GB2356400A - Drosophila CED-6 homologue and its uses - Google Patents

Abstract

A nucleic acid (as shown in Fig. 1, preferably from nucleotide 132 to 1682) encoding a Drosophila CED-6 (dCED-6) protein with the amino acid sequence shown in Fig. 2 is claimed. Also claimed are expression vectors and transgenic organisms harbouring this sequence, antibodies to epitopes of dCED-6. Methods of identifying compounds that inhibit or enhance phagocytosis of apoptotic cells using dCED-6 or mutated forms thereof are also claimed.

Description

2356400 DrosMhila CED-6 homolocrue The present invention relates to the

field of programmed cell death or apoptosis, and in particular to the phenomenon whereby apoptotic cells are rapidly phagocytosed or engulfed by other cells.

Specifically, the invention provides an insect homologue of a gene previously identified in humans and in the nematode C. elegans which has been implicated in the engulfment of apoptotic cells by phagocytes.

During development and maintenance of living tissues a large number of cells undergo programmed cell death or apoptosis. This phenomenon is observed in both vertebrates and invertebrates. Lysis of apoptotic cells is potentially harmful since their contents may cause toxic damage to the surrounding tissues. It has been observed that this harmful effect is avoided because apoptotic cells are engulfed and subsequently degraded by other cells. In higher organisms, such as mammals or insects, the engulfing cells may be professional or semi-professional phagocytes such as neutrophils or macrophages or they may be neighbours of the dying cells.

A key feature of the process of programmed cell death, or apoptosis, is the efficiency with which dying cells are recognized and engulfed by phagocytes (Savill, J. et al. (1993) Immunol Today, 14: 131-136). Apoptosis triggers a distinct sequence of events characterized by the expression of phosphatidylserine on the cell surface, DNA fragmentation or laddering and the release of membrane-bound cell fragments called apoptotic blebs and bodies (Cohen, J. J.et. al, Annu Rev Immunol, 10:267-293, 1992.;Kerr J.F.R.et. al, Br J Cancer, 26:239, 1972.). Apoptotic cells and bodies are phagocytosed via various receptors that recognize pho, sphat idyl serine and other undefined ligands unique to the surface of apoptotic material (Savill, J. S.et. al, J Clin Invest, 83:865-875, 1989.;Fadok, V. A.et. al, J Immunol, 148:2207-2216, 1992.;Savill, J.et. al, Nature, 343:170-173, 1990.) In this way, apoptotic cells which contain potentially inflammatory factors are rapidly cleared by neighboring cells acting as semi-professional phagocytes or voracious experts of the macrophage line without inducing an inflammatory response (Fadok, V. A. et. al, J Clin Invest, 101:890-898, 1998.).

The process of apoptosis has been associated with a number of human diseases, including cancer, autoimmune diseases, various neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis, Huntingdon's disease and Alzheimer's disease, stroke, myocardial infarction and AIDS (Thompson, CB, Science 267, pp 1456-1462). Thus, much attention has been focused on elucidating the mechanism of apoptosis and the genes controlling it with a view to developing new therapeutic strategies for these diseases.

In addition, particular diseases have also been associated with an impairment of phagocytosis of apoptotic bodies. Examples of such diseases include autoimmune diseases such as systemic lupus erythematosus, (Herrmann, M.et. al, Arthritis Rheum, 41:1241-1250, 1998.), AIDS (Zocchi, M. R.et. al, AIDS, 11:1227-1235, 1997.), acute pulmonary infections (Cox, G.et. al, Am.J Respir.Cell Mol.Biol., 12:232-237, 1995.) and allergy (Ying, S.et. al, Proc Assoc Am Physicians, 109:42-50, 1997.). It is clear that modulation of phagocytosis of apoptotic cells by drugs is a promising strategy for future therapies.

A swift engulfment of apoptotic cells is observed in the hermaphrodite C. elegans and this nematode has provided a useful tool for study of the engulfment process. The C. elegans ced-6 has been identified as being involved in the engulfment of apoptotic cells by phagocytes, as described in the applicant's co-pending application, serial number WO 99/37770. Two human homologues of C. elegans ced-6 have also been identified (see also WO 99/37770). These are designated hlced-6 and h2-ced-6, h2ced-6 being a splice variant of hlced-6 and thought to be a dominant negative version thereof.

