Having isolated a nucleic acid molecule comprising a sequence coding for the polypeptide that is capable of complementing the phenotypic expressions of the anx/anx genotype, it becomes possible to provide for recombinant production of such a polypeptide and to utilise the polypeptide as such or substances inhibiting or enhancing the biological activi- ties of the polypeptide as body weight controlling agents. Based on the provision of the isolated nucleic acid and the polypeptide of the invention and/or substances inhibiting or enhancing its activities, diagnostic means for detecting genetic and physiological disor- ders become readily accessible.

TECHNICAL BACKGROUND AND PRIOR ART Inappropriate or abnormal food intake patterns resulting in clinically recognisable disor- ders such as anorexia and obesity constitute an increasing medical health problem, in particular in the Western world. However, in spite of major research efforts, little is known about the genetic and biochemical mechanisms that control eating behaviour. The regula-

tion of food intake is a complex and poorly understood process. In this connection, an im- portant aspect is the interactions between the central nervous system (CNS) and food in- take and it is generally believed that appetite, energy balance and body weight gain are regulated or modulated by several neurochemical and neuroendocrine signals from differ- ent organs in the body and diverse regions in the brain. It is known that the hypothalamus has an important function in these processes, acting through a variety of systems that in- volve a close interaction between nutrients, amines, neuropeptides and hormones (Lei- bowitz, Trends in Neurosciences, 1992,15: 491-497).

One approach to gaining understanding of the regulation of food intake and energy output is by genetic analysis of animals carrying mutant genes. The first recessive obesity muta- tion, the obese mutation (ob) was described by Ingall et al., 1950, J. Hered. 41: 317-318.

Subsequently, several single-gene mutations in mice have been found to produce an obese phenotype as described by Friedman et al., 1990, Cell 69: 217-220. The mouse obese gene and its human homologue have been cloned as described by Zhang et al., 1994, Nature 372: 425. In WO 96/35787 is described the production of an ob polypeptide using recombinant DNA technology and pharmaceutical compositions comprising ob polypeptides for the control of obesity. Another approach to regulating obesity is sug- gested in WO 97/48806 based upon delivery of an obesity regulating gene, preferably a gene coding for leptin or a leptin receptor, to a mammal.

Anorexia (anx) is a spontaneous recessive lethal mutation located on mouse Chromo- some 2 that causes starvation in mice including mice in the preweanling period (Maltais et al., 1984, J. Hered. 75: 468-472). It was first discovered in 1976 at the Jackson Laborato- ries, U. S. A. in the F2 generation of DW/J x (M. m. poschiavinus x Swiss). These mutant animals of the anx/anx genotype appear normal at birth but they develop symptoms of growth failure and emaciation as well as head weaving, body tremors and uncoordinated gait. The animals die prematurely. No abnormalities in respect of histology and anatomy of the gastrointestinal tract or in respect of biochemical parameters in the blood have been reported. Mutant mice (anxlanx) are characterised by poor appetite with reduced stomach contents compared to normal littermates as from postnatal day 5 (Maltais et al., 1984). Interestingly, the pattern of food intake, measured as daily changes in stomach content, for anx/anx mice is very similar to that observed in normal littermates from birth to 20 days of age (Maltais et al., 1984). These data indicate that anx/anx mice fail to properly regulate the amount of food consumed rather than failing to eat for other reasons.

The neurological symptoms involved in the phenotype caused by the anx/anx genotype, i. e. head weaving, body tremors and uncoordinated gait can be partially suppressed by serotonin antagonists. Increasing the serotonin levels by injecting serotonin agonists pro- duces severe neurological symptoms both in anx/anx mice and healthy litter mates (Mal- tais et al., 1984). It has been reported that mutant mice show hyperinnervation of seroton- ergic fibres in several structures in the brain including the hippocampus, cortex, olfactory bulb and cerebellum (Son et al., 1994, Mol. Brain Res. 25: 129-134).

It is known that the hypothalamus plays an important role in regulation of food intake that is effected by a range of neurochemical messenger molecules that stimulate food intake such as neuropeptide Y (NPY), the agouti gene-related protein (AGRP), the melanin con- centrating hormone (MCH) and orexin (ORX), and molecules having an inhibiting effect on food intake such as leptin, the cocaine and amphetamine regulated transcript (CART) and the a-melanocyte stimulating hormone (a-MSH).

In recent studies, the distribution of several of such neurochemical messenger molecules (including NPY and its Y1, Y2 and Y5 receptors, cholecystokinin, galanin, serotonin, so- matotostatin, acetylcholinesterase, AGRP, the pro-opiomelanocortinergic (POMC) pep- tides and CART), in mice that are homozygous for the anorexia mutation has been stu- died by using immunohistochemistry and in situ hybridisation (Broberger et al., 1997, 1998,1999 and Johansen et al., 2000). Among the significant findings in these studies are: NPY-like immunoreactivity is increased markedly in arcuate nucleus cell bodies and decreased in terminals of the arcuate nucleus and other hypothalamic regions of anx/anx mice as compared to normal litter mates. Similar observations were made in respect of AGRP. No differences in mRNA expression for NPY and AGRP, respectively was found in the arcuate nucleus of the anx/anx mice. However, a decreased number of POMC mRNA-expressing neurons in the anx/anx arcuate nucleus was observed and, in parallel, decreased mRNA levels for the Y1 and Y5 receptors for NPY which are expressed in POMC-neurons. Additionally, anx/anx mice were found to have significantly decreased levels of CART mRNA label and peptide-immunoreactive cell bodies and fibres in the ar- cuate nucleus and a lower number of detectable CART-expressing cells in the dorsome- dial hypothalamic nucleus/lateral hypothalamic area.

The results of the above studies clearly indicate that the anx/anx genotype confers phe- notypic traits that are directly or indirectly involved in food intake regulation. A primary

objective of the present invention is to provide the gene product (s) of the gene or genes which is/are mutated in the anx/anx genotype or similar genotypes conferring phenotypic traits similar to the anx/anx genotype as the basis for a novel approach to regulating food intake and weight gain.

SUMMARY OF THE INVENTION Accordingly, the present invention pertains in a first aspect to an isolated nucleic acid molecule comprising a nucleotide sequence derived from a vertebrate animal, said nu- cleotide sequence codes for a polypeptide that is capable of at least partially comple- menting the phenotype of an animal having the anx/anx genotype as described in Maltais et al., J. Hered., 1984,75: 468-472, or a similar genotype.

In further aspects the invention provides an expression vector comprising the nucleic acid molecule as defined above and a host cell comprising such a vector.

In a still further aspect the invention relates to a method of producing a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, the method comprising the steps of: (i) providing a nucleic acid molecule as defined above, (ii) intro- ducing the nucleic acid molecule into a host cell that is capable of expressing the nucleo- tide sequence coding for the phenotype complementing polypeptide, (iii) culturing the host cell under conditions permitting expression of the nucleotide sequence coding for the phenotype complementing polypeptide, and (iv) harvesting the polypeptide.

In other aspects there is provided a method of inducing, in a vertebrate animal, the pro- duction of a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984) or a similar genotype, the method comprising administering to the animal the nucleic acid molecule as defined herein; and an isolated polypeptide that is capable of at least partially comple- menting the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, said polypeptide is obtainable by the above method for producing the polypeptide.

In yet further aspects the invention pertains to methods of regulating food or feed intake or weight gain in a vertebrate animal, the method comprising administering to the animal a food or feed intake regulating amount of the polypeptide as defined herein.

There is also provided a method of regulating food or feed intake in a vertebrate animal, the method comprising administering to the animal an effective amount of a molecule that blocks or inhibits or enhances the biological activity of the polypeptide according to the invention.

In still further aspects, the invention relates to a pharmaceutical composition comprising a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, or a pharmaceutical active part thereof, and a pharmaceutical acceptable carrier and to a pharmaceutical composition comprising a molecule that blocks or inhibits or en- hances the biological activity of a polypeptide that is capable of at least partially comple- menting the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, said molecule is selected from the group consisting of an antibody capable of binding to the polypeptide, a receptor capable of binding to the polypeptide, an antagonist that prevents the polypeptide from binding to its receptor and any other molecule that is capable of binding to the polypeptide or a pharmaceutically ac- tive part thereof, and a pharmaceutically acceptable carrier.

In yet another aspect there is provided a method of isolating a molecule that is capable of interacting with a polypeptide that is capable of at least partially complementing the phe- notype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, the method comprising the steps of: (i) providing the polypeptide of the invention, (ii) permitting the polypeptide to react with cells or fragments or extracts thereof to form a binding pair, and (iii) separating from the binding pair a molecule that binds to the polypeptide.

There is also provided an antibody comprising at least one binding site that is capable of binding to the polypeptide encoded by the above nucleotide sequence derived from a vertebrate animal, said nucleotide sequence codes for a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, and a method of detecting the

presence of a polypeptide that is capable of at least partially complementing the pheno- type of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, or a homologue of said polypeptide, the method comprising contacting an antibody capable of binding to the polypeptide with a sample suspected of containing the polypeptide or the homologue thereof to allow formation of a polypeptide/antibody complex and detecting the complex.

In yet another aspect the invention relates to an isolated molecule that interacts with a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, said isolated molecule is provided by the above method of isolating such a molecule.

In another further aspect there is provided a method of producing an antibody to the above isolated molecule that interacts with a polypeptide that is capable of at least par- tially complementing the phenotype of an animal having the anx/anx genotype as de- scribed in Maltais et al. (1984), or a similar genotype, the method comprising administer- ing an immunologically effective amount of the molecule to an animal and collecting from the animal serum containing antibodies against the receptor or spleen cells for the pro- duction of monoclonal antibodies against the molecule.

