US20030149995A1

US20030149995A1 - Compound screening methods

Info

Publication number: US20030149995A1
Application number: US10/371,101
Authority: US
Inventors: Richard Zwaal; Jose Groenen; Thierry Bogaert
Original assignee: Devgen NV
Current assignee: Devgen NV
Priority date: 1999-04-15
Filing date: 2003-02-21
Publication date: 2003-08-07
Also published as: US6540996B1

Abstract

The invention provides methods of screening for compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA), using the nematode worm C. elegans.

Description

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 09/549,872 filed Apr. 14, 2000, now pending. This application claims priority under Title 35 U.S.C. §119(e), of U.S. provisional application No. 60/129,596, filed Apr. 15, 1999, entitled COMPOUND SCREENING METHODS, and foreign priority benefits under Title 35, U.S.C., §119(a)-(d) or §365(a),(b) of foreign patent application. Nos. GB 9908670.4, filed Apr. 15, 1999, & GB 9912736.7, filed Jun. 1, 1999, the entire contents of which are incorporated herein by reference.[0001]

SPECIFICATION

The invention is concerned with methods for use in the identification of compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca ²+ATPase (SERCA).

In most animal cells and plant cells, the normal concentration of free cytosolic Ca ²⁺ is 50 to 100 nM. Since Ca²⁺ acts as a major intracellular messenger, elevating these levels affects a wide range of cellular processes including contraction, secretion and cell cycling (Dawson, 1990, Essays Biochem. 25:1-37; Evans et al., 1991, J. Exp. Botany 42:285-303). Intracellular Ca²⁺ stores hold a key position in the intracellular signalling. They allow the rapid establishment of Ca²⁺ gradients, and accumulate and release Ca²⁺ in order to control cytosolic Ca²⁺ levels. Moreover, lumenal Ca²⁺ intervenes in the regulation of the synthesis, folding and sorting of proteins in the endoplasmic reticulum (Brostrom and Brostrom, 1990, Ann. Rev. Physiol. 52:577-590; Suzuki et al., 1991, J. Cell. Biol. 114:189-205; Wileman et al., 1991, J. Biol. Chem. 266:4500-4507). Furthermore it controls signal-mediated and passive diffusion through the nuclear pore (Greber and Gerace, 1995, J. Cell. Biol. 128:5-14).

Three genes that code for five different isoforms of the sarco/endoplasmic reticulum Ca ²⁺ ATPase (SERCA) are known in vertebrates, SERCA1a/b, SERCA2a/b and SERCA3. The SERCA isoforms are usually tagged to the endoplasmic reticulum (ER) or ER subdomains like the sarcoplasmic reticulum, although the precise subcellular location is often not known. The SERCA proteins belong to the group of ATP-driven ion-motive ATPases, which also includes, amongst others, the plasma membrane Ca²⁺-transport ATPases (PMCA), the Na+-K+-ATPases, and the gastric H+-K+-ATPases. The SERCA Ca²⁺-transport ATPases can be distinguished from their plasma membrane counterparts like PMCA by the specific SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)-1,4-benzohydroquinone (Thastrup et al., 1990, PNAS 87:2466-2477; Seidler et al., 1989, J. Biol. Chem. 264:17816-17823; Oldershaw and Taylor, 1990, FEBS Lett. 274:214-216). In view of the diverse role of Ca²⁺ in the cell and the fact that Ca²⁺ is stored in diverse organelles, the diversity in Ca²⁺-accumulation pump isoforms is not surprising.

SERCA1 is only expressed in fast-twitch skeletal muscle fibres. The gene encodes two different isoforms; SERCA1b which is the neonatal isoform and SERCA1a the adult isoform (Brandl et al., 1986, Cell 44:597-607; Brandl et al., 1987, J. Biol. Chem. 262:3768-3774). The difference between the two isoforms is the result of an alternative splice. As a consequence, the neonatal isoform contains a highly charged carboxyl-terminal extension (Korczak et al., 1988, J. Biol. Chem. 263:4813-4819). The reason for this alternative splicing is as yet unknown; the functional significance of this extension is not yet clear. When expressed in COS cells, SERCA1a and SERCA1b exhibit nearly identical maximal Ca ²⁺-turnover rate, Ca²⁺-affinity and ATP-dependency of Ca²⁺ transport (Maruyama and MacLennan, 1988, PNAS 85:3314-3318). The human SERCA1 gene is mapped on chromosome 16P12.1 and is about 26 kb long (MacLennan et al., 1987, Somatic Cell Mol. Genet. 13:341-346; Callen et al., 1991, Am. J. Hum. Genet. 49:1372-1377).

SERCA2 is expressed in muscle and non-muscle cells. The human SERCA2 gene maps to chromosome 12q23-q24.1 (Otsu et al., 1993, Genomics 17:507-509). Partial sequence analysis suggests that the same exon/intron layout is conserved between SERCA1 and SERCA2. mRNA of SERCA2 can be divided in 4 different classes; class 1 encodes SERCA2a and is mainly expressed in muscle, the other classes encode SERCA2b and are mainly expressed in non-muscle tissues. SERCA2b harbors a 49 amino acid extension, which contains a highly hydrophobic stretch. As with SERCA1, no functional difference can be measured between the two SERCA2 isoforms when expressed in COS cells (Campbell, 1991, J. Biol. Chem. 266:16050-16055). However, differences in Ca²⁺ affinity and turnover rate of the phosphoprotein intermediate have been observed (Lytton et al., 1992, 267:14483-14489; Verboomen et al., 1992, J. Biochem. 286:591-596). Both isoforms are expressed in a tissue-dependent pattern, both qualitatively and quantitatively (Eggermont et al., 1990, J. Biochem. 271:649-653). Cardiac muscle expresses 5- to 20-fold higher levels of SERCA2 than smooth muscle. Slow-twitch skeletal and cardiac muscle only express SERCA2a, while SERCA2b (referred to as the “housekeeping” isoform) is expressed in all non-muscle tissue, and represents about 75% of the Ca²⁺-transporting ATPase activity in smooth-muscle tissue. Different protein-to-message ratios for SERCA2a and SERCA2b have been observed. Cardiac muscle expresses 70 times more protein and only 7 times more SERCA2a mRNA compared to stomach smooth muscle which expresses SERCA2b (Khan et al., 1990, J. Biochem. 268:415-419).

SERCA3 is considered to be the non-muscle SERCA isoform. SERCA3 lacks the putative interacting domain for phospholamban, and hence, does not respond to this modulator (Toyofuku et al., 1993, J. Biol. Chem. 268:2809-2815). When expressed in COS cells, SERCA3 shows approximately 5-fold lower activity for Ca ²⁺ and a slightly higher pH optimum (Toyofuku et al., 1992, J. Biol. Chem. 267:14490-14496). In platelets, mast cells and lymphoid cells SERCA3 is co-expressed with SERCA2b (Wuytack et al., 1994, J. Biol. Chem. 269:1410-1416; Wuytack et al., 1995, Bioscience Rep. 15:299-306). Expression has also been observed in some arterial endothelial cells, in early developing rat heart, in some secretory epithelial cells of endodermal origin and in cerebellar Purkinje neurons.

In slow-twitch skeletal muscle, cardiac muscle and smooth-muscle tissues, SERCA2 activity is modulated by phosphorylation of the regulatory protein phospholamban (PLB) (see Fuji et al., 1991, FEBS Lett. 273:232-234). In cardiac muscle, in vivo phosphorylation of PLB by cAMP- or Ca ²⁺/Calmodulin-dependent protein kinase has a positive effect on the Ca²⁺ transport (Le Peuch et al., 1997, Biochemistry 18:5150-5157; Tada et al., 1979, J. Biol. Chem. 254: 319-326; Davis et al., 1983, J. Biol. Chem. 258:13587-13591; Wegener et al., 1989, J. Biol. Chem. 264:11468-11474). In order to determine the exact in vivo role of phospholamban, PLB-deficient mice have been generated (Luo et al., 1994, Circ. Res. 75:401-409). A marked effect is observed on Ca²⁺ uptake, whereas no effect is measured in Vmax. The ablation of the PLB gene in mice is associated with increased myocardial contractility, and a loss of the positive inotropic response to δ-adrenergic stimulation. The precise molecular mechanism underlying the modulation of SERCA by PLB is not apparent. An electrostatic mechanism has been proposed, as a direct interaction between PLB and SERCA, in which the unphosphorylated PLB inhibits the SERCA pump (Kirchberger et al., 1986, Biochemistry 25:5484-5492; Chiesi and Schwaller, 1989, FEBS Lett. 244:241-244; Xu and Kirchberger, 1989, J. Biol. Chem. 264:16644-16651). Alternatively, PLB and the SERCA Ca²⁺ pump are able to interact and phosphorylation of PLB alters its properties, as confirmed by cross-linking experiments (James et al., 1989, Nature 342:90-92). In some experiments, inhibitory effects of PLB have been observed on co-transfection of PLB and SERCA2a in COS-1 cells (Fuji et al. 1990, FEBS Lett. 273:232-234). Several models have been proposed to explain the regulatory effect of PLB on Ca²⁺ ATPases. These include the aggregation of SERCA2 around a pentameric form of PLB (Voss et al., 1994, Biophys J. 67:190-196). Another explanation starts from the electrostatic inhibition of Ca²⁺ binding due to the SERCA-PLB interaction (Toyofuku et al., 1994, J. Biol. Chem. 269:3088-3094). The interaction between PLB and SERCA2a has been studied in more detail, revealing a putative PLB binding domain that is also present SERCA1 but not in SERCA3. This has been further confirmed by expression studies in COS-1 cells (Toyofuku et al., 1993, J. Biol. Chem., 268:2809-2815). Such a finding is remarkable as SERCA1 and PLB are never co-expressed in vivo.

Direct phosphorylation of SERCA by Ca ²⁺/CaM kinase II results in a 2-fold higher maximal velocity Xu and Kirchberger, 1989, J. Biol. Chem. 264: 16644-16651. This CaM kinase phosphorylation is specific for SERCA2 and may act synergistically with the phosphorylation of phospholamban.

Sarcolipin (SLN) is a peptide of 33 amino acids in length that co-purifies with SERCA1. The human gene encoding SLN was mapped to chromosome 11q22-q23. The protein sequence shows some homology to phospholamban, especially in the lumenal part of the protein. In rabbits SLN is highly expressed in fast-twitch skeletal muscle, as is SERCA1 (Odermatt et al., 1997, Genomics 45:741-553). In co-expression studies in HEK-293 T-cells, a decrease of SERCA1 affinity for Ca ²⁺ was observed, but maximal Ca²⁺ uptake rates were stimulated. Mutational analysis provided evidence for different mechanisms of interaction of both SLN and PLB with the SERCA molecules (Odermatt, et al., 1998, J. Biol. Chem. 273:12360-12369).

SERCA plays an important role in regulating Ca ²⁺ levels, and hence in pathologies related to abnormal Ca²⁺ concentrations and regulation. For instance, abnormal cytosolic free Ca²⁺ levels are involved in different muscle pathologies (Morgan, 1991, N. Engl. J. Med. 325:625-632; Perreault et al., 1993, Circulation 87 Suppl. VII:31-37). Other major pathologies in which SERCA may play a role include cardiac hypertrophy, heart failure, and hypertension (Arai et al., 1994, Circ. Res. 74:555-564; Lompré et al., 1994, J. Mol. Cell. Cardiol. 26:1109-1121).

Cardiac hypertrophy is an adaptive response of the cardiac muscle to a hemodynamic overload, in which diastolic dysfunction is one of the earliest signs of pathological hypertrophic response. In animal models, where most studies are performed, a highly significant positive correlation has been obtained between end-diastolic cytosolic Ca ²⁺ levels and diastolic relaxation abnormalities. After aortic binding, SERCA2 mRNA and protein levels are decreased, as is the sarcoplasmic reticulum Ca²⁺ uptake (Komuro et al., 1989, J. Clin Invest. 83:1102-1108; de la Bastie et al., 1990, Circ. Res. 66:554-564). This effect was only found in cases of severe hypertrophy, and was only observed when heart failure occurs. In moderate hypertropy and in cases of compensated hypertrophy no changes in the level of SERCA mRNA were observed (de la Bastie et al, ibid; Feldman et al., 1993, Circ. Res. 73:184-192).

In humans, most studies report a decrease in SERCA2 mRNA, SERCA2 protein levels and decreased Ca ²⁺ uptake in a failing heart (Arai et al., 1993, Circ. Res. 72:463-469; Hasenfuss et al., 1994, Circ. Res., 75: 434-442). The decreased levels of SERCA2 expression are accompanied by decreased expression of phospholamban, cardiac ryanodine receptor and dihydropyridine receptor (Vatner et al., 1994, Circulation 90:1423-1430; Go et al., 1995, J. Clin. Invest. 95:888-894; Takahashi et al., 1992, J. Clin. Invest. 90:927-935). These human heart failure data are confirmed in different animal models. In a hypertrophic animals, SERCA2 expression levels are decreased; in a dilated strain Ca²⁺ uptake is decreased with increasing age (Kuo et al., 1992, Biochem Biophys acta 1138:343-349; Whitmer et al., 1988, Circ. Res. 62:81-85). Most striking in both humans and animal models is the strong positive correlation between SERCA2 and phospholamban mRNA levels. Examples in literature that do not confirm these data are most likely the result of various pathogenic mechanisms that can lead to heart failure.

Blood vessels from hypertensive animals have an increased wall thickness and show altered contractile properties. Several lines of evidence indicate that diminished Ca ²⁺ pump activities might contribute to elevation in cytoplasmic Ca²⁺ levels in hypertension. However, increased expression of SERCA2 has also been observed. Further study is required to resolve these contradictory results.

Darier-White disease is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. In several patients mutations have been found in SERCA2, demonstrating the role of SERCA and Ca ²⁺-signalling pathway in the regulation of cell-to-cell adhesion and differentiation of the epidermis (Sakuntabhai et al., 1999, Nature Genetics 21:271-277).

Although little is known about the involvement of SERCA in skeletal muscle disorders, deficiency in the Ca ²⁺-transport ATPase activity has been found in Brodys disease (Benders et al., 1994, J. Clin. Res. 94:741-748). The disorder is characterized by exercise-induced impairment of muscle relaxation. Normal levels of SERCA1 protein were detected, but the SERCA activity was decreased by about 50% in patients suffering from the disease. In other research, SERCA1 in fast-twitch fibers of Brody patients could not be detected immunologically (Danon et al., 1988, Neurology 38:812-815). However, three Brody patients show no defects in their SERCA1 gene, indicating pleiotropic mechanisms underlying Brody disease (Zhang et al., 1995, Genomics 30:415-424).

The underlying mechanism of non-insulin-dependent diabetes mellitus (NIDDM) is still unknown. In islets of Lagerhans from db/db mice (a NIDDM model), glucose-induced initial induction and subsequent oscillations of intracellular Ca ²⁺ concentrations were absent. Further analysis showed that SERCA3 was almost entirely lacking from the db/db islets. These results and thapsigargin experiments implicate SERCA3 in the defective insulin secretion associated with NIDDM (Roe et al., 1994, J. Biol. Chem. 269:18279-28282). A significant reduction of SERCA3 expression was also found in Goto-Kakizaki rats, a non-obese model of NIDDM (Varadi et al., 1996, J. Biochem. 319:521-527) Interactions have been reported between different SERCAs (SERCA1 and SERCA2) and different Insulin Receptor Substrates (IRS-1 and IRS-2). This interaction was dependant on insulin (Algenstaedt et al., 1997, J. Biol. Chem. 272:23696-23702). Inactivation of IRS-2 has recently been shown to resemble certain aspects of type 2 diabetes (Withers et al., 1998, Nature 391:900-904).

In mammals, there are three genes encoding different SERCA isoforms. In contrast, the nematode worm Caenorhabditis elegans (C. elegans) has only a single homologue of the mammalian SERCA protein, which was identified by the C. elegans genome-sequencing consortium (see Science issue 282, 1998). The C. elegans SERCA gene is located on chromosome III on a cosmid named K11D9. On a physical level, the gene consists of six exons that span an Open Reading Frame of 3.2 kb, resulting in a predicted protein of 1059 amino acids. The consensus alternative splice site that is present in the C-terminal end of mammalian SERCA genes is present in the worm as well. This leads to a second isoform consisting of 7 exons that span an ORF of 3.0 kb, resulting in a protein of 1004 amino acids. This may indicate a functional conservation of this domain of the protein, e.g. in regulating the activity of the SERCA pump.

C. elegans is a small roundworm that has a life span of only three days, allowing rapid accumulation of large quantities of individual worms. The cell-lineage is fixed, allowing identification of each cell which has the same position and developmental potential in each individual animal. C. elegans is extremely amenable to genetic approaches and a large collection of mutants have been isolated that are defective in embryonic development, behaviour, morphology, neurobiology etc. There is also a large cosmid collection covering almost the whole C. elegans genome, which is used to determine the complete genomic sequence of the worm.

These characteristics of C. elegans make it the organism of choice for use as a tool in the drug discovery process. In particular, C. elegans may be used in the development of high throughput live animal compound screens, useful in the development of potential candidate drugs, in which worms are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded. The present inventors have developed a number of C. elegans-based screening methods which may be used to identify compounds which modulate the activity of SERCA, either directly or via the SERCA/PLB interaction. Compounds identified as modulators of SERCA activity using these screening methods may be useful as pharmaceuticals in the treatment of the wide range of diseases with which the SERCA genes have been associated.

Accordingly, in a first aspect the invention provides a method of identifying compounds which are capable of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:

contacting C. elegans which exhibit reduced SERCA ATPase activity compared to wild type C. elegans in one or more cell types or tissues with a compound under test; and

detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity in the one or more cell types or tissues which exhibit reduced SERCA activity in the absence of the compound.

The method of the invention, which will be hereinafter referred to as the ‘up-regulation assay’ is performed using a C. elegans strain which exhibits reduced SERCA ATPase activity in one or more cell types or tissues, as compared to the SERCA ATPase activity in wild-type C. elegans. It has been observed that worms which exhibit reduced SERCA activity compared to wild-type worms manifest a variety of phenotypic and behavioural defects. The basis of the up-regulation assay is therefore to take worms which exhibit defects due to reduced SERCA activity, contact these worms with the compound under test and screen for phenotypic, behavioural or biochemical changes indicating a reversion towards wild-type SERCA activity. For example, worms with reduced SERCA activity often show a reduction in the rate of pharynx pumping. In this case, screening for an increase in the rate of pharynx pumping in the presence of a test compound would indicate a reversion towards wild-type SERCA activity due to the ability of the compound to enhance or up-regulate SERCA. For comparison purposes, an example of a C. elegans strain which exhibits ‘wild-type’ SERCA activity is the N2 strain (this strain can be obtained from CGC, University of Minnesota, USA). The N2 strain has been particularly well characterised in the literature with respect to properties such as pharynx pumping rate, growth rate and egg laying capacity (see Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern biological analysis of an organism, ed. by Henry F. Epstein and Diane C. Shakes, 1995 Academic Press; The nematode Caenorhabditis elegans, ed. by William Wood and the community of C. elegans researchers., 1988, Cold Spring Harbor Laboratory Press; C. elegans II, ed. by Donald L. Riddle, Thomas Blumenthal, Barbara J. Meyer and James R. Priess, 1997, Cold Spring Harbor Laboratory Press.).

C. elegans which exhibit reduced SERCA activity in one or more cell types or tissues can be obtained in several different ways. In a first embodiment, worms with reduced SERCA activity are obtained by treating a culture of worms with a chemical inhibitor of SERCA such as, for example, thapsigargin. As will be demonstrated in the examples given herein, treatment of C. elegans with thapsigargin results in recognisable phenotypic and behavioural changes such as paleness, reduced growth, pharynx pumping defects and production of very few progeny which are sick and grow very slowly. Accordingly, reversion of any one of these characteristics towards wild-type can provide an indication of a reversion towards wild-type SERCA activity.

In another embodiment, worms with reduced SERCA activity can be produced by specifically down-regulating the expression of SERCA in one or more tissues using antisense techniques or double stranded RNA inhibition. This can be achieved by transfection of C. elegans with a vector that expresses either an antisense C. elegans SERCA RNA or double stranded C. elegans SERCA RNA. Specific down-regulation of SERCA expression in different cell types or tissues of the worms can be achieved by incorporating into the vector an appropriate tissue-specific promoter to drive expression of the antisense RNA or double stranded RNA in the required tissues. SERCA expression will be specifically down-regulated only in those tissues which express the antisense RNA or double stranded RNA. By way of example, the promoter region of the C. elegans SERCA gene itself (see the examples given below) can be used to direct expression of an antisense RNA or double stranded RNA in all the cells and tissues which express SERCA. The C. elegans myo-2 promoter can be used to direct expression in the pharynx. The C. elegans myo-3 promoter can be used to direct expression in the body wall muscles. The use of antisense and double stranded RNA inhibition will be further understood with reference to the Examples included herein.

Alternative RNAi techniques which may be used to inhibit SERCA activity are described in the applicant's co-pending International patent application No. WO 00/01846. These techniques, which are based on delivery of dsRNA to C. elegans by feeding with an appropriate dsRNA or feeding with food organisms which express an appropriate dsRNA, may lead to a more stable RNAi phenotype than results from injection of dsRNA.

In a still further embodiment, the C. elegans exhibiting reduced SERCA ATPase activity in one or more cell types or tissues may be a mutant strain in which SERCA activity is reduced but not eliminated i.e. a reduction-of-function mutant. The mutation may give rise to reduced SERCA activity through a down-regulation of SERCA expression in one or more cell types or tissues or through a defect in the SERCA protein itself or a defect in regulation of the activity of the SERCA protein.

A reduction-of-function mutant or a knock-out mutant can be isolated using a classical non-complementation screen, starting with a heterozygote C. elegans strain carrying a mutant SERCA allele on one chromosome and a recessive marker close to the wild-type SERCA allele on the other chromosome. The worms are subjected to mutagenesis using standard techniques (EMS or UV-TMP are suitable for this purpose) and the progeny is screened by eye for defects, especially in tissues which express SERCA. Since the screening is performed in the F1 generation, mutations will only give rise to a phenotype if the mutation occurs in the SERCA gene (due to non-complementation) or if the mutation is dominant, which does not occur frequently. These two possibilities can be distinguished in subsequent generations. A newly introduced SERCA mutation should be linked to the recessive marker. As a further control, DNA sequencing can be performed to determine the nature of the mutation.

The step of ‘detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity’ may be performed in several different ways. The method of choice is generally dependent upon the phenotype/behavioural characteristics of the starting worm strain, which is in turn generally dependent upon the nature of the cell types or tissues in which SERCA activity is reduced.

Inhibition experiments, for example the RNAi experiments and thapsigargin experiments described herein, demonstrate that SERCA is a vital protein for C. elegans. Moreover, reduction of SERCA activity results in a variety of phenotypes that can be used as basis of an assay to isolate compounds that alter the activity of SERCA. The main defects, and hence phenotypes, associated with reduced SERCA activity are related to muscle function e.g pharyngeal muscle, body wall muscle, vulva muscle, anal repressor muscle, and anal sphincter muscle. Screens based on reversion of defects in these muscles to wild-type can be used to identify compounds and genes that alter the activity of SERCA. Moreover, other phenotypes, such as paleness, reduced growth, reduced progeny, protruding vulva and protruding rectum can be used to identify compounds and genes that alter the function of SERCA.

In one embodiment, particularly suitable for use when the starting worm strain exhibits defects in pharynx pumping due to reduced SERCA activity in the pharynx (as compared to wild-type C. elegans) the up-regulation assay can be based on detection of changes in the pharynx pumping efficiency. If the starting worm strain exhibits a reduced rate of pharynx pumping due to reduced SERCA activity in the pharynx, then an increase in the rate of pharynx pumping in the presence of a test compound can be used as an indicator of a reversion towards wild-type SERCA activity in the pharynx.

C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and internalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up of liquid and subsequently spitting it out, requiring contraction and relaxation of muscles, is called pharyngeal pumping or pharynx pumping.

