EP1212620A1 - Genes essentiels et recherches relatives a ceux-ci - Google Patents

Genes essentiels et recherches relatives a ceux-ci

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Publication number
EP1212620A1
EP1212620A1 EP00958829A EP00958829A EP1212620A1 EP 1212620 A1 EP1212620 A1 EP 1212620A1 EP 00958829 A EP00958829 A EP 00958829A EP 00958829 A EP00958829 A EP 00958829A EP 1212620 A1 EP1212620 A1 EP 1212620A1
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EP
European Patent Office
Prior art keywords
est
protein
geno
gene
activity
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Legal status (The legal status 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 status listed.)
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EP00958829A
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German (de)
English (en)
Inventor
Roger Wayne University of Glasgow DAVIES
Kim Uni. of Glasgow IBLS Div. ofMol. Gen. KAISER
Ming Yao University of Cambridge YANG
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University of Glasgow
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University of Glasgow
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates in part to target based screening assays, particularly for pesticides, based on the identification and use of essential genes/proteins, as well as novel genes/proteins themselves and compounds identified by such assays which may have a modulatory effect on the protein. It also relates to screening assays for compounds with therapeutic use in cancer therapy or other proliferative diseases.
  • Pest destruction by organisms such as insects results in a considerable economic loss and serious reduction in productivity.
  • Chemical pesticides are typically used in order to control the pests and reduce crop loss.
  • pesticide development has generally been less than controlled or focussed, such that the biochemical or genetical functions of the pesticide have not been a major concern, but rather simply whether or not the pesticide was effective ie. killed the pests.
  • the present inventors have used genetic techniques in order to study a model "pest", namely Dro ⁇ ophila . As a result of these studies, the present inventors have identified a considerable number of essential genes/proteins, which may be used in assays based on the functional activity of the protein and/or ligand binding assays for screening for modulators of protein activity which have potential use for example as pesticides.
  • the present invention provides a screening assay for identifying compounds which have a physiological or biochemical effect on an organism, the assay comprising the steps of: a) reacting a test compound with a protein encoded by an essential gene comprising a sequence selected from the group consisting of SEQ ID Nos. 1-902, specific fragment thereof, or homologue thereof, from the organism; and b) detecting any modulatory effect the compound has on the protein.
  • a modulatory effect is one which alters the function and/or activity of the protein.
  • negative modulation refers to a reduction in the level and/or activity of an essential gene product (eg. polypeptide) relative to the level and/or activity of the essential gene product in absence of the modulatory treatment.
  • the reduction in the level and/or activity may be taken to be for example less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the level and/or activity of the essential gene product in the absence of the modulatory treatment.
  • Positive modulation refers to an increase in the level and/or activity of the essential gene product, relative to the level and/or activity of the gene product in the absence of the modulatory treatment. For example greater than 150%, 200%, 300%, 400% increase with respect to the relative level in the absence of the modulatory treatment.
  • the assay is designed to be used to screen for compounds which effect the physiology or biochemistry in a manner which is harmful or biocidal eg. pesticidal, to the organism.
  • the assay may be used to screen for other effects, such as beneficial or therapeutic effects.
  • the assay may be used to screen for compounds which are effective against cancer or other proliferative diseases. In this manner it may be possible to identify proteins which are negatively or positively modulated in a patient suffering from for example cancer. Consequently the assay would be used to identify compounds which may reduce or substantially eliminate such negative or positive modulation.
  • a lethal phenotype is defined as a phenotype characterised by organism death due to cellular or system failure at some developmental stage.
  • a semi-lethal phenotype is for example characterised by low fecundity (the tendency to produce none or only few offspring, eg. less than 50%, 40%, 30%, 20%, 10%, 5% or 1% compared to wildtype) , and frequently by short lifespan, or a behavioural modification which leads to reduced ability to generate offspring.
  • fly lines in the model organism, Drosophila , which display a lethal or semi-lethal phenotype.
  • Fly lines which display such a lethal or semi- lethal phenotype have been generated using the technique of P-element transposon-tagged insertion (1) Torok, T,G. Tick, M. Alvarado and I. Kiss, 1993 P-lacW Insertional Mutagenesis on the second chromosome of Drosophila melanogaster: Isolation of lethals with different overgrowth phenotypes. Genetics 135: 71 - 80; (2) Deak, P., M.M. Omar, R.D.C. Saunders, M. Pal, O. Komonyi, J.
  • SEQ ID Nos. 1-902. The partial sequences of regions surrounding the P-element insertion site from distinct fly lines are identified herein as SEQ ID Nos. 1-902. It is immediately evident to one skilled in the art how to use this information to clone a larger portion of nucleic acid containing the complete gene and thereafter express the encoded protein. Such techniques are disclosed for example in Sambrook et al (1989) . It will be appreciated that the P-element may be inserted within a particular gene, or in regulatory sequences associated with a gene, such that P- element insertion affects expression of the gene, resulting in the lack of the expressed protein or expression of a dysfunctional form (ie.
  • essential gene refers to the gene sequence itself as well as associated regulatory sequences which may be necessary for functional protein expression.
  • each of the genes thus identified are shown to be potentially essential for cell or organism survival and/or reproduction based on the evidence of the effect of a complete loss of gene function caused by a mutation.
  • Those skilled in the art will use a series of confirmatory steps to demonstrate that the transposon insertion site from which the identifiable gene sequence derives is indeed the site associated with lethality. Such confirmatory steps may comprise: repeated crossing to a Wild-type strain (eg.
  • the present invention provides a polynucleotide fragment comprising nucleotides capable of encoding or partially encoding an essential gene for use in assays of the present invention.
  • the nucleotide fragment may be selected from any of the sequences identified by SEQ. ID. Nos. 1-902, or fragment thereof as described herein or other species homologue.
  • the present invention provides means for obtaining essential proteins as encoded by the essential genes defined herein for use in assays of the present invention.
  • Polynucleotide fragment refers to a chain of nucleotides such as deoxyribose nucleic acid (DNA) and transcription products thereof, such as RNA. Naturally, the skilled addressee will appreciate the whole naturally occurring Drosophila or other "pest" species genome is not included in the definition of polynucleotide fragment.
  • DNA deoxyribose nucleic acid
  • RNA transcription products thereof
  • polynucleotide fragment can be isolated in the sense that it is substantially free of biological material with which the whole genome is normally associated in vivo .
  • the isolated polynucleotide fragment may be cloned to provide a recombinant molecule comprising the polynucleotide fragment.
  • polynucleotide fragment includes double and single stranded DNA, RNA and polynucleotide sequences derived therefrom, for example, subsequences of said fragment and which are of any desirable length. Where a nucleic acid is single stranded then both a given strand and a sequence or reverse complementary thereto is within the scope of the present invention.
  • the term "expression product” refers to both transcription and translation products of said polynucleotide fragments.
  • the expression product is a "polypeptide” (i.e. a chain or sequence of amino acids displaying a biological activity substantially similar (eg. 98%, 95%, 90%, 80%, 75% activity) to the biological activity of an essential protein) , it does not refer to a specific length of the product as such.
  • polypeptide encompasses inter alia peptides, polypeptides and proteins.
  • the polypeptide if required, can be modified in vivo and in vitro, for example by glycosylation, amidation, carboxylation, phosphorylation and/or post-translational cleavage.
  • the present invention further provides an isolated polynucleotide fragment capable of specifically hybridising to a related polynucleotide sequence from another species.
  • the present invention provides probes and/or primers for use in ex vivo and/or in situ detection and expression studies.
  • Typical detection studies include polymerase chain reaction (PCR) studies, hybridisation studies, or sequencing studies.
  • PCR polymerase chain reaction
  • any specific polynucleotide sequence fragment from the identified sequences may be used in detection and/or expression studies.
  • a specific fragment is a fragment of the sequence which is of sufficient length, generally greater than 10, 12, 14, 16 or 20 nucleotides in length, to bind specifically to the sequence, under conditions of high stringency, as defined herein, and not bind to unrelated sequences, that is sequences from elsewhere in the genome of the organism other than an allelic form of the sequence or non- homologous sequences from other organisms.
  • Capable of specifically hybridising is taken to mean that said polynucleotide fragment preferably hybridises to a related or similar polynucleotide sequence in preference to unrelated or dissimilar polynucleotide sequences.
  • the invention includes polynucleotide sequence (s) which are capable of specifically hybridising to an essential polynucleotide sequence or to a part thereof without necessarily being completely complementary or reverse complementary to said related polynucleotide sequence or fragment thereof. For example, there may be at least 50%, or at least 75%, at least 90%, or at least 95% complementarity. Of course, in some cases the sequences may be exactly reverse complementary (100% reverse complementary) or nearly so (e.g. there may be less than 10, typically less than 5 mismatches) . Thus, the present invention also provides anti-sense or complementary nucleotide sequence(s) which is/are capable of specifically hybridising to the disclosed polynucleotide sequence.
  • the polynucleotide must be capable of hybridising to related nucleic acid and capable of initiating chain extension from 3 ' end of the polynucleotide, but not able to correctly initiate chain extension from unrelated sequences.
  • a polynucleotide sequence of the present invention is to be used in hybridisation studies to obtain a related sequence from another organism the polynucleotide sequence should preferably remain hybridised to a sample polynucleotide under stringent conditions.
  • the test or sample polynucleotide may be immobilised.
  • the test polynucleotide sequence is at least 10, 14, 20 or at least 50 bases in length. It may be labelled by suitable techniques known in the art.
