AU7557201A - Porcine leptin protein, nucleic acid sequences coding therefor and uses thereof - Google Patents

Description

S&F Ref: 450956D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicants: Purdue Research Foundation 1060 Hovde Hall West Lafayette Indiana 47907 United States of America Purina Mills, Inc.

1401 South Hanley Rd St. Louis Missouri 63144 United States of America 9*- *9 Actual Inventor(s): Address for Service: Christopher A. Bidwell, Michael E. Spurlock Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Porcine Leptin Protein, Nucleic Acid Sequences Coding Therefor and Uses Thereof Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c PORCINE LEPTIN PROTEIN, NUCLEIC ACID SEQUENCES CODING THEREFOR AND USES THEREOF BACKGROUND OF THE INVENTION 1. Field of the Invention: This invention relates to the regulation of energy intake and metabolism in growing, finishing, lactating or nonlactating, and gestating swine. More specifically, it relates to a specific porcine polypeptide termed leptin which is secreted by adipocytes or other cell types and which influences energy intake and metabolism, fat deposition, and weight gain in swine. In addition, this invention relates to the nucleotide sequences encoding the porcine leptin polypeptide, the antibodies directed against the porcine leptin polypeptide, and methods to determine susceptibility to fat deposition, alter energy intake, and minimize excessive fat deposition in swine.

2. Description of the Background Art: -Obesity has been declared a public health hazard by the National Institutes of Health and has prompted the food animal industry to seek methods of limiting fat deposition in food animals. Additionally, the energetic cost of having food animals convert feed energy to fat rather than lean tissue provides considerable incentive to develop technology to facilitate the efficient production of leaner meat products and to accurately match the nutrient content of the diet to the nutrient needs of the animal.

To combat these health and production problems, both prophylactic and therapeutic approaches are necessary. For prophylactic purposes, it would be useful to be able to predict and measure the propensity or susceptibility to excessive fat deposition. For S t I therapeutic purposes, it would be of great benefit to improve current methods of minimizing the deposition of feed energy as fat in the adipocyte. Currently, neither of these desired objectives has been achieved completely.

Proteins from genes expressed only (or predominantly) in adipose tissue and for which the level of expression can be related to fat deposition serve as prime targets for approaches directed toward prediction of fat accretion potential and the control of fat deposition. For example, a mammalian adipocyte-specific polypeptide, termed p154, was reported in USP 5,268,295 to Serrero, which is incorporated in its entirety herein by reference, as being expressed in high quantities in adipogenic cell lines after cell differentiation and is abundant in the fat pads of normal and genetically obese mice. To date, however, there have been no reports of adipocyte-specific proteins expressed at different levels in fat swine as compared with normal controls.

Leptin, the protein produced by the leptin (ob) gene, is possibly related to fat deposition in swine because research has shown that mutations in genetically (oblob) obese mice resulting in excessive fat deposition are associated with altered expression of the leptin gene. Furthermore, at least one restriction fragment length polymorphism (RFLP) has been identified and related to the fat phenotype (Zhang et al., 1994, Nature 371:425). The leptin gene is expressed specifically in the terminally differentiated adipocyte (Maffei et al., 1995, Proc. Natl. Acad. Sci. 92:6957; Leroy et al., 1996, J. Biol. Chem. 271(5):2365). Additionally, leptin is a regulator of feed intake (Pellymounter et al., 1995, Sci. 269:540; Halaas et al., 1995, Sci. 269:543; Campfield et al., 1995, Sci. 269:546).

Although the murine leptin gene has been positionally cloned and a cDNA sequence reported (Nature 371:425), neither the porcine leptin cDNA or genomic sequence is available. Thus, the insights obtained with respect to porcine metabolism is not accessible to porcine systems. Furthermore, the biologically active purified porcine protein leptin) has not been obtained.

SUMMARY OF THE INVENTION The present invention provides gene sequences, peptides, antibodies, and methods of using them which permit the prediction and modulation of fat deposition and regulation of feed intake appetite) in the porcine species.

In one aspect, this invention is directed to a porcine adipocyte polypeptide, the porcine leptin protein, substantially free of other porcine proteins, or functional derivatives thereof. The present invention includes a porcine adipocyte polypeptide of at least about 8 amino acids of the amino acid sequence depicted in Figure 1, preferably the amino acid sequence-depicted in Figure 2, still more preferably, the amino acid sequence depicted in Figure 3, or functional derivatives thereof.

•The present invention is also directed to a single or double stranded DNA molecule or an RNA molecule consisting essentially of a nucleotide sequence that encodes the above polypeptide or functional derivatives thereof, the DNA or RNA molecule being substantially free of other porcine DNA or RNA sequences. The DNA molecule is preferably a single or double stranded DNA molecule having a nucleotide sequence consisting essentially of at least about 20 nucleotides of the 3 nucleotide sequence depicted in Figure 1, preferably, the nucleotide sequence depicted in Figure 2, still more preferably the nucleotide sequence depicted in Figure 3 or a sequence complementary to the nucleotide sequences depicted in Figures 1-3, substantially free of other porcine DNA sequences. The RNA molecule is preferably an mRNA sequence encoding the above porcine adipocyte polypeptide, or functional derivatives thereof.

