EP1212620A1 - Essential genes and assays relating thereto - Google Patents

Essential genes and assays relating thereto

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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)
French (fr)
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

The present invention relates to screening assays for compounds which have a physiological (e.g. harmful or beneficial) effect on proteins identified as being essential. The inventors have identified nucleic acid sequence from genes encoding proteins which are thought to be essential, the lack of expression of which leads to a lethal or semi-lethal phenotype and have described assays which may be conducted to screen for suitable compounds. The present invention also relates to novel sequence per se.

Description

ESSENTIAL GENES AND ASSAYS RELATING THERETO
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.
Crop destruction by organisms (eg. pests) 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. However 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.
However, increasing environmental concerns and development of resistance to existing pesticides has led to a more rational approach to pesticide development being voiced.
It is amongst the objects of the present invention to provide a more rational approach to pesticide development by providing pesticide screening assays based on the identification and use of genes/proteins which are considered to be essential to the pest.
The present inventors have used genetic techniques in order to study a model "pest", namely Droεophila . 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. Thus, in a first aspect 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. For example, negative modulation, as used herein, 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, as used herein, 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.
Typically 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. However, the assay may be used to screen for other effects, such as beneficial or therapeutic effects. For example 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.
An essential gene is one for which it has been determined that the expression of a functional protein is necessary in order to avoid a lethal or semi-lethal phenotype. 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.
Studies carried out by the present inventors have identified many distinct 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. Szidonya, P. Maroy, Y. Zhang, M. Ashburner, P. Benos, C. Savakis, I. Siden-Kiamos, C. Louis, V.N. Bolshakov, F.C. Kafatos, E. Madueno, J. Modolell and D.M. Glover, 1998 P element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: Correlation of physical and cytogenetics maps in chromosomal region 86E- 87F. Genetics 14: 1697 - 1722). Using the technique of plasmid rescue, it has been possible to determine the nucleotide sequence surrounding the site of transposon insertion. 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. a protein with no activity or reduced activity such as a protein displaying less than 75%, 50%, 40%, 30%, 20%, 10%, 5% or 1% activity with respect to normal protein function) . It is to be understood therefore that for the purposes of the present invention the term "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. Canton S) with investigation of the maintenance of a genetic linkage between the lethal locus and the transposon insertion site (cantonisation) ; reversion studies to show a complete correlation of lethal phenotype reversal and loss of the transposon; accurate genetic co-localisation of the lethal locus and the transposon insertion site using a set of deficiency (deletion) mutations covering the genomic region concerned. Since the action of a protein encoded by such genes is thought to be essential for cell and/or organism survival or reproduction, it follows that chemical interference with the action or production of the protein will mimic the effect of mutation and result in the death of cells and the organism (or in the case of semi-lethality failure to reproduce or reduction in offspring) . Thus, chemical interference with the action of each potentially essential gene and protein represents a way to kill cells and organisms, and specifically pests such as insects, arachnids, etc.
In a further aspect 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. Typically 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. More particularly 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" as used herein 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.
The 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. Thus, "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.
In general, the term "expression product" refers to both transcription and translation products of said polynucleotide fragments. When 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. Thus, the skilled addressee will appreciate that "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. In this manner, 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. In principle any specific polynucleotide sequence fragment from the identified sequences may be used in detection and/or expression studies. The skilled addressee understands that 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. If a specific polynucleotide is to be used as a primer in PCR and/or sequencing studies, 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.
If 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. If desired, either the test or sample polynucleotide may be immobilised. Generally 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. Preferably the test polynucleotide sequence is at least 200 bases in length and may even be several kilobases in length. Thus, either 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) . This can be followed by rinsing of the support at the same temperature but with a buffer having a reduced SSC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such 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. Thus, 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.
The term "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.
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. Typically 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 4βC 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.
When oligonucleotides of generally less than 30 bases in length are used in sequencing and/or PCR studies, 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. In the case of PCR studies 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.
It is possible when conducting PCR studies to predict an expected size or sizes of PCR product (s) obtainable using an appropriate combination of two or more oligonucleotides, based on where they would hybridise to the sequences SEQ ID Nos. 1-902. If, on conducting such a PCR on a sample of DNA, a fragment of the predicted size is obtained, then this is predictive that the DNA encodes a homologous sequence from a test organism.
The partial sequences of the essential genes, identified as SEQ ID Nos. 1-902, 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. Finally, some 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.
Additionally the class heading "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.
Additionally a number of sequences represented in Table 1 have now been found to relate to sequences which were previously published as being associated with sequences from genes known to be essential. These sequences are now shown in bold in Table 1. Genes described herein generally fall into two classes:
A. Genes encoding proteins with recognizable similarity to proteins of known functional class e.g. protein kinases, neurotransmitter receptors; and
B. Genes encoding proteins of unknown function.
Among this set of genes are included genes with exact or a high degree of homology to known Drosophila Expressed Sequence Tags (ESTs) 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.
The 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:
1. GENES ENCODING RECEPTORS AND ASSOCIATED PROTEINS
SEQ ID Nos. 2. 3. 38, 157, 468, 471, 532, 538, 670 and 860.
2. GENES ENCODING CHANNELS
SEQ ID Nos. 39, 524, 798, 854, 864 and 896.
3. GENES ENCODING MEMBRANE PROTEINS (GENERAL; excluding receptors and channels
SEQ ID Nos. 6, 11, 13, 31, 199, 253, 299, 441, 448, 450, 559, 579, 581, 604, 717, 804, 813 and 856
4. GENES ENCODING KINASES including protein kinases
SEQ ID Nos. 62, 65, 66, 147, 185, 287, 332, 379, 393, 471, 486, 497, 547, 551, 583, 638, 685, 707 and 888.
5. GENES ENCODING PHOSPHATASES SEQ ID Nos. 297, 420, 659 and 720. 6. GENES ENCODING GENERAL CELL SIGNALLING PROTEINS
SEQ ID Nos. 1, 22, 23, 69, 85, 102, 132, 159, 183, 229, 234, 313, 414, 421, 548, 625, 696, 715, 770, 771 and 885.
7. GENES ENCODING NTPases, including ATPases and GTPases SEQ ID Nos. 11, 21, 24, 31, 40, 117, 227, 296, 356, 515 and 803.
8. GENES ENCODING INTRACELLUR STRUCTURAL, ORGANELLAR (except mitochondrial) AND SECRETORY SYSTEM PROTEINS
SEQ ID Nos. 29, 51, 55, 71, 195, 263, 301, 305, 333, 457, 560, 637, 743 and 753.
9. GENES ENCODING HEAT SHOCK PROTEINS
SEQ ID Nos. 41, 45, 49, 52, 64, 426 and 649.
10. GENES ENCODING DNA AND RNA ASSOCIATED PROTEINS (except transcriptional activators and repressors)
SEQ ID Nos. 5, 10, 20, 37, 72, 217, 274, 301, 303, 328, 364, 369, 394, 404, 466, 469, 516, 523, 549, 635, 636, 698, 725, 767, 802, 816, 818, 862, 893 and 898.
11. GENES ENCODING TRANSCRIPTION FACTORS, ACTIVATORS, REPRESSORS AND ASSOCIATED PROTEINS
SEQ ID NOS. 58, 61, 93, 411, 444, 451, 456, 492, 554, 633, 648, 684, 729, 831, 867 and 890.
12. GENES ENCODING PROTEIN SYNTHESIS, PROTEIN HANDLING AND ASSOCIATED PROTEINS
SEQ ID Nos. 15, 46, 47, 127, 243, 261, 321, 360, 367, 460, 558, 560, 759, 845 and 850.
13. GENES ENCODING PROTEIN MODIFICATION PROTEINS SEQ ID Nos. 67, 244, 484, 776 and 873. 14. GENES ENCODING GENERAL PROTEIN DEGRADATION PROTEINS (except proteases)
SEQ ID Nos. 90, 116, 309, 326, 405, 611 and 765.
15. GENES ENCODING PROTEASES
SEQ ID Nos. 231, 346, 441, 485 and 742.
16. GENES ENCODING PROTEINS INVOLVED IN METABOLISM
SEQ ID Nos. 27, 34, 56, 83, 89, 108, 114, 121, 148, 188,
193, 196, 223, 230, 248, 278, 311, 312, 329, 353, 387, 416,
428, 435, 479, 521, 543, 564, 595, 597, 619, 631, 655, 677,
711, 716, 724, 726, 727, 738, 763, 773, 810, 825, 849, 862, 863, 866 and 892.
17. GENES ENCODING MITOCHONDRIAL AND ASSOCIATED PROTEINS SEQ ID Nos. 12, 16, 32, 35, 40, 75, 118, 225, 514 and 842.
18. MISCELLANEOUS GENES CORRESPONDING OR HOMOLOGOUS TO KNOWN GENES OF DROSOPHILA OR OTHER ORGANISMS (other than the genes corresponding to the SEQ ID Nos listed for categories 1 to 17 above) .
SEQ ID Nos. 4, 7, 8, 14, 18, 19, 26, 28, 42, 43, 44, 48, 54, 57, 60, 63, 68, 70, 73, 74, 78, 80, 87, 92, 95, 111, 134, 154, 161, 162, 168, 209, 213, 239, 246, 275, 276, 281, 295, 300, 304, 320, 322, 334, 335, 348, 358, 371, 372, 381, 389, 390, 410, 419, 442, 470, 499, 500, 566, 568, 621, 624, 632, 639, 654, 693, 695, 700, 709, 713, 730, 736, 745, 761, 766, 770, 790, 796, 829, 848, 853, 858, 863, 874, 881 and 901.
19. SEQ ID Nos corresponding to ESTs of Drosophila or other organisms for which no other information is available
SEQ ID NOS. 76. 77, 79, 81, 82, 84, 86, 88, 94, 96, 99, 101, 103, 104, 105, 106, 107, 110, 112, 113, 115, 119, 120, 122, 123, 124, 125, 126, 128, 129, 130, 131, 133, 135 to 146, 149 to 153, 160, 165, 166, 167, 169-181, 184, 186, 189 to 192, 194, 197, 198, 200, 201, 205 to 208, 211, 212, 214 to 216, 219 to 222, 224, 226, 228, 232, 235 to 238, 241, 242, 245, 247, 250 to 252, 254 to 260, 262, 264 to 273, 277, 280, 282 to 285, 288 to 294, 306 to 308, 310, 316 to 319, 323 to 325, 327, 330, 331, 336 to 345, 347, 349 to 352, 354, 355, 357, 359, 361 to 363, 365, 366, 368, 370, 373 to 376, 382 to 386, 388, 392, 395 to 403, 406 to 409, 412, 413, 415, 419, 422 to 424, 427, 429, 443 and 462.
20. ALL SEQ IDs NOT IN CATEGORIES 1 - 19 ABOVE CORRESPOND TO DROSOPHILA GENOMIC SEQUENCES WITHIN OR ADJACENT TO NEW GENES WITH NO CURRENT INFORMATION ON FUNCTION OR GENE EXPRESSION.
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.
Assays for proteins of unknown function typically rely on assessment of ligand binding only, but other assays based on disturbance of chemical levels are well known to those of skill in the art.
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. Thus, where the purpose of the assay is selection of insecticides, chemicals will be sought that interfere with the protein to modulate activity of the protein.
For proteins of known function with available functional assays, application of these assays will rapidly select a set of chemicals having the desired effect on the protein in the appropriate assay system.
In a second step, each member of the set of chemicals may then be tested directly for killing activity on insects. Drosophila itself is a convenient assay insect. In 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" 10M) may be used. After a defined treatment, flies are returned to normal conditions and observed. Rate of killing and percentage lethality are the parameters assessed.
In a third step, compounds with very effective killing activity on Drosophila may then be tested on pest species or accepted model pest related insects. Such 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) . For example, 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. As another example, lepidopteran pest larvae may be maintained on artificial media or plant leaves, which are treated with defined doses of chemicals, and survival is assessed.
The provision of candidate chemicals for use in the present invention are well known to those skilled in the art. For example 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.
For proteins of unknown function, 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. The use of 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.
An alternative or additional procedure is to use a cellular killing assay as an intermediate step. For example, 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. In many cases, 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. Alternatively, permanent cell lines of any suitable origin may be transfected, and lines expressing the protein permanently selected. In many cases, 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 . To enable effective purification of the protein, a vector may be used that incorporates an epitope tag (or other "sticky" extension such as His6) onto the protein on synthesis. A number of such vectors and purification systems are commercially available. 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) .
It will be understood that for the particular polypeptides embraced herein, natural variations such as may occur due to polymorphisms, can exist between individuals or between members of the family. These variations may be demonstrated by (an) amino acid difference (s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives showing the recognised modulatory activity are included within the scope of the invention. For example, for the purpose of the present invention conservative replacements may be made between amino acids within the following groups:
(I) Alanine, serine, threonine;
(II) Glutamic acid and aspartic acid;
(III) Arginine and leucine;
(IV) Asparagine and glutamine;
(V) Isoleucine, leucine and valine;
(VI) Phenylalanine, tyrosine and tryptophan
Moreover, recombinant DNA technology may be used to prepare nucleic acid sequences encoding the various derivatives outlined above.
As is well known in the art, the degeneracy of the genetic code permits substitution of bases in a codon resulting in a different codon which is still capable of coding for the same amino acid, e.g. the codon for amino acid glutamic acid is both GAT and GAA. Consequently, it is clear that for the expression of polypeptides from nucleotide sequences shown in SEQ. ID Nos. 1-902 or fragments thereof, use can be made of a derivative nucleic acid sequence with such an alternative codon composition different from the nucleic acid sequences shown in SEQ. ID Nos. 1-902.
The polynucleotide fragments of the present invention are preferably linked to regulatory control sequences. Such 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. Thus, 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. Such hybrid molecules are preferably derived from, for example, plasmids or from nucleic acid sequences present in bacteriophages or viruses and are termed vector molecules.
Specific vectors which can be used to clone nucleic acid sequences according to the invention are known in the art (e.g. Rodriguez, R.L. and Denhadt, D.T., Edit., Vectors: a survey of molecular cloning vectors and their uses, Butterworths, 1988, or Jones et al . ,Vectors: Cloning Applications: Essential Techniques (Essential techniques series) , John Wiley & Son. 1998) .
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", as used herein, 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 present invention will now be described by way of non-limiting example and with reference to the attached sequence listing.
Example 1 - Generation of fly lines
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 : β-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.
In addition to the reporter gene, 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 β-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; Ε>{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. Omar, R.D.C. Saunders, M. Pal, O. Komonyi, J. Szidonya, P. Maroy, Y. Zhang, M. Ashburner, P. Benos, C. Savakis, I. Siden-Kiamos, C. Louis, V.N. Bolshakov, F.C. Kafatos, E. Madueno, J. Modolell and D.M. Glover, 1998 P element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: Correlation of physical and cytogenetics maps in chromosomal region 86E-87F. Genetics 14: 1697 - 1722).
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 ι (CS) strain was derived by backcrossing w1118 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.
Example 2 - Plasmid Rescue and cDNA Cloning
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 3P-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, 100μg/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.
Example 3 - DNA Sequencing
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-2μg 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)
2) 51 primer 5 ' -GCCACCTGACGTCTAAGAAACC-3 • sequences the rescued chromosomal DNA from a point in the P-element vector 5' to the EcoRI site, in a reverse orientation to Primer 1 (ie the 3' end of the chromosomal DNA)
NB. Sequence obtained from Primer 2 is only included in those sequences where the combined sequence runs yielded the complete insert of a particular clone.
The reactions were run on 5% polyacrylamide sequencing gels in 373A STRETCH PE Biosystems automated sequencer. Greater than 900 separate lethal/semi-lethal fly lines were identified by sequencing. The sequence obtained from these fly lines is represented in SEQ 10 Nos. 1-902.
DNA sequence analysis/storage was performed using Genejockey II and BLAST (Biosoft, Cambridge, UK)
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.
Example 4 - X-Gal Staining of Tissues
The procedure for X-gal staining of embryo is essentially as described by O'Kane (1998) . 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. in PBS and 0.1% Triton X-100, and resuspended in staining buffer with 0.2% X-gal for 1-2 hours at 37°C. After staining, staining solution are removed and about 400μl of mixture solution (Glycerol : staining buffer = 2:1) are replaced. Embryos can then be mounted on a slide in a coverslip chamber.
For whole-mounts, 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) .
To obtain sections, flies were mounted in "fly collars" (modified from Heisenberg and Bδhl 1979) , soaked in OCT embedding medium (Miles, USA) for 10 min and then embedded in the OCT medium. 12 μm 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.
Example 5 - In situ Hybridisation to Polytene Chromosome
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.
Tagging the genes with a 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)/H202. After hybridization, the slides were stained with Giemsa and mounted using DPX which is a sliding mount commonly used by those skilled in the art.
As mentioned previously, the genes comprising any of the sequences disclosed herein may be cloned to allow expression of the associated protein and testing in an assay.
There now follows a non-exhaustive list of the types of assays which may be employed to test the ability of a candidate pesticide or therapeutic agent. In addition to the assays mentioned herein, the skilled addressee will be immediately aware of general texts such as Methods in Enzymology which is incorporated herein by reference. Additionally unless specifically mentioned the assays may be conducted at between 0°C-40°C, such as 4°C-35°C, or 20°C- 30°C. Additionally the assays may be conducted at or around physiological pH.
1. Binding assays for proteins of unknown function
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.
In binding assays 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. After incubation to allow molecular interaction, and a wash step, 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"10M, such as 10"3 - 10"8M
In the assay taught herein, 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. Alternatively, the protein may for example be biotinylated, and the same support materials derivatised with streptavidin (which has a very high affinity for biotin) used. Further, 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. However, 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. In such an assay, chemicals are assessed for displacement or reduction in binding of the antibody. Remaining bound antibody is detected by a standard fluorescently labeled second antibody.
2. Competitive Binding assays for proteins
For proteins belonging to families for which chemical or peptide ligands can be predicted, 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. In general, 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. In a parallel tube, 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
1. Kinases
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.
Step 1 can also be carried out with either the enzyme or the substrate immobilized on a solid support. For example, 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) . Typical assay of protein kinase activity
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. For example in a reaction volume of lOOμl containing 30 mM HEPES (pH 7.5), 5 μM MgCl2, 40 μg of histone, lOOμM CaCl2, 10 μM [γ-32P] ATP and 1.25 mg/ml phosphatidylserine. 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. After the addition of Laemmli sample buffer the sample is resolved on a 10% SDS-PAGE gel; the gel is dried and then autoradiographed. The amount of incorporated nucleotide is then determined using either autoradiography or phosphoimaging. It may be advantageous to add co-factors known to those skilled in the art that activate a particular kinase e.g. Calcium-dependent kinases would require calcium within the assay.
Such an assay is described for example in Wilkinson SE, Parker P and Nixon JS (1993) Biochem. J. , 294, 335-337. Further reference is made to Methods in Enzymology, 200:pp85-158,1991. 2. Protein Phosphatases
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.
Typical assay for protein phosphatase activity
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.
3. Adenylyl cyclases - cAMP formation from ATP
Measurement of intracellular cAMP.
Cyclic adenosine 3', 5' monophosphate (cAMP) can be measured in tissue slices, dissociated tissue, cultured cells and membrane preparations.
Two procedures are currently used for measurement of cAMP: (1) radioimmunoassay and (2) the cAMP binding protein method
Radioimmunoassay uses antibody raised to acetylated cAMP and involves competition between cAMP in the sample and 125I-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 3H-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. The Biotrack™ assay (Amersham Pharmacia Biotech) is an enzyme immunoassay in which the sample cAMP and peroxidase-linked cAMP compete for binding to antibody raised against acylated cAMP.
Measurement of adenylyl cyclase activity.
Adenylyl cyclase catalyses the formation of cAMP from ATP in the presence of Mg2+. 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.
Assay for adenylyl cyclase using ot32P-labelled ATP: Radioactively labelled cAMP produced from α32P-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. Problems with 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 3P-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 [3H]cAMP as a recovery label allows correction for differences in the performance of the individual chromatography columns. To isolate cAMP from the adenylate cyclase reactions 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 [2P] 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 [32P] ATP binds to the alumina and the labelled cAMP is eluted using imidazole buffer. Samples are counted in 32P and 3H channels using a scintillation counter. Measurement of total [32P] 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 . Reactions contain ATP, Mg2* and/or Mn2+ , 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.
See also Methods in Enzy ology 195:pp3-21; and 288:pp326- 4. Guanylyl cyclases - cGMP formation from GTP
Measurement of αuanylate cyclase activity.
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. 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.
Assay for guanylate cyclase using [32P] GTP: This method depends on the separation of labelled-cGMP from unreacted substrate [32P] GTP by sequential chromatography (Karczewski, P. and Krause, E.G. (1978). Acta Biol . Med.
Ger. , 37, 961.). Dowex 50 cation exchange columns and alumina columns are prepared and calibrated in exactly the same way as for the separation of cAMP except that the Dowex columns should be longer. cGTP is eluted from the alumina column with 0.2M ammonium formate buffer.
Non-labelled guanylate cyclase reactions. Reactions contain GTP, Mn2+, 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) .
See also Methods in Enzymology, 195:pp345-354.
5. Phosphodiesterases - cAMP/cGMP hydrolysis
Assay of cyclic nucleotide phosphodiesterase activity.
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.
See also Methods in Enzymology, 159:pp457-470; and 159:pp685-701.
6. ATPases - hydrolysis of ATP to ADP
Assay for adenosine 5 ' -triphosphatase
Adenosine 5 '-triphosphatases (ATPases) catalyse the hydrolysis of ATP to ADP and inorganic phosphate in the presence of Mg2+, Na+ and K+. The colorimetric assay quantifies the inorganic phosphate released from ATP by measuring the Aβ60nm 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) .
7. GTPases - hydrolysis of GTP to GDP
Assays essentially the same as for ATPases (see Sections 6 and 17) . Commercial kits available.
8. Proteases
General Assay for Proteolytic activity Proteases
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.
Example of a specific protease assay - Assay for the serine protease Chymotrypsin.
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.
9. Assays for secretion and import of proteins
These assays fall into three groups
A) Reconstitution in cell-free extracts
B) Reconstitution in semi-intact perforated cells
C) Assays for Endocytosis
A) Reconstitution in cell free extracts
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.
Example:- The detection of endocytic vesicle fusion in vitro using an assay based on the avidin-biotin association reaction.
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 β-galactosidase, see for example William A. Braell in "Methods in Enzymology" 219, 12-21 Academic Press inc. 1992.
B) Reconstitution using Semi-intact/perforated cells
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.
Example:- Transport of a Protein between the Endoplasmic Reticulum and Golgi compartments.
This assay is based on the expression and transport of the Vesicular stomatitis virus (VSV) G protein. 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).
C) Assays for Endocytosis
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 125I-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. Upon vesicle fusion 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)
10. Ribo/deoxyribo-nucleases - endo/exo-nuclease activity
Deoxyribonuclease
An endonuclease with preference for DNA. Pancreatic DNAse I yields di- and oligo-nucleotide 5' phosphates, pancreatic DNAse II yields 3' phosphates. In 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
Widely distributed type of enzyme that cleaves RNA. May act as endonucleases or exonucleases depending upon the type of enzyme. Generally recognise target by tertiary structure rather than sequence. 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.
