AU2006200422B8

AU2006200422B8 - Novel nucleic acid molecules

Info

Publication number: AU2006200422B8
Application number: AU2006200422A
Authority: AU
Inventors: Marilyn Anne Anderson; David James Craik; Cameron Victor Jennings
Original assignee: Hexima Ltd
Current assignee: University of Queensland UQ; Hexima Ltd
Priority date: 1999-11-05
Filing date: 2006-02-01
Publication date: 2010-04-01
Anticipated expiration: 2020-11-03
Also published as: AU2006200422A1; AU783525C; AU783525B2; AU1120401A; AU2006200422B2

Description

AUSTRALIAN DIVISIONAL PATENT APPLICATION APPLICANTS: The University of Queensland and LaTrobe University TITLE: Novel Nucleic Acid Molecules - 1 NOVEL NUCLEIC ACID MOLECULES FIELD OF THE INVENTION 5 The present invention relates generally to a novel nucleic acid molecule encoding an amino acid sequence wherein said amino acid sequence or a derivative form thereof is capable of fonning a cyclic structure. Cyclization may occur, for example, within a cell or cell membrane or linear forms of the molecules may be circularized or at least partly circularized in vitro using, for example, isolated enzyme systems or chemical means. The 10 cyclised amino acid sequence is generally in the form of a stabilized folded structure such as a cyclic knotted peptide, polypeptide or protein or its functional equivalent. The present invention is further directed to cyclized molecules and in particular cyclic peptides, polypeptides or proteins, linear forms thereof including non-cyclic structural homologues of the cyclic peptides, polypeptides and proteins and precursor or derivative forms thereof 15 encoded by the subject nucleic acid molecules. The nucleic acid molecules and cyclic and linear peptides, polypeptides and proteins of the present invention are useful inter alia in the generation of molecules having animal or plant therapeutic properties as well as in a range of diagnostic, industrial and agricultural including horticultural applications. Of particular importance is the use of these molecules in the protection of plants such as crop 20 plants from pest and/or pathogen infestation. The cyclic and linear peptides, polypeptides and polypeptides may be naturally occurring or may be modified by the insertion or substitution of heterologous amino acid sequences. The therapeutic properties may be inherent in the naturally occurring cyclic or linear molecules and/or may be associated with the heterologous amino acid sequence. The present invention further provides microbial, 25 plant and animal cell systems as well as in vitro systems capable of cyclizing linear forms of the peptides, polypeptides and proteins of the present invention. The present invention also extends to the peptide, polypeptide or protein sequences which are capable of cyclizing in the absence of any other exogenous factor and more specifically capable of circularizing through a catalytic process being an inherent activity of the peptides, 30 polypeptides or proteins.

-2 BACKGROUND OF THE INVENTION Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. 5 Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country. 10 A number of macrocyclic peptides with diverse biological activities have been discovered in plants in the Rubiaceae and Violaceae families. These include kalata B1 (1), the circulins (2), cyclopsychotride (3) and several peptides from Viola species (4-6). They range in size from 29-31 amino acids and contain six conserved Cys residues. These macrocyclic peptides differ from classical proteins in that they have no free N- or C 15 terminus due to their amide-circularized backbone. They also incorporate a cystine knot in which an embedded ring in the structure formed by two disulfide bonds and their connecting backbone segments is threaded by a third disulfide bond. These combined features of the cyclic cystine knot (CCK) produce a unique protein fold that is topologically complex and has exceptional chemical and biological stability. 20 Small cyclic peptides are also known in nature, particularly as antibiotics of microbial origin, and appear to have advantages of improved stability and biological activity over their non-cyclic counterparts. Because of their favourable properties, cyclic peptides (or mimics of them) have had pharmaceutical applications. One example is the 25 immunosuppressent, cyclosporine. These classical cyclic peptides invariably comprise fewer than 15 amino acids, usually lack disulfide bonds and generally do not have well defined three dimensional structures. Such peptides are not gene products but are thought to be biosynthesized, non-ribosomally, via peptide synthetases. 30 In work leading up to the present invention, the inventors investigated the genetic basis of the macrocyclic peptides. In contrast to small cyclic peptides, the macrocyclic peptides, -3 referred to herein as "cyclotides", are encoded for by gene sequences and exhibit folding structures characteristic of true proteins. The elucidation of the genetic basis behind the cyclotides enables their expression and manipulation in transgenic plant, animal and microbial cells. Being cyclic, the cyclotides have a range of potential therapeutic, 5 diagnostic, industrial and agricultural including horticultural applications. The cyclizing enzyme or enzymes themselves also have utility in the development of in vivo or in vitro systems for cyclizing target peptides, polypeptides and proteins. Furthermore, the present invention permits the generation of linear structural homologues of peptides, polypeptides and proteins.

-4 SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the 5 inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims. 10 One aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic 15 backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone. Another aspect of the present invention present is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its 20 complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic knotted peptide, polypeptide or protein. A further aspect of the present invention provides a nucleic acid molecule comprising a 25 sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure: C[Xi ... X] C[X 1 ... XIb] C[X"H 1 ... X"eC C[Xl" ... X 1 1 1 d] C[Xli ... Xle] C[X't ... X"'r 30 wherein C is cysteine; 5 each of [Xi ... Xa], [X', ... X'], [X", ... X"j, [X"'I ... X"'], [Xw" ... X"e] and [Xv, ... Xvt] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each 10 respective sequence and each of a to f may be the same or different and range from I to about 20. Still another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes 15 an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure: C[XI ... Xa] C[XI ... X's] C[X" 1 ... X"c] C[Xi ... X"I] C[Xw 1 ... X"e] C[XV I ... X d] 20 wherein C is cysteine; 25 each of [Xi ... X.], [X'I ... X',], [X"I ... X"1, [X" 1 I ... X 1 1 d], [XVi ... X'] and [Xv, ... X'r] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each 30 respective sequence and each of a to f may be the same or different and range from 1 to about 10.

-6 Even still another aspect of the present invention provides a nucleic acid molecule comprising A sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the 5 cyclic backbone comprises the structure: C[Xi ... Xa] C[X' 1 ... XIb] C[X" ... X 1 T C] C[X"i ... X"lc] C[Xw 1 ... X'e] C[XVI ... XVr] 10 wherein C is cysteine; each of (Xi ... Xa], [X' ... X'b], [X"i ... X"c], [X"'I ... X"'d], [XW 1 ... X"e] and 15 [Xv 1 ... Xv] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is from about 3 to about 6, b is from about 3 to about 5, 20 c is from about 2 to about 7, d is about 1 to about 3, e is about 3 to about 6 and f is from about 4 to about 9. Yet another aspect of the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of 25 nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure: 30 C[X ... X.] C[X'i ... X'b] C[Xu% ... X" 0 ] C[X"'I ... X11 ] C[XI ... XVe] C[X'S x . X -7 wherein C is cysteine; 5 each of [Xi ... Xa], [X'i ... X'i], [XII ... X"c], [X'"' 1 ... X"'], [XF'I ... X"e] and [Xv 1 ... Xvr] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and 10 wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a is about 3, b is about 4, c is about from 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7. Even yet another aspect of the present invention provides a nucleic acid molecule an 15 isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure: 20 C[X, ... Xa] C[X ... X's) C[X i ... X"j C[XIi ... XUI] C[Xv . XFV] C(XV ... v wherein 25 C is cysteine; each of [XI ... Xa], [X 1 ... X' 1 , ], [X 1 1 - X'IJ, [X'i ... X"'dj, [XWI ... Xv'] and [Xv 1 ... Xr] represents one or more amino acid residues wherein each one or more amino 30 acid residues within or between the sequence residues may be the same or different; and -8 wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is about 6, b is about 5, c is about 3, d is about 1, e is about 5 and f is about 8. 5 A further aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein 10 said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three-dimensional structure of said backbone wherein said backbone comprises free amino and carboxy termini. Another aspect of the present invention contemplates a method of identifying nucleic acid 15 molecules which encode one or more enzymes required for cyclization of an amino acid sequence said method comprising obtaining a nucleic acid molecule which encodes a precursor form of an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein; introducing or fusing to said nucleic acid molecule, a nucleotide sequence which encodes a reporter molecule capable of providing a detectable signal 20 wherein said nucleotide sequence is inserted or fused to a portion of the nucleic acid molecule which is cleaved off prior to or during cyclization; introducing said nucleic acid molecule comprising the nucleotide sequence encoding the reporter molecule into a bank of cells carrying a DNA library comprising all or part of genomic DNA or cDNA from a plant which carries the enzyme or enzymes required for cyclization of an amino acid 25 sequence; screening for and selecting cells which do not synthesize the reporter molecule. A further aspect of the present invention contemplates a genetically modified cell or cells or a plant or animal comprising said genetically modified cells, said cells comprising a nucleic acid molecule having a nucleotide sequence or complementary nucleotide 30 sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein.

-9 Still another aspect of the present invention contemplates a method of incorporating an amino acid sequence conferring a particular trait into a cyclic peptide, polypeptide or protein, said method comprising fusing or introducing a nucleotide sequence encoding said 5 amino acid sequence to or into a second nucleotide sequence wherein said second nucleotide sequence encodes a peptide, polypeptide or protein which peptide, polypeptide or protein or a derivative therefor is capable of being cyclized into a knotted peptide, polypeptide or protein. 10 Even still another aspect of the invention contemplates an isolated nucleic acid molecule comprising the. following nucleotide sequence: [XI...Xbnln 2 ... na]y'.. .yid 15 wherein [nin2 ... na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and 20 X 1 .. Xb and y...ye represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d. Yet another aspect of the invention contemplates an isolated nucleic acid molecule 25 comprising the following nucleotide sequence: ji ... je[Xi...Xajnin2 ... n.]y1...yelql ... qr wherein 30. [nIn2 ... n 2 ] represents a nucleotide sequence encoding an amino acid sequence -10 capable of being cyclized to a knotted peptide or polypeptide or protein; X,... Xb and y... ye represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the 5 amino acid sequence may be unique for each integer of d; and i-. .je and qi...qr represent nucleotide sequences encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle where a, b, c, d, e and f may be any number, where d is >1, the amino acid 10 sequence may be unique for each integer of d. Even yet another aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence: 15 [XI ... Xbfnin2 ... (ki ... k6)" ~yi ... yc~d wherein [nn 2 ... n,] represents a nucleotide sequence encoding an amino acid sequence 20 capable of being cyclized to a knotted peptide or polypeptide or protein;