Included in the invention is a DNA molecule as described above which is cDNA or genomic DNA, preferably in the form of an expressible vehicle or plasmid.

The present invention is also directed to hosts transformed or transfected with the above DNA molecules, including a prokaryotic host, preferably a bacterium, a eukaryotic host such as a yeast cell, or a mammalian cell.

The present invention also provides a process for preparing a porcine adipocyte polypeptide or functional derivatives as described above, the process comprising the steps of: culturing a host capable of expressing the polypeptide under culture conditions; expressing the polypeptide; and recovering the polypeptide from the culture.

Also included in the present invention is a method for detecting the presence of a nucleic acid molecule having the sequence of the DNA molecule described above, or a complementary sequence, in a nucleic acid-containing sample, the method comprising: contacting the sample with an oligonucleotide probe complementary to the sequence of interest under hybridizing conditions; and measuring the hybridization of the probe to the nucleic acid molecule, thereby detecting the presence of the nucleic acid molecule. The above method may additionally comprise before step selectively amplifying the number of copies of the nucleic acid sequence.

Another embodiment of this invention is an antibody specific for an epitope of the porcine adipocyte polypeptide, or functional derivatives thereof, either polyclonal or monoclonal. Also intended is a method for detecting the presence or measuring the quantity of the porcine adipocyte polypeptide leptin in a biological sample, comprising contacting the sample with the above antibody and detecting the binding of the antibody to an antigen in the sample, or measuring the quantity of antibody bound.

The present invention includes methods for determining the susceptibility of swine to fat deposition which comprises removing a biological sample from a pig and measuring therein the amount of the polypeptide or mRNA coding therefor, where the amount of the polypeptide or mRNA is related to susceptibility. The present invention also includes methods for determining the susceptibility of a subject to fat deposition which comprises removing a biological sample, extracting the DNA, digesting the DNA with restriction endonucleases, probing the sample with an oligonucleotide probe, separating the resulting fragments by gel electrophoresis, and relating the number of bands (banding pattern) generated by restriction enzyme digestion to fat deposition RFLP techniques).

Another method provided herein is for evaluating the efficacy of a drug (or other agent) directed to the regulation of fat deposition and feed intake which comprises contacting the drug being tested with an adipocyte culture in vitro and measuring the amount of the porcine adipocyte polypeptide or mRNA that is produced by the adipocyte, the efficacy of the drug or agent being related to changing the production of the polypeptide or mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the nucleotide sequence of the porcine leptin gene and the amino acid translation of the porcine leptin coding sequences.

FIG. 2 depicts the nucleotide sequence and the amino acid translation of the coding region of the entire porcine leptin cDNA signal peptide and secreted protein).

FIG. 3 depicts the nucleotide sequence and the amino acid translation of the porcine leptin cDNA corresponding to the secreted porcine leptin protein.

FIG. 4 shows a comparison of the porcine leptin cDNA sequence corresponding to the entire porcine leptin protein with the murine and human sequences.

FIG. 5 depicts the Northern blot analysis of porcine leptin mRNA.

FIG. 6 depicts the isolation of a genomic DNA clone for porcine leptin.

FIG. 7 depicts a polyacrylamide gel electrophoresis of porcine leptin protein induction and purification in Escherichia coli.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is directed to DNA and RNA molecules that encode a porcine adipocyte polypeptide, termed "leptin," or a functional derivative thereof, and the porcine leptin protein itself, or a functional derivative thereof. The porcine leptin protein is useful for regulation of feed intake, energy metabolism, and fat deposition in swine. Such objectives can be achieved by administering recombinant or purified porcine leptin, altering the expression of the porcine leptin gene or administering an antibody directed against the porcine leptin protein to achieve neutralization, depending on the desired result. The porcine leptin DNA, RNA, and protein, or functional derivatives thereof, and antibodies specific for the protein are used in assays to predict the potential for fat deposition in swine. These molecules can also be utilized in the development of commercially valuable technology for altering feed intake and regulating fat deposition in swine, and for matching the nutrient content of the diet to the nutrient needs of the pig.

In its first aspect, the present invention provides a porcine adipocyte polypeptide termed "leptin". The term "polypeptide" as used herein is intended to include not only the porcine leptin protein, and functional derivatives thereof, but also amino acid sequences having additional components, amino acid sequences having additional components such as a sugar, as in a glycopeptide, or other modified protein structures known in the art.

The polypeptide of this invention has an amino acid sequence as depicted in Figures 1 and 2, and preferably as depicted in Figure 3. Also intended within the scope of the present invention is any peptide having at least about 8 amino acids present in the above-mentioned sequence. Sequences of this length are useful as antigens and for making immunogenic conjugates with carriers for the production of antibodies specific for various epitopes of the entire protein. Such peptides are also useful in screening such antibodies and in the methods of the present invention directed to detection of the leptin protein in biological samples. It is well-known in the art that peptides of about 8 amino acids are useful in generation of antibodies to larger proteins of biological interest.

