WO2002033405A1 - Procedes d'identification de composes pesticides - Google Patents

Procedes d'identification de composes pesticides Download PDF

Info

Publication number
WO2002033405A1
WO2002033405A1 PCT/IB2001/002391 IB0102391W WO0233405A1 WO 2002033405 A1 WO2002033405 A1 WO 2002033405A1 IB 0102391 W IB0102391 W IB 0102391W WO 0233405 A1 WO0233405 A1 WO 0233405A1
Authority
WO
WIPO (PCT)
Prior art keywords
serca
activity
elegans
protein
indicator
Prior art date
Application number
PCT/IB2001/002391
Other languages
English (en)
Inventor
Richard Zwaal
Titus Kaletta
Marc Van Den Craen
Marc Logghe
Elke Smits
Wim Van Creikinge
Thierry Bogaert
Original Assignee
Devgen Nv
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0025576A external-priority patent/GB0025576D0/en
Application filed by Devgen Nv filed Critical Devgen Nv
Priority to AU2002218457A priority Critical patent/AU2002218457A1/en
Publication of WO2002033405A1 publication Critical patent/WO2002033405A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates

Definitions

  • the invention is concerned with methods for use in the identification of compounds having potential utility as pesticides.
  • the invention relates to methods for use in identifying compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) .
  • SERCA sarco/endoplasmic reticulum Ca 2+ ATPase
  • the insecticide resistance committee (IRAC) , reports regularly on the emergence of new resistance of insects against insecticides. The results of a resistance survey carried out in 1996, published in "The Pest Manual, 11th edition, ed CDS Tomlin", by the British Crop Protection Council, 49 Downing Street, Farnham, Surrey, GU9 7PH, UK, indicating the problems that exist with insect resistance and hence the need to develop new insecticides.
  • the Fungicide resistance action committee has already indicated that well known fungi have already developed resistance to well known fungicides such as benzimidazoles, dicarboximides, phenyla ides, sterol biosynthesis inhibitors.
  • well known fungicides such as benzimidazoles, dicarboximides, phenyla ides, sterol biosynthesis inhibitors.
  • the stobilurins and the anilinopyrimidines were introduced on the market as novel fungicides.
  • no resistance has been observed against those to classes of compounds, but one may expect that in the near future fungi will also develop resistance against these fungicides.
  • HRAC herbicide resistance action committee
  • SERCA proteins belong to the group of ATP-driven ion- motive ATPases, which also includes, amongst others, the plasma membrane Ca 2+ -transport ATPases (PMCA) , the Na + -K + -ATPases, and the gastric H + -K + -ATPases .
  • SERCA proteins are present in all higher organisms, including pest species.
  • the evolutionary conservation of SERCA proteins identifies these proteins as an interesting target for pesticidal intervention.
  • inhibition or deletion of SERCA activity in a variety of organisms results in lethality, or at least a marked reduction in the vitality of the organism.
  • the present inventors have shown that inhibition of SERCA activity in the nematode C. elegans results in lethality.
  • Inhibition of SERCA activity, and hence depletion of endoplasmic reticulum calcium stores also results in a lowering of muscle relaxation and hence immobility and/or respiration deficiency.
  • SERCA inhibitors are potential pesticides or can be considered as basic compounds for the development of pesticides such as herbicides, insecticides and nematocides. It has been shown that SERCA function is essential in the intracellular trafficking of the
  • Notch receptor in drosophila (Periz et al., 1999 EMBO J; 5983-5993) .
  • This study and others indicate that SERCA is an interesting target for pesticidal intervention.
  • the inventors have developed generic screening methods which may be used to identify compounds which down-regulate SERCA activity and may therefore have the potential to kill pests.
  • Several of these screens are performed in microscopic nematode worms such as Caenorhabdi tis elegans .
  • C. elegans is a small roundworm that has a life cycle of only three days, allowing rapid accumulation of large quantities of individual nematodes.
  • elegans may be used in the development of high throughput live animal compound screens in which nematodes are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded.
  • the present inventors have developed a number of C. e-Ze ans-based screening methods which may be used to identify compounds which modulate the activity of SERCA.
  • these C. elegans based screening methods may also be used to identify compounds which modulate the activity of other proteins in the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell.
  • the invention provides a method of identifying compounds having pesticidal activity, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the microscopic nematode worm in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.
  • the method of the invention may be used to identify compounds which have pesticidal activity because they directly or indirectly affect the activity of the SERCA protein.
  • the invention further provides a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises: providing microscopic nematode worms expressing a pest SERCA protein, said protein being derived from a pest species, other than the C. elegans SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the microscopic nematode worm in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.
  • the preferred microscopic nematode species for use in the screening methods of the invention is Caenorhabd.i t Is elegans . It will, however, be appreciated that the methods may be carried out with other nematodes and in particular with other microscopic nematodes, preferably microscopic nematodes belonging to the genus Caenorhabdi tis including C. briggsae.
  • the term "microscopic" nematode encompasses nematodes of approximately the same size as C. elegans, being of the order 1mm long in the adult stage. Microscopic nematodes of this approximate size are extremely suited for use in mid- to high-throughput screening as they can easily be grown in the wells of a multi-well plate of the type generally used in the art to perform such screening.
  • C. elegans occurs naturally in the soil but can be easily grown in the laboratory on nutrient agar inoculated with bacteria, preferably E. coll , or in liquid culture. Each worm grows from an embryo to an adult worm of about 1 mm long in three days or so. As it is fully transparent at all stages of its life, cell divisions, migrations and differentiation can be seen in live animals. Furthermore, although its anatomy is simple its somatic cells represent most major differentiated tissue type including muscles, neurons, intestine and epidermis. Accordingly, differences in phenotype which represent a departure from that of wild-type C. elegans are relatively easily observed and many phenotypic, physiological or biochemical characteristics of the nematode submit to quantitative measurement.
  • pest SERCA protein encompasses any SERCA protein derived from a pest species.
  • pest species encompass species recognised as such by one skilled in the art. Pest species include, but are not necessarily limited to, arthropods such as insects, ticks, mites, spiders and nematodes (excluding C. elegans for the purposes of this application) and also fungi, plants and rodents.
  • pest species also encompasses parasitic pest species, including human parasites, and the term “compounds having pesticidal activity” is to be interpreted accordingly as encompassing compounds having anti-parasitic activity which may have utility in the pharmaceutical and/or veterinary fields.
  • a non-exhaustive list of pest species is included in the accompanying Examples. Further lists of pest species can be found in "The Pest Manual”, ed CDS Tomlin, BCPC.
  • compounds having pesticidal activity is to be interpreted as encompassing compounds which are lethal to one or more pest species as hereinbefore defined or lethal to the progeny of such a pest species. As aforesaid, this definition encompasses compounds having anti-parasitic activity.
  • SERCA protein derived from a pest species is intended to encompass any SERCA protein naturally expressed by a pest species, including naturally occurring splice variants, allelic variants and isoforms. Many species express more than one SERCA isoform and the scope of the invention is not restricted to any particular isoform.
  • SERCA protein derived from a pest species is also intended to encompass specific mutant versions of naturally occurring pest SERCA proteins, including, for example, mutant proteins engineered by directed mutagenesis techniques. Specific mutant pest SERCA proteins will advantageously retain near wild- type SERCA ATPase activity.
  • Further examples of "SERCA proteins derived from a pest species" within the scope of the invention are chimeric proteins created by in-frame fusion of fragments of two or more SERCA proteins, at least one of which is a SERCA protein derived from a pest species. Chimeric proteins included within this definition include fusions of a pest SERCA protein and a C. elegans SERCA protein (see accompanying Examples) .
  • the microscopic nematode worm expressing the pest SERCA protein may, advantageously, be a transgenic worm containing a transgene comprising nucleic acid encoding the pest SERCA protein operably linked to a promoter.
  • transgene refers to a DNA construct comprising a promoter operably linked to a DNA sequence encoding the pest SERCA protein.
  • the construct may contain additional DNA sequences in addition to those specified above.
  • the transgene may, for example, form part of an expression vector, such as plasmid vector.
  • operably linked it is to be understood that the promoter is positioned to drive transcription of the protein-encoding DNA fragment.
  • transgenic C. elegans including C. elegans carrying multiple transgenes