X

1 ... X, and y. ,.ye represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d, 6 and X may be any number and when d or X is > 1, the amino acid sequence may be unique for each integer of d and X; 25 k 1 ...ka represent a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait. Another aspect of the invention contemplates an isolated nucleic acid molecule comprising 30 the following nucleotide sequence:- - II [nin2 ... (nl'n2'... n...a1 wherein 5 [nin 2 ... n.] and (n, n 2 ... nQ') represent polynucleotide sequences encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and y and a and m may be any number and when m is >1, the amino acid sequence may 10 be unique for each integer of m. A further aspect of the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence: 15 jil... je[XI -... Xb[nin2 ... (nI'n2'...(k, ... ka))#1a )mna]yi ... ye-1qj ... qr. wherein [nn 2 ... n.] represents a nucleotide sequence encoding an amino acid sequence 20 capable of being cyclized to a knotted peptide or polypeptide or protein; X. .Xb and yi.. .y represents a polynucleotide sequence capable of encoding an amino acid sequence where a and b and c and d and e may be any number and when d is >1, the amino acid sequence may be unique for each integer of d; 25 ji.. . and q, ... qf represents a nucleotide sequence encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle; 30 k,... k 6 represents a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait; - 12 X and m and d may be any number and when X and m and d are each >1, the amino acid sequence may be unique for each integer of X, m and d. 5 Yet another aspect of the present invention further contemplates a genetically modified plant which comprises a nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein and which confers on said plant a trait not present in the same species or variety of plant prior to genetic modification. 10 Even yet another aspect of the present invention provides the use of a nucleic acid molecule encoding an amino acid sequence, which amino acid sequence or a derivative or precursor form thereof is capable of being cyclized into a knotted peptide, polypeptide or protein, in the manufacture of a transgenic or genetically modified plant capable of 15 producing said cyclic knotted peptide, polypeptide or protein. Still yet another aspect of the present invention relates to an immunointeractive molecule specific for a peptide, polypeptide or protein when in cyclic form and encoded by a nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its 20 complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic -backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone. 25 Another aspect of the present invention is directed to an immunointeractive molecule specific for a peptide, polypeptide or protein encoded by a nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or 30 a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein - 13 said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone wherein said backbone comprises free amino and carboxy termini.

- 14 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a representation showing (A) the amino acid sequence of Kalata B . The Kalata BI peptide is composed of 29 amino acids and has a cyclic peptide backbone; (B) 5 the primers used in the PCR reactions. Primers Kall (<400>29) and Kal2 (<400>30) correspond to amino acid residues 12 to 17 or 29 to 5 (see Figure LA). I represents inosine, Y represents C or T, and R represents A, C, T or G. The encoded amino acids are represented in single letter code and the introduced restriction enzyme sites are in italics; and (C) the PCR products. cDNA prepared from 0. affinis leaf RNA was amplified with 10 primers Kall and oligo dT-HindIII (lane 1) or primers Kal2 and oligo-dT-HindI (lane 2) [<400>32]. The amplified fragments were separated on a 2% w/v agarose gel and stained with ethidium bromide. Five major fragments were obtained, two from primer Kal 1 (1-2) and three from primer Kal2 (3-5). Fragments 1 and 5 were subcloned and sequenced. 15 Figure 2 is a presentation of the sequence of the 412 bp DNA fragment amplified from 0. affinis cDNA with primers complementary to the Kalata BI sequence (Figure IC, fragment 5). The 412 bp fragment has an open reading frame (<400>7) that encodes the entire 29 amino acids of Kalata B1 [<400>8] together with an additional four amino acids at the C terminus. The 3' untranslated region is shown in <400>9. The primer sequences are shown 20 in italics, stop codons are indicated by "*" and a region corresponding to the sequence of the mature peptide is underlined. Figure 3 is a photographic representation showing gel bot analysis of RNA from 0. affinis leaves. (A) The RNA blot. The Kalata B I cDNA (see Figure 2) hybridized to a single 25 RNA transcript of -750 bases. (B) Identical gel to (A) stained with ethidium bromide to reveal the rRNA bands. Size markers were the 0.28-6.58 kb RNA markers from Promega. Figure 4 is a representation of nucleotide sequence (<400>4) and predicted amino-acid sequence (<400>5) of Oakl, the cDNA encoding Kalata BI from 0. affinis. The 5' and 3' 30 ends of the DNA are shown in <400>1 and <400>6, resepctively. The nucleotide sequence encoding the signal peptide and the corresponding amino acid -sequence is shown in - 15 <400>3 or <400>4, respectively. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code is numbered beginning with 1 for the predicted first amino acid in the precursor protein. The putative signal peptide is indicated by negative numbers. An amino acid 5 sequence subjected to processing to give Kalata B 1 is shaded. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata BI domain (NT conserved) is highly conserved in other Oak clones (see Figures 8A and 8B). Figure 5 is a representation of the nucleotide sequence (<400>13) and predicted amino 10 acid sequence (<400>14) of Oak2, the cDNA encoding Kalata B3 and B6 from 0. affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code, is numbered beginning with 1 for the predicted first amino acid in the precursor protein. The putative signal peptide is indicated by negative numbers (<400>12) and is encoded by the 15 nucleotide sequence set forth in <400>11. An amino acid sequence subjected to processing to give Kalata B3 and B6 is shaded. Dark and light shading respectively highlights the sequence of Kalata B3 and B6. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B3 and B6 domains (NT-conserved) is highly conserved in other Oak clones (see Figures 8A and 8B). The untranslated 5' and 3' ends of 20 the DNA is shown in <400>10 and <400>15, respectively: Figure 6 is a representation of the nucleotide sequence (<400>19) and predicted amino acid sequence (<400>20) of Oak3, the cDNA encoding Kalata B7 from O.affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the 25 sequence. The amino acid sequence, shown in single letter code, is numbered beginning with i for the first predicted amino acid in the precursor protein. The putative signal peptide (<400>18) is indicated by negative numbers and is encoded by the nucleotide sequence set forth in <400>17. An amino acid sequence subjected to processing to give Kalata B7 is shaded. Arrows indicate potential processing sites. The underlined region at 30 the N-terminus of the Kalata B7 domain (NT-conserved) is highly conserved in other Oak -16 clones (see Figures 8A and 8B). The untranslated 5' and 3' ends of the DNA is shown in <400>16 and <400>21, respectively. Figure 7 is a representation of the nucleotide sequence (<400>25) and predicted amino 5 acid sequence (<400>26 and <400>27) of Oak4, the cDNA encoding Kalata B2 from O.affinis. Only one strand with the polarity of the mRNA is shown. Nucleotides are numbered above the sequence. The amino acid sequence, shown in single letter code, is numbered beginning with I for the first predicted amino acid in the precursor protein. The putative signal peptide (<400>24) is indicated by negative numbers and is encoded by 10 <400>23. An amino acid sequence subjected to processing to give Kalata B2 (shaded) is repeated three times. Arrows indicate potential processing sites. The underlined region at the N-terminus of the Kalata B2 domain (NT-conserved) is highly conserved in other Oak clones (see Figures 8A and 8B). The untranslated 5' and 3' ends of the DNA is shown in <400>22 and <400>28, respectively. 15 Figure 8 is a schematic diagram of (A) of the precursor proteins predicted from the Oakl,2, 3 and 4 clones showing the signal peptide, the regions corresponding to the mature kalata peptides (shaded), the region of 17 conserved amino acids on the N-terminal side of the kalata peptide sequence (N-T conserved, hatched), and (B) the sequence around the 20 potential processing sites. The mature cyclic peptide retains one copy of the GIy-Leu-Pro sequence, which may be derived entirely from one of the two flanking elements (shaded), or partially from both depending on the initial cleavage sites. Figure 9 is a diagrammatic representation showing the structure of Kalata B2 and a 25 comparison with Kalata B 1. (A) shows the circular backbone and cross-linking disulfide bonds of Kalata B2. The Cys residues making up these disulfide bonds are labelled I-VI. The arrows represent regions of beta strands. The side chains of two aromatic residues which are located on proximate turns are highlighted. (B) shows a superimposition of the backbone residues of Kalata B2 and Kalata B] and demonstrates the similarity of their 30 three dimensional structures.

-17 Figure 10 is a photographic representation of gel blot analysis of genomic DNA from O.affinis. Gel blot analysis of genomic DNA digested with HinduI, BamHl, Nde I and EcoRV and probed with radiolabelled Oakl cDNA (Figure 4). All enzymes gave at least twelve hybridizing bands. The Oak clones appear to belong to a multigene family with up 5 to twelve members. Figure 11 is a representation showing bacterial expression of the precursor protein encoded by the Oakl clone. (A) Total cell lysates were prepared at various time points (0-5 hr) post-induction with IPTG by removing 100 p1 of cell culture which was lysed in SDS 10 sample buffer. The proteins were separated on a 12% w/v SDS polyacrylamide gel and stained with silver. A band of approximately the expected size (arrowed) appeared after IPTG induction. Lane numbers indicate hours after induction and broad range kaleidoscope markers (BioRad) were used. IMAC is induced protein purified by immobilized metal affinity chromatography (IMAC). Full-length Oakl was expressed 15 from the pQE.30 vector in an K coli MIS cell line. B. RP-HPLC of the material that bound to the metal affinity column. The protein in the peak had the same mass as the protein predicted by the Oakl cDNA together with the hexahistidine tag and henceforth will be called Kalata BI precursor. 20 Figure 12 is a representation showing immunoblot analysis with the antibody raised to the Kalata BI precursor. (A) Purified kalata BI (1 pig), Kalata BI precursor (1 pg) and buffer soluble proteins from O.afinis leaves (100 pg) fractionated on a precast 4-12% bis-tris protein gel (Novex, San Diego, CA, USA) and stained with silver. Markers are the SeeBlue (trademark) Pre-Stained standard from Novex. (B) Proteins in an identical gel 25 after transfer to nitrocellulose (0.2 R) and immunoblotting with antiserum (1:1000) raised against the bacterially expressed Kalata Bi precursor (see Figure 11). Antibody raised to the Kalata B 1 precursor recognizes both the precursor and cyclic Kalata B 1. Figure 13 is a graphical representation showing the effect of Kalata Bl on growth and 30 development of H. punctigera larvae. (A) Survival of larvae fed on Haricot bean artificial diet containing Kalata B1 and the control diet. (B) Average mean weight of larvae fed on - 18 Kalata BI and control diet. (C) Size of larvae after 16 days on artificial diet containing Kalata B 1 (-5 mm) or control diet (-30 mm). Figure 14 illustrates the effect of cyclic and linear Kalata B 1 on growth of H armigera 5 larvae. (A) Graphical representation of the growth of larvae fed on a cotton leaf artificial diet containing Kalata BI at 0.15% w/v of diet (high cyclic), Kalata B 1 at 0.03% w/v (low, cyclic), linear Kalata B 1 at 0.15% w/v or no added Kalata B I (control). (B) Relative size of larvae after 12 days. 1. Cyclic Kalata B1 (0.15%w/v), 2. Cyclic Kalata BI (0.03%w/v), 3. Linear Kalata B 1 (0.1 5%w/v), 4. Control. 10 Figure 15 is a representation of the sequence of the 311 bp fragment amplified from Viola odorata cDNA with the primers Kal2 and oligo dT-HindHI (see Figure 1). The 311 bp fragment has an open reading frame (<400>32) that encodes 26 amino acids (<400>33) of Kalata S (underlined) a cyclotide isolated from V. odorata (12). An additional four amino 15 acids are located at the C-tenninus of the Kalata S sequence. The primer sequences are shown in italics, the stop codon is indicated by "*" and the coding region is underlined. The untranslated 3' end of the DNA is shown in <400>34. Figure 16 is a photographic representation showing gel blot analysis of RNA from roots, 20 leaves and stems of 0. affinis. (A) The RNA blot. The OaklcDNA (see Figure 4) hybridized to a broad band of about 750 bases. (B) Identical gel to (A) stained with ethidium bromide to reveal the rRNA bands. Size markers were the 1 Kb Plus DNA Ladder (trademark) from Gibco BRL. 25 Table 1 is a summary of single and three letter abbreviations used throughout the specification are defined in Table 1. Table 2 is a summary of amino acid and nucleotide sequence identifiers.