Endonuclease
One of a large group of enzymes that cleave nucleic acids at positions within the chain. Some act on both 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.
Nuclease
An enzyme capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids.
Restriction Endonuclease
Class of bacterial enzymes that cut DNA at specific sites. In bacteria their function is to destroy foreign DNA, such as that of bacteriophages (host DNA is specifically modified at these sites) . 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. Because 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. For example 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.
Ref: Definitions taken from the Dictionary of Cell Biology (Second Edition), Academic Press.
General Assay
All of the above 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.
11. DNA metabolism - ligase, topoisomerase, etc
DNA glycosidase
Class of enzymes involved in DNA repair. They recognise altered bases in DNA and catalyse their removal by cleaving the glycosidic bond between the base and the deoxyribose sugar. At least 20 such enzymes occur in cells. DNA l igase
Enzyme involved in DNA replication. 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.
DNA methylation
Process by which 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/RNA synthesis
DNA polymerase and RNA polymerase are enzymes involved in template-directed synthesis of DNA from deoxyribonucleotide triphosphates and RNA from ribonucleotide triphosphates.
Repair nuclease
Class of enzymes involved in DNA repair. It 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
An enzyme capable of altering the degree of supercoiling of double-stranded DNA molecules. Various topoisomerases can increase or relax supercoiling, convert single-stranded rings to intertwined double-stranded rings, tie and untie knots in single stranded and duplex rings, catenate and decatenate duplex rings. Topoisomerase II of E. coli is commonly known as gyrase.
general Assay
All of the above act to modify the structure of DNA. For each enzyme involved in DNA metabolism, a corresponding assay is available commercially.
12. Transcription factors
Transcription Factor Assays
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 β-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.). Of the many different strategies available for using genetic reporters, 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 above is an in vivo transcription factor assay requiring the transfection of an appropriate cell line with the reporter vector. However, an in vitro method for transcription/DNA binding factor analysis also exists.
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 32P 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.
The process of 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.
In the early stages of apoptosis, changes occur at the cell surface and plasma membrane. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, where PS becomes exposed at the external surface of the cell. 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. Active 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. The biochemical hallmark of apoptosis is the fragmentation of genomic DNA, an irreversible event that commits the cell to die and occurs before changes in plasma membrane permeability.
In concert with increased understanding of the physiological events that occur during apoptosis, a number of assay methods have been developed for its detection. These assay methods can measure one of the following apoptotic parameters:
1. Fragmentation of DNA in populations of cells or in individual cells, in which apoptotic DNA breaks into different length pieces.
2. Alterations in membrane asymmetry. PS translocates from the cytoplasmic to the extracellular side of the cell membrane.
3. Activation of apoptotic caspases.
4. Release of cytochrome C into the cytoplasm by mitochondria.
Each provides the researcher with a different level of information as each of these events occurs at a different stage in apoptosis.
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.
See also general reference Methods in Enzymology 322 pp3- 522.
14. Calcium
Calcium dynamics
In a multicellular organism, cell communication is essential to regulate the different activities of specialised tissues. In all animal cells, there are conserved intracellular second messenger pathways. For many of these, calcium is an important second messenger. In nerve cells, muscle and other cells, modulation of intracellular calcium activity from typical resting levels of 100 nM regulates many short and long-term processes. Measurement of calcium can thus be of great utility in following the responses of transgene products to applied pharacological agents including insecticides.
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.
For example, 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.
Additionally, fluorescent probes (such as fura-2, indo- 1, quin-2) show a spectral response upon binding calcium and it is then possible to detect changes in intracellular free calcium concentrations using 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 called "cameleons" 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. 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.
Additionally, 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).
15. CAMP
Effects on dynamics of intracellular cAMP as reported by appropriate dyes or reporter constructs (eg. aequorin) . See section 13.
16. Voltage
Analysis of 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.
Patch clamping
In brief, 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. It is also possible to isolate single ion channels, providing information on the unitary conductance and kinetic behaviour of individual channels, and allow the factors which alter these properties to be studied in exquisite detail (Crawley et al., 1997 Neurophysiology Current protocols in neuroscience Volume 1 [John Wiley and Sons, Inc.] ). Patch Clamp techniques are widely used and cited throughout scientific literature (Siegel M.S and Isacoff E.Y (1997) Neuran 19_, 735 - 741; Sensi S., Canzoniero L.M, Yu S.P, Ying H.S, Koh J.Y, Kerchner G.A, Choi D.W. (1997) J. Neuroscience 12, 9554 - 9564; Piller S.C, Jans P., Gage P.W, Jans D.A (1998) Proc. Natl. Acad. Sci. USA, 9_5_, 4595 - 4600; Marie D. , Marie I., Wen X., Fritschy J.M, Sieghart W. , Barker J.L, Serafini R. , (1999) J. Neuroscience 19 , 4921 - 4937) . An example of how this kind of analysis may be used is outlined below.
Whole cell patch clamp recording to study the effects of a viral protein on whole cell currents of cultured hippocampal neurons .
Whole cell currents represent the integrated channel activity over the whole cell. Cultured cells on coverslips were perfused with bath solution (140 mM NaCl, 5 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM glucose, 10 mM TES [pH7.3]) at room temperature (23 to 28C) . Pipettes made from boroscilate glass were fire polished and filled with pipette solution normally containing 150 mM NaCl, 0.5 mM CaCl2, 2 mM MgCl2, 5 mM EGTA and 10 mM TES [pH7.3]. 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 β 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 . 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. 273f 18230 - 18234).
In the study by Salvador et al., 1998, transmembrane electrical potential was measured by analysis of the differential absorption (625 - 603 nm) of 2 μM oxonol using dual wavelength spectrophotometry. The medium comprised Pipes buffer, pH7.1; 0.42 μg/ml calmodulin; 5mM MgCl2 and 2 μM Oxonol VI. The callibration was performed by several additions 130 mM KCl to the medium in the presence of 1 μM 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.
Dall'Asta et al., 1997 visualize changes in membrane potential using Bis-oxonol. 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.
17. Receptors/ion channels
Ion Channels/Receptors
Neuronal signaling depends on rapid changes in the electrical potential difference across nerve cell membranes. These rapid changes in potential are made possible by 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.
Several major classes of ion channels have now been identified. Primary sequence information has been used to suggest the structure of different channel proteins. Efforts to determine secondary structure rely on X-ray crystalography. However, additional information can be obtained by comparing the primary amino acid sequence of related channels from different species and identifying regions of sequence homology, suggesting the importance of such regions in channel structure and function. Further insight into structure-function relationships can be obtained from sequence homologies among different, but related, channels. Such homologous regions are likely to underlie a common biophysical function shared by the different channels, i.e. Voltage-gated versus transmitter- gated channels.
The flux of ions through ion channels is passive, requiring no expenditure of metabolic energy. The direction and eventual equilibrium for this flux is determined not by the channel itself, but rather by the electrochemical driving force across the membrane. 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*, Ca2*, and Mg2*. However, most cation-selective channels are more selective; each one is permeable primarily to a single type of ion, either Na*, K*, or Ca2*. All known types of anion-selective channel are permeable to Cl". Note that the Ca2+ influx controlled by channels can alter many metabloic processes within cells, leading to activation of various enzymes and proteins. Ca2+ influx also acts as a trigger for the release of neurotransmitter.
The activity of 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.
Molecular analysis: starting with an unknown chemical for which no information is available, and depending on the size of the starting molecule, 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. And Barnard E.A (1987) Nature 328, 221 - 227) . From peptide sequence, it is possible, by back-translation, to identify the nucleotide sequences from which the peptide may be translated, and based on the codon usage of a particular organism, best-guess oligonucleotides can be synthesized and used to screen species-specific cDNA libraries. Any cDNA clones identified can then be sub- cloned, sequenced and the primary sequence analysed for known sequence homologies with BLAST database searches. The full-length sequence, cDNA and corresponding expressed protein, can then be subjected to standard biochemical and molecular characterisation procedures.
Functional analysis : 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.
Two main gene families;
1. Voltage gated channels: Na*, K* and Ca2*
2. Ligand-gated ion-channel receptors: cation: (nACh) , either Integral (pore forming, 5HT, glutamate, Ion gating) or Second messenger anion: GABA systems (associated) Glycine
Methods
1. cloning: sequence analysis primary and secondary
2. structure: crystalography, in situ, immunocyto, immunohisto, immunoEM
3. function: electrophysiology: slice culture/patch clamp transfection/patch clamp Voltage-Gated Channels
In nerve cells at rest (membrane potential: -65mV) , the steady Na* influx through non-gated channels is balanced by steady K* efflux, so that the membrane potential is constant. This steady state balance changes when the cell is sufficiently depolarised to trigger an action potential. A transient depolarising potential, such as excitatory synaptic potential, causes some voltage-gated Na* channels to open, and the resultant increase in membrane Na* permeability allows Na* influx to outstrip the K* efflux. Thus, a net influx of positive charge flows through the membrane, and positive charges accumulate inside the cell, causing further depolarisation. The increase in depolarisation causes more voltage-gated Na* channels to open, resulting in a greater influx of positive charge, which accelerates the depolarisation further.
This regenerative, positive feedback cycle develops explosively, driving the membrane potential toward the Na* equilibrium potential of +55mV. Because K* efflux continues through the K* channels, the membrane potential never actually reaches the equilibrium potential of sodium. A slight diffusion of Cl" into the cell also counteracts the depolarising tendency of the Na* influx.
As depolarisation continues, it slowly turns off, or inactivates, the voltage-gated Na* channels. That is, the Na* channels have two types of gating mechanisms: activation, which rapidly opens the channel in response to depolarisation, and inactivation, which slowly closes the channel if depolarisation is maintained. The second repolarising process results from the delayed opening of voltage-gated K* channels. The delayed increase in K* efflux combines with a decreased Na* influx to produce a net efflux of positive charge from the cell, which continues until the cell has repolarised to its resting membrane potential. Intracellular recording: this technique uses two glass capilliary electrodes full of an ionic conductor solution (usually 3M KCl) . To measure the resting membrane potential, 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) .
Ligand-Gated Channels - Integral Channel
(e.g., nicotinic acetylcholine (nACh) receptor, 5 hydroxytryptamine (serotonin) (5HT3) receptor, glutamate, GABAA, 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 107 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. There are many examples of these in the literature, and of the differences in receptor characteristics when different combinations of receptor subtypes are expressed. Initial assays established by the inventors will focus on the most clinically relevant subtype (s) of each receptor. With these permanent cell lines, 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.
Lioand-Gated G-protein linked Receptors
(e.g., mACh, 5HT, GABAB, Glutamate, Dopamine, etc)
Many cell surface receptors are coupled to 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) .
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.
18. 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.
Heterotrimeric G proteins are a large family of GTPases which consist of an , a β 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 αβy 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 βy complex also has also been show to have some regulatory activity.
Small (p21) GTPases
These proteins consist of a single subunit similar to the α subunit of heterotrimeric G proteins. These include the RAS family of proteins the abnormal activity of which can contribute to tumour formation.
Other GTP binding proteins
Other members of the Guanosine nucleotide binding protein superfamily include GTP binding translation elongation factors and members of the Dynamin family of proteins.
Use of Recombinant G proteins
Expression of recombinant 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.
Active recombinant 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 35S methionine to the in-vitro translation reaction results in the production of specifically labelled protein.
It is also possible to express the proteins in cultured cells and look for whole cell effects such as increased cell proliferation, increased DNA synthesis or changes in the activity of various enzymes. The use of cell lines lacking G protein subunits.
Several cell lines have been isolated, or made using gene-disruption techniques, which lack particular G protein subunits. The most widely used of these is the eye" variant of the S49 mouse lymphoma cell line lack the Gs subunit. It is possible to add back recombinant or purified G proteins to investigate their function. Purified or in- vitro translated protein can be added back to membrane preparations from the cell lines or the cells can be transfected with plasmid constructs which express the protein.
GTP binding assays
The nonhydrolyzable GTP analogue 35SγGTP 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 35SγGTP and the reaction products passed through a nitrocellulose filter. Protein bound 5SγGTP will be retained on the filter and the activity measured (Carty D.J. and Iyengar R. (1994). Methods in Enzymology. 237: 38-45.) .
GTPvS activation
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 35SγGTP 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.
35SγGTP 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.
As an alternative to measuring the binding of 35SγGTP 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. γ32P GTP is added to a reaction mix containing a membrane preparation of cells and the amount of 32P released from the labelled GTP is measured.
See also Methods in Enzymology, 195:ppl71-474. SEQ ID NO. IDNumber Class Chr Feature AccNo Name of match
1 NPS1 GNL 2 4-133 X54648 Frizzeled gene
2 NPS2 GNL 2 68-345 D17389 Ryanodyne receptor
3 NPS3 GNL 2 1 -354 M23412 Muscarinic acetylcholine receptor. Genomic AC006938 intron.
4 NPS4 GNL 2 9-277 U91980 Tpr homolgue
5 NPS5 GNL 2 1 -587 X61209 Type II topoisomerase
6 NPS6 GNL 2 270-408 U22439 Neuron surface antigen 2
7 NPS7 GNL 2 30-461 INV Y13272 Indora
8 NPS8 GNL 2 1 -267 L03209 GDP dissociation inhibitor homologue
9 NPS9 GNL 2 345-583INV L17340 germlinβ transcription factor gene
10 NPS10 GNL 2 1 -480 AB003784 Histone H3
1 1 NPS1 1 GNL 2 179-360 AA699128 EST matching 5' of V-ATPase C subunit
12 NPS13 GNL 2 226-409 U94702 MtPolB
13 NPS14 GNL 2 1 10-191 L13305 AND 396-472,integrin beta subunit {beta neu)
14 NPS15 GNL 2 1 -432 X57484 tra-2 gene
15 NPS16 GNL 2 61 -276 X15805 EF2 Translation factor
16 NPS18 GNL 2 1 -532 X15008 49bp upstream of TU-36B gene, cytochrome b related protein.
17 NPS19 GNL 2 250-536 M29602 GO protein alpha subunit homolog class II
18 NPS20 GNL 2 81 -476 L13255 Lachesin
19 NPS21 GNL 2 1 19-457inv U63556 larval serum protein 1 beta subunit
20 NPS22 GNL 2 121 -417 AF027300 418-481 intron,481 -577exon.Positive transcription elongation factor b
21 NPS23 GNL 2 1 -577 X84681 organellar-type Ca-ATPase gene.
22 NPS26 GNL 2 1 -534 X71866 GTP-binding protein.
23 NPS27 GNL 2 1-523 M23094 Intron of G protein alpha subunit gene
24 NPS28 GNL 2 19-215 AF0 1048 AA246996 est match.EST matches CD39-like NTPase gene
25 NPS30 GNL 2 387-473inv AF071 17 phosphatidylinositol 4-phosphate 5-kinase, sktH
26 NPS31 GNL 2 1 -319 S55886 rbp9
27 NPS32 GNL 2 1 -493 L34276 manganese superoxide dismutase (mnSOD)
28 NPS33 GNL 2 233-377inv D84313 rab2
29 NPS34 GNL 2 1 -63inv AF003826 myosin V
30 NPS35 GNL 2 325-528 U09369 ribonucleoside-diphosphate reductase large subunit gene
31 NPS36 GNL 3 234-271 U95821 transmembrane GTPase (fzo)
32 NPS38 GNL 3 322-450 U00669 mitochondrial single-stranded DNA-binding protein
33 NPS39 GNL 3 14-385 X52846 RM62
34 NPS40 GNL 3 1 -422inv AF069297 pterin-4a-carbinolamine dehydratase gene
35 NPS41 GNL 3 329-346 Y09065 330-414 intron, 415-51 1 exon.cytochrome c oxidase subunit Va preprotein
36 NPS42 GNL 3 1 -283 M22428 Ubiquitin
NPS44 GNL 3 1 -380 M17719 Intron of Rhodopsm 4 and Ml 7730
NPS45 GNL 3 1 -449 U27561 TipE
NPS46 GNL 3 463-528 X99665 mitochondrial ATPase coupling factor 6. Match on EST AI405330
NPS47 GNL 3 1 -246 K01294 heat shock locus 87C1 : proximal gene, 3' end.
NPS48 GNL 3 221-318 U73160 AA440389 EST matching Dros fas gene
NPS49 GNL 3 15-95 M32141 AI297861 1 st EST in 8 contig matches 49-kilodalton phosphoprotein gene
NPS50 GNL 3 231-293 M2 159 Tcp-1
NPS51 GNL 3 1 -349 V00213 Hsp70
NPS52 GNL 3 1 -241 U59923 glutamyl-prolyl-tRNA synthetase gene,
NPS53 GNL 3 225-237 D16257 238-333 intron, 334-499 exon ribosomal protein S4
NPS54 GNL 3 1 -462 X73216 Rlb1
NPS55 GNL 3 1 -164inv U62005 Rel/NF-kappa B homolog (Relish)
NPS56 GNL 3 1 -207inv X0731 1 HSP2
NPS57 GNL 3 15-438 X54061 205K microtubule-associated protein (MAP)
NPS58 GNL 3 1 -80inv J01 102 HSP68
NPS59 GNL 3 1 -450 Y10015 anon-66Da gene
NPS60 GNL 3 56-187 M63792 RAD6
NPS61 GNL 3 391 -465 U28966 Septin 2
NPS62 GNL 3 1 -514 M98351 fructose 1 ,6 bisphosphate aldolase gene,
NPS63 GNL 3 46-251 inv U01035 Bottleneck gene
NPS64 GNL 3 49-450 U38238 HLH106
NPS65 GNL 3 328-581 AB004232 DAD gene
NPS66 GNL 3 1 -436 U22176 15bp upstream of Brother gene on AC005557
NPS67 GNL 3 46-176 M90755 Transcriptional repressor protein Aef-1
NPS68 GNL 3 224-298 Y07908 Match to EST AI292767. This then matches serine/threonine protein kinase.
NPS69 GNL 3 1 -531 M3231 1 Fascilin 1
NPS70 GNL 3 1 -421 inv X03889 HSP23
NPS71 GNL 3 548-882inv Y12861 bifunctional ATP sulfurylase/APS 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
NPS75 GNL 3 468-539 U23485 Guanylate cyclase. Match found via EST AA392994
NPS76 GNL 3 1 -547 Y1 1349 UbcD4
NPS77 GNL 3 1 -163 U09374 SNAP
NPS78 GNL 3 1 -104inv U62388 chromatin assembly factor 1 p55 subunit
NPS79 GNL 3 374-518inv AB007692 Elongin B
NPS80 GNL 3 1 -231 L06861 232-401 intron, 402-590 exon matching TAF1 10
76 NPS82 EST 2 509-591 AA202837 hypothetical yeast/arabidopsis/prot and mouse EST
77 NPS83 EST 2 166-393 AI293734
78 NPS84 EST 2 261 -377inv AA202757 Match to Human EST
79 NPS86 EST 2 1 -247 AA696498
80 NPS87 EST 2 100-646 AA950073
81 NPS89 EST 2 1 -50inv AA695104
82 NPS91 EST 2 1 -427 AA942153
83 NPS92 EST 2 42-334 AA540352
84 NPS93 EST 2 1 15-162 AI238523
85 NPS97 EST 2 1 -69inv AI260872 EST matches mouse signalling factor U29156
86 NPS98 EST 2 5-77bp AA801728
87 NPS99 GNL 2 228-675 AF053083 Drosophila SMT3 gene
88 NPS100 EST 2 1 -210inv AA439866
89 NPS105 EST 2 31 -590 AA820803 Poss related to human aldolase
90 NPS106 EST 2 30-478 AA803545 AA697132 match to frog/human MSS1
91 NPS108 EST 2 76-178 AA438591
92 NPS109 EST 2 1 -169 AA979551 also AA567400
93 NPS1 1 1 EST 2 138-414 AA439261 Match to Rat EST
94 NPS1 13 EST 2 7-354 AM 07509
95 NPS1 14 EST 2 1 -48bp AA540348
96 NPS1 15 EST 2 1 -31 1 inv AA735555 match to AC005646. 26bp 5' to EST match area. AI542218/AI25740 765bp. Def SEC61
97 NPS1 18 EST 2 1 -582 AI064020 homologue
98 NPS119 EST 2 7-170 AA263700 also AA978721
99 NPS120 EST 2 364-583 AA941785 also AA695548
100 NPS121 EST 2 1-260 and 562-645 AA802928 also AA817115
101 NPS122 EST 2 1 -395 AA539001
102 NPS123 EST 2 1 -35inv AA735863 Poss. related to human death assoc prot 3 X83544
68-195 and
103 NPS125 EST 2 AA941860 475-621
104 NPS127 EST 2 1 -210inv AA246460
105 NPS128 EST 2 66-593 AA141928
106 NPS131 EST 2 1 -332 AA979014
107 NPS134 EST 2 52-475 AA817254
108 NPS137 EST 2 1 -37bp AA536262 1209bp EST contig.AA948897. AA539274, AA392320. Poss glycogen synthase
109 NPS139 GNL 2 35-86 and 475-581 AF1 13612 Drosophila Aspartate ligase
1 10 NPS140 EST 2 368-636 AA390587
NPS142 EST 2 31 -460 AA341353
NPS143 EST 2 65-299 AA201303 also AA541066
NPS144 EST 2 538-581 inv AA6981 19 Mate to Human gycerol-3-phosphate dehydrogenase
NPS145 EST 2 1 1 1 -549 AA696174
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
Also AA820473. (AF034644) putative cytochrome bc-1 complex core protein [Haematobia
NPS149 EST 2 1 -107inv AI1 14218 irritans irritans]
19-102 and
NPS150 EST 2 A AAA997788444499 Also AA940834. 103-1 14 gap of unknown length 1 15-485
NPS152 EST 2 182-362 AA802905
235-279 and Also AI296787. Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase
NPS154 EST 2 AI259166 376-452 complex precursor (human)P10515
NPS155 EST 2 1 -238inv AA951 193
NPS156 EST 2 326-482 AA696743 Also AA803977
NPS157 EST 2 1 1 -512 AA990758 Also AA246427. 975bp contig
NPS158 EST 2 1 -406 AA697797
NPS159 EST 2 1 -29inv AA802206 1341 contig. AA202662, AA801949, AA942041
NPS160 EST 2 344-592 AA978904
101 -223 and
NPS161 EST 2 AA202366 292-551
NPS162 EST 2 103-468 AA950164
NPS163 EST 2 23-98 and 102-602 AA952159 99-101 gap of unknown length. Match to mouse EST
NPS166 EST 2 102-512 AA392519 also AA695318 and AA441243.758bp contig.