  • a typical approach involves the construction of a plasmid-based expression vector in which a protein-encoding DNA of interest is cloned downstream of a promoter having the appropriate tissue or cell-type specificity.
  • the plasmid vector is then introduced into C. elegans of the appropriate genetic background, for example using microinjection.
  • a second plasmid carrying a selectable marker may be co-injected with the experimental plasmid.
  • Plasmid vectors are usually maintained in cells of transgenic C. elegans in the form of an extrachromosomal array. Although plasmid vectors are relatively stable as extrachromosomal arrays they can alternatively be stably integrated into the C. elegans genome using standard technology, for example, using gamma ray-induced integration of extrachromosomal arrays (methods in Cell Biology, Vol 48 page 425-480) .
  • the DNA sequence encoding the pest SERCA protein may be any DNA sequence comprising the complete open reading frame of the corresponding pest SERCA gene, such as, for example, a fragment of genomic DNA or cDNA.
  • a number of pest SERCA cDNA sequences are available from publicly accessible sequence databases such as the GenBank database.
  • the inventors have developed an approach to isolate SERCA cDNAs from various other pest species, in particular pest species for which no or limited sequence data is available through database sources.
  • the inventors' method is generally applicable and comprises the following steps:
  • the promoter part of the transgene may be any promoter which is capable of directing gene expression in the nematode.
  • the DNA encoding the pest SERCA protein is operably linked to the promoter region of a SERCA gene.
  • the promoter region of the C. elegans sca-1 gene is used.
  • the term promoter region' as used herein refers to a fragment of the upstream region of a given gene which is capable of directing a pattern of gene expression substantially identical to the natural pattern of expression of the given gene.
  • the promoter may, advantageously, be the promoter region of a C. elegans gene and may be a tissue-or cell type-specific promoter.
  • the pest SERCA protein can be expressed in all the cells of C. elegans, in a given type of tissue (i.e. all muscles), in a single organ or tissue (for example, the pharynx or the vulva) , in a subset of cell types, in a single, cell type or even in a single cell.
  • Tissue-specific C. elegans promoters which may be used in accordance with the invention include the myo-2 promoter which directs gene expression in the pharynx, the myo-3 promoter which directs gene expression in the body wall muscles, the egl-15 and ceh-24 promoters which direct gene expression in vulva muscles.
  • Other tissue-specific C. elegans promoters are well known to persons skilled in the art.
  • C. elegans has a single SERCA gene, which was identified by the C. elegans genome-sequencing consortium (see Science issue 282, 1998) .
  • the C. elegans SERCA gene, designated sca -1 is located on chromosome III on a cosmid named K11D9.
  • the gene On a physical level, the gene consists of seven exons that span an Open Reading Frame of 3.2 kb, resulting in a predicted protein of 1059 amino acids.
  • the consensus alternative splice site that is present in the C-terminal end of mammalian SERCA genes is also present in C. elegans . This leads to a second isoform consisting of eight exons that span an ORF of 3.0kb, resulting in a protein of 1004 amino acids.
  • ⁇ activity' used in relation to a SERCA protein refers to the calcium ATPase activity of the protein, unless otherwise stated.
  • the activity of the endogenous nematode SERCA protein can be substantially reduced or abolished. In one embodiment, this is achieved by introducing the transgene encoding the pest SERCA protein into a mutant strain which exhibits no or substantially reduced activity of the endogenous SERCA protein in one or more tissues or cell types.
  • This mutant strain may carry a knock-out or loss-of-function mutation in the chromosomal SERCA gene.
  • the mutation may abolish/reduce SERCA activity through a down-regulation of SERCA expression in one or more cell types or tissues or a defect in regulation of the activity of the SERCA protein.
  • C. elegans having a reduction-of-function mutation or a knock-out mutation in the sca-1 gene can be isolated using a classical non-complementation screen, starting with a heterozygote C. elegans strain carrying a mutant sca-1 allele on one chromosome and a recessive marker close to the wild-type sca-1 allele on the other chromosome.
  • the nematodes are subjected to mutagenesis using standard techniques (EMS or
  • UV-TMP are suitable for this purpose) and the progeny is screened by eye for defects, especially in tissues which express SERCA. Since the screening is performed in the Fl generation, mutations will only give rise to a phenotype if the mutation occurs in the sca-1 gene (due to non-complementation) or if the mutation is dominant, which does not occur frequently. These two possibilities can be distinguished in subsequent generations. A newly introduced sca-1 mutation should be linked to the recessive marker. As a further control, DNA sequencing can be performed to determine the nature of the mutation.
  • C. elegans strain which carries a knock-out mutation in the sca-1 gene is strain okl 90, described in the accompanying Examples.
  • a protocol for introducing a pest SERCA transgene onto an sca-1 knock-out genetic background is included in the accompanying examples.
  • activity of the endogenous nematode SERCA protein can be reduced by specifically down-regulating the expression of the SERCA protein in one or more tissues using antisense techniques or double-stranded RNA inhibition (RNAi) .
  • RNAi double-stranded RNA inhibition
  • This can be achieved by transfection of the nematode, preferably C. elegans, with a vector that expresses either an antisense SERCA RNA or a double-stranded SERCA RNA.
  • the antisense or double-stranded SERCA RNA should be capable of selectively inhibiting expression of the endogenous nematode SERCA protein but not the pest SERCA protein.
  • SERCA expression will be specifically down-regulated only in those tissues which express the antisense RNA or double stranded RNA.
  • the promoter region of the C. elegans sca-1 gene itself can be used to direct expression of an antisense RNA or double stranded RNA in all the cells and tissues of C. elegans which express endogenous SERCA.
  • the C. elegans myo-2 promoter can be used to direct expression in the pharynx.
  • the C. elegans myo-3 promoter can be used to direct expression in the body wall muscles.
  • RNAi technology is well known in the C. elegans field as a tool for inhibiting expression of a specific target gene in C. elegans , as described by Fire et al . , Nature 391:801-811 (1998) and Timmins and Fire, Nature 395:854 (1998).
  • the standard approach is based on injection of dsRNA directly into the worm.
  • Alternative RNAi techniques which may be used to inhibit SERCA activity are described in the applicant's International patent application No. WO 00/01846. These techniques, which are based on delivery of dsRNA to C. elegans by feeding with an appropriate dsRNA or feeding with food organisms which express an appropriate dsRNA, may lead to a more stable RNAi phenotype than results from injection of dsRNA.
  • a pest SERCA-specific screen may be performed by using transgenic C. elegans expressing a pest SERCA protein which is resistant to a chemical inhibitor of SERCA activity, such as thapsigargin.
  • the pest SERCA protein may be variant carrying a mutation in the thapsigargin binding site The mutation Phe259Val renders C. elegans SERCA resistant to inhibition with thapsigargin.
  • Equivalent mutations may be introduced into transgenes encoding pest SERCA proteins using standard site-directed mutagenesis .
  • An alignment of SERCA amino acid sequences such as that shown in Figure 2, may be used to locate the amino acid residue in the pest SERCA protein which is equivalent to residue Phe 259 of C. elegans SERCA.
  • Applying the SERCA inhibitor, for example thapsigargin to transgenic C. elegans which express a resistant mutant pest SERCA will result in inhibition of the endogenous C. elegans SERCA only.
  • the screen will be specific for the pest SERCA.
  • the invention also encompasses an embodiment of the screening method in which the pest SERCA protein is specifically expressed in a tissue or cell type of the nematode which exhibits no or only minor background activity of the endogenous C. elegans SERCA protein. In this case it is not necessary to reduce/abolish activity of the endogenous nematode SERCA protein in order to screen selectively on the pest SERCA protein.
  • nematode tissue which exhibits little or no SERCA activity is the neurons.
  • the screen is performed using transgenic C. elegans in which expression of a pest SERCA protein is driven by a neuron-specific promoter.
  • neuron-specific promoters which may be used in this embodiment of the invention are the unc- 119, ser-1, eat-18, acm-1, acm-3 and avr-14 promoters.
  • Other suitable neuron-specific C. elegans promoters are known in the art.
  • the screening methods of the invention rely on detection of an indicator of SERCA activity in the presence or absence of a test compound.
  • an indicator of SERCA activity in the presence or absence of a test compound.
  • the inventors have observed that a reduction in SERCA activity in nematodes such as C. elegans results in various phenotypic and behavioural defects. Many of these defects can be used as basis of an assay to isolate compounds that alter the activity of SERCA, and also compounds which affect the activity of other components of the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell.
  • the main defects, and hence phenotypes, associated with reduced SERCA activity are related to muscle function e.g pharyngeal muscle, body wall muscle, vulva muscle, anal repressor muscle, and anal sphincter muscle, as illustrated by the RNAi experiments and thapsigargin inhibition experiments described in the accompanying examples.