-19 TABLE 1 Single and three letter amino acid abbreviations Amino Acid Three-letter One-letter 5 Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N 10 Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G 15 Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M 20 Phenylalanine Phe . F Proline Pro P Serine Ser S Threonine The T Tryptophan Trp W 25 Tyrosine Tyr Y Valine Val V Any residue Xaa X -20 TABLE 2 SEQUENCE IDENTIFIER DESCRIPTION <400>1 Untranslated 5' end of DNA encoding Kalata B1 <400>2 Nucleotide sequence encoding signal peptide of Kalata BI <400>3 Amino acid sequence of signal peptide of Kalata BI <400>4 Nucleotide sequence of cDNA encoding Kalata B1 <400>5 Amino acid sequence of Kalata BI encoded by <400>4 <400>6 Untranslated 3' end of DNA encoding Kalata B1 <400>7 Partial nucleotide sequence of a 400 bp DNA fragment amplified from 0. affins CdNA <400>8 Partial amino acid sequence encoded by <400>7 <400>9 Untranslated 3' end of 400 bp DNA fragment from 0. affins cDNA <400>10 Untranslated 5' end of DNA encoding Kalata B3 and B6 <400>11 Nucleotide sequence encoding signal peptide of Kalata B3 and B6 <400>12 Amino acid sequence of signal peptide of Kalata B3 and B6 <400>13 Nucleotide sequence of cDNA encoding Kalata B3 and B6 <400>14 Amino acid sequence of Kalata B3 and B6 <400>15 Untranslated 3' end of DNA encoding Kalata B3 and B6 <400>16 Untranslated 5' end of DNA encoding Kalata B6 <400>17 Nucleotide sequence encoding signal peptide of Kalata B7 <400>18 Amino acid sequence of signal peptide of Kalata B7 <400>19 Nucleotide sequence of cDNA encoding Kalata B7 <400>20 Amino acid sequence of Kalata B7 <400>21 Untranslated 3' end of DNA encoding Kalata B6 <400>22 Untranslated 5' end of DNA encoding Kalata B26 <400>23 Nucleotide sequence encoding signal peptide of Kalata B2 <400>24 Amino acid sequence of signal peptide of Kalata B2 -21 SEQUENCE IDENTIFIER DESCRIPTION <400>25 Nucleotide sequence of cDNA encoding Kalata B2 <400>26 and <400>27 Amino acid sequence of Kalata B2 <400>29 Untranslated 3' end of DNA encoding Kalata B2 <400>29 Nucleotide sequence of Kall primer <400>30 Nucleotide sequence of Ka12 primer <400>31 Oligo-dT-HindIII nucleotide sequence <400>32 Nucleotide sequence of coding region of 311 bp fragment amplified by Viola odorata cDNA <400>33 Amino acid sequence encoded by <400>33 <400>34 Untranslated 3' end of 311 bp fragment amplified from Viola odorata cDNA -22 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on the elucidation of the genetic basis behind the production of macrocyclic peptides. 5 Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell 10 to form a cyclic backbone wherein said cyclic backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone. The term "knotted" is not to be limited by any mathematical or geometrical definition of 15 the term "knot". The knots contemplated by the present invention are such due to their similarity to a mathematical knot and/or by virtue of the intertwined folding of the molecule which results. Preferably, the stabilized folded structure contains a knotted topology. Accordingly, the 20 present invention is directed, therefore, to an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell to form a cyclic knotted peptide, polypeptide or protein. The amino acid sequence may also be cyclizable in 25 an in vitro system comprising, for example, cyclizing enzymes or the chemical means for cyclization. An "isolated nucleic acid molecule" is a nucleic acid molecule which has undergone at least one purification step from a biological sample. Purification steps include inter alha 30 precipitation, centrifugation, chromatography, electrophoresis and/or filtration. The nucleic acid molecule may be single or double stranded RNA or DNA or an RNA:DNA hybrid, - 23 The nucleic acid molecule may comprise naturally occurring nucleotide bases or the bases may be synthetic or chemical analogues of bases or be chemically modified such as a C-5 propyne or phosphorothiolate modification. 5 An "amino acid sequence" generally means a sequence of two or more amino acid residues. In terms of the cyclotides of the present invention, generally the amino acid sequence comprises at least from about 10 to about 150 amino acid residues, preferably from about 15 to about 100 amino acid residues and even more preferably from about 15 to about 50 amino acid residues, when in cyclic form. The amino acid sequence referred to 10 herein may be considered a peptide or polypeptide and these terms are used interchangedly in the subject specification. A polypeptidee' may also be considered a "protein". However, the nucleic acid molecule of the present invention may first encode a precursor, peptide, polypeptide or protein, generally in linear form. Such a precursor may comprise 15 from about 50 to about 1000 amino acid residues or from about 50 to about 500 amino acid residues or from about 50 to about 300 amino acid residues. The precursor amino acid sequence is derivatized to a smaller amino acid sequence which is then cyclized. Alternatively, the cyclization process may include a derivatization step. 20 The cyclization step may also occur in vitro using isolated enzyme systems or using chemical means. Reference to a "derivative" of the amino acid sequence includes the derivatization of a precursor sequence to a cyclizable sequence. 25 The present invention extends to the nucleic acid molecule encoding the cyclotide or its linear precursor. Furthermore, the 'cyclotide" may be linear in the sense that it has free amino acid and carboxy termini but still folds into a knot arrangement. Such a linear form is regarded as a structural homologue of the cyclotide. 30 Accordingly, another aspect of the present invention contemplates an isolated nucleic acid -24 molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of forming a structural homologue of a cyclic peptide, polypeptide or protein within a cell or a membrane of a cell to form a backbone wherein 5 said backbone comprises sufficient disulfide bonds to confer a stabilized folded structure on the three dimensional structure of said backbone wherein said backbone comprises free amino and carboxyl termini. Reference herein to a "cyclic backbone" preferably includes a molecule comprising a 10 sequence of amino acid residues or homologues thereof without free amino acid and carboxy and amino termini. The cyclic backbone encoded by the nucleic acid molecule of the present invention comprises sufficient disulfide bonds, or chemical equivalents thereof, to confer a stabilized 15 folded structure on the three dimensional structure of the cyclic backbone. Preferably, the stabilized folded structure comprises a knotted topology. In a preferred embodiment, the cyclic backbone comprises a cystine knot. A cystine knot 20 occurs when a disulfide bond passes through a closed cyclic loop formed by two other disulfide bonds and the amino acids in the backbone. Such a cystine knot is referred to herein as "cyclic cystine knot" or "CCK". Reference herein, however, to a cyclic cystine. knot or a CCK includes reference to structural equivalents thereof which provide similar constraints to the three dimensional structure of the cyclic backbone. For example, 25 appropriate turns and loops in the cyclic backbone may also be achieved by engineering suitable covalent bonds or other forms of molecular associations. All such modifications to the cyclic backbone which retains the three-dimensional knotted topology conferred by the cyclic cystine knot are encompassed by the present invention including such modifications to the nucleic acid molecule which encodes a modified cyclotide. Furthermore, although a 30 cyclic cystine knot is characterized by a knot formed on three disulfide bonds, the present invention extends to molecules comprising only two disulfide bonds. In such a case, the -25 cyclic peptide, polypeptide or protein may need to be further stabilized using other means or the molecule may retain suitable activity despite a change in three-dimensional structure caused by the absence of a third disulfide bond. Reference herein to a "knotted topology" is not to be construed as limiting the invention to such a topology alone since the instant 5 invention extends to any stabilizing folded structure. Furthermore, the cyclic backbone may comprise more than three disulfide bonds such as occurring in a double or multiple cystine knot arrangement or in a single cystine knot arrangement supplemented by one or two additional disulfide bonds. 10 Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure: 15 C[X1 ... Xa} C[X11 ... X'b] C[X" ... X1c] C[X111 ... X1la] C[X"V ... X[VC] C[X ... X d wherein 20 C is cysteine; each of [Xi ... X.], [X 1 I ... Xb),. [X ... X"J, [X"'m ... Xmd], [XwI ... X'e] and [Xv, ... Xvr] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and 25 wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and each of a to f may be the same or different and range from 1 to about 20. 30 Preferably, each of a to f ranges from I to about 10.