Alterations in SERCA activity influence the pharyngeal pumping rate. In particular, inhibition of SERCA using thapsigargin causes a reduction in the rate of pharynx pumping. Measurement of the pumping rate of the C. elegans pharynx is hence a method to determine the activity of SERCA. The pharynx pumping efficiency can be conveniently measured by placing the nematodes in liquid containing a fluorescent marker molecule precursor, such as calcein-AM. Calcein-AM present in the medium is taken up by the nematodes and the AM moiety is cleaved off by the action of esterases present in the C. elegans gut, resulting in the production of the fluorescent molecule calcein. As the quantity of calcein-AM that is delivered in the gut is dependent of the pumping rate of the pharynx, and hence of the activity of SERCA, the fluorescence measured in the gut of the formed calcein is a quantitative and qualitative measurement of the SERCA activity. It would be readily apparent to one skilled in the art that other types of marker molecule precursor which are cleavable by an enzyme present in the gut of C. elegans to generate a detectable marker molecule could be used instead of calcein-AM with equivalent effect.

In further embodiments, particularly suitable for use when the starting worm strain exhibits reduced SERCA activity in the vulva muscles, the up-regulation assay can be based on detection of changes in the egg laying behaviour of the C. elegans or on detecting changes in the amount of progeny produced by the C. elegans.

Defects associated with reduced SERCA activity in the vulva muscles include defects in the production and laying of eggs and hence a reduction in the number of progeny produced. Typically, worms with reduced SERCA expression in the vulva are not able to lay their eggs. The eggs thus hatch inside the mother, which then dies. These mothers are easy to recognize under the dissection microscope. As a consequence of the egg laying defect, these worms produce less progeny, and hence the culture as a whole grows much more slowly. Defects associated with reduced SERCA activity have also been observed in the gonad, including the sheath cells and the spermatheca. These defects also result in reduced egg formation and hence a reduced egg laying phenotype.

One convenient way in which the egg production and egg laying behaviour of the worms can be monitored is by counting the number of resultant offspring produced. A variety of different techniques can be used for this purpose. For example, the offspring can be measured directly using the growth rate assay and/or the movement assay described below. Alternatively, specific antibodies and fluorescent antibodies can be used to detect the offspring. Any specific antibody that only recognizes eggs, or L1 or L2 or L3 or L4 stage worms, will only recognize offspring, such a specific antibody that recognizes an antigen on the L1 surface has been described by Donkin and Politz, W13G 10(2):71. Finally, the number of eggs or offspring in each well can be counted directly using a FANS device. The FANS device is a ‘worm dispenser apparatus’ having properties analogous to flow cytometers such as fluorescence activated cell scanning and sorting devices (FACS) and is commercially available from Union Biometrica, Inc, Somerville, Mass., USA. The FANS device, also designated a nematode flow meter, can be the nematode FACS analogue, described as fluorescence activated nematode scanning and sorting device (FANS). The FANS device enables the measurement of nematode properties, such as size, optical density, fluorescence, and luminescence and the sorting of worms based on these properties.

In a still further embodiment, particularly suitable for use when the starting worm strain exhibits reduced SERCA activity in the anal sphincter or the anal repressor, the up-regulation assay can be based on detection of a change in the defecation behaviour of the C. elegans.

A reduction in the SERCA activity in the anal sphincter and/or the anal repressor, for example following treatment with thapsigargin, results in worms which are constipated and also in worms with a protruding rectum. Changes in the defecation rate of the worms can therefore also serve as an indicator of SERCA activity.

Defecation rate can be measured using an assay similar to that described above for the measurement of pharynx pumping efficiency, but using a marker molecule which is sensitive to pH. A suitable marker is the fluorescent marker BCECF. This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to worms growing in liquid medium the worms will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF. BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH<6) the compound exhibits no or very low fluorescence. As a result of the defecation process the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent. The level of BCECF fluorescence in the medium (measured using a fluorimeter on settings Ex/Em=485/550) is therefore an indicator of the rate of defecation of the nematodes.

Defecation can also be measured using a method based on the luminescent features of the chelation of terbium by aspirin. The method requires two pre-loading steps, first the wells of a multi-well plate are pre-loaded with aspirin (prior to the addition of the nematode worms) and second, bacteria or other nematode food source particles are pre-loaded with terbium using standard techniques known in the art. C. elegans are then placed in the wells pre-loaded with aspirin and are fed with the bacteria pre-loaded with terbium.

The terbium present in the pre-loaded bacteria added to the wells will result in a low level of background luminescence. When the bacteria are eaten by the nematodes the bacterial contents will be digested but the terbium will be defecated back into the medium. The free terbium will then be chelated by the aspirin which was pre-loaded into the wells resulting in measurable luminescence. The luminescence thus observed is therefore an indicator of nematode defecation.

It has been observed that a reduction in SERCA activity, for example using inhibition by thapsigargin or double stranded RNA inhibition, results in a reduction in the growth rate of a C. elegans culture. Growth rate of the culture as a whole is reduced because the worms produce fewer progeny and also because the few progeny that are produced show poor/delayed growth. Cultures of worms which produce many healthy progeny grow faster than cultures of worms with few and/or sick progeny. Hence measurement of the growth rate of a culture of C. elegans is in indication of the activity of SERCA in the individual worms of the culture.

Growth rate can be monitored by measuring the number of eggs or the number offspring present in the culture, by measuring the total fluorescence in the culture (this can be autoflourescence, or fluorescence caused by a transgene encoding a flourescent or luminescent protein), but can also be measured using the movement screen described below. Alternatively, the growth rate of a culture of C. elegans can also be assayed by measuring the turbidity of the culture. In order to perform this ‘turbidity assay’ the worms are grown in liquid culture in the presence of E. coli or other suitable bacterial food source. As the culture of worms grows the food source bacteria will be consumed. The greater the number of worms in the culture, the more food source bacteria will be digested. Hence, measurement of the turbidity or optical density of the liquid culture will provide an indirect indication of the number of worms in the culture. By taking sequential measurements over a period of time it is possible to monitor the growth rate of the whole C. elegans culture.

As an alternative to the above-described methods, the growth rate and amount of progeny can be measured on a plate. Slow growing nematodes, nematodes with vulva defects and nematodes with gonad defects will produce less progeny within a certain time compared to nematodes which do not have these defects. Preferentially, the amount of offspring produced is scored on day five and on day eight. In experiments where the amount of offspring is reduced very drastically due to severe defects in the vulva, gonad or growth rate reduction, the offspring can also be scored at later time intervals.

In a still further embodiment, the up-regulation assay can be performed by detecting changes in the movement behaviour of C. elegans. As is illustrated by the examples included herein, SERCA is widely expressed in the muscles of C. elegans, including the muscles of the body wall. A reduction of SERCA activity in the body wall muscles gives rise to worms with movement defects. These strains can be used as the basis of an assay in which the worms are contacted with a compound under test and any changes in the movement behaviour of the worms are observed. Compounds which cause the defective movement to revert towards wild-type movement behaviour are scored as compounds capable of enhancing/up-regulating the activity of SERCA.

Changes in the movement behaviour of the worms can obviously be detected by visual inspection, but as an alternative a number of non-visual approaches for analysing the movement behaviour of worms have been developed which can be performed in a multi-well plate format and are therefore suitable for use in high-throughput screening. Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematode worms that move and nematodes that do not move. Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing a liquid culture of nematodes that move normally as compared to wells containing a liquid culture of nematodes that are defective for movement. For the wells containing the nematodes that move normally, a low level of autofluorescence will be observed, whilst a high level of autofluorescence can be observed in the wells that contain the nematodes that are defective in movement.

In an adaptation of the movement assay, autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.

As an alternative to the above-described embodiments of the up-regulation assay which are all based on the observation of changes in phenotypic and/or behavioural characteristics of the C. elegans as an indicator of SERCA activity, the inventors have developed a method of analysing SERCA activity in a given cell type or tissue which is based upon the use of the marker molecule apoaequorin which is sensitive to changes in intracellular Ca²⁺.

Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria. Recombinant apoaequorin, which is luminescent in the presence of calcium but not in the absence of calcium, is most useful in determining intracellular calcium concentrations and even calcium concentrations in sub-cellular compartments. Expression vectors suitable for expressing recombinant apoaequorin and, in addition, vectors expressing apoaequorin proteins which are targeted to different sub-cellular compartments, for example the nucleus, the mitochondria or the endoplasmic reticulum are available commercially (see below).

As SERCA is a endoplasmic reticulum-localized calcium pump, an apoaequorin that is targeted to the endoplasmic reticulum (hereinafter referred to as erAEQ) is particularly useful for developing assays for SERCA activity. Such apoaequorin is available from Molecular probes (Eugene, Oreg., USA). The vector erAEQ/pcDNAI (Molecular Probes) contains an Igγ2b heavy chain gene from mouse, an HA1 epitope and a recombinant apoaequorin in fusion. The mouse gene targets the aequorin to the endoplasmic reticulum, and the aequorin is mutated to make it less sensitive to calcium, as the concentrations of this ion are relatively high in the endoplasmic reticulum. Although apoaequorin is the calcium sensor of choice, it would be apparent to persons skilled in the art that any other calcium sensor localized in the endoplasmic reticulum could be used with equivalent effect

Plasmid expression vectors which drive expression of the ER-localized apoaequorin in C. elegans can be easily constructed by cloning nucleic acid encoding erAEQ downstream of a promoter capable of directing gene expression in one or more tissues or cell types of C. elegans, such that the promoter and the erAEQ-encoding sequence are operatively linked. As used herein the term “operatively linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In a typical cloning procedure, the apoaequorin gene in fusion with the signals to locate the resulting protein to the endoplasmic reticulum was isolated from erAEQ/pcDNAI by EcoRI digestion and cloned into pBlue2SK. The erAEQ was then isolated as an EcoRI/Acc65I fragment by partial digestion and cloned in the vector pGK13 digested with the same enzymes.

Suitable promoters include the pharynx-specific promoter myo-2, the C. elegans SERCA promoter which directs expression in a wide range of muscle tissues and the body wall muscle-specific promoter myo-3. The vectors can then be used to construct transgenic C. elegans according to the standard protocols known to those of ordinary skill in the art. Expression of erAEQ allows for the determination of the calcium levels in the endoplasmic reticulum of various C. elegans cells and tissues, using the protocols of the manufacturer of erAEQ, or minor modifications thereof. Alterations in SERCA activity influence the concentration of calcium in the endoplasmic reticulum as SERCA functions as an endoplasmic reticulum calcium pump. Hence the apoaequorin luminescence measured in the assay is directly related to SERCA activity.

The basic ‘up-regulation assay’ methodology can also be adapted to perform a genetic screen in order to identify C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of SERCA. Accordingly, the invention also provides a method of identifying C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:

subjecting a population of C. elegans with wild-type SERCA activity to random mutagenesis;

allowing the mutagenized C. elegans to grow for one or two generations;

treating the mutagenized C. elegans to reduce the activity of the SERCA ATPase in one or more cell types or tissues; and

scoring a phenotypic, biochemical or behavioural characteristic of the C. elegans as an indicator of SERCA ATPase activity in the C. elegans in the said one or more cell types or tissues.

This genetic screen differs from the ‘up-regulation’ assay used to identify compounds in that the C. elegans are subjected to a random mutagenesis step before they are treated to reduce the activity of the SERCA ATPase. The random mutagenesis step can be performed using any of the techniques known in the art. EMS and UV-TMP mutagenesis, both of which are well known in the art (see Methods in Cell biology Vol. 48, 1995, ed. by Epstein and Shakes, Academic press) are preferred. After mutagenesis the worms are grown for one or two generations before they are treated to reduce the activity of SERCA. After one generation, the worms are heterozygous for any mutation, after two generations they may be homozygous or heterozygous for any mutation. Therefore growth for one generation leads to isolation of dominantly acting suppressors, growth for two generations yields both recessively and dominantly acting suppressors.

The step of treating the C. elegans to reduce the activity of the SERCA ATPase preferably comprises either treating the worms with a chemical inhibitor of SERCA, for example thapsigargin, or specifically down-regulating the expression of SERCA using antisense or double-stranded RNA inhibition.

When thapsigargin is added to worms in plate or liquid culture few progeny are produced and these don't grow as well as wild-type worms. To perform a genetic screen based on thapsigargin inhibition wild-type worms are first subjected to standard mutagenesis protocols (using EMS or UV/TMP or any other mutagen). F1 or F2 progeny of the mutagenized worms are distributed individually to standard growth medium with bacteria, to which 10 to 50 mM thapsigargin is added. After 4-8 days the cultures are inspected for growth of progeny, either by eye or using the ‘turbidity assay’, as described above. Wild-type C. elegans with an integrated transgenic array causing general expression of a reporter protein such as GFP can also be used. In this case, cultures are inspected for growth of progeny either by eye or by detecting expression of the reporter protein.

Thapsigargin causes a short term pharynx pumping defect. Hence, the genetic screen can also be performed by measuring changes in the pharynx pumping efficiency. Wild-type worms are mutagenized and grown on solid media according to standard techniques known in the art. Adults are washed off the plates and put in buffer with calcein-AM and thapsigargin (an assay buffer of 40 mM NaCl, 6 mM KCl, 1 mM CaCl ₂, 1 mM MgCl₂can be used for this purpose). After two hours the worms are viewed under a fluorescence microscope and individual worms that show far brighter gut fluorescence than the other worms are selected, placed individually onto fresh plates and grown for an additional generation. Calcein-AM uptake in the presence of thapsigargin is then re-checked.

Inhibition of SERCA by antisense or double stranded-RNA inhibition will result in the same phenotypes as described above for the up-regulation assay and hence the same screens can be used to select for mutants that enhance or up-regulate SERCA activity. The precise nature of the screen used depends on the tissue in which the antisense or double stranded SERCA RNA is expressed.

An analogous genetic screen can also be performed using a reduction-of-function mutant C. elegans strain which exhibits reduced C. elegans activity in one or more cell types or tissues. Accordingly, in a further aspect the invention provides a method of identifying C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises the steps of:

subjecting a population of mutant C. elegans which exhibit reduced SERCA activity in one or more cell types or tissues to random mutagenesis;

allowing the mutagenized C. elegans to grow for one or two generations; and

A suitable reduction-of-function mutant strain can be isolated as described above.

The basis of the above-described genetic screens is to screen for mutations that have the effect of enhancing or up-regulating SERCA activity and thus suppress the inhibitory effect of thapsigargin treatment, antisense or double stranded RNA inhibition of SERCA expression or a reduction-of-function mutation. Mutations likely to be identified using the method of the invention include mutations in genes involved in transcription and/or translation of SERCA, mutations that influence Ca ²⁺ cycling between the ER and cytoplasm, mutations that influence Ca²⁺ buffering and mutations that influence the activity of Ca²⁺ binding proteins. Once a mutant worm has been identified using a genetic screen it is a matter of routine to identify the mutated gene using techniques commonly used in the art.

In summary, the up-regulation assay which may be used to identify compounds which enhance the activity and/or expression of SERCA is based on the use of C. elegans worms in which the activity or expression of the C. elegans SERCA protein is reduced. This may be achieved in at least three different ways. First, mutants can be selected that show reduced SERCA activity. Second, wild-type, mutant, or transgenic C. elegans strains can be treated with compounds that inhibit SERCA activity, such as thapsigargin. Third, RNAi technology can be applied to wild-type, mutant or transgenic C. elegans to reduce the SERCA activity. In each case, screening can be performed to select for compounds that enhance SERCA activity. Such screens may be based on the pharynx pumping rate, egg laying or movement. In a particular example of the up-regulation assay, wild-type, mutant or transgenic strains can be made transgenic for apoaequorin or another calcium marker. These markers may be expressed in the various tissues, such as the pharynx, the body wall muscles, the oviduct, vulva-muscles etc, for which specific promoters are known in the art. Apoaequorin may also be expressed more generally in C. elegans, for instance under the control of the SERCA promoter. The apoaequorin may further be fused to a specific signal peptide translocating the apoaequorin to the endoplasmic reticulum. Selecting compounds that enhance the activity or the expression of SERCA will enhance calcium uptake, and hence increase the bio-luminescence of the apoaequorin located in the lumen of the endoplasmic or sarcoplasmic reticulum.

In a second aspect the invention provides a method of identifying compounds which modulate the interaction between a sarco/endoplasmic reticulum calcium ATPase and phospholamban, which method comprises:

exposing transgenic C. elegans which contains a first transgene comprising nucleic acid encoding a vertebrate PLB protein and which expresses a SERCA protein to a compound under test; and

detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating an increase in the activity of the SERCA protein.

The vertebrate phospholamban (PLB) protein used in this second method of the invention, hereinafter referred to as the ‘interaction assay’ can be any vertebrate PLB protein. Most preferred are pig PLB (GenBank P07473) or human PLB (GenBank P26678) or a humanized pig PLB (see below). Mutant PLB proteins which exhibit stronger or weaker inhibition of SERCA relative to the wild-type protein may also be used.

The SERCA protein expressed by the C. elegans may be a C. elegans SERCA protein, a vertebrate SERCA protein, a fusion between a vertebrate SERCA protein and C. elegans SERCA protein or a mutant SERCA protein, for example a mutant which exhibits greater sensitivity to PLB.

The vertebrate SERCA protein can be any vertebrate SERCA isoform. Preferred isoforms are pig SERCA2a (GenBank P11606), human SERCA1a (GenBank AAB 53113), human SERCA1b (GenBank AAB 53112), human SERCA2a (GenBank P16614) and human SERCA2b (GenBank P16615). Human and pig SERCA2a are most preferred.

Various types of fusion proteins between C. elegans SERCA and vertebrate SERCA proteins which may be used in the method of the invention are described in the accompanying Examples. For example, the fusion might comprise the N-terminal part of C. elegans SERCA and the C-terminal part of a vertebrate SERCA.

It is essential that a ‘functional’ combination of SERCA and PLB is chosen i.e. that the SERCA protein and the PLB protein are able to interact with each other such that the activity of SERCA can be inhibited by the PLB, mimicking the regulatory interaction occurring in vertebrates.

In the context of this application the term “transgene” refers to a DNA construct comprising a promoter operatively linked to a protein-encoding DNA fragment. The construct may contain additional DNA sequences in addition to those specified above. The transgene may, for example, form part of a plasmid vector. By the term “operatively linked” it is to be understood that the promoter is positioned to drive transcription of the protein-encoding DNA fragment.

Methods of preparing transgenic C. elegans, including worms carrying multiple transgenes, are well known in the art and are particularly described by Craig Mello and Andrew Fire, Methods in Cell Biology, Vol 48, Ed. H. F. Epsein and D. C. Shakes, Academic Press, pages 452-480. A typical approach involves the construction of a plasmid-based expression vector in which a protein-encoding DNA of interest is cloned downstream of a promoter having the appropriate tissue or cell-type specificity. The plasmid vector is then introduced into C. elegans of the appropriate genetic background, for example using microinjection. In order to facilitate the selection of transgenic C. elegans a second plasmid carrying a selectable marker may be co-injected with the experimental plasmid.

The plasmid vector is maintained in cells of the transgenic C. elegans in the form of an extrachromosomal array. Although plasmid vectors are relatively stable as extrachromosomal arrays they can alternatively be stably integrated into the C. elegans genome using standard technology, for example, using gamma ray-induced integration of extrachromosomal arrays (methods in Cell Biology, Vol 48 page 425-480).

The DNA fragment encoding the SERCA protein or the PLB protein may be a fragment of genomic DNA or cDNA. Preferably the DNA encoding the vertebrate SERCA protein is operatively linked to the promoter region of a SERCA gene. Most preferably the promoter region of the C. elegans SERCA gene is used. The term ‘promoter region’ as used herein refers to a fragment of the upstream region of a given gene which is capable of directing a pattern of gene expression substantially identical to the natural pattern of expression of the given gene.

Provided that a functional combination is chosen, wherever the SERCA protein and the vertebrate PLB are co-expressed the two proteins will interact such that PLB inhibits the activity of SERCA. The aim of the interaction assay is to identify compounds which directly or indirectly disrupt the SERCA/PLB interaction, leading to an increase in SERCA activity. The increase in SERCA activity is monitored indirectly, by detecting phenotypic, biochemical or behavioural changes in the C. elegans which are indicative of an increase in SERCA activity. Advantageously, the nucleic acid encoding PLB is operatively linked to a tissue-specific promoter. With the use of a promoter of appropriate specificity, the vertebrate PLB can be expressed in all the cells of C. elegans , in a given type of tissue (i.e. all muscles), in a single organ or tissue (for example, the pharynx or the vulva), in a subset of cell types, in a single cell type or even in a single cell.

By restricting the expression of PLB to certain tissues it is possible to specifically down-regulate SERCA activity in these tissues and thus to influence the phenotype of the resultant transgenic worms. For example, when PLB is expressed in the pharynx, the resultant inhibition of SERCA activity in the pharynx results in a reduction in the rate of pharynx pumping. When PLB is expressed in the vulva muscles, the resultant inhibition of SERCA activity in the vulva results in an egg laying defect.

Although the interaction assay may be performed using functional combinations of C. elegans SERCA (especially mutant versions thereof, as discussed below) and vertebrate PLB, it is preferred to use functional combinations of vertebrate SERCA and vertebrate PLB. In order to ensure that the interaction assay can be used to identify compounds which specifically modulate the vertebrate SERCA/vertebrate PLB interaction it is preferred to use a transgenic strain which has been modified such that expression of the endogenous C. elegans SERCA protein is abolished or substantially reduced down to background levels. This may be achieved by introducing the transgenes encoding the vertebrate SERCA and PLB into a mutant strain having a knock-out or loss-of-function mutation in the chromosomal C. elegans SERCA gene (e.g. strain ok190 described in the accompanying Examples). A protocol for isolating a suitable knock-out mutant strain is given in the examples included herein. In a variation of this approach, expression of the endogenous C. elegans SERCA gene may be abolished/reduced using RNAi technology, as described hereinbefore. In this case, the genetic background of the transgenic C. elegans may be wild-type.

In a further embodiment, a vertebrate-specific interaction assay may be achieved by using transgenic C. elegans expressing a mutant version of the vertebrate SERCA protein which is resistant to a chemical inhibitor of SERCA activity, such as thapsigargin. The mutation Phe259Val renders C. elegans SERCA resistant to inhibition with thapsigargin. Equivalent mutations may be introduced into transgenes encoding the vertebrate SERCA proteins using standard site-directed mutagenesis. Applying the SERCA inhibitor, e.g. thapsigargin, to transgenic C. elegans which express a resistant mutant vertebrate SERCA and a vertebrate PLB will result in inhibition of the endogenous C. elegans SERCA only. Thus, if the inhibitor is added to the interaction assay in addition to the test compound, the screen will be specific for the interaction between the vertebrate SERCA and the vertebrate PLB.

A particular variant of the interaction assay uses a mutant version of the C. elegans SERCA protein which is more sensitive to vertebrate PLB proteins, such as, for example, a C. elegans SERCA containing the KDDKPV (SEQ ID NO:39) insertion. As illustrated in the accompanying Example 9, introduction of the amino acid sequence KDDKPV (SEQ ID NO:39) into the C. elegans SERCA protein results in a more efficient interaction between the mutant SERCA and vertebrate PLB. Therefore, double transgenic C. elegans strains containing a first transgene encoding a vertebrate PLB protein and a second transgene encoding a C. elegans SERCA KDDKPV (SEQ ID NO:39) insertion mutant may be used in the interaction assay.

In order to provide specificity for the mutant SERCA/PLB interaction, it is preferred that the double transgenic is also modified such that expression of the endogenous C. elegans SERCA gene is abolished or substantially reduced. As described above, this may be achieved by using a mutant C. elegans genetic background having a knock-out or loss-of-function mutation in the chromosomal SERCA gene or by using RNAi technology to inhibit SERCA expression. Alternatively, it is possible to engineer the mutant SERCA so that in addition to the KDDKPV (SEQ ID NO:39) insertion it also carries a further mutation which renders it resistant to a SERCA inhibitor other than PLB, e.g. the thapsigargin resistance mutation Phe259Val. Addition of the SERCA inhibitor, e.g. thapsigargin, to the assay will result in specific inhibition of the endogenous C. elegans SERCA protein but not the resistant mutant.