  • Screens based on the detection of phenotypic characteristics associated with reduced SERCA activity in these muscles can be used to identify compounds and genes that alter the activity of SERCA.
  • other phenotypes such as paleness, reduced growth, reduced progeny, protruding vulva and protruding rectum can be used to identify compounds and genes that alter the function of SERCA.
  • the assay can be based on detection of pharynx pumping efficiency as an indicator of SERCA activity. If the starting nematode strain exhibits a near wild-type rate of pharynx pumping, then a decrease in the rate of pharynx pumping in the presence of a test compound can be used as an indicator of reduction of SERCA activity in the pharynx.
  • the pest SERCA protein In order to use pharynx pumping efficiency as an indicator of the activity of the pest SERCA protein, the pest SERCA protein must be expressed in at least the muscles of the pharynx. Activity of the endogenous nematode SERCA protein should also be abolished or substantially reduced in the pharynx muscles in order to confer specificity for the pest SERCA protein.
  • C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up of liquid and subsequently spitting it out, requiring contraction and relaxation of muscles, is called pharyngeal pumping or pharynx pumping.
  • food e.g. bacteria
  • inhibition of SERCA using thapsigargin causes a reduction in the rate of pharynx pumping.
  • Measurement of the pumping rate of the C. elegans pharynx is hence a method to determine the activity of SERCA.
  • Pharynx pumping efficiency can be conveniently measured by placing the nematodes in liquid containing a fluorescent marker molecule precursor, such as calcein-AM. Calcein-AM present in the medium is taken up by the nematodes and the AM moiety is cleaved off by the action of esterases present in the C. elegans gut, resulting in the production of the fluorescent molecule calcein.
  • calcein fluorescence measured in the gut is a quantitative and qualitative measurement of the SERCA activity. It would be readily apparent to one skilled in the art that other types of marker molecule precursor which are cleavable by an enzyme present in the gut of C. elegans to generate a detectable marker molecule could be used instead of calcein-AM with equivalent effect.
  • the assay can be based on detection of changes in the egg laying behaviour of the nematode or on detecting changes in the amount of progeny produced by the nematode as indicators of SERCA activity.
  • the nematode should express the pest SERCA protein in at least the vulva muscles.
  • Activity of the endogenous nematode SERCA protein should be abolished or substantially reduced in the vulva muscles in order to confer specificity for the pest SERCA protein.
  • Defects associated with reduced SERCA activity in the vulva muscles include defects in the production and laying of eggs and hence a reduction in the number of progeny produced.
  • nematodes with reduced SERCA expression in the vulva are not able to lay their eggs. The eggs thus hatch inside the mother, which then dies. These mothers are easy to recognize under the dissection microscope. As a consequence of the egg laying defect, less progeny are produced and hence the culture as a whole grows much more slowly.
  • Defects associated with reduced SERCA activity have also been observed in the gonad, including the sheath cells and the spermatheca. These defects also result in reduced egg formation and hence a reduced egg laying phenotype.
  • the offspring can be measured directly using the growth rate assay and/or the movement assay described below.
  • specific antibodies and fluorescent antibodies can be used to detect the offspring. Any specific antibody that only recognizes eggs, or Ll or L2 or L3 or L4 stage nematodes, will only recognize offspring.
  • an antibody that recognizes an antigen on the surface of C. elegans Ll larvae has been described by Hemmer et al . , (1991) J Cell Biol , 115(5): 1237-47.
  • the FANS device is a worm dispenser apparatus' having properties analogous to flow cytometers such as fluorescence activated cell scanning and sorting devices (FACS) and is commercially available from Union Biometrica, Inc,
  • the FANS device also designated a nematode flow meter, can be the nematode FACS analogue, described as fluorescence activated nematode scanning and sorting device (FANS) .
  • FANS fluorescence activated nematode scanning and sorting device
  • the FANS device enables the measurement of nematode properties, such as size, optical density, fluorescence, and luminescence and the sorting of nematodes based on these properties.
  • the assay can be based on detection of a change in the defecation behaviour of the nematode as an indicator of SERCA activity.
  • This embodiment is particularly suitable for use when the nematode expresses the pest SERCA protein in the anal sphincter or the anal repressor. In this case, activity of the endogenous nematode
  • SERCA protein should be abolished/reduced in the anal sphincter or anal repressor in order to confer specificity for the pest SERCA protein.
  • Defecation rate can be measured using an assay similar to that described above for the measurement of pharynx pumping efficiency, but using a marker molecule which is sensitive to pH.
  • a suitable marker is the fluorescent marker BCECF.
  • This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to nematodes growing in liquid medium the nematodes will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF.
  • BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH ⁇ 6) the compound exhibits no or very low fluorescence.
  • the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent.
  • the level of BCECF fluorescence in the medium is therefore an indicator of the rate of defecation of the nematodes.
  • Defecation can also be measured using a method based on the luminescent features of the chelation of terbium by aspirin.
  • the method requires two preloading steps, first the wells of a multi-well plate are pre-loaded with aspirin (prior to the addition of the nematodes) and second, bacteria or other nematode food source particles are pre-loaded with terbium using standard techniques known in the art. C. elegans are then placed.in the wells pre-loaded with aspirin and are fed with the bacteria pre-loaded with terbium. The terbium present in the pre-loaded bacteria added to the wells will result in a low level of background luminescence. When the bacteria are eaten by the nematodes the bacterial contents will be digested but the terbium will be defecated back into the medium.
  • the assay may be based on the use of growth rate as an indicator of SERCA activity.
  • Growth rate can be monitored by measuring the number of eggs or the number offspring present in the culture, by measuring the total fluorescence in the culture (this can be autofluorescence, or fluorescence caused by a transgene encoding a flourescent or luminescent protein) , but can also be measured using the movement screen described below.
  • the growth rate of a culture of C. elegans can also be assayed by measuring the turbidity of the culture. In order to perform this turbidity assay' the nematodes are grown in liquid culture in the presence of E. coli or other suitable bacterial food source. As the culture of nematodes grows the food source bacteria will be consumed.
  • the growth rate and amount of progeny can be measured on a plate.
  • Slow growing nematodes, nematodes with vulva defects and nematodes with gonad defects will produce less progeny within a certain time compared to nematodes which do not have these defects.
  • the amount of offspring produced is scored on day five and on day eight. In experiments where the amount of offspring is reduced very drastically due to severe defects in the vulva, gonad or growth rate reduction, the offspring can also be scored at later time intervals.
  • the assay may be based on detecting changes in the movement behaviour of C. elegans as an indicator of SERCA activity.
  • This embodiment is particularly suitable for use when the nematodes express the pest SERCA protein in at least the body wall muscles.
  • activity of the endogenous nematode SERCA protein should also be abolished/reduced in at least the body wall muscles in order that the assay is specific for the pest SERCA protein .
  • SERCA is widely expressed in the muscles of C. elegans, including the muscles of the body wall. A reduction of SERCA activity in the body wall muscles gives rise to nematodes with movement defects. Thus, movement defects can be used as the basis of an assay in which the nematodes are contacted with a compound under test and any changes in the movement behaviour of the nematodes are observed as an indication of SERCA activity. Compounds which cause defective movement behaviour are scored as compounds capable of down-regulating the activity of SERCA.
  • Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematodes that move and nematodes that do not move.
  • Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing a liquid culture of nematodes that move normally as compared to wells containing a liquid culture of nematodes that are defective for movement.
  • autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.
  • the assay may be based on detection of intracellular Ca 2+ levels as an indicator of SERCA activity in a given cell type or tissue. This may be accomplished using a marker molecule which is sensitive to changes in intracellular Ca 2+ such as, for example, apoaequorin.
  • Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea ictoria.
  • Recombinant apoaequorin which is luminescent in the presence of calcium but not in the absence of calcium, is most useful in determining intracellular calcium concentrations and even calcium concentrations in sub- cellular compartments.
