Novel insecticidal toxins from the venom of the spider Segestria florentina.

Three insecticidal polypeptide toxins (F5.5, F5.6, F5.7) with molecular masses 4973, 4993 and 5159Da were isolated from the venom of the central Asian spider Segestria florentina. These toxins caused the complete flaccid paralysis of Heliothis virescens (Lepidoptera: Noctuidae) larvae (LD(50) 4-10 microg/g), whereas they were inactive upon intravenous injections into mice. On the basis of N-terminal amino acid sequences a family of eight genes encoding highly homologues polypeptides (SFI1-SFI8) was revealed, some of which encode polypeptides actually demonstrated to be present in S. florentina venom. All deduced polypeptides consist of 46 amino acids residues. Comparison of primary structures of SFI1-SFI8 with other spider toxins suggests that this family might share structural and functional relationships with other small spider neurotoxins, several of which are known to be highly selective agonists/antagonists of different voltage-dependent Ca(2+) channels.

Characterization of the ryanodine receptor-Ca2+ release channel from the thoracic tissues of the lepidopteran insect Heliothis virescens.

The existence of invertebrate forms of the RyR has recently been confirmed (Takeshima et al., 1994, Puente et al., 2000). However, information on the functional properties of this insect RyR is still limited. We report the functional characterization of a RyR from the thoracic muscle of H. virescens (Scott-Ward et al., 1997). A simple purification protocol produced membranes from homogenized prefrozen H. virescens thoracic muscle with a [3H]-ryanodine binding activity of 1.19 +/- 0.21 pmol/mg protein (mean +/- SE; n = 4). [3H]-Ryanodine binding to the H. virescens receptor was dependent on the ryanodine concentration in a hyperbolic fashion with a KD of 3.82 nM (n = 4). [3H]-ryanodine binding was dependent on [Ca2+] in a biphasic manner and was stimulated by 1 mM ATP. Millimolar caffeine did not stimulate [3H]-ryanodine binding to H. virescens membranes in the presence of either nanomolar or micromolar Ca2+. A protein of at least 400 KDa was recognized in H. virescens membrane proteins by a specific anti-H. virescens RyR antibody. Discontinuous density sucrose gradient fractionation of microsomal membranes produced vesicles suitable for single-channel studies. Ca2+-sensitive, Ca2+-permeable channels were successfully inserted into artificial lipid bilayers from H. virescens membrane vesicles. The H. virescens RyR-channel displayed a Ca2+ conductance of approximately 110 pS and underwent a persistent and characteristic modification of ion handling and gating following addition of 100 nM ryanodine. The gating of H. virescens channels was sensitive to ATP and ruthenium red in a manner similar to mammalian RyR. This is the first report to describe the single channel and [3H]-ryanodine binding properties of a native insect RyR.

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni.

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.

Cloning, heterologous expression and co-assembly of Mpbeta1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae.

Nicotinic acetylcholine receptors (nAChRs) play a major role in excitatory synaptic transmission in insects and are also the target site for chloronicotinyl insecticides such as imidacloprid. Here we report the cloning and characterization of a novel nAChR beta subunit, Mpbeta1, from the aphid Myzus persicae, an economically important pest species. Sequence analysis has identified an open reading frame of 509 amino acids with features typical of nAChR subunits. The Mpbeta1 gene is expressed as a single major transcript of 4.6 kb, considerably larger than the predicted length of the Mpbeta1 open reading frame (1527 bp). By heterologous expression in Drosophila S2 cells, the Mpbeta1 subunit has been shown to co-assemble with the previously cloned nAChR subunits Mpalpha1 and Mpalpha2. In contrast, no co-assembly of Mpbeta1 could be detected with either Mpalpha3 or Mpalpha4. With the aim of gaining a clearer insight into the influence of subunit composition upon assembly, the ability of M. persicae nAChR subunits to co-assemble with vertebrate nAChR subunits has also been examined.

Identification of a polymorphic ryanodine receptor gene from Heliothis virescens (Lepidoptera: noctuidae).

cDNAs encoding the C-terminal 1172 amino acids of a ryanodine receptor (RyR) from the lepidopteran pest Heliothis virescens (Hv-RyR) have been cloned and characterised. Sequence comparisons, organisational studies on corresponding genomic regions and a genetic segregation analysis provide evidence for two polymorphic alleles of the Hv-RyR locus. Comparison of the Hv-RyR C-terminal amino acid sequence with equivalent regions of other RyRs reveals a high level of overall amino acid homology (74% identity with D. melanogaster and between 47.9 and 50.1% with vertebrate isoforms). Homologies are however not uniformly distributed, though regions of high and low similarity are consistent with patterns in other RyR isoforms. The structural similarity of Hv-RyR with other RyRs is also indicated by comparison of hydropathy profiles and other previously described functional domains. Such results are consistent with this region of Hv-RyR containing the Ca(2+) channel itself and being intimately involved in RyR regulation. Potential uses of the cDNAs described in the discovery and development of novel ryanodine like insecticides are discussed.