  • the test polynucleotide sequence is at least 200 bases in length and may even be several kilobases in length.
  • a denatured sample or test sequence can be first bound to a support.
  • Hybridization can be effected at a temperature of between 50 and 70°C in double strength SSC (2xNaCl 17.5g/l and sodium citrate (SC) at 8.8g/l) buffered saline containing 0.1% sodium dodecyl sulphate (SDS) .
  • SSC double strength SSC
  • SC sodium citrate
  • SDS sodium dodecyl sulphate
  • This can be followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration.
  • reduced concentration buffers are typically single strength SSC containing 0.1%SDS, half strength SSC containing 0.1%SDS and one tenth strength SSC containing 0.1%SDS.
  • Sequences having the greatest degree of similarity are those the hybridisation of which is least affected by washing in buffers of reduced concentration. It is most preferred that the sample and inventive sequences are so similar that the hybridisation between them is substantially unaffected by washing or incubation in standard sodium citrate (0.1 x SSC) buffer containing 0.1%SDS.
  • Oligonucleotides may be designed to specifically hybridise to essential nucleic acid. They may be synthesised, by known techniques and used as primers in PCR or sequencing reactions or as probes in hybridisations designed to detect the presence of related material in a sample.
  • the oligonucleotides may be labelled by suitable labels known in the art, such as, radioactive labels, chemiluminescent labels or fluorescent labels and the like.
  • suitable labels known in the art, such as, radioactive labels, chemiluminescent labels or fluorescent labels and the like.
  • the present invention also provides oligonucleotide probes and primers for use in detecting essential genes from other organisms and which may be used in screening assays for pesticides.
  • oligonucleotide is not meant to indicate any particular length of sequence and encompasses nucleotides of preferably at least 10b (e.g. 10b to lkb) in length, more preferably 12b-500b in length and most preferably 15b to 100b.
  • the oligonucleotides may be designed with respect to any of the sequences shown in SEQ ID Nos. 1-902 and may be manufactured according to known techniques. They may have substantial sequence identity (e.g. at least 50%, at least 75%, at least 90% or at least 95% sequence identity) with one of the strands shown therein or an RNA equivalent, or with a part of such a strand. Preferably such a part is at least 10, at least 30, at least 50 or at least 200 bases long. It may be an open reading frame (ORF) or a part thereof.
  • ORF open reading frame
  • Oligonucleotides which are generally greater than 30 bases in length should preferably remain hybridised to a sample polynucleotide under one or more of the stringent conditions mentioned above. Oligonucleotides which are generally less than 30 bases in length should also preferably remain hybridised to a sample polynucleotide but under different conditions of high stringency.
  • the melting temperature of an oligonucleotide less than 30 bases may be calculated according to the formula of; 2°C for every A or T, plus 4 ⁇ C for every G or C, minus 5°C. Hybridization may take place at or around the calculated melting temperature for any particular oligonucleotide, in 6 x SSC and 1% SDS.
  • Non specifically hybridised oligonucleotides may then be removed by stringent washing, for example in 3 x SSC and 0.1% SDS at the same temperature. Only substantially similar matched sequences remain hybridised i.e. said oligonucleotide and corresponding test nucleic acid.
  • the melting temperature may be calculated in the same manner as described above.
  • the oligonucleotide may then be allowed to anneal or hybridise at a temperature around the oligonucleotides calculated melting temperature.
  • the annealing temperature should be around the lower of the calculated melting temperatures for the two priming oligonucleotides. It is to be appreciated that the conditions and melting temperature calculations are provided by way of example only and are not intended to be limiting. It is possible through the experience of the experimenter to vary the conditions of hybridisation and thus anneal/hybridise oligonucleotides at temperatures above their calculated melting temperature. Indeed this can be desirable in preventing so-called non-specific hybridisation from occurring.
  • the partial sequences of the essential genes have been analysed in order to ascertain if there is any homology to previously sequences contained in nucleotide sequence databases such as the GENBANK and EMBL databases.
  • Database searching has ascertained that a number of the nucleotide sequences show homology to sequences deposited in such databases. Some sequences show homology to sequences to which have been ascribed a function. However, the function of the gene/protein associated with the sequence may not have been suggested to be an essential gene/protein, the modulation of which may result in a lethal/semi-lethal phenotype. Other sequences show homology only to sequences for which no putative function has been ascribed.
  • sequences appear to show little or non-significant homology to sequences deposited in databases at the time of filing the priority application. Nevertheless, because of sequencing projects, such as the human and Drosophila genome sequencing projects many sequences have been deposited in such databases in the priority year. Thus, sequences which displayed little or no homology to sequences in the databases on filing the priority application, may now show homology to sequences. However, the majority of such newly deposited sequences have no function ascribed to the sequence, or for that matter are known to relate or be associated with essential gene sequence.
  • Tables 1 and 2 summarise details of the sequences identified by the present invention. Those sequences which display homology to previously identified Drosophila gene sequences are identified under the class heading as GNL. Sequences which show a match with expressed sequence tags (ESTs) are represented under the class heading as EST. Sequences which show homology to genomic sequences are shoen under the class heading as GENO.
  • IDNumber is not of relevance to the present application; "Chr” relates to the chromosome from which the sequence was obtained; "feature” relates to the portion of sequence showing a match with the identified feature shown under “Name of match; and "AccNo” relates to the database accession number of the matching sequence.
  • Table 1 also refers to sequences identified by the present inventors (ie. SEQ ID Nos. 430-783 and 899-902) which do not show a clear match or homology to sequences in the database.
  • Table 2 is essentially Table 1, updated, once information, such as the Drosophila genome sequencing project information had been submitted to a database. Most of the sequences which did not show a match or homology to sequences in the database, as shown in Table 1, as at the priority date, are shown in Table 2 as having homology to Drosphila sequences since this information became available during the priority year.
  • genes with exact or a high degree of homology to known Drosophila Expressed Sequence Tags which provides evidence that they correspond to unknown genes that are expressed as messenger RNA.
  • Other genes in this class have strong homology to ESTs from other organisms. All the remaining sequences are recognised as Drosophila genes on the basis of the genomic sequence.
  • genes may be grouped on the basis of predicted related functions. This may be summarised as shown below, where the numbers correspond to SEQ ID Nos. disclosed herein:
  • GENES ENCODING NTPases including ATPases and GTPases SEQ ID Nos. 11, 21, 24, 31, 40, 117, 227, 296, 356, 515 and 803.
  • Assays for protein activities of known function are known in the art. Generally such assays are termed functional assays and may be conducted in vitro in a cell free or cell based system. A list of typical assays for some of the major classes of protein that are estimated to represent likely targets is exemplified herein. Where a functional assay is available, it is to be preferred to a ligand binding assay.
  • the typical purpose of the assays described herein is to select for pesticides/insecticides, though in some cases lead compounds may have therapeutic activity, such as in inducing cell death which may be applicable in cancer therapy and other proliferative diseases.
  • a relative specificity of action based on species groups or species may be achieved based on differences in protein sequence and structure, differences in protein expression, variations in development role and/or variations in degree of redundancy with related proteins.
  • the information disclosed herein teaches that the loss of the function of any of the proteins encoded by the genes comprising the partial sequences identified by SEQ ID Nos. 1-902, causes death of insects at some point during development, or causes severe physiological effects or reproductive failure.
  • An insecticidal chemical compound will therefore be a compound that strongly modulates, either agonistically or antagonistically, the activity of such a protein.
  • chemicals will be sought that interfere with the protein to modulate activity of the protein.
  • each member of the set of chemicals may then be tested directly for killing activity on insects.
  • Drosophila itself is a convenient assay insect.
  • a typical fly killing assay young flies are kept without fluid for a time, then transferred to vials containing filter paper dosed with a solution of the chemical to be tested.
  • a range of chemical concentrations eg. 10 "2 - 10 " 10 M) may be used.
  • flies are returned to normal conditions and observed. Rate of killing and percentage lethality are the parameters assessed.
  • compounds with very effective killing activity on Drosophila may then be tested on pest species or accepted model pest related insects.
  • pests include Dictyoptera (cockroaches) ; Isoptera (termites) ; Orthoptera (locusts, grasshoppers and crickets) ; Diptera (house flies, mosquito, tsetse fly, crane-flies and fruit flies) ; Hymenoptera (ants, wasps, bees, saw-flies, ichneumon flies and gall-wasps) ; Anoplura (biting and sucking lice) ; Siphonaptera (fleas) ; and Hemiptera (bugs and aphids) , as well as arachnids such as Acari (ticks and mites) .
  • aphid species may be maintained on isolated lettuce plants: the time of death and the numbers of aphids falling dead onto paper traps beneath the plants after spraying with defined doses of the candidate chemical is assessed.
  • lepidopteran pest larvae may be maintained on artificial media or plant leaves, which are treated with defined doses of chemicals, and survival is assessed.
  • candidate chemicals for use in the present invention are well known to those skilled in the art.
  • libraries of compounds can be easily synthesised and tested. This is well described for example in: Applications of combinatorial technologies to drug discovery, 2. Combinatorial organic synthesis, library screening techniques, and future direction, J. Med. Chem. ,1994,37,1385-1401.
  • the ligand binding assays outlined herein will also define a group of candidate chemicals. However, this group is likely to be large, since binding may occur to a number of different sites on the exposed surface of the protein, and binding alone does not predict the effect of ligand binding on the activity of the protein. Stringent selection among the candidate chemicals for those with the greatest affinity will define a set of chemicals small enough to be tested for insect killing.