  • Expression vectors suitable for expressing recombinant apoaequorin and, in addition, vectors expressing apoaequorin proteins which are targeted to different sub-cellular compartments, for example the nucleus, the mitochondria or the endoplasmic reticulum are available commercially (e.g. from Molecular probes, Eugene, OR, USA) .
  • an apoaequorin that is targeted to the endoplasmic reticulum (hereinafter referred to as erAEQ) is particularly useful for developing assays for SERCA activity.
  • the vector erAEQ/pcDNAI (Molecular Probes) contains an Ig ⁇ 2b heavy chain gene from mouse, an HA1 epitope and a recombinant apoaequorin in fusion.
  • the mouse gene targets the apoaequorin to the endoplasmic reticulum, and the apoaequorin is mutated to make it less sensitive to calcium, as the concentrations of this ion are relatively high in the endoplasmic reticulum.
  • apoaequorin is the calcium sensor of choice, it would be apparent to persons skilled in the art that any other calcium sensor localized in the endoplasmic reticulum could be used with equivalent effect .
  • Plasmid expression vectors which drive expression of the ER-localized apoaequorin in C. elegans can be easily constructed by cloning nucleic acid encoding erAEQ downstream of a promoter capable of directing gene expression in one or more tissues or cell types of C. elegans, such that the promoter and the erAEQ- encoding sequence are operably linked.
  • the apoaequorin gene in fusion with the signals needed to locate the resulting protein to the endoplasmic reticulum was isolated from erAEQ/pcDNAI by EcoRI digestion and cloned into pBlue2SK.
  • pGK13 is a plasmid vector containing a 2915bp fragment of the upstream region of the C. elegans sca-1 gene.
  • Suitable promoters which may be included into an expression vector to drive erAEQ expression include the pharynx-specific promoter myo-2, the C. elegans sca-1 promoter which directs expression in a wide range of muscle tissues and the body wall muscle- specific promoter myo-3.
  • the vectors can then be used to construct transgenic C. elegans according to the standard protocols known to those of ordinary skill in the art.
  • erAEQ allows for the determination of the calcium levels in the endoplasmic reticulum of various C. elegans cells and tissues, using the protocols of the manufacturer of erAEQ, or minor modifications thereof.
  • Alterations in SERCA activity influence the concentration of calcium in the endoplasmic reticulum as SERCA functions as an endoplasmic reticulum calcium pump.
  • the apoaequorin luminescence measured in the assay is directly related to SERCA activity.
  • nematodes are exposed to a variety of test compounds and compounds are selected which induce a change in the chosen indicator of SERCA activity.
  • a plurality of tests may be run in parallel containing different concentrations of the test compound.
  • a negative control zero concentration of test compound
  • Automated measuring allows the assay to be performed in mid-to-high throughput format.
  • concentration of the candidate compound to be tested in the screening method may vary according to the nature of the compound and such factors as solubility etc. It is advantageous to test a range of concentrations of the candidate compound. Concentrations in the range of about 5 ⁇ M to about 2000 ⁇ M are generally observed to be suitable.
  • the chosen concentration need not necessarily an amount which would be considered a pesticidal' dose.
  • Screens can also be performed using nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein.
  • Compounds which inhibit the endogenous nematode SERCA protein may also inhibit SERCA proteins from pest species.
  • the invention provides a further nematode-based screening method which does not require the use of a pest-derived SERCA protein.
  • This method comprises steps of: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has the potential to kill pests.
  • This method may also be used to identify compounds which have pesticidal activity because they directly or indirectly affect the activity of the SERCA protein. Therefore, according to this aspect of the invention there is also provided a method of identifying compounds capable of down-regulating the activity of a sarco/endoplas ic reticulum calcium ATPase, which method comprises: providing microscopic nematodes which exhibit wild-type activity of the endogenous nematode SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the nematodes in the presence or absence of test compounds ; and thereby identifying compounds capable of down- regulating the activity of SERCA.
  • C. elegans strain which exhibits wild-type SERCA activity is the N2 strain, available from the C. elegans Genetic Center (CGC) at the University of Minnesota, St Paul, Minnesota, USA.
  • CGC C. elegans Genetic Center
  • the screening method may be carried out using the N2 strain.
  • the N2 strain has been particularly well characterised in the literature with respect to properties such as pharynx pumping rate, growth rate and egg laying capacity (see Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern biological analysis of an organism, ed. by Henry F. Epstein and Diane C. Shakes, 1995 Academic Press; The nematode Caenorhabditis elegans, ed. by William Wood and the community of C.
  • the screening methods may also be carried out using a C. elegans strain other than N2 which exhibits similar SERCA activity to N2. This may be a mutant strain or a transgenic strain.
  • a range of C. elegans mutants may be obtained from the C. elegans mutant collection at the C. elegans Genetic Center, University of Minnesota.
  • mutants may be generated by standard methods known in the art. Suitable methods are described by J. Sutton and J. Hodgkin in "The Nematode Caenorhabditis elegans", Ed. by William B. Wood and the Community of C. elegans researchers CSHL, 1988 594-595; Zwaal et al, "Target - Selected Gene Inactivation in Caenorhabditis elegans by using a Frozen Transposon Insertion Mutant Bank" 1993, Proc. Natl. Acad. Sci. USA 90 pp 7431 -7435.
  • a population of nematodes can be subjected to random mutagenesis by using EMS, TMP-UV or radiation (Methods in Cell Biology, Vol 48, ibid) .
  • EMS Electromase
  • TMP-UV Thermal Imaging Protocol
  • radiation Methods in Cell Biology, Vol 48, ibid
  • Several selection rounds of PCR may then be performed to select a mutant with a deletion in a desired gene.
  • the screening methods may be carried out using a constitutive pharynx pumping strain of C. elegans .
  • Phenotypic, behavioural or biochemical indicators of the activity of the endogenous nematode SERCA protein which can be used as the basis of the screening method include pharynx pumping efficiency, egg laying behaviour, mating behaviour, defecation behaviour, growth rate, movement behaviour, life/death of the nematode and intracellular Ca 2+ concentration. The methods described above for the measurement of these characteristics are equally applicable to this second aspect of the invention.
  • the invention provides a method of identifying compounds having pesticidal activity which is carried out in cultured cells as opposed to whole organisms.
  • This method comprises steps of: providing cultured cells expressing a SERCA protein; and detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence 1 or absence of test compounds; wherein a reduction in SERCA activity in the presence of a compound is taken as an indication that the compound has pesticidal activity.
  • a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic ' reticulum calcium ATPase comprises: providing cultured cells expressing a SERCA protein; detecting a phenotypic, biochemical or behavioural indicator of SERCA activity in the cells in the presence or absence of test compounds; and thereby identifying compounds capable of down- regulating the activity of SERCA.
  • the cultured cells may be cells derived from a pest species which express the endogenous pest SERCA protein. This may be a cultured primary cell line or a continuous, transformed cell line. The cell line will be capable of growth in culture, preferably monolayer or suspension culture.
  • suitable cell lines derived from pest species are known in the art. Many of these are derived from insect species, for example Heliothis virescens (Lynn, Development and characterisation of insect cell lines, Cytotechnology, 20: 3-11, 1996).
  • cell lines derived from a pest species allows screening on the endogenous pest SERCA protein expressed in the cell line.
  • the cell culture assays may be based on the use of cultured cells which have been engineered to express a pest SERCA protein.
  • the assays may be carried out using eukaryotic host cells containing an expression vector comprising nucleic acid encoding the pest SERCA protein.
  • Suitable expression vectors will include a sequence of deoxynucleotides encoding the pest SERCA protein, including a start codon (usually AUG) and a termination codon for detachment of the ribosome, and also regulatory elements required for expression of the encoded SERCA protein in a eukaryotic host cell.
  • regulatory elements may include a promoter region, preferably one which is recognised by RNA polymerase II, optionally one or more additional transcriptional regulatory elements (e.g. enhancer elements) and also a terminator sequence and downstream polyadenylation signal.
  • the vector may also possess an origin of replication allowing replication in prokaryotic cells and one or more selectable markers, such as a gene for antibiotic resistance.
  • the expression vector will preferably be a plasmid vector, although virus and phage-based vectors designed for protein expression in eukaryotic host cells may also be used.
  • the eukaryotic host cells may be a cell line capable of growing in monolayer or suspension culture and will preferably not express high levels of an endogenous SERCA protein (i.e. the SERCA protein encoded in the genome of the host cell) . Fibroblast cell lines or epithelial cell lines are most preferred.