In a further aspect the invention pertains to a method for detection of a molecule that is capable of binding to a polypeptide capable of at least partially complementing the phe- notype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, the method comprising the steps of: (i) providing the antibody pro- duced by the above method of producing an antibody to the above isolated molecule that interacts with a polypeptide that is capable of at least partially complementing the pheno- type of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, (ii) reacting the antibody with cells or fragments or extracts thereof to form a binding pair, and (iii) detecting the presence of the binding pair.

In further aspects the invention provides a kit for detection of a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx geno- type as described in Maltais et al. (1984), or a similar genotype, said kit comprising an an- tibody comprising at least one binding site that is capable of binding to the polypeptide encoded by the above nucleotide sequence derived from a vertebrate animal, said nu-

cleotide sequence codes for a polypeptide that is capable of at least partially comple- menting the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype; a kit for detection of a receptor for a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, said kit comprising said polypeptide ; and a kit for detection of antibodies to a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, said kit comprising the polypeptide.

The invention pertains in a still further aspect to a method of identifying in a sample a nu- cleotide sequence that codes for a putative polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype. In presently preferred embodiments, the method comprises the steps of: (i) providing the coding nucleotide sequence as defined above or its complementary strand, or a part thereof, (ii) contacting the sample with said sequence or part thereof under hybridising conditions to form a binding pair, and (iii) detecting the presence of the binding pair. In other useful embodiments, the coding sequence is identified by PCR or other similar nucleic acid amplification methods.

In a still further aspect the invention relates to a method of controlling in an animal the ex- pression of a nucleotide sequence coding for a polypeptide that is capable of at least par- tially complementing the phenotype of an animal having the anx/anx genotype as de- scribed in Maltais et al. (1984), or a similar genotype, the method comprising the steps of: (i) providing a molecule that inhibits the translation of a transcript of the coding sequence, and (ii) administering to the animal a translation inhibiting effective amount of said mole- cule.

DETAILED DISCLOSURE OF THE INVENTION It is a primary objective of the present invention to provide a nucleic acid molecule that comprises a nucleotide sequence coding for a polypeptide that is capable of at least par- tially complementing the phenotype of an animal having the anx/anx genotype as de- scribed in Maltais et al. (1984), or a similar genotype. In this context, the expression"nu-

cleic acid molecule"refers to DNA, cDNA or RNA and"coding nucleotide sequence"re- fers to a DNA or a RNA that encodes a specific amino sequence, or their complementary strand. As used herein, the expression"capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype"implies that the polypeptide, when it is introduced into or produced in an animal having the anxlanx genotype or a similar genotype, is capable of eliminating or reducing at least some of the phenotypic traits associated with that genotype. Such phenotypic traits include not only those described in Maltais et al. (1984), which is incor- porated herein by reference, but they include any other traits, whether they have been discovered or will be discovered in the future, which can be associated with the anx/anx genotype, or with a similar genotype. Accordingly, the phenotypic traits include any de- tectable behavioural, biochemical, histological and physiological changes, relative to an animal not having the genotype, including an animal of the same species such as a litter mate, in an animal having the anx/anx genotype or the similar genotype such as those changes published by Broberger et al. (1997,1998 and 1999) and Johansen et a/. (2000), which publications are also incorporated herein by reference. As used herein, the term "animal"include any species of vertebrate animals including rodents such as a mouse or a rat, carnivores such as canine and feline animals, ruminants, pigs, horses, birds, fish, and humans.

Accordingly, the phenotypic traits as referred to above include the following (as compared to an animal not having the genotype): 1. Traits described in Maltais et al. (1984) (i) growth failure and emaciation; (ii) blood parameters including total RBC, haematocrits, haemoglobin, mean cell volume within normal ranges; (iii) higher serum cholesterol level ; (iv) lower blood glucose level ; (v) elevated alkaline phosphatase level ; (vi) abnormal behaviour including head weaving, body tremors, uncoordinated gait and hyperactivity, which can be diminished by injection of 5,7-dihydroxytryptamine;

2. Traits described in Broberger et a/. (1997,1998 and 1999) and Johansen et al.

(2000) (viii) Increased agouti gene-related protein (AGRP) immunoreactivity in arcuate nucleus cell bodies and decreased immunoreactivity in hypothalamic terminals, no difference in AGRP mRNA expression in the arcuate nucleus (Broberger et al., 1998); (ix) increase of neuropeptide Y-like immunoreactivity in hypothalamic arcuate cell bodies and decrease of said immunoreactivity in terminals in the arcuate nucleus, but essentially no difference in neuropeptide Y (NPY) mRNA in the arcuate nucleus (Broberger et al., 1997); (x) decreased numbers of Pro-Opiomelanocortin (POMC) mRNA-expressing neurons in the arcuate nucleus (Broberger et al., 1999); (xi) decreased mRNA levels for NPY Y1 and Y5 receptors in POMC neurons (Broberger et al., 1999); (xii) decreased immunoreactivities in the hypothalamus for adrenocorticotropic hormone (marker for POMC cell bodies), a-melanocyte-stimulating hormone (marker for axonal projections) and NPY Y1 receptor (marker for dendritic arborisations) (Broberger et al., 1999) ; (xiii) cocaine and amphetamine regulated transcript (CART) in the arcuate nucleus and serum leptin levels are reduced and a decreased number of CART expressing cells in the dorsomedial and lateral hypothalamic areas.

3. Further, unpublished phenotypic traits (xiv) the Na+, K+-ATPase activity is upregulated in the striatum and striatal dopamine-and dopamine metabolite levels are lower ; (xv) the insulin content in pancreatic (3-cells is increased at a statistically significant level and insulin is not secreted.

As used herein, the expression"similar genotype"refers to a genetic disorder in a verte- brate animal that confers one or more of the above phenotypic traits such as e. g. having at least half of the traits (i) to (vi), optionally combined with at least one of the above traits (vii) to (xv) such as at least two of these traits including at least three, four, five or six of such traits (vii) to (xv).

It will be appreciated that in the present context"a genetic disorder"in an anx/anx geno- type or in a similar genotype can be a mutation in a coding sequence resulting in the ex- pression of a gene product having, relative to the wildtyp product, a reduced biological activity, a mutation in a coding sequence that results in a reduced expression or even no expression of a biologically active polypeptide or a mutation in a nucleic acid sequence that codes for a gene product having a regulatory effect on the expression of a coding se- quence e. g. causing a down regulation of the expression of a biologically active polype- tide such as the Tyro 3 gene product. When the genetic disorder is one that results in lower or higher expression level, relative to a wildtyp individual, of a gene product, it can e. g. be detected by comparing the level of mRNA species in total mRNA determined in vitro or in vivo in anx/anx genotype animals and wildtyp litter mates and identifying mRNA polymorphisms. An example of such an approach is described in Example 3 below. The genetic disorder may also be one that results in a changed activity of the gene product.

As a matter of clarification, it should be noted that the expression"capable of at least par- tially complementing the phenotype of an animal having the anx/anx genotype as de- scribed in Maltais et al. (1984), or a similar genotype"as defined above is used herein to characterise the effects and functionalities of the polypeptide expressed by the nucleotide sequence and this use does not imply any limitations in respect of the use of the polype- tide as can be appreciated from the following description of the invention.

The nucleic acid molecule comprising the nucleotide sequence that codes for a polype- tide as defined herein can be derived from any vertebrate animals. One preferred ap- proach to providing the nucleic acid molecule is to derive it from an animal having the anxlanx genotype or a similar genotype as defined above by isolating a nucleic acid molecule from such an animal comprising one or more mutations as defined above that confer (s) the phenotype as defined above and isolating and characterising the coding se- quence (s) or the regulatory sequence (s) containing the mutation or mutations. The coding sequence for the putative polypeptide as defined above can then be derived synthetically e. g. by modifying the mutant gene to its wild type sequence, or the isolated mutant se- quence or a part hereof can be used as a hybridisation probe to identify the corresponding wildtyp sequence in any vertebrate species or such a wild type sequence can alterna- tively be identified by comparing the mutant sequence with published gene sequence data.

It will be appreciated that the expression"nucleic acid molecule that comprises a nucleo- tide sequence coding for a polypeptide"includes a nucleic acid molecule that consists only of the nucleotide sequence coding for a polypeptide as defined herein. However, the nucleic acid may also comprise further nucleotides including nucleotide sequences that have a regulatory effect on the expression of the polypeptide and may be of a size which is typically in the range of 1 kilobase (kb) to 1 megabase, such as in the range of 10 kb to 0.75 megabase including the range of 50 kb to 0.5 megabase.

The nucleic acid molecule of the invention can, as it is mentioned above, comprise a regulatory nucleotide sequence that regulates the expression of the nucleotide sequence coding for the phenotype complementing polypeptide. As used herein, the expression "regulatory nucleotide sequence"includes any expression control sequence, i. e. a se- quence that is conventionally used to affect expression of a nucleotide sequence or a gene that encodes a polypeptide and includes one or more components that affect (s) ex- pression, including transcription and translation signals. Such a sequence includes e. g. one or more of the following : a promoter sequence, an enhancer sequence, an upstream activation sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5'leader sequence to optimise initiation of translation in mammalian host cells, a secretion leader sequence that provides for secretion of the phenotype complementing polypeptide upon expression of the nucleotide sequence coding for the polypeptide and a Shine-Delgarno sequence. Appropriate regulatory nucleotide sequences differ depending on the host system in which the polypeptide is to be expressed. E. g. in prokaryotes, such a regulatory sequence can include one or more of a promoter sequence, a ribosomal binding site and a transcription termination sequence. In eukaryotes such a sequence can include a promoter sequence and a transcription termination sequence. In accordance with the present invention, any appropriate regulatory nucleotide sequence can be a se- quence that is naturally associated with the sequence coding for the polypeptide as de- fined herein, but it is also contemplated to use such sequences that are not naturally as- sociated with the nucleotide sequence coding for the phenotype complementing polype- tide. Use of viral promoters to regulate the expression of the polypeptide of the invention is also contemplated.

