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WO1997035981A1 - Genes de neuropeptides d'insectes et peptides - Google Patents

Genes de neuropeptides d'insectes et peptides Download PDF

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Publication number
WO1997035981A1
WO1997035981A1 PCT/GB1997/000843 GB9700843W WO9735981A1 WO 1997035981 A1 WO1997035981 A1 WO 1997035981A1 GB 9700843 W GB9700843 W GB 9700843W WO 9735981 A1 WO9735981 A1 WO 9735981A1
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WIPO (PCT)
Prior art keywords
leu
gly
tyr
phe
peptide
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PCT/GB1997/000843
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English (en)
Inventor
Alan Thorpe
Hanne Duve
Anders Holten Johnsen
Peter East
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Btg International Limited
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Priority to EP97914442A priority Critical patent/EP0923645A1/fr
Priority to AU21688/97A priority patent/AU2168897A/en
Priority to JP09534145A priority patent/JP2001501806A/ja
Publication of WO1997035981A1 publication Critical patent/WO1997035981A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies

Definitions

  • the present invention relates to novel DNA isolated from the blowfly C. vomi toria which encodes novel peptides of the callatostatin class of insect peptides.
  • the novel peptides have also been isolated and purified from C. vomi toria .
  • the DNA also encodes the other known Leu-callatostatin peptides of C. vomitoria.
  • the homologue of this gene has also been isolated from the Australian sheep blowfly Lucilia cuprina and peptides of this class have also been shown to exist in the codling moth, Cydia pomonella and the armyworm Helicoverpa armigera , both potential targets for insecticides based on these peptides. Variants of these peptides have been identified from the large willow sawfly Pteronida salici s .
  • the first group of callatostatins to be isolated from Calliphora vomi toria were shown to have some sequence homology to cockroach allatostatins (Duve et al in Proc . Na t ' l . Acad . Sci . USA 90: 2456-2460) and the peptides have the following sequences:
  • the first four peptides are known as Leu-callatostatins, now designated Leu-callatostatins 1, 2, 3 and 4.
  • the fifth peptide is known as Met-callatostatin or Met-cast (Duve et al Cell Ti ssue Res . 276 367-379 (1994)) .
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • three further Met-callatostatins have been isolated (Duve et al J. Biol . Chem. 269 21059-21066 (1994) ; Duve et al Regul .
  • Met-callatostatinpeptides Two of the additional Met-callatostatinpeptides are post-translationally modified by hydroxylation at either the Pro 2 or Pro 3 amino acid residues of the parent Met-callatostatin.
  • the third Met- callatostatin is an N-terminally truncated des-Gly 1 -Pro 2 variant.
  • the peptides have the following sequences:
  • Pro-Tyr-Asp-Phe-Gly-Met-NH 2 (designated Met-callatostatin 3-8 or des-Gly-Pro-Met-callatostatin) .
  • the Leu-callatostatin sub-family of peptides appears to be characterised by the possession of the C-terminal sequence - Tyr-Xaa-Phe-Gly-Leu-NH 2 (where Xaa is Gly or Ser) .
  • the four Met-callatostatins differ from the Leu-callatostatins by having Asp in position Xaa and Met-NH 2 at the C-terminus.
  • This new class of insect peptides may be related to the allatostatin class of peptides isolated from other unrelated insect species.
  • the allatostatin class of insect neuropeptides in cockroaches have been shown capable of inhibiting the production of juvenile hormone (JH) by the corpus allaturn.
  • the corpus allatum (CA) is a classical endocrine gland of insects situated in close proximity to the brain with which it has nervous connection.
  • Members of the allatostatin class of neuropeptides have now been isolated and identified in five species (other than C. vomi toria) , from four different orders of insects (other than the order Diptera) .
  • Diploptera punctata Order: Blattodea
  • five allatostatins ranging in size from 8 to 18 amino acids have been characterised (Pratt et al in Proc . Nat ' l . Acad. Sci USA
  • the significance of the allatostatins lies in the fact that " in vi tro” they have been shown, with the exception of the allatostatin from the tobacco hornworm moth, to inhibit the production of juvenile hormone (JH) by the corpus allatum in cockroaches.
  • the allatostatin molecule isolated from the tobacco hornworm moth inhibits JH production in the species in which it is found i.e. Manduca sexta but not in cockroaches.
  • the Callatostatins are of interest because they are potent inhibitors of JH synthesis and release in cockroaches, but not in the blowfly, the species from they originate.
  • the callatostatins are also of interest because of their effect on normal gut physiology in insects.
  • Juvenile hormone plays a crucial role in insect development by controlling metamorphosis, adult sexual maturity and reproduction. Interference with juvenile hormone biosynthesis and release through exploitation of the allatostatins and callatostatins may lead to insect control strategies that do not damage the environment .
  • Use of the DNA encoding the callatostatin peptides and the novel callatostatin peptides of the present invention may also provide an additional insect control strategy which relies on interfering with the normal gut physiology in larvae and/or adults of major lepidopteran pest species, such as the codling moth Cydia pomonella and the armyworm Helicoverpa armigera .
  • the peptides may also have utility as insecticides against other insect species.
  • the present invention is based on the discovery and potential practical commercial application of the DNA encoding one or more of four novel peptides identified in the blowfly Calliphora vomi toria and the other known Leu-callatostatins 1 to 4 in C. vomitoria, and the isolation and purification of the novel peptides, designated callatostatins 5, 6, 7 and 8.
  • the present invention also extends to the homologous prohormone genes from the Australian sheep blowfly Lucilia cuprina and Helicoverpa armigera and to the peptides coded for by these genes. Variants of the peptides have also been isolated from Cydia pomonella and Pteronidia salicis .
  • a recombinant or isolated DNA sequence encoding the amino acid sequence shown in Figure 2 , in Figure 4 , in Figure 18, or an amino acid sequence which is substantially homologous thereto.
  • a third aspect of the present invention provides a recombinant or isolated DNA sequence comprising the protein coding region of the DNA sequence shown in Figure 2, in Figure 4 or in Figure 17.
  • FIG. 2 Particularly preferred coding sequences are shown in Figure 2 for the C. vomi toria Leu-callatostatin peptides gene, in Figures 4 and Figure 17 for the homologous L. cuprina and Helicoverpa armigera Leu-callatostatin peptides genes respectively, as will subsequently be described in the examples.
  • Figures 4 and Figure 17 Particularly preferred coding sequences are shown in Figure 2 for the C. vomi toria Leu-callatostatin peptides gene, in Figures 4 and Figure 17 for the homologous L. cuprina and Helicoverpa armigera Leu-callatostatin peptides genes respectively, as will subsequently be described in the examples.