NPS168 EST 2 304-541 AI515517 also AI404462. Poss Ras related protein
NPS169 EST 2 191 -387 AA698481
NPS170 EST 2 451 -606inv AA803082 2166bp EST contig. Poss. Alt splice. AA941565, AA820668, AA978815 and AA697381
NPS1067 EST 2 1 -570 AI405762 Seq.sim to hypothetical prots from arabidopsis and C. elegans
NPS1 3 EST 2 1-38inv AA391495 1135bp contig. AA439145 and AA949325. Match to mouse EST
NPS174 EST 2 353-476 AA942305
NPS178 EST 2 72-391 AA951839 also AA979603
NPS1 9 EST 2 1 -1 12 AI386817 also AI404737
NPS1 80 EST 2 435-475 AA438658
NPS181 EST 2 31 -212 Al 106794 also AI107315
NPS1068 EST 2 1 -228 AI403747
NPS188 EST 2 1 -272 AA802791 also AA390699
NPS189 EST 2 1-190 AA949990 also AA246423
146 NPS191 EST 2 202-472 AA9/B32/ poss. ur i i p nomoiog iπomu sapieπsj 147 NPS192 EST 2 84 -318 AA541084 also AA538937 148 NPS195 EST 2 390-509 AA951890 RIR2_mouse ribonucleoside- diphosphate reductase m2 chain 149 NPS198 EST 2 1 -140 AA439230 150 NPS199 EST 2 3-522 AA948907 also AA942191 151 NPS200 EST 2 25-76inv AA802379 also AA246624
9-100 AND
152 NPS1069 EST AI404485
179-41 1
153 NPS1070 EST 2 60-449 AM 08647 154 NPS1071 EST 2 1 -49inv AA951902 other ESTs inc AA949796 155 NPS204 GNL 2 1-489 AF143860 Drosophila RanGap gene 156 NPS205 EST 2 1-278 AA940865 Xenopus/ human chromosomal assembly protein(U1367) 157 NPS206 EST 2 1 17-263 AA803314 also AA941391. Human B-cell receptor associated protein. 158 NPS207 EST 2 209-405 AA201448 856bp contig.AA438721 and AA247046 159 NPS209 EST 2 37-243 AA696343 also AA696180. Match to human/ C. elegans calponin 160 NPS210 EST 2 261 -580inv AA540783
26-267 and also AA698310. FKB4_RABIT P59 PROTEIN
161 NPS211 EST AA695850
336-459
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. 168 NPS220 EST 2 1 -184 AA390646 1705bp contig with AA440523 and AA696470 169 NPS225 EST 2 104 and 310-467 AI064169 also AA816652 170 NPS226 EST 2 1-288 AA439345 802contig with AA949877 and AA439626 171 NPS227 EST 2 1 -350 AA979503 181 bp upstream of EST Genomic AC005452 172 NPS228 EST 2 -93 and 170-446 AI293141 173 NPS229 EST 2 12-244 AM 07445 also AA390813 174 NPS233 EST 2 12-478 AA390942
1 1 -103 and
175 NPS235 EST AA802688 Poss 10k HSP
296-389
176 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
181 NPS243 EST 2 186-593 AA441247 also AA820771
1942bp contig with All 08811 , AA950029, AA202725, AA440491 , and AA697007. Match 182 NPS244 EST 2 318-431 AA202196 mouse EST also AA263284. Match to human androgen induced prostate proliferative shutoff assoc.
183 NPS245 EST 83-319inv AI064123 protein.
184 NPS247 EST 2 1 -89bp AA441 173 185 NPS250 EST 2 65-414inv AA440852 also AA541034 186 NPS251 EST 2 2-131 AI062640 187 NPS252 EST 2 1 -77inv AA695507 Poss. match to Rat cytochrome C 188 NPS254 EST 2 89-251 AA736186 also AA801973. Poss. match to horse Thioredoxin 189 NPS255 EST 2 1 -417 AA697603 also AA801716 190 NPS256 EST 2 1 -528 AA950741 191 NPS257 EST 2 1 -53bp AI063204 887bp contig with AA697347 and AA201878 192 NPS258 EST 2 1 -44bp AA441029
1 141 bp contig with AA949325, AA735675 and AA391495. Poss.match to human GMP
193 NPS259 EST 1 -157 AM 14266 synthase
194 NPS260 EST 1 -562 AA951648 1340bp contig with AA539581 , AA802940 and AA263326
26-137 and
195 NPS261 EST AA391 135 Match to SEC61 , different area to NPS 1 18
360-422
196 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
215 NPS290 EST 2 378-471 AA3&UUB4 alSO AA3 /BtJt>»
20-236 and 216 NPS291 EST 2 AI062945
292-439
2293bp EST contig. Poss. human cleavage and polyadenylation specificity factor, 160 kd
217 NPS293 EST 1 -312 AA440345 subunit.AA201536,AA539993,AA942332, AA979174 ,AA202096
218 NPS294 EST 2 10-501 AA696930
219 NPS295 EST 2 8-437inv AA440135 220 NPS296 EST 2 75-157 AI063979 also AA802032 221 NPS297 EST 2 1 -144 AA699194 222 NPS298 EST 3 507-547 AA441233 also AA392152 223 NPS299 EST 3 1 -79inv AA438352 33% over 1 13 AA Plant oxygenase 224 NPS300 EST 3 480-534 AI455428 225 NPS301 EST 3 1 1 -190inv AA246916 Rat Mitochondrial import receptor 226 NPS302 EST 3 233-348 AA392258 227 NPS304 EST 3 1 -41 inv AI296848 Prob. 40-kDa V-ATPase subunit (mam) 228 NPS305 EST 3 255-354 AI388389 229 NPS306 EST 3 335-448inv AA441471 also AA540182. 52% over 107 AA like Bov/Hum/Mouse RHO GDP-dissoc. ihibitor 1 230 NPS307 EST 3 22-242 AA439855 also AA567284.
1 -141 and
231 NPS308 EST AA941606
397-446inv
232 NPS310 EST 3 209-435 AA392324 233 NPS311 EST 3 1-393inv AA264796
234 NPS312 EST 3 1 -152 AA540030 Poss rat calcium binding prot. 235 NPS313 EST 3 85-596 AH 09898 236 NPS314 EST 3 365-473 AI259723 237 NPS316 EST 3 1 -141 AI294469 238 NPS317 EST 3 145-325 AA 140945 239 NPS318 EST 3 1 -331 AI259816 Related to Epsin (Hum) 240 NPS322 EST 3 209-433 AA141103 241 NPS323 EST 3 1 -98inv AA246767 also AA141059 242 NPS324 EST 3 1 180239inv AA441468 also AA142226. 42% over 128 AA like C. elegans prot.Z66496 243 NPS327 EST 3 1 -82inv AA247070 1366bp contig with AA567381 , AA568013, AA540724. C. elegans prot/human EST 244 NPS328 EST 3 433-469 AA802401 Prob. Alg2, glycosyltransferase horn./ Mouse MER 5 245 NPS330 EST 3 1 -96inv AH 35263 Alt splice 246 NPS331 EST 3 243-489 AA695904 247 NPS334 EST 3 1 -317 AA246386 also AA541060 248 NPS335 EST 3 31 1 -427 AA264961 57% over 82AA like mouse/ human Thioredoxin
74-276 and
250 NPS338 EST AA263803
344-438
251 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
1 -47 and 2631 bp contig.
261 NPS349 EST AA201223
145-317ivn AA538867/AA439491/AA390780/AA390983/AA201661 /AA391700/AA202007. Human 8
262 NPS351 EST 537-687 AI454966
715bpcontig with AA201231 and AA392823.
NPS352 EST 31 % over 129AA like Rat Nup84 and Human
263 10-441 AA202767 KDa nucleopore complex
264 NPS353 EST 3 3-40inv AA201212 265 NPS354 EST 3 1 -33inv AI404994 And AI260898. Alt splice 266 NPS356 EST 3 1 -292 AA539914 1042bp contig with AA201959 267 NPS357 EST 3 36-454 AA440953 268 NPS359 EST 3 145-253 AA264591 269 NPS360 EST 3 47-380 AA539491 270 NPS361 EST 3 202-381 inv AI403737 271 NPS362 EST 3 270-443inv AA5671 1 272 NPS363 EST 3 1 -478 AH 34670 273 NPS364 EST 3 413-535inv AA263763 274 NPS365 EST 3 1 -99bp AA56801 1 275 NPS367 EST 3 64-449 AH 07456 276 NPS370 GNL 3 212-414 AF074957 Drosophila Karγopherin alpha 277 NPS371 EST 3 1 -146 AI295205 and AA141054. Alt splice 278 NPS372 EST 3 8-382 AA567704
74-224 and
279 NPS373 EST AA539252
297-344
280 NPS374 EST 3 1 -347inv AI260759 281 NPS375 EST 3 1 -77inv AI260646 282 NPS377 EST 3 160-306 AA202424 and AA264609.
284 NPS380 EST 3 322-573 AA802438 I030bp contig with AiυbcSbBϊ
285 NPS381 EST 3 34-470inv AA438500
14-153 and 216-
286 NPS382 EST 3 AI456286 348 ,419-445
287 NPS383 EST 3 41 -56 and 223-353 AI062265 1475 contig. AA694862 and AI064128.UNC51 ser/thr kinase (C.elegans)
288 NPS384 EST 3 1 -429inv AA247020
289 NPS385 EST 3 1 -143 AA264635
290 NPS387 EST 3 58-491 AA201749 877bp contig with AA803278/ human hypothetical gene
291 NPS388 EST 3 1 -162 AA392551
EST contig1200bp, ORF at 5' end. Matches Human ERF 1. AA201773, AA263752 and
292 NPS389 EST 3 1-379 AA438539 AA439563
293 NPS390 EST 3 297-447 AA141715
294 NPS392 EST 3 80-161 AA695862
295 NPS393 EST 3 2-132 AA201517
296 NPS394 EST 3 176-239 AA202297 Vertebrate vacuolar ATPase
297 NPS395 EST 3 1 -33bpinv AA567483
298 NPS396 EST 3 1 -209 and 271 -468 AA817479
ATG orf hits Rat (and other) sodium dependant dicarboxylate transporter AB001321 58% ove
299 NPS397 EST 3 17-139 AA441327 74 AA
300 NPS398 EST 3 1 -391 AA69801 1
301 NPS399 EST 3 67-207 AA951986 nucleolar protein p40 [Homo sapiens]
2018contig with AI258429, AA696170, AI109519, AA391348. MouseAPG-1 , hsp/osmotic
302 NPS400 EST 3 1-186 AI295731 shock gene
303 NPS402 EST 3 1 -82bp AA201430
304 NPS403 GNL 3 15-54bp AF132912 Drosophila ARP gene. Match to EST matching ARP
305 NPS404 EST 3 1 -140 AA541045 May be distantly related to cystatin
306 NPS406 EST 3 392-501 AA390337
1 -202 and 273- Matches mouse/human ESTs
307 NPS407 EST 3 AA141555 440inv
308 NPS408 EST 3 158-252 AA263730 A little like yeast hypothetical protein YOL124c
324-370 and
309 NPS409 EST 3 AI259832 and AA990765. Human Ubiquitin conjugating enzyme 12 448-545
310 NPS410 EST 3 75-483 AI514268
20bp 5' to EST on AC006562. Part of ORF similar to molybdenum cofactor biosynthesis
31 1 NPS41 1 EST 3 1 -435 AI293256 protein A[Homo sapiens]
312 NPS412 EST 3 1 -71 and 148-435 AA201987 Poss Asparaginase
313 NPS413 EST 3 12-408 AA540020
315 NPS416 EST 3 15-404 AAzυi at)/ Mouse ta i s 316 NPS417 EST 3 1 -353 AA695344 317 NPS418 EST 3 1 -450 AA441018 318 NPS419 EST 3 301-334inv AA202301 319 NPS420 EST 3 245-393 AA735819 320 NPS421 EST 3 1 -147 AA440886 also AA695395. matches UMP kinase from C.elegans and bacteria 321 NPS422 EST 3 76-217 AA803640 859bp with AA803683 and AA803676. Human Ribosomal L28 protein
472-786 and
322 NPS424 EST AI257267 chick glycine cleavage system h protein
842-1073
323 NPS425 EST 3 1-75bp AA539327 324 NPS426 EST 3 419-468 AI530922 325 NPS427 EST 3 92-265 AI402854 326 NPS428 EST 3 1 -222 and 291 -354 AA441362 40% like human/mouse proteasome subunit HsN3 327 NPS429 EST 3 1 -219 AA202487 A little like hypothetical yeast protein YEY6 328 NPS430 EST 3 328-455 AA263590 39% over 61 AA like human hRNP F 329 NPS431 EST 3 50-1 13 AA201496 57% over 50 AA like human oxoglutarate dehydrogenase 330 NPS432 EST 3 281 -510 AA391430 331 NPS434 EST 3 1 -50 inv AI292722 also AI534704 332 NPS435 EST 3 1 -65inv AA439393 333 NPS436 EST 3 299-512inv AA820797 also AA438876 334 NPS437 EST 3 1 -52inv AA697891 Homologue of Bovine gamma COP 335 NPS438 EST 3 1 -31 inv AA696845 336 NPS439 EST 3 1-384 AI259031 337 NPS440 EST 3 1 -82bp AA803464 may match human hypothetical protein KIAA0258 338 NPS441 EST 3 169-489 AA539974 339 NPS442 EST 3 1 -46 and 432-524 AA941993 340 NPS443 EST 3 43-431 AA803074 341 NPS444 EST 3 1-197 and 268-534 AA695870 Dog/rat/ Yeast signal peptidase 18kd subunit 342 NPS445 EST 3 775-91 1 AA433251 343 NPS446 EST 3 718-1007 AI297203 1122bp contig with AA438815 and AI455195 344 NPS448 EST 3 l-106inv AA694869 1632bp with AA735812, AA568063 and AA695306. mammalian transketolasβ 345 NPS449 EST 3 1 -99inv AA392932 346 NPS451 EST 3 1 -260 and 310-510 AA391707
45-141 AND
347 NPS452 EST AI294564 Match to mouse EST
445-582
348 NPS453 GNL 345-460 AF152928 Drosophila karyophylin alpha 3. 349 NPS454 EST 1 -177 AA540743 1 129bp with AI064582, AI519458 and AA568024
351 NPS457 EST 3 1-73bp AI062939
352 NPS458 EST 3 927-1070 AH09224
353 NPS459 EST 3 43-146 AA696728 Poss. isopentyl pyrophosphate isomerase
354 NPS460 EST 3 59-533 AI518328
355 NPS461 EST 3 43-432 AA263622
356 NPS463 EST 3 1-164 AA539661 Matches Human Proton ATPase like protein
357 NPS464 EST 3 1-152 and 214-579 AI388964
358 NPS465 EST 3 365-462 AA438987 also AA264877. FXR1 mental retardation gene, Human
359 NPS466 EST 3 6-257 AA392117
45-120 and
360 NPS468 EST 3 AA821194 987bp with AA736168. human 40s ribosomal protein s29 545-591
361 NPS469 EST 3 12-469 AA539752
362 NPS473 EST 3 1-382 and 456-484 AA803203
363 NPS476 EST 3 105-154 AA802887 also AA820871. Hypothetical C. elegans prot B0336.1 1
364 NPS477 EST 3 22-177 AA817394 V. similar to Dead box familly of DNA helicases (initiation factors)
365 NPS479 EST 3 1-77inv AI064638
366 NPS480 EST 3 1-37bp AA736157 also AA140746. Match tomouseEST
367 NPS482 EST 3 7-369 AA820427
368 NPS483 EST 3 1-533 AA391736 1692bp with AA202259 and AA820861.
369 NPS484 EST 3 158-470 AA567184
370 NPS486 EST 3 1-122 AA735277 1 176bp with AA697907
371 NPS487 GNL 3 514-616 AF129080J Drosophila COP9 complex homolog subunit 1 -2 DCH1 -2
372 NPS489 EST 3 140-189 AA202581 Match to human EST
373 NPS490 EST 3 41-377 AA390775
374 NPS491 EST 3 169-488 AA539898
375 NPS492 EST 3 1-127 AA390453
376 NPS493 EST 3 1-321inv AA568061 1356bp with AA264532 and AA441674
377 NPS495 EST 3 102-311 AA141908 794bp with AA802528
378 NPS496 EST 3 1-100inv AA539224
379 NPS497 EST 3 431-622inv AA246367 Hum ribosome S6 PK
380 NPS499 EST 3 46-319 AA817295
381 NPS501 EST 3 35-383 AA439743
382 NPS503 EST 3 1-264 AA441568
337-408 and
383 NPS504 EST 3 AA247082 479-568
384 NPS505 EST 3 1-321 inv AA201685 868bp contig with AA540405
385 NPS506 EST 3 83-218 AA540693 1450bp with AA441321 , AA440080 andAA392794. Match to mouse EST
387 NPS508 EST 3 2-339 AA439667
388 NPS509 EST 3 12-394 AA539198
83-227 and
389 NPS510 EST 3 AA696927 Match to human citrin 498-518
390 NPS512 EST 3 360-501 AA438961 Human KIAA0160 gene
391 NPS513 EST 3 53-260iπv AA735138
392 NPS514 EST 3 1 -237inv AI064414
393 NPS515 EST 3 1 -67inv AA540712 809bp with AA440879 and AA440431. human cyclin G assoc. Kinase.
394 NPS516 GNL 3 596-682inv AF132145 Drosophila damage-specific DNA binding protein DDBa p127 subunit
395 NPS517 EST 3 1 -513 AF007159
396 NPS518 EST 3 13-235inv AI51 1691
397 NPS519 EST 3 1-61 inv AA264883 also AA392712. Alt splice.
398 NPS520 EST 3 30-451 AA438399 821 bp contig with AA439438
399 NPS521 EST 3 600-627 AA440272 1324bp contig with AA438941
400 NPS526 EST 3 65-483 AA264865
401 NPS527 EST 3 1 -246 AA263693
137-160 and
402 NPS528 EST 3 AA698620 379-475
403 NPS529 EST 3 1 -51 inv AA391350
404 NPS530 EST 3 100-499 AA392183 DNA J homologue
149-348 and
405 NPS531 EST 3 AA696390 mouse/human yeast/ub fusion protein 1 412-457
406 NPS532 EST 3 1 -267inv AA802961 816bp with AA817584
407 NPS533 EST 3 1 -259inv AA699045 Poss. Slug CDNA25
408 NPS534 EST 3 1-99bp AA952055 1205bp with AA202358, AA202625 and AA951416. Siah binding protein 1 (human)
409 NPS535 EST 3 1 -610inv AA142266
410 NPS536 EST 3 52-534 AA696974 Matches Human CGI-37 protein
503-626 and
41 1 NPS537 EST 3 AI532170 1565bp with AI544333 and AI062662. Definite transcription factor, MTF-1 734-1069
412 NPS538 EST 3 442-569 AA567128 Match to mouse EST
413 NPS540 EST 3 1 -79bp AA950480
414 NPS541 EST 3 2-360 AA950161 1217bp with AA950864 and AA950181
30-194 and
415 NPS542 EST 3 AA539625 882bp with AA202440 and AA390927 291 -366
416 NPS543 EST 3 1 -255 AA951297
417 NPS544 EST 3 20-95 and 479-654 AA948996 781 bp with AA541068 and AA950730
418 NPS545 EST 3 1-378 AA941568
421 NPS548 EST 3 1-279 AA697191 1002bp with AA392404 and AA438791
422 NPS549 EST 3 650-689 AI518422
423 NPS1065 EST 3 27-689 AI535025
424 NPS551 EST 3 8-563 AA950826
425 NPS553 EST 3 48-150 AA949934 Mam. Casein kinase
426 NPS555 EST 3 411-582 AI109292
427 NPS556 EST 3 1-279inv AA202259 1693bp with AA391736 and AA820861
428 NPS557 EST 3 131-647 AA142065 poss. succinate semialdehyde dehydrogenase
429 NPS558 EST 3 76-559 AA536402 0-783 NPS559-920 I Unknown
784 NPS921 GENO. 2 1-537INV AC006073 In intron of gene coding for 246AA protein at 1663-44895 . No database matches
785 NPS922 GENO. 2 1-720 AC004299 In space before Drosophila Homologue of Human C-TAK 1 ser/thr kinase
786 NPS924 GENO. 2 1-599 AC004115 In space before gene coding for 372AA protein.16297-18074. No database matches.
787 NPS925 GENO. 2 1-581 AC004 16 In intron of gene coding for 355AA protien. 51777-83843bp. No database matches
788 NPS926 GENO. 2 1-628 AC005889 No good predicted exons in this area. space before gene coding for 401 AA protein. 75943-77148bp. Sequence similarity to
789 NPS927 GENO. 2 1-536 L49408
Mammlian glia maturation factor.
In space before gene coding for 878AA protein. 39765-47183bp. Sequence similarity to Mous
790 NPS928 GENO. 2 1-86inv AC004296
G-protein
791 NPS929 GENO. 2 1-573inv AC004306 No good predicted exons in this area.
In intron of gene coding for 1876AA protein at 62506-79351. Seq similarity hypothetical
792 NPS1077 GENO. 2 1-648 AC006472 proteins from human and yeast
793 NPS931 GENO. 2 1 -463inv AC006092 In space before gene represented by ESTs AA990657 and AI294791.
794 NPS932 GENO. 2 1-519 AC006073 No good predicted exons in this area.
In gene coding for 566AA protein.85106-110350bp(complement). Possible transcription factor
795 NPS933 GENO. 2 1-704inv AC007176
In space before gene coding for a 702AA prootein at 47585-59400bp. Sequence similarity to
796 NPS935 GENO. 2 1-307inv AC004423
Xenopus DNA repair protein XPGC
Part of gene coding for 422AA protein at 34104-35373(complement). Strong sequece
797 NPS336 GENO. 2 1-412inv AC005448 similarity to Drosophila Septin 2
Possibly in 3'UTR of gene coding for 355AA protein at 39720-40727.Weak sequence similarit
798 NPS937 GENO. 2 1-478 AC004313 to potassium channel gene
In intron of gene coding for 402AA protein at 101233-1531 10bp. Strong sequence similarity t
799 NPS938 GENO. 2 1-489 AC004641
Xenopus FLAP endonuclease.
800 NPS1078 GENO. 2 1-558inv AC004306 No good predicted exons in this area.
ΠΓO^TU similarity to Mouse serine C-palmitoyltransferase
ESTs matching at 74650 (AA803646, AI518976, AH 081 14) Sequence similarity to U5 snRN
802 NPS941 GENO. 2 1 -544 AC005334
In intron of gene coding for a 292AA protein at 12210-30840bp. Sequence similarity to huma
803 NPS942 GENO. 2 1 -201 AC004154 geranylgeranyltransferase
In intron of gene coding for a 1442AA protein at 40620-93241 (complement). Sequence
804 NPS943 GENO. 2 1 -524 AC004766 similarity to hypothetical C.elegans gene ZK1 128.2.
805 NPS944 GENO. 2 1 -621 AC004361 No good predicted exons in this area.
806 NPS945 GENO. 2 1 -569inv AC007185 No good predicted exons in this area.
In space before gene coding for 401 AA protein at 75943-77148bp. Sequence similarity to
807 NPS946 GENO. 2 1-462 L49408
Mammlian glia maturation factor. in intron of gene coding for an 1813AA protein at 51 190-77775 (complement). Sequence
808 NPS947 GENO. 2 1 -233inv AC005750 similarity to Rat CPG2 protein
809 NPS948 GENO. 2 1-525 AC005269 No good predicted exons in this area.
In intron of gene coding for 2355AA gene at 16134-35638. Sequence simlarity to Rat Fatty
810 NPS949 GENO. 2 1 -531 AC005554 acid synthase.
81 1 NPS951 GENO. 2 1-443inv AC004758 No good predicted exons in this area.