The polypeptide of this invention is sufficiently large to comprise an •antigenically distinct determinant, or epitope, which can be used as an immunogen to produce antibodies against porcine leptin or a functional derivative thereof, and to test such antibodies. The polypeptide of this invention may also exist covalently or noncovalently bound to another molecule. For example it may be fused a fusion protein) to one or more other polypeptides via one or more peptide bonds.

One embodiment includes the polypeptide substantially free of other porcine peptides. The polypeptide of the present invention may be biochemically or immunochemically purified from cells, tissues, or a biological fluid. Alternatively, the polypeptide can be produced by recombinant means in a prokaryotic or eukaryotic host cell.

"Substantially free of other porcine polypeptides" reflects the fact that because the gene for the porcine adipocyte polypeptide of interest can be cloned, the polypeptide can be expressed in a prokaryotic or eukaryotic organism, if desired.

Methods are also well known for the synthesis of polypeptides of a desired sequence on solid phase supports and their subsequent separation from the support.

Alternatively, the protein can be purified from tissue or fluids of the swine in which it naturally occurs so that it is purified away from at least 90 percent (on a weight basis), and from even at least 99 percent if desired, of other porcine polypeptides and is therefore substantially free of them. That can be achieved by subjecting the tissue or fluids to standard protein purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the protein. Alternatively, the purification from such tissue or fluids can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, and ion exchange chromatography.

As alternatives to a native purified or recombinant porcine adipocyte 20 polypeptide molecule, functional derivatives of the porcine adipocyte polypeptide may be used. As used herein, the term "functional derivative" refers to any "fragment", "variant", "analog", or "chemical derivative" of the porcine adipocyte polypeptide that retains at least a portion of the function of the porcine adipocyte polypeptide which permits its utility in accordance with the present invention.

A "fragment" of the porcine adipocyte polypeptide as used herein refers to any subset of the molecule, that is, a shorter peptide.

A "variant" of the porcine adipocyte polypeptide as used herein refers to a molecule substantially similar to either the entire peptide or a fragment thereof.

Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide,- using methods well-known in the art. Alternatively, amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide (again using methods well-known in the art). Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structures.

An "analog" of the porcine adipocyte polypeptide as used herein refers to a non-natural molecule substantially similar to either the entire molecule or a fragment 20 thereof.

A "chemical derivative" of the porcine adipocyte polypeptide or peptide as used herein contains additional chemical moieties not normally a part of the polypeptide. Covalent modifications are included within the scope of this invention.

Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.

The polypeptide of the present invention is encoded by a nucleic acid molecule, one strand of which has the nucleotide sequence shown in Figure 1, preferably as shown in Figure 2, and still more preferably as shown in Figure 3. The present invention is directed to a DNA sequence encoding the polypeptide, or a functional derivative thereof, substantially free of other porcine DNA sequences.

Such DNA may be single stranded sense strand, antisense strand or cDNA sequence) or double stranded. The DNA sequence should preferably have about 20 or more nucleotides to allow hybridization to another polynucleotide. In order to achieve higher specificity of hybridization, characterized by the absence of hybridization to S' sequences other than those encoding the polypeptide or a functional derivative thereof, a length of at least about 50 nucleotides is preferred.

The present invention is also directed to an RNA molecule comprising a mRNA sequence encoding the polypeptide of this invention, or a functional derivative thereof.

The present invention is further directed to the above DNA molecules which are functional in recombinant expression systems utilizing as hosts transfected or transformed with the vehicles and capable of expressing the polypeptide. Such hosts may be prokaryotic or eukaryotic. The DNA can be incorporated into the host organism by transformation, transduction, transfection, or a related process known in the art.

In addition to a DNA and mRNA sequence encoding the porcine adipocyte polypeptide molecule, this invention provides methods for expression of the nucleic acid sequences. Further, the genetic sequences and oligonucleotides of the invention allow the identification and cloning of additional, yet undiscovered adipocyte polypeptides having sequence homology to the adipocyte polypeptide described herein.

The recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, DNA or RNA synthesis, or more preferably, by application of recombinant DNA techniques. Techniques for synthesizing such molecules are disclosed by, for example, Wu, et al., Prog.

Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978), which is incorporated herein by reference. Procedures for constructing recombinant molecules in accordance with the above-described method are disclosed by Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is herein incorporated by reference.

Oligonucleotides representing a portion of the porcine adipocyte polypeptide a are useful for screening for the presence of genes encoding such proteins and for the cloning of porcine adipocyte polypeptide genes. Techniques for synthesizing such oligonucleotides are disclosed by, for example, Wu, et al.. Prog. Nucl. Acid.

Res. Molec. Biol. 21:101-141 (1978).

A suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the porcine adipocyte polypeptide gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified, synthesized, and hybridized by means well known in the art, against a DNA or, more preferably, a cDNA preparation derived from cells which are capable of expressing the porcine adipocyte polypeptide gene. Single stranded oligonucleotide molecules complementary to the "most probable" porcine adipocyte polypeptide-encoding sequences can be synthesized using procedures which are well known to those of ordinary skill in the art (See USP 5,268,295). Additionally, DNA synthesis may be achieved through the use of automated synthesizers. Techniques of nucleic acid hybridization are disclosed by Sambrook et al. (supra).

SIn an alternative way of cloning the porcine adipocyte polypeptide gene, a library of expression vectors is prepared by cloning DNA or, more preferably, cDNA (from a cell capable of expressing the porcine adipocyte polypeptide) into an expression vector. The library is then screened for members capable of expressing a protein which binds to anti-porcine-adipocyte polypeptide antibody, and which has a nucleotide sequence that is capable of encoding polypeptides that have the same amino acid sequence as the porcine adipocyte polypeptide, or fragments thereof. In this 20 embodiment, DNA, or more preferably cDNA, is extracted and purified from a cell which is capable of expressing the porcine adipocyte polypeptide protein. The purified cDNA is fragmentized (by shearing, endonuclease digestion, etc.) to produce a pool of DNA or cDNA fragments. DNA or cDNA fragments from this pool are then cloned into an expression vector in order to produce a genomic library of expression vectors whose members each contain a unique cloned DNA or cDNA fragment.

An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) is capable of expressing a DNA (or cDNA) molecule which has been cloned into the vector and of thereby producing a polypeptide or protein. Expression vectors of the present invention may be either prokaryotic or eukaryotic. Examples of suitable prokaryotic expression vectors include pASK75 (Biometra) or pET 21a-d (Novagen). Examples of suitable eukaryotic expression vectors include pcDNA3 or pRc/RSV (In Vitrogen, Inc.).

A DNA sequence encoding the porcine adipocyte polypeptide of the present invention, or its functional derivative, may be recombined with vector DNA in *4 So°: accordance with conventional techniques such as those disclosed by Sambrook, et al.

(supra).

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage

S

in which the regulatory DNA sequences and the DNA sequence sought to be o• expressed are connected in such a way as to permit gene expression.

The precise nature of the regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis. A promoter is a double-stranded DNA or RNA molecule which is capable of binding RNA polymerase and promoting the transcription of the "operably linked" nucleic acid sequence. The promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral. Strong promoters are, however, preferred. Suitable promoters are repressible, or more preferably, constitutive. Examples of suitable prokaryotic promoters include the tetracycline (TetA) promoter for pASK75 and T71ac for pET21. Examples of suitable eurkaryotic promoters include alpha actin or beta actin. Examples of suitable viral promoters include Rous sarcoma or cyotmegala.

The present invention is also directed to an antibody specific for an epitope of the porcine adipocyte polypeptide, and the use of such antibody to detect the presence 00096: of, or measure the quantity or concentration of the polypeptide, a functional derivative 0 thereof, in a cell, a cell or tissue extract, or a biological fluid. As used herein, the term "epitope" refers to that portion of any molecule capable of being bound by an S. ~antibody which can also be recognized by that antibody. Epitopes or "antigenic o2*. 20 determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.

The porcine adipocyte polypeptide of the present invention, or a functional derivative thereof, preferably having at least about 8 amino acids is used as an antigen for induction of a polyclonal antibody or monoclonal antibody (mAb). As used herein, an "antigen" is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.

The term "antibody" is meant to include polyclonal antibodies, monoclonal S- antibodies (mAbs), and chimeric antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigenic epitopes. MAbs may be obtained by methods known to those skilled in the art. (See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; de St. Groth, S. F. et al.. J.

Immunol. Methods, 35:1-21 (1980); and Hartlow, E. et al., Antibodies: A Laboratory i 20 Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988).

*e Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having a variable region derived from a porcine mAb and a murine immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad.

Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Neuberger et al., Nature 314:268-270 (1985); Liu et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Better et al., Science 240:1041-1043 (1988)). These references are hereby incorporated by reference.

The term "antibody" is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 which are capable of binding antigen. Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).

The reaction of the antibodies and the polypeptides of the present invention are detected by immunoassay methods well known in the art (See, for example, Hartlow et al. supra). The antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells 20 which express the porcine adipocyte polypeptide protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody coupled with microscopy, flow cytometric, or fluorimetric detection.

The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of the porcine adipocyte polypeptide. In situ detection may be accomplished by removing a histological specimen from a pig, and providing a labeled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the porcine adipocyte polypeptide but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

*o SSuch assays for porcine adipocyte polypeptide typically comprise incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested or cultured cells containing adipogenic cells or adipocytes, in the presence of a detectably S labeled antibody capable of identifying the porcine adipocyte polypeptide, and detecting the antibody by any of a number of techniques well-known in the art, such as enzyme immunoassays (EIA or ELISA) or radioimmunoassays (RIA).

20 The antibody molecules of the present invention may also be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay.

In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support any support capable of binding antigen or antibodies) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

The binding activity of a given lot of antibody to the porcine adipocyte polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

Antibodies can be used in an immunoaffinity column to purify the porcine adipocyte polypeptide of the invention by a one step procedure, using methods known in the art.