  • Suitable cell lines include COS1, BHK21, L929, PC12, CV1, SWISS3T3, HT144, IMR32, HEPG2, MDCK, MCF7, HEK293, Hela, A549, SW48 and G361. However, this list is not exhaustive.
  • transfecting expression vectors into eukaryotic host cells are well known in the art (see ⁇ Current Protocols in Molecular Biology' , Ed Ausubel et al., John Wiley & Sons, Inc) . Most preferably the host cell will be stably or permanently transfected with the expression vector such that it is retained through many cell divisions. However, it is also within the scope of the invention to use cells which are transiently transfected with the expression vector.
  • the cell culture assays rely on detection of an indicator of SERCA activity in the presence or absence of a test compound.
  • Suitable indicators of SERCA activity in cultured cells include intracellular Ca 2+ levels, in particular Ca 2+ levels in the endoplasmic reticulum, and cell death or apoptosis.
  • Suitable methods for the measurement of intracellular Ca 2+ levels in cultured cells are based on fluorescent calcium indicators excited by ultraviolet light, such as fura-2, indo-2, quin-2 or visible light such as fluo-3 and rhod-2 that are available from Molecular Probes, Eugene, USA.
  • the acetoxymethyl esters can passively diffuse across cell membranes to avoid the use of invasive loading techniques. Once inside the cells, these esters are cleaved by intracellular esterases to yield cell-impermeant fluorescent calcium indicators.
  • the test compound is usually added directly prior to the fluorescent indicator, but the screen, can also be performed by pre-incubating the cells with the test compound for an incubation time of, for example, 1 min, 5 min, 10 min or 30 min.
  • Fluorescence can be measured directly after the addition of the indicator, but it is preferred to take fluorescence measurements over a period of time, for example every 10 minutes for one hour. Fluorescence data from typical experiments show that measurements after 10 to 15 minutes are generally sufficient to determine the calcium levels in the cell.
  • Quantitative determination of Ca 2+ levels in the endoplasmic reticulum of cultured eukaryotic cells can be carried out using the bioluminescent calcium indicator aequorin, or the recombinant form apoaequorin, available from Molecular Probes, Eugene, OR, USA.
  • bioluminescent calcium indicator aequorin or the recombinant form apoaequorin, available from Molecular Probes, Eugene, OR, USA.
  • cultured cell lines may be transiently or stably transfected with an aequorin expression vector containing the aequorin structural gene.
  • cells Once cells have been transfected with aequorin, they are incubated in a medium containing the cell-permeant coelenterazine or one of its analogs that are available from Molecular Probes, Eugene, USA in order to reconstitute the aequorin complex. After formation of the active aequorin complex, intracellular Ca 2+ levels are measured by assaying cells for light production using a luminometer.
  • aequorin Screens based on the use of aequorin may be performed in multiwell plates.
  • the test compound can be added prior to the addition of the aequorin substrate, but can also be added in time intervals before or after the addition of the substrate.
  • Luminescence can be measured directly after the addition of the substrate, but preferentially luminescence measurements are performed over time ranges every 10 minutes for one hour after addition of the substrate. Luminescence data from typical experiments show that measurements after 10 to 15 minutes are sufficient to determine Ca 2+ levels in the endoplasmic reticulum.
  • Yet another method for measuring intracellular Ca 2+ levels is by use of green fluorescent based calcium indicator "cameleon". This method is described by Tsien et al, WO98/40477.
  • the cameleon calcium indicator can be transiently or stably expressed in mammalian, plant, insect or other pest cell lines and fluorescence ratio imaging of cameleon allows time-dependent measurements of intracellular calcium levels (Allen GJ et al . , 1999. Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J. , 19:735-47).
  • Cameleon fluorescence can be measured directly after the addition of the test compound or fluorescence measurements can be taken at various time intervals after addition of the test compound.
  • the cell culture assays may also be based on the use of cell death or apoptosis as an indicator of SERCA activity in the cell. Methods to determine cell death are well described by Barile Frank in Introduction to in vitro cytotoxicology: mechanisms and methods .1994. ISBN 0849386594. Most of the methods described therein can be performed using standard kits which are commercially available, for example from Molecular Probes or Boehringer Mannheim. Inhibition of SERCA activity leads to apoptosis which can easily measured using specific apoptotic labels as has been described by Smits et al. in WO 99/64586.
  • a variation on the cell culture assay may be based on measurement of calcium levels in isolated microsomes rather than intact cultured cells.
  • Techniques for isolation of microsomes are known to those skilled in the art. Isolated microsomes are placed in a solution containing radioactively labelled calcium and ATP. After 10 minutes incubation the amount of radioactivity inside the microsomes is measured in a beta-counter. This approach has been described by Dode, L. et al . , 1998. Structure of the human sarco/endoplasmic reticulum Ca 2+ -ATPase 3 gene. Promoter analysis and alternative splicing of the SERCA3 pre-mRNA. J. Biol. Chem. 273: 13982-13994. Once again the test compound can be added at several time intervals after the isolation of the microsomes, and prior or after the addition of the radioactive calcium.
  • the cell culture assays will preferably be carried out in multi-well plates of the type well known in the art for use in mid-to-high-throughput screening.
  • non-transfected host cells may also be exposed to the test compounds in order to control for expression of the endogenous host SERCA protein, i.e. to determine the selectivity of the assay for the pest SERCA protein.
  • the non-transfected control cells may also be used to assess general toxicity of the test compounds.
  • concentration of the candidate compound to be tested in the screening method may vary according to the nature of the compound and such factors as solubility etc.
  • An initial test may be performed using a single concentration of 10 ⁇ M.
  • Interesting compounds may then be re-tested to establish a dose-response curve, for example using concentrations of 300 ⁇ M, 10.0 ⁇ M, 30 ⁇ M, 10 ⁇ M, 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M, 0.01 ⁇ M and 0.003 ⁇ M and a zero concentration negative control.
  • concentrations between 300 ⁇ M and 0.001 ⁇ M is sufficient.
  • the above-described screening methods of the invention may all be used to identify compounds which have pesticidal activity because of their ability to down-regulate the activity of SERCA proteins, particularly SERCA proteins derived from pest species. Included within the category of ⁇ compounds which down-regulate SERCA activity' may be compounds which act directly on the SERCA protein, including SERCA inhibitors and antagonists.
  • the screens may also identify compounds which act indirectly to down-regulate SERCA activity, for example by affecting regulation of SERCA activity or expression of the SERCA protein.
  • the screens may also identify compounds that modulate the activity of other proteins in the SERCA pathway, such as proteins involved in the calcium homeostasis of the cell.
  • Test compounds may include compounds having a known pharmacological or biochemical activity, compounds having no such identified activity and completely new molecules or libraries of molecules such as might be generated by combinatorial chemistry.
  • Compounds which are DNA, RNA, PNA, polypeptides or proteins are not excluded.
  • Compounds identified as having pesticidal activity using the nematode-based assay, particularly the assays which do not involve a target pest SERCA protein may be re-tested in a cell culture assay, for example to assess toxicity of the compound or to assess the specificity of the compound for a pest SERCA protein.
  • the invention further provides compounds identified as having the potential to kill pests using the methods of the invention.
  • Such compounds are potential pesticides or can be considered as lead compounds for the development of novel pesticides, including insecticides, herbicides, nematocides and rodenticides .
  • compounds identified as having pesticidal activity against parasitic pest species using the screening methods described herein may have potential utility as anti-parasitic agents or as lead compounds in the development of anti-parasitic agents useful in the treatment of parasitic infections in humans and animals.
  • Figure 1 is an alignment of SERCA cDNA sequences from plant species, indicating consensus sequences and primer locations.
  • Figure 2 is a general alignment of SERCA cDNA sequences, indicating consensus sequences and primer locations.
  • Figure 3 shows the complete nucleotide sequence of a plasmid construct comprising the Arabidopsis SERCA cDNA in the vector pcDNA3.
  • Figure 4 shows the complete nucleotide sequence of a plasmid construct comprising the Heliothis SERCA cDNA in the vector pcDNA3.
  • Figure 5 shows the complete nucleotide sequence of a plasmid construct comprising the Heliothis
  • Figure 6 shows the complete nucleotide sequence of a plasmid construct comprising the Arabidopsis SERCA cDNA cloned in the vector pDW2600 (containing the sca-1 promoter) .
  • Figure 7 shows the complete nucleotide sequence of the plasmid pDW2700.
  • Figure 8 shows the complete nucleotide sequence of the plasmid pDW2800.
  • Figure 9 shows the complete nucleotide sequence of the plasmid pDW2400.
  • Figure 10 shows the complete nucleotide sequence of the plasmid pDW2422.
  • Figure 11 shows the complete nucleotide sequence of the plasmid pDW2721, comprising DNA encoding GFP cloned into pDW2700.