As used herein the term"polypeptide"includes any polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as de- scribed in Maltais et a/. (1984), or a similar genotype. Accordingly, the term includes the

putative polypeptide (s) encoded by the wild type of the gene or genes carrying the anx mutation in a mouse such as is described in the above reference and any phenotype complementing functional homologue of such a polypeptide, including both a mature polypeptide and a polypeptide provided with a signal peptide sequence and any trunca- tions, variants, alleles, analogues and derivatives thereof. In this context, the expression "phenotype complementing functional homologue"indicates a polypeptide that is capable of complementing at least one of the phenotypic traits conferred by the anx/anx genotype in mice, or a similar genotype.

The term"polypeptide"is not limited to a specific length of the product of the coding se- quence. Thus, polypeptides that are identical or are at least 60%, preferably at least 70% such as at least 80% including at least 90% identical to the polypeptide encoded by the mutated gene in an anx/anx genotype animal or to its wild type, irrespective of the human or non-human source from which it is derived, are included within the definition of the polypeptide of the invention. Also included are therefore alleles and variants of the pro- duct of the gene carrying the anx mutation that contain amino acid substitutions, deletions or insertions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acid residues such as to alter a glyco- sylation site, a phosphorylation site, an acetylation site, or to alter the folding pattern by altering the position of cysteine residues that are not necessary for function. Conservative amino acid substitutions are those that preserve the general charge, hydropho- bicity/hydrophilicity and/or steric bulk of the amino acid substituted, e. g. substitutions be- tween the members of the following groups are conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln and Phe/Trp/Tyr. Furthermore, the term"polypep- tide"as used herein does not exclude post-expression modifications of the polypeptide such as e. g. glycosylations, acetylations and/or phosphorylations. It will be appreciated that a polypeptide as defined herein can be a component of a fusion protein where it is combined with one or more polypeptides or part (s) hereof.

In one useful embodiment, the nucleic acid molecule of the invention is derived from a ro- dent such as a mouse, including a mouse having the genotype anx/anx, e. g. as it is de- scribed in Maltais et a/. (1984). The molecule may consist only of the gene which carries the anx-associated mutation (s) or it may comprise further nucleotides and be of a size as it is defined above. In a specific embodiment, the nucleic acid molecule is a molecule which is derived from a fragment of the mouse Chromosome 2 that is delimited by:

(i) the locus D2Mit133 (SEQ. ID. NO : 207) that is published in: http ://carbon. wi. mit. edu: 8000/cgi-bin/mouse/sequenceinfo ? database=&sts=D2Mit133, and by (ii) the locus D2Jojo5 (SEQ. ID. NO : 208) (see Figure 1). The size of this fragment is about 0.2 centiMorgan or 400 kilobase. This fragment, which is further described in the following examples, is further characterised in that it comprises a nucleotide sequence selected from the group consisting of the sequences D2Dcr14 (accession No. G36398) (SEQ. ID. NO : 209), D2Mit104 (SEQ. ID. NO : 210) published in: http ://carbon. wi. mit. edu: 8000/cgi-bin/mouse/sequenceinfo ? database=&sts=D2Mit 104, D2Mit395 (SEQ. ID. NO : 211) published in http ://carbon. wi. mit. edu: 8000/cgi-bin/mouse/sequenceinfo ? database=&sts=D2Mit395, Ltk (accession Nos. Mus. musculus : X52621; X07984) (SEQ. ID. NOS : 161-180), Tyro 3A (accession No. Mus. musculus : U18342) (SEQ. ID. NO : 212), Tyro 38 (accession No.

Mus. musculus : U18343) (SEQ. ID. NO : 213) and Tyro3 (accession Nos. Mus. musculus : X78103 (SEQ. ID. NO : 214); AH006738; U23718 (SEQ. ID. NO : 215); U 23719 (SEQ. ID. NO : 216); U 23720 (SEQ. ID. NO : 217); U23721 (SEQ. ID. NO : 218)).

The sequence of the above nucleic acid molecule and any other nucleic acid molecule as defined herein that codes for the phenotype complementing polypeptide according to the invention can e. g. be obtained by determining the nucleotide sequence of the molecule and subsequently searching for ORFs against known genes in databases or by using computer software that predicts genes in novel sequences. Examples of useful databases include Genscan (http://CCR-081. mit. edu/GENSCAN. html) and FGENE (http://dot. imgen. bcm. tmc. edu: 9331/gene-finder/gf. html).

In a further aspect, the invention provides an expression vector comprising the nucleic acid molecule as described above. Such vectors can be provided by ligating the nucleic acid molecule of the invention to an appropriate plasmid vector containing an appropriate

promoter for expression in the selected host expression system. Depending on the ex- pression system, the promoter is preferably a prokaryotic cell promoter, a eukaryotic cell promoter or a viral promoter. Expression plasmids with various promoters are currently available commercially. One example of a suitable plasmid is plasmid pET23 that can be purchased from Novagen (Madison, WI). This plasmid utilises a T7 promoter sequence for expression in bacterial cells. Commercially available mammalian expression plasmids can also be used for the present purposes. Accordingly, the expression vector of the invention includes a vector further comprising a regulatory nucleotide sequence as defined herein- before that regulates the expression of the nucleotide sequence coding for the phenotype complementing polypeptide of the invention. Such a regulatory nucleotide sequence can be one that is naturally associated with the nucleotide sequence coding for the phenotype complementing polypeptide, but is preferably a nucleotide sequence that is not naturally associated with said sequence. As it is generally preferred that the phenotype comple- menting polypeptide, when produced industrially, is secreted into the growth medium for the host cell comprising the expression vector, the vector is preferably one that further comprises a nucleotide sequence encoding a secretion leader sequence that provides for secretion of the phenotype complementing polypeptide upon expression of the nucleotide sequence coding for the polypeptide.

In accordance with the above description of the nucleic acid of the invention, the expres- sion vector is in one embodiment a vector wherein the nucleotide sequence expressing a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, is derived from a vertebrate animal including mammals such as rodents, e. g. a mouse in- cluding a mouse having the genotype anx/anx, or humans, fish or birds. In one specific embodiment, the expression vector comprises a nucleotide sequence expressing a poly- peptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, which is derived from a fragment of the murine Chromosome 2 that is delimited by locus D2Mit133 (SEQ. ID. NO : 207) and by locus D2Jojo5 (SEQ. ID. NO : 208) and which optionally comprises a nucleotide sequence selected from D2Dcr14 (SEQ. ID. N0 : 209), D2Mit104 (SEQ. ID. NO : 210), D2Mit395 (SEQ. ID. NO : 211), Tyro 3A (SEQ. ID. NO : 212), Tyro 3B (SEQ. ID. NO : 213) and Tyro 3 (SEQ. ID. NOS : 188-206, 214-218), as defined above and any of the further genes within the fragment delimited by locus D2Mit133 (SEQ. ID. NO : 207) and by locus D2Jojo5 (SEQ. ID. NO : 208) as described in the following. If

appropriate, the expression vector may further comprise a nucleotide sequence coding for a selective marker.

In another aspect, the invention relates to a host cell comprising the above vector, i. e. to an expression system for the phenotype complementing polypeptide. The host is prefe- rably selected from a prokaryotic cell and a eukaryotic cell. In preferred embodiments, the host cell expression system is a bacterial expression system. Control elements for use in bacterial host cells include promoters, optionally containing operator sequences, and ribo- some binding sites. Useful promoters include sequences derived from sugar metabolising enzymes such as galactose, lactose (lac) and maltose metabolising enzymes. Further ex- amples include promoter sequences derived from biosynthetic enzymes such as the trp, the ß-lactamase (bla) promoter systems, bacteriophage APL and T7 promoters. In addition, synthetic promoters such as the tac promoter can be used.

Useful host cells can be selected from gram negative bacteria and gram positive bacteria.

Examples of gram negative host cells include E. coli, Pseudomonas spp., Salmonella spp. and Serratia spp and examples of gram positive bacteria that may be used in the inven- tion include Bacillus spp., Streptomyces spp and lactic acid bacterial species such as Streptococcus spp. and Lactococcus spp. Methods for introducing exogenous DNA into such host cells typically include the use of CaCI2 or other agents such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation, nuclear injection or protoplast fusion as described generally in Sambrook et aL (1989). Preferably, the host cell should secret minimal amounts of proteolytic enzymes. Alternatively, in vitro methods of cloning, e. g. PCR or other nucleic acid polymerase reactions are suitable.

Prokaryotic cells used to produce the phenotype complementing polypeptide of the inven- tion are cultured in suitable media as described generally in Sambrook et a/., supra.

Suitable eukaryotic host cells can also be selected from fungal cells including yeast cells, mammalian cells and insect cells.

Expression vectors and transformation vectors, either extrachromosomal replicons or in- tegrating vectors, have been developed for transformation into yeast species including Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris and Schizosaccharomyces pombe, and into species

of filamentous fungi such as Aspergillus spp., Trichoderma spp., Neurospora spp. or Penicillium spp.