The present inventors have now identified a ced-6 homologue from the dipteran insect Drosophila melanogaster. The Drosophila CED-6 protein (herein denoted dCED-6 and being encoded by the Drosophila ced-6 gene also referred to herein as dced-6) and its encoding nucleic acid are useful for carrying out assays as described herein to identify compounds which are inhibitors or enhancers of signal transduction pathways which promote phagocytosis of apoptotic cells. Such compounds may be useful in the development of therapeutic agents for use in the treatment of some of the aforementioned diseases.

In a first aspect, the invention provides a nucleic acid molecule encoding a dCED-6 protein, said protein comprising the sequence of amino acids illustrated in Figure 2.

In a preferred embodiment, the nucleic acid molecule of the invention comprises the sequence of nucleotides from position 132 to 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence illustrated in Figure 1.

In accordance with the present invention, a defined nucleic acid includes not only nucleic acid molecules comprising the identical sequence of nucleotides but also any minor base variations, including in particular base substitutions which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degeneracy of the genetic code. The term "nucleic acid" or "nucleic acid molecule" includes single or double stranded RNA, single or double stranded DNA (encompassing both genomic DNA or cDNA and also recombinant DNA molecules), synthetic forms and mixed polymers, both sense and antisense strands.

Furthermore, the nucleic acid molecule of the invention may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases as will be readily appreciated by those skilled in the art. Possible modifications include, for example, the addition of isotopic or non-isotopic labels, substitution of one or more of the naturally occurring nucleotide bases with an analog, internucleoLide modifications such as uncharged is linkages (e.g. methyl phosphonates, phosphoamidates, carbamates, etc.) or charged linkages (e.g.

phosphorothioates, phosphorodithioates, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence, for example via hydrogen bonding.

Such molecules are known in the art and include, for example, so-called peptide nucleic acids (PNAs) in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

2S Libraries of Drosophila chromosomal or cDNA fragments may be screened as sources of the nucleic acids of the present invention. Alternatively, nucleic acid sequences according to the invention may be produced using recombinant or synthetic means, for example by PCR amplification of sequences resident in chromosomal DNA or cloned fragments thereof or by RT PCR amplification starting from total or poly A+ RNA, preferably isolated from a tissue which is known to express dCED-6. Generally such techniques are well known in the art (see Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labcratory Press; F. M. Ausubel et al. (eds.

Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)).

Also provided by the invention are nucleic acid molecules which are capable of hybridising to nucleic acid molecules according to the invention under conditions of high stringency. A nucleic acid molecule is "capable of hybridising" to another nucleic acid molecule, such as a fragment of DNA or an RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and ionic strength, which conditions would be well known to those skilled in the art (See Sambrook et al. or Ausubel et al., supra). Stringent temperature is conditions will generally include temperatures in excess of 30'C, typically in excess of 370C, and preferably in excess of 45'C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM and preferably less than 200 mM.

The nucleic acid capable of hybridising to a nucleic acid molecule according to the invention under high stringency conditions will generally be at least 80%, preferably at least 90% and more preferably at least 95% homologous to the nucleotide sequences according to the invention.

In a second aspect, the invention provides an isolated dCED-6 protein comprising the sequence of amino acids illustrated in Figure 2.

In a preferred embodiment, the isolated dCED-6 protein is encoded by a nucleic acid molecule according to the first aspect of the invention. Most preferably, the dCED-6 protein of the invention is encoded by a nucleic acid molecule comprising the 3S sequence of nucleotides; from position 132 to position 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence - 6 illustrated in Figure 1.

In accordance with the present invention, a defined dCED-6 protein or polypeptide includes proteins which are substantially homologous but have one or more amino acid changes, including naturally occurring allelic variants, or in vivo or in vitro chemical or biochemical modifications (e. g. acetylation, carboxylation, phosphorylation, glycosylation etc) which are conservative of biological function. In this context, a N'substantially homologous" sequence is regarded as a sequence which has at least 80 or 90% and more preferably at least 95% amino acid homology with the dCED-6 protein of the invention. The term "biological function" is defined herein to mean the ability to regulate or affect phagocytosis of apoptotic cells. Amino acid changes which are "conservative" are those which permit biological function to be retained although it may be less than or greater than the level of biological function of the wild-type Drosophila dCED-6 protein. The choice of amino acids for making conservative changes will be well-known to those skilled in the art.