-26 In a particularly preferred embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides which encodes or is complementary to a sequence which encodes an amino acid sequence capable of forming a cyclic backbone wherein the cyclic backbone comprises the structure: 5 C[XI Xa] C[X', X'j C[X". X"r] CX"'. X 1 1 j1 C[XW, ... X'eJ C[XVI ... XVdr I ~I wherein 10 C is cysteine; each of [XI ... Xa's),' [Xb], ... X"c], [Xm', ... X"], [Xw 1 ... X'\] and [Xv 1 ... Xvr] represents one or more amino acid residues wherein each one or more amino 15 acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is from about 3 to about 6, b is from about 3 to about 5, c is from about 2 to about 7, d is about 1 to about 3, e is about 3 to about 6 and f is from 20 about 4 to about 9. In an even more particularly preferred embodiment, the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino 25 acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure: C[XI Xa] C[X' 1 ... X'b] C[X", . X 1 1 c] C[XI" ... X" 1 li] C[XwI ... XIe] C[Xv X 1 30 -27 wherein C is cysteine; 5 each of [Xi ... X. [XI ... X'b], [X"I ... X"j], [XI ... X"mdI [X'w 1 ... X'\] and [Xv, ... Xvr] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and wherein a, b, c, d, e and f represent the number of amino acid residues in each 10 respective sequence and each of a is about 3, b is about 4, c is about from 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7. In still an even more particularly preferred embodiment, the present invention provides a nucleic acid molecule an isolated nucleic acid molecule comprising a sequence of 15 nucleotides, which sequence of nucleotides, or its complementary form, encodes an amino acid sequence wherein the amino acid sequence or a derivative form thereof is capable of being cyclized within a cell or a membrane of a cell in an in vitro cyclizing system to form a cyclic backbone wherein the cyclic backbone comprises the structure: 20 C[X ... Xa] C[X'. ... X 1 e] CX 1 1 .. 1 C[X"I ... X 111 ] C(Xwi ... X~e] C[X VI ... X v d wherein 25 C is cysteine; each of [Xi ... X 0 ], [X 1 ... XIb], [X"I ... X"c), [XI" ... Xm 1 d, [XW, ... X"Q} and [Xv, ... Xvf] represents one or more amino acid residues wherein each one or more amino acid residues within or between the sequence residues may be the same or different; and 30 -28 wherein a, b, c, d, e and f represent the number of amino acid residues in each respective sequence and wherein a is about 6, b is about 5, c is about 3, d is about 1, e is about 5 and f is about 8. 5 The invention extends to and includes peptide, polypeptide and protein sequences which have not been acted upon by an enzyme system separate from the molecule itself. For example, the present invention extends to autocatalytic cyclization. The cyclization of the amino acid sequence may occur in a cell such as a plant cell or cell 10 membrane which naturally contains the cyclization enzyme or enzymes. An in vitro system may also be employed with isolated enzyme(s) capable of cyclizing target molecules. Chemical means may also be employed to facilitate cyclization. Alternatively, cells may be engineered to express the genes encoding the cyclization enzyme or enzymes. In relation to the former, preferred cells are whole plants, callus or cell lines or membranous 15 preparations of cells from the Rubiaceae, Violaceae or Cucurbitaceae plant families. However, any other plant or part of a plant which contains the requisite cyclization enzymes are encompassed by the present invention. In relation to engineered cells, these may be plant, animal, insect, fungal, yeast or microbial cells. The cyclization enzyme(s) may be encoded by genetic sequences resident on a plasmid or vector or the genetic 20 sequences may be integrated into the chromosome of the cells. A genetic construct may also be introduced comprising nucleotide sequences encoding an amino acid sequence to the cyclized and the enzyme(s) required for cyclization. The gene or genes required to encode the cyclization enzymes may be isolated by any 25 number of means including differential display, yeast two hybrid systems, immunological screening techniques and a variety of visual display techniques. In one example, a nucleic acid molecule of the present invention is manipulated to include a nucleotide sequence which encodes a reporter molecule capable of giving an identifiable signal. The reporter gene sequence is inserted into or fused to a nucleotide sequence encoding a precursor form 30 of the cyclotide. Examples of suitable reporter molecules include luciferase and (- - 29 galactosidase. The reporter molecule may also encode an amino acid sequence for which an antibody, labelled with a reporter molecule, may interact. The modified nucleic acid molecule can then be transferred by, for example, 5 transformation, electroporation, conjugation or Agrobacterium-mediated transfer to, for example, a bank of cells carrying a genomic library from a plant known to contain the cyclising enzyme or enzymes. When a modified nucleic acid molecule comprising a reporter gene sequence is introduced into a cell comprising the cyclizing enzymes, the precursor sequence is processed and the reporter molecule-encoding portion is cleaved off. 10 As a result, the cells would not produce a detectable signal. Such cells would then be selected for further analysis. Agrobacterium-mediated transformation may be via embryonic or organogenic callus. Many other approaches may be used to screen for and select clones encoding the cyclizing 15 enzymes or to directly identify the enzyme(s). For example, the cyclizing enzyme(s) may be identified using fluoro or colormetric substrates designed based on knowledge of the precursor sequence described herein. In one embodiment, linear peptides are produced which comprise a cleaveable sequence with a colored reagent (e.g. PNA) that would get released and be detectable on treatment with plant-extract containing the enzyme(s). This 20 provides a very powerful selection protocol for plant material containing cyclizing enzyme(s). Accordingly, another aspect of the present invention contemplates a method of identifying nucleic acid molecules which encode one or more enzymes required for cyclization of an 25 amino acid sequence said method comprising obtaining a nucleic acid molecule which encodes a precursor form of an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein; introducing or fusing to said nucleic acid molecule, a nucleotide sequence which encodes a reporter molecule capable of providing a detectable signal wherein said nucleotide sequence is inserted or fused to a portion of the 30 nucleic acid molecule which is cleaved off prior to or during cyclization; introducing said nucleic acid molecule comprising the nucleotide sequence encoding the reporter molecule - 30 into a bank of cells carrying a DNA library comprising all or part of genomic DNA or cDNA from a plant which carries the enzyme or enzymes required for cyclization of an amino acid sequence; screening for and selecting cells which do not synthesize the reporter molecule. 5 In a preferred embodiment, the plant is from the Rubiaceae, Violaceae or Cucurbitaceae family. The cyclizing enzyme(s) may also be useful in cyclizing smaller peptides and are useful, 10 for example, in the generation of combinatorial chemistry libraries of small (e.g. 5-30 amino acids) cyclic peptides. These may have a range of applications such as pharmaceutical applications. The present invention further contemplates a genetically modified cell or cells or a plant or 15 animal comprising said genetically modified cells, said cells comprising a nucleic acid molecule having a nucleotide sequence or complementary nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein. The peptide, polypeptide or protein may be cyclized in vivo or in vitro. 20 The cells may also be in the form of cells or cell lines or cell cultures. The present invention permits the manipulation of the nucleic acid molecules to introduce particular functional traits into the cyclized molecules or their linear forms or their 25 precursor forms. A "trait" includes an activity, molecule interacting ability or some other attribute which has the capacity to alter a phenotype. For example, the peptides, polypeptides or proteins may be manipulated to introduce modulating activity of, for example, calcium channel-binding useful in the treatment of pain or a stroke, C5a binding activity useful as an anti-inflammatory agent, proteinase inhibitor activity in plants or 30 animals, antibiotic activity, viral activity (e.g. of HIV or hepatitis virus), microbial activity, fungal activity, cytokine binding and blood clot inhibiting ability amongst other properties.

-31 Alternatively, the cyclic molecules themselves comprise useful traits such as being active against plant pests or pathogens. A plant pest or pathogen includes an insect, arachnid, microorganism, virus and a fungus. The term "pathogen" refers to any biological agent capable of interfering with biological function of said crop plants such that potential 5 agronomic output of said crop plants is reduced. Accordingly, the present invention contemplates a method of incorporating an amino acid sequence conferring a particular trait into a cyclic peptide, polypeptide or protein, said method comprising fusing or introducing a nucleotide sequence encoding said amino acid 10 sequence to or into a second nucleotide sequence wherein said second nucleotide sequence encodes a peptide, polypeptide or protein which peptide, polypeptide or protein or a derivative therefor is capable of being cyclized into a knotted peptide, polypeptide or protein. 15 The present invention extends to an antibody that can be used to identify and detect said peptide which is cyclized or not. The invention further extends to the use of the antibody as a primary antibody to detect the said peptide sequence after gel electrophoresis and Western blot transfer. 20 The present invention comprises a peptide sequence that can be processed from a larger polypeptide sequence. More specifically, the excised polypeptide sequence is flanked by the amino acid sequence triplet GLP at the N-terminal end of the peptide to be cyclized and at the C-terminal end of the peptide to be cyclized. More specifically, the invention refers to a peptide sequence which can be cleaved at the peptide bond immediately prior to 25 either glycine, leucine, proline or valine in either of the GLP sequences flanking the peptide to be excised (see Figure 8B). All these processing sites are encompassed by the present invention. In particular, the present invention contemplates a range of processing sites which may occur naturally or be introduced. For example, different processing sites may be introduced such that depending on the organelle or tissue targeted, the peptide, 30 polypeptide or protein may be differently processed.

- 32 The introduced amino acid sequence is referred to herein as a "heterologous" amino acid sequence. Another another aspect of the invention contemplates an isolated nucleic acid molecule 5 comprising the following nucleotide sequence: [Xi .. .Xb[nin2 ... nlyj .-YcId wherein 10 - [nin2 ... n.] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; and XI... Xb and yi... yc represent polynucleotide sequences capable of encoding an 15 amino acid sequence where a and b and c and d may be any number and when d is >1, the amino acid sequence may be unique for each integer of d. The nucleic acid molecule according to this aspect of the present invention may be regarded as a hybrid nucleotide sequence comprising the structure: 20 ji...j,[X ... Xa[nin 2 ... naylI...ycJdqi...qr wherein 25 [nin 2 ... n] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein;

X

1 .. .X and yI...y, represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d may be any number and when d is >1, the 30 amino acid sequence may be unique for each integer of d; and - 33 ji. .je and qi...qr represent nucleotide sequences encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle where a, b, c, d, e and f may be any number, where d is >1, the amino acid sequence may be unique for each integer of d. 5 In a related embodiment, there is provided an isolated nucleic acid molecule comprising the following nucleotide sequence: [Xi...Xb[njn2 ... (kI -...k6)Xna. .Y Ycl 10 wherein [nin2 ... n.] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; 15 X1... Xb and y... y represent polynucleotide sequences capable of encoding an amino acid sequence where a and b and c and d, 5 and X may be any number and when d or X is >1, the amino acid sequence may be unique for each integer of d and X; 20 kl...k 8 represent a nucleotide sequence encoding an amino acid sequence conferring a particular activity or other trait. In yet another related embodiment, the present invention is directed to an isolated nucleic acid molecule comprising the following nucleotide sequence: 25 [nIn 2 ... (ni n21.. .n')...na]m wherein 30 [nin2 ... n.] and (ni'n2.. .n 7 ) represent polynucleotide sequences encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or - 34 protein; and y and a and m may be any number and when m is >1, the amino acid sequence may be unique for each integer of m. 5 Further, the invention contemplates an isolated nucleic acid molecule comprising the following nucleotide sequence: ji...je[Xi...Xb[nin2 ... (ni in21...(ki ... ksnal)mn.]yi ... ycIdql... qr 10 wherein [nin2 ... na] represents a nucleotide sequence encoding an amino acid sequence capable of being cyclized to a knotted peptide or polypeptide or protein; 15