Control sequences for yeast vectors include as examples promoter regions from genes such as alcohol dehydrogenase (ADH), endolase, glucokinase, glucose-6-phosphate iso- merase and pyruvate kinase. Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions are available. Yeast enhancers are also advantageously used with yeast promoters. E. g. can upstream activating sequences (UAS) of one yeast promoter be joined with the transcription activation regions of another yeast promoter to create a synthetic hybrid promoter. Furthermore, a yeast promoter may include naturally occurring promoters of non-yeast origin having the ability to bind yeast RNA polymerase and initiate transcription. Other control elements that may be included in yeast expression vectors are terminators and leader sequences which encode signal se- quences for secretion such as e. g. the leader sequence derived form the yeast invertase gene and the a-factor gene.

Methods of introducing exogenous DNA into yeast hosts are well known in the art and typically include transformation of spheroplasts or intact yeast cells treated with alkali cations. The introduction can be carried out by transformation, nuclear injection, electro- poration or protoplast fusion as described generally in Sambrook et al., supra.

For yeast secretion of the polypeptide of the invention, the native signal sequence can be substituted by another leader such as the yeast invertase, a-factor or acid phosphatase leaders. For intracellular production of the polypeptide in yeast, a sequence encoding a yeast protein can be linked to the coding sequence for the phenotype complementing polypeptide to produce a fusion protein that can be cleaved intracellularly upon expres- sion.

Particularly useful insect cell expression systems are based upon baculovirus expression vectors (BEVs), recombinant insect viruses in which the coding sequence for a foreign gene is inserted behind a baculovirus promoter in place of a viral gene, e. g. polyhedrin such as it is described in US Patent No. 4,745,051. A typical useful insect cell expression vector includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, followed downstream by an insect signal DNA sequence capable

of directing secretion of a desired polypeptide, and a site for insertion of the foreign gene encoding the foreign polypeptide and the foreign gene placed under transcriptional control of a baculovirus promoter, the foreign gene herein being the nucleotide sequence coding for the phenotype complementing polypeptide of the invention.

Useful promoters for an insect cell expression system can be derived from any baculovi- rus infecting cells such as e. g. a baculovirus immediate-early gene IEI or IEN promoter or a strong polyhedrin promoter of baculovirus. The insect expression vector for use herein may also include the polyhedrin polyadenylation signal and a selective marker. DNA en- coding suitable signal sequences may also be included such as e. g. the signal sequence of the baculovirus polyhedrin gene or mammalian signal sequences.

The phenotype complementing polypeptide of the invention may also be expressed in mammalian cells such as e. g. adipocytes, using promoters and enhancers that are func- tional in such cells. Typical promoters for mammalian cell expression include as exam- ples, the SV40 early promoter, the CMV promoter, the mouse mammary tumour virus LTR promoter, the adenovirus late promoter and the herpes simplex virus promoter. Mamma- lian expression may be either constitutive or regulated (inducible).

Mammalian cell lines available as hosts for expression are also known and include many immortalised cell lines available from the ATCC, including Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells, human embryonic kidney cells, human lung cells and hu- man liver cells.

It is another objective of the invention to provide a method of producing a polypeptide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et aL (1984), or a similar genotype. The method comprises as a first step that a nucleic acid molecule as described herein is provided, fol- lowed by introducing the nucleic acid molecule into a host cell that is capable of express- ing the phenotype complementing polypeptide and culturing the host cell under conditions permitting expression of the nucleotide sequence coding for the phenotype complement- ing polypeptide, and harvesting the polypeptide. The method of introducing the nucleic acid molecule into the host cell will, as those of skill in the art will readily recognise, de-

pend on the selected host cell system and the expression vector used as it is described hereinbefore, Likewise, the method of culturing the host cells including the composition of the cultivation medium and temperature conditions, and the harvesting processes, will depend on the host cell type and can be determined by those of skill in the art.

Whereas the polypeptide of the invention can be used as such for pharmaceutical pur- poses as the active component of pharmaceutical compositions, its therapeutic use by genetic therapy is also contemplated. Accordingly, the invention relates in one aspect to a method of inducing, in a vertebrate animal, the production of a polypeptide that is capa- ble of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype, the method com- prising administering to the animal the nucleic acid molecule or at least the sequence coding for the complementing polypeptide as described herein.

Accordingly, the nucleic acid molecule that comprises the sequence coding for the phe- notype complementing polypeptide, with or without a signal sequence, can be used for regulation of food or feed intake and/or for regulating body weight, such as for treatment of obesity, by administration thereof via gene therapy. Gene therapy strategies for delivery of the nucleic acid molecule construct can utilise viral or non-viral vectors in in vivo or ex vivo modality. Expression of the coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or inducible.

For delivery of the nucleic acid molecule comprising the coding sequence using viral vec- tors, any of a number of viral vectors may be used including those described in Jolly, Cancer Gene Therapy, 1994,1: 51-64. The coding sequence can be inserted into plas- mids designed for expression in retroviral vectors, adenoviral vectors, adeno-associated viral vectors or sindbis vectors. Promoters that are suitable for use with these vectors in- clude as examples the Moloney retroviral LTR, CMV promoter and the mouse albumin promoter. Replication competent free virus can be produced and injected directly into the animal or human or by transduction of an autologous cell ex vivo, followed by injection in vivo.

The coding sequence can also be inserted into a non-viral delivery system, such as a plasmid, for expression of the polypeptide in vivo or ex vivo. For in vivo administration the coding sequence can be delivered by direct injection or by intravenous infusion. Promo- ters suitable for use in this manner include endogenous and heterologous promoters such as e. g. CMV. The coding sequence can be injected in a formulation comprising buffers or other agents that can stabilise the coding sequence and facilitate transduction thereof into cells and/or provide targeting.

After it has been expressed and harvested according to method described hereinabove, the phenotype complementing polypeptide can be purified to provide an isolated polypep- tide that is capable of at least partially complementing the phenotype of an animal having the anx/anx genotype as described in Maltais et al. (1984), or a similar genotype. The pu- rification and, optionally, folding of the polypeptide can be carried out using conventional methods of isolating and purifying proteins including, but not limited to, precipitation, chromatography, filtration, ultrafiltration and reverse osmosis procedures. If required, the purification of the polypeptide may imply a degree of purity where the polypeptide prepa- ration is essentially free of other polypeptides.

The isolated polypeptide can be used in a variety of ways as it will be described in the following. For example, the polypeptide, optionally in conjunction with suitable carriers, can be administered to animals and humans intravenously, subcutaneously or orally in a therapeutical or prophylactically effective amount in methods of regulating food or feed intake or body weight including weight gain inhibition, in a vertebrate animal, the method comprising administering a food or feed intake regulating amount of the polypeptide ac- cording to the invention. The specific amount to be administered depends on the condition to be treated.

The expression"therapeutically or prophylactically effective amount"is generically the amount of the polypeptide that will provide a desired therapeutic or prophylactic result ei- ther in respect of regulating food or feed intake or in respect of a desired control of body weight, i. e. weight gain inhibition or, optionally weight gain enhancement. The precise in- hibitory amount varies depending on the health and physical conditions of the individual to be treated, the capacity of the individual's ability to adjust to the change in regulation of food intake and metabolism and body size, the formulation and other relevant factors.

As an alternative to administering the polypeptide of the invention as described above, it is also contemplated that food or feed intake in a vertebrate animal can be regulated by administering to the animal an effective amount of a molecule that blocks or inhibits or enhances the biological activity of the phenotype complementing polypeptide of the in- vention. Such a blocking, inhibiting or enhancing molecule can be selected from an anti- body capable of binding to the polypeptide, a receptor capable of binding to polypeptide, an antagonist that prevents the polypeptide from binding to its receptor, an agonist sub- stance that enhances the biological activity of the polypeptide, and any other molecule that blocks, inhibits or enhances the biological activity of the polypeptide. In the present context, the term"receptor"indicates a structure, generally a protein, located on or in a cell, e. g. in the membrane, that specifically recognises a sequence of amino acids of the phenotype complementing polypeptide so as to bind to it with a higher affinity than to a random polypeptide. Such an interaction between the polypeptide and the receptor is generally expected to trigger an intracellular response.

As it is mentioned above, the polypeptide of the invention can be used in a pharmaceuti- cal composition comprising the polypeptide as defined herein, and a pharmaceutical ac- ceptable carrier. The expression"pharmaceutically acceptable carrier"refers to a carrier for administration of a therapeutic or prophylactic agent, such as the polypeptide of the invention or antibodies, and refers to any pharmaceutical carrier that does not in itself in- duce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity effects. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus par- ticles. Such carriers are well known to those of ordinary skill in the art. The carrier may be a pharmaceutically acceptable salt such as e. g. mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates and the like, and the salt of organic acids such as acetates, propionates, malonate, benzoates and the like. A discussion of pharmaceuti- cally acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Publ. Co., N. J. 1991). Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary sub- stances such as wetting or emulsifying agents, pH buffering substances and the like, may be present in such vehicles. Typically, the therapeutic compositions of the invention are provided as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be used.

The pharmaceutical acceptable carrier as defined above can also be used in pharmaceutical compositions that comprise a molecule that blocks, inhibits or enhances the biological activity of the polypeptide according to the invention where the molecule is selected from the group consisting of an antibody capable of binding to the polypeptide, a receptor capable of binding to the polypeptide, an antagonist that prevents the polype- tide from binding to its receptor, an agonist molecule that enhances the biological activity of the polypeptide, and any other molecule that is capable of binding to the polypeptide or a pharmaceutically active part thereof and which blocks, inhibits or enhances its biological activity.

In a further aspect, the invention provides a method of isolating a molecule that is capable of interacting with the phenotype complementing polypeptide as defined herein. This method comprises as the first step that the polypeptide is provided as described above followed by permitting the polypeptide to react with cells or fragments or extracts thereof to form a binding pair, and separating from the binding pair the molecule member that binds to the polypeptide. As used herein, the term"binding pair"refers to a pair of mole- cules such as e. g. a protein/protein pair, a protein/RNA pair or a protein/DNA pair in which the components of the pair bind specifically to each other with a higher affinity than to a random molecule such that upon binding, e. g. in case of a ligand/receptor interaction.

Specific binding indicates a binding interaction having a low dissociation constant, which distinguishes specific binding from non-specific, background binding. The molecules which can be isolated using the method include an antibody capable of binding to the polypeptide or a fragment hereof, a receptor capable of binding to the polypeptide or to a fragment of the polypeptide, an antagonist that prevents the polypeptide from binding to its receptor, e. g. an antibody to the receptor, an agonist molecule that enhances the biological activity of the polypeptide, and any other molecule that is capable of binding to the polypeptide.

The phenotype complementing polypeptide of the invention can be used to generate monoclonal or polyclonal antibodies. Accordingly, the invention provides in a further as- pect a monoclonal or a polyclonal antibody comprising at least one binding site that is ca- pable of binding to the polypeptide encoded by the nucleotide sequence as defined above. Such antibodies to the polypeptide can be prepared by conventional methods, e. g. by immunising a suitable animal such as a mouse, rat, rabbit or goat. Immunisation is

generally performed by mixing or emulsifying the polypeptide in saline, preferably in com- bination with an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion. The immunisation may be boosted 2-6 weeks with one or more injections.

Like any other antibodies, the resulting antibodies, whether polyclonal or monoclonal, can be labelled using conventional techniques. Suitable labels include chromophores, fluoro- phores, radioactive labels, electron-dense reagents, enzymes and ligands having specific binding partners. In this context,"specific binding partner"refers to a protein capable of binding a ligand molecule with high specificity such as in the case of an antigen and an antibody specific herefor. Other specific binding partners include biotin, avidin or strepta- vidin, IgG and protein A.

The antibodies generated in this manner can be used in any conventional applications including for diagnostic and therapeutic purposes. E. g., as a diagnostic, they can be used in an immunoassay for identification or detection of a polypeptide of the invention or a homologue thereof in a sample suspected of containing the polypeptide. For this purpose, the antibodies can be labelled with a suitable marker, such as a radioactive label, and al- lowed to react with the sample. After an appropriate reaction time, the sample is exam- ined for the presence of specific binding pairs. Presence of specific binding suggests that a polypeptide of the invention is present in the sample. The antibodies, when used for di- agnostic purposes, can be a part of kits for detection of the phenotype complementing polypeptide of the invention.

Additionally, the antibodies to the polypeptide can be used for therapeutic purposes for blocking the in vivo biological activity of the polypeptide. When used as therapeutics, the antibodies are preferably compatible with the host subjected to such treatment. E. g. when used for humans, the antibodies can be human antibodies or humanised antibodies, as this term is generally known in the art. The humanised antibodies can be made by any conventional methods for that purpose. e. g. by complementary determining region graft- ing, veneering, phage library display or by use of xeno-mice.

As mentioned above, the invention pertains in one aspect to a method of detecting the presence of a polypeptide as defined herein, or a homologue of said polypeptide, the method comprising contacting an antibody capable of binding to the polypeptide with a sample suspected of containing the polypeptide or the homologue thereof to allow forma-

tion of a polypeptide/antibody complex, i. e. a binding pair as defined hereinbefore, and detecting the complex. The immunoassays which can be used for that purpose can be designed to meet specific requirements, e. g. determined by the nature of the sample to be assayed. Generally, protocols for the immunoassay can e. g. be based on competition, direct reactions for sandwich type assays. Protocols may also include the use of solid supports or they may be by immunoprecipitation. The assays which can be used in accor- dance with the invention may involve the use of labelled antibodies to the polypeptide or labelled target polypeptides. Useful labels include fluorescent, chemiluminescent, radio- active or dye molecules. Assays which amplify the signals from the probe can also be used. Examples hereof include assays which utilise biotin or avidin and enzyme labelled and mediated immunoassays such as ELISA assay. The immunoassays can, if desired, be designed so as to determine the amount of polypeptide antigen that is present in a sample.

An immunoassay utilising the antibodies to the phenotype complementing polypeptide can be used for any biological sample, such as a blood sample, a serum sample, a tissue sample including as examples a sample of brain tissue or a sample of pancreas tissue.

In yet another aspect the invention relates to an isolated molecule that interacts with a polypeptide according to the invention including such a polypeptide that is produced by the above method of producing the polypeptide. Such a molecule includes a monoclonal or a polyclonal antibody as described above, and a receptor molecule as also defined above.

A receptor molecule can e. g. be identified and isolated by reacting cells, cell membranes or extracts with a suitably labelled polypeptide of the invention. The mixture is then ex- amined for presence of specific binding to the labelled polypeptide and the binding pairs formed can be separated by conventional techniques, such as by use of solvents or de- naturing reagents for by passage through a column that selectively binds one member of the binding pair, and eluting the opposite member, i. e. the receptor molecule, with an ap- propriate solvent. The receptor molecule can be purified by conventional techniques and the amino acid sequence thereof can then be determined. Based on the amino acid se- quence identified, an oligonucleotide probe can be made to probe a cDNA or genomic li- brary and clones that hybridise to the probe can be amplified and sequenced. A cDNA clone that encodes a full length receptor can be used for recombinant production of large

quantities of the receptor, useful for further studies into the mechanism of regulation of food intake and/or body weight and to obtain agonists and antagonists thereto.

In a useful aspect of the present invention there is provided a method of producing an an- tibody to the above molecule that is capable of interacting with the polypeptide of the in- vention, including an antibody or a receptor molecule. Such an antibody can be produced by any conventional method as it is described above including that an immunologically effective amount of the molecule is administered to an animal and that sera containing antibodies against the receptor or spleen cells for the production of monoclonal antibodies against the molecule are collected from the immunised animals.

Such antibodies, whether monoclonal or polyclonal can be used in immunoassays, such as it is described above, for detecting molecules that are capable of binding to the poly- peptide of the invention. Generally, an immunoassay for such purposes involves that the above antibody is reacted with cells or fragments or extracts thereof to form a binding pair, and detecting the presence of the binding pair.

A particularly interesting use of antibodies to a receptor for the polypeptide of the inven- tion is to block, reduce or inhibit the biological activity of the polypeptide in vivo. Accor- dingly, such antibodies can be used as therapeutic agents and be provided in formula- tions or pharmaceutical compositions that are suitable for that purpose.

The polypeptide of the invention, optionally in a form where it is provided with a detectable label including those mentioned above, can be provided as a part of a kit for detection of a receptor for the polypeptide or for detection of antibodies to the polypeptide.

It is a further objective of the invention the provide a method of identifying in a sample a target nucleotide sequence that codes for a putative phenotype complementing polypep- tide as defined herein. For that purpose any DNA-or RNA-containing sample can be col- lected from an animal of interest including a human. The method involves that a nucleotide sequence as defined hereinbefore or a complementary strand hereof, or a part or fragment thereof, which is preferably provided with a detectable label, e. g. a radioactive label, is contacted with the sample to be tested under hybridising conditions to form a binding pair and subsequently detecting the presence of the binding pair. The members of the binding pair can then be separated e. g. by a denaturing treatment. The isolated target

member can be used as the starting point for a recombinant production of a polypeptide according to the invention.

It is also, as mentioned above, an objective of the invention to provide a method of con- trolling in an animal the expression of a nucleotide sequence coding for a phenotype complementing polypeptide of the invention. This method comprises as a first step that a molecule that inhibits the translation of a transcript of the coding sequence is provided, followed by administering to the animal a translation inhibiting effective amount of said molecule. In accordance with the invention, presently preferred translation inhibiting mole- cules include antisense RNA and PNA.

The invention is further described in the following examples and the drawings wherein: Fig. 1 illustrates genetic linkage maps of the anx interval of mouse Chromosome 2. The percentage recombination 1 SE is indicated for each genetic interval. Centromere at the top (filled circle) and telomere at the bottom; Fig. 2 is a physical map spanning the anx interval on mouse Chromosome 2 as described and defined in Example 1. The map is not drawn to scale. The relative positions of genes and markers, shown above the map, were deduced form the genetic linkage map and their presence and absence in clones. Chromosome centromere to the left and telomere to the right. Clones are identified by type of clone and number. Open box indicates internal deletion within the clone. AChartier et a/. (1990) Nature Genet. 1,132; Bhttp ://carbon. wi. mit. edu : 8000/cgi-in/mouse/ yacinfo ? yac=281 _H_8&database=mouserelease ; CP 1 library derived from E14 (129/Ola) ES cells ; DBAC library RPCI-23, segment two (BACPAC Resources, Oakland, CA); EPAC library RPCI-21 (BACPAC Resources, Oakland, CA). Genes identified : Ip3k (SEQ. ID. NOS : 181-187) (clone ID P1-Ltk) ; EMBL No. baa92641 (SEQ. ID. NOS : 137-160) (clone ID P1 Ltk); DII4, EMBL No. aaf76428 (SEQ. ID. NOS : 2-12) (clone ID 356H20); Vps18, EMBL No. baa95999 (SEQ. ID. NOS : 13-18) (clone ID 356H20); Epa6, EMBL No. (SEQ. ID. NOS : 19-20) epab mouse (clone ID 356H20); EMBL No. mmaa11531 (SEQ. ID. NOS : 21-24) (clone ID 356H20); EMBL No. q9ulg1 (SEQ. ID. NOS : 25-58) (clone ID 356H20); EMBL No. q9vci9 (SEQ. ID. NOS : 59-61) (clone ID 356H20); Chp, EMBL No. q9z1yO (SEQ. ID. NOS : 62-64) (clone ID 356H20); S27-1, EMBL No. aad56582 (clone ID 356H20); Fabe, EMBL No. (SEQ. ID. NO : 65) fabemouse