  • Drosophila as a test organism enables large numbers of compounds to be assessed. Therefore the same procedure may be used as for proteins for which functional assays are available.
  • a cellular killing assay as an intermediate step.
  • a gene of unknown function can be examined for location and timing of gene expression in tissues throughout development.
  • the primary sites of tissue death may be determined by apoptosis assays or direct observation.
  • particular cell types e.g. nerve cells, can be defined as subject to death when the protein is not expressed or inhibited.
  • Appropriate cell types can be isolated from the appropriate tissue and developmental stage of Drosophila or a larger insect. Effects of candidate chemicals from the binding assay screen on survival of these cells in culture may then be ascertained, using commercially available live/dead cell assessment methods.
  • a further alternative or additional procedure is to express the protein target in a cell which has been manipulated genetically to contain a sensor for calcium ions, cyclic AMP or other components of cell signaling pathways. This may be achieved, for example, by generating transgenic Drosophila containing the gene encoding the protein with its expression driven by a promoter that is utilized in the cell type of choice.
  • permanent cell lines of any suitable origin may be transfected, and lines expressing the protein permanently selected.
  • expression of an unknown protein will cause a shift in the level of cell signaling components, which will be detected by the sensor and can be read, for example, as a fluorescent or luminescent signal.
  • the difference between the protein-expressing cells and control cells forms the basis of the assay. Effects of chemicals on the difference between protein expressing and control lines are assessed.
  • Proteins for all the assays described can be produced by cloning the gene for example into plasmid vectors that allow high expression in a system of choice e.g. insect cell culture, yeast, animal cells, bacteria such as Escherichia coli .
  • a vector may be used that incorporates an epitope tag (or other "sticky" extension such as His6) onto the protein on synthesis.
  • epitope tag or other "sticky" extension such as His6
  • the polynucleotide fragment can be molecularly cloned into a prokaryotic or eukaryotic expression vector using standard techniques and administered to a host. The expression vector is taken up by cells and the polynucleotide fragment of interest expressed, producing protein.
  • the cloning and expression of a recombinant essential polynucleotide fragment also facilitates in producing anti- essential antibodies and fragments thereof (particularly monoclonal antibodies) .
  • recombinant DNA technology may be used to prepare nucleic acid sequences encoding the various derivatives outlined above.
  • polynucleotide fragments of the present invention are preferably linked to regulatory control sequences.
  • control sequences may comprise promoters, operators, inducers, enhancers, silencers, ribosome binding sites, terminators etc.
  • Suitable control sequences for a given host may be selected by those of ordinary skill in the art.
  • a polynucleotide fragment according to the present invention can be ligated to various expression controlling sequences, resulting in a so called recombinant nucleic acid molecule.
  • the present invention also includes an expression vector containing an expressible nucleic acid molecule.
  • the recombinant nucleic acid molecule can then be used for the transformation of a suitable host.
  • hybrid molecules are preferably derived from, for example, plasmids or from nucleic acid sequences present in bacteriophages or viruses and are termed vector molecules.
  • the methods to be used for the construction of a recombinant nucleic acid molecule according to the invention are known to those of ordinary skill in the art and are inter alia set forth in Sambrook, et al. (Molecular Cloning: a laboratory manual Cold Spring Harbour Laboratory, 1989) .
  • the present invention also relates to a transformed cell containing the polynucleotide fragment in an expressible form.
  • Transformation refers to the introduction of a heterologous polynucleotide fragment into a host cell.
  • the method used may be any known in the art, for example, direct uptake, transfection transduction or electroporation (Current Protocols in Molecular Biology, 1995. John Wiley and Sons Inc.).
  • the heterologous polynucleotide fragment may be maintained through autonomous replication or alternatively, may be integrated into the host genome.
  • the recombinant nucleic acid molecules preferably are provided with appropriate control sequences compatible with the designated host which can regulate the expression of the inserted polynucleotide fragment, e.g. tetracycline responsive promoter, thymidine kinase promoter, SV-40 promoter and the like.
  • Suitable hosts for the expression of recombinant nucleic acid molecules may be prokaryotic or eukaryotic in origin.
  • Hosts suitable for the expression of recombinant nucleic acid molecules may be selected from bacteria, yeast, insect cells and mammalian cells.
  • the construct of the P ⁇ lacW ⁇ element used below is a defective P-eleraent.
  • a defective P-element is one which cannot transpose itself without the provision of a transposase enzyme from another source. Thus, once inserted into a site in the genome, a defective P-element will remain in position and will not distribute copies of itself.
  • the reporter gene in P ⁇ lacW ⁇ is an E . Col : ⁇ -gal lacZ gene under the control of a weak promoter. This weak promoter, however, responds to enhancer elements in the neighbourhood of the insertion site to give a pattern of lacZ expression that is related, to a variable extent, to the pattern of expression of the gene targeted. This provides temporal and/or tissue expression patterns which may be useful in deciding whether a gene/protein could be a potentially valuable target for insecticide or therapeutic development.
  • P ⁇ lacW ⁇ carries a mini-white eye colour gene to identify flies that contain insertions.
  • P ⁇ lacW ⁇ also contains a bacterial origin of replication and the ⁇ -lactamase gene coding for ampicillin resistance at its 3' end. This feature permits easy cloning of DNA flanking the insertion site of P ⁇ lacW ⁇ and further clone relevant genes (Bire, E., H. Vaessin, S. Shepherd, K. Lee, K. Mccall, S. Barbel, L. Ackermam, R. Carretto, T. Uemura, E. Grell, L.Y. Jan and Y.N. Jan, 1989 Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes and Development 3: 1273 - 1287) .
  • the mutant flies, in which P ⁇ lacW ⁇ was inserted on the second chromosome are on the y w; ⁇ > ⁇ lacZ,w + ⁇ CyO genotype (Torok, T,G. Tick, M. Alvarado and I. Kiss, 1993 P-lacW Insertional Mutagenesis on the second chromosome of Drosophila melanogaster: Isolation of lethals with different overgrowth phenotypes. Genetics 135: 71 - 80) .
  • the mutant flies, in which P ⁇ lacW ⁇ was inserted on the third chromosome are of the y w; P ⁇ lacZ,w + ⁇ TM3, sb ser genotype (Deak, P., M.M.
  • the genetic background of the w/w;P(lacW) mutants was equilibrated with that of the wild-type (Canton-S) strain by repeatedly backcrossing heterozygous w/w;P(lacW) /+ females (which carried the w + eye-color marker) to w(CS) males for more than five generations.
  • the ⁇ (CS) strain was derived by backcrossing w 1118 flies to wild-type (Canton-S) flies for 10 generations; the w(isoCJl) strain was derived from w(CS) and carries isogenic X, 2nd and 3rd chromosomes.
  • Genomic sequences flanking the P-element were cloned by plasmid rescue using standard techniques (Drosophila: A practical Approach, the 2nd ed. 1998) . Briefly, genomic DNA was digested with .EcoRI, followed by ligation to form a rescue plasmid, which was propagated in E. coli . The rescue fragment then was 3 P-radiolabeled by random priming and used to screen plaques from a Drosophila genomic bacteriophage lambda library. The lambda genomic fragment was subcloned into the plasmid vector pBluescript, radiolabeled and used to probe a Drosophila adult head cDNA library and a Northern blot of adult whole fly polyA + RNA, etc.
  • Southern blotting was carried out essentially as described by Sambrook et al . (1989). Hybridization was carried out at 64°C in 6xSSC, SxDenhardt's reagent, 0.5% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA. Filters were washed in lxSSC and 0.1%SDS for 15 min, and then in 0.1XSSC and 0.1%SDS for 30 min.
  • rescued plasmids Prior to DNA sequencing, rescued plasmids were quantified by restriction digestion with EcoRI to linearise followed by electrophoresis on a 1% agarose gel, comparisons being made to a Bacteriophage lambda lkb marker ladder. For DNA sequencing 500ng-2 ⁇ g of rescued plasmid was used in each sequencing reaction. Sequencing was carried out using a BigDye dideoxy terminator kit (Perkin- Elmer) with the following sequencing primers:- 1) 3' primer 5 • -CGCACTTATTGCAAGCATACG-3 • sequences into the rescued chromosomal DNA immediately 3 ' to the point of insertion (5* end of the chromsomal DNA insert)
  • Table 1 shows in summary details of the sequences obtained from the 902 distinct fly lines.
  • Table 2 shows in summary an updated version of Table 1 including references to sequences now contained in databases, but which were not disclosed until after the priority date of the present invention.
  • Embryos are collected from yeasted apple/grape juice agar plates into a container with a nylon mesh screen at the bottom, dechorionated by dipping into 50% bleach for 4 minutes and washed thoroughly with water. Embryos are placed into an Eppendorf tube containing a mixture of 0.35ml fix solution (1% glutaraldehyde in PBS) and 0.7ml n-heptane and fixed for 15 minutes at room temperature on a rotating mixer. After removing heptane and fix solution from tube, embryos are washed three times for 10 min.
  • larval, pupal and adult brains were dissected in PBS, and fixed in 4% paraformaldehyde for 20 min. They were then washed three times for 20 min in PBS, and stained with staining buffer and 2% X-gal for 1-2 h at 37°C (Ashburner, Drosophila. A Laboratory Manual, Plainview N.Y.: CSH Lab. Press 1989). They were then washed for 20 min in PBS, cleared overnight at 4°C with PBS/12.5% hydrogen peroxide, washed for 10 min with PBS, dehydrated through graded ethanol, and mounted in glycerol gelatin (Sigma) .