According to the present invention, a pig that is susceptible to fat deposition is treated with the porcine adipocyte protein to limit such fat deposition. This treatment may be performed in conjunction with other anti-adipogenic therapies. A typical regimen for treating swine with a propensity for fat deposition comprises administration of an effective amount of the porcine adipocyte polypeptide administered over a period of time.

20 The porcine adipocyte polypeptide of the present invention may be administered by any means that achieve its intended purpose, preferably to alter feed intake or limit fat deposition in a subject. For example, administration may be by various parenteral routes including, but not limited to, subcutaneous, intravenous, intradermal, intramuscular, and intraperitoneal routes. Alternatively, or concurrently, administration may be by the oral route which may be accomplished by the use of genetically-altered feedstuffs, in which the porcine leptin gene has been inserted and expressed. Parenteral administration can be by bolus injection or by gradual perfusion over time such as by implant of osmotic delivery device. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which may contain auxiliary agents or excipients which are known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods.

It is understood that the dosage of porcine adipocyte polypeptide administered S" may be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. The total dose required for each treatment may be administered by multiple doses or in a single dose. The porcine adipocyte polypeptide of the present invention may be administered alone or in conjunction with other therapeutics directed toward the regulation of fat deposition.

In a preferred embodiment, the concentration of the porcine adipocyte 20 polypeptide or mRNA of this invention is measured in a cell preparation, tissue extract or biological fluid of a subject as a means for determining the susceptibility or the propensity of the subject for fat deposition. The susceptibility of the subject to fat deposition is related to the level of the porcine adipocyte polypeptide or its mRNA.

Additionally, restriction fragment length polymorphisms in the porcine adipocyte gene will be used to predict fat deposition potential.

Another embodiment of the invention is evaluating the efficacy of a drug or other agent, directed to the increase or decrease of feed intake by measuring the ability of the drug or agent to stimulate or suppress the production of the porcine adipocyte polypeptide, or mRNA of this invention by a cell or cell line capable of producing such polypeptides or mRNAs. Preferred cells are cells of an adipogenic cell line. The antibodies, cDNA probe or riboprobe of the present invention are useful in the method for evaluating these drugs or other agents in that they can be employed to determine the amount of the porcine adipocyte polypeptide or mRNAs S .•_using one of the above-mentioned inmunoassays.

*o An additional embodiment of the present invention is directed to assays for measuring the susceptibility of a pig to fat deposition based on measuring in a tissue or fluid from the subject the amount of the mRNA sequences present that encode the porcine adipocyte polypeptide, or a functional derivative thereof, preferably using an RNA-DNA hybridization assay. The susceptibility to fat deposition is related to the o •amount of such mRNA sequences present. For such assays, the source of the mRNA sequences is preferably a pig's adipogenic cells. The preferred technique for measuring the amount of mRNA is a hybridization assay using RNA .o Ribonuclease Protection Assay) or DNA Northern or Slot Blot Assays) of complementary base sequence.

Nucleic acid detection assays, especially hybridization assays, can be predicated on any characteristic of the nucleic acid molecule, such as its size, sequence, susceptibility to digestion by restriction endonucleases, etc. The sensitivity of such assays may be increased by altering the manner in which detection is reported or signaled to the observer. Thus, for example, assay sensitivity can be increased through the use of detectably labeled reagents. A wide variety of such labels have been used for this purpose. Kourilsky et al. Pat. No. 4,581,333) describe the use of enzyme labels to increase sensitivity in a detection assay. Radioisotopic labels are disclosed by Falkow et al. Pat. No. 4,358,535), and by Berninger (U.S.

Pat. No. 4,446,237). Fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.

4,563,417), modified bases (Miyoshi et al., EP 119448), etc. have also been used in an effort to improve the efficiency with which detection can be observed.

1 5 One method for overcoming the sensitivity limitation of nucleic acid concentration is to selectively amplify the nucleic acid whose detection is desired prior to performing the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or 20 RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al.

Pat. No. 4,237,224), Maniatis, et al., etc.

Recently, an in vitro enzymatic method has been described which is capable of increasing the concentration of such desired nucleic acid molecules. This method has been referred to as the "Polymerase Chain Reaction" or "PCR" (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 424; EP 84,796, EP 258,017, EP 237,362; Mullis, EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. No. 4,683,194). The polymerase chain reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.

Having now generally described the invention, the same will be more readily S"understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

20 EXAMPLE I ISOLATION OF PORCINE LEPTIN cDNA The putative secreted portion of porcine leptin gene product was amplified from adipose tissue mRNA using reverse transcriptase-polymerase chain reaction.