(clone ID 348 L16); Mga, EMBL No. q9qxj5 (SEQ. ID. NOS : 66-88) (clone ID 348 L16); JNK (SEQ. ID. NOS : 89-119), EMBL No. q9r010 (clone ID 348 L16); CPLA2-beta (SEQ. ID. NOS : 120-136), EMBL No. q9ukv7 (clone ID 348 L16); p22, EMBL No. rn39875 (clone ID 365N11) ; SEQ. ID. NO : 1 is the sequence of gene S27-1, EMBL No aad56582, exon sequences are indicated with letters in bold (also in the following figures); SEQ. ID. NOS : 2-12 is the sequence of gene DII4, EMBL No. aaf76428; SEQ. ID. NOS : 13-18 is the sequence of gene Vps18, EMBL No. baa95999; SEQ. ID. NOS : 19-20 is the sequence of gene Epa6, EMBL No. epabmouse ; SEQ. ID. NOS : 21-24 is the sequence of gene EMBL No. mmaa11531 ; SEQ. ID. NOS : 25-58 is the sequence of gene EMBL No. q9ulg1 ; SEQ. ID. NOS : 59-61 is the sequence of gene EMBL No. q9vci9 ; SEQ. ID. NOS : 62-64 is the sequence of gene Chp, EMBL No. q9z1yO ; SEQ. ID. NO : 65 is the sequence of gene Fabe, EMBL No. fabemouse ; SEQ. ID. NOS : 66-88 is the sequence of gene Mga, EMBL No. q9qxj5; SEQ. ID. NOS : 89-119 is the sequence of gene JNK, EMBL No. q9r010 ; SEQ. ID. NO : 120-136 is the sequence of gene CPLA2-beta, EMBL No. q9ukv7; SEQ. ID. NOS : 137-160 is the sequence of gene EMBL No. baa92641; SEQ. ID. NOS : 161-180 is the sequence of gene Ltk; SEQ. ID. NOS : 181-187 is the sequence of gene Ip3k ; and

SEQ. ID. NOS : 188-206 is the sequence of gene Tyro 3.

EXAMPLE 1 Identifying the anx interval in mouse Chromosome 2 Material and methods.

1. Markers and primers All markers were simple sequence length polymorphisms (SSLPs) and were tested by PCR. The D2Mit markers were obtained from Research Genetics, Huntsville, AL. The markers were chosen based on the chromosomal map position reported in the Chr2 : lntegrated MIT SSLP and Copeland/Jenkins RLFP Genetic Maps (http ://carbon. wi. edu: 8000/cgi-bin/mouse/sts_by_chrom). The D2Jojo primer pairs, with predicted annealing temperatures of 55°C or higher, were designed around simple re- peats located in the clones covering the anx interval.

The forward primer was labelled with y-32P-dATP as follows : F primer (6, 7µM), 0. 125 µl/sample ; 10X PNK buffer, 1.6 pLI ; T4 PNK (10U/µl), 1 pI/16 pl reaction; y-32P (6000 Ci), 0.03 pI/sample ; H20 to 16 pI final volume.

2. PCR protocol The PCR was performed according to the following protocol.

Labelling reaction mixture, 16 pLI ; R primer (6,7 µM), 0.125 pI/sample, dNTPs (5 mM), 0.4 /sample ; 10X PCR buffer (0.5M KCI, 15mM MgCl2, 100mM Tris, 0. 1% gelatin), 1 Ill/sample ; Taq (5U/pI), 0.1 ti/sample ; DNA, 2 111 ; H20 to 10 pI final volume.

95°C, 5 minutes; 95°C, 30 seconds; 50-60°C, 30 seconds (temperature optimised for each marker); 72°C, 30 seconds; 35 cycles ; 72°C, 10 minutes; 4°C.

After the PCR had been carried out, the products were separated on a 6% denaturing polyacrylamide gel, National Diagnostics, Atlanta, GE, at 90V for approximately 1.5 hrs.

The gel was then transferred to an X-ray film that was exposed for a period of time in the range of two hours to a couple of days.

3. DNA preparation DNA was prepared from 2-5 mm mouse tail snips as follows: Add 1 ml of Tail Solubilisation Buffer (TSB) (1XSET [10XSET: 10% SDS, 50 mM EDTA, 100 mM Tris (pH 8.0)], 100mM NaCI, 200 llg/ml proteinase K) to the tail snip.

Incubate for about 4 hrs at 55°C to digest tail.

Add 400 pI Tail Salts [4. 21M Nacl, 0.63M KCI, 10mM Tris (pH 8.0)] and vortex tho- roughly. Incubate overnight at 4°C to precipitate proteins.

'Centrifuge the tubes for 10 minutes at maximum speed in a microfuge.

# Remove 100 pI supernatant and precipitate the DNA with 200 VI 95% EtOH for 20 minutes at-70°C or 4 hrs at-20°C.

Pellet the DNA in a microfuge at maximum speed for 20 minutes.

# Remove supernatant and resuspend the DNA in 100 pI water or TE.

2µl of DNA suspension was used in a PCR reaction.

4. Breeding and mapping scheme and results The anx mutation arose at The Jackson Laboratory (TJL), Bar Harbor, ME, in 1976 in the F2 generation of a cross between strain DW/J and an inbred strain derived from a cross of M. m. poschiavinus to a Swiss stock. The anx mutation has since then been kept on a

nonagouti hybrid background referred to as B6C3-a/a F1. A general treatise of mouse ge- netics can be found in Mouse Genetics, Lee M. Silver, Oxford University Press, New York, 1995.

When starting a new linkage study in mice, two questions must be addressed. First, what strains should be used and second, what type of breeding scheme is suitable. To map a mutationally defined locus it is required to start with one strain that carries the mutation.

This strain is then crossed with another strain that should be selected based on the ge- netic distance between the two strains-the larger the distance, the bigger chance is there to identify polymorphisms at DNA marker loci. The choice of breeding scheme is usually limited to one of two approaches: backcross or intercross. The nature of the phe- notype may limit this choice even further. With both approaches, the first mating will al- ways be an outcross between the two parental strains. Once F1 hybrid animals have been generated, it must be decided whether to backcross them to one of the parental strains or to intercross them with each other. There are disadvantages and advantages with both breeding scheme approaches. In this study it was decided to use the intercross approach to map the anx gene. The intercross has two major advantages over the back- cross approach. The first is that it can be used to map recessive lethal mutations, such as the anx mutation, since both heterozygous F1 parents will be normal. The second advan- tage is that informative meiotic events will occur in both parents leading to twice as much information on a"per animal"basis as compared to the backcrossing. anx breeding mice (B6C3-a/a-a +/+ anx) were obtained from TJL. The anx gene had al- ready been mapped to Chromosome 2 and two intercrosses were set up to narrow the anx interval further. Cross 1 comprises 2050 F2 progeny (4100 meioses) of an (B6C3Fe- anx A/+ a x B6C3H F1) intercross. Cross 2 was set up between B6C3Fe-anx A/+ a and CAST/Ei and 372 F2 progeny (744 meioses) from this cross were analysed.

The mapping project was initiated by identifying polymorphic markers in the area to which the anx locus had been mapped, approximately 20cM proximal of the agouti locus on mouse Chromosome 2 (Maltais et al., 1984). Approximately 100 markers from Research Genetics, Huntsville, AL, were tested and about 20% of these turned out to be polymor- phic. The polymorphic markers were tested on the F2 progeny from both of the above crosses. As all of the anorexic mice had to have the genotype anx/anx at the mutation site, it was possible to work out how closely linked to the mutation the markers were,

based on how many anorexic mice were recombined (showed an anx/+ or +/+ genotype) at those specific loci. A marker whose variant co-segregates with the anorexic phenotype is close to the gene controlling the phenotype. The order of the markers closest to the mutation is shown in figure 1. Three animals showed recombination between markers D2Jojo5 and anx (Fig. 1) thus defining D2Jojo5 as the distal flanking marker.

One F2 progeny from cross 2 identified D2Mit133 as the proximal flanking marker. The anx mutation was found to co-segregate with the markers D2Mit104, D2Mit395 and D2Jojo8. Recombination frequencies and standard errors were calculated as described by Green (1981). Genetic distances are shown in figure 1 as percentage recombination + 1 SE.

These results are backed up by a large number of recombinations on either side of the interval. All markers within the interval are non-recombinant and closely linked to the mu- tation.

EXAMPLE 2 Establishing a contig covering the identified anx interval.

The distal end of the anx interval was covered by a Yeast Artificial Chromosome (YAC) spanning from Capn3 (distal to D2Jojo5) to D2Mit395, including Ltk and Tyro3 (Aamir Zu- beri, The Jackson Laboratory, personal communication). When searching the Whitehead database (http ://carbon. wi. mit. edu: 8000/cgi-bin/mouse/index) for YACs containing the other loci known to map within or close to the anx interval, one YAC was identified that not only spans the proximal end of the anx interval, but also overlaps with the distal YAC (Fig.

2).

In order to further characterise the interval, three libraries (a P1 library derived from E14 (129/Ola) ES cells, PAC-RPCI-21 and BAC-RPCI-23) were screened for clones spanning the anx interval. The P1 library was screened by successive rounds of PCR with primers specific for D2Mit133, D2Mit104, D2Mit395 and Ltk. Four P1 clones were recovered (one for each locus). These clones were partially sequenced to obtain STS markers to be used as probes in the screening of the two RPCI-libraries. The clones were assembled in the order established from the genetic linkage map. The clones were linked together with STS

markers generated from the ends of the clone inserts. In some cases the libraries were rescreened to find overlapping clones. Figure 2 shows the relative order of markers, STS loci and the clones that span the interval between D2Mit133 and Capn3. Total DNA was isolated from clones using a CsCI gradient ultra-centrifugation or a modified Qiagen midi- prep protocol (Sambrook et al., 1989; Pinkel et al., 1998).