  • Those skilled in the art will with the information given in this specification, be able to identify with sufficient precision the coding regions and to isolate and/or recombine DNA containing them.
  • the gene encoding the novel Leu- callatostatin peptides 5, 6, 7 and 8 and the known Leu ⁇ callatostatin peptides 1, 2, 3 and 4 of C. vomi toria , L . cuprina and H. armigera will be referred to as the Leu ⁇ callatostatin gene.
  • the Leu-callatostatin genes shown in Figure 2, Figure 4 and Figure 17 contain coding regions and the invention therefore also extends to the prohormone sequences coded for by the Leu ⁇ callatostatin genes.
  • the coding region contains a number of putative peptide sequences which would be excised after translation of the mRNA for the gene.
  • the putative coding sequences correspond with the peptides already identified and the novel peptides of the present invention.
  • amino acid sequences of the peptides according to this invention are as follows :-
  • Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu (designated Leu ⁇ callatostatin 7) , or
  • Leu-callatostatin 8 Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu.
  • the invention embraces Leu-callatostatins 5 to 8 defined above when isolated and/or when substantially purified and essentially free of other peptide material.
  • the present invention also extends to variants of the peptides of the present invention.
  • An example of a variant of the present invention is a Leu-callatostatin peptide as defined above, apart from the substitution of one or more amino acids with one or more other amino acids.
  • the skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. Additionally, different amino acids as substituents may enhance the activity of a particular peptide.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains) .
  • amino acids having aliphatic side chains amino acids having aliphatic side chains
  • valine, methionine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic) .
  • amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains) ; lysine, arginine and histidine (amino acids having basic side chains) ; aspartate and glutamate (amino acids having acidic side chains) ; asparagine and glutamine (amino acids having amide side chains) ; and cysteine and methionine (amino acids having sulphur containing side chains) . Substitutions of this nature are often referred to as “conservative" or “semi- conservative" amino acid substitutions.
  • Amino acid deletions or insertions may also be made relative to the amino acid sequences of the peptides of the present invention given above.
  • amino acids which do not have a substantial effect on the activity of the peptides of the present invention, or at least which do not eliminate such activity may be deleted.
  • Such deletions can be advantageous since the overall length and the molecular weight of a peptide can be reduced whilst still retaining activity. This can enable the amount of peptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.
  • Amino acid insertions relative to the sequence of the peptides of the present invention can also be made. This may be done to alter the properties of a substance of the present invention, for example to assist in identification, purification or expression in relation to production of fusion proteins.
  • Amino acid changes relative to the sequences of the peptides of the present invention given above can be made using any suitable technique e.g. by using site-directed mutagenesis.
  • amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present. However, D- amino acid substitutions are known, for example D-phenylalanine in certain molluscan neuropeptides and the present invention is not limited in this regard.
  • preferred peptides of the present invention have at least 50% sequence identity with a peptide according to the present invention, more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or at least 95% are most preferred. The degree of amino acid sequence identity can be calculated using a program such as "bestfit” (Smith and Waterman, Advances in Appli ed Mathematics, 482-489 (1981) ) to find the best segment of similarity between any two sequences.
  • the alignment is based on maximising the score achieved using a matrix of ammo acid similarities, such as that described by Schwarz and Dayhof (Atlas of Protein Sequence and Structure, Dayhof, M.O., ed. 353-358 (1979)) . Where high degrees of sequence identity are present there will be relatively few differences in ammo acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences .
  • the present invention also extends to variants of the peptides of the present invention and these variants include the peptides isolated from Cydia pomonella, Helicoverpa armigera and Pteronidia salicis when isolated and/or when substantially purified and essentially free of other peptide material.
  • the peptides isolated from C. pomonella are as follows:
  • Lys-Met-Tyr-Asp-Phe-Gly-Leu (designated cydiastatin 7)
  • the peptides isolated from H. armigera are as follows:
  • Glu-Arg-Asp-Met-His-Arg-Phe-Ser-Phe-Gly-Leu (designated helicostatin 9) .
  • the peptides with the above sequences from C. vomitoria, L. cuprina, H. armigera, C. pomonella and P. salicis are amidated at the carboxy terminus.
  • the peptide Leu-callatostatin 5 isolated from C. vomitoria has the sequence ARPYSFGL-NH 2 and this sequence is shared by the peptides isolated from C. pomonella and H. armigera. In P. salicis the sequence is altered by the substitution of a leucine residue m place of the prolme at position 3, i.e. the peptide has the sequence ARLYSFGL-NH 2 .
  • C. pomonella and H. armigera share a peptide sequence SRPYSFGL-NH 2 which differs from Leu-callatostatm 5 by the substitution of a serine residue in place of the alanine residue at position 1.
  • Leu-callatostatin 6 from C. vomitoria has the peptide sequence RPYSFGL-NH 2 which represents a deletion of the N-terminal alanine residue.
  • the peptide Leu-callatostatin 7 from C. vomi toria has the sequence VERYAFGL-NH 2 and this sequence is shared in part by the peptide GGEDFGHRYAFGL-NH 2 from P. salicis .
  • the valine and glutamic acid residues at positions 1 and 2 in Leu ⁇ callatostatin 7 are replace by a glycine and a histidine residue respectively with the addition of prior pentapeptide sequence.
  • Leu-callatostatin 8 from C. vomi toria has the sequence LPVYNFGL-NH 2 and this sequence is shared by peptides isolated from C. pomonella and H. armigera .
  • C. pomonella peptides have also been isolated which have the valine residue at position 3 replaced by either an isoleucine or a leucine residue.
  • This sequence is also shared by peptides isolated from P. salicis .
  • Further changes include the replacement of the valine at position 3 by a methionine residue in H. armigera and the replacement of the leucine and proline residue at positions 1 and 2 by alanine and arginine respectively in P. salicis .
  • C. pomonella and H. armigera also share the peptide sequence AYSYVSEYKRLPVYNFGL-NH 2 which represents the sequence of Leu-callatostatin 8 with a prior decapeptide sequence.
  • the lepidopteran species C. pomonella and H. armigera also share some sequence homology in the newly isolated peptides which have aspartic acid as the post-tyrosyl residue.
  • the peptides ARGYDFGL-NH 2 and KMYDFGL-NH 2 in C. pomonella differ in that the residues 1 to 3 of the first peptide being alanine, arginine and glycine are replaced by lysine and methionine in the second peptide.
  • the peptide sequence is altered by the replacement of the glycine at position 3 in the first C. pomonella peptide by an alanine residue.
  • the invention also embraces insecticidal compositions which contain one or more of the above peptides described above, which may be a liposomal formulation, and methods of killing or controlling insects which involve applying to the insects or their environment such msecticidal compositions.
  • the insecticidal compositions may contain besides at least one msecticidally effective peptide described above, a suitable carrier, diluent or excipient therefor.
  • the formulations according to the present invention may be administered in the form of a spray.
  • Methods of making insecticidal compositions are also embraced by the present invention which comprises admixing one or more the said Leu-callatostatins, helicostatins, pteridiostat s or cydiastat s described above with a suitable carrier, diluent or excipient therefor.
  • DNA according to the present invention can be synthesised by standard techniques for the preparation of genetic material.
  • the process may comprise ligating together successive nucleic acid residues and/or oligonucleotides to produce DNA.
  • peptides described above can be synthesized by the skilled worker by routine peptide synthesis by selecting the appropriate ammo acids and reaction conditions.
  • Such peptides as an insecticide is also within the scope of the present invention which may comprise the inhibition of gut motility.
  • the use of such secticidal compositions may be specific for the codling moth Cydia pomonella or for the army worm Helicoverpa armigera .
  • the codling moth Cydia pomonella , is a major worldwide pest of apples and pears and causes the greatest economic damage of any lepidopteran pest the UK. Resistance is developing to chemicals currently being used for its control and there is a strong desire to reduce chemical inputs, particularly on unprocessed food crops.
  • the army worm, Helicoverpa armigera is reported to be the most important insect pest known to agricultur .
  • the invention also extends to vector systems comprising a DNA sequence which encodes a peptide as described above for expression m a host cell .
  • Suitable vectors are preferably specific for the target organism and may be baculovirus based.
  • Baculoviruses are currently being developed as pest control agents (Payne, C. C, in Biological Plant and Heal th Protection, ed. Franz, J.M. (Fort suitse der Zoologie 32) , G. Fischer Verlag, Stuttgart, New York (1986) ) and several commercial products based on these viruses are now available (W stanley et al in Exploitation of Microorganisms, ed. D. G. Jones, 105-136, Chapman & Hall, London (1993)) . Viruses are, however, slower to kill insects than chemical insecticides and feeding may continue for some time after application of the msecticidal agent. It has been possible to reduce this damage by using genetically-engineered viruses that express arachnid toxins (Stewart et al Nature 352 85-88
  • a more appropriate baculovirus would be Cydia pomonella granulosis virus (CpGV) .
  • CpGV Cydia pomonella granulosis virus
  • This virus has been isolated in the UK, is very effective against the codling moth C. pomonella , which is a ma or U.K. pest.
  • This baculovirus has a narrow host range which includes only a few closely related tortricid species, and recombinant strains can be produced.
  • CpGV is already produced commercially, improved strains, capable of faster killing, could also be used to reduce further the crop damage.
  • Expression of the callatostatin peptides at high levels by baculoviruses would have the highly desirable effect of inhibiting feeding in infected larvae.
  • the heterologous genes of the blowfly, Calliphora vomi toria , of the sheep blowfly, Lucillia cuprina , or of Helicoverap armigera may be used in such a baculovirus.
  • the present invention also comprehends the use of a synthetic gene based on identified peptide sequences from C. pomonella or the actual endogenous gene of C. pomonella .
  • a major advantage of the latter approach is that CpGV can be engineered to carry a gene that is found only in the host larvae, thus minimising any possible risk to the environment. Similar approaches may also suitable with respect to the development of insecticides against H. armigera .
  • the present invention has now demonstrated that similar peptides to the C.
  • vomitoria peptides are expressed in these pest species and in some cases the amino acid sequence is identical e.g. Leu-callatostatin 8 which has the structure LPVYNFGL-NH 2 and this peptide is also found in both C. pomonella and H. armigera .
  • CpGV was the most promising agent available, though the relatively slow rate of kill by the virus allows significant crop damage to be caused by the pest. Very recently this virus has gone into commercial production, for example Carpovirusine TM or GranupomTM.
  • the peptides described by the present invention are the third group of the callatostatin class of compounds to be isolated from this major order of insects, the Diptera.
  • the first and second groups were also from this species, the blowfly Calliphora vomi toria .
  • FIGURE 1 shows the design of an oligonucleotide callatostatin gene probe of low degeneracy for C. vomi toria library screening.
  • (c) shows the sequence of a 12-fold degenerate oligonucleotide probe PCRCastl based on the sequences of the four PCR amplicons.
  • I and N indicate that either inosine or all our nucleotides respectively were used at that position in the oligonucleotide.
  • FIGURE 2 shows the sequence of the C. vomi toria Dra I fragment showing two open reading frames.
  • Each of the putative peptides (boxed) is flanked by a pair of basic amino acid residues (bold underline) and has a glycine residue (italicised) at its C-terminal end.
  • Proposed intron splice sites are marked with a vertical bar and the region of the putative intron is indicate.
  • FIGURE 3a shows a dot matrix comparison of L. cuprina and C. vomi toria genomic DNA sequences.
  • the black bars indicate the position of the two open reading frames encoding callatostatin-like peptides in the C. vomi toria sequence.
  • FIGURE 3b shows a sequence comparison of the prohormone amino acid sequences identified in C. vomi toria and L. cuprina .
  • the symbol "•••” represents an identical amino acid residue and the symbol "-" represents an insertion of a residue relative to the C. vomi toria sequence.
  • FIGURE 4 shows L. cuprina cDNA and the deduced prohormone sequence. Putative peptides are boxed and numbered with Roman numerals. Pairs of basic residues proposed as sites of proteolytic cleavage are underlined in bold and the C-terminal glycine substrates for carboxylamidation are marked in italics. The translation termination codon is indicated by an asterisk (*) and vertical arrows mark the positions where introns occur in the genomic DNA. Locations of primers used for RT-PCR analysis are indicated.
  • FIGURE 5 shows a diagrammatic representation of the dipteran Leu-callatostatin prohormone deduced from L. cuprina genomic and cDNA and C. vomi toria genomic DNA clones.
  • the positions of the callatostatin peptides are marked with diagonal lines and numbered I-V according to the nomenclature of Table 1. Sites of endoproteolytic cleavage are indicated by solid black boxes and the proposed signal peptide domain is marked by vertical bars.
  • FIGURE 6 shows the organisation of the dipteran Leu ⁇ callatostatin prohormone gene showing sites of two introns. Numbers refer to nucleotide coordinates of the L cuprina cDNA. The positions of translation initiation (ATG) and termination (TAA) codons are indicated and the prohormone open reading frame is shaded.
  • FIGURE 7 shows nested reverse transcriptase PCR analysis of mRNA pools prepared from L. cuprina adult head and midgut .
  • Lane M contains molecular weight markers (lOObp ladder) .
  • the head and midgut (Gut) cDNA samples are indicated and "1", "2" are the first and second (nested) PCR reaction products respectively.
  • Lane "Gen” contains the amplification product from L. cuprina genomic DNA with the primer pair AST-2/AST-3.
  • FIGURE 8 shows a flow diagram of the purification of Leu-callatostatin 7 and Leu-callatostatin 8 from C. vomi toria .
  • M r * indicates the mass of the peptide after methylation.
  • FIGURE 9 shows a flow diagram of the purification of Leu-callatostatins 4, 5 and 6 from C. vomi toria [Leu- callatostatin 4 being a comparative example] . Chromatography being carried out according to conditions detailed in step 6. M r * indicates the mass after methylation.
  • FIGURE 10 shows dose responses for inhibition of spontaneous muscle contractions of the rectum (colon and rectal pouch) of C. vomi toria by Leu-callatostatins 5 and 8. Percentage inhibition calculated relative to basal spontaneous contraction movements. Each point represents the mean of 5 to 10 measurements from a number of different vitellogenic flies.
  • FIGURE 11 shows a chromatographic profile of an extract of 100 heads of adult blowflies of C. vomi toria assayed with five different callatostatin RIAs specific for five different types of peptides, shown in the figure, ending C-termmally as follows:
  • FIGURE 12 shows a chromatographic profile of an extract of 1000 Cydia pomonella larvae chromatographed and assayed in an identical manner to C. vomi toria as shown in Figure 11.
  • FIGURE 13 shows dissected C. pomonella 5th star larvae :
  • Figure 13A shows a normal larvae
  • Figure 13B shows a larvae fixed and stained with X-gal to show the presence of recombinant CpGV expressing ⁇ - galactosidase (blue-black coloration) .
  • This demonstrates the successful engineering of C. pomonella granulosis virus.
  • FIGURE 14 shows a drawing of the gut of C. pomonella to show the regions 1 to 5 from which Figures 15-1 to 15-5 are taken.
  • Figure 15-1 shows frontal ganglion containing 4 callatostatin-immunoreactive cells of which the two most anterior cells give rise to Leu-callatostatin- immunoreactive material in and around the valve between the foregut and the midgut (see Figure 15- 4) .
  • Figure 15-2 shows the dorsal part of the brain with Leu-callatostatin immunoreactivity in certain of the median neurosecretory cell groups.
  • Figure 15-3 shows callatostatin immunoreactive axons in the gastric nerve. These have their origins from cells of both the frontal ganglion and the brain.
  • Figure 15-4 shows accumulation of Leu-callatostatin immunoreactive material in the muscles of the valve separating the foregut from the midgut.
  • Figure 15-5 shows Leu-callatostatin immunoreactivity in posteriorly located midgut endocrine cells immediately anterior to the valve between the midgut and the hindgut .
  • FIGURE 16 shows dose-response for the inhibition of peristaltic contractions of the foregut of 5th instar larvae of C. pomonella by the blowfly callatostatin neuropeptide Leu-callatostatin 3 (Ala-Asn-Arg-Tyr-Gly- Phe-Gly-Leu-NH 2 ) .
  • the percentage inhibition is relative to basal spontaneous peristaltic contractions.
  • FIGURE 17 shows the cDNA sequence of the helicostatin gene of Helicoverpa armigera .
  • FIGURE 18 shows the deduced amino acid sequence of the open reading frame from the cDNA of the helicostatin gene of Helicoverpa armigera .
  • Insect rearing and tissue dissection C. vomitoria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) .
  • the flies were fed sugar, beef heart and water for 1-3 weeks after which they were anaesthetized with C0 2 , frozen and stored at -20°C prior to use.
  • Larval L. cuprina were reared under a 12:12 hour light :dark cycle, on fresh minced sheep liver supplemented with meat meal, fish meal and cotton lint.
  • Adult L. cuprina were maintained on water and a protein biscuit comprising a solidified mixture of sugar, egg powder, milk powder and yeast.
  • Tissues for RNA extraction were dissected under sterile, ice-cold phosphate- buffered saline (139 mM NaCl, 10 mM sodium phosphate, pH 7.2) , transferred to a microfuge tube and snap frozen in liquid nitrogen. Tissues were stored frozen at -80°C until use.
  • Example 1 Preparation of a callatostatin gene probe
  • oligonucleotide primers based on the ammo- and carboxyl-terminal sequences of the hexadecapeptide Leu ⁇ callatostatin 1 were synthesised (Pharmacia Gene Assembler) and used to amplify a fragment of the callatostatin gene from C. vomi toria genomic DNA using the polymerase chain reaction (Pratt et al Proc . Nat ' l . Acad. Sci . USA 88 2412-2416 (1991)) .
  • the structures of the sense (ClSl) and antisense (ClAl) primers are shown m Figure 1.
  • PCR reactions consisted of 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl 2 , 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of each primer, 1 ⁇ g of C. vomi toria genomic DNA, 2.5 units Tag DNA polymerase and H 2 0 to a final volume of 50 ⁇ l .
  • Example 2 Callatostatin gene cloning Approximately 1.5 x 10 5 recombinant bacteriophages from a C. vomi toria genomic D ⁇ A library were plated on the E. coli host strain KW251 and plaque D ⁇ A was transferred to nitrocellulose filters according to standard techniques (Sambrook et al Molecular Cloning: A Laboratory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989)) .
  • the oligonucleotide PCRCastl was radiolabelled with ⁇ - 32 P-dATP using the Klenow fragment of E. coli D ⁇ A polymerase (Sambrook et al Molecular Cloning: A Labora tory Manual 2nd edition.
  • the L. cuprina callatostatin homologue was isolated from a recombinant genomic DNA library using a 936 bp Dra I restriction enzyme fragment containing the C. vomi toria prohormone gene as probe.
  • Hybridisation probes were prepared by random-primed synthesis using the NEBlot kit (New England Biolabs) and ⁇ - 32 P-dATP (NEN-DuPont) .
  • cuprina random-primed head cDNA library were screened using a lkb Hind III fragment containing the L. cuprina callatostatin gene as probe. Hybridisation and wash conditions were as described for the L. cuprina genomic library screen. The cDNA insert from hybridising phages was excised with Eco Rl and subcloned into pBluescript SK(+) for sequencing.
  • First strand cDNA was synthesised from 1 ⁇ g of poly(A) + RNA in a reaction consisting of: 50 mM Tris-HCL (pH 8.3) , 75 mM Kcl , 3 mM MgCl 2 , 1 mM DTT, 15 mM each of dATP, dCTP, dGTP and dTTP, 20 units RNasin (Promega Biotec) , 25 ng oligo(dT) primer, 1 ⁇ g mRNA, 200 units of Superscript II reverse transcriptase
  • lcap-1 5' -CTCAACTAGAGGATAAAAGC-3 '
  • lcap-2 5' -CGTTAGCCTTTTGATGTTGG-3'
  • lcap-3 5' -CGACGTCCTAAACCAAAGC-3 '
  • lcap-4 5' -GGAATTATTGGCTGGATAGTG-3'
  • the first PCR reaction contained; 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl 2 , 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of the primer pair lcap-l/lcap-4, 10 ⁇ l of cDNA and H 2 0 to a final volume of 50 ⁇ l .
  • Amplification conditions were; 95°C for 5 min, 50°C for 1 min with addition of 2 ⁇ l Taq DNA polymerase, followed by 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, for 30 cycles; then 1 cycle of 72°C for 5 min to end the reaction.
  • the nested PCR reaction conditions were identical to those above, except that the primer pair lcap-2/lcap-3 was used and the template was 1 ⁇ l of the first PCR reaction.
  • Double-stranded plasmid DNA for sequencing was prepared by a standard alkaline lysis mini-prep procedure (Sambrook et al
  • Example 5 Isolation of the C. vomi toria callatostatin gene Since the amino acid sequences of the four Leu-callatostatin peptides were not favourable for the design of oligonucleotides suitable for library screening, a PCR approach was used to amplify a short region of the callatostatin gene to provide a basis for probe design.
  • vomi toria genomic library were screened, from which a single positive ( ⁇ Cvastl)was obtained.
  • Restriction enzyme analysis of ⁇ Cvastl identified a 3.2 kb Bam El/Xba I fragment that hybridised to the oligonucleotide probe.
  • Partial sequencing of this fragment identified a 936 bp Dra I restriction enzyme fragment that contained the sequence of the oligonucleotide probe.
  • the Dra I fragment was subcloned and sequenced on both strands. Sequence analysis identified likely protein coding regions in two distinct open reading frames, that together encoded five separate putative Leu-callatostatin peptides, each of which was flanked by pairs of basic amino acids that constitute potential endoproteolytic cleavage sites, shown in Figure 2. Each of these peptides had a single glycine residue at the C-terminal end that provides a substrate for peptidyl glycine a-amidating monooxygenase, consistent with the observation that all previously identified callatostatin peptides are amidated at the C-terminus (Duve et al Proc. Nat'l. Acad. Sci. USA 90 2456-2460 (1993)) . The sequences of the five putative callatostatins are given in Table 1 in order of their appearance in the gene.
  • Peptide IV corresponds to Leu-callatostatin 4 of Duve et al ⁇ Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993)) and resolves the uncertainties in the original sequence data for that peptide.
  • Peptide V corresponds to Leu-callatostatin 1.
  • the putative peptides I-III were not identified in the previous purification studies (Duve et al Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993) , Duve et al J. Biol. Chem. 269 21059-21066
  • Peptide III is identical to one of the peptides encoded on the cockroach prohormone gene (Donly et al Proc. Nat'l. Acad. Sci. USA 90 8807-8811 (1993) ) .
  • the 936 bp Dra I fragment containing the callatostatin gene was radiolabelled and used to screen a L. cuprina genomic DNA library. Two hybridising clones were purified and found to contain a 4.3 kb Xba I restriction enzyme fragment that hybridised to the C. vomi toria probe. A 2.6 kb Cla I/Xba I fragment was subcloned from the Xba I fragment and partially sequenced to identify the region homologous to the callatostatin prohormone gene.
  • Figure 3 shows a dot matrix comparison of the L. cuprina sequence with that of the C. vomi toria Dra I fragment. The two sequences were 84% identical over this region and similarity was greatest m the two domains corresponding to the open reading frames identified in C. vomi toria indicating that gene structure was conserved between the two species.
  • a 1 kb Hind III fragment that encompassed the peptide coding region of the L. cuprina gene was used to probe a cDNA library prepared from adult head mRNA. A single positive was isolated from a sample of approximately 3 x 10 5 clones, suggesting that transcripts are relatively rare.
  • the cDNA clone was sequenced and found to contain a single open reading frame encoding a Lucilia Leu-callatostatin prohormone 179 amino acids m length, shown m Figure 4. Since the clone was isolated from a random-primed library it cannot represent a full length cDNA.
  • the open reading frame is flanked by 341 nucleotides of 5' and 271 nucleotides of 3' sequence, each of which contains multiple translation stop codons in all three reading frames.
  • the cDNA encodes all of the L. cuprina callatostatin pre-prohormone.
  • the first in-frame methionine residue, and proposed translation initiation site occurs at nucleotide 342 and the translation termination codon is at nucleotide 879.
  • the putative pre-prohormone begins with a hydrophobic domain of 19 amino acids that is a probable signal peptide. The most likely site of cleavage of this signal sequence is at residue 20 (von Heijne, G. Nucl . Acids Res . 14 4683-4690 (1986)) .
  • the deduced prohormone sequence contains five Leu-callatostatin- like peptides that are identical to those identified from the C. vomi toria genomic clone, shown in Figure 2 and in Table 1.
  • the proposed structure of the dipteran callatostatin prohormone is shown diagrammatically in Figure 5.
  • the first three peptides are tandemly arrayed, separated only by the residues required for proteolytic processing and carboxyamidation.
  • the other two peptides occur as a tandem pair at the carboxyl end of the prohormone. This structure is absolutely conserved in the two blowfly species.
  • L . cuprina Leu-callatostatin gene is composed of at least three exons, as shown in Figure 6. Introns are located between nucleotides 289/290 and 777/778 of the L . cuprina cDNA clone, as seen in Figure 5.
  • the first intron is located in the 5' untranslated region of the mRNA and although no attempt was made to identify exon 1 in genomic DNA, this intron must be at least 2 kb long, based on hybridisation of the cDNA to existing clones (not shown) .
  • the second intron is very short, 63 bp in length, and occurs within the prohormone open reading frame in the region between the two blocks of peptides. Direct comparison of the L. cuprina genomic and cDNA sequences confirmed the prohormone structure proposed from the C. vomi toria genomic analysis of Figure 2.
  • Example 8 Expression of the callatostatin gene Reverse transcriptase-PCR was used to investigate expression of the callatostatin prohormone gene in cDNA pools prepared from whole adult head, representing an enriched source of brain and sub-oesophageal ganglion tissue, and from isolated midgut. To provide maximum sensitivity a 'nested' PCR design was used (McPherson et al in M.J. McPherson, P. Quirke, P. and
  • the amplicon generated from genomic DNA was slightly larger than the corresponding product from the cDNA pools as expected, due to the 63 bp intron, shown in Figure 7.
  • This genomic PCR result demonstrated that the RT-PCR products were bona fide cDNA products and not artifacts produced from contaminating genomic DNA.
  • a genomic clone encoding the sequences of the peptides Leu- callatostatin 1 and Leu-callatostatin 4 was isolated from a C. vomi toria library. This clone contained two open reading frames, each of which contained Leu-callatostatin sequences.
  • Three related, but structurally distinct putative Leu ⁇ callatostatin peptides were encoded on the first open reading frame. These three putative peptides all possessed the C- terminal pentapeptide sequence -Tyr-Xaa-Phe-Gly-Leu characteristic of the callatostatin/ allatostatin peptide family.
  • the octapeptide Leu-callatostatin 4 contained two uncertain residue assignments from the Edman peptide sequencing. The first (N-terminal) residue was identified as either Asp or Asn and the fifth amino acid was tentatively identified as Ser. The prohormone gene sequence has allowed unequivocal assignment of these residues as Asn at position 1 and Ser at position 5.
  • Leu-callatostatins 1-3 were isolated, it was noted that the hexadecapeptide Leu-callatostatin 1 contained a pair of basic (Arg) residues at positions 7 and 8 which, if used as a proteolytic cleavage site during prohormone maturation, would give rise to the octapeptide Leu ⁇ callatostatin 3.
  • Leu-callatostatin 2 was identical to Leu-callatostatin 1 with the two amino- terminal residues, Asp-Pro, removed. At this time, it was not clear whether these three peptides were each encoded independently on the callatostatin prohormone, or if Leu- callatostatins 2 and 3 were derived by proteolytic processing of Leu-callatostatm 1.
  • the prohormone and deduced peptide sequences are identical in both blowfly species.
  • This absolute conservation of peptide structure might reflect a functional constraint on sequence evolution in the Leu-callatostatin peptide family, since at least one other multi-member neuropeptide family, the CalliFMRFamides, does not have the same degree of conservation in these two species (Duve et al in Perspectives in Comparative Endocrinology, 91-96, eds. K.G. Davey, R.E. Peter, and S.S. Tobe, National Research Council of Canada: Ottawa (1994) ) .
  • the allatostatin prohormones of the cockroaches D. puncta ta and P. ameri cana are also highly conserved (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) . However, there is very little similarity in prohormone organisation between the cockroaches and flies.
  • the cockroach prohormone is approximately 370 amino acids long and encodes 13 and 14 allatostatin peptides in D . punctata (Donly et al Proc . Na t ' l . Acad . Sci . USA 90 8807-8811 (1993)) and P. americana (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) respectively, with the peptides occurring in several distinct blocks separated by acidic spacer regions.
  • dipteran prohormone is approximately 180 residues in length and contains only five Leu-callatostatin peptides distributed in two blocks, separated by an acidic spacer.
  • the octapeptide Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH 2
  • the second putative peptide of the dipteran prohormone (peptide II) is which may be a structural homologue of the allatostatin ASB2 isolated by Pratt et al ( Proc . Na t ' l . Acad . Sci . USA 88 2412- 2416 (1991) ) , which is also the second peptide in the cockroach prohormone (Donly et al Proc . Nat ' l . Acad . Sci . USA 90 8807-8811 (1993)) .
  • Leu- callatostatin 1 is several orders of magnitude less potent than its C-terminal derivative Leu-callatostatin 3.
  • the allatostatins are also myoinhibitory in cockroaches (Lange et al Arch . Insect . Biochem . Physiol . 24 79-92 (1993) , Lange et al J. Insect Physiol . 41 581-588 (1995) , Duve et al Physiol . Ento ol . 20 33-44 (1995)) . All thirteen peptides encoded on the D .
  • Leu-callatostatin/allatostatin peptides appear to be quite old in evolutionary terms.
  • the presence of allatostatin immunoreactivity and biological activity of allatostatin peptides in a crab (Skiebe et al J. Exp . Biol . 194 195-208
  • Radioimmunoassay The two putative octapeptides deduced from the Leu- callatostatm gene sequence (East et al Regul . Pept . [m press] (1996)) , Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH 2 and Val- Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH 2 , together with the octapeptide Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH 2 (assuming an enzymatic cleavage at the dibasic pair of residues the longer putativepeptideAla-Tyr-Thr-Tyr-Thr-Asn-Gly-Gly-Asn-Gly-Ile- Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH 2 ) , were synthesized (by Affiniti Research
  • nb Results are expressed as a % of the reactivity shown by the peptide against which the antiserum was raised, in the same RIA
  • concentration of standards in RIAs used to monitor HPLC elution profiles ranged from 015nM to lOnM
  • Calliphora vomi toria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) .
  • the flies were fed sugar, ox heart and water for 1-3 weeks after which they were anaesthetized with C0 2 , frozen and stored at -20°C prior to use. Approximately 40,000 heads were collected by shaking the frozen flies in a plastic bag containing solid C0 2 followed by sieving. They were ground in batches of 2000 in a mortar and pestle in solid C0 2 and homogenized further in a Waring blender m 400 ml methanol/acetic acid/water (87:5:8) at 4°C.
  • the extract was left overnight at 4°C after which it was centrifuged at 3000 x g for 30 min.
  • the pellet was re- extracted with 1 1 of extraction fluid.
  • the combined supernatants were concentrated to approximately 300 ml on a rotary evaporator at 35°C under vacuum.
  • the concentrate was allowed to stand overnight at 4°C before being filtered through Whatman No. 4 paper and re-centrifuged at 15000 x g for 10 min prior to purification by HPLC.
  • the HPLC system for Steps 1-4 comprised two Waters 6000M pumps and a Waters 741 detector.
  • a Waters 625 system was used at room temperature, and for some Step 6 procedures (61) and all of Step 7 a Hewlett-Packard model 1090 HPLC system at 50°C was used. Details of the reversed-phase HPLC columns, solvent systems, flow rates, gradients and their rates of change, used in the purification are as follows.
  • Step 2
  • Step 1 The same column and the same basic running conditions were used as in Step 1 except that 10 mM ammonium acetate (pH 6.5) replaced 0.1%TFA.
  • the pooled material from Step 1 was aliquotted to give 25 separate runs, each containing the equivalent of 1600 heads.
  • Radioimmunoassays of the profiles showed separation into five different areas: Gly/Leu together with Asp/Met material (i.e. those peptides previously identified and designated Leu- callatostatins 1-3 and the various forms of Met-callatostatin (Duve et al Proc . Na t ' l . Acad . Sci . USA 90 2456-2460 (1993) , Duve et al J. Biol . Chem . 269 21059-21066 (1994) , Duve et al Regul . Pept .
  • Step 3 Bio-Rad Hi-Pore C 4 column (250 x 4.6 mm, 5 ⁇ m, 300 A), linear gradient 0 - 80% CH 3 CN/10 mM ammonium acetate (pH 6.5) in 200 min, flow rate 1.5ml/min.
  • Step 4 Kromasil C 1B column (250 x 4.6 mm, 5 ⁇ m, 300 A) , gradient CH 3 CN/0.1% TFA 0 - 15% in 10 min; 15 - 50% in 140 min, flow rate 1 ml/min.
  • Step 5 Kromasil C IB column (250 x 3.2 mm, 5 ⁇ m, 300A) , gradient CH 3 CN/0.1% TFA 0 - 15% in 15 min; 15 - 50% in 175 min, flow rate 0.5 ml/min.
  • Vydac C 18 column (150 x 2.1 mm, 5 ⁇ m, 300 A) , gradient CH 3 CN/0.1% TFA (5 - 15% in 5 min; 15 - 25 % in 50 min, flow rate 0.2ml/min. [Ser/Leu Ilai and Ser/Leu Ilaii] Column temperature 50°C.
  • Step 7
  • Vydac C 18 column (150 x 2.1 mm, 5 ⁇ m, 300 A) gradient CH 3 CN/0.1% TFA (for Asn/Leu material 5 - 17% in 5 min; 17 - 27 in 50 min; for Ser/Leu I, Ser/Leu Ilbi and Ser/Leu Ilbii 5 - 11% in 5 min; 11 - 18% in 35 min, flow rate 0.2 ml/min.
  • the flow charts in Figure 8 and Figure 9 give the % acetonitrile at which immunoreactive material eluted, and details of the points in the procedures where M r determinations and amino acid sequencing were carried out (nb.
  • the abbreviations for the various types of peptide being isolated are those used in Table 2) .
  • the immunoreactive fractions at Step 6 or 6f were analyzed by matrix-assisted laser desorption mass spectrometry using a Biflex (Bruker- Franzen) instrument. For this, 0.5 ⁇ l was mixed with 0.5 ⁇ l of a solution of 33mM ⁇ -cyano-4-hydroxycinnamic acid in acetonitrile/ methanol (Hewlett-Packard) . After drying, the mixture was analyzed in the linear mode at 15 kV. The method has an accuracy of 0.1%. If a single molecular mass was recorded, the material was subjected to amino acid sequence analysis and definitive mass spectrometry. If more than one peptide was seen to be present in the sample, it was chromatographed further.
  • amino acid sequencing The amino acid sequences of the purified peptides (5 - 50 pmol) were determined with an automated protein sequencer (Applied Biosystems, Procise 494A) equipped with an on-line system for the detection of the amino acid phenylthiohydantoin derivatives. All reagents and solvents were from Applied Biosystems.
  • the third peptide with the sequence Asn-Arg-Pro-Tyr-Ser-Phe-Gly-Leu- NH 2 , previously identified as Asx-Arg-Pro-Tyr- (Ser) -Phe-Gly- NH 2 and designated Leu-callatostatin 4 (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993)) , proved impossible to separate from a lesser amount of Leu-callatostatin 6, shown in Figure 9 as Ser/Leu I and Ser/Leu Ilbi) . The major, unambiguous sequence, however, was that of Leu-callatostatin 4.
  • the M r values of the peptide before and after methylation were 952.8 and 953.7 respectively, thereby confirming carboxyamidation and the fact that the first residue was Asn and not Asp.
  • the final yields of the three peptides were Leu ⁇ callatostatin 4, 28 pmols ; Leu-callatostatin 5, 14 pmols; and Leu-callatostatin 6, 120 pmols.
  • Table 3 shows the sequences of the peptides isolated, together with their M r values, before and after methylation. Table 3: Amino acid sequences and M r values of Leu-callatostatin neuropeptides isol ated from C. vomitoria.
  • Leu-callatostatin I s D-P-L-N-E-E-R R-A-N-R-Y-G-F-G-L-NH 2 1906 1 19039 1949 1
  • Leu-callatostatin 3 s A-N-R-Y-G-F-G-L-NH 2 8960 8968 -
  • Leu-callatostatin 8 L-P-V-Y-N-F-G-L-NH 2 921 1 9214 9223
  • Leu-callatostatins 5 and 8 showed them consistently to have a potent, reversible, inhibitory effect on the spontaneous contractile activity of the rectum of C. vomi toria as shown in Figure 10.
  • Leu-callatostatin 5 effected complete inhibition at 10 "9 M, 90% inhibition at 10 "12 M and 50% inhibition at 10 ⁇ 14 M.
  • Leu-callatostatin 8 which showed a biphasic dose-response curve similar to that previously observed with members of the Met-callatostatin family (Duve et al J. Biol . Chem. 269 21059-21066 (1994) , Duve et al Regul . Pept .
  • Leu-callatostatin 1 which appears as a putative peptide on the gene, and is present in extracts together with small amounts
  • C. voinitoria possesses a peptide (ARPYSFGL-NH 2 ) identical to one of D. punctata (Leu-callatostatin 5 and dipstatin 6) .
  • vomitoria occurs as the C-terminal sequence preceded by a dibasic pair of residues Lys-Arg in the octadecapeptide designated dipstatin 2 in D. punctata.
  • Leu-callatostatin 3 the truncated octapeptide variant of Leu-callatostatin 1, which can now be seen to occur as a result of post-translational processing of the precursor by enzymatic cleavage at the dibasic pair of residues (Arg-Arg) , gives complete, reversible inhibition of the ileum at concentrations as low as 10 "15 M.
  • the 'parent' hexadecapeptide is also effective on this in vitro preparation, although with a reduced potency (maximum inhibition at 10 '10 M) .
  • Leu-callatostatin immunoreactivity in nerves on the surface of the rectum in the form of either Ser/Leu or Asn/Leu peptides, may provide the morphological basis for the potent effects of the Ser/Leu (Leu-callatostatin 5) and Asn/Leu (Leu-callatostatin 8) peptides seen. Although contractions of the ileum are inhibited by these two peptides, the effect appears to be irreversible, unlike that seen for Leu-callatostatins 1 and 3.
  • Examples 12 to 15 Improved control of the codling moth Cydia pomonella by baculoviruses expressing insect neuropeptides
  • Example 12 Insertion of the C. vomi toria callatostatin (cal) gene into CpGV
  • the cal gene may be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter.
  • This vector has already been used with the lacZ gene and demonstrated high levels of expression of ⁇ - galactosidase in larvae infected with the recombinant virus, as shown in Figure 13A and Figure 13B.
  • the vector pCpDNl is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this vector may be suitable for studying the peptides that are expressed, for detailed studies on larval gut paralysis and for measurements of LT 50 values, it will be preferable to produce an occluded recombinant virus for more extensive assays. For this, a second transfer vector could be constructed to allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
  • Example 13 Purification of the callatostatin peptide homologues of C. pomonella
  • Example 15 Physiological and degradation studies of the callatostatin-like peptides in C. pomonella
  • a possible methodology for inserting the callatostatin gene or DNA encoding one or more of the peptides into a suitable delivery vehicle for use as an insecticide against a pest such as C. pomonella is as follows.
  • C. pomonella for the construction of a synthetic C. pomonella gene and, eventually, for producing oligonucleotide probes for the isolation of the endogenous callatostatin gene of C. pomonella .
  • the cal gene will be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter.
  • This is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this will be suitable for studying the peptides that are expressed and for detailed studies on larval gut paralysis and measurements of LT 50 values, it would also be desirable to produce an occluded recombinant virus for more extensive assays.
  • a second transfer vector will be constructed which will allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
  • the non-occluded recombinant virus will be used to study the extent of paralysis in C. pomonella larvae and compare LT 50 values with wild-type virus. This will give a very good indication of how effective the cal gene is. Subsequently, accurate measurements of LD 50 values will be performed with an occluded recombinant CpGV using both C. pomonella and Cryptophlebia leucotreta (false codling moth) . In the laboratory, C. leucotreta can be infected with wild-type CpGV but only at a 1000-fold higher dose than is required for C. pomonella . Bioassays with both these species would indicate whether there is any change in infectivity or host range of CpGV due to insertion of the cal gene.
  • the cal gene will be cloned into pAcUW2B to give pAcUW- cal .
  • This transfer vector places the cal gene under the control of the high level plO promoter and also contains the polyhedrin gene.
  • Cotransfection of pAcUW-cal with linearized polyhedrin- negative AcNPV will give rise to polyhedrin-positive recombinants expressing callatostatins.
  • the production of normal occlusion bodies (polyhedra) by recombinant virus will allow accurate bioassays in both neonate and later instars and is the form of the virus which is used in formulations for insecticidal application.
  • Bioassays to determine the biological effect of insertion of the cal gene into the virus will be done in H. virescens using neonate and later instars. Precise dosing will be achieved by droplet feeding of the larvae and LD 50 and LT 50 values will be compared. AcNPV is able to infect a large number (>40) different moth species and less detailed assays with other species will also be carried out.
  • Cp-cal The DNA sequence of a synthetic C. pomonella callatostatin gene (Cp-cal) will be derived from the amino acid sequence (s) of the most active peptide (s) using codon preferences obtained from an analysis of eight CpGV genes previously characterized. A C-terminal Gly will be added for amidation and the gene will be preceded by a signal peptide sequence e.g. from chorion and adipokinetic hormone genes which have been used successfully with the baculovirus expression system. The gene will be flanked by appropriate restriction enzyme sites for cloning into the CpGV transfer vector and subsequent insertion into the CpGV genome. Studies on expression and pathogenicity will be performed as in (2) and (3) above.
  • Example 16 Identification and sequencing of helicostatin gene of Heli coverpa armicrera
  • the gene encoding the helicostatin peptides of Helicoverpa armigera was similarly identified and sequenced.
  • the cDNA sequence of the helicostatin gene of H. armigera is shown in Figure 17.
  • the cDNA is 838 nucleotides long and includes the entire protein open reading frame (nucleotides 100 to 781 inclusive) .
  • the deduced amino acid sequence of the open reading frame of the cDNA of the gene is shown in Figure 18.
  • the sequence is the putative peptide precursor sequence and contains 228 amino acid residues.
  • Example 17 Isolation of peptides from Cydia pomonella , Heli coverpa armigera and Pteronidia salici s
  • Table 6 shows the amino acid sequences and M r values of the Leu-callatostatin neuropeptides or cydiastatins isolated from C. pomonella .
  • the M r of those peptides lacking an acidic residue remains the same as the M r before methylation, thus showing that the carboxyl group at the C-terminus is amidated during processing.
  • M r shows an increase of approximately 30 following methylation. This indicates that (a) the Met residue has been oxidised during the methylation process, (b) that the Asp residue has been methylated, and that (c) that the C-terminal Leu residue is amidated.
  • Table 7 shows the amino acid sequences and M r values of the Leu-callatostatin neuropeptides or helicostatins isolated from H. armigera .
  • ° - M r shows an increase of approximately 16 following methylation. This result indicates that the Met residue has been oxidised during the methylation process and that the C-terminus is amidated.

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Abstract

On a isolé sur la mouche à viande Calliphora vomitoria de nouveaux peptides de codage de l'ADN appartenant à la classe callatostatine de neuropeptides d'insectes. L'ADN qui code ces nouveaux peptides code aussi les autres peptides de Leu-callatostatine qui ont déjà été isolés sur C. vomitoria. Les homologues de ce gène ont été également isolés sur Helicoverpa armigera et sur Lucilia cuprina, autrement dit la lucilie. Les peptides inhibent la motilité du tube digestif de C. vomitoria et pourraient produire des effets allatostatiques chez les cafards. Les peptides peuvent servir d'insecticides. L'ADN qui code ces peptides peut être introduit dans un baculovirus pour servir d'insecticide contre l'espèce nuisible Cydia pomonella ou Helicoverpa armigera ou contre d'autres organismes. Des variantes de ces peptides ont également été isolées sur Cydia pomonella, Helicoverpa armigera et Pteronida salicis.
PCT/GB1997/000843 1996-03-26 1997-03-26 Genes de neuropeptides d'insectes et peptides WO1997035981A1 (fr)

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WO2003014150A2 (fr) 2001-08-08 2003-02-20 University Of Durham Proteines hybrides adaptees a la desinsectisation
US6664371B1 (en) * 1998-11-13 2003-12-16 The United States Of America As Represented By The Secretary Of Agriculture Mimetic insect allatostatin analogs for insect control
US9804116B2 (en) 2014-12-26 2017-10-31 Kabushiki Kaisha Toshiba Method and device for detecting sample

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CN116250548A (zh) * 2022-08-30 2023-06-13 华南师范大学 抑咽侧体神经肽在防治美洲大蠊中的应用

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WO1994020530A2 (fr) * 1993-03-12 1994-09-15 Queen Mary And Westfield College Neuropeptides et leur utilisation comme insecticides
WO1995001991A1 (fr) * 1993-07-05 1995-01-19 The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Allatostatines et leur utilisation

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US6664371B1 (en) * 1998-11-13 2003-12-16 The United States Of America As Represented By The Secretary Of Agriculture Mimetic insect allatostatin analogs for insect control
WO2003014150A2 (fr) 2001-08-08 2003-02-20 University Of Durham Proteines hybrides adaptees a la desinsectisation
WO2003014150A3 (fr) * 2001-08-08 2003-10-30 Univ Durham Proteines hybrides adaptees a la desinsectisation
US9804116B2 (en) 2014-12-26 2017-10-31 Kabushiki Kaisha Toshiba Method and device for detecting sample

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AU2168897A (en) 1997-10-17

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