The dCED-6 protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant.

The invention further provides functional fragments of the dCED-6 protein, the term "functional fragment" referring to an isolated sub-region, domain or fragment of a dCED-6 protein or a sequence of amino acids that, for example, comprises a functionally distinct region of the protein. The human and C. elegans CED-6 homologues have been shown to contain a phosphotyrosine domain and a serine/proline rich region. Accordingly, the invention provides isolated fragments which correspond to the equivalent regions of the Drosophila dCED-6, the boundaries of which may be determined based on an optimal alignment of the amino acid sequences of the CED-6 species homologues.

Functional fragments of the dCED-6 protein of the invention may be produced by recombinant DNA techniques or chemical synthesis or may be derived from a full-length dCED-6 protein, for example by chemical or enzymatic cleavage, recombinant techniques being the most preferred.

Also within the scope of the invention are fusion proteins /polypeptides comprising a dCED-6 protein according to the invention or a functional fragment thereof. The dCED-6 protein of the invention may be fused either N-terminally or C-terminally to heterologous protein or peptide fragments, for example an epitope tag to facilitate identification and/or purification of the fusion protein or the product of a reporter gene. Fusion proteins will typically be made by recombinant nucleic acid techniques or may be chemically synthesized. A number of vectors especially designed for the expression of recombinant fusion proteins in which an epitope tag is added to the N- or C-terminus of a protein of interest are available commercially.

In a further aspect, the invention provides an expression vector comprising a sequence of nucleotides which encodes a dCED-6 protein comprising the amino acid sequence illustrated in Figure 2 or an amino acid sequence which differs therefrom only in amino acid changes which are conservative of biological function.

In a preferred embodiment, the expression vector is one in which the sequence of nucleotides which encodes the dCED-6 protein comprises the sequence from position 132 to 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence illustrated in Figure 1.

As would be readily appreciated by one skilled in 8 the art, the expression vectors described above will comprise not only nucleic acid encoding the Drosophila dCED-6 protein, or a variant thereof having equivalent biological function, but also regulatory sequences operably linked to said nucleic acid, that are capable of effecting expression of the said nucleic acid. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

Regulatory sequences required to effect gene expression generally include promoter sequences to position RNA polymerase at the transcription start site and to direct an appropriate frequency of transcription initiation at this site and also translation initiation sequences for ribosome binding. As would be readily understood by one skilled in the art, the precise nature of the regulatory sequences required to effect expression of the dCED-6 protein will vary according to the nature of the host cell.

For expression in a prokaryotic host cell (e.g. the bacterium E.coli) the expression vector would include a promoter, such as the lac promoter, to drive transcription and for translation initiation the Shine-Dalgarno and a translation initiation codon (usually AUG). For expression in eukaryotic host cells, the expression vector may include a heterologous or homologous promoter region, preferably one which is recognised by RNA polymerase II and optionally one or more additional transcriptional regulatory elements (e.g. enhancer elements), also a terminator sequence and downstream polyadenylation signal, a start codon (usually AUG) and a termination codon for detachment of the ribosome. Such vectors may be obtained commercially or may be assembled from the elements described by methods well known in the art.

Examples of expression vectors according to the invention are plasmids, viral or phage vectors and also vectors based on mobile genetic elements. Such vectors will normally possess an origin of replication and one or more selectable markers, such as a gene for antibiotic resistance.

In a preferred embodiment, the expression vector of the invention is one which is suitable for driving expression of the dCED-6 protein, or a fragment thereof, in a dipteran insect such as Drosophila melanogaster. The most preferred expression vectors for this purpose are based on the Drosphila P elements, which are transposable DNA elements which occur naturally in the genome of P (paternally contributing) strains of Drosophila. Expression vectors derived from P elements are well known in the art (see, for example, R.W. Old and S.B. Primrose, Principles of Gene Manipulation, Blackwell Science, Ltd; Mockett, R.J. et al. (1999) FASEB J. 13: 173342), as are procedures for the construction of transgenic Drosophila using such vectors.