  • flies were mounted in "fly collars" (modified from Heisenberg and B ⁇ hl 1979) , soaked in OCT embedding medium (Miles, USA) for 10 min and then embedded in the OCT medium.
  • 12 ⁇ m serial sections of head or body were cut in a cryostat (Anglia Scientific) at -18°C. The sections were stained and mounted as described by Yang et al . (1995). Thereafter sections were examined and photographed on a Nomarski optical microscope.
  • In situ hybridisation to polytene chromosomes localises a DNA sequence (such as a gene, or am inserted P-element) on the physical DNA map of Drosophila, and may be related to the genetic map. For those insertion mutations which affect genes of known function, localisation of the P-element to the site where the gene mutated is known to reside is evidence that lethality does in fact result from insertion of the P-element in this gene.
  • PlacW transposon allowed its immediate localization in situ to a precise cytological region using P element DNA as a probe.
  • the procedure for in situ hybridization to third larval instar polytene chromosomes was essentially as described by Pardue (1986) .
  • pBluescript (Strategene, USA) were labelled with Bio-16-dUTP by nick-translation.
  • Hybridization was detected using 3,3' diaminobenzidine (DAB)/H 2 0 2 .
  • DAB 3,3' diaminobenzidine
  • the slides were stained with Giemsa and mounted using DPX which is a sliding mount commonly used by those skilled in the art.
  • genes comprising any of the sequences disclosed herein may be cloned to allow expression of the associated protein and testing in an assay.
  • Ligands for any protein may be discovered by direct binding assays. In order to select true lead chemicals for insecticide or therapeutic development, these must be followed by insect killing assays or other functional assays as mentioned herein.
  • one partner molecule is immobilized, and the other is labeled in some fashion (e.g. using a fluorescent tag, or by the incorporation of a radioactive isotope) and added free in solution.
  • the amount of bound ligand is measured using an appropriate detection system. This may be used in a qualitative mode at first. Ligands showing significant binding may then be studied further by ensuring that the protein is in excess, and carrying out experiments with a dilution series of the ligand at a set of known concentrations, typically from 10 "2 - 10 "10 M, such as 10 "3 - 10 "8 M
  • the protein encoded by the essential gene is identified, and the chemical ligand is unknown. Therefore the protein may be purified using an affinity system and immobilized. The chemical ligands will be labeled, incubated with the immobilized protein, washed, and the amount of retained label assessed.
  • Proteins may conveniently be immobilized using an epitope or other affinity tags provided by the expression vector (see above) , on a support material to which the appropriate antibody or binding agent for the tag is attached.
  • the support material may be nitrocellulose membrane, Sephadex or other type of protein purification column support, or specialized beads such as those commercially available from Dynal or Promega.
  • the protein may for example be biotinylated, and the same support materials derivatised with streptavidin (which has a very high affinity for biotin) used.
  • proteins may be modified chemically in a variety of ways, and covalently attached to support materials.
  • Nucleic acid or peptide ligands may conveniently be radioactively labeled by standard procedures. Organic chemical ligands may also be provided in radioactively labeled form.
  • a more convenient labeling system for large scale screening by binding assays is the use of chemicals that are tagged with oligonucleotide sequence labels, or by other means. This allows many chemicals to be tested together initially, since each can be identified by the use of a PCR based detection system.
  • Monoclonal antibodies raised against a particular protein may be used to select chemicals that bind to particular regions of the protein - the epitope recognised by the antibody.
  • chemicals are assessed for displacement or reduction in binding of the antibody. Remaining bound antibody is detected by a standard fluorescently labeled second antibody.
  • binding assays may be employed in a ligand-competition mode. This measures chemical interaction with the site on the protein at which the natural ligand binds, and is thus going to give a higher rate of significant hits. This type of assay is also more quantitative.
  • Examples of typical known ligands which would be labeled (typically radioactively) and used in displacement assays are: pharmacological agonists and antagonists, activators and inhibitors, neurotransmitters, growth factors and cytokines, cAMP, cGMP, enzyme cofactors such as NAD and FAD, regulatory polypeptides (e.g. calmodulin) and other subunits of multicomponent proteins.
  • a typical assay relies on the generation of purified protein as discussed above.
  • binding assays rely on labeled ligand, usually radiolabeled, to enable competition for the binding site to be detected.
  • a set concentration (enough to saturate the binding site) of labeled ligand is incubated with a purified sample containing the purified protein.
  • the test chemical is also added.
  • Bound ligand/protein complexes are washed (to remove free ligand) , precipated e.g. by TCA, collected with a cell harvester (for example) and the level of radioactivity measured. Displacement can be observed as a reduction in the amount of radioactivity detected in the assay. Enhancement of binding can also be observed in this type of assay, where radioactivity levels are increased - this indicates activity of the test chemical near to but not competing with the site of ligand interaction. Examples of some possible functional assays
  • Protein kinases are enzymes that transfer the terminal phosphate group of ATP and/or GTP to their substrate molecule. These enzymes have been shown to be involved in many cell processes including signal transduction, apoptosis and regulation of the cell cycle. Protein kinases are the largest known protein family and have been characterised in mammals, plants, fungi and microorganisms.
  • An assay of kinase activity generally requires two distinct steps: (1) transfer of the (labeled) terminal phosphoryl group of the nucleoside triphosphate donor to the substrate and (2) separation of the phosphorylated product from unutilized nucleotide.
  • Step 1 is generally carried out in solution, with both the enzyme and the substrate in the liquid phase.
  • Step 2 is usually accomplished by trichloroacetic acid (TCA) precipitation, by sodium dodecyl sulphate (SDS) gel electrophoresis, or by binding the labeled product to a solid support such as phosphocellulose paper or nitrocellulose membrane. These steps are then followed by detection of the amount of labeled phosphoryl that has been transferred to the substrate.
  • TCA trichloroacetic acid
  • SDS sodium dodecyl sulphate
  • Step 1 can also be carried out with either the enzyme or the substrate immobilized on a solid support.
  • complex protein mixtures can be fractionated by SDS gel electrophoresis, blotted onto membrane, and then tested as potential substrates by incubating the membrane with a non-specific blocking agent followed by the desired protein sample plus labeled ATP.
  • Another variation on this type of assay involves detection of the phosphorylated form of a protein using a monoclonal antibody directed to the phosphorylated form. The amount of phosphorylation may then be assayed in an enzyme- linked immunosorbent assay (ELISA) .
  • ELISA enzyme- linked immunosorbent assay
  • the kinase activity of a particular sample or protein can be assayed using histone H-l (or other convenient protein kinase substrate) as a substrate to which the kinase transfers phosphate.
  • histone H-l or other convenient protein kinase substrate
  • Assays are started by the addition of 2.5m-units (arbitrary units, dilution series) of sample, incubated at 30°C for 10 minutes and terminated either by spotting on to P81 paper (Whatman) or by the addition of Laemmli buffer (Laemmli U.K., 1970 Nature 227, 680-685., or for more recent methods see Current Protocols in Molecular Biology Chapter 10, 1994-1997, eds Ausbel F.M. et al. , Wiley) . Spotting onto Whatman paper is followed by extensive washing in 75 mM orthophosphoric acid. The papers are then washed in ethanol, dried and incorporated radioactivity determined either by autoradiography, scintillation spectroscopy or phosphoimaging.
  • Protein phosphorylation provides one means of regulating cellular processes. Protein dephosphorylation by protein phophatases plays an equally important role. Phosphatases are involved in the removal of the phosphoryl group from proteins that have been phosphorylated by kinases. See for example Methods in Enzymology 201:pp389- 468.
  • Assays for phosphatase activity can be carried out in the same way as a kinase assay. This would involve the pre- phosphorylation of for example histone by a kinase in the presence of radioactive ATP, followed by desphorylation by the test protein. The sample is then spotted onto P81 paper and the amount of radioactive ATP still incorporated is measured as previously described.
  • Cyclic adenosine 3', 5' monophosphate can be measured in tissue slices, dissociated tissue, cultured cells and membrane preparations.
  • Radioimmunoassay uses antibody raised to acetylated cAMP and involves competition between cAMP in the sample and 125 I-labelled cAMP (Steiner, A.L. , Wehmann, R.E., Parker, C.W. and Kipnis, D.M. (1972). Adv. Cyc . Nucleotide Res . , 2, 51.). Following an overnight incubation, unbound cAMP is removed using charcoal. cAMP levels are quantified by comparison with a cAMP standard curve and expressed relative to protein content of sample. This method is sensitive in the femtomolar range if the sample cAMP and the standard curve cAMP are acetylated before assay. Kits are available commercially (Amersham) .
  • the cAMP binding protein method is based on competition between 3 H-labelled cAMP and sample cAMP for binding sites on the regulatory subunit of cAMP-dependent protein kinase (Gilman, A.G. (1970). Proc. Natl . Acad. Sci . USA, 67, 305.). The procedure is analogous to radioimmunoassay but is more rapid because competition equilibrium is achieved in a 2 hour incubation. cAMP-dependent protein kinase preparation (Sigma) and binding protein assay kits (Amersham) are available commercially.
  • Enzyme immunoassay for cAMP is an enzyme immunoassay in which the sample cAMP and peroxidase-linked cAMP compete for binding to antibody raised against acylated cAMP.
  • Adenylyl cyclase catalyses the formation of cAMP from ATP in the presence of Mg 2+ .
