GENE SCREENING METHODS AND RELATED ASSAYS

Abstract: The present invention relates to cell death genes, which are genes required for programmed cell death; mutant organisms, in which ectopic programmed cell death occurs in embryos to a greater than normal extent and agents which alter the ability to modulate programmed cell death of cells. A mutant form of Daktl, Daktl?q? in a Drosophila melanogaster has been identified. Daktl and its mammalian homolog, PKB are known to protect cells from undergoing apoptosis. A genetic screen for identifying genes related to programmed cell death by their ability to interact genetically with the mutant form of Daktl is described.

Description: GENE SCREENING METHODS AND RELATED ASSAYS CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from TJ S provisional application Ser No 60/083,077, filed Apπl 27. 1998, the contents of which is hereby incorporated m its entirety by reference FIELD OF THE INVENTION The present invention relates generally to the fields of genetics and molecular biollogy and, m particular, to genetic control of programmed cell death (PCD), or apoptosis m insect and human cells BACKGROUND OF THE INVENTION The decision between survival and death is an important aspect of cellular regulation during development and mahgnancs Central to this regulation is the process of programmed cell death (PCD) or apoptosis. which is conserved in multi -cellular organisms (Jacobson MD, et al Cell 1997, 88 347-354 ) While there has been rapid progress in elucidation of the cellular apoptotic processes such as the caspases. the mechanisms by which this critical process is regulated are not clearly understood (Harrington et al EMBO J 1994. 13 3286-3295 ) A variety of signalling cascades has been implicated in modulation of PCD including the phosphatidyhnositol 3 ' kmase(PI3 'K) Activation of PI3'K. a lipid kinase, protects against PCD, whereas it's inhibition enhances PCD (Yao R, et al Science 1995, 267 2003-2006 Kauffmann-Zeh et al Nature 1997 385 544-548 Kennedy et al Genes Dev 1997. 11 701-713 Kulik et al Mol Cell Biol 1997 17 1595-1606 Dudek et al Science 1997, 275 661-665 Marte et al TIBS 1997, 22 355-358 ) Recently, the protective effects of PI3'K in mammalian cells have been linked to its activation of the protein kinase termed PKB or AKT (Bellacosa et al Science 1991. 254. 274-277) The PKB/AKT gene was identified as the human homologue of a retrσs iral oncogene named v-Akt. which was isolated from the acute transforming retrovirus. AKT8 (Bellacosa et al Science 1991, 254, 274-277) PKB has been shown to be expressed m brain, thymus, heart and lung It has also been reported that PKB is overexpressed in some mammalian pancreatic, prostate, ovarian and breast cancers as well as ghoblastomas and leukaemia It is known that defects in the msulm signalling pathway are associated w ith mutations in PKB Such diseases may include hyperglycemia, particularly non-insulin dependent daibetes mellitus (NIDDM). by related disorders, which may include obesity, hypertension, hypercholesterolemia, hypertrigryceπdemia, etc , proliferation disorders, e g , cancer, restenosis. rheumatoid arthritis, or by loss of bone density, e g osteoporosis PKB has also been implicated in angiogenesis Much work has focused on the signalling pathway for the acuvation and function of PKB/AKT in mammals, and despite intense research surrounding the downstream actions of this enzyme, few in vivo phosphorylation targets have been identified While the activation of PKB/AKT in mammals has been linked to protection against PCD, a definitive role for this kinase in PCD has to be established It is also possible that PKB/AKT has other as yet unknown physiological roles not related to PCD The use of knockout mice is a powerful tool for finding information about the function of a gene in vivo In this method a selected gene is inactivated while the remaining genome is unaffected There are several dιsad\ antages to generaung knockout mice Besides being time consuming and expensive, mouse genetic analysis is slow and complicated Also, the strategy for generating knockout mice will in many cases delete a gene critical for early embryonic development In such cases the pups carrying the knocked out gene will die in utero In the particular case of PKB/AKT, the application of knockout strategy is further hampered by the fact that PKB/AKT already exist as three isoforms encoded by three different genes termed PKB /AKTl. PKBβ/AKT2 and PKBγ/AKT3 The proteins encoded by these genes have high homology with each other Because of the difficulties of studying functions of genes in mammals, many researchers have focused their efforts on model systems that are genetically more amenable to manipulation Drosophila geneUc studies have been a powerful tool in dissecting signalling cascades and regulatory pathways Recombinant DNA expeπments with Drosophila have assisted the clarification of principles and ideas established by decades of classical genetic and embπ ological studies There is evidence suggesting that the physiology of PCD is quite highly conserved between mammals and arthropods The morphological changes associated with programmed cell death are similar in both vertebrates and invertebrates (Kerr, et al Br J Cancer, 26 239-257) A number of viral proteins that suppress PCD in their hosts e g , p35 and crmA, exhibit potent anu-apoptotic activity in a range of heterologous species (Hay et al Development 120 2121-2129, Pronk, et al Science 271 808-810) A number of functional homologues of mammalian PCD factors have been identified in Drosophila For example, counterparts to the caspases are represented as are the inhibitor of apoptosis (lap) proteins (Harrington et al EMBO J 1994, 13 3286-3295. Sanson et al Xature 1996. 383 627-633, Heitzler et al Genetics 1996, 143 1271-1286, Chou et al Genetics 1996, 144 1673-1679) A Drosophila homologue of PKB/AKT (Daktl) has also been biochemically characteπzed howe\er corresponding mutants have not been identified (Franc et al Immunity 1996. 4 431-443. Grether et al Genes De\ 1995 9 1694-1708) The Dakt I gene spans approximately 5 6Kb of DNA and maps to cytological position 89B on the right arm of the third chromosome of Drosophila melanogaster Like its mammalian homologue. Daktl contains an N-terminal PH domain, a central catalytic kinase domain and the two activation sites at Thr 342 and Ser 505 Daktl has 75% homology to mammalian PKB at the ammo acid level and 64% identity at the DNA le\el Specifically , the N-termmal PH domain is 71% homologous and the catalytic domain shows 86% homology with conserved motifs for ATP binding and for defining the Ser/Thr specificity of the kinase activity Both transcnpuon and tire intrinsic kinase activity are differentially regulated throughout embryogenesis. larval development. pupanation and between the sexes in adults There are clinical implications to understanding the biology of PCD In normal development of a multicellular organism, many more cells are produced than are needed in the mature or adult tissue Therefore, the process of cell birth, mitosis, must be counterbalanced by a process for reducing cell number There is a \\ ide range of diseases in which a disruption of the normal cell death pathway may play a role In nervous system disorders such as stroke and head trauma, neurons die due to external death tπggers such as lack of oxygen Recent studies suggest that at least a portion of this neuronal death ma} occur by PCD In other neuronal diseases, such as Alzheimer s disease, and amyotrophic lateral sclerosis, neurons die as part of a degenerauve process whose tπggers have not yet been identified but which could involve PCD Bv contrast, in disorders such as cancer and hypenmmune diseases, cells live where they would normally die This results in tumors (cancer) or an overactive immune system (autoimmune diseases) Thus, the ability to intervene pharmacologically in the cell death pathway provides the possibility of a new set of therapeautic agents useful in a wide range of human disease Despite its importance in animal development, a great deal remains unknown about the genetic and molecular mechanisms involved in PCD There is thus a need for methods of identifying genes and their products controlling PCD in cells that have a disrupted PCD function by restoration of PCD genes Results obtained from the studv of Drosophila can produce insight into other species including mammalian and even human cells SUMMARY OF THE INVENTION One aspect of the present invention is a method of determining whether a mutation capable of enhancing programmed cell death is present in a Drosophila melanogaster fly The method includes providing a mutant first fly having a recessive first mutation, the presence of which mutation is known to induce programmed cell death in an embryo of a fly homozygous for the mutation A second mutant flv is provided This second fly has a mutation which and the method is applied to determine whether such a mutauon is capable of enhancing programmed cell death The first and second filed are crossed with each other It is then determined whether there are embryos obtained from the crossing of the flies w hich have undergone ectopic programmed cell death If it is determined that there are embryos that have undergone ectopic cell death, this indicates that a mutation capable of enhancing programmed cell death is present in the second mutated fly According to a particular method, described in detail below, the determination of whether embryos have undergone ectopic programmed cell death includes determimng whether at least about 5 percent of the total number of embryos formed have undergone ectopic cell death In making this determination it must be taken into account that some embryos develop and go on to hatch In any case, the higher the percentage of embryos that are found to undergo ectopic cell death, the greater the certainty that the second mutant fly has a mutation capable of enhancing programmed cell death The observed percentage can be 6 percent, 7 percent, or greater The total observable percentage would be 25 percent, based on the statistics of genetic crossings, but this maximum would rarely, if ever, be observed In an illustrated embodiment, the first fly used in the method has been characteπzed to the extent that it is known to have a mutation in its DAktl gene and the mutation is known to induce programmed cell death in an embryo of a fly homozygous for the mutation The mutation has been observed to result in loss of the kinase activity of the protein encoded by the gene, the "gene product" In particular, the mutation results in a phenylalanine to isoleucine substitution at position 327 of the protein In terms of the gene itself, this mutation has been determined is given below In another illustrated embodiment, the first fly is known to have at least one mutation in a DPP2A gene known to induce programmed cell death DPP2A is known to encode a protein having phosphatase activity, so it is thought that the gene contains a mutation which results m loss of phosphatase activity of the protein encoded thereby The second fly . the fly having a mutation for which it is to be determined whether a gene mutation capable of enhancing programmed cell death is present, can be obtained by conventional methods Particularly, a mutant fly can be obtained bv exposing a wild-type fly to ionizing radiation, by chemical mutagenesis of a wild-type fly. or by inserting P-elements in the genome of a wild-type fly In a method of the invention, illustrated in detail below, embryos obtained from the crossing of the flies are initially visually examined (under a microscope) Dead embryos displaying loss of cuticle are selected for determination of whether they have undergone ectopic programmed cell death Embryos displaying such cuticle loss are treated to degrade or remove the choπon of the embryos This permits staining of embryonic cells inteπor of the choπon for visual determination of the presence of ectopic programmed cell death The treated embryos are thus treated, as with a dy e. and they are visually inspected to determine whether they have undergone programmed cell death Particular embryonic treatments include terminal deoxy-transferase mediated hapten-labeled-dUTP nick end labeling (TUNEL) or with acπdine orange staining According to the method, once a mutant fly is determined to have a mutaϋon capable of enhancing programmed cell death, the chromosomal location of mutations in the fly can be determined Once the location of a mutation is determined, the nucleotide sequence of the mutation can be determined Sequence information obtained from the mutant can be compared with wild type sequences, as possibly contained in a sequence database, to determine wild type sequence(s) corresponding to the mutant sequence From tlus, potentially counterpart sequences could be located If a function is known of a protein encoded by a wild type sequence potentially counterpart to that determined for the mutant, the a protein based on the mutant sequence could be created and tested for activity corresponding to that of the potential counterpart Also, the wild type fly sequence on which the mutant sequence is based, could be determined In another broad aspect, the invention is a method of determimng whether a mutation capable of disrupting development is present in a Drosophila melanogaster fly The method includes (a) providing a first mutant fly having a recessive first mutation, the presence of which mutation is known to induce programmed cell death m an embryo of a fly homozygous for the mutation, (b) providing a second mutant fly for determination, (c) crossing the flies in (a) and (b), (d) examimng dead embryos obtained in step (c) to determine whether dead embryos of a phenotype different from either the first or second mutant flies are present, (e) wherein, if a said different phenotype is observed then a mutation capable of disrupting development is present in the second mutant fly Disrupted, or abnormal, development is evidenced in an embryo by the presence of embryos which fail to experience normal cell fates In a particular embodiment, illustrated below, step (d) includes visually examining embryos obtained in step (c) to determine whether trachea are missing In another broad aspect, the invention is a method of determimng whether a mutation capable of disrupung development is present m a Drosophila melanogaster fly, the method compnsing (a) providing a first mutant fly having a recessive first mutation, the presence of which mutation is know n to disrupt development m an embryo of a fly homozygous for the mutation, (b) providing a second mutant fly for determination. (c) crossing the flies in (a) and (b), (d) examining dead embryos obtained in step (c) to determine whether dead embry os of a phenotype different from either the first or second mutant flies are present, (e) wherein, if a said different phenotype is present then a mutation capable of disrupting development is present in the second mutant fly In a particular embodiment, illustrated below, the first fly is known to e at least one mutaϋon in a trachealess gene known to disrupt development in an embryo of a fly homozygous for the mutation It is possible, for convenience, to introduce a marker into the chromosome of the mutant second fly at a location suitable to permit identification of a mutation introduced into the second fly . and wherein the method further comprises the step of identifying the mutation This permits identifying the mutation by sequencing the chromosome of the f in the vicinity of said marker and comparing the sequence obtained with a wild type sequence to determine the wild type sequence corresponding to tire mutant sequence In another aspect, the invention is a method of inducing programmed cell death in a cell by mhibiung the kinase activity of DAktl protein In particular, the method can include admimstenng a nucleic acid molecule sufficiently complementary such as to inhibit translation of DAktl mRNA present in a cell In another aspect, the invention is a method of inducing programmed cell death in a cell by inhibmng the phosphatase acuvity of a PP2A protein In particular, the metliod can include admimstenng a nucleic acid molecule sufficiently complementary such as to inhibit translation of PP2A mRNA present in a cell In another broad aspect, the invention is an assay for determimng whether a substance is potentially suitable for usage as an active ingredient in inducing programmed cell deatli or in inhibiting cell deatli, w hich assay compπses the following steps exposing DAkt having kinase activity to the substance. quantitaUvely assaying the effect of the substance on the kinase activity, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or m inhibiting programmed cell death Particularly, a said substance is potentially suitable as an active ingredient in inducing programmed cell death if determined to quanutatively inhibit the kinase activity Alternatively a said substance is potentially suitable as an active ingredient in inhibiting programmed cell deatli if determined to quantitaUvely enhance the kinase activity The assay can further include exposing embryos of Drosophila melanogaster fly to the substance. quantitatively assaying the effect of the substance on ectopic cell death in the embryos, and determining based on the results of said assay the potential suitability of the substance as an actι\ e ingredient in inducing programmed cell death or inhibiting programmed cell death In another aspect, the invention is an assay for determining whether a substance is potentially suitable for usage as an acuve ingredient m inducing programmed cell deatli or inhibiting programmed cell death, w hich compnses the following steps exposing PP2A having phosphatase acuvity to the substance. quanϋtatively assaying the effect of the substance on the phosphatase activity, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or in inhibiting programmed cell death Particularly, such a substance is potenuallv suitable as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit the phosphatase acUvity Particularly, such a substance is potentially suitable as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance the phosphatase activity Such an assay can also include exposing embπ os of Drosophila melanogaster fly to the substance, quanUtatively assaying the effect of the substance on the ectopic cell death in the embryos, and determining based on the results of said assay the potential suitability of the substance as an ac e ingredient in inducing programmed cell deatli or in inhibiting programmed cell death Another assay of the invention is for determining whether a substance is potentially suitable for usage as an active ingredient in arresting development of an organism, wherein the assay includes the following steps exposing a trachealess protein capable of initiating transcription to the substance. quantitaUvely assaying the effect of the substance on the ability of the protein to initiate transcription, and determining based on the results of said assay the potential suitability of the substance as an active ingredient m arresting development of an organism The assay can further include the following steps exposing embryos of Drosophila melanogaster fly to the substance. quantitatively assaying the effect of the substance on development of the embryos, and determining based on the results of said assay the potential suitability of the substance as an acuve ingredient in arresung development of an organism Assaying tlie effect of the substance on development of the embryos can include determining tlie effect of the substance on tracheal development The invention includes a method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for disrupted development, the method comprising the steps of providing a sample of genetic material of the subject, determining whether the genetic material includes a nucleotide sequence encoding a mutant trachealess protein incapable of initiating transcπption. wherein the presence of said nucleotide sequence indicates the presence of a potential to confer on its offspπng a predisposition for abnormal development In a particular embodiment of this aspect of the mvenUon. the method also includes determining whether the genetic material includes a nucleotide sequence encoding a trachealess protein capable of initiating transcπption In a particular aspect, the trachealess protein capable of initiating transcπption includes the amino sequence identified as SEQ ID NO 1 In another aspect, the invenuon is a method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for abnormal development, the method comprising the steps of providing a sample of genetic material of the subject, determining whether the genetic material includes a nucleotide sequence encoding a mutant PP2A protein lacking phosphatase activity, wherein the presence of said nucleotide sequence indicates said potential predisposition In another aspect, the invenuon is a method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for abnormal development, the method comprising the steps of providing a sample of genetic material of the subject, determining hether the genetic material includes a nucleotide sequence encoding a mutant Daktl /PKB protein having kinase activity lower than the activity of wild type dakT/PKB, wherein the presence of said nucleotide sequence indicates said potential predisposition This invention includes a method of inducing programmed cell death m a cell, the method comprising inhibiting the activity of PP2A in the cell Inhibiting the activity of PP2A in the cell can include admimstenng to the cell an effective amount of an antisense nucleic acid molecule sufficiently complementary to an mRNA encoding PP2A to bind thereto so as reduce translation thereof Inhibiting the activity of PP2A in tlie cell can include admimstenng to the cell an effective amount of a phosphatase inhibitor The inhibitor can be selected from the group consisting of microcystin LR and calicu n In another important aspect, the invention is an assay for determimng whether a substance is potentially suitable for usage as an acuve ingredient in inducing programmed cell deatli or in inhibiting programmed cell death, which assay compnses the following steps providmg a cell m which a reporter gene is operably linked to a promoter/enhancer which is acted upon by trachealess, wherein the reporter gene is one which, in nature, is not operablv linked to the sequence promoter/enhancer, exposing the cell to the substance. quantitatively assaying the effect of the substance on expression of the reporter gene, and determining based on the results of said assay tlie potential suitability of the substance as an acUve ingredient in inducing programmed cell death or in inhibiting programmed cell death The cell is preferably a cell of a Drosophila melanogaster fly The promoter/enhancer, in a particular embodiment, includes the nucleotide sequence identified as SEQ ID NO 3 In particular embodiments, the determination step includes comparing the effect of the substance on the expression of the reporter gene with the expression of the reporter gene in the absence of the substance The substance being assayed can be a kinase inhibitor or a phosphatase inhibitor According to a certain aspect of this assay of the invention, a said substance is potentially suitable for usage as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit expression of the reporter gene Alternauvely. a said substance is potentially suitable for usage as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance expression of the reporter gene In another aspect, the invention is an assay for determining whether a substance is potentially suitable for usage as an active ingredient in inducing programmed cell death or in inhibiting programmed cell death, which assay comprises tlie following steps exposing a cell to the substance. quantitatively assaying the effect of the substance on expression of the trachealess gene, and deteπrumng based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or in inhibiUng programmed cell death Such an assav can include determimng the amount of RNA encoding trachealess produced by the cell in the presence of the substance and companng the amount with the amount of RNA encoding trachealess produced by the cell in the absence of the substance Determining the amount of RNA encoding trachealess can include amplifying the RNA in the presence of pπmers complementary thereto A said substance is potentially suitable as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit said expression A said substance is potentially suitable as an active ingredient m inhibiting programmed cell death if determined to quantitatively enhance said expression In this specification, percent homology between ammo acid sequences is determined using GCG software using the "best fit " command BRIEF DESCRIPTION OF THE FIGURES FIG. 1 Schemetic representation of the genetic screen performed to identify mutations that interact with Daktl FIG. 2 Analysis of Daktl protein expression and activity in wild-type an q larvae FIG. 3 The phenotype of Daktl germ line clone (GLC) mutants FIG. 4 Momtoπng PCD markers in Daktl GLC embryos FIG. 5 Phosphorylation of trachealess by PKB (I) FIG. 6 Phosphorylation of trachealess by PKB (II) FIG. 7 PKB specifically phosphorylates trachealess at Senne 665 FIG. 8 Schemetic representation of the trachealess/dARNT (tango) hetero- imer transactivation of the Drosophila breathless gene and promotion of trachea development FIG. 9 The regulation of trachealess/άARNT transactivation via S665 of trachealess DETAILED DESCRIPTION OF THE FIGURES FIG. 1 The screen focuses on the dominant enhancement of the zygotic Daktl phenotype by second site lethal mutations The source of these lethals can be selected from mutant stocks that can be generated either by exposing wild-type flies to ionizing radiation, chemical mutagenesis or P-element insertion into their genome This screemng approach benefits from the observation that the Daktl mutant is a zygotic larval lethal that does not die during embryogenesis The cross is performed with heterozygous Daktll+ females to the various recessive lethal mutant fly lines Progeny will be analyzed for failure to hatch Only interacting loci will generate dead embryos which will then be prepared for cuucle analysis For example, fly lines with P-element mutations are tested for their ability to cause enhanced embryonic lethality in Daktl mutant heterozygous embryos that are expected to develop normally (embryonic lethality of 0-1%) The P-elements and their induced mutants are stabilized on their respective chromosomes and maintained as stocks by being kept in trans to balancer chromosomes The heterozygous embryos have only half the normal amount of maternal Daktl (due to expression being limited to only one gene) and would also be carrying only one wild type copy of both Daktl and the P-element mutant gene If the mutant gene had a wild-type function in Daktl signalling, it could be expected that loss of half of the maternally- contnbuted Daktl, combined with the loss of half the normal zygotic expression of the mutant gene, might result m some lethality in the .rαns-heterozygotes Enhancement is scored when a significant number of embryos fail to hatch and produce a phenotype FIG. 2 (A) Results of Daktl protein expression and activity in wild-type and q larvae (B) Immunoblot analysis of Daktl protein from wild-type and q larvae using aπti-Daktl antibody (C) Schematic diagram of Daktl amino acid sequence showing the phenylalamne to isoleucine (F→I) change at ammo acid 327 in the q allele of Daktl and the sequence of the relevant region m the bovine and human PKB homologs (D) Coπesponding sequence change of F293 to I m bovine PKB creates a protein lacking kinase activity Testing of the kinase activity and expression of PKB(F293I) mutant and PKB wt m HeLa S3cells "+" denotes addition of pervanadate FIG. 3 (A) First instar cuticle of wild-type larva (B) Phenotype of Daktl GLC embryo in the absence of zygotic Daktl activity showing almost complete loss of cuticle (C) Phenotype of Daktl GLC embryo with zygotic Daktl activity showing complete loss of head and dorsal structures Only a portion of the ventral cuticle remains and does not show extensive defects in segmentation (D) Phenotype of Daktl GLC embryo rescued with HsDaktl induced maternally and zygoticall Note the reappearence of most cuticle structures demonstrating a functional rescue of the phenotype (E) Acπdine Orange (AO) satining of wild-type late stage 12 embryo Note the signal present in the amnioserosa and in the regions of the head (F) AO staining of stage 12 Daktl GLC embry o showing ubiquitous AO signal FIG. 4 (A) TUNEL signal in a stage 11 wild-type embryo Only a few cells are positive for the TUNEL signal at this stage (B) TUNEL signal m a stage 8 Daktl GLC embryo Even by stage 8, extensive DNA fragmentation as assayed by TUNEL has begun in the absence of Daktl (C) A stage 11 wild-type embryo showing the presence of Crq-expressing macrophages near sites of apoptosis (e g presumptive amnioserosa) (D,E) A stage 11 Daktl GLC embryo showing the extensive and widespread presence of macrophages as assayed by Crq signal (F) Close-up of (E) The expression of Rpr is not appreciably altered in (G) stage 10 q GLC embroys compared to (H) wild-type stage 10 embryos (I) The cuticle phenotype of Daktl GLC embryos homozygous for H99 is similar to that of Daktl mutants (J) Cuticle phenotype of Daktl GLC embryos expressing ectopic baculoviral p35 from hs-p35 transgene This indicates suppression of the Daktl GLC phenotype by p35 but not the H99 deficiency FIG. 5 293 cells were transfected with nucleic acid sequences encoding either HA tagged PKB inactive kinase (Δ. lane 1 and 4). or HA-tagged PKB wild-type (WT, lane 2,3.5. and 6) 48 hr after transfection. cells were treated with 50 ng/ml of IGF-1 (activation "+", lanes 1,3,4 and 6), or 100 nmol of wortmannin (activation "-" lanes 2 and 5) for 15 mm HA tagged PKB's were immunoprecipitated from cell lystaes with anti-HA 12CA5 antibody Both trachealess proteins. Trh N-term (1-175 aa) and Trhl (477-728 aa, the region that contains the two putative PKB phosphc— seπnes. Ser 571 and Ser 665), were expressed in E coli strain BL21 DE3 and punfied as 6X Histidine fusion proteins over a Ni-agarose column Trachealess proteins were subjected to PKB kinase assays in the presence of [γ- 32 P] ATP The reaction mixtures were resolved on a 15% SDS-PAGE and γ- 32 P incorporation visualized on a Molecular Dynamics Phosphoπmager FIG. 6 Phosphorylation of trachealess protein by PKB was performed essentially as descπbed in FIG. 5 In this experiment, another fragment of the trachealess protein, Trh II, was tested as a substrate for PKB The nucleic acid sequence for Trhll also encodes a fragment encompassing tlie two putatne senne residues for PKB phosphorylation (ammo acids 546-728) Trhll was also phosphorylated by PKB kinase activity (lane 6 top panel) The 32 P incorporated Trhll can be stπpped off by the addition of protein seπne/threonme phosphatase (lane 7) Tlie bottom panel is the Commassie Blue stain of the gel, showing that equal amount of trachealess protein (trhl trhll) was added to each assay FIG. 7 Site specific mutations at Ser 571->Alanιne (lanes 4-6), Ser 665->Alamne (lanes 7-9), or a double mutation (AA) at Ser 571-»Alanme and Ser 665->Alanιne (lanes 10-12) of trachealess protein were made by methods well known in the art (Top panel) Phosphorylation of these substrates by active PKB was assayed as described above and compared with wild-type trachealess protein (lanes 1-3) Phosphorylation by PKB was abolished by Ser 665— >Alanιne and not surpπsingly by the double mutation as well, establishing that PKB targets trachealess on Ser 665 The Bottom panel shows equal loading of the trachealess substrates FIG. 8 Genetic evidence from Drosophila analysis indicates that treacheless/dARNT heterodimer recognizes Drosophila breathless promoter/enhancer sequence and transactivates the expression of breathless gene Hence, the transactivation activity of treacheless. either by itself or by the partnership with άARNT. can be evaluated with the transactivation from breathless enhancer/promoter (B123) FIG. 9 SL-2 cells were transfected with trachealess and/or άARNT (as indicated), together with a breathless promoter-luciferase reporter 48 hr after transfection, cell lysates were subjected to luciferase assays Both trachealess wild-type and Ser665→Asp (S665-»D) mutant (negatively charged Asp residue partially mimmicks phosphoseπne and gives constitutive activity of trachealess), as well as άARNT transactivate from breathless promoter This transactivation is synergistically elevated by trachealess/άARNT, partnership However. treacheless, either alone or in partnership with dARNT, contnbutes no transactivation acuvity to the reporter when the targeted Seπne is mutated to Alarune (S665->A), an inactive mutation DETAILED DESCRIPTION OF THE INVENTION The invention herein descπbes a method for obtaining mutant Drosophila melanogaster flies with a mutation in a nucleic acid sequence known to disrupt PCD with an object of initiating a geneuc analysis of the regulatory components of the PCD pathway The invention herein further descnbes a genetic screen using a mutant fly to screen for other genes involved in the PCD pathway m Drosophila and to their encoded RNA and protein products By extension, the mammalian counterparts to these genes will be components of PKB signalling m mammals and can be manipulated for the process of drug discovery for PKB related diseases or processes Methods for obtaining and characterizing tlie nucleic acid sequence of Drosophila melanoga<;ter mutants of Dakt Drosophila mutants of Daktl, can be identified using a lethal Drosophila fly line for failure to complement a large deletion at cytological position 89B but which can be rescued by a Dakt transgene The lethal fly line is generated bv exposing wild-type flies to either ionizing radiation, chemical mutagenesis or by P-element transformation Since molecular analysis has placed Daktl within Df(3R)sbd* screening a collection of mutants mapping to Df(3R)sbd 4i can identify larval lethal mutations which can be subsequently rescued by a Daktl transgene Mutations in the nuclei acid sequence are thus identified by sequencing the coding region from genomic DNA isolated from the mutant larva and compared with the wild type sequence Methods for sequencing nucleic acids are well known m the art and are described in Current Protocols in Molecular Biology 1995 Alternatively, once known, one of ordinary skill in the art can engineer the mutation in the nucleic acid sequence using site-directed mutagenesis and by cloning into a pUAST transformation vector as descnbed by Brand et al Methods Cell Biol 44 635-654 After P-element transformation, the transgenic flies are to be crossed into a background of a transcπpt-null Daktl mutant in combination with a ubiquitous promoter-G /,4 transgene The transcript-null Daktl mutant is generated using X-ray mutagenesis and assayed using Southern blotting The Drosophila mutant of Daktl descnbed herein can also be obtained if mutations other than the one descnbed herein of Daktl are isolated and characterized as kinase dead point mutants Such mutants should also provide a comparable allele to the one utilized herein Mutagenesis Chemical mutagens include ethylmethanesulfonate (EMS), methylmethanesulfonate (MMS), methylnitrosoguanidine (NTG), 4-mtroquιnolιne-l -oxide (NQO), 2-ammopunne, 5-bromouracιl. ICR 191 and other acndme deπvatives, sodium bisulfite, ethidium bromide, nitrous acid, hydroxylamine, N-methyl-N'-mtroso-N- mtroguamdine. and alkylating agents Physical mutagens include ultraviolet radiation and x-rays Mutagenesis is accomplished according to methods well-known in the art (see. e g , Current Protocols in Molecular Biology 1995. Vol 2. Section 13 3, wherein all the cited references are dated 1990 or earlier) Conditions for mutagenesis such as concentration (chemical mutagenesis) or intensity (e g , ultraviolet mutagenesis) and duration are preferably optimized to produce a high rate of mutation while minimizing the amount of killing among the exposed flies In general, mutagenesis is performed at a temperature that is below the optimal growing temperature for the organism, because the sub-optimal temperature has been found to decrease cell killing P-elements are transposable genetic elements that generate mutations by insertion into different parts of the Drososphila genome The P-elements and their induced mutants are stabilized on their respective chromosomes and maintained as stocks by being kept in trans to balancer chromosomes Methods for generating mutant flies with P-element insertions are well known m the art and are described in (Drosophila, a Laboratory Handbook, Michael Ashburner. Cold Spnng Harbor Laboratory Press. 1989) The genetic screen The screen focuses on the dominant enhancement of the zygotic Daktl phenotype by second site lethal mutations or "lethals " The source of these lethals can be selected from mutant stocks that can be generated either by exposing wild-type flies to ionizing radiation, chemical mutagenesis or P-element insertion into their genome Generally, embryonic screens are not undertakne due to the success of eye screens (Doyle et al Genes & Dev 1. 633-646. Verheyen et al Genetics 144, 1127-1141, Kaπm et al Genetics 143, 315-329, Dickson et al Genetics 142. 163-171. Ma et al Genetics 142. 1199-1213) However, to date, it has not been possible to generate a Daktl overexpression or dominant-negative phenotype in the eye which can be easily scored in a screen Thus, the embryonic scheme is the only one possible at this time This screemng approach benefits from tlie observation that the Daktl mutant is a zygotic larval lethal that does not die during embryogenesis This is because in Daktl homozygous mutant embryos the maternal contπbution of Daktl from the heterozygous mother is only half the amount that would be contributed by a wild-type mother, but the homozygous embryos still have enough maternalh -derived Daktl to rescue them to 1st or 2nd instar larvae before dying Pπor to this point dunng development, the requirement for zygotically-expressed Daktl has been masked by the presence of the maternally- derived enzyme It is. presumably, at this stage of development when zygotic expression becomes necessary, possibly due to a gradual loss of the maternally derived enzyme during development Heterozygous embryos are viable and develop into normal larvae, pupae and adults The cross is performed with heterozygous Daktll+ females to the vaπous recessive lethal mutant lines generated by exposing wild-type flies to ionizing radiation, chemical mutagenesis or by P-element insertions The progeny will be analyzed for failure to hatch (see Figure 1 for screen schematic) Only interacting loci will generate dead embryos which will then be prepared for cuticle analysis The inventors have observed that the extent of development of cuticle is a good first indicator for ectopic PCF Staverley et al Current Biology, 1998 8 599-602 For example, fly lines with P-element mutations are tested for their ability to cause enhanced embryonic lethality in Daktl mutant heterozygous embryos that are expected to develop normally (embryonic lethality of 0-1%) The heterozygous embryos have only half the normal amount of maternal Daktl (due to expression being limited to only one gene) and would also be carrying only one wild type copy of both Daktl and the P-element mutant gene If the mutant gene had a wild-type function in Daktl signalling, it could be expected that loss of half of the maternally- contnbuted Daktl. combined with the loss of half the normal zygotic expression of the mutant gene, might result in some lethality in the .rαπs-heterozygotes Enhancement is scored when a significant number of embrvos fail to hatch and produce a phenotype Mutants which are positive in the screen are analyzed as follows Mutants are mapped (meiotically or with deficiencies) and organized according to polytene chromosomal location P-element induced alleles ill be the first to be analyzed molecularly However, with the rapid progress of the Berkeley Drosophila Genome Project (BDGP), many of the chemically induced mutants may be within molecular markers and thereby analyzable at the molecular level by searching the Berkeley Blast Server using the BLASTN algonthm Altschul et al J Mol Biol (1990) 215 403-10 For each new mutant complementation group, a series of genetic is earned out (1) Interactions outlined m the screens are repeated to confirm the results (2) Additional alleles can be identified or generated (3) Tlie embryonic phenotype of each mutant is assessed for possible involvement in development and ectopic apoptosis by terminal deoxy-transferase mediated hapten-labelled-dUTP nick end labelling (TUNEL) and acπdme orange staimng (4) GLC analysis is used to assess the maternal effect phenotype of the mutants (5) Where possible, genetic epistatic tests can be performed with Daktl Enhancers of Daktl can be tested ith activated Daktl (e g CAAX and DD mutants) and suppressors can be tested with the Daktl mutants Biological Functional Equivalents Modification and changes may be made in the structure of the encoded polypeptides used m the vectors and DNA segments of the present invention and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics The follow ing is a discussion based upon changing the ammo acids of a protein to create an equivalent, or even an improved, second-generation molecule The ammo acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table TABLE 1 Amino Acid 3 Letter 2 Letter Code Codons Code Alanme Ala A GCA GCC GCG GCU Cysteme Cys C UGC UGU Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine He I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methiomne Met M AUG Asparagme Asn N AAC AAU Prolme Pro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginme Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG Threomne Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp w UGG Tvrosine Y UAC UAU For example, certain ammo acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as binding sites on substrate molecules Since it is the interactive capacity and nature of a protein that determines that protein's biological functional activity, certain ammo acid sequence substitutions can be made in a protein sequence, and. of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the apoptotic proteins, or coπesponding DNA sequences which encode said proteins with retention of biological utility or activity In making such changes, the hydropathic index of amino acids mav be considered The importance of tlie hydropathic ammo acid index in confemng interactive biologic function on a protein is generally understood in tlie art (Kyle and Doolittle. 1982) It is accepted that the relative hydropathic character of the ammo acid contributes to the secondan' structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors. DNA. antibodies, antigens, and the like Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge charactenstics (Kyte and Doolittle. 1982). these are isoleucine (+4 5), vahne (+4 2), leucme (+3 8), phenylalanine (+2 8), cysteine/cystine (+2 5), methionme (+1 9). alanine (+1 8), glycine (-0 4), threomne (-0 7), serine (-0 8), tryptophan (-0 9), tyrosme (- 1 3), proline (-1 6). histidine (-3 2), glutamate (-3 5), glutamine (-3 5), aspartate (-3 5). asparagine (-3 5). ly sine (- 3 9), and arginme (-4 5) It is known in the art that certain amino acids maλ be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity . 1 e . still obtain a biological functionally equivalent protein In making such changes, the substitution of amino acids whose hydropathic indices are within +- 2 is prefeπed, those which are within +- 1 are particularly prefeπed, and those within +- 0 5 are e\en more particularly preferred It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity U S Pat No 4,554, 101. incorporated herein by reference, states that the greatest local average hvdrophilicity of a protein, as governed by the hydrophilicit of its adjacent amino acids, coπelates with a biological property of the protein As detailed in U S Pat No 4.554,101, tlie following hydrophilicity values have been assigned to ammo acid residues arginme (+3 0), lysine (+3 0), aspartate (+3 0 +- 1), glutamate (+3 0 +- 1). serine (+0 3). asparagine (+0 2), glutamme (+0 2). glycine (0), threomne (-0 4), proline (-0 5 +- 1), alanine (-0 5), histidine (-0 5), cysteine (- 1 0), methionme (-1 3). vahne(-l 5), leucine (-1 8). isoleucine (-1 8), tyrosine (-2 3), phenylalanine (-2 5) tryptophan (-3 4) It is understood that an ammo acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein In such changes, the substitution of amino acids whose hydrophilicity values are within +- 2 is preferred, those which are within +- 1 are particularly preferred, and those within +- 0 5 are even more particularly prefeπed Since it is tlie interactive capacity and nature of a protein that defines that protein's biological functional activity . certain ammo acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like (agonistic) properties Equally, tlie same considerations may be employed to create a protein or polypeptide with counterveihng (e g , antagonistic) properties It is thus contemplated by the inventors that vanous changes may be made m the sequence of the Daktl, treachealess or DPP2A protein or peptide (or underlying DNA) without appreciable loss of biological utility or activity Nucleic Acid Compositions The scope of the invention with respect to nucleic acid compositions includes, but is not necessaπly limited to. nucleic acids having a sequence set forth in any one of the sequences demonstrated by the present methods to be active in enhancing PCD. nucleic acids that hybridize the provided sequences under stnngent conditions, genes corresponding to the provided nucleic acids, vaπants of the provided nucleic acids and their coπesponding genes, particularly those vaπants that retain a biological activity of the encoded gene product Other nucleic acid compositions contemplated by and within the scope of the present invention will be readily apparent to one of ordinary skill in the art when provided with the disclosure here The nucleic acids of the invention also include nucleic acids having sequence similarity or sequence identity Nucleic acids having sequence similarity are detected by hybridization under low stringency conditions, for example, at 50°C and 1 OXSSC (0-9 M NaCl/0 09 M sodium citrate) and remain bound when subjected to washing at 55°C in 1 OXSSC Sequence identity can be determined by h bridization under stnngent conditions, for example, at 50°C or higher and 0 1XSSC (9 mM NaCl/0 9 mM sodium citrate) Hybridization methods and conditions are well known m the art Nucleic acids that are substantially identical to the provided nucleic acid sequences, e g allehc vanants, genetically altered versions of the gene, etc , bind to the provided nucleic acid sequences under stnngent hybridization conditions By using probes, particularly labelled probes of DNA sequences, one can isolate homologous or related genes The source of homologous genes can be any species Preferably, hybridization is performed using at least 15 contiguous nucleotides of at least one of the PCD enhancing sequence The probe will preferentially hybridize with a nucleic acid or mRNA compnsing the complementary sequence, allowing the identification and retneval of the nucleic acids of the biological material that uniquely hybridize to the selected probe Probes of more than 15 nucleotides can be used, e g probes of from about 18 nucleotides to not more than about 100 nucleotides, but 15 nucleotides generally represents sufficient sequence for unique identification The nucleic acids of the invention also include naturally occurnng variants of the nucleotide sequences, e g degenerate variants, allehc vaπants. etc Vaπants of the nucleic acids of the invention are identified by hybridization of putative variants with nucleotide sequences disclosed herein, preferably by hybridization under stnngent conditions For example, by using appropπate wash conditions, variants of the nucleic acids of the invention can be identified where the allehc vanant exhibits at most about 25-30% base pair mismatches relative to tlie selected nucleic acid probe In general, allehc vaπants contain 5-25% base pair mismatches, and can contain as little as even 2-5%, or 1-2% base pair mismatches, as well as a single base-pair mismatch The invention also encompasses homologs corresponding to the provided nucleic acids, where the source of homologous genes can be any related species within the same genus or group Within a group, homologs have substantial sequence similanty, e g at least 75% sequence identity, usually at least 90%. more usually at least 95% between nucleotide sequences Sequence similanty is calculated based on a reference sequence, which may be a subset of a larger sequence, such as a conserved motif coding region, flanking region, etc A reference sequence will usually be at least about 18 contiguous nucleotides long, more usually at least about 30 nucleotides long, and may extend to the complete sequence that is being compared Algoπthms for sequence analysis are know n in the art. such as BLAST, described in Altschul et al . J Mol Biol (1990) 215 403-10 In general, vanants of the invention have a sequence identity greater than at least about 65%, preferably at least about 75%. more preferably at least about 85%, and can be greater than at least about 90% or more as determined by the Smith Waterman homology search algorithm as implemented in MPSRCH program (Oxford Molecular) For the purposes of this invention, a preferred method of calculating percent identity is the Smith- Waterman algorithm, using the following, Global DNA sequence identity must be greater than 65% as determined bv the Smith-Waterman homology search algorithm as implemented m MPSIRCH program (Oxford Molecular) using an affine gap search with the following search parameters gap open penalty. 12 and gap extension penalty, 1 The subject nucleic acids can be cDNAs or genomic DNAs, as well as fragments thereof, particularly fragments that encode a biologically active gene product and/or are useful in tlie methods disclosed herein The term "cDNA" as used herein is intended to include all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species where sequence elements are exons and 3' and 5' non-coding regions Normally mRNA species have contiguous exons. with the intervening introns, when present, being removed by nuclear RNA splicing, to create a continuous open reading frame encoding a polypeptide of the invention A genomic sequence of interest comprises the nucleic acid present between the initiation codon and the stop codon, as defined in the listed sequences, including all of the introns that are normally present in a native chromosome It can further include the 3 and 5 untranslated regions found in the mature mRNA It can further include specific transcπptional and translational regulatory sequences, such as promoters, enhancers, etc . including about 1 kb. but possibly more, of flanking genomic DNA at either the 5 and 3 end of the transcribed region The genomic DNA can be isolated as a fragment of 100 kbp or smaller, and substantially free of flanking chromosomal sequence The genomic DNA flanking the coding region, either, 3' and 5', or internal regulatory sequences as sometimes found in introns. contains sequences required for expression The nucleic, acid compositions of the subject invention can encode all or a part of the subject differentially expressed polypeptides Double or single stranded fragments can be obtained from tlie DNA sequence by chemically synthesizing oligonucleotides in accordance with conventional methods, by restπction enzyme digestion by PCR amplification, etc Isolated nucleic acids and nucleic acid fragments of the invention compnse at least about 15 up to about 100 contiguous nucleotides, or up to the complete sequence provided For tlie most part, fragments will be of at least 16 nt. usually at least 18 nt or 26 nt, and up to at least about 50 contiguous nt in length or more Probes specific to the nucleic acids of the invention can be generated using the nucleic acid sequences disclosed, and the fragments as descnbed above The probes can be synthesized chemically or can be generated from longer nucleic acids using restnction enzymes The probes can be labelled, for example w ith a radioactive biotinylated. or fluorescent tag, Preferably, probes are designed based upon an identifying sequence of a nucleic acid More preferably, probes are designed based on a contiguous sequence of one of the subject nucleic acids that remain unmasked following application of a masking program for masking low complexity (e g . XBLAST) to the sequence , I e one would select an unmasked region, as indicated by the nucleic acids outside the poly-n stretches of the masked sequence produced by the masking program The nucleic acids of the subject invention are isolated and obtained in substantial puπty generally as other than an intact chromosome Usually, the nucleic acids, either as DNA or RNA, will be obtained substantially free of other naturally-occumng nucleic acid sequences, generally being at least about 50%. usually at least about 90% pure and are typically "recombinant". e g , flanked by one or more nucleotides with which it is not normally associated on a naturally occurnng chromosome The nucleic acids of the invention can be provided as a linear molecule or within a circular molecule They can be provided within autonomously replicating molecules (vectors) or within molecules without replication sequences They can be regulated by their own or by other regulatory sequences, as is known in the art The nucleic acids of the invention can be introduced into suitable host cells using a variety of techmques which are available in the art. such as transferπn polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, hposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion viral infection, electroporation, gene gun, calcium phosphate-mediated transfection. and the like The subject nucleic acid compositions can be used to, for example, produce polypeptides. as probes for the detection of mRNA of the invention in biological samples (e g , extracts of cells) to generate additional copies of the nucleic acids, to generate ribozymes or antisense oligonucleotides, and as single stranded DNA probes or as triple-strand forming oligonucleotides The probes described herein can be used to. for example, determine the presence or absence of the nucleic acid sequences or variants thereof in a sample The probes secπbed herein can also be used for detection of genomic nucleic acid sequences and complementary DNA sequences from genomic nucleic acid and complementary DNA branes Methods of probing and generating such hbranes are well known in the art and are described in Cuπent Protocols in Molecular Biology 1995 Programmed Cell Death Assay The assay for PCD can be accomplished by methods well known in the art Tlie vital dy es acndine orange ( AO), a fluorescent dy e and Nile Blue (NB) can be used to visualize programmed cell deaths in In e Drosophila embryos The dyes have a selective affinity for dying cells and are able to penetrate the tissues of the embryo The dyes have been found to stain apoptotic cell corpses inside engulfing phagocytes, without staining the phagocytes themselves The assay requires preliminary removal of the embryonic choπon with bleach, followed by permeabihzation of the waxy coat with heptane or octane The stained cells are then visualized bv conventional microscopy and Nomarski optics, in tlie case of NB. or fluorescent microscopy, in the case of AO Tissue sections can also be prepared from the stained embryos to obtain higher resolution of the cell structures AO was found to selectively stain apoptotic cells, whereas NB stains both apoptotic and necrotic cells Since the vast majoπty of programmed cell deaths are apoptotic. both vital dyes were found to be useful for visualizing programmed cell deaths Using this assay , it was possible to determine the extent and pattern of ectopic PCD occurnng throughout Drosophila embryogenesis. including those affecting tlie development of certain organs and body sy stems so as to quantitatively assay which embryos have undergone ectopic PCD Recombinant Host Cells Vectors and their Promoters/Enhancers As used herein, the term "engineered" or "recombinant" cell is intended to refer to a cell into which a recombinant nucleic acid sequence, such as a nucleic acid sequence encoding a protein identified by the genetic screen descnbed above, has been introduced Therefore, engineered cells are distinguishable from naturally occuπing cells which do not contain a recombinantly introduced gene Recombmantly introduced nucleic acid sequences w ill either be in the form of a cDNA nucleic acid sequence (I e , they will not contain introns). a copy of a genomic nucleic acid sequence, or will include nucleic acid sequences positioned adjacent to a promoter not naturally associated with tlie particular introduced nucleic acid sequence The term promoter is used herein to refer to a group of transcπptionai control modules that are clustered around the initiation site for RNA polymerase II Much of the thinking about how promoters are organized deπves from analyses of several viral promoters, including those for the HSV thymidme kinase (tk) and SV40 early transcπption umts These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA. and containing one or more recognition sites for transcriptional activator proteins At least one module in each promoter functions to position the start site for RNA synthesis The best known example of this is the TATA box. but in some promoters lacking a TATA box. such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the S V 40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation Additional promoter elements regulate the frequency of transcπptional initiation Typically these are located m the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well The spacing between elements is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another In the tk promoter, the spacing between elements can be increased to 50 bp apart before activity begins to decline Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA This ability to act over a large distance had little precedent in classic studies of prokaryotic transcnptional regulation Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters That is, they are composed of many individual elements, each of which binds to one or more transcπptional proteins The basic distinction between enhancers and promoters is operational An enhancer region as a whole must be able to stimulate transcnption at a distance, this need not be true of a promoter region or its component elements On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities Aside from tins operational distinction, enhancers and promoters are very similar entities They ha\e tlie same general function of activating transcription in the cell They are often overlapping and contiguous, often seeming to have a very similar modular organization Taken together, these considerations suggest that enhancers and promoters are homologous entities and that the transcriptional activator proteins bound to these sequences may interact with tlie cellular transcriptional machinery in fundamentally the same way Generally speaking, it may be more convenient to employ as the recombinant nucleic acid sequence a cDNA v ersion of the nucleic acid sequence It is believed that the use of a cDNA version will provide advantages in that the size of the nucleic acid sequence will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic nucleic acid sequence, which will typically be up to an order of magnitude larger than the cDNA nucleic acid sequence However, the inventors do not exclude the possibility of employing a genomic version of a particular nucleic acid sequence where desired The engineeπng of DNA segment(s) for expression in a prokaryotic or eukaryotic system may be performed by techniques generally known to those of skill in recombinant expression It is believed that virtually any expression system may be employed in the expression of the PCD nucleic acid sequence identified In general. plasmid vectors containing rephcon and control sequences which are derived from species compatible w ith the host cell are used in connection with these hosts The vector ordmanly caπies a replication site, as well as marker sequences which are capable of providing phenotypic selection in transformed cells For example, E coli is typically transformed using pBR322, a plasmid deπved from an E coli species (Bolivar et al 1977) pBR322 contains nucleic acid sequences for ampicilhn and tetracychne resistance and thus provides easy means for identifying transformed cells The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own proteins In addition, phage vectors containing rephcon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts For example, the phage lambda GEM TM -11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as E co LE392 Those promoters most commonly used in recombinant DNA construction include tlie B-lactamase (penicilhnase) and lactose promoter systems (Chang et al 1978, Itakura et al , 1977 Goeddel et al , 1979) and a tryptophan (tip) promoter system (Goeddel et al , 1980, EPO Appl Publ No 0036776) While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (EPO Appl Publ No 0036776) In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available For expression in Saccharomyces. tlie plasmid YRp7. for example. is commonly used (Stinchcomb et al . 1979, Kingsman et al . 1979, Tschemper et al 1980) Tlus plasmid already contains the trpl nucleic acid sequence which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No 44076 or PEP4-1 (Jones, 1977) The presence of the trpl lesion as a characteπstic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al , 1980) or other glycolytic enzymes (Hess et al . 1968, Holland et al . 1978), such as enolase. glyceraldehyde-3 -phosphate dehydrogenase, hexokinase. pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase. 3-phosphoglycerate mutase, pyruvate kinase. tnosephosphate isomerase, phosphoglucose isomerase. and glucokmase In constructing suitable expression plasmids. the termination sequences associated with these nucleic acid sequences are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination Other promoters, which ha\ e the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, lsocytochrome C, acid phosphatase. degradative enzymes associated with nitrogen metabolism, and tlie aforementioned glyceraldehyde-3 -phosphate dehydrogenase. and enzymes responsible for maltose and galactose utilization Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable In addition to microorganisms, cultures of cells denved from multicellular organisms may also be used as hosts In pπnciple. any such cell culture is workable, whether from vertebrate or invertebrate culture However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973) Examples of such useful host cell lines are VERO and HeLa cells. Chinese hamster ovary (CHO) cell lines, and W138, BHK. COS-7. 293 and MDCK cell lines Expression vectors for such cells ordinarily include (if necessary) an ongm of replication, a promoter located in front of the nucleic acid sequence to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences DNA Dehven Following the identification of novel regulatory components by the method of the invention descnbed herein, it is envisioned that the present invention may generally next be used to direct the introduction an exogenous nucleic acid sequence, such as a cDNA or gene, into a recipient cell to create a transformed cell The frequency of occuπence of cells receiving exogenous nucleic acid sequences is believed to be low Moreover, it is most likely that not all recipient cells receiving nucleic acid sequences will result m a transformed cell wherein the nucleic acid is stably integrated into the animal genome and or expressed Some may show only initial and transient gene expression However, certain eukaryotic cells may be stably transformed, and these cells developed into transgenic animals, or arthropods, preferably insects There are many methods for introducing transforming nucleic acid sequences into cells, but not all are suitable for dehveπng nucleic acid sequences to eukaryotic cells Suitable methods are believed to include virtually any method by which nucleic acid sequences can be introduced into a cell, such as by direct delivery of nucleic acid sequences, by desiccation inhibition-mediated nucleic acid sequences uptake, by electroporation. by agitation with silicon carbide fibers, by acceleration of nucleic acid sequences coated particles, by chemical transfection. by hpofection or hposome-mediated transfection. by calcium chloride-mediated nucleic acid sequences uptake, etc In certain embodiments, acceleration methods are preferred and include, for example, microprojectile bombardment Pharmaceutical Assay s and Methods for Identifying Drug Candidates This invention also provides pharmaceutical assays and methods for identifying drug candidates, I e chemical compounds hich may be useful as drugs for the treatment of PCD related diseases Such assays and methods involve testing compounds for their effects on the activity of the regulatory component identified by the method of the invention described herein, the mammalian counterparts of which will be components of PKB signalling in mammals Examples of effects for which testing may be conducted include inhibition. acti\ ation or enhancement of enzymatic activity, e g kinase activity of Daktl or phosphatase activity of DPP2A Thus, drugs that elevate expression of wild-type Daktl or DPP2A or their mammalian counterparts. PKB or PP2A. inhibit expression of a mutated or altered form of these proteins or inhibit a protein that antagonizes the activity of these proteins may be useful in treating diseases in which it is preferred that PCD be inhibited Further. RNA or DNA probes may be used to detect chemical agents that alter expression or activity of the proteins Moreover, the Daktl or DPP2A nucleic acids may also be used to engineer cells that would be useful for the detection of chemicals that alter expression or activity of the proteins Most preferably, the Daktl or DPP2Λ protein may be used in direct biochemical assays for agents that activate or alter its activity If one of the components identified bv the genetic screen descnbed herein is a transcription factor, reporter assays can be designed for identifying agents which can modulate Dakt 1 expression For example, host cells known to contain Daktl or PKB can be transfected with a plasmid reporter gene, wherein the reporter gene (E coli lac Z, or luciferase) is fused to the promoter/enhancer module of the transcnption factor known to be m the Dakt //PKB pathway The cells are next exposed to an agent for a period of time, and the level of reporter gene product expressed is compared the level of reporter gene product expressed in cells not exposed to the drug (reference cell) If the reporter gene product expressed in the cells exposed to the agent is higher when compared to the level of the reporter gene product in the reference cells, then the agent is able to enhance expression of Daktl /PKB or maintain Daktl/FKB in a constitutively active state Such agents will be useful in treating diseases where it is desirable to inhibit PCD Conversely, identification of agents capable of inhibiting expression of tlie reporter gene product can also be assayed by this method for the identification of agents for inducing PCD An alternative method for determining the effect of an agent on the activity of Daktl /PKB is to assav for the level of product of the natural gene fused to the promoter/enhancer module to which the transcπption factor binds and initiates transcπption For example, it is known that the protein product of trachealess initiates transcription of the breathless gene by contacting the B123 promoter/enhancer module Instead of engmeenng a reporter gene construct, the effect of an agent on the activity of trachealess can be determined by assaying the level of breathless DNA by RT-PCR produced in the cell exposed to the agent for a penod of time and comparing it to the level of breathless DNA produced by RT-PCR in