  • Figure 12 illustrates the nucleotide sequence of the genomic fragment of C. elegans SERCA bounded by primers SERCA P4 and SERCA P8. Exon IV and exon V are shown in capitals, intron IV in lower case. The fragment deleted in okl90 is underlined.
  • Figure 13 shows the nucleic acid sequence of a 732bp EcoRI-Hindll fragment of C. elegans SERCA exon 5. This fragment was cloned into pGEM3 for use in RNA inhibition experiments.
  • Figure 14 shows the nucleic acid sequence of a 11207bp Spel-Mlul fragment of cosmid K11D9. This fragment contains the complete C. elegans SERCA gene with 5631bp of upstream sequence, the entire coding region and 1088bp of downstream sequence. The fragment was cloned into pUCl8 to give plasmid pGKJ .
  • Figure 15 shows the nucleic acid sequence of a 5026 bp fragment of the upstream region of C. elegans SERCA, up to and including A of the initiating ATG.
  • Figure 16 shows the nucleic acid sequence of a 2915bp fragment of the upstream region of C. elegans SERCA, as found in plasmid pGK13.
  • Figure 17 shows the nucleic acid sequence of a 6612bp fragment of the C. elegans SERCA gene containing 5637bp of upstream sequence and ending in exon 4.
  • Figure 18 shows the nucleic acid sequence of the long isoform of the C. elegans SERCA cDNA.
  • Figure 19 shows the nucleic acid sequence of the C. elegans myo-2 promoter.
  • Figure 20 shows the nucleic acid sequence of the C. elegans myo-3 promoter.
  • Figure 21 shows the nucleic acid sequence of the C. elegans vulval muscle enhancer. This is an enhancer element from ceh-24 that directs gene expression in the vulval muscles (Harfe and Fire, 1998, Developmental 125: 421-429)
  • Figure 22 shows a dose-response curve for thapsigargin produced using a liquid culture assay.
  • Figure 23 shows a dose response curve for thapsigargin produced using a plate assay.
  • Manipulations of C. elegans worms may be performed using techniques described in Methods in
  • Vectors pDW2700 general cloning vector containing C. elegans myo-2 promoter Figure 7 .
  • pDW2400 general cloning vector containing C. elegans egl -15 promoter ( Figure 9) .
  • pDW2422 general cloning vector containing C. elegans ceh-24 promoter ( Figure 10) .
  • pDW2721 cloning vector comprising DNA encoding GFP cloned into in pDW2700.
  • Pest SERCA cDNA sequences are available in databases such as GenBank. Further sequences can be cloned using standard PCR technology. Pest SERCA cDNAs can be cloned into standard expression vectors to enable expression in C. elegans or in cultured mammalian cells. By way of example, the complete nucleotide sequences of plasmids that enable the expression of Heliothis insect SERCA and
  • Arabidopsis plant SERCA in C. elegans are shown in the accompanying Figures . These plasmids contain SERCA- encoding DNA cloned under the control of the C. elegans SERCA (sca-2) promoter. The complete nucleotide sequences of plasmid constructs comprising the
  • Heliothis SERCA cDNA and the Arabidopsis SERCA cDNA in the vector pcDNA3 are also shown.
  • Isolate full length cDNA sequence for example using 3' or 5 ' RACE or by hybridisation techniques, e.g. cDNA library screening, using labelled cDNA fragments as probes.
  • C. elegans the latter being a SERCA mutant such as okl90 or a wild-type strain where the endogenous SERCA is inhibited, for example by RNAi technology
  • RNAi RNAi
  • a fusion protein may be constructed that has sufficient properties of the C. elegans SERCA for rescue of the mutant phenotype, and has those pest SERCA properties sufficient in a screen to select for compounds that alter the pest SERCA activity.
  • At least four types of fusion proteins are contemplated: 1) A fusion protein harboring the - terminal end of the C. elegans SERCA and the C-terminal part of a pest SERCA .
  • Such fusion proteins can easily be constructed using standard molecular biology techniques.
  • the expression levels of SERCA in C. elegans can be specifically reduced by using antisense technology or double stranded RNA inhibition.
  • antisense technology to specifically reduce expression of a given protein is well known.
  • antisense RNA in the worm the non-coding strand of a fragment of the sca-2 gene can be expressed under the control of the sca-1 , myo-2 or myo-3 promoter or any other promoter.
  • the expression of the antisense SERCA RNA will result in the inhibition of expression of SERCA.
  • Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion or the mature protein sequence, which encodes for the SERCA protein is used to design an antisense RNA oligonucleotide of from 10 to 50 base pairs in length.
  • the antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the protein (Okano, J.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix - see Lee et al . Nucl. Acids Res., 6:3073 (1979); Cooney et al . , Science, 241:456 (1988); and Dervan et al . , Science,
  • an EcoRI-Hind III fragment of SERCA exon 5 was cloned antisense under the control of the myo-2 promoter, the myo-3 promoter, the SERCA promoter or the ceh-24 enhancer and injected into C. elegans. These vectors result in the expression of an antisense SERCA RNA, and hence in inhibition of SERCA activity.
  • the expression of a given gene in a cell can also be specifically reduced by introducing into the cell double stranded RNA corresponding to a region of the transcript transcribed from the gene.
  • Double stranded RNA can be prepared by cloning an appropriate fragment into a plasmid vector containing opposable promoters.
  • a suitable example is the pGEM® series of vectors from Promega Corporation, Madison, WI, USA, which contain opposable promoters separated by a multiple cloning site.
  • RNA will be transcribed from each of the promoters.
  • the vector contains two promoters oriented in the opposite sense, complementary sense and antisense transcripts will be transcribed which will combine to form double stranded RNA.
  • the injection of double stranded RNA in C. elegans has previously been described (Fire et al, Potent and Specific Genetic Interference by Double- Stranded RNA in C. elegans 1998, Nature 391, 860-811) .
  • the generated double stranded RNA was injected into C. elegans (see Fire at al., 1998, Nature 391:806-811). This resulted in the following phenotypes: 50% of the progeny of the injected animals were embryonic lethal, while the other 50% were early larval lethal. This indicates that SERCA function is vital for C. elegans .
  • inhibition of the expression of SERCA in all tissues results in embryonic or early larval lethality of the nematode. Inhibition of SERCA using RNAi feeding technology
  • RNAi methods which lead to more stable RNAi phenotypes exist and are described, for example in International patent application No. WO 00/01846. More particularly, an RNAi technology has been developed and tested in which dsRNA can be delivered by feeding the nematode dsRNA or by feeding nematodes with DNA.
  • pGN4 was constructed by cloning the Hindlll - EcoRI fragment of SERCA cloned in vector pGNl using these same restriction sites. This is the same fragment as was used for in vi tro transcription and dsRNA injection, described above.
  • HT115(DE3) bacteria (Fire A, Carnegie Institution, Baltimore, MD) were transfected with pGN4 (and controls with pGNl) and seeded on plates containing IPTG and ampicillin resulting in a high expression of dsRNA by the bacteria.
  • N2 and nuc-1 (el392) adult nematodes were put on these plates and allowed to lay eggs and the progeny was followed over time. The progeny mostly looked healthy during the larval stages, but the adults (and some of the L4) had a starved appearance (nuc-1 more pronounced then N2) . Pharynx pumping was irregular and slower then normal, and the growth rate was somewhat reduced.
  • RNAi phenotype useful in assay development and compound screening can be developed using feeding.
  • other possibilities and variants can be used to create a C. elegans SERCA RNAi phenotype.
  • RNAi technology allows the production of C. elegans strains in which the activity of the endogenous SERCA protein is abolished/substantially reduced without the construction of a C. elegans SERCA mutant.
  • E. coli HT115 has the following characteristics which make it a useful host cell for high level expression of dsRNA: HT115 (DE3) : F- mcrA mcrb I (rrnD-rrnE) 1 ⁇ - rncl 4 : : trl O (DE3 lysogen: lacUV5 promoter-T7 polymerase) ; host for IPTG inducible T7 polymerase expression; Rnaselll-.
  • the following strategy may be used to isolate a nematode that is mutated in the sca -1 gene, using standard selection procedures well known in the art.
  • a population of nematodes are mutagenized, preferentially using UV-TMP, and grown for two generations.
  • the mutagenized worms are distributed per 500 over approximately 1152 plates and grown for an additional two generations.
  • DNA is isolated from a fraction of the worms from each of these plates and used as a template for PCR selection to select for an sca-2 gene that has a deletion. From a plate with worms, of which some have been demonstrated to contain an sca-1 deletion, new plates are started with fewer worms. Further rounds of PCR selection finally result in the isolation of a heterozygote C. elegans carrying a mutation in the sca-1 gene (see Jansen et al., 1997, Nature Genetics 17:119-121).
  • C. elegans strain okl90 which is mutated in the sca-1 gene was kindly provided by R. Barstead (Oklahoma, USA) .
  • This strain can be purchased from the same supplier or from the C. elegans Genetic Center, Minnesota, USA (see above) .
  • Heterozygous animals show no defect, but their homozygous progeny die as Ll .
  • the lethal phenotype can be rescued by reintroduction of the C. elegans gene by injection of pGK7.
  • a nested PCR was performed on C. elegans genomic DNA using the following primer pairs:
  • the starting C. elegans strain is an sca-1 (okl90) /qCl heterozygotic strain.
  • the heterozygous strain is used as an okl90/qCl strain is viable, whilst both okl90/okl90 and qCl/qCl are lethal.
  • the qCl allele is a balancer, and is well known in the area of C. elegans genetics .
  • GFP marker cassette eg. pDW2721 (GFP) or rol-6.
  • GFP marker cassette the myo-2 promoter is chosen to prevent interference with the read out in the pharynx pumping assay. Using this promoter GFP is only expressed in the pharynx.
  • the pest SERCA and marker cassettes are transformed into the worm using standard C. elegans techniques.
  • the starting C. elegans strain may be wild-type C. elegans (N2 strain) or a selected mutant strain.
  • SERCA/C. elegans SERCA chimera which is resistant to inhibition by thapsigargin under the control of the C. elegans SERCA (sca-2) promoter.
  • the screen is performed in the presence of a lethal dose of thapsigargin.
  • the strain In the presence of thapsigargin the strain exhibits substantially wild-type pharynx pumping, movement, egg laying, defecation, mating etc. These characteristics can therefore be used as indicators of SERCA activity to perform screens on the pest SERCA target, based on detection of changes in these phenotypes.
  • the starting C. elegans strain may be wild-type
  • C. elegans N2 strain
  • a selected mutant strain N2 strain
  • the strain exhibits ectopic expression of a pest SERCA protein or pest SERCA/C. elegans SERCA chimera in one or more neurons of C. elegans .
  • the phenotype is evaluated and a characteristic selected to form the basis of a screen.
  • Example 5 Inhibition of endogenous C. elegans SERCA by compounds :
  • the optimal concentration of compounds that inhibit the activity SERCA has been determined.
  • the first assay is designated the drop or plate assay in which the nematodes are fed E. coli strains pre-loaded with the compound.
  • the compound is administrated to the worm in liquid culture.
  • a standard plate drop assay is performed according to the following protocol. 4ml NGM agar
  • test compound dissolved in DMSO or other suitable solvent is pipetted onto the bacterial lawn so that the lawn is covered completely.
  • one C. elegans (L4 stage) per plate is put onto the bacterial lawn. Plates are incubated at 21°C and checked after some hours. Plates are checked again after 4 days for phenotypes of the FI progeny (control shows all stages up to gravid hermaphrodites) .
  • Thapsigargin at various concentrations causes the nematode to stop pharynx pumping within 10 min.
  • the worms restart pumping, although at a low level.
  • the worms are pale and thin and have a slow and irregular movement, with an increased amplitude. No plate drop response is observed, and the worms show poor backing, reduced pumping and strong constipation.
  • the worms have a defective gonad with only very few eggs, and a protruding vulva. Some worms also have a protruding rectum. Progeny reaches L2 stage only after four days, and the brood size is very small. Lower concentrations of thapsigargin (0.5 ⁇ M, 0.25 ⁇ M, 0.125 ⁇ M) still cause reduced brood size.
  • 2,5-di-tert butylhydroquinone at a concentration of 500 ⁇ M resulted in pale, starved, thin worms with slow movement, defective gonad, constipated and reduced brood size.
  • Cyclopiazonic acid at a concentration of 500 ⁇ M resulted in nematodes that lay still or move slowly after one hour.
  • the worms showed strong avoidance and after 24 hours they look starved, pale and thin, with only a few eggs in the body, a defective gonad, and reduced brood size. A delayed growth of the FI generation was observed.
  • Thapsigargin-epoxide did not result in a clear observable effect, even at the highest concentration tested (1 mM drop, 5 ⁇ M end concentration) .
  • Liquid culture assay Thapsigargin at 100, 50 and 20 ⁇ M resulted in small worms which show slow and loopy movement. They had a protruding vulva, and no progeny (or no progeny that grows up) were observed. At lower concentrations of 10 ⁇ M and 5 ⁇ M a reduced number of progeny and delayed growth could be observed.
  • 2,5-di-tert butylhydroquinone at a concentration of ImM resulted in progeny exhibiting delayed growth and the worms were observed to be thinner than ⁇ normal' worms.
  • Cyclopiazonic acid at a concentration of ImM resulted in pale, thin worms with a slow movement and a very strongly reduced brood size. At lower concentrations of 0.5mM, growth delay was observed.
  • synchronized worms are used.
  • the production of large amounts of synchronized worms has been described in (Methods in cell biology, Vol. 48, ibid) .
  • an assay buffer 40mM NaCl, 6mM Kcl, ImM CaCl 2 , ImM MgCl 2
  • worms are then diluted and resuspended in semi-soft agar (final concentration of 0.25% low melting agarose in M9 buffer) .
  • This procedure results in an equal, homogenous and stabilised suspension of the nematodes.
  • Other polymers than low melting agarose can be used in this procedure.
  • the presence of a homogenous worm suspension facilitates the equal distribution of the worms in the multi-well plates, but is not essential. Any other method that results in a homogenous distribution of the nematodes worms over the wells will be useful. More specifically, the use of a worm dispenser will result in even a better, and hence a more equal distribution of the worms over the wells of the multi-well plate.
  • the worms are distributed in the multi-well plates using electronic 8 channel pipettes.
  • 40 +/- 5 worms are added to every well of the microtiter plate.
  • DMSO methyl methacrylate
  • Any other solvent can be used for this purpose, but most selected compounds appear to be soluble in DMSO.
  • the compounds are added in the wells at various concentrations.
  • the concentration of the DMSO should not be too high and preferentially should not exceed 1%, more preferentially the concentration of the DMSO should not exceed 0.5% and even more preferentially, the concentration of the DMSO is lower than 0.3%.
  • C. elegans strains can be used. Screens to select for compounds inhibiting the pumping rate of the C. elegans pharynx are preferably performed with mutant C. elegans strains which have a constitutively pumping pharynx. Wild-type worms can also be used in this screen, but the mutants worms are preferred. Other C. elegans mutants can be used in this screen to select for inhibitors of pumping. The selected mutant C. elegans with the constitutively pumping pharynx pumps medium into the gut at a constant rate and reduction/rescue of this phenotype can easily be scored, which facilitates the detection and selection of compounds.
  • the pumping rate of the pharynx is measured indirectly by adding a marker molecule precursor such as calcein-AM to the medium and measuring the formation of marker dye in the C. elegans gut.
  • Calcein-AM is cleaved by esterases present in the C. elegans gut to release calcein, which is a fluorescent molecule.
  • the pumping rate of the pharynx will determine how much medium will enter the gut of the worm, and hence how much calcein-AM will enter the gut of the worm. Therefore by measuring the accumulation of calcein in the nematode gut, detectable by fluorescence, it is possible to determine the pumping rate of the pharynx.
  • a concentration of between 1 and lOO M calcein-AM is added into the medium.
  • Preferably 5 to lO ⁇ M calcein-AM is used.
  • This measurement of the pharynx pumping rate by detecting the accumulation of a marker molecule is not limited to calcein-AM.
  • Other precursors can be used and thus the assay as described here can be changed to be suitable for other precursors.
  • the precursor can be cleaved by esterases, but could also be a substrate for other enzymes in the nematode gut.
  • the marker molecule should not necessary be a fluorescent molecule, but can be a molecule detectable by other methods. Most of these precursor substances are commercially available or could be synthesized according to methods known in the art. Some examples are:
  • FDP Fluorescein diphospate
  • DNAses Other target enzymes present in the gut for which substrates can be found or developed are DNAses,
  • COS cells have been transfected with erAEQ/pCDNAi provided by Molecular probes, to investigate the influence of calcium modulation of thapsigargin in these cells.
  • Transfection was performed using the Lipofectamine Plus reagent (Life technloglogies, Inc) according to the standard protocol supplied by the manufacturer.
  • Cell lysis was performed as described in "The Molecular Sampler Kit” provided by Molecular Probes, to determine the best substrate.
  • coelentazine hep is the best substrate (data not shown) . For other cells the most suitable substrate would need to be determined by experiment.
  • Tests were repeated in multiwell plates, without cell lysis.
  • the transfected cells were treated with thapsigargin and aequorin fluorescence was measured directly. A clear variation was observed between cells treated with thapsigargin and cells that have not been treated. Measurements in a high throughput format can be made from 5 minutes to at least 45 minutes after contacting the cells with the appropriate test compound.
  • Aspergillus niger AAF37300.
  • Bacillus halodurans BAB06234.
  • Bacillus subtilis 034431.
  • Bos taurus AAF64433.
  • Candida albicans CAB87245.
  • Gallus gallus Q9YGL9 ,ATC1_CHICK, B40812.
  • Homo sapiens 060900 ,ATC1_HUMAN.
  • Leishmania mexicana 009489. Lycopersicon esculentum: Q42883.
  • Makaira nigricans ATC1_MAKNI .
  • thermoautotrophicum 027560
  • Mus musculus Q64517 ,ATC2_M0USE.
  • Mycobacterium tuberculosis CTPF_MYCTU.
  • Neurospora crassa Q9UUY0.
  • Oryctolagus cuniculus ATC2_RABIT.
  • Oryza sativa BAA90510 ,004938.
  • Patinopecten yessoensis 096039. Placopecten magellanicus : 077070.
  • Plasmodium falciparum ATC_PLAFK.
  • Schistosoma mansoni 096527 , 0.21119.
  • Synechocystis sp. Q59999.
  • Trichomonas vaginalis Q95060.
  • Trypanosoma brucei ATC_TRYBB.
  • Ureaplasma urealyticu AE002123_6.
  • Zea mays AAF73985.
  • Rhizoglyphus callae R robini Acari: Acaridae Rhizoglyphus
  • Tribolium castaneum Coleoptera Tribolium

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention traite de procédés permettant d'identifier des composés présentant une utilité potentielle comme pesticides. En particulier, l'invention traite de procédés permettant d'identifier des composés qui affectent l'activité d'une pompe à calcium physiologiquement importante, le réticulum endoplasmique/sarcoplasmique Ca2+ ATPase (SERCA).
PCT/IB2001/002391 2000-10-18 2001-10-15 Procedes d'identification de composes pesticides WO2002033405A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002218457A AU2002218457A1 (en) 2000-10-18 2001-10-15 Methods for identifying pesticidal compounds

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US24150200P 2000-10-18 2000-10-18
US60/241,502 2000-10-18
GB0025576A GB0025576D0 (en) 2000-10-18 2000-10-18 Methods for identifying pesticidal compounds
GB0025576.0 2000-10-18

Publications (1)

Publication Number Publication Date
WO2002033405A1 true WO2002033405A1 (fr) 2002-04-25

Family

ID=26245175

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2001/002391 WO2002033405A1 (fr) 2000-10-18 2001-10-15 Procedes d'identification de composes pesticides

Country Status (2)

Country Link
AU (1) AU2002218457A1 (fr)
WO (1) WO2002033405A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008125378A (ja) * 2006-11-17 2008-06-05 Sumitomo Chemical Co Ltd 昆虫由来のベシクルフュージングａｔｐアーゼ活性に関わる有害生物の生理状態に変化を与える薬剤
WO2008095969A1 (fr) * 2007-02-09 2008-08-14 Basf Plant Science Gmbh Compositions et procédés d'utilisation de l'interférence d'arn de gènes analogues à sca1 pour la lutte contre les nématodes
CN102565015A (zh) * 2011-12-24 2012-07-11 浙江大学 一种快速定量测定水样中磺酰脲类除草剂残留的方法
US8759611B2 (en) 2005-09-16 2014-06-24 Monsanto Technology Llc Methods for genetic control of insect infestation in plants and compositions thereof
US8946510B2 (en) 2004-04-09 2015-02-03 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009096A2 (fr) * 1989-02-03 1990-08-23 Cambridge Neuroscience Research, Inc. Procede de tirage et de classification de composes
WO2000034438A2 (fr) * 1998-12-07 2000-06-15 Devgen Nv Methode de construction de bibliotheques de profils phenotypiques
GB2349217A (en) * 1999-04-15 2000-10-25 Devgen Nv Screening methods for compounds affecting SERCA activity

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009096A2 (fr) * 1989-02-03 1990-08-23 Cambridge Neuroscience Research, Inc. Procede de tirage et de classification de composes
WO2000034438A2 (fr) * 1998-12-07 2000-06-15 Devgen Nv Methode de construction de bibliotheques de profils phenotypiques
GB2349217A (en) * 1999-04-15 2000-10-25 Devgen Nv Screening methods for compounds affecting SERCA activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MAHANEY JAMES ET AL: "Phospholamban reduces cardiac Ca-ATPase sensitivity to thapsigargin and cyclopiazonic acid.", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol. 372, no. 2, 15 December 1999 (1999-12-15), pages 408 - 413, XP001055979, ISSN: 0003-9861 *
PERIZ, G. AND FORTINI, M.E.: "Ca2+-ATPase function is required for intracellular trafficking of the Notch receptor in Drosophila", EMBO, vol. 18, no. 21, 1999, pages 5983 - 5993, XP001061694 *
WAGGONER JASON R ET AL: "Fluorescence studies of the cardiac Ca-ATPase expressed in insect cells: Effect of phospholamban on Ca-ATPase conformational states.", BIOPHYSICAL JOURNAL, vol. 80, no. 1 Part 2, January 2001 (2001-01-01), 45th Annual Meeting of the Biophysical Society;Boston, Massachusetts, USA; February 17-21, 2001, pages 432a - 433a, XP001057572, ISSN: 0006-3495 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9340797B2 (en) 2004-04-09 2016-05-17 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US11685930B2 (en) 2004-04-09 2023-06-27 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
US11492638B2 (en) 2004-04-09 2022-11-08 Monsanto Technology, Llc Compositions and methods for control of insect infestations in plants
US10787680B2 (en) 2004-04-09 2020-09-29 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US10167484B2 (en) 2004-04-09 2019-01-01 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US8946510B2 (en) 2004-04-09 2015-02-03 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US9238822B2 (en) 2004-04-09 2016-01-19 Monsanto Technology Llc Compositions and methods for control of insect infestations in plants
US9695439B2 (en) 2005-09-16 2017-07-04 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US8759611B2 (en) 2005-09-16 2014-06-24 Monsanto Technology Llc Methods for genetic control of insect infestation in plants and compositions thereof
US10538783B2 (en) 2005-09-16 2020-01-21 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US11312975B2 (en) 2005-09-16 2022-04-26 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
US11939589B2 (en) 2005-09-16 2024-03-26 Monsanto Technology Llc Methods for genetic control of insect infestations in plants and compositions thereof
JP2008125378A (ja) * 2006-11-17 2008-06-05 Sumitomo Chemical Co Ltd 昆虫由来のベシクルフュージングａｔｐアーゼ活性に関わる有害生物の生理状態に変化を与える薬剤
EP2336154A3 (fr) * 2006-11-17 2011-08-31 Sumitomo Chemical Company, Limited Agent modulant l'état physiologique d'organismes nuisibles et jouant un rôle dans l'activité de l'atpase de fusion de vésicules d'insectes
WO2008095969A1 (fr) * 2007-02-09 2008-08-14 Basf Plant Science Gmbh Compositions et procédés d'utilisation de l'interférence d'arn de gènes analogues à sca1 pour la lutte contre les nématodes
CN102565015A (zh) * 2011-12-24 2012-07-11 浙江大学 一种快速定量测定水样中磺酰脲类除草剂残留的方法

Also Published As

Publication number Publication date
AU2002218457A1 (en) 2002-04-29

Similar Documents

Publication Publication Date Title
Perry et al. The biology of insecticidal activity and resistance
DE60034059T2 (de) Zielgerichtete Verfahren des Wirkstoff-Screenings unter Verwendung von Co-Kultivierungsverfahren
Boivin et al. Constraints on adaptive mutations in the codling moth Cydia pomonella (L.): measuring fitness trade-offs and natural selection
EP2067787A1 (fr) Procédé pour le contrôle de populations d'insectes
Jindra et al. A decade with the juvenile hormone receptor
AU780574B2 (en) Compound screening method
Mottet et al. Assessment of the dominance level of the R81T target resistance to two neonicotinoid insecticides in Myzus persicae (Hemiptera: Aphididae)
Denecke et al. The wiggle index: An open source bioassay to assess sub-lethal insecticide response in Drosophila melanogaster
US6664441B1 (en) Compound assay
Chen et al. Transgenic overexpression of P450 genes confers deltamethrin resistance in the fall armyworm, Spodoptera frugiperda
Heinick et al. Caenorhabditis elegans P5B-type ATPase CATP-5 operates in polyamine transport and is crucial for norspermidine-mediated suppression of RNA interference
Cioci et al. Transgenic strains of the nematode Caenorhabditis elegans as biomonitors of metal contamination
Wang et al. CRISPR/Cas9 mediates efficient site‐specific mutagenesis of the odorant receptor co‐receptor (Orco) in the malaria vector Anopheles sinensis
WO2002033405A1 (fr) Procedes d'identification de composes pesticides
Guest et al. Studies of an insecticidal inhibitor of acetyl-CoA carboxylase in the nematode C. elegans
US20030154501A1 (en) Compound screening method
US6787125B1 (en) Compound screening method
EP1171628A2 (fr) Procedes de criblage de composes
GB2359627A (en) Screening etc. methods using nematodes
Banfield et al. Protein-repair and hormone-signaling pathways specify dauer and adult longevity and dauer development in Caenorhabditis elegans
US20040250299A1 (en) Compound screening method
Markovic et al. Drosophila lamin mutations cause melanotic mass formation and lamellocyte differentiation
US20050229265A1 (en) Insect ammunition vectors and methods of use to identify pesticide targets
Holbrook-Smith Chemical Genetic Interrogation of Strigolactone Receptors in Arabidopsis thaliana and Striga hermonthica
Peng et al. Reverse genetic study reveals the molecular targets of chordotonal organ TRPV channel modulators

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP