825 Gly wall gin Thr Ala Gly same time Ser Cys Ala wall wall Asp Gly Ala Ala 260 265 270 275 GCG GCT TTG GTG GCT CGA GAG TCG TCT GCG ACA CAG CCG GTC TTG GCT 1873 Ala Ala Leu wall Ala Arg Glu Ser Ser Ala Thr gin for wall Leu Ala 280 285 290 AGG ATA CTG GCT ACC TCC GTA GTC GGG ATC GAG CCC GAG CAT ATG GGG 1921 Arg Ile Leu Ala Thr Ser wall wall Gly Ile Glu for Glu His Met Gly 295 300 305 CTC GGC CCT GCG CCC GCG ATT CGC CTG CTG CTT GCG CGT AGT GAT CTT 1969 Leu Gly for Ala for Ala Ile Arg Leu Leu Leu Ala Arg Ser Asp Leu 310 315 320 AGT TTG AGG GAT ATC GAC CTC TTT GAG ATA AAC GAG GCG CAG GCC GCC 2017 Ser Leu Arg Asp Ile Asp Leu Phe Glu Ile same time Glu Ala gin Ala Ala 325 330 335 CAA GTT CTA GCG GTA CAG CAT GAA TTG GGT ATT GAG CAC TCA AAA CTT 2065 gin wall Leu Ala wall gin His Glu Leu Gly Ile Glu His Ser Lys Leu 340 345 350 355 AAT ATT TGG GGC GGG GCC ATT GCA CTT GGA CAC CCG CTT GCC GCG ACC 2113 same time Ile Trp Gly Gly Ala Ile Ala Leu Gly His for Leu Ala Ala Thr 360 365 370 · ··· · · · · · · · · · -45 · · · · · · · · · · · · · · · · · · · · · · · · · · · GGA Gly TTG Leu CGT CTC TGG ATG ACC CTC GCT CAC CAA TTG CAA GCT AAT AAC 2161 Arg Leu Cys Met 375 Thr Leu Ala 380 His gin Leu gin Ala Asn Asn 385 TTT CGA TAT GGA ATT GCC TCG GCA TGC ATT GGT GGG GGA CAG GGG ATG 2209 Phe Arg Tyr Gly Ala Ala Ser Ala Cys Ile Gly Gly Gly Gin Gly Met 390 395 400 GCG GTT CTT TTA GAG AAT CCC CAC TTC GGT TCG TCC TCT GCA CGA AGT 2257 Ala wall Leu Leu Glu Asn Pro His Phe Gly Ser Ser Ser Ala Arg Ser 405 410 415 TCG ATG ATT AAC AGA GTT GAC CAC TAT CCA CTG AGC TAA CGGGCATCTC 2306 Ser Met Ile Asn Arg Val Asp His Tyr for Leu Ser 420 425 430 431 CTTTGTTGCT TTGAGGTGGC GCACGAAGGA GGGCTCGAAA ATCTCTGCTA AAAACAAGAA 2366 GAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAAGTTTAGG AGTCCAGGCT 2426 AAACTTGCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT 2486 TTCTACAGTG TACATACCTT GTCAGGGTTG GTGGGAATTC 2526 FIG. 2p: • · · · -46 ·· ··· · · · · · · · · · · · · · GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT Met Ser Trp Ser 15 10 GAC Asp ACT GCG TTG GTT GCG GCA CCC TGG ATT GAT TGC GGG Gly 160 Thr Ala 15 Leu Val Ala Ala Val Arg Thr for Suffering 25 Asp Cys GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208 Gly Ala Leu Ser Leu wall Ser for Ile Asp Leu Gly wall Lys wall Ala 30 35 40 CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC . 256 Arg Glu wall Leu Met Arg Ala Ser Leu Glu for gin Met wall Asp Ser 45 50 55 60 GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304 wall Leu Ala Gly Ser Met Ala gin Ala Ser Phe Asp Ala Tyr Leu Leu 65 70 75 CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352 for Arg His Ile Gly Leu Tyr Ser Gly wall for Lys Ser wall for Ala 80 85 90 TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400 Leu Gly wall gin Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg gin 95 100 105 GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448 Ala Gly Glu gin Ile Ser gin Gly Ala Asp His wall Leu Cys wall 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 same time for 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 for wall 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 for Ala for Gly Leu Asp Met Ile Ala Thr Ala Glu 160 ÄAC CTG GGGGAGAGGC GGTTTGCGTA TTGGGCGCAT GCATAAAAAC TGTTGTAATT 648 Asn Leu 174 CATTAAGCAT TCTGCCGACA TGGAAGCCAT CACAAACGGC ATGATGAACC TGAATCGCCA 708 GCGGCATCAG CACCTTGTCG CCTTGCGTAT AATATTTGCC CATGGACGCA CACCGTGGAA 768 ACGGATGAAG GCACGAACCC AGTTGACATA AGCCTGTTCG GTTCGTAAAC TGTAATGCAA 828 GTAGCGTATG CGCTCACGCA ACTGGTCCAG AACCTTGACC GAACGCAGCG GTGGTAACGG 888 -47 ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· · CGCAGTGGCG GTTTTCATGG CTTGTTATGA CTGTTTTTTT GTACAGTCTA TGCCTCGGGC 948 ATCCAAGC AGCAAGCGCG TTACGCCGTG GGTCGATGTTTG ATGTTATGGA GCAGCAACG 1007 ATG TTA CGC AGC Ser AGC AAC GAT GTT ACG CAG 1055 Met Leu Arg Ser 5 same time Asp Val Thr gin 10 gin Gly Ser Arg Pro Lys 15 ACA AAG TTA GGT GGC TCA AGT ATG GGC ATC ATT CGC ACA TGT AGG CTC 1103 Thr Lys Leu Gly Gly Ser Ser Met Gly Ile Ile Arg Thr Cys Arg Leu 20 25 30 GGC CCT GAC CAA GTC AAA TCC ATG CGG GCT GCT CTT GAT CTT TTC GGT 1151 Gly for Asp gin wall Lys Ser Met Arg Ala Ala Leu Asp Leu Phe Gly 35 40 45 CGT GAG TTC GGA GAC GTA GCC ACC TAC TCC CAA CAT CAG CCG GAC TCC 1199 Arg Glu Phe Gly Asp wall Ala Thr Tyr Ser gin His gin for Asp Ser 50 55 60 GAT TAC CTC GGG AAC TTG CTC CGT AGT AAG ACA TTC ATC GCG CTT GCT 1247 Asp Tyr Leu Gly same time Leu Leu Arg Ser Lys Thr Phe Ile Ala Leu Ala 65 70 75 80 GCC TTC GAC CAA GAA GCG GTT GTT GGC GCT CTC GCG GCT TAC GTT CTG 1295 Ala Phe Asp gin Glu Ala wall wall Gly Ala Leu Ala Ala Tyr wall Leu 85 90 95 CCC AGG TTT GAG CAG CCG CGT AGT GAG ATC TAT ATC TAT GAT CTC GCA 1343 for Arg Phe Glu gin for Arg Ser Glu Ile Tyr Ile Tyr Asp Leu Ala 100 105 110 GTC TCC GGC GAG CAC CGG AGG CAG GGC ATT GCC ACC GCG CTC ATC AAT 1391 wall Ser Gly Glu His Arg Arg gin Gly Ile Ala Thr Ala Leu Ile same time 115 120 125 CTC CTC AAG CAT GAG GCC AAC GCG CTT GGT GCT TAT GTG ATC TAC GTG 1439 Leu Leu Lys His Glu Ala same time Ala Leu Gly Ala Tyr wall Ile Tyr wall 130 135 140 CAA GCA GAT TAC GGT GAC GAT CCC GCA GTG GCT CTC TAT ACA AAG TTG 1487 gin Ala Asp Tyr Gly Asp Asp for Ala wall Ala Leu Tyr Thr Lys Leu 145 150 155 160 GGC ATA CGG GAA GAA GTG ATG CAC TTT GAT ATC GAC CCA AGT ACC GCC 1535 Gly Ile Arg Glu Glu wall Met His Phe Asp Ile Asp for Ser Thr Ala 165 170 175 AAA TGG ATT GAC CAA GAG ATC GTG GCT GTG ACAT GAT Lys Trp Ile Asp Gin Glu White Val Ala Val Thr Asp 205 210 ACC TAA CAATTCGTTC AAGCCGAGATGGCTTCCCA TTG AGG GCG CAA GAG GAG 1589 Thr Leu Arg Glu Glu Glu 177,197 200 GAA CAG TTC GAT 1637 Glu Gin Phe Asp 215 -48 ·· ························ B · · I · ··· B · · · B · · · ·· ·· TTA GAG GAC TAC AAC AGT CGA GCA AAA Lys 1685 Leu Glu 220 Gly Tyr Asn Ser Arg 225 Ala Ile Glu Leu Pro 230 Arg Lys Ala TTG TTG ATC GTG ACA GTC ATC CGC GGC CTA GCA GTC TTT GAA GCC CTT 1733 Leu Leu Ile wall Thr wall Ile Arg Gly Leu Ala wall Phe Glu Ala Leu 235 240 245 250 TCC CGA TTG AAG CCT GTT CAT TCT GGC GGG GTG CAG ACT GCG GGC AAC 1781 Ser Arg Leu Lys for wall His Ser Gly Gly wall gin Thr Ala Gly same time 255 260 265 AGC TGT GCC GTA GTG GAC GGC GCC GCG GCG GCT TTG GTG GCT CGA GAG 1829 Ser Cys Ala wall wall Asp Gly Ala Ala Ala Ala Leu wall Ala Arg Glu 270 275 280 TCG TCT GCG ACA CAG CCG GTC TTG GCT AGG ATA CTG GCT ACC TCC GTA 1877 Ser Ser Ala Thr gin for wall Leu Ala Arg Ile Leu Ala Thr Ser wall 285 290 295 GTC GGG ATC GAG CCC GAG CAT ATG GGG CTC GGC CCT GCG CCC GCG ATT 1925 wall Gly Ile Glu for Glu His Met Gly Leu Gly for Ala for Ala Ile 300 305 310 CGC CTG CTG CTT GCG CGT AGT GAT CTT AGT TTG AGG GAT ATC GAC CTC 1973 Arg Leu Leu Leu Ala Arg Ser Asp Leu Ser Leu Arg Asp Ile Asp Leu 315 320 325 330 TTT GAG ATA AAC GAG GCG CAG GCC GCC CAA GTT CTA GCG GTA CAG CAT 2021 Phe Glu Ile same time Glu Ala gin Ala Ala gin wall Leu Ala wall gin His 335 340 345 GAA TTG GGT ATT GAG CAC TCA AAA CTT AAT ATT TGG GGC GGG GCC ATT 2069 Glu Leu Gly Ile Glu His Ser Lys Leu same time Ile Trp Gly Gly Ala Ile 350 355 360 GCA CTT GGA CAC CCG CTT GCC GCG ACC GGA TTG CGT CTC TGC ATG ACC 2117 Ala Leu Gly His for Leu Ala Ala Thr Gly Leu Arg Leu Cys Met Thr 365 370 375 CTC GCT CAC CAA TTG CAA GCT AAT AAC TTT CGA TAT GGA ATT GCC TCG 2165 Leu Ala His gin Leu gin Ala same time same time Phe Arg Tyr Gly Ile Ala Ser 380 385 390 GCA TGC ATT GGT GGG GGA CAG GGG ATG GCG GTT CTT TTA GAG AAT CCC 2213 Ala Cys Ile Gly Gly Gly gin Gly Met Ala wall Leu Leu Glu same time for 395 400 405 410 CAC TTC GGT TCG TCC TCT GCA CGA AGT TCG ATG ATT AAC AGA GTT GAC 2261 His Phe Gly Ser Ser Ser Ala Arg Ser Ser Met Ile same time Arg wall Asp (+420) 415 420 425 CAC TAT CCA CTG AGG TAA CGGGCATCTC CTTTGTTGCT TTGAGGTGGC His Tyr Pro Leu Ser 430 431 2309 ·· ··························· · ···· ·· ·· ·· ·· · GCACGAAGGA GGGCTCGAAA ATCTCTGCTA AAAACAAGAA GAAGGAACAG GGAACÄTGAT 2369 TAGTTTCGCT CGTATGGCAG AAAGTTTAGG AGTCCAGGCT AAACTTGCCC TTGCCTTCGC 2429 ACTCGTATTA TGTGTCGGGC TGATTGTTAC CGGCACGGGT TTCTACAGTG TACATACCTT 2489 GTCAGGGTTG GTGGGAATTC 2509 FIG. 2Q: -50 ·· ·· ·· ·· ··························································································· · ···· ·· ·· ·· ·· · GAATTCCGCG GTCGGCGAAA GTTGATGCGC TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GTGACGAGGC CACACT GTG AGT TGG TCA GGG GGG GCT Met Ser Trp Ser 15 10 GAC Asp ACT GCG TTG GTT GCG GCA TGC GGG Cys Gly 160 Thr Ala 15 Leu Val Ala Ala Val Arg Thr Pro Trp Ile 25 Asp GGT GCC CTG TCG CTG GTG TCG CCT ATC GAC TTA GGG GTA AAG GTC GCT 208 Gly Ala Leu Ser Leu wall Ser for Ile Asp Leu Gly wall Lys wall Ala 30 35 40 CGC GAA GTT CTG ATG CGT GCG TCG CTT GAA CCA CAA ATG GTC GAT AGC 256 Arg Glu wall Leu Met Arg Ala Ser Leu Glu for gin Met wall Asp Ser 45 50 55 60 GTA CTC GCA GGC TCT ATG GCT CAA GCA AGC TTT GAT GCT TAC CTG CTC 304 wall Leu Ala Gly Ser Met Ala gin Ala Ser Phe Asp Ala Tyr Leu Leu 65 70 75 CCG CGG CAC ATT GGC TTG TAC AGC GGT GTT CCC AAG TCG GTT CCG GCC 352 for Arg His Ile Gly Leu Tyr Ser Gly wall for Lys Ser wall for Ala 80 85 90 TTG GGG GTG CAG CGC ATT TGC GGC ACA GGC TTC GAA CTG CTT CGG CAG 400 Leu Gly wall gin Arg Ile Cys Gly Thr Gly Phe Glu Leu Leu Arg gin 95 100 105 GCC GGC GAG CAG ATT TCC CAA GGC GCT GAT CAC GTG CTG TGT GTC GCG 448 Ala Gly Glu gin Ile Ser gin Gly Ala Asp His wall Leu Cys wall 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 same time for 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 for wall 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 for Ala for Gly Leu Asp Met Ile Ala Thr Ala Glu 160 AAC CTG GCG CGC A TTG AGG GCG CAA GAG Gn Glu Glu Glu Glu Glu Glu 175 176 197 200 205 ' ATC Ile GTG GCT GTT Val Ala Val 210 ACG GAT GAA CAG TTC GAT TTA AAC AGT 689 Thr Asp Glu 215 gin Phe Asp Leu Glu 220 Gly Tyr same time Ser CGA GCA ATT GAA CTG CCT CGG AAG GCA AAA TTG TTG ATC GTG ACA GTC 737 Arg Ala Ile Glu Leu for Arg Lys Ala Lys Leu Leu Ile wall Thr wall 225 230 235 240 ·· ·· • · -51 ············ · · · ATC Ile CGC GGC CTA GCA GTC TTT GAA GCC CTT TCC CGA TTG 785 Arg Gly Leu Ala 245 wall Phe Glu Ala Leu 250 Ser Arg Leu Lys for 255 wall CAT TCT GGC GGG GTG CAG ACT GCG GGC AAC AGC TGT GCC GTA GTG GAC 833 His Ser Gly Gly wall gin Thr Ala Gly same time Ser Cys Ala wall wall Asp 260 265 270 GGC GCC GCG GCG GCT TTG GTG GCT CGA GAG TCG TCT GCG ACA CAG CCG 881 Gly Ala Ala Ala Ala Leu wall Ala Arg Glu Ser Ser Ala Thr gin for 275 280 285 GTC TTG GCT AGG ATA CTG GCT ACC TCC GTA GTC GGG ATC GAG CCC GAG 929 wall Leu Ala Arg Ile Leu Ala Thr Ser wall wall Gly Ile Glu for Glu 290 295 300 CAT ATG GGG CTC GGC CCT GCG CCC GCG ATT CGC CTG CTG CTT GCG CGT 977 His Met Gly Leu Gly for Ala for Ala Ile Arg Leu Leu Leu Ala Arg 305 310 315 320 AGT GAT CTT AGT TTG AGG GAT ATC GAC CTC TTT GAG ATA AAC GAG GCG 1025 Ser Asp Leu Ser Leu Arg Asp Ile Asp Leu Phe Glu Ile same time Glu Ala 325 330 335 CAG GCC GCC CAA GTT CTA GCG GTA CAG CAT GAA TTG GGT ATT GAG CAC 1073 gin Ala Ala gin wall Leu Ala wall gin His Glu Leu Gly Ile Glu His 340 345 350 TCA AAA CTT AAT ATT TGG GGC GGG GCC ATT GCA CTT GGA CAC CCG CTT 1121 Ser Lys Leu same time Ile Trp Gly Gly Ala Ile Ala Leu Gly His for Leu 355 360 365 GCC GCG ACC GGA TTG CGT CTC TGC ATG ACC CTC GCT CAC CAA TTG CAA 1169 Ala Ala Thr Gly Leu Arg Leu Cys Met Thr Leu Ala His gin Leu gin 370 375 380 GCT AAT AAC TTT CGA TAT GGA ATT GCC TCG GCA TGC ATT GGT GGG GGA 1217 Ala same time same time Phe Arg Tyr Gly Ile Ala Ser Ala Cys Ile Gly Gly Gly 385 390 395 400 CAG GGG ATG GCG GTT CTT TTA GAG AAT CCC CAC TTC GGT TCG TCC TCT 1265 gin Gly Met Ala wall Leu Leu Glu same time for His Phe Gly Ser Ser Ser 405 410 415 GCA CGA AGT TCG ATG ATT AAC AGA GTT GAC CAC TAT CCA CTG AGC TAA 1313 Ala Arg Ser Ser Met Ile same time Arg wall Asp His Tyr for Leu Ser 420 425 430 431 CGGGCATCTC CTTTGTTGCT TTGAGGTGGC GCACGAAGGA GGGCTCGAAA ATCTCTGCTA 1373 AAAACAAGAA GAAGGAACAG GGAACATGAT TAGTTTCGCT CGTATGGCAG AAAGTTTAGG 1433 AGTCCAGGCT AAACTTGCCC TTGCCTTCGC ACTCGTATTA TGTGTCGGGC TGATTGTTAC 1493 CGGCACGGGT TTCTACAGTG TACATACCTT GTCAGGGTTG GTGGGAATTC 1543 FIG. 2r: · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · <· • · · · · · -52 ·· Sequence 1 CTGCAGCCAG GGCTGAÄAAG GAGGGATTCA GGCTCCAATT GCTCGATGGC GCCGCGATTG GCTAGGGAGA TAAATTTGCT GGCCATGGTG CATTCTGCAT CATGAAATTC ATGAAATCAT AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCATGGAAAT GGCATGAGCT TTGCTGGATA TGGAAGCACG ATTCCTCGCC CGGTAGAGCG AAAGAGCATG CAACTGACCA ACAAGAAAAT TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CATGCCGAGC CTGACTCTGG ATGCTTTCGT CGATAAGGCC ATCGGGACAG CAAGCGAACC GGTTGGGAAG CCCTGCAAAG 7ÄAACTGGAT CAGGGGATCA AGATCTGATC AAGAGACAGG TGGATTGCAC GCAGGTTCTC CGGCCGCTTG ACAACAGACÄ ATCGGCTGCT CTGATGCCGC 007707777? GTCAAGACCG ACCTGTCCGG GCGGCTA7CG TGGCTGGCCA CGACGGGCG7 TGAAGCGGGA AGGGACTGGC TGCTATTGGG 7CACCTTGCT CCTGCCGAGA AAGTATCCAT GCTTGATCCG GCTACCTGCC CATTCGACCA TACTCGGATG GAAGCCGGTC TTGTCGATCA CGCGCCAGCC GAACTGTTCG CCAGGCTCAA CGTGACCCA7 GGCGATGCCT GCTTGCCGAA ATTCATCGAC TGTGGCCGGC TGGGTGTGGC CCGTGATATT GCTGAAGAGC TTGGCGGCGA TATCGCCGCT CCCGATTCGC AGCGCATCGC AGCGGGACTC TGGGGTTCGA AATGACCGAC TCA7GTG7GC TGAGGAGTCA CGTTGGATCA TGGCATCGAC CTACGTGTAA GTTCGTGGAC AGCAGCTGAA AGCAGCTTTG GTTTTGA7CG TAAATAATAA AGGATTCTTG TGAAGCTTTA GGAATGATAT GAAAGCAAGT AGATCAGTCT CCATTTA7GC AGCGCTGCCA ATGTCAGCTG CGGATTGGAC GGTGCGTTGG GGACAACACC GAGCAAGACT CAAGTCTGAC AAATGCGCCG GTCAGGGTGA TCGTAACTTT GACCGGGGGC GCAG GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480 CGGCGCCACA GTGATTGGCG TAGATCGCAA 540 TCAGGCTGAC CTGAGCCATC CTGAAGGCAT 600 GGAATTGCCA GCTGGGGCGC CCTCTGGTAA 660 GGCTTTCTTG CCGCCAAGGA TCTGATGGCG 720 ATGAGGATCG TTTCGCATGA TTGAACAAGA 780 GGTGGAGAGG CTATTCGGCT ATGACTGGGC 840 CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC 900 TGCCCTGAAT GAACTGCAGG ACGAGGCAGC 960 TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC 1020 CGAAGTGCCG GGGCAGGATC TCCTGTCATC 1080 CATGGCTGAT GCAATGCGGC GGCTGCATAC 1140 CCAAGCGAAA CATCGCATCG AGCGAGCACG 1200 GGATGATCTG GACGAAGAGC ATCAGGGGCT 1260 GGCGCGCATG CCCGACGGCG AGGATCTCGT 1320 TATCATGGTG GAAAATGGCC GCTTTTCTGG 1380 GGACCGCTAT CAGGACATAG CGTTGGCTAC 1440 ATGGGCTGAC CGCTTCCTCG TGCTTTACGG 1500 CTTCTA7CGC CTTCTTGACG AGTTCTTCTG 1560 CAAGCGACGC CCTGGCCGCG GTGATTG CAT 1620 ACGGCA7AAA TATTCCAGTG GACGGAGGTT 1680 GCCCTTTGCA CGCGCACTAT ATCTCTATGC 1740 GAGGTAGCGG GCGGAAAGGT GCAGAATGTC 1800 GTTGTCCGTÄ AACGAAAATA AAAATAAAGA 1860 GCACTTTCAA AATAGCTACC CTGGCAGGCG 1920 CAAACTCGAT GCAGCTGGAT GTAGGTAGCT 1980 CTCAAGTATA GCCTTGCCTC TCGCCTGAAT 2040 ACTGTCAATG GTTATATCCG GATATTCAAA 2100 TTGGTATCCA ATCGTCTCGA TATTCTGGCT 2160 2164 • · ·· • · -53 · · 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 53 Sekyencia 2 CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 GGCTCCAATT GCTCGATGGC GCCGCGATTG AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCTAGGGAGA TAAATTTGCT GGCCATGGTG GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CATTCTGCAT CATGAAATTC ATGAAATCAT CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 GCATGGAAAT GGCATGAGCT TTGCTGGATA TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 TGGAAGCACG ATTCCTCGCC CGGTAGAGCG GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 AAAGAGCATG CAACTGACCA ACAAGAAAAT CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480 TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CGGCGCCACA GTGATTGGCG TAGATCGCAA 540 CATGCCGAGC CTGACTCTGG ATGCTTTCGT TCAGGCTGAC CTGAGCCATC CTGAGGGGAG 600 AGGCGGTTTG CGTATTGGGC GCATGCATAA AAACTGTTGT AATTCATTAA GCATTCTGCC 660 GACATGGAAG CCATCACAAA CGGCATGATG AACCTGAATC GCCAGCGGCA TCAGCACCTT 720 GTCGCCTTGC GTATAATATT TGCCCATGGA CGCACACCGT GGAAACGGAT GAAGGCACGA 780 ACCCAGTTGA CATAAGCCTG TTCGGTTCGT AAACTGTAAT GCAAGTAGCG TATGCGCTCA 840 CGCAACTGGT CCAGAACCTT GACCGAACGC AGCGGTGGTA ACGGCGCAGT GGCGGTTTTC 900 ATGGCTTGTT ATGACTGTTT TTTTGTACAG TCTATGCCTC GGGCATCCAA GCAGCAAGCG 960 CGTTACGCCG TGGGTCGATG TTTGATGTTA TGGAGCAGCA ACGATGTTAC GCAGCAGCAA 1020 CGATGTTACG CAGCAGGGCA GTCGCCCTAA AACAAAGTTA GGTGGCTCAA GTATGGGCAT 1080 CATTCGCACA TGTAGGCTCG GCCCTGACCA AGTCAAATCC ATGCGGGCTG CTCTTGATCT 1140 TTTCGGTCGT GAGTTCGGAG ACGTAGCCAC CTACTCCCAA CATCAGCCGG ACTCCGATTA 1200 CCTCGGGAAC TTGCTCCGTA GTAÄGACATT CATCGCGCTT GCTGCCTTCG ACCAAGAAGC 1260 GGTTGTTGGC GCTCTCGCGG CTTACGTTCT GCCCAGGTTT GAGCAGCCGC GTAGTGAGAT 1320 CTATATCTAT GATCTCGCAG TCTCCGGCGA GCACCGGAGG CAGGGCATTG CCACCGCGCT 1380 CATCAATCTC CTCAAGCATG AGGCCAACGC GCTTGGTGCT TATGTGATCT AC3TGCAAGC 1440 AGATTACGGT GACGATCCCG CAGTGGCTCT CTATACAAAG TTGGGCATAC GGGAAGAAGT 1500 GATGCACTTT GATATCGACC CAAGTACCGC CACCTAACAA TTCGTTCAAG CCGAGATCGG 1560 CTTCCCTGAT TGCATTCATG TGTGCTGAGG AGTCACGTTG GATCAACGGC ATAAATATTC 1620 CAGTGGACGG AGGTTTGGCA TCGACCTACG TGTAAGTTCG TGGACGCCCT TTGCACGCGC 1680 ACTATATCTC TATGCAGCAG CTGAAA GCAG CTTTGGTTTT GATCGGAGGT AGCGGGCGGA 1740 AAGGTGCAGA ATGTCTAAAT AATAAAGGAT TCTTGTGAAG CTTTAGTTGT CCGTAAACGA 1800 AAATAAAAAT AAAGAGGAAT GATATGAAAG CAAGTAGATC AGTCTGCACT TTCAAAATAG 1860 CTACCCTGGC AGGCGCCATT TATGCAGCGC TGCCAATGTC AGCTGCAAAC TCGATGCAGC 1920 TGGATGTAGG TAGCTCGGAT TGGACGGTGC GTTGGGGACA ACACCCTCAA GTATAGCCTT 1980 GCCTCTCGCC TGAATGAGCA AGACTCAAGT CTGACAAATG CGCCGACTGT CAATGGTTAT 2040 ATCCGGATAT TCAAAGTCAG GGTGATCGTA ACTTTGACCG GGGGCTTGGT ATCCAATCGT 2100 CTCGATATTC TGGCTGCAG 2119 • • • • -54 · · · · · · · · · · · · · · · · · · · · · · · · · Sequences 3 CTGCAGCCAG GGCTGAAAAG GAGGGATTCA GGCTCCAATT GCTCGATGGC GCCGCGATTG GCTAGGGAGA TAAATTTGCT GGCCATGGTG CATTCTGCAT CATGAAATTC ATGAAATCAT AAGGTTGCTA GGAGAGTGCA TTGCTCGTAA GCATGGAAAT GGCATGAGCT TTGCTGGATA TGGAAGCACG ATTCCTCGCC CGGTAGAGCG AAAGAGCATG CAACTGACCA ACAAGAAAAT TGCCGAAACT GCCCGCGTTC TGCGCTCTCA CATGCCGAGC CTGACTCTGG ATGCTTTCGT CGATCAACGG CATAAATATT CCAGTGGACG GTGGACGCCC TTTGCACGCG CACTATATCT TGATCGGAGG TAGCGGGCGG AAAGGTGCAG GCTTTAGTTG TCCGTAAACG AAAATAAAAA CAGTCTGCAC TTTCAAAATA GCTACCCTGG CAGCTGCAAA CTCGATGCAG CTGGATGTAG AACACCCTCA AGTATAGCCT TGCCTCTCGC GCGCCGACTG TCAATGGTTA TATCCGGATA GGGGGCTTGG TATCCAATCG TCTCGATATT GTGAGGTCAT GAAGGGAGGG GACGGCGCCT 60 AGTGTCTTGG GCGCGGTCTT GGAGAGTTCG 120 GCGGCCCCTG ATGGGTTGGA TGATTTTCTG 180 CACTTTTCGG GGGGTGGGTG CACGGGATTG 240 GCCCAGGAAG CACGCGGGTT TCAGGATGGT 300 TGATTAGAGA CATTAACTAT TTTGGCGGAA 360 GTAACCGCGA CATTCAGGAC CGTAAAAAGG 420 CGTCGTCACC GGAGTGTCCT CCGGTATCGG 480 CGGCGCCACA GTGATTGGCG TAGATCGCAA 540 TCAGGCTGAC CTGAGCCATC CTGAAGGCAT 600 GAGGTTTGGC ATCGACCTAC GTGTAAGTTC 660 CTATGCAGCA GCTGAAAGCA GCTTTGGTTT 720 AATGTCTAAA TAÄTAÄAGGA TTCTTGTGAA 780 TAAAGAGGAA TGATATGAAA GCAAGTAGAT 840 CAGGCGCCAT TTATGCAGCG CTGCCAATGT 900 GTAGCTCGGA TTGGACGGTG CGTTGGGGAC 960 CTGAATGAGC AAGACTCAAG -55 · · · 55 * 55 55 55 55 55 55 55 55 55 55 Sequence 4 GAATTCCGCG TATCGCCCGG TTCTATCAGC GGTAGGGTCT TTTTCTTGGC CATGCTTGTT TGCGTTTGCC GCTTCGCTTC GCGATGAACC TTAACTCGCG TAAGCATTCT GTCATTTTTT GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CGATTAAGAT AATTAAAATA AGGAAACCGC CTCCAGCTCA AGGGCAATTT TTGGGCTATT AGAATAACAA TTGACTCCTC AGGAGGTCAG CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC AGGGGCCTGC AAACTTGGAG CTGCGTCTGA TGGAAAATCG TGAAGCAATT GCCGACGCGG AGCAAACACT GCTTTGCGAC ATTGCTGGCT ACGTGGCCAA ATGGATGGAG CCCGAACATC GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG TACTGGCCTT TGGGCCGCTG GCCGGCATAT CGTCCGAGCT TACCCCGCGG ACTTCTGCCC ATGAAACTGA GCTGACTACA GTGCTGGGCG AGCCTTTCGA TCATCTGATC TTCACCGGCG CCGCGGCGGA TAACCTAGTG CCCGTTACCC TTTCCCGCAG TGCAGATATG GCGGACGTTG ATGCCGGGCA AATCTGTCTG GCACCGGACT AACCGGAATT GCCAGCTGGG GCGCCCTCTG GGATGGCTTT CTTGCCGCCA AGGATCTGAT CAGGATGAGG ATCGTTTCGC ATGATTGAAC CTTGGGTGGA GAGGCTATTC GGCTATGACT CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC CCGGTGCCCT GAATGAACTG CAGGACGAGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CCATCATGGC TGATGCAATG CGGCGGCTGC ACCACCAAGC GAAACATCGC ATCGAGCGAG ATCAGGATGA TCTGGACGAA GAGCATCAGG TCAAGGCGCG CATGCCCGAC GGCGAGGATC CGAATATCAT GGTGGAAAAT GGCCGCTTTT TGGCGGACGG CTATCAGGGG GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480 TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600 TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900 TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 ATGTGCTGCT GCCGGAAGGG ACAGCAAGCG 1260 GTAAGGTTGG GAAGCCCTGC AAAGTAAACT 1320 GGCGCAGGGG ATCAAGATCT GATCAAGAGA 1380 AAGATGGATT GCACGCAGGT TCTCCGGCCG 1440 GGGCACAACA GACAATCGGC TGCTCTGATG 1500 GCCCGGTTCT TTTTGTCAAG ACCGACCTGT 1560 CAGCGCGGCT ATCGTGGCTG GCCACGA CGG 1620 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT 1680 CATCTCACCT TGCTCCTGCC GAGAAAGTAT 1740 ATACGCTTGA TCCGGCTACC TGCCCATTCG 1800 CACGTACTCG GATGGAAGCC GGTCTTGTCG 1860 GGCTCGCGCC AGCCGAACTG TTCGCCAGGC 1920 TCGTCGTGAC CCATGGCGAT GCCTGCTTGC 1980 CTGGATTCAT CGACTGTGGC CGGCTGGGTG 2040 CTACCCGTGA TATTGCTGAA GAGCTTGGCG 2100 ACGGTATCGC CGCTCCCGAT TCGCAGCGCA 2160 · · · · · • • • · · · · · · · · · · · · · -56TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG ACTCTGGGGT TCGAAATGAC 2220 CGACCAAGCG ACGCCCGCCA TGCCAAGCCT GTTCTCGTGC AAAGTCCTGT GGGTGAGTCG 2280 AACTTGGCGA TGCGCGCACC CTACGGAGAA GCGATCCACG GACTGCTCTC TGTCCTCCTT 2340 TCAACGGAGT GTTAGAACCG TTGGTAGTGG TTTTGGACGG GCCCAGGAGC ATGCGCTTCT 2400 GGGCCCGTTT CTTGAGTATT CATTGGATAG TCACGCGTGG TAGCTTCGAG CCTGCACAGC 2460 TGATGAGCAC CCTGGAAGGC GCGCTGTACG CGGACGACTG GGTTCATCTT CGCCATTCAT 2520 GACGGAACTC CGTTCCCCAG TACCGCGATG ACTATTTTGC CTCTTCCGAT GTCCGATTCC 2580 ACGCCGCCTG ACGCTAAGCG GGGGCGGGGG CGCCCGCATC CCAGCCCAGA CAGCAACAAA 2640 TGAGTAGGCT CTTGGATGCC GCGGCGGCTG AGATTGGTAA CGGCAATTTC GTCAATGTGA 2700 CGATGGATTC GATTGCCCGT GCTGCCGGCG TCTCAAAAAA AACGCTGTAC GTCTTGGTGG 2760 CGAGCAAGGA AGAACTCATT TCCCGGTTAG TGGCTCGAGA CATGTCCAAC CTTGAGGAAT 2820 TC 2822 • · · · · -57 ·········· Sequence 5 GAATTCCGCG TATCGCCCGG TTCTATCAGC GGTAGGGTCT TTTTCTTGGC CATGCTTGTT TGCGTTTGCC GCTTCGCTTC GCGATGAACC TTAACTCGCG TAAGCATTCT GTCATTTTTT GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CGATTAAGAT AATTAAAATA AGGAAACCGC CTCCAGCTCA AGGGCAATTT TTGGGCTATT AGAATAACAA TTGACTCCTC AGGAGGTCAG CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC AGGGGCCTGC AAACTTGGAG CTGCGTCTGA TGGAÄAATCG TGAAGCAATT GCCGACGCGG AGCAAACACT GCTTTGCGAC ATTGCTGGCT ACGTGGCCAA ATGGATGGAG CCCGAACATC GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG TACTGGCCTT TGGGCCGCTG GCCGGCATAT CGTCCGAGCT TACCCCGCGG ACTTCTGCCC ATGAAACTGA GCTGACTACA GTGCTGGGCG AGCCTTTCGA TCATCTGATC TTCACCGGCG CCGCGGCGGA TAACCTAGTG CCCGTTACCC TTTCCCGCAG TGCAGATATG GCGGACGTTG ATGCCGGGCA AATCTGTCTG GCACCGGACT GGGCGCATGC ATAAAAACTG TTGTAATTCA CAAACGGCAT GATGAACCTG AATCGCCAGC TATTTGCCCA TGGACGCACA CCGTGGAAAC CCTGTTCGGT TCGTAAACTG TAATGCAAGT CCTTGACCGA ACGCAGCGGT GGTAACGGCG GTTTTTTTGT ACAGTCTATG CCTCGGGCAT GATGTTTGAT GTTATGGAGC AGCAACGATG GGCAGTCGCC CTAAAACAAA GTTAGGTGGC CTCGGCCCTG ACCAAGTCAA ATCCATGCGG GGAGACGTA G CCACCTACTC CCAACATCAG CGTAGTAAGA CATTCATCGC GCTTGCTGCC GCGGCTTACG TTCTGCCCAG GTTTGAGCAG GCAGTCTCCG GCGAGCACCG GAGGCAGGGC CATGAGGCCA ACGCGCTTGG TGCTTTCGG GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480 TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 GTAGGCTGGA TCGTGCGATT GCAATGCTTC 600 TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TCGCAGCAGG TAATCGCGCC ATGCTCAAGC 900 TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 ATGTGCTGGG GGAGAGGCGG TTTGCGTATT 1260 TTAAGCATTC TGCCGACATG GAAGCCATCA 1320 GGCATCAGCA CCTTGTCGCC TTGCGTATAA 1380 GGATGAAGGC ACGAACCCAG TTGACATAAG 1440 AGCGTATGCG CTCACGCAAC TGGTCCAGAA 1500 CAGTGGCGGT TTTCATGGCT TGTTATGACT 1560 CCAAGCAGCA AGCGCGTTAC GCCGTGG GTC 1620 TTACGCAGCA GCAACGATGT TACGCAGCAG 1680 TCAAGTATGG GCATCATTCG CACATGTAGG 1740 GCTGCTCTTG ATCTTTTCGG TCGTGAGTTC 1800 CCGGACTCCG ATTACCTCGG GAACTTGCTC 1860 TTCGACCAAG AAGCGGTTGT TGGCGCTCTC 1920 CCGCGTAGTG AGATCTATAT CTATGATCTC 1980 ATTGCCACCG CGCTCATCAA TCTCCTCAAG 2040 ATCTACGTGC AAGCAGATTA CGGTGACGAT 2100 ATACGGGAAG AAGTGATGCA CTTTGATATC 2160 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · -58GACCCAAGTA CCGCCACCTA ACAATTCGTT CAAGCCGAGA TCGGCTTCCC TGCAAAGTCC 2220 TGTGGGTGAG TCGAACTTGG CGATGCGCGC ACCCTACGGA GAAGCGATCC ACGGACTGCT 2280 CTCTGTCCTC CTTTCAACGG AGTGTTAGAA CCGTTGGTAG TGGTTTTGGA CGGGCCCAGG 2340 AGCATGCGCT TCTGGGCCCG TTTCTTGAGT ATTCATTGGA TAGTCACGCG TGGTAGCTTC 2400 GAGCCTGCAC AGCTGATGAG CACCCTGGAA GGCGCGCTGT ACGCGGACGA CTGGGTTCAT 2460 CTTCGCCATT CATGACGGAA CTCCGTTCCC CAGTACCGCG ATGACTATTT TGCCTCTTCC 2520 GATGTCCGAT TCCACGCCGC CTGACGCTAA GCGGGGGCGG GGGCGCCCGC ATCCCAGCCC 2580 AGACAGCAAC AAATGAGTAG GCTCTTGGAT GCCGCGGCGG CTGAGATTGG TAACGGCAAT 2640 TTCGTCAATG TGACGATGGA TTCGATTGCC CGTGCTGCCG GCGTCTCAAA AAAAACGCTG 2700 TACGTCTTGG TGGCGAGCAA GGAAGAACTC ATTTCCCGGT TAGTGGCTCG AGACATGTCC 2760 AACCTTGAGG AATTC 2775 · · · · • · • · • · • · • · · · · -59 ··· ··· ·· ··· • fl ·· Sequence 6 GAATTCCGCG TATCGCCCGG TTCTATCAGC GGTAGGGTCT TTTTCTTGGC CATGCTTGTT TGCGTTTGCC GCTTCGCTTC GCGATGAACC TTAACTCGCG TAAGCATTCT GTCATTTTTT GTCTCGCCCT TTGAGGCCGA TTCTTGGGCG CGATTAAGAT AATTAAAATA AGGAAACCGC CTCCAGCTCA AGGGCAATTT TTGGGCTATT AGAATAACAA TTGACTCCTC AGGAGGTCAG CGGTCGGAGC TGAGCAGCTG GGCTCGGCTC AGGGGCCTGC AAACTTGGAG CTGCGTCTGA TGGAAAATCG TGAAGCAATT GCCGACGCGG AGCAAACACT GCTTTGCGAC ATTGCTGGCT ACGTGGCCAA ATGGATGGAG CCCGAACATC GCGTTGAGTT TCAGCCGCTG GGTGTCGTTG TACTGGCCTT TGGGCCGCTG GCCGGCATAT CGTCCGAGCT TACCCCGCGG ACTTCTGCCC ATGAAACTGA GCTGACTACA GTGCTGGGCG AGCCTTTCGA TCATCTGATC TTCACCGGCG CCGCGGCGGA TAACCTAGTG CCCGTTACCC TTTCCCGCAG TGCAGATATG GCGGACGTTG ATGCCGGGCA AATCTGTCTG GCACCGTGGG CGGAGAAGCG ATCCACGGAC TGCTCTCTGT GTAGTGGTTT TGGÄCGGGCC CAGGAGCATG TGGATAGTCA CGCGTGGTAG CTTCGAGCCT CTGTACGCGG ACGACTGGGT TCATCTTCGC CGCGATGACT ATTTTGCCTC TTCCGATGTC GCGGGGGCGC CCGCATCCCA GCCCAGACAG GCGGCTGAGA TTGGTAACGG CAATTTCGTC GCCGGCGTCT CAAAAAAAAC GCTGTACGTC CGGTTAGTGG CTCGAGACAT GTCCAACCTT GGGCCGCTTT CGAAAGTCAT GGTGTTAGCC 60 GCCTGAACCT TCGTTGACAT AGGGCAGAGG 120 GCATCGAGAT GCTGAGGTCA GGATTTTTCC 180 TGGTGGCTTT GAACAGCCTG ATGAAAGGTG 240 CTTGGCGGCG TCGAAGCGAT GCTCCACTAC 300 ATGGTTTCTT ATGTGAATTT GTCTGGCATA 360 GGCTGAGCAG TTGCCTCTAT ATGGTTATTC 420 CGATGAGCAT TCTTGGTTTG AATGGTGCCC 480 TTGATCGCAT GAAGAAGGCG CACCTGGAGC 540 GTAGGCTGGA TCGTGCGATT GCÄATGCTTC 600 TTTCTGCTGA CTTTGGCAAT CGCAGCCGTG 660 CGGTGGCAAG CCTGAAGGAT AGCCGCGAGC 720 ACAAGGCGAT GTTTCCAGGG GCGGAGGCAC 780 GGGTCATTAG TCCCTGGAAC TTCCCTATCG 840 TCGCAGCAGG TAATCGCGCC A7GCTCAAGC 900 TGCTTGCGGA GCTAATTGCT CGTTACTTCG 960 ACGCTGAAGT CGGTGCGCTG TTCAGTGCTC 1020 GCACTGCCGT GGCCAAGCAC ATCATGCGTG 1080 TGGAATTGGG TGGCAAATCG CCGGTGATCG 1140 CACAACGGGT GTTGACGGTG AAAACCTTCA 1200 TGAGTCGAAC TTGGCGATGC GCGCACCCTA 1260 CCTCCTTTCA ACGGAGTGTT AGAACCGTTG 1320 CGCTTCTGGG CCCGTTTCTT GAGTATTCAT 1380 GCACAGCTGA TGAGCACCCT GGAAGGCGCG 1440 CATTCATGAC GGAACTCCGT TCCCCAGTAC 1500 CGATTCCACG CCGCCTGACG CTAAGCGGGG 1560 CAACAAATGA GTAGGCTCTT GGATGC CGCG 1620 AATGTGACGA TGGATTCGAT TGCCCGTGCT 1680 TTGGTGGCGA GCAAGGAAGA ACTCATTTCC 1740 GAGGAATTC 1779 Sequence 7 CTGCAGCCGA GCATCGATTG AGCACTTTAC CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 CCCGCGGCAC TATCCAATCT AAATCGATCT TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 CTCGCCTCAT TTCAATCTCT AACTTGATAA AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AGAGTACAGT GAACGCCGAG TCCACATTGC 240 AACCGCAGGC ATCATCATGC TCTGCTCAGC CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 CTCCGGTTGA GGTTACGCAA GACGCTGGAG GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360 TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420 AACAAACCTG CGTTGCTGCC AGGGCGGCAA ATGGGGAATG GCGTCGTATC AGCTACGCGG 480 AAATGTTCCA CAACGTCCGC GCCATCGCAC AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540 AGCGTCCGCT GCTTATCGTC TCTGGAAATG ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600 CTATGTATGC GGGCATTCCC TATTGCCCGG TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660 ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720 ATGCAGCACC TTTCCAGGGG ACAGCAAGCG AACCGGAATT GCCAGCTGGG GCGCCCTCTG 780 GTAAGGTTGG GAAGCCCTGC AAAGTAAACT GGATGGCTTT CTTGCCGCCA AGGATCTGAT 840 GGCGCAGGGG ATCAAGATCT GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC 900 AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT 960 GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC 1020 GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG 1080 CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG 1140 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT 1200 CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC 1260 ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG 1320 CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG 1380 GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC 1440 TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT 1500 CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG 1560 CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT 1620 ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT 1680 TCTGAGCGGG ACTCTGGGGT TCGAAAT GAC CGACCAAGCG ACGCCCCTGT TTTGCAATGG 1740 CGGTCGGCGA AAGTTGATGC GCTGTATCGT GGTGAAGATC AATCCATGCT GCGTGACGAG 1800 GCCACACTGT GAGTTGGTCA GGGGGGGCTT ACTCGGCGTT TTCCGACACT GCGTTGGTTG 1860 CGGCAGTGCG CACCCCCTGG ATTGATTGCG GGGGTGCCCT GTCGCTGGTG TCGCCTATCG 1920 ACTTAGGGGT AAAGGTCGCT CGCGAAGTTC TGATGCGTGC GTCGCTTGAA CCACAAATGG 1980 TCGATAGCGT ACTCGCAGGC TCTATGGCTC AAGCAAGCTT TGATGCTTAC CTGCTCCCGC 2040 GGCACATTGG CTTGTACAGC GGTGTTCCCA AGTCGGTTCC GGCCTTGGGG GTGCAGCGCA 2100 TTTGCGGCAC AGGCTTCGAA CTGCTTCGGC AGGCCGGCGA GCAGATTTCC CAAGGCGCTG 2160 ATCACGTGCT GTGTGTCGCG GGCTGCAG 2188 ·· ··· ··· -61 ·· ·· ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Sequence 8 CTGCAGCCGA GCATCGATTG AGCACTTTAC CCCGCGGCAC TATCCAATCT AAATCGATCT CTCGCCTCAT TTCAATCTCT AACTTGATAA CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AACCGCAGGC ATCATCATGC TCTGCTCAGC CTCCGGTTGA GGTTACGCAA GACGCTGGAG TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC AACAAACCTG CGTTGCTGCC AGGGCGGCAA AAATGTTCCA CAACGTCCGC GCCATCGCAC ÄGCGTCCGCT GCTTATCGTC TCTGGAAATG CTATGTATGC GGGCATTCCC TATTGCCCGG ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC ATGCAGCACC TTTCCAGGGG GAGAGGCGGT TGTAATTCAT TAAGCATTCT GCCGACATGG ATCGCCAGCG GCATCAGCAC CTTGTCGCCT CGTGGAAÄCG GATGAAGGCA CGAACCCAGT AATGCAAGTA GCGTATGCGC TCACGCAACT GTAACGGCGC AGTGGCGGTT TTCATGGCTT CTCGGGCATC CAAGCAGCAA GCGCGTTACG GCAACGATGT TACGCAGCAG CAACGATGTT TTAGGTGGCT CAAGTATGGG CATCATTCGC TCCATGCGGG CTGCTCTTGA TCTTTTCGGT CAACATCAGC CGGACTCCGA TTACCTCGGG CTTGCTGCCT TCGACCAAGA AGCGGTTGTT TTTGAGCAGC CGCGTAGTGA GATCTATATC AGGCAGGGCA TTGCCACCGC GCTCATCAAT GCTTATGTGA TCTACGTGCA AGCAGATTAC AAGTTGGGCA TACGGGAAGA AGTGATGCAC CAATTCGTTC AAGCCGAGAT CGGCTTCCCC TGCGCTGTAT CGTGGTGAAG ATCAATCCAT TCAGGGGG GG CTTACTCGGC GTTTTCCGAC TGGATTGATT GCGGGGGTTGC CCTGTCGCTG GCTCGCGAAG TTCTGATGCG TGCGTCGCTT GGCTCTATGG CTCAAGCAAG CTTTGATGCT CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 AGAGTACAGT GAACGCCGAG TCCACATTGC 240 CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360 gtctcgagca TTGGGCTAAG ACCCGTCCAG 420 ATGGGGAATG GCGTCGTATC AGCTACGCGG 480 AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540 ACCTGGAACA TCTTCÄGCTG GCATTTGGGG 600 TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660 TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720 TTGCGTATTG GGCGCATGCA TAAAAACTGT 780 AAGCCATCAC AAACGGCATG ATGAACCTGA 840 TGCGTATAAT ATTTGCCCAT GGACGCACAC 900 TGACATAAGC CTG7TCGGTT CGTAAACTGT 960 GGTCCAGAAC CTTGACCGAA CGCAGCGGTG 1020 GTTATGACTG TTTTTTTGTA CAGTCTATGC 1080 CCGTGGGTCG ATGTTTGATG TTATGGAGCA 1140 ACGCAGCAGG GCAGTCGCCC TAAAACAAAG 1200 ACATGTAGGC TCGGCCCTGA CCAAGTCAAA 1260 CGTGAGTTCG GAGACGTAGC CACCTACTCC 1320 AÄCTTGCTCC GTAGTAAGAC ATTCATCGCG 1380 GGCGCTCTCG CGGCTTACGT TCTGCCCAGG 1440 TATGÄTCTCG CAGTCTCCGG CGAGCACCGG 1500 CTCCTCAAGC ATGAGGCCAA CGCGCTTGGT 1560 GGTGACGATC CCGCAGTGGC TCTC Tataca 1620 T7TGATATCG ACCCAAGTAC CGCCACCTAA 1680 TGTTTTGCAA TGGCGGTCGG CGAAAGTTGA 1740 GCTGCG7GAC GAGGCCACAC TGTGAGTTGG 1800 ACTGCGTTGG TTGCGGCAGT GCGCACCCCC 1860 GTGTCGCCTA TCGACTTAGG GGTAAAGGTC 1920 GAACCACAAA TGGTCGATAG CGTACTCGCA 1980 TACCTGCTCC CGCGGCACAT TGGCTTGTAC 2040 GGGGTGCAGC GCATTTGCGG CACAGGCTTC 2100 TCCCAAGGCG CTGATCACGT GCTGTGTGTC 2160 2171 ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · -62 · · · · · · · · · · · · · · · · · · · · · · · · · · · Sequence 9 CTGCAGCCGA GCATCGATTG AGCACTTTAC CCCGCGGCAC TATCCAATCT AAATCGATCT CTCGCCTCAT TTCAATCTCT AACTTGATAA CAAGGCCAGT CGCGGAGAGT CTCGAAGAGG AACCGCAGGC ATCATCATGC TCTGCTCAGC CTCCGGTTGA GGTTACGCAA GACGCTGGAG TTCTTCCCTT CCCGGGTCGA ATTCTTGAGC AACAAACCTG CGTTGCTGCC AGGGCGGCAA AAATGTTCCA CAACGTCCGC GCCATCGCAC AGCGTCCGCT GCTTATCGTC TCTGGAAATG CTATGTATGC GGGCATTCCC TATTGCCCGG ATTTGGCGAA GCTGCGTCAC ATCGTAGGTC ATGCAGCACC TTTCCAGCGC GCTGTTTTGC ATCGTGGTGA AGATCAATCC ATGCTGCGTG GGCTTACTCG GCGTTTTCCG ACACTGCGTT TTGCGGGGGT GCCCTGTCGC TGGTGTCGCC AGTTCTGATG CGTGCGTCGC TTGAACCACA GGCTCAAGCA AGCTTTGATG CTTACCTGCT TCCCAAGTCG GTTCCGGCCT TGGGGGTGCA TCGGCAGGCC GGCGAGCAGA CCAGCTGCGC TGGCTGACCA TTCAGAATGG 60 TCGGGCGCCG CGGGCATCAT GCCCGCGGCG 120 AAACAGAGCT GTTCTCCGGT CTTGGTGGAT 180 AGAGTACAGT GAACGCCGAG TCCACATTGC 240 CACGCTACCG CAGTGTGTCG ATTGGTCATC 300 GTATTGTCCG GATGCGTTCT CTCGAGGCGC 360 GTCTCGAGCA TTGGGCTAAG ACCCGTCCAG 420 ATGGGGAATG GCGTCGTATC AGCTACGCGG 480 AGAGCTTGCT TCCTTACGGA CTATCGGCAG 540 ACCTGGAACA TCTTCAGCTG GCATTTGGGG 600 TGTCTCCTGC TTATTCACTG CTGTCGCAAG 660 TTCTGCAACC GGGACTGGTC TTTGCTGCCG 720 AATGGCGGTC GGCGAAAGTT GATGCGCTGT 780 ACGAGGCCAC ACTGTGAGTT GGTCAGGGGG 840 GGTTGCGGCA GTGCGCACCC CCTGGATTGA 900 TATCGACTTA GGGGTAAAGG TCGCTCGCGA 960 AATGGTCGAT AGCGTACTCG CAGGCTCTAT 1020 CCCGCGGCAC ATTGGCTTGT ACAGCGGTGT 1080 GCGCATTTGC GGCACAGGCT TCGAACTGCT 1140 CGCTGATCAC GTGCTGTGTG TCGCGGGCTG 1200 1203 ·· · · · · · · · · · · · · · · · · · · · · -63 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Sequences 10 GAATTCCCCT GGCGACGAAA GGGCGGCAGG GCTTGCGTTA ATCGTTAACC GTTTGAAATT GGGTACGCCT TTCCGTGCGC TTTGATCTGC AATTGACAGA ACTATAGGTT CGCAGTAGCT GGTGCACGAT GAATAGCTAC GATGGCCGTT GTATCGCTTG GGTCACGCTG AACCGCCCGG ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TTCTGACTGG TGCAGGCGAA TCCTGGACCG AGACCGATGC TGGCCCCGAA ATTCTGCAAG GGAATTGCCA GCTGGGGCGC CCTCTGGTAA GGCTTTCTTG CCGCCAAGGA TCTGATGGCG ATGAGGATCG TTTCGCATGA TTGAACAAGA GGTGGAGAGG CTATTCGGCT ATGACTGGGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC TGCCCTGAAT GAACTGCAGG ACGAGGCAGC TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC CGAAGTGCCG GGGCAGGATC TCCTGTCATC CATGGCTGAT GCAATGCGGC GGCTGCATAC CCAAGCGAAA CATCGCATCG AGCGAGCACG GGATGATCTG GACGAAGAGC ATCAGGGGCT GGCGCGCATG CCCGACGGCG AGGATCTCGT TATCATGGTG GAAAATGGCC GCTTTTCTGG GGACCGCTAT CAGGACATAG CGTTGGCTAC ÄTGGGCTGAC CGCTTCCTCG TGCTTTACGG CTTCTATCGC CTTCTTGACG AGTTCTTCTG CAAGCGACGC CCCGAGCAGG GCATGAAGCA CTTGCAGACC TACAAGCGCT GATAAATGCG AATAATGACA ATAATGAGGA GTGCCCAATG CCTTGTTCAG CATCTGATGA GCGCACCTTC GTATCGCGCG TCGCTGCTGC CAGTTTGGAA GCTGCGTTT C CTGAATGGGC GGCGCTTGCT GCGGCGGATC TTCTAGAGGA CCGTTCTTCC GCAGCGGGAA ACTGGTATGG GTTTAACGTT C CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300 AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360 TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420 CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480 AGAAGATTCG TCGGGGACAG CAAGCGAACC 540 GGTTGGGAAG CCCTGCAAAG TAAACTGGAT 600 CAGGGGATCA AGATCTGATC AAGAGACAGG 660 TGGATTGCAC GCAGGTTCTC CGGCCGCTTG 720 ACAACAGACA ATCGGCTGCT CTGATGCCGC 780 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG 840 GCGGCTATCG TGGCTGGCCA CGACGGGCGT 900 TGAAGCGGGA AGGGACTGGC TGCTATTGGG 960 TCACCTTGCT CCTGCCGAGA AAGTATCCAT 1020 GCTTGATCCG GCTACCTGCC CATTCGACCA 1080 TACTCGGATG GAAGCCGGTC TTGTCGATCA 1140 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA 1200 CGTGACCCAT GGCGATGCCT GCTTGCCGAA 1260 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC 1320 CCGTGATATT GCTGAAGAGC TTGGCGGCGA 1380 TATCGCCGCT CCCGATTCGC AGCGCATCGC 1440 AGCGGGACTC TGGGGTTCGA AATGACCGAC 1500 GTTCCTTGAC GAGAAAAGCA TCAAGCCGGG 1560 CCGGGGCCCT CGCTGCGCCC CCGGCCT TCC 1620 TTTCACGTGC CCCTGCTTAT TGGTGGTAAG 1680 GAGCGTCGTA GCCCGCTGAC CGGAGAAGTG 1740 GATGCGGACG CCGCAGTGGC 1981 -64Sequence 11 GAATTCCCCT GGCGACGAAA GGGCGGCAGG GCTTGCGTTA ATCGTTAACC GTTTGAAATT GGGTACGCCT TTCCGTGCGC TTTGATCTGC AATTGACAGA ACTATAGGTT CGCAGTAGCT GGTGCACGAT GAATAGCTAC GATGGCCGTT GTATCGCTTG GGTCACGCTG AACCGCCCGG ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TTCTGACTGG TGCAGGCGAA TCCTGGACCG AGACCGATGC TGGCCCCGAA ATTCTGCAAG GTATTGGGCG CATGCATAAA AACTGTTGTA CATCACAAAC GGCATGATGA ACCTGAATCG TATAATATTT GCCCATGGAC GCACACCGTG ATAAGCCTGT TCGGTTCGTA AACTGTAATG CAGAACCTTG ACCGAACGCA GCGGTGGTAA TGACTGTTTT TTTGTACAGT CTATGCCTCG GGGTCGATGT TTGATGTTAT GGAGCAGCAA AGCAGGGCAG TCGCCCTAAA ACAAAGTTAG GTAGGCTCGG CCCTGACCAA GTCAAATCCA AGTTCGGAGA CGTAGCCACC TACTCCCAAC TGCTCCGTAG TAAGACATTC ATCGCGCTTG CTCTCGCGGC TTACGTTCTG CCCAGGTTTG ATCTCGCAGT CTCCGGCGAG CACCGGAGGC TCAAGCATGA GGCCAACGCG CTTGGTGCTT ACGATCCCGC AGTGGCTCTC TATACAAAGT ATATCGACCC AAGTACCGCC ACCTAACAAT AGGGCATGAA GCAGTTCCTT GACGAGAAAÄ GCTGATAAAT GCGCCGGGGC CCTCGCTGCG GGAGTGCCCA ATGTTTCACG TGCCCCTGCT TGAGCGCACC TTCGAGCGTC GTAGCCCGCT TGCCAGTTTG GAAGATGCGG ACGCCGCAGT GGCGGCGCT T GCTCCGAGCG AACGCCGTGC GGACCGTTCT TCCGAGTTCA CCGCCGCAGC TGGGTTTAAC GTTTACCTGG CGGCGGGCAT CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 CCTTGCCÄAA TTTCGGCGAG AGAATCATGC 120 GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300 AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360 TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420 CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480 AGAAGATTCG TCGGGGGAGA GGCGGTTTGC 540 ATTCATTAAG CATTCTGCCG ACATGGAAGC 600 CCAGCGGCAT CAGCACCTTG TCGCCTTGCG 660 GAAACGGATG AAGGCACGAA CCCAGTTGAC 720 CAAGTAGCGT ATGCGCTCAC GCAACTGGTC 780 CGGCGCAGTG GCGGTTTTCA TGGCTTGTTA 840 GGCATCCAAG CAGCAAGCGC GTTACGCCGT 900 CGATGTTÄCG CAGCAGCAAC GA7GTTACGC 960 GTGGCTCAAG TATGGGCATC ATTCGCACAT 1020 TGCGGGCTGC TCTTGATCTT TTCGGTCGTG 1080 ATCAGCCGGA CTCCGATTAC CTCGGGAACT 1140 CTGCCTTCGA CCAAGAAGCG GTTGTTGGCG 1200 AGCAGCCGCG TAGTGAGATC TATATCTATG 1260 AGGGCATTGC CACCGCGCTC ATCAATCTCC 1320 ATGTGATCTA CGTGCAAGCA GATTACGGTG 1380 TGGGCATACG GGAAGAAGTG ATGCACTTTG 1440 TCGTTCAAGC CGAGATCGGC TTCCCCGAGC 1500 GCATCAAGCC GGGCTTGCAG ACCTACAAGC 1560 CCCCCGGCCT TCCAATAATG ACAAT And ATG 1620 TATTGGTGGT AAGCCTTGTT CAGCATCTGA 1680 GACCGGAGAA GTGGTATCGC GCGTCGCTGC 1740 GGCCGCTGCA CAGGCTGCGT TTCCTGAATG 1800 CCGACTGCTG CGAGCGGCGG ATCTTCTAGA 1860 GAGTGAAACT GGCGCAGCGG GAAACTGGTA 1920 GTTGCGGGGA ATCC 1964 · · · · · · · · • · • · • · • · · · · · -65 ·· ·· ·· ·. Sequence 12 GAATTCCCCT GGCGACGAAA GGGCGGCAGG CCGCATGGCC ACGGCTGGGC GGTAACTGAT 60 GCTTGCGTTA ATCGTTAACC GTTTGAAATT CCTTGCCAAA TTTCGGCGAG AGAATCATGC 120 GGGTACGCCT TTCCGTGCGC TTTGATCTGC GCTTCCGTGC CTTGAATCAG AAAAATAGTT 180 AATTGACAGA ACTATAGGTT CGCAGTAGCT TTTGCTCACC CACCAAATCC ACAGCACTGG 240 GGTGCACGAT GAATAGCTAC GATGGCCGTT GGTCTACCGT TGATGTGAAG GTTGAAGAAG 300 GTATCGCTTG GGTCACGCTG AACCGCCCGG AGAAGCGCAA CGCAATGAGC CCAACTCTCA 360 ATCGAGAGAT GGTCGAGGTT CTGGAGGTGC TGGAGCAGGA CGCAGATGCT CGCGTGCTTG 420 TTCTGACTGG TGCAGGCGAA TCCTGGACCG CGGGCATGGA CCTGAAGGAG TATTTCCGCG 480 AGACCGATGC TGGCCCCGAA ATTCTGCAAG AGAAGATTCG TCGCGAGCAG GGCATGAAGC 540 AGTTCCTTGA CGAGAAAAGC ATCAAGCCGG GCTTGCAGAC CTACAAGCGC TGATAAATGC 600 GCCGGGGCCC TCGCTGCGCC CCCGGCCTTC CAATAATGAC AATAATGAGG AGTGCCCAAT 660 GTTTCACGTG CCCCTGCTTA TTGGTGGTAA GCCTTGTTCA GCATCTGATG AGCGCACCTT 720 CGAGCGTCGT AGCCCGCTGA CCGGAGAAGT GGTATCGCGC GTCGCTGCTG CCAGTTTGGA 780 AGATGCGGAC GCCGCAGTGG CCGCTGCACA GGCTGCGTTT CCTGAATGGG CGGCGCTTGC 840 TCCGAGCGAA CGCCGTGCCC GACTGCTGCG AGCGGCGGAT CTTCTAGAGG ACCGTTCTTC 900 CGAGTTCACC GCCGCAGCGA GTGAAACTGG CGCAGCGGGA AACTGGTATG GGTTTAACGT 960 TTACCTGGCG GCGGGCATGT · TC 992 · · -66 ··· • ················· Sequence 13 GAATTCCAAT AATGACAATA ATGAGGAGTG CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 TGGTAAGCCT TGTTCAGCAT CTGATGAGCG CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 » TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 GCTGCGAGCG GCGGATCTTC TAGAGGACCG TTCTTCCGAG TTCACCGCCG GGAAGCCGCG GCCATGACCA CACAGATTCA GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420 TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCTCCGGTA ATCCTTGGCG TACGGGCTGT TGCGATGCCG TTGGCATGCG GCAATACCGT 540 GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTGGTG GAGCGACTGA TTGCAAATCC TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 GACCCACGTT GGACGGATCA TTGGTGAGCT GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780 GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840 CGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TCTGATTGTG ACAGCAGTCG CAGACGCCTT TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ACTGCGTGCT GGCGATCCTA ATGATCCGCA ATCGGTCTTG GGTTCGTTGA TTGATGCCAÄ 1020 TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TGATGCGCTC GGGGACAGCA AGCGAACCGG 1080 AATTGCCAGC TGGGGCGCCC TCTGGTAAGG TTGGGAAGCC CTGCAAAGTA AACTGGATGG 1140 CTTTCTTGCC GCCAAGGATC TGATGGCGCA GGGGATCAAG ATCTGATCAA GAGACAGGAT 1200 GAGGATCGTT TCGC ATGATT GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG 1260 TGGAGAGGCT ATTCGGCTAT GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG 1320 TGTTCCGGCT GTCAGCGCAG GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG 1380 CCCTGAATGA ACTGCAGGAC GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC 1440 CTTGCGCAGC TGTGCTCGAC GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG 1500 AAGTGCCGGG GCAGGATCTC CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA 1560 TGGCTGATGC AATGCGGCGG CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC 1620 AAGCGAAACA TCGCATCGAG CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG 1680 ATGATCTGGA CGAAGAGCAT CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG 1740 CGCGCATGCC CGACGGCGAG GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA 1800 TCATGGTGGA AAATGGCCGC TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG 1860 ACCGCTATCA GGACATAGCG TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT 1920 GGGCTGACCG CTTCCTCGTG CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT 1980 TCTATCGCCT TCTTGACGAG TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA 2040 AGCGACGCCC GGCCCAGCGC GTCGATTCGG GCATTTGCCA TATCAATGGA CCGACTGTGC 2100 ATGACGAGGC TCAGATGCCA TTCGGTGGGG TGAAGTCCAG CGGCTACGGC AGCTTCGGCA 2160 · · · · · · · · · · · · · -67GTCGAGCATC GATTGAGCAC TTTACCCAGC TGCGCTGGCT GACCATTCAG AATGGCCCGC 2220 GGCACTATCC AATCTAAATC GATCTTCGGG CGCCGCGGGC ATCATGCCCG CGGCGCTCGC 2280 CTCATTTCAA TCTCTAACTT GATAAAAACA GAGCTGTTCT CCGGTCTTGG TGGATCAAGG 2340 CCAGTCGCGG AGAGTCTCGA AGAGGAGAGT ACAGTGAACG CCGAGTCCAC ATTGCAACCG 2400 CAGGCATCAT CATGCTCTGC TCAGCCACGC TACCGCAGTG TGTCGATTGG TCATCCTCCG 2460 GTTGAGGTTA CGCAAGACGC TGGAGGTATT GTCCGGATGC GTTCTCTCGA GGCGCTTCTT 2520 CCCTTCCCGG GTGGAATTC 2539 • · • · · • · · -68 · · · · · · · · · · · · · · · · · · · · · · · · · · · Sequence 14 GAATTCCAAT AATGACAATA ATGAGGAGTG TGGTAAGCCT TGTTCAGCAT CTGATGAGCG AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTGCGAGCT GCGGGATCTC GGAAGCCGCG GCCATGACCA CACAGATTCA TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGCTCCGGTA ATCCTTGGCG TACGGGCTGT GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGCGGTGGTG GAGCGACTGA TTGCAAATCC GACCCACGTT GGACGGATCA TTGGTGAGCT GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCTGATTGTG ACAGCAGTCG CAGACGCCTT ACTGCGTGCT GGCGATCCTA ATGATCCGCA TGCAGGTCAA CGCATCCAGG TGGGGAGAGG CTGTTGTAAT TCATTAAGCA TTCTGCCGAC CTGAATCGCC AGCGGCATCA GCACCTTGTC ACACCGTGGA AACGGATGAA GGCACGAACC CTGTAATGCA AGTAGCGTAT GCGCTCACGC GGTGGTAACG GCGCAGTGGC GGTTTTCATG ATGCCTCGGG CATCCAAGA GCAAGCGCGT AGCAGCAACG ATGTTACGCA GCAGCAACGA AAAGTTAGGT GGCTCAAGTA TGGGCATCA CAAATCCATG CGGGCTGCTC TTGATCTTTT CTCCCAACAT CAGCCGGACT CCGATTACCT CGCGCTTGCT GCCTTCGACC AAGAAGCGGT CAGGTTTGAG CAGCCGCGTA GTGAGATCTA CCGGAGGCAG GGCATTGCCA CCGCGCTCAT TGGTGCTTAT GTGATCTACG TGCAAGCAGA TACAAAGTTG GGCATACGGG AAGAAGTGAT CTAACAATTC GTTCAAGCCG AGATCGGCTT TTTGCCATAT CAATGGACCG ACTGTGCATG AGTCCAGCGG CTACGGCAGC TTCGGCÄGTC CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180 GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300 TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360 GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420 TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCGATGCCG TTGGCATGCG GCAATACCGT 540 TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780 CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840 TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ATCGGTCTTG GGTTCGTTGA TTGATGCCAÄ 1020 CGGTTTGCGT ATTGGGCGCA TGCATAAAAA 1080 ATGGAAGCCA TCACAAACGG CATGATGAAC 1140 GCCTTGCGTA TAATATTTGC CCATGGACGC 1200 CAGTTGACAT AAGCCTGTTC GGTTCGTAAA 1260 AACTGGTCCA GAACCTTGAC CGAACGCAGC 1320 GCTTGTTATG ACTGTTTTTT TGTACAGTCT 1380 TACGCCGTGG GTCGATGTTT GATGTTATGG 1440 TGTTACGCAG CAGGGCAGTC GCCCTAAAAC 1500 TCGCACATGT AGGCTCGGCC CTGACCAAGT 1560 CGGTCGTGAG TTCGGAGACG TAGCCA CCTA 1620 CGGGAACTTG CTCCGTAGTA AGACATTCAT 1680 TGTTGGCGCT CTCGCGGCTT ACGTTCTGCC 1740 TATCTATGAT CTCGCAGTCT CCGGCGAGCA 1800 CAATCTCCTC AAGCATGAGG CCAACGCGCT 1860 TTACGGTGAC GATCCCGCAG TGGCTCTCTA 1920 GCACTTTGAT ATCGACCCAA G7ACCGCCAC 1980 CCCAATTGGC CCAGCGCGTC GATTCGGGCA 2040 ACGAGGCTCA GATGCCATTC GGTGGGGTGA 2100 GAGCATCGAT TGAGCACTTT ACCCAGCTGC 2160 I · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · -69GCTGGCTGAC CATTCAGAAT GGCCCGCGGC ACTATCCAAT CTAAATCGAT CTTCGGGCGC 2220 CGCGGGCATC ATGCCCGCGG CGCTCGCCTC ATTTCAATCT CTAACTTGAT AAAAACAGAG 2280 CTGTTCTCCG GTCTTGGTGG ATCAAGGCCA GTCGCGGAGA GTCTCGAAGA GGAGAGTACA 2340 GTGAACGCCG AGTCCACATT GCAACCGCAG GCATCATCAT GCTCTGCTCA GCCACGCTAC 2400 CGCAGTGTGT CGATTGGTCA TCCTCCGGTT GAGGTTACGC AAGACGCTGG AGGTATTGTC 2460 CGGATGCGTT CTCTCGAGGC GCTTCTTCCC TTCCCGGGTG GAATTC 2506 • · • · • · • · -70 ··· · · ···· · · · · · · Sequence 15 GAATTCCAAT AATGACAATA ATGAGGAGTG TGGTAAGCCT TGTTCAGCAT CTGATGAGCG AGAAGTGGTA TCGCGCGTCG CTGCTGCCAG TGCACAGGCT GCGTTTCCTG AATGGGCGGC GCTGCGAGCG GCGGATCTTC TAGAGGACCG AACTGGCGCA GCGGGAAACT GGTATGGGTT GGAAGCCGCG GCCATGACCA CACAGATTCA TAGCTTTGCC ATGGCGGTTC GACAGCCATG TGCTCCGGTA ATCCTTGGCG TACGGGCTGT GGTGTTGAAA AGCTCTGAGC TGAGTCCCTT TGATGCTGGT CTGGGGGATG GCGTGGTGAA TGCGGTGGTG GAGCGACTGA TTGCAAATCC GACCCACGTT GGACGGATCA TTGGTGAGCT GGAATTAGGT GGTAAGGCTC CGTTCTTGGT CGAAGCGGCG GCCTTTGGTG CCTACTTCAA TCTGATTGTG ACAGCAGTCG CAGACGCCTT ACTGCGTGCT GGCGATCCTA ATGATCCGCA TGCAGGTCAA CGCATCCAGG TTCTGGTCGA TTGGCCCAGC GCGTCGATTC GGGCATTTGC GCTCAGATGC CATTCGGTGG GGTGAAGTCC TCGATTGAGC ACTTTACCCA GCTGCGCTGG CCAATCTAAA TCGATCTTCG GGCGCCGCGG AATCTCTAAC TTGATAAAAA CAGAGCTGTT GGAGAGTCTC GAAGAGGAGA GTACAGTGAA ATCATGCTCT GCTCAGCCAC GCTACCGCAG TACGCAAGAC GCTGGAGGTA TTGTCCGGAT GGGTGGAATT C CCCAATGTTT CACGTGCCCC TGCTTATTGG 60 CACCTTCGAG CGTCGTAGCC CGCTGACCGG 120 TTTGGAAGAT GCGGACGCCG CAGTGGCCGC 180 GCTTGCTCCG AGCGAACGCC GTGCCCGACT 240 TTCTTCCGAG TTCACCGCCG CAGCGAGTGA 300 TAACGTTTAC CTGGCGGCGG GCATGTTGCG 360 GGGCGATGTC ATTCCGTCCA ATGTGCCCGG 420 TGGCGTGGTG CTCGGTATTG CGCCTTGGAA 480 TGCGATGCCG TTGGCATGCG GCAATACCGT 540 TACCCATCGC CTGATTGGTC AGGTGTTGCA 600 TGTCATCAGC AATGCCCCGC AAGACGCTCC 660 TGCGGTACGT CGAGTGAACT TCACCGGTTC 720 GTCTGCGCGT CATCTGAAGC CTGCTGTGCT 780 CTTGGACGAT GCCGACCTCG ATGCGGCGGT 840 TCAGGGTCAA ATCTGCATGT CCACTGAGCG 900 TGTTGAAAAG CTGGCGAGGA AGGTCGCCAC 960 ATCGGTCTTG GGTTCGTTGA TTGATGCCAA 1020 TGATGCGCTC GCAAAAGGCG CGCAATGGAÄ 1080 CATATCAATG GACCGACTGT GCATGACGAG 1140 AGCGGCTACG GCAGCTTCGG CAGTCGAGCA 1200 CTGACCATTC AGAATGGCCC GCGGCACTAT 1260 GCATCATGCC CGCGGCGCTC GCCTCATTTC 1320 CTCCGGTCTT GGTGGATCAA GGCCAGTCGC 1380 CGCCGAGTCC ACATTGCAAC CGCAGGCATC 1440 TGTGTCGATT GGTCATCCTC CGGTTGAGGT 1500 GCGTTCTCTC GAGGCGCTTC TTCCCTTCCC 1560 1571 I -71 ·· ······························· Sequence 16 GAATTCCGCG GTCGGCGAAA GTTGATGCGC GTGACGAGGC CACACTGTGA GTTGGTCAGG GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT GCCTATCGAC TTAGGGGTAA AGGTCGCTCG ACAAATGGTC GATAGCGTAC TCGCAGGCTC GCTCCCGCGG CACATTGGCT TGTACAGCGG GCAGCGCATT TGCGGCACAG GCTTCGAACT AGGCGCTGAT CACGTGCTGT GTGTCGCGGC GTATACACAC CGGGGCGGGT TCCGCCTCGG GGAGGCATTG TTTGATCCTG CTCCAGGACT GACAGCAAGC GAACCGGAAT TGCCAGCTGG CAAAGTAAAC TGGATGGCTT TCTTGCCGCC TGATCAAGAG ACAGGATGAG GATCGTTTCG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC GACCGACCTG TCCGGTGCCC TGAATGAACT GGCCACGACG GGCGTTCCTT GCGCAGCTGT CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA CGAGAAAGTA TCCATCATGG CTGATGCAAT CTGCCCATTC GACCACCAAG CGAAACATCG CGGTCTTGTC GATCAGGATG ATCTGGACGA GTTCGCCAGG CTCAAGGCGC GCATGCCCGA TGCCTGCTTG CCGAÄTATCA TGGTGGAAAA CCGGCTGGGT GTGGCGGACC GCTATCAGGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TTCGAAATGA CCGACCAAGC GACGCCCATT AGAGATCGTG GCTGTTACGG ATGAACAGTT TGAACTGCCT CGGAAGGCAA AATTGTTGAT TGAAGCCCTT TCCCGATTGA AGCCTGTTCA CTGTGCCGT A GTGGACGGCG CCGCGGCGGC GCCGGTCTTG GCTAGGATAC TGGCTACCTC GCTCGGCCCT GCGCCCGCGA TTCGCCTGCT TATCGACCTC TTTGAGATAA ACGAGGCGCA ATTGGGTTT TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420 AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 CGACATGATC GCTACCGCAG AAAACCTGGG 600 GGCGCCCTCT GGTAAGGTTG GGAAGCCCTG 660 AAGGATCTGA TGGCGCAGGG GATCAAGATC 720 CATGATTGAA CAAGATGGAT TGCACGCAGG 780 CGGCTATGAC TGGGCACAAC AGACAATCGG 840 AGCGCAGGGG CGCCCGGTTC TTTTTGTCAA 900 GCAGGACGAG GCAGCGCGGC TATCGTGGCT 960 GCTCGACGTT GTCACTGAAG CGGGAAGGGA 1020 GGATCTCCTG TCATCTCACC