a cell not exposed to the agent In vitro studies may use purified target macromolecules to screen large compound libraries for inhibitory drugs, or the punfied target molecule may be used for a rational drug design program, which requires first determining the structure of the macromolecular target or. the structure of the macromolecular, target in association with its customary substrate or hgand This information is then used to design inhibitory compounds which must be synthesized and tested further Test results are used to refine tlie molecular models and drug design process in an iterative fashion until a lead compound emerges Kinases. particularly protein kinase B and homologs thereof arc involved in multiple biologically important processes Pharmacological agents designed to affect only specific kinase activities are of particular interest Presently available compounds tend to be non-specific and elicit both positive and negative responses, thereby reducing climcal efficacy The subject mutations may be used in in vitro and in vivo models to test tlie specificity of novel compounds, and of analogs and derivatives of compounds known to act on kinases Numerous pharmacological agents have profound affects on kinase activity Of particular interest are screening assays for agents that have a low toxicity for human cells A wide variety of assays may be used for this purpose, including momtoπng cellular excitation and conductance, labelled in vitro protem-protem binding assays, electrophoretic mobility shift assay s. immunoassays for protein binding, and the like The punfied protein may also be used for determination of three-dimensional crystal structure, which can be used for modelling intermolecular interactions The term "substance " or "agent" as used herein describes anv molecule, e g protein or pharmaceutical With the capability of inhibiting, alteπng or mimicking the kinase activities associated with tlie screened gene products Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations sen'es as a negative control, l e at zero concentration or below the level of detection Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more then 50 and less than about 2.500 daltons Candidate agents compnse functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbanyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups The candidate agents often compnse cyclical carbon or heteroc chc structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups Candidate agents are also found among biomolecules including peptides. sacchaπdes. fatty acids, steroids, purines. pyπmidines. derivatives, structural analogs or combinations thereof Candidate agents are obtained from a wide variety of sources including hbranes of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide vanety of organic compounds and biomolecules. including expression of randomized oligonucleotides and ohgopeptides Alternatively, hbranes of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readilv modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation. alkylation, esteπfication, amidification. etc, to produce structural analogs Where the screening assay is a binding assay, one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal Vaπous labels include radioisotopes. fluorescence, chemiluminescence . enzymes, specific binding molecules, particles, e g magnetic particles, and the like Specific binding molecules include pairs, such as biotm and streptavidin, digoxm and antidigoxin etc For die specific binding members, the complementary member would normally be labelled with a molecule that provides for detection, in accordance ith known procedures A vanety of other reagents may be included in the screening assay These include reagents like salts, neutral proteins, e g albumin, detergents, etc that are used to facilitate optimal protem-protem binding and/or reduce non-specific or background interactions Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc may be used The mixture of components are added in any order that provides for the requisite binding, Incubations are performed at any suitable temperature, typically between 4 and 40°C Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screemng Typically between 0 1 and 1 hours will be sufficient Example 1 Molecular analyses localized Daktl to within ~30 kb of the stubbloid (sbd) gene (Franke et al Oncogene 1994, 9 141-148, Andjelkovic et al J B ol Chem 1995, 270 4066-4075) placing Daktl withm Df(3R)sbd i (Appel et al Proc Natl Acad Sci USA1993. 90 4937-4941) Thus, a hunt for a Daktl mutant focused on lethals that failed to complement a large deletion at cytological position 89B on the right arm of the third chromosome of Drosophila melanogaster As Daktl is ubiquitously expressed (Franke et al Oncogene 1994, 9 141-148, Andjelkovic et al J Biol Chem 1995, 270 4066-4075), a GAL4 transgene dπven by the armadillo promoter (arm-GAL4) was used to allow expression of Daktl in a fairly ubiquitous pattern using an upstream activating sequence Daktl (UAS-Daktl) transgene Screening a collection of mutants mapping to DfβRlsbd 45 , one larval lethal mutation, l(3)89Bq' was identified that could be rescued by the combination of the arm-GAL4 and UAS-Daktl transgenes, arm-Daktl (% rescue=34 1) As PKB/AKT and Daktl have a similanty of 76 5% at tlie amino-acid level [(Franke et al Oncogene 1994, 9 141-148, Andjelkovic et al J Biol Chem 1995, 270 4066-4075, Coffer et al Eur J Biochem 1991, 201 475-481). bovine PKB/AKT was tested for its ability to rescue l(3)89Bq' mutants using a UAS PKB/AKT transgene The arm-PKB/AKT combination was effective at rescuing 3)89Bq' albeit with a lower efficiency (% rescue=17 8), demonstrating that l(3)89Bq 1 or simply 'q ' encodes Daktl. the functional homolog of PKB/AKT Rescue was scored as the percentage of flies with a homozygous q chromosome For Both Daktl bovine PKB, and PKB DD mutant cDNAs were cloned into pUAST P-element transformation vector and injected into M W " S embryos to generate transformants The heat-shock mducible UsDaktl line was generated using the pHS Casper P-element vector For the rescue experiments, the following crosses were made (A) q/TM6B=q' 'TM6B Tb e Hu (XXJ) (B) (arm-Daktl) q/TM6B=armGAL4, q'/TM6B Tb e Hu X UASDaktl/CyO, q' TM6B Tb e Hu The results of cross B were coπected for the CvO chromosome where CyO progeny (which would not carry UASDaktl) were removed from the data (C) (arm-PKB) q/TM6B=GAL4, q/TM6B Tb e Hu X UASPKB, q'/TM6B Tb e Hu (D) (HsDaktl) q/TM6B=HsDaktl q' TM6B Tb e Hu (KXJ) For heat-induced rescue, flies were heat-shocked 30 min daily after egg lying in glass vials GLC analysis for 3R using FRT S2β was performed as descnbed in (Chou Et al Genetics 1996, 144 1673- 1679) For determining the extent of zygotic/paternal rescue, l(3)89Bq' GLC females were crossed to wild-type, 3)89Bq', TM3, Sb, l(3)89B q 2 and DfβRJsb t 5 males and the embryonic phenotypes compared As l(3)89Bq' and Df(3R)sbd 15 chromosomes behaved identically, it is believed that l(3)89Bq' is a genetically null allele of Daktl Also a second Daktl allele (termed q 2 ) was also used for these expeπments However, as q was induced on TM3 chromosome, it was not possible to perform GLC experiments using this allele Df(3L)H99 FRT 3213 1(3) 89B q ! chromosome was constructed using standard recombination techniques well known m the art and then crossed to FRT S2/1 ovoD males for the induction of GLCs GLC females ere then crossed to the Df(3L)H99 FRT 820 1(3) 89 B q' chromosome and the progeny analyzed A 1 kb BamUl fragment of pBSPS contaimng the entire coding region of Autographica californica P35 (Clem et al Mol Cell Biol 1994, 14 5215-5222) was cloned into tlie Bglll site of pHS Casper and transformed For the epistasis experiment, q' GLC females were crossed to HsP35, q'/TM3 males and the progeny collected onto nylon sieves Embryos were then staged (stages 10-12), heat-schocked at 37°C in a water bath for 8 mm and allowed to recover at 25°C Embryos were then allowed to develop cuticle and analyzed For ectopic expression of activated PKB (PKB DD), armGAL4 females were crossed to UAS PKB DD males to generate arm-PKB DD embryos Embryos were then collected and either treated for Acπdine Orange (AO) staining or for antibody staimng Example 2 Antisera against Daktlweτe raised against a histidine tagged Daktl fusion protein (His-Daktl) Uis-Daktl was constructed by cloning a 1590 bp fragment of the Daktl complete coding region (530 ammo acids) into the BamHl site of the pET 15b vector (Novagen) Fusion proteins were produced in E coli strain (BL21) and punfied from bacterial lysates through binding to a mckel-chelating resin according to the manufacturer ' s instructions (Novagen) Rabbits were immunized subcutaneously with punfied recombinant proteins in complete Freund ' s adjavunt followed by booster injections at 4 week intervals For kinase assays. Daktl and its mutant, Daktl 9 . was lmmunoprecipitated using the antibody to Daktl generated as described above from lysates of embryos prepared by lysing the embryos in Gentle Soft buffer (20mM PIPES pH 7 4. lOmM NaCl. 0 5% NP-40, 5mM EDTA. 0 05% 2- mercaptoethanol, 5mg/ml leupeptin. ImM benzamidine, 0 5 mM NaF and 100 mM Na vanadate) Larvae homozygous for q (expressing Daktl q ) were identified by following the TM6B balancer chromosome Lysates were normalized for total protein before lmmunoprecipitation 5 ml of rabbit polyclonal antiserum were added to the cell lysates for 2 h at 4°C Immunocomplexes prebound to protein A-Sepharose (Sigma) were ashed five times with lysis buffer pnor to performing the kinase assays In vitro Daktl, Daktl q (FIG 1 (A)). HA tagged bovine wild-type PKB and kinase inactive HA-PKB F293I (FIG 1 (D)) kinase assays were measured by the capacity of the kinases to phosphorylate a GSK3 peptide substrate corresponding to the sequence in GSK3 β suπounding the Ser9 site that is phosphorylated by PKB The Daktl, Daktl 11 and HA tagged bovine PKB immunecomplexes were incubated at 30°C with 30mM GSK3 peptide m the presence of 50 mM [γ- 32 P] ATP m 50 mM PIPES. pH 7 4, 10 mM MgCl 2 and 1 mM EGTA The phosphorylated peptides were separated from unincorporated [γ— 32 P] ATP by Tπcine-SDS-PAGE and quantified by analysis on a Molecular Dynamics Phosphonmager For immunoblot analysis, lyastes were prepared as descnbed above and normalized for total protein before separation by SDS-PAGE for lmmunoblotting Daktl and its mutant. Daktl q protein was visualized using the ECL system (Amersham) (FIG 1 (B)) Example 3 A genetic screen as described above was performed against a collection of P-element induced mutant f lines as well as other lethals with the intention of identifying genes that interact with Daktl The inventors discovered that a mutant fly line, P1747, resulted m an average of 7 4% lethality while in trans to Daktl This mutant has a P-element insertion in the 5 ' untranslated region of the trachealess (trh) gene (SEQ ID No 1) at cytological region 61C of chromosome three Surprisingly no ectopic PCD was associated with the dead embryos from the trh/Daktl cross when assayed for PCD by the standard methods descnbed above This suggested that DAkt's involvement with the trh protein (SEQ ID No 2) may be separate from its role in PCD and follows that Daktl may be involved in fr ?-medιated pathways as well Inspection of the am o acid sequence of trh protein showed that it contains two consensus sequences for PKB phosphorylation The first is ammo acid sequence RGRGRS^'AA in which Ser 571 may be phosphorylated The second is amino acid sequence RSRLPS ofo IV in which Ser 665 may be phosphorylated To determine if treacheless protein is a substrate for PKB kinase activity, a kinase assay was performed as described in Example 2 and in the detailed description of FIG. 5 with PKB as the kinase and treacheless protein or fragments thereof as the test substrates FIG. 5 shows that only Trhl. containing the two putative serine residues is phosphorylated by active PKB and not kinase inactive PKB or wild-type PKB inactivated by the PI3'K inhibitor wortmannin These data validate trachealess protein as a substrate for PKB The data presented in FIG. 6 and FIG. 7 confirm PKB phosphorylation on Ser 665 of the trachealess protein fragment substrates In summary, these data show an unexpected result obtained from the genetic screen described above The identification of dead embryos not undergoing ectopic PCD was a complete surpπse considering that the genetic screen descnbed above is designed to identify enhancers of the zygotic Daktl phenotype. I e. enhanced PCD by second site lethal mutations Although the embryos scored, were dead, ectopic PCD was not observed Instead it was determined that death was a result of abnormal embryonic development, thus indicating tliat DAkt 's involvement with trachealess is separate from its role in apoptosis Such a role for DAkt or its mammalian homolog. PKB. has not been identified in the pnor art Trachealess is a master regulatory gene that induces differentiation of the embryonic tracheal system from ectodermal precursor cells in Drosophila At stage 10 (4 hours after egg lay [AEL]) of embryonic development, clusters of cells on both sides of each of the ten posterior parasegments (T2-A8) assume a tracheal-specific fate Duπng stages 10 and 11 (4-5h AEL). each cluster of precursor cells undergoes two postblastoderm mitoses, tlie first mitosis event establishes a region in the ectoderm called the tracheal placode, and the second mitosis event is coincident with imagination of the tracheal placode and formation of the tracheal pit Subsequent formation of the tracheal system occurs by cell migration and shape changes without further cell divisions Tracheation occurs in two general stages, pπmary and terminal tracheation Primary tracheation (stages 11-16 [5h AEL-hatchmg]) is the establishment of the stereotypical branching pattern of the ma mulucellular and unicellular tracheae that make up each tracheal metamere Terminal tracheation begins duπng stage 15 (l l-13h AEL) and continues into the larval period This overlapping stage is characterized by the development of fine unicellular and subcellular tracheae that branch from or are continuations of, the main components of the tracheal system The terminal branches are extensive arrays of subcellular tubes that reach individual target cells or tissues in the embryo The pattern of pπmary branching is stereotypical but the pattern of terminal branching is highly variable and most likely dependent on the oxygen needs of target tissues The entire tracheal network in the embryo is continuous and supplied by a single opemng, the posterior spiracle, which forms during stage 11 (5h AEL) by fusion of the tracheal pit and posterior spiracle in parasegment A8 Coincident with the establishment of tracheal cell fate at stage 10 is the expression of Trh and Vvl (Ventral Veinless/Dπfter) in tlie tracheal precursor cells Immunostaining has shown that Trh protein is localized to the nuclei of the tracheal precursors and that, once initiated, it is expressed for the remainder of embryogenesis and throughout most of la al development The Trh protein is required for the initial invagination of the tracheal placodes. tubulogenesis and the expression of tracheal-specific genes Despite its early co-expression with Trh. the Vvl POU domain-containing transcription factor is needed later in development for migration of the pπman branches and the expression of some tracheal-specific markers, but it is not required for any initiation events The segment polanty genes ssg (wingless) and dpp (decapentaplegic) appear to have an effect on the localization of the Trh signal in the parasegment. and the homeotic genes may also play a role in determimng tlie initial spatial expression of Trh since this gene is only expressed in ten of the fourteen parasegments Trh is a member of the bHLH-PAS family of transcπption factors The 949 amino acid protein contains a bHLH (basic hehx-loop-helix), PAS (homology to Per, Arnt and Sim) and transcπption activation domains, as well as a nuclear localization signal (NLS) The bHLH domain determines the protein's ability to contact DNA and form dimers. while no function has been attributed to the PAS domain Once Trh expression is initiated, the protein must heterodimeπze with the Drosophila Arnt (DArnt)ΛTango (Tgo) gene product in order to translocate to the nucleus and activate transcription of nuclear target genes, including itself Trh must initiate an autoregulatory feedback loop in order to amplify the initial signal and then maintain that level of expression for efficient activation of transcription of downstream targets Tgo, also a bHLH-PAS protein, is the homologue of the mammalian Arnt gene which functions as a dimeπzation partner for the aromatic hydrocarbon receptor (Ahr) transcription factor m mammals Once heterodimeπzed and inside tlie nucleus, the Trh Tgo transcriptional activator recognizes a cw-regulatory CNS midhne element (CME) in the target genes that are destined for tracheal-specific transcription Trh is needed for expression of many tracheal-specific genes including the breathless (Btl) FGF receptor and the tracheae defective (tdf) bZIP transcπption factor Btl binds the branchless (bnl) ligand and is required for the non-directionally-specific, chemotactic outgrowth of the pπmary branches towards the sites of highest bnl expression The expression of other genes such as thick veins and rhomboid, which are transcπptionally activated by Vvl, and the expression of their target genes (dpp and spitz, respectively) acts to determine the directional migratory fate of the pnmary branches The sproutv gene appears to be activated in response to Btl/Bnl activity and acts to limit branching activity to the region of highest btl activation Example 4 A genetic screen as described above is performed against a collection of P-element induced mutant fly lines as well as other lethals with the intention of identifying genes that interact w lt Daktl A mutant fly line, PI 193 is determined to result in an average of 7 6% lethality by ectopic PCD while in trans to Daktl This mutant is a P-element insertion in the 5' untranslated region of the DPP2A gene at cytological region 61C of chromosometwo Ectopic PCD is associated with the dead embryos from tlie DPP2Λ'Daktl cross when assayed for PCD by the standard methods descnbed above To determine if DPP2A protein is a substrate for PKB/AKT/Daktl kinase activity, a kinase assay is performed as described in Example 2 and in the detailed description of FIG. 5 with PKB as the kinase and treacheless protein or fragments thereof as the test substrates All citations referred to in the specification are incorporated herein in their entirety as though their relevant contents had been directly reproduced herein

Claims: CLAIMS 1 A method of determining whether a mutation capable of enhancing programmed cell death is present in a Drosophila melanogaster fly, the method compnsing (a) providing a mutant first fly having a recessive first mutation, the presence of which mutation is known to induce programmed cell death in an embryo of a fly homozygous for the mutation, (b) providing a second mutant fly for determination, (c) crossing the first and second flies (d) determining whether embryos obtained in step (c) have undergone ectopic programmed cell deatli. (e) wherein if the embryos obtained in step (d) have undergone ectopic cell death then a mutation capable of enhancing programmed cell death is present in the second fly 2 The method of claim 1, wherein the first fly is known to have at least one mutation in a DAktl gene known to induce programmed cell death in an embryo of a fly homozygous for the mutation 3 The method of claim 2, wherein the at least one mutation in the DAktl gene results in loss of kinase activity of the protein encoded thereby 4 The method of claim 3, wherein the at least one mutation results in a phenylalanine to isoleucine substitution at position 327 of the said protein 5 The method of claim 4 wherein the mutation is a TTC→ATC 6 The method of claim 1, wherein the first fly is known to have at least one mutation in a DPP2A gene known to induce programmed cell death 7 The method of claim 6, wherein the at least one mutation in the DPP2A gene results in loss of phosphatase activity of the protein encoded thereby 8 The method of any preceding claim, wherein the second fly is obtained by exposing a wild-type fly to ionizing radiation, by chemical mutagenesis of a wild-type fly, or by inserting P-elements in the genome of a wild- type fly 9 The method of anv of claims 1 to 8. wherein step (d) includes visually examimng emb os obtained in step (c) for cuticle loss 10 The method of claim 9, further comprising the step of treating embryos having cuticle loss by degrading or removing the choπon of said embryos to permit staining of embryonic cells intenor of the chonon 11 The method of claim 10, further comprising the step of staimng the treated embryos with a dye and determining whether said embryos have undergone programmed cell death by visual inspection of said embryos 12 The method of claim 11, further comprising determimng whether said treated embryos have undergone programmed cell death by assaying by terminal deoxy-transferase mediated hapten-labeled-dUTP nick end labeling or with acπdine orange 13 The method of any preceding claim, further comprising the step of determimng the chromosomal location a mutation in tlie second fly 14 The method of claim 13 , further comprising the step of determining the nucleotide sequence of the located mutation 15 The method of claim 14, further comprising tlie step of sequencing the chromosome of the fly in the vicinity of said mutation and comparing tlie sequence obtained with a wild type sequence to determine tlie wild type sequence coπesponding to the mutant sequence 16 The method of claim 15, further comprising tlie step of, if the wild type sequence is a known sequence, determining whether a function of the wild type sequence is known 17 The method of claim 16. further comprising the step of determimng the coding portion of the wild type sequence 18 The method of claim 17. further comprising the step of companng the coding portion w ith know n sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded by the fly wild type sequence 19 The method of claim 15, further comprising the step of, if the wild type sequence is not a known sequence. companng the sequence of the mutant fly in the vicinity of said mutation with sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded bv the fly wild type sequence 20 The method of claim 19. further comprising the steps of creating a nucleic acid molecule for use as a probe and screening a library of nucleic acid molecules with tlie probe 21 The method of any preceding claim, further comprising the step of determimng tlie phenotype of the dead embryos after step (e) 22 The method of any preceding claims, wherein step (c) includes determimng whether at least 5 about percent, more preferably at least about 6 percent, more preferably at least about 7 percent, of embryos have undergone ectopic cell death 23 A method of determining whether a mutation capable of disrupting development is present in a Drosophila melanogaster fly. the method comprising (a) providing a first mutant fly having a recessive first mutation, the presence of which mutation is known to induce programmed cell death m an embryo of a fly homozygous for the mutation, (b) providing a second mutant fly for determination, (c) crossing the flies in (a) and (b). (d) examimng dead embryos obtained in step (c) to determine whether dead embryos of a phenotype different from either the first or second mutant flies are present, (e) wherein, if a said different phenotype is observed then a mutation capable of disrupting development is present in the second mutant fly 24 The method of claim 23, wherein the first fly is known to have at least one mutation in a DAktl gene known to induce programmed cell death m an embryo of a fly homozygous for the mutation 25 The method of claim 24, wherein the at least one mutation in the DAktl gene results in loss of kinase activity of the protein encoded thereby 26 The method of claim 25. wherein the at least one mutation results in a phenylalanine to isoleucine substitution at position 327 of the said protein 27 The method of claim 26, wherein the mutation is a TTC— >ATC 28 The method of claim 23, wherein the first fly is known to have at least one mutation in a DPP2A gene known to induce programmed cell death 29 The method of claim 28, wherein the at least one mutation in the DPP2A gene results in loss of phosphatase activity of the protein encoded thereby 30 The method of any of claims 23 to 29. wherein the second fly is obtained by exposing a wild-type fly to ionizing radiation, by chemical mutagenesis of a wild-type fly, or by inserting P-elements in the genome of a wild- type fly 31 The method of any of claims 23 to 30, wherein step (d) includes visually examining embryos obtained in step (c) to determine whether trachea are missing from the embryos 32 The method of anv of claims 23 to 31. further compπsing the step of determimng the chromosomal location a said mutation in the second fly 33 The method of claim 32, further comprising the step of determimng tlie nucleotide sequence of the located mutation 34 The method of claim 33 , further comprising the step of sequencing the chromosome of the fly in the vicinity of said mutation and comparing the sequence obtained with a wild type sequence to determine the wild type sequence corresponding to the mutant sequence 35 The method of claim 34, further comprising the step of, if the wild type sequence is a known sequence, determining whether a function of the wild type sequence is known 36 The method of claim 35, further comprising the step of determimng tlie coding portion of the wild type sequence 37 The method of claim 36, further comprising the step of companng the coding portion with known sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded by the fly wild type sequence 38 The method of claim 34, further comprising the step of, if the wild type sequence is not a known sequence, companng the sequence of the mutant flv in the vicinity of said mutation with sequences of other species and selecting a sequence which potentiallv encodes a protein having a function in common w ith a protein encoded by the fly wild type sequence 39 The method of claim 38, further comprising the steps of creating a nucleic acid molecule for use as a probe and screening a library of nucleic acid molecules with the probe 40 The method of any of claims 23 to 39. further comprising the step of determimng the phenotype of the dead embryos after step (e) 41 The method of any of claims 23 to 40. wherein step (c) includes determining whether at least 3 about percent, more preferably at least about 4 percent, more preferably at least about 5 percent, more preferably at least about 6 percent, of dead embrvos have a phenotype in common with each other and different from either the first or second mutant flies 42 A method of determining hether a mutation capable of disrupting development is present in a Drosophila melanogaster fly, the method comprising (a) providing a first mutant fly having a recessive first mutation, the presence of which mutation is known to disrupt development in an embryo of a fly homozygous for the mutation. (b) providing a second mutant fly for determination, (c) crossing the flies in (a) and (b), (d) examimng dead embryos obtained m step (c) to determine whether dead emb os of a phenotype different from either the first or second mutant flies are present, (e) wherein, if a said different phenotype is present then a mutation capable of disrupting development is present in the second mutant fly 43 Tlie method of claim 42, wherein the first fly is known to have at least one mutation in a trachealess gene known to disrupt development in an embryo of a fly homozygous for the mutation 44 The method of claim 43, wherein the mutation comprises a a P-element insertion in the 5 ' untranslated region of the trachealess gene at cytological region 61C of chromosome three 45 The method of any of claims 42 to 44, wherein the second fly is obtained by exposing a wild-type fly to ionizing radiation, by chemical mutagenesis of a wild-type fly, or by inserting P-elements in the genome of a wild- type fly 46 The method of any of claims 42 to 44, wherein step (d) includes visually examining embryos obtained in step (c) to determine whether embryos missing trachea are present 47 The method of any of claims 23 to 46, further comprising the step of determimng the cliromosomal location a said mutation in the second fly 48 The method of claim 47, further comprising tlie step of determimng the nucleotide sequence of the located mutation 49 The method of claim 48, further comprising the step of sequencing the chromosome of the fly in the vicinity of said mutation and comparing the sequence obtained with a wild type sequence to determine the wild type sequence coπesponding to the mutant sequence 50 The method of claim 49. further compπsing the step of, if the wild type sequence is a known sequence, determining whether a function of the wild type sequence is known 51 The method of claim 50. further comprising the step of determimng the coding portion of the wild type sequence 52 The method of claim 51 , further comprising the step of companng the coding portion with known sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded by the fly wild type sequence 53 The method of claim 49, further comprising the step of. if the wild type sequence is not a known sequence, companng the sequence of the mutant fly in the vicinity of said mutation with sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded by the fly wild type sequence 54 The method of claim 53. further comprising the steps of creating a nucleic acid molecule for use as a probe and screening a library of nucleic acid molecules with the probe 55 The method of anv of claims 42 to 54. further comprising the step of determimng the phenotype of the dead embryos after step (e) 56 The method of any of claims 42 to 55. wherein step (c) includes determining whether at least 5 about percent, more preferably at least about 6 percent, more preferably at least about 7 percent, of dead embryos have a phenotype m common with each other and different from either the first or second mutant flies 57 The method of any of claims 1 to 12, 23 to 32, 42 to 44 or 46, wherein a marker has been introduced into the chromosome of the mutant second fly at a location suitable to permit identification of a mutation introduced into the second fly . and wherein the method further compπses the step of identifying the mutation 58 The method of claim 57. wherein the step of identifying the mutation includes sequencing the chromosome of the fly in the vicinity of said marker and companng the sequence obtained with a ild type sequence to determine the wild type sequence corresponding to the mutant sequence 59 The method of claim 58, further comprising the step of determining whether a function of the w Ud type sequence is known 60 The method of claim 58. further comprising the step of determimng tlie coding portion of the w lid type sequence 61 The method of claim 60. further comprising the step of companng tlie coding portion with known sequences of other species and selecting a sequence which potentially encodes a protein having a function in common with a protein encoded by the fly wild type sequence 62 A method of inducing programmed cell death in a cell by inhibiting the kinase activity of DAktl protein 63 The method of claim 62, including admimstenng a nucleic acid molecule sufficiently complementary such as to inhibit translation of DAktl mRNA present in a cell 64 A method of inducing programmed cell death in a cell by inhibiting the phosphatase activity of a PP2A protein 65 The method of claim 64. including admimstenng a nucleic acid molecule sufficiently complementary such as to inhibit translation of PP2 A mRNA present in a cell 66 An assay for determining whether a substance is potentially suitable for usage as an active ingredient in inducing programmed cell death or in inhibiting cell death, which assay comprises the following steps exposing DAkt having kinase activity to the substance, quantitativelv assaying the effect of the substance on the kinase activity, and determining based on the results of said assay the potential suitability of the substance as an acuve ingredient in inducing programmed cell death or in inhibiting programmed cell death 67 The assay of claim 66. wherein a said substance is potentially suitable as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit the kinase activity 68 The assay of claim 66, wherein a said substance is potentially suitable as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance the kinase activity 69 The assay of claim 66, further comprising the following steps exposing embrvos of Drosophila melanogaster fly to the substance, quantitatively assaying the effect of the substance on ectopic cell death in the embryos, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or inhibiting programmed cell death 70 An assay for determining whether a substance is potentially suitable for usage as an active ingredient in inducing programmed cell deatli or inhibiting programmed cell death, which assay comprises the following steps exposing PP2A having phosphatase activity to the substance, quantitatively assaying the effect of the substance on the phosphatase activity, and determining based on the results of said assay the potential suitability of the substance as an activ e ingredient in inducing programmed cell death or in inhibiting programmed cell death 71 The assay of claim 70, wherein a said substance is potentially suitable as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit the phosphatase activity 72 The assay of claim 70. wherein a said substance is potentially suitable as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance the phosphatase activity 73 The assay of claim 70, further compπsing the following steps exposing embryos of Drosophila melanogaster fly to the substance, quantitatively assaying tlie effect of the substance on the ectopic cell death m the embryos, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or in inhibiting programmed cell death 74 An assay for determining whether a substance is potentially suitable for usage as an active ingredient m aπesting development of an organism, which assay compπses the following steps exposing a trachealess protein capable of initiating transcription to the substance, quantitatively assaying the effect of the substance on the ability of the protein to initiate transcription, and determining based on the results of said assay the potential suitability of the substance as an active ingredient m arresting development of an organism 75 The assay of claim 74. further compnsing the following steps exposing embryos of Drosophila melanogaster fly to the substance, quantitatively assaying the effect of the substance on development of the embry os, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in arresting development of an organism 76 Tlie assay of claim 75, wherein assaying the effect of the substance on development of the embryos includes determining the effect of the substance on tracheal development 77 A method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for abnormal development, tlie method compnsing the steps of providing a sample of genetic material of the subject, determining whether the genetic material includes a nucleotide sequence encoding a mutant trachealess protein incapable of initiating transcnption, wherein the presence of said nucleotide sequence indicates the presence of a potential to confer on its offspnng a predisposition for abnormal development 78 The method of claim 77. further comprising the step of determining whether the genetic material includes a nucleotide sequence encoding a trachealess protein capable of initiating transcπption 79 The method of claim 77. wherein the trachealess protein capable of initiating transcnption includes the amino sequence identified as SEQ ID NO 1 80 A method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for abnormal development the method compnsing the steps of providing a sample of genetic material of the subject, determining whether the genetic material includes a nucleotide sequence encoding a mutant PP2A protein lacking phosphatase activity, wherein the presence of said nucleotide sequence indicates said potential predisposition 81 A method of predicting in a subject the presence of a potential to confer on its offspring a predisposition for abnormal development the method compnsing the steps of providing a sample of genetic material of the subject, alternativeh determimng whether the genetic matenal includes a nucleotide sequence encoding a mutant D AkT/PKB protein having kinase activity lower than the activity of the coπesponding wild type DAkT/PKB of the subject, wherein] the presence of said nucleotide sequence indicates said potential predisposition 82 A method of inducing programmed cell death in a cell, the method comprising inhibiting the activity of PP2A m the cell 83 The method of claim 82, wherein inhibiting the activity of PP2A in the cell includes admimstenng to the cell an effective amount of an antisense nucleic acid molecule sufficiently complementary to an mRNA encoding PP2A to bind thereto so as reduce translation thereof 84 The method of claim 82 v herein inhibiting the activity of PP2 A in tlie cell includes admimstenng to the cell an effective amount of a phosphatase inhibitor 85 The method of claim 84 w herein the inhibitor is selected from the group consisting of microcystin LR and calicuhn 86 An assay for determining whether a substance is potentially suitable for usage as an active ingredient in inducing programmed cell death or in inhibiting programmed cell death, which assay comprises tlie following steps providing a cell m which a reporter gene is operably linked to a promoter/enhancer which is acted upon by trachealess. wherein the reporter gene is one which, in nature, is not operably linked to the sequence promoter/enhancer, exposing the cell to the substance, quantitatively assaying the effect of the substance on expression of the reporter gene, and determining based on the results of said assay the potential suitability of the substance as an activ e ingredient m inducing programmed cell death or in inhibiting programmed cell death 87 The assay of claim 86, wherein the cell is a cell of a Drosophila melanogaster fly 88 The assay of claim 87, wherein the promoter/enhancer includes the nucleotide sequence SEQ ID NO 3 89 The assay of any of claims 86 to 88, wherein die determination step includes companng the effect of the substance on the expression of the reporter gene with the expression of the reporter gene in the absence of the substance 90 The assay of any of claims 86 to 89, wherein the substance is a kinase inhibitor or a phosphatase inhibitor 91 The assay of any of claims 86 to 90, wherein a said substance is potentially suitable for usage as an active ingredient m inducing programmed cell death if determined to quantitatively inhibit expression of the reporter gene 92 The assay of anv of claims 86 to 91, wherein a said substance is potentially suitable for usage as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance expression of the reporter gene 93 An assay for determining whether a substance is potentially suitable for usage as an active ingredient m inducing programmed cell death or in inhibiting programmed cell death, which assay compnses the following steps exposing a cell to the substance, quantitatively assaying the effect of the substance on expression of the trachealess gene, and determining based on the results of said assay the potential suitability of the substance as an active ingredient in inducing programmed cell death or m inhibiting programmed cell death 94 The assay of claim 93. wherein the assaying step includes determining the amount of RNA encoding trachealess produced by the cell in the presence of the substance and companng the amount with the amount of RNA encoding trachealess produced by the cell in the absence of the substance 95 The assay of claim 94 wherein determimng the amount of RNA encoding trachealess includes amplifying the RNA in the presence of pπmers complementary thereto 96 The assay of claim 94, wherein a said substance is potentially suitable as an active ingredient in inducing programmed cell death if determined to quantitatively inhibit said expression 97 The assay of claim 94, wherein a said substance is potentially suitable as an active ingredient in inhibiting programmed cell death if determined to quantitatively enhance said expression

Inventors: JING JIN, MANOUKIAN ARMEN S, WETSCH BEN, WOODGETT JAMES R, JING, JIN, MANOUKIAN, ARMEN, S., WETSCH, BEN, WOODGETT, JAMES, R.
Applicants: JING JIN, MANOUKIAN ARMEN S, TORONTO HOSPITAL, WETSCH BEN, WOODGETT JAMES R, JING, JIN, MANOUKIAN, ARMEN, S., THE TORONTO HOSPITAL, WETSCH, BEN, WOODGETT, JAMES, R.
Application year: 1999
Main classification: C12Q1
IPC codes: A01K67, C12N5, C12N15, C12Q1