Materials and methods.

1. Partial sequencing of clone inserts Partial sequences from the clones were generated using appropriate primers using the following reaction mixture: 2 pg DNA; 8 pI BIG Dye (Amersham); 20 pmol primer; H20 to 20 pI final volume.

The sequencing reaction was performed during 100 cycles of ramping at temperatures in the range of 50°C to 96°C as follows : 96°C,30 seconds 1°C/sec to 50°C ##100x 50°C, 5 seconds 1°C/secto60°C 60°C, 4 minutes 4°C 2. Purification of samples The samples were then purified according to the below protocol using Sephadex G50 mi- crotiter filter plates (Millipore). Sephadex G50 (Pharmacia Biotech), 10 g/130 ml.

Fill the wells with Sephadex. Place a collection microtiter plate under the filter plate.

Centrifuge at 1500 rpm for 2 minutes. Empty the collection plate.

Fill the wells again and repeat centrifugation.

Add the sequencing reactions. Place a clean collection plate under the filter plate.

'Centrifuge as above.

Vacuum dry the samples collected and store at-20°C until needed.

Add loading dye and load the samples onto the ABI gel.

3. Providing labelled probes The STS loci generated were PCR amplified and used as probes when screening the PAC-RPCI-21 and BAC-RPCI-23 libraries (BACPAC Resources, Oakland, CA).

The probes were labelled using a standard Random Priming protocol (Current Protocols in Molecular Biology, 3.5.9-3.5.10). Finally, the probes were purified using QiaQuick Nu- cleotide removal kit (Qiagen, Venlo, the Netherlands).

4. Hybridization The following hybridisation conditions were used: The filters containing the libraries were pre-hybridised in hybridisation solution at 42°C for 2 hrs. Then new hybridisation solution including labelled probes (denatured at 95°C, 5 minutes) was added and the filters were left to hybridise overnight. The filters were washed once for 15 minutes at room temperature (2XSSC, 0.1% SDS) followed by an- other wash for 15 minutes (0. 1XSSC, 0.1 % SDS). If the filters were still very hot the last wash was repeated.

Hybridisation solution: 2% SDS, 5XSSC, 10X Denhardt's solution, 50mM P-buffer (pH 6.5), 50% formamide, salmon sperm DNA.

The filters were wrapped in saran wrap and applied onto an X-ray film that was exposed overnight and optionally for several days. The positive clones were obtained from Roswell Park, Buffalo, NY. Which clone had which marker was determined as follows:

Each clone was inoculated on a Nylon N+ membrane on top of a LB + 50 !/m ! kanamycin plate. The clones were allowed to grow at 37°C overnight. Then the membrane was treated as follows : 1.0.5M NaOH, 1.5 M NaCI for 15 minutes, then dry.

2.0.5M Tris pH 7.5,1.5M NaCI for 10 minutes, then dry.

3.0.5M Tris pH 7.5,1.5M NaCI, 50 pg/ml proteinase K for 30 minutes, then dry.

4.2XSSC for 10 minutes, scrape off the colonies, then dry.

5. Stratalink.

The filters were then hybridised (conditions see above) with each of the probes separately to determine which clone corresponded with the different probes. The other markers within the interval were tested on the new PACs by PCR. The results are summarised in Fig. 2.

EXAMPLE 3 Showing that Tyro3 is down regulated in anxlanx mice 3.1. Summary A differential display analysis was performed on total RNA extracted from whole brain from newborn anx/anx (n=2) and +/+ (n=3) mice, and three weeks old anxlanx (n=3) and +/+ (n=3) mice. The analysis was done by Lark Technologies, Inc. using the Hieroglyph mRNA Profile Kit (Beckman Coulter, Inc., Fullerton, CA) and the genomyx LR System (Beckman Coulter, Inc., Fullerton, CA) to generate and display large amplified fragments.

Differently expressed bands were excised from the gel, reamplified and, when necessary, subcloned prior to sequencing by Lark Technologies, Inc.

The differential display analysis identified 102 bands differing in expression levels be- tween anx/anx and +/+ mice of one or both ages. When analyzing the bands it was dis- covered that one band that was missing in anx/anx mice of both ages was identical to the 3'UTR of Tyro3. To confirm this, a Northern blot and mRNA in situ hybridization was per- formed on brain tissue from both anx/anx and +/+ mice. These experiments confirmed a down regulation of Tyro3 mRNA in cerebellum of anx/anx mice.

3.2. Materials and Methods 1. Northern blotting The Northern blot was performed using the following protocols : (i) Providing a total RNA preparation Total RNA was isolated from whole brain using Trizol (LifeTechnologies) according to the manufacturer's protocol.

(ii) Northern blotting gel In the Northern blotting a 1,5% agarose gel was used. 200 ml of the gel was prepared by adding 3 g agarose to 150 ml DEPC-treated water followed by boiling to dissolve the aga- rose. To the solution 40 ml 5xMOPS (see below) was added and when cooled to 50°C, 10.8 ml formaldehyde was added. Prior to blotting the RNA, the gel was treated by rinsing in DEPC-treated water at least two times, in 50mM NaOH for 20 minutes followed by rinsing in DEPC-treated water and in 20xSSC for 45 minutes.

(iii) Northern blotting procedure The gel was prerun for 5 minutes at 100V in 1xMOPS. Total RNA samples were prepared by adding 10-12 pg of total RNA in 5 pI transfer buffer and vacuum dried if necessary, fol- lowed by adding 10 ut loading buffer. The resulting sample mixtures were denatured at 65°C for 3-5 minutes.

The samples and an RNA ladder (3 g) were loaded on the side leaving at least two empty lanes between ladder and samples and the gels were run at 40-50V. The BPB band was permitted to run approximately 10 cm. After running, the ladder was cut off, washed in 1xMOPS followed by staining with EtBr for 20-30 minutes and destaining in 0.2M NH40ac for 2x30 minutes.

Prior to blotting the RNA, the gel was treated by rinsing in DEPC-treated water at least twice in 50 mM NaOH for 20 minutes followed by rinsing in DEPC-treated water and in 20xSSC for 45 minutes.

Set up the blot (see Maniatis). Use 7,5mM NaOH as transfer buffer and Hybond N+ membrane. Let it sit for at least 4,5 hours.

Rinse the membrane in 2xSSC, 0.1 % SDS.

Stratalink.

(iv) Reagents used 5xMOPS buffer: The buffer contains 0.1 M MOPS (pH 7.0), 40mM NaOAc, 5mM EDTA (pH 8.0) and is pre- pared as follows : For 1 litre 20,6 g MOPS is added to 800 ml DEPC-treated NaOAc. pH is adjusted to 7.0 with 2M NaOH. 10 ml DEPC-treated EDTA is added. Bring to 1 litre with DEPC-treated water. Filter sterilise (Nalgene 0.2 m) and store at room temperature pro- tected from light.

Loading buffer: The loading buffer used had the following composition: 7.2 ml deionized formamide, 3.2 ml 5xMOPS, 2.6 ml 37% formaldehyde, 1 ml 80% glycerol, 800 pI saturated bromphenol blue, 200p1 water.

2. Northern hybridization (i) Preparation of probe The probe was labelled using a standard Random Priming protocol (Current Protocols in Molecular Biology, 3.5.9-3.5.10). Finally, the probe was purified using OiaQuick Nucleo- tide removal kit (Qiagen, Venlo, the Netherlands).

(ii) Hybridization procedure The hybridisation solution used contained per 100 ml : 8 ml 25% SDS, 20 ml 25xSSC, 10 ml 100xDenhardt solution, 5 ml 1 M phosphate buffer (pH 6,5), 50 ml formamide, 50% 1 ml ssDNA (denatured) and 6 ml water.

Pre-hybridisation of the sample was carried out for at least 1 hour at 42°C (6 hours if the membrane has never been used before). Use a Tupperware container and just enough solution to cover the membrane. The hybridisation solution was replaced and the probe previously denatured at 95°C for 5 minutes was added. Hybridisation was done overnight.

Following hybridisation, the membranes were rinsed in 2xSSC, 0,1% SDS followed by washing in 2xSSC, 0,1% SDS for 15 minutes at room temperature and in 0, 1 xSSC, 0,1% SDS for 15 minutes at room temperature. If the membrane is still very hot a further was in 0,1xSSC, 0,1% SDS at 65°C for 5-15 minutes may be carried out.

After a final wash in 2xSSC for 10-30 minutes, the membrane is wrapped in saran wrap and an autoradiography film exposed overnight with the membrane.

3. mRNA in situ hybridization Brains to be tested were collected on postnatal day 20-22. Both male and female mice were analyzed. For in situ hybridization, the brains were rapidly dissected from decapi- tated mice and immersed in phosphate-buffered saline (PBS). Brains were mounted on chucks and rapidly frozen at-70°C.

A 45-mer antisense oligonucleotide, complementary to nucleotides 2411-2455 of Tyro 3 (Acc. no. U18343) was used. The oligonucleotide was subsequently labeled with 35S-dATP (NEG 034H, NEN DuPont, Zaventem, Belgium) at the 3'end using terminal de- oxydinucleotidyl transferase to a specific activity of 2 x 109 cpm/g and purified using QlAquick Nucleotide Removal Kit (Qiagen, Venlo, the Netherlands).

The frozen brains were cut in 14 um serial sections at-17°C in a cryostat (Jung CM 3000, Leica Instruments GmbH, Nussloch, Germany). The sections were thaw-mounted onto ProbeOn slides (Fisher Biotech, Springfield, N. J.), and processed for in situ hybridization

as described elsewhere (Schalling et al., 1988). In brief, the sections were incubated at 42°C for 15-18 hr with 106 cpm of labeled probe per 100 pl of a solution containing 50% formamide, 4 x SSC (1xSSC : 0.15 M NaCI, 0.015 M sodium citrate), 1xDenhardt's solu- tion (0.02% polyvinyl pyrrolidone, 0.02% bovine serum albumin and 0.02 % Ficoll), 1 % sarcosyl, 0.02 M sodium phosphate (pH 7.0), 10% dextran sulfate, 500 ug/ml sonicated salmon sperm DNA and 200 mM dithiothreitol. The sections were subsequently rinsed in 1 x SSC at 55°C for one hour with five changes of buffer, following which they were dried and exposed to Hyperfilm R-max X-ray film (Amersham International, Little Chalfront, UK) for seven days before development and fixation. Some of the sections were subsequently dipped in NTB 2 nuclear track emulsion (Kodak, Rochester, NY), followed by two weeks of exposure, developed in Kodak D19, fixed in Kodak 3000A and analyzed using a micro- scope equipped for darkfield illumination (Axiophot, Carl Zeiss, Oberkochen, Germany).

Pictures from these emulsion radiographs were taken using T-MAX 100 film (Kodak).

4. Mutation analysis Mutation analysis was performed by exon sequencing of genomic DNA. In short: each exon was PCR amplified and cycle-sequenced using an ABI 377 equipment. No muta- tions could be found in any of the exons. The promoter sequence was also searched for mutations but without success.

5. Conclusions Tyro3 is down regulated in anxlanx mice compared to normal litter mates. While no muta- tions were found in this gene it is still possible that its down regulation contributes to the phenotype of anx/anx mice.

EXAMPLE 4 The ANKT gene (SEQ. ID. NO. 219), encoding a nucleolar protein, was cloned in liver lymphocytes from a C57B/6 mouse. It is described as a novel nucleolar protein expressed in proliferating cells. Nothing has been published regarding this novel gene that is submitted to Genbank under accession number AF305710.

When sequencing the ANKT gene, several interesting aspects of intron 3 were found.

Intron 3 is a direct repeat of the 30 last bases of exon 3, and instead of a traditional GT donor site it has a CT site, a phenomenon that has not previously been reported for any other gene. Intron 3 of the ANKT gene has the following sequence: Ctg aga tac acg tta gca gtg agg agc aag (SEQ. ID. NO. 220) Accordingly, the ANKT gene subsequence bridging exons 3 and 4 (Intron 3 is underlined) has the following sequence: Ctg aga tac acg tta gca gtg agg agc aag ctq aqa tac acq tta aca qta aqq age aaq ctg aga cgg aat cag gtg gtc acg tca cca aaac (SEQ. ID. NO. 221) CDNA sequencing revealed that anx/anx mice (age 0 days (n=1) and 3 weeks (n=2)) do not splice out the 30 bp long intron 3 in ANKT mRNA from brain. +/+ mice splice out the intron at the age of 3 weeks (n=2) but seem to have both variants when newborn (n=1).

No mutations could be found in neither exon 3, exon 4 nor intron 3 in anx/anx mice with exon sequencing, suggesting that the splicing is regulated by regulatory element (s) elsewhere.

The ANKT mRNA subsequence bridging exons 3 and 4 of +/+ mice has the following sequence: ctg aga tac acg tta gca gtg agg agc aag ctg aga cgg aat cag gtg gtc acg tca cca aaac (SEQ. ID. NO. 222) On the contrary the ANKT mRNA subsequence bridging exons 3 and 4 of anx/anx mice has the following sequence: Ctg aga tac acg tta gca gtg agg agc aag ctg aga tac acg tta gca gtg agg agc aag ctg aga cgg aat cag gtg gtc acg tca cca aaac (SEQ. ID. NO. 221) 16 mouse ESTs spanning this interval has been found in Genbank. Nine of these did not splice out intron 3 whereas seven of them did. Of the seven that did splice out the intron, three were derived from spleen from mice that were 6 days old, one from 7 days old

cerebellum, one from 9.5 d embryo, one from 10 d embryo and one from a thymus cell line. Seven of the nine ESTs that did not splice out the intron were derived from various cell lines and one was from 14.5 d embryo. The last non-spliced EST was derived from a pooled library containing pancreas from embryonic day 10.5,12.5,16.5, newborn and adult. The ESTs are presented in the following table: EST acc. no. SEQ. ID. NO. Origin of EST Intron 3 spliced (yes/no) BB844288 223 6 days old spleen yes BB844227 224 6 days old spleen yes BB843956 225 6 days old spleen yes BB639871 226 7 days old cerebellum yes BB610909 227 10 days old embryo yes BB643025 228 9.5 days old embryo yes BE631551 229 Thymus 2NbMT yes BB866888 230 Pooled cell lines no BB867381 231 Pooled cell lines no BB864718 232 Colon cell line no BB859168 233 Pooled cell lines no BB867081 234 Pooled cell lines no B1658798 235 Mammary tumor cell line no BB863588 236 Mammary tumor cell line no BG806540 237 14.5 days old embryo retina no BM125859 238 Mixed pancreas no Consequently, the non-spliced version of ANKT mRNA may be an isoform needed in late embryonic development as it has been found in embryo day 14.5, but not earlier. The presence of both variants in newborn +/-mice and absence of the non-spliced variant in 6,7, and 21 days old mice suggest that the non-spliced variant is down-regulated and the spliced variant is up-regulated after birth. In anxlanx mice, this regulatory shift seems to be disturbed so that they continue to express only the non-spliced variant. Wheather it is expressed in early anx/anx embryo remains to be elucidated.

No mutations have been found in intron 3 or the surrounding exons and it is believed that this alternative splicing may be regulated by the promoter region. It is possible that this

gene has two or more alternative promoters regulating the expression of the different splice variants. It is believed that the anx mutation lies within one or several of the alternative ANKT promoters, thereby causing the splice defect observed in anx/anx mice and the anx phenotype as a consequence.

It is believed that the anx/anx genotype is caused by the ANKT variant protein encoded by the above non-spliced ANKT mRNA. From now on, this ANKT variant protein is referred to herein as anx-ANKT (SEQ. ID. NO. 240). anx-ANKT differs from the normal ANKT protein (SEQ. ID. NO. 239) in that the amino acid sequence ala-glu-ile-his-val-ser-ser-glu- glu-gin is inserted after amino acid 73 of the normal ANKT protein.

Presently, it is believed that the phenotype of an animal having the anx/anx genotype is caused by expression of anx-ANKT instead of ANKT. anx-ANKT is then believed to interact/not interact with one or more other components in the cell resulting in the phenotype of an animal having the anx/anx genotype. It is therefore postulated that there are at least one molecule affecting food intake patterns interacts differently with anx- ANKT compared with ANKT. ANKT and anx-ANKT can therefore be used in screening methods for finding molecules affecting food intake patterns.

Included in the invention is therefore the use of ANKT and anx-ANKT for finding proteins with which ANKT and anx-ANKT interacts. This can be carried out by e g a two-hybrid system such as those described by Fields et al., Nature, 1989,340,245, or in Current Protocols in Molecular Biology 1999, John Wiley & Sons, NY and Ausubel, F.

M. et al. 1992, Short protocols in molecular biology, fourth edition, Greene and Wiley- interscience, NY. Kits are available from Clontech, Palo Alto, CA (Matchmaker Two- Hybrid System 3).

REFERENCES 1. Broberger, C., J. Johansen, M. Schalling and T. Hökfelt (1997). Hypothalamic Neuro- histochemistry of the Murine anorexia (anxlanx) Mutation: Altered Processing of Neuro- peptide Y in the Arcuate Nucleus, J. Comp. Neurol. 387: 124-135.

2. Broberger, C., J. Johansen, C. Johansson, M. Schalling and T. Hökfelt (1998). The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice, Proc. Natl. Acad. Sci. USA 95: 15043-15048.

3. Broberger, C., J. Johansen, H. Brismar, C. Johansson, M. Schalling and T. Hökfelt (1999). Changes in Neuropeptide Y Receptors and Pro-Opiomelanocortin in the Anorexia (anxlanx) Mouse Hypothalamus, J. Neurosci 19: 7130-7139.

4. Green, M. C. (1981). Gene mapping, In"The mouse in Biomedical Research" (H. L.

Foster, J. D. Small and J. D. Fox, Eds.), pp. 105-117, Academic Press, New York.

5. Johansen J. E., C. Broberger, C. Lavebratt, C. Johansson, M. J. Kuhar, T. Hökfelt, M.

Schalling (2000). Hypothalamic CART and serum leptin levels are reduced in the anorec- tic (anx/anx) mouse. Mol. Brain Res. 84: 97-105.

6. Pinkel, D., R. Segraves, D. Sudar, S. Clark, 1. Poole, D. Kowbel, C. Collins, W. L. Kuo, C. Chen, Y. Zhai, S. H. Dairkee, B. M. Ljung, J. W. Gray, D. G. Albertson (1998). High reso- lution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nature Genet. 20: 207-211.

7. Sambrook, J., E. F. Fritsch, T. Maniatis. Molecular cloning ; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

8. Schalling, M., K. Seroogy, T. Hokfelt, S-Y. Chai, H. Hallman, H. Persson, D. Larham- mar, A. Ericsson, L. Terenius, J. Graffi, J. Massoulie and M. Goldstein (1988). Neuropep- tide tyrosine in the rat adrenal gland-Immunohistochemical and in situ hybridization studies, Neuroscience, 24: 337-349.