  • the main methods are: (1) the measurement of ⁇ P-labelled cAMP formed from ⁇ P-labelled ATP and (2) the measurement of cAMP formed in a non-labelled reaction using either the radioimmunoassay or the binding protein assay.
  • Radioactively labelled cAMP produced from ⁇ 32 P-ATP in an in vitro reaction is separated from unreacted substrate and radioactive contaminants by sequential chromatography steps on Dowex and alumina columns and measured by liquid scintillation counting (Salomon, Y. , Londos, C. , and Rodell, M. (1974). Anal .Biochem. , 58, 541.). Crude or partially purified adenylate cyclase samples may contain contaminating activities that interfere with the assay.
  • nucleoside triphosphatase are minimised using a high substrate concentration in the adenylate cyclase reaction and by including phosphoenol pyruvate and pyruvate kinase as an ATP regenerating system.
  • Degradation of 3 P-labelled cAMP can be prevented by including a high concentration of unlabelled cAMP in the reaction.
  • the enzymatic reaction is terminated by addition of unlabelled ATP and by boiling for 2 minutes. Addition of [ 3 H]cAMP as a recovery label allows correction for differences in the performance of the individual chromatography columns.
  • the samples are first layered on a column of Dowex AG 50 WX 4 resin (200-400 mesh, H * form) equilibrated in water.
  • the cAMP has a greater affinity for the resin than ATP so the bulk of the [ 2 P] ATP can be washed of the column with water before eluting the cAMP directly onto an alumina column equilibrated with 0.1 M imidazole HCl, pH 7.5.
  • the remaining [ 32 P] ATP binds to the alumina and the labelled cAMP is eluted using imidazole buffer. Samples are counted in 32 P and 3 H channels using a scintillation counter.
  • Measurement of total [ 32 P] ATP and f ⁇ ] cAMP allows calculation of pmols of cAMP present in the sample.
  • Adenylate cyclase enzymatic activities are expressed as pmol cAMP formed per min per mg protein in the sample.
  • the Dowex and alumina columns must be calibrated before use to determine elution profiles of ATP and cAMP but they may be regenerated after each assay and used repeatedly.
  • the assay is sensitive, relatively simple and may be completed in one day. Apparatus for the double chromatography should be constructed from perspex to reduce risk from exposure to radioactivity.
  • Non-IaJelled adenylate cyclase reactions contain ATP, Mg 2 * and/or Mn 2+ , an ATP regenerating system and an inhibitor of cAMP phosphodiesterase such as 3-isobutyl-l- methylxanthine (IBMX) . Reactions are terminated by boiling and cAMP formed is measured by radioimmunoassay or cAMP protein binding assay.
  • IBMX 3-isobutyl-l- methylxanthine
  • Guanylate cyclase catalyses the hydrolysis of guanosine triphosphate (GTP) to cyclic guanosine 3', 5' monophosphate (cGMP) in a reaction analogous to that of adenylate cyclase.
  • GTP guanosine triphosphate
  • cGMP cyclic guanosine 3', 5' monophosphate
  • Methodology used in the assay of guanylate cyclase activity is essentially the same as that for adenylate cyclase.
  • Manganese is required as a cofactor for guanylate cyclase activity. Reactions are terminated by addition of HCl and boiling for 3 minutes.
  • Non-labelled guanylate cyclase reactions contain GTP, Mn 2+ , a GTP regenerating system and IBMX. Reactions are terminated by boiling and cGMP formed is measured by radioimmunoassay using antibody against acetylated cGMP. Kits are available commercially (Amersham) .
  • Cyclic nucleotide phosphodiesterase catalyses the hydrolysis of the 3 ' ,5 '-phosphodiester bond of the cyclic nucleotides, cAMP and cGMP.
  • the radioactive assay uses ⁇ -labelled cAMP or cGMP and involves quantification of the reaction product (5'- nucleotide monophosphate) (Thompson,, W.J. and Appleman, M.M. (1971). Biochemistry, 10, 311.).
  • the labelled nucleotide mono-phosphate (NMP) formed in the first reaction is converted to 5 '-nucleotide in a second reaction by a 5'- nucleotidase present in snake venom (Alomone Labs, Jerusalem, Isreal) .
  • the labelled 5 ' -nucleotide is isolated by Dowex-1-chloride anion exchange chromatography and quantified by liquid scintillation counting.
  • Adenosine 5 '-triphosphatases catalyse the hydrolysis of ATP to ADP and inorganic phosphate in the presence of Mg 2+ , Na + and K + .
  • the colorimetric assay quantifies the inorganic phosphate released from ATP by measuring the A ⁇ 60 nm following treatment of the enzyme reaction with TCA and Taussky-Shorr Colour Reagent (Bonting, S.L., Simon, K.A. , and Hawkins, N.M. (1961) Arch . Biochem . Biophys . , 95, 416-423. Tausky, HH and Shorr, E. (1953) J. Biol . Chem. , 202, 675-685.). Similar methods are used to assay guanosine 5 ' -triphosphatases (GTPases) .
  • This assay is based on the proteolytic digestion of casein and the spectrophotometric detection of released aromatic amino-acids. Briefly, casein is incubated with the suspected protease and then acid precipitated. The solution is then filtered and the absorbance of the acid soluble phase is measured at 280-290nm. See for example W. Rick in "Methoden der Enzymatichen Analyse", (H.U. Bergmeyer ed.) 3rd edition, 1046 and 1056. Verlag Chemie, Weinheim.
  • Endpoint titration with the fluorescent molecule 4- methyllumbelliferyl p-(N,N,N-triethylammonium) cinnamate.
  • This compound is sensitive to 10 "11 moles of enzyme with a 2 min reaction time, see for example G.W. Jameson, D.V. Roberts, R.W. Adams, W.S.A Kyle and D.T. Elmore. (1973) Biochem. J. , 131, 107. See also Methods in Enzymology, 248:pp3-782.
  • the general principle of this type of assay is based on the detection of membrane fusion events and/or the delivery of protein contents using purified membrane compartments.
  • the detection methods include immunodetection, fluorescence and release of chromogenic substances.
  • the assay involves the use of two different populations of vesicles, each containing a different molecular probe conjugated to a marker protein. Upon fusion the probes bind to one another to generate a detectible signal, in this case the binding of avidin to biotin. Complexes are detected by an ELISA protocol (detecting the biotinylated protein e.g. transferrin) and fluorescent detection of avidin conjugated ⁇ -galactosidase, see for example William A. Braell in "Methods in Enzymology" 219, 12-21 Academic Press inc. 1992.
  • Semi-intact/perforated cells are those which have lost a part of their plasma membrane by physical perforation. These assays can be done in Yeast or mammalian cells. Though lacking many soluble cytoplasmic factors, these cells retain their internal membrane and organellar structure and can efficiently reconstitute vesicular transport between compartments. They are also accessible to exogenously added factors such as antibodies and inhibitors.
  • VSV Vesicular stomatitis virus
  • This viral glycoprotein has two Aspargine linked oligosaccharide chains which undergo extensive modifications as the protein transverses the ER and Golgi compartments. Oligosaccharide processing intermediates confer different electrophoretic mobilities on the VSV polypeptide, these intermediates can therefore be detected by SDS PAGE, see for example C.J.M. Beckers, D.S. Keller and W.E. Balch. Cell. 50, 523 (1987).
  • Assays for the endocytic pathway include those for detection of the binding of proteins to cell surface receptors, formation of clathryn coated endocytic vesicles, transport to the endosome, uncoating of the vesicles, delivery of the vesicle contents and recycling to the plasma membrane.
  • Example:- Detection of Functional Clathryn Coated Vesicles This assay involves the preparation of two vesicle fractions i) The "donor" population containing 125 I-labelled transferrin. ii) The "acceptor" vesicles, these being the clathryn coated vesicles under test. The acceptor vesicles contain internalised Anti-transferrin antibody.
  • the donor and acceptor populations are mixed in a solution containing cytosol and an ATP cocktail.
  • a radiolabelled immunocomplex is formed.
  • the vesicles are then solubilised and the mix passed through a Staphlococcus aureus column to isolate the immunocomplexes, which are then eluted from the column and the radioactivity measured, see for example P.G. Woodman and G. Warren in "Methods in Enzymolgy" , 219, 251 (1992)
  • pancreatic DNAse I yields di- and oligo-nucleotide 5' phosphates
  • pancreatic DNAse II yields 3' phosphates.
  • chromatin the sensitivity of DNA to digestion by DNAse I depends on its state of organization, transcriptionally active genes being much more sensitive than inactive genes.
  • Ribonuclease E is an RNase involved in the formation of 5S riboso al RNA from pre-rRNA.
  • Ribonuclease F is stimulated by interferons and cleaves viral and host RNAs and thus inhibits protein synthesis.
  • Ribonuclease H specifically cleaves an RNA base-paired to a complementary DNA strand.
  • Ribonuclease P is an endonuclease that generate t-RNAs from their precursor transcripts.
  • Ribonuclease T is an endonuclease that removes the terminal AMP from the 3' CCA end of a non-aminoacylated tRNA.
  • RNase Tl cleaves RNA specifically at guanosine residues.
  • RNase III cleaves double-stranded regions of RNA molecules.
  • RNA and DNA eg. SI nuclease, EC.3.1.30.1, that is specific for single stranded molecules
  • Ribonucleases such as pancreatic, Tl etc. are specific for RNA
  • Deoxyribonucleases for DNA.
  • Bacterial restriction endonucleases are crucial in recombinant DNA technology for their ability to cleave double-stranded DNA at highly specific sites.
  • An enzyme capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids.
  • Type I restriction endonucleases occur as a complex with the methylase and a polypeptide that binds to the recognition site on DNA.
  • Type II restriction endonucleases are the classic experimental tools. They have very specific recognition and cutting sites. The recognition sites are short, 4-8 nucleotides, and are usually palindro ic sequences.
  • both strands have the same sequence running in opposite directions the enzymes make double-stranded breaks, which, if the site of cleavage is off-centre, generates fragments with short single-stranded tails; these can hybridise to the tails of other fragments and are called sticky ends.
  • They are generally named according to the bacterium from which they were isolated (first letter of genus name and the first two letters of the specific name) .
  • the bacterial strain is identified next and multiple enzymes are given Roman numerals.
  • the two enzymes isolated from the R strain of E. coli are designated Eco RI and Eco RII.
  • the more commonly used restriction endonucleases are known to those skilled in the art, but may be found in manufacturers catalogues, such as New England Biolabs, USA.
  • nucleases cleave DNA and/or RNA, therefore a general assay would be to incubate unknown/test protein/chemical with a known quantity and type of DNA or RNA for a given time, and separate the products using gel electrophoresis along with a known set of standards. Any nuclease activity will be readily visible on the gel. Once nuclease activity has been detected, direct comparisons can be made with the DNA cleavage patterns generated by known nucleases in order to identify the type of nuclease involved.
  • the DNA ligase of E. coli seals nicks in one strand of double-stranded DNA, a reaction required for linking precursor fragments during discontinuous synthesis on the lagging strand.
  • Nicks are breaks in the phosphodiester linkage that leave a free 3 ' -OH and 5 • -phosphate.
  • the ligase from phage T4 has the additional property of joining two DNA molecules having completely base-paired ends. DNA ligases are crucial in joining DNA molecules and preparing radioactive probes (by nick translation) in recombinant DNA technology.
  • methyl groups are added to certain nucleotides in genomic DNA. This affects gene expression, as methylated DNA is not easily transcribed. The degree of methylation is passed on to daughter strands at mitosis by maintenance DNA methylases. Accordingly, DNA methylation is thought to play an important developmental role in sequentially restricting the transcribable genes available to distinct cell lineages. In bacteria, methylation plays an important role in the restriction systems, as restriction enzymes cannot cut sequences with certain specific methylations.
  • DNA polymerase and RNA polymerase are enzymes involved in template-directed synthesis of DNA from deoxyribonucleotide triphosphates and RNA from ribonucleotide triphosphates.
  • Class of enzymes involved in DNA repair includes endonucleases that recognise a site of damage or an incorrect base pairing and cut it out, and exonucleases that remove neighbouring nucleotides on one strand. These are then replaced by a DNA polymerase.
  • Topoisomerase II of E. coli is commonly known as gyrase.
  • Transcription factor activity lies in the centre of a signalling cascade that begins at the cell surface by the activation of a receptor. Intracellular signal transduction events activate or repress specific transcription factors, which in turn regulate the expression of specific genes.
  • the activity of a transcription factor can be assessed by linking the appropriate regulatory sequence to a reporter gene encoding among other reporters ⁇ -galactosidase, Chloramphenicol acetyl transferase (CAT) , luciferase and green fluorescent protein (GFP) in an engineered plasmid vector.
  • This vector is used to transfect a cell line and the activity of the transcription factor of interest analysed by measuring the amount of reporter activity (Brannon, M. et al (1997) Gen. Dev. 11, 2359.).
  • luciferase offers the most ideal situation because the reporter measurements are nearly instantaneous, exceptionally sensitive and there is little or no endogenous activity in the host cells to interfere with quantitation.
  • Firefly luciferase (Ow, D et al (1986) Science 234, 856.) is by far the most commonly used of bioluminescent reporters.
  • the enzyme catalyses a two-step oxidation reaction to yield light at 550-570nm that can be detected by the use of a luminometer.
  • the assay can be adapted for use with single or multiple samples depending on the type of luminometer available, i.e. tube or plate.
  • the gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting sequence- specific binding proteins, such as transcription factors (Ausubel, F.M. et al. (1989) In: Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, New York.).
  • the assay is based upon the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double stranded oligonucleotides.
  • the gel shift assay is performed by incubating a purified protein or a complex mixture of proteins such as a nuclear extract preparation with a 32 P labelled DNA fragment containing the putative binding site. The reaction products are then analysed on a nondenaturing polyacrylamide gel.
  • the specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest. 13. Apoptosis
  • Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes, e.g. tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system and endocrine-dependent tissue atrophy.
  • Apoptosis involves a cascade of cytoplasmic and nuclear events that result in a series of morphological changes and eventually cause the demise of the cell. Apoptosis can be initiated by a variety of different stimuli that lead to a convergence of biochemical signalling pathways into a common collection of executioner molecules.
  • phosphatidylserine PS
  • PS phosphatidylserine
  • Mitochondrial physiology is disrupted in cells undergoing apoptosis. Permeability is altered and specific protease activators are released. Specifically, the discontinuity of the outer mitochondrial membrane results in the redistribution of cytochrome C to the cytosol followed by subsequent depolarisation of the inner mitochondrial membrane. Cytochrome C release further promotes apoptosis by the activation of the caspases, cysteine proteases.
  • caspases participate in a cascade of cleavage events, which disable key homeostatic and repair enzymes and bring about a systematic structural disassembly of dying cells.
  • the biological substrates of caspases include poly (ADP ribose) polymerase (PARP) , DNA-dependent protein kinase (DNA-PK) , lamins, topoisomerases, Gas2, protein kinase C (PKC) , sterol regulatory element binding proteins (SREBP) , Ul-70kDa protein and Huntingtin protein.
  • PARP poly (ADP ribose) polymerase
  • DNA-PK DNA-dependent protein kinase
  • lamins lamins
  • topoisomerases Gas2, protein kinase C (PKC)
  • PKC protein kinase C
  • SREBP sterol regulatory element binding proteins
  • Ul-70kDa protein Huntingtin protein.
  • PS translocates from the cytoplasmic to the extracellular side of the cell membrane.
  • the early proteolytic events of apoptosis can be monitored using an adaptation of the absorbance-based assay originally devised by Thornberry, N.A. (1994) Interleukin-1 beta converting enzyme. Meth.Enzymol. 244, 615.
  • the colorimetric substrate (Ac-DEVD-pNA) is labelled with the chromophore p-nitroaniline(pNA) .
  • pNA is released from the substrate upon cleavage by DEVDase.
  • Free pNA produces a yellow colour that is monitored by a photometer at 405nm. The amount of yellow colour that is produced upon cleavage is proportional to the amount of DEVDase activity present in the sample.
  • the potent, irreversible, pan-caspase inhibitor benzoxycarbonyl-val-ala-asp fluromethyl ketone (Z-VAD-FMK) , (Zhou Q, Krebs JF, Snipas SJ, Price A, Alnemri ES, Tomaselli KJ, Salvesen GS Biochem 37 10757 (1998) can be used as a negative control and it is suggested that apoptosis be induced by the addition of Fas or TNF agonist antibodies.
  • the protocol can be used to test multiple samples by performing the assay in a total volume of 100ml using cells cultured in 96 well plates. The absorbance produced by each sample is read using a plate reader.
  • Calcium dynamics may be detected directly or indirectly by a range of methods; including but not restricted to: a) transgenic apoaequorin, a calcium-sensitive luminescent protein; b) other methods that monitor intracellular calcium concentration; c) other methods that monitor the operation of intracellular calcium signalling pathways; d) methods that monitor the operation of other types of signalling pathway; e) methods that monitor neuronal electrical potentials.
  • transgenic apoaeqorin has been used to monitor calcium dynamics in the intact Drosophila renal system and the intact Drosophila brain (Rosay et al (1997) J. Cell. Sci. 110, 1683-1692; O'Donnell et al. (1998) Am. J. Physiol. 43(4), R1039-R1049.) . It has also been used to provide a bioluminescent assay for agonist activity against G protein coupled receptors (Stables et al. (1997) Anal. Biochem. 252, 115-126).
  • Transgenic apoaequorin can thus be used to assess the effect of an exogenous gene on intracellular calcium dynamics, the method comprising detecting a pattern of calcium dyma ics in cells, tissues or organisms expressing the exogenous gene, and comparing said pattern with a pattern of calcium dynamics in cells, tissues or organisms without said exogenous gene.
  • fluorescent probes such as fura-2, indo- 1, quin-2
  • fluorescence microscopy flow cytometry and fluorescence spectroscopy.
  • Most of these fluorescent indicators are variations of the nonfluorescent calcium chelators EGTA and BAPTA (Cobbold and Rink (1987) Biochem. J. , 248, 313.).
  • New fluorescent indicators for calcium may also be used and are genetically encoded without cofactors and are targetable to specific intracellular locations.
  • These so-called “cameleons” consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP) , calmodulin, the calmodulin-binding peptide M13 , and an enhanced green- or yellow-emitting GFP.
  • GFP green fluorescent protein
  • Binding of calcium makes calmodulin wrap around the M13 domain, increasing (Miyawaki et al., (1997) Nature, 388, 882-887.) or decreasing (Romoser et al., (1997) JBC, 272, 13270-13274.) the fluorescence resonance energy transfer between flanking GFPs.
  • potentiometric optical probes may be used. Potentiometric optical probes measure membrane potential in organelles and in cells. In conjunction with imaging techniques, these probes can be employed to map variations in membrane potential along neurons and among cell populations with high spatial resolution and sampling frequency (Rohr and Salzberg (1994) Biophys. J., 67, 1301.). Additionally, GFP-based reporter genes that monitor intracellular cAMP dynamics may be used, and to monitor intracellular pH changes (Miesenbock et al. (1998) Nature 394, 192-5).
  • transmembrane potential permits study of the elements which mediate electrical behaviour of cells. This form of study may be undertaken in a number of ways, including: voltage (patch) - clamping and the use of voltage sensitive dyes.
  • this involves sealing a blunt micropipette tip to a cell membrane.
  • This is termed a gigaseal.
  • the gigaseal electrically isolates the whole cell or a patch of the membrane allowing detection of picoampere, ionic currents while accurately controlling the voltage.
  • This form of analysis may be utilised in the study of cultured cells, tissue slices or recombinant ion channels expressed post DNA transfection in heterologous cells.
  • Whole cell recording measures the activity of the full complement of active channels in a cell; typically specific populations of channels are isolated using channel-blocking agents.
  • Reversal potentials were determined experimentally by altering the holding potential until currents reversed direction and the potential for zero current was recorded. Cells were routinely clamped at - 60 mV. Whole cell currents were recorded both before and after the addition of purified viral protein by using an Axopatch 200A. Viral protein ⁇ 0.6 nM in bath solution was applied directly onto patched cells through gravity fed drug delivery tubing, whole-cell currents were filtered at 5 or 10 kHz, digitized at 44 kHz, and stored on videotape. For data analysis currents were replayed through the same system and digitized using an A to D converter interfaced with an IBM- compatible computer. Inward currents are depicted as downward deflections from the zero current level.
  • Electrical potential may also be measured using voltage sensitive dyes .
  • voltage sensitive dyes e .g. Oxonol VI / Bis-oxonol (Dall'Asta V., Gahi R. , Orlandini G., Rossi P.A, Rotoli B.M, Sala R. , Bussolati O. , Gazzola G.C, (1997) Experimental Cell Research 231, 260 - 268; Salvador J.M, Inesi G, Rigaud J.L, Mata A.M, (1998) J. Biol. Chem. 273 f 18230 - 18234).
  • transmembrane electrical potential was measured by analysis of the differential absorption (625 - 603 nm) of 2 ⁇ M oxonol using dual wavelength spectrophotometry.
  • the medium comprised Pipes buffer, pH7.1; 0.42 ⁇ g/ml calmodulin; 5mM MgCl2 and 2 ⁇ M Oxonol VI.
  • the callibration was performed by several additions 130 mM KCl to the medium in the presence of 1 ⁇ M of the K + ionophore valinomycin and in the absence of ATP.
  • Absorption changes were standardized using the Nernst equation. Within a range 0 - 40 mV Absorption by oxonol demonstrates a linear increase with increasing membrane potential. This proportionality permits straightforward assay of changes in membrane potential.
  • Bis-oxonol is a fluorescent dye which distributes across biological membranes according to the membrane potential and binds to hydrophobic components: since the quantum yield of the dye increases with binding, the fluorescence of the cells incubated in a medium containing the dye increases with depolarization and decreases with hyperpolarization.
  • Ion channels a class of integral membrane proteins that traverse the cell membrane. These channels have three important properties: (1) they conductions, (2) they recognise and select among specific ions, and (3) they open and close in response to specific electrical, mechanical, or chemical signals. [Principles of Neural Science, (Kandel and Schwartz) , Chapter 5 Ion Channels] . Ion channels are large integral membrane glycoproteins, which have a central aqueous pore that spans the entire width of the membrane. Many ion channels are made up of two or more subunits, which may be identical or distinct.
  • Three major signals gate ion channels voltage (voltage- gated channels) , chemical transmitters (transmitter-gated channels) , and pressure or stretch (mechanically-gated channels) .
  • Gating involves a conformational change of the channel in response to the above stimuli.
  • Ion channels select the type of ions that they allow to cross the membrane through physio-chemical interaction between the ion and various amino acid residues that line the walls of the channel pore (on the basis of ionic charge) , allowing either cations or anions to permeate.
  • Some cation-selective channel types are relatively non- selective, passing Na * , K*, Ca 2* , and Mg 2 *.
  • channels can be modified by cellular metaboloic reactions, including protein phosphorylation, by various channel blockers, toxins, poisons, and drugs.
  • Channels are important targets in various diseases, eg myasthenia gravis and cystic fibrosis.
  • peptide sequence can be obtained either directly or by using the chemical bound to a column to purify the target molecule in the cell (e.g., benzodiazepine affinity chromatography purification columns were used to isolate and identify the first cDNA clones encoding GABA receptor subunits in 1987 - Schofield P.R, Darlison M.G, Fujita N. , Burt D.R., Stephenson F.A, Rodriguez H. , Rhee L.M, Ramachandran J. , Reale V., Glencorse T.A, Seeburg P.
  • single-channel recording can measure the activity of a single protein molecule (electrophysiology) .
  • the patch clamp technique has made it possible to measure directly the activity of single ion channel molecules by recording the unit current flow through single open channels.
  • Expression of cRNAs in the Xenopus oocyte system, cDNA in transfected cell lines or whole tissue slice cultures can be used.
  • Example of cRNA expression in Xenopus oocyte pure mRNA, produced by in vitro transcription from cDNA, is microinjected into Xenopus oocytes and pulses of known compounds (eg gamma amino butyric acid (GABA) , glutamate, etc) can be superfused over the oocyte while recording membrane currents under voltage-clamp conditions. Current response to applied compound can be measured.
  • GABA gamma amino butyric acid
  • Ligand-gated ion-channel receptors cation: (nACh) , either Integral (pore forming, 5HT, glutamate, Ion gating) or Second messenger anion: GABA systems (associated) Glycine
  • an intracellular electrode is inserted into the nerve cell (grown in culture or via slice culture) - the pipette acts as a salt bridge, providing electrical connection between the cytoplasm and a metal electrode that is connected to the electronic apparatus.
  • the second extracellular electrode can be used to confirm resting potential and/or stimulate the cell. Both electrodes are connected to a voltage amplifier, which in turn is connected to an oscilloscope that displays the amplitude of the membrane potential (-65mV at rest) .
  • nACh nicotinic acetylcholine
  • 5HT 3 5 hydroxytryptamine receptor
  • glutamate glutamate
  • GABA A Glycine
  • a transmembrane ion channel whose permeability is increased by the binding of a specific ligand, typically a neurotransmitter at a chemical synapse.
  • the permeability change is often drastic; such channels let through effectively no ions when shut, but allow passage at up to 10 7 ions s -1 when a ligand is bound.
  • These receptors have been found to share considerable sequence homology, implying that there may be a family of structurally related ligand-gated ion channels.
  • Ion channel receptors are composed of 4 or 5 subunits, which may be the same or different, each of which contains 4 or 5 membrane-spanning -helical regions. These - helices are thought to align to form the pore of the channel, through which ions can flow. The characteristics of each channel is determined by the type of subunits that are present in each receptor subtype. Annals of the New York Academy of Sciences (1999) Volume 868) Current flow depends on the number of open channels, the concentration of the transmitter, channel conductance and membrane potential.
  • Receptor specific assays will have to be created for each receptor/ion channel under investigation.
  • the best/easiest way to do this is to create permanent cell lines expressing a particular combination of receptor subunits in order to form particular receptor subtypes.
  • Initial assays established by the inventors will focus on the most clinically relevant subtype (s) of each receptor.
  • functional assays can be used to investigate the effects of any chemical on the receptor characteristics e.g., electrophysiology (patch-clamp single-channel recording) , binding assays (see section 1) , etc.
  • G-proteins GTP-binding protein
  • G-protein-coupled receptors are thought to have seven membrane spanning domains, and have been divided into 2 subclasses: those in which the binding site is in the extracellular domain e.g. receptors for glycoprotein hormones, such as thyroid stimulating hormone (TSH) and follicle stimulating hormone (FSH) , and those in which the ligand-binding site is likely to be in the plane of the 7 transmembrane domains e.g. rhodopsin and receptors for small neurotransmitters (nACh, 5HT, glutamate-NMDA, GABA, Glycine) and hormones. All transduce their signal by conformational change activation of an associated G-protein (see section 17) .
  • TSH thyroid stimulating hormone
  • FSH follicle stimulating hormone
  • G proteins There are two main classes of G proteins, the heterotrimeric G proteins that associate with receptors of the seven transmembrane domain superfamily and are involved in signal transduction, and the small cytoplasmic G proteins.
  • the small G proteins are a diverse group of monomeric GTPases that include ras, rab, rac and rho and that play an important part in regulating many intracellular processes including cytoskeletal organisation and secretion.
  • Their GTPase activity is regulated by activators (GAPs) and inhibitors (GIPs) that determine the duration of the active state, (see section 17) , see for example Principles of Neural Science, (Kandel and Schwartz), Third Edition 1991.
  • G-proteins (GTP binding proteins)
  • GTP binding proteins are a superfamily of related proteins which bind to guanosine nucleotides (Kaziro Y., Itoh H. , Kozasa T. , Nakafuku M. and Satoh T. Ann. Rev. Biochem. (1991) . They are found in an inactive form which is bound to GDP and an active form which is bound to GTP. Other proteins such as ligand bound receptors promote the exchange of GDP with GTP, activating the protein. G proteins are inactivated by hydrolysis of the GTP to GDP. This reaction is catalysed by the G protein itself but the rate of GTP hydrolysis can be influenced by interaction with other proteins. Activated G proteins regulates the activities of a large number of target proteins including adenylate cyclase, phospholipase C and ion-channels.
  • Heterotrimeric G proteins are a large family of GTPases which consist of an , a ⁇ and a y subunit. They are involved in signal transduction from receptor proteins in the plasma membrane to second messenger systems within the cell receptors that activate. Activation of a receptor (e.g. by ligand binding) activates the G protein by promoting the exchange of bound GDP with GTP. The presence of GTP in the active site causes the dissociation of the subunit from the ⁇ y complex. The free subunits is most active. Different subunit subtypes interact with a wide variety of different target proteins including adenylate cyclase, phospholipase C and ion-channels. The free ⁇ y complex also has also been show to have some regulatory activity.
  • These proteins consist of a single subunit similar to the ⁇ subunit of heterotrimeric G proteins. These include the RAS family of proteins the abnormal activity of which can contribute to tumour formation.
  • Guanosine nucleotide binding protein superfamily include GTP binding translation elongation factors and members of the Dynamin family of proteins.
  • G proteins allows the biochemical properties of proteins identified by DNA sequencing to be studied and allows the isolation of large amounts of the proteins for structural and biochemical studies. It also allows the production of mutant proteins produced by site directed modification of cDNA sequences.
  • G proteins have been expressed in large amounts in bacterial and insect-cell/baculovirus systems. Expression of G proteins in cell free translation systems is a convenient way of producing small amounts of protein for biochemical studies. The addition of 35 S methionine to the in-vitro translation reaction results in the production of specifically labelled protein.
  • the nonhydrolyzable GTP analogue 35 S ⁇ GTP will bind to most GTP binding proteins in the absence of any activator molecule.
  • Purified or in-vitro translated G protein can be incubated with 35 S ⁇ GTP and the reaction products passed through a nitrocellulose filter. Protein bound 5 S ⁇ GTP will be retained on the filter and the activity measured (Carty D.J. and Iyengar R. (1994). Methods in Enzymology. 237: 38-45.) .
  • Conformational changes in G proteins and changes in subunit interaction can be studied by incubating the G protein with GTPyS which binds to, and irreversibly activates, the protein. Conformational changes in subunits and changes in subunit interaction alter the sites available for degradation by trypsin.
  • the tryptic fragments of radio-labelled protein can be run on a SDS PAGE gel and visualised by autoradiography. Subunit interaction can also be studied by looking as sedimentation rates during ultra centrifugation and by using chemical crosslinking agents (Audigier Y. (1994) . Methods in Enzymology. 237: 239-254. Activation of other proteins as a result of G protein activation
  • G proteins in cell extracts can be activated by incubation with GTPyS and the activities of possible downstream target proteins such as adenylate cyclase and phospholipaseC measured.
  • Receptor stimulated GTP binding and GTP hydrolysis.
  • Receptor stimulated binding of the radio-labelled non- hydrolyzable GTP analog 35 S ⁇ GTP can be used to show if the addition of a receptor ligand leads to the activation of a G protein (Wieland T and Jakobs K.H (1994) Methods in Enzymology 237, 3 - 13) . It is possible to study the activation of endogenous G proteins or to use a membrane preparation lacking particular G proteins and add back a purified or recombinant G protein.
  • 35 S ⁇ GTP is added to a reaction mix containing a membrane preparation of the cells being studied. After incubation at 37°C for an appropriate length of time the reaction is stopped. The reaction mix is then passed through a filter which binds protein of membrane. The amount of radioactivity incorporated into the protein/membrane fraction is then measured. The amount of radioactivity incorporated in the presence and absence of candidate receptor ligand molecules can then be compared.
  • GTPase activity As an alternative to measuring the binding of 35 S ⁇ GTP it is possible to measure GTPase activity. Activation of a G protein by a ligand bound receptor results in an increase in GTP hydrolysis activity. This is more often a result of increasing the rate of exchange of GDP with GTP rather than an increasing the rate of hydrolysis of bound GTP. ⁇ 32 P GTP is added to a reaction mix containing a membrane preparation of cells and the amount of 32 P released from the labelled GTP is measured.
  • NPS47 GNL 3 1 -246 K01294 heat shock locus 87C1 proximal gene, 3' end.
  • NPS66 GNL 3 1 -436 U22176 15bp upstream of Brother gene on AC005557
  • NPS68 GNL 3 224-298 Y07908 Match to EST AI292767. This then matches serine/threonine protein kinase.
  • NPS72 GNL 3 83-135 U12010 putative serine/threonine protein kinase (nemo)
  • NPS73 GNL 3 1 -357 U20554 UDP-glucose:glycoprotein glucosyltransferase mRNA
  • NPS74 GNL 3 1 -20bp U87925 Cbl gene confirmed by match to EST AA441040
  • NPS146 EST 2 107-243 AI064230 Also AA263288. Match to Mouse proteasome subunit
  • NPS1 7 EST 2 1 -212 and 276-382 Al 106957 1756bp EST contig. Also AA391 125, AA567307, AA735971
  • NPS1 3 EST 2 1-38inv AA391495 1135bp contig. AA439145 and AA949325. Match to mouse EST
  • NPS1 80 EST 2 435-475 AA438658
  • 162 NPS212 EST 2 1 -224 AA441346 also AA390646 and AA696470.
  • 1677 contig 163 NPS213 EST 2 1 -514 AI064375 164 NPS216 EST 2 181-299 AA540197 also AA695503 and AA941503.732bp contig 165 NPS217 EST 2 167-212inv AA979442 also AA392418 166 NPS218 EST 2 89-159 AA536378 also AA949458 167 NPS219 EST 2 1 -570 AI515537 Genomic AC004345. Also AI062109. 50bp upstream of EST.
  • NPS236 EST 2 1 -414 AA392415 177 NPS239 EST 2 1 -22bp AA695619 178 NPS240 EST 2 399-542 AA142132 179 NPS241 EST 2 366-520 AA536537
  • NPS262 EST 2 1 -124 AA696531 C.elegans pro7, Z66519/ mouse EST 197 NPS265 EST 2 442-549 AM 24332 198 NPS266 EST 2 52-382 AA949873 199 NPS1073 EST 2 1 -167 AI133902 see also AC006562 poss phosphate transporter 200 NPS269 EST 2 1-550 AI403609 Genomic AC005129, 420bp upstream of EST 201 NPS271 EST 2 299-375 AA391470 202 NPS272 GNL 2 37-77bp AF085601 Drosophila inorganic pyrophosphatase NURF-38 203 NPS273 EST 2 1 -76inv AA696584 204 NPS275 EST 2 1-319 AA439099 1132bp contig with AA949325 and AA940848 poss.
  • GMP synthase (human) 205 NPS276 EST 2 21 -377 AA695424 206 NPS277 EST 2 152-590inv AA440949 207 NPS278 EST 2 132-312 AI062455 also AA440915 208 NPS279 EST 2 68-31 1 AA816432 209 NPS281 EST 2 1 -258 AA979191 Match to human CGI-28 210 NPS283 EST 2 2-318 AA391495 211 NPS285 EST 2 1 -89bp AA441636 AA820540 and AA817484.
  • Alt splice 212 NPS1075 EST 2 59-488 AI295363 213 NPS288 EST 2 51 -170 AH 14059 also AA941565
  • NPS312 EST 3 1 -152 AA540030 Poss rat calcium binding prot.
  • NPS339 EST 3 3-166inv AA202200 also AA202128 252 NPS340 EST 3 1 -48 inv AA439530 253 NPS341 EST 3 28-207 AH 09459 Poss GPI-anchored protein(human) 254 NPS342 EST 3 471 -506inv AH 09779 255 NPS343 EST 3 147-247 AA141054 256 NPS1061 EST 3 65-1 18inv AA141365 257 NPS345 EST 3 144-549 AI063643 258 NPS346 EST 3 1-148 AH 07445 also AA390813 259 NPS347 EST 3 1 -75bp AI297362 260 NPS348 EST 3 96-230inv AA392916
  • NPS352 EST 31 % over 129AA like Rat Nup84 and Human
  • NPS404 EST 3 1 -140 AA541045 May be distantly related to cystatin
  • Mammlian glia maturation factor Mammlian glia maturation factor.
  • KIAA0596 protein Homo sapiens
  • NPS0052 GNL 3 1 -241 U59923 glutamyl-prolyl-tRNA synthetase gene
  • NPS0068 GNL 3 224-298 Y07908 Match to EST AI292767. This then matches serine/threonine protein kinase.
  • NPS0080 GNL 3 1-231 L06861 232-401 intron, 402-590 exon matching TAF1 10
  • NPS0081 EST 2 1 -314inv AI259618 From genomic data.40bp upstream Prob. cytochrome B5. AC005641

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Abstract

La présente invention concerne des recherches concernant des composés qui entraînent un effet physiologique (nuisible ou bénéfique) sur des protéines identifiées comme étant essentielles. Les inventeurs ont identifié une séquence d'acide nucléique issue de gènes codant pour des protéines dont on pense qu'elles sont essentielles, et dont la carence d'expression conduit à un phénotype létal ou semi-létal. Ces inventeurs on décrit des essais qui peuvent être conduits pour rechercher des composés convenables. Cette invention concerne aussi des séquences nouvelles en soi.
EP00958829A 1999-09-07 2000-09-06 Genes essentiels et recherches relatives a ceux-ci Withdrawn EP1212620A1 (fr)

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