Four separate cDNA synthesis reactions were carried out using 1-2 pg of porcine adipose tissue total RNA or 1-2 yg of poly A+ mRNA, 150 pmol of random hexamer oligonucleotides, 500 nM dNTP, 200 U of MMLV RNase H- reverse transcriptase (Life Technologies, Inc.) in 20 ul of the supplied buffer. The reactions were incubated for 1 h at 37 "C and terminated by heating to 70 'C for 10 min. The leptin cDNA product was amplified by PCR using the following degenerate oligonucleotide primers with restriction site linkers for BamHI/Bsa I and EcoRI/Eco47 III, respectively: Sense strand: GTGCC(C/T)ATCCA(A/G)AAAGTCC-3' Antisense strand: 5'-GAATTCAGCGCT GCA(C/T)(C/T)CAGGGCT(G/A)A(G/C)(G/A)TC- 3' These oligonucleotide primers were designed from a multiple sequence alignment of the mouse and human cDNA sequences. Approximately 100 ng of adipose tissue cDNA was added as template to 50 pl PCR reactions made in the manufacturers buffer with 100 pmol of each primer and 2.5 U of Taq DNA polymerase (Life Technologies, Inc.). A three stage amplification was carried out under the following conditions; Stage 1- 95 "C, 3 min; 52 1 min, 72 'C 1 min, 1 cycle; Stage 2- 94 45s; 52 45s, 72 1 min, 4 cycles; Stage 3- 94 45 s; 55 30 s, 72 'C 1 min, 28 cycles. Template 20 cDNA from three out of four cDNA reactions produced a 466 bp product.

The PCR products were prepared for ligation into the protein expression vector (Biometra Inc.) by complete digestion with Eco47III and partial digestion with Bsa I. The restriction enzyme digested PCR products were purified by electrophoresis in low melting point agarose and a 437 bp product was excised from the gel and ligated into the vector. The ligations were transformed in E. coli XL1-Blue (Stratagene Inc.) and plated on LB plates containing 50 jg/ml ampicillin for plasmid selection. Twelve E. coli colonies were isolated that contained the porcine leptin cDNA, and plasmid DNA was isolated for DNA sequencing.

The nucleotide sequence of the porcine leptin gene comprising 5917 base pairs, and the amino acid translation of the leptin coding sequences are depicted in Figure 1.

The nucleotide sequence and the amino acid sequence of the entire porcine leptin cDNA signal peptide and secreted proteins) comprising 501 base pairs and 167 amino acids are depicted in Figure 2. The nucleotide sequence and the amino acid sequence of the porcine leptin cDNA corresponding to the secreted protein alone and comprising 435 base pairs and 145 amino acids are depicted in Figure 3.

There was an 83% identity between the pig and human cDNA sequence and a 76% identity between the pig and mouse cDNA sequence as depicted in Figure 4.

EXAMPLE II ISOLATION OF mRNA CORRESPONDING TO PORCINE LEPTIN cDNA 20 The porcine leptin cDNA was used as a probe for detection of the full length mRNA. A northern blot containing porcine adipose and bovine adipose poly A+ mRNA as well as ob/ob mouse adipose total RNA was provided by Dr. M. Spurlock of Purina Mills Inc. The blot was hybridized with an [32p] dCTP labeled porcine leptin cDNA in hybridization solution (HY; 0.9 M NaC1, 0.09 M sodium citrate, 0.05% ficoll, 0.05% polyvinylpyrolidone, 0.05% BSA, 0.5% SDS, 0.1% sodium pyrophosphate, 10 mM EDTA and 100 mg/ml sonicated salmon sperm DNA at 60 'C for 15 h. The blot was washed to a final stringency of 0.2X SSC (0.03M NaCI, 0.003 M sodium citrate), 0.1% SDS at 60 'C and exposed to X-ray film. A 3,090 bp leptin mRNA was detected in porcine and bovine adipose tissue and a 3,240 bp leptin mRNA was detected on oblob mouse adipose tissue. As shown in Figure 5, lanes 1 and 2 contain the porcine adipose poly A+mRNA, lane 3 contains the adipose total RNA from a control mouse and lanes 4 and 5 contain the adipose total RNA from an ob/ob mouse, and lane 6 contains the bovine adipose poly A+mRNA.

EXAMPLE III ISOLATION OF GENOMIC DNA CLONE CORRESPONDING TO PORCINE LEPTIN The porcine leptin cDNA was also used to screen a porcine genomic DNA library.

15 Specifically, a porcine genomic library containing 4.64 X 105 recombinants was previously constructed in SuperCos 1 (Stratagene, Inc.) and screened for porcine leptin.

Specifically, two sets of replica filters were prehybridized for 2 h at 60'C. Filters were hybridized overnight with 32 P] dCTP labeled probe at 5 X 105 cpm per ml of hybridization solution at 65'C. Filters were sequentially washed in 2X SSC (0.3.M NaCI, 0.03 M sodium citrate), 0.5% SDS; IX SSC, 0.5% SDS; and 0.2X SSC S SDS with each wash at 60'C for 30 min. Positive clones that showed signals on both replica filters were recovered from the agar plates and individual colonies were isolated by a second low density replica plating and hybridization step. A cosmid designated Obg- 361 was isolated that hybridized to the porcine ob cDNA probe and had essentially the same restriction enzyme digestion pattern as found in porcine genomic DNA.

Figure 6 illustrates the isolation of the cosmid Obg-361. Specifically, lanes 1-4 are an agarose gel containing Kb ladder molecular mass markers (lane cosmid Obg- 361 digested with Eco RI (lane 2) and Hind III (lane 3) and biotinylated lambda/Hind III molecular mass markers (lane 4).

Southern blot analysis of the gel in lanes 2-4 were probed with the porcine leptin cDNA indicate that the EcoRI fragments (lane 5) and the Hind III fragments (lane 6) contain leptin sequences. Lane 7 is lambda/Hind III molecular mass markers.

i Porcine genomic DNA digested with BAM HI (lane EcoRI (lane 9) and Hind III (lane 10) and hybridized with a Bsa I fragment (300 bp) of the porcine leptin cDNA showed equivalent bands that contain leptin sequences indicating that the porcine leptin gene was isolated in cosmid Obg-361.

The 5917 bp Hind III fragment was subcloned into Bluescript II SK+ (Stratagene, Inc.). Both strands of the sequence was determined using progressive nested deletions using Exonuclease III and Mung Bean nuclease. Sequencing reactions were carried out 20 with Sequenase V2.0. This sequence was 5917 bp in length and contains the entire o00 coding region in two exons (Figure There was 78.6% nucleotide identity between the pig and human as well as 71.2% nucleotide identity between pig and mouse coding sequences. The splice junctions for the two exons were confirmed by the cDNA sequence. The cDNA sequence of the protein coding region is shown in Figure 2. The 501 bp sequences encodes 166 amino acid residue leptin polypeptide with a predicted molecular mass of 18,334 Da.

A clone was obtained using the process described above, Obg H3-15, was deposited with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852-1776, on July 11, 1996, and has been designated ATCC No.

97653. This microorganism was deposited under the conditions of the Budapest Treaty on the International Recognition of Deposit of Microorganisms for the purpose of Patent Procedure. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. This deposit will be maintained for a time period of 30 years from the date of deposit or 5 years after the last request for the material, whichever is longer.

*S*

S

EXAMPLE IV PURIFICATION OF THE PORCINE LEPTIN GENE PRODUCT •The polypeptide sequence encoded by the porcine leptin cDNA was synthesized and purified using the Strep-Tag system (Biometra, Inc.). The pASK plasmid contains the ompA leader sequence for secretion of the protein into the periplasmic space of E.

@o coli as well as a ten amino acid carboxyl terminus that binds to strepavidin for affinity 20 chromatography. Synthesis of the porcine leptin protein by E. coli strain XL1-Blue was induced with 200 pg/1 of anhydrotetracycline and the cells harvested after 3 h. The proteins in the periplasmic space were isolated by osmotic shock by suspending the cells in 100 mM Tris-HCl pH 8.0, 500 mM sucrose, 1 mM EDTA and 0.02% NaN 3 for 30 m at 4 The cells were removed by centrifugation and the porcine leptin protein was purified from the periplasmic proteins by strepavidin affinity chromatography as depicted in Figure Specifically, Figure 7 shows the polyacrylamide gel electrophoresis of porcine leptin protein induction and purification in E. coli. Molecular mass markers are located in lane 1. Lane 2 contains total protein from XL-1 Blue and an pASK/Ob cell line before (lane 3) and after (lane 4) anhydrotetracycline induction. Affinity purified porcine leptin protein is located in lane 6.

EXAMPLE V ANTIBODIES TO PORCINE LEPTIN PROTEIN AND THEIR USE TO DETECT PORCINE LEPTIN IN ADIPOGENIC CELLS ,'Polyclonal and/or monoclonal antibodies are produced with the recombinant porcine leptin protein. The techniques used for producing, screening, detecting, and/or 0000 quantifying antibodies for porcine leptin are discussed extensively in "Antibodies: a Laboratory Manual" (Harlow et al., 1988, Cold Spring Harbor laboratory). All media or medium components, mouse or cell strains BALB/C mouse, sp2/0 myeloma cells, JA744A.1 macrophages etc.) are commercially available.

A. Immunization of Animals 1. Rabbits: Purified porcine leptin protein is injected into rabbits for production of polyclonal antibodies. Specifically, each rabbit receives repeated subcutaneous injections with antigen in Freund's complete adjuvant followed by at least 1 booster injection of about 200 /g to 1 mg. When the serum titer of the immunized rabbits is sufficiently high when tested using the porcine leptin as antigen, rabbit serum is harvested as the polyclonal 29 antiserum for porcine leptin.

2. BALB/C mice (4-week old): Purified porcine leptin protein is injected into BALB/C mice for production of monoclonal antibodies. Specifically, each mouse is injected with about 50 ,g porcine leptin protein with Ribi's S-TDCM adjuvants (RIBI ImmunoChem Research, Inc., Hamilton, Montana). The number of injections depends on the titer of the antibody in the serum of immunized mice as determined by ELISA using porcine leptin as the antigen. In the course of producing monoclonal antibodies against porcine leptin protein, the spleens of immunized mice are used to prepare spleenocytes. Hybridoma cells are made by fusing the spleenocytes with sp2/0 myeloma cells (treated with 8-Azaguanine containing medium) in the presence of 50% PEG-1500. Hybridoma cells are incubated in selection HAT (hypoxanthine, aminopterine, and thymidine) medium. Subsequent screening for positive clones uses the recombinant porcine leptin as antigen in ELISA methodology.

Positive clones that produce strong anti-porcine-leptin antibody are characterized for specificity, subtype, affinity, binding sites, etc.

When large quantities of purified antibody are needed, the positive clones are cultured in large scale and antibody purified from the culture supernatant, or injected into 20 the intraperitoneal cavity of BALB/C mice for production of ascites. The latter procedure requires about 1-2x10 6 hybridoma cells per mouse, and usually takes about 7-14 days.

Large quantities of antibody is then purified from ascites by ammonium sulphate precipitation and ion exchange chromatography DEAE-Trisacryl M).

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Page(s) are claims pages they appear after the sequence listing Sequence Listing (iii) NUMBER OF SEQUENCES: 8 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 5917 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: COS LOCATION: join(942..1085, 3400..3753) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: AAGCTTTCTT GGCCCCTAAC AGCAACCAOA TTATACTCTT ACTGGCTATT CCTTGGC CAATACCCAG OOOAGGGGAC OCCTCTTOOA GGGAGOOOOG OTTGTACTOO TGAGATG TGTCOTTCTT GCAGAGCTCT TOCTOAOGGC ATCGGGAOGG OGGTTCACCO TTTTGCCT CCGGATAAAC TGTMAGOTAC TTGAGAGCAG AGAACATCCA TTGTTCGCTG TGGCATC( GGTAOOTAGC ACGGCATCTG ACATATTATO AGATCTTOCA OAAAGGOCAG TTTACGGI AATGCCOGTT GAATTCAGGC TCCCAGTGGG AGAGOGAGGA AGTAATAAAG OOGGTGA ATGCCGCCGT GGAGACAOOA GOGGGCTGOO GTGAGACTAA TGGAGAGGAC AGTAACG TOTOTAATGC GAGGGTGGTT ATAGAGTACA TTTCATAACA CCTTTAAAGC TOTTTOAC GCATTATCCA ATTTGATCCT OATAAAAGCO TGGAGATGTG TATATTGTGG TGGATGG GGGAGTOTTT AGCAGTTATG GGATATGCCT GAAGTCGTGC AGCTAGTAAA TGGCTGG CAAAOOAGAO OTCAAAAGCC TGCCTGTTTG CTCATGOOOC OTGOOOCGAO TGOOOAC GTGGCOAOA GOACAACTCA COGTOGOTTT OTTGATOOGT TTTCTTGATO OGGOTOT OTOOOCAAGG AATGCTTTTC ATTAAOATAT GTOTAGGTAA TGAATTATCT TGACTCT( GAGGCCATAG OAOATGOOGT AAOGCGACAG OTOCTTTGAT CTGCATCTGA GGOTGTG GGTAAOGGGC GTGGGGAGGG GGCGTTCGOT GAGACCOCAG GGACACGCCA TGTGTGG OOTOTGTTTO CAGGCOCAG AAGOAOATOC OGGAAAGGAA A ATG OGO TGT Met Arg Cys 000 OTG TGC CGA TTC CTG OTG GOT TTG GOC TAT OTG TOO TAO GTT Pro Leu Oys Arg Phe Leu Leu Ala Leu Ala Tyr Leu Ser Tyr Val 5 10 15 CTT TOA 120

CT

CGT

FTG

TMA

TTA

;AC

AGG

ATT

TOT

GOT

3AG

GOT

TTO

GGA

31y

GMA

Glu 180 240 300 360 420 480 540 600 660 720 780 840 900 953 1001 GOC GTG 000 ATO TGG AGA GTO CAG Ala Val Pro Ile Trp Arg Val Gin AOG ATT GTO ACC AGG ATO AGT GAO Thr Ilie Val Thr Arg Ilie Ser Asp GAT GAO ACC AMA ACC OTO ATO MAG 1049 Asp Asp Thr Lys Thr Leu Ilie Lys ATT TOA CAC ATG GTAGGGMAGG Ile Ser His Met 1095 OOOSGGGGGA GGAGGGGTAO OGGAOOTOAG 1155

OOTGGGAGAO

AGGTTGGOGG

OAGOTGOOTT

TOOOAGAOTG

TGOOAGGTGO

TTOTMATOTG

AOTGOTAGTO

TOTOTOMATG

OTOAGOTGAG

OOOTOTOTGT

CATOCAGATA

GGAGOTOMAG

OAGGMAGGOA

TOTAOGGAGG

OTTOTTOOCA

TAGGGGTGOT

OOAOTOTTTO

TOACAGMAAT

MAGGTOGMOC OTGTGGOOAG AGGTGGGMAG GGTOGGOGGT

TGOTOOTOOG

GMATOOTGAT

GOAGACOCOC

TAGGMATTOT

OTGOOOATTG

TOOOOAGGGT

GