PROTEINS FROM THE WEBS OF NEPHILENGYS CRUENTATA, AVICULARIA JURUENSIS AND PARAWIXIA BISTRIATA SPIDERS ISOLATED FROM BRAZILIAN BIODIVERSITY

Abstract

ates to molecules isolated from the nucleic acid that encodes spider web proteins or fragments of these or other derivatives of these. The invention also refers to a chimerical gene and an expression vector containing molecules isolated from the nucleic acid that codes for proteins related to the webs of Nephilengys, Cruentata, Avicularia Juruensis and Parawixia Bistriata spiders. Another embodiment of the present invention are transformed cells containing a chimerical gene or an expression vector of the present invention. Yet another embodiment of the present invention relates to a method for obtaining genetically modified organisms containing inventive chimerical genes or expression vectors and a method for obtaining recombinant proteins from the silks of Nephilengys, Cruentata, Aviculana Juruensis and Parawixia Bistriata spiders. Finally, the invention describes products, such as biofilaments and compositions, using the recombinant proteins of the present invention. The discovery of new spider silk proteins, as well as their characterisation and expression in different heterologous systems shall be of great use in numerous areas, such as medicine and industry.

Description

FIELD OF THE INVENTION

[0001]The present invention relates to molecules isolated from the nucleic acid that encodes proteins related to spider webs, fragments of these or other of their derivates. The invention also refers to a chimerical gene and an expression vector containing molecules isolated from the nucleic acid that encode proteins related to the webs of Nephilengys Cruentata, Avicularia Juruensis and Parawixia Bistriata spiders. Another embodiment of the present invention are transformed cells containing a gene construct or an expression vector of the present invention.

[0002]Yet another embodiment of the present invention relates to a method for obtaining genetically modified organisms containing inventive gene constructs or expression vectors and a method for obtaining recombinant proteins from the silks of Nephilengys Cruentata, Avicularia Juruensis and Parawixia Bistriata spiders. Finally, the invention describes products, such as biofilaments and compositions, composed from the recombinant proteins of the present invention.

BACKGROUND OF THE INVENTION

[0003]Industry has recently demonstrated great interest in obtaining synthetic or natural fibres that simultaneously provide high resistance, low weight, and overall versatility. Most of the synthetic fibres currently used, such as Nylon or Kevlar, present high production costs as well as some other undesirable characteristic such as high density or restricted fields of application.

[0004]Amongst the natural fibres, silk provided by the silkworm (Bombyx mori) has been used for over 5.000 years in the textile industry (Hyde, N. 1984. The queen of textiles. Natl. Geogr. 165, 3-49). The fibres of this egg sac are composed of two continuous filament of silk, heavy-(≈350 kDa) and light-chain fibroin (≈25 kDa), linked by adhesive proteins termed sericins (Jin H. J., Kaplan D. L. (2003). Mechanism of silk processing in insects and spiders. Nature 424:1057-1061). Commercially, sericin is removed from the egg sacs by immersion in hot water and soap, which yields between 300 and 1200 m of usable fibre (fibroin) per egg sac.

[0005]Different from the silkworm, spiders have not yet been domesticated for textile applications. This basic difference is the result of the difficulty in obtaining large spider populations due to their solitary and predatory nature; furthermore, spider silk is produced in small quantities and cannot be gathered into skeins like simpler fibres, in the manner of the silkworm coccon. However, the physical characteristics presented by the silks produced by spiders are far superior to that of the silk from B. mori (Dickinson M. H. (1999). Bionics: Biological insight into mechanical design. Proc. Natl. Acad. Sci. USA 96:14208-14209). Due to its great elasticity and resistance, the silk from spider's webs has not only aroused much interest in the textile industry but also in other industries from the most diverse sectors, such as the cosmetic industry (US20050019297).

[0006]Spiders are amongst the organisms that present the greatest diversity and abundance on Earth. The order Araneae is the second largest group among the arachnids and the seventh among arthropods, with over 39.000 species included in 110 families (Selden P. A. 1989. Orb-weaving spiders in the early Cretaceous. Nature, 340: 711-712; Shear W. A., Palmer J. A., Coddington J. A., Bonamo P. M. 1989. a Devonian Spinnert: early evidence of spiders and silk use. Science 246: 479-481; Platnick, N. I. 2006. The world spider catalog, version 6.5. American Museum of Natural History). It is estimated that Brazil alone is home to between 4.000 and 10.000 species of spiders [Brescovit, A. D. 1999. Araneae. In: Biodiversidade do Estado de Sao Paulo, Brazil. Joly, C. A. & C.E.M. Bicudo (orgs.). Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo, SP].

[0007]The fibres obtained from the proteins of spider silks are up to five times more resistant than steel and 30% more flexible than Nylon. They may be used in in the manufacture of ropes and cables, fishing lines, bullet-proof vests, parachute materials, amongst other uses. Furthermore, as they are composed of biodegradable matter, spider silks may have medical applications such as in the manufacture of sutures and surgical dressings, bandages, atificial tendons and ligaments, matrix for drug carriers, etc. (WO2004016651; Gosline, J. M.; P. A Guarette; C. S. Ortlepp & K. N. Savage, 1999. The mechanical design of spider silks: from fibroin sequence to mechanical function. The Journal of Experimental Biology, 202: 3295-3303; Heslot, 1998. Artificial fibrous proteins: a review. Biochimie, 80: 19-31).

[0008]Silks produced by spiders are synthesised in glands located in the region of the abdomen and polymerised through a series of spinnerts that convert the water soluble silk proteins of high molecular weight into non-water soluble fibres (Benito B., 2002. Synthesizing spider silk. Trends Biotechnol. 20:189). The types and nature of fibres are several and they depend on the spider's species [Denny, M. W., 1980. Silks--their properties and functions. In: Mechanical properties of Biological Materials. Vincent, J. F. V., Currey, J. D. (Eds.), Cambridge University Press, Cambridge, pp. 247-272]. Spiders possess seven silk producing glands: the aciniform gland responsible for producing the silks used to encapsulate insects, the cylindrical gland that produces the silk forming the egg sac where the eggs are deposited, and the flagelliform, "major ampullate", "minor ampullate", pyriform and coronata glands, that produce the silks that form the orb web. But, however, no known family of spiders possesses all seven glands.

[0009]Among the different silks produced by spiders is the dragline synthesised by the "major ampullate" gland which is extremely rigid and has a tensile strength similar to that of Kevlar (4×109 N/m²) associted to good viscoelasticity (dragline 35%, Kevlar 5%) (Oroudjev E., Soares J., Arcidiacono S., Thompson J. B., Fossey A. S., Hansma H. G. (2002). Segment nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy. Proc. Natl. Acad. Sci. USA 99:6460-6465). The dragline silk is used by spiders to escape from predators and as a frame for the production of silks. The silk produced by the "minor ampullate" gland, used as a reinforcement when building the web, has a tensile strength similar to that of the dragline, but with less elasticity (Colgin M. A., Lewis R. V. 1998. Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like `spacer regions`. Protein Sci. 7:667-672; Hayashi C. Y., Blackledge T. A., Lewis R. V. 2004. Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family. Mol. Biol. Evol. 21:1950-1959). Spider silks are biopolymers that present exrtaordinary physical properties (Cunniff P. M., Fossey S. A., Auerbach M. A., 1994a. Mechanical and thermal properties of dragline silk from the spider Nephila clavipes. Poly. Adv. Technol. 5:401-410, Cunniff P. M., Fossey S. A., Auerbach M. A., 1994b. Mechanical properties of major ampullate gland silk fibers extracted from Nephila clavipes spiders. In: Kaplan, D. L., Adams, W. W., Farmer, B., Viney, C. (Eds.). Silk Polymers: Materials science and Biotechnology, American Chemical Society Symposium Series, 544, pp. 234-251; Ko F. K., Jovicic J., 2004. Modelling of mechanical properties and structural design of spider web. Biomacromolecules 5:780-785), but there is only limited knowledge about the composition of the different silks produced by a specific species of spider. The different silk proteins contain repetitive amino acids that vary depending on the purpose of the silk and thus confer different mechanical properties to the biopolymers (Gosline J. M., Guerette P. A., Ortlepp C. S., Savage K. N. (1999). The mechanical design of spider silks: from fibroin sequence to mechanical function. The J. Exp. Biol. 202:3295-3303). Depending on environmental conditions and requirements, the composition of the silk amino acids may vary considerably, not just between different spiders but for the same spider on different days (Work R. W., Young C. T., 1987. The amino acid compositions of major and minor ampullate silks of certain orb-web-building spiders (Araneae, Araneidae). J. Arachnol. 15:65-80; Volltrah F. 1999. Biology of spider silk. Int. J. Biol. Macromol. 24:81-88; Craig C. L., Riekel C., Herberstein M. E., Weber R. S., Kaplan D., Pierce N. E., 2000. Evidence for diet effects on the composition of silk proteins produced by spiders. Mol. Biol. Evol. 17:1904-1913). This fact raises questions concerning the genomic sequences and the organisation of the genes that encode these proteins.

[0010]The first studies intending the industrial use of these silks were mainly directed at the protein analysis of two species: Nephila clavipes and Araneus diadematus. The dragline silk isolated from these two species is the most studied of all the fibres synthesised by spiders. The dragline is formed from two types of proteins produced by the "major ampullate" gland, termed MaSp1 and MaSp 2 (Major Ampullate Spidroin) in N. clavipes, and ADF-3 and ADF-4 in A. diadematus (Araneus Diadematus Fibroin) (Hinman M. B., Lewis, R. V. 1992. Isolation of a clone coding a second dragline silk fibroin, Nephila clavipes dragline silk is a two protein fiber. J. Biol. Chem. 267:19320-19324; Guerette P., Ginzinger D., Weber B., Gosline J. 1996. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 272:112-115; Beckwitt R., Arcidiacono S. 1994. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae). J. Biol. Chem. 269:6661-6663; Beckwit R., Arcidiacono S., Stote R. 1998. Evolution of repetitive proteins: spider silks from Nephila clavipes (Tetragnathidae) and Araneus bicentarius (Araneidae). Insect. Biochem. Molec. 28:121-130). The dragline proteins have a molecular mass between 180 kDa and 720 kDa, depending on the analysis conditions (Mello C. M., Senecal K. Yeung B., Vouros P., Kaplan D. I. 1994. Initial characterization of Nephila clavipes dragline protein. In: Kaplan D. L. Adams W. W Farmer B. Viney C. (Eds.). Silk Polymers Materials Science and Biotechnology. American Chemical Society Symposium Series. 544:67-79). The amino acid composition of these proteins tends to indicate that the molecular ratio between MaSp1 and MaSp2, and between ADF-4 and ADF-3 is of approximately 3:1 in the dragline fibre (Hinman M. B., Lewis, R. V. 1992. Isolation of a clone coding a second dragline silk fibroin, Nephila clavipes dragline silk is a two protein fiber. J. Biol. Chem. 267:19320-19324; Lombardi S. J., Kaplan D. L. 1990. The amino acid composition of major ampullate gland silk (dragline) of Nephila clavipes (Araneae, Tetragnathidae). J. Arachnol. 18:297-306; Guerette P., Ginzinger D., Weber B., Gosline J. 1996. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 272:112-115). Despite being produced by two different species, the proteins of the "major ampullate" gland comprise a high number of repetitions of the same amino acids. In MaSp2 and ADF-3, for example, glycine, alanine, proline, serine and tyrosine are responsible for up to 99% of the amino acids present in their structure (Hayashi C. Y., Lewis R. V. 1998. Evidence from flagelliform silk. cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Biol. 275:773-784).

[0011]Several works have been developed over the characteristics and possible applications for the dragline silk of Nephila clavipes [U.S. Pat. No. 6,268,169; U.S. Pat. No. 6,412,261; WO9116351; Beckwitt R. & Arcidiacono S., 1994. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae). J. Biol. Chem. 269, 6661-6663; Arcidiacono S., Mello, C., Kaplan D. L., Cheley, S., Bayley, H., 1998. Purification and characterization of recombinant spider silk expressed in Escherichia coli. Appl. Microbiol. Biotechnol. 49, 31-38].

[0012]Apart from the proteins of the silk produced by the "major ampullate" gland, another frequently studied silk of the N. clavipes is that produced by the "minor ampullate" gland. Just as in the case of the silk produced by the former gland, this one is also formed from two peptides (MiSP1 and MiSP2) composed by imperfect repetitions of amino acid sequences (U.S. Pat. No. 5,733,771).

[0013]In 1998, Hayashi and Lewis (Hayashi C. Y., Lewis R. V., 1998. Evidence from flagelliform silk. cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Bio, 275: 773-784), sequenced the protein of the silk produced by the flagelliform gland of N. clavipes (Gosline, J. M.; P. A Guarette; C. S. Ortlepp & K. N. Savage, 1999. The mechanical design of spider silks: from fibroin sequence to mechanical function. The Journal of Experimental Biology, 202: 3295-3303). Similar results have been published for the species Araneus diadematus, involving the "major ampullate" that produces proteins ADMAG1 and ADMAG2 (Guerette P., Ginzinger D., Weber B Gosline J., 1996. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science, 272:112-115).

[0014]Based on DNA analysis, it is possible to affirm that all proteins comprising spider silks are formed by repetitive peptide units. These may be grouped into four major groups: GPGXX (where X frequently represents Q), alanine rich sequences (An or (GA)n), GGX (where X=A, Y, L or Q) and the spacers. A fifth category is represented by non-repetitive regions at the N- and C-terminal ends of the proteins and are normally chains constituted of 100 or more amino acids (Xu M., Lewis R. V., 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. USA 87:7120-7124; Hinman M. B., Lewis, R. V. 1992. Isolation of a clone coding a second dragline silk fibroin, Nephila clavipes dragline silk is a two protein fiber. J. Biol. Chem. 267:19320-19324; Colgin M. A., Lewis R. V. 1998. Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like `spacer regions`. Protein Sci. 7:667-672; Hayashi C. C. Y., Shipley N. H., Lewis R. V. 1999. Hypothesis that correlate the sequence, structure and mechanical properties of spider silk proteins. Int. Biol. Macromol. 24:271-275; Oroudjev E., Soares J., Arcidiacono S., Thompson J. B., Fossey A. S., Hansma H. G. 2002. Segment nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy. Proc. Natl. Acad. Sci. USA 99:6460-6465; Tai P. L., Hwang G. Y., Tso I. M., 2004. Inter-specific sequence conservation and intra-individual sequence variation in a spider silk gene. Inter. J. Biol. Macromolecules 34:295-301).

[0015]In accordance with different studies, the majority of the repetitive units present in spider silks present specific structural properties (Xu M., Lewis R. V., 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. USA 87:7120-7124; Hayashi C. Y., Lewis R. V. 1998. Evidence from flagelliform silk. cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Biol. 275:773-784; Van Beek J. D., Hess S., Vollrath F., Meier B. H., 2002. The molecular structure of spider dragline silk: Folding and orientation of the protein backbone. Proc. Natl. Acad. Sci. USA 99:10266-10271; Bini E., Knight D. P., Kaplan D. L. 2004. Mapping domain structures in silks from insects and spiders related to protein assembly. J. Mol. Biol. 335:27-40; Scheibel T. 2004. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3:14; Stantcheva N. N. P., Mason S. J. M. 2004. Molecular studies of a novel dragline silk from nursery web spider, Euprosthenops sp (Psauridae). Comp. Biochem. Phisiol. 138:371-376). The GPGXX module is responsible for the formation of the β-spiral structures, and probably confers elasticity to the silk. The flagelliform silk, produced by the flagelliform gland, possesses an elasticity of over 200% and comprises at least 43 GPGXX modules in each repetitive unit (Hayashi C. Y., Lewis R. V. 2000. Molecular architecture and evolution of a modular of spider silk protein gene. Science 287:1477-1479). In conformity with the low elasticity of dragline silk, the latter only presents nine repetitions of this motive before being interrupted by another module. Alaninee rich modules are normally constituted of 6-9 residues of this amino acid with these being responsible for the formation of the β-sheets that provide rigidity to the fibre. The silks produced by the "major" and "minor ampullate" glands are both very strong and present An or (GA)n motives but, however, these motives are not encountered in flagelliform silks (Gatesy J., Hayashi C., Motriuk D., Woods J., Lewis R. 2001. Extreme diversity, conservation, and convergence of spider silk fibroin sequences. Science 291:2603-2605). In turn, GGX, which is a 310 helix, forms an amorphous matrix that connects the crystalline regions and confers elasticity to the fibre, probably in conjunction with GPGXX. This motive may be encountered in all the flagelliform, "major" and "minor ampullate" glands. The spacer regions are constituted of charged groups that separate the glycine rich regions (Colgin M. A., Lewis R. V. 1998. Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like `spacer regions`. Protein Sci. 7:667-672; Hayashi C. C. Y., Shipley N. H., Lewis R. V. 1999. Hypothesis that correlate the sequence, structure and mechanical properties of spider silk proteins. Int. Biol. Macromol. 24:271-275) but, however, its structural purpose remains unknown. The non-repetitive terminations are common in all the fibres produced by spiders of the Araneidae family, with the C-terminal sequences being highly conserved among the species (Bini E., Knight D. P., Kaplan D. L. 2004. Mapping domain structures in silks from insects and spiders related to protein assembly. J. Mol. Biol. 335:27-40; Hayashi C. Y., Blackledge T. A., Lewis R. V. 2004. Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family. Mol. Biol. Evol. 21:1950-1959; Stantcheva N. N. P., Mason S. J. M. 2004. Molecular studies of a novel dragline silk from nursery web spider, Euprosthenops sp (Psauridae). Comp. Biochem. Phisiol. 138:371-376; Tian M., Liu C., Lewis R., 2004. Analysis of major ampullate silk cDNA from two non-orb-weaving spiders. Biomacromolecules 5:657-660). Recent studies conducted with ADF-3 and 4 revealed an α-helix structure formed by the C-terminal region, which raises the hypothesis that this region has an important role in the polymerisation of the fibre (Huemmerich D., Scheibel T., Vollrath F., Cohen S., Gat U., Ittah I. 2004. Novel assembly properties of recombinant spider dragline silk proteins. Curr. Biol. 14:472-476).

[0016]The spinning mechanism, or, in other terms, the polymerisation of water soluble proteins into insoluble fibres, is a process that commences with an increase in the concentration of the protein in the glandular lumen forming a "spinning solution". In the major ampullate gland, for example, the proteins of dragline silk are present in a concentration over 50% (m/v) (Artkins E. 2003. Silk's secrets. Nature 424:1010; Scheibel T. 2004. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3:14). The increased concentration of MaSp causes a structural modification to these proteins, which change from a coil to a β-helix structure and increase their stability (Dicko C., Knight D., Kenney J. M., Vollrath F. 2004. Structural conformation of spidroin in solution: A synchrotron radiation circular dichroism study. Biomacromolecules 5:758-767). In this manner, the spider maintains a relatively high concentration of protein in an aqueous solution, without leading to the formation of insoluble β-sheets. The polymerisation of the proteins occurs when the "spinning solution" passes through the glandular duct, concommitantly with the extraction of water, sodium and chloride. Hydrogen and potassium ions are secreted which reduces the pH from 6.9 to 6.3 (Chen X., Knight D. P., Shao Z., Vollrath F. 2002. Conformation transition in silk protein films monitored by time-resolved fourier transform infrared spectroscopy: Effect of potassium ions on Nephila spidroin films. Biochemistry 41:14944-14950; Dicko C., Vollrath F., Kenney J. M. 2004. Spider silk protein refolding is controlled by changing pH. Biomacromolecules 5:704-710). Such alterations trigger the alignment of the proteins in the distal part of the duct and while their poly-A hydrophobic sequences align and come closer they are exposed to an increasingly hydrophobic environment which most probably instigates the structural conversion of these proteins to β-sheets (Scheibel T. 2004. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3:14), and, consequently, the polymerisation of the fibre.

[0017]The high organisation of the fibre structures, extensive hydrogen bonds and Van der Walls interactions induce the expulsion of water from the regions between the β-sheets. Spider silks are insoluble in water, dilute acids and bases, chaotropic agents such as urea and guanidine hydrochloride as well as the majority of organic solvents (Lombardi S. J., Kaplan D. L. 1990. The amino acid composition of major ampullate gland silk (dragline) of Nephila clavipes (Araneae, Tetragnathidae). J. Arachnol. 18:297-306). The silks are also resistant to the majority of proteolytic enzymes. The silks dissolved slightly in saline solutions of lithium bromide, lithium thiocyanate, calcium chloride and other calcium salts. High concentrations of a propionic/hydrochloridric acid mixture as well as formic acid may also be used (Mello C. M., Senecal K. Yeung B., Vouros P., Kaplan D. I. 1994. Initial characterization of Nephila clavipes dragline protein. In: Kaplan D. L. Adams W. W Farmer B. Viney C. (Eds.). Silk Polymers: Materials Science and Biotechnology. American Chemical Society Symposium Series. 544:67-79 Lewis R. V., Hinman M., Kothakota S., Fournier M. J. 1996. Expression and purification of a spider silk proteins: A new strategy for producing repetitive proteins. Express. Prif 4:400-406).

[0018]Different parties have attempted to process the silk artificially using different types of diluents. Most efforts have centred around the liquid processing used for B. mori. Silk recombinant proteins have been processed using solvents such as hexafluoroisopropanol (WO 9429450) as diluents or protein solutions diluted in concentrated solutions of formic acid [Lewis R. V., Hinman M., Kothakota S., Fournier M. J., 1996. Expression and purification of a spider silk protein: A new strategy for producing repetitive proteins. Express. Prif (4): 400-406]. However, in both above cases the mechanical properties of natural silk were not efficiently reproduced. (Fahnestock S. R., Bedzyk L. A, 1997. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl. Microbiol. Biotechnol, 47: 23-32).

[0019]Other essays succeeded in solubilising the spider silks through immersion of the fibres in saline concentrations such as lithium bromide, lithium thiocyanate, calcium chloride and other calcium salts. High concentrations of a propionic/hydrochloridric acid mixture as well as formic acid may also be used [Mello C. M., Senecal K., Yeung B., Vouros P., Kaplan D. I., 1994. Initial characterization of Nephila clavipes dragline protein. In: Silk Polymers: Materials Science and Biotechnology. American Chemical Society Symposium Series. Kaplan D. L., Adams W. W, Farmer B., Viney C. (Eds.), 544 pp].

[0020]Initially, the process that leads to the high hydrophobicity of the spider silk proteins triggers the formation of repetitive crystalline sequences. In silkworm, the process is accompanied by changes in physiological conditions such as pH and salt concentrations in the glands and presumably help to maintain solubility. The physical break generated during the spinning process of the soluble silk seems to be, in large part, responsible for the conversion of the soluble protein into the insoluble fibre in the natural processing sequence. [Ilzuka, E., 1985. Silk: an overview. J. Appl. Polymer. Sci. Jpn. 41: 163-171; Ilzuka, E., 1985. Silk thread: Mechanism of spinning and its mechanical properties. J. Appl. Polymer Sci Jpn. 41: 173-185; Magoshi J., Magoshi Y., Nakamura S., 1985. Crystallization, liquid crystal, and fiber formation of silk fibroin. J. Appl. Polymer Sci. 41: 187-204; Magoshi J., Magoshi Y., Nakamura S., 1994. Mechanism of fiber formation of silkworm. In: Silk Polymers: materials Science and Biotechnology, American Chemical Society Symposium Series, Kaplan D. L., Adams W. W., Farmer B., Viney C. (Eds.), 544 pp].

[0021]The large scale production of spider silk fibres would enable the production of a new generation of biomaterials with high rates of biodegradability that would have practical applications in diverse fields of the industrial sector. The inability to domesticate spiders in order to produce sufficient quantities of proteins for their adequate study and commercial use has induced the development of studies to make the production of silk proteins viable through large scale heterologous expression systems. Recent successes cloning cDNAs and synthetic genes and the expression of spider silk recombinant proteins in different systems have been vital to developing a better understanding of the structure, processing and purpose of these proteins, and of their important mechanical properties (Kaplan D. L., Adams W. W., Farmer B., Viney C. 1994. Silk Polymers: Materials Science and Biotechnology. American Chemical Society Symposium Series Volume 544; Kaplan D. L., Mello C. M., Arcidiacono S., Fossey S., Senecal K., Muller W., 1998. Silk. In: McGrath, K., Kaplan. D. L. (Eds.), Protein Based Materials. Birkhauser, Boston).

[0022]Studies are presently underway to increase available knowledge concerning these processes. However, the highly repetitive nature of these genes, the specific codons used by the spiders and the uncommon secondary structure adopted by the mRNA results in an inefficient translation of the proteins and limits the size of the fibre capable of being produced (Fahnestock S. R., Bedzyk L. A. 1997. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl. Microbiol. Biotechnol. 47:23-32; Scheibel T. 2004. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3:14). Due to the repetitive feature of the sequences, initial research performed on mRNAs collected from the "major ampullate" gland of N. clavipes were not successfully translated in vitro (Candelas G. C., Cintron J. J. 1981. A spider fibroin and its synthesis. J. Exp. Zool. 216:1-6; Candelas G. C., Lopez, F. 1983. Synthesis of fibroin in the cultured glands of Nephila clavipes. Comp. Biochem. Physiol. 74:637-641; Candelas G. C., Candelas T., Ortiz A., Rodriguez O. 1983. Translation pauses during a spider fibroin synthesis. Biochem. Biophys. Res. Commun. 116:1033-1038).

[0023]Different heterologous expression systems are being used in the attempt to produce spider fibres. Recent studies using constructs made from partial cDNAs of the dragline genes produced recombinant proteins in E. coli (Arcidiacono S., Mello, C., Kaplan D. L., Cheley, S., Bayley, H. 1998. Purification and characterization of recombinant spider silk expressed in Escherichia coli. Appl. Microbiol. Biotechnol. 49:31-38), in MAC-T (bovine) and BHK (hamster) cell cultures (Lazaris A., Arcidiacono S., Huang Y., Zhou J. F, Duguay F., Chretien N., Welsh E. A., Soares J. W., Karatzas C. N. 2002. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 295:472-476), and in cell lines of the Spodoptera frugiperda insect using the baculovirus expression system (Huemmerich D., Scheibel T., Vollrath F., Cohen S., Gat U., Ittah I. 2004. Novel assembly properties of recombinant spider dragline silk proteins. Curr. Biol. 14:472-476). Several studies used constructs containing cDNA of genes that encode proteins of the "minor ampullate" and flagelliform glands of spiders, such as in the case of patent documents U.S. Pat. No. 576,677 and U.S. Pat. No. 5,994,099. Document U.S. Pat. No. 5,728,810 describes the expression of Spidroin sequences 1 and 2 of N. clavipes in microorganisms. Documents U.S. Pat. No. 6,608,242, US20050010035 and WO0194393 report a method for producing synthetic proteins of spider silks in plants and constructs expressing synthetic proteins of the silk derived from Nephila clavipes and other species of spiders. The documents of patent CN1380418 describe the synthetic construct of spider web "Spidroin" protein for expression in cotton plants. Studies have shown the expression of spider silks proteins in animals, as in the case of patent documents WO9947661 and US2001042255, that describe methods for the recombinant production of biofilaments in the milk and/or urine of transgenic animals.

[0024]Synthetic genes based on the MaSp sequence of N. clavipes and Araneus gemmoides have also been used for the expression of heterologous proteins in E. coli (Fahnestock S. R., Bedzyk L. A. 1997. Production of synthetic spider dragline silk protein Pichia pastoris. Appl. Microbiol. Biotechnol. 47:23-32), Pichia Pastoris (Fahnestock S. R., Bedzyk L. A. 1997. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl. Microbiol. Biotechnol. 47:23-32) and plants (US20040210956; Scheller J., Gurhuns K. H., Grosse F., Conrad U. 2001. Production of spider silk proteins in tobacco and potato. Nat. Biotechnol. 19:573-577; Piruzian E. S., Bogush, V. G., Sidoruk K. V., Goldenkova I. V., Mysiychuk K. A., Debabov V. G. 2003. Construction of synthetic genes for analogues of spider silk spidroin 1 and their expression in Tabacco plants. Mol. Biol. 27:554-560; Scheller J., Henggeler D., Viviani A., Conrad U. 2004. Purification of spider-elastin from transgenic plants and application for human chondrocyte proliferation. Transg. Res. 13:51-57). Unfortunately, no MaSp gene has been completely cloned as yet and the data available refers to partial cDNA clones initiated by the 3' termini of the dragline silk genes of N. clavipes, A. diadematus and other species (Xu M., Lewis R. V., 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. USA 87:7120-7124; Hinman M. B., Lewis, R. V. 1992. Isolation of a clone coding a second dragline silk fibroin, Nephila clavipes dragline silk is a two protein fiber. J. Biol. Chem. 267:19320-19324; Beckwitt R., Arcidiacono S. 1994. Sequence conservation in the C-terminal region of spider silk proteins (Spidroin) from Nephila clavipes (Tetragnathidae) and Araneus bicentenarius (Araneidae). J. Biol. Chem. 269:6661-6663 Guerette P., Ginzinger D., Weber B., Gosline J. 1996. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 272:112-115 Hayashi C. Y., Lewis R. V. 1998. Evidence from flagelliform silk. cDNA for the structural basis of elasticity and modular nature of spider silks. J. Mol. Biol. 275:773-784). An explanation for such results may be the possible degradation of the mRNA during its extraction from the silk producing glands when constructing cDNA libraries since the longer mRNAs are more sensitive to enzymatic degradation (Stantcheva N. N. P., Mason S. J. M. 2004. Molecular studies of a novel dragline silk from nursery web spider, Euprosthenops sp (Psauridae). Comp. Biochem. Phisiol. 138:371-376). The production of spider silk proteins in heterologous systems and the manipulation of the primary structures of these proteins using modular structure engineering has been based on the available knowledge of natural spider silks (Cappello J., Crissman J., Dorman M. 1990. Genetic engineering of structural protein polymers. Biotech. Prog. 6:198-202; Scheibel T. 2004. Spider silks: recombinant synthesis, assembly, spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3:14; Kang W. J., Cho S. S., Huh H., Chung D. T 1997. Identification of dynamic behavior of sheet metals for an auto-body with tension split Hopkinson bar. Trans. KSME 21:2209-2219, Kang W. J., Cho S. S., Huh H., Chung D. T. 1999. Modified Johnson-Cook model for dynamic behaviour of sheet metals for auto-body Crash-worthiness. Int. J. Vehicle Design, 21:424-435).

[0025]The possibility of producing proteins from spider silks in heterologous systems on a large scale with the intended kinetics and functions shall allow their application in numerous medical products such as dressings and suture microfilaments for neurosurgery. These high-performance fibres could have diverse technical and industrial applications. The silks may be used in ropes and special fishing nets, parachutes, ballistic applications (bullet-proof vests, etc.) sporting products, textile industries, cosmetic industry and as a low-weight raw material for aerospace construction. An additional benefit would be the use of spider silk proteins in the manufacture of microbiocides and defensins against diseases and pests in the areas of agriculture, livestock and human health.

[0026]There is therefore a need for identifying new spider silk proteins, expressing them in different systems and developing other methods that afford solutions to the existing problems in the area described above.

[0027]The present invention describes new spider silk proteins extracted from the Nephilengys cruentata, Avicularia juruensis and Parawixia bistriata as well as the expression of these proteins in recombinant systems. The present invention further describes the expression of the silk proteins in plant, animal and fibre producing microorganism cells in order to produce new fibrous biomaterials with enhanced characteristics.

SUMMARY OF THE INVENTION

[0028]The discovery of new spider silk proteins, as well as their characterisation and expression in different heterologous systems shall be of great use in numerous fields, such as the medical and industrial sectors.

[0029]The proteins from spider silks may be obtained through synthetic polypeptides having amino acid sequences substantially similar to a consensus sequence of the silk protein or through polypeptides expressed from nucleic acid sequences coding a protein of a natural or engineered silk, or derivates of these. Depending on the application for which the silk protein is required, it may be useful to form fibres from a single spider web protein or from a combination of different spider web proteins.

[0030]One aspect of the invention provides isolated molecules of spider nucleic acid characterised by comprising: [0031]a) sequences substantially similar to any of the sequences selected from the group identified as SEQ ID N. 1-19; [0032]b) complements of the sequences described in SEQ ID N. 1-19; [0033]c) reverse complements of the sequences described in SEQ ID N. 1-19; [0034]d) reverse sequences of the sequences described in SEQ ID N. 1-19;

[0035]A second aspect of the invention provides a chimerical gene characterised by comprising: [0036]a) a promoter optionally linked to a leader sequence and operationally linked to; [0037]b) a coding sequence substantially similar to any of the sequences identified as SEQ ID N. 1-19.

[0038]Another aspect of the present invention provides an expression vector characterised by comprising: [0039]a) a promoter optionally linked to a leader sequence and operationally linked to; [0040]b) a coding sequence substantially similar to any of the sequences identified as SEQ ID N. 1-19 operationally linked to; [0041]c) a termination signal; [0042]d) an origin of replication; [0043]e) a selective marker; and [0044]f) a cloning site.

[0045]A fourth embodiment of the present invention relates to molecules isolated from the spider silk protein characterised by comprising sequences substantially similar to any of the sequences selected from the group identified as SEQ ID N. 20-38.

[0046]Yet another aspect of the invention provides host cells comprising at least one of the spider silk proteins encoded by nucleic acids. These host cells include, but are not limited to, bacterial cells, fungus cells, insect cells, mammal cells and plant cells. Host cells over expressing one or more spider silk proteins encoded by the nucleic acid of the present invention provide useful reagents for diverse purposes including, but not limited to, the production of silk fibres comprising at least one silk protein that may be incorporated within a material to modulate the structural properties of that material.

[0047]The present invention also describes a method for producing a genetically modified organism characterised by the fact of comprising the following steps: [0048]a) transforming a cell, tissue, organ or embryo with a chimerical gene in accordance with any of the claims 3 to 11 or an expression vector in accordance with any of the claims 12 to 23; [0049]b) selecting transformed cells, cell calluses, embryos or seeds; [0050]c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b); [0051]d) selecting the mature plants, mature embryos or microorganisms cells of stage (c) containing the chimerical gene or expression vector with the nucleotide sequences that encode the spider silk protein.

[0052]The present invention also describes a method for the production of recombinant spider silk proteins in prokaryote and eukaryote cells characterised by the fact of comprising the following steps: [0053]a) transforming a cell, tissue, organ or embryo with an expression vector in accordance with any of the claims 12 to 23; [0054]b) selecting transformed cells, callus cells, embryos or seeds; [0055]c) regenerating mature plants, mature embryos or microorganisms having transformed cells, callus cells, embryos or seeds selected in stage (b); [0056]d) selecting the mature plants, mature embryos or microorganisms cells of stage (c) containing the expression vector with the nucleotide sequences that encode the spider silk protein; [0057]e) extracting the recombinant spider silk protein produced in the organisms selected in stage (d).

[0058]The invention also includes recombinant proteins having microbicide, defensin and dermatological activity, as well as dermatological compositions for pharmaceutical use and microbicide compositions for agricultural use.

[0059]The invention further relates a dermatological composition characterised by the fact of comprising: [0060]a) A recombinant protein in accordance with claim 42; [0061]b) A pharmaceutically acceptable vehicle.

[0062]The invention also describes a microbicide composition characterised by the fact of comprising: [0063]a) A recombinant protein in accordance with claim 43; [0064]b) An agriculturally acceptable vehicle and, optionally, [0065]c) Additives.Lastly, the invention describes biopolymers produced from the recombinant proteins of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0066]FIG. 1--Alignment between SEQ ID N. 1 and gi|7106229. The sequences inserted in the rectangles with a dark background highlight the amino acids identical to both sequences. The numbers over the sequence identify the position in the alignment and do not correspond to the position in either sequence. The difference is due to the insertion of gaps to maintain alignment.

[0067]FIG. 2--Vectors containing signal peptide and beta-conglycinin promoter used for bombardment in transformation and co-transformation systems for soybean and cotton plants.

[0068]FIG. 3--Vector pAG1 used for bombardment in transformation and co-transformation systems for soybean and cotton plants.

[0069]FIG. 4--Vector pET19b: SEQs. 1-19 used in the transformation of E. coli for the expression of silk proteins.

[0070]FIG. 5--Vector pCMV-Script containing sequences 1 to 19 for expression in mammal cells. MCS: multiple cloning site; cloning site containing 15 different enzymes with unique sites.

[0071]FIG. 6--Vector pBC 1 containing sequences 1 to 19 for expression of web proteins in the milk of transgenic animals.

[0072]FIG. 7--Model of the protein structure obtained for SEQ ID N. 1 (Product of the Gene: Nephilengys cruentata--NCFlag). A. represents a horizontal view of the structure and B. represents a view of the upper end from one of the extremities.

[0073]FIG. 8--Silk produced in vitro

DETAILED DESCRIPTION OF THE INVENTION

[0074]The following definitions are provided for better understanding the present invention:

[0075]The term "isolated nucleic acid molecule" is used in reference to the nucleic acids of the present invention. This term, when applied to DNA, refers to the DNA molecule that is separate from the directly contiguous sequences (in directions 5' and 3') that occur naturally in the genome of the organism from which they were derived. For example, an "isolated nucleic acid molecule" may be inserted in a vector, such as a plasmid or a virus vector, or incorporated within the genomic DNA of a prokaryote or eukaryote. An "isolated nucleic acid molecule" may also comprise a cDNA molecule. An isolated nucleic acid molecule inserted in a vector is also sometimes referred to herein as a recombinant nucleic acid molecule. The term "isolated nucleic acid molecule" may also be applied to RNA molecules transcribed from an isolated DNA molecule as described above. Alternatively, the term may also refer to a RNA molecule that has been sufficiently separated from the RNA molecules to which it was formerly associated in its natural state (i.e. in cells or tissues).

[0076]The definition of the terms "complement", "reverse complement" and "reverse sequence" as used herein may be illustrated by the following example: for the sequence 5'AGTGAAGT3', the complement is 3'TCACTTCA5', the reverse complement is 3'ACTTCACT5' and the reverse sequence is 5'TGAAGTGA3'.

[0077]"Coding sequence" refers to the DNA sequence that encodes a specific protein and excludes the non-coding sequence. An "interrupted coding sequence" means a sequence that acts as a separator (e.g. one or more introns linked by junctions). An "intron" is the sequence of a nucleotide that is transcribed and is present in the pre-mRNA but is subsequently removed by cleavage and re-linking of the mRNA within the cell which generates a mature mRNA that may be translated into a protein. Examples of introns include, but are not limited to, intron pdk2, castor oil catalase intron, Delta 12 cotton desaturase intron, Delta 12 Arabidopsis desaturase, maize ubiquitin intron, SV40 intron, malate synthase gene introns.

[0078]A "gene construct" is a gene comprising a promoter and an coding region of different origins. In the case of the present invention, the gene construct comprises the polynucleotides of the present invention linked either in an isolated or associated form to expression regulating regions, such as promoters and termination signals.

[0079]The methods for obtaining gene constructs comprising promoters linked to nucleic acids is known in the state-of-the-art and may be found in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).

[0080]The term "vector" refers to a replicon, such as a plasmid, cosmid, BAC, phage or virus, in which other genetic sequences or elements (whether DNA or RNA) may be linked to be replicated together with the vector. Preferentially the virus derived vector is selected from the bacteriophage, vaccinia, retrovirus or the bovine papillomavirus. An "expression vector" is a specialized vector that contains a gene with the regulatory regions necessary for the expression of a host cell. Such vectors may be obtained commercially, including Clontech Laboratories, Inc (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Mass.) and Promega (Madison, Wis.). Some examples of the vectors used in the present invention are, but are not limited to, pMAC/PS, pCMV-Gal and pGFP/NEO. The term "operationally linked" means that the regulatory sequences necessary for the expression of the coding sequence are placed in the DNA molecule in appropriate positions in relation to the coding sequence for the purpose of its expression. This same definition is sometimes applied to the arrangements of the coding sequences and transcription controlling elements (e.g. promoters, enhancers and termination elements) in the expression vector. An exogenous coding region is typically flanked by operationally linked regulatory regions that regulate the expression of the exogenous coding region in a transformed cell (which may be microorganism, plant or animal). A typical regulatory region operationally linked to an exogenous coding region includes a promoter, as such, a fragment of nucleic acid that may cause transcription of the exogenous coding regions, positioned in the 5' region of the exogenous coding region. The present invention is not limited to the use of any particular promoter and a broad variety of promoters are known in the state-of-the-art. These promoters may be, but are not limited to, inducible, constitutive and tissue-specific. Preferentially, the promoter of the present invention is selected from the group of promoters for the cotton fibre genes and may be, but are not limited to, E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexin, FbL2A and actin 2.

[0081]In one of the aspects of the invention, the promoter is a constitutive promoter. In another aspect of the invention, promoter activity is stimulated by external factors such as, but without being limited to, hormones, chemical compositions, mechanical impulses, and biotic or abiotic stress conditions. Promoter activity may also be regulated in a temporal and spatial manner (such as, for example, tissue-specific promoters and promoters regulated during development).

[0082]The promoter may contain "enhancer" elements. An "enhance" is a DNA sequence capable of stimulating promoter activity. It can be an element innate to the promoter or a heterologous element inserted to increase the promoter's tissue-specificity and/or intensity. "Constitutive promoters" refer to those promoters that direct genic expression to all tissues in a constant manner. "Tissue-specific promoters" or "development-specific promoters" are those that direct genic expression almost entirely to specific tissues, such as leaves, roots, stems, flowers, fruits or seeds, or only during specific stages of development of a tissue, such as at the beginning or end of embryogenesis.

[0083]In one of the aspects of the invention, the promoter is a promoter expressed in plants. As used herein, the term "promoter expressed in plants" means a DNA sequence capable of initiating and/or controlling transcription in a plant cell. This includes any promoter of plant origin; Any promoter of non-plant origin capable of directing expression in a plant cell, for example, promoters of viral or bacterial origin such as 19S and 35S of CaMV (such as mentioned in patent application US20030175783, Hapster et al, 1988 Mol. Gen. Genet. 212, 182-190), bacteriophage promoter T7 and gene promoters present in T-DNA of Agrobaterium; tissue-specific or organ-specific promoters including, but not limited to, seed-specific promoters (WO8903887), primary organ specific promoters (such as those mentioned in patent application US20030175783, An et al., 1996 The Plant Cell 8, 15-30), stem specific promoters (such as those mentioned in patent application US20030175783, Keller et al., 1988 EMBO J. 7: 3625-3633), leaf specific promoters (such as those mentioned in patent application US20030175783, Hudspeth et al., 1989 Plant Mol Biol 12:579-589), mesophyll specific promoters, root specific promoters (such as those mentioned in patent application US20030175783, Keller et al., 1989 Genes Devel. 3:1639-1646), tubercle specific promoters (such as those mentioned in patent application US20030175783, Keil et al., 1989 EMBO J. 8: 1323:1330), vascular tissue specific promoters (such as those mentioned in patent application US20030175783, Peleman et al., 1989 Gene 84: 359-369), stamen specific promoters (WO8910396, WO9213956), dehiscence specific promoters (WO9713865); and the similar. Apart from the specific promoters, other endogenous plant promoters exist. These include, but are not limited to, the promoter of the small subunit of ribulose 1.6 biphosphate (RUBP), beta-conglycinin promoter, beta-phaseolin promoter, γ-kafirin promoter, beta-amylase, maize alcohol dehydrogenase, cruciferine (seed-specific), rubisco, RD2 tobacco gene, SAG Arabidopsis gene (leaves), polygalacturonase (fruit), patatin (tubercles), barley hordein, napin, rice actin, maize ubiquitin promoter, ADH promoter, GPAL2 promoter, GPAL3 promoter and thermal shock protein promoters, amongst others. The expression of silk in fibre-producing plants such as cotton, sisal, rush, palm, jute, cane, bamboo, agave and hemp, amongst others, may use the beta-tubulin, A1, A2, A4 and MYB (MYB-like transcription factor) cellulose synthetase gene promoters, amongst others (U.S. Pat. No. 6,608,242). The invention preferentially includes cotton fibre gene promoters that include, but are not limited to, the E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexin, FbL2A and actin 2 gene promoters.

[0084]In one of the aspects of the invention, the promoter is a promoter expressed in animals. As used herein, the term "promoter expressed in animals" means a DNA sequence capable of initiating and/or controlling transcription in an animal cell. This includes any promoter of animal origin and any promoter of non-animal origin capable of directing expression in an animal cell, for example, the milk beta-casein promoter (Invitrogen). The preferred promoters used in the invention direct the transcription of a protein in milk producing cells, such as, but not limited to, the promoters of the following genes: whey acid protein (WAP), alpha casein S1, alpha casein S2, beta casein, kappa casein, beta lactoglobulin, alpha lactalbumin, amongst others. Further preferred promoters of the invention direct the transcription of a protein in urine producing cells (e.g. a uroepithelial cell or a cell of the same nature); such promoters include, but are not limited to, the uroplakin gene promoter. Yet other preferred promoters of the invention direct transcription of a protein in an embryo cell.

[0085]Apart from the promoters described above, one of the embodiments of the present invention refers to the promoters expressed in bacteria, fungus and insects. As used herein, the term "promoter expressed in bacteria" means a DNA sequence capable of initiating and/or controlling transcription in a bacterial cell. As used herein, the term "promoter expressed in fungus" means a DNA sequence capable of initiating and/or controlling transcription in a fungal cell. As used herein, the term "promoter expressed in insects" means a DNA sequence capable of initiating and/or controlling transcription in an insect cell.

[0086]A "leader sequence" or "signal sequence" in the present invention means a sequence of nucleic acid that, when operationally linked to a molecule of nucleic acid, allows the secretion of the product of the nucleic acid molecule. The leader sequence is preferentially located in the 5' region of the nucleic acid molecule. Preferentially, the leader sequence is obtained from the same gene than the promoter used to direct the transcription of the nucleic acid molecule, or is obtained from the same gene from which the nucleic acid molecule was derived. Preferentially, the present invention uses the signal sequence of α-coixin.

[0087]The transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to, the SV40 termination signal, the HSV TK adenylation signal, the termination signal of the nopaline synthetase gene (NOS) of Agrobacterium tumefaciens, the octopine synthetase gene termination signal, the termination signal of the 19S and 35S genes of CaMV, the maize alcohol dehydrogenase gene termination signal, the manopine synthetase gene termination signal, the beta-phaseolin gene termination signal, the ssRUBISCO gene termination signal, the sucrose synthetase gene termination signal, the termination signal of the virus that attacks the Trifolium subterranean (SCSV), the termination signal of the trpC gene of Aspergillus nidulans, and other similars.

[0088]As described above, the term "expression vectors" may comprise an inducible promoter operationally linked to a nucleic acid sequence encoding a spider web protein. "Inducible" promoters may direct the expression the expression of a polynucleotide with which they are operationally linked, in a tissue or specific stage of development or in response to environmental conditions. In one of the aspects of the invention, expression vectors comprise a strongly regulated inducible vector operationally linked to a nucleic acid molecule coding a spider web protein. This expression vector may further comprise a selection marker gene (e.g. a gene coding a protein that confers resistance to antibiotics) operationally linked to a constitutive promoter or a strongly regulated inducible promoter. Depending on the purpose, it may benefit the expression of a nucleic acid sequence coding a spider web protein through a pathogen inducible promoter. These promoters include those promoters derived from proteins related to pathogenesis (PR proteins) which are induced through infections by a pathogen, such as, for example, PR proteins, SAR proteins, beta glucanase 1.3, chitinase, etc. In an aspect of the present invention, it may be advantageous to use promoters that are expressed locally or close to the infection site of the pathogen. Furthermore, since many pathogens enter plants through wounds that are often the result of insect damage, a wounding inducible promoter may be included amongst the expression vectors of the invention. Wound inducible promoters include, but are not limited to, the potato proteinase inhibitor gene (pinII) promoter, win 1 and win 2 gene promoters, systemine gene promoter, MPI gene promoter.

[0089]The transcriptional activity of inducible promoters may also be regulated by various environmental factors including, but not limited to, temperature, anaerobic stress and light. Examples of inducible promoters include the Adh1 promoter (induced by hypoxia or cold stress), Hsp70 promoter (induced by heat stress) and PPDK promoter (induced by light).

[0090]As used herein, the term "variant" or "substantially similar" comprises sequences of amino acids or nucleotides different from the specifically identified sequences, in which one or more nucleotides or amino acid residues are deleted, substituted or added. The variants may be allelic variants occurring naturally or variants of non-natural origin. The variant or substantially similar sequences refer to fragments of nucleic acids or peptides that may be characterized by the percentage of the identity of their nucleotide or amino acid sequences with the nucleotide (SEQ ID Ns 1-19) or amino acid (SEQ ID Ns 20-38) sequences described herein, as determined by common algorithms used in the state-of-the-art. The preferred fragments of nucleic acids or peptides are those having a sequence of nucleotides or amino acids with at least around 40 or 45% of sequence identity, preferentially around 50% or 55% of sequence identity, more preferentially around 60% or 65% of sequence identity, more preferentially around 70% or 75% of sequence identity, more preferentially around 80% or 85% of sequence identity, yet more preferentially around 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity when compared to the sequence of reference. The percentage of identity is determined by the alignment of the two sequences to be compared, ascertaining the number of identical residues in the aligned portion, dividing this number by the total number of residues in the sequence being assessed and multiplying the result by 100. This alignment may be done using software tools in the public domain, one of which is BLASTN, available at the National Center for Biotechnology Information/NCBI (www.ncbi.nlm.nih.gov) homepage. The sequence alignment and identity percentage calculation of the present invention have been performed and the sequences deposited in the Gene Bank, through integration of the web browser.

[0091]The term "specifically hybridizing" refers to the association between two molecules of single chain nucleic acid possessing sufficiently complementary sequences to allow such hybridization under pre-determined conditions generally described in the state-of-the-art (sometimes referred to as "substantially complementary" in the present invention). More particularly, the term refers to the hybridisation of an oligonucleotide with a substantially complementary sequence containing a molecule of single chain DNA or RNA of the present invention. The appropriate conditions necessary to enable the specific hybridisation between single chain nucleic acid molecules of variable complementariness are well described in the state-of-the-art. The following formula is commonly used for calculating the required conditions of stringency for hybridisation between nucleic acid molecules to occur (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)-0.63 (% formamide)-600/pb in duplex (probe)

[0092]As can be seen by the above formula, using [Na+]=[0.368] and 50% formamide, with a GC content of 42% and an average probe size of 200 bases, the Tm shall be 57° C.

[0093]The term "oligonucleotide" refers herein to `primers` and `probes` of the present invention, and is defined as a nucleic acid molecule comprising one or more ribo- or deoxyribonucleotides, preferentially more than three. The exact size of the oligonucleotides shall depend on various factors as well as the particular application and use of the oligonucleotides. The preferred oligonucleotides comprise 15-50 consecutive bases.

[0094]The term "probe" when used in the present invention refers to an oligonucleotide, polynucleotide or nucleic acid, being RNA or DNA, when occurring naturally such as in the digestion of a purified or synthetically produced restriction enzyme and that is capable of either annealing or specifically hybridizing with a nucleic acid containing complementary sequences of the probe. A probe may further be single or double chain. The exact length of the probe will depend on numerous factors, including temperature, the probe's origin and use of the method. For example, depending on the complexity of the target sequence, the oligonucleotide probe may typically contain 15-25 or more nucleotides, although it might actually contain less nucleotides. The probes herein are selected so as to be complementary in order to discern the chains of a particular nucleic acid sequence. This means that the probe may be sufficiently complementary to be capable of "specifically hybridising" or annealing with its respective target-chains under a series of pre-determined conditions. Consequently, the probe sequence does not necessarily exactly reflect the target complementary sequence. For example, a non-complementary nucleotide fragment may be linked to the 5' or 3' extremity of the probe, with the remaining sequence of the probe being complementary to the target chain. Alternatively, non-complementary bases or long sequences may be interspersed within the probe provided the latter is sufficiently complementary with the target nucleic acid sequence to anneal specifically with it.

[0095]The term "primer" as used herein refers to an oligonucleotide, being RNA or DNA, single or double chain, derived from a biological system and generated by the digestion of a purified or synthetically produced restriction enzyme that, when placed in an appropriate environment, is capable of functionally acting as the initiator of a template-dependent nucleic acid synthase. When in the presence of an appropriate nucleic acid template, suitable nucleoside triphosphates precursors for nucleic acids, a polymerase enzyme, adequate cofactors and conditions such as temperature and suitable pH values, the primer may extend at its 3' terminal by the addition of nucleotides through the action of polymerase or some similar activity to produce a first extension of the product. The `primer` may vary in length depending on particular conditions and application requirements. For example, for diagnostic applications, the oligonucleotide `primer` typically contains 15-25 or more nucleotides in length. The `primer` must sufficiently complementary with the intended template to initiate the extension synthase of the intended product. This does not mean that the `primer` must represent the intended template exactly. For example, a non-complementary nucleotide sequence may be linked to the 5' extremity of a complementary `primer`. Alternatively, non-complementary bases or long sequences may be interspersed within the oligonucleotide sequence of the `primer` provided the latter is sufficiently complementary with the intended template sequence to functionally provide a template-primer for the extension synthase of the product. The description of the primers used in the present invention can be found in the section of the examples where these primers are required (e.g. PCR reactions).

[0096]The term "isolated protein" or "isolated and purified protein" is occasionally used in the present invention. This term refers to a protein produced by the expression of an isolated nucleic acid molecule of the present invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins to which it may be naturally associated, such as when existing in its "substantially pure" form. The term "isolated" does not exclude synthetic or artificial mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity of that protein. These may be present, for example, following incomplete purification or the addition of stabilisers, and also combined within immunogenic preparations or pharmaceutically acceptable preparations. Pharmaceutically acceptable preparations may be used in the production of fibres and synthetic polymers, for example, and may be incorporated to numerous medical implements, including, but not being limited to, sutures, wound dressings and implants.

[0097]The term "pharmaceutically acceptable vehicle" refers to solutions in which a spider web protein or a nucleic acid coding sequence of a spider web protein may be maintained without any alteration to the functional properties of the spider web molecule described herein for pharmaceutical purposes. For administration to mammals, for example, a spider web protein or a nucleic acid coding sequence of a spider web protein may be suspended in any pharmaceutically acceptable vehicle, such as, for example, the "HEPES" saline buffer with an approximate pH of 7.8. Other useful pharmaceutically acceptable vehicles include, but are not limited to, glycerol, water, saline solution, ethane and other pharmaceutically acceptable saline solutions such as phosphates and organic acid salts. Examples of these and other pharmaceutically acceptable vehicles are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0098]The term "agriculturally acceptable vehicle" refers to solutions in which a spider web protein or a nucleic acid coding sequence of a spider web protein may be maintained without any alteration to the functional properties of the spider web molecule described herein for agricultural purposes. The vehicles used for the present invention may be liquids or solids. The liquid vehicles that may be used to form compositions using the recombinant protein of the present invention may be, but are not limited to, water or organic solvents, such as polyalcohols, esters, methylene chloride, alcohol or plant oils. Other components that may be incorporated to the formulation include humectants, preservatives, thickeners, antimicrobial agents, antioxidants, emulsifiers, film forming polymers and mixtures of these. The humectants may include polyalcohols, sugars (such as molasses) glycols and hygroscopic salts. Vitreous membranes and film forming polymers include rosin gum, latex, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyethylene, polyvinyl acetate and mixtures of these. Further optional additives include methyl, methacrylate and mixtures of these.

[0099]The term "mature protein" or "mature polypeptide" mean a polypeptide possessing an amino acid sequence after any processing event that normally occurs to the polypeptide during its generation, such as the proteolytic processing of a polyprotein precursor. When designating the sequence or limits of a mature protein, the first amino acid of the mature protein's sequence is designated as amino acid residue 1. In the case of the present invention, any amino acid residue associated to a mature protein not naturally encountered in association to the protein preceding amino acid 1 are designated amino acid -1, -2, -3, etc. In the case of recombinant expression systems, the methionine initiator codon is frequently used when intending efficient translation. As used herein, this methionine residue in the resulting polypeptide must be in the -1 position of the sequence of the mature protein.

[0100]The term "peptidic analogue" means a natural or mutant analogue of a protein, comprising a series of linear or discontinuous fragments of that protein and which may have one or more amino acids replaced with other amino acid(s). It may also have its biological activity altered, enhanced or diminished compared to the parent or non-mutant protein.

[0101]The term "biological activity" refers to a function or group of functions performed by a molecule in a biological context (i.e. in an organism or in vitro substitute or some similar model). In the case of spider web proteins, biological activity is characterised by their physical properties (e.g. tensile strength and elasticity) as described herein.

[0102]The term "substantially pure" refers to preparations comprising at least 50-60% of the weight of the component of interest (e.g. nucleic acid, oligonucleotide, polypeptide, protein, etc.). More preferentially, the preparation comprises at least 75% of the weight, and yet more preferentially, 90-99% of the weight of the component of interest. Purity shall be measured by methods appropriate to the component of interest (e.g. chromatography methods, HPLC analysis, mass spectrometry and the similar).

[0103]The term "vector" refers to a replicon, such as a plasmid, cosmid, bacmid, phagus or virus, in which other genetic sequences or elements (whether DNA or RNA) may be linked to be replicated together with the vector. Preferentially the virus derived vector is selected from the bacteriophages, vaccinias, retrovirus or bovine papillomavirus. An "expression vector" is a specialized vector that contains a gene with the regulatory regions necessary for the expression of a host cell. Such vectors may be obtained commercially, including Clontech Laboratories, Inc (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), Invitrogen (Carlsbad, Calif.), New England Biolabs (Beverly, Mass.) and Promega (Madison, Wis.). Some examples of the vectors used in the present invention are, but are not limited to, pMAC/PS, pCMV-Gal and pGFP/NEO. The term "operationally linked" means that the regulatory sequences necessary for the expression of the coding sequence are placed in the DNA molecule in appropriate positions in relation to the coding sequence for the purpose of its expression. This same definition is sometimes applied to the arrangements of the coding sequences and transcription controlling elements (e.g. promoters, enhancers and termination elements) in the expression vector. An exogenous coding region is typically flanked by operationally linked regulatory regions that regulate the expression of the exogenous coding region in a transformed cell (which may be microorganism, plant or animal). A typical regulatory region operationally linked to an exogenous coding region includes a promoter, as such, a fragment of nucleic acid that may cause transcription of the exogenous coding regions, positioned in the 5' region of the exogenous coding region. The present invention is not limited to the use of any particular promoter and a broad variety of promoters are known in the state-of-the-art. These promoters may be, but are not limited to, inducible, constitutive and tissue-specific.

[0104]In one aspect of the invention, the promoter is a constitutive promoter. In another aspect of the invention, promoter activity is stimulated by external factors such as, but without being limited to, hormones, chemical compositions, mechanical impulses, and biotic or abiotic stress conditions. Promoter activity may also be regulated in a temporal and spatial manner (such as, for example, tissue-specific promoters and promoters regulated during development).

[0105]The promoter may contain "enhancer" elements. An "enhancer" is a DNA sequence capable of stimulating promoter activity. It can be an element innate to the promoter or a heterologous element inserted to increase the promoter's tissue-specificity and/or intensity. "Constitutive promoters" refer to those promoters that direct gene expression to all tissues in a constant manner. "Tissue-specific promoters" or "development-specific promoters" are those that direct gene expression almost entirely to specific tissues, such as leaves, roots, stems, flowers, fruits or seeds, or only during specific stages of development of a tissue, such as at the beginning or end of embryogenesis.

[0106]In one of the aspects of the invention, the promoter is a promoter expressed in plants. As used herein, the term "promoter expressed in plants" means a DNA sequence capable of initiating and/or controlling transcription in a plant cell. This includes any promoter of plant origin and any promoter of non-plant origin capable of direction expression in a plant cell, for example, promoters of viral or bacterial origin such as 19S and 35S of CaMV (such as mentioned in patent application US20030175783, Hapster et al, 1988 Mol. Gen. Genet. 212, 182-190), bacteriophage promoter T7 and gene promoters present in T-DNA of Agrobaterium; tissue-specific or organ-specific promoters including, but not limited to, seed-specific promoters (WO8903887), primary organ specific promoters (such as those mentioned in patent application US20030175783, An et al., 1996 The Plant Cell 8, 15-30), stem specific promoters (such as those mentioned in patent application US20030175783, Keller et al., 1988 EMBO J. 7: 3625-3633), leaf specific promoters (such as those mentioned in patent application US20030175783, Hudspeth et al., 1989 Plant Mol Biol 12:579-589), mesophyll specific promoters, root specific promoters (such as those mentioned in patent application US20030175783, Keller et al., 1989 Genes Devel. 3:1639-1646), tubercle specific promoters (such as those mentioned in patent application US20030175783, Keil et al., 1989 EMBO J. 8: 1323:1330), vascular tissue specific promoters (such as those mentioned in patent application US20030175783, Peleman et al., 1989 Gene 84: 359-369), stamen specific promoters (WO8910396, WO9213956), dehiscence specific promoters (WO9713865); and the similar. Apart from the specific promoters, other endogenous plant promoters exist. These include, but are not limited to, the promoter of the small subunit of ribulose 1.6 biphosphate (RUBP), beta-conglycinin promoter, beta-phaseolin promoter, γ-kafirin promoter, beta-amylase, maize alcohol dehydrogenase, cruciferine (seed-specific), rubisco, RD2 tobacco gene, SAG Arabidopsis gene (leaves), polygalacturonase (fruit), patatin (tubercules), barley hordein, napin, rice actin, maize ubiquitin promoter, ADH promoter, GPAL2 promoter, GPAL3 promoter and thermal shock protein promoters, amongst others. The expression of silk in fibre-producing plants such as cotton, sisal, rush, palm, jute, cane, bamboo, agave and hemp, amongst others, may use the beta-tubulin, A1, A2, A4 and MYB (MYB--like transcription factor) cellulose synthetase gen promoters, amongst others (U.S. Pat. No. 6,608,242). The invention preferentially includes cotton fibre gene promoters that include, but are not limited to, the E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexin, FbL2A and actin 2 gene promoters.

[0107]In one of the aspects of the invention, the promoter is a promoter expressed in animals. As used herein, the term "promoter expressed in animals" means a DNA sequence capable of initiating and/or controlling transcription in an animal cell. This includes any promoter of animal origin and any promoter of non-animal origin capable of directing expression in an animal cell, for example, the milk beta-casein promoter (Invitrogen). The preferred promoters used in the invention direct the transcription of a protein in milk producing cells, such as, but not limited to, the promoters of the following genes: whey acid protein (WAP), alpha casein S1, alpha casein S2, beta casein, kappa casein, beta lactoglobulin, alpha lactalbumin, amongst others. Further preferred promoters of the invention direct the transcription of a protein in urine producing cells (e.g. a uroepithelial cell or a cell of the same nature); such promoters include, but are not limited to, the uroplakin gene promoter. Yet other preferred promoters of the invention direct transcription of a protein in an embryo cell.

[0108]Apart from the promoters described above, one of the embodiments of the present invention refers to the promoters expressed in bacteria, fungus and insects. As used herein, the term "promoter expressed in bacteria" means a DNA sequence capable of initiating and/or controlling transcription in a bacterial cell. As used herein, the term "promoter expressed in fungus" means a DNA sequence capable of initiating and/or controlling transcription in a fungal cell. As used herein, the term "promoter expressed in insects" means a DNA sequence capable of initiating and/or controlling transcription in an insect cell. A "leader sequence" or "signal sequence" in the present invention means a sequence of nucleic acid that, when operationally linked to a molecule of nucleic acid, allows the secretion of the product of the nucleic acid molecule. The leader sequence is preferentially located in the 5' region of the nucleic acid molecule. Preferentially, the leader sequence is obtained from the same gene than the promoter used to direct the transcription of the nucleic acid molecule, or is obtained from the same gene from which the nucleic acid molecule was derived. Preferentially, the present invention uses the signal sequence of α-coixin.

[0109]The transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to, the SV40 termination signal, the HSV TK adenylation signal, the termination signal of the nopalin synthetase gene (NOS) of Agrobacterium tumefaciens, the octopin synthetase gene termination signal, the termination signal of the 19S and 35S genes of CaMV, the maize dehydrogenase alcohol gene termination signal, the manopine synthetase gene termination signal, the beta-phaseolin gene termination signal, the ssRUBISCO gene termination signal, the sucrose synthetase gene termination signal, the termination signal of the virus that attacks the Trifolium subterranean (SCSV), the termination signal of the trpC gene of Aspergillus nidulans, and other similar.

[0110]As described above, the term "expression vectors" may comprise an inducible promoter operationally linked to a nucleic acid sequence coding a spider web protein. "Inducible" promoters may direct the expression the expression of a polynucleotide with which they are operationally linked, in a tissue or specific stage of development or in response to environmental conditions. In one of the aspects of the invention, expression vectors comprise a strongly regulated inducible vector operationally linked to a nucleic acid molecule coding a spider web protein. This expression vector may further comprise a selection marker gene (e.g. a gene coding a protein that confers resistance to antibiotics) operationally linked to a constitutive promoter or a strongly regulated inducible promoter. Depending on the purpose, it may benefit the expression of a nucleic acid sequence coding a spider web protein through a pathogen inducible promoter. These promoters include those promoters derived from proteins related to pathogenesis (PR proteins) which are induced through infections by a pathogen, such as, for example, PR proteins, SAR proteins, beta glucanase 1.3, chitinase, etc. In an aspect of the present invention, it may be advantageous to use promoters that are expressed locally or close to the infection site of the pathogen. Furthermore, since many pathogens enter plants through wounds that are often the result of insect damage, a wound inducible promoter may be included amongst the expression vectors of the invention. Wound inducible promoters include, but are not limited to, the potato proteinase inhibitor gene (pinII) promoter, win 1 and win 2 gene promoters, systemine gene promoter, MPI gene promoter.

[0111]The transcriptional activity of inducible promoters may also be regulated by various environmental factors including, but not limited to, temperature, anaerobic stress and light. Examples of inducible promoters include the Adh1 promoter (induced by hypoxia or cold stress), Hsp70 promoter (induced by heat stress) and PPDK promoter (induced by light).

[0112]The construction of vectors comprising promoters linked to nucleic acids is known in the state-of-the-art and may be found in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).

[0113]Expression vectors comprising spider web protein coding nucleic acid sequences are included in the scope of the present invention. The following are also included in the present invention: plant cells, recombinant seeds, recombinant plant embryos, recombinant plants, animal cells, recombinant animal embryos, recombinant animals, insect cells, recombinant insects and recombinant microorganisms comprising expression vectors coding the spider web proteins described herein.

[0114]A "transfected cell" or a "transformed cell" means a cell in which a molecule of the nucleic acid coding a polypeptide of the present invention has been inserted using recombinant DNA techniques. The cells may be from a host organism that include, but are not limited to, bacterial cells, fungus cells, insect cells, plant cells and animal cells. Preferentially, the cell is a eukaryote cell of a multicellular organism (e.g. plants and animals).

[0115]The expression vectors may be inserted into the genome of the intended host plant by a variety of conventional techniques. For example, they may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and the microinjection of plant cell protoplasts or, otherwise, the expression vector may be directly introduced to the plant tissue using ballistic methods, such as the bombardment of DNA-coated particles.

[0116]Micro-injection techniques are known in the state-of-the-art and well described in scientific and patent literature. The introduction of expression vectors using polyethylene glycol precipitations is described by Paszkowski et al. Embo J. 3:2717-2722, 1984 (as mentioned in patent application US20020152501). The techniques of electroporation are described by From et al. Proc. Natl. Acad. Sci. USA 82:5824, 1985 (as mentioned in patent application US20020152501). Ballistic transformation techniques are described by Klein et al. Nature 327:70-73, 1987 (as mentioned in patent application US20020152501).

[0117]Alternatively, the expression vectors containing the recombinant nucleic acid molecule may be combined to appropriate T-DNA flanker regions and introduced in the conventional host vector Agrobacterium tumefaciens. The virulence function of the Agrobacterium tumefaciens host will direct the insertion of the recombinant nucleic acid molecules and the adjacent marker inside the DNA of the plant cell when this cell is infected by the bacteria. Transformation techniques mediated by Agrobacterium tumefaciens, including disarmament and the use of binary vectors, are well described in scientific literature (as mentioned in patent application US 20020152501, Horsch et al. Science 233:496-498, 1984; and Fraley et al. Proc. Natl. Acad. Sci. USA 80:4803, 1983).

[0118]The cells of transformed plants that are derived through any of the transformation techniques described above may be cultivated to regenerate an entire plant possessing a transformed genotype and thus the intended phenotype for the production of spider web proteins. These regeneration techniques rely on the manipulation of certain phytohormones in tissue culture growth medium and typically containing a biocide and/or herbicide marker that must be introduced together with the intended sequence of nucleotides. Preferentially, the present invention uses selective markers chosen from the antibiotic and herbicide resistant genes such as kanamycin, neomycin, ampicillin, chloranphenicol, streptomycin, hygromycin, geneticin, phosphinotrycin, glyphosate, gluphosinate ammonium, amongst others. The present invention also uses reporter genes to assess the transformation potential of the genes, such as AHAS, BAR and GUS. Regeneration of plants from protoplast cultures is described by Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985 (as mentioned in patent application US20020152501). Regeneration may also be obtained from the callus of plants, explants, organs, or parts of these. Such regeneration techniques are described overall by Klee et al., Ann. Ver. of Plant Phys. 38:467-486, 1987 1985 (as mentioned in patent application US20020152501) and may also be found in Clark, 1997 (Clark, M. S. eds., 1997. Plant Molecular Biology A laboratory Manual. Springer-Verlag, Berlin, Heidelberg); Maliga et al., 1995 (Maliga, P.; D. F. Flessing, A. R. Cashmore, W. Cruissem, J. E. Varner, eds., 1995. Methods in Plant Molecular Biology, A Laboratory Course Manual. Cold Spring Harbor Laboratory Press) and Martinez-Zapater & Salinas, 1998 (Martinez-Zapater, J. M. & J. Salinas, eds., 1998. Methods in Molecular Biology, v. 82: Arabidopsis Protocols. Humana Press, Totowa, N.J.).

[0119]The methodology for the maintenance and growth of microorganism cultures (bacteria, fungus, yeasts) is known to those versed in the matter. The description of such techniques may also be found in related technical manuals such as those by Gerhardt et al., 1994 (Gerhardt, P.; R. G. E. Murray; R. N. Costilow; E. W. Nester; W. A. Wood; N. R. Krieg & G. B. Phillips eds. Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, D.C) or Brock, 1989 (Brock, T. D. 1989. Biotechnology: A Textbook of Industrial Microbiology. Second edition, Sinauer Associates, Inc., Sunderland, Mass.).

[0120]An "embryonary cell" means a cell capable of being progenitor to all the cells of the somatic and germinative line of an organism. Examples of embryonary cells include trunk cells (ES cells) and fertilised ovocytes. Preferentially, the embryo cells of the invention are embryo cells of mammals.

[0121]"Germinative cell line" means a progenitor eukaryote cell, or the similar of a progenitor cell, that is the product of a meiotic cell division.

[0122]A "clone" or a "clonal cell population" is a population of cells derived from a simple cell or common ancestry through mitosis.

[0123]A "cell line" is a clone of a primary cell or cell population capable of stable in vitro growth for many generations.

[0124]"Plants" refers to photosynthetic organisms, both eukaryote or prokaryote, whereby the term "developed plants" refers specifically to eukaryote plants. The nucleic acid of the invention may be used to confer desirable traits to basically any plant. Thus, the invention is useful to various species of plants, including species of the genera Anacardium, Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

[0125]"Animals" refers to eukaryote organisms that may either belong to the phyla of vertebrates or invertebrates, whereby the term "superior animals" refers to the phyla of vertebrate animals. The nucleic acid of the invention may be used to confer desirable traits to basically any animal. Thus, the invention is useful to various species of vertebrate animals, including species of mammals that include, but are not limited to, primates, cetaceans, insectivores, dermopters, chiropters, rodents, lagomorphs, carnivores, perssodactyls, hyracoid, proboscides, artiodactyls, xenarthrans, folidotes, tubulidentatas, sirenias, marsupials and monotremates. Preferentially, the present invention concerns the use of the nucleic acids of the present invention in the species of mammals that include, but are not limited to the groups Afrotheria, Euarchontoglires, Laurasiatheria and Xenarthra. Preferentially, the present invention concerns the mammals selected among mice, bovines, ovines, caprines and equines.

[0126]The term "inferior animals" refers to the phyla of invertebrate animals. The invention is useful to various species of invertebrate animals, including species of arachnids that include, but are not limited to, Acari, Amblypygi, Araneae, Opiliones, Palpigradi, Pseudoscorpiones, Ricinulei, Scorpiones, Solifugae, Uropygi. Preferentially, the present invention concerns the use of the nucleic acids of the present invention in the species of spiders that include, but are not limited to the groups Araneomorphae, Mesothelae and Mygalomorphae.

[0127]"Microorganisms" refers to microscopic organisms such as bacteria, viruses, fungus and protozoa. Preferentially, the microorganisms of the present invention include the organisms selected from the bacteria and fungus groups. "Bacteria" refers to prokaryote organisms, with exception the cyanophyta. The nucleic acid of the invention may be used to confer desirable traits to basically any bacteria. Thus, the invention is useful to various species of bacteria that include, but are not limited to, the groups Actinobacteria, Aquificae, Bacteroidetes/group Chlorobi, Chlamydiae/group Verrucomicrobia, Chloroflexi, Chrysiogenetes, Cyanobacteria, Gloeobacteria, Nostocales, Oscillatoriales, Pleurocapsales, Prochlorales, Stigonematales, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres/group Acidobacteria, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfobacteria and Thermotogae. "Fungus" refers to organisms of the Fungi kingdom, that may either be unicellular or multicellular. The nucleic acid of the invention may be used to confer desirable traits to basically any fungus. Thus, the invention is useful to various species of funguses that include, but are not limited to, the groups Ascomycota and Basidiomycota. Preferentially, the present invention uses the species of microorganisms selected from the genus Aspergillus, Bacillus, Escherichia, Pichia, Saccharomyces or Streptomyces.

[0128]An "immune response" means any reaction occurring in response to an antigen, such as a viral antigen, in a host having a functional immune system. Immune responses may be humoral "in nature" (i.e. involving the production of immunoglobulins or antibodies) or cellular "in nature" that involve various types of "B" and "T" lymphocytes, dendritic and macrophage cells, antigen-bearing cells and the similar, or both types of responses. The immune response may also involve the production or generation of several effecter molecules such as cytokines, lymphokines and the similar. Immune responses may be assessed in vitro or in animal cells and systems. These immune responses may be important to protect the host against diseases and may be used prophylactically and therapeutically.

[0129]A "derivate" of a spider web protein or fragment thereof means a polypeptide modified by a variation in the amino acid sequence of the protein (e.g. through the manipulation of the nucleic acids coding the protein or by an alteration to the protein itself). Such derivations of the natural amino acid sequence may involve the insertion, addition, deletion or substitution of or more amino acids and may or not alter the essential activity of the spider web protein.

[0130]The term "native or natural spider web protein" refers to those proteins that are present in the webs produced by spiders. These proteins may be derived from the web itself through dissolution or from the specific web silk gland located in the abdomen of the spider before the silk is spun. The term may also be applied to spider web proteins produced using a variety of expression systems but which substantially comprise the same amino acid sequence as that produced by the spider.

[0131]The term "synthetic spider web protein" refers to a protein produced by an expression system having a sequence that may be based on the natural spider web protein sequence or an artificially produced nucleic acid sequence that encodes amino acid motives of spider web proteins.

[0132]The term "biofilament" means a fibrous protein that is normally produced and secreted by any of a variety of insects and arachnids. Biofilaments are composed of alternate crystalline and amorphous regions. Examples of biofilaments include spider webs, an externally woven fibrous protein secretion found in numerous arachnids (e.g. Nephilengys cruentata, Avicularia juruensis and Parawixia bistriata), and fibroin, an externally spun fibrous protein secretion found in a variety of insects (e.g. Bombyx mori). Preferably, when the biofilament is secreted in the form of a secretion subject to spinnert action and mechanical extension, it will have a polyalanine segment forming a crystal domain that undergoes a transition from helix to beta-sheet thus forming a (beta) crystal that stabilises this structure. Preferentially, the biofilament's amorphous domain forms a beta-type sheet where the spaces between the sheets are between 3 Ångstroms and 8 Ångstroms, and preferentially between 3.5 Ångstroms and 7.5 Ångstroms.

[0133]Preferentially, the biofilament has a C-terminal portion with a repeated amino acid motive being between 20 to 40 amino acids in length, more preferentially, being between 34 amino acids in length, and a consensus sequence between 35 to 55 amino acids in length, more preferentially, being between 47 amino acids in length. Preferentially, the biofilament has a repeated amino acid motive (creating both the amorphous and crystalline domains) having a sequence at least 50% identical to the sequences selected from the group SEQ ID N. 19-34), more preferentially, at least 70% identical, and yet more preferentially, at least 90% identical, to the sequences identified as SEQ ID N. 19-34). "Culture medium" means a medium that surrounds the cell and is responsible for its survival. If the cell is secreting a protein (e.g. a biofilament), the cell's culture medium shall contain the protein secreted by this cell.

[0134]The discovery of new spider silk proteins, as well as their characterisation and expression in different heterologous systems shall be of great use in numerous areas, such as medicine and industry.

[0135]Spider silks proteins may be obtained through synthetic polypeptides having amino acid sequences substantially similar to a consensus unit of the silk protein or through polypeptides expressed from nucleic acid sequences coding a natural or engineered silk protein, or derivates of these. Depending on the application for which the silk protein is required, it may be useful to form fibres from a single spider web protein or from a combination of different spider web proteins.

[0136]One aspect of the invention provides nucleic acid sequences coding new spider web proteins. The nucleic acid sequences of the present invention comprise SEQ ID Ns. 1-19.

[0137]One particular aspect of the invention provides nucleic acid sequences coding silk proteins principally related, but not limited to the Major Ampullate gland. Examples of the nucleic acid sequences related to this gland comprise SEQ ID Ns. 3, 17 and 18.

[0138]One particular aspect of the invention provides nucleic acid sequences coding silk proteins principally related, but not limited to the Minor Ampullate gland. Examples of the nucleic acid sequences related to this gland comprise SEQ ID Ns. 4, 5, 6 and 16.

[0139]One particular aspect of the invention provides nucleic acid sequences coding silk proteins principally related, but not limited to the Flagelliform gland. Examples of the nucleic acid sequences related to this gland comprise SEQ ID Ns. 1 and 15.

[0140]One particular aspect of the invention provides nucleic acid sequences coding silk proteins principally related, but not limited to the Tubuliform gland. An example of a nucleic acid sequence related to this gland comprises SEQ ID N. 2.

[0141]One particular aspect of the invention provides nucleic acid sequences coding silk proteins principally related, but not limited to the Aciniform gland. An example of a nucleic acid sequence related to this gland comprises SEQ ID N. 14.

[0142]The nucleic acid molecules coding polypeptides of the present invention may be prepared through two overall methods: either the artificial synthesis of nucleotides that encode the spider web protein or through the isolation of nucleotides originating from the spiders themselves. Both methods use protocols well described in the state-of-the-art. The information from the nucleotide sequence, such as the DNA sequences coding a synthetic or natural spider web protein, may be prepared from an isolated nucleic acid molecule of the invention through the synthesis of the oligonucleotide. The synthesis of oligonucleotides may be prepared by the phosphoramide method used by the DNA Synthesizer of Applied Biosystems 38A or similar equipment. The resulting construct may be used directly or purified in accordance with methods commonly used in the state-of-the-art, such as liquid chromatography (HPLC).

[0143]In accordance with the present invention, nucleic acids having appropriate sequence homology rates with the sequences coding a spider web protein may be identified through hybridization conditions and appropriate stringency wash. Such methods are useful for numerous purposes, including the triage of libraries comprising mutant sequences of nucleic acid coding a spider web protein. Hybridisations may be performed according to the methodologies described in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), Cold Spring Harbor Laboratory Press).

[0144]The invention also refers to molecules of new spider silk proteins. In a particular aspect6 of the invention, the spider silk proteins comprise the sequences SEQ ID Ns. 20-38.

[0145]One particular aspect of the invention provides amino acid sequences of the silk principally related, but not limited to the Major Ampullate gland. Examples of the amino acid sequences related to this gland comprise SEQ ID Ns. 22, 35 and 36.

[0146]One particular aspect of the invention provides amino acid sequences principally related, but not limited to the Minor Ampullate gland. Examples of the amino acid sequences related to this gland comprise SEQ ID Ns. 23, 24, 25 and 37.

[0147]One particular aspect of the invention provides amino acid sequences principally related, but not limited to the Flagelliform gland. Examples of the amino acid sequences related to this gland comprise SEQ ID Ns. 20 and 34.

[0148]One particular aspect of the invention provides amino acid sequences principally related, but not limited to the Tubuliform gland. An example of the amino acid sequence related to this gland comprises SEQ ID N. 21.

[0149]One particular aspect of the invention provides amino acid sequences principally related, but not limited to the Aciniform gland. An example of the amino acid sequence related to this gland comprises SEQ ID N. 33.

[0150]Another aspect of the present invention provides an isolated molecule of nucleic acid having a sequence selected from the group of SEQ ID Ns.: 1-19 and whereby the expression is controlled by means of specific or constitutive promoters and terminators having a polyadenylation region.

[0151]The present invention also describes a method for the production of bio filaments from spider silk proteins produced in prokaryote and eukaryote cells.

[0152]Yet another aspect of the invention provides host cells comprising at least one of the spider silk proteins encoded by nucleic acids. These host cells include, but are not limited to, bacterial cells, fungus cells, insect cells, mammal cells and plant cells. Host cells super expressing one or more spider silk proteins encoded by nucleic acids of the present invention provide useful reagents for diverse purposes including, but not limited to, the production of silk fibres comprising at least one silk protein that may be incorporated within a material to modulate the structural properties of that material.

[0153]The invention further relates to dermatological compositions characterized by comprising: [0154]a) A recombinant spider silk protein; [0155]b) a pharmaceutically acceptable vehicle.

[0156]The invention also describes microbicide compositions characterized by comprising: [0157]a) A recombinant spider silk protein; [0158]b) an agriculturally acceptable vehicle and, optionally, [0159]c) additives.

[0160]Another object of the present invention is to provide prokaryote cells and prokaryote organisms containing DNA molecules of the present invention that may be any of the identified sequences from the group SEQ ID N. 1-19, or cells containing gene constructs capable of producing the proteins of the present invention (SEQ ID N. 20-38), or variants of these. The gene constructs may be stably incorporated in the genome of the prokaryote organism cells.

[0161]Another object of the present invention is to provide eukaryote cells and eukaryote organisms containing DNA molecules of the present invention that may be any of the identified sequences from the group SEQ ID N. 1-19, or cells containing gene constructs capable of producing the proteins of the present invention (SEQ ID N. 20-38), or variants of these. The gene constructs may be stably incorporated in the genome of the eukaryote organism cells.

[0162]In another aspect of the invention, the gene constructs may be provided with a DNA molecule capable of replicating in an autonomous manner in the cells of eukaryote organisms, such as viral vectors. The gene construct may also be arranged in a transitory manner in the cells of eukaryote organisms.

[0163]The present invention also describes a method for producing a genetically modified organism characterized by the fact of comprising the following stages: [0164]a) transforming a cell, tissue, organ or embryo with a gene construct in accordance with any of the claims 3 to 11 or an expression vector in accordance with any of the claims 12 to 23; [0165]b) selecting transformed cells, cell calluses, embryos or seeds; [0166]c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b); [0167]d) selecting the mature plants, mature embryos or microorganisms cells of stage (c) containing the gene construct or expression vector with the nucleotide sequences that encode the spider silk protein.

[0168]The present invention also describes a method for the production of recombinant protein characterized by the fact of comprising the following stages: [0169]a) transforming of a cell, tissue, organ or embryo with an expression vector in accordance with any of the claims 12 to 23; [0170]b) selecting of transformed cells, cell calluses, embryos or seeds; [0171]c) regenerating of mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b); [0172]d) selecting of the mature plants, mature embryos or microorganisms cells of stage (c) containing the expression vector with the nucleotide sequences that encode the spider silk protein. [0173]e) extracting of the recombinant spider silk protein produced in the organisms selected in stage (d).

[0174]The production of large quantities of spider web proteins in viable prokaryote or eukaryote systems becomes feasible with the possibility of coding spider web proteins with nucleic acid molecules. For example, part or all of at least one DNA molecule coding a natural or synthetic spider web protein, such as a sequence of nucleic acid selected from the group of SEQ ID N. 1-19, may be inserted in a plasmidial vector adapted for the expression of bacteria cells, such as E. coli. Such vectors comprise regulatory elements necessary for the expression of the DNA in a host cell positioned in such a manner as to allow the expression of the DNA in that host cell. Such regulatory elements required for expression include promoter sequences, transcription initiation sequences and, optionally, enhancer sequences. Such methods may be used to assess constructs for the expression of spider web proteins, for example, in a bacterial system, thus providing a fast and real triage technique.

[0175]The spider web proteins produced through gene expression in a recombinant prokaryote or eukaryote system may be purified following methods known in the state-of-the-art. Preferentially, a commercially viable secretion/expression system may be used, whereby a recombinant protein is expressed and subsequently secreted by the host cell, in order to facilitate purification in a culture medium. If expression/secretion vectors are not used, an alternative technique involves purifying the recombinant protein of the lysed cells derived from the prokaryote or eukaryote cells from which the protein was expressed. Methods to handle such cell lysines are well known in the state-of-the-art. Recombinant proteins may be purified by affinity separation, such as through the immunological interaction with antibodies that specifically bind to the recombinant proteins or nickel columns for isolating the recombinant proteins tagged with 6-8 histidine residues at the N-terminal or C-terminal. Alternative tags consist either of FLAG epitope or hemaglutinin epitope. Such methods are well described in the state-of-the-art and are widely used by experts in the field.

[0176]Alternatively, standard purification strategies designed to isolate silk proteins differentially from plant homogenates may also be used to advantage. The purification of spider web proteins expressed in plants may be made easier due to their extreme stability under conditions that normally denature typical proteins such as, for example, high temperatures and low pH values. Protein purification strategies may generally be adapted to optimise the purification of spider web proteins from leaves. Above ground parts of transgenic plants may be picked and dried by normal methods. These dehydrated plant parts may be homogenised in an appropriate buffer followed by several treatments designed to eliminate contaminants differentially. The silk proteins recovered may be optimised following treatments in which the plant extracts are subjected to one or more combinations of the following steps: 1) boiling, either in the presence or absence of detergent; 2) differential centrifugation; 3) progressive decrease of pH, and; 4) precipitation with variable concentrations of urea or ammonium sulphate. These steps may vary in accordance with the intended optimisation of production and the purification efficiency of the spider web proteins in plants.

[0177]The spider web protein level may be determined by immunoblotting while the purity and concentration are determined by analysis of the amino acids. Purified spider web protein may be analysed through it's mechanical properties so as to ascertain that the recombinant protein possesses the intended characteristics. The spider web proteins prepared as described above may be analysed in accordance to standard procedures. For example, these proteins may be subjected to analyses of the amino acid sequences in accordance with known methods.

[0178]The spider web proteins of the present invention may be used as microbiocides against viral replication; as defensins against insects and pests; as cosmetics or dermatological compositions; in combination with other materials. They may also be introduced in cotton plants to be expressed jointly with cotton fibres in order to increase the resistance and flexibility of the fibre. The proteins of the present invention are also associated to the generation of new variations of the silks naturally produced by spiders and the production of new proteins, peptides and polypeptides having different physical and chemical properties.

EXAMPLES

[0179]The present invention is further defined by the following examples. It should be understood that while these examples indicate a part of the invention, they are merely provided in an illustrative form, and do not therefore place any limitation on the scope of the present inventions.

[0180]Common molecular biology techniques such as the transformation of bacteria and the electrophoresis of nucleic acids in agarose gel are referred to in the terms by which they are usually described. Details of the practices of these techniques, all well known in the state-of-the-art, are described in Sambrook, et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. 1989, Cold Spring Harbor Laboratory Press). Several solutions used in the experimental manipulations are referred to by their common names such as "lysing solution", "SSC", "SDS", etc. The composition of these solutions may be found in the above mentioned reference (Sambrook, et al.).

Example 1

Collection and Classification of Spiders and their Webs

[0181]Silks of the species of spiders Argiope sp., Ephebopus sp., Nephila clavipes, Nephilengys cruentata, Avicularia juruensis and Parawixia bistriata were collected from the Brazilian biodiversity, mainly from the Amazon region, Atlantic rainforest and corral. The silks were dried at ambient temperature and analysed through infrared microscopy (FTIR). Significant differences were noted in the results for the alpha- and beta-sheets, mainly associated to the flexibility and resistance of the silks in relation to Nephila clavipes. The FTIR has recently been used as a method for determining the secondary structure of proteins in solid state and has proved most viable, especially for insoluble proteins such as those of the spider webs. The secondary structures of the proteins were quantified using the recognition of standards method developed by Forato et al., 1998. The spectrums of the spider webs and products of transgenic expression were obtained from samples prepared in KBr tablets, and was used for quantifying amide band I, between 1600 and 1800 cm-1.

TABLE-US-00001 TABLE 01 Percentage of secondary structures found through infra-red analysis of the different species of spiders collected. α helix β-sheet Coils Others Species (%) (%) (%) (%) Argiope 7 56 27 13 Nephila clavipes 16 43 24 13 Ephebopus 2 57 37 13 Nephylengys cruentata 6 53 32 11 Parawixia bistriata 17 47 32 12 Avicularia juruensis 5 58 32 12

Example 2

Obtaining Polynucleotide Sequences: Construction of cDNA Libraries, Sequencing

[0182]After collection, the silk producing glands of the spiders were isolated in laboratory, immediately frozen in liquid nitrogen and maintained at a temperature of -70° C. Following pulverisation, extraction of the Total RNA was performed using the reagent TRIZOL (Invitrogen), in accordance with the manufacturer's instructions. The Oligotex kit (Qiagen) was used for the purification of the mRNA used for the synthesis of the cDNA, preferentially through the use of the "SUPERSCRIPT II Plasmid System with GATEWAY Technology for cDNA Synthesis and Cloning" Kit (Invitrogen), following the manufacturer's guidelines. After synthesis and fractioning by size in chromatography columns, using Sepharose CL-2B resin (Pharmacia). Both large (1-5 Kb) and small (0.5-2 Kb) cDNA fragments were inserted in appropriate vectors such as pSPORT-1 (Life), pCMV-SPORT 6 or pTrueBlue (Genetix). The libraries thus obtained were introduced into host cells, preferentially by electroporation (25mF, 200W, 1.8 KV) in DH5α, bacteria (Invitrogen).

[0183]The transformed bacteria were cultivated on 7.5 cm Petri plates containing LB (Luria-Bertani) medium with an appropriate selective agent (Sambrook et al., 1989). Those presenting an insert (white) were transferred to 96 well plates. A copy of each 96 well plate was made by means of a replicator and both replicas were maintained at a temperature of -70° C.

[0184]The DNA for sequencing was prepared from inoculums from one of the replica plates using the alkaline lysis method (Sambrook et al., 1989) modified for use with 96 well plates. The sequencing reactions were performed using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). The primers used in the sequencing reactions were chosen in accordance with the vector in which the library was constructed. All clones were sequenced from the original 5' extremity of the mRNA molecule of the insert, and part of them were also sequenced from the 3' extremity

[0185]The sequencing reactions were read in Applied Biosystems 3700 automatic sequencers. The resulting electropherograms were transferred to a centralised Data Base, located at the Laboratorio de Bioinformatica da Embrapa Recursos Geneticos e Biotecnologia [Bioinformatics Laboratory of Embrapa Genetic Resources and Biotechnology], for processing and analysis.

[0186]The sequences produced were deposited in the GenBank (Benson et al. 1999) and transferred to the BCCC (http://www.bcccenter.fcav.unesp.br) where they are at the disposal of the international scientific community. These sequences are also described in the Sequences List, SEQ. ID. N. 1 to SEQ. ID. N. 19.

[0187]The techniques of genetic engineering described herein are known to experts in the field and are also described in Sambrook et al., 1989 (Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual--volumes 1, 2 and 3. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), Silhavy et al., 1984 (Silhavy, T. J.; M. L. Bennan & L. W. Enquist. 1984. Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel, et al., 1987. (Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience).

Example 3

Mechanical Analysis of the Silks from the Different Spiders Species

[0188]Collection of silk--Five samples of each species with lengths of approximately 5 cm were positioned on a mechanical trial card, as formerly described by Stauffer et al. (Stauffer S L, Coguil S L, Lewis R V. Comparison of physical properties of three silks from Nephila clavipes and Araneus gemmoides. The journal of Arachnology. 1994; 22:5-11).

[0189]Measurement of fibre diameters--The fibres were analysed using a Nikon Eclipse E200 microscope equipped with a camera. Each silk was observed magnified 800 times and the images were visualised with the SPOT Basic software. The diameters were determined using the ImageJ software, version 1.32 (http://rsb.info.nih.gov/ij/), and the final value was obtained from the average of 5 measurements taken along the length of the fibres.

[0190]Mechanical Test--Each fibre was tested with a Synergie 100 Mechanical Testing System (MTS), using a custom 10-g load cell. The fibres were stretched at a rate of 2 mm/min and the data was collected at a frequency of 35 Hz. Data collection was done using the Testworks 4 software (MTS Systems Corporation, Cary, N.C.). Stress×strain graphs were constructed using Microsoft Office Excel 2003 software.

[0191]The equations below were used to calculate the stress, strain and stiffness values.

σ(stress)=F/A, where F is the force applied and A the transversal section area.

ε(strain)=ΔL/L0, where ΔL is the change in length of the fibre and L0 is the initial length.

Y(stiffness or Young's modulus)=σ/ε

TABLE-US-00002 DIAMETER STRAIN STRESS STIFFNESS SPECIES (μm) (%) (GPa) (GPa) Avicularia juruensis 9.62 ± 3.92 7.54 ± 5.71 0.07 ± 0.03 0.017 ± 0.018 Nephilengys cruentata 4.82 ± 0.61 11.88 ± 2.97 0.81 ± 0.23 0.253 ± 0.306 Parawixia bistriata 7.56 ± 1.21 21.54 ± 5.01 0.76 ± 0.14 0.069 ± 0.024 Nephila clavipes* ? 22.89 ± 3.58 9.53 ± 0.06 0.062 ± 0.019 Araneus diadematus* ? 28.00 ± 4.00 1.08 ± 0.16 6.90 ± 1.22 Argiope aurantia*** ? 24.14 ± 4.73 1.36 ± 0.58 0.202 ± 0.205 Lactrodectus geometricus** 2.78 ± 0.24 14.00 ± 6.00 0.83 ± 0.19 12.91 ± 7.38 *Brooks AE, Steinkraus HB, Nelson SR, Lewis RV. An investigation of the divergence of major ampullate silk fibers from Nephila clavipes and Argiope aurantia. Biomacromolecules. 2005 Nov-Dec; 6(6): 3095-9. **Motriuk-Smith D, Lewis RV. Brown Widow (Latrodectus geometricus) major ampullate silk protein and its material properties. Biomed Sci Instrum. 2004; 40: 64-9. ***Madsen B, Shao ZZ, Vollrath F. Variability in the mechanical properties of spider silks on three levels: interspecific, intraspecific and intraindividual. Int J Biol Macromol. 1999 Mar-Apr; 24(2-3): 301-6.

Example 4

Comparative of the Sequences of the Present Invention with the Existing Sequences in the State-of-the-Art

[0192]In relation to SEQ ID N. 1 (Product of Gene: Nephilengys cruentata--NCFlag) a search in GenBank using the BLASTP software revealed the following 10 protein sequences as being the most similar described:

1. gi|7106228|gb|AAF36091.12. gi|7106224|gb|AAF36090.13. gi|2833649|gb|AAC38847.14. gi|13561982|gb|AAK30594.15. gi|2833647|gb|AAC38846.16. gi|70913024|gb|AAZ15322.17. gi|13562004|gb|AAK30605.18. gi|93138993|gb|ABE99838.19. gi|7106229|gb|AAF36092.110. gi|89276819|gb|ABD66603.1

[0193]As may be verified referring to Table 1, sequence number 9 (gi|7106229, highlighted by an asterisk on the table) is the one having the smallest percentage of amino acid discrepancies when aligned with SEQ ID N. 1. Of the total amino acids aligned between these two protein sequences, 23% are divergent. It is important to note that amino acids aligned with gaps are not included in this calculation, and that the alignment contemplated by this analysis is the multiple alignment between all eleven proteins. Observing the message "Error: Reference source not found" it is possible to note that the discrepancy would be even greater if the gaps were included in the calculation. The Table also confirms that several other previously described sequences (underlined values) present greater similarity levels between themselves rather than that observed between SEQ ID N. 1 and the previously described sequences.

TABLE-US-00003 TABLE 1 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 1. SI 1 1 2 3 4 5 6 7 8 9 1 44 2 39 20 3 25 46 40 4 42 47 43 43 5 42 10 27 45 47 6 52 54 52 50 53 53 7 52 54 52 50 53 53 0 8 53 57 55 52 55 55 16 16 9 23* 40 41 8 43 37 51 51 54 10 59 65 62 60 59 62 58 58 61 60 "SI 1" indicates the SEQ ID N. 1. The numbers that identify lines and columns correspond to the numbers on the list of the sequences previously described in the state-of-the-art. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 1 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 1. Only half of the Table is filled out because the values are reciprocal.

[0194]FIG. 1 indicates an alignment between SEQ ID N. 1 and sequence number 9 (gi|7106229) which highlights identical amino acids between the two sequences. The numbers over the sequence identify the position in the alignment. The difference is due to the insertion of gaps to maintain the alignment.

[0195]In order to calculate the percentage of discrepancies, the sequence of the present invention was used in a comparative search of the GenBank CDS non-redundant protein sequences bank (Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., Wheeler, D. L. (2006). GenBank. Nucleic Acids Res. 34 (Database issue): D16-20). The search was conducted without using a low complexity filter, with size 3 words, using a BLOSUM62 matrix, an 11 point penalty for the opening of gaps and 1 point per extended amino acid. The sequences with the 10 highest "scores" were used in a multiple alignment with the ClustalX (Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. and Higgins, D. G. (1997). The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 24: 4876-4882). The multiple alignments used a Gonnet 250 matrix penalising a gap opening by 10 points and according 0.20 to each extended amino acid. The pair by pair alignment used the "slow--precise" approach by lowering the gap extension penalty to 0.10 points. The result of the final multiple alignment was used in a pair by pair distance comparative, with the aid of the MEGA3 software (Kumar, S., Tamura, K. and Nei, M. (2004). MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in Bioinformatics 5: 150-163). The percentage of discrepancies was calculated by dividing the number of different amino acids by the total number of amino acids compared. The gaps in the alignment were retained in the analysis and eliminated pair by pair. The figure of the alignment was generated from an alignment between the two sequences under consideration, using the same parameters as for the single alignment. The alignment result was formatted using the ESPript software (Gouet, P., Courcelle, E., Stuart, D. I. and Metoz, F. (1999). ESPript: multiple sequence alignments in PostScript. Bioinformatics. 15: 305-8. http://espript.ibcp.fr/ESPript/ESPript/).

[0196]In relation to SEQ ID N. 2 (Product of Gene: Nephilengys cruentata--NCTuSp), the 10 most similar protein sequences already described are:

1. gi|837584272. gi|683425013. gi|893657764. gi|893657745. gi|630543296. gi|613872447. gi|709276548. gi|630543279. gi|6138723710. gi|61387234

TABLE-US-00004 TABLE 2 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 2. SI 2 1 2 3 4 5 6 7 8 9 1 33 2 33 7 3 36 45 45 4 37 48 45 11 5 38 48 48 11 13 6 37 22 20 47 47 49 7 47 51 51 40 40 41 53 8 46 51 50 39 39 41 53 1 9 27* 47 44 23 24 22 47 33 34 10 39 46 45 10 06 14 47 47 47 21 "SI 2" indicates the SEQ ID N. 2. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 2 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 2. Only half of the Table is filled out since the values are reciprocal.

[0197]In relation to SEQ ID N. 3 (Product of Gene: Nephilengys cruentata--NCMaSp), the 10 most similar protein sequences already described are:

1. gi|837584272. gi|683425013. gi|893657764. gi|893657745. gi|630543296. gi|613872447. gi|709276548. gi|630543279. gi|6138723710. gi|61387234

TABLE-US-00005 TABLE 3 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 3. SI 3 1 2 3 4 5 6 7 8 9 1 7* 2 15 7 3 18 9 11 4 24 9 14 2 5 22 10 13 2 4 6 17 10 10 1 1 1 7 17 9 14 2 2 2 2 8 16 7 14 9 9 7 8 9 9 22 10 13 2 3 2 2 2 10 10 11 8 5 1 1 0 0 1 6 1 "SI 3" indicates the SEQ ID N. 3. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 3 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 3. Only half of the Table is filled out since the values are reciprocal.

[0198]In relation to SEQ ID N. 4 (Product of Gene: Nephilengys cruentata--NCMaSp), the 10 most similar protein sequences already described are:

1. gi|2605800|2. gi|856808993. gi|2605798|4. gi|503631435. gi|503631416. gi|503631457. gi|2911274|8. gi|503631379. gi|1356199210. gi|50363139

TABLE-US-00006 TABLE 4 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 4. SI 4 1 2 3 4 5 6 7 8 9 1 25* 2 41 34 3 50 34 8 4 45 50 54 54 5 50 51 59 58 2 6 50 52 55 55 19 21 7 51 53 59 57 4 7 19 8 47 52 57 55 1 1 18 4 9 50 52 58 58 29 34 37 38 30 10 49 54 51 50 2 2 17 2 2 33 "SI 4" indicates the SEQ ID N. 4. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 4 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 4. Only half of the Table is filled out since the values are reciprocal.

[0199]In relation to SEQ ID N. 5 (Product of Gene: Nephilengys cruentata--NCMiSp 06A01), the 10 most similar protein sequences already described are:

1. gi|26057982. gi|85720613. gi|7653234. gi|272289595. gi|470079236. gi|470079637. gi|135619928. gi|1087077649. gi|8927681710. gi|2914731

TABLE-US-00007 TABLE 5 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 5. SI 5 1 2 3 4 5 6 7 8 9 1 13* 2 39 52 3 39 47 2 4 46 47 49 49 5 46 56 49 49 57 6 45 53 36 39 54 53 7 53 58 60 60 58 64 59 8 47 51 48 48 56 53 52 59 9 53 57 62 61 56 66 61 37 58 10 54 53 60 60 54 67 57 36 58 22 "SI 5" indicates the SEQ ID N. 5. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 5 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 5. Only half of the Table is filled out since the values are reciprocal.

[0200]In relation to SEQ ID N. 6 (Product of Gene: Nephilengys cruentata--NCMiSp 11F12), the 10 most similar protein sequences already described are:

1. gi|856808992. gi|2605798|3. gi|891139924. gi|135620185. gi|630543336. gi|630543537. gi|630543298. gi|613872379. gi|1356199410. gi|89365776

TABLE-US-00008 TABLE 6 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 6. SI 6 1 2 3 4 5 6 7 8 9 1 67 2 64* 8 3 74 50 54 4 89 83 84 87 5 82 77 77 78 78 6 87 76 80 76 79 60 7 83 78 81 80 77 54 43 8 85 77 81 80 75 53 47 22 9 77 74 76 81 75 63 74 69 69 10 83 79 81 80 80 54 42 11 22 69 "SI 6" indicates the SEQ ID N. 6. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 6 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 6. Only half of the Table is filled out since the values are reciprocal.

[0201]In relation to SEQ ID N. 7 (Product of Gene: Nephilengys cruentata--NCfibroin), the 10 most similar protein sequences already described are:

1. gi|630543312. gi|6984160|3. gi|709056424. gi|893657765. gi|709056416. gi|630543297. gi|709056438. gi|149732699. gi|1356201810. gi|8885520|

TABLE-US-00009 TABLE 7 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 7. SI 7 1 2 3 4 5 6 7 8 9 1 80 2 77 72 3 78 73 68 4 81 58 77 75 5 78 73 68 3 75 6 81 61 73 74 11 74 7 78 72 72 25 78 25 74 8 76* 70 45 61 72 61 72 68 9 84 77 78 72 79 72 79 74 72 10 79 72 51 65 75 65 74 66 33 76 "SI 7" indicates the SEQ ID N. 7. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 7 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 7. Only half of the Table is filled out since the values are reciprocal.

[0202]In relation to SEQ ID N.8 (Product of Gene: Nephilengys cruentata--NCdefensin), the most similar protein sequences already described are:

1. gi|895121212. gi|410194633. gi|410194654. gi|771580115. gi|901923686. gi|333488507. gi|622757808. gi|577925079. gi|4945804610. gi|49458052

TABLE-US-00010 TABLE 8 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 8. SI 8 1 2 3 4 5 6 7 8 9 1 63* 2 66 62 3 67 63 11 4 67 62 1 11 5 63 62 49 51 50 6 65 61 2 12 2 50 7 64 60 0 10 0 48 0 8 67 63 17 15 17 51 18 15 9 66 61 18 18 18 51 18 18 13 10 65 61 15 13 15 50 15 15 1 13 "SI 8" indicates the SEQ ID N. 8. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 8 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 8. Only half of the Table is filled out since the values are reciprocal.

[0203]In relation to SEQ ID N. 9 (Avicularia juruensis AJFibroin 1A), the 10 most similar protein sequences already described are:

1. gi|175369632. gi|505484833. gi|765808834. gi|524282735. gi|522096736. gi|714163557. gi|892768198. gi|135620189. gi|7165476010. gi|109511662

TABLE-US-00011 TABLE 9 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 9. SI 9 1 2 3 4 5 6 7 8 9 1 84 2 86 87 3 82 83 86 4 82 83 86 1 5 82 83 86 1 1 6 80 84 87 77 77 77 7 76* 81 83 87 87 87 80 8 76* 85 87 86 86 86 84 84 9 79 84 88 76 76 76 6 79 83 10 82 86 83 81 81 81 79 84 88 78 "SI 9" indicates the SEQ ID N. 9. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 9 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 9. Only half of the Table is filled out since the values are reciprocal.

[0204]In relation to SEQ ID N. 10 (Avicularia juruensis AJFibroin 1B), the 10 most similar protein sequences already described are:

1. gi|681715642. gi|175369633. gi|892768194. gi|381977455. gi|135620206. gi|381977437. gi|837584298. gi|714163559. gi|7091302210. gi|1263289

TABLE-US-00012 TABLE 10 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 10. SI 10 1 2 3 4 5 6 7 8 9 1 88 2 83 91 3 75 91 81 4 75 86 85 45 5 76 90 84 76 70 6 74 88 85 44 0 68 7 77 88 84 85 87 84 85 8 82 89 84 78 81 85 80 84 9 76 88 83 41 50 72 50 82 78 10 73* 88 83 30 45 70 45 85 78 49 "SI 10" indicates the SEQ ID N. 10. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 10 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 10. Only half of the Table is filled out since the values are reciprocal.

[0205]In relation to SEQ ID N. 11 (Product of Gene: Avicularia juruensis AJFibroin 2), the 10 most similar protein sequences already described are:

1. gi|14053872. gi|381977493. gi|381977514. gi|381977455. gi|503091996. gi|381977557. gi|381977598. gi|381977479. gi|5042356310. gi|38197757

TABLE-US-00013 TABLE 11 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 11. SI 11 1 2 3 4 5 6 7 8 9 1 66 2 41 76 3 42 76 1 4 55 77 1 0 5 68 83 83 83 86 6 33 75 8 10 10 81 7 42 76 2 2 2 81 9 8 42 76 1 1 1 83 9 2 9 76 86 87 88 89 68 86 86 87 10 32* 77 17 17 17 82 8 17 18 84 "SI 11" indicates the SEQ ID N. 11. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 11 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 11. Only half of the Table is filled out since the values are reciprocal.

[0206]In relation to SEQ ID N. 12 (Product of Gene: Avicularia juruensis AJNegProtein 1), the 10 most similar protein sequences already described are:

1. gi|871332392. gi|871332413. gi|175393084. gi|720113705. gi|709130246. gi|683650427. gi|498711018. gi|1107564879. gi|8511170510. gi|39973263

TABLE-US-00014 TABLE 12 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 12. SI 12 1 2 3 4 5 6 7 8 9 1 73* 2 80 32 3 89 90 93 4 91 92 89 93 5 86 40 43 93 91 6 87 92 91 89 94 90 7 84 79 77 91 92 83 88 8 90 93 93 92 91 93 91 92 9 93 90 89 93 94 92 93 92 93 10 90 88 93 92 92 92 91 91 92 93 "SI 12" indicates the SEQ ID N. 12. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 12 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 12. Only half of the Table is filled out since the values are reciprocal.

[0207]In relation to SEQ ID N. 13 (Product of Gene: Avicularia juruensis AJNegProtein 2), the 10 most similar protein sequences already described are:

1. gi|220742922. gi|150214223. gi|887131134. gi|555931565. gi|825394046. gi|1094670827. gi|32363708. gi|66778179. gi|7199204810. gi|62175305|

TABLE-US-00015 TABLE 13 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 13. SI 13 1 2 3 4 5 6 7 8 9 1 72* 2 72* 0 3 74 59 59 4 85 90 90 87 5 90 91 91 91 94 6 83 85 85 83 64 93 7 83 84 84 83 92 92 90 8 84 87 87 82 64 93 24 90 9 75 58 58 56 88 87 84 80 84 10 80 77 77 80 90 92 90 87 90 72 "SI 13" indicates the SEQ ID N. 13. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 13 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 13. Only half of the Table is filled out since the values are reciprocal.

[0208]In relation to SEQ ID N. 14 (Product of Gene: Parawixia bistriata--PBAciniform), the 10 most similar protein sequences already described are:

1. gi|498711012. gi|891140103. gi|449806334. gi|407873725. gi|588648996. gi|829361547. gi|167413978. gi|72431039. gi|1159614410. gi|45439370

TABLE-US-00016 TABLE 14 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 14. SI 14 1 2 3 4 5 6 7 8 9 1 28* 2 75 74 3 89 90 91 4 89 89 91 90 5 89 91 94 89 93 6 89 91 94 89 93 1 7 91 93 96 89 93 0 0 8 89 91 94 89 93 1 2 1 9 89 91 94 89 92 1 2 1 0 10 89 91 94 89 92 1 2 1 0 0 "SI 14" indicates the SEQ ID N. 14. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 14 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 14. Only half of the Table is filled out since the values are reciprocal.

[0209]In relation to SEQ ID N. 15 (Product of Gene: Parawixia bistriata--PBFlag), the 10 most similar protein sequences already described are:

1. gi|624655892. gi|272289573. gi|135619804. gi|1095000955. gi|476068456. gi|59211937. gi|519752458. gi|32363709. gi|10948747210. gi|47219204

TABLE-US-00017 TABLE 15 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 15. SI 15 1 2 3 4 5 6 7 8 9 1 20* 2 20* 0 5 88 72 93 72 91 6 83 85 92 85 86 92 7 94 93 92 93 92 90 93 8 82 82 93 82 91 89 88 94 9 92 93 91 93 93 94 91 90 92 10 46* 82 94 82 86 91 84 90 86 94 "SI 15" indicates the SEQ ID N. 15. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 15 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 15. Only half of the Table is filled out since the values are reciprocal.

[0210]In relation to SEQ ID N. 16 (Product of Gene: Parawixia bistriata--PBMiSp), the 10 most similar protein sequences already described are:

1. gi|14053872. gi|881757013. gi|175078794. gi|556195495. gi|710265776. gi|509020807. gi|668052918. gi|714081389. gi|5093179510. gi|55274106

[0211]Table 16: Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 16. "SI 16" indicates the SEQ ID N. 16. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 16 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 16. Only half of the Table is filled out since the values are reciprocal.

[0212]In relation to SEQ ID N. 17 (Product of Gene: Parawixia bistriata--PBMaSp1), the 10 most similar protein sequences already described are:

1. gi|475692342. gi|328156713. gi|493298924. gi|519752465. gi|427827896. gi|1095000957. gi|552741048. gi|552740849. gi|5036314510. gi|1263285

TABLE-US-00018 TABLE 17 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 17. SI 17 1 2 3 4 5 6 7 8 9 1 73 2 14* 81 3 74 14 81 4 74 15 80 14 5 74 8 81 12 14 6 88 85 79 86 86 85 7 20 83 20 83 83 84 78 8 23 80 19 80 79 81 84 2 9 33 76 21 76 77 76 87 8 9 10 29 74 14 74 73 74 87 25 25 30 "SI 17" indicates the SEQ ID N. 17. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 17 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 17. Only half of the Table is filled out since the values are reciprocal.

[0213]In relation to SEQ ID N. 18 (Product of Gene: Parawixia bistriata--PBMaSp2), the 10 most similar protein sequences already described are:

1. gi|709130222. gi|328156713. gi|552741044. gi|552740805. gi|552740926. gi|552741367. gi|552741288. gi|552740869. gi|3819774510. gi|55274082

TABLE-US-00019 TABLE 18 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 18. SI 18 1 2 3 4 5 6 7 8 9 1 55 2 17* 45 3 22 42 23 4 31 46 21 2 5 27 45 26 4 6 6 39 52 23 2 1 6 7 31 47 21 2 2 6 3 8 26 44 21 2 0 2 1 0 9 19 57 25 31 38 35 45 37 33 10 34 46 23 4 2 8 3 4 2 40 "SI 18" indicates the SEQ ID N. 18. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 18 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 18. Only half of the Table is filled out since the values are reciprocal.

[0214]In relation to SEQ ID N. 19 (Product of Gene: Parawixia bistriata--silk gland spidroin), the 10 most similar protein sequences already described are:

1. gi|13999452. gi|709130223. gi|175078794. gi|552740805. gi|552740866. gi|552741367. gi|552741288. gi|552741129. gi|5527413810. gi|55274092

TABLE-US-00020 TABLE 19 Percentage of amino acid discrepancies between the pair by pair alignments of 11 sequences relating to SEQ ID N. 19. SI 19 1 2 3 4 5 6 7 8 9 1 78 2 54 79 3 78 0 79 4 48 79 37 79 5 47* 77 37 77 0 6 48 77 37 77 1 1 7 47* 80 38 80 2 0 3 8 49 78 37 78 1 1 1 1 9 49 78 37 78 2 2 2 2 1 10 51 80 42 80 8 8 8 8 6 7 "SI 19" indicates the SEQ ID N. 19. The numbers that identify lines and columns correspond to the numbers on the above list. The number found at the intersection of a line with a column represents the percentage of discrepancy observed in the alignment of the two sequences. The asterisk denotes the greatest similarity encountered between SEQ ID N. 19 and the previously described sequences. The underlined values represent similarities between the previously described sequences greater than those found for SEQ ID N. 19. Only half of the Table is filled out since the values are reciprocal.

Example 5

Construction of Expression Vectors Containing Genes of Spider Silk Proteins in Plants

[0215]The expression vectors used in the present invention contain at least one promoter sequence and one coding sequence for a spider silk protein selected from the group SEQ ID N. 1-19 and one polyadenilation sequence. The expression vector may further contain regulatory sequences responsible for the post-transcriptional processing and compartmentalisation of the heterologous proteins.

[0216]Expression vectors were constructed using standardised recombinant DNA manipulation methodologies (Sambrook, J. Molecular Cloning: A Laboratory Manual (3-Volume Set) Cold Spring Harbor Laboratory Press; 3rd edition, 2001). Basically, the coding region relating to the fragments: in the case of soy bean, the coding sequences of the silk proteins were cloned under the control of the beta-conglycinin peptide signal and promoter (previously cloned in the Laboratorio de Transfer ncia de Genes [Gene Transfer Laboratory]--EMBRAPA) (FIG. 2). The intended alterations aimed adapting the fragment for the addition of the coding sequence of the plant peptide signal, thus allowing it to properly process the recombinant proteins. In the case of cotton, the coding sequences of the silk proteins were cloned under the control of the actin2 peptide signal and promoter of Arabidopsis (Aragao F. J. L., Vianna G. R., Carvalheira S. B. R., Rech E. L. (2005) Germ line genetic transformation in cotton (Gossypium hirsutum L.) by selection of transgenic meristematic cells with an herbicide molecule. Plant Sci. 168: 227-1233).

[0217]The gene and protein sequences of the present invention may be modified according to their intended use and still remain within the scope of the invention. For example, when it is intended that the fibre should have low elasticity, the sequence of alanine (Ala) repetitions should be removed and, to the contrary, when a high rate of elasticity is sought, the size of the poly-Ala portion may be increased. Furthermore, the constructs may possibly only include the repetitive modules of the sequences presented herein and, also, a combination of the different protein modules in order to achieve the intended characteristic.

Example 6

Production of Soybean and Cotton Plants Containing Gene Sequences of Spider Silk Proteins

[0218]The expression vectors obtained were used in transformation and co-transformation experiments with vector pAG1 (FIG. 3) containing the coding sequence of the ahas gene under control of the ahas gene promoter and NOS terminator. This vector allows the selection of transgenic soybean and cotton plants in vitro. When necessary, the GUS marker gene under control of the 35SCaMV promoter and nos terminator were cloned in pAG1. Transgenic soybean and cotton plants were developed through the bioballistic system developed by EMBRAPA and protected under patent PI9714887-3.

[0219]The transgenic plants produced were analysed by PCR (Dellaporta, S. L., Wood, J. and Hicks, J. B. (1983). A plant DNA minipreparation: version II. Plant Molecular Biology Reports 1:19-21; Aragao, F. J. L., Barros, L. M. G., Brasileiro, A. C. M., Ribeiro, S. G., Smith, F. D., Sanford, J. C. Rech E. L. (1996). Inheritance of foreign genes in transgenic bean (Phaseolus vulgaris L.) co-transformed via particle bombardment. Theor Appl Genet. 93:142-150) and Southern blot (Dellaporta, S. L., Wood, J. and Hicks, J. B. (1983). A plant DNA minipreparation: version II. Plant Molecular Biology Reports 1:19-21; Sambrook J., Fritsch E. F., Maniatis T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) to detect the transgenes as well as by bio-chemical analyses and bioassays. Histochemical analyses were performed to detect the integration of heterologous genes.

Example 7

Use of Agroinfiltration and Viral Vectors for the Expression of Spider Proteins

[0220]The transitory expression of the spider genes was assessed by inoculation in planta of Agrobacterium tumefaciens strains containing the spider genes cloned in the binary vector pCambia 1300 (www.cambia.org), or in a viral vector based on Potato virus X (PVX). This viral vector, based on the vector pGR107 (Chapman S, Kavanagh T and Baucombe D (1992) Potato virus X as a vector for gene expression in plants. Plant J 2:549-557), presents a duplication of the protein sheath promoter and restriction sites for the cloning of exogenous genes. The sequence corresponding to PVX is cloned in a binary vector, under the control of the CaMV35S promoter, thus allowing inoculation through Agrobacterium (agroinoculation). The original vector, pGR107, was modified by the addition of the conversion cassette from the Gateway® cloning system (Invitrogen), at the site SmaI. The resulting vector, PVXGW, is therefore compatible with the Gateway® cloning system (Invitrogen), which allows the cloning of genes in a much faster and efficient manner when compared to the usual methods of restriction and linking. Agrobacterium tumefaciens strains with either binary or viral vectors containing the spider genes were inoculated on leaves of Nicotiana benthamiana and the transient expression was assessed after 4-6 days.

Example 8

Construction of Expression Vectors Containing Genes of Spider Silk Proteins in Bacterial Vectors and Production of these Proteins in the Bacterial Expression System

[0221]The expression vectors for bacteria used in the present invention were constructed using the pET system (Novagen). Repetitive modules of the spider silk protein coding sequences selected from the group SEQ ID N. 1-19 were synthesised and cloned in vector in vector pUC19 associated to restriction sites that allow multiplication of the repetitive units N times, according to the strategy described by Lewis et al. (Lewis R V, Hinman M, Kothakota S, Fournier M I Expression and purification of a spider silk protein: A new strategy for producing repetitive proteins. PROTEIN EXPRESSION AND PURIFICATION 7 (4): 400-406 JUN 1996). The cassettes containing the repetitive modules were transferred to the pET19b vector (Novagen) (FIG. 4) under the control of promoter T7 and fused to a tail of N-terminal histidines. These resulting vectors were used to transform competent E. coli cells (strains BL21(DE3) and BL21(DE3)pLysS) by thermal shock. The recombinant bacteria containing the expression vector were inoculated in a culture medium containing the appropriate antibiotic and grown to a OD.sub.600nm between 0.8 and 0.9. Protein production was induced with IPTG at a concentration of 1 mM during 3 h at 37° C. under agitation. The culture was then centrifuged at 1500 g for 15 min and resuspended in a lyse buffer, as described by the system's manufacturer. Under these experimental conditions, cells BL21 (DE3) and BL21(DE3)pLysS induced the expression of the genes cited in this heterologous system, and the lysed extract was used to perform the purification by column chromatography using Ni-NTA His-Bind Resin (Qiagen) charged with 50 mM of NiSO4 and 5 mM of imidazole. The fraction containing the recombinant protein was eluted using an elution buffer containing 100 mM of imidazole, dialysed in distilled water over 2 days and then freeze-dried. The recuperation of the purified protein remained at between 0.2 mg/g and 10 mg/g of the dry cell weight.

Example 9

Construction of Expression Vectors Containing the Genes of the Spider Silk Proteins in a Vector of Mammal Cells and Production of these Proteins in the Mammal Expression System

[0222]At least one of the sequences was used for cloning in the pCMV-script® vector (Stratagene). This vector is for expression in mammal cells in culture, CHO (Chinese hamster ovary), using the Citomegalovirus promoter and the SV40 polyadenylation site, which allows large constitutive expression rates. The products are expressed from the purified culture supernatant and rested.

[0223]The sequence of the silk protein coding sequences selected from the group SEQ ID N. 1-19, as well as the modularly manipulated versions of these sequences, was inserted in a vector based on the early Cytomegalovirus promoter. The resulting vectors (FIG. 5) were used in the co-transfection of hamster ovary cells (CHO--Chinese hamster ovary) with the use of lipofectin and calcium phosphate, together with the reporter vectors pCMV-Gal (Promega) and pGFP/NEO (Promega). The integrity of the silk proteins under reducer or non-reducer conditions was assessed by Western Blotting. This technique is intended to detect proteins after separation by electrophoresis in polyacrylamide gel and transfer to nitrocellulose or nylon membranes. Detection is through antibodies that specifically react with the epitopes of the intended protein, followed by colorimetric or radiographic reactions.

[0224]The production of transgenic mice containing expression vectors having the spider web protein gene sequences was performed using the technique of pronuclear micro-injection. This technique is used for generating transgenic animals by addition. The technique further allows the introduction of long DNA sequences from different species in the genome of mammals conferring high expression levels and integration of the transgene in the germinative cells. Using a micromanipulator coupled to a high-resolution microscope, copies of the expression vectors containing the spider web protein gene sequences selected from the group SEQ ID N. 1-19 were directly injected in a freshly fertilised embryo pronucleus collected from the oviduct of superovulated female donors. The pronucleus is the maternal and paternal nuclei originating in the ovule and the spermatozoid, respectively, before they unite to become a single nucleus containing the genome of the new individual. Following the micro-injection, the embryo were transferred to the oviduct of a pseudo pregnant receptive female earlier mated with a vasectomised male that will bring to term the litter of possible transgenics later genotyped for the presence of the exogenous gene. Integration of the transgene by pronuclear micro-injection occurs in a random way in the genome and all the animal's cells are genetically modified, including the germinative ones which thus transmit the alteration to its descendants. The entire positive transgenic animal originating from a micro-injected embryo was classified as the founder of a single transgenic line that differs from another founder as to the insertion location and number of copies of the transgene in the genome. The detailed protocol for the manipulation of animals for addition of a gene is well described in the state-of-the-art and may be found in manuals and revisions in the literature (Hogan, B., Beddington, R., Costantini, F., Lacy, E. (1994). Manipulating the mouse embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor; Godard, A. L. B., Guenet, J. (1999) Genetica de Camundongos. Modelos animais de doenccas humanas. Biotecnologia, Ci ncia & Desenvolvimento 9:96-100).

[0225]The production of transgenic bovines was used for the actual production of spider silk proteins in milk. The expression vectors used were derived from pBC1 vector (Invitrogen) for the expression of recombinant proteins in the milk of transgenic animals. Genes from the spider web proteins selected from the group SEQ ID N. 1-19 were cloned at site XhoI of these vectors (FIG. 6) with constitutions containing promoters that direct the expression of milk proteins, such as the beta-casein promoter and constitutive (Iguma L. T., Lisauskas S. F. C., Melo E. O., Franco M. M., Pivato I., Vianna G. R., Sousa R. V., Dode M. A. N., Aragao F. J. L., Rech E. L., Rumpf R. (2005) Development of bovine embryos reconstructed by nuclear transfer of transfected and non-transfected adult fibroblast cells. Genet. Mol. Res. 4: 55-66; Oliveira R. R., Carvalho D. M. de, Lisauskas S., Mello E., Vianna G. R., Dode M. A. N., Rumpf R., Aragao F. J. L., Rech E. L. (2005) Effectiveness of liposomes to transfect livestock fibroblasts. Genet. Mol. Res. 4:185-196).

Example 10

Synthesis of the Spider Web Proteins

[0226]The spider web proteins of the present invention were synthesised using the technique of automatic synthesis in F-moc solid-phase in a Perseptive Biosystems Pioneer Synthesiser. The proteins were purified in Shimadzu Class VP and Akta Explorer liquid chromatographers using reverse phase columns. Proteins and peptides were analysed by MALDI-TOF mass spectroscopy and sequenced by MS/MS using Voyager DE STR spectrometers, 4700 Proteomics Analyser and Q-TOF, respectively. The liposomes of variable phospholipidic composition were prepared in accordance with the instructions of the commercially available GIBCO BRL (USA) kits.

Example 11

Analysis of the Spider Web Fibres by Electronic Scan Microscopes

[0227]A study with electronic scan microscopes was conducted to ascertain the ultrastructural details (thickness, length, external layout, disposal of protein layers, etc.) of the spider web fibres. For such, fibre samples were fixed, dehydrated to critical point, mounted on stub and metallised with different metal alloys, after which they were observed using a Zeiss DSM 962 microscope in accordance with the methodology adapted from Bozzola & Russel (Bozzola, J. J.; Russel, L. D. (1999). Electron microscopy: principles and techniques for biologists. Jones and Bartllet Publishers, Inc. (eds). London, UK. pp. 670-6'78).

Example 12

Isolation and Characterisation of the Spider Web Proteins by High Powered Liquid Chromatography (HPLC) and by Capillary Liquid Chromatography Coupled to a Mass Spectroscope (CapLC/Q-TOF/MS/MS; TOF-TOF/MS/MS)

[0228]The protein extracts of interest were fractioned in a HPLC system and the fractions obtained were enzymatically digested, submitted to capillary liquid chromatography coupled to a mass spectroscope. This process provided the internal sequences of the isolated proteins which were then identified, characterised physically and chemically and used for searches in data bases (Pegah R., Dass J. C. (2004). Proteome analysis in the bovine adrenal medulla using liquid chromatography with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 18:1877-1884).

Example 13

Evaluation and Quantification of the Spider Silk Protein Purity

[0229]The purity of the proteins was evaluated using HPLC and amino acid composition. The identity of the proteins was confirmed by Western Blot.

[0230]The purified material was quantified using the extinction and coefficient method (at 280 nm) and ELISA, with polyclonal antibodies.

Example 14

Preparation and Polymerisation of the Biopolymers

[0231]Samples of spider web gland proteins expressed in E. coli, purified, dialysed and freeze-dried were prepared for polymerisation and extrusion. Between 60-90 mg of protein were solubilised in 200 μl of hexafluoroisopropanol (HFIP), isopropanol or ethanol and maintained overnight under constant agitation at ambient temperature. The insoluble material was removed by centrifugation and the supernatant was placed in a syringe adapted to a tube used for HPLC having a diameter of 4-300 μl. After removal of all air from the syringe apparatus, an extrusion was made in a coagulation bath in a container holding isopropanol/methanol or ethanol and other organic solvents, which produced fibres similar to the natural fibres (see example in FIG. 8: silk produced in vitro). This process was adapted from the methodology described by Seidel (Seidel A. (1998) Artificial spinning of spider silk. Macromolecules 31:6733-6736).

Example 15

Test of the Biopolymers

[0232]A quasi static mechanical test was performed using Instron 55R4201 equipment, at 23° C. and 50% relative humidity. The mechanical properties were determined using Instron series IX software specific for the test of materials. The high load tests used the Hopkinson Tension equipment which was defined as being the most appropriate for the sensitivity analyses of the different materials (Shim V. P. W. (2001) Dynamic mechanical properties of fabric armor. Int. J. of Impact Eng. 25:1-15; Huh H., Kang W. J., Han S. S. (2002). A tension split Hopkinson bar for investigating dynamic behaviour of sheets metals. Exp. Mechanics 42:8-17). The filaments were aligned and pre-tensioned to ensure uniformity of distribution during the trials.

Example 16

Recombinant Proteins of the Silk Producing Gland of Spiders Used as Defensins, Antimicrobial Peptides and Microbicides

[0233]Recombinant proteins of spider silks were used to inhibit the replication of several viruses and other pathogenic microorganisms in plants and animals. This may occur due to various mechanisms such as the linking of negatively charged recombinant proteins of the silk gland to charges of the protein sheath of different viruses, thus inhibiting their replication and acting as microbiocides (Scordi-Bello I. A., Mosoian A., He C., Chen Y., Jarvis, Marla G. A., Keller J., Hogarty K., Waller D. P., Profy A. T., Herold B. C., Klotman M. E. 2005. Candidate Sulfonated and Sulfated Topical Microbicides: Comparison of Anti-Human immunodeficiency Virus activities and Mechanisms of Action. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 49: 3607-3615), defensins (Thevissen K., Francois I. E. J. A., Alberts A. M., Cammue B. P. A. (2005) Fungal sphingolipids as targets for the development of selective antifungical therapeutics. Current Drugs Targ. 6:923-928) and antimicrobial peptides or polypeptides (Prates M. V., Sforca M. L., Regis W. C. B., Leite J. R. S. A., Silva L. P., Pertinhez H. A., Araujo A. L. T., Azevedo R. B., Spisni A., Bloch C. Jr. (2004) The NMR-derived Solution Structure of a New Cationic Antimicrobial Peptide from the Skin Secretion of the Anuran Hyla punctata. J. Biol. Chemistry 279:13018-13026). Therefore, the proteins of the silk producing glands were expressed and tested for this type of activity, showing positive results for the inhibition of viral, fungal and bacterial growth.

Example 17

Molecular Modelling of the Spider Web Proteins

[0234]Prediction of the three-dimensional structures of the spider web glands was done by homology modelling or "comparative protein modelling", which is based on the observation that the homology between amino acid sequences implies structural and functional similarities and that homologous proteins present conserved internal regions (mainly formed by secondary structure α-helix and β-sheet elements).

[0235]The modelling of these proteins by homology basically followed four successive steps: [0236]Identification and selection of template proteins with a known three-dimensional structure directly from the PDB (Protein Data Bank). A systematic search was conducted using BLAST³⁶ for one or more adequate using the protein primary sequence as bait in a data base of primary structures derived from proteins with confirmed tertiary structures deposited in the PDB. [0237]Alignment of the amino acid residue sequences. The objective of the alignment is to align structurally equivalent residues taking into account common structural characteristics, such as secondary structure elements. It thus becomes possible to recognise structurally conserved regions and variable regions. [0238]Model construction. This stage deals with modelling the structurally conserved regions, modelling the loop regions and modelling the lateral chains. This was done using the Blue Star Sting software (Goran Neshich, Ivan Mazoni, Stanley R. M. Oliveira, Michel E. B. Yamagishi, Paula R. Kuser-Falcao, Luiz C. Borro, Douglas U. Morita, Kassyus R. R. Souza, Gustavo V. Almeida, Diego N. Rodrigues, Jose G. Jardine, Roberto C. Togawa, Adauto L. Mancini, Roberto H. Higa, Sergio A. B. Cruz, Fabio D. Vieira, Edgard H. dos Santos, Raquel C. de Melo and Marcelo M. Santoro. The Star STING server: A multiplatform environment for protein structure analysis. Genet. Mol. Res. 5 (2) 2006). [0239]Model Validation. An adequately modelled protein should have a satisfactory tertiary structure. Its quality depends on the protein selected as a template and the alignment calculated. It is important to verify if any major unexplained conformance differences exist between the secondary structure elements (conserved regions) of the template-structure and the model-structure. This example was validated using the PROCHECK software (Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M.; J. Appl. Crystallogr. 1993, 26, 283).

[0240]These steps were followed for SEQ ID N. 1 (Product of Gene: Nephilengys cruentata--NCFlag), and the model obtained may be seen in FIG. 7.

[0241]As the silk proteins and modular structures have no known atomic structure as yet, the geometries shall be constructed ab initio (prediction of the three-dimensional structures of the proteins derived from their primary structures), based on the contents of the secondary structures and the hypotheses relating to the doubling of the proteins. Portions such as the N-terminus and C-terminus that do not contain modular sequence blocks shall have their structure determined by x-ray crystallography. The purified proteins shall be crystallised through optimisation of the matrix results. As they possess characteristics of new proteins, the structures shall be determined using either multiple isoform replacements (MIR) after treatment of the atoms or through anomalous dispersion of the crystals containing selenomethionine.

Sequence CWU 1

4013277DNANephilengys cruentata 1agcgggaggt tcaggtggaa caacagtcat agaagatttg gacataacag ttaatggtcc 60aggaggcccg ataacaatct cagaagagct aacaattggt ggtccaggtg ctggaggttc 120aggacctggt ggtgctggac caggaggcgc aggacccggt ggtgcaggac caggaggcgc 180aggacctggt ggtgcaggac caggaggagt aggacctggt ggtgctggag gccctggtgg 240tgctggagga cctttcggtc caggtggttc cggacccgga ggtgcaggcg gcgctggagg 300accttatgga cctggtggag cttacggacc tggtggacct ggagggcctg gtggtcctgg 360aggacccggt tctggcggac cttacggacc tggtggtgct tacggacctg gtggtgctta 420cggacctggt ggtgcttatg gtcctggtgg agctggtgga ccaggtggag ctggtggacc 480atatggaccc ggtggacctt acggaccagg tggtccatac ggacctggtg gagctggtgg 540accaggtggt gctggtggac cttatggacc aggaggtgct ggacctggcg gatacggacc 600tggaggcgct ggacctggtg gatacggacc tggcggttct ggacctggag gcgctggacc 660tggtggatac ggacctggcg gttctggacc tggtggtgct ggacccggtg gatatggacc 720tggtggtgct ggacccggtg gatacggacc tggtggtgct ggacctggcg gtgctggacc 780tggtggtgct ggtcctggtg gatacggacc tggcggttct ggaccaggtg gtcctggatc 840tggtggccca ggcggagcgg gaggttcagg tggaacaaca gtcatagaag atttggacat 900aacagttaat ggtccaggag gcccgataac aatctcagaa gagctaacag ttggtggtcc 960aggtgctgga ggttcaggac ctggtggtgc tggaccagga ggcgcaggac ccggtggtgc 1020aggaccagga ggagtaggac ctggtggtgc tggaggccct ggtggtgctg gaggaccttt 1080cggtccaggt ggttccggac ccggaggtgc aggcggcgct ggaggacctt atggacctgg 1140tggagcttac ggacctggtg gacctggagg gcctggtggt cctggaggac ccggttctgg 1200cggaccttac ggacctggtg gtgcttacgg acctggtggt gcttacggac ctggtggtgc 1260ttatggacct ggtggagctg gtggaccata tggacccggt ggaccttacg gaccaggtgg 1320tccatacgga cctggtggag ctggtggacc aggtggtgct ggtggacctt atggaccagg 1380aggtgctgga cctggcggat acggacctgg aggcgctgga cctggtggat acggacctgg 1440cggttctgga cctggaggcg ctggacctgg tggatacgga cctggcggtt ctggacctgg 1500tggtgctgga cccggtggat atggacctgg tggtgctgga cccggtggat acggacctgg 1560tggtgctgga cctggcggtg ctggacctgg tggtgctggt cctggtggat acggacctgg 1620cggttctgga ccaggtggtc ctggatctgg tggcccaggc ggagcgggag gttcaggtgg 1680aacaacagtc atagaagatt tggacataac agttaatggt ccaggaggcc cgataacaat 1740ctcagaagag ctaacagttg gtggtccagg tgctggaggt tcaggacctg gtggtgctgg 1800accaggaggc gcaggacccg gtggtgcagg accaggaggc gcaggacctg gtggtgcagg 1860accaggagga gtaggacctg gtggtgctgg aggccctggt ggtgctggag gacctttcgg 1920tccaggtggt tccggacccg gaggtgcagg cggcgctgga ggaccttatg gacctggtgg 1980agcttacgga ccaggtggac ccggaggacc tggagggcct ggtggtcctg gaggacccgg 2040ttctggcgga ccttacggac ctggtggtgc ttacggacct ggtggtgctt acggacctgg 2100tggtgcttat ggacctggtg gagctggtgg accaggtgga gctgctggac catatggacc 2160cggtggacct tacggaccag gtggtccata cggacctggt ggagctggtg gaccaggtgg 2220tgctggtgga ccttatggac caggaggtgc tggacctggc ggatacggac ctggaggcgc 2280tggacctggt ggatacggac ctggcggttc tggacctgga ggcgctggac ctggtggata 2340cggacctggc ggttctggac ctggtggtgc tggacccggt ggatatggac ctggtggtgc 2400tggacccggt ggatacggac ctggtggtgc tggacctggc ggtgctggac ctggtggtgc 2460tggtcctggt ggatacggac ctggcggttc tggaccaggt ggtcctggat ctggtggccc 2520aggcggagcg ggaggttcag gtggaacaac agtaatagaa gatttggaca taacacttaa 2580tggtccagga ggcccgataa caatctcaga agagctaaca gttggtggtc caggtgctgg 2640aggttcagga cctggtggtg ctggaccagg aggcgcagga cccggtggtg caggaccagg 2700aggcgcagga ccaggaggag taggacctgg tggtgctgga ggaccttatg gttctggtgg 2760tttcggattc ggaggtgcag gcggctctgg aggaccttat gtacctggtg gagcatatgg 2820agctggttct ggtacaccat cttatagtgg atctcgtgtt cctgatttgg tgaatggtat 2880aatgcgttcg atgcaaggct ctggtttcaa ctatcaaatg tttggcaaca tgttatcgaa 2940atatgcctcc ggatcaggtg catgcaattc aaatgatgtt aatgttttaa tggatgctct 3000tcttgcggct ttgcactgtc tcagtagcca tggatcccca tcatttgggt cttctccaac 3060cccttctgca atgaatgcat attccaactc tgttcgaaga atgttccaat tctaaggtta 3120tactccttta aacttgaatt tattttcaaa tcattttgat gaaccttagt tactcatttg 3180aagaaaaaaa taaatatctt tttagcagaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3240aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 327721636DNANephilengys cruentata 2gcaagccaga gcgctagcag cagcagtgct tcggcctctg cattcgcaca acagtcctct 60gcttcccttg cagcctcctc ttctttcagc caggccttcg cttcggccgc ttccgcctct 120gccgtcggaa acgttgctta ccagctaggc ttatccgcag cacaatctct cggaatagcc 180aatgctggag cactcgctag tgctttagct cagtctgttt cttctgtagg cgttggagcc 240agttcaagtg cctacgccaa tgcagtcgcc ggtgccgttg gacagttctt agccaatcag 300ggtattttga acacaggcaa tgcatcttcc ctagcctcct cgttctccag tgccctctcc 360gcctcggcag cagccgcgca atcccaatca ttcgcacaga gtcaagcagc agcttcggcc 420ttccaacaag cagcatcaca gagtgctagc cagagtgctg cccaatctgg ttctcagtcc 480tcttccacca ctaccaccac ctcggcctca ggaagtcaat ccgcaagcca gagcgctagc 540agcagcagtg cttcggcctc tgcattcgca caacagtcct ctgcttccct tgcagcctcc 600tcttctttca gccaggcctt cgcttcggcc gcttccgcct ctgccgtcgg aaacgttgct 660taccagctag gcttatccgc agcacaatct ctcggaatag ccaatgctgg agcactcgct 720agtgctttag ctcagtctgt ttcttctgta ggcgttggag ccagttcaag tgcctacgcc 780aatgcagtcg ccggtgccgt tggacagttc ttagccaatc agggtatttt gaacacaggc 840aatgcatctt ccctagcctc ctcgttttct aatgcgcttt cgtcatccgc cgctaattca 900gttggttctg gattgttatt gggtccttca caatacgttg gaagtattgc tccaagtata 960ggaggtgctg ctggaatatc aatcgctggt cctggaattt tatcatactt acctcctgtt 1020tctccgctga atgcacagat aatctcctct ggtttacttg cttctttggc accagtatta 1080tcatcttccg gcttagcatc atccagtgcg acttctagag ttggcagttt agctcaatct 1140ttggcatccg cattgcaatc ttcgggaggt acactggatg tttcgacctt cttgaatctt 1200ctgtctccca tttctacaca aattcaagcc aatacttctc taaatgcatc acaggcgatt 1260gtccaagttt tacttgaagc tgtagctgct ctgctgcaaa ttatcaacgg agctcaaata 1320acttctgtca attttggcag tgtctccagc gtaaacacag ccttggcaac tgctctcgct 1380ggttgatttt tatgtccctt ttgaagaatt ctttgcctac tggaaactta taaaatgtat 1440aatctttatt ttatttctga tttcaactca atattgcatt cgttatcttt gcttgtctgt 1500gcaattattg atttttaaaa ttatattatg aaccctgaaa ttttcctgat aataaatatt 1560cttgaatgcc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620aaaaaaaaaa aaaaaa 163631700DNANephilengys cruentatamodified_base(1616)..(1616)a, c, t, g, unknown or other 3gacaaggtgc cggagcagca gcagcagcag cagccgcagg tggagctgga caaggcggat 60atggaggtct tggtggccaa ggagctggag ccgcagctgc agcagctggt ggtgctggac 120aaggaggata tggaggtcaa ggtgctggac aaggtgcagc cgcagcagca gctagtggtg 180ccggacaagg aggatatgaa ggtccaggtg ccggacaagg tgcaggtgca gccgcagcag 240cagctggtgg tgccggacaa ggaggatatg gaggtcttgg tggccaagga gctggacaag 300gagctggagc cgcagctgca gcagctggtg gtgccggaca aggaggatat ggaggtcttg 360gtggccaagg agctggacaa ggagctggag ccgcagctgc agcagctggt ggtgccggac 420aaggaggata tggaggtcaa ggtgctggac aaggtgcagc agcagcagca gctggtggtg 480ctggacaagg aggatatgga ggcctaggtt ctggacaagg cggatatggt agacaaggtg 540ccggagcagc agcagcagca gcagccgcag gtggagctgg acaaggcgga tatggaggtc 600ttggtggcca aggagctgga gccgcagctg cagcagctgg tggtgctgga caaggaggat 660atggaggtca aggtgctgga caaggtgcag ccgcagcagc agctagtggt gccggacaag 720gaggatatgg aggtccaggt gccggacaag gtgcaggtgc agccgcagca gcagctggtg 780gtgccggaca aggaggatat ggaggtcttg gtggccaagg agctggacaa ggagctggag 840ccgcagctgc agcagctggt ggtgccggac aaggaggata tggaggtcaa ggtgctggac 900aaggtgcagc agcagcagca gctggtggtg ccggacaagg aggatatgga ggcctaggtt 960ctggacaagg cggatatggt ggacaaggtg ccggagcagc agcagccgca ggtggagctg 1020gacaaggcgg atatggaggt cttggtggcc aaggagctgg acaaggtgct ggagccgcag 1080ctgcagctgc tggtggttcc ggaagaggag gatatggaag tcaaggtgct ggacaaggag 1140cagcagcagc agcagctggt ggtgcaggtc aaggtggata tggtggtgca ggttctggag 1200ctgctgcggc ctctgcagct gcttcccgtt tgtcttctcc tgaagctagt tcgagagttt 1260catctgcagt ttctaatttg gtttcaagtg gtcctactaa ttcggctgcc ttgtcgaata 1320ccatcagtag tgttgtctcc caaattagcg caagcaatcc tggtctctct ggatgtgatg 1380tccttgttca agctcttttg gaagtcgttt ctgctcttat ccatattttg ggatcttcta 1440gcatcggccc agttaactat ggctcagcta gccaatccac tcaaatcgtt ggtcaatcgg 1500tttaccaagc tctaggttaa ttatgaaatc aaatttcctc aaaattattt tgatagaatt 1560actaagtttt tgtaataatt ttgtaaaatt ggttttcaat aaatagtatg catatnanaa 1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1680aaaaaaaaaa aaaaaaaaaa 170043487DNANephilengys cruentata 4gagctggtgg tgctggagga tatggcgttg gacaaggcta tggtgcaggt gcaggagctg 60gggctgccgc cggtgcagga gctggtggtg ctggaggata tggcgctgga caaggctatg 120gtgcaggtgc aggagttggt gctgccgccg ctgctggagc aggtgcagga gttggtggtg 180ctggaggtta cggaagaggt gctggagccg gagctggagc tggagctgga gctgctgccg 240gagctggagc tggagctgct gctggagcag gagctggtgg tgctggagga tatggcgctg 300gacaaggcta tggtgcaggt gcaggagttg gtgctgccgc cgctgctgga gcaggtgcag 360gagttggtgg tgctggaggt tacggaagag gtgctggagc tggagctggt gctggtgctg 420gtggtgcagg aggttacgga agaggtgctg gtgctggagc tggagctggt gcaggagctg 480gtggtgctgg aggatatggc gctggacaag gctatggtgc aggtgcagga gctggtgcag 540ccgccgctgc tggagccggt gcaggtgctg gtggtgctgg aggttacgga agaggtgctg 600gtgctggagc tggagctggt gcaggagctg gtggtgctgg aggatatggc gctggacaag 660gctatggtgc aggtgcagga gctggtgcag ccgccgctgc tggagccggt gcaggtgctg 720gtggtgctgg aggttacgga agaggtgctg gtgctggagc tggagctggt gcaggagctg 780gtggtgctgg aggatatggc gctggacaag gctatggtgc aggtgcagga gctggtgcag 840ccgccgctgc tggagccggt gcaggtgctg gtggtgctgg aggttacgga agaggtgctg 900gtgctggagc tggagctggt gcaggtgctg gtggtgctgg aggttacgga agaggtgctg 960gagctggagc tggggctgga gctgctgcag gagcaggagc tggtggtgct ggacgatatg 1020gcgctggaca aggctatggt gcaggtgcag gagctggggc tgccgccggt gcaggagcag 1080gtggtgctgg aggatatggc gctggacaag gctatggtgc aggtgcagga gctggtgctg 1140ccgccgctgc tggagcaggt gcaggagttg gtggtgctgg aggttacgga agtggtgctg 1200gagccggagc tggagctgga gctggagctg cttctggagc tgctgctgga gctgctgctg 1260gagcaggagc tggtggtgct ggaggatatg gcactggaca aggctatggt gcaggtgcag 1320gtgctggagc tggtgctggt gctggtggtg caggaggtta cggaagaggt gctggagctg 1380gagctggtgc aggagctggt ggtgctggag gatatggcgc tggacaaggc tatggtgcag 1440gtgcaggagc tggtgcagcc gccgctgctg gagacggtgc aggtgctggt ggtgctggag 1500gttacggaag aggtgctgga gctggagctg gggctggagc tgctgcagga gcaggagctg 1560gtggtgctgg aggatatggc gctggacaag gctatggtgc aggtgcagga gctggggctg 1620ccgccggtgc aggagcaggt ggtgctggag gatatggcgc tggacaaggc tatggtgcag 1680gtgcaggagc tggtgctgcc gccgctgctg gagcaggtgc aggagttggt ggtgctggag 1740gttacggaag aggtgctgga gccggagctg gagctgctgc cggagctgga gctggagctg 1800ctgctggagc aggagctggt ggtgctggag gatatggcac tggacaaggc tatggtgcag 1860gtgcaggtgc tggagctggt gctggtggtg caggaggtta cggaagaggt gctggtgctg 1920gagctggagc tggtgcagga gctggtggtg ctggagaata tggcgctgga caaggctatg 1980gtgcaggtgc aggagctggt gcagccgccg ctgctggagc cggtgcaggt gctggaggtg 2040ctggaggtta cggaagaggt gctggagctg gagctggggc tggagctgct gcaggagcag 2100gagctggtgg tgctggagga tatggcgcta gacaaggcta tggtgcaggt gcaggagctg 2160gggctgccgc cggtgcagga gctggaggtg ctggaggata tggcgctgga caaggctatg 2220gtgcaggtgc aggagctggt gcagccgccg ctgctggagc cggtgcaggt gctggtggtg 2280ctggaggtta cggaagaggt gctggagctg gagctggagc tggggctgga gctgctgcag 2340gacaaggcta tggttcaggt gcaggtgctg gagctggtgc cagtgctggt ggtgcaggaa 2400gttacggaag aggtgccgga gctggtgctg ccgccgcttc tggagccggt gctggaggat 2460atggcgctgg acaaggctat ggtgcaggtg caggagctgt tgcttctgcc gctgctggag 2520ccggttcagg agctggtggt gctggaggtt acggaagagg tgccgttgct ggttctggag 2580ctggtgccgg agcaggagct ggtggtgctg gaggatatgg tgcaggagct ggtgctggtg 2640ccgctgctgg agcagttgct ggtggttctg gaggatatgg cggcagacaa ggcggttata 2700gcgcaggtgc gggagctggt gcggcggctg ctgctggagc aggtgcaggt ggaactggag 2760gctacggaag aggttctggt gctggagccg cagctggtgc tgctgctgga gctggtgctg 2820ctggaggata tggtggctat ggcgcaggtg ctggagctgg tgccggtggt gctagaggtt 2880acggaggagg tgctggtgct ggagcaggtg ccgctgctgg aggttatgga agaagagcag 2940gtggatccat tgtaggaact ggaataagtg caatttcttc tggaactggt tctagctatt 3000ccgtttcttc cggcggttac gcctctgcgg gtgtaggtgt tggatccact gttgcatcta 3060ccacatctcg tttgagttca gcacaagcat cttctagaat atctgctgct gcttctactt 3120taatatctgg aggttacttg aatacatctg ccttaccatc agtcatttct gatttgtttg 3180cccaagtcag tgcttcatcc cctggtgtat cagatagtga agttttgatt caagttttgt 3240tggaaattgt ttcttccctt atccatattc tcagttcttc cagtgtaggg caagttgact 3300tcaattctgt tggttcgtct gctgcagctg ttggacagtc catgcaagtt gtcatgggtt 3360aattaaaatg gctgtctctc cccaattaat tctttaaata cagttaagca tttaaaaata 3420aaaaataatg taaaattttc tgcataaata aaaatatttt cctgcttgga aaaaaaaaaa 3480aaaaaaa 348751163DNANephilengys cruentata 5ggaaccagct ccagcacctg cgccatagcc accatatcct ccagcagcac cagctccagc 60agcagcacca gctgcggctc cagcaccaga acctcttccg tagcctccag ttccaccagc 120acctgctcca gcagccgccg ccgcaccagc tcccgcacct gcgctataac caccttgtct 180gccgccatat cctccagaac caccagcacc tactccagca gcggcaccag ctcctgcacc 240atatcctcca gcaccaccag ctcctgctcc ggcaacagca ccggcacctc ttccgtaacc 300tccagcacca ccagctcctg aaccggctcc agcagcggca gaagcaacag ctcctgcacc 360tgcaccatag ccttgtccag cgccatatcc tccagcaccg gctccagaag cggcggcagc 420accagctccg gcacctcttc cgtaacctcc tgcaccacca gcacgggcac cagctccagc 480acctgcacct gaaccatagc cttgtctggc gccatatcct ctagcaccac cagctcctgc 540accggcggca gccccagctc ctgcacctgc accatagcct tgtccagcgc catatcctcc 600agcaccacca gctcctgctc ctgcagcagc tccagcccca gccccagctc cagcacctct 660tccgtaacct ccagcaccac cagcacctgc accggctcca gcagcggcgg ctgcaccagc 720tcctgcacct gcaccatagc cttgtccagc gccatatcct cctgcaccag ctccagctcc 780agcaccagca cctcttccgt aacctcctgc accaccagca ccaacaccag ctccagcacc 840tgcaccttca ccatagcctt gtccagtgcc atatcctcca gcaccaccag ctcctgctcc 900agcagcagct ccagctccag ctccggcagc agctccagct ccagctccag cacctcttcc 960gtaacctcca gcaccaccag cacctgcccc ggctccagca gcggcagcag caccagctcc 1020tgcaccatag ttttgtccac cgccatatcc tccagcacct ccagctcctg caccagctcc 1080ggctccagca cctacacctc ttccgtaacc tccagcacca ccagtaccgg caccagctcc 1140tgcacctgca ccatagcctt gtc 116361292DNANephilengys cruentata 6ggaaccagct ccagcacctg cgccataacc accttgtctg ccgccatatc ctccagaacc 60accagctcct gctccagcag ccgcaccagc accagctcct gcaccatatc ctccagcacc 120accagctcca gcaccacttc cgtatcctcc agcgccacct gcacctgctc cagcagcggc 180tgcagcacca gctccagcac ctcttccgta aaatacggct ggcccataag atgtcaatgt 240tgttgtaacc gacgaagcag cactcgaagc agcggaagat gcggtacttt cattaataga 300accagtgtta gctaaaacat tgccaatagc tgaagatatt gcatttgcat aagctgcagc 360atctgcaaca gcaccaccca atgatgaaac atacccacca acagcagcca gtaaattgtt 420catagcattg gcatcgaggc caagttgatt tccaacattt tgagcaacgc tcgttgcaac 480gctcacagcc tgatcagtag aagttgtggt ggagatcatt tgaacaaaat ctcctgatga 540gaggagattg gatgagaggg attgtgcgaa agcgtttcca gctgcacttc ctgctccagc 600accagctcca gcacctgcac catagccttg tccgacgcca tatcctccag caccaccagc 660tcctgctccg gccccagctc cagcaccagc acctcttccg taacctccag caccaccagc 720accggcacca gctccagcac ctgcaccagc tccagcacct gcaccatagc cttgtcctgc 780accatatcct ccagcaccac cagttcctgc accggctcca gcagcggcgg cagcaccagc 840tcctgcacct gcaccatagc cttgtccagc gccatatcct ccagcaccac cagctcctgc 900tccagcagcg gcggcagcac cagctccggc tccagcacca gcacctcttc cgtaacctcc 960agcaccacca gcaccggcac cagctccagc agcggcggct gcaccagctc ctgcacctgc 1020accatagcct tgtccagcgc catatcctcc agcaccacca gctcctgctc cggcggcagc 1080tccagcacca gctccggctc cagcaccagc acctcttccg aaacctccag caccaccagc 1140acctgcaccg gctccagcag cggcggctgc accagctcct gcacctgcac catagccttg 1200tccagcgcca tatcctccag caccaccagc tcctgctccg gcggcagctc cagcaccagc 1260tccggctcca gcaccagcac ctcttccgta ac 129272172DNANephilengys cruentata 7tgctcctcgt cctctaccag ctcctgctcc tcgtcctcta ccagctcctg ctcctcgtcc 60cctaccagca cctttgccag ctcctcttcc aagaccacgc ccagcaccca tagtttctca 120agtgcaacag gcatccgctc tacaggcaca gtcacaacag tctgcttttg ctcaatccca 180acaatcgtcc attgcacaat ctcagcaagc ctctgtcgcc caatcccaac gagcctccgt 240ctctcaatcc cagcaatcta gtaatgcgtt ttcttctgca gcatcatttg gagcttctag 300cgtagcatcc agtgcttcga cttactttaa ttcgggaata gtacaaagta gcatcgcgtc 360gtcgttgcag tcttccagtg ctctcagttc cattgcctac ggccagacaa ccgcttctat 420caacgatata gcatcggcag tcgctggcag cattgcaaat tcaatcggac tctcgcaaca 480aaccgttcaa agtattatta gtcaacaact agccagtgca ggatccggag catctgctca 540aacattggct tcattgatat ccagcgcagt ttcctccttg gttcaacaat ctggatcggt 600atcagccgga caagaacaga gtatttcgca agcactttcc agttctatct cgagttcttt 660gaatcaattg gtagctgcaa gacctctacc agctcctgct cctcgtcccc taccagcacc 720tttgcctgct cctcttccaa gaccacgccc agcacccata gtttctcaag tgcagcaggc 780atccgctcta caggcacagt cacaacagtc tgcttttgct caatcccaac agtcgtccat 840tgcacagtct cagcaagcct ctgtcgccca atcccaacaa tcctccatct cccaatccca 900acaagcctcc gtctctcaat cccagcaatc tagtaatgcg ttttcttctg cagcatcttc 960tggagcttct agcgtagcat ccagtgcttc gacttacttc aattcgggca tagtacaaag 1020cagcatcgcg tcgtcgttgc agtcttccag tgctctcagt tccattgcct acggccagac 1080aaccgcttct atcagcgata tagcatcggc agtcgctggc agcattgcaa attcaatcgg 1140actctcgcaa caaaccgttc aaagtgttat tagtcaacaa ctagccagtg caggatccgg 1200agcatctgct caaacattgg cttcattgat atccagcgca gtttcctcct tggttcaaca 1260atctggatcg gtatcagccg gacaagaaca gagtatttcg caagcacttt ccagttctat 1320ctcgagttct ttgaatcagt tggtagccgc aagacctcta ccagcccctg ctcctcgtcc 1380cctaccagca cctttaccag ctcctctttc aagacctcga ccagttccag tccaaagacc 1440tcaacccgta ttttcaccca gtcccgctcc tgcctatgct cctgccccat tcactcagca 1500gtcgactttt gctcagtctc aacaagcttc tcttgctcaa tctcaacaac aagcatctat 1560cgctcgatcc caacaagcgt ctctagcgca atctcaacaa tcggcttttg ctcaatccca 1620acaagtagct acagcacaat ctcagcaatc ttctggtgga ttctccacat catctactgg 1680agcttctcaa atcagttctt cagccataag tacttcttcg ggatctgcat tggctaattc 1740cgcacaacaa ctcacatcac ctgcagcctc tcaaagaata tctcagttat ccaattccct 1800agcatctgca gtttctggtg gacaggtcaa ctatgcagcc ttatctaatt ctattgctag 1860tgctgcaagt caaattggag gtggatctgg attatccaaa acggaagttc taattgaaac 1920tctcttagaa accctggctg ctttattgga atctctttct cttcctggat cagctagtgg 1980tggaagtcaa ttcgctcaag caatgcttgc agctcttgca taaaatgtgg

ttaataacaa 2040ataattttgt tggaatgctt atgaatattt ttaggggaat atatgatata cactttaaat 2100gaataatatt gtataacttt ttgttaatgg gaaataaaat ttattagcaa gcatcaaaaa 2160aaaaaaaaaa aa 21728924DNANephilengys cruentata 8atgtggaatc gacaagtctt accaatatat attttggtaa ttgtctcgct agcgatactt 60actacgcatg tttccacgtc gaaacaacgt cctttttata taatgggaca catggtaaac 120agtatcgaag aaatatcgga attcctagaa agaggatcca acgttttgga atcagatgtt 180caattctttt caaacggatc tgtaaaagca gtccgtcatg gatttccttg cgattgtggt 240agattttgcg agaacacagc caatctggcg gattacttgc agagtgttcg atacatcact 300gatccagata cacctgatag ttattacaac caactggtac tgcagttctt tgatttgaag 360ctgagtacgt ccgaaaataa aagacaatct ggacgagaga tagctcacca tgttctggat 420tatttatggg gtgaagaagg cgaaagagag aaagagatcc gagttgtaat ttacttcgaa 480aagcttgaag agaaggatgt aatccttgga tttatggacg tattcaaact ccgaaaccaa 540acatcgcgtc tcagagatgt cggttttgac ggtggaactg gaaacatttc agatatcgct 600agaatgttct ccaaatttaa tataaaagat aatatttggc ttggagatgg tgcaacaaat 660tgttttgaac cttttaaatc atttgtgcgt ctaaagaatg caatagacaa ccgagattcc 720aggaaaggtt ttgtttcaaa aatttatcaa tggactaatg atataaaaac aacaatgatg 780cgttccctaa gacttggagt ggatgggatg atcactaaca aacctgagag actcctggag 840gttctgcaag aacccgaatt tgcgaaggat ttcagattag caacaattta cgacgatcct 900ttcgaatact tctgtgacga gtga 92492070DNAAvicularia juruensis 9cgtactctct agcaagctcc attgcaagcg ctgcatcctc gagtgcatct tcggcagcag 60cagcggcgtc atcttcttcc gcagcagcag gagcagccgc ggcttcggaa gcagcagctt 120ctgccgccgc cacttccacg acaacaacaa caagtacttc tcgtgccgca gcagcagcat 180ccgccgcagc cgcggcctct gcctcgggag ccgccggcgc agcgggagca gcatcagccg 240ctagcgctgc ttcagcttct tcgtccttgc aacagtctct gggatctgcc ttagcacaaa 300gtagctcatt tgcagcagcc ttcgcccaag caagtagcgc tgcttctgca gcagccatag 360catatgctct tgcacagacc gtggcgaatc aaatcggttt ctcttcctac tcctcagctt 420tcgcaagagc agcttcatca gccgtataca gcataggggg cttggcttct gcatctgcat 480atgcctttgc ttttgccagc gccttttcac aagttctctc aaattacggt ttacttaaca 540taaataacgc gtactctcta gcaagctcca ttgcaagcgc tgcatcctcg agtgcatctt 600cggcagcagc agcagcggcg tcatcttctt ccgcagcagc aggagcagcc gcggcttcag 660gtacagcagc ttctgccgcc gccacttcca ccaccacaac aacaagtact tctagagccg 720ctgcagcagc atccgccgca gccgcggcct ctgcctcggg agccgccgac gcagcgggag 780cagcatcagc cgctagcgct gcttcagctt cttcgtcctt gcaacaatct ctgggatctg 840ccttagcaca aagtagctca tttgcagcag ccttcgccca agcaaatagc gctgcttctg 900cagcagccat agcatatgct cttgcacaga ccgtggcaaa tcaaatcggt ttctcttcct 960actcctcagc tttcgcaagc gcagcttctt cagccgtatc cagcttaggg ggcttcgctt 1020ctgcatctgc atatgccttt gcttttgcca gcgccttttc acaagttctc tcaaattacg 1080gtttacttaa cataaataac gcctactctc tagcaagctc cattgcaagc gctgcatcct 1140cgagtgcatc ttcggcagca gcagcggcat catattcctt ctcagcaaca ggagcagcct 1200cttcggcagc agtaggtgcg gcagcgacat ctggtgcagc gacatctggt gcagcgactt 1260cctctagctc tgcgacgggt gtcggaggaa gtgtctcctc tggagcatca cccgcttccg 1320ctggaactgc aacaggtggc ggtatctcat ttctacctgt ccagacacaa cgtggtttcg 1380ggcttgtgcc ctctccttca ggtaatattg gtgcaaattt tcctggttct ggtgaatttg 1440gtccatcacc tttgacatca ccagtttatg gtccgggtat tcttggccct gggcttgtcg 1500tgccctcatt acaggggctg ttgccacctt tatttgtttt accatcgaat tcggcaactg 1560aaagaatttc gtccatggta tcgtctttgt tgtccgcagt ttcttccaat ggattggatg 1620cttcttcttt tggtgatacc atagcttccc tggtttcgca gatatccgtg aataattccg 1680atctttcttc gtcacaagtc ttgcttgagg cgctccttga aattttgtct ggaatggtac 1740aaatcctttc ttatgctgaa gtcgggactg ttaatacgaa gaccgtgagt tcaacttccg 1800ctgctgtggc tcaagctatc tcttcggctt tttcgggaaa tcagaattct tgagctgcct 1860aatgaaggtt ttttttcatc aaatattttt aaaatattat gacccactga tttaattttt 1920attactatca atattggaag tgaaatttaa taggtgttgt tatttctgct gtgtaatgtt 1980ggtaatggtt gtaaatgtaa ctagtatggt attggtaaaa aaaaaaaaaa aaaaaaaaaa 2040aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2070101925DNAAvicularia juruensis 10acaacaagca cttctacagc cgcagcagca gccgcagcag cggacttcgg ctcgggagcc 60gcccgcgcag cgcaaacagc atcagccgct agcgctgctt cagcttcttc gtccttgcaa 120cagtctttgg gatctgcctt agcacaaagt agctcatttg cagcagcctt cgaccaagca 180aatagcgctg cttctgcagc agccatagca tacgctcttg cacagtccgc ggcgaatcaa 240gtcggtttgt cttcctactc cgcagctatc tcaaacgcag ctgcagcagc cgtaggaagc 300gtaggtggct acgcttctgc atctgcctat gcctttgctt ttgccagcgg cgtttcacaa 360gttctatcaa attacggttt aattaaccta agtaacgcct tatttttagc aagttcgata 420gcaaacgctg catcggcgag tgcatcttcg gcagcagcag cggcgtcatc ttcttccgca 480gcaacaggag cagccgcggc tttgggaggc gctggttctg ccgccgccac ttccaccacc 540acaataacaa gcacttctac agccgtagca gcagcctctg gctcgggagc cgcccgcgca 600gcgcaaacag catcagccgc tagcgctgct tcagcttctt cgtcccttgc acagtctttg 660ggatctgcct tagcacaaag tagctcattt gcagcagcct tcgaccaagg caatagcgct 720gcttctgcag cagccatagc atacgttctt gcacagtccg cggcgaataa agtcggtttg 780tcttcctact ccgcagctat ctcaaacgct gcttcagcag ccgtagaaag cgtaggtggc 840tacgcttccg catctgccca tgcctttgct tttgccagcg ccgtttcaca agttctatca 900aattacggtt taattaacct aagtaacgcc ttgtccctag caagttcgat agcaaacgct 960gtatcggcga gtgcatcttc ggcagcagct gtgtcatctg ctgcagcagc aacaggtgca 1020acctcttcgg cagcagtagg tgcagcagcg acatgtgggg cagcgacttc cgctagttct 1080gcgacgggcg tcggagaaac tgttgcctgt gcaacatcgc ccgcgtccac tggaaccgcg 1140gcaggtggcg gtatctcatc tttacctgtt cagacacaac ctggttttgg gtttttgctc 1200tctccctcag gtaatattgg tccaagtgtt tctggttctg gtgggtttgg tccatcacct 1260ttgccatctc cagcttctga cggatttagc ccatcgcctt tgccatcaca agtttatggt 1320cctggtattc ttggtcccgg tctcgtcgca ccttcgttag aagggctgtt gccaccttta 1380tcaattttgc catcggattc tgcaaatgaa agaatttcgt ctgtagtatc ttctttgttg 1440gccgccgttt cttccaatgg attggatgct tcttctcttg gcgataactt agcttcactg 1500gtttcgcaga tatccgcgaa taatgccgat ctttcttcgt cacaagttat ggttgaggct 1560cttcttgaag ttttgtctgg aatagttcag atcctttctt atgctgaagt tggggctgtt 1620aatacggaaa ccgtaagttc aacttcctct gctgtggctc aagctatttc ttcggctgtt 1680ttgggataat caaaattctt gagctgccta atgaaactgt ttttttttta acaaatattt 1740taaaaatatt atggcccact gatttaattt tcattagtat caatgttgga agtgggaatt 1800taatatgttt tgtttatttc tgctgtgtaa tgttgttaat ggttgtatat gtaactagta 1860tggtattggt aataaaaaca ttgcatttga aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920aaaaa 1925111874DNAAvicularia juruensis 11gttcgggatc tggttctgga agtggagcag ggtctggtgg aggaagtggt gcgggatctg 60gttctggaag cggagcagga gcaggatctg gtagtggctc aggttcagga agtggagcag 120gatctggtag tggctcaggt tcaggaagtg gagcaggttc aggtagtggc tcaggttcag 180gaagtggagc acgatctggt agtggctcag gttcaggaag tggagcaggt tcgggtagtg 240gctcaggttc aggaagtggg gcaggtgcag gtagtggttc aggttcagga agtggagcag 300gttcaggtag tggagcaggt tcaggtagtg gctcaggttc aggaagtggg gcaggttcag 360gtagtggctc aggttcagga agtggggcag gcgcaggtag tggctcaggt tcaggacgtg 420gagcaggttc aggtagtggt tcaggttcag gaagtggagc aggttcaggt agtggctcag 480gttcaggacg tggagcaggt tcaggtagtg gctcaggttc aggaagtggg gcaggtgcag 540gtagtggctc aggttcagga agtggagcag gttcaggtag tggctcaggt tcaggaagtg 600gagcaggttc aggtagtggc tcaggttcag gaagtggagc tggatctggt agtggctcag 660gttcaggaag tggagcagga tctggtagtg gctcaggttc aggaagtgga gcaggttcag 720gtagtggctc aggttcagga agtggagcag gttcaggtag tggctcaggt tcaggaagtg 780gagcaggctc aggtagtggc tcaggttcag gaagtggagc aggagctggt agtggttcag 840gttcatgtag aaaagatgca ggtggtcatg atggcggata tgggaaaaag cttggttttg 900aattcggtac gcctgcagca gcagctgtta cccttggacc tggagctgga caacaaggcc 960caggtggagc tggacaacaa ggaccaggag gccaaggacc atatggacca gttgctagcg 1020ccgccgcagc tgttgctgga ggttatggac ctggagcttt accacaagga ccagcacgcc 1080aaggaccttc cggtcctgtt tcttcagcac cagttgcatc ggcagctgct gctcgccttt 1140cttctcctca ggctagttct agagtatctt cagctttttt ttctttggta tcaagtggtc 1200caactagtcc tggtgcactt tctaatgcca tcagtagtgt tgtttcacaa gttagtgcaa 1260gcaatccagg tctctctggt tgcgatgtac tcgtgcaagc attgctggaa attgtatccg 1320cccttgtatc tatccttgcg tcatctagta tcgggcaaat caactatgga gcttccgctc 1380agtatgcctc tttggttggc caatctgtaa atcaagctct tcgttattaa tttagcaaat 1440gatttgcaaa cttttttcaa tgttactaac acatactttt aaaatttctc aataaatttg 1500aagcatatta tatttcctct tgtgttattt atttgttaca tgcggagatg aacattgatc 1560ctgttacaaa tttatattta aaattatttc tttaaataat cgaaagtgga ttaaaagtac 1620ttttacaaaa ctttgcattt agatttcatg aaaaaatatt tgtttcagcg ttagtaaacg 1680ataaacattt ttggtcctat caattattaa ttttttttat aatcttttga ttgccatatt 1740tataatttct ttaaaattat ttacgatttc tcctacattt tcttttttaa atccattttc 1800atgtgtcttt caagaatttt gtgattaaat gtggtatttt ttcatgataa aaaaaaaaaa 1860aaaaaaaaaa aaaa 1874121922DNAAvicularia juruensis 12aacaaacttt aagcatgact tcgtttaccc aaatcgcaac aacgcttttt tttgttttcg 60tgggggttgc aatcgccagg gacaacaatc tttctccaaa cgttccatgg ataactcagg 120aagatggcga aagttttttg ttagcctttg aagaagccat atctgaaaaa atgcaacccg 180atgggataag tgacttagaa tttctgtttg gaagcttgct gtcattaata cctcagagat 240cggggagtct gtctgtcgct aggttacaag cacttaatat ggctttggca tctattatcg 300ccgaaattgt aagaatagat gggagaggat ccatggaaga aaaaattgaa ttcgttcgtg 360aggggctcat aaaggcattc ctagcaacat caggcttcgt caatacagca cttataaagg 420aagttttaag tatgattcga cttttctatg aggaagagga aggtgataac acaatagacc 480agaacttccc tcagcaggaa tacccagaag ttacttccca attcgactcg gcgggtaaat 540tcgaaacctt tgattcagta gctggtcaac aagcctcaac agaagcaagt cagtcttcat 600cgactgtgtc cacaacgaca tcgactagtc agacatactc tgaacaaacg gccagctcat 660cggatgttgc ctccacagca gcaacatcag aagcatcttc ccgttttaca caatacgtga 720cgtctttctt gctgcaggat ttagaatttg ttgatcagta taacaccata gcatcttcag 780gtatagctag tacgttagca tccgcatctg ctgaagctgt agcatattca atcggtcaag 840gcagtatcgc gtcagctata gcatctgctg tttctcaggc tacagcaaat atctcctttg 900tgactgtacc ttttgttttc gtccatgctt ttgcatctgc ggtgtcagag accctttctg 960cttttggtgt gttgaacttg gacaatgtaa atacactagc aagtgaattc gcaaattctc 1020tgtttaatgc tatattaaca gcttcagctt cttctacgac atcagcttca gcttcttcta 1080cgacatcagc ttcagcttct tctacgacat cagcttcagc ttcttctacg acatcagctt 1140cagcttcttc tgagacatca gcttcagctg catcagcttc aacagcacta caaacagatt 1200ctactgctgc aggatcctta gcttcgtccg gaacttcaag tgcgaactac ggaccatcgt 1260ttggtattga aagtccgttt tcccctgctt ttggggctgg aagtgggccg aacacttttg 1320atttcctcac tccatcgcca agcattcctg ctcttccaac caatccagaa ctttctcgct 1380actcaccatt aatatctgag ctgttacagt ccccttctgg tttaaagtct cctgcagcag 1440acgaaagaat tgcttcttca gtaccactgc ttgctttagc ggtaacaaat ggctttaatc 1500cttctctctt ctcagttgtt ttgtcttcgt tagtttctca gatttctcaa agctctagtt 1560ttacgtcatc tcaggttctc attgaagcaa ttttggagat aatatccggt atgttgaaca 1620tcctaacttc agcacaactt ggtttggtga gtacagcttc actggctgca acagtttctt 1680ctattgtcca gtctatttcc agttcaataa ttgcatgaat caacatgtta aagtatcata 1740tgaaaaaaaa aagcagaaac tgtagaaaaa ttaaatttga ctttcaggct aagctgacgc 1800aaaaaaaaaa aaaaaatatg catttatata tacgtttaag taacaaatca tgtagcctca 1860tttacctaaa atgcaaactg tactaaaata aaaaatcttt ctttttctcc aaaaaaaaaa 1920aa 1922132588DNAAvicularia juruensis 13acggcactgg tggcagacat gatgaggatg ataagggacg aactggtgaa agacatgatg 60aaggttccaa aggaggaact aacggaagac atggtgaaag ttccagagaa ggccctgacg 120gaagacatgg tgaacgtccc agaggaggag tagacggaag acatggtgaa ggttgcagag 180aaggcgctga cggaagacat agtgaaggtt ccagaggagg ttctggcgaa agacatggtg 240aaggttccag aggaggagta gacggaagac atggtgaagg ttgcagagaa ggcgctgacg 300gaagacatag taaaggttcc agaggaggcg ctggcgaaag acatggtgaa ggttccagag 360gaggagtaga cggaagacat ggtgaaggtt gcagagaagg ctctgaaaga agacacggtg 420aaggttccag aggagacgca gacggaaggc acggtgaagg ttacaaagga ggctctgaaa 480gaagacatgg tgaaggtttc agaagaggag tagacggaag acatggtgaa ggttgcagag 540gaggccctga aagaagacat ggtgaaggat ccagaatagg aggtgacgga aaacatggtg 600aaggttccag agaaggcgct catggagggc atggtgaagg gcccagagaa ggagtagacg 660gaggacatgg tgaacgttcc agaggaggag tagacggaag acatggtgaa ggttccagag 720aaggagctga tggaggacat ggtgaaggtt caagagaagg agtagatgga agacatggcg 780aaggttcgag aggaggagtg gacggaagac atggcgaagg ttcgagagga ggttttgacg 840gaagacatgg tgaaggttgc aaaggaggcc ctgaaagaag acatggtgaa ggatccagaa 900taggaggtga cggaaaacat ggtgaaggtt ccagagaagg cgctcacgga gggcatggtg 960aagggcccag agaaggagta gacggaggac atggtgaacg ttccagagga gaagtagatg 1020gaggacatgg tgaaggtacc agagaaggcg ctgacggagg atatggtgaa ggttccagag 1080aaggcgatga cggaagacat ggtgaacgtc ccagaggagg agtagacgga agacatggtg 1140aaggttgcag agaaggcgct gacggaagac atagtgaagg ttccagagga ggcgctggcg 1200aaagacatgg tgaaggttcc agaggaggag tagacggaag acatggtgaa ggttgcagag 1260aaggcgctga cggaagacat agtgaaggtt ccagaggagg cgctggcgaa agacatggtg 1320aaggttccag aggaggagta gacggaagac atggtgaagg ttgcagagaa ggctatgaaa 1380gaagacatgg tgcaggttgt agaggaggaa tatacagaag acatgatgaa ggttacaaag 1440gaggctctga aagaagacat ggtgaaggtt tcagaagagg agtagacgaa agacatggtg 1500aaggttgcag aggaggccct gaaagaagac atggtgaagg atccagaata ggaggtgacg 1560gaaaacatgg tgaaggttcc agagaaggcg ctcacggaag gcatggtgaa gggcccagaa 1620aaggagtaga cggaggacat ggtgaagggc ccagagaagg agtagacgga ggacatggtg 1680aacgttccag aggaggagta gacggaagac atggtgaagg ttccagagaa ggcgctgacg 1740gaggtcatgg tgaaggttcc agagaaggag tagatggaag acatggcgaa ggttcgagag 1800gaggagtgga cggaagacat ggcgaaggtt cgagaggagg ttttgacgga agacatggtg 1860aaggcagaga aggcgctgac ggaggacatg gtgaaggttc cagagaagga gctgagggag 1920gatatgggga aggttccaga ggaggagtag acggaggaca tggtgaaggt tccagagaag 1980gcgtagacag aggccatggt gaaggttcca gagaagacgc tgacggagga tctgctgaag 2040gttccagaga aggcgatgac ggaaaacgtg gtggtgacgc tggtggtgat gcaaaagtcg 2100cctttgaaag cgacagtgga tggaaagggt atcaacagtc atggggatat gaagaccgtt 2160atagttttgg aaaattaaat ggacatgatg ctagtggaaa ttaaaaccgt agggacaacg 2220accacggaag tgtcgctaga aaagtaagtt acaatggaat tgacattaaa aaacaaatcg 2280gattttcagt agaaggagaa aaacgtttct gatatatttc ctttgacgta ctcttagtcc 2340aattagtttg tgacactctc tgcaagcatt tggaacgaat gtatttacat gatgctattg 2400gaattttgga attgacatta aatatttctg ccttatgaac ttaacaatat gacaaaagca 2460cgtgttttta ccttataatg aatctttcaa atgtgcaaca tcattgcttg cagaattaat 2520ctaataaata aaggatgttc tggaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2580aaaaaaaa 2588142090DNAParawixia bistriata 14gccgttcaag cattgtctag ttctctcggc atcgacggaa ataacttggc aagaatagcg 60tcacaaacaa tccttagagt tcccgcaggt tcagacactt ctgcatacgc tcaagcattc 120tctactgctt tgttcaattc tggagttctg aatgcaagta acgttaatac attgggatca 180caagtcgtgt caacactttt aagaggaata tcaagtacgg cacaaggcct tggcttaaac 240gtagacgctg gaagtgtaca gagcgacatt agttctagca gcagcttcct atccacaagc 300tcttcatcga ccagttcctc tcagacaact gctgcttcga catctggatt cacaggagcc 360tcataccccg gacctcaagt ctcacaacca gcaccatttg gcgttggacc tcagcctggt 420ggagcacttc ccggtttcgg ccaagtaagc ggcgcgcaaa gtgccctgat ctccagaata 480gcaaacgcat tgggaaatac agcaacaatg agagcggttc ttagaagcgg tgtttcccaa 540cagattgtct cgaacgtagt acaaggagcc gttcaagcat tgtctagttc tctcggcatc 600gacggaaata acttggcaag aatagcgtca caaacaatcc ttagagttcc cgcaggttca 660gacacttctg catatgctca agcattctct actgctttgt tcaattctgg agttctgaat 720gcaagtaacg ttaatacatt gggatcacaa gtcgtgtcaa cacttttaag aggaatatca 780agtacggcac aaggccttgg cttaaacgta gacgctggaa gtgtacagag cgacattagt 840tctagcagca gcttcctatc cacaagctct tcatcgacta gttcctctca gacaactgct 900gcttcgacat ctggattcac aggagcctca taccccggac ctcaagtctc acaaccagca 960ccatttggcg ttggacctca gcctggtgga gcacttcccg gtttcggcca agtaagcggc 1020gcgcaaagtg ccctgatctc cagaatagca aacgcattgg gaaatacagc aacaatgaga 1080gcggttctta gaagcggtgt ttcccaacag attgtctcga acgtagtaca aggagccgtt 1140caagcattgt ctagttctct cggcatcgac ggaaataact tggcaagaat agcgtcacaa 1200acaatcctta gagttcccgc aggttcagac acttctgcat acgctcaagc attctctact 1260gctttgttca attctggagt tctgaatgca agtaacgtta atacattggg atcacaagtc 1320gtgtcaacac ttttgagagg aatatcaaat acggcacaag gccttggctt aaacgtagac 1380gctggaagtg tacagagcga cattagttct agcagcagct tcctatccac aagctcttca 1440tcgactagtt cctctcagac aactgctgct tcgacatctg gattcgcaag agcatacact 1500ggacctcaaa tctcacaacc tgcacctttg ggcgttggac ctcaggtctc acaacctcga 1560cctttaggcg ttgctcctca gacttctggg gcaaggcctt ttggtggagt aactgggccg 1620tcggctggaa tttctttagg atctgccctt aattcaccga ttggactgag atctggtttg 1680gcagcagcta gaattagcca actgacatca tctctaggga atgccatcac cccctatggc 1740gttgatgcta atgctcttgc cagcagtcta caagcaagtt tctcaactct tcaaagttcc 1800ggtatgtctg caagcgatgc taaaatcgaa gttttgttag aaactatagt aggactgctt 1860caactcttga gcaacactca gattcgtgga gtgaacatgg ctacggcgtc ttctgtggcg 1920agttctgctg ccaaatcatt tgaattagtt ttatcttaaa gtttttgatc ttttttcagt 1980cgcgtaaaat tttattttcc gatatgtaaa ttacagatga aatttttgtt caagcacaat 2040aaaaagcatt ttttcaacgc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2090151479DNAParawixia bistriata 15aggagcccca ggcggagcag gaggtggtgt tggaccagga ggcggtgctg gtggaacctc 60tggaggtgct agcggatcag gtccagtatc tgtctctact gctgtaaatg tcggcggtgc 120tggcggacca ggtgcaggtg gaccaggagc tggtggcgtc ggaccgggtg ttgtaggccc 180tggaggactt ggaggtcccg gtggattcgg tggaccaggc ggtcctggag gaccaggcgg 240ccctggggca ccaggcggcg ctggaggaat gttcggtcct ggaggtgctg gaggcatgta 300cggacctgga ggtgctggag gcatgtacgg acctggtggt gctggacgag gtccaggagg 360agctggtgca cctggagctc cgggaggacc aggaggtccc ggaggaccag gaggattcgg 420aggtggagca ggagctggtg gtatggttcc tggtggagcc tctagaggtc ctggcggatc 480aggcccagta acagttacag agacagttac agttggagga gccggaggac caggacctgg 540tggaatcggt ggatcgtcag gtcctggagc aggcggtgca ccaggtggat tcggtggtcc 600tggaggtcct ggtggacctg gaggacccgg aggtccaggt ggtgcagccg gaggaccagg 660agctggtggt gcaggtcctg gaggatctgg tccggcaact gtttcttctt ctgtaactgt 720tgttggcgct ggaggacccg gtggaccagg agctggtgga atcgttccag gaggtattta 780cggtccagga ggagctggtg gtgtcgtacc gggcggtatt tacggtccag gaggagtacc 840tagtggacca ggaggtccag gtggacctgt tggtccaggt ggttacggag ctcctggtgg 900attaggtgtt ggtattttac ctgggactgc tagtgctgga acttctggcc

caacaactgt 960cacagaagtt gtttccatta atgttagtgg tggtcaatca tcaagtggtg tccgacccgg 1020aaatagctac actcctgcag ctggaggatc cgcaagatta ccttctctta ttaacggtgt 1080catgagttct atgcaaggag gtggatttaa ttaccagaat ttcggcaatg ttctctcgca 1140gtttgctact ggatccggaa cttgcaatag caatgatata aatctgttaa tggatgctct 1200ttttgcggct ctccataccc taagctatca aggacagtcc tctgttccaa catatccttc 1260gcccgctgca atgtcctctt attcgcaatc tgttcgaggg tgctttggat attaattgag 1320tttttatgat gtttgaatag tctcaaattc ttattatgca ttgtttgaaa ataagttttt 1380tgtaaatgtt ttgtttttaa gttctcataa aactaattaa taagataata aattattatt 1440gtgcaagcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1479161709DNAParawixia bistriata 16ggaggatacg gcgcaggagc tggtgccggt gcaggtgcag ccgctgctgc tgctgctgga 60gctggtgctg gaggcggata tggtggagga tacggcgcag gaggtggtgc cggtgcaggt 120gctggagcag gtgcaggtgc aggtgctgga gcaggagccg gacgtggagg tgccggtgga 180tatggcgctg gagctggtgc cggtgcaggt gcagcagctg ccgccgctgc tggagctggt 240gctggaggcg gatatggtgg aggatacggt gcaggagttg gtgccggtgc aggagccgga 300gctggtgctg gaggcggata tggtggagga tacggcgcag gagctggtgc cggtgcaggt 360gcagccgctg ctgccgctgc tggagctggt gctggaggcg gatatggtgg aggatacggc 420gcaggaggtg gtgccggtgc aggtgctgga gcaggtgcag gtgcaggtgc tggagcagga 480gccggacgag gaggtgccgt tggatatgga gctggtgccg gtgcaggtgc agcagctgcc 540gccgctgctg gagctggtgc tggaggtgga tatggtggag gatatggtgc aggaggtggt 600gccggtgcag gagccggagc tggtgctgga ggcggatatg gtggaggata cggcgcagga 660ggtggtgccg gtgcaggtgc aggtgctgga gcaggagccg gacgaggagg tgccggtgga 720tatggcgctg gagccggtgc cgctgcaggt gcagcagctg ccgccgctgc tggagctggt 780gctggaggcg gatatggtgg aggatacggt gcaggaggtg gtgctggtgc aggtgctggt 840gcagcggctg gcgccggagc tggagctgga cgaggaggtg ccggtggata tggcgctgga 900gctggtgccg gtgcaggtgc agctgctgct gctggagctg gtgctggagg cggatatggt 960ggaggataca gcgcaggagg tggtgctggt gctggttcag gtgctgcggc aggagccgga 1020gctggacgag gaggtgccgg tggatatagc gctggagctg gtaccggtgc aggtgcagct 1080gctggagctg gtacagcagg cggatatagt ggaggatacg gtgccggagc ttcttcaagt 1140gctggaagca gtttcatttc ttcaagttcg atgagctcat ctcaagcaac tggatacagt 1200tcctcaagtg gatatggagg tggagctgcg agtgctgccg ctggtgcagg agctgctgca 1260ggcggatatg gtggaggtta cggagcagga gctggtgctg gtgcagccgc tgcttcaggt 1320gccactggca gagtagcaaa tagtcttgga gcaatggctt ctggtggaat taatgcctta 1380cctggtgtat tttcaaacat cttctcacaa gtaagtgctg cttcgggtgg tgcctctggt 1440ggtgcagttc tagttcaggc tttgacagag gttattgcct tgcttcttca tatattaagc 1500agtgcctcta tcggtaatgt tagttcacaa ggattagaag gctcaatggc tattgctcaa 1560caggccatag gagcttacgc tggttgagtc agatgacagt ctctctcatt taaaagtata 1620acttttgtct actagcttct atgtctatga ttgtatttaa tacaagcaaa taaatttagc 1680attctgaaaa aaaaaaaaaa aaaaaaaaa 1709171754DNAParawixia bistriata 17gccgccacag catcagcagc tggtggactt ggaggacagg gaggacttgg tggattaggt 60tcacaaggag ctggtctagg aggatacgga caaggaggag ccggtcaagg tggtgccgcc 120gccgcagcag cagcagctgg tggacttgga ggacaaggcg gacgaggtgg attaggttca 180caaggagctg gccaaggagg atatggacaa ggaggagccg gtcaaggtgg tgccgccgcc 240gcagcagcag cagctggtgg acttggagga caaggaggac ttggtgcatt aggttcacaa 300ggagctggtc aaggaggagc tggccaagga ggatacggac aaggaggagc cgcagcagca 360gcagctggtg gacttggagg acaaggagga cttggtggat taggttcaca aggagctgga 420caaggaggat acggacaagg aggagccggt caaggtggtg ccgccgccgc cgcagcagca 480gctggtggac ttggaggaca gggaggactt ggtggattag gttcacaagg agctggtcca 540ggaggatacg gacaaggagg agccggtcaa ggtggtgccg ccgccgcagc agcagcagct 600ggtggacttg gaggacaagg aggacttggt gcattaggtt cacaaggagc tggtcaagga 660ggatacggac aaggaggagc tggtcaaggt ggtgccgccg ccgcagcagc tgctggtgga 720cttggaggac aaggaggact tggtgcatta ggttcacaag gagctggcca aggaggatat 780ggacaaggag gttcacaagg agctggccaa ggaggatacg gacaaggagg agccgccgct 840gccgcagcag cagctggtgg acttggaggc caaggaggac taggtggatt aggttcacaa 900ggagctggcc aaggaggata tggacaagga ggttcacaag gagctggcca aggaggagcc 960gccgccgcag cagcagcagc tggtggactt ggaggacaag gaggatttgg tggattaggt 1020tcacaaggag ctggtcaagg aggatacgga caaggaggag ccggtcaagg tggtgccgcc 1080gccgcagcag cagcagctgg tgtacttgga ggacaaggag gacttggtgg attaggttca 1140caaggagctg gtcaaggtgg atacggacaa ggaggagctg gtcaaggtgg tgccgccgcc 1200gccgcagcag cagcagcagc tggtggactt ggaggacaag gaggacgagg tggattaggt 1260tcacaaggag ctggccaagg gggatacgga caaggaggag ctggtgcctc atccgctgct 1320gctgcgtctg ctgctgcttc tcgtctgtca tccgcaagtg ctgcttctag ggtctcgtct 1380gccgtttcat ctttagtatc aagtggtgga ccaactaatt ctgcagcatt gtcgagtacc 1440attagcaatg ttgtatctca agttagcgca agcaaccctg gtctttctgg ttgtgatgtt 1500ctcgtccagg cgctacttga aatcgtttca gcattggttc atattcttgg atcctctagt 1560attggtcaag ttaactataa tgccgctggt caatcagcgt cagttgtcgg acagtctttt 1620taccaagctc ttgcttaaga aagtcatgtg aacccttctg aattttcttt ttctttaata 1680gtcttgtttt gtatatgttc tttaaaataa atttttgcat gattgaaaaa caaaaaaaaa 1740aaaaaaaaaa aaaa 1754181481DNAParawixia bistriatamodified_base(1364)..(1364)a, c, t, g, unknown or other 18caaggaccag gagcaggtca acaaggacca ggagcaggtc aacaaggacc atatggacca 60agtgccgccg ccgcagcagc tgccgctgga ggttatggac caggaggtgc aggacaacaa 120ggaccaggag caggtcaaca aggaccaggt agtcaaggac aatctggacc tggtgctacc 180gtcgccgcag ctgccgctgg aggttatgga ccaggagggg caggacaaca aggaccagga 240gcaggtcaac aaggtcaagg tagccaagga ccatatggac cagctgccac cgccgccgca 300gccgccgctg gaggttatgg accaggatct ggacaacagg gaccaggagc aggtcaacaa 360ggaccaggag gtcaaggacc atatggacca agtgccgccg ccgcagcagc tgccgctgga 420ggttatggac caggatctgg acaacaagga ccaggagcag gtccacaagg cccaggtagt 480caaggacctt atggaccagg tgccgccgca gcagcagccg ccgttggagg ttatggacca 540ggatctggac aacaaggacc aggagcaggt caacaaggac caggaggtca aggaccatat 600ggaccaagtt ccgccgccgc agcagctgcc gctggaggtt atggaccagg aggtgcagga 660caacaagtac caggagcagg tcaacaaggt ccaggtaacc aaggaccatc tggaccaggt 720gccgccgccg cagcagctgc cgctggaggt tatggaccag gaggtgcagg acaacaagga 780ccagcagcag gtcaacaagg tccaggtagt caaggatcat atggaccagg tgccgccgct 840gcagcagctg ctgctggagg ttatggacca ggatctggac aacaaggacc aggaggagct 900ggtcaacaag gacctggagg tcaaggacct tatggacctg gctcttcttc agcagcagcc 960gtcggaggtt atggaccaag ttctggatta caaggaccag caggtcaagg gccttatgga 1020cctggtgcag ctgcttccgc agcagcagcc gctggggctt ctcgcctttc ttctccacag 1080gccagttcca gagtttcatc tgctgtatct tctttggtat caagcggtcc tacgaattcc 1140gctgcactta ccaataccat tagtagcgtt gtatcacaaa ttagtgcaag taatccaggt 1200ctctctggtt gcgatgtact tatacaagcg ttattggaaa ttgtatctgc ccttgtacac 1260attcttggat attctagtat cggccaaatc aactatgatg ctgccgcaca gtatgcgtca 1320ttggttggtc agtctgtagc tcaagccctt gcttgatgca ttancactgg atttgcaatt 1380ttttgttaaa ttactttaat ataattttaa attttctcaa taaactgnan cattnnnngn 1440nnnnnaaaan nnnaannnnn nnnnnnaaan aannnnnnnn n 148119807DNAParawixia bitriata 19ggtggtgcca atggcgcttc cgctgcagca gcttcagctg gaggtgcagg aggatatgga 60agtgatggag gatatggtca aggtggacaa ggagctggag gcgatggctc tgctgccgcc 120gcagccgcag cagcatccgg tggacgaggt ggacaaggcg gatttggttc tcaaggagca 180ggtggtagag gtctaggagg atctgcacga ggtggtgctg gtggtacttc cgctgcagct 240gcatcagctg gtggtgcaag aggatatgga ggtgacggag gatatggtca aggtggatct 300ggacgaggtg gtgctggtag tgcttccgca gcagcggctt cagctggagg tgcaggagga 360tatggaggtg acggaggata tggtgaaggt ggacaaggag caggaggcga tggagtcgca 420acttcttctg ctgcttcccg tctgtcatct ccctcttcta tacgaagaat atcggaagtt 480gtatctacat tctcagatga tgactttgga aattcagctt ctttttcaaa tgtttataac 540agtgtggctt ctggaattac atcatccaat cctggtctct ctggatgcga tgttcaaatt 600caaacgttac ttgaaatgaa ttcggcattg cttgctttac tttatggatt tgatgcttat 660tcgtcggctg ctttagtaaa cgatttcgtt aatcaacctc attaatgagc gatataactt 720ttctttttaa aatttttcaa ttgtaatatg taaatcttac taaataaaat tatgcaatga 780taaaaaaraa aaaaaaaaaa aaaaaaa 807201037PRTNephilengys cruentata 20Ala Gly Gly Ser Gly Gly Thr Thr Val Ile Glu Asp Leu Asp Ile Thr1 5 10 15Val Asn Gly Pro Gly Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Ile 20 25 30Gly Gly Pro Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly 35 40 45Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly 50 55 60Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Gly Gly65 70 75 80Ala Gly Gly Pro Phe Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly 85 90 95Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly 100 105 110Pro Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Ser Gly Gly Pro Tyr 115 120 125Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly 130 135 140Ala Tyr Gly Pro Gly Gly Ala Gly Gly Pro Gly Gly Ala Gly Gly Pro145 150 155 160Tyr Gly Pro Gly Gly Pro Tyr Gly Pro Gly Gly Pro Tyr Gly Pro Gly 165 170 175Gly Ala Gly Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly 180 185 190Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr 195 200 205Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly 210 215 220Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro225 230 235 240Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly 245 250 255Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly 260 265 270Ser Gly Pro Gly Gly Pro Gly Ser Gly Gly Pro Gly Gly Ala Gly Gly 275 280 285Ser Gly Gly Thr Thr Val Ile Glu Asp Leu Asp Ile Thr Val Asn Gly 290 295 300Pro Gly Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Val Gly Gly Pro305 310 315 320Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 325 330 335Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly 340 345 350Pro Gly Gly Ala Gly Gly Pro Phe Gly Pro Gly Gly Ser Gly Pro Gly 355 360 365Gly Ala Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Tyr Gly 370 375 380Pro Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Ser Gly385 390 395 400Gly Pro Tyr Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly Ala Tyr Gly 405 410 415Pro Gly Gly Ala Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro 420 425 430Gly Gly Pro Tyr Gly Pro Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly 435 440 445Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Pro 450 455 460Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly465 470 475 480Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly 485 490 495Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala 500 505 510Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 515 520 525Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro 530 535 540Gly Gly Pro Gly Ser Gly Gly Pro Gly Gly Ala Gly Gly Ser Gly Gly545 550 555 560Thr Thr Val Ile Glu Asp Leu Asp Ile Thr Val Asn Gly Pro Gly Gly 565 570 575Pro Ile Thr Ile Ser Glu Glu Leu Thr Val Gly Gly Pro Gly Ala Gly 580 585 590Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly 595 600 605Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Val 610 615 620Gly Pro Gly Gly Ala Gly Gly Pro Gly Gly Ala Gly Gly Pro Phe Gly625 630 635 640Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro Tyr 645 650 655Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly 660 665 670Pro Gly Gly Pro Gly Gly Pro Gly Ser Gly Gly Pro Tyr Gly Pro Gly 675 680 685Gly Ala Tyr Gly Pro Gly Gly Ala Tyr Gly Pro Gly Gly Ala Tyr Gly 690 695 700Pro Gly Gly Ala Gly Gly Pro Gly Gly Ala Ala Gly Pro Tyr Gly Pro705 710 715 720Gly Gly Pro Tyr Gly Pro Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly 725 730 735Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Pro 740 745 750Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly 755 760 765Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly 770 775 780Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala785 790 795 800Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 805 810 815Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro 820 825 830Gly Gly Pro Gly Ser Gly Gly Pro Gly Gly Ala Gly Gly Ser Gly Gly 835 840 845Thr Thr Val Ile Glu Asp Leu Asp Ile Thr Leu Asn Gly Pro Gly Gly 850 855 860Pro Ile Thr Ile Ser Glu Glu Leu Thr Val Gly Gly Pro Gly Ala Gly865 870 875 880Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly 885 890 895Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ala 900 905 910Gly Gly Pro Tyr Gly Ser Gly Gly Phe Gly Phe Gly Gly Ala Gly Gly 915 920 925Ser Gly Gly Pro Tyr Val Pro Gly Gly Ala Tyr Gly Ala Gly Ser Gly 930 935 940Thr Pro Ser Tyr Ser Gly Ser Arg Val Pro Asp Leu Val Asn Gly Ile945 950 955 960Met Arg Ser Met Gln Gly Ser Gly Phe Asn Tyr Gln Met Phe Gly Asn 965 970 975Met Leu Ser Lys Tyr Ala Ser Gly Ser Gly Ala Cys Asn Ser Asn Asp 980 985 990Val Asn Val Leu Met Asp Ala Leu Leu Ala Ala Leu His Cys Leu Ser 995 1000 1005Ser His Gly Ser Pro Ser Phe Gly Ser Ser Pro Thr Pro Ser Ala 1010 1015 1020Met Asn Ala Tyr Ser Asn Ser Val Arg Arg Met Phe Gln Phe 1025 1030 103521461PRTNephilengys cruentata 21Ala Ser Gln Ser Ala Ser Ser Ser Ser Ala Ser Ala Ser Ala Phe Ala1 5 10 15Gln Gln Ser Ser Ala Ser Leu Ala Ala Ser Ser Ser Phe Ser Gln Ala 20 25 30Phe Ala Ser Ala Ala Ser Ala Ser Ala Val Gly Asn Val Ala Tyr Gln 35 40 45Leu Gly Leu Ser Ala Ala Gln Ser Leu Gly Ile Ala Asn Ala Gly Ala 50 55 60Leu Ala Ser Ala Leu Ala Gln Ser Val Ser Ser Val Gly Val Gly Ala65 70 75 80Ser Ser Ser Ala Tyr Ala Asn Ala Val Ala Gly Ala Val Gly Gln Phe 85 90 95Leu Ala Asn Gln Gly Ile Leu Asn Thr Gly Asn Ala Ser Ser Leu Ala 100 105 110Ser Ser Phe Ser Ser Ala Leu Ser Ala Ser Ala Ala Ala Ala Gln Ser 115 120 125Gln Ser Phe Ala Gln Ser Gln Ala Ala Ala Ser Ala Phe Gln Gln Ala 130 135 140Ala Ser Gln Ser Ala Ser Gln Ser Ala Ala Gln Ser Gly Ser Gln Ser145 150 155 160Ser Ser Thr Thr Thr Thr Thr Ser Ala Ser Gly Ser Gln Ser Ala Ser 165 170 175Gln Ser Ala Ser Ser Ser Ser Ala Ser Ala Ser Ala Phe Ala Gln Gln 180 185 190Ser Ser Ala Ser Leu Ala Ala Ser Ser Ser Phe Ser Gln Ala Phe Ala 195 200 205Ser Ala Ala Ser Ala Ser Ala Val Gly Asn Val Ala Tyr Gln Leu Gly 210 215 220Leu Ser Ala Ala Gln Ser Leu Gly Ile Ala Asn Ala Gly Ala Leu Ala225 230 235 240Ser Ala Leu Ala Gln Ser Val Ser Ser Val Gly Val Gly Ala Ser Ser 245 250 255Ser Ala Tyr Ala Asn Ala Val Ala Gly Ala Val Gly Gln Phe Leu Ala 260 265 270Asn Gln Gly Ile Leu Asn Thr Gly Asn Ala Ser Ser Leu Ala Ser Ser 275 280 285Phe Ser Asn Ala Leu Ser Ser Ser Ala Ala Asn Ser Val Gly Ser Gly 290 295 300Leu Leu Leu Gly Pro Ser Gln Tyr Val Gly Ser Ile Ala Pro Ser Ile305 310 315 320Gly Gly Ala Ala Gly Ile Ser Ile Ala Gly Pro Gly Ile Leu Ser Tyr 325 330 335Leu Pro Pro Val Ser Pro Leu

Asn Ala Gln Ile Ile Ser Ser Gly Leu 340 345 350Leu Ala Ser Leu Ala Pro Val Leu Ser Ser Ser Gly Leu Ala Ser Ser 355 360 365Ser Ala Thr Ser Arg Val Gly Ser Leu Ala Gln Ser Leu Ala Ser Ala 370 375 380Leu Gln Ser Ser Gly Gly Thr Leu Asp Val Ser Thr Phe Leu Asn Leu385 390 395 400Leu Ser Pro Ile Ser Thr Gln Ile Gln Ala Asn Thr Ser Leu Asn Ala 405 410 415Ser Gln Ala Ile Val Gln Val Leu Leu Glu Ala Val Ala Ala Leu Leu 420 425 430Gln Ile Ile Asn Gly Ala Gln Ile Thr Ser Val Asn Phe Gly Ser Val 435 440 445Ser Ser Val Asn Thr Ala Leu Ala Thr Ala Leu Ala Gly 450 455 46022505PRTNephilengys cruentata 22Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly1 5 10 15Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala Ala 20 25 30Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala 35 40 45Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala Gly Gln Gly Gly 50 55 60Tyr Glu Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala65 70 75 80Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly 85 90 95Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly 100 105 110Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala Gly Gln Gly Ala 115 120 125Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly 130 135 140Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala145 150 155 160Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gly Gln Gly Gly Tyr Gly 165 170 175Arg Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala 180 185 190Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala Gly Ala Ala 195 200 205Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly 210 215 220Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala Gly Gln Gly225 230 235 240Gly Tyr Gly Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala 245 250 255Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln 260 265 270Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala 275 280 285Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala 290 295 300Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser305 310 315 320Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala 325 330 335Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala 340 345 350Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ser Gly Arg 355 360 365Gly Gly Tyr Gly Ser Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala 370 375 380Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Ala Gly Ser Gly Ala385 390 395 400Ala Ala Ala Ser Ala Ala Ala Ser Arg Leu Ser Ser Pro Glu Ala Ser 405 410 415Ser Arg Val Ser Ser Ala Val Ser Asn Leu Val Ser Ser Gly Pro Thr 420 425 430Asn Ser Ala Ala Leu Ser Asn Thr Ile Ser Ser Val Val Ser Gln Ile 435 440 445Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val Gln Ala 450 455 460Leu Leu Glu Val Val Ser Ala Leu Ile His Ile Leu Gly Ser Ser Ser465 470 475 480Ile Gly Pro Val Asn Tyr Gly Ser Ala Ser Gln Ser Thr Gln Ile Val 485 490 495Gly Gln Ser Val Tyr Gln Ala Leu Gly 500 505231119PRTNephilengys cruentata 23Ala Gly Gly Ala Gly Gly Tyr Gly Val Gly Gln Gly Tyr Gly Ala Gly1 5 10 15Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly 20 25 30Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Val Gly Ala Ala 35 40 45Ala Ala Ala Gly Ala Gly Ala Gly Val Gly Gly Ala Gly Gly Tyr Gly 50 55 60Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly65 70 75 80Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly 85 90 95Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Val Gly Ala Ala 100 105 110Ala Ala Ala Gly Ala Gly Ala Gly Val Gly Gly Ala Gly Gly Tyr Gly 115 120 125Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly 130 135 140Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly145 150 155 160Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly 165 170 175Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala 180 185 190Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 195 200 205Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly 210 215 220Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly225 230 235 240Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly 245 250 255Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly 260 265 270Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly 275 280 285Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly 290 295 300Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly305 310 315 320Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala 325 330 335Gly Arg Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly 340 345 350Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly 355 360 365Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly 370 375 380Ala Gly Ala Gly Val Gly Gly Ala Gly Gly Tyr Gly Ser Gly Ala Gly385 390 395 400Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ser Gly Ala Ala Ala Gly 405 410 415Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Thr Gly 420 425 430Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 435 440 445Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly 450 455 460Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly465 470 475 480Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Asp Gly Ala Gly Ala Gly 485 490 495Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly 500 505 510Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly 515 520 525Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly 530 535 540Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly545 550 555 560Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Val Gly 565 570 575Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Ala 580 585 590Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala 595 600 605Gly Gly Tyr Gly Thr Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly 610 615 620Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly625 630 635 640Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Glu Tyr Gly Ala Gly 645 650 655Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly 660 665 670Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly 675 680 685Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala 690 695 700Gly Gly Tyr Gly Ala Arg Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly705 710 715 720Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly 725 730 735Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly 740 745 750Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly 755 760 765Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Gln Gly Tyr Gly 770 775 780Ser Gly Ala Gly Ala Gly Ala Gly Ala Ser Ala Gly Gly Ala Gly Ser785 790 795 800Tyr Gly Arg Gly Ala Gly Ala Gly Ala Ala Ala Ala Ser Gly Ala Gly 805 810 815Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala 820 825 830Val Ala Ser Ala Ala Ala Gly Ala Gly Ser Gly Ala Gly Gly Ala Gly 835 840 845Gly Tyr Gly Arg Gly Ala Val Ala Gly Ser Gly Ala Gly Ala Gly Ala 850 855 860Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala865 870 875 880Ala Ala Gly Ala Val Ala Gly Gly Ser Gly Gly Tyr Gly Gly Arg Gln 885 890 895Gly Gly Tyr Ser Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly 900 905 910Ala Gly Ala Gly Gly Thr Gly Gly Tyr Gly Arg Gly Ser Gly Ala Gly 915 920 925Ala Ala Ala Gly Ala Ala Ala Gly Ala Gly Ala Ala Gly Gly Tyr Gly 930 935 940Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Arg Gly Tyr945 950 955 960Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Gly Tyr Gly 965 970 975Arg Arg Ala Gly Gly Ser Ile Val Gly Thr Gly Ile Ser Ala Ile Ser 980 985 990Ser Gly Thr Gly Ser Ser Tyr Ser Val Ser Ser Gly Gly Tyr Ala Ser 995 1000 1005Ala Gly Val Gly Val Gly Ser Thr Val Ala Ser Thr Thr Ser Arg 1010 1015 1020Leu Ser Ser Ala Gln Ala Ser Ser Arg Ile Ser Ala Ala Ala Ser 1025 1030 1035Thr Leu Ile Ser Gly Gly Tyr Leu Asn Thr Ser Ala Leu Pro Ser 1040 1045 1050Val Ile Ser Asp Leu Phe Ala Gln Val Ser Ala Ser Ser Pro Gly 1055 1060 1065Val Ser Asp Ser Glu Val Leu Ile Gln Val Leu Leu Glu Ile Val 1070 1075 1080Ser Ser Leu Ile His Ile Leu Ser Ser Ser Ser Val Gly Gln Val 1085 1090 1095Asp Phe Asn Ser Val Gly Ser Ser Ala Ala Ala Val Gly Gln Ser 1100 1105 1110Met Gln Val Val Met Gly 111524387PRTNephilengys cruentata 24Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Thr Gly Gly Ala1 5 10 15Gly Gly Tyr Gly Arg Gly Val Gly Ala Gly Ala Gly Ala Gly Ala Gly 20 25 30Ala Gly Gly Ala Gly Gly Tyr Gly Gly Gly Gln Asn Tyr Gly Ala Gly 35 40 45Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala 50 55 60Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly65 70 75 80Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly 85 90 95Tyr Gly Thr Gly Gln Gly Tyr Gly Glu Gly Ala Gly Ala Gly Ala Gly 100 105 110Val Gly Ala Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly 115 120 125Ala Gly Ala Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala 130 135 140Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Ala145 150 155 160Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala 165 170 175Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala 180 185 190Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala 195 200 205Gly Ala Gly Gly Ala Arg Gly Tyr Gly Ala Arg Gln Gly Tyr Gly Ser 210 215 220Gly Ala Gly Ala Gly Ala Gly Ala Arg Ala Gly Gly Ala Gly Gly Tyr225 230 235 240Gly Arg Gly Ala Gly Ala Gly Ala Ala Ala Ala Ser Gly Ala Gly Ala 245 250 255Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Val 260 265 270Ala Ser Ala Ala Ala Gly Ala Gly Ser Gly Ala Gly Gly Ala Gly Gly 275 280 285Tyr Gly Arg Gly Ala Gly Ala Val Ala Gly Ala Gly Ala Gly Gly Ala 290 295 300Gly Gly Tyr Gly Ala Gly Ala Gly Ala Ala Ala Gly Val Gly Ala Gly305 310 315 320Gly Ser Gly Gly Tyr Gly Gly Arg Gln Gly Gly Tyr Ser Ala Gly Ala 325 330 335Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Thr Gly 340 345 350Gly Tyr Gly Arg Gly Ser Gly Ala Gly Ala Ala Ala Gly Ala Ala Ala 355 360 365Gly Ala Gly Ala Ala Gly Gly Tyr Gly Gly Tyr Gly Ala Gly Ala Gly 370 375 380Ala Gly Ser38525430PRTNephilengys cruentata 25Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala1 5 10 15Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly 20 25 30Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly 35 40 45Ala Gly Ala Gly Gly Ala Gly Gly Phe Gly Arg Gly Ala Gly Ala Gly 50 55 60Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala65 70 75 80Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly 85 90 95Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly 100 105 110Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala 115 120 125Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly 130 135 140Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly145 150 155 160Ala Gly Thr Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly 165 170 175Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 180 185 190Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala Gly 195 200 205Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Val Gly Gln Gly Tyr Gly 210 215 220Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Ala Ala Gly Asn Ala225 230 235 240Phe Ala Gln Ser Leu Ser Ser Asn Leu Leu Ser Ser Gly Asp Phe Val 245 250 255Gln Met Ile Ser Thr Thr Thr Ser Thr Asp Gln Ala Val Ser Val Ala 260 265 270Thr Ser Val Ala Gln Asn Val Gly Asn Gln Leu Gly Leu Asp Ala Asn 275 280 285Ala Met Asn Asn Leu Leu Ala Ala Val Gly Gly Tyr Val Ser Ser Leu 290 295 300Gly Gly Ala Val Ala Asp Ala Ala Ala Tyr Ala Asn Ala Ile Ser Ser305 310 315 320Ala Ile Gly Asn Val Leu Ala Asn Thr Gly Ser Ile

Asn Glu Ser Thr 325 330 335Ala Ser Ser Ala Ala Ser Ser Ala Ala Ser Ser Val Thr Thr Thr Leu 340 345 350Thr Ser Tyr Gly Pro Ala Val Phe Tyr Gly Arg Gly Ala Gly Ala Gly 355 360 365Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly 370 375 380Ser Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Ala Gly385 390 395 400Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ser Gly Gly Tyr Gly 405 410 415Gly Arg Gln Gly Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ser 420 425 43026673PRTNephilengys cruentata 26Ala Pro Arg Pro Leu Pro Ala Pro Ala Pro Arg Pro Leu Pro Ala Pro1 5 10 15Ala Pro Arg Pro Leu Pro Ala Pro Leu Pro Ala Pro Leu Pro Arg Pro 20 25 30Arg Pro Ala Pro Ile Val Ser Gln Val Gln Gln Ala Ser Ala Leu Gln 35 40 45Ala Gln Ser Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Ser Ser Ile 50 55 60Ala Gln Ser Gln Gln Ala Ser Val Ala Gln Ser Gln Arg Ala Ser Val65 70 75 80Ser Gln Ser Gln Gln Ser Ser Asn Ala Phe Ser Ser Ala Ala Ser Phe 85 90 95Gly Ala Ser Ser Val Ala Ser Ser Ala Ser Thr Tyr Phe Asn Ser Gly 100 105 110Ile Val Gln Ser Ser Ile Ala Ser Ser Leu Gln Ser Ser Ser Ala Leu 115 120 125Ser Ser Ile Ala Tyr Gly Gln Thr Thr Ala Ser Ile Asn Asp Ile Ala 130 135 140Ser Ala Val Ala Gly Ser Ile Ala Asn Ser Ile Gly Leu Ser Gln Gln145 150 155 160Thr Val Gln Ser Ile Ile Ser Gln Gln Leu Ala Ser Ala Gly Ser Gly 165 170 175Ala Ser Ala Gln Thr Leu Ala Ser Leu Ile Ser Ser Ala Val Ser Ser 180 185 190Leu Val Gln Gln Ser Gly Ser Val Ser Ala Gly Gln Glu Gln Ser Ile 195 200 205Ser Gln Ala Leu Ser Ser Ser Ile Ser Ser Ser Leu Asn Gln Leu Val 210 215 220Ala Ala Arg Pro Leu Pro Ala Pro Ala Pro Arg Pro Leu Pro Ala Pro225 230 235 240Leu Pro Ala Pro Leu Pro Arg Pro Arg Pro Ala Pro Ile Val Ser Gln 245 250 255Val Gln Gln Ala Ser Ala Leu Gln Ala Gln Ser Gln Gln Ser Ala Phe 260 265 270Ala Gln Ser Gln Gln Ser Ser Ile Ala Gln Ser Gln Gln Ala Ser Val 275 280 285Ala Gln Ser Gln Gln Ser Ser Ile Ser Gln Ser Gln Gln Ala Ser Val 290 295 300Ser Gln Ser Gln Gln Ser Ser Asn Ala Phe Ser Ser Ala Ala Ser Ser305 310 315 320Gly Ala Ser Ser Val Ala Ser Ser Ala Ser Thr Tyr Phe Asn Ser Gly 325 330 335Ile Val Gln Ser Ser Ile Ala Ser Ser Leu Gln Ser Ser Ser Ala Leu 340 345 350Ser Ser Ile Ala Tyr Gly Gln Thr Thr Ala Ser Ile Ser Asp Ile Ala 355 360 365Ser Ala Val Ala Gly Ser Ile Ala Asn Ser Ile Gly Leu Ser Gln Gln 370 375 380Thr Val Gln Ser Val Ile Ser Gln Gln Leu Ala Ser Ala Gly Ser Gly385 390 395 400Ala Ser Ala Gln Thr Leu Ala Ser Leu Ile Ser Ser Ala Val Ser Ser 405 410 415Leu Val Gln Gln Ser Gly Ser Val Ser Ala Gly Gln Glu Gln Ser Ile 420 425 430Ser Gln Ala Leu Ser Ser Ser Ile Ser Ser Ser Leu Asn Gln Leu Val 435 440 445Ala Ala Arg Pro Leu Pro Ala Pro Ala Pro Arg Pro Leu Pro Ala Pro 450 455 460Leu Pro Ala Pro Leu Ser Arg Pro Arg Pro Val Pro Val Gln Arg Pro465 470 475 480Gln Pro Val Phe Ser Pro Ser Pro Ala Pro Ala Tyr Ala Pro Ala Pro 485 490 495Phe Thr Gln Gln Ser Thr Phe Ala Gln Ser Gln Gln Ala Ser Leu Ala 500 505 510Gln Ser Gln Gln Gln Ala Ser Ile Ala Arg Ser Gln Gln Ala Ser Leu 515 520 525Ala Gln Ser Gln Gln Ser Ala Phe Ala Gln Ser Gln Gln Val Ala Thr 530 535 540Ala Gln Ser Gln Gln Ser Ser Gly Gly Phe Ser Thr Ser Ser Thr Gly545 550 555 560Ala Ser Gln Ile Ser Ser Ser Ala Ile Ser Thr Ser Ser Gly Ser Ala 565 570 575Leu Ala Asn Ser Ala Gln Gln Leu Thr Ser Pro Ala Ala Ser Gln Arg 580 585 590Ile Ser Gln Leu Ser Asn Ser Leu Ala Ser Ala Val Ser Gly Gly Gln 595 600 605Val Asn Tyr Ala Ala Leu Ser Asn Ser Ile Ala Ser Ala Ala Ser Gln 610 615 620Ile Gly Gly Gly Ser Gly Leu Ser Lys Thr Glu Val Leu Ile Glu Thr625 630 635 640Leu Leu Glu Thr Leu Ala Ala Leu Leu Glu Ser Leu Ser Leu Pro Gly 645 650 655Ser Ala Ser Gly Gly Ser Gln Phe Ala Gln Ala Met Leu Ala Ala Leu 660 665 670Ala27306PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 27Met Tyr Asn Arg Gln Val Leu Pro Ile Tyr Ile Leu Val Ile Val Ser1 5 10 15Leu Ala Ile Leu Thr Thr His Val Ser Thr Ser Lys Gln Arg Pro Phe 20 25 30Tyr Ile Met Gly His Met Val Asn Ser Ile Glu Glu Ile Ser Glu Phe 35 40 45Leu Glu Arg Gly Ser Asn Val Leu Glu Ser Asp Val Gln Phe Phe Ser 50 55 60Asn Gly Ser Val Lys Ala Val Arg His Gly Phe Pro Cys Asp Cys Gly65 70 75 80Arg Phe Cys Glu Asn Thr Ala Asn Leu Ala Asp Tyr Leu Gln Ser Val 85 90 95Arg Tyr Thr Asp Pro Asp Thr Pro Asp Ser Tyr Tyr Asn Gln Leu Val 100 105 110Leu Gln Phe Phe Asp Leu Lys Leu Ser Thr Ser Glu Asn Lys Arg Gln 115 120 125Ser Gly Arg Glu Ile Ala His His Val Leu Asp Tyr Leu Tyr Gly Glu 130 135 140Glu Gly Glu Arg Glu Lys Glu Ile Arg Val Val Ile Tyr Phe Glu Lys145 150 155 160Leu Glu Glu Lys Asp Val Ile Leu Gly Phe Met Asp Val Phe Lys Leu 165 170 175Arg Asn Gln Thr Ser Arg Leu Arg Asp Val Gly Phe Asp Gly Gly Thr 180 185 190Gly Asn Ile Ser Asp Ile Ala Arg Met Phe Ser Lys Phe Asn Ile Lys 195 200 205Asp Asn Ile Tyr Leu Gly Asp Gly Ala Thr Asn Cys Phe Glu Pro Phe 210 215 220Lys Ser Phe Val Arg Leu Lys Asn Ala Ile Asp Asn Arg Asp Ser Arg225 230 235 240Lys Gly Phe Val Ser Lys Ile Tyr Gln Tyr Thr Asn Asp Ile Lys Thr 245 250 255Thr Met Met Arg Ser Leu Arg Leu Gly Val Asp Gly Met Ile Thr Asn 260 265 270Lys Pro Glu Arg Leu Leu Glu Val Leu Gln Glu Pro Glu Phe Ala Lys 275 280 285Asp Phe Arg Leu Ala Thr Ile Tyr Asp Asp Pro Phe Glu Tyr Phe Cys 290 295 300Asp Glu30528616PRTAvicularia juruensis 28Tyr Ser Leu Ala Ser Ser Ile Ala Ser Ala Ala Ser Ser Ser Ala Ser1 5 10 15Ser Ala Ala Ala Ala Ala Ser Ser Ser Ser Ala Ala Ala Gly Ala Ala 20 25 30Ala Ala Ser Glu Ala Ala Ala Ser Ala Ala Ala Thr Ser Thr Thr Thr 35 40 45Thr Thr Ser Thr Ser Arg Ala Ala Ala Ala Ala Ser Ala Ala Ala Ala 50 55 60Ala Ser Ala Ser Gly Ala Ala Gly Ala Ala Gly Ala Ala Ser Ala Ala65 70 75 80Ser Ala Ala Ser Ala Ser Ser Ser Leu Gln Gln Ser Leu Gly Ser Ala 85 90 95Leu Ala Gln Ser Ser Ser Phe Ala Ala Ala Phe Ala Gln Ala Ser Ser 100 105 110Ala Ala Ser Ala Ala Ala Ile Ala Tyr Ala Leu Ala Gln Thr Val Ala 115 120 125Asn Gln Ile Gly Phe Ser Ser Tyr Ser Ser Ala Phe Ala Arg Ala Ala 130 135 140Ser Ser Ala Val Tyr Ser Ile Gly Gly Leu Ala Ser Ala Ser Ala Tyr145 150 155 160Ala Phe Ala Phe Ala Ser Ala Phe Ser Gln Val Leu Ser Asn Tyr Gly 165 170 175Leu Leu Asn Ile Asn Asn Ala Tyr Ser Leu Ala Ser Ser Ile Ala Ser 180 185 190Ala Ala Ser Ser Ser Ala Ser Ser Ala Ala Ala Ala Ala Ala Ser Ser 195 200 205Ser Ser Ala Ala Ala Gly Ala Ala Ala Ala Ser Gly Thr Ala Ala Ser 210 215 220Ala Ala Ala Thr Ser Thr Thr Thr Thr Thr Ser Thr Ser Arg Ala Ala225 230 235 240Ala Ala Ala Ser Ala Ala Ala Ala Ala Ser Ala Ser Gly Ala Ala Asp 245 250 255Ala Ala Gly Ala Ala Ser Ala Ala Ser Ala Ala Ser Ala Ser Ser Ser 260 265 270Leu Gln Gln Ser Leu Gly Ser Ala Leu Ala Gln Ser Ser Ser Phe Ala 275 280 285Ala Ala Phe Ala Gln Ala Asn Ser Ala Ala Ser Ala Ala Ala Ile Ala 290 295 300Tyr Ala Leu Ala Gln Thr Val Ala Asn Gln Ile Gly Phe Ser Ser Tyr305 310 315 320Ser Ser Ala Phe Ala Ser Ala Ala Ser Ser Ala Val Ser Ser Leu Gly 325 330 335Gly Phe Ala Ser Ala Ser Ala Tyr Ala Phe Ala Phe Ala Ser Ala Phe 340 345 350Ser Gln Val Leu Ser Asn Tyr Gly Leu Leu Asn Ile Asn Asn Ala Tyr 355 360 365Ser Leu Ala Ser Ser Ile Ala Ser Ala Ala Ser Ser Ser Ala Ser Ser 370 375 380Ala Ala Ala Ala Ala Ser Tyr Ser Phe Ser Ala Thr Gly Ala Ala Ser385 390 395 400Ser Ala Ala Val Gly Ala Ala Ala Thr Ser Gly Ala Ala Thr Ser Gly 405 410 415Ala Ala Thr Ser Ser Ser Ser Ala Thr Gly Val Gly Gly Ser Val Ser 420 425 430Ser Gly Ala Ser Pro Ala Ser Ala Gly Thr Ala Thr Gly Gly Gly Ile 435 440 445Ser Phe Leu Pro Val Gln Thr Gln Arg Gly Phe Gly Leu Val Pro Ser 450 455 460Pro Ser Gly Asn Ile Gly Ala Asn Phe Pro Gly Ser Gly Glu Phe Gly465 470 475 480Pro Ser Pro Leu Thr Ser Pro Val Tyr Gly Pro Gly Ile Leu Gly Pro 485 490 495Gly Leu Val Val Pro Ser Leu Gln Gly Leu Leu Pro Pro Leu Phe Val 500 505 510Leu Pro Ser Asn Ser Ala Thr Glu Arg Ile Ser Ser Met Val Ser Ser 515 520 525Leu Leu Ser Ala Val Ser Ser Asn Gly Leu Asp Ala Ser Ser Phe Gly 530 535 540Asp Thr Ile Ala Ser Leu Val Ser Gln Ile Ser Val Asn Asn Ser Asp545 550 555 560Leu Ser Ser Ser Gln Val Leu Leu Glu Ala Leu Leu Glu Ile Leu Ser 565 570 575Gly Met Val Gln Ile Leu Ser Tyr Ala Glu Val Gly Thr Val Asn Thr 580 585 590Lys Thr Val Ser Ser Thr Ser Ala Ala Val Ala Gln Ala Ile Ser Ser 595 600 605Ala Phe Ser Gly Asn Gln Asn Ser 610 61529562PRTAvicularia juruensis 29Thr Thr Ser Thr Ser Thr Ala Ala Ala Ala Ala Ala Ala Ala Asp Phe1 5 10 15Gly Ser Gly Ala Ala Arg Ala Ala Gln Thr Ala Ser Ala Ala Ser Ala 20 25 30Ala Ser Ala Ser Ser Ser Leu Gln Gln Ser Leu Gly Ser Ala Leu Ala 35 40 45Gln Ser Ser Ser Phe Ala Ala Ala Phe Asp Gln Ala Asn Ser Ala Ala 50 55 60Ser Ala Ala Ala Ile Ala Tyr Ala Leu Ala Gln Ser Ala Ala Asn Gln65 70 75 80Val Gly Leu Ser Ser Tyr Ser Ala Ala Ile Ser Asn Ala Ala Ala Ala 85 90 95Ala Val Gly Ser Val Gly Gly Tyr Ala Ser Ala Ser Ala Tyr Ala Phe 100 105 110Ala Phe Ala Ser Gly Val Ser Gln Val Leu Ser Asn Tyr Gly Leu Ile 115 120 125Asn Leu Ser Asn Ala Leu Phe Leu Ala Ser Ser Ile Ala Asn Ala Ala 130 135 140Ser Ala Ser Ala Ser Ser Ala Ala Ala Ala Ala Ser Ser Ser Ser Ala145 150 155 160Ala Thr Gly Ala Ala Ala Ala Leu Gly Gly Ala Gly Ser Ala Ala Ala 165 170 175Thr Ser Thr Thr Thr Ile Thr Ser Thr Ser Thr Ala Val Ala Ala Ala 180 185 190Ser Gly Ser Gly Ala Ala Arg Ala Ala Gln Thr Ala Ser Ala Ala Ser 195 200 205Ala Ala Ser Ala Ser Ser Ser Leu Ala Gln Ser Leu Gly Ser Ala Leu 210 215 220Ala Gln Ser Ser Ser Phe Ala Ala Ala Phe Asp Gln Gly Asn Ser Ala225 230 235 240Ala Ser Ala Ala Ala Ile Ala Tyr Val Leu Ala Gln Ser Ala Ala Asn 245 250 255Lys Val Gly Leu Ser Ser Tyr Ser Ala Ala Ile Ser Asn Ala Ala Ser 260 265 270Ala Ala Val Glu Ser Val Gly Gly Tyr Ala Ser Ala Ser Ala His Ala 275 280 285Phe Ala Phe Ala Ser Ala Val Ser Gln Val Leu Ser Asn Tyr Gly Leu 290 295 300Ile Asn Leu Ser Asn Ala Leu Ser Leu Ala Ser Ser Ile Ala Asn Ala305 310 315 320Val Ser Ala Ser Ala Ser Ser Ala Ala Ala Val Ser Ser Ala Ala Ala 325 330 335Ala Thr Gly Ala Thr Ser Ser Ala Ala Val Gly Ala Ala Ala Thr Cys 340 345 350Gly Ala Ala Thr Ser Ala Ser Ser Ala Thr Gly Val Gly Glu Thr Val 355 360 365Ala Cys Ala Thr Ser Pro Ala Ser Thr Gly Thr Ala Ala Gly Gly Gly 370 375 380Ile Ser Ser Leu Pro Val Gln Thr Gln Pro Gly Phe Gly Phe Leu Leu385 390 395 400Ser Pro Ser Gly Asn Ile Gly Pro Ser Val Ser Gly Ser Gly Gly Phe 405 410 415Gly Pro Ser Pro Leu Pro Ser Pro Ala Ser Asp Gly Phe Ser Pro Ser 420 425 430Pro Leu Pro Ser Gln Val Tyr Gly Pro Gly Ile Leu Gly Pro Gly Leu 435 440 445Val Ala Pro Ser Leu Glu Gly Leu Leu Pro Pro Leu Ser Ile Leu Pro 450 455 460Ser Asp Ser Ala Asn Glu Arg Ile Ser Ser Val Val Ser Ser Leu Leu465 470 475 480Ala Ala Val Ser Ser Asn Gly Leu Asp Ala Ser Ser Leu Gly Asp Asn 485 490 495Leu Ala Ser Leu Val Ser Gln Ile Ser Ala Asn Asn Ala Asp Leu Ser 500 505 510Ser Ser Gln Val Met Val Glu Ala Leu Leu Glu Val Leu Ser Gly Ile 515 520 525Val Gln Ile Leu Ser Tyr Ala Glu Val Gly Ala Val Asn Thr Glu Thr 530 535 540Val Ser Ser Thr Ser Ser Ala Val Ala Gln Ala Ile Ser Ser Ala Val545 550 555 560Leu Gly30476PRTAvicularia juruensis 30Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Gly Gly Ser1 5 10 15Gly Ala Gly Ser Gly Ser Gly Ser Gly Ala Gly Ala Gly Ser Gly Ser 20 25 30Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser 35 40 45Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala 50 55 60Arg Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser65 70 75 80Gly Ser Gly Ser Gly Ser Gly Ala Gly Ala Gly Ser Gly Ser Gly Ser 85 90 95Gly Ser Gly Ala Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser 100 105 110Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 115 120 125Gly Ala Gly Ala Gly Ser Gly Ser Gly Ser Gly Arg Gly Ala Gly Ser 130 135 140Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser145 150 155 160Gly Ser Gly Arg Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 165 170 175Gly Ala Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser 180 185

190Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser 195 200 205Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 210 215 220Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser225 230 235 240Gly Ser Gly Ser Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser 245 250 255Gly Ser Gly Ser Gly Ala Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 260 265 270Gly Ala Gly Ala Gly Ser Gly Ser Gly Ser Cys Arg Lys Asp Ala Gly 275 280 285Gly His Asp Gly Gly Tyr Gly Lys Lys Leu Gly Phe Glu Phe Gly Thr 290 295 300Pro Ala Ala Ala Ala Val Thr Leu Gly Pro Gly Ala Gly Gln Gln Gly305 310 315 320Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 325 330 335Pro Val Ala Ser Ala Ala Ala Ala Val Ala Gly Gly Tyr Gly Pro Gly 340 345 350Ala Leu Pro Gln Gly Pro Ala Arg Gln Gly Pro Ser Gly Pro Val Ser 355 360 365Ser Ala Pro Val Ala Ser Ala Ala Ala Ala Arg Leu Ser Ser Pro Gln 370 375 380Ala Ser Ser Arg Val Ser Ser Ala Phe Phe Ser Leu Val Ser Ser Gly385 390 395 400Pro Thr Ser Pro Gly Ala Leu Ser Asn Ala Ile Ser Ser Val Val Ser 405 410 415Gln Val Ser Ala Ser Asn Pro Gly Leu Ser Gly Cys Asp Val Leu Val 420 425 430Gln Ala Leu Leu Glu Ile Val Ser Ala Leu Val Ser Ile Leu Ala Ser 435 440 445Ser Ser Ile Gly Gln Ile Asn Tyr Gly Ala Ser Ala Gln Tyr Ala Ser 450 455 460Leu Val Gly Gln Ser Val Asn Gln Ala Leu Arg Tyr465 470 47531571PRTAvicularia juruensis 31Gln Thr Leu Ser Met Thr Ser Phe Thr Gln Ile Ala Thr Thr Leu Phe1 5 10 15Phe Val Phe Val Gly Val Ala Ile Ala Arg Asp Asn Asn Leu Ser Pro 20 25 30Asn Val Pro Tyr Ile Thr Gln Glu Asp Gly Glu Ser Phe Leu Leu Ala 35 40 45Phe Glu Glu Ala Ile Ser Glu Lys Met Gln Pro Asp Gly Ile Ser Asp 50 55 60Leu Glu Phe Leu Phe Gly Ser Leu Leu Ser Leu Ile Pro Gln Arg Ser65 70 75 80Gly Ser Leu Ser Val Ala Arg Leu Gln Ala Leu Asn Met Ala Leu Ala 85 90 95Ser Ile Ile Ala Glu Ile Val Arg Ile Asp Gly Arg Gly Ser Met Glu 100 105 110Glu Lys Ile Glu Phe Val Arg Glu Gly Leu Ile Lys Ala Phe Leu Ala 115 120 125Thr Ser Gly Phe Val Asn Thr Ala Leu Ile Lys Glu Val Leu Ser Met 130 135 140Ile Arg Leu Phe Tyr Glu Glu Glu Glu Gly Asp Asn Thr Ile Asp Gln145 150 155 160Asn Phe Pro Gln Gln Glu Tyr Pro Glu Val Thr Ser Gln Phe Asp Ser 165 170 175Ala Gly Lys Phe Glu Thr Phe Asp Ser Val Ala Gly Gln Gln Ala Ser 180 185 190Thr Glu Ala Ser Gln Ser Ser Ser Thr Val Ser Thr Thr Thr Ser Thr 195 200 205Ser Gln Thr Tyr Ser Glu Gln Thr Ala Ser Ser Ser Asp Val Ala Ser 210 215 220Thr Ala Ala Thr Ser Glu Ala Ser Ser Arg Phe Thr Gln Tyr Val Thr225 230 235 240Ser Phe Leu Leu Gln Asp Leu Glu Phe Val Asp Gln Tyr Asn Thr Ile 245 250 255Ala Ser Ser Gly Ile Ala Ser Thr Leu Ala Ser Ala Ser Ala Glu Ala 260 265 270Val Ala Tyr Ser Ile Gly Gln Gly Ser Ile Ala Ser Ala Ile Ala Ser 275 280 285Ala Val Ser Gln Ala Thr Ala Asn Ile Ser Phe Val Thr Val Pro Phe 290 295 300Val Phe Val His Ala Phe Ala Ser Ala Val Ser Glu Thr Leu Ser Ala305 310 315 320Phe Gly Val Leu Asn Leu Asp Asn Val Asn Thr Leu Ala Ser Glu Phe 325 330 335Ala Asn Ser Leu Phe Asn Ala Ile Leu Thr Ala Ser Ala Ser Ser Thr 340 345 350Thr Ser Ala Ser Ala Ser Ser Thr Thr Ser Ala Ser Ala Ser Ser Thr 355 360 365Thr Ser Ala Ser Ala Ser Ser Thr Thr Ser Ala Ser Ala Ser Ser Glu 370 375 380Thr Ser Ala Ser Ala Ala Ser Ala Ser Thr Ala Leu Gln Thr Asp Ser385 390 395 400Thr Ala Ala Gly Ser Leu Ala Ser Ser Gly Thr Ser Ser Ala Asn Tyr 405 410 415Gly Pro Ser Phe Gly Ile Glu Ser Pro Phe Ser Pro Ala Phe Gly Ala 420 425 430Gly Ser Gly Pro Asn Thr Phe Asp Phe Leu Thr Pro Ser Pro Ser Ile 435 440 445Pro Ala Leu Pro Thr Asn Pro Glu Leu Ser Arg Tyr Ser Pro Leu Ile 450 455 460Ser Glu Leu Leu Gln Ser Pro Ser Gly Leu Lys Ser Pro Ala Ala Asp465 470 475 480Glu Arg Ile Ala Ser Ser Val Pro Leu Leu Ala Leu Ala Val Thr Asn 485 490 495Gly Phe Asn Pro Ser Leu Phe Ser Val Val Leu Ser Ser Leu Val Ser 500 505 510Gln Ile Ser Gln Ser Ser Ser Phe Thr Ser Ser Gln Val Leu Ile Glu 515 520 525Ala Ile Leu Glu Ile Ile Ser Gly Met Leu Asn Ile Leu Thr Ser Ala 530 535 540Gln Leu Gly Leu Val Ser Thr Ala Ser Leu Ala Ala Thr Val Ser Ser545 550 555 560Ile Val Gln Ser Ile Ser Ser Ser Ile Ile Ala 565 57032733PRTArtificial SequenceDescription of Artificial Sequence Synthetic polypeptide 32Gly Thr Gly Gly Arg His Asp Glu Asp Asp Lys Gly Arg Thr Gly Glu1 5 10 15Arg His Asp Glu Gly Ser Lys Gly Gly Thr Asn Gly Arg His Gly Glu 20 25 30Ser Ser Arg Glu Gly Pro Asp Gly Arg His Gly Glu Arg Pro Arg Gly 35 40 45Gly Val Asp Gly Arg His Gly Glu Gly Cys Arg Glu Gly Ala Asp Gly 50 55 60Arg His Ser Glu Gly Ser Arg Gly Gly Ser Gly Glu Arg His Gly Glu65 70 75 80Gly Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu Gly Cys Arg Glu 85 90 95Gly Ala Asp Gly Arg His Ser Lys Gly Ser Arg Gly Gly Ala Gly Glu 100 105 110Arg His Gly Glu Gly Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu 115 120 125Gly Cys Arg Glu Gly Ser Glu Arg Arg His Gly Glu Gly Ser Arg Gly 130 135 140Asp Ala Asp Gly Arg His Gly Glu Gly Tyr Lys Gly Gly Ser Glu Arg145 150 155 160Arg His Gly Glu Gly Phe Arg Arg Gly Val Asp Gly Arg His Gly Glu 165 170 175Gly Cys Arg Gly Gly Pro Glu Arg Arg His Gly Glu Gly Ser Arg Ile 180 185 190Gly Gly Asp Gly Lys His Gly Glu Gly Ser Arg Glu Gly Ala His Gly 195 200 205Gly His Gly Glu Gly Pro Arg Glu Gly Val Asp Gly Gly His Gly Glu 210 215 220Arg Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu Gly Ser Arg Glu225 230 235 240Gly Ala Asp Gly Gly His Gly Glu Gly Ser Arg Glu Gly Val Asp Gly 245 250 255Arg His Gly Glu Gly Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu 260 265 270Gly Ser Arg Gly Gly Phe Asp Gly Arg His Gly Glu Gly Cys Lys Gly 275 280 285Gly Pro Glu Arg Arg His Gly Glu Gly Ser Arg Ile Gly Gly Asp Gly 290 295 300Lys His Gly Glu Gly Ser Arg Glu Gly Ala His Gly Gly His Gly Glu305 310 315 320Gly Pro Arg Glu Gly Val Asp Gly Gly His Gly Glu Arg Ser Arg Gly 325 330 335Glu Val Asp Gly Gly His Gly Glu Gly Thr Arg Glu Gly Ala Asp Gly 340 345 350Gly Tyr Gly Glu Gly Ser Arg Glu Gly Asp Asp Gly Arg His Gly Glu 355 360 365Arg Pro Arg Gly Gly Val Asp Gly Arg His Gly Glu Gly Cys Arg Glu 370 375 380Gly Ala Asp Gly Arg His Ser Glu Gly Ser Arg Gly Gly Ala Gly Glu385 390 395 400Arg His Gly Glu Gly Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu 405 410 415Gly Cys Arg Glu Gly Ala Asp Gly Arg His Ser Glu Gly Ser Arg Gly 420 425 430Gly Ala Gly Glu Arg His Gly Glu Gly Ser Arg Gly Gly Val Asp Gly 435 440 445Arg His Gly Glu Gly Cys Arg Glu Gly Tyr Glu Arg Arg His Gly Ala 450 455 460Gly Cys Arg Gly Gly Ile Tyr Arg Arg His Asp Glu Gly Tyr Lys Gly465 470 475 480Gly Ser Glu Arg Arg His Gly Glu Gly Phe Arg Arg Gly Val Asp Glu 485 490 495Arg His Gly Glu Gly Cys Arg Gly Gly Pro Glu Arg Arg His Gly Glu 500 505 510Gly Ser Arg Ile Gly Gly Asp Gly Lys His Gly Glu Gly Ser Arg Glu 515 520 525Gly Ala His Gly Arg His Gly Glu Gly Pro Arg Lys Gly Val Asp Gly 530 535 540Gly His Gly Glu Gly Pro Arg Glu Gly Val Asp Gly Gly His Gly Glu545 550 555 560Arg Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu Gly Ser Arg Glu 565 570 575Gly Ala Asp Gly Gly His Gly Glu Gly Ser Arg Glu Gly Val Asp Gly 580 585 590Arg His Gly Glu Gly Ser Arg Gly Gly Val Asp Gly Arg His Gly Glu 595 600 605Gly Ser Arg Gly Gly Phe Asp Gly Arg His Gly Glu Gly Arg Glu Gly 610 615 620Ala Asp Gly Gly His Gly Glu Gly Ser Arg Glu Gly Ala Glu Gly Gly625 630 635 640Tyr Gly Glu Gly Ser Arg Gly Gly Val Asp Gly Gly His Gly Glu Gly 645 650 655Ser Arg Glu Gly Val Asp Arg Gly His Gly Glu Gly Ser Arg Glu Asp 660 665 670Ala Asp Gly Gly Ser Ala Glu Gly Ser Arg Glu Gly Asp Asp Gly Lys 675 680 685Arg Gly Gly Asp Ala Gly Gly Asp Ala Lys Val Ala Phe Glu Ser Asp 690 695 700Ser Gly Tyr Lys Gly Tyr Gln Gln Ser Tyr Gly Tyr Glu Asp Arg Tyr705 710 715 720Ser Phe Gly Lys Leu Asn Gly His Asp Ala Ser Gly Asn 725 73033652PRTParawixia bistriata 33Ala Val Gln Ala Leu Ser Ser Ser Leu Gly Ile Asp Gly Asn Asn Leu1 5 10 15Ala Arg Ile Ala Ser Gln Thr Ile Leu Arg Val Pro Ala Gly Ser Asp 20 25 30Thr Ser Ala Tyr Ala Gln Ala Phe Ser Thr Ala Leu Phe Asn Ser Gly 35 40 45Val Leu Asn Ala Ser Asn Val Asn Thr Leu Gly Ser Gln Val Val Ser 50 55 60Thr Leu Leu Arg Gly Ile Ser Ser Thr Ala Gln Gly Leu Gly Leu Asn65 70 75 80Val Asp Ala Gly Ser Val Gln Ser Asp Ile Ser Ser Ser Ser Ser Phe 85 90 95Leu Ser Thr Ser Ser Ser Ser Thr Ser Ser Ser Gln Thr Thr Ala Ala 100 105 110Ser Thr Ser Gly Phe Thr Gly Ala Ser Tyr Pro Gly Pro Gln Val Ser 115 120 125Gln Pro Ala Pro Phe Gly Val Gly Pro Gln Pro Gly Gly Ala Leu Pro 130 135 140Gly Phe Gly Gln Val Ser Gly Ala Gln Ser Ala Leu Ile Ser Arg Ile145 150 155 160Ala Asn Ala Leu Gly Asn Thr Ala Thr Met Arg Ala Val Leu Arg Ser 165 170 175Gly Val Ser Gln Gln Ile Val Ser Asn Val Val Gln Gly Ala Val Gln 180 185 190Ala Leu Ser Ser Ser Leu Gly Ile Asp Gly Asn Asn Leu Ala Arg Ile 195 200 205Ala Ser Gln Thr Ile Leu Arg Val Pro Ala Gly Ser Asp Thr Ser Ala 210 215 220Tyr Ala Gln Ala Phe Ser Thr Ala Leu Phe Asn Ser Gly Val Leu Asn225 230 235 240Ala Ser Asn Val Asn Thr Leu Gly Ser Gln Val Val Ser Thr Leu Leu 245 250 255Arg Gly Ile Ser Ser Thr Ala Gln Gly Leu Gly Leu Asn Val Asp Ala 260 265 270Gly Ser Val Gln Ser Asp Ile Ser Ser Ser Ser Ser Phe Leu Ser Thr 275 280 285Ser Ser Ser Ser Thr Ser Ser Ser Gln Thr Thr Ala Ala Ser Thr Ser 290 295 300Gly Phe Thr Gly Ala Ser Tyr Pro Gly Pro Gln Val Ser Gln Pro Ala305 310 315 320Pro Phe Gly Val Gly Pro Gln Pro Gly Gly Ala Leu Pro Gly Phe Gly 325 330 335Gln Val Ser Gly Ala Gln Ser Ala Leu Ile Ser Arg Ile Ala Asn Ala 340 345 350Leu Gly Asn Thr Ala Thr Met Arg Ala Val Leu Arg Ser Gly Val Ser 355 360 365Gln Gln Ile Val Ser Asn Val Val Gln Gly Ala Val Gln Ala Leu Ser 370 375 380Ser Ser Leu Gly Ile Asp Gly Asn Asn Leu Ala Arg Ile Ala Ser Gln385 390 395 400Thr Ile Leu Arg Val Pro Ala Gly Ser Asp Thr Ser Ala Tyr Ala Gln 405 410 415Ala Phe Ser Thr Ala Leu Phe Asn Ser Gly Val Leu Asn Ala Ser Asn 420 425 430Val Asn Thr Leu Gly Ser Gln Val Val Ser Thr Leu Leu Arg Gly Ile 435 440 445Ser Asn Thr Ala Gln Gly Leu Gly Leu Asn Val Asp Ala Gly Ser Val 450 455 460Gln Ser Asp Ile Ser Ser Ser Ser Ser Phe Leu Ser Thr Ser Ser Ser465 470 475 480Ser Thr Ser Ser Ser Gln Thr Thr Ala Ala Ser Thr Ser Gly Phe Ala 485 490 495Arg Ala Tyr Thr Gly Pro Gln Ile Ser Gln Pro Ala Pro Leu Gly Val 500 505 510Gly Pro Gln Val Ser Gln Pro Arg Pro Leu Gly Val Ala Pro Gln Thr 515 520 525Ser Gly Ala Arg Pro Phe Gly Gly Val Thr Gly Pro Ser Ala Gly Ile 530 535 540Ser Leu Gly Ser Ala Leu Asn Ser Pro Ile Gly Leu Arg Ser Gly Leu545 550 555 560Ala Ala Ala Arg Ile Ser Gln Leu Thr Ser Ser Leu Gly Asn Ala Ile 565 570 575Thr Pro Tyr Gly Val Asp Ala Asn Ala Leu Ala Ser Ser Leu Gln Ala 580 585 590Ser Phe Ser Thr Leu Gln Ser Ser Gly Met Ser Ala Ser Asp Ala Lys 595 600 605Ile Glu Val Leu Leu Glu Thr Ile Val Gly Leu Leu Gln Leu Leu Ser 610 615 620Asn Thr Gln Ile Arg Gly Val Asn Met Ala Thr Ala Ser Ser Val Ala625 630 635 640Ser Ser Ala Ala Lys Ser Phe Glu Leu Val Leu Ser 645 65034437PRTParawixia bistriata 34Gly Ala Pro Gly Gly Ala Gly Gly Gly Val Gly Pro Gly Gly Gly Ala1 5 10 15Gly Gly Thr Ser Gly Gly Ala Ser Gly Ser Gly Pro Val Ser Val Ser 20 25 30Thr Ala Val Asn Val Gly Gly Ala Gly Gly Pro Gly Ala Gly Gly Pro 35 40 45Gly Ala Gly Gly Val Gly Pro Gly Val Val Gly Pro Gly Gly Leu Gly 50 55 60Gly Pro Gly Gly Phe Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly65 70 75 80Pro Gly Ala Pro Gly Gly Ala Gly Gly Met Phe Gly Pro Gly Gly Ala 85 90 95Gly Gly Met Tyr Gly Pro Gly Gly Ala Gly Gly Met Tyr Gly Pro Gly 100 105 110Gly Ala Gly Arg Gly Pro Gly Gly Ala Gly Ala Pro Gly Ala Pro Gly 115 120 125Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly Phe Gly Gly Gly Ala Gly 130 135 140Ala Gly Gly Met Val Pro Gly Gly Ala Ser Arg Gly Pro Gly Gly Ser145 150 155 160Gly Pro Val Thr Val Thr Glu Thr Val Thr Val Gly Gly Ala Gly Gly 165 170 175Pro Gly Pro Gly Gly Ile Gly Gly Ser Ser Gly Pro Gly Ala Gly Gly 180 185 190Ala Pro Gly Gly Phe Gly Gly Pro Gly Gly Pro Gly Gly Pro Gly Gly 195 200 205Pro Gly

Gly Pro Gly Gly Ala Ala Gly Gly Pro Gly Ala Gly Gly Ala 210 215 220Gly Pro Gly Gly Ser Gly Pro Ala Thr Val Ser Ser Ser Val Thr Val225 230 235 240Val Gly Ala Gly Gly Pro Gly Gly Pro Gly Ala Gly Gly Ile Val Pro 245 250 255Gly Gly Ile Tyr Gly Pro Gly Gly Ala Gly Gly Val Val Pro Gly Gly 260 265 270Ile Tyr Gly Pro Gly Gly Val Pro Ser Gly Pro Gly Gly Pro Gly Gly 275 280 285Pro Val Gly Pro Gly Gly Tyr Gly Ala Pro Gly Gly Leu Gly Val Gly 290 295 300Ile Leu Pro Gly Thr Ala Ser Ala Gly Thr Ser Gly Pro Thr Thr Val305 310 315 320Thr Glu Val Val Ser Ile Asn Val Ser Gly Gly Gln Ser Ser Ser Gly 325 330 335Val Arg Pro Gly Asn Ser Tyr Thr Pro Ala Ala Gly Gly Ser Ala Arg 340 345 350Leu Pro Ser Leu Ile Asn Gly Val Met Ser Ser Met Gln Gly Gly Gly 355 360 365Phe Asn Tyr Gln Asn Phe Gly Asn Val Leu Ser Gln Phe Ala Thr Gly 370 375 380Ser Gly Thr Cys Asn Ser Asn Asp Ile Asn Leu Leu Met Asp Ala Leu385 390 395 400Phe Ala Ala Leu His Thr Leu Ser Tyr Gln Gly Gln Ser Ser Val Pro 405 410 415Thr Tyr Pro Ser Pro Ala Ala Met Ser Ser Tyr Ser Gln Ser Val Arg 420 425 430Gly Cys Phe Gly Tyr 43535528PRTParawixia bistriata 35Gly Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala1 5 10 15Ala Ala Ala Gly Ala Gly Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly 20 25 30Ala Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly 35 40 45Ala Gly Ala Gly Ala Gly Arg Gly Gly Ala Gly Gly Tyr Gly Ala Gly 50 55 60Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Gly Ala Gly65 70 75 80Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly Ala Gly Val Gly Ala Gly 85 90 95Ala Gly Ala Gly Ala Gly Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly 100 105 110Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Gly 115 120 125Ala Gly Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly Ala Gly Gly Gly 130 135 140Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly145 150 155 160Ala Gly Arg Gly Gly Ala Val Gly Tyr Gly Ala Gly Ala Gly Ala Gly 165 170 175Ala Ala Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Gly Tyr Gly 180 185 190Gly Gly Tyr Gly Ala Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly 195 200 205Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly Ala Gly Gly Gly Ala Gly 210 215 220Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Arg Gly Gly Ala Gly Gly225 230 235 240Tyr Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Ala Ala Ala Ala Ala 245 250 255Ala Gly Ala Gly Ala Gly Gly Gly Tyr Gly Gly Gly Tyr Gly Ala Gly 260 265 270Gly Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly 275 280 285Ala Gly Arg Gly Gly Ala Gly Gly Tyr Gly Ala Gly Ala Gly Ala Gly 290 295 300Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Gly Tyr Gly305 310 315 320Gly Gly Tyr Ser Ala Gly Gly Gly Ala Gly Ala Gly Ser Gly Ala Ala 325 330 335Ala Gly Ala Gly Ala Gly Arg Gly Gly Ala Gly Gly Tyr Ser Ala Gly 340 345 350Ala Gly Thr Gly Ala Gly Ala Ala Ala Gly Ala Gly Thr Ala Gly Gly 355 360 365Tyr Ser Gly Gly Tyr Gly Ala Gly Ala Ser Ser Ser Ala Gly Ser Ser 370 375 380Phe Ile Ser Ser Ser Ser Met Ser Ser Ser Gln Ala Thr Gly Tyr Ser385 390 395 400Ser Ser Ser Gly Tyr Gly Gly Gly Ala Ala Ser Ala Ala Ala Gly Ala 405 410 415Gly Ala Ala Ala Gly Gly Tyr Gly Gly Gly Tyr Gly Ala Gly Ala Gly 420 425 430Ala Gly Ala Ala Ala Ala Ser Gly Ala Thr Gly Arg Val Ala Asn Ser 435 440 445Leu Gly Ala Met Ala Ser Gly Gly Ile Asn Ala Leu Pro Gly Val Phe 450 455 460Ser Asn Ile Phe Ser Gln Val Ser Ala Ala Ser Gly Gly Ala Ser Gly465 470 475 480Gly Ala Val Leu Val Gln Ala Leu Thr Glu Val Ile Ala Leu Leu Leu 485 490 495His Ile Leu Ser Ser Ala Ser Ile Gly Asn Val Ser Ser Gln Gly Leu 500 505 510Glu Gly Ser Met Ala Ile Ala Gln Gln Ala Ile Gly Ala Tyr Ala Gly 515 520 52536545PRTParawixia bistriata 36Ala Ala Thr Ala Ser Ala Ala Gly Gly Leu Gly Gly Gln Gly Gly Leu1 5 10 15Gly Gly Leu Gly Ser Gln Gly Ala Gly Leu Gly Gly Tyr Gly Gln Gly 20 25 30Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly 35 40 45Leu Gly Gly Gln Gly Gly Arg Gly Gly Leu Gly Ser Gln Gly Ala Gly 50 55 60Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala65 70 75 80Ala Ala Ala Ala Ala Gly Gly Leu Gly Gly Gln Gly Gly Leu Gly Ala 85 90 95Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Ala Gly Gln Gly Gly Tyr 100 105 110Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Gly Gly Leu Gly Gly Gln 115 120 125Gly Gly Leu Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr 130 135 140Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala145 150 155 160Ala Gly Gly Leu Gly Gly Gln Gly Gly Leu Gly Gly Leu Gly Ser Gln 165 170 175Gly Ala Gly Pro Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly 180 185 190Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Leu Gly Gly Gln Gly Gly 195 200 205Leu Gly Ala Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gln 210 215 220Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Gly Gly225 230 235 240Leu Gly Gly Gln Gly Gly Leu Gly Ala Leu Gly Ser Gln Gly Ala Gly 245 250 255Gln Gly Gly Tyr Gly Gln Gly Gly Ser Gln Gly Ala Gly Gln Gly Gly 260 265 270Tyr Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Leu 275 280 285Gly Gly Gln Gly Gly Leu Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln 290 295 300Gly Gly Tyr Gly Gln Gly Gly Ser Gln Gly Ala Gly Gln Gly Gly Ala305 310 315 320Ala Ala Ala Ala Ala Ala Ala Gly Gly Leu Gly Gly Gln Gly Gly Phe 325 330 335Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gln Gly 340 345 350Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Val 355 360 365Leu Gly Gly Gln Gly Gly Leu Gly Gly Leu Gly Ser Gln Gly Ala Gly 370 375 380Gln Gly Gly Tyr Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala385 390 395 400Ala Ala Ala Ala Ala Ala Ala Gly Gly Leu Gly Gly Gln Gly Gly Arg 405 410 415Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gln Gly 420 425 430Gly Ala Gly Ala Ser Ser Ala Ala Ala Ala Ser Ala Ala Ala Ser Arg 435 440 445Leu Ser Ser Ala Ser Ala Ala Ser Arg Val Ser Ser Ala Val Ser Ser 450 455 460Leu Val Ser Ser Gly Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser Thr465 470 475 480Ile Ser Asn Val Val Ser Gln Val Ser Ala Ser Asn Pro Gly Leu Ser 485 490 495Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu 500 505 510Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Asn Ala 515 520 525Ala Gly Gln Ser Ala Ser Val Val Gly Gln Ser Phe Tyr Gln Ala Leu 530 535 540Ala54537451PRTParawixia bistriata 37Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly1 5 10 15Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 20 25 30Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln Gly 35 40 45Pro Gly Ser Gln Gly Gln Ser Gly Pro Gly Ala Thr Val Ala Ala Ala 50 55 60Ala Ala Gly Gly Tyr Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly65 70 75 80Ala Gly Gln Gln Gly Gln Gly Ser Gln Gly Pro Tyr Gly Pro Ala Ala 85 90 95Thr Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ser Gly Gln 100 105 110Gln Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 115 120 125Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 130 135 140Gly Ser Gly Gln Gln Gly Pro Gly Ala Gly Pro Gln Gly Pro Gly Ser145 150 155 160Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly 165 170 175Gly Tyr Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Ala Gly Gln Gln 180 185 190Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ser Ala Ala Ala Ala 195 200 205Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gly Ala Gly Gln Gln Val Pro 210 215 220Gly Ala Gly Gln Gln Gly Pro Gly Asn Gln Gly Pro Ser Gly Pro Gly225 230 235 240Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Gly Ala 245 250 255Gly Gln Gln Gly Pro Ala Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 260 265 270Ser Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 275 280 285Gly Pro Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly 290 295 300Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ser Ser Ser Ala Ala Ala305 310 315 320Val Gly Gly Tyr Gly Pro Ser Ser Gly Leu Gln Gly Pro Ala Gly Gln 325 330 335Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ser Ala Ala Ala Ala Ala Gly 340 345 350Ala Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala 355 360 365Val Ser Ser Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Thr 370 375 380Asn Thr Ile Ser Ser Val Val Ser Gln Ile Ser Ala Ser Asn Pro Gly385 390 395 400Leu Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile Val Ser 405 410 415Ala Leu Val His Ile Leu Gly Tyr Ser Ser Ile Gly Gln Ile Asn Tyr 420 425 430Asp Ala Ala Ala Gln Tyr Ala Ser Leu Val Gly Gln Ser Val Ala Gln 435 440 445Ala Leu Ala 45038234PRTParawixia bistriata 38Gly Gly Ala Asn Gly Ala Ser Ala Ala Ala Ala Ser Ala Gly Gly Ala1 5 10 15Gly Gly Tyr Gly Ser Asp Gly Gly Tyr Gly Gln Gly Gly Gln Gly Ala 20 25 30Gly Gly Asp Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ser Gly Gly 35 40 45Arg Gly Gly Gln Gly Gly Phe Gly Ser Gln Gly Ala Gly Gly Arg Gly 50 55 60Leu Gly Gly Ser Ala Arg Gly Gly Ala Gly Gly Thr Ser Ala Ala Ala65 70 75 80Ala Ser Ala Gly Gly Ala Arg Gly Tyr Gly Gly Asp Gly Gly Tyr Gly 85 90 95Gln Gly Gly Ser Gly Arg Gly Gly Ala Gly Ser Ala Ser Ala Ala Ala 100 105 110Ala Ser Ala Gly Gly Ala Gly Gly Tyr Gly Gly Asp Gly Gly Tyr Gly 115 120 125Glu Gly Gly Gln Gly Ala Gly Gly Asp Gly Val Ala Thr Ser Ser Ala 130 135 140Ala Ser Arg Leu Ser Ser Pro Ser Ser Ile Arg Arg Ile Ser Glu Val145 150 155 160Val Ser Thr Phe Ser Asp Asp Asp Phe Gly Asn Ser Ala Ser Phe Ser 165 170 175Asn Val Tyr Asn Ser Val Ala Ser Gly Ile Thr Ser Ser Asn Pro Gly 180 185 190Leu Ser Gly Cys Asp Val Gln Ile Gln Thr Leu Leu Glu Met Asn Ser 195 200 205Ala Leu Leu Ala Leu Leu Tyr Gly Phe Asp Ala Tyr Ser Ser Ala Ala 210 215 220Leu Val Asn Asp Phe Val Asn Gln Pro His225 230398PRTArtificial SequenceDescription of Artificial Sequence Synthetic His tag 39His His His His His His His His1 540626PRTNephila inaurata 40Ser Gly Gly Ser Gly Gly Thr Thr Val Ile Glu Asp Leu Asp Ile Thr1 5 10 15Ile Asp Gly Ala Asp Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Ile 20 25 30Ser Gly Ala Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly 35 40 45Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly 50 55 60Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly65 70 75 80Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro Gly 85 90 95Gly Ala Tyr Gly Pro Gly Gly Ser Gly Gly Pro Gly Gly Ala Gly Gly 100 105 110Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro Tyr 115 120 125Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro 130 135 140Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr145 150 155 160Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly 165 170 175Pro Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Thr Gly Pro Gly Gly 180 185 190Tyr Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser 195 200 205Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly 210 215 220Pro Gly Gly Ser Gly Pro Gly Gly Phe Gly Pro Gly Gly Ser Gly Pro225 230 235 240Gly Gly Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly 245 250 255Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly 260 265 270Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser 275 280 285Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 290 295 300Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Pro305 310 315 320Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Pro Gly 325 330 335Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Pro Gly Gly 340 345 350Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Arg Gly Gly Ala 355 360 365Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ser Gly Gly 370 375 380Ala Gly Gly Ser Gly Gly Thr Thr Val Ile Glu Asp Leu Asp Ile Thr385 390 395 400Ile Asp Gly Ala Asp Gly Pro Ile Thr Ile Ser Glu Glu Leu Thr Ile 405 410 415Gly Gly Ala Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ser Gly Pro 420 425 430Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly 435 440 445Gly Leu Gly Ser Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly 450

455 460Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser465 470 475 480Gly Gly Leu Tyr Gly Pro Gly Ser Tyr Gly Pro Gly Gly Ser Gly Val 485 490 495Pro Tyr Gly Ser Ser Gly Thr Tyr Gly Ser Gly Gly Gly Tyr Gly Pro 500 505 510Gly Gly Ala Gly Gly Ala Tyr Gly Pro Gly Ser Pro Gly Gly Ala Tyr 515 520 525Gly Pro Gly Ser Gly Gly Ser Tyr Tyr Pro Ser Ser Arg Val Pro Asp 530 535 540Met Val Asn Gly Ile Met Ser Ala Met Gln Gly Ser Gly Phe Asn Tyr545 550 555 560Gln Met Phe Gly Asn Met Leu Ser Gln Tyr Ser Ser Gly Ser Gly Ser 565 570 575Cys Asn Pro Asn Asn Val Asn Val Leu Met Asp Ala Leu Leu Ala Ala 580 585 590Leu His Cys Leu Ser Asn His Gly Ser Ser Ser Phe Ala Pro Ser Pro 595 600 605Thr Pro Ala Ala Met Ser Ala Tyr Ser Asn Ser Val Gly Arg Met Phe 610 615 620Ala Tyr625

Claims

1. Isolated molecules of spider nucleic acid, comprising:a) sequences substantially similar to any of the sequences selected from the group identified as SEQ ID N. 1-19;b) complements of the sequences described in SEQ ID N. 1-19;c) reverse complements of the sequences described in SEQ ID N. 1-19;d) reverse sequences of the sequences described in SEQ ID N. 1-19;

2. Isolated molecules of spider nucleic acid in accordance with claim 1, wherein the sequences are isolated from Nephilengys cruentata, Avicularia juruensis and Parawixia bistriata.

3. A chimeric gene comprising the isolated molecule of claim 1.

4. A chimeric gene comprising:a) a promoter optionally linked to a leader sequence and operationally linked to;b) a coding sequence substantially similar to any of the sequences set forth in SEQ ID N. 1-19.

5. A chimeric gene in accordance with claim 4 wherein said promoter is selected from the group consisting of constitutives, inducibles and tissue-specific.

6. A chimeric gene in accordance with claim 5 wherein the tissue-specific promoter is selected from cotton fibre gene promoters.

7. A chimeric gene in accordance with claim 6 wherein said cotton fibre gene promoters are selected from the group consisting of E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexine, FbL2A and actin 2.

8. A chimeric gene in accordance with claim 3 wherein the promoter contains enhancer elements.

9. A chimeric gene in accordance with claim 4 wherein said promoter may be expressed in plants, animals, fungus or insects.

10. A chimeric gene in accordance with claim 4 wherein the leader sequence is obtained from the same gene as the promoter used to direct transcription of the isolated nucleic acid molecule.

11. Expression vector comprising a chimeric gene in accordance with claim 3.

12. An expression vector comprising:a) a promoter optionally linked to a leader sequence and operationally linked to;b) an coding sequence substantially similar to any of the sequences identified as SEQ ID N. 1-19 operationally linked to;c) a termination signal;d) an origin of replication;e) a selective marker; andf) a cloning site.

13. Expression vector in accordance with claim 12 wherein the promoter is selected from the group consisting of constitutives, inducibles and tissue-specific.

14. An expression vector in accordance with claim 13 wherein the tissue-specific promoter is selected from cotton fibre gene promoters.

15. An expression vector in accordance with claim 14 wherein the cotton fibre gene promoters are selected from the group consisting of E6, H6S, Rac13, LTP, ACP, expansin, CAP, anexin, FbL2A and actin 2.

16. An expression vector in accordance with claim 11 wherein the promoter contains enhancer elements.

17. An expression vector in accordance with claim 12 wherein said leader sequence is obtained from the same gene as the promoter used to direct transcription of the isolated nucleic acid molecule.

18. An expression vector in accordance with claim 12 wherein the promoter may be expressed in plants, animals, fungus or insects.

19. An expression vector in accordance with claim 12 wherein the transcription termination signal and the polyadenylation region of the present invention includes, but is not limited to, the SV40 termination signal, the HSV TK adenylation signal, the termination signal of the nopaline synthetase gene (NOS) of Agrobacterium tumefaciens, the octopine synthetase gene termination signal, the termination signal of the 19S and 35S genes of CaMV, the maize alcohol dehydrogenase gene termination signal, the manopine synthetase gene termination signal, the beta-phaseolin gene termination signal, the ssRUBISCO gene termination signal, the sucrose synthetase gene termination signal, the termination signal of the virus that attacks the Trifolium subterranean (SCSV), the termination signal of the trpC gene of Aspergillus nidulans, and other similar.

20. An expression vector in accordance with claim 12 wherein the selective marker is selected from the sequences that confer resistance to antibiotics or visual markers.

21. An expression vector in accordance with claim 20 wherein said selective marker is chosen from the gene coding sequences conferring resistance to kanamycin, neomycin, ampicillin, chloranphenicol, streptomycin, hygromycin, geneticin, phosphinotrycin, glyphosate, ammonium gluphosinate, AHAS, BAR and GUS.

22. Isolated molecules of spider silk protein comprising sequences substantially similar to any one of the sequences selected from the group identified as SEQ ID N. 20-38:

23. Isolated molecules of spider silk protein in accordance with claim 22 wherein said sequences are isolated from Nephilengys cruentata, Avicularia juruensis and Parawixia bistriata.

24. Transformed cell containing a chimeric gene in accordance with claim 3.

25. Transformed cell containing an expression vector in accordance with claim 11.

26. Transformed cell containing an isolated protein molecule in accordance with claim 22.

27. Transformed cell in accordance with claim 24 wherein the cell originated from any one of the groups consisting of bacteria, fungus, insects, mammals and vegetables.

28. Plant, or a part thereof, or a propagule or progeny thereof, comprising a chimeric gene in accordance with claim 3.

29. Plant, or a part thereof, or a propagule or progeny thereof, comprising an expression vector in accordance with claim 11.

30. Plant, or a part thereof, or a propagule or progeny thereof, comprising an isolated protein molecule in accordance with claim 22.

31. Plant, or a part thereof, or a propagule or progeny thereof, in accordance with claim 30 wherein the protein is expressed in seeds.

32. Animal, or a part thereof, or a progeny thereof, comprising a chimeric gene in accordance with claim 3.

33. Animal, or a part thereof, or a progeny thereof, comprising an expression vector in accordance with claim 11.

34. Animal or a part thereof, or a progeny thereof, comprising an isolated protein molecule in accordance with claim 22.

35. Animal, or a part thereof, or a progeny thereof, in accordance with claim 34 wherein the protein is expressed in the mammary glands.

36. Microorganism, or a part thereof, comprising a chimeric gene in accordance with claim 3.

37. Microorganism, or a part thereof, comprising an expression vector in accordance with claim 11.

38. Method for producing a genetically modified organism comprising the following stages:a) transforming a cell, tissue, organ or embryo with a chimeric gene in accordance with claim 3 or an expression vector in accordance with claim 11;b) selecting transformed cells, cell calluses, embryos or seeds;c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b);d) selecting mature plants, mature embryos or microorganisms cells of stage (c) containing the chimeric gene or expression vector with the nucleotide sequences that encode the spider silk protein.

39. Method for the production of recombinant protein comprising the following stages:a) transforming a cell, tissue, organ or embryo with an expression vector in accordance with claim 11;b) selecting transformed cells, cell calluses, embryos or seeds;c) regenerating mature plants, mature embryos or microorganisms of the transformed cells, cell calluses, embryos or seeds selected in stage (b);d) selecting mature plants, mature embryos or microorganisms cells of stage (c) containing the expression vector with the nucleotide sequences that encode the spider silk protein.e) extracting of the recombinant spider silk protein produced in the organisms selected in stage (d).

40. Recombinant protein obtained by the method described in claim 39.

41. Recombinant protein in accordance with claim 40 characterised by the fact the protein presents microbicide activity.

42. Recombinant protein in accordance with claim 41 wherein the microbicide activity is against viral replication.

43. Recombinant protein in accordance with claim 40 wherein said protein presents defensin activity.

44. Recombinant protein in accordance with claim 43 wherein said defensin activity is against pests and insects.

45. Recombinant protein in accordance with claim 40 wherein said the protein presents dermatological activity.

46. Dermatological composition comprising:a) A recombinant protein in accordance with claim 40;b) a pharmaceutically acceptable vehicle.

47. Dermatological composition in accordance with claim 46 wherein the pharmaceutically acceptable vehicle is selected from the group consisting of glycerol, water, saline solution, ethane, solutions with phosphates and organic acid salts.

48. Microbicide composition comprising:a) A recombinant protein in accordance with claim 41;b) an agriculturally acceptable vehicle and, optionally,c) additives.

49. Microbicide composition in accordance with claim 48 wherein the agriculturally acceptable vehicle is selected from the group consisting of water, organic solvents, humectants, preservatives, thickeners, antimicrobial agents, antioxidants, emulsifiers, film forming polymers and mixtures of these.

50. Microbicide composition in accordance with claim 48 wherein the additives are selected from the group consisting of rosin gum, latex, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl chloride, polyethylene, polyvinyl acetate and mixtures of these. Further optional additives include methyl, methacrylate and mixtures of these.

51. Biopolymers produced from the recombinant protein obtained in accordance with the method described in claim 39.

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