X

1 ... X and yi... yc represents a polynucleotide sequence capable of encoding an amino acid sequence where a and b and c and d and e may be any number and when d is >1, the amino acid sequence may be unique for each integer of d; 20 ji...je and qi.. .q represents a nucleotide sequence encoding a peptide, polypeptide or protein capable of directing the peptide, polypeptide or protein to a cellular compartment or organelle; k 1 ... ke represents a nucleotide sequence encoding an amino acid sequence 25 conferring a particular activity or other trait; X and m and d may be any number and when X and m and d are each >1, the amino acid sequence may be unique for each integer of X, m and d. 30 The "activity" may inter alia be a therapeutic activity, an enzymic activity or an activity useful as a laboratory reagent. Protease inhibitor activities, for example, may be used to - 35 generate plants (e.g. crops) resistant to pathogens. Alternatively, protease activities may be used to produce stable molecules useful in industrial grade washing compositions. In a particularly preferred embodiment, the activity confers protection to a plant or animal 5 cell from pathogen infestation. Examples of pathogens include insects, spiders, and other arachnids, microorganisms, viruses and fungi. Such an activity may also be exhibited by the cyclic peptide, polypeptide or protein without need for the introduction of an additional amino acid sequence. The present invention further extends to linear forms and precursor forms of the peptide, polypeptide or protein which may also have activity or other utilities. 10 Various agricultural applications of the cyclic molecules or their linear forms are particularly contemplated by the present invention. For example, the present invention extends to engineering crop plants (such as cotton) to be resistant to pathogens (e.g. insects). 15 The present invention further provides genetic constructs comprising the nucleic acid molecules of the present invention. Such a genetic construct is particularly useful for expressing the nucleic acid molecule to produce linear or precursor forms of the cyclizable amino acid sequence. In this case, the genetic construct also comprises one or more promoters operably linked to the nucleotide sequences. Genetic constructs suitable for use 20 in plants are particularly preferred. The genetic constructs may encode linear forms only of the peptides which are then subsequently circularized in vitro using, for example, enzyme(s) or chemical means. Alternatively, cell or cell membrane systems may be employed. 25 The genetic construct of the present invention may comprise a sequence of nucleotides or be complementary to a sequence of nucleotides which comprise one or more of the following: a promoter sequence, a 5' non-coding region, a cis-regulatory region such as a functional binding site for a transcriptional regulatory protein or a translational regulatory protein, an upstream activator sequence, an enhancer element, a silencer element, a TATA 30 box motif, a CCAAT box motif, an open reading frame, a transcriptional start site, a translational start site, and/or nucleotide sequence which encodes a leader sequence. The -36 genetic construct of the present invention also encodes cyclizable peptide, polypeptide or protein. Furthermore, the genetic construct may comprise a cassette which may be used to insert a nucleotide sequence to be inserted into or fused to a cyclized peptide, polypeptide or protein. Furthermore, the nucleotide sequence to be inserted may consist of 5 polynucleotide units called codons. A codon consists of three nucleotide bases wherein the sequence of the three nucleotide bases defines a specific amino acid. The invention extends to the use of any codon, triplet or polynucleotide sequence known to encode a specific amino acid. Furthermore, the invention extends to any polynucleotide sequence that defines a peptide sequence or polypeptide that can be fused to the inserted sequence for the 10 purpose of targeting, transporting or regulating the expression of the polypeptide sequence to which it is fused. In one particular embodiment, a vacuole or other ceullar organelle is targeted. The term "5' non-coding region" is used herein in its broadest context to include all 15 nucleotide sequences which are derived from an upstream region of an expressible gene, other than those sequences which encode amino acid residues which comprise the polypeptide product of the gene, wherein the 5' non-coding region confers or activates or otherwise facilitates, at least in part, expression of the gene. 20 The term "gene"' is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product. A gene includes any sequence of nucleotides which may be transcribed into a mRNA molecule. Use of the term "gene" is not to place any structural or functional constraints on the scope 25 of the present invention. As used herein, the term "cis-acting sequence" or "cis-regulatory region" or similar term shall be taken to mean any sequence of nucleotides which is derived from an expressible genetic sequence wherein the expression of the first genetic sequence is regulated, at least 30 in part, by said sequence of nucleotides. Those skilled in the art will be aware that a cis regulatory region may be capable of activating, silencing, enhancing, repressing or -37 otherwise altering the level of expression and/or cell-type-specificity and/or developmental specificity of any structural gene sequence. Reference herein to a "promoter" is to be taken in its broadest context and includes the 5 transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type-specific manner. A promoter is 10 usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. In the present context, the term "promoter" is also used to describe a synthetic or fusion 15 molecule, or derivative which confers, activates or enhances expression of a structural gene or other nucleic acid molecule, in a cell. Preferred promoters according to the invention may contain additional copies of one or more specific regulatory elements to further enhance expression in a cell and/or to alter the timing of expression of a structural gene to which it is operably connected. 20 The term "operably connected" or "operably linked" in the present context means placing a structural gene under the regulatory control of a promoter which then controls expression of the gene. Promoters and the like are generally but not necessarily positioned 5' (upstream) to the genes which they control. In the construction of heterologous 25 promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting, i.e., the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be 30 accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is -38 defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. The genetic construct(s) of the present invention may be introduced into a cell by various 5 techniques known to those skilled in the art. The technique used may vary depending on the known successful techniques for that particular cell. Techniques for introducing recombinant DNA into cells such as plant cells include, but are not limited to, transformation using CaC 2 and variations thereof, direct DNA uptake into 10 protoplasts, PEG-mediated uptake to protoplasts, electroporation, microinjection of DNA, microparticle bombardment of tissues or cells, vacuum-infiltration of tissue with nucleic acid, and T-DNA-mediated transfer from Agrobacterium to the plant tissue. For microparticle bombardment of cells, a microparticle is propelled into a cell to produce 15 a transformed cell. Any suitable ballistic cell transformation methodology and apparatus can be used in performing the present invention. Exemplary apparatus and procedures are disclosed by Stomp el aL (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed. 20 Examples of microparticles suitable for use in such systems include 0.1 to 10 pm and more particularly 1 to 5 pm tungsten or gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation. 25 In a particularly preferred embodiment, the nucleotide sequence encoding the cyclizable amino acid sequence or its precursor comprises the amino acid sequence substantially as set forth in <400>2, <400>4 or <400>6 or an amino acid sequence having at least 60% similarity thereto. 30 Preferably, the nucleotide sequence is substantially as set forth in <400>1, <400>3 or <400>5 or a sequence having at least 60% similarity thereto or capable of hybridizing -39 thereto under low stringency conditions at 42*C. The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, 5 "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, 10 nucleotide and sequence comparisons are made at the level of identity rather than similarity. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", 15 "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide 20- sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 25 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, 30 FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best - 40 alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (7). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (8). 5 The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two 10 optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the 15 window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the 20 reference manual accompanying the software. Similar comments apply in relation to sequence similarity. Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about I M to at least about 2 M salt for 25 hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30"C to about 42"C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v 30 to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing -41 conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formanide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)% (9). 5 However, the Tm of a duplex DNA decreases by 1*C with every increase of 1% in the number of mismatch base pairs (10). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42*C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20*C to 65*C; high stringency is 10 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65*C. The present invention further contemplates a genetically modified plant which comprises a nucleotide sequence which encodes an amino acid sequence capable of being cyclized into a knotted peptide, polypeptide or protein and which confers on said plant a trait not present 15 in the same species or variety of plant prior to genetic modification. The plant may also comprise one or more nucleotide sequences which encode one or more cyclizing enzymes. The genetically modified plants of this aspect of the present invention include plants which are resistant to certain pathogens. Crop plants resistant to pathogens are particularly 20 preferred. Crop plants include but are not limited to cotton, cereal crops, vegetable crops, seed crops and flowering crops. Yet another aspect of the present invention provides the use of a nucleic acid molecule encoding an amino acid sequence, which amino acid sequence or a derivative or precursor 25 form thereof is capable of being cyclized into a knotted peptide, polypeptide or protein, in the manufacture of a transgenic or genetically modified plant capable of producing said cyclic knotted peptide, polypeptide or protein. The present invention is further described by the following non-limiting Examples. 30 -42 EXAMPLE 1 Plant material Oldenlandia affinis DC and Viola odorata were grown under standard glasshouse 5 conditions. EXAMPLE 2 RNA isolation 10 RNA was prepared from various tissues of 0. affinis using TRIzol (trademark) reagent and the protocol from Gibco BRL (see Gibco BRL form #3796, TRIzol (trademark) Reagent Total RNA Isolation Reagent). EXAMPLE 3 15 Methods for isolating a partial cDNA clone encoding Kalata B1 Single stranded cDNA was prepared from 0. affinis leaf RNA using the Gibco BRL RT PCR kit and an oligo-dT primer according to the manufacturer's instructions. The cDNA produced was amplified by the polymerase chain reaction using one of two degenerate 20 primers and oligo-dT (Bresatec). The two degenerate primers and the encoded protein sequence are shown in Figure 1B. The oligonucleotides were dissolved in milliQ water to a final concentration of 200 yM: The PCR reaction was performed using a profile of 30 cycles of 94"C (3 min), 37"C (3 min) and 70*C (3 min). After 30 cycles, there was a final extension step at 72*C for 10 min. PCR products were separated on 2% w/v agarose gels in 25 TBE (45 mM Tris Borate, I mM EDTA). The QIAGEN Gel extraction kit was used to purify the amplified fragments which were subsequently cloned into the pBluescript SK vector (Stratagene) and sequenced by SUPAMAC (Sydney University and Prince Albert Macromolecular Analysis Centre, Sydney, Australia), using T3 and T7 primers.

- 43 EXAMPLE 4 Preparation and screening of the 0. affinis cDNA library Total RNA (1 mg) was prepared from leaves and stem and mRNA was separated using the 5 PolyATract (trademark) I (Promega) mRNA isolation system. Five microgram of mRNA was used to produce the cDNA library in a Lambda-Zap vector using the Stratagene ZAP cDNA and Gigapack cloning kits according to manufacturer's instructions. The amplified library was screened using a 3 2 P-labelled DNA fragment corresponding to bases 7-105 of the partial Kalata BI clone (Figure 2) as probe. After a second round of screening, 10 hybridizing clones were chosen for sequence analysis. Excised phagemids were transformed into XLI-Blue (Stratagene) E. coli cells via electroporation using a GenePulsar electroporation apparatus (BioRad). Plasmid was isolated using the alkaline lysis protocol (11). Sequence analysis was performed by SUPAMAC (Sydney University and Prince Albert Macromolecular Analysis Centre, Sydney, Australia), using T3 and T7 15 primers. Analysis of DNA sequences was performed using SeqEd (Applied Biosystems). The Oak] and Oak 2 clones were isolated as described above. The Oak 3 and Oak 4 clones were isolated using an identical procedure except the full length Oak] cDNA was used as probe. 20 EXAMPLE 5 RNA blots Total RNA (10 pg) was fractionated on 1.2% w/v agarose gels in the presence of formaldehyde and transferred to HyBond N* (Amersham) as described by Sambrook et at 25 (11). Prehybridization (at 42*C) and hybridization (16 hr at 42*C) was performed in 5 x SSPE (0.9 M NaCI, 50 mM NaH 2

PO

4 .2H 2 0, 5 mM EDTA), 1% w/v SDS, 5 x Denhardt's solution [0.1% w/v Ficoll, 0.1% w/v BSA fraction V, Sigma], 0.1% w/v polyvinylpyrrolidone, 50% w/v deionised formamide and 100 pg/ml Herring sperm DNA. The membrane was probed with either the Kalata Bl clone (Figure 2) or the Oakl cDNAO 30 (Figure 4), unbound probe was removed by washing three times with 2 x SSC and 1% w/v SDS at 42*C for 10 min. Hybridizing RNA was visualized after exposure to a -44 phosphoimager screen for 15 hr using a model 400B Phosphorimager (Molecular Dynamics) and ImageQuant (trademark) software. EXAMPLE 6 5 DNA blots Genomic DNA was isolated from fresh leaf material (1.5 g) as described in the QIAGEN Genomic Tip protocol. Genomic DNA (10 pg) was digested with restriction enzymes; HindII, BamHl, Ndel and EcoRV (Promega, 5 units) and separated on a 0.7% agarose gel 10 in the presence of ethidium bromide and TBE buffer (11). The DNA was transferred to an N* nitrocellulose membrane as described (11) and the blot was probed with the full-length Oakl cDNA. The blot was prehybridized, hybridized and washed as described for the RNA blots. Hybridizing DNA was visualized using a model 400B Phosphorimager (Molecular Dynamics) and ImageQuant (trademark) software. 15 EXAMPLE 7 Bacterial expression ofthe protein encoded by Oakl The cDNA encoding the Kalata B1 precursor protein was PCR amplified from the 20 OaklcDNA using oligonucleotide primers complementary to bases 61-75 (forward primer) and 361-372 (reverse primer). The amplified fragment was cloned into the pGEM-T-Easy vector (Promega) before it was excised and subcloned into pQE-30 (QIAGEN) to create pQKBl. Eschericia coli m15 cells containing the pREP-4 plasmid were transformed with pQKBI and grown in Luria broth containing ampicillin (100 pg/ml) and kanamycin (12.5 25 pig/ml) before induction with IPTG (1 mM). Cells were then pelleted by centrifugation and resuspended in sample buffer (12) and heated before analysis by SDS-PAGE. Alternatively, the cells were suspended in a lysis buffer (50 mM Tris-HCI pH 8.5, 2 mM EDTA, 50 pg/ml lysozyme and 10%v/v Triton X-100; 5 ml of lysis buffer/g of cells) before incubation at 37*C for 15 min. Cell lysate was then mixed with MgCl 2 (10 mM 30 final concentration) and DNase 1 (Roche, 10 pg/ml final concentration) and insoluble material was collected by centrifugation (13,000 rpm, 15min, 4*C). The insoluble protein -45.

pellet was washed several times with 0.5% Triton X-100, 10 mM EDTA before a final wash in distilled water. The proteins in the pellet were dissolved in denaturing lysis buffer (10 mM Tris-HCI pH 8.5, 100 mM NaCl, 8 M urea) before chromatography on an immobilized metal affinity column (TALON (registered trademark) metal affinity resin, 5 Clontech, Palo Alto, CA, USA) according to the procedure described by the manufacturer for Batch/Gravity-Flow Column Purification (Clontech user manual Protocol #PT1320-1, version #PR96975). Protein eluted from the column was analyzed by SDS-PAGE, RP HPLC (reversed phase high performance liquid chromatography) or ESMS (electrospray ionization mass spectrometry). 10 EXAMPLE S Reverse Phase High Pressure Liquid Chromatography (RP-HPLC) and mass spectrometry 15 RP-HPLC was performed on a Brownlee RP300 C8 analytical column (4.6 x 100 nm) using a Waters model 510 pump and a Waters model 481 UV detector. Samples were applied in 0.1% v/v trifluoroacetic acid (Buffer A) and were eluted with 60% v/v acetonitrile, 0.089% v/v trifluoroacetic acid (Buffer B) according to a gradient of 0-100% Buffer B over 30 min with a flow rate of I ml/min. Eluted protein was detected by 20 absorbance at 215 nm. The molecular mass of the RP-HPLC purified protein was determined by electrospray ionization mass spectrometry (ESMS) using a Perkin-Elmer Sciex API-300 triple quadrupole mass spectrometer fitted with a micro-ionspray ion source. 25 EXAMPLE 9 Bioassays with artificial diets Helicoverpa punctigera larvae were raised on artificial diets based on Haricot beans (Teakle et al (13)). One litre of diet was composed of 234 g Haricot beans, 14 g agar, 700 30 ml water, 35 g Tortula yeast, 50 g wheatgerm, 3.5 g ascorbic acid, 1.1 g sorbic acid, 2.2 g p-hydroxybenzoic acid methyl ester, 0.2 g ampicillin, 0.2 g streptomycin, 16 mg - 46 prochloraz. The beans were soaked overnight in water, drained and homogenized to a fine paste. Wheatgerm, yeast and 300 ml of water were added. The agar was dissolved in 400 ml of boiling water and added to the mixture. The mixture was cooled to 50"C before the addition of the remaining ingredients. The blended diet was poured into trays and after 5 setting was used immediately or stored at -20*C for no longer than two weeks. The test diet was supplemented with the Kalata BI peptide (0.825 pmol/g of diet). Twenty newly emerged neonates were added to each diet and mortality was recorded every two days. Weight gain was recorded at the sixth day and then every second day thereafter. The larvae were reared in 1.5 ml eppendorfs microfuge tubes (one larva/tube) until day eight when 10 they were transferred to individual plastic containers with lids (Solo (trademark) plastic portion cups, 28 ml) at the eight day. Larvae were fed small amounts of diet (40 mg) initially that was replaced as required to provide a continuous supply. The larvae were kept in a temperature controlled room at 25 +1"C, 16:8 (L:D). 15 Helicoverpa armigera larvae were raised on an artificial diet based on cotton leaves. One hundred ml of cotton leaf artificial diet was composed of 3 g cotton leaf powder (see below), 2 g Tortula yeast, 2.4 g wheat germ, 3.2 g ascorbic acid, 0.08 g sorbic acid, 0.16 g paraben (mould inhibitor), 0.08 ml linseed oil, 0.16 ml wheatgerm oil, 0.028 g ampicillin, 0.028 g streptomycin, 3.2 g agar and 80 ml water. Cotton leaf powder was prepared from 20 freshly picked young, healthy cotton leaves which had been rapidly frozen in liquid nitrogen and freeze dried before they were ground to a fine powder using a mortar and pestle. Test diets contained the cyclic Kalata BI peptide at 0.15% w/v of diet (high kalata) or 0.03% w/v of diet (low kalata), linear peptide (backbone chain opened between residues 7 and 8 [see Figure 1A] at 0.15% w/v of diet. The control diet contained casein at 0.15% 25 w/v of diet. The diet was mixed and dispensed as described for the Haricot bean diet and the bioassay was conducted in the same manner.

- 47 EXAMPLE 10 Isolation of a partial cDNA encoding Kalata B As Kalata B 1 is a cyclic protein of only 29 amino acids and the N-terminus was unknown, 5 two degenerate primers were designed to amplify part of the encoding DNA (see Figure 1). Five PCR amplified products were obtained using these primers in combination with the oligo-dT-HindIII primer (Figure 1C). The 412 bp fragment produced from primer Kal2 (fragment 5, Figure IC) has a 3' untranslated region of 267 bp and a poly A tail of 32 bp together with the complete coding sequence of Kalata B 1 (Figure 2). When used as a probe 10 on RNA blots containing 0. affinis leaf RNA, the partial Kalata BI clone hybridised to an RNA transcript of about 750 bases, suggesting the cyclic peptide is derived from a larger precursor protein (Figure 3). EXAMPLE 11 15 Isolation of a full length cDNA clone for Kalata Bl and a second cDNA clone encoding two Kalata related peptides The cDNA library prepared from leaf and stem mRNA was screened using the partial Kalata BI cDNA as probe. Two full length clones were obtained, the first designated 20 OaK1 (for 0. affinis Kalata BI) was 725 bp long and encodes a predicted protein of 124 amino acids (Figure 4). The 29 amino acid Kalata B 1 sequence is embedded in a precursor protein which has a typical endoplasmic reticulum (ER) signal sequence of 20 amino acids. It is likely that the precursor enters the secretory pathway where folding and disulphide bond formation occurs prior to the cyclization and cleavage events that release the mature 25 cyclic peptide. The B1 sequence is preceded by about 70 amino acids at the N-terminus and four to seven amino acids at the C-terminus. All six cysteines in the precursor are located in the B 1 sequence. The predicted precursor has no potential N-glycosylation sites and, hence, has an expected mass of 11.18 kDa. 30 The second cDNA clone, designated Oak2 has an insert of 843 bp and unlike the first clone is predicted to encode two Kalata BI related sequences which we have called B3 and B6 -48 (Figure 5). The predicated protein also has typical ER signal sequence of 20 amino acid which is followed by a 46 to 49 amino acid sequence before the first Kalata sequence (B6) is encountered. This peptide is separated from the Kalata B3 sequence by about 25 amino acids. The B3 sequence is flanked four amino acids at the C-terminus (SAAA) which are 5 similar to those which flank Bl(SLAA) in the protein encoded by the Oakl clone. Like the precursor encoded by the Oakl clone, the precursor encoded by the Oak2 clone has no potential N-glycosylation sites and all cysteine residues are confined to the Kalata peptide sequences. After removal of the potential ER signal sequence the precursor encoded by the Oak2 clone has a predicted mass of 14.56 kDa. The size of both clones is consistent with 10 the size of the hybridizing transcript detected in the Northern analysis of leaf RNA (Figure 3). The third clone designated Oak3 was 677 bp long (Figure 6) and encodes a predicted protein of Ill amino acids. It has only one Kalata Bl related sequence that has been called 15 B7. The fourth clone designated Oak4 has an insert of 993 bp and encodes a predicted protein of 210 amino acids (Figure 7). This protein has three identical sequences that are related to Kalata B 1. This sequence has been called Kalata B2. 20 A schematic diagram of the precursor proteins predicted from the Oak 1, 2,3 and 4 clones is given in Figure 8A. Cyclic peptides with the same sequence as Kalata B 1, B2 and B3 have been isolated from the leaves of the 0. affinis plant (14) (Example 12). The inventors conclude that these peptides are derived by proteolytic cleavage of a precursor protein and 25 formation of a new peptide bond. That is, it is likely that Bi, B2 and B3 cyclotides are produced from precursor proteins encoded by the Oak], Oak4 and Oak2 clones respectively. Each of the BI, B3, B6 and B7 peptides in the predicted precursor proteins (Figure 8B) is 30 flanked on both sides by the highly conserved sequence -1-Gly-2-Leu-3-Pro-4-. The circularization process thus involves specific ligation at the same cleavage site within both -49 flanking sequences (one of the four peptide bonds shown) and ligation of the new N- and C-termini. The mature cyclic peptide retains one copy of the Gly-Leu-Pro sequence, which may be derived entirely from one of the original flanking elements, or partially from both depending on the initial cleavage sites (Figure 8A). 5 The protein encoded by the Oak4 clone offers further insight into the potential processing site. Unlike the proteins encoded by the other three clones, the Oak4 protein has three copies of a Kalata like sequence. This sequence (B2) is flanked by Gly-Leu-Pro at the N terminus and Ser-Leu-Pro at the C-terminus (Figure 8B). The B2 peptide isolated from the 10 plant (14) (Example 12) has retained the Gly-Leu-Pro sequence. Processing thus appears to have occurred at the peptide bonds preceding the glycine and the seine (see Figure 8B). EXAMPLE 12 Isolation and structure determination of Kalata B2 15 Kalata B2 was isolated from aerial parts of 0 affinis by extraction with dichloromethane/methanol (50:50 v/v) and purified using reverse phase HPLC (Vydac C18 column). Gradients of CH3CN in H 2 0 (0.1% trifluoroacetic acid, v/v) were employed in the purification. The purified Kalata B2 was reduced with an excess of tris-carboxyethyl 20 phosphine, TCEP, and alkylated with maleimide. The reduced and alkylated peptide was cleaved with Endo-Glu C in ammonium acetate buffer at pH 7.7 for 2 hours and then purified by reverse phase HPLC. The cleaved peptide was N-terminally sequenced using Edman degradation on an Applied Biosystems 477A Protein Sequencer. 25 The structure of Kalata B2 was determined using NMR spectroscopy and simulated annealing calculations. Samples for 'H NMR measurement contained -1.5 mM peptide in 90% H20/10% D 2 0 (v/v) at pH 3.6. Spectra were recorded at 290K, 298K and 305K on a Bruker ARX-500 spectrometer equipped with a shielded gradient unit and on a Bruker DRX-750 spectrometer. The following homonuclear 2D NMR spectra were recorded in 30 phase-sensitive mode using time-proportional phase incrementation for quadrature detection in the ti: TOCSY using a MLEV- 17 spin lock sequence with an isotropic mixing -50 period of 80 ms; NOESY with mixing times of 200 ms, 250 ms and 300 ms; double quantum filtered DQF-COSY and E-COSY. For DQF-COSY and E-COSY spectra solvent suppression was achieved using selective low-power irradiation of the water resonance during a relaxation delay of 1.8 ms. Water suppression for NOESY and TOCSY 5 experiments was achieved using a modified WATERGATE pulse sequence. Spectra were acquired over 6024 Hz with 4096 complex data points in F2 and 512 increments in the F1 dimension, with 16 to 64 scans per increment. Spectra were processed on a Silicon Graphics Indigo workstation using UXNMR (Bruker) software. The t dimension was zero-filled to 2048 real data points and 90* phase-shifted sine bell window functions were 10 applied prior to Fourier transformation. Chemical shifts were referenced to DSS at 0.00 ppm. Distance restraints were derived from the 250 ms and 300 ms NOESY spectra recorded at 290 K, 298 K and 300 K. Inter-proton distance restraints were assigned upper-distance 15 bounds of 2.76 A, 3.50 A or 5.00 A corresponding to strong, medium or weak cross-peak volumes, respectively. Pseudoatom corrections were applied where necessary to methylene and methyl protons. Backbone dihedral angle restraints were measured from either ID NMR spectra or the anti-phase cross-peak splitting in a high digital resolution 2D DQF COSY spectrum. Stereospecific assignment of methylene protons and xi dihedral angle 20 restraints were derived from coupling constants measured from an E-COSY spectrum in combination with HN-Hp, HN-HO, Ha-H, and Ha-He NOE intensities. Slow exchanging amide protons were detected after the sample was lyophilized and reconstituted in 99.99% 2