TTGCTCCTGC 1080 GCGGCGGCTG CATACGCTTG ATCCGGCTAC 1140 CATCGAGCGA GCACGTACTC GGATGGAAGC 1200 AGAGCATCAG GGGCTCGCGC CAGCCGAACT 1260 CGGCGAGGAT CTCGTCGTGA CCCATGGCGA 1320 TGGCCGCTTT TCTGGATTCA TCGACTGTGG 1380 CATAGCGTTG GCTACCCGTG ATATTGCTGA 1440 CCTCGTGCTT TACGGTATCG CCGCTCCCGA 1500 TGACGAGTTC TTCTGAGCGG 1560 GAGGGCGCAA GAGGAGAAAT GGATTGA CCA 1620 CGATTTAGAG GGCTACAACA GTCGAGCAAT 1680 CGTGACAGTC ATCCGCGGCC TAGCAGTCTT 1740 TTCTGGCGGG GTGCAGACTG CGGGCAACAG 1800 TTTGGTGGCT CGAGAGTCGT CTGCGACACA 1860 CGTAGTCGGG ATCGAGCCCG AGCATATGGG 1920 GCTTGCGCGT AGTGATCTTA GTTTGAGGGA 1980 GGCCGCCCAA GTTCTAGCGG TACAGCATGA 2040 TTGGGGCGGG GCCATTGCAC TTGGACACCC 2100 GACCCTCGCT CACCAATTGC AAGCTAATAA 2160 • · • · · · • · ···· • · ··· · · · · · · · · · · · · · · · · · · · · · · · · -72CTTTCGATAT AGAGAATCCC CTATCCACTG TCGAAAATCT TGGCAGAAAG TCGGGCTGAT GAATTC GGAATTGCCT CGGCATGCAT TGGTGGGGGA CAGGGGATGG CGGTTCTTTT 2220 CACTTCGGTT CGTCCTCTGC ACGAAGTTCG ATGATTAACA GAGTTGACCA 2280 AGCTAACGGG CATCTCCTTT GTTGCTTTGA GGTGGCGCAC GAAGGAGGGC 2340 CTGCTAAAAA CAAGAAGAAG GAACAGGGAA CATGATTAGT TTCGCTCGTA 2400 TTTAGGAGTC CAGGCTAAAC TTGCCCTTGC CTTCGCACTC GTATTATGTG 2460 TGTTACCGGC ACGGGTTTCT ACAGTGTACA TACCTTGTCA GGGTTGGTGG 2520 2526 ·· -73 ··· ··· · ····························· · · · · · · · · · · Sequences 17 GAATTCCGCG GTCGGCGAAA GTTGATGCGC GTGACGAGGC CACACTGTGA GTTGGTCAGG GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT GCCTATCGAC TTAGGGGTAA AGGTCGCTCG ACAAATGGTC GATAGCGTAC TCGCAGGCTC GCTCCCGCGG CACATTGGCT TGTACAGCGG GCAGCGCATT TGCGGCACAG GCTTCGAACT AGGCGCTGAT CACGTGCTGT GTGTCGCGGC GTATACACAC CGGGGCGGGT TCCGCCTCGG GGAGGCATTG TTTGATCCTG CTCCAGGACT GGAGAGGCGG TTTGCGTATT GGGCGCATGC TGCCGACATG GAAGCCATCA CAAACGGCAT CCTTGTCGCC TTGCGTATAA TATTTGCCCA ACGAACCCAG TTGACATAAG CCTGTTCGGT CTCACGCAAC TGGTCCAGAA CCTTGACCGA TTTCATGGCT TGTTATGACT GTTTTTTTGT AGCGCGTTAC GCCGTGGGTC GATGTTTGAT GCAACGATGT TACGCAGCAG GGCAGTCGCC GCATCATTCG CACATGTAGG CTCGGCCCTG ATCTTTTCGG TCGTGAGTTC GGAGACGTAG ATTACCTCGG GAACTTGCTC CGTAGTAAGA AAGCGGTTGT TGGCGCTCTC GCGGCTTACG AGATCTATAT CTATGATCTC GCAGTCTCCG CGCTCATCAA TCTCCTCAAG CATGAGGCCA AAGCAGATTA CGGTGACGAT CCCGCAGTGG AAGTGATGCA CTTTGATATC GACCCAAGTA TCGGCTTCCC ATTGAGGGCG CAAGAGGAGA CGGATGAACA GTTCGATTTA GAGGGCTACA CAAAATTGTT GATCGTGACA GTCATCCGCG TGAAGCCTGT TCATTCTGGC GGGGTGCAGA GCGCCGCGGC GGCTTTGGTG GCTCGAGAGT TACTGGCTAC CTCCGTAGTC GGGATCGAGC CGATTCGCCT GCTGCTTGCG CGTAGTGATC TAAACGAGGC GCAGGCCGCC CAAGTTCTAG CAAAACTTAA TATTTGGGGCC GGGGGCTG TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420 AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 CGACATGATC GCTACCGCAG AAAACCTGGG 600 ATAAAAACTG TTGTAATTCA TTAAGCATTC 660 GATGAACCTG AATCGCCAGC GGCATCAGCA 720 TGGACGCACA CCGTGGAAAC GGATGAAGGC 780 TCGTAAACTG TAATGCAAGT AGCGTATGCG 840 ACGCAGCGGT GGTAACGGCG CAGTGGCGGT 900 ACAGTCTATG CCTCGGGCAT CCAAGCAGCA 960 GTTATGGAGC AGCAACGATG TTACGCAGCA 1020 CTAAAACAAA GTTAGGTGGC TCAAGTATGG 1080 ACCAAGTCAA ATCCATGCGG GCTGCTCTTG 1140 CCACCTACTC CCAACATCAG CCGGACTCCG 1200 CATTCATCGC GCTTGCTGCC TTCGACCAAG 1260 TTCTGCCCAG GTTTGAGCAG CCGCGTAGTG 1320 GCGAGCACCG GAGGCAGGGC ATTGCCACCG 1380 ACGCGCTTGG TGCTTATGTG ATCTACGTGC 1440 CTCTCTATAC AAAGTTGGGC ATACGGGAAG 1500 CCGCCACCTA ACAATTCGTT CAAGCCGAGA 1560 AATGGATTGA CCAAGAGATC GTGGCTG TTA 1620 ACAGTCGAGC AATTGAACTG CCTCGGAAGG 1680 GCCTAGCAGT CTTTGAAGCC CTTTCCCGAT 1740 CTGCGGGCAA CAGCTGTGCC GTAGTGGACG 1800 CGTCTGCGAC ACAGCCGGTC TTGGCTAGGA 1860 CCGAGCATAT GGGGCTCGGC CCTGCGCCCG 1920 TTAGTTTGAG GGATATCGAC CTCTTTGAGA 1980 CGGTACAGCA TGAATTGGGT ATTGAGCACT 2040 CACTTGGACA CCCGCTTGCC GCGACCGGAT 2100 TGCAAGCTAA TAACTTTCGA TATGGAATTG 2160 ·· • · ······ -74 ·· CCTCGGCATG CATTGGTGGG GGACAGGGGA TGGCGGTTCT TTTAGAGAAT CCCCACTTCG 2220 GTTCGTCCTC TGCACGAAGT TCGATGATTA ACAGAGTTGA CCACTATCCA CTGAGCTAAC 2280 GGGCATCTCC TTTGTTGCTT TGAGGTGGCG CACGAAGGAG GGCTCGAAAA TCTCTGCTAA 2340 AAACAAGAAG AAGGAACAGG GAACATGATT AGTTTCGCTC GTATGGCAGA AAGTTTAGGA 2400 GTCCAGGCTA AACTTGCCCT TGCCTTCGCA CTCGTATTAT GTGTCGGGCT GATTGTTACC 2460 GGCACGGGTT TCTACAGTGT ACATACCTTG TCAGGGTTGG TGGGAATTC 2509 I ·· ·· • · · I · · -75 · · · 75 75 75 75 75 75 75 75 75 75 75 Sequence 18 GAATTCCGCG GTCGGCGAAA GTTGATGCGC GTGACGAGGC CACACTGTGA GTTGGTCAGG • GTTGGTTGCG GCAGTGCGCA CCCCCTGGAT GCCTATCGAC TTAGGGGTAA AGGTCGCTCG ACAAATGGTC GATAGCGTAC TCGCAGGCTC »GCTCCCGCGG CACATTGGCT TGTACAGCGG GCAGCGCATT TGCGGCACAG GCTTCGAACT AGGCGCTGAT CACGTGCTGT GTGTCGCGGC GTATACACAC CGGGGCGGGT TCCGCCTCGG GGAGGCATTG TTTGATCCTG CTCCAGGACT GCGCATTGAG GGCGCAAGAG GAGAAATGGA AACAGTTCGA TTTAGAGGGC TACAACAGTC TGTTGATCGT GACAGTCATC CGCGGCCTAG CTGTTCATTC TGGCGGGGTG CAGACTGCGG CGGCGGCTTT GGTGGCTCGA GAGTCGTCTG CTACCTCCGT AGTCGGGATC GAGCCCGAGC GCCTGCTGCT TGCGCGTAGT GATCTTAGTT AGGCGCAGGC CGCCCAAGTT CTAGCGGTAC TTAATATTTG GGGCGGGGCC ATTGCACTTG TCTGCATGAC CCTCGCTCAC CAATTGCAAG CATGCATTGG TGGGGGACAG GGGATGGCGG CCTCTGCACG AAGTTCGATG ATTAACAGAG CTCCTTTGTT GCTTTGAGGT GGCGCACGAA GAAGAAGGAA CAGGGAACAT GATTAGTTTC GCTAAACTTG CCCTTGCCTT CGCACTCGTA GGTTTCTACA GTGTACATAC CTTGTCAGGG TGTATCGTGG TGAAGATCAA TCCATGCTGC 60 GGGGGCTTAC TCGGCGTTTT CCGACACTGC 120 TGATTGCGGG GGTGCCCTGT CGCTGGTGTC 180 CGAAGTTCTG ATGCGTGCGT CGCTTGAACC 240 TATGGCTCAA GCAAGCTTTG ATGCTTACCT 300 TGTTCCCAAG TCGGTTCCGG CCTTGGGGGT 360 GCTTCGGCAG GCCGGCGAGC AGATTTCCCA 420 AGAGTCCATG TCGCGTAACC CCATCGCGTC 480 TGCGCCCGTT GAGTTCAAGG ATTTTTTGTG 540 CGACATGATC GCTACCGCAG AAAACCTGGC 600 TTGACCAAGA GATCGTGGCT GTTACGGATG 660 GAGCAATTGA ACTGCCTCGG AAGGCAAAAT 720 CAGTCTTTGA AGCCCTTTCC CGATTGAAGC 780 GCAACAGCTG TGCCGTAGTG GACGGCGCCG 840 CGACACAGCC GGTCTTGGCT AGGATACTGG 900 ATATGGGGCT CGGCCCTGCG CCCGCGATTC 960 TGAGGGATAT CGACCTCTTT GAGATAAACG 1020 AGCATGAATT GGGTATTGAG CACTCAAAAC 1080 GACACCCGCT TGCCGCGACC GGATTGCGTC 1140 CTAATAACTT TCGATATGGA ATTGCCTCGG 1200 TTCTTTTAGA GAATCCCCAC TTCGGTTCGT 1260 TTGACCACTA TCCACTGAGC TAACGGGCAT 1320 GGAGGGCTCG AAAATCTCTG CTAAAAACAA 1380 GCTCGTATGG CAGAAAGTTT AGGAGTCCAG 1440 TTATGTGTCG GGCTGATTGT TACCGGCACG 1500 TTGGTGGGAÄ TTC 1543 I 1/3 · «« «3 3 3 3 3 3 3 3 3 3 3 3 1/ 3 · · · · · · · / · / / ÄQKm Fig. 1a ca / ÄQGm Fig. 1b ca / ΑΔ Fig. 1c ca / SQKm Fig. 1d ca / SQGm Fig. 1e calBň psn BMTBal 31 Deletion 539 bp SCORE SoAl '/ Bal 31 SmaV jj'-CnKm-Elémen:; SmaV BcAI7Bal3i V, ecoRi EcoRI SoJlITBal 31 Smal Smal * ΒσΛΓ / Bal 31 BcoRI fcoRI Bglll / Bal 31 fcoRI Deletion 586 bp Fig. 1f ·· ··············· 2/3 ································· Fig. 1g fcsQGm Fig. 1h tesá Fig. 1i ecóQKm Fig. 1j ecríQGm Fig. 1k echá — un · c ^ jai '' inkm-Elemenl Deletion 1290 bp HCOR NnA 'Laugh O SmaT NnA 'O ^with -TOKm-Element ecoRi EccRl NnA'Smaľ Smáľ NnA ' EeoRl NnA EedK ECCF Deletion 483 bp Fig. 11 ·· 3/3 Smal * SssHII · *;.? * ΓΛΚπϊ-εΐβπΐβπΐ * - ' ^: -j |||| p ·· ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Fig. 1m y E / jQGm Fig. 1n vdhň Fig. 1o aa / QKm Fig. 1p aaíQGm Fig. 1q t. AATA SssHII'Real £ coRI Deletion 210 bp £ csRi SssHIS / nal · _ Smal · SssHII Network - 'Éiemefs £ coRl SssHirSmall V Delete SssHII · £ cpRI £ coRI SssHII £ «πι Deletion 59 bp Fig. 1r