As with the ‘up-regulation assay’ described above, the step of “detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating an increase in the activity of SERCA” can be performed in several different ways.

In one embodiment, particularly suitable for use when the transgenic C. elegans expresses PLB in the pharynx, the method is performed by detecting changes in the pharynx pumping efficiency. The rate of pharynx pumping can be measured using a marker molecule precursor such as calcein-AM, as described above for the up-regulation assay.

In still further embodiments, particularly suitable for use when the transgenic C. elegans expresses PLB in the vulva, the method can be performed by detecting changes in the egg laying behaviour of the C. elegans or by detecting changes in the number of progeny produced by the C. elegans. The number of progeny produced by the C. elegans can, as described above in connection with the up-regulation assay, be directly counted or can be measured indirectly using a growth assay or a turbidity assay.

In a still further embodiment, again particularly suitable for use when the transgenic C. elegans expresses PLB in the pharynx, SERCA activity in cells of the C. elegans pharynx can be monitored using apoaequorin luminescence. To achieve this the C. elegans are transfected with a third transgene which comprises nucleic acid encoding an apoaequorin protein, preferably ER-targeted apoaequorin, operatively linked to promoter capable of directing gene expression in the C. elegans pharynx. The construction of suitable expression vectors comprising such a transgene has been described hereinbefore.

In summary, the basic SERCA-PLB interaction screen to select for compounds that inhibit the interaction between SERCA and PLB is based on the construction of transgenic C. elegans expressing PLB. The PLB may be of any vertebrate origin, such as human or pig. The PLB may be expressed ubiquitously or in specific tissues, such as the pharynx, the body wall muscles, the oviduct, vulva muscles etc, for which specific promoters are known in the art. Preferred configurations of the interaction assay are summarised below, however, this is not intended to be limiting to the scope of the invention:

Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a vertebrate SERCA; expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,

Double transgenic C. elegans , first transgene encoding a vertebrate PLB, second transgene encoding a fusion between C. elegans SERCA and a vertebrate SERCA; expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,

Double transgenic C. elegans , first transgene encoding a vertebrate PLB, second transgene encoding a mutant vertebrate SERCA which is resistant to a SERCA inhibitor other than PLB, e.g. thapsigargin; wild-type genetic background; inhibitor is added to the assay in addition to the compound under test to specifically inhibit endogenous C. elegans SERCA expression,

Double transgenic C. elegans , first transgene encoding a vertebrate PLB, second transgene encoding a mutant C. elegans SERCA which is more sensitive to inhibition by vertebrate PLB (e.g. KDDKPV (SEQ ID NO:39) insertion); expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,

Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a double mutant C. elegans SERCA which is (i) more sensitive to inhibition by vertebrate PLB (e.g. KDDKPV (SEQ ID NO:39) insertion) and (ii) resistant to inhibition by a SERCA inhibitor such as thapsigargin (e.g. Phe259Val); wild-type genetic background; inhibitor is added to the assay in addition to the compound under test to specifically inhibit endogenous C. elegans SERCA expression.

In a third aspect the invention provides a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:

exposing transgenic C. elegans containing a transgene comprising nucleic acid encoding a SERCA protein operatively linked to a promoter capable of directing gene expression to a sample of the compound under test; and

detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a decrease in the activity of the SERCA protein.

The SERCA protein used in this third aspect of the invention, hereinafter referred to as the ‘down-regulation assay’ can be any SERCA isoform from any species. Preferably the SERCA protein is C. elegans SERCA, pig SERCA2a, or a human SERCA isoform, most preferably human SERCA 2A.

Preferably the nucleic acid encoding the SERCA protein is operatively linked to a tissue-specific promoter. Most preferably, the tissue-specific promoter is the C. elegans myo-2 promoter which directs tissue-specific expression in the pharynx.

In a preferred embodiment the transgenic C. elegans further contain a second transgene comprising nucleic acid encoding a reporter protein operatively linked to a promoter which is capable of directing gene expression in one or more cell types or tissues of C. elegans. The reporter protein is preferably an autonomous fluorescent protein, for example, a green fluorescent protein or a blue fluorescent protein or a luminescent protein.

Transgenic C. elegans over-expressing SERCA are generally observed to be starved and show delayed growth. Compounds which reduce or down-regulate the activity of SERCA will cause a reversion or reduction of this phenotype towards a wild-type phenotype. Accordingly, these worms can be used as a basis of a screen to identify compounds capable of reducing or down-regulating the activity of SERCA, by bringing the worms into contact with the compound under test and then detecting a reversion of the over-expression phenotype reflecting a decrease in the activity of the SERCA transgene.

The step of “detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a decrease in the activity if the SERCA protein” can be performed in several different ways. As mentioned above, transgenic C. elegans which overexpress the SERCA protein exhibit delayed growth. Accordingly, it is possible to look for a reversion of the overexpression phenotype by comparing the growth rate of the transgenic C. elegans in the presence and the absence of the compound under test. Compounds which increase the growth rate of the C. elegans culture are scored as compounds which are capable of reducing or down-regulating SERCA activity. Any of the growth assay methods described in connection with the ‘up-regulation’ assay could be used for this purpose.

Transgenic C. elegans which overexpress SERCA also exhibit altered egg laying behaviour and reduced pharynx pumping. Hence, the down-regulation assay can also be performed by detecting changes in the egg laying behaviour or the pharynx pumping efficiency, as described previously.

In summary, the basic down-regulation assay consists of introducing extra SERCA into C. elegans and screening for a compound that inhibits SERCA activity. The SERCA introduced into C. elegans may be C. elegans SERCA or a SERCA of any vertebrate origin, such as human or pig. The SERCA protein may be expressed ubiquitously or in specific tissues such as the pharynx, the body wall muscles, the oviduct, the vulva muscles etc, for which appropriate tissue or cell type-specific promoters are known in the art.

The above-described methodology for the down-regulation assay can be adapted to perform a genetic screen to identify C. elegans carrying a mutation having the effect of reducing or down-regulating SERCA activity. Thus, in a further aspect the invention provides a method of identifying C. elegans which carry a mutation having the effect of reducing or down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises the steps of:

providing a transgenic C. elegans strain containing a first transgene comprising nucleic acid encoding a SERCA protein operatively linked to a promoter capable of directing gene expression in one or more cell types or tissues of C. elegans;

subjecting a population of the said C. elegans strain to random mutagenesis;

allowing the mutagenized C. elegans to grow for one or more generations; and

The genetic screen is preferably carried out using transgenic C. elegans containing an integrated transgene harboring 20-50 ng/μl pGK7 (containing the C. elegans genomic SERCA gene, including the promoter region, see examples given below) and a general GFP expressing construct. These worms are starved and show general growth delay. The same results are obtained using a vertebrate SERCA, such as the human or pig SERCA.

Alternatively, the screen can be performed using transgenic nematodes containing an integrated transgene harboring the genomic C. elegans SERCA gene operatively linked to the myo-2 promoter, and a general GFP expressing construct. These worms are also starved and show growth delay.

The worms are grown and subjected to random mutagenesis according to standard techniques known in the art. The mutagenized worms then are distributed individually to standard growth medium with supplemented with food source bacteria. After 4-8 days the cultures are inspected for growth of progeny, either by eye, by using any of the growth assay techniques mentioned previously in connection with the up-regulation assay, using the turbidity assay or by counting the numbers of progeny produced.

The basis of these genetic screens is that mutations having the effect of reducing or down-regulating SERCA activity will suppress the effect of SERCA over-expression. Mutations identified using this screen may include mutations in genes involved in transcription and/or translation of SERCA, mutations that influence Ca ²⁺ cycling between the ER and the cytoplasm, mutations that influence Ca²⁺ buffering and mutations that influence the activity of Ca²⁺ binding proteins.

In the field of human pharmaceuticals, compounds identified as modulators of SERCA activity using the screening methods of the invention may be useful leads in the development of pharmaceuticals for the treatment of the wide range of diseases with which the SERCA genes have been associated, such as cardiac hypertrophy, heart failure, hypertension, NIDDM, Darier-White disease, Brody's disease.

Outside the pharmaceutical field, compounds identified as modulators of SERCA activity may find important applications as pesticides, particularly insecticides, herbicides or nematocides. Maintaining high calcium concentrations in the ER is important for the proper synthesis of protein, including translation, folding, glycosylation, processing and transport. Treatment of living organisms with chemicals that inhibit the activity of SERCA will hence have a negative effect on the welfare of these organisms. As such, SERCA inhibitors are potential pesticides or can be considered as basic compounds for the development of pesticides such as herbicides, insecticides and nematocides. It has been shown that SERCA function is essential in the intracellular trafficking of the Notch receptor in drosophila (Periz et al., 1999 EMBO J; 5983-5993). This studies and others indicate that SERCA is an interesting target for pesticidal intervention. Accordingly, the screening methods described herein could be applied to screen for pesticides.

The invention will be further understood with reference to the following experimental Examples together with the accompanying Figures in which: [0120]
FIG. 1 shows a dose-response curve for thapsigargin produced using a liquid culture assay. [0121]
FIG. 2 shows a dose-response curve for thapsigargin produced using a plate assay. [0122]
FIG. 3 illustrates the growth of [0123] C. elegans strain UG530 (strain harboring plasmid pGK28) on different concentrations of thapsigargin. The stage of the progeny was determined 5 days after adults were put on the plates. Since the mothers carry the pGK28 containing plasmid on an extrachromosomal transgene, part of the progeny inherited it and part of the progeny did not. These were differentiated based on a GFP marker also present on this transgene.
FIG. 4 illustrates the nucleotide sequence of the genomic fragment of [0124] C. elegans SERCA bounded by primers SERCA P4 and SERCA P8. Exon IV and exon V are shown in capitals, intron IV in lower case. The fragment deleted in ok190 is underlined.
All Molecular biology work was performed as described by Sambrook et al. Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, or using minor modifications of the methods described therein. [0125]
All manipulations of [0126] C. elegans worms were performed using techniques described in Methods in Cell Biology, vol 84; Caenorhabditis elegans: modern biological analysis of an organism, ed. Epstein and Shakes, academic press, 1995, or using minor modifications of the methods described therein.
Transgenic [0127] C. elegans strains were constructed by injection of plasmid DNA into worms using standard techniques known in the art (see Methods in Cell Biology, vol 84 as mentioned above).

EXAMPLE 1

Inhibition of Expression of [0128] C. elegans SERCA Using RNAi.
732 bp EcoRI-HindIII fragment from [0129] C. elegans SERCA exon 5 (SEQ ID NO: 1) was PCR amplified and cloned into the vector pGEM3 (PROMEGA corporation, Madison, Wis., USA). RNA was in vitro transcribed from both strands using standard procedures. The generated double stranded RNA was injected into C. elegans (see Fire at al., 1998, Nature 391:806-811). This resulted in the following phenotypes: 50% of the progeny of the injected animals were embryonic lethal, while the other 50% were early larval lethal. This indicates that SERCA function is vital for C. elegans. In conclusion, inhibition of the expression of SERCA in all tissues results in embryonic or early larval lethality of the nematode.
Inhibition of SERCA Using RNAi Feeding Technology [0130]
Although injection of SERCA dsRNA results in a clear phenotype, useful in the development of assays to select for compounds that alter the SERCA activity, or that alter the activity of partners in the SERCA pathway, or that alter the activity proteins involved in calcium metabolism, a more stable RNAi phenotype would be more efficient. Improved RNAi methods which lead to more stable RNAi phenotypes exist and are described, for example in International patent application No. WO 00/01846. More particularly, an RNAi technology has been developed and tested in which dsRNA can be delivered by feeding the nematode dsRNA or by feeding nematodes with DNA. [0131]
pGN4 was constructed by cloning the HindIII-EcoRI fragment of SERCA cloned in vector pGN1 using these same restriction sites. This is the same fragment as was used for in vitro transcription and dsRNA injection, described above. [0132]
HT115(DE3) bacteria (Fire A, Carnegie Institution, Baltimore, Md.) were transfected with pGN4 (and controls with pGN1) and seeded on plates containing IPTG and ampicillin resulting in a high expression of dsRNA by the bacteria. N2 and nuc-1 (e1392) adult nematodes were put on these plates and allowed to lay eggs and the progeny was followed over time. The progeny mostly looked healthy during the larval stages, but the adults (and some of the L4) had a starved appearance (nuc-1 more pronounced then N2). Pumping was irregular and slower then normal, and the growth rate was somewhat reduced. This example indicates that a stable RNAi phenotype useful in assay development and compound screening can be developed using feeding. As described in co-pending application No. WO 00/01846, other possibilities and variants can be used to create a [0133] C. elegans SERCA RNAi phenotype. The use of RNAi technology allows the development of screens for compounds that alter SERCA activity or that alter the SERCA pathway, without the construction of a C. elegans SERCA mutant. E. coli HT115 has the following characteristics which make it a useful host cell for high level expression of dsRNA: HT115 (DE3): F-mcrA mcrb IN(rrnD-rrnE) 1 γ-rnc14::tr10 (DE3 lysogen: lacUV5 promoter-T7 polymerase); host for IPTG inducible T7 polymerase expression; RnaseIII-. Other host strains suitable for expression of dsRNA could be used with equivalent effect.

EXAMPLE 2

Overexpression of [0134] C. elegans SERCA.
A 11207 bp SpeI-MluI fragment from the cosmid K11D9 (SEQ ID NO:2) was cloned into the vector pUC18 (Messing, J., 1998, Methods in Enzymol. 101: 20), resulting in the plasmid pGK7. This genomic fragment contains the complete SERCA gene with 5631 bp upstream sequences, the complete coding region and 1088 bp downstream sequences. Transfection of [0135] C. elegans with this vector using standard technology resulted in various results. Transfection with high concentrations of DNA (80-200 ng/μl) induced embryonic lethality. At lower concentrations of DNA (20-50 ng/μl) worms were generally sick; also they were constipated, showed a starved appearance, and had pharynx pumping defects. These experiments indicate the importance of fine-tuning the expression level of SERCA in the nematode C. elegans. High levels of overexpression of SERCA is lethal, as is inhibition of expression. Furthermore, intermediate levels of overexpression of SERCA results in defects all over the worm, affecting almost all vital functions.

EXAMPLE 3

Expression Pattern of SERCA in [0136] C. elegans.
A 5026 bp fragment of the upstream region of the [0137] C. elegans SERCA gene, starting 5026 bp upstream of the translation initiation codon and continuing up to and including the A of the ATG initiation codon (SEQ ID NO:3), was cloned into the vector pPD95.79 (described in Fire et al. (1990) Gene, 93: 189-198)in fusion with a GFP fluorescent protein, resulting in vector pGK10. The cloned fragment can be considered as the promoter region of the C. elegans SERCA. The vector was injected into C. elegans, using standard methodology well known to persons skilled in the art, and the expression of the GFP was monitored applying standard fluorescent techniques. GFP expression was observed all over the early embryo of the worm, although expression was faint in some tissues. In a later stage of development, from mid-embryo stage, through larval stage to adult stage, strong GFP expression could be observed in all muscle tissue, including the pharyngeal muscles, the body wall muscles, the anal depressor and the anal sphincter. In adults staining was seen in the vulva muscles, the uterine muscles, the spermatecae and the proximal myoepithelial sheath cells of the gonad.
A construct containing a smaller promoter fragment, including A of the initiating ATG codon and extending 2915 bp upstream (SEQ ID NO:4), fused to a GFP gene was generated by a PstI deletion of the plasmid pGK10. This plasmid was designated pGK13. Transfection of the nematode with pGK13 resulted in the same pattern of GFP expression as was observed with pGK10. [0138]
Finally, a third construct was made containing a 6612 bp fragment of the [0139] C. elegans SERCA gene in the plasmid pPD95.75 (described in Fire et al. (1990) Gene, 93: 189-198). The resultant plasmid was designated pGK12. This 6612 bp fragment contains 5637 bp of upstream sequences an ends in exon 4 of the C. elegans SERCA gene (SEQ ID NO:5). The fragment was cloned as a SalI-BglII fragment isolated from pGK7, and cloned in fusion to GFP. This fragment contains two transmembrane domains of SERCA. Transfection of C. elegans with this construct resulted in the same pattern of GFP expression as was observed with pGK10 and pGK13, i.e. GFP expression could be localized to the muscle tissues of C. elegans. Detailed analysis of the expression pattern in the muscles showed clearly that the GFP protein was localized to the endoplasmic reticulum and the dense bodies.
These expression studies clearly demonstrate that the SERCA protein of [0140] C. elegans is expressed in all muscle tissue, and that it is localized in the endoplasmic reticulum, indicating that the C. elegans SERCA probably has analogous function to the vertebrate SERCAs.
In addition to pGK10, pGK12 and pGK13, several further constructs have been used to analyse the pattern of SERCA expression in [0141] C. elegans. These are summarised as follows:
pGK26 contains GFP inserted directly after the CDS of [0142] C. elegans SERCA isoform A using overlap PCR, also containing the SERCA downstream region.
pGK27 contains GFP inserted directly after the CDS of the [0143] C. elegans SERCA isoform B using overlap PCR, also containing the SERCA downstream region.
pGK26 was constructed by the following strategy: Three separate PCR reactions were done to yield three PCR fragments that are joined in consequent overlap PCR. The first fragment is made with the oligonucleotides oGK25 and oGK26 and contains the region upstream of where GFP is inserted. The primer oGK26 is an overlap primer and contains the last 21 nt of SERCA until but not including the stop codon followed by the first 15 nt of GFP. The second fragment contains the complete ORF of GFP including the stop codon. The third fragment is made with oGK27 and oGK28 and contains the region downstream of where GFP is inserted. The primer oGK27 is an overlap primer and contains the last 15 nt of GFP including the stop codon and the first 22 nt of the 3′ UTR. The end result after overlap PCR is a “recombination” of these three fragments in which GFP is inserted exactly after and in-frame with the SERCA coding region such that the fragment encodes a SERCA::GFP fusion protein. This PCR fragment is cloned into pGK7 to replace the normal C-terminus of the gene using unique restriction sites in the SERCA coding region and 3′ UTR (ApaI in the first fragment and PacI in the third fragment). [0144]
pGK27 was constructed the same way, using primers oGK21 and oGK22 instead of oGK25 and oGK26, and primers oGK23 and oGK24 instead of primers oGK27 and oGK28. [0145]
Sequence of primers: [0146]
oGK21: TGGACTCATCTCTGGATGGCTC (SEQ ID NO:17) [0147]
oGK22: CTTCTCCTTTACTCATCAATTCGTTATGTAACTTGTCGG (SEQ ID NO:18) [0148]
oGK23: GAACTATACAAATAGTTGAAGTTCTTCTAACCCCC (SEQ ID NO:19) [0149]
oGK24: GCGTTTATCCTTGATTGGAGCTTC (SEQ ID NO:20) [0150]
oGK25: GAATGGATCGCCGTGTTGAAG (SEQ ID NO:21) [0151]
oGK26: TTCTCCTTTACTCATGTCGCGTTTATCCTTGATTGG (SEQ ID NO:22) [0152]
oGK27: GAACTATACAAATAGAAATGACAGTGCTCCCTCAATC (SEQ ID NO:23) [0153]
oGK28: GTGGGATCCTGGTTTGTTCTGAG (SEQ ID NO:24) [0154]
When the various constructs were injected into wild-type [0155] C. elegans the following expression patterns were observed:
Expression was observed in all muscle types, such as the body wall muscle, the pharynx, the vulva muscle, the uterine muscles etc, but expression was also observed in the anal depressor, the gut, the gonad sheath cells etc. (see Table 1). This is not unexpected, due to the importance of SERCA in the calcium metabolism of [0156] C. elegans , as has also been observed in other organisms. Moreover the various constructs give different expression patterns, indicating complex regulation of SERCA expression as suggested.

The expression patterns of the constructs, and hence of the endogenous SERCA, indicate that various assays can be developed. These include assays based on body wall muscle function and hence on movement, assays on pharyngeal function and hence on the pumping rate, assays on vulva muscle function and hence on egg laying, assays on anal repressor function and hence on defecation, assays on the gonad sheath cell, uterine muscle and uterine sheath cell function and hence on egg laying.

TABLE 1


expression patterns for SERCA constructs in wild-type C. elegans.

	pGK10	pGK26	pGK27

body wall/head muscle	Yes	Yes	Yes
ER	No	Yes	Yes
dense bodies	No	Yes	Yes
muscle arms	Yes	No	No
pharynx	Yes	Yes	Yes
TB	Yes	Yes	Yes
isthmus	Yes	Yes	Yes
metacorpus	No	No	No
procorpus	No	No	No
vulva muscles	Yes	Yes	Yes
anal depressor	Yes	Yes	Yes
gonad sheath cells	Yes	Yes	Yes
gut	Yes	Yes	Yes
uterine muscles	Yes	?	?
uterine sheath cells	No	No	Yes
spermatheca	Yes	No	Yes
tailspike	Yes	No	Yes
coelomocytes	No	No	Yes
excretory canal	No	No	Yes

EXAMPLE 4

Expression of Mammalian SERCA in [0158] C. elegans.
Further constructs were made in which the pig SERCA2a cDNA was cloned under the regulation of the [0159] C. elegans SERCA promoter. Suitable constructs can easily be made by replacing the GFP sequences in pGK10 or pGK13 with the coding region of the pig SERCA2a cDNA. The sequence of the pig SERCA2a cDNA is shown in SEQ ID NO:7. C. elegans were transfected with plasmid pGK101, harboring the pig SERCA2a cDNA under the control of the worm SERCA promoter derived from pGK 10 by injection of the plasmid at a concentration of 100 ng/μl, resulting in the overexpression of the pig SERCA2a in all C. elegans muscles. The overexpression of this vertebrate SERCA protein results in embryonic lethality, L1 arrest and growth delay, effects which are quite analogous to the overexpression of C. elegans SERCA.
The pig SERCA2a was also expressed in [0160] C. elegans under the control of the myo-2 promoter (pGK201), which is specific for induction of expression in the pharyngeal muscles. Overexpression of SERCA2a in the pharyngeal muscles resulted in apparently normal healthy lines, although a slight growth delay was observed. In a pharynx pumping assay, with the fluorescent dye precursor calcein-AM, it was shown that the nematode pumps with a slightly lower efficiency than a wild-type strain.
Expression in [0161] C. elegans of the pig SERCA2a under the regulation of the myo-3 promoter, which directs gene expression in the body wall muscles, resulted in apparently normal, healthy lines, with no apparent movement defects.

EXAMPLE 5

Expression of Mammalian Phospholamban (PLB) in [0162] C. elegans.
Human, humanized and pig PLB fused and not fused to GFP were expressed under the myo-2 promoter in the pharyngeal muscles. The transfected nematodes appeared sick, showed a reduced growth and a clearly reduced pharynx pumping phenotype. Further generations of offspring seem to be healthier and perform in a pharynx pumping assay as wild-type worms. [0163]
Expression of the PLB-GFP fusion protein in the body wall muscles, was done under the regulation of the myo-3 promoter. Expression of the fusion protein could clearly be localized to the endoplasmic reticulum and the dense bodies, but no clear phenotype could be observed. [0164]

EXAMPLE 6

Construction of a Mutated SERCA [0165] C. elegans.
[0166] Strategy 1
The following strategy may be used to isolate a nematode that is mutated in the SERCA gene, using standard selection procedures well known in the art. A population of nematodes are mutagenized, preferentially using UV-TMP, and grown for two generations. The mutagenized worms are distributed per 500 over approximately 1152 plates and grown for an additional two generations. DNA is isolated from a fraction of the worms from each of these plates and used as a template for PCR selection to select for a SERCA gene that has a deletion. From a plate with worms, of which some have been demonstrated to contain a SERCA deletion, new plates are started with fewer worms. Further rounds of PCR selection finally result in the isolation of a heterozygote [0167] C. elegans carrying a mutation in the SERCA gene (see Jansen et al., 1997, Nature Genetics 17:119-121). As the above-mentioned experiments have shown that the expression level of SERCA is important for the survival of the nematode it is possible that this strategy may result only in the isolation of partial knock-out mutations as heterozygote C. elegans carrying a severe knock-out mutation in the SERCA gene may not viable. In this situation, strategy 1 based on extrachromosomal expression can be used to isolate severe knock-out mutations.
Strategy 2 [0168]
Although primary RNAi experiments indicate that the level of expression the SERCA protein needs to be fine-tuned for the survival of the [0169] C. elegans nematode, strains in which the level of SERCA activity is reduced, in particular strains in which SERCA activity is reduced in a single tissue, are probably still viable. Due to the sensitivity of C. elegans to the level of SERCA activity this could result in a recognisable phenotype, such as reduced pharyngeal pumping, vulva muscle defects, and hence egg laying defects, anal repressor and anal sphincter defects, and hence defecation defects, and body wall muscle defects, and hence movement defects. Such strains can be used as the basis of screens to identify compounds capable of enhancing or up-regulating the activity of SERCA.
The expression levels of SERCA in [0170] C. elegans can be specifically reduced by using antisense technology or double stranded RNA inhibition. The use of antisense technology to specifically reduce expression of a given protein is well known. For the expression of antisense RNA in the worm, the non-coding strand of a fragment of the SERCA gene can be expressed under the control of the SERCA, myo-2 or myo-3 promoter or any other promoter. The expression of the antisense SERCA RNA will result in the inhibition of expression of SERCA.
Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion or the mature protein sequence, which encodes for the SERCA protein, is used to design an antisense RNA oligonucleotide of from 10 to 50 base pairs in length. The antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the protein (Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix—see Lee et al. Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991), thereby preventing transcription and the production of the protein. [0171]
In order to perform an antisense experiment in [0172] C. elegans, an EcoRI-Hind III fragment of SERCA exon 5 was cloned antisense under the control of the myo-2 promoter, the myo-3 promoter, the SERCA promoter or the ceh-24 enhancer and injected into C. elegans. These vectors result in the expression of an antisense SERCA RNA, and hence in inhibition of SERCA activity.
As an alternative to the antisense approach, the expression of a given gene in a cell can also be specifically reduced by introducing into the cell double stranded RNA corresponding to a region of the transcript transcribed from the gene. Double stranded RNA can be prepared by cloning an appropriate fragment into a plasmid vector containing opposable promoters. A suitable example is the pGEM7 series of vectors from Promega Corporation, Madison, Wis., USA, which contain opposable promoters separated by a multiple cloning site. When the plasmid vector is transformed or transfected into a host cell or organism which expresses the appropriate polymerases, RNA will be transcribed from each of the promoters. As the vector contains two promoters oriented in the opposite sense, complementary sense and antisense transcripts will be transcribed which will combine to form double stranded RNA. The injection of double stranded RNA in [0173] C. elegans has previously been described (Fire et al, Potent and Specific Genetic Interference by Double-Stranded RNA in C. elegans 1998, Nature 391, 860-811).

EXAMPLE 7

Analysis of a [0174] C. elegans Mutant (Designated ok190)
A [0175] C. elegans strain mutated in the SERCA gene was kindly provided by R. Barstead (Oklahoma, USA). Heterozygous animals show no defect, but their homozygous progeny die as L1. The lethal phenotype can be rescued by reintroduction of the C. elegans gene by injection of pGK7.
Using standard PCR protocols the genomic region of ok190 around the deleted area was cloned in the following way: [0176]
A nested PCR was performed on [0177] C. elegans genomic DNA using the following primer pairs:
Outer: SERCA P2: CGAAGAGCACGAAGATCAGACAG (SEQ ID NO:25) SERCA P8: GAGAGGCGGTTGGTTTGGG (SEQ ID NO:26) [0178]
Inner: SERCA P4: CCGTTCGTCATCCTTCTCATTC (SEQ ID NO:27) SERCA P7: CGACAGATGGACCGACGAGC (SEQ ID NO:28) [0179]
Analysis of the nested PCR product by agarose gel electrophoresis showed that the PCR product in the ok190 strain harbors a deletion of 1.7 kbp. (The wild-type PCR product from SERCA P4-SERCA P7 would be 3.4 kbp but the observed ok190 PCR product was only 1.7 kbp). [0180]
To enable detailed analysis of the deleted region the PCR product was cloned into the pCR-XL-TOPO vector (Invitrogen, The Netherlands). The resulting plasmid was designated pKO4. This cloned fragment was then sequenced revealing the exact coordinates of the deleted region. One of the breakpoints of the deletion occurred in the intron between exon IV and exon V, the other in exon V, deleting a total of 1702 bp of which 1690 bp represent coding sequence. [0181]
The nucleotide sequence of the genomic fragment of [0182] C. elegans SERCA bounded by primers SERCA P4 and SERCA P8 is shown in FIG. 4 and as SEQ ID NO: 16. Exon IV and exon V are shown in capitals, intron IV in lower case. The fragment deleted in ok190 is underlined.

EXAMPLE 8

Construction of a Thapsigarzin Resistant SERCA. [0183]
A mutated [0184] C. elegans SERCA gene which encodes mutant protein resistant to thapsigargin inhibition has been constructed. The mutation is TTC□GTC, which results in a Phe258Val substitution. This is analogous to the substitution Phe256Val in hamsters, which was shown to be 40-fold resistant to thapsigargin inhibition (Yu et al., 1999, Arch. Biochem. Biophys. 15:225-232).
The mutation was introduced in the gene with the QuickChange Site-Directed Mutagenesis Kit (Stratagene, Calif., USA). PCR was performed on pGK7, according to the instructions supplied by the manufacturer, with the following primers: [0185]
oGK33F256V (CAACAGAAGTTGGACGAAGTCGGAGAGCAACTTTC) (SEQ ID NO:29) [0186]
oGK34F256V (GAAAGTTGCTCTCCGACTTCGTCCAACTTCTGTTG) (SEQ ID NO:30) [0187]
The resulting mutation was screened by EcoRI digestion, as the mutation resulted in the disruption of the EcoRI restriction site. The new vector was sequenced, and the vector was transfected into [0188] C. elegans. The resulting vector was designated pGK28.
Test Sensitivity of Phe259Val SERCA Mutation [0189]
Several [0190] C. elegans transgenic lines where constructed that carry the thapsigargin resistant SERCA mutant by standard injection of pGK28 into the gonad.
The effect of thapsigargin on worms carrying a pGK28 transgene was measured in the following way: 10 μl of thapsigargin dissolved in DMSO (5, 2.5, 1, 0.5, 0.25, 0.1, and 0.05 mM respectively) was added onto a drop of [0191] E. coli strain OP50cs2 in 12-well plates. The wells with compounds were placed at 10□C. overnight, after which 1 to 10 young adults were added to the wells. The pharynx pumping rate and movement behaviour was scored for the ten worms after 10 minutes and after one hour (short term effect). Furthermore the wells were scored for protruding vulva and rectum, production of progeny (few eggs in body) after one day (mid-term-effects), and for progeny after four days (long term effect)
Cold-sensitive [0192] E. coli strain OP50cs2 was deposited on Mar. 25, 1999 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in the Belgian Coordinated Collections of Microorganisms (BCCM)/Laboratorium voor Microbiologie-Bacterienverzameling (LMG) bacteria collection, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000, Gent, Belgium under accession number LMG P-18934.
Conclusions [0193]
The results from this experiment confirmed that the Phe259Val pGK28 lines tested are more resistant to thapsigargin. The clearest indication for resistance was that a large fraction of the GFP-animals (the animals harboring pGK28) grew up to adult or L4 while most non-GFP (not harboring or expressing the mutated SERCA) arrested at L1 and L2 and never grew up further than L3 (see FIG. 3). Furthermore it was observed that nematodes harboring the Phe259Val SERCA mutant had slightly more progeny than wild-type and that the protruding vulva and rectum-phenotype was very often observed in the wild-type nematodes treated with thapsigargin, whereas these phenotypes were not or were only occasionally observed in strain UG530 (strain harboring plasmid pGK28), treated with thapsigargin. [0194]

EXAMPLE 9

Construction of a [0195] C. elegans SERCA Mutant cDNA with a PLB Recognition Site.
Phospholamban is known to interact with the KDDKPV (SEQ ID NO:39) site in mammalian SERCA1 and SERCA2 (Toyofuku et al., J. Biol Chem. 1994, 269:22929-22932). SERCA3 does not contain this sequence and does not bind phospholamban. SERCA3 does bind phospholamban when the KDDKPV (SEQ ID NO:39) sequence is introduced, while SERCA2 can no longer bind phospholamban when this sequence is mutated (Toyofuku et al., J. Biol Chem. 1994, 269:22929-22932). Phospholamban also interacts with transmembrane helix TM6, which is identical in all three mammalian SERCA genes (Asahi et al., J. Biol. Chem. 1999, 274:32855-32862). [0196]
The [0197] C. elegans SERCA gene does not contain the KDDKPV (SEQ ID NO:39) sequence, but the TM6 domain is identical to mammalian SERCA. A variant of C. elegans SERCA containing the KDDKPV (SEQ ID NO:39) recognition site was constructed using standard site directed mutagenesis technology (QuikChange Site-Directed Mutagenesis Kit of Stratagene). The primers oGK118 and oGK119 and plasmid pGK28 were used to obtain a plasmid designated pGK115 containing thapsigargin-resistant C. elegans SERCA with the KDDKPV (SEQ ID NO:39) site.
oGK118:GCCAGTCGGAAAGGTTTCCA[0198] AGGACGACAAGCCAGTTAACCCAGCT GCTGGAGAATT (SEQ ID NO:31)
oGK119:AATTCTCCAGCAGCTGGGTT[0199] AACTGGCTTGTCGTCCTTGGAAACCTTT CCGACTGGC (SEQ ID NO:32)
(Nucleotides leading to the mutated SERCA are underlined.) [0200]
Plasmid pGK115 was introduced into [0201] C. elegans using standard techniques. Introduction of this mutant SERCA into C. elegans results in a more efficient interaction between the C. elegans SERCA (here the mutant) and vertebrate PLB. Introduction of a KDDKPV (SEQ ID NO:39) mutant into a C. elegans strain which is mutant for SERCA, such as the ok190 strain, results in a strain which is directly useful for performing screens to select for compounds that alter the interaction of SERCA with PLB and hence that alter the activity of SERCA. Since pGK115 also contains the thapsigargin resistance mutation, it can also be expressed in a wild-type C. elegans for use in screens to select for compounds that alter the interaction of SERCA with PLB and hence that alter the activity of SERCA. In such a screen, thapsigargin should be added to differentiate between activity of the endogeneous SERCA and the introduced double mutant SERCA.

EXAMPLE 10

Construction of Fusion Proteins between [0202] C. elegans SERCA and Vertebrate SERCA.
The introduction of vertebrate SERCA in [0203] C. elegans, the latter being a SERCA mutant such as ok190 or a wild-type strain where the endogenous SERCA is inhibited for example by RNAi technology, will result in rescue of the mutant phenotypes, but maybe not to the full extent. This could be due, for example, to different kinetic properties of C. elegans and vertebrate SERCA, or to interaction with other partners than phospholamban. Using fusion proteins will overcome this problem. A fusion protein may be constructed that has sufficient properties of the C. elegans SERCA for rescue of the mutant phenotype, and has those vertebrate SERCA properties sufficient in a screen to select for compounds that alter the vertebrate SERCA activity.
At least four types of fusion proteins are contemplated: [0204]
1) A fusion protein harboring the N-terminal end of the [0205] C. elegans and the C-terminal part of a vertebrate SERCA
2) A fusion protein harboring the N-terminal part of a vertebrate SERCA and the C-terminal part of the [0206] C. elegans SERCA
3) A fusion protein harboring the C- and N-terminal part of the [0207] C. elegans SERCA and an internal part of a vertebrate SERCA. This construction can be considered as a variant of the KDDKPV (SEQ ID NO:39) mutation described above.
4) A fusion protein harboring the C- and N-terminal part of a vertebrate SERCA and an internal part of the [0208] C. elegans SERCA
Such fusion proteins can easily be constructed using standard molecular techniques. [0209]
A SERCA fusion protein of [0210] type 1 has been made in the following way:
A PCR reaction was performed on plasmid pGK110 (harboring the pig thapsigargin resistant SERCA2A) using primers oGK108 and oGK109. [0211]
oGK108: GACCGTACGAAATTTTCAGGAAAGGAATGCAGAAAATGCC (SEQ ID NO:33) [0212]
oGK109: CCCCGGCCGGCCTTACTCCAGTATTGCAGGTTCCAGG (SEQ ID NO:34) [0213]
The resulting 2701 bp PCR fragment was digested with BsiWI and EagI and cloned in the 10131 bp fragment of pGK8 (containing genomic [0214] C. elegans SERCA) cut at the same sites. The resulting vector was designated pGK114.

EXAMPLE 11

Construction of Phospholamban Mutants that Show Altered Interaction with SERCA. [0215]
The interaction of phospholamban with SERCA has been described very extensively in the literature. Furthermore mutants of PLB have been described that have an enhanced or a diminished interaction with SERCA and hence have a stronger or weaker inhibitory effect on SERCA (Toyofuku et al., J. Biol. Chem 1993, 269:3088-3094; Kimura et al., J. Biol. Chem. 1996, 271:21726-21731; Kimura et al., J. Biol. Chem. 1997, 272:15061-15064; Kimura et al., J. Biol. Chem. 1998, 273:14238-14241). [0216]
Introduction of a PLB mutant with an altered inhibition of SERCA in [0217] C. elegans can improve the basic screen to select for compounds that alter the interaction of PLB and SERCA in such a way that the parameters of the screen can be fine-tuned exactly as is most useful, allowing screening for more specific compounds directed to the PLB SERCA interaction.
PLB mutations can easily be made using standard site directed mutagenesis techniques as described above, and as known in the art. [0218]
One phospholamban mutant of particular interest is Ser16Ala. In intact beating hearts or isolated cardiac myocytes, serine16 becomes phosphorylated by cAMP-dependent protein kinase upon stimulation with isoproterenol. This leads to increased cardiac relaxation due to decreased inhibition. A phospholamban mutant for this phosphorylation site thus lacks cAMP-dependent protein kinase-mediated regulation (Simmerman et al., J. Biol. Chem. 1986, 261:13333-13341; Wegener et al., J. Biol. Chem. 1989, 264:11468-11474; Kuschel et al., Am. J. Physiol. 1999, 276:H1625-H1633). [0219]

EXAMPLE 12

Cloning of Pig PLB, Construction of Humanized Pig PLB. [0220]
Pig PLB cDNA was cloned from pGEM7PigPLB (Wuytack, personal gift) by PCR amplification using the primer combinations listed below. PCR amplification was performed using standard procedures (PCR, A practical approach, ed. by M. J. McPherson, P. Quirke and G. R. Taylor, 1993, Oxford University Press. Summary of primer combinations: [0221]
oGK51-oGK55: pg PLB, including stop codon [0222]
oGK51-oGK56: pig PLB, excluding stop codon (open ended) [0223]
oGK52-oGK55: humanized pig PLB, including stop codon [0224]
oGK52-oGK56: humanized pig PLB, excluding stop codon (open ended) [0225]
oGK51 and oGK52 contain an XbaI site for cloning [0226]
oGK55 and oGK56 contain an Asp718 site for cloning [0227]
oGK52 contains T-to-G point mutation compared to pig PLB cDNA so as to introduce a D-to-E amino acid substitution at position 2 of PLB. Since this is the only difference between the human and pg PLB proteins, the resultant polypeptide is the same as the human PLB sequence (NB the point mutated cDNA does not have the same sequence as the human PLB cDNA but encodes a protein having identical amino acid sequence to human PLB, hence it is referred to as a humanized pig PLB cDNA). Sequences of the primers are as follows: [0228]
oGK51: GCTCTAGATGGATAAAGTCCAATACCTCAC (SEQ ID NO:35) [0229]
oGK52: GCTCTAGATGGAGAAAGTCCAATACCTCAC (SEQ ID NO:36) [0230]
oGK55: GGGGTACCTCAGAGAAGCATCACGATGATG (SEQ ID NO:37) [0231]
oGK56: GGGGTACCATGAGAAGCATCACGATGATGCAAATC (SEQ ID NO:38) [0232]
pGK202 was constructed by cloning the oGK51-oGK55 PCR fragment digested with XbaI and Asp718 into pPd96.48 digested with the same enzymes. The vector expresses pig PLB under the control of the myo-2 promoter. [0233]
pGK204 was constructed by cloning the oGK51-oGK56 PCR fragment digested with XbaI and Asp718 into pGK203 digested with the same enzymes. The vector expresses the pig PLB fused to GFP under the control of the myo-2 promoter. [0234]
pGK205 was constructed by cloning the oGK52-oGK55 PCR fragment digested with XbaI and Asp718 into pPD96.48 digested with the same enzymes. The vector expresses the humanized pig PLB under the control of the myo-2 promoter. [0235]
pGK206 was constructed by cloning the oGK52-oGK56 PCR fragment digested with XbaI and Asp718 into pGK203 digested with the same enzymes. The vector expresses the humanized pig PLB fused to GFP under the control of the myo-2 promoter. [0236]
pGK302 was constructed by cloning the oGK51-oGK55 PCR fragment digested with XbaI and Asp718 into pPD96.52 digested with the same enzymes. The vector expresses the pig PLB under the control of the myo-3 promoter. [0237]
pGK304 was constructed by cloning the oGK51 -oGK56 PCR fragment digested with XbaI and Asp718 into pGK303 digested with NheI-Asp718. The vector expresses the pig PLB fused to GFP under the control of the myo-3 promoter. [0238]
pGK305 was constructed by cloning the oGK52-oGK55 PCR fragment digested with XbaI and Asp718 intointo pPD96.52 digested with NheI-Asp718. The vector expresses the humanized pig PLB under the control of the myo-3 promoter. [0239]
pGK306 was constructed by cloning the oGK52-oGK56 PCR fragment digested with XbaI and Asp718 into pGK303 digested with NheI-Asp718. The resulting vector expresses the humanized pig PLB fused to GFP under the regulation of the myo-3 promoter. [0240]

EXAMPLE 13

Inhibition of SERCA by Compounds. [0241]
Several compounds are known to inhibit the function of SERCA, such as cyclopiazonic acid, cyproheptadine, thapsigargin, 2,5-di (tert-butyl)-1,4-benzohydroquinone, 2.4-benzoquinone, and vanadate. Other compounds are known to activate the activity of SERCA, such as diethylether, gingerol, and 1-(3,4-dimethoxyphenyl)-3-dodecanone. Still other compounds have a dual activity, they stimulate SERCA at low concentrations, but inhibit at high concentrations, such as phenothiazines, and pentobarbital. [0242]
Using two kinds of assays, the optimal concentration of compounds that inhibit the activity SERCA has been determined. The first assay is designated the drop or plate assay in which the nematodes are fed [0243] E. coli strains pre-loaded with the compound. In a second assay, the compound is administrated to the worm in liquid culture.
Plate Assay [0244]
A standard plate drop assay is performed according to the following protocol. 4 ml NGM agar (see “The nematode [0245] C. elegans” Ed. by William B. Wood and the Community of C. elegans Researchers, CSHL Press, 1988, pg589) is into 3 cm plates and seeded with approximately 5 μl of an E. coli overnight culture and grown preferably for one week at room temperature. Approximately 10 μl of test compound dissolved in DMSO or other suitable solvent is pipetted onto the bacterial lawn so that the lawn is covered completely. After overnight soaking in or compound, one C. elegans (L4 stage) per plate is put onto the bacterial lawn. Plates are incubated at 21° C. and checked after some hours. Plates are checked again after 4 days for phenotypes of the F1 progeny (control shows all stages up to gravid hermaphrodites).
Thapsigargin at various concentrations (5 μM, 2.5 μM and 1.25 μM) causes the nematode to stop pharynx pumping within 10 min. Within an hour the worms restart pumping, although at a low level. The worms are pale and thin and have a stow and irregular movement, with an increased amplitude. No plate drop response is observed, and the worms show poor backing, reduced pumping and strong constipation. The worms have a defective gonad with only very few eggs, and a protruding vulva. Some worms also have a protruding rectum. Progeny reaches L2 stage only after four days, and the brood size is very small. Lower concentrations of thapsigargin (0.5 μM, 0.25 μM, 0.125 μM) still cause reduced brood size. [0246]
2,5-di-tert butylhydroquinone at a concentration of 500 μM resulted in pale, starved, thin worms with slow movement, defective gonad, constipated and reduced brood size. [0247]
Cyclopiazonic acid at a concentration of 500 μM resulted in nematodes that lay still or move slowly after one hour. The worms showed strong avoidance and after 24 hours they look starved, pale and thin, with only a few eggs in the body, a defective gonad, and reduced brood size. A delayed growth of the F1 generation was observed. [0248]
Thapsigargicin at 500 μM, 125 μM, 31 μM, 10 μM, 5 μM resulted in nematodes with similar phenotypes to those described above for thapsigargin at 5 μM, 2.5 μM, 1.25 μM. Lower concentrations of thapsigargicin (3 μM and 1.5 μM) caused a slightly reduced brood size. [0249]
Thapsigargin-epoxide did not result in a clear observable effect, even at the highest concentration tested (1 mM drop, 5 μM end concentration). [0250]
1,4-benzoquinone did not result in a clear observable effect, even at the highest concentration tested (100 mM drop, 500 μM end concentration). [0251]
Liquid [0252]
Thapsigargin at 100, 50 and 20 μM resulted in small worms which show slow and loopy movement. They had a protruding vulva, and no progeny (or no progeny that grows up) were observed. At lower concentrations of 10 μM and 5 μM a reduced number of progeny and delayed growth could be observed. [0253]
2,5-di-tert butylhydroquinone at a concentration of 1 mM resulted in progeny exhibiting delayed growth and the worms be observed to be thinner than ‘normal’ worms. [0254]
Cyclopiazonic acid at a concentration of 1 mM resulted in pale, thin worms with a slow movement and a very strongly reduced brood size. At lower concentrations of 0.5 mM, growth delay was observed. [0255]
Thapsigargicin at 1000 μM, 250 μM, 62.5 μM and 16 μM concentrations resulted in small worms with slow and loopy movement, a protruding vulva, and no progeny (or no progeny that grows up) were observed. At lower concentrations of 10 μM, delayed growth and reduced progeny were observed. [0256]
The effect of thapsigargin on progeny of wild-type strains was tested with the liquid assay: On an average of 12 worms, the number of progeny for the different concentrations is summarized in FIG. 1. [0257]
The effect of thapsigargin was also tested on progeny of wild-type strains using the plate assay: On an average of 12 worms the number of progeny at different concentrations is summarized in FIG. 2. [0258]
The effect of thapsigargin on the production of progeny was determined for a number of different [0259] C. elegans strains. The numbers of progeny produced following thapsigargin treatment was counted for an average of 15 animals, the results are summarised as follows:
unc-31: control: 132 0.5 mM: 35 1 mM: 5,6 [0260]
srf-3: control: 50 1 mM :18,3 [0261]
The effect of thapsigargin on pharynx pumping behaviour was also determined. In wild-type worms, all animals stopped pumping after 10 minutes. In mutant strain unc-31 at a concentration of 1 mM thapsigargin, all worms stopped pumping after 10 minutes, some start again after half an hour, but pumping is only one third of normal speed. [0262]
In summary, the above experiments demonstrate that inhibition of [0263] C. elegans SERCA activity using thapsigargin or other chemical inhibitors of SERCA results in worms with recognisable phenotypic characteristics, including paleness, reduced growth, reduced rate of pharynx pumping and reduced numbers of progeny.