An expression vector according to the invention can be used to express the protein encoded therefrom in a suitable host cell or organism. Thus, in a further aspect, the invention provides a process for preparing a dCED-6 protein according to the invention which comprises cultivating a host cell, comprising an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the dCED-6 protein, and recovering the expressed dCED-6 protein. Procedures for incorporation of a cloned DNA into a suitable expression vector, introduction of the expression vector into a host cell, selection of transformed cells harboring the vector, culture of the host cells and recovery of the expressed protein are well known to those skilled in the art, as provided by Sambrook et al. (1989), Molecular Cloning: A Laborator_y Manual, Cold Spring Harbor Laboratory Press or F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In a further aspect, the invention also provides a non-human transgenic organism comprising a transgene capable of expressing a dCED-6 protein according to the invention.

The term "transgene capable of expressing" as used herein means a suit able nucleic acid sequence which leads to expression of a Drosophila dCED-6 protein or a protein having the same biological function, as described herein. The transgene, may include, for example, the complete dCED-6 cDNA is (illustrated in Figure 1) or the coding region thereof operably linked to a promoter region and optionally one or more additional transcription regulatory elements (e.g. enhancer elements). In one embodiment, the promoter (or promoter plus enhancer elements) may direct a tissue-specific or cell type-specific pattern of gene expression.

In a preferred embodiment, the transgenic organism is a dipteran insect, such as, for example, Drosophila melanogaster, and the transgene is present in a vector derived from a Drosophila P element.

In a further aspect, the invention provides an antibody directed against an epitope of a dCED-6 protein comprising the sequence of amino acids illustrated in Figure 2 or a functional fragment thereof. Antibodies to an epitope of dCED-6 can be prepared by techniques which are known in the art.

For example, polyclonal antibodies may be prepared by inoculating a host animal, such as a rabbit, with an immunogenic preparation comprising dCED-6 or a fragment thereof as the challenging antigen and recovering immune serum. Monoclonal antibodies may be prepared according to known techniques, first described by Kohler R. and Milstein C., Nature (1975) 256, 495-497, which would be well known to persons skilled in the relevant art.

In accordance with a still further aspect, the invention provides a method for determining whether a compound is an inhibitor or an enhancer of a signal transduction pathway which promotes phagocytosis of apoptotic cells, which method comprises determining the rate of uptake of apoptotic corpses by phagocytic cells of transgenic Drosophila comprising a transgene capable of expressing dCED-6 in at least said phagocytic cells in the presence or absence of the compound.

This first assay method of the invention may be conveniently referred to herein as the "dCED-6 overexpression assay".

The invention further provides a method for determining whether a compound is an inhibitor or an enhancer of a signal transduction pathway which promotes phagocytosis of apoptotic cells, which method comprises determining rate of uptake of apoptotic corpses by phagocytic cells of a mutant Drosophila having a deletion, disruption or loss-of-function mutation in the dced-6 gene in the presence or absence of the compound.

This second assay method of the invention may be conveniently referred to herein as the ''dCED-6 mutant assay".

The present inventors have observed that both overexpression of dCED-6 and disruption of the dced-6 gene results in an alteration in the ability of the macrophages in Drosophila embryos to phagocytose apoptotic corpses. This effect, which is detectable as a change in the "mean phagocytic index" of the embryos in the assay system of Franc et al., (1999) Science, 284: 1991-1994, forms the basis of both the dCED-6 overexpression assay and the dCED-6 mutant assay.

Both methods of the invention are preferably s carried out using embryonic mutant or transgenic Drosophila, as described in the Examples included herein. The phagocytic cells are preferably macrophages which are primarily responsible for clearance of apoptotic cells in Drosophila embryos.

The dCED-6 overexpression assay requires a transgenic Drosophila strain comprising a transgene capable of expressing dCED-6 in at least the phagocytic cells which will be examined to determine the rate of uptake of apoptotic corpses in the is presence or absence of the test compound. Accordingly, the "transgene capable of expressing a dCED-6 protein" should comprise nucleic: acid encoding a dCED-6 protein, as defined herein, operably linked to transcription regulatory sequences (typically a promoter region with optionally one or more enhancer elements) which are capable of directed gene expression in at least the said phagocytic cells. In a preferred embodiment, the nucleic acid encoding the dCED-6 protein will comprise the complete dCED-6 cDNA 2S (having the sequence of nucleotides illustrated in Figure 1) or the coding region thereof.