In intron gene coding for 196AA protein at 57158-63906bp. Weak sequence similarity to rat
812 NPS952 GENO. 2 1 -498inv AC005894 metalloprotease.
In intron of gene codning foe a 156AA protein at 20899-39698bp. Sequence similarity to
813 NPS954 GENO. 2 1 -320 AC004564
Arabidopsis Immunophilin
In intron of gene coding for 732 AA protein at 63422-80946bp. Sequence similarity to Rat
814 NPS956 GENO. 2 1 -429 AC005716 follistatin
In intron of gene coding for 21 AA protein at 81722-84603 (complement). No database
815 NPS958 GENO. 2 1 -71 bp AC007180 matches
In intron of gene coding for a 945AA protein at 87648-1 13518. Strong sequence similarity to
816 NPS1079 GENO. 2 1 -75bp AC004758
Human retinoblastoma binding protein 2
In intron of gene coding for a 286AA protein at 49143-60866bp (complement). Is Dros Wing
817 NPS962 GNL. 2 1-116inv AC001661 blister gene
In intron of gene coding for a 945AA protein at 87648-1 13518. Strong sequence similarity to
818 NPS963 GENO. 2 1 -512inv AC004758
Human retinoblastoma binding protein 2
819 NPS964 GENO. 2 1-54bp AC004334 In space before gene coding for 433AA proetein at 21602-22903. No database matches.
820 NPS966 GENO. 2 1 -557inv AC005149 In intron of gene coding for 424AA protein at 70149-97938. No database matches.
821 NPS968 GENO. 2 1 -202 AC005333 No good predicted exons in this area.
822 NPS970 GENO. 2 1 -534inv AC005334 In intron of gene coding for 309AA protein at 64276-77888bp. No database matches
823 NPS971 GENO. 2 1 -438 AC006421 No good predicted exons in this area.
824 NPS972 GENO. 2 1 -524INV AC005443 No good predicted exons in this area.
——.—. mammalian Undine phophorylase.
826 NPS974 GENO. 2 1-535 AC005889 No good predicted exons in this area.
In Intron of gene coding for 826AA protein at 3650 -13339bp (complement) ESTs AA949050
827 NPS975 GENO. 2 1-47bp AC005130 and AA817663 come from this gene. Sequence similarity to Helix-loop-helix genes
In intron of gene coding for 2355AA gene at 16134-35638. Sequence simlarity to Rat Fatty
828 NPS976 GENO. 2 1-551 AC005554 acid synthase.
829 NPS9 7 GNL 2 1-100, 146-499 AF097364 Drosophila Drongo gene
Part of gene coding for 2355AA gene at 16134-35638. Sequence simlarity to Rat Fatty acid
830 NPS978 GENO. 2 1-580 AC005554 synthase.
Space before gene coding for 834AA protein at 33470-40630. Sequence similarity to
831 NPS979 GENO. 2 1-256inv AC004722 bromodomain containing proteins.
832 NPS980 GENO. 2 1-406 AC003054 In intron of gene coding for a 822AA protein at 9312-46969. No database matches.
833 NPS982 GENO. 2 1-460 AC004280 No good predicted exons in this area.
834 NPS983 GENO. 2 1-99bp AC004722 In intron of gene coding for a 289AA protein at 43804-61450bp. No database matches
In intron of gene coding for 300AA protein at 30647-46841. Weak sequence similarity to
835 NPS985 GENO. 2 1-178inv AC001661
Mouse surfeit gene
836 NPS986 GENO. 2 1-602 AC005269 No good predicted exons in this area.
837 NPS987 GENO. 2 1-562 AC004362 No good predicted exons in this area.
In intron of gene coding for 1277AA protein at 40819-69834 (complement). Sequemce
838 NPS988 GENO. 2 1-521 AC004370 similarity to human nuclear transport receptor.
839 NPS989 GENO. 2 1-619 L49408 No good predicted exons in this area.
840 NPS991 GENO. 2 1-535inv AC005447 In intron of gene coding for a 802AA protein at 28001 -49228bp. No database matches.
841 NPS992 GENO. 2 1-342 AC004120 No good predicted exons in this area.
In space before gene coding for a 399AA protein at 66560-68732bp. Sequence similarity to
842 NPS993 GENO. 2 1-512 AC005454 mitochondrial carrier protein genes.
843 NPS994 GENO. 2 1-515inv AC005130 No good predicted exons in this area.
844 NPS995 GENO. 2 1-499bp AC005439 No good predicted exons in this area.
845 NPS997 GENO. 2 1-565inv AC005127 No good predicted exons in this area.
3bp overlap with gene coding for1365AA protein at 67351 -74867bp. ESTs AH 06939 and
846 NPS998 GENO. 2 1-568 AC005889
AI296430 come from this gene
In intron of gene coding for 1277AA protein at 40819-69834bp (complement). Sequemce
847 NPS999 GENO. 2 1-503 AC005558 similarity to human nuclear transport receptor.
In gene coding for 676 AA protein at 3451 1 -37955bp. Sequence similarity to mouse LUN gene.
848 NPS1000 GENO. 2 1-620 AC004351
In Intron of gene coding for 1467AA protein at 867-18363bp. Sequence similarity to Drosophil
849 NPS1001 GENO. 2 1-519inv AC004766 Lipase 3.
850 Nfjji υυ IjEI U. i. l -OUII IV
Valyl tRNA synthetase.
In intron of gene coding for a 1208AA protein at 56208-83122bp (complement). No database
851 NPS1003 GENO. 2 1 -370inv AC005129 matches.
852 NPS1004 GENO. 2 1 -748inv AC005894 No good predicted exons in this area.
Part of gene coding for 239AA protein at 507-13551 bp (complement). Sequence similarity to
853 NPS1005 GENO. 2 1 -535inv AC005447
C. elegans gene ace. no. AF002196
In intron of gene coding for 242AA protein at 33006-40459bp (complement, incomplete
854 NPS1006 GENO. 2 1 -581 inv AC005643 sequencce). No database matches.
Part of gene coding for 2355AA gene at 16134-35638. Sequence simlarity to Rat Fatty acid
855 NPS1007 GENO. 2 1-342 AC005554 synthase.
In intron of gene coding for 1 145AA protein at 5993-19843. Sequence similarity to C.elegans
856 NPS1009 GENO. 2 1 -77inv AC004532
AF067608.
Space before gene coding for 351 AA protein at 1 10375-11 1625bp. Sequence similarity to
857 NPS1010 GENO. 2 1 -496inv AC007186
Human YL gene.
In intron of gene coding for 566AA protein at 84105-109350 (complement). Sequence
858 NPS101 1 GENO. 2 1 -582 AC007176 similarity to zinc finger transcription factors.
In intron of gene coding for 604AA protein at 3052-881 Obp.Sequence similarity to C.elegans
859 NPS1012 GENO. 2 1 -483 AC004423
AL021481 gene.
860 NPS1013 GENO. 2 1 -560 AC00581 1 No good predicted exons in this area.
In intron of gene coding for 528AA at 40963-70180bp. Sequence similarity to C.elegans
861 NPS1016 GENO. 2 1 -596 AC005653
U40420
Part of gene coding for 1730AA protein at 50171 -62324bp. Sequence similarity to C.elegans
862 NPS1017 GENO. 2 1 -539 AC004516
UNC89
In intron of gene coding forl 142AA protein at 1 16605-128877bp. Sequence similarity to
863 NPS1019 GENO. 2 1-505inv AC005285
Guanine nucleotide exchange genes.
864 NPS1021 GENO. 2 1 -504inv AC007137 No good predicted exons in this area.
865 NPS1022 GENO. 2 1 -191 inv AC005643 No good predicted exons in this area.
In intron of gene coding for 1296AA protein at 1037-28442(complement). Sequence similarity
866 NPS1023 GENO. 2 1 -468 AC004642 to putative lysophosphatidic acid acyltransferase [Mus musculus]
In intron of gene coding for 1481 AA protein at 159-1 1694bp. Sequence similarity to
867 NPS1024 GENO. 2 1 -578inv AC005749
KIAA0596 protein (Homo sapiens]
868 NPS1025 GENO. 2 1 -598 AC007185 No good predicted exons in this area.
In space before gene coding for 864AA protein at 20219-29453 (complement). Good sequenc
869 NPS1027 GENO. 2 1 -634 AC004340 similarity to Human sec24 homologue
Space before gene coding for 450AA protein at 45629-48055bp (complement). Sequence
870 NPS1028 GENO. 2 1-415 AC005456 similarity to Human GMP synthase.
871 NPS1029 GENO. 2 1 -198 AC004375 No good predicted exons in this area.
ΓVM I i /αeaτn assoc. proτ
Space before gene coding for 292AA protein at 12210-30840bp. Sequence similarity to huma
873 NPS1031 GENO. 2 1 -495inv AC004154 rab geranylgeranyl transferase
In intron of gene coding for 1063AA protein at 63470-78557(complement). Has been predicte
874 NPS1032 GENO. 2 1 -1 16 AC004328 from Dros genomic AL03531 1 and has similarity to mouse BOP1
In intron of gene coding for 40 AA protein at 3680-49217(complement). No database matche
875 NPS1033 GENO. 2 1 -581 inv AC0051 12
876 NPS1034 GENO. 2 1 -506inv AC004367 Space before gene coding for 387AA protein at 3496-35348bp. No database matches
1st exon of gene coding for 626AA protein at 20859-27089bp. Sequence similarity to human
877 NPS1036 GENO. 2 1 -41 1 AC005472
N AT 1 /death assoc. prot
878 NPS1080 GENO. 2 1-492 AC007121 gene coding for 365AA protein at 22511-56594 (complement). No database matches
In intron of gene codning for 665AA protein at 632-41 87 (complement). Sequence similarity
879 NPS1038 GENO. 3 1 -291 L49405 to human hypothetical SBBI03 protein
880 NPS1039 GENO. 3 1 -454 AC004658 In intron of gene coding for 394AA protein (complement). No database matches
881 NPS1062 GENO. 3 1 -376 AC007757 Also matches Dros. EST AA951801. Poss. Transcription factor.
In intron of gene coding for 564AA protein at 74249-97818bp. Sequence similarity to
882 NPS1044 GENO. 3 1 -597 AC006091
YII3_Yeast hypothetical protein.
883 NPS1045 GENO. 3 1 -498 AC005720 No good predicted exons in this area.
Space before gene coding for 562AA protein at 60626-68675. Sequence similarity to Rat
884 NPS1046 GENO. 3 1-375 AC005814
NAB1.
In intron of gene coding for 575AA protein at 10435-29297. Weak sequence similarity to CD3
885 NPS1049 GENO. 3 1 -486 AC004713 genes.
886 NPS1050 GENO. 3 1 -544 AC005813 No good predicted exons in this area.
887 NPS1051 GENO. 3 1 -549 AC006936 No good predicted exons in this area.
888 NPS1052 GENO. 3 1 -306inv AC005425 No good predicted exons in this area.
889 NPS1053 GENO. 3 1-579inv AC005720 In intron of gene coding for 394AA protein at 61401 -93968. No database matches
In intron of gene coding for 931AA at 52045-70222bp (complement). Sequencw similarity to
890 NPS1056 GENO. 3 1 -191 inv AC004266
C. elegans Zinc finger protein. Drosophila EST AI259457 comes from this gene
891 NPS1059 GENO. 3 1 -264 AC006936 No good predicted exons in this area.
892 NPS1063 GNL 3 488-536 AI062190 EST comes from Dros ferrochelatase
893 NPS1064 GENO. 3 AF 104256 Sequence similarity to Human transcriptional co-activator CRSP150
894 NPS1076 EST 2 101 -597 AI388606 1249bp contig with AI258281 and AI258326
895 NPS1081 GENO. 2 1 -491 L49408 No good predicted exons in this area.
896 NPS1082 GENO. 2 1 -475inv AC005714 space before 390AA orf at 162294-163466. Horn to hum. death assoc prot 3
897 NPS1083 GENO. 2 1 -461 AC004375 No good predicted exons in this area.
898 NPS1084 GENO. 2 12-419 AF000177 translational sequence similarity to human CaSm protein
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Z CLO CαO. CαO, CαO. CαO. C-O. CiO CiO CαΛ. CQO. C-O. CaO, C-O. CθOL C-O. CαO. CιO CαO. CαO. CLO CiιO. CiO CαO. CiO CiO CuO. CLO CαO. C-O, CDO. CαO, CαO. CαO, CaO. CιiO Q Z 2 Z Z Z Z Z Z Z Z Z Z Z Z 22222 Z 22222 Z 222222222
Ω _ ^«- ro mι > τ^* ιuΛ> iιjnj rn^» mι« 0^ ^_ ^^ w^ ^^ Λ^ ^^ ∞^ ^n θC ^C CCM| CCVJ f^s( CCθ rr^N| OfvO| OT(N {'- CMM <<ytf3- ιMΛ rc^ OrtO ccn θ_t «,-_t. α O NPS0048 GNL 3 221-318 U73160 AA440389 EST matching Dros fas gene
NPS0049 GNL 3 15-95 M32141 AI297861 1 st EST in 8 contig matches 49-kilodalton phosphoprotein gene
NPS0050 GNL 3 231 -293 M21 159 Tcp-1
NPS0051 GNL 3 1-349 V00213 Hsp70. Poss EMPTY?
NPS0052 GNL 3 1 -241 U59923 glutamyl-prolyl-tRNA synthetase gene,
NPS0053 GNL 3 225-237 D16257 238-333 intron, 334-499 exon ribosomal protein S4
NPS0054 GNL 3 1 -462 X73216 Rlb1
NPS0056 GNL 3 1 -207inv X0731 1 HSP2
NPS0057 GNL 3 15-438 X54061 205K microtubule-associated protein (MAP)
NPS0058 GNL 3 1 -80inv J01 102 HSP68
NPS0060 GNL 3 56-187 M63792 RAD6
NPS0061 GNL 3 391 -465 U28966 Septin 2
NPS0062 GNL 3 1 -514 M98351 fructose 1 ,6 bisphosphate aldolase gene,
NPS0063 GNL 3 46-251 inv U01035 Bottleneck gene
NPS0064 GNL 3 49-450 U38238 HLH106
NPS0066 GNL 3 1 -436 U22176 15bp upstream of Brother gene on AC005557
NPS0067 GNL 3 46-176 M90755 Transcriptional repressor protein Aef-1
NPS0068 GNL 3 224-298 Y07908 Match to EST AI292767. This then matches serine/threonine protein kinase.
NPS0069 GNL 3 1 -531 M3231 1 Fascilin 1
NPS0070 GNL 3 1 -421 inv X03889 HSP23
NPS0071 GNL 3 548-882inv Y12861 bifunctional ATP sulfurylase/APS kinase.
NPS0072 GNL 3 83-135 U12010 putative serine/threonine protein kinase (nemo)
NPS0073 GNL 3 1 -357 U20554 UDP-glucose:glycoprotein glucosyltransferase mRNA
NPS0074 GNL 3 1 -20bp U87925 Cbl gene confirmed by match to EST AA441040
NPS0075 GNL 3 468-539 U23485 Guanylate cyclase. Match found via EST AA392994
NPS0076 GNL 3 1 -547 Y1 1349 UbcD4
NPS0077 GNL 3 1 -163 U09374 SNAP
NPS0078 GNL 3 1 -104inv U62388 chromatin assembly factor 1 p55 subunit
NPS0079 GNL 3 374-518inv AB007692 Elongin B
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
NPS0082 EST 2 509-591 AA202837 hypothetical yeast/arabidopsis/prot and mouse EST
NPS0083 EST 2 166-393 AI293734 824bp EST contig with AI293545.
NPS0084 GNL 2 42-137inv AJ249466 Dros DXI6 gene
NPS0086 EST 2 1-247 AA696498
882bp EST contig with AI542046 and AI533154.Sequence similarity to Human PDI related
NPS0087 EST 2 100-646 AA950073 protein
81 NPS0089 EST 2 1 -50inv AA695104 689bp EST contig with AA802812 and AI294978.
82 NPS0091 EST 2 1 -427 AA942153 868bp EST contig with AA803815 and AI519003.
83 NPS0092 EST 2 42-334 AA540352 Also AH 08950. poss. dehydrogenase
84 NPS0093 EST 2 1 15-162 AI238523
85 NPS0097 EST 2 1 -69inv AI260872 EST matches mouse signalling factor U29156
86 NPS0098 EST 2 5-77bp AA801728 830bp contig with AA263864 and forms part of mRNA AF184228.
87 NPS0099 GNL 2 228-675 AF053083 Drosophila SMT3 gene
88 NPS0100 EST 2 1 -210inv AA439866
89 NPS0105 EST 2 31 -590 AA820803 Other ESTs. evidence of Alt splice. Poss. aldose reductase
90 NPS0106 GNL 2 30-478 AF145307 Matches EST matching Dros. 26S proteasome regulatory complex subunit p48B
91 NPS0108 EST 2 76-178 AA438591 1 199bp contig with AM 07768 and AA697679.
952bp EST contig AA979551 /AA567400. Matches Dros Nebula gene. (EP line AQ254719-letha
92 NPS0109 GNL 2 1 -169 AF147700
?)
93 NPS01 1 1 EST 2 138-414 AA439261 1254bp contig with AI519697 and AA951893(polyA). Poss. zinc finger prot.
94 NPS01 13 EST 2 7-354 AM 07509
95 NPS01 14 GNL 2 1 -48bp AJ242855 ESTs AA735265 and AA540348. Match to Dros. amphiphysin. EP line AQ073590-lethal ?
96 NPS01 15 EST 2 1 -31 1 inv AA735555 1422bp contig with AI386481 and AI296915.
99 NPS0120 EST 2 364-583 AA941785 1 1 15bp EST contig with AA695548 and AI513251 (polyA).
101 NPS0122 EST 2 1 -395 AA539001
102 NPS0123 EST 2 1 -35inv AA735863 Poss. related to human death assoc prot 3 X83544 (EP line AQ73716-lethal7)
103 NPS0125 EST 2 68-195 and 475-621 AA941860
104 NPS0127 EST 2 1 -210inv AA246460 Other ESTs, evidence of Alt. splice
105 NPS0128 EST 2 66-593 AA1 1928 ORF from MGD3 retrotransposon X95908
106 NPS0131 EST 2 1 -332 AA979014
107 NPS0134 EST 2 52-475 AA817254
108 NPS0137 EST 2 1 -37bp AA536262 2350bp EST contig. Poss glycogen synthase
1 10 NPS0140 EST 2 368-636 AA390587 988bp EST contig with AI543996
1 1 1 NPS0141 EST 2 55-1 10inv AA979454 sim to human REC1 L protein. Ace. X57303
1 12 NPS0142 EST 2 31 -460 AA941359
1 13 NPS0143 EST 2 65-299 AA201303 also AA541066 Other ESTs, evidence of Alt. splice.
1 14 NPS0144 EST 2 538-581 inv AA6981 19 799bp EST contig. Match to Human gycerol-3-phosphate dehydrogenase
1 15 NPS0145 EST 2 1 1 1 -549 AA696174
922bp contig with AA263288 andA1109084 . Match to fish proteasome subunit (EP line
1 16 NPS0146 EST 2 107-243 AI064230
AQ73732-lethaL?)
1 17 NPS01 7 EST 2 1 -212 and 276-382 Al 106957 1906bp EST contig. 328ATG ORF/PolyA. Poss ATPase
Also AA820473. (AF034644) putative cytochrome bc-1 complex core protein (Haematobia
1 18 NPS0149 EST 2 1 -107inv AM 14218 irritans irritans]
119 NPS0150 EST 2 19-102 and 115-485 AA978449 Also AA940834. 103-1 14 gap of unknown length. Matches Dros mRNA AF 145655
120 NPS0152 EST 2 182-362 AA802905 EP line AQ74042- = lethal?
Also AI296787. Dihydrolipoamide acetyltransf erase component of pyruvate dehydrogenase
121 NPS0154 EST 2 35-279 and 376-45 AI259166 complex precursor (human)P10515
122 NPS0155 EST 2 1-238inv AA951193 EP line AQ72909 lethal?
123 NPS0156 EST 2 326-482 AA696743 Also AA803977
124 NPS0157 EST 2 11-512 AA990758 Also AA246427. 975bp contig. Matches Dros mRNA AF145681 (EP line AQ73763-lethal?)
125 NPS0158 EST 2 1-406 AA697797 1 1 13bp EST contig with AI260878 and AI946139.
126 NPS0159 EST 2 1-29inv AA802206 1626bp EST contig.
127 NPS0160 GNL 2 1-222and 422-592 AF035251 Dros. Tripeptidyl peptidase. Tppll (FBgn0020370)
128 NPS0161 EST 2 01-223 and 292-55 AA202366 Dros. Unknown gene AF132158 (EP line AQ254870-lethal?)
129 NPS0162 EST 2 103-468 AA950164
130 NPS0163 EST 2 23-98 and 102-602 AA952159 99-101 gap of unknown length.Match to mouse EST
131 NPS0166 EST 2 102-512 AA392519 also AA695318 and AA441243.758bp contig.
132 NPS0168 EST 2 304-541 AI515517 also AI404462. Poss Ras related protein
133 NPS0169 EST 2 191-387 AA698481 961 bp EST contig with AI402698. Sim. to other hypothetical proteins. EP line AQ73580-lethal?
134 NPS0170 GNL 2 451-606inv AA803082 2097bp EST contig. Poss. Alt splice. EST match Dros Akap200 dif, insert point to NPS0288
135 NPS1067 EST 2 1-570 AI405762 Seq.sim to hypothetical prots from arabidopsis and C. elegans
137 NPS0174 EST 2 353-476 AA942305 EP line AQ73548-lethal?
138 NPS0178 EST 2 72-391 AA951839 979bp EST contig with AA979603 and AI519882.
139 NPS0179 EST 2 1-112 AI386817 also AI404737
140 NPS0180 EST 2 435-475 AA438658 951 bp EST contig with AI546582
142 NPS1068 EST 2 1-228 AI403747
143 NPS0188 EST 2 1-272 AA802791 also AA390699
145 NPS0190 EST 2 1-202 AA201161 Same EST contig as NPS180, diiferent insertion points
146 NPS0191 EST 2 202-472 AA978927 Also AA978887. poss. PCF1 1 p homolog (Homo sapiens]
147 NPS0192 EST 2 84-318 AA541084 930bp EST contig withAA538937. Poss. choline kinase
148 NPS0195 GNL 2 390-509 U09370 Matches EST matching Dros. ribonϋcleoside diphosphate reductase
149 NPS0198 EST 2 1-140 AA439230
151 NPS0200 EST 2 25-76inv AA802379 1 135bp EST contig with AA246624 and AI294568.
152 NPS1069 EST 2 9-100 AND 179-411 AI404485
153 NPS1070 EST 2 60-449 AM 08647
154 NPS1071 EST 2 1 -49inv AA951902 other ESTs inc AA949796. Sequence sim. to human ARP2/3 protein compex subunit 34
157 NPS0206 EST 2 117-263 AA803314 1505bp EST contig. Evidence of alt. splice. Human B-cell receptor associated protein.
159 NPS0209 EST 2 37-243 AA696343 also AA696180. Match to human/ C. elegans calponin
160 NPS0210 EST 2 261-580inv AA540783 659bp EST contig with AI456854.
161 NPS0211 EST 2 26-267 and 336-459 AA695850 1305bp EST contig with AA698310 and AI516896. FKB4_RABIT P59 PROTEIN
162 NPS0212 GNL 2 1-224 AF149795 Dros Drep2-no flybase
163 NPS0213 EST 2 1-514 AI064375
164 NPS0216 EST 2 181-299 AA540197 also AA695503 and AA941503.732bp contig. Poss sialomucin
165 NPS0217 EST 2 167-212inv AA979442 732bp contig with AA392418 and AI543860.
166 NPS0218 EST 2 89-159 AA536378 2393bp EST contig. Poss arsenate resistance protein ARS2(human)
167 NPS0219 EST 2 1-570 AI515537 Genomic AC004345. Also AI062109. 50bp upstream of EST.fEP line AQ073614-lethal?)
168 NPS0220 GNL 2 1-184 AF149795 Dros Drep2-no flybase
169 NPS0225 EST 2 1-104 and 310-467 AI064169 also AA816652
170 NPS0226 EST 2 1-288 AA439345 802contig with AA949877 and AA439626
171 NPS0227 EST 2 1-350 AA979503 806bp EST contig with AI512942.181 bp upstream of EST Genomic AC005452
172 NPS0228 EST 2 1-93 and 170-446 AI293141
173 NPS0229 EST 2 12-244 AM07445 955bp EST contig with AA390813
174 NPS0233 EST 2 12-478 AA390942
175 NPS0235 EST 2 11-103 and 296-389 AA802688 Also AH 34220. Poss 10k HSP
176 NPS0236 EST 2 1-414 AA392415
177 NPS0239 EST 2 1-22bp AA695619
178 NPS0240 EST 2 399-542 AA142132 Dros mRNA AB010264
179 NPS0241 EST 2 366-520 AA536537 EP line AQ073930-lethal?
180 NPS0242 EST 2 26-303 AA264253 Also AH 06910. Poss. SNF7 homologue
181 NPS0243 EST 2 186-593 AA441247 also AA820771
183 NPS0245 EST 2 83-319inv AI064123 also AA263284. Match to human androgen induced prostate proliferative shutoff assoc. protein.
184 NPS0247 EST 2 1-89bp AA441173
185 NPS0250 GNL 2 1-347inv AF168467 Dros dual specificity kinase DYRK2(no flybase)
186 NPS0251 EST 2 2-131 AI062640 1 190bp EST contig with AI063780. EP line AQ074008-lethal?
187 NPS0252 EST 2 1-77inv AA695507 1392bp EST contig. Poss.Alt splice. Poss. RNA binding prot.
1270bp EST contig with AA801973 and AI294493 . Poss. match to horse Thioredoxin. EP line
188 NPS0254 EST 2 89-251 AA736186
AQ073432-lethal?
189 NPS0255 EST 2 1-417 AA697603 also AA801716. EP line AQ073594
190 NPS0256 EST 2 1-528 AA950741
191 NPS0257 EST 2 1-53bp AI063204 1079bp EST contig.
192 NPS0258 EST 2 1-44bp AA441029
EP line AQ073622-lethal? 1 141 bp contig with AA949325, AA735675 and AA391495.
193 NPS0259 EST 2 1-157 AH 14266
Poss.match to human GMP synthase
194 NPS0260 EST 2 1-562 AA951648 1340bp contig with AA539581 , AA802940 and AA263326
195 NPS0261 EST 2 26-137 and 360-422 AA391135 Match to SEC61 , different area to NPS1 18. EP line AQ072909 lethal?
196 NPS0262 EST 2 1-124 AA696531 Sim. to 2-hydroxyphytanoyl-CoA lyase (Human)
197 NPS0265 EST 2 442-549 AH24332 198 NPS0266 EST 2 52-382 AA949873 199 NPS1073 EST 2 1-167 AH 33902 see also AC006562 poss phosphate transporter 200 NPS0269 EST 2 1-550 AI403609 Genomic AC005129, 420bp upstream of EST 201 NPS0271 EST 2 299-375 AA391470 203 NPS0273 EST 2 1-76inv AA696584 205 NPS0276 EST 2 21-377 AA695424 Also AI295527 206 NPS0277 EST 2 152-590inv AA440949 207 NPS0278 EST 2 132-312 AI062455 also AA440915 208 NPS0279 EST 2 68-311 AA816432 209 NPS0281 EST 2 1-258 AA979191 Match to human CGI-28 211 NPS0285 EST 2 1-89bp AA441636 AA820540 and AA817484. Alt splice 212 NPS1075 EST 2 59-488 AI295363 213 NPS0288 GNL 2 64-170 AF132884 Dros Akap200 also ep line AQ074077 lethal ?(no flybase) 214 NPS0289 EST 2 3-355 and 443-479 AA801691 also AA441008 215 NPS0290 EST 2 378-471 AA950084 1473bp EST contig. 216 NPS0291 EST 2 20-236 and 292-439 AI062945 798bp EST contig with AI405532 and AA264809. Some similarity to Human p47 protein.
2293bp EST contig. Poss. human cleavage and polyadenylation specificity factor, 160 kd
217 NPS0293 EESSTT 2 1-312 AA440345 subunit.AA201536,AA539993,AA942332, AA979174 ,AA202096
219 NPS0295 EST 2 8-437inv AA440135 EP line AQ073560-lethal? 220 NPS0296 EST 2 75-157 AI063979 also AA802032 221 NPS0297 EST 2 1-144 AA699194 222 NPS0298 EST 3 507-547 AA441233 also AA979182 223 NPS0299 EST 3 1-79inv AA438352 33% over 1 13 AA Plant oxygenase 224 NPS0300 EST 3 480-534 AI455428 225 NPS0301 EST 3 11-190inv AA246916 Also AI532444. Rat Mitochondrial import receptor 226 NPS0302 EST 3 233-348 AA392258 Also AI259457 227 NPS0304 EST 3 1-41inv AI296848 Prob. 40-kDa V-ATPase subunit (mam) 228 NPS0305 EST 3 255-354 AI388389 229 NPS0306 EST 3 335-448inv AA441471 also AA540182. 52% over 107 AA like Bov/Hum/Mouse RHO GDP-dissoc. ihibitor 1
2093bp EST contig. Evidence of Alt splice, seq. sim. to Human HYDROXYMETHYLBILANE
230 NPS0307 EESSTT 3 22-242 AA439855
SYNTHASE
231 NPS0308 EST 3 -141 and397-446in AA941606 also AA978838. Seq. sim to serine proteinase [Anopheles gambiae] 232 NPS0310 EST 3 209-435 AA392324 EP line AQ073800 lethal? 234 NPS0312 EST 3 1-152 AA540030 Also AI517519. Poss rat calcium binding prot. EP line AQ073037 lethal? 235 NPS0313 EST 3 85-596 AH 09898 236 NPS0314 EST 3 365-473 AI259723
237 NPS0316 EST 3 1-141 AI294469
238 NPS0317 EST 3 145-325 AA140945 Dros Fex retrotransposon
239 NPS0318 GNL 3 1-331 AF160975 Dros. Liquid facets gene. Epsin homologue. No flybase.
241 NPS0323 EST 3 1-98inv AA246767 also AA141059
242 NPS0324 EST 3 1180239inv AA441468 1 167bp EST contig.
243 NPS0327 EST 3 1-82inv AA247070 2486bp EST contig. Poss. translation factor.
244 NPS0328 EST 3 433-469 AA802401 Also AA802218. Prob. Alg2, glycosyltransferase horn./ Mouse MER 5
245 NPS0330 EST 3 1-96inv AH 35263 1514bp EST contig or 1 1 19AS contig, ATG ORFS, no good hits.
246 NPS0331 GNL 3 243-489 Y14998 Dros Bip1 gene (no flybase)
247 NPS0334 EST 3 1-317 AA246386 1392bp EST contig with AA541060 and AI294651
248 NPS0335 EST 3 311-427 AA264961 1083bp EST contig with AI402225. 57% over 82AA like mouse/ human Thioredoxin
250 NPS0338 EST 3 74-276 and 344-438 AA263803
251 NPS0339 EST 3 3-166inv AA202200 1329bp EST contig. EP line AQ073952 lethal?
252 NPS0340 EST 3 1-48 inv AA439530 Also AA951823
253 NPS0341 EST 3 28-207 AH09459 764bp EST contig with AA695738. Poss GPI-anchored protein(human)
254 NPS0342 EST 3 471-506inv AH09779
255 NPS0343 EST 3 147-247 AA141054
256 NPS1061 EST 3 65-118inv AA141365
257 NPS0345 EST 3 144-549 AI063643
258 NPS0346 EST 3 1-148 AH07445 1662bp EST contig. Sequence sim. to yeast proteins.
259 NPS0347 EST 3 1-75bp AI297362
260 NPS0348 EST 3 96-230inv AA392916 EP line AQ025069-lethal? Evidence of Alt. splice
261 NPS0349 EST 3 1-47 and 145-317iv AA201223 4020bp EST contig (1 1 ESTs). Probable translation initiation factor
262 NPS0351 EST 3 537-687 AI454966
901 bpcontig with AI387427,AA201231 and AA392823. 31 % over 129AA like Rat Nup84 and
263 NPS0352 EST 3 10-441 AA202767
Human 88 KDa nucleopore complex
264 NPS0353 EST 3 3-40inv AA201212 Also AI237918
265 NPS0354 EST 3 1-33inv AI404994 And AI260898. Alt splice
266 NPS0356 EST 3 1-292 AA539914 1500bp EST contig. Some sim. to human nudix (nucleoside diphosphate linked moiety X)
267 NPS0357 EST 3 36-454 AA440953 705bp EST contig with AI518505
268 NPS0359 EST 3 145-253 AA264591 EP line AQ073779 -lethal?
270 NPS0361 EST 3 202-381 inv AI403737
271 NPS0362 EST 3 270-443inv AA567141 2042bp EST contig. Some seq. sim. to C.elegans hypothetical genes
272 NPS0363 EST 3 1-478 AH 34670 Also AH 08248(Alt. splice)
273 NPS0364 EST 3 413-535inv AA263763 Also AA696172, some seq. sim. to mouse EX070
274 NPS0365 GNL 3 1-99bp AF190745 EST AA56801 1 matches Dros. PolyU binding splicing factor.No flybase.
275 NPS0367 GNL 3 64-449 AF145627 Dros Scratch gene scrt (no flybase)
276 NPS0370 GNL 3 212-414 AF074957 Drosophila Karyopherin alpha
277 NPS0371 EST 3 1-146 AI295205 and AA141054. Alt splice
278 NPS0372 EST 3 8-382 AA567704 1466bp EST contig. EP line AQ025097 lethal?Seq. sim to mouse malate oxidoreductase.
280 NPS0374 EST 3 1-347inv AI260759
281 NPS0375 GNL 3 1-199inv AF170829 Dros homolgue of mouse NP15.6 (neuronal)
282 NPS0377 EST 3 160-306 AA202424 1650bp EST contig.Matches full length mRNA AF184229. EP line AQ073843 lethal?
283 NPS0379 EST 3 300-379 AA802555 Also AI064221 , evidence of Alt. splice.
284 NPS0380 EST 3 322-573 AA802438 1030bp contig with AI063681
285 NPS0381 EST 3 34-470inv AA438500 Also AA7351 1 1. EP line AQ025197-lethal?
287 NPS0383 EST 3 41-56 and 223-353 AI062265 1475 contig.AA694862 and AI064128.UNC51 ser/thr kinase (C.elegans)
288 NPS0384 EST 3 1-429inv AA247020
289 NPS0385 EST 3 1-143 AA264635 Also AA816470.
290 NPS0387 EST 3 58-491 AA201749 877bp contig with AA803278/ human hypothetical gene
291 NPS0388 EST 3 1-162 AA392551 Also AA951287. EP line AQ072985
293 NPS0390 EST 3 297-447 AA141715
294 NPS0392 EST 3 80-161 AA695862
295 NPS0393 EST 3 2-132 AA201517 1 104bp EST contig. Seq. sim. to Dros minidiscs gene
296 NPS0394 EST 3 176-239 AA202297 788bp contig matching complete mRNA AF132183. Seq. sim. toVertebrate vacuolar ATPase
297 NPS0395 GNL 3 1-33bpinv AA567483 EST matches Dros protein tyrosine phosphatase L1 1253.
298 NPS0396 EST 3 1-209 and 271-468 AA817479
ATG orf hits Rat (and other) sodium dependant dicarboxylate transporter AB001321 58% over
299 NPS0397 EST 3 17-139 AA441327
74 AA
300 NPS0398 GNL 3 1-391 AA698011 EST matches Dros Melted gene, AF205831
301 NPS0399 EST 3 67-207 AA951986 nucleolar protein p40 [Homo sapiens]
303 NPS0402 EST 3 1-82bp AA201430 848bp EST contig with AI256930. seq. sim. to yeast mitosis protein.
304 NPS0403 GNL 3 15-54bp AF132912 Drosophila ARP gene. Match to EST matching ARP
305 NPS0404 EST 3 1-140 AA541045 1850bp EST contig. May be distantly related to cystatin
306 NPS0406 EST 3 392-501 AA390337 Also AA990810. EP line AQ254714 lethal?
307 NPS0407 EST 3 -202 and 273-440in AA141555 Matches mouse/human ESTs
308 NPS0408 EST 3 158-252 AA263730 Also AI531907(Alt splice). A little like yeast hypothetical protein YOL124c
916bp EST contig with AI946646 and AA990765. Good Seq. sim. toHuman Ubiquitin
309 NPS0409 EST 3 24-370 and 448-54 AI259832 conjugating enzyme 12
310 NPS0410 EST 3 75-483 AI514268
20bp 5' to EST on AC006562. Part of ORF similar to molybdenum cofactor biosynthesis protein
311 NPS0411 EST 3 1-435 AI293256
A[Homo sapiens]
312 NPS0412 EST 3 1-71 and 148-435 AA201987 Also AI293141. Poss Asparaginase
313 NPS0413 GNL 3 14-408 AF104357 Dros DRONC Nedd2 like caspase
314 NPS415 EST 1-38bp AA201670
315 NPS416 EST 15-404 AA201957 Mouse ESTs 316 NPS0417 EST 1-353 AA695344
Also AA264070. Matches lethal P-element insertion.AFI 746829 7/12/99 poss. in dalmation, (n
317 NPS0418 EST 1-450 AA441018 sequence)
319 NPS0420 EST 3 245-393 AA735819 320 NPS0421 GNL 3 1-147 AA440886 EST matches Dros Dak1 AB025924 (no flybase) 321 NPS0422 EST 3 76-217 AA803640 672bp with AA803683 and AA803676. Human Ribosomal L28 protein
722bp EST contig with AI947051. Match to Dros pumpless gene AF203725. Lethal known
322 NPS0424 GNL 3 72-786 and 842-107 AI257267
Dec 1999
323 NPS0425 EST 3 1 -75bp AA539327 653bp EST contig with AI294865. 324 NPS0426 EST 3 419-468 AI530922 325 NPS0427 EST 3 92-265 AI402854 326 NPS0428 EST 3 1 -222 and 291-354 AA441362 1218bp EST contig.40% like human/mouse proteasome subunit HsN3 327 NPS0429 EST 3 1 -219 AA202487 1 122bp EST contig with AA696163 and AA803708. A little like hypothetical yeast protein 328 NPS0430 EST 3 328-455 AA263590 1222bp EST contig. Seq. sim. to human hRNP F 329 NPS0431 EST 3 50-1 13 AA201496 2556bp EST contig. Seq sim to human oxoglutarate dehydrogenase 330 NPS0432 EST 3 281 -510 AA391430 787bp EST contig with AA802523 331 NPS0434 EST 3 1 -50 inv AI292722
1828bp EST contig. Evidence of alt. splice. Part of Dros nemo gene. Different insert point to
332 NPS0435 GNL 3 1 -65inv AA439393 nps0072.
333 NPS0436 EST 3 299-512inv AA820797 1851 bp EST contig. Seq. sim. to human SEC63 334 NPS0437 GNL 3 1 -52inv AF191563 Dros coatomer protein. No flybase.
875bp EST contig with AI456186.Matches Dros Klar. gene AF157066. EP line AQ254737 -
335 NPS0438 GNL 3 1 -31 inv AA696845 lethal?
336 NPS0439 EST 3 1 -384 AI259031 337 NPS0440 EST 3 1-82bp AA803464 Also AI064211 (alt. splice). Seq. sim. to human hypothetical protein KIAA0258 338 NPS0441 EST 3 169-489 AA539974 842bp EST contig (evidence of alt. splice). EP line AQ025134-lethal? 339 NPS0442 EST 3 1 -46 and 432-524 AA941993 340 NPS0443 EST 3 43-431 AA803074 Also AI517810. Match to Dros mRNA(complete) AF145665 342 NPS0445 EST 3 775-91 1 AA433251 345 NPS0449 EST 3 1 -99inv AA392932 819bp EST contig with AA392017. 346 NPS0451 EST 3 1 -260 and 310-510 AA391707 1 165bp EST contig with AI546088. Some similarity to human MSP2 protease. 347 NPS0452 EST 3 5-141 AND 445-58 AI294564 Also AI51 1708. Match to mouse EST 348 NPS0453 GNL 3 345-460 AF152928 Drosophila karyophγlin alpha 3. 349 NPS0454 EST 3 1 -177 AA540743 1 129bp with AI064582, AI519458 and AA568024 350 NPS0456 EST 3 1 -325 AA539054 Match to rat EST
J i Nι"SU4b/ EST 3 1-73bp AI062939 Some sequence similarity to human ataxia 2
352 NPS0458 EST 3 927-1070 AH09224
353 NPS0459 EST 3 43-146 AA696728 Poss. isopentyl pγrophosphate isomerase
354 NPS0460 EST 3 59-533 AI518328
355 NPS0461 EST 3 43-432 AA263622 Also AI135048.Match to complete mRNA AF132157. EP line AQ254717-lethal?
356 NPS0463 EST 3 1-164 AA539661 1304bp EST contig. Matches Human Proton ATPase like protein
357 NPS0464 EST 3 1-152 and 214-579 AI388964
358 NPS0465 GNL 3 365-462 AF205597 Dros Fragile X related gene, no flybase.
359 NPS0466 EST 3 6-257 AA392117 Also AA392507
360 NPS0468 EST 3 45-120 and 545-591 AA821194 1599bp EST contig . human 40s ribosomal protein s29
361 NPS0469 EST 3 12-469 AA539752 813bp EST contig with AI386513(alt splice).
362 NPS0473 EST 3 1-382 and 456-484 AA803203 1218bp EST contig.
363 NPS0476 EST 3 105-154 AI297317 Evidence of Alt splice
364 NPS0477 EST 3 22-177 AA817394 Also AA263804. V. similar to Dead box familly of DNA helicases (initiation factors)
365 NPS0479 EST 3 1-77inv AI064638
366 NPS0480 EST 3 1-37bp AA736157 1462bp EST contig with AI109785 and AH 14024. Seq. sim to 54TMp [Homo sapiens]
367 NPS0482 EST 3 7-369 AA820427 Seq sim. to 3-oxoacyl carrier protein synthase II [Arabidopsis thaliana]
368 NPS0483 EST 3 1-533 AA391 36 2188bp EST contig.
1425bp EST contig.Evidence of Alt. splice. Good seq. sim. to human spliceosomal protein SAP
369 NPS0484 EST 3 158-470 AA567184
130
370 NPS0486 EST 3 1-122 AA735277 Same ESTs as NPS0361 , different point of insertion
371 NPS0487 GNL 3 514-616 AF129080J Drosophila C0P9 complex homolog subunit 1 -2 DCH1 -2
372 NPS0489 EST 3 140-189 AA202581 1210bp EST contig. Match to AMMECR1 protein [Homo sapiens]
373 NPS0490 EST 3 41-377 AA390 75 Also AI292888
374 NPS0491 EST 3 169-488 AA539898
375 NPS0492 EST 3 1-127 AA390453 Also AA951634
376 NPS0493 EST 3 1-321 inv AA568061 1356bp with AA264532 and AA441674
379 NPS0497 GNL 3 431-837inv AF142061 Dros prot. kinase JIL-1
380 NPS0499 EST 3 46-319 AA817295 Seq. similarity to bestrophin [Homo sapiens]. EP line AQ073104-lethal?
381 NPS0501 EST 3 35-383 AA439743 Also AI403533. Evidence of alt splice.
382 NPS0503 EST 3 1-264 AA441568 1 164bp EST contig. EP line AQ025280-lethal?
383 NPS0504 EST 3 37-408 and 479-56 AA247082
384 NPS0505 EST 3 1-321inv AA201685 868bp contig with AA540405 and AI513192
386 NPS0507 EST 3 452-1044 AI295950
387 NPS0508 EST 3 2-339 AA439667 also AI54395. Sequence similarity to human spermidine aminopropyltransferase.
388 NPS0509 EST 3 12-394 AA539198
389 NPS0510 GNL 3 83-227 and 498-518 AA696927 1 141 bp contig with AI107574 and AA801718.Match to Dros ARALAR 1 Y18197
390 NPS0512 EST 3 360-501 AA438961 Human KIAA0160 gene
392 NPS0514 EST 3 1 -237inv AI064414
393 NPS0515 EST 3 1 -67inv AA540712 809bp with AA440879 and AA440431. human cyclin G assoc. Kinase.
394 NPS0516 GNL 3 596-682inv AF132145 Drosophila damage-specific DNA binding protein DDBa p127 subunit
395 NPS0517 EST 3 1 -513 AF007159 Dros CDNA AF007159
396 NPS0518 EST 3 13-235inv AI51 1691
397 NPS0519 EST 3 1 -61 inv AA264883 also AA392712. Alt splice. EP line AQ254720-lethal ?
398 NPS0520 EST 3 30-451 AA438399 821 bp contig with AA439438. EP line AQ254604-lethal?
399 NPS0521 EST 3 600-627 AA440272 1324bp contig with AA438941. see also genomic AC013237
400 NPS0526 EST 3 65-483 AA264865 720bp EST contig with AA439894 and AI061812(end .Alt splice).
401 NPS0527 EST 3 1 -246 AA263693 Dros Unknown AF132150
402 NPS0528 EST 3 37-160and, 379-47 AA698620
403 NPS0529 EST 3 1 -51 inv AA391350
404 NPS0530 EST 3 100-499 AA392183 1 194bp EST contig. Seq. sim. to human DNA J
405 NPS0531 EST 3 49-348 and 412-45 AA696390 Also AI387907. Seq. sim. to mammalian ubiquitin degradation fusion protein 1
406 NPS0532 EST 3 1 -267inv AA802961 816bp with AA817584 .Match to Dros. complete mRNA AF145690. EP line AQ073773-lethal?
407 NPS0533 EST 3 1 -259inv AA699045 1989bp EST contig. random slug cDNA25 protein
408 NPS534 EST 3 1 -99bp AA952055 1205bp with AA202358, AA202625 and AA951416. Siah binding protein 1 (human)
409 NPS0535 EST 3 1 -610inv AA142266
684bp EST contig with AA736106. See also genomic AC014104. Matches Human CGI-37
410 NPS0536 EST 3 52-534 AA696974 protein
41 1 NPS0537 EST 3 03-626 and 734-106 AI532170 1565bp with AI544333 and AI062662. Definite transcription factor, MTF-1
412 NPS0538 EST 3 442-569 AA567128 Match to mouse EST.EP line AQ025072
413 NPS0540 EST 3 1 -79bp AA950480
414 NPS0541 GNL 3 1 -360 AJ006772 Dros cyclin B3
415 NPS0542 EST 3 30-194 and 291 -366 AA539625 882bp with AA202440 and AA390927. Dros Unknown AF132164
416 NPS0543 EST 3 1 -255 AA951297 Also AH 08147. Seq. sim. to mouse lysyl hydroxylase 3
417 NPS0544 EST 3 20-95 and 479-654 AA948996 1516bp EST contig. Seq. sim. to ZNF127-Xp [Homo sapiens]
419 NPS0546 EST 3 228-522 AA801928
420 NPS0547 EST 3 14-98 AA951 147 960bp EST contig with AA695598, AH 09677 and AA540269. Poss. protein phosphatase.
421 NPS0548 EST 3 1 -279 AA697191 1002bp with AA392404 and AA438791 . Seq sim. to mitogen inducible gene mig-2 - human
422 NPS0549 EST 3 650-689 AI518422
423 NPS1065 EST 3 27-689 AI535025
424 NPS0551 EST 3 8-563 AA950826
426 NPS0555 EST 3 41 1 -582 AH 09292 Poss heat shock protein
427 NPS0556 EST 3 1 -279inv AA202259 Same ESTs as NPS0483, different insertion point.
428 NPS0557 EST 3 131 -647 AA142065 Also AH 09234. poss. succinate semialdehyde dehydrogenase
« O OHW«-
430 NPS0559 GENO. 2 1-217 AC020266 At 18.6-19.2kb. Poss in space before gene coding for 669AA ORF.
431 NPS0560 GENO. 2 1-606 AC015232 At 2.5-3k. Intron of gene at 2-22k. 180AA ORF.
432 NPS0561 GENO. 2 1-169inv ACO13001 At 2.2-2.4k. 1.8kb before gene at 3-5kb. 183AAORF.
434 NPS0563 GENO. 2 1-849inv AC014142 At 37.7-38.6k(odd gaps?). No good exons
435 NPS0564 GENO. 2 1-585 A ACC0011 557788 6.3-6.8k. Space before EST contig coding for ORF with Seq sim to mam. NADH dehydrogenase
436 NPS0565 GENO. 2 1-505inv AC015232 At 10.2-10.7k. Intron of gene at 2-22k. 180AA ORF.
At 8.6-9.2k. In intron gene at 4-18k. 244AA ORF. No database matches. Large ORFS on Rev
437 NPS0566 GENO. 2 1-581 AC015178 too.
438 NPS0567 GENO. 2 1-637 ACO17945 At 1 -1.5k. ESTs inc AA949804 at 1.5-3.9k no matches
440 NPS0570 GENO. 2 1-662inv ACO15222 At 1 15.2-1 15.9K.lntron of gene represented by mRNA AF145688 on reverse strand. At 9.4-9.9kb. In intron of gene coding for a 1 121 AA ORF. Seq. sim to membrane type
441 NPS0571 GENO. 2 1 -496inv AC019859 metalloproteases.
442 NPS0572 GNL 2 1-559inv AC007299 At 92145-92705. 348AA ORF at 104-91 k.lntron of Dros GDI (see NPS0008) at 90658-93408
443 NPS0573 EST 2 50-397 AL062213 Only 1000bp long/EST AI532704 1 158 EST contig matching 2303cDNA with 537AAORF
444 NPS0574 GENO. 2 1-479inv AC020183 At 74.5-75kb. In intron of gene represented by ESTs inc.AA264274. Poss transcription factor.
445 NPS0575 GENO. 2 1-182inv AC007257 At 132883-133065. EST AA735518 at 132600bp
446 NPS0576 GENO. 2 1-370 AC017289 At 6.1 -6.5k. No good exons in this area. ESTs at 2.5k no matches.
447 NPS0577 GENO. 2 1-435 AC019995 At 12-12.4kb. In intron of gene at 22-4k. 946AA ORF. some sim. to other hypo, genes.
448 NPS0578 GNL 2 1-235 AC017330 At 7.3-7.6kb. 4bp overlap with Dros clathryn light chain AF055900
449 NPS0579 GENO. 2 1-328 ACO13001 At 4.7-5k. Part of gene at 3-5k coding for 183AA ORF.
At 14.5kb. In intron of gene coding for 948AA ORF. Seq. sim to Mam. Microsomal triglyceride
450 NPS0580 GENO. 2 1-110 AC017678 transport protein. ESTs confirm.
At 13.1 -13.6. In intron of gene on rev. coding for 671 AA ORF. Seq. sim to mouse sex-
451 NPS0581 GENO. 2 1-472 AC019893 determination protein homolog Fern 1 a
452 NPS0582 GENO. 2 1-790 AC020017 At 18-18.8kb. In intron of gene at 25-5k. 369AA ORF.
453 NPS0584 GENO. 2 1-404 AC014987 At21.5-22k. 1 kb 5' of gene represented by ESTs inc. AI257570
455 NPS0586 GENO. 2 1-518 AC017529 At 1.3-1.8kb. Poss. in space before gene coding for 360 AAORF at 3-19k (ESTs at 16-18k)
456 NPS0587 GENO. 2 1-324 AC015180 At 31.3-31.6kb. ORF on Rev. good match to transcription factors.
At 10-1.4kb. In intron of gene at 4-16kb(complement).1 173AA predicted ORF. Good homology
457 NPS0588 GENO. 2 1-325 AC015179 to human SEC24
459 NPS0590 GENO. 2 1-571inv AC015233 At 9.3-9.9kb. No matches this area.
At 7.9-8.4kb.ln intron of gene coding for 427AA protein. Similarity to alpha-amidating enzymes.
460 NPS0591 GENO. 2 1-455inv AC014124 ESTs at 7Kb
462 NPS0593 EST 2 1-295inv AI532704
464 NPS0595 GENO. 2 1-470 AC014326 At 1.5-2kb. Short sequence, no exons found.
4bb NKSUbSB J. 1 -bU/ A J 15424 At 1 15.4-I l5.9kb. In intron of gene at 101 -129k, 1267AA ORF. EST matches at 119k
466 NPS0597 GENO. 2 1 -260inv AC018184 At 1 17.4-1 17.7kb.Space before 367AA ORF at 1 18k. Poss DNA binding protein.
468 NPS0599 GNL 2 1 -615 U34383 At 2.6-3.2kb in AC017782. In intron of Dros Dopamine receptor DopR99B.
469 NPS0600 GENO. 2 1 -27bp ACO 14368 At 40kb. In intron of gene coding for 530AA ORF. Poss. DNA binding protein
At 1246-1804bp. In intron of gene represented by ESTs at 600bp-9kb.349AA ORF v. good sim.
470 NPS0601 GENO. 2 1 -551 AC014496 to coronin proteins.
At 60-60.5kb. Also AC006247. In intron of gene on rev. 574AA ORF. seq.sim to receptor
471 NPS0603 GENO. 2 1 -465inv ACO 15064 tγrosine kinase.
472 NPS0604 GENO. 2 1 -215 AC008370 At 56841 -57049bp.1 b downstream of Dros mutL gene
473 NPS0605 GENO. 2 1 -412inv AC017529 At 8.7-9.1. In intron of gene at 3-19k, 360AA ORF. ESTs at 16-19k.
AT 28.7k-29.2k. Space before ESTs at 21 -28k coding for 31 1 AA ORF on reverse strand
474 NPS0606 GENO. 2 1 -559 AC017138 poss.retinaldehyde-binding protein
475 NPS0607 GENO. 2 1 -474inv AC020031 At 7.7-8.1 kb. In intron of gene coding ofr 952AA ORF. Poss. WD repeat protein. ESTs confirm
477 NPS0609 GENO. 2 1-157 AC020227 At 1 16kb. Poss. iin space bewtween Dros. H3.3 gene and ESTs at 1 17k
At 31.1 -31 .5kb. In intron of gene coding for 1 152AA ORF. Seq. sim to mam. sphingosine
479 NPS061 1 GENO. 2 1 -485inv AC020007 phosphate lyase 1. ESTs confirm.
480 NPS0612 GENO. 2 1 -1 145inv AC014142 Same area as NPS0563. Diff. insert point. No good exons
482 NPS0614 GENO. 2 1 -522inv ACO 14423 At 55.1 -55.6kb. Gene at 51 k on rev , gene at 67k. nothing in between.
483 NPS0615 GENO. 2 1 -325 AC020320 At 42.5-42.8kb. No good exons/ESTs found
At 5.6-6kb. in intron of gene coding for 800AA ORF. Good similarity histone
484 NPS0616 GENO. 2 1 -426inv ACO 14073 acetyltransferase.(human)ESTs at 3k.
At 90-90.5k. In intron of gene at 81 -104k. 720AA ORF. Seq sim to carboxypeptidases. EST
485 NPS0617 GENO. 2 1 -527 AC019753 confirms
At 60.6-61.1 kb. Intron of gene coding for 1514AA ORF. Seq. sim to mammalianMAP/ERK kinas
486 NPS0618 GENO. 2 1 -504inv AC017381 kinase 4. ESTs confirm
491 NPS0623 GENO. 2 1 -333inv AC015399 At 44.2-44.6. Space before gene represented by EST AI517547
At 5.7k. In intron of gene at 1-8k. 397AA ORF. Seq sim to mouse zinc finger protein 40. ESTs
492 NPS0624 GENO. 2 1 -91 bp AC017666 confirm
494 NPS0626 GENO. 2 1 -548 ACO 15064 At 9.8-10.4kb. In intron of gene coding for 185AA ORF. No matches /ESTs
495 NPS0627 GENO. 2 1 -120inv AC017502 At 1 166-1285bp. Space before gene represented by EST AA695901
496 NPS0628 GNL 2 1 -408 AF132145 Same as NPS0516. diferent insert, point.
497 NPS0629 GENO. 2 1 -559inv AC007146 At 134370-135130bp. Full length cDNA at 135140-140020.ie space before this gene
498 NPS0630 GENO. 2 1-592 AC017643 At 40.8-41.4kb. In intron of gene at 32-45kb. 1058AA ORF. ESTs at 34 and 39k.
499 NPS0631 GNL 2 1 -108inv AF216532 AT 14kb in AC017347. In intron of Dros Migraine gene.
At 15.5-15. β.lntron of gene at 1 -3k. Coding for 449AA ORF. Seq sim to Sad1 unc-84 domain
500 NPS0632 GENO. 2 1 -284 AC020443 protein 1 [Homo sapiens]. ESTs confirm
502 NPS0634 GENO. 2 1 -522inv ACO 19961 At 600bp. No good exons in this area. Dros tetraspanin 1 at 8-14k
503 NPS0635 GENO. 2 1 -676bp AC014142 At 24.8-25.5k. No good exons in this area. ESTs at 17k and 27k.
504 NPS0636 GENO. 2 1 -541 AC017396 At 1 1.1-1 1.6kb. In intron of gene at 28-9k. coding for 378AA ORF. ESTs confirm.
505 NPS0637 GENO. 2 1 -59inv AC009197 At 90836-90894bp. 436AA orf at 86000-89400bp. EST AI513240 at 90600bp
506 NPS0638 UA 2 Matches various genomic sequences only one section not whole length?
507 NPS0639 GENO. 2 1 -234 AC017928 At 85kb.ln intron of gene at 95-70k coding for 1 160AA ORF.
At 44kb. In intron of gene represented by ESTs at 42-45k. Diff. insert point to NPS0105, same
508 NPS0640 GENO. 2 1 -31 inv ACO 14497
ESTs.
509 NPS0641 GENO. 2 1 -892inv ACO 14076 Strange Gap in match. Poss. not correct genomic?
51 1 NPS0643 GENO. 2 1 -197inv AC020308 At 2-2.2k. In intron of gene at 2-16k.456AA ORF ESTs confirm.
512 NPS0644 GENO. 2 1 -305 ACO 15089 At 36.1 -36.4kb. Space before gene represented by EST AI543228 on the reverse strand.
513 NPS0645 GENO. 2 1 -313 AC015089 At 35.7-36.1.15bp overlap with NPS0644
At 18.8-19.3kb In intron of gene at 1 -21 k coding for 915AA ORF. Some sim to mito carrier
514 NPS0646 GENO. 2 1 -530inv AC017981 proteins.
515 NPS0647 GENO. 2 1 -516 AC007452 At 98781 -99300bp. in Intron of gene represented by EST AI517398. V.good sim. to ATPases
516 NPS0648 GNL 2 1 -583inv AC012981 At 14-14.5k. In intron of Dros DDP-1 DNA binding protein AJ238847. No flybase
517 NPS0649 GENO. 2 1 -436 AC018631 At 52.6-53(n's) Incomplete sequence
518 NPS0650 GENO. 2 1 -442 AC017382 At 1-1.4k. V close to previously published lethal insert which overlaps an EST.
519 NPS0651 GNL 3 1 -536 AC014121 At 41.8-42.3kb. Space before Dros glut-1 on reverse strand,
At 45-45.4kb. In intron of gene represented by EST AI516046. Matches full length cDNA
520 NPS0652 GENO. 3 1 -469inv AC010120
AF160941.
521 NPS1066 GENO. 3 1 -417inv AC013107 At 26.6-27kb. In intron of gene coding for 928AA ORF with seq sim to luciferases. ESTs confir
523 NPS0655 GENO. 3 1 -510 AC020444 At 7.7-8.2kb. gene on rev. v. sim to human splicing factor 2 at 9k onwards (ESTs confirm)
At 76.4-76.9kb. Space before gene coding for 573AA ORF with good sim. to potassium chann
524 NPS0656 GENO. 3 1 -527inv AC020018 genes.
At 16.8-17.2kb. Predicted gene at 23- 20k (EST confirms) on rev. No matches in area of our
526 NPS0658 GENO. 3 1 -408inv AC012939 insert.
529 NPS0661 GENO. 3 1 -348inv AC014810 At 45.4-45.7kb. 3bp overlap with EST AI534852. In gene coding for 1354AA ORF.
530 NPS0662 GENO. 3 1 -463inv AC018338 At 9.6-10.1 kb. In intron of gene represented by ESTs inc. AA950503.
531 NPS0663 GENO. 3 1 -150inv AC013963 At 43.1 -43.2kb. ESTs at 46k and 47k. No other matches.
At 109930-1 10370bp. 875AA orf on rev. Poss. hormone receptor. ESTs match this orf inc.
532 NPS0664 GENO. 3 1-439 AC010662
AA735548
535 NPS0667 GENO. 3 1 -461 AC018284 At 41.8-42.3kb. No good exons/ESTs here.
537 NPS0669 GENO. 3 1 -544bp AC013201 At 34.7-35.2kb. 400bp 51 to EST AI258331. No matches.
538 NPS0670 GENO. 3 1 -530inv AC019785 At 61.4-61.9kb.2kb 3' to the start of TOLLO receptor on rev.strand.
541 NPS0673 GENO. 3 1 -513 ACO 15427 At 13-13.5kb. No exons/ESTs this area.
543 NPS0675 GENO. 3 1 -61 1 AC015159 At 20.3-20.9kb. No good exons/ESTs in this area.
545 NrbUb// hsr a 455-586inv AI944858 And AI946620 alt. splice. Genomic AC014457
At 57.3-57.7kb. Space before gene coding for 1514AA ORF. Seq, sim to mammalian MAP/ERK
547 NPS0679 GENO. 3 1 -371 inv AC017381 kinase kinase 4. ESTs confrim
548 NPS0680 GENO. 3 1 -539inv AC020108 At 7.8-8.3kb. No good exons /ESTs in this area.
At 9.4-9.9kb. 27bp overlap with EST AA264916(part of cluster at 9.5-8.3k). 287AA ORF with
549 NPS0682 GENO. 3 1 -449 AC015358
Good Horn to Rat tip49. TATA binding protein.
551 NPS0684 GENO. 3 1-480 AC017581 At 76.4-76.9kb. In intron of gene at 50-77k.2216AA ORF. Good sim to Mouse A-RAF kinase. 553 NPS0686 GENO. 3 1 -515 AC014262 At 7.4-7.9kb. Probably in intron of gene represented by ESTs at 7 and 8K. No other matches.
At 50.7-51 kb. In intron of gene coding for 1075AA ORF with some sim. to Human inhibitor of
554 NPS0687 GENO. 1 -357 AC015138 kappa light polypeptide gene enhancer in B-cells. EST at 48k.
555 NPS0688 EST 3 10-462, 518-end. in AI541626 159AAorf, no hits. Genomic AC018224 557 NPS0690 GENO. 3 1 -203 AC019753 At 50.8-51 kb. No predicted genes in this area. No ESTs.
558 NPS0691 GENO. 3 1 -202 AC019694 At 77.9-78.1 kb. In gene on rev(ESTs at77 and 79) with good seq. sim to ribosomal protein L31. At 21.3-21.6kb. Poss in intron of gene matching a yeast probable membrane protein. EST at 559 NPS0692 GENO. 3 1 -31 1 AC017810
21.7k
560 NPS0693 GENO. 1-51 1 AC013175 At 6.1-6.6kb. Probably in first intron of TCP-1-eta chaperonin homologue. ESTs at 7K onwards.
562 NPS0695 GENO. 3 1 -505 AC014850 At 1.8-2.3kb. No exons/ ESTs found. 563 NPS0697 GENO. 3 1 -406 AC013939 At 35.4-35.8kb. In intron of gene represented by ESTs Inc AA817130.
564 NPS0698 GENO. 3 1 -368inv AC020332 At 32-32.3k In intron of gene coding for 445 AA ORF. Seq. sim. to NADH oxidoreductase genes
565 NPS0699 GENO. 3 1 -278 AC020248 At 1 1.2-1 1.5kb. In gene represented by ESTs at 1 1.2k AA979097.
At 49.5-49.8kb. Poss part of gene coding for a 377AA protein with some sim. to human CGI-88
566 NPS0700 GENO. 3 1-290inv AC014875
ESTs at 53-58k also, may large gene).
568 NPS0702 GENO. 3 1 -766inv AC015159 At 5.4-6.2kb. Poss in intron of FTZ-F1 gene.
NPS0705 has no EC0R1 site yet doesn't match along whole length, first 168bp are unrelated to
571 NPS0705 GENO. 3 168-497 AC009457
AC009457(or other matching clones)
573 NPS0707 GENO. 3 1 -1306 AC019746 At 14.7-16k. Poss. on intron of gene coding for 991 AA ORF. ESTs at 10k and 23k. 575 NPS0709 EST 3 47-125 AI531 164 And AI518916.Genomic AC009344 576 NPS0710 GENO. 3 1 -375 AC014674 At 20.2-20.5kb. In space before gene represented by EST at 19 AI454966. 577 NPS0711 GENO. 3 1-322 AC013945 At 38.5-38.8kb. No exons/ESTs in this region 578 NPS0712 GENO. 3 1 -67inv ACO 14444 At 32.8-33.1 kb. ESTs upto 30k. None in this area. 579 NPS0713 GNL 3 1 -348inv AF055900 Matches AC009383. Dros Clathryn matches AF055900, different insert point to NPS0578 580 NPS0 14 GENO. 3 1 -316inv AC014674 At 15.2-15.5kb. EST AI297184 upto 15k 581 NPS0715 GENO. 3 1 -51 1 ACO 10028 At 22575-2308bp 199AA orf. poss peptidoglycan recognition protein
At 36.2-36.7kb. In intron of gene represented by ESTAI260669. ORF on rev, 350AA poss s-
583 NPS0717 GENO. 3 1 -490 ACO 14872 phase kinase.
At 61.5-61.9kb. In intron of gene coding for 1243AA ORF. ESTs matching whole ORF. Inc.
584 NPS0718 GENO. 3 1 -409 AC019531
AA941806
585 NPS0719 GENO. 3 1 -705 AC015426 At 9.7-10.4kb. No exons/ESTs in this area
588 NPS0722 GENO. 3 1 -480 ACO 13946 At 2.6-3.1 kb. No good exons/ESTs
589 NPS0723 GENO. 3 1 -1 14inv ACO 14946 At 29.4-29.7kb. No good exons/ ESTs in this area.
590 NPS0724 GENO. 3 1 -460inv ACO 17270 At 88.2-88.6kb. Poss. in first intron ofgene coding for 1382AA orf on rev. ESTs confirm.
592 NPS0726 GENO. 3 1 -300 AC019588 At 27.4-27.7kb. No exons/ESTs in this area.
Also AC015336 and AC013186. Part of gene represented by ESTs inc AA201355. Match to
593 NPS0727 GNL 3 1 -184inv AC009391
Dros PTB. AF21 1 191
594 NPS0728 GENO. 3 1 -866inv ACO 19542 At 31.9-32.8kb. 23bp 5" to EST AI404673.No other hits
At 24892-25196bp. orf matching ESTs AA735710 and AA942459. at 25220bp. V.like manno
595 NPS0729 EST 3 57-352inv AC009537
1 -phosphate guanyltransferase
At 1.2-1.6kb. In intron of gene represented by EST AI515487. V.good match to 2-oxoglutarate
597 NPS0731 GENO. 3 1 -443inv AC019528 deH.
598 NPS0732 GENO. 3 1-352inv AC017582 At 15.5-15.9kb. In intron of gene coding for a 645AA ORF. No ests/matches.
599 NPS0733 GENO. 3 1 -513inv AC015353 At 12.9-13.4kb. ESTs at 1 1 and 18k. None in this area.
600 NPS0734 GENO. 3 1 -600 ACO 14473 At 64.9-65.6k 900bρ from start of gene represented by complete cDNA AF132183 on rev.
At 20.4-20.7kb ESTs at 19 and 21 k. Probably in gene represented by these. 2050bp EST
603 NPS0737 GENO. 3 1 -371 AC02021 1 contig.471AA ORF.
At 44.5-45kb.Sρace before gene coding for 485AA ORF on reverse. Poss. membrane prot. ESTs
604 NPS0738 GENO. 3 1 -488 AC015395 at 40-41 k.
606 NPS0740 GENO. 3 1 -387 AC013175 At 18.6-19kb. Probably in space before gene represented by ESTs at 21 k
607 NPS0741 GENO. 3 1 -322inv ACO 17856 At 14.4-14.8kb. no good exons here. ESTs at 1 1 and Dros ribosomal s12 gene at 19k.
608 NPS0742 GENO. 3 1 -590inv AC017582 At 34.9-35.5kb. gene upto 31 k. EST at 37k. nothing at 31 .5-37k.
609 NPS0743 GENO. 3 1 -416 AC013197 At 13.2-13.6kb. Part of gene at 13-7k on rev. 558AA. No matches.
610 NPS0744 GENO. 3 1 -504inv AC019750 At 74.3-74.8kb. No good exons/ESTs in this area.
61 1 NPS0745 GNL 3 1 -879inv AC017132 At 35.5-36.4kb. 300 bp from start of Dros Proteasome gene X70304.
612 NPS0746 GENO. 3 1 -443inv AC018338 At 6.1 -6.5kb. A few bps 5' to EST AA801928.
613 NPS0747 EST 3 1 -231 AI517830 Also genomic AC014084
614 NPS0748 GENO. 3 1 -473 ACO 14209 At 3.6-4kb. 20bp 5" to EST AI404170. No other matches.
615 NPS0749 GENO. 3 1 10-188 AC02021 1 At 17.4-17.6kb. just after a gene on plus strand. No ESTs until 19k
616 NPS0750 GENO. 3 266-439 ACO 19754 At 6-6.4kb. No good exons/ESTs in this area
618 NPS0752 GENO. 3 1 -410 AC019753 At 122.4-122.8kb. No exons/ESTs this area.
619 NPS0753 GENO. 3 1 -531 inv AC020031 At 57.3-57.8kb 200bp 5' to EST(AI402026) with seq. sim to rolyl 4-hydroxylase, alpha subunit.
620 NPS0754 EST 3 407-546inv AI546104 Also genomic AC007577
621 NPS0755 GNL 3 1 -462 AC014889 At 23.3-23.7kb. In intron of transforming acidic coiled-coil containing protein (tacc)AF 146700.
At 73.1 -73.3kb. 300bp 5" to cDNA AF181626 coding for 1429AA protein, no database
622 NPS0756 GNL 3 1 -145inv AC017685 matches.
624 NPS0758 GNL 3 1 -249 ACO 14071 From 1.7-1.9kb. In intron of Dros E78B, U01088.
At 9.7-102kb. ESTs at 5-8kand 17k. Nothing in this area.EST at 8k. has apoptosis gene
625 NPS0759 GENO. 3 1 -534inv AC019869 similarities.
626 NPS0760 GENO. 3 1 -557 ACO 19742 At 24.2-24.7kb. ESTs inc. AI297362 at 23.5-24.1 kb.
628 NPS0762 GENO. 3 1 -408 AC014368 At 6.8-7.2kb. 1.7kb 5' to EST AA392444
629 NPS0763 GENO. 3 1 -566inv AC01781 1 At 36.5-37.1 kb. No good exons/ESTs this area.
630 NPS0764 GENO. 3 1 -570 ACO 12807 At 14.7-15.3kb. No good exons/ESTs this area
At 40.8-41.3kb.in intron of gene for 500AA ORF. Good seq sim to Succinyl-COA:3-Ketoacid-
631 NPS0765 GENO. 3 1 -579 AC017241
Coenzyme A Tranf erase precursor. Many ESTs confirm.
632 NPS0766 GNL 3 1 -51 1 AC0141 10 At 2.5-3.1 kb. In first intron of Dros Barbu gene AF132987.
633 NPS0767 GENO. 3 1 -505 ACO 15229 At 55.8-56.3kb. Gene predicting 1429AA ORF at 40-70kb. Seq sim. to FAT tumour supressor.
At 61.8-62.1 k. 15bp 3' to ESTs inc. AA952007. Part of gene coding for 591 AA ORF on rev.
635 NPS0769 GENO. 3 1 -210inv AC020260
Seq sim to P.walti rnp associated protein 55.
636 NPS0770 GENO. 3 1 -317 AC009904 At 66179-66500. 1 1 1 AA ORF on Rev, good match to Human DNA dir. RNA pol II NP_002687.1
Also genomic AC019950, at 126.4-126.6kb. EST doesn't match but may be in intron of
637 NPS0771 EST 3 1 -170 AI534756 tropomyosin II. lethal known 1989.
At 84.3-84.6kb. Space before ESTs on rev. at 82k. Good seq. sim to mouse nuclear body
638 NPS0772 GENO. 3 1 -317inv ACO 19758 associated kinase 1 b
639 NPS0774 GNL 3 109-606inv ACO 19824 At 1 1.2-1 1.8kb. In intron of dros Bab-1 gene, AJ252082. No flybase.
At 19.3-19.7kb. In intron of gene coding for 859AA protein. Sim to other hypothetical genes.
640 NPS0775 GENO. 3 1 -375inv ACO 13046
ESTs at 15-21 k confirm.
641 NPS0776 GENO. 3 1 -435inv ACO 13969 At 3.3-3.8kb. ESTs at 7kb no hits in this area.
642 NPS0777 GENO. 3 1 -790inv AC018284 Overlap NPS0667 Diff. insert point.
643 NPS0778 GENO. 3 1 -565 AC012986 Not a complete match.
At 13-13.5kb. In intron ofgene coding for 505AA ORF on rev. Good seq. sim to other
644 NPS0779 GENO. 3 1 -51 1 inv AC018173 hypothetical proteins. ESTs confirm.
645 NPS0780 GENO. 3 1 -558 ACO 19746 At 2.9-3.4kb. No good exons/ESTs in this area.
646 NPS0781 EST 3 482-573bp AI946243 No further hits. V. poor sequence
647 NPS0782 GENO. 3 1 -507 AC014870 At 4.9-5. δkb.ln intron of gene represented by ESTs inc. AH 14091.
648 NPS0783 GENO. 3 1 -30inv AC019806 At 30.5kb. 2bp 5' to EST AA950675. Probable transcription factor.
At 54.9-55.3kb. Poss. in space before gene coding for 1796AA ORF with good seq.sim to
649 NPS0784 GENO. 3 1 -412 ACO 19585 endoplasmins/HSPs. ESTs confirm.
650 NPS0785 GENO. 3 1 -492inv AC015425 At 9.9-10.4kb. No good exons/ ESTs in this area.
651 NPS0786 GENO. 3 1 -582 AC020454 At 3.3.5kb. No ESTs/exons this area.
bb-J NFSU/B / ϋblMU. ά 1 -518 ACO 15075 At 4.8-5.3kb. No ESTs/ Exons in this area
653 NPS0788 GENO. 3 1 -446 AC014674 At 34.6-35.1 kb. 40bp 3' to ESTs inc.AA949585.
654 NPS0789 GNL 3 1 -403 AF221066 At 18.6-19kb in AC015332. In intron ofDros Kurtz arrestin gene.
At 28.1-28.6kb.Prob. in intron of gene on rev.from 42-23k.good seq sim to branched chain
655 NPS0790 GENO. 3 1 -525 AC020081 ketoacid dehydrogenases. ESTs confirm
At 19.7-20.3kb. ESTAA141 1 18 at 19.3 K may be in intron of gene represented by this EST (en
656 NPS0791 GENO. 3 1 -589inv AC013164 of genomic clone).
657 NPS0792 GENO. 3 1 -528inv AC019758 At 83.3-83.8kb. In space beffore gene represented by EST at 82.2k AI533899.
At 33.2-34kb. In intron of gene coding for 256AA orf with good similarity to myosin
659 NPS0794 GENO. 3 1 -756 ACO 14423 phosphatase regylatory subunit.
660 NPS0795 GENO. 3 1 -630 ACO 19651 At 13.9-14.5kb. 1 kb 3' to ESTs inc. AA141648.
661 NPS0796 GENO. 3 1 -162 AC015227 At 10.5 -10.6kb. In intron of gene represented by EST AA820684.No good matches.
662 NPS0797 GENO. 3 1 -509inv ACO 13024 At 1.1 -1.5kb. No ESTs/ exons in this area
At 15.6-15.8kb. Space before Dros receptor like guanylate cyclase, U23485. Diff. insert point to
663 NPS0798 GNL 3 1 -182inv AC018013
NPS0075.
664 NPS0799 GENO. 3 1 -541 AC019758 At 1 1.4-1 1.9kb. No good exons/ESTs in this area.
665 NPS0800 GENO. 3 1 -633inv AC012837 At 8.9-9.6kb. In space before gene at 7k coding for 250 AA ORF. ESTs confirm.
666 NPS0801 GENO. 3 1 -460inv ACO 10052 At 8.3-8.7kb. 14bp before cDNA AF16091 1. Sim to dead box proteins.
667 NPS0802 GENO. 3 1 -443 ACO 17671 At 26.3-26.8kb. ESTs at 25k Large ORF at 27.4k?
At 86-600bp. Plus 5k from AC019567. 4bp 5' to ESTAA142181 part of gene coding for 350A
668 NPS0803 GENO. 3 1 -524inv ACO 19498
ORF.
669 NPS0804 GNL 3 1 -537inv AC018013 At 17-17.5kb. Space before HLH gene U38238. Diff. insert point to NPS0064.
670 NPS0805 GENO. 3 1 -459inv AC013958 At 27.6-28kb. Either in 3'region or Dros NMDA receptor, or in space before Na/K cotransporter,
671 NPS0806 GENO. 3 1 -371 ACO 14084 At 48.8-49.1kb. Dros proteasome gene at 44k, ESTs at 52k, nothing in this area.
672 NPS0807 GENO. 3 1 -551 inv AC017890 At 42.1 -42.6k. cDNA ending at 43.7k. ORFs/ESts at 37k. No hits this area.
674 NPS0809 GENO. 3 1 -515inv ACO 14087 At 2.5-3kb. 300bp 5' to EST AI062268(no db matches.)
At 10.4-10.9kb,. In intron of gene represented by ESTs inc AI456639.2770bp contig no db
675 NPS0810 GENO. 3 1 -513 AC018127 matches.
676 NPS081 1 GENO. 3 1 -549inv AC017731 At 1 1.7-12.3kb EST at 12.9k incAH 08782, no DB matches
At 70.3-70.6kb. In intron of gene represented by ESTs at 71 -68k. Poss ATPsynthase. EP line
677 NPS0812 GENO. 3 1 -339inv ACO 14784 too, lethal?
678 NPS0813 GENO. 3 1 -582inv AC014994 At 5.1-5.6kb. ESTs at 7-12k. no other exons/matches.
679 NPS0814 GENO. 3 1 -323 AC017623 At 16.68-17kb. Poss. in intron of gene represented by ESTAI063960 at 16641 bp.
680 NPS0815 GENO. 3 small genomic fragment
681 NPS0816 GENO. 3 1 -722inv AC006402 At 43.6-44.4kp. 34bp 5* to ESTAI109098. No db matches
682 NPS0817 GENO. 3 1 -860inv ACO 15221 At 5.9-6.7kb. No exons/ESTs this area.
683 NPS0818 GENO. 3 1 -570inv AC017270 At 82.4-83kb. In intron of gene coding for 1382 AA ORF on rev. ESTs confirm
At 40.2-40.7kb. In intron of gene coding for 612AA orf, ESTs at 41 -44k.Poss transcription
684 NPS0819 GENO. 3 AC020026 factor.
685 NPS0820 GNL 3 1 -22inv AC018284 At 32k. 200bp before LK6 knase U76378.
686 NPS0821 GENO. 3 1-378inv AC015351 At 12-12.3kb. No exons / ESTs in this area
687 NPS0822 GENO. 3 1 -541 inv AC020316 At 6.4-6.9kb. ESTs at 9.3k, none in this area.
688 NPS0823 GENO. 3 1 -427inv ACO 12807 At 7.7-8.1 kb. No good exons/ESTs this area
689 NPS0824 GENO. 3 1 -157inv AC020316 At 7.1-7.2kb. ESTs at 9.3k, none in this area.
690 NPS0825 GENO. 3 154-408inv AC020539 At 45.6-46kb. EST at 44k, no other matches.
691 NPS0826 GENO. 3 1 -441 inv AC017691 At 48-48.4kb. No exons/ESTs in this area.
692 NPS0827 GENO. 3 1 -686 AC014819 At 19.1 -19.8kb. 100bp 3' to EST AI294326, part of gene coding for 389AA ORF on rev.
693 NPS0828 GENO. 3 1 -927inv ACO 14840 AT 54.7-55.6kb. 1 kb 3' to start of dros spazle gene on rev. strand.
695 NPS0830 GENO. 3 1 -201 AC017740 At 99.2-99.4kb. 1 kb from start of dros Dofl in rev. strand. AJ010642.
At 48-48.1 kb. Poss. in intron of gene at 47-73k.898AA ORF.ESTs from 65k, prob.
696 NPS0831 GENO. 3 1 -1 14inv AC020304 phospholipase.
At 7.3-8k.ln gene on rev. coding for 310AA ORF.Seq. Sim. to HumanTATA binding protein
698 NPS0833 GENO. 3 1 -786 AC015358 interacting protein 49 kDa. EST cluster confirm this.
699 NPS0834 GENO. 3 1 -574inv AC012776 At 9.9-10.5kb. No exons/ESTs this area.
700 NPS0835 GNL 3 1 -621 inv AC020154 At 34.8-35.4kb. In intron of Dros FAX gene, U21685. EP lines known.
702 NPS0837 GENO. 3 1 -469 AC017267 At 21.1 -21.6kb. 1 kb 5' to anothre p-element claiming to be in Hearty, no match on AC014595.
703 NPS0838 GENO. 3 1 -963 AC012791 At 12.1 -13kb. In intron of gene represented by EST AA439616. No db matches.
705 NPS0840 GENO. 3 1 -754 AC020432 At 3.4-4.2. EST at 2.3kb. no other hits.
At 27.5-28.4kb. In intron of gene represented by ESTs from 20-37k. V. good sim. to rat casein
707 NPS0842 GENO. 3 1 -989inv AC019820 kinase.
At 96.1 -96.3kb. In intron of gene represented by EST at 95-97k.AI546104. poor match to WD-
708 NPS0843 GENO. 3 1 -183inv AC020006
40 repeat prot.
At 396-709bp. 1 bp overlap with gene at 708-1538. Good seq. sim. to phosducin like
709 NPS0844 GENO. 3 1 -304inv ACO 19806 genes. ESTs confirm.
710 NPS0845 GENO. 3 1 -855 AC015138 At 63.3-64.1 kb. Large ORF in this area, no DBmatches. No ESTs.
71 1 NPS0846 GENO. 3 1 -825inv AC020332 At 29.2-30kb. In gene coding for 445 AA ORF. Seq. sim. to NADH oxidoreductase genes.
712 NPS0847 GENO. 3 1 -798 AC015342 At 42.1 -42.9kb. No good exons/ESTs in this area.
713 NPS0848 GNL 3 1 -797inv AC013201 At 7.4-8.2kb. 500bp 5' to Dros SIN mRNA.AF151390.
714 NPS0849 GENO. 3 1 -491 inv AC017905 At 52.9-53.4kb. No exons/ESTs in this area.
At 71.8-72.8kb. In area of predicted gene with sim. to cγtosotic phospholipase A2 beta. EST at
715 NPS0850 GENO. 3 1 -1013 ACO 17721
74k.
At 122.6-123.5kb. In intron of gene coding for 320AA ORF . V. good seq. sim to human
716 NPS0851 GENO. 3 1 -902 AC014376 phosphoribosyl pyrophosphate synthetase. ESTs confirm.
At 26.8kb. 60bp 5' to EST AI542698. Other ESTs from 27-41 k(introns). may be part of
717 NPS0852 GENO. 3 1-64bp AC020327 bumetanide-sensitive Na-K-2CI eotransporter
At 120.3-120.4kb. In space before gene coding for 286AA ORF on rev. Seq. sim. to Protein
720 NPS0855 GENO. 3 1-110inv AC019562
Phosphatase2C
At 15-1 δkb.Either space before Dros MPCP gene on rev or before gene represented by EST at
721 NPS0856 GENO. 3 1-1070inv AC018185
18k.
722 NPS0857 GENO. 3 1-765 ACO13945 At 36.3-37.1 kb. No good exons/ESTs in this area
723 NPS0858 GENO. 3 1-568 AC017811 At 41.4-41.9kb. No good exons/ESTs this area.
At 17.4-18kb. In intron of gene at 21 -9kb. 432AA ORF, seq. sim. to mouse nucleotide binding
724 NPS0859 GENO. 3 1-580 ACO14351 protein 2.
725 NPS0860 GNL 3 1-403inv AC012937 At 38.9-39.3kb. Probably in intron of Dros polypyrimidine tract binding protein (PTB).AF21 1 191 .
726 NPS0861 GNL 3 1-416 AC017132 At 23.2-23.7kb. In intron of Dros fatty acid desaturase.U73160. diff. insert point to NPS0048.
727 NPS0862 GENO. 3 1-52bp ACO10070 At 900bp. Part of gene represented by EST AI515487. V.good match to 2-oxoglutarate deH.
728 NPS0863 GENO. 3 1-490 AC020332 At 33.6-34.1 kb. No exons/ESTs this area.
729 NPS0864 GENO. 3 1-1153inv AC019826 At 12.4-13.5kb. In space after EST at 10k or before Dros Brahma at 14.8k
730 NPS0865 GNL 3 1-1144 AC017132 At 22.6-23.7. Intron of FAD U73160.Diff insert point to NPS 0861 /NPS0048.
At 26.6-27.4kb. In 1 st intron of gene coding for342AA ORF on rev. Seq. Sim. to yeast
731 NPS0866 GENO. 3 1-858 ACO15441 autophageosis prot. ESTs confirm.
732 NPS0867 GENO. 3 1-832inv AC018100 At 36.3-37.2kb. In intron of gene coding for 354AA ORF. EST at 39k confirms.
733 NPS0868 GENO. 3 1-532 AC012947 At 99.4-99.9kb.No good EST/Exons in this area.
735 NPS0870 GENO. 3 1-1145 AC017676 At 45-46kb. AC017592 matches end of AC017676. 1 kb 3' to EST at 44k.
736 NPS0871 GNL 3 1-447inv AC015301 At 4.2-4.7kb. Probably in first intron/promoter of Dros SF1 gene on rev.AJ243904.
At 141 .3-142.2kb. Just 3' to ESTs at 140-141 k inc.AI387574. Seq. sim. to human UDP-glucos
738 NPS0873 GENO. 3 1-885 AC019753
4-epimerases.
739 NPS0875 GENO. 3 1-1083 AC012873 At 30.7-31.8kb. No exons/ESTs this area.
740 NPS0876 GENO. 3 1-1796 ACO14884 At 9.9-1 1.7kb. No exons/ESTs this area.
741 NPS0877 GENO. 3 1-819 ACO15220 At 1.5-2.3kb. No exons/ESTs this area.
At 16.8-17.9kb. Probably in promoter of gene represented by cDNA AF132153. Probable
742 NPS0878 GENO. 3 1-1003 AC014889 endopeptidase.
743 NPS0879 GENO. 3 1-384 AC019683 At 385-768bp. EST /Gene sequence at 2kb. not perfect Match. Cytoplasmic dynein/annexin 9.
745 NPS0881 GENO. 3 1-519 AC012727 At 52-52.5kb. In intron of Dros anon 1 A3 at 53.3kb. AF005843. ESTAA439438 confirms.
747 NPS0883 GENO. 3 1-99bp AC019746 At 14.7kb. No exons/ESTs this area.
748 NPS0884 GENO. 3 1-423 AC020248 At 9.9-10.4kb. In intron of gene represented by ESTs at 1.6kb. inc AI517276. No db matches.
749 NPS0887 GENO. 3 1-495 ACO10059 And start of AC015427. At 1.1-1.6kb. No exons/ESTs this area.
At 81.2-81.7kb. Poss. in intron of gene represented by ESTs at 80 and 82k. Some seq. sim. to
753 NPS0889 GENO. 3 1-556inv AC017585 nucleoporins.
At 26.7-27.6kb(end). 17bp overlap with EST AC018045.1435bp EST contig, no good db
754 NPS0890 EST 3 1-925 ACO18045 matches.
755 NPS0891 GENO. 3 1-1125 AC017250 At 26-27kb. 300bp 5' to EST AI944859.
756 NPS0892 GENO. 3 1-1137 ACO14884 same area as NPS0876, diff. insert point
757 NPS0893 GENO. 3 1-534 ACO13945 At 965-158 I bp. No good exons/ESTs this area.
758 NPS0894 GENO. 3 1-527inv AC019746 At 7.9-8.4kb. No exons/ESTs in this area.
759 NPS0896 GENO. 3 1-646 ACO10005 At 37123-37769bp. ORF related to Human polypeptide chain release factor ERF1
760 NPS0897 GENO. 3 1-93bp ACO15358 At 5.8-5.9kb. Intron of gene represented by EST AI257536.
761 NPS0898 GENO. 3 1-1044 ACO10005 At 80366-81430bp. ORF related to FRG1 from Fugufmuscular dystrophy related)
762 NPS0899 GENO. 3 1-1345 AC015076 At 25.6-26.9kb. 23bp overlap with EST AA141054, same as NPS0371 , diff. insert point.
At 124.8-125.4kb. In intron of Dros gene represented by cDNA AF145694. 447AA ORF. Prob.
763 NPS0900 GENO. 3 1-597inv ACO14084 adenylosuccinate synthetase 1
764 NPS0901 GENO. 3 1-577 AC019995 At 63.9-64.5kb. In intron of gene represented by EST AI402854. no db matches.
At 2-3kb. In intron of gene on rev, 402AA. Good seq. sim. to Ubiquitin carrier proteins. EST
765 NPS0902 GENO. 3 1-940 AC020333 confirm.
766 NPS0903 GNL 3 1-1131 AC017132 At 24.3-24.4kb. 100bp 3" to start of Dros FAD .U73160. Diff. insert point to NPS0048.
At 60-4-61.1 kb. In intron of gene represented by ESTs at 57-61.8kb. Part of gene coding for
767 NPS0904 GENO. 3 1-687inv AC020260
591 AA ORF on rev. Seq sim to P.walti rnp associated protein 55.
768 NPS0905 GENO. 3 1-510 AC017784 At 69.7-70.2kb. ESTs at 65 and 75k. No hits in this area.
At 4.1 -4.2kb. 200bp 5' to ESTs inc. AI542840. V. good seq. sim to myeloid/lymphoid or mixed
770 NPS0907 GENO. 3 1-113inv AC014365 lineage-leukemia translocation to 10 homolog. MAF10.
At 73.3-74.4kb.ln area of predicted gene with sim. to cytosolic phospholipase A2 beta. EST at
771 NPS0908 GENO. 3 1-1116inv AC017721
74k.
At 22.9-23.6kb. May be in intron of gene on rev. coding for 1586 AA ORF with seq. sim. to ATP
773 NPS0910 GENO. 3 1-727inv AC014325 binding proteins.
774 NPS0911 GENO. 3 1-1010 AC015160 At 28.6-29.6kb. No good exons/EST in this area.
775 NPS0912 GENO. 3 1-1426 AC019772 At 0.6-2kb. No exons/ESTs in this area.
776 NPS0913 GNL 3 1-403 ACO19850 At 86-86.4kb. In intron of dros protein disulphide isomerase gene.UI 8973.
777 NPS0914 GENO. 3 1-1111inv AC014588 At 19.3-20.5kb. 35bp 5' to ESTAI543592.
778 NPS0915 GENO. 3 1-499 AC017495 At 23.9-24.4kb. ESTs at 21 k, none in this area.
779 NPS0916 GENO. 3 1-371inv AC014994 At 600-900bp.ESTs at 7-12k. no other exons/matches.
781 NPS0918 EST 3 AI514396 also genomic AC013006. At 4.5-5.5kb.
At 15.5-15.6kb. Space before Dros Guanylate cyclase at 14.2kb on rev. U23485. Diff. insert
782 NPS0919 GNL 3 1-118 AC018013 point to NPS0075.
At 33.8-33.9kb. In intron of gene coding for 1500AA ORF from 25k with good sim. to UNC51
783 NPS0920 GENO. 3 1-176 AC017329 kinase. EST confirmfsee NPS0383)
784 NPS0921 GENO. 2 1-537INV AC006073 At 32.8-33.3kb. In intron of gene coding for 246AA. No db hits, no ESTs.
785 NPS0922 GENO. 2 1 -720 AC004299 At 83.9-84.7kb.ln intron of gene represented by EST AI533754
786 NPS0924 GENO. 2 1-599 AC004115 At 13.6-14.2kb. In gene represented by ESTs AI520524 and AI945841. No db matches.
At 72.4-73kb.ln intron of gene coding for 355AA protein. 51777-83843bp. No database
787 NPS0925 GENO. 2 1 -581 AC004716 matches
788 NPS0926 GENO. 2 1 -628 AC005889 At 36-36.6kb. No exons/ ESTs in this area.
790 NPS0928 GNL 2 1 -86inv AF165153 In intron of Dros DGP-1 , developmental gene. See genomic AC004296
With AC004766inv. In intron of gene represented by ESTs AI238344, AH 08292 and AI533605
791 NPS0929 GENO. 2 1 -573inv AC004306
At 52k-67k
At 71.6-72.2kb.ln intron of gene represented by EST AI542461 , part of 1506AA ORF. Poss.
792 NPS1077 GENO. 2 1 -648 AC006472 transcription factor.
At 67-67.4kb. In Intron of gene represented by ESTs inc AA941489.1647bp EST contig. No
793 NPS0931 GENO. 2 1 -463inv AC007175 good db matches.
794 NPS0932 GENO. 2 1 -519 AC006073 At 40.7-41.2kb. No exons/ ESTs in this area.
796 NPS0935 GNL 2 1 -307inv AC004423 In intron of Dros AKAP see also NPS288
797 NPS0936 GNL 2 1 -412inv AF167578 Space before Dros Septin 5. See genomic AC005448
At 39-39.4kb. Poss. in 3'UTR of gene coding for 355AA protein at 39.7-40.7.Weak seq. simi. t
798 NPS0937 GENO. 2 1 -478 AC004313 potassium channel gene
799 NPS0938 GENO. 2 1 -489 AC004641 At 125.4-125.8kb. No exons/ESTs in this area
800 NPS1078 GENO. 2 1 -558inv AC004306 At 57.4-57.9kb. In intron of gene of gene represented by ESTs AI533605 and AI238344.
At 73.3.73.8kb. ESTs at 74650bp (AA803646, AI518976, AH 081 14) Sequence similarity to
802 NPS0941 GENO. 2 1 -544 AC005334
U5 snRNP
803 NPS0942 GNL 2 1 -201 AC00 154 Part of Dros NTPase different insert point from NPS28
804 NPS0943 GNL 2 1 -524 AC004766 At 60.5-61 kb. 130bp 3' to start of Dros Igloo gene on rev.S72579.Gap 43 like protein.
805 NPS0944 GENO. 2 1 -621 AC004361 At 21.4-22kb. 338AA ORF at 19-32k, no db hits.
806 NPS0945 GENO. 2 1 -569 AC020283 At 16.6-17.1 kb. ESTs at 15k and 21k none in this region.
808 NPS0947 GENO. 2 1 -233inv AC005750 At 71.8-72kb. 630AA orf at 60-77k no hits. No ESTs either.
At 43.8-44.3kb. 274AA ORF at 40-50k,complement.Seq. sim to human TGFB inducible early
809 NPS0948 GENO. 2 1 -525 AC005269 growth response 2
At 19.8-20.3kb. Part of gene represented by EST AI402921. Part of large ORF matching
810 NPS0949 GENO. 2 1 -531 AC005554 mammalian fatty acid synthase.
812 NPS0952 GENO. 2 1 -498inv AC005894 At 63-63.4kb. No good exons/ESTs in this area
At 34.6-34.9kb. ESTs at 30k match Dros syndecan,U03282.ln large intron going into
813 NPS0954 GNL 2 1 -320 AC004564
AC019924.
814 NPS0956 GENO. 2 1 -429 AC005716 At 72.2-72.6kb.EST at 69-71 k, probably part of this gene.
815 NPS0958 GENO. 2 1-71 bpinv AC01 1662 At 21 1.6kb.10O bp 5' to ESTA1108521 at 211 -226k.
In intron of gene coding for a 945AA protein at 87648-1 13518. Strong sequence similarity to
816 NPS1079 GENO. 2 1 -75bp AC004758
Human retinoblastoma binding protein 2
At 90-90.5k. In intron of gene coding for 1079AA ORF with good seq. sim. Human
818 NPS0963 GENO. 2 1-512inv AC004758 retinoblastoma binding protein 2. EST at 94k confirms.
819 NPS0964 GENO. 2 1-54bp AC004334 At 19.8kb.ln space b/n ESTs at 18k and those at 21 k.
820 NPS966 GENO. 2 1-557inv AC005149 In intron of gene coding for 424AA protein at 70149-97938. No database matches.
821 NPS0968 GENO. 2 1-202 AC005333 At 94.2-94. kb. Also AC018171. No good exons/ESTs this area.
822 NPS970 GENO. 2 1-534inv AC005334 In intron of gene coding for 309AA protein at 64276-77888bp. No database matches
823 NPS0971 GENO. 2 1-438 AC006421 At 7-7.4kb. Also AC005449. No good exons/ESTs this area.
824 NPS0972 GENO. 2 1-524INV AC005443 At 23.3-23.8kb. No good exons/ESTs this area.
At 5.8-6.2kb. Poss. in intron of gene coding for 2245AA ORF represented by ESTAI947230 at
825 NPS0973 GENO. 2 1-492 AC005650
6.6k. Seq. sim to human uridine phosphorylase.
826 NPS0974 GENO. 2 1-535 AC005889 At 18.8-19.3kb. No good exons/ESTs this area.
827 NPS0975 GENO. 2 1 -47bpinv AC005130 At 13.6kb. In Intron of gene represented by ESTs at 7-28k inc AA949050(2072bp contig).
829 NPS0977 GNL 2 1-100, 146-499 AF097364 Drosophila Drongo gene
At 32.6-32.9kb.Space before gene coding for 834AA protein at 33470-40630. Sequence
831 NPS0979 GENO. 2 1-256inv AC004722 similarity to bromodomain containing proteins.ESTs confirm.
832 NPS0980 GENO. 2 1-406 AC003054 At 25.2-25.6kb. Poss. in intron of gene at 14-46k, 538AA ORF. EST at 18-44k.
834 NPS0982 GENO. 2 1-460 AC004280 At 48.5-48.9kb. ESTs at 47.8k and 49.5k. No db matches.
In intron of gene coding for 300AA protein at 30647-46841. Weak sequence similarity to Mouse
835 NPS985 GENO. 2 1-178inv AC001661 surfeit gene
836 NPS0986 EST 2 1-602 AI533769 See also genomic AC005269. Other ESTs no DBmatches.
837 NPS987 GENO. 2 1-562 AC004362 No good predicted exons in this area.
838 NPS0988 GENO. 2 1-521 AC004370 At 54.6-55.1 kb. 3bp 3* to EST AI124332 on rev. No db hits.
840 NPS0991 GENO. 2 1-535inv AC005447 At 34.1 -34.7kb. In intron of gene coding for a 802AA protein at 28-49kb.EST at 33, 40 and 48k.
841 NPS0992 GENO. 2 1-342 AC019901 1 19.6-1 19.9kb. No good exons/ESTs in this area
At 65.6-66. I kb. In space before gene coding for a 399AA protein at 66-68kbp. ESTs confirm.
842 NPS0993 GENO. 2 1-512 AC005454
Seq. sim. to mitochondrial carrier protein genes.
843 NPS0994 GENO. 2 1-515inv AC005130 At 42.5-43kb. No good exons/ESTs in this area
At 62.4-62.7. Space before gene represented by ESTs inc. AI294250. Seq. sim. to ribosomal
845 NPS0997 GENO. 2 1-565inv AC012753 protein L30.
AT 66.7-67.3kb. 3bp overlap with gene coding for1365AA protein at 67-74kb. ESTs AH 06939
846 NPS0998 GENO. 2 1-568 AC005889 and AI296430 confirm.
847 NPS0999 GENO. 2 1-503 AC004370 AT 52.9-53.4kb.ln intron of gene represented by EST AH 24332.
At 36.3-36.9. In gene coding for 676AA protein at 3451 1 -37955bp. Sequence similarity to
848 NPS1000 GENO. 2 1-620 AC004351 mouse LUN gene.
At 1 1.2-1 1.7kb.ln Intron of gene coding for 1467AA protein at 0.8-18kbp. 3 bp overlap with
849 NPS1001 GENO. 2 1-519inv AC004766
EST at 1 1.7. Seq. sim. to Drosophila Lipase 3.
In gene coding for 805AA protein at 160506-163420bp. Sequence similarity to Mammalian Valyl
850 NPS1002 GENO. 2 1 -80inv AC006247 tRNA synthetase.
At 13.7-14kb. In intron of gene coding for a 1 131AA protein at 20-1 k. No database matches.
851 NPS1003 GENO. 2 1 -370inv AC006574
ESTs at 16-18k.
853 NPS1005 GENO. 2 1 -535inv AC005447 At 0.3-0.8kb. Also AC019827. 1 kb 3' to Dros Anillin and ESTs.
At 30.5-31 kb.ln intron of gene coding for 593AA protein at 39-20kbp. Poss. calcium channel.
854 NPS1006 GENO. 2 1 -581 inv AC005643
EST at 31.5-33k.
At 13.8kb. 20bp Overlap with EST AI2601 1 1. 889bp EST contig. Seq. sim. to multivitamin
856 NPS1009 GENO. 2 1 -77inv AC004532 transporter.
857 NPS1010 GENO. 2 1 -496inv ACO 19704 At 1 1.5-12kb. Between ESTs at 10k and ESTs at 13k.
At 4.5-5kb.ln intron of gene coding for 604AA protein at 3-8.8k.Sequence similarity to C.elegan
858 NPS1012 GENO. 2 1 -483 AC004423
AL021481 gene.ESTs confirm gene
860 NPS1013 GNL 2 1 -560 AC00581 1 Intron of Dros Steroid receptor L06423 FTZ-F1 B
861 NPS1016 GENO. 2 1 -596 AC005653 At 46.8-47.4kb.EST upto 43k. full length cDNA at 51 k, nothing in between.
862 NPS101 GENO. 2 1 -539 AC004516 At 60-60.5k. Part of gene at 63-47k coding for 732AA ORF. Poss. exonuclease. EST at 54k.
At 1 19.7-120.2kb. In intron of gene coding forl 142AA protein at 1 16605-128877bp. Sequenc
863 NPS1019 GENO. 2 1 -505inv AC005285 similarity to Guanine nucleotide exchange genes.
At 96.6-97.1 kb. In intron of gene coding for 395AA ORF at 91 -1 18k. Seq. sim . to inwardly
864 NPS1021 GENO. 2 1 -504inv AC020029 rectifying calcium channels.
865 NPS1022 GENO. 2 1 -191 AC005643 At 1 1.5-1 1.7kb. In intron of gene represented by ESTs inc. AI238833.
At 22.8-23.2kb. 100bp 3' to ESTAI519819. Part of gene on rev.1 159AA ORF.Poss.
866 NPS1023 GENO. 2 1 -468 AC004642 lysophosphatidic acid acyltransferase.
At 1 1.3-1 1.8kb. Part of gene coding for 1481AA protein at 159-1 1694bp. Good Seq. sim. to
867 NPS1024 GENO. 2 1 -578inv AC005749
JNK-binding protein JNKBP1 Mouse. ESTs confirm.
868 NPS1025 GENO. 2 1 -598 AC020254 At 2.6-3.2kb. ESTs at l and 8k.
869 NPS1027 EST 2 1-40bp AI257015 661 bp EST contig. See also genomic AC004340.
871 NPS1029 GENO. 2 1 -198 AC004375 At 30.5-30.7kb. ESTs at 32-34k. No exons/ESTs at 30k area.
872 NPS1030 GENO. 2 1 -316 AC005472 At 78.5-78.8kb.lntron of gene represented by ESTs at 78-81 k inc. AI258704. No db hits.
873 NPS1031 GNL 2 1 -495inv AC004154 At 1 1.6-12.1 kb. 21 bp overlap with Dros. geranylgeranyl transferase type ll,AF133269.
At 22.6-22.7kb.ln intron of gene coding for 1218AA protein at 2-28k. Has been predicted from
874 NPS1032 GENO. 2 1 -1 16 AC004328
Dros genomic AL03531 1 and has similarity to mouse BOP1. Many ESTs confirm.
At 33.7-34.2k. In intron of gene coding for 407AA protein at 3680-49217(complement). No
875 NPS1033 GENO. 2 1 -581 inv AC0051 12 database matches
At 2.8-3.3kb Space before gene coding for 387AA protein at 3496-35348bp. No database
876 NPS1034 GENO. 2 1 -506inv AC004367 matches. ESTs at 7k
At 78.9-79.3kb.lntron of gene represented by ESTs at 78-81 k inc. AI258704(different point of
877 NPS1036 GENO. 2 1 -41 1 AC005472 insertion to NPS 1030)
υtivυ. 1 "i31 L*3»ua AT .sb.j-.5b.bKD. No good exons/ESTs this area.
880 NPS1039 EST 3 1-250 AI543199 1 127bp contig with AI544292.See also genomic AC004658.
881 NPS1062 GNL 3 1-376 AC007757 Also matches Dros. EST AA951801. Part of Dros Elongin B (diff. insert point to NPS79)
At 102.6-103.2kb. In intron of gene represented by EST AA246373 at 100-104k. No db
882 NPS1044 GENO. 3 1-597 AC006091 matches.
883 NPS1045 GENO. 3 1-498 AC005720 At 163.5-164kb. In intron of gene represented by ESTs AI260814 and AI947168.
At 16.4-16. βkb.ln intron of gene coding for 575AA protein at 10435-29297. EST at 12-15k
885 NPS1049 GENO. 3 1-486 AC004713
AH 08650. Weak similarity to CDC36 genes.
886 NPS1050 GENO. 3 1-544 AC005813 No good predicted exons in this area.
887 NPS1051 GENO. 3 1-549 AC006936 At 95.5-96kb. No good exons/ESTs in this area
888 NPS1052 GNL 3 1-306inv AJ236864 Dros. putative adenosine kinase. Homozygous deletion has no phenotype.
889 NPS1053 GENO. 3 1-579inv AC005720 At 79.4-79.9kb.ln intron of gene represented by ESTs at 78-83k.AI544292 and AI543199.
AT 68.4-68.6kb. In intron of gene coding for 931 AA at 70-50kb. Seq. sim. to C. elegans Zinc
890 NPS1056 GENO. 3 1-191inv AC004266 finger protein. ESTs AI259457 and AI519745 at 61 -67k.
891 NPS1059 GENO. 3 1-264 AC006936 At 132.3-132.5kb. No ESTs/Exons this area.
892 NPS1063 GNL 3 488-536 AI062190 EST comes from Dros ferrochelatase
At 30-31 k.Part of gene at 35-29k. 900 AA ORF.Seq sim to Human transcriptional co-activator
893 NPS1064 GENO. 3 1-1068 AC019758
CRSP150, AF 104256. ESTs confirm.
894 NPS1076 EST 2 101-597 AI388606 1249bp contig with AI258281 and AI258326
895 NPS1081 EST 2 1-491 AI533993 AC005714
At 99630-1001 Obp. 1065AA ORF at 95-1 18k. EST AI295594. Similarity to mammalian RAD50
896 NPS1082 GENO. 2 1-475inv AC005714 poss. channel
897 NPS1083 GENO. 2 1-461 AC004375 At 31.1 -31.5kb. different insert point NPS1029.Closer to EST at 32k.
At 16.3-16.8kb. Between ORF at 15k (seq. sim. to pre-mRNA splicing SR protein rA4) or Dros
898 NPS1084 GENO. 2 1-507inv AC018075
ERCC2 at 17.4k. EST at 15.8k.
899 NPS1085 GENO. 2 74-502 AC007413 At 22(n's)-22.4kb. Area of transposon insertions.
900 NPS1086 GENO. 2 225-525 AC015186 At 33.2-33.5kb. No good exons/EST in this area.
At 3.3-3.8kb. In gene on rev coding for335AA ORF. Mouse polycystic kidney disease-related
901 NPS1087 GENO. 2 1-521 AC017673 protein.
At 35.2-35.5kb. In intron of gene represented by ESTs at 32-40k, inc AI542779. 2023bp EST
902 NPS1088 GENO. 2 1-378 AC017676 contig . 666AA ORF. No db matches.

Claims

C AIMS
1. A screening assay for identifying compounds which have a physiological 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.
2. The screening assay according to claim 1 wherein the sequence is selected from the group consisting of SEQ ID Nos. 430-783 and 899-902.
3. The screening assay according to any preceding claim wherein the effect on the protein is a negative modulation.
4. The screening assay according to any preceding claim wherein the assay is a ligand binding assay for detecting the effect the compound has on the ligand binding of the protein
5. The screening assay according to any one of claims 1 to 4 wherein the assay is a functional activity assay for detecting the effect the compound has on the functional activity of the protein.
6. The screening assay according to claim 5 wherein the functional activity assay is selected from the group consisting of kinase assays; protein phosphatase assays; adenyl cyclase assays; guanylyl cyclase assays; phosphodiesterase assays; nucleosidease assays; protease assays; protein secretion and/or import assays; nuclease assays; DNA metabolism assays; transcription factor assays; apoptosis assays; calcium utilisation assays; receptor/ion channel assays; and G protein assays.
7. A compound having modulatory activity on a protein encoded by an essential gene, as identified by an assay according to any preceding claim.
8. A pesticidal formulation comprising a compound according to claim 7, together with a pesticidally acceptable excipient.
9. Use of a compound having modulatory activity on a protein encoded by an essential gene as identified by an assay according to any one of claims 1 to 6 or derivative or analogue thereof as a pesticide.
10. A pesticidally active compound identified by an assay according to any one of claims 1 to 6 and further tested for its ability to kill pests.
11. Use of a pesticidally active compound according to claim 10, in conjunction with other pesticides, herbicides and agriculturally usual auxiliaries as crop protection material.
12. A method of selectively modulating activity, in an organism, of a protein encoded by an essential gene comprising a sequence selected from the group consisting of SEQ ID Nos. 1-902 or a specific fragment thereof, or homologue thereof, comprising administering a compound that selectively modulates activity of the protein in the organism.
13. The method according to claim 12, wherein the selective modulation in activity of the protein has the result of substantially eliminating or severely reducing the activity of the protein, as compared to the activity of the protein without modulation.
14. The method according to claims 12 or 13 , wherein the compound modulates the activity of the protein and has a minimal modulatory effect on other proteins of the organism.
15. The method according to any of claims 12 to 14, wherein the modulation in activity of the protein has the effect of being lethal or semi-lethal to the organism.
16. A method of modulating activity, in an organism, of a protein encoded by an essential gene comprising a sequence selected from the group consisting of SEQ ID Nos. 1-902 or a specific fragment thereof, or homologue thereof, comprising administering a compound, that selectively modulates activity of the protein, to an organism, and wherein the ability of the protein to modulate the activity of the protein is determined by:
- exposing the protein which has been produced by a genetically engineered cell expressing the protein, with the compound for a period of time;
- measuring the activity of the exposed protein using a ligand binding or functional activity assay; and
- comparing the activity of the exposed protein with an activity of a control protein which has not been exposed to the compound, so that compounds that modulate the protein activity are identified.
17. The method according to claim 16, for selectively modulating activity, in an organism, of a protein, further comprising the steps of:
- exposing a further cellular protein(s) of the organism to the compound for a period of time;
- measuring the activity of said further protein(s) using an assay(s) appropriate for such a purpose; and
- comparing the activity of said exposed further cellular protein(s) with an activity of a control protein(s) which has not been exposed to the compound, so that compounds that substantially do not, or minimally modulate said further cellular protein(s) activity, are identified.
18. A method of identifying compounds having a potentially pesticidal activity caused by modulation of a protein encoded by an essential gene comprising a sequence selected from the group consisting of SEQ ID Nos. 1-902 or a specific fragment thereof, or homologue thereof, which comprises;
- obtaining the protein by heterologous expression of the essential gene in a host cell;
- employing the protein in an assay according to any one of claims 1 to 6 for detecting a compound which displays modulatory activity on the protein; and
- testing the compound which displays modulatory activity on the protein for its pesticidal activity on an organism.
19. A compound identified by the method according to claim 17 as having pesticidal activity.
20. Use of a compound according to claim 19 as a pesticide.
21. A pesticidal formulation comprising a compound according to claim 19 identified as having pesticidal activity, together with a pesticidally acceptable excipient
22. A method for the production of a pesticidal composition comprising identifying a compound that displays pesticidal activity using the method according to claim 18 and mixing the compound identified, or a derivative, or an analogue thereof, with a pesticidally acceptable carrier.
23. An isolated polynucleotide fragment comprising a sequence selected from the group consisting of SEQ ID Nos.430-783 and 899-902, a fragment thereof, or a homologue thereof.
24. An essential gene comprising a sequence selected from the group consisting of SEQ ID Nos.430-783 and 899- 902, a fragment thereof, or a homologue thereof.
25. An isolated polynucleotide which hybridises under stringent conditions to a polynucleotide fragment selected from the group consisting of SEQ ID Nos. 430-783 and 899- 902 or a fragment thereof.
26. Use of an isolated polynucleotide fragment comprising a sequence selected from the group consisting of SEQ ID Nos. 430-783 and 899-902, a fragment thereof, or a homologue thereof to identify and facilitate isolation of an essential gene.
27. Use of a polynucleotide fragment selected from the group consisting of SEQ ID Nos.430-783 and 899-902 or a fragment thereof, to identify and facilitate isolation of homologous sequences from other organisms.
28. Use of a polynucleotide fragment selected from the group consisting of SEQ ID Nos.430-783 and 899-902 or a fragment thereof, to identify and facilitate isolation of genes, from other organisms comprising homologous sequences.
29. An essential gene comprising a sequence selected from the group consisting of SEQ ID Nos.430-783 and 899- 902, a fragment thereof, or a homologue thereof.
30. An expression vector comprising the essential gene according to claim 29.
31. An expression vector according to claim 30 comprising one or more control sequences capable of directing the replication and/or expression of an operatively linked essential gene.
32. A prokaryotic or eukaryotic host cell comprising the expression vector according to either of claims 30 or 31.
33. A method of producing a polypeptide comprising culturing a host cell according to claim 32 under conditions permitting expression of the polypeptide.
34. A polypeptide produced by the method of claim 33.
35. Use of the polypeptide according to claim 34 in an assay for detecting compounds which modulate activity of the protein.
36. Use of a polypeptide expressed by an essential gene comprising a sequence selected from the group consisting of SEQ ID Nos. 1-902, or a fragment, or a homologue thereof, in a pesticide screening assay for identifying a compound which modulates activity of the polypeptide.
EP00958829A 1999-09-07 2000-09-06 Essential genes and assays relating thereto Withdrawn EP1212620A1 (en)

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