EXAMPLE 14

Screening for Antagonists of a Compound (Thapsigargin) [0264]
The compound thapsigargin is known to inhibit the activity of SERCA. The SERCA protein pumps calcium into the sarco/endoplasmic reticulum and provides the cell with an internal storage of calcium. The internal storage of calcium is important for muscle activity. In [0265] C. elegans, inhibiting SERCA activity by applying thapsigargin to the worm results in a decrease in the pharynx pumping rate. Another feature observed by the action of thapsigargin on the nematode worm C. elegans is decreased movement, which is a result of the inhibition of SERCA activity of the body wall muscles.
A pharynx pumping screen has been developed to screen for chemical substances that suppress the activity of thapsigargin on SERCA. In this screen the pumping rate of the pharynx is measured indirectly by adding a marker molecule precursor such as calcein-AM to the medium and measuring the formation of marker dye in the [0266] C. elegans gut. Calcein-AM is cleaved by esterases present in the C. elegans gut to release calcein, which is a fluorescent molecule. The pumping rate of the pharynx will determine how much medium will enter the gut of the worm, and hence how much calcein-AM will enter the gut of the worm. Therefore by measuring the accumulation of calcein in the nematode gut, detectable by fluorescence, it is possible to determine the pumping rate of the pharynx.
A standard pharynx pumping screen may be carried out as follows: [0267]
1) Dispense substantially equal numbers of [0268] C. elegans nematodes into the wells of multi-well assay plates. A ‘worm dispenser’ apparatus, e.g. the device commercially available from Union Biometrica, Inc, Somerville, Me., USA which has properties analogous to flow cytometers, such as fluorescence activated cell scanning and sorting devices (FACS), may be used for this purpose. Typically, 40+/−5 worms are added to each well of the microtiter plate.
2) Thapsigargin is added to the worms at an inhibitory concentration and calcein-AM is added at a concentration of 5-10 μM. [0269]
3) The chemical substances to be selected are added. Control wells are also set up containing thapsigargin alone with no second chemical substance. The chemical substances are typically made up in DMSO. Any other solvent can be used for this purpose, but most selected chemical substances appear to be soluble in DMSO. The chemical substance is added in the wells at various concentrations. but preferentially a concentration between 3 to 30 μM is chosen as this gives the clearest results. It possible to screen for dosage effects by varying the concentration of the chemical substance from less than 1 μM up to 100 μM. [0270]
The concentration of the DMSO should not be too high and preferentially should not exceed 1%, more preferentially the concentration of the DMSO should not exceed 0.5% and even more preferentially, the concentration of the DMSO is lower than 0.3%. [0271]
4) Fluorescence intensity is measured using a multi-well plate reader (e.g. Victor2, Wallac Oy, Finland) with following settings: Ex/Em=485/530. [0272]
Wells harboring a chemical substance where the measured fluorescence is higher than in the control wells containing no chemical substance are scored. These wells harbor a chemical substance that is an antagonist of the thapsigargin activity, as the inhibitory activity of thapsigargin is suppressed. Chemical substances thus identified may inhibit directly the activity of thapsigargin, or stimulate the activity of SERCA, or have an enhancer activity on the SERCA pathway, and hence on the calcium biology of the organism. [0273]
Chemical substances selected in this screen as antagonists of thapsigargin are considered as potential therapeutics, or as hits for the further development of therapeutics in the disease areas which are the cause of a malfunction of the calcium biology of the organism. Examples of disease areas for which these therapeutics are useful are cardiac hypertrophy, cardiac failure, arterial hypertension, Type 2 diabetes and Brody disease. [0274]
In the example given above, thapsigargin is used as an example of a compound having a defined phenotypic effect on [0275] C. elegans as a result of inhibition of SERCA activity. It will be appreciated that other SERCA inhibitors which have an inhibitory activity on the pharynx pumping rate may be used in analogous screens with equivalent effect.

EXAMPLE 15

Screening for Chemical Substances in Transgenic, Mutant and Humanized Animals (SERCA-PLB) [0276]
An increase of the internal storage of calcium is general considered to be important for the strength of muscle contraction, and consequently an improvement or increase of this muscle contraction can be realized by enhancing SERCA activity. Chemical substances that enhance SERCA activity or inhibit the SERCA-PLB interaction are considered as potential therapeutics, or as hits for the further development of therapeutics in the disease areas which are the cause of a malfunction of the calcium biology of the cell or organism. Examples of disease areas where an increase of SERCA activity may be beneficial are cardiac hypertrophy, cardiac failure, arterial hypertension, Type 2 diabetes and Brody disease. [0277]
The different SERCA genes and isoforms which are associated with different types of diseases; SERCA2 and PLB are associated with cardio-vascular diseases, SERCA1 and sarcolipin are associated with skeletal-muscle diseases, and three SERCA genes have been associated with non-insulin-dependent diabetes mellitus. [0278]
In order to perform screens to identify chemical substances which modulate the activity of SERCA pathways SERCA genes and PLB have been expressed in [0279] C. elegans. The expression of these genes can be regulated under the control of several specific promoters with the following activities:
a) The [0280] C. elegans myo-2 promoter which promotes expression in the pharynx
b) The [0281] C. elegans SERCA promoter which promotes expression in the C. elegans muscles, including the pharynx, the vulva muscles and the body wall muscles.
The following transgenics were constructed: [0282]
a) pig and/or human SERCA under the SERCA and/or myo-2 promoter. [0283]
b) pig and/or human SERCA under the SERCA and/or myo-2 promoter in a [0284] C. elegans mutated for the C. elegans SERCA (Knock-outs and selected mutants).
c) pig and/or human PLB under the SERCA and/or the myo-2 promoter. [0285]
d) pig and/or human PLB under the SERCA and/or the myo-2 promoter in a [0286] C. elegans mutated for the C. elegans SERCA (Knock-out and selected mutants).
e) pig and/or human PLB-GFP fusion under the SERCA and/or the myo-2 promoter. [0287]
f) pig and/or human PLB-GFP fusion under the SERCA and/or the myo-2 promoter in a [0288] C. elegans mutated for the C. elegans SERCA (Knock-outs and selected mutants).
g) pig and/or human SERCA under the SERCA promoter and pig and/or human PLB under the myo-2 promoter. [0289]
h) pig and/or human SERCA under the SERCA promoter and pig and/or human PLB under the myo-2 promoter in a [0290] C. elegans mutated for the C. elegans, SERCA (Knock-out and selected mutants).
i) pig and/or human SERCA under the SERCA promoter and pig and/or human PLB-GFP under the myo-2 promoter. [0291]
j) pig and/or human SERCA under the SERCA promoter and pig and/or human PLB-GFP under the myo-2 promoter in a [0292] C. elegans mutated for the C. elegans SERCA (Knock-out and selected mutants).
Some of these constructed transgenic and mutant worms show a clear change in pharynx pumping rate as can be measured by the fluorescence of calcein in the gut using the calcein-AM pharynx pumping assay. Some of these strains were considered to be useful for further screen development. To perform the pharynx pumping assay, the transgenic and mutant animals were placed in the wells of multi-well plates. Calcein-AM and chemical substances under test were then added. The fluorescence of the calcein formed in the gut was measured in a multi-well plate reader set to measure fluorescence. Chemical substances that altered the properties of the pharynx pumping rate, and hence altered the function and activity of the SERCA pathway were selected for further analysis, and can be considered as potential compounds for therapeutic use, or as hits for the further development of therapeutics. [0293]
A analogous experiment can be performed with the SERCA1 gene and its regulator Sarcolipin (SLN), to detect chemical substances that alter their activity and/or regulation. [0294]

EXAMPLE 16

Construction of Plasmids. [0295]
The ‘pPD’ series of vectors were all obtained from the laboratory of Andrew Fire, see Fire A, Harrison S. W., and Dixon D. A modular set of LacZ fusion vectors for studying gene expression in [0296] Caenorhabditis elegans. 1990. Gene 93:189-198. The sequences of these vectors are freely available at ftp://stein.cshl.org/pub/elegans_vector/).
pGK301 was constructed by cloning a 3181 bp fragment of pERIItA (F. Wuytack, personal communication) into pPD96.52 digested with the same restriction enzymes. pGK301 expresses the SERCA2a cDNA under the regulation of the myo-3 promoter. [0297]
pGK201 was constructed by cloning a 480009 bp NheI/SpeI fragment of pGK301 in pPD96.48 digested with the same enzymes. The vector expresses pig SERCA2a under the regulation of the moy-2 promoter. [0298]
pGK101 was constructed by cloning a 4828bp NheI/ApaI fragment of pGK201 into plasmid pDW2600 digested with the same enzymes. The vector expresses the pig SERCA2a cDNA under the regulation of the worm SERCA promoter. [0299]
pDW2600 was constructed by cloning a 5046bp SphI-SmaI fragment of pGK10 in pPD49.26. [0300]
pGK203 was constructed by cloning the AccI/SpeI fragment of pPD95.79 into pPD96.48 digested with the same enzymes. This vector contains the myo-2 promoter, GFP and unc-54 3′UTR. [0301]
pGK303 was constructed by cloning the Asp718-ApaI fragment of pPD95.79 into pPD96.52 digested with the same enzymes. This vector contains the myo-2 promoter, GFP and unc-54 3′UTR. [0302]
List of Genbank Accession Numbers for SERCA and PLB cDNA Sequences: [0303]
pig SERCA2a GenBank P11606 [0304]
human SERCA1a GenBank AAB 53113 [0305]
human SERCA1b GenBank AAB 53112 [0306]
human SERCA2a GenBank P16614 [0307]
human SERCA2b GenBank P16615 [0308]
human SERCA3 GenBank Q93084 [0309]
pig PLB GenBank P07473 [0310]
human PLB GenBank P26678 [0311]
Sequence Listing [0312]
SEQ ID NO:1 is the nucleic acid sequence of a 732 bp EcoRI-HindII fragment of [0313] C. elegans SERCA exon 5. This fragment was cloned into pGEM3 for use in RNA inhibition experiments.
SEQ ID NO:2 is the nucleic acid sequence of a 11207 bp SpeI-MluI fragment of cosmid K11D9. This fragment contains the complete [0314] C. elegans SERCA gene with 5631 bp of upstream sequence, the entire coding region and 1088 bp of downstream sequence. The fragment was cloned into pUC18 to give plasmid pGK7.
SEQ ID NO:3 is the nucleic acid sequence of a 5026 bp fragment of the upstream region of [0315] C. elegans SERCA, up to and including A of the initiating ATG. This fragment was cloned into pPD95.79, in fusion with GFP, to give plasmid pGK10.
SEQ ID NO:4 is the nucleic acid sequence of a 2915 bp fragment of the upstream region of [0316] C. elegans SERCA, as found in plasmid pGK13.
SEQ ID NO:5 is the nucleic acid sequence of a 6612 bp fragment of the [0317] C. elegans SERCA gene containing 5637 bp of upstream sequence and ending in exon 4, as cloned in pPD95.75, resulting in pGK12.
SEQ ID NO:6 is the nucleic acid sequence of the long isoform of the [0318] C. elegans SERCA cDNA.
SEQ ID NO:7 is the nucleic acid sequence of the pig SERCA2a cDNA. [0319]
SEQ ID NO:8 is the nucleic acid sequence of the human SERCA2a cDNA. [0320]
SEQ ID NO:9 is the nucleic acid sequence of the pig phospholamban cDNA. [0321]
SEQ ID NO:10 is the nucleic acid sequence of the [0322] C. elegans myo-2 promoter.
SEQ ID NO:11 is the nucleic acid sequence of the [0323] C. elegans myo-3 promoter.
SEQ ID NO:12 is the nucleic acid sequence of the [0324] C. elegans vulval muscle enhancer. This is an enhancer element from ceh-24 that directs gene expression in the vulval muscles (Harfe and Fire, 1998, Developmental 125: 421-429)
SEQ ID NO:13 is the nucleic acid sequence of humanized pig PLB cDNA. [0325]
SEQ ID NO:14 is the amino acid sequence of pig PLB. [0326]
SEQ ID NO:15 is the amino acid sequence of human PLB and humanized pig PLB. [0327]
SEQ ID NO:16 is the nucleotide sequence of a genomic fragment of [0328] C. elegans SERCA covered by primers SERCA P4 and SERCA P8.
SEQ ID Nos: 17-38 are primers used in the accompanying Examples. [0329]
All references disclosed herein are incorporated by reference in their entirety. [0330]
1 39 1 732 DNA Caenorhabditis elegans 1 gaattcgaat cactcaccga gttggccatg atctgcgcta tgtgcaatga ttcatctgtt 60 gattacaatg agaccaagaa gatctacgag aaagtcggag aagccactga aactgctctt 120 atcgttcttg ctgagaagat gaatgttttc ggaacctcga aagccggact ttcaccaaag 180 gagctcggag gagtttgcaa ccgtgtcatc caacaaaaat ggaagaagga gttcacactc 240 gagttctccc gtgatcgtaa atccatgtcc gcctactgct tcccagcttc cggaggatct 300 ggagccaaga tgttcgtgaa gggagcccca gaaggagttc tcggaagatg cacccacgtc 360 agagttaacg gacaaaaggt tccactcacc tctgccatga ctcagaagat tgttgaccaa 420 tgcgtgcaat acggaaccgg aagagatacc cttcgttgtc ttgccctcgg aaccatcgat 480 accccagtca gcgttagcaa catgaacctc gaagactcta cccaattcgt caaatacgaa 540 caagacatca catttgtcgg agtcgtcgga atgcttgacc ccccaagaac tgaagtttcg 600 gactcgatca aggcttgtaa ccacgctgga atccgtgtca tcatgatcac cggagacaac 660 aagaacaccg ctgaggctat cggaagaaga atcggactct tcggagagaa cgaggatacc 720 actggaaagc tt 732 2 11207 DNA Caenorhabditis elegans 2 ctagttttga aatccaaaaa aaaaacaaag ttcaataaaa tgttacccaa ttgtgcgatt 60 tttgctttaa aaatacggta cccggtctcg atgcggcaat tgtttggtaa atgtaaaagg 120 gtgtgcgcct ttaaagagta ctgtaatttc aatcttccga cactgctgaa tttttattga 180 ctttttgttc attaatttta tatatgattt attggtatgt taaaaaaaca cccattttca 240 aatctattaa aattccacaa caacaaaagt tcgagattac agtacttttt agaggcgcac 300 atcctttttg ggatactaaa caattgtcgc gtcgagacca ggtaccatat ttccaaaaca 360 caatttcgcg tgtaaataaa aaatatcaac ataataattt ccatttttcg aaatttaaag 420 ttaatcactt tttggtttag attatgattt cacacgtttt tttccttcta gttctctttt 480 ttttgttatt tgcctgaaaa atggtctgaa aacttaggca atcagcaatg tgtcacataa 540 tttctcccag agaaatccct ttcaacaaaa tctcccggat tgacctgtgt gctcgacctt 600 gataaattgg ttggcagctc tctggcttat cttttgagag gaaaaagatc caacaaattt 660 ttatctccct tatccctttt tctcttcatc actaccaata ataatagttt tttttttcgt 720 cgcggaagca aaatggcgaa caagtgttgg aataagagta ctccagggat ttaagggctg 780 aaagccagtg atttatgagc tccaattttt cagatgtttt ttcctccatc gcgtatttgt 840 ctaaacattc gattttcttc ctgcttccca acttttcaaa tcgaaataaa agagcatctg 900 tcgcttttta tcgatgtgct tctgtgagac taaagaacta ctcgttttca ctcgttctct 960 ctctctcaac tatcaaagtt ttgttgattg cgtgtgtcag cttccttctt tttattatca 1020 tcttttcatt ggaggaaaaa aataacttct gaagagcaaa agaactaact tcggggaata 1080 cagagaaaat tcctgtaaaa atctggaaat tttttcgctt aactcgaaat atttagtttt 1140 tcactgtgat ttctgggaaa aatcaagaaa tatttgccta aaacacgagt tttcacatga 1200 aaaatgaatt atttattgat tttttatgga gattacaaaa aagacacacg tgaaactact 1260 gctaccgtag ttgtgtaaac gtagtgttct ctattttaga cctgtttaat gtattttttt 1320 gcagttgaaa atttttaaaa atattttagt tatttttaaa aatatttaat ttacaaaata 1380 attagcctga acccatgaaa agatacgtta tatttaattt ttaccgtaag actttcaaga 1440 tcgttgcgag acccggcgcc taggtcaaag agcctccctt taaacccatc aacacgtttt 1500 gcctttttca tcgatttttt gcagttcttt tcttctttcc aactgatttt tcttcatttt 1560 taaagttttt ttcctcattt ttcccatttg aaattattta aacacgtgca accagctggt 1620 aacatgtgtc acatgccgtt atctaacttc aaaacagtac atttccgatc acacgtcccc 1680 cgcgccgagt tttatagttt cattaataac ttttcggttt ttgataatac taattgagtt 1740 ttattaattg tttccatatt catctagcac tttgacctgt ccttcttcga attctcaaat 1800 atttgcactc tgggtttagg tgtgaaaaga attgtcgtca ttaagcgggg catccggggc 1860 accgaaaaaa gccctccgat tttaacgaat ttgagataaa gttggagaga gagcccagtg 1920 tttgcttgcc caagatatat atcttggatt tatcaattac tgtttgtcaa cctgtcgccg 1980 gcgccccctt tttgctcttg ctcccacgcc ccgagattga atttcaattt tatttcgaag 2040 taagtctctt gattgtttcg aaaatccgat gacagttttc attacttttt tgtctgttga 2100 ttttgtaggg aaacattgaa atttttctga tctttctttg atcttatgat ttttcattta 2160 ttccaattaa aaaaaattag cgcattcaga accagagtga agcttgagat gttgtaggtt 2220 tatcaaaaga tcaaaatctc gaattccttc gaaatgtttt tagttttcga cttccgtgtg 2280 atttctagcg atcctgacag agatcactga attttaatgt tatcgagatt gttgtgtagg 2340 ctccatctcc tctctgaagc ttctgatttt gccgaaagtc tagttacttg ccgactgctg 2400 acactaggat atcccactac cgtacccatt gttggatccg tactctgctg cgacttcttc 2460 tctgtttcac gtgaacctcc gggatcgtcg gtaagccccg cccgttatct gtgccaactt 2520 gtcttcgtgc cctcgagcga cgagctcatt caatcacgcc acgacctccg tctggacaga 2580 tgctctcatt gtctctgcgt ctccaagtat tcgtcacact atctcatgca ttctattcaa 2640 aacgcgagag aaagcgcggg aacgagagag agttcagaca gatcgaactt gtttttatcc 2700 ccccccccct cgtccggctg cagagcaaaa aaatactgct tttccttgca aaattcggtg 2760 ctttcttcaa agagaaactt ttgaagtcgg cgcgagcatt tccttctttg acttctctct 2820 ttccgccaaa aagcctagca tttttattga taatttgatt acacacactc agagttcttc 2880 gacatgataa agtgtttcat tggcactcgc cctaacagta catgacaagg gcggattatt 2940 atcgatcgat attgaagaca aactccaaat gtgtgctcat tttggagccc cgtgtggggc 3000 agctgctctc aatatattac tagggagacg aggaggggga ccttatcgaa cgtcgcatga 3060 gccattcttt cttctttatg cactctcttc actctctcac acattaatcg attcatagac 3120 tcccatattc cttgatgaag gtgtgggttt ttagcttttt ttcccgattt gtaaaaggaa 3180 gaggctgacg atgttaggaa aaagagaacg gagccgaaaa aacatccgta gtaagtcttc 3240 cttttaagcc gacacttttt agacagcatt cgccgctagt tttgaagttt aaattttaaa 3300 aaataaaaat tagtttcaat tttttttaat tactaaatag gcaaaagttt tttcaagaac 3360 tctagaaaaa ctagcttaat tcatgggtac tagaaaaatt cttgttttaa atttaatatt 3420 tatcttaaga tgtaattacg agaagctttt ttgaaaattc tcaattaaaa gaatttgccg 3480 atttagaata aaagtcttca gaaatgagta aaagctcaaa ttagaagttt gtttttaaag 3540 gaaaaacacg aaaaaagaac actatttatc ttttcctccc cgcgtaaaat tagttgttgt 3600 gataatagtg atccgctgtc tatttgcact cggctcttca caccgtgctt cctctcactt 3660 gacccaacag gaaaaaaaaa catcacgtct gagacggtga attgccttat caagagcgtc 3720 gtctctttca cccagtaaca aaaaaaattt ggtttcttta ctttatattt atgtaggtca 3780 caaaaaaaaa gtgatgcagt tttgtgggtc ggttgtctcc acaccacctc cgcctccagc 3840 agcacacaat catcttcgtg tgttctcgac gattccttgt atgccgcggt cgtgaatgca 3900 ccacattcga cgcgcaacta cacaccacac tcactttcgg tggtattact acacgtcatc 3960 gttgttcgta gtctcccgct ctttcgtccc cactcactcc tcattattcc ccttggtgta 4020 ttgatttttt ttaaatggta caccactcct gacgtttcta ccttcttgtt ttccgtccat 4080 ttagatttta tctggaaatt tttttaaaat tttaggccag agagttctag ttcttgttct 4140 aaaagtctag gtcagacata cattttctat ttctcatcaa aaaaaaagtt gataaagaaa 4200 actggttatt cagaaagagt gtgtctcgtt gaaattgatt caaaaaaaaa ttcccacccc 4260 tcgcttgttt ctcaaaatat gagatcaacg gattttttcc ttctcgattc aattttttgc 4320 tgcgctctgt ctgccaaagt gtgtgtgtcc gagcaaaaga tgagagaatt tacaaacaga 4380 aatgaaaaaa agttggccaa ataatgaagt tttatccgag attgatggga aagatattaa 4440 tgttctttac ggtttggagg ggagagagag atagattttc gcatcaaact ccgcctttta 4500 catgtctttt agaatctaaa atagattttt ctcatcattt ttaatagaaa atcgagaaat 4560 tacagtaatt tcgcaatttt cttgccaaaa atacacgaaa tttgtgggtc tcgccacgat 4620 ctcggtctta gtggttcatt tggtttaaaa gtttataaaa tttcaaattc tagtgtttaa 4680 tttccgcata attggaccta aaatgggttt ttgtcatcat tttcaacaag aaatcgtgaa 4740 aatcctgttg tttcgcaatt ttcttttcaa aaatacacga aatatatggt aatttcccga 4800 aatattgagg gtctcgccac gatttcagtc acagtggcca ggatttatca cgaaaaaagt 4860 tcgcctagtc tcacatttcc ggaaaaccga atctaaatta gttttttgtc atcattttga 4920 acaaaaaatc gagacatccc tatagtttcg caattttcgt cgcttttctc tccaaaaatg 4980 acagtctaga attaaaattc gctggaactg ggaccatgat atcttttctc cccgtttttc 5040 attttatttt ttattacact ggattgacta aaggtcacca ccaccgccag tgtgtgccat 5100 atcacacaca cacacacaca caatgtcgag attttatgtg ttatccctgc ttgatttcgt 5160 tccgttgtct ctctctctct attcatcttt tgagccgaga agctccagag aatggagcac 5220 acaggatccc ggcgcgcgat gtcgtcggga gatggcgccg cctgggaagc cgccgagaga 5280 tatcagggaa gatcgtctga tttctcctcg gatgccacct catctctcga gtttctccgc 5340 ctgttactcc ctgccgaacc tgatatttcc cgttgtcgta aagagatgtt tttattttac 5400 tttacaccgg gtcctctctc tctgccagca cagctcagtg ttggctgtgt gctcgggctc 5460 ctgccaccgg cggcctcatc ttcttcttct tcttctctcc tgctctcgct tatcacttct 5520 tcattcattc ttattccttt tcatcatcaa actagcattt cttactttat ttattttttt 5580 caattttcaa ttttcagata aaaccaaact acttgggtta cagccgtcaa catggaggac 5640 gcgcatgcca aagacgccaa tgaggtactt ttatagtttt taaattttag tttttaatac 5700 aatttatttt ccaggtgtgc aaattcttcg gaacgggtcc ggagggattg actccacagc 5760 aagttgaaac attgaggaac aaatatggag aaaatggttg gttttttaca tggatttctc 5820 attaaaaatt gaattttttc cagaaatgcc cgccgaagag ggaaaatcac tgtgggagct 5880 gattctcgag caattcgacg atcttctcgt caagattctc ctcctcgccg ccatcatctc 5940 gtttgtgctc gcccttttcg aagagcacga agatcagaca gaagcagtga cggcgttcgt 6000 cgaaccgttc gtcatccttc tcattcttat tgccaacgcg accgtcggag tgtggcaggt 6060 aggaacaaca cagacaggcg cacgcgctga aagaaaataa gaagaagaag aaaaagcaca 6120 gttgttttct gtgtttttgt agatcaaaag aaaggaacta ggagtgattg cacagagaga 6180 gagagagaga aataatgtct ttttgacttg tttttgttgg tgagagagat agggaaaaag 6240 agtccctaaa gaaaaaatag tgtaacgggc ggtccggaag aaatgctctt tgcgccgaaa 6300 agtttttgaa aaaagaagaa aatgatgaag gaaaggcgtg cgtcatgagc ttcgcattta 6360 cgtacgcaaa aagtgaggga tatgtgaaaa agatattggg tgatagaata gttgatggat 6420 tgggctgcac tatttgcctc aatttgccac aaatttccat ctaatttgtc ataattttcc 6480 aggaacgaaa tgctgaatcg gccatcgaag cgctcaagga atacgaacca gaaatggcca 6540 aggtcatccg atccggacac cacggaattc agatggttcg cgctaaggaa ctcgtgccag 6600 gagatcttgt cgaagtttca ggttagcaaa aacttttttt tttaactttc aaattttaaa 6660 ccatatattt ttcagtcgga gacaagatcc cagccgatct ccgtcttgtg aagatctact 6720 ccaccaccat ccgtatcgat cagtccatcc tcaccggaga atctgtgtct gttatcaagc 6780 acaccgactc tgtgccagat ccacgcgctg ttaaccagga caagaagaat tgtctgttct 6840 cgggaaccaa tgtcgcatct ggaaaggctc gtggaatcgt cttcggaacc ggattgacca 6900 ctgaaatcgg aaagatccgt accgaaatgg ctgagaccga gaatgagaag acaccacttc 6960 aacagaagtt ggacgaattc ggagagcaac tttccaaggt tatctctgtt atttgcgttg 7020 ctgtttgggc tatcaacatt ggacatttca acgatccagc tcacggtgga tcatgggtta 7080 agggagcaat ctactacttc aaaatcgccg ttgctcttgc cgtcgctgct attccagaag 7140 gacttccagc tgtcatcacc acgtgccttg ccctcggaac tcgccgtatg gccaagaaga 7200 acgctattgt aagatccctt ccatccgtcg aaactcttgg atgcacatct gttatctgct 7260 ctgacaagac tggaactctc accaccaacc agatgtctgt gtcaaagatg ttcatcgctg 7320 gacaagcttc tggagacaac atcaacttca ccgagttcgc catctccgga tccacctacg 7380 agccagtcgg aaaggtttcc accaatggac gtgaaatcaa cccagctgct ggagaattcg 7440 aatcactcac cgagttggcc atgatctgcg ctatgtgcaa tgattcatct gttgattaca 7500 atgagaccaa gaagatctac gagaaagtcg gagaagccac tgaaactgct cttatcgttc 7560 ttgctgagaa gatgaatgtt ttcggaacct cgaaagccgg actttcacca aaggagctcg 7620 gaggagtttg caaccgtgtc atccaacaaa aatggaagaa ggagttcaca ctcgagttct 7680 cccgtgatcg taaatccatg tccgcctact gcttcccagc ttccggagga tctggagcca 7740 agatgttcgt gaagggagcc ccagaaggag ttctcggaag atgcacccac gtcagagtta 7800 acggacaaaa ggttccactc acctctgcca tgactcagaa gattgttgac caatgcgtgc 7860 aatacggaac cggaagagat acccttcgtt gtcttgccct cggaaccatc gataccccag 7920 tcagcgttag caacatgaac ctcgaagact ctacccaatt cgtcaaatac gaacaagaca 7980 tcacatttgt cggagtcgtc ggaatgcttg accccccaag aactgaagtt tcggactcga 8040 tcaaggcttg taaccacgct ggaatccgtg tcatcatgat caccggagac aacaagaaca 8100 ccgctgaggc tatcggaaga agaatcggac tcttcggaga gaacgaggat accactggaa 8160 aagcttacac tggacgtgaa tttgacgatc ttccaccaga gcaacaatct gaagcctgcc 8220 gcagagctaa gcttttcgcc cgtgtcgagc catctcacaa gtccaagatt gtcgatatcc 8280 ttcaatccca gggagagatt actgctatga ccggagacgg agtcaacgac gctccagctt 8340 tgaagaaggc cgaaatcgga atttctatgg gatcaggaac tgctgtcgcc aagtctgcat 8400 ctgaaatggt tcttgctgac gataacttcg catccattgt gtctgctgtc gaagaaggac 8460 gtgctattta caacaacatg aaacaattca tcagatatct catctcatct aacgtcggag 8520 aagtcgtctc catcttcatg gtcgccgcac tcggaattcc agaggctctc attccagttc 8580 aacttctctg ggttaacttg gtcactgacg gtcttccagc cactgctctc ggattcaatc 8640 caccagatct tgacattatg gacagacatc cacgttcagc caacgatgga ctcatctctg 8700 gatggctctt cttcagatat cttgctgtcg gaagtacgtt taaaaaattc ccctaaaaaa 8760 gtataattct aaaattgaaa ttttccagcc tacgtcggag ttgccaccgt cggagcctca 8820 atgtggtggt tcttgttgta cgaggaggga ccacagatca cctactacca gctcactcac 8880 tggatgagat gtgaaatcga gccagacaac tttgccgatc ttgactgcgc cgtattcgag 8940 gacaatcacc cgaacgccat ggctctgtcc gtgcttgtca ccattgagat gctcaacgcc 9000 atcaactcac tttccgagaa tcaatcgctt ttagtgatgc caccatggaa gaacatctgg 9060 ctgatggccg ccatttccct ttcgatgtct cttcactttg tcattctcta cgttgacatc 9120 atggccacca tcttccaggt atcacaatta atcatatatt aatcgaaaca tctaattcaa 9180 atcttcagat cacccctctc aactgggtcg aatggatcgc cgtgttgaag atctcactgc 9240 cagtgctcct tctcgatgaa attctcaagt tcatcgccag aaactacatc gacggtaagc 9300 cggagacggt cggcgcgaag gcacgtagtg ccatctcgct gctcgcctgg gtgtctgtga 9360 cgctcgccta ctttgcgtgg atgttgggcc cgtacgccga gctcattaac catgcgctcg 9420 tcggtccatc tgtcgatccg tcgaaattcg acgcggttgt cacgcccgac aagttacata 9480 acgaattgtg attgaagttc ttctaacccc caaaccaacc gcctctcaaa caacttgtga 9540 tgatttctct ttattttctc tctctttctt gttctaatca ttttgggcct ttttcccttt 9600 ttctctctgc agtgtgttaa ctgatccata atccttcgtg taaacccccc tctccctact 9660 tttaggattt cttcctcgtt gctcattgta ttttgtccaa atcgccacaa tttccctaca 9720 aatatatatg ttttttttgc taattttttg tgtttccctt ccttcttgtc cactgaaagt 9780 tctacgtctc tcgctctcca catccccatt gttctcccct tttttcataa taatttatta 9840 ttatcctttt tttaaattaa tttttgttgc gtgtgaatct attaggagct cacaaataaa 9900 agtgatcctt taaaaaacct tacttccttc tgttttttct ctaacctaac caatgtgtct 9960 gttcagggag tgcctctttt ctttaccgaa tggtgtgcaa ttttgtcgac tgtcgatctc 10020 gtccatggca atgcaggatt tgaaactaaa tttccctgga aaaagaaata attttggtga 10080 ttttcagttg aagctccaat caaggataaa cgcgactaaa aatgacagtg ctccctcaat 10140 cagagtgagc ccagccgccg cccatctcat ttttcagact ctttcatatt ttctaagttt 10200 tccaattttt tttcttttgt agtgcgatcg ttttcgtttc gagacccgaa atcgaaagga 10260 tctcttttag agatctttag gatctttttt ctttgctcaa ctcatcattc tttgtttttt 10320 cttctatatc ctcttgttga cggtgatcag acaaatttgt tagaaatatt attacatttc 10380 ctttaggttt cttctattaa aaaaaaagaa aacttctgct aaattcgtgt acgttgtctc 10440 tcccatttct cattaaaaat cgatattaat tgtaattttt ggtttgtcct ccagtgtcgt 10500 gtgcgccatc gatggaaata aaaaagtttc aaaactatta tagcttttct ttttatgaaa 10560 aatataaaat acaattagaa tgtttttgtt aaatgcgata cggtgtgcgc ctttaaagag 10620 tagagtactg tagttccaaa attttgttgg tgcgggattt tcattgattt ttcatcgttt 10680 ttcgacaaaa atatatttat ttattgaaaa aagttaaata aaactattaa aaacacagaa 10740 tttttaacaa attgtgaaaa acacatgaaa aatcgatgac aattctacag taacgaacat 10800 tttgaattac agtaatcttt aaaggcgcgc acacgtttgc atttaattaa aatgtgtcgt 10860 gtcgagaccg actaccacgt ctcgttatta tagaacgagc aattacagcc taacatcaac 10920 tcagaacaaa ccaggatccc acgcaagttt aaaggagcat gctgggtatc acaacgattt 10980 tttgaagaga acgaggcccc acgaaacggg gagcagaacg aaaaggggat ctgcaaaaag 11040 gggatctgca aaaaggggat ctgcgaaaag gggagatatg aaaaggggag atacgaaaag 11100 gggagctggc actgtgccaa acgcacaaaa cgcaattttt ctcacgcaac gcacgttgat 11160 ttttgaaatt ttcttctaga agatacgctt aacaacacgc gacgcgg 11207 3 5026 DNA Caenorhabditis elegans 3 ttggttggca gctctctggc ttatcttttg agaggaaaaa gatccaacaa atttttatct 60 cccttatccc tttttctctt catcactacc aataataata gttttttttt tcgtcgcgga 120 agcaaaatgg cgaacaagtg ttggaataag agtactccag ggatttaagg gctgaaagcc 180 agtgatttat gagctccaat ttttcagatg ttttttcctc catcgcgtat ttgtctaaac 240 attcgatttt cttcctgctt cccaactttt caaatcgaaa taaaagagca tctgtcgctt 300 tttatcgatg tgcttctgtg agactaaaga actactcgtt ttcactcgtt ctctctctct 360 caactatcaa agttttgttg attgcgtgtg tcagcttcct tctttttatt atcatctttt 420 cattggagga aaaaaataac ttctgaagag caaaagaact aacttcgggg aatacagaga 480 aaattcctgt aaaaatctgg aaattttttc gcttaactcg aaatatttag tttttcactg 540 tgatttctgg gaaaaatcaa gaaatatttg cctaaaacac gagttttcac atgaaaaatg 600 aattatttat tgatttttta tggagattac aaaaaagaca cacgtgaaac tactgctacc 660 gtagttgtgt aaacgtagtg ttctctattt tagacctgtt taatgtattt ttttgcagtt 720 gaaaattttt aaaaatattt tagttatttt taaaaatatt taatttacaa aataattagc 780 ctgaacccat gaaaagatac gttatattta atttttaccg taagactttc aagatcgttg 840 cgagacccgg cgcctaggtc aaagagcctc cctttaaacc catcaacacg ttttgccttt 900 ttcatcgatt ttttgcagtt cttttcttct ttccaactga tttttcttca tttttaaagt 960 ttttttcctc atttttccca tttgaaatta tttaaacacg tgcaaccagc tggtaacatg 1020 tgtcacatgc cgttatctaa cttcaaaaca gtacatttcc gatcacacgt cccccgcgcc 1080 gagttttata gtttcattaa taacttttcg gtttttgata atactaattg agttttatta 1140 attgtttcca tattcatcta gcactttgac ctgtccttct tcgaattctc aaatatttgc 1200 actctgggtt taggtgtgaa aagaattgtc gtcattaagc ggggcatccg gggcaccgaa 1260 aaaagccctc cgattttaac gaatttgaga taaagttgga gagagagccc agtgtttgct 1320 tgcccaagat atatatcttg gatttatcaa ttactgtttg tcaacctgtc gccggcgccc 1380 cctttttgct cttgctccca cgccccgaga ttgaatttca attttatttc gaagtaagtc 1440 tcttgattgt ttcgaaaatc cgatgacagt tttcattact tttttgtctg ttgattttgt 1500 agggaaacat tgaaattttt ctgatctttc tttgatctta tgatttttca tttattccaa 1560 ttaaaaaaaa ttagcgcatt cagaaccaga gtgaagcttg agatgttgta ggtttatcaa 1620 aagatcaaaa tctcgaattc cttcgaaatg tttttagttt tcgacttccg tgtgatttct 1680 agcgatcctg acagagatca ctgaatttta atgttatcga gattgttgtg taggctccat 1740 ctcctctctg aagcttctga ttttgccgaa agtctagtta cttgccgact gctgacacta 1800 ggatatccca ctaccgtacc cattgttgga tccgtactct gctgcgactt cttctctgtt 1860 tcacgtgaac ctccgggatc gtcggtaagc cccgcccgtt atctgtgcca acttgtcttc 1920 gtgccctcga gcgacgagct cattcaatca cgccacgacc tccgtctgga cagatgctct 1980 cattgtctct gcgtctccaa gtattcgtca cactatctca tgcattctat tcaaaacgcg 2040 agagaaagcg cgggaacgag agagagttca gacagatcga acttgttttt atcccccccc 2100 ccctcgtccg gctgcagagc aaaaaaatac tgcttttcct tgcaaaattc ggtgctttct 2160 tcaaagagaa acttttgaag tcggcgcgag catttccttc tttgacttct ctctttccgc 2220 caaaaagcct agcattttta ttgataattt gattacacac actcagagtt cttcgacatg 2280 ataaagtgtt tcattggcac tcgccctaac agtacatgac aagggcggat tattatcgat 2340 cgatattgaa gacaaactcc aaatgtgtgc tcattttgga gccccgtgtg gggcagctgc 2400 tctcaatata ttactaggga gacgaggagg gggaccttat cgaacgtcgc atgagccatt 2460 ctttcttctt tatgcactct cttcactctc tcacacatta atcgattcat agactcccat 2520 attccttgat gaaggtgtgg gtttttagct ttttttcccg atttgtaaaa ggaagaggct 2580 gacgatgtta ggaaaaagag aacggagccg aaaaaacatc cgtagtaagt cttcctttta 2640 agccgacact ttttagacag cattcgccgc tagttttgaa gtttaaattt taaaaaataa 2700 aaattagttt caattttttt taattactaa ataggcaaaa gttttttcaa gaactctaga 2760 aaaactagct taattcatgg gtactagaaa aattcttgtt ttaaatttaa tatttatctt 2820 aagatgtaat tacgagaagc ttttttgaaa attctcaatt aaaagaattt gccgatttag 2880 aataaaagtc ttcagaaatg agtaaaagct caaattagaa gtttgttttt aaaggaaaaa 2940 cacgaaaaaa gaacactatt tatcttttcc tccccgcgta aaattagttg ttgtgataat 3000 agtgatccgc tgtctatttg cactcggctc ttcacaccgt gcttcctctc acttgaccca 3060 acaggaaaaa aaaacatcac gtctgagacg gtgaattgcc ttatcaagag cgtcgtctct 3120 ttcacccagt aacaaaaaaa atttggtttc tttactttat atttatgtag gtcacaaaaa 3180 aaaagtgatg cagttttgtg ggtcggttgt ctccacacca cctccgcctc cagcagcaca 3240 caatcatctt cgtgtgttct cgacgattcc ttgtatgccg cggtcgtgaa tgcaccacat 3300 tcgacgcgca actacacacc acactcactt tcggtggtat tactacacgt catcgttgtt 3360 cgtagtctcc cgctctttcg tccccactca ctcctcatta ttccccttgg tgtattgatt 3420 ttttttaaat ggtacaccac tcctgacgtt tctaccttct tgttttccgt ccatttagat 3480 tttatctgga aattttttta aaattttagg ccagagagtt ctagttcttg ttctaaaagt 3540 ctaggtcaga catacatttt ctatttctca tcaaaaaaaa agttgataaa gaaaactggt 3600 tattcagaaa gagtgtgtct cgttgaaatt gattcaaaaa aaaattccca cccctcgctt 3660 gtttctcaaa atatgagatc aacggatttt ttccttctcg attcaatttt ttgctgcgct 3720 ctgtctgcca aagtgtgtgt gtccgagcaa aagatgagag aatttacaaa cagaaatgaa 3780 aaaaagttgg ccaaataatg aagttttatc cgagattgat gggaaagata ttaatgttct 3840 ttacggtttg gaggggagag agagatagat tttcgcatca aactccgcct tttacatgtc 3900 ttttagaatc taaaatagat ttttctcatc atttttaata gaaaatcgag aaattacagt 3960 aatttcgcaa ttttcttgcc aaaaatacac gaaatttgtg ggtctcgcca cgatctcggt 4020 cttagtggtt catttggttt aaaagtttat aaaatttcaa attctagtgt ttaatttccg 4080 cataattgga cctaaaatgg gtttttgtca tcattttcaa caagaaatcg tgaaaatcct 4140 gttgtttcgc aattttcttt tcaaaaatac acgaaatata tggtaatttc ccgaaatatt 4200 gagggtctcg ccacgatttc agtcacagtg gccaggattt atcacgaaaa aagttcgcct 4260 agtctcacat ttccggaaaa ccgaatctaa attagttttt tgtcatcatt ttgaacaaaa 4320 aatcgagaca tccctatagt ttcgcaattt tcgtcgcttt tctctccaaa aatgacagtc 4380 tagaattaaa attcgctgga actgggacca tgatatcttt tctccccgtt tttcatttta 4440 ttttttatta cactggattg actaaaggtc accaccaccg ccagtgtgtg ccatatcaca 4500 cacacacaca cacacaatgt cgagatttta tgtgttatcc ctgcttgatt tcgttccgtt 4560 gtctctctct ctctattcat cttttgagcc gagaagctcc agagaatgga gcacacagga 4620 tcccggcgcg cgatgtcgtc gggagatggc gccgcctggg aagccgccga gagatatcag 4680 ggaagatcgt ctgatttctc ctcggatgcc acctcatctc tcgagtttct ccgcctgtta 4740 ctccctgccg aacctgatat ttcccgttgt cgtaaagaga tgtttttatt ttactttaca 4800 ccgggtcctc tctctctgcc agcacagctc agtgttggct gtgtgctcgg gctcctgcca 4860 ccggcggcct catcttcttc ttcttcttct ctcctgctct cgcttatcac ttcttcattc 4920 attcttattc cttttcatca tcaaactagc atttcttact ttatttattt ttttcaattt 4980 tcaattttca gataaaacca aactacttgg gttacagccg tcaaca 5026 4 2915 DNA Caenorhabditis elegans 4 ctgcagagca aaaaaatact gcttttcctt gcaaaattcg gtgctttctt caaagagaaa 60 cttttgaagt cggcgcgagc atttccttct ttgacttctc tctttccgcc aaaaagccta 120 gcatttttat tgataatttg attacacaca ctcagagttc ttcgacatga taaagtgttt 180 cattggcact cgccctaaca gtacatgaca agggcggatt attatcgatc gatattgaag 240 acaaactcca aatgtgtgct cattttggag ccccgtgtgg ggcagctgct ctcaatatat 300 tactagggag acgaggaggg ggaccttatc gaacgtcgca tgagccattc tttcttcttt 360 atgcactctc ttcactctct cacacattaa tcgattcata gactcccata ttccttgatg 420 aaggtgtggg tttttagctt tttttcccga tttgtaaaag gaagaggctg acgatgttag 480 gaaaaagaga acggagccga aaaaacatcc gtagtaagtc ttccttttaa gccgacactt 540 tttagacagc attcgccgct agttttgaag tttaaatttt aaaaaataaa aattagtttc 600 aatttttttt aattactaaa taggcaaaag ttttttcaag aactctagaa aaactagctt 660 aattcatggg tactagaaaa attcttgttt taaatttaat atttatctta agatgtaatt 720 acgagaagct tttttgaaaa ttctcaatta aaagaatttg ccgatttaga ataaaagtct 780 tcagaaatga gtaaaagctc aaattagaag tttgttttta aaggaaaaac acgaaaaaag 840 aacactattt atcttttcct ccccgcgtaa aattagttgt tgtgataata gtgatccgct 900 gtctatttgc actcggctct tcacaccgtg cttcctctca cttgacccaa caggaaaaaa 960 aaacatcacg tctgagacgg tgaattgcct tatcaagagc gtcgtctctt tcacccagta 1020 acaaaaaaaa tttggtttct ttactttata tttatgtagg tcacaaaaaa aaagtgatgc 1080 agttttgtgg gtcggttgtc tccacaccac ctccgcctcc agcagcacac aatcatcttc 1140 gtgtgttctc gacgattcct tgtatgccgc ggtcgtgaat gcaccacatt cgacgcgcaa 1200 ctacacacca cactcacttt cggtggtatt actacacgtc atcgttgttc gtagtctccc 1260 gctctttcgt ccccactcac tcctcattat tccccttggt gtattgattt tttttaaatg 1320 gtacaccact cctgacgttt ctaccttctt gttttccgtc catttagatt ttatctggaa 1380 atttttttaa aattttaggc cagagagttc tagttcttgt tctaaaagtc taggtcagac 1440 atacattttc tatttctcat caaaaaaaaa gttgataaag aaaactggtt attcagaaag 1500 agtgtgtctc gttgaaattg attcaaaaaa aaattcccac ccctcgcttg tttctcaaaa 1560 tatgagatca acggattttt tccttctcga ttcaattttt tgctgcgctc tgtctgccaa 1620 agtgtgtgtg tccgagcaaa agatgagaga atttacaaac agaaatgaaa aaaagttggc 1680 caaataatga agttttatcc gagattgatg ggaaagatat taatgttctt tacggtttgg 1740 aggggagaga gagatagatt ttcgcatcaa actccgcctt ttacatgtct tttagaatct 1800 aaaatagatt tttctcatca tttttaatag aaaatcgaga aattacagta atttcgcaat 1860 tttcttgcca aaaatacacg aaatttgtgg gtctcgccac gatctcggtc ttagtggttc 1920 atttggttta aaagtttata aaatttcaaa ttctagtgtt taatttccgc ataattggac 1980 ctaaaatggg tttttgtcat cattttcaac aagaaatcgt gaaaatcctg ttgtttcgca 2040 attttctttt caaaaataca cgaaatatat ggtaatttcc cgaaatattg agggtctcgc 2100 cacgatttca gtcacagtgg ccaggattta tcacgaaaaa agttcgccta gtctcacatt 2160 tccggaaaac cgaatctaaa ttagtttttt gtcatcattt tgaacaaaaa atcgagacat 2220 ccctatagtt tcgcaatttt cgtcgctttt ctctccaaaa atgacagtct agaattaaaa 2280 ttcgctggaa ctgggaccat gatatctttt ctccccgttt ttcattttat tttttattac 2340 actggattga ctaaaggtca ccaccaccgc cagtgtgtgc catatcacac acacacacac 2400 acacaatgtc gagattttat gtgttatccc tgcttgattt cgttccgttg tctctctctc 2460 tctattcatc ttttgagccg agaagctcca gagaatggag cacacaggat cccggcgcgc 2520 gatgtcgtcg ggagatggcg ccgcctggga agccgccgag agatatcagg gaagatcgtc 2580 tgatttctcc tcggatgcca cctcatctct cgagtttctc cgcctgttac tccctgccga 2640 acctgatatt tcccgttgtc gtaaagagat gtttttattt tactttacac cgggtcctct 2700 ctctctgcca gcacagctca gtgttggctg tgtgctcggg ctcctgccac cggcggcctc 2760 atcttcttct tcttcttctc tcctgctctc gcttatcact tcttcattca ttcttattcc 2820 ttttcatcat caaactagca tttcttactt tatttatttt tttcaatttt caattttcag 2880 ataaaaccaa actacttggg ttacagccgt caaca 2915 5 6612 DNA Caenorhabditis elegans 5 tcgactctag ttttgaaatc caaaaaaaaa acaaagttca ataaaatgtt acccaattgt 60 gcgatttttg ctttaaaaat acggtacccg gtctcgatgc ggcaattgtt tggtaaatgt 120 aaaagggtgt gcgcctttaa agagtactgt aatttcaatc ttccgacact gctgaatttt 180 tattgacttt ttgttcatta attttatata tgatttattg gtatgttaaa aaaacaccca 240 ttttcaaatc tattaaaatt ccacaacaac aaaagttcga gattacagta ctttttagag 300 gcgcacatcc tttttgggat actaaacaat tgtcgcgtcg agaccaggta ccatatttcc 360 aaaacacaat ttcgcgtgta aataaaaaat atcaacataa taatttccat ttttcgaaat 420 ttaaagttaa tcactttttg gtttagatta tgatttcaca cgtttttttc cttctagttc 480 tctttttttt gttatttgcc tgaaaaatgg tctgaaaact taggcaatca gcaatgtgtc 540 acataatttc tcccagagaa atccctttca acaaaatctc ccggattgac ctgtgtgctc 600 gaccttgata aattggttgg cagctctctg gcttatcttt tgagaggaaa aagatccaac 660 aaatttttat ctcccttatc cctttttctc ttcatcacta ccaataataa tagttttttt 720 tttcgtcgcg gaagcaaaat ggcgaacaag tgttggaata agagtactcc agggatttaa 780 gggctgaaag ccagtgattt atgagctcca atttttcaga tgttttttcc tccatcgcgt 840 atttgtctaa acattcgatt ttcttcctgc ttcccaactt ttcaaatcga aataaaagag 900 catctgtcgc tttttatcga tgtgcttctg tgagactaaa gaactactcg ttttcactcg 960 ttctctctct ctcaactatc aaagttttgt tgattgcgtg tgtcagcttc cttcttttta 1020 ttatcatctt ttcattggag gaaaaaaata acttctgaag agcaaaagaa ctaacttcgg 1080 ggaatacaga gaaaattcct gtaaaaatct ggaaattttt tcgcttaact cgaaatattt 1140 agtttttcac tgtgatttct gggaaaaatc aagaaatatt tgcctaaaac acgagttttc 1200 acatgaaaaa tgaattattt attgattttt tatggagatt acaaaaaaga cacacgtgaa 1260 actactgcta ccgtagttgt gtaaacgtag tgttctctat tttagacctg tttaatgtat 1320 ttttttgcag ttgaaaattt ttaaaaatat tttagttatt tttaaaaata tttaatttac 1380 aaaataatta gcctgaaccc atgaaaagat acgttatatt taatttttac cgtaagactt 1440 tcaagatcgt tgcgagaccc ggcgcctagg tcaaagagcc tccctttaaa cccatcaaca 1500 cgttttgcct ttttcatcga ttttttgcag ttcttttctt ctttccaact gatttttctt 1560 catttttaaa gtttttttcc tcatttttcc catttgaaat tatttaaaca cgtgcaacca 1620 gctggtaaca tgtgtcacat gccgttatct aacttcaaaa cagtacattt ccgatcacac 1680 gtcccccgcg ccgagtttta tagtttcatt aataactttt cggtttttga taatactaat 1740 tgagttttat taattgtttc catattcatc tagcactttg acctgtcctt cttcgaattc 1800 tcaaatattt gcactctggg tttaggtgtg aaaagaattg tcgtcattaa gcggggcatc 1860 cggggcaccg aaaaaagccc tccgatttta acgaatttga gataaagttg gagagagagc 1920 ccagtgtttg cttgcccaag atatatatct tggatttatc aattactgtt tgtcaacctg 1980 tcgccggcgc cccctttttg ctcttgctcc cacgccccga gattgaattt caattttatt 2040 tcgaagtaag tctcttgatt gtttcgaaaa tccgatgaca gttttcatta cttttttgtc 2100 tgttgatttt gtagggaaac attgaaattt ttctgatctt tctttgatct tatgattttt 2160 catttattcc aattaaaaaa aattagcgca ttcagaacca gagtgaagct tgagatgttg 2220 taggtttatc aaaagatcaa aatctcgaat tccttcgaaa tgtttttagt tttcgacttc 2280 cgtgtgattt ctagcgatcc tgacagagat cactgaattt taatgttatc gagattgttg 2340 tgtaggctcc atctcctctc tgaagcttct gattttgccg aaagtctagt tacttgccga 2400 ctgctgacac taggatatcc cactaccgta cccattgttg gatccgtact ctgctgcgac 2460 ttcttctctg tttcacgtga acctccggga tcgtcggtaa gccccgcccg ttatctgtgc 2520 caacttgtct tcgtgccctc gagcgacgag ctcattcaat cacgccacga cctccgtctg 2580 gacagatgct ctcattgtct ctgcgtctcc aagtattcgt cacactatct catgcattct 2640 attcaaaacg cgagagaaag cgcgggaacg agagagagtt cagacagatc gaacttgttt 2700 ttatcccccc ccccctcgtc cggctgcaga gcaaaaaaat actgcttttc cttgcaaaat 2760 tcggtgcttt cttcaaagag aaacttttga agtcggcgcg agcatttcct tctttgactt 2820 ctctctttcc gccaaaaagc ctagcatttt tattgataat ttgattacac acactcagag 2880 ttcttcgaca tgataaagtg tttcattggc actcgcccta acagtacatg acaagggcgg 2940 attattatcg atcgatattg aagacaaact ccaaatgtgt gctcattttg gagccccgtg 3000 tggggcagct gctctcaata tattactagg gagacgagga gggggacctt atcgaacgtc 3060 gcatgagcca ttctttcttc tttatgcact ctcttcactc tctcacacat taatcgattc 3120 atagactccc atattccttg atgaaggtgt gggtttttag ctttttttcc cgatttgtaa 3180 aaggaagagg ctgacgatgt taggaaaaag agaacggagc cgaaaaaaca tccgtagtaa 3240 gtcttccttt taagccgaca ctttttagac agcattcgcc gctagttttg aagtttaaat 3300 tttaaaaaat aaaaattagt ttcaattttt tttaattact aaataggcaa aagttttttc 3360 aagaactcta gaaaaactag cttaattcat gggtactaga aaaattcttg ttttaaattt 3420 aatatttatc ttaagatgta attacgagaa gcttttttga aaattctcaa ttaaaagaat 3480 ttgccgattt agaataaaag tcttcagaaa tgagtaaaag ctcaaattag aagtttgttt 3540 ttaaaggaaa aacacgaaaa aagaacacta tttatctttt cctccccgcg taaaattagt 3600 tgttgtgata atagtgatcc gctgtctatt tgcactcggc tcttcacacc gtgcttcctc 3660 tcacttgacc caacaggaaa aaaaaacatc acgtctgaga cggtgaattg ccttatcaag 3720 agcgtcgtct ctttcaccca gtaacaaaaa aaatttggtt tctttacttt atatttatgt 3780 aggtcacaaa aaaaaagtga tgcagttttg tgggtcggtt gtctccacac cacctccgcc 3840 tccagcagca cacaatcatc ttcgtgtgtt ctcgacgatt ccttgtatgc cgcggtcgtg 3900 aatgcaccac attcgacgcg caactacaca ccacactcac tttcggtggt attactacac 3960 gtcatcgttg ttcgtagtct cccgctcttt cgtccccact cactcctcat tattcccctt 4020 ggtgtattga ttttttttaa atggtacacc actcctgacg tttctacctt cttgttttcc 4080 gtccatttag attttatctg gaaatttttt taaaatttta ggccagagag ttctagttct 4140 tgttctaaaa gtctaggtca gacatacatt ttctatttct catcaaaaaa aaagttgata 4200 aagaaaactg gttattcaga aagagtgtgt ctcgttgaaa ttgattcaaa aaaaaattcc 4260 cacccctcgc ttgtttctca aaatatgaga tcaacggatt ttttccttct cgattcaatt 4320 ttttgctgcg ctctgtctgc caaagtgtgt gtgtccgagc aaaagatgag agaatttaca 4380 aacagaaatg aaaaaaagtt ggccaaataa tgaagtttta tccgagattg atgggaaaga 4440 tattaatgtt ctttacggtt tggaggggag agagagatag attttcgcat caaactccgc 4500 cttttacatg tcttttagaa tctaaaatag atttttctca tcatttttaa tagaaaatcg 4560 agaaattaca gtaatttcgc aattttcttg ccaaaaatac acgaaatttg tgggtctcgc 4620 cacgatctcg gtcttagtgg ttcatttggt ttaaaagttt ataaaatttc aaattctagt 4680 gtttaatttc cgcataattg gacctaaaat gggtttttgt catcattttc aacaagaaat 4740 cgtgaaaatc ctgttgtttc gcaattttct tttcaaaaat acacgaaata tatggtaatt 4800 tcccgaaata ttgagggtct cgccacgatt tcagtcacag tggccaggat ttatcacgaa 4860 aaaagttcgc ctagtctcac atttccggaa aaccgaatct aaattagttt tttgtcatca 4920 ttttgaacaa aaaatcgaga catccctata gtttcgcaat tttcgtcgct tttctctcca 4980 aaaatgacag tctagaatta aaattcgctg gaactgggac catgatatct tttctccccg 5040 tttttcattt tattttttat tacactggat tgactaaagg tcaccaccac cgccagtgtg 5100 tgccatatca cacacacaca cacacacaat gtcgagattt tatgtgttat ccctgcttga 5160 tttcgttccg ttgtctctct ctctctattc atcttttgag ccgagaagct ccagagaatg 5220 gagcacacag gatcccggcg cgcgatgtcg tcgggagatg gcgccgcctg ggaagccgcc 5280 gagagatatc agggaagatc gtctgatttc tcctcggatg ccacctcatc tctcgagttt 5340 ctccgcctgt tactccctgc cgaacctgat atttcccgtt gtcgtaaaga gatgttttta 5400 ttttacttta caccgggtcc tctctctctg ccagcacagc tcagtgttgg ctgtgtgctc 5460 gggctcctgc caccggcggc ctcatcttct tcttcttctt ctctcctgct ctcgcttatc 5520 acttcttcat tcattcttat tccttttcat catcaaacta gcatttctta ctttatttat 5580 ttttttcaat tttcaatttt cagataaaac caaactactt gggttacagc cgtcaacatg 5640 gaggacgcgc atgccaaaga cgccaatgag gtacttttat agtttttaaa ttttagtttt 5700 taatacaatt tattttccag gtgtgcaaat tcttcggaac gggtccggag ggattgactc 5760 cacagcaagt tgaaacattg aggaacaaat atggagaaaa tggttggttt tttacatgga 5820 tttctcatta aaaattgaat tttttccaga aatgcccgcc gaagagggaa aatcactgtg 5880 ggagctgatt ctcgagcaat tcgacgatct tctcgtcaag attctcctcc tcgccgccat 5940 catctcgttt gtgctcgccc ttttcgaaga gcacgaagat cagacagaag cagtgacggc 6000 gttcgtcgaa ccgttcgtca tccttctcat tcttattgcc aacgcgaccg tcggagtgtg 6060 gcaggtagga acaacacaga caggcgcacg cgctgaaaga aaataagaag aagaagaaaa 6120 agcacagttg ttttctgtgt ttttgtagat caaaagaaag gaactaggag tgattgcaca 6180 gagagagaga gagagaaata atgtcttttt gacttgtttt tgttggtgag agagataggg 6240 aaaaagagtc cctaaagaaa aaatagtgta acgggcggtc cggaagaaat gctctttgcg 6300 ccgaaaagtt tttgaaaaaa gaagaaaatg atgaaggaaa ggcgtgcgtc atgagcttcg 6360 catttacgta cgcaaaaagt gagggatatg tgaaaaagat attgggtgat agaatagttg 6420 atggattggg ctgcactatt tgcctcaatt tgccacaaat ttccatctaa tttgtcataa 6480 ttttccagga acgaaatgct gaatcggcca tcgaagcgct caaggaatac gaaccagaaa 6540 tggccaaggt catccgatcc ggacaccacg gaattcagat ggttcgcgct aaggaactcg 6600 tgccaggaga tc 6612 6 3180 DNA Caenorhabditis elegans 6 atggaggacg cgcatgccaa agacgccaat gaggtgtgca aattcttcgg aacgggtccg 60 gagggattga ctccacagca agttgaaaca ttgaggaaca aatatggaga aaatgaaatg 120 cccgccgaag agggaaaatc actgtgggag ctgattctcg agcaattcga cgatcttctc 180 gtcaagattc tcctcctcgc cgccatcatc tcgtttgtgc tcgccctttt cgaagagcac 240 gaagatcaga cagaagcagt gacggcgttc gtcgaaccgt tcgtcatcct tctcattctt 300 attgccaacg cgaccgtcgg agtgtggcag gaacgaaatg ctgaatcggc catcgaagcg 360 ctcaaggaat acgaaccaga aatggccaag gtcatccgat ccggacacca cggaattcag 420 atggttcgcg ctaaggaact cgtgccagga gatcttgtcg aagtttcagt cggagacaag 480 atcccagccg atctccgtct tgtgaagatc tactccacca ccatccgtat cgatcagtcc 540 atcctcaccg gagaatctgt gtctgttatc aagcacaccg actctgtgcc agatccacgc 600 gctgttaacc aggacaagaa gaattgtctg ttctcgggaa ccaatgtcgc atctggaaag 660 gctcgtggaa tcgtcttcgg aaccggattg accactgaaa tcggaaagat ccgtaccgaa 720 atggctgaga ccgagaatga gaagacacca cttcaacaga agttggacga attcggagag 780 caactttcca aggttatctc tgttatttgc gttgctgttt gggctatcaa cattggacat 840 ttcaacgatc cagctcacgg tggatcatgg gttaagggag caatctacta cttcaaaatc 900 gccgttgctc ttgccgtcgc tgctattcca gaaggacttc cagctgtcat caccacgtgc 960 cttgccctcg gaactcgccg tatggccaag aagaacgcta ttgtaagatc ccttccatcc 1020 gtcgaaactc ttggatgcac atctgttatc tgctctgaca agactggaac tctcaccacc 1080 aaccagatgt ctgtgtcaaa gatgttcatc gctggacaag cttctggaga caacatcaac 1140 ttcaccgagt tcgccatctc cggatccacc tacgagccag tcggaaaggt ttccaccaat 1200 ggacgtgaaa tcaacccagc tgctggagaa ttcgaatcac tcaccgagtt ggccatgatc 1260 tgcgctatgt gcaatgattc atctgttgat tacaatgaga ccaagaagat ctacgagaaa 1320 gtcggagaag ccactgaaac tgctcttatc gttcttgctg agaagatgaa tgttttcgga 1380 acctcgaaag ccggactttc accaaaggag ctcggaggag tttgcaaccg tgtcatccaa 1440 caaaaatgga agaaggagtt cacactcgag ttctcccgtg atcgtaaatc catgtccgcc 1500 tactgcttcc cagcttccgg aggatctgga gccaagatgt tcgtgaaggg agccccagaa 1560 ggagttctcg gaagatgcac ccacgtcaga gttaacggac aaaaggttcc actcacctct 1620 gccatgactc agaagattgt tgaccaatgc gtgcaatacg gaaccggaag agataccctt 1680 cgttgtcttg ccctcggaac catcgatacc ccagtcagcg ttagcaacat gaacctcgaa 1740 gactctaccc aattcgtcaa atacgaacaa gacatcacat ttgtcggagt cgtcggaatg 1800 cttgaccccc caagaactga agtttcggac tcgatcaagg cttgtaacca cgctggaatc 1860 cgtgtcatca tgatcaccgg agacaacaag aacaccgctg aggctatcgg aagaagaatc 1920 ggactcttcg gagagaacga ggataccact ggaaaagctt acactggacg tgaatttgac 1980 gatcttccac cagagcaaca atctgaagcc tgccgcagag ctaagctttt cgcccgtgtc 2040 gagccatctc acaagtccaa gattgtcgat atccttcaat cccagggaga gattactgct 2100 atgaccggag acggagtcaa cgacgctcca gctttgaaga aggccgaaat cggaatttct 2160 atgggatcag gaactgctgt cgccaagtct gcatctgaaa tggttcttgc tgacgataac 2220 ttcgcatcca ttgtgtctgc tgtcgaagaa ggacgtgcta tttacaacaa catgaaacaa 2280 ttcatcagat atctcatctc atctaacgtc ggagaagtcg tctccatctt catggtcgcc 2340 gcactcggaa ttccagaggc tctcattcca gttcaacttc tctgggttaa cttggtcact 2400 gacggtcttc cagccactgc tctcggattc aatccaccag atcttgacat tatggacaga 2460 catccacgtt cagccaacga tggactcatc tctggatggc tcttcttcag atatcttgct 2520 gtcggaacct acgtcggagt tgccaccgtc ggagcctcaa tgtggtggtt cttgttgtac 2580 gaggagggac cacagatcac ctactaccag ctcactcact ggatgagatg tgaaatcgag 2640 ccagacaact ttgccgatct tgactgcgcc gtattcgagg acaatcaccc gaacgccatg 2700 gctctgtccg tgcttgtcac cattgagatg ctcaacgcca tcaactcact ttccgagaat 2760 caatcgcttt tagtgatgcc accatggaag aacatctggc tgatggccgc catttccctt 2820 tcgatgtctc ttcactttgt cattctctac gttgacatca tggccaccat cttccagatc 2880 acccctctca actgggtcga atggatcgcc gtgttgaaga tctcactgcc agtgctcctt 2940 ctcgatgaaa ttctcaagtt catcgccaga aactacatcg acggtaagcc ggagacggtc 3000 ggcgcgaagg cacgtagtgc catctcgctg ctcgcctggg tgtctgtgac gctcgcctac 3060 tttgcgtgga tgttgggccc gtacgccgag ctcattaacc atgcgctcgt cggtccatct 3120 gtcgatccgt cgaaattcga cgcggttgtc acgcccgaca agttacataa cgaattgtga 3180 7 2994 DNA Sus sp. 7 atggagaacg cgcacacaaa gacggtggag gaggtgctgg gccacttcgg cgtcaacgag 60 agcacggggc tgagcctgga gcaggtcaag aagctcaagg agagatgggg ctccaacgag 120 ttaccggctg aagaagggaa aaccttgctg gaacttgtga ttgagcagtt tgaagactta 180 ctcgttagaa ttttattgtt ggcagcatgt atatcttttg ttttggcttg gtttgaagaa 240 ggcgaagaaa caattacagc ctttgtagaa ccctttgtaa ttttacttat attagtagcc 300 aatgcaattg tgggtgtatg gcaggaaagg aatgcagaaa atgccatcga agcccttaag 360 gagtatgagc ctgaaatggg caaagtgtat cgacaggaca ggaagagtgt acaacgaatt 420 aaagctaaag acatagttcc tggtgatatt gtagaaattg ctgttggtga caaagttcct 480 gctgatataa gattaacgtc catcaaatct actactctaa gagttgacca gtcaattctc 540 acaggtgagt ctgtctctgt catcaagcac accgaccctg tccctgaccc acgggctgtc 600 aaccaagata agaagaacat gctcttttct ggtacaaaca tagcagctgg caaagccatg 660 ggagtggtgg tggcaactgg agttaacact gaaattggca agatccggga tgaaatggta 720 gcaacggaac aggagagaac acccctccag cagaaactag atgagtttgg ggaacagctt 780 tccaaagtca tctcccttat ttgcattgca gtctggatca taaacattgg gcacttcaat 840 gacccggttc atggaggctc ctggatcaga ggtgctattt attactttaa aattgcagtg 900 gccctggctg tagcagccat tcctgaaggc ctgcctgctg tcattaccac ctgcctggct 960 cttggaactc gtagaatggc aaagaaaaat gccattgttc gaagtctccc ttctgtggaa 1020 acccttggtt gcacttccgt tatctgctca gacaagactg gtacacttac aacaaaccag 1080 atgtcagtct gcaggatgtt cattctggac aaagttgaag gtgatacttg ttccctgaat 1140 gagtttacca taactggatc aacatatgct cctattggag aagtccataa agatgataaa 1200 ccagtaaagt gtcatcaata tgatggtctt gtggaattgg caacaatttg tgctctctgt 1260 aatgactctg ctttggatta caatgaggca aagggtgtgt atgaaaaagt tggagaagct 1320 acagagactg ctctcacttg cctggtagag aagatgaatg tctttgatac tgagttaaag 1380 ggtctttcta aaatagaacg agcaaatgcc tgcaactcgg tcattaaaca attgatgaaa 1440 aaggaattta ctctagagtt ttcacgtgat agaaaatcaa tgtcagttta ttgtacacca 1500 aacaaaccaa gccggacatc gatgagcaaa atgtttgtga agggtgctcc cgaaggtgtc 1560 attgacaggt gtacccacat tcgagttgga agtactaaag tccccatgac tcctggcgtc 1620 aaacagaaga tcatgtctgt cattcgggaa tggggcagtg gcagcgacac actgcgatgc 1680 ctggctctgg ccactcatga caacccgatg agaagagaag aaatgaacct tgaggattct 1740 gccaacttta ttaaatacga gaccaatctg actttcgttg gctgtgtggg catgctggac 1800 cctccaagaa tcgaagtggc ctcctctgtg aagctgtgcc ggcaggcagg catccgggtc 1860 attatgatca caggcgacaa caagggtacc gctgtggcca tctgccgtcg cattggcatc 1920 tttgggcagg acgaggatgt gacgtcaaag gcttttacag gtcgggagtt tgatgagctc 1980 aatccttcag cccagagaga agcctgcctg aatgcccgct gtttcgctcg agttgaacct 2040 tcccacaagt ctaaaattgt agaatttctt cagtcttttg atgagattac agctatgact 2100 ggggacggtg tgaatgatgc tcctgctctg aagaagtctg agatcggcat tgccatgggc 2160 tctggcaccg cggtggctaa aactgcctcc gagatggtcc tggctgatga caacttctcc 2220 accattgtgg ctgctgtgga ggagggacgg gcaatataca acaacatgaa gcagttcatt 2280 cgctacctca tctcgtccaa cgtgggagaa gttgtctgta ttttcctgac agcagccctt 2340 ggatttcctg aggctttaat tcctgtccag ctgctctggg tcaatctggt gacagatggc 2400 ctgcctgcca ctgcactggg gttcaatcct cctgatctgg acattatgaa caaaccaccc 2460 cggaacccaa aggaaccact gatcagtggg tggctctttt tccgctacct ggctattggc 2520 tgttacgttg gtgctgctac tgtgggtgct gctgcgtggt ggttcattgc tgccgatggt 2580 ggtccgagag tgaccttcta ccagctgagt catttcctac agtgtaaaga ggacaaccca 2640 gactttgagg gagtggattg tgcagtcttt gaatcccctt acccaatgac aatggcgctg 2700 tctgttctag tcaccataga gatgtgtaac gccctcaaca gtttgtcgga aaaccagtcc 2760 ctgctaagga tgccaccttg ggagaacatt tggctcgtgg gctccatctg cctgtccatg 2820 tcactccact tcctaatcct ctatgtggaa cccctgccac ttatcttcca gatcacaccg 2880 ctgaatttga cccagtggct gatggtgctg aaaatctcct tgcctgtgat tctaatggat 2940 gagaccctca agtttgtggc ccgcaactac ctggaacctg caatactgga gtaa 2994 8 2994 DNA Homo sapiens 8 atggagaacg cgcacaccaa gacggtggag gaggtgctgg gccacttcgg cgtcaacgag 60 agtacggggc tgagcctgga acaggtcaag aagcttaagg agagatgggg ctccaacgag 120 ttaccggctg aagaaggaaa aaccttgctg gaacttgtga ttgagcagtt tgaagacttg 180 ctagttagga ttttattact ggcagcatgt atatcttttg ttttggcttg gtttgaagaa 240 ggtgaagaaa caattacagc ctttgtagaa ccttttgtaa ttttactcat attagtagcc 300 aatgcaattg tgggtgtatg gcaggaaaga aatgctgaaa atgccatcga agcccttaag 360 gaatatgagc ctgaaatggg caaagtgtat cgacaggaca gaaagagtgt gcagcggatt 420 aaagctaaag acatagttcc tggtgatatt gtagaaattg ctgttggtga caaagttcct 480 gctgatataa ggttaacttc catcaaatct accacactaa gagttgacca gtcaattctc 540 acaggtgaat ctgtctctgt catcaagcac actgatcccg tccctgaccc acgagctgtc 600 aaccaagata aaaagaacat gctgttttct ggtacaaaca ttgctgctgg gaaagctatg 660 ggagtggtgg tagcaactgg agttaacacc gaaattggca agatccggga tgaaatggtg 720 gcaacagaac aggagagaac accccttcag caaaaactag atgaatttgg ggaacagctt 780 tccaaagtca tctcccttat ttgcattgca gtctggatca taaatattgg gcacttcaat 840 gacccggttc atggagggtc ctggatcaga ggtgctattt actactttaa aattgcagtg 900 gccctggctg tagcagccat tcctgaaggt ctgcctgcag tcatcaccac ctgcctggct 960 cttggaactc gcagaatggc aaagaaaaat gccattgttc gaagcctccc gtctgtggaa 1020 acccttggtt gtacttctgt tatctgctca gacaagactg gtacacttac aacaaaccag 1080 atgtcagtct gcaggatgtt cattctggac agagtggaag gtgatacttg ttcccttaat 1140 gagtttacca taactggatc aacttatgca cctattggag aagtgcataa agatgataaa 1200 ccagtgaatt gtcaccagta tgatggtctg gtagaattag caacaatttg tgctctttgt 1260 aatgactctg ctttggatta caatgaggca aagggtgtgt atgaaaaagt tggagaagct 1320 acagagactg ctctcacttg cctagtagag aagatgaatg tatttgatac cgaattgaag 1380 ggtctttcta aaatagaacg tgcaaatgcc tgcaactcag tcattaaaca gctgatgaaa 1440 aaggaattca ctctagagtt ttcacgtgac agaaagtcaa tgtcggttta ctgtacacca 1500 aataaaccaa gcaggacatc aatgagcaag atgtttgtga agggtgctcc tgaaggtgtc 1560 attgacaggt gcacccacat tcgagttgga agtactaagg ttcctatgac ctctggagtc 1620 aaacagaaga tcatgtctgt cattcgagag tggggtagtg gcagcgacac actgcgatgc 1680 ctggccctgg ccactcatga caacccactg agaagagaag aaatgcacct tgaggactct 1740 gccaacttta ttaaatatga gaccaatctg accttcgttg gctgcgtggg catgctggat 1800 cctccgagaa tcgaggtggc ctcctccgtg aagctgtgcc ggcaagcagg catccgggtc 1860 atcatgatca ctggggacaa caagggcact gctgtggcca tctgtcgccg catcggcatc 1920 ttcgggcagg atgaggacgt gacgtcaaaa gctttcacag gccgggagtt tgatgaactc 1980 aacccctccg cccagcgaga cgcctgcctg aacgcccgct gttttgctcg agttgaaccc 2040 tcccacaagt ctaaaatcgt agaatttctt cagtcttttg atgagattac agctatgact 2100 ggcgatggcg tgaacgatgc tcctgctctg aagaaagccg agattggcat tgctatgggc 2160 tctggcactg cggtggctaa aaccgcctct gagatggtcc tggcggatga caacttctcc 2220 accattgtgg ctgccgttga ggaggggcgg gcaatctaca acaacatgaa acagttcatc 2280 cgctacctca tctcgtccaa cgtcggggaa gttgtctgta ttttcctgac agcagccctt 2340 ggatttcccg aggctttgat tcctgttcag ctgctctggg tcaatctggt gacagatggc 2400 ctgcctgcca ctgcactggg gttcaaccct cctgatctgg acatcatgaa taaacctccc 2460 cggaacccaa aggaaccatt gatcagcggg tggctctttt tccgttactt ggctattggc 2520 tgttacgtcg gcgctgctac cgtgggtgct gctgcatggt ggttcattgc tgctgacggt 2580 ggtccaagag tgtccttcta ccagctgagt catttcctac agtgtaaaga ggacaacccg 2640 gactttgaag gcgtggattg tgcaatcttt gaatccccat acccgatgac aatggcgctc 2700 tctgttctag taactataga aatgtgtaac gccctcaaca gcttgtccga aaaccagtcc 2760 ttgctgagga tgcccccctg ggagaacatc tggctcgtgg gctccatctg cctgtccatg 2820 tcactccact tcctgatcct ctatgtcgaa cccttgccac tcatcttcca gatcacaccg 2880 ctgaacgtga cccagtggct gatggtgctg aaaatctcct tgcccgtgat tctcatggat 2940 gagacgctca agtttgtggc ccgcaactac ctggaacctg caatactgga gtaa 2994 9 159 DNA Sus sp. 9 atggataaag tccaatacct cactcgctct gctattagaa gagcttcaac cattgaaatg 60 cctcaacaag cacgtcaaaa ccttcagaac ctatttatca atttctgtct catcttaata 120 tgcctcttgc tgatttgcat catcgtgatg cttctctga 159 10 837 DNA Caenorhabditis elegans 10 aaagtgaata atcgaccaaa taataactca ctttggtatt tattcctgtc ttataatgtt 60 atgtatgaat taaattcata tgcatatggc tcactctgac aaaaaaaaat aatcttccag 120 atcaatattg actaccgatg cgggtggtct tttgctttga attctgctga actttacacc 180 ccgaacagca atgtgtgctt cagctaaaaa aaagtaagtg tgttaatcag tccccccgat 240 tcttcatttt ttgcccctct ctcccgtttc gtcggcaaaa gaagagaaaa taaagataag 300 tctcaagata ggttggtaat cgctaaagtg gttgtgtgga taagagtagc aaaatggcag 360 gaagagcact ttgcgcgcac acactgtact cattgttctg gataaaattc tctcgttgtt 420 tgccgtcgga tgtctgcctc tctgccattg agccggcttc ttcactatct ttagttaacc 480 taaaatgccg tttcttttct cgtatcccac tatccgttga ggttctctgc tctcttcgct 540 cccttaccgc cagcgagcaa ctatccgtgg gggcgccttg ctcggaagat gggggggaag 600 aaagaagatt tttgctattt gcacttgaga aagagacttt tcctgcgtcg atggttagag 660 aacagtgtgc agacactttt cagctaccta gatacatgga tatccccgcc tcccaatcca 720 cccacccagg gaaaaagaag ggctcgccga aaaatcaaag ttatctccag gctcgcgcat 780 cccaccgagc ggttgacttc tctccaccac ttttcatttt aaccctcggg gtacggg 837 11 2396 DNA Caenorhabditis elegans 11 agcttgggct gcaggtcggc tataataagt tcttgaataa aataattttc ccgacaaaac 60 atgagtattt ctttcgaaaa taaaagtgca ggctaattag agattattct gtaattaact 120 gcataatttg tcacgtgcca tagttttaca ttccactacg tcatagttct taaaatacta 180 atctcctgaa aatagaagta ggtgaagaaa gtttaattat cagttctaaa atgacaattg 240 atctttggaa tatgttctga aactaccgat cattgaacag atgctatttg aatgatatag 300 aattgtatat ttgcaatttc tgaaacgcgt tcttaaaggc acacagatta attcaaaagg 360 gtctggccgc aaaaaggttt atggtggccg attttgagtt ttgtgtgtga ttgctttttc 420 acaatcagtg ttttcaggat tatgtgatga actagatctt caagtttcgt tacatttcat 480 atgttttcgg aactcacgaa gtacatattg ggtattgtgc tcaaaaaatt cagcaatcag 540 cttcgctccg ctgactttag aacccaaaaa aatagtatgg ccaaactgac tgtgttacga 600 tcatttcaat ttttcaatac atatttaaga tttctaagag taagaaggtc aaaaactgtt 660 ctggaataca tatatatttt tcaggttaca attagtcaaa aagtgcactg aaatatacgt 720 tttaatttca cgaataaccc aattagttca atgtattttt ggtcaaccaa cgttaaagtt 780 tggcttccaa ccaattatca tttctgatca accacaatgt tttttcttta tctgcaagtt 840 aattttttat ttttatccag atgtttggca tatttttcaa ttcttcacta gcgcccactt 900 cttgcacttc cggcgccctg aatctaatgc atctgttgca agaattgaaa gaccaatcaa 960 cacattgttt tcttcacgag atactgaaga aaatgaataa aaacagagaa aaagagccat 1020 gtgattagtg acaactgttg ctaacagata ccatagcttg gacttggtac gtgatggcaa 1080 cgtatgggtc aacaaaaatg attgcagagg gggtgcaaaa cagtcaagtc gagaaaatat 1140 gaaaaacaga aaacaaagaa cagaaaaatg ggtttgagag tcagtataat ttataaaaga 1200 aaaattgtac atagaaatta accatttttg tagaagaagt tatttttcaa gcatcgttaa 1260 aaattattca aagcacctta tttcatattt aattttaaac atggttaaat gaacaacacg 1320 gtgcgcaatc aggaaaactt gaaatctgaa actgttgttg tgatcttctt cgcaactgtt 1380 cagatagcac tagtgtaatg ttaagagtgc gcgaatataa tggaatataa tggatcacac 1440 ctcctgccat caggtaaacg tctctgttat cacatatttc caactattaa atttttacct 1500 tttacagttt tacatttttt tgaaaaaagt aactttttgt cttcaaaatc cctgacgaaa 1560 atatcaaata ttttaatcga gactgcagag gaaccgattg atgatttgga aaatccagct 1620 ttacctgtgt aagaactgaa aagtttcata accctagggt attcccagtt acattcccca 1680 ctggctaaca atagcaccca gtttttcatc acctttcttc aaatttctcg gcgatttgtt 1740 aaaaacaaaa tttgtgtccc ttctctgata tctctatgtc tctaaacaca agttcatcgg 1800 aaaacgaagg agggtaggtg ttggttgggc tcccgaagtg aaaatagaag agcaagaata 1860 gaatattaga gagagagtgc agagagggcg ggatagctcc cgggattccg ttttcttctt 1920 ctttatcttc aacgatgatg tgtgtgcgtg ttgtatagat tctgttgctc ccccacaact 1980 cgctccgaag gctcaataca attcaattga tattggagga gagcctaccg gagtgggagg 2040 ataagaagaa acataagaag aagaagaaga agaagcatgc ttctggtttt tgatgctatg 2100 aaaacggcac aaaaagatga ttgaggtccc ttttcaatac cttctctcat ctttcaaatc 2160 ccattgaaac ctaaaacttc tcaccacgct ttaccattgt tctccaaaaa cttatagcaa 2220 tgtctataac ttttttatct ctgaaaagca gtgttccatt tttctttttc ctattttatt 2280 tcaattgttt ctcacatttc gtttggattc tttgcttgtc aaccagcttc ttcttccact 2340 tttaccgtct aattttcagg gcagggagcc atcaaaccca cgaccactag atccat 2396 12 45 DNA Caenorhabditis elegans 12 ccttctcgat ttcaaaatgt caactaaaca tatgcaacat atgtg 45 13 159 DNA Artificial Sequence Description of Artificial SequenceHUMANIZED PIG PLB cDNA 13 atggagaaag tccaatacct cactcgctct gctattagaa gagcttcaac cattgaaatg 60 cctcaacaag cacgtcaaaa ccttcagaac ctatttatca atttctgtct catcttaata 120 tgcctcttgc tgatttgcat catcgtgatg cttctctga 159 14 52 PRT Sus sp. 14 Met Asp Lys Val Gln Tyr Leu Thr Arg Ser Ala Ile Arg Arg Ala Ser 1 5 10 15 Thr Ile Glu Met Pro Gln Gln Ala Arg Gln Asn Leu Gln Asn Leu Phe 20 25 30 Ile Asn Phe Cys Leu Ile Leu Ile Cys Leu Leu Leu Ile Cys Ile Ile 35 40 45 Val Met Leu Leu 50 15 52 PRT Homo sapiens 15 Met Glu Lys Val Gln Tyr Leu Thr Arg Ser Ala Ile Arg Arg Ala Ser 1 5 10 15 Thr Ile Glu Met Pro Gln Gln Ala Arg Gln Asn Leu Gln Asn Leu Phe 20 25 30 Ile Asn Phe Cys Leu Ile Leu Ile Cys Leu Leu Leu Ile Cys Ile Ile 35 40 45 Val Met Leu Leu 50 16 2251 DNA Caenorhabditis elegans 16 gaacgaaatg ctgaatcggc catcgaagcg ctcaaggaat acgaaccaga aatggccaag 60 gtcatccgat ccggacacca cggaattcag atggttcgcg ctaaggaact cgtgccagga 120 gatcttgtcg aagtttcagg ttagcaaaaa cttttttttt taactttcaa attttaaacc 180 atatattttt cagtcggaga caagatccca gccgatctcc gtcttgtgaa gatctactcc 240 accaccatcc gtatcgatca gtccatcctc accggagaat ctgtgtctgt tatcaagcac 300 accgactctg tgccagatcc acgcgctgtt aaccaggaca agaagaattg tctgttctcg 360 ggaaccaatg tcgcatctgg aaaggctcgt ggaatcgtct tcggaaccgg attgaccact 420 gaaatcggaa agatccgtac cgaaatggct gagaccgaga atgagaagac accacttcaa 480 cagaagttgg acgaattcgg agagcaactt tccaaggtta tctctgttat ttgcgttgct 540 gtttgggcta tcaacattgg acatttcaac gatccagctc acggtggatc atgggttaag 600 ggagcaatct actacttcaa aatcgccgtt gctcttgccg tcgctgctat tccagaagga 660 cttccagctg tcatcaccac gtgccttgcc ctcggaactc gccgtatggc caagaagaac 720 gctattgtaa gatcccttcc atccgtcgaa actcttggat gcacatctgt tatctgctct 780 gacaagactg gaactctcac caccaaccag atgtctgtgt caaagatgtt catcgctgga 840 caagcttctg gagacaacat caacttcacc gagttcgcca tctccggatc cacctacgag 900 ccagtcggaa aggtttccac caatggacgt gaaatcaacc cagctgctgg agaattcgaa 960 tcactcaccg agttggccat gatctgcgct atgtgcaatg attcatctgt tgattacaat 1020 gagaccaaga agatctacga gaaagtcgga gaagccactg aaactgctct tatcgttctt 1080 gctgagaaga tgaatgtttt cggaacctcg aaagccggac tttcaccaaa ggagctcgga 1140 ggagtttgca accgtgtcat ccaacaaaaa tggaagaagg agttcacact cgagttctcc 1200 cgtgatcgta aatccatgtc cgcctactgc ttcccagctt ccggaggatc tggagccaag 1260 atgttcgtga agggagcccc agaaggagtt ctcggaagat gcacccacgt cagagttaac 1320 ggacaaaagg ttccactcac ctctgccatg actcagaaga ttgttgacca atgcgtgcaa 1380 tacggaaccg gaagagatac ccttcgttgt cttgccctcg gaaccatcga taccccagtc 1440 agcgttagca acatgaacct cgaagactct acccaattcg tcaaatacga acaagacatc 1500 acatttgtcg gagtcgtcgg aatgcttgac cccccaagaa ctgaagtttc ggactcgatc 1560 aaggcttgta accacgctgg aatccgtgtc atcatgatca ccggagacaa caagaacacc 1620 gctgaggcta tcggaagaag aatcggactc ttcggagaga acgaggatac cactggaaaa 1680 gcttacactg gacgtgaatt tgacgatctt ccaccagagc aacaatctga agcctgccgc 1740 agagctaagc ttttcgcccg tgtcgagcca tctcacaagt ccaagattgt cgatatcctt 1800 caatcccagg gagagattac tgctatgacc ggagacggag tcaacgacgc tccagctttg 1860 aagaaggccg aaatcggaat ttctatggga tcaggaactg ctgtcgccaa gtctgcatct 1920 gaaatggttc ttgctgacga taacttcgca tccattgtgt ctgctgtcga agaaggacgt 1980 gctatttaca acaacatgaa acaattcatc agatatctca tctcatctaa cgtcggagaa 2040 gtcgtctcca tcttcatggt cgccgcactc ggaattccag aggctctcat tccagttcaa 2100 cttctctggg ttaacttggt cactgacggt cttccagcca ctgctctcgg attcaatcca 2160 ccagatcttg acattatgga cagacatcca cgttcagcca acgatggact catctctgga 2220 tggctcttct tcagatatct tgctgtcgga a 2251 17 22 DNA Artificial Sequence Description of Artificial Sequence primer oGK21 17 tggactcatc tctggatggc tc 22 18 39 DNA Artificial Sequence Description of Artificial Sequence primer oGK22 18 cttctccttt actcatcaat tcgttatgta acttgtcgg 39 19 35 DNA Artificial Sequence Description of Artificial Sequenceprimer oGK23 19 gaactataca aatagttgaa gttcttctaa ccccc 35 20 24 DNA Artificial Sequence Description of Artificial Sequence primer oGK24 20 gcgtttatcc ttgattggag cttc 24 21 21 DNA Artificial Sequence Description of Artificial Sequence primer oGK25 21 gaatggatcg ccgtgttgaa g 21 22 36 DNA Artificial Sequence Description of Artificial Sequence primer oGK26 22 ttctccttta ctcatgtcgc gtttatcctt gattgg 36 23 37 DNA Artificial Sequence Description of Artificial Sequence primer oGK27 23 gaactataca aatagaaatg acagtgctcc ctcaatc 37 24 23 DNA Artificial Sequence Description of Artificial Sequence primer oGK28 24 gtgggatcct ggtttgttct gag 23 25 23 DNA Artificial Sequence Description of Artificial Sequence primer SERCA P2 25 cgaagagcac gaagatcaga cag 23 26 19 DNA Artificial Sequence Description of Artificial Sequence primer SERCA P8 26 gagaggcggt tggtttggg 19 27 22 DNA Artificial Sequence Description of Artificial Sequence primer SERCA P4 27 ccgttcgtca tccttctcat tc 22 28 20 DNA Artificial Sequence Description of Artificial Sequence primer SERCA P7 28 cgacagatgg accgacgagc 20 29 35 DNA Artificial Sequence Description of Artificial Sequence primer oGK33F256V 29 caacagaagt tggacgaagt cggagagcaa ctttc 35 30 35 DNA Artificial Sequence Description of Artificial Sequence primer oGK34F256V 30 gaaagttgct ctccgacttc gtccaacttc tgttg 35 31 57 DNA Artificial Sequence Description of Artificial Sequence primer oGK118 31 gccagtcgga aaggtttcca aggacgacaa gccagttaac ccagctgctg gagaatt 57 32 57 DNA Artificial Sequence Description of Artificial Sequence primer oGK119 32 aattctccag cagctgggtt aactggcttg tcgtccttgg aaacctttcc gactggc 57 33 40 DNA Artificial Sequence Description of Artificial Sequence primer oGK108 33 gaccgtacga aattttcagg aaaggaatgc agaaaatgcc 40 34 37 DNA Artificial Sequence Description of Artificial Sequence primer oGK109 34 ccccggccgg ccttactcca gtattgcagg ttccagg 37 35 30 DNA Artificial Sequence Description of Artificial Sequence primer oGK51 35 gctctagatg gataaagtcc aatacctcac 30 36 30 DNA Artificial Sequence Description of Artificial Sequence primer oGK52 36 gctctagatg gagaaagtcc aatacctcac 30 37 30 DNA Artificial Sequence Description of Artificial Sequence primer oGK55 37 ggggtacctc agagaagcat cacgatgatg 30 38 35 DNA Artificial Sequence Description of Artificial Sequence primer oGK56 38 ggggtaccat gagaagcatc acgatgatgc aaatc 35 39 6 PRT Caenorhabditis elegans 39 Lys Asp Asp Lys Pro Val 1 5

Claims

1. A method of identifying compounds which modulate the interaction between a sarco/endoplasmic reticulum calcium ATPase and phospholamban, which method comprises:

exposing transgenic C. elegans which contains a first transgene comprising nucleic acid encoding a vertebrate phospholamban protein and which expresses a SERCA protein to a compound under test; and

2. A method as claimed in claim 1 wherein the vertebrate phospholamban protein is pig phospholamban or human phospholamban.

3. A method as claimed in claim 1 or claim 2 wherein the first transgene comprises nucleic acid encoding a vertebrate phospholamban protein operatively linked to a tissue-specific promoter.

4. A method as claimed in claim 3 wherein the tissue-specific promoter is capable of directing gene expression in cells of the C. elegans pharynx, preferably pharynx muscle cells or capable of directing gene expression in cells of the C. elegans vulva, preferably vulva muscle cells or capable of directing gene expression in the cells of the C. elegans body wall muscles.

5. A method as claimed in any one of claims 1 to 4 wherein the transgenic C. elegans contains a second transgene comprising nucleic acid encoding the said SERCA protein.

6. A method as claimed in claim 5 wherein the second transgene comprises nucleic acid encoding the said SERCA protein operatively linked to the promoter region of a SERCA gene.

7. A method as claimed in claim 6 wherein said promoter region is the promoter region of the C. elegans SERCA gene.

8. A method as claimed in any one of claims 5 to 7 wherein the SERCA protein is a vertebrate SERCA protein.

9. A method as claimed in claim 8 wherein the vertebrate SERCA protein is pig SERCA1a, pig SERCA1b, pig SERCA2a, pig SERCA2b, Pig SERCA3, human SERCA1a, human SERCA1b, human SERCA2a, human SERCA2b or human SERCA3.

10. A method as claimed in any one of claims 5 to 7 wherein the SERCA protein is a fusion between a C. elegans SERCA protein and a vertebrate SERCA protein.

11. A method as claimed in any one of claims 1 to 10 wherein the vertebrate phospholamban protein is expressed in at least cells of the pharynx and preferably the pharynx muscle cells of said transgenic C. elegans.

12. A method as claimed in claim 11 wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating an increase in the activity of the vertebrate SERCA protein comprises detecting a change in the pharynx pumping efficiency of the C. elegans.

13. A method as claimed in claim 11 wherein the transgenic C. elegans contains a further transgene comprising a promoter which is capable of directing gene expression in the muscles of the C. elegans pharynx operatively linked to nucleic acid encoding an apoaequorin protein.

14. A method as claimed in claim 13 wherein the promoter is the C. elegans myo-2 promoter or the C. elegans SERCA promoter.

15. A method as claimed in claim 13 or claim 14 wherein the step of detecting a phenotypic, biochemical or behavioural change indicating an increase in the activity of the vertebrate SERCA protein comprises comparing the level of apoaequorin luminescence in the absence of the compound under test and the level of apoaequorin luminescence in the presence of the compound under test.

16. A method as claimed in any one of claims 1 to 10 wherein the vertebrate phospholamban protein is expressed in at least cells of the C. elegans vulva and preferably vulva muscle cells of said transgenic C. elegans.

17. A method as claimed in claim 16 wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a change in the activity of the vertebrate SERCA protein comprises comparing the growth rate of the C. elegans in the absence of the compound under test and the growth rate of the C. elegans in the presence of the compound under test.

18. A method as claimed in claim 16 wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a change in the activity of the vertebrate SERCA protein comprises comparing the turbidity of the C. elegans in the absence of the compound under test and the turbidity of the C. elegans in the presence of the compound under test.

19. A method as claimed in claim 16 wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a change in the activity of the vertebrate SERCA protein comprises detecting a change in the egg laying behaviour of the C. elegans.

20. A method as claimed in claim 16 wherein the step of detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a change in the activity of the vertebrate SERCA protein comprises detecting a change in the number of progeny produced by the C. elegans.