When, in accordance with the preferred embodiment of the invention, the assay is to be carried out using embryonic Drosophila, the transcription regulatory sequences should be capable of directing gene expression in at least the embryonic macrophages. Advantageously, the transcription regulatory sequences may comprise a "constitutive" or "housekeeping" type promoter which directs ubiquitous gene expression in all cell types. Alternatively, the promoter may be a tissue-specific or cell type-specific promoter which is capable of directing gene expression in cells of the macrophage lineage.

A transgenic Drosophila suitable for use in the dCED-6 overexpression method may be constructed in accordance with techniques known in the art.

The dCED-6 mutant assay requires a mutant Drosophila strain having a deletion, disruption or loss-of -function mutation in the dced-6 gene. Suitable strains may be deleted for a whole or a part of the dced-6 locus, such that the dCED-6 protein is no longer expressed or that the dCED-6 protein which is expressed is non-functional. Alternatively, the mutant strain may be disrupted in the c1ced-6 gene, typically by the insertion of a stretch of foreign DNA into the genome within the dced-6 locus, again resulting in no expression or expression of a nonfunctional dCED-6 protein. As exemplified herein, the dced-6 gene could be disrupted with the use of a P element. In a still further embodiment, the mutant strain may carry a loss-of -function mutation other than a disruption or a deletion which again results in no expression or, more typically, expression of a nonfunctional protein.

In preferred embodiments of the above-described assay methods of the invention the mutant or transgenic Drosophila strain, as appropriate is a mutant or transgenic strain of Drosophila melanogaster.

It will be appreciated that a wide variety of compounds can be tested to see whether they are inhibitors or enhancers of signal transduction pathways which promote phagocytosis of apoptotic cells. The compound may be of any chemical formula, a polymer or a monomer. The compound may be one of known biological or pharmacological activity, a known compound without such activity or a novel molecule such as might be present in a combinatorial library of compounds.

Any compound identified as an inhibitor or an enhancer of phagocytosis of apoptotic cells by the assays described above can be further tested to establish whether the effect is medicated through dCED-6. Such further testing can be carried out be determining the rate of uptake of apoptotic corpses by phagocytic cells of wild-type Drosophila in the presence or absence of the compound.

The invention will be further understood with reference to the following non-limiting experimental examples, together with the accompanying Figures, in which:

Figure 1 illustrates the nucleotide sequence of the Drosophila dCED-6 cDNA. The complete open reading frame, from position 132-1682, is underlined.

Figure 2 illustrates the amino acid sequence of the Drosophila dCED-6 protein.

Example 1-Evaluation of the role of ced-6 in phagocytosis of apoptotic cells in Drosophila embryos.

In Drosophila embryos, the clearance of apoptotic cells is primarily mediated by macrophages. In order to evaluate the role of dCED-6 in this process, mutant Drosophila embryos were engineered in which dCED-6 expression was either increased, i.e. by overexpression, or abrogated, i.e by disruption of the Drosophila ced-6 gene.

Gene disruption of the Drosophila CED-6 gene was - is - carried out by P-element insertion, as previously described (Ballinger and Benzer, 1989, Proc Natl Acad USA, 86:9402-9406; Spradling et al., 1999, Genetics 153:135-177). Both polymerase chain reaction (PCR) on single embryos and immunostaining can be used to confirm the success of gene disruption in the mutant strains.

Overexpression of the dCED-6 cDNA was mainly done using previously described methods using a vector derived from a Drosophila P element (Rubin and Spradling, 1982, Science 218:348-353; Mockett et al., 1999, Arch. Biochem Biophys. 371:260-269; Mockett et al., 1999, FASEB J 13:1733-1742; Minowanda et al.,1999, Development 20:4465-4475).

The efficiency of engulfment in the mutant and overexpressing Drosophila embryos was quantified by counting the number of engulfed corpses per macrophage in at least five fields of four embryos under Nomarsky microscope, according to the procedure described in Franc, NC, Heitzler, P, Ezekowitz, AB, White K (1999) Requirement for Croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284:1991-1994. Using this procedure, a phagocytic index that is the mean number of engulfed corpses per macrophage can be calculated for each embryo.

Both disruption of the dced-6 gene and overexpression of dCED-6 clearly resulted in an altered phagocytic index, as compared to wild-type Drosophila embryos. This indicates that the dCED-6 gene has a function in the phagocytosis of apoptotic particles in D-rosophila, confirming that CED-6 has a similar function in Drosophila as in C. elegans (Liu et al, 1999, W099/37770).

Example 2-Assays to screen for compounds that alter the phagocytosis of apolptotic particles in Drosophila.

Procedures for the detection and measurement of the level of phagocytosis of apoptotic particles in Di-osophila are described by Franc et al., 1999, Science 284:1991-1994. As described therein, phagocytosis of apoptotic corpses is best observed in Drosophila embryos. To select for compounds that alter the phagocytic properties in Drosophila, and more particularly to detect compounds that reduce or enhance the phagocytic properties in Drosophila associated with dCED-6, the following assay method was developed. Wild-type Drosophila, Drosophila mutants disrupted in the ced-6 locus and Drosophila transgenics overexpressing dCED-6 (these disrupted and overexpressing strains having been prepared as described in Example 1) were subjected to compounds in the early embryonic stage for various time intervals.

The embryos were then allowed to further develop, either in the compound or after a shorter contact with the compound. At the appropriate developmental stage, the embryos were prepared for microscopy (as described by Franc et al., ibid.). A mean phagocytic index was determined for every compound for the wild-type, mutant and transgenic Drosophila. This allowed selection of compounds that either enhance or inhibit the phagocytosi4s of apoptotic particles. Moreover, due to the presence of dced-6 mutants and transgenes, this assay method allows selection for compounds that interact specifically with dCED-6 or that act in the dCED-6 pathway.

Claims (19)

Claims:

1. A nucleic acid molecule encoding a dCED-6 protein, said protein comprising the sequence of amino acids illustrated in Figure 2.

2. A nucleic acid molecule according to claim 1 which comprises the sequence of nucleotides from position 132 to 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence illustrated in Figure 1.

3. A nucleic acid molecule which is capable of hybridising to the nucleic acid molecule of claim 1 or claim 2 under conditions of high stringency.

4. A nucleic acid molecule as claimed in any one of claims 1 to 3 which is a DNA molecule.

5. An isolated protein comprising the sequence of amino acids illustrated in Figure 2.

6. An isolated dCED-6 protein which is encoded by a nucleic acid molecule as defined in any one of claims 1 to 3.

7. An expression vector comprising a sequence of nucleotides which encodes a dCED-6 protein comprising the amino acid seque nce illustrated in Figure 2 or an amino acid sequence which differs therefrom only in amino acid changes which are conservative of biological function.

8. An expression vector as claimed in claim 7 wherein the sequence of nucleotides encoding a dCED-6 protein comprises the sequence of nucleotides from position 132 to position 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence illustrated in Figure 1.

9. A host cell comprising the expression vector of claim 7 or claim 8.

10. A non-human transgenic organism comprising a transgene capable of expressing a dCED-6 protein according to claim 5 or claim 6.

11. A transgenic organism as claimed in claim 10 which is a dipteran insect.

12. A transgenic organism as claimed in claim 11 which is of the genus Drosophila.

13. A transgenic organism as claimed in claim 12 wherein the transgene is present in a vector derived from a Drosophila P element.

14. A transgenic organism as claimed in any one of claims 10 to 13 wherein the transgene comprises the sequence of nucleotides from position 132 to position 1682 of the nucleic acid sequence illustrated in Figure 1 or the complete nucleic acid sequence illustrated in Figure 1.

15. An antibody directed against an epitope of a dCED-6 protein comprising the sequence of amino acids illustrated in Figure 2 or a functional fragment thereof.

16. An antibody as claimed in claim 15 which is a monoclonal antibody.

17. A method for determining whether a compound is an inhibitor or an enhancer of a signal - 19 transduction pathway which promotes phagocytosis of apoptotic cells, which method comprises determining the rate of uptake of apoptotic corpses by phagocytic cells of transgenic Drosophila comprising a transgene capable of expressing dCED-6 in at least said phagocytic cells in the presence or absence of the compound.

18. A method for determining whether a compound is an inhibitor or an enhancer of a signal transduction pathway which promotes phagocytosis of apoptotic cells, which method comprises determining rate of uptake of apoptotic corpses by phagocytic cells of a mutant Drosophila having a deletion, disruption or loss-of -function mutation in the dced-6 gene in the presence or absence of the compound.

19. A method as claimed in claim 17 or claim 18 wherein the phagocytic cells are macrophages.