H

2 0, and were later used to check for consistency of hydrogen bonding interactions in the calculated structures. 25 Based on the NMR constraint data the three-dimensional structure of Kalata B2 was calculated using a dynamic simulated annealing protocol in the program X-PLOR version 3.1. The procedure was based on that described by Saether et al, 1995 (1). After an initial simulated annealing calculation of a family of 50 structures the ensemble of structures was 30 checked for violations in NOE restraints and ambiguous cross-peaks were resolved on the basis of inter-proton distances. Finally, each member of the ensemble was energy - 51 minimized for 1000 cycles using the conjugate gradient Powell algorithm and a refined force field based on the program CHARMm. A schematic representation of the structure of Kalata B2 is shown in Figure 9 (left panel). The right panel shows an overlay of the structures of Kalata B1 (1) and Kalata B2. 5 EXAMPLE 13 Genes encoding Kalata like peptides belong to a multigene family in 0. affinis Genomic DNA was digested with HindIII, Bam HI, Ndel and EcoRV and subjected to 10 DNA blot analysis using Oakl cDNA as probe. About twelve hybridizing bands were obtained in all the digests (Figure 10) suggesting the cyclotides are derived from a multigene family with up to 12 related genes. EXAMPLE 14 15 Bacterial expression of the Kalata B1 precursor encoded by the Oak) clone The Oakl cDNA was subcloned into the pQE-30 vector for bacterial expression. A protein of the expected size was induced after addition of IPTG and was purified by immobilized metal affinity chromatography (IMAC) (Figure 11). This protein has a mass of 12,938 20 1.2 Da which is consistent with the predicted mass of the protein encoded by the Oakl clone together with the hexahistidine tag. Furthermore, the six cysteine residues had formed into three disulphide bonds. The metal affinity purified protein produced a single, sharp peak on reversed phase HPLC (Figure 11B) indicating that the protein had folded to a single conformation. 25 EXAMPLE 15 Antibody to the bacterially expressed Kalata B) precursor recognizes the cyclic peptide 30 The bacterially expressed Kalata BI precursor (Figure 11) which had been purified by metal affinity chromatography and reversed phase HPLC (Figure 11B) was used to - 52 immunize a rabbit to generate polyclonal antibodies. The protein (1 mg/ml) in phosphate buffered saline (11) was emulsified with an equal volume of complete Freund's adjuvant (Gibco/BRL) and 100 Ig of protein was injected intramuscularly into a rabbit. The first bleed (15 ml) was taken 14 days after injection and serum was collected after incubation of 5 the blood at 37"C for 1 hr and at 4"C overnight. The clotted blood was collected by centrifugation (13000 rpm for 20 min at 4"C) and serum was collected, divided into 200 pi aliquots and stored at -80"C. A booster injection (prepared as described previously except incomplete Freund's adjuvant was used in place of complete Freund's adjuvant) was administered 4, 8 and 12 weeks after the initial injection. Serum prepared from blood 10 collected two weeks after the third boost was used on the immunoblot shown in Figure 12 at a 1:1000 dilution. Purified kalata BI (1 pg), Kalata BI precursor (1 pg) and buffer soluble proteins from 0. affinis leaves (100 pg) were subjected to SDS-PAGE on a precast 4-12% bis-tris protein 15 gel (Novex, San Diego, CA, USA) and transferred to a nitrocellulose membrane 0.2 Im pore size) Micron Separations, Inc., Westborough, MA, USA) in transfer buffer (48 mM Tris-HCI, 192 mM glycine and 20% [w/v] methanol) by using a Novex transfer apparatus at lOOV for 1 hr at 4 0 C. After transfer blots were fixed in isopropanol for I min followed by 2% w/v glutaraldehyde for 20 min. The blot was then washed for 5 min in TBS (20 mM 20 Tris-HCI and 150 mM NaCl, pH 7.5) before it was blocked by incubation in 5% (w/v) skim milk powder in TBS for 1 hr at room temperature. The blot was rinsed in TBS for 5 min before incubation for I hr at room temperature with the primary antibody diluted (1:1000) in TBST (0.1% [v/v] Tween 20 in TBS) containing 5% w/v skim milk powder. The blot was washed again in TBST before the addition of goat anti-rabbit IgG-HRP 25 conjugate (Amersham, 5 x 103 dilution in TBST ) containing 5% w/v skim milk powder. After 1 hr at room temperature the membrane was washed in TBST and bound HRP was detected by the enhanced chemiluminescence (ECL) detection system of Amersham as described by the manufacturer. 30 The antibody raised to the bacterially expressed precursor protein encoded by Oakl recognized purified Kalata BI and the bacterially expressed precursor (Figure 12). This -53 antibody also bound to a peptide with the same mobility as the Kalata B 1 cyclotide in buffer soluble extracts from 0. affinis leaves. This antibody has application in location of the Kalata BI cyclotide in plant cells and as a tool to monitor processing of the precursor and thus to assist in the identification of processing enzymes. 5 EXAMPLE 16 Effect of Kalata B) on the growth and development of H. punctigera larvae Kalata B 1 had a significant effect on the development of larvae. No mortality was 10 observed in the first six days, although 50% failed to survive past day 16 (Figure 13A). After 16 days none of the 12 survivors had progressed past the first instar stage of development. Most larvae on the control diet, however, had achieved fifth instar (Figures 13B, C). 15 EXAMPLE 17 Effect of Kalata B) on the growth and development of H.armigera larvae Newly hatched larvae were fed on an artificial diet based on cotton leaves, with and without added Kalata B1. After 12 days on the diet the neonates on the Kalata Bl diet had 20 failed to progress past the first instar stage of development, whereas larvae on the control diet had achieved third to fourth instar. Larvae on the Kalata B 1 diet were about 18% of the size of those fed on the control diet. EXAMPLE 18 25 Activity of a linear form of Kalata B) in insect bioassays Kalata peptides isolated from leaves are normally cyclic. The inventors sought to determine whether cyclization was essential for biological activity against insects. Newly hatched H.armigera larvae were added to an artificial diet based on cotton leaves which 30 had been supplemented with cyclic Kalata B1 at either 0.15% w/v of diet or 0.03% w/v of diet. Growth of these larvae was compared to growth of larvae on diet with no added -54 peptide or with added linear Kalata BI at 0.15% w/v of the diet. Linear Kalata BT is identical to cyclic Kalata Bi except the backbone chain is open between residues 7 and 8 [see Figure IA]. 5 In contrast to cyclic Kalata B1 the linear form of Kalata B1 had no significant effect on growth for the first nine days of the feeding trial. After 12 days larvae fed on the linear peptide were only about 50% the size of control larvae. The cyclic form of Kalata BI had a more significant effect on growth even when used at one fifth the concentration of the linear form. 10 EXAMPLE 19 Cyclotide genes are expressed in roots, leaves and stems of 0. affinis The Oak 1 cDNA was used as a probe on RNA blots containing 0. affinis root, shoot and 15 leaf RNA. The Oak I clone hybridized to RNA transcripts of about 750 bases which are most abundant in leaves and shoots but are also produced by roots (see Figure 16). EXAMPLE 20 Cyclotide genes are conserved outside the Rubiaceae: isolation of a 20 partial cDNA encoding a cyclotide from Viola odorata Viola odorata produces cyclic peptides that are closely related to Kalata Bl and the other cyclotides produced by 0. affinis (12). The inventors sought to determine whether the genes that encode these peptides are conserved in both Viola and Oldenlandia species. 25 Single stranded cDNA was prepared from Viola odorata leaf RNA and cDNA encoding a cyclic peptide was amplified using the procedure and oligonucleotide primers described in Example 3 for 0. affinis. A 311 bp fragment was amplified using the Kal2 and oligo-dT HindIII primers (Figure 15). This fragment has a 3' untranslated region of 203 bp and a poly A tail of 18 bp. The predicted peptide encoded by nucleotides 1-78 is identical in 30 sequence to 26 of the 29 amino acid residues of Kalata S, a cyclic peptide that has been extracted from Viola odorata plants (12). The three amino acids not covered by this open - 55 reading frame are Gly-Leu-Pro. This indicates that the sequence Gly-Leu-Pro is located at the N-terminus of the mature Kalata sequence in the precursor protein encoded by the full length gene. Thus the potential cleavage site in the predicted V odorata precursor occurs before the Glycine at the N-terminus and after the asparagine residue at the C-terminus 5 (last residue in the underlined sequence Figure 15). The inventors have shown that cyclotides and their encoding genes are conserved in two plant families, Rubiaceae (0. affinis) and Violaceae (V. odorata) that come from two taxonomically distinct subclasses (Asteridae and Rosidae) in the eudicot lineage of 10 flowering plants. This suggests that the cyclotide genes may have arisen early in the evolution of flowering plants and are likely to be conserved in other plant families. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood 15 that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

- 56 BIBLIOGRAPHY 1. Saether et al., Biochemistry 34: 4147-4158 (1995). 2. Gustafson et al., J. Am. Chem. Soc. 116: 9337-9338 (1994). 3. Witherup et al., Journal of Natural Products 57: 1619-1625 (1994). 4. Schopke et al., Sci. Pharm. 61: 145-153 (1993). 5. Claeson et al., Journal ofNatural Products 61: 77-81 (1998). 6. Goransson et al., Journal of Natural Products 62: 283-286 (1999). 7. Altschul et al., NucL Acids Res. 25: 3389 (1997) 8. Ausubel et al., "Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15. 9. Marmur &.Doty, J. Mo. Biol.5: 109 (1962). 10. Bonner & Laskey, Eur. J Biochem 46: 83'(1974). 11. Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, C. Nolan ed. (Cold Spring Harbor, New York; Cold Spring Harbor Laboratories Press). 12. Laemmli Nature 227: 680-685 (1970). 13. Teakle et al., Journal ofInvertebrate Pathology 46: 166-173, (1985). 14. Craik et al., J. Mol. Biol. 294: 1327-1336 (1999).

Claims

1. An isolated, nucleic acid molecule comprising a sequence of nucleotides encoding a precursor form kf a cystine knot polypeptide operably linked to a heterologous promoter, wherein the aminb acid sequence of said precursor form comprises the sequence of at least one cystine knot pol peptide that in its mature form comprises the structure: C 1 (X 1 . X 8 )C 2 (X 1 X'b)C 3 (Xt',X"c)C4(XmI .Xmd)C 5 (X"v vX)C 6 (X I.X loop 1 oop 2 loop 3 loop 4 loop 5 wherein C 1 to C 6 are cysteine residues; wheren each of C 1 and C 4 , C 2 and C 5 , and Ca and C 6 are connected by a disulfide bohd to form a cystine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues may be the same or different; wherein d is about 1-2; wherein for a, b, c, e, and f, and i) a may be any number from 3-10, and ii) b, c, e, and f may be any number from I to 20.

2. The isolated nucleic acid of Claim 1, wherein a is from about 3 to 6, b is from about 3 to about 5, c is from bout 2 to about 7, e is from about 3 to about 6 and f is from about 4 to about 9.

3. The isolated nucleic acid of Claim 1, wherein a is about 3, b is about 4, c is from about 4 to about 7, d is about 1, e is about 4 or 5 and f is from about 4 to about 7.

4. The isolated nucleic acid molecule of Claim 1, wherein the amino acid sequence of said precursor form comprises sequences of a plurality of cystine knot polypeptides.

5. The isolated ihucleic acid of Claim 1, wherein in said amino acid sequence of said precursor form, the sequnce of said at least one cystine knot polypeptide is flanked by amino acid triplets selected frbm the group consisting of GLP and SLP. 58

6. The isolate nucleic acid of Claim 5, wherein the second amino acid of a flanking amino acid triplet is leucine.

7. The isolated nucleic acid molecule of Claim 1, wherein said cystine knot polypeptide is selected from the group consisting of a Kalata B1 polypeptide, a Kalata B2 polypeptide, a Kalata B3 polypeptide, a Kalata B6 polypeptide, and a Kalata B7 polypeptide.

S. The isolate nucleic acid molecule of Claim 1, wherein said nucleic acid sequence encoding a precursor form of a cystine knot polypeptide is from a plant family selected from the group consisting of Rubiaceae and Violaceae.

9. The isolated, nucleic acid molecule of Claim 1, wherein said nucleic acid sequence encoding a precurs r form of a cystine knot polypeptide is from Oldenlandia affinis DC or Viola odorata.

10. The isolated' nucleic acid molecule of Claim 1, wherein said sequence of nucleotides encoding a precisor form of a cystine knot polypeptide comprises a sequence encoding a signal peptide, herein said signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 11, 17 and 21.

11. An isolated ncleic acid molecule according to Claim 1, wherein the cystine knot polypeptide comprises a least two disulfide bonds.

12. A method for producing a cystine knot polypeptide, comprising: transforming a host cell with a vector con prising a nucleic acid molecule according to Claim 1, wherein said precursor form of said at least one cystine knot polypeptide is expressed. 59

13. A method for producing a cyclic cystine knot polypeptide, comprising: transforming a host cell with a vector comprising a nucleic acid molecule according to Claim 1, wherein a precursor fotm is expressed and processed to form a cyclic cystine knot polypeptide having the structure: CI(X 1 ...Xa)C 2 (XII.. b)C3(X 1...Xc)C4(X1 m.XIId)C5(XI1. XIVe)CXVI XVt 9 ; loop I loop 2 loop 3 loop 4 loop 5 loop 6

14. The method of Claim 13, wherein in the amino acid sequence of said precursor form of said at leapt one cystine knot polypeptide, the sequence of said at least one cystine knot polypeptide is flanked by amino acid triplets that are the sane or different and that are selected from the grqup consisting of GLP and SLP.

15. The method! of any one of Claims 12 to 14, wherein said host cell is a bacterial cell.

16. The method of any one of Claims 12 to 14, wherein said host cell is a plant cell.

17. The method of Claim 16, wherein said plant cell is from a plant family selected from the group consi ting of Rubiaceae, Violaceae or Cucurbitaceae.

18. The method cf any one of Claims 12 to 14, wherein said host cell carries an enzyme for processing said precursor form of said cystine knot polypeptide to produce a cyclic cystine knot polypeptide.

19. A method for roducing a cystine knot polypeptide, comprising: a) amplifying by polymerase chain reaction a nucleic acid encoding a precursor fort Kalata cyclic polypeptide, said amplifying comprising contacting cDflA encoding a precursor Kalata cyclic polypeptide with forward and reverse priners such that said contacting results in amplification of said cDNA to produce an amplified product comprising DNA encoding said precursor form [Kalata cyclic polyp eptide, wherein; 60 i) said forward primer is hybridizable to a sequence complementary to nucleotides 214-231 of SEQ ID NO:4, and ii) sTd reverse primer is complementary to nucleotides 272-288 of SEQ D NO:6, b) constructing a vector comprising said DNA encoding said precursor form Kalata cyclic polypeptide, and c) transtprming a host cell with said vector, wherein said precursor form of said cystide knot polypeptide is expressed.

20. A method of incorporating an amino acid sequence conferring a particular trait into a cyclic peptide, polypeptide or protein, said method comprising fusing or introducing a nucleotide sequence encoding said amino acid sequence to or into a second nucleotide sequence wherein said nucleotide sequence encodes a polypeptide that comprises a cyclisable amino acid sequence as defied in any one of Claims I to 10.

21. A method accordin to Claim 20, wherein two or more amino acid sequences are inserted or otherwise grafted! onto the backbone.

22. A method according' to Claim 21, wherein the inserted or otherwise grafted amino acid residues are a single residue, or a linear sequence of 2 residues to 30 residues.

23. A method according to Claim 21 to Claim 22, wherein the inserted or otherwise grafted amino acid residues' are a single residue or a linear sequence of 2 residues to 10 residues.

24. An isolated nucleic acid molecule according to Claim 5 wherein said molecule comprises the following nucl otide sequence: e...je[X...X[nin2 . n y.nqi...qf wherein 61 [nin 2 ... na] represeIts a nucleotide sequence encoding a cyclisable amino acid sequence which, when flanked by amino acids derived from one or more of said flanking sequences which together niake up one copy of a triplet GLP when in cyclic form, is capable of being cyclised to a knotted peptide, polypeptide or protein; X 1 ... X and y1... yc represent polynucleotide sequences capable of encoding an amino acid sequence where a and and c and d may be any number and when d is > 1, the amino acid sequence may be uniq4 e for each integer or d where X.. .X 10 encodes said N-terminal flanking triplet GLP and y. .y, encodes said C-terminal flanking triplet GLP or SLP; and jf.. .je and qi...q repesent nucleotide sequences encoding a peptide, polypeptide or protein capable of directing ,he peptide, polypeptide or protein to a cellular compartment or organelle where a, b, c, d, and f may be any number, where d is > 1, the amino acid sequence may be unique for each integer of d.

25. An isolated nucleic 4cid molecule according to Claim 5, wherein said molecule comprises the following nucleotide sequence: [XI...Xb[In2 ... (ki. k8Ahna..yc Yv wherein [nin 2 . .n] represents a nucleotide sequence encoding a cyclisable amino acid sequence which, when flanked by amino acids derived from one or more of said flanking sequences which together male up one copy of a triplet GLP when in cyclic form, is capable of being cyclised to a knotted teptide, polypeptide or protein; XI.. .X and yj... ye rep esent polynucleotide sequences capable of encoding an amino acid sequence where a and b afid c and d, 8 and X may be any number and when d or X is > 1, the amino acid sequence may e unique for each integer of d and k wherein XI... Xb encodes said N-terminal flanking triplet GLP and y1.. yc encodes said C-terminal flanking triplet GLP or SLP; and k, ... k represents a nuc leotide sequence encoding an amino acid sequence conferring a particular activity or other trait. 62

26. An isolated nucleic 'cid of Claim 24 or Claim 25, wherein the amino acid sequence is cyclisable in an in vitro system comprising cyclising enzymes or chemical means for cyclisation.

27, A genetically modifi d plant which comprises a nucleotide sequence which encodes a cyclisable amino acid seque;1ce as defined in any one of Claims I to I1 which is capable of being cyclised into a knoite4 peptide, polypeptide or protein and which confers on said plant a trait not present in the same species or variety of plant prior to genetic modification.

28. A genetically modified plant as claimed in Claim 27 wherein said nucleotide sequence is as defined in any one of Cktims 1 to 11, or Claims 24 to 25,

29. A genetically modified plant according to Claim 27 or Claim 28, wherein said trait is protection against a plant patlogen.

30. A genetically modified plant according to Claim 29, wherein the pathogen is an insect, arachnid, microorganism, virus, nematode or fungus.

31. Use of an isolated nuceiec acid according to any one of Claims I to 11, or Claims 24 to 25 for the protection of a plint against a pathogen.

32. Use of an isolated nucleic acid molecule according to any one of Claims 1 to 11 or Claims 24 to 25 in the manufacture of a genetically modified plant having protection against a plant pathogen.

33. Use according to Claim 31 or Claim 32, wherein the pathogen is an insect, arachnid, microorganism, virus, nematode or fungus.