Purification and cDNA cloning of an insecticidal protein from the venom of the scorpion Orthochirus scrobiculosus.

Injection of crude venom from the scorpion Orthochirus scrobiculosus into larvae of Heliothis virescens (Lepidoptera: Noctuidae) caused trembling and uncoordinated movement before development of a progressive and prolonged flaccid paralysis. The isolation of the toxin (OsI-1) responsible for this effect of O. scrobiclosus venom is described. The molecular mass of OsI-1 toxin was 6994 Da, as determined by desorption mass spectroscopy. The complete primary structure of OsI-1 was deduced from the sequence of cDNA clones obtained by rapid amplification of cDNA ends (RACE) PCR. Comparison of the deduced amino acid sequence of OsI-1 with those of other insecticidal scorpion toxins indicates that it is a sodium (Na+) channel active depressant insect-selective toxin. The analysis of amino acid sequence of the toxin in conjunction with mass spectroscopy data indicates post-translational modification in maturation with the removal of 3 C-terminal amino acids and amidation of the C-terminus.

Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristeroneA.

Ecdysteroids play an important role during insect development. We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor. Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster. Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR. Genomic Southern blots showed that the HvEcR is encoded by a single copy gene. Two lines of evidence towards the functional activity of the HvEcR are presented. In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP. Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.

Molecular characterization and imidacloprid selectivity of nicotinic acetylcholine receptor subunits from the peach-potato aphid Myzus persicae.

The recent introduction of the chloronicotinyl insecticide imidacloprid, targeting insect nicotinic acetylcholine receptors (nAChRs), emphasises the importance of a detailed molecular characterisation of these receptors. We are investigating the molecular diversity of insect nAChR subunit genes in an important agricultural pest, the peach-potato aphid Myzus persicae. Two M. persicae alpha-subunit cDNAs, Mp alpha1 and Mp alpha2, have been cloned previously. Here we report the isolation of three novel alpha-subunit genes (Mp alpha3-5) with overall amino acid sequence identities between 43 and 76% to characterised insect nAChR subunits. Alignment of their amino acid sequences with other invertebrate and vertebrate nAChR subunits suggests that the insect alpha subunits evolved in parallel to the vertebrate neuronal nAChRs and that the insect non-alpha subunits are clearly different from vertebrate neuronal beta and muscle non-alpha subunits. The discovery of novel subtypes in M. persicae is a further indicator of the complexity of the insect nAChR gene family. Heterologous co-expression of M. persicae nAChR alpha-subunit cDNAs with the rat beta2 in Drosophila S2 cells resulted in high-affinity binding of nicotinic radioligands. The affinity of recombinant nAChRs for [3H]imidacloprid was influenced strongly by the alpha subtype. This is the first demonstration that imidacloprid selectively acts on Mp alpha2 and Mp alpha3 subunits, but not Mp alpha1, in M. persicae.

In vitro host range of Autographa californica nucleopolyhedrovirus recombinants lacking functional p35, iap1 or iap2.

We have examined the host range in different insect cell lines of Autographa californica nucleopolyhedrovirus (AcMNPV) recombinants lacking p35, iap1 or iap2. These genes encode, or are predicted to encode, anti-apoptotic proteins. Abrogation of p35 reduced the ability of AcMNPV to replicate in permissive cell lines derived from Spodoptera frugiperda insects by inducing apoptosis. In semi-permissive cell lines, such as Lymantria dispar and Spodoptera littoralis cells, we observed cytopathic effects after infection with AcMNPV but little virus production. Infection of these cells by AcMNPV lacking p35 resulted in apoptosis. However, p35-deficient viruses were still able to replicate normally in Trichoplusia ni, Mamestra brassicae and Panolis flammea cell lines. Disruption of AcMNPV iap1 and iap2 was found not to affect virus replication in any of the cell lines. It was also possible to disrupt both iap1 and iap2 in the same virus without loss of infectivity. A virus without iap1 and p35 demonstrated identical growth characteristics and host range to a virus lacking p35. We conclude that in cells which respond to AcMNPV infection by initiating programmed cell death, the p35 gene product alone is sufficient to inhibit apoptosis. Removal of iap1 or iap2 has no effect on virus replication, even in cell lines which do not undergo apoptosis in response to AcMNPV infection. Our results with two semi-permissive cell lines further indicate that whilst p35 is important in blocking block apoptosis, other factors are involved in restricting AcMNPV replication within these cells.

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae.

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.

Small binding proteins selected from a combinatorial repertoire of knottins displayed on phage.

Knottins are a group of small, disulphide-bonded proteins that bind with high specificity to their target molecules. These proteins appear to use different faces of the protein for their interactions with different targets. Here, we attempted to create knottins with novel binding activities based on the cellulose-binding domain of the fungal enzyme cellobiohydrolase I. Variation was introduced to the face of the protein that binds cellulose. Seven residues, which are located in two regions of the polypeptide chain and form a patch of about 400 A2 on the protein surface, were simultaneously varied by random mutation of the gene. The repertoire was cloned for display on filamentous bacteriophage (5.5 x 10(8) clones), and selected for binding to cellulose or to one of three enzymes (alpha-amylase, alkaline phosphatase and beta-glucuronidase). We thereby isolated variant knottins against cellulose (differing in sequence from the parent knottin) and also against alkaline phosphatase. The binding to (glycosylated) alkaline phosphatase was highly specific with an affinity of about 10 microM, required the presence of disulphide bonds and was mediated through protein (rather than carbohydrate) contacts. Knottin scaffolds therefore appear to be a promising architecture for the creation of small folded proteins with binding activities, with the potential for improvement of binding affinities by mutation, or of using other faces of the protein to provide greater structural diversity in the primary repertoire.

Cloning and expression of an insect Ca(2+)-ATPase from Heliothis virescens.

A complementary DNA for the Tobacco Budworm, Heliothis virescens, sarco(endo)plasmic reticulum-type Ca(2+)-ATPase (HVSERCA) has been cloned and sequenced. cDNA fragments of adult rabbit fast-twitch muscle Ca(2+)-ATPase (SERCA1a) were used as heterologous probes to isolate a partial cDNA clone coding for a protein with high homology to the Ca(2+)-ATPase from Drosophila melanogaster (DRSERCA) and vertebrate ER/SR Ca2+ pumps. The entire cDNA clone contains an ORF encoding a protein of 1000 amino acids which shares the characteristic motifs of a P-type ATPase. HVSERCA shares 89% identity with DRSERCA, 80% identity with the Artemia Ca(2+)-ATPase and 72% identity with avian and mammalian SERCAs. An insect Ca(2+)-ATPase-specific polyclonal antiserum has been raised against a fusion protein containing sequence from the cytoplasmic domain of HVSERCA. Heterologous expression of the insect pump in COS-7 cells has been demonstrated by immunocytochemistry and the reticular pattern of staining is consistent with an ER localisation. However, the expressed enzyme from COS-7 cells does not appear to be active.

Photoaffinity labeling of the ryanodine receptor/Ca2+ release channel with an azido derivative of ryanodine.

Ryanodine receptors/Ca2+ release channels play an important role in regulating the intracellular free calcium concentrations in both muscle and nonmuscle cells. Ryanodine, a neutral plant alkaloid, specifically binds to and modulates these Ca2+ release channels. In the work described here, we characterize the interaction of a tritium-labeled, photoactivable derivative of ryanodine (3H-labeled 10-O-[3-(4-azidobenzamido)propionyl]ryanodine ([3H]ABRy)) with the ryanodine receptor of skeletal, cardiac, and brain membranes. Scatchard analysis demonstrates that this ligand binds to a single class of high affinity sites in skeletal muscle triads. Furthermore, competition binding assays of [3H]ryanodine with skeletal, cardiac, and brain membranes in the presence of increasing concentrations of unlabeled ABRy illustrate that this azido derivative of ryanodine is able to specifically displace [3H]ryanodine from its binding site(s). Analysis of the effects of Ca2+, ATP, and KCl on [3H]ABRy binding in triad membranes shows a similar modulation of binding to that seen in these membranes with [3H]ryanodine. Photoaffinity labeling of triads with [3H]ABRy resulted in specific and covalent incorporation of [3H]ABRy into a 565-kDa protein that was shown to be the skeletal muscle ryanodine receptor. Digestion of the labeled ryanodine receptor revealed a [3H]ABRy-labeled 76-kDa tryptic fragment that was identified with an antibody directed against the COOH-terminal of the receptor. These results demonstrate that the 76-kDa COOH-terminal tryptic fragment contains the high affinity binding site for ryanodine.

Radioimmunoassay for the calcium release channel agonist ryanodine.

A novel photo-activatable derivative of ryanodine, 9-hydroxy-21-(4-azidobenzoyloxy)-9-epiryanodine, has been synthesized and conjugated to keyhole limpet hemocyanin for the production of antibodies with high affinity and specificity to ryanodine. The anti-ryanodine antibodies reacted specifically on immunoblots with the azido-ryanodine compound covalently conjugated to bovine serum albumin. A radioimmunoassay specific for ryanodine was developed using the anti-ryanodine antibodies, and a dissociation constant for ryanodine of 1 nM was determined. Half-maximal inhibition constants (IC50) for various ryanodine derivatives were found to range between 3.2 and 200 nM. These IC50 values correlated very well with the IC50 values obtained for the compounds binding to the skeletal muscle membrane receptor. These antibodies should be useful for the characterization of the ryanodine binding site on the sarcoplasmic reticulum Ca2+ release channel.

ALS and SAD-like nicotinic acetylcholine receptor subunit genes are widely distributed in insects.

Segments of nicotinic acetylcholine receptor alpha subunit genes have been isolated from a panel of insect species by polymerase chain reaction, using degenerate oligonucleotide primers designed to recognize conserved regions of the Drosophila melanogaster ALS and SAD genes. The amplified segments encode elements of typical alpha-subunits anticipated to play roles in ligand binding and ion channel formation. Each is also clearly either ALS or SAD-like. The predicted protein sequences display extremely high levels of conservation (over 85% for each subtype) even though derived from very distantly related insect species.

Identification of a functional allele of a human interferon-alpha gene previously characterized as a pseudogene.

Three recombinant phage lambda L47 clones containing 4 alpha interferon (IFN) genes have been isolated from a newly constructed human genomic library. Each gene is an allele of a previously described IFN gene, three being only minor variants. The fourth gene SMTIII.1A is a functional allele of the psi LeIF-L gene which previously has been described only as a pseudogene. Therefore, it appears likely that other variant alleles may remain to be described and that the IFN system may be able to tolerate some degeneracy as a consequence of the large number of members of the family.

Molecular cloning of cDNAs from androgen-independent mRNA species of DBA/2 mouse sub-maxillary glands.

cDNA libraries have been constructed from mRNAs isolated from mature male DBA/2 mouse submaxillary glands. Several recombinant plasmids have been assigned to particular mRNA species and their in vitro translation products by HART and hybrid selection. Clones containing copies of two abundant mRNA species that showed no sexual dimorphism were selected for detailed characterisation. Nucleotide sequences determined from one series of clones define an 850 nucleotide mRNA encoding a polypeptide of 16.5 kd having an N-terminal signal sequence, an acidic core and four glycosylation sites. A second family of clones correspond to an mRNA of 800 nucleotides, the sequence of which can be interpreted as coding for an intracellular protein of 14.7 kd. Computer searches of protein and nucleic acid sequences have not revealed the identity of either of these submaxillary gland products.

Mapping of murine interferon-alpha genes to chromosome 4.

A cDNA library was constructed from polysomal poly(A)+RNA from Newcastle disease virus (NDV)-induced mouse C243 cells, and screened with a human interferon-alpha (HuIFN-alpha) cDNA probe. A cDNA clone for one of the murine interferon-alpha (MuIFN-alpha) genes was isolated, and sequencing analysis revealed that it was a partial copy which is almost identical to the published sequence for the MuIFN-alpha 2 gene. This partial cDNA clone represents a virus-induced message as seen by Northern blot analysis of RNA from NDV-induced C243 cells, and Southern blot analysis of DNA from BALB/c mouse revealed the presence of a multiple IFN-alpha gene family. The MuIFN-alpha genes were mapped to chromosome 4 by Southern blot analysis of hamster/mouse somatic cell hybrid DNAs.

The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.

An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.

Molecular cloning of partial cDNA copies of two distinct mouse IFN-beta mRNAs.

Two cDNA libraries were constructed, using respectively the 12S and the 16S sucrose gradient fractions of polysomal poly (A)+ RNA from mouse C243 cells induced with Newcastle disease virus. Screening of a part of both libraries by mRNA selection hybridization assays revealed the presence of two plasmids hybridizing to an mRNA, whose translation product was characterized as mouse IFN-beta. Blot analysis of RNA indicated that mRNA hybridizing to the DNA from both plasmids could be detected in induced but not in uninduced C243 cells. The two cDNA inserts did not cross hybridize and had distinct restriction maps. Sequencing revealed that both inserts represented the end of the coding region and the entire 3' non coding region of two district mRNAs. Although different, the putative 39 AA and 65 AA carboxy termini of both Mu IFN-beta s display some homology to human IFN-beta 1. Thus there are at least two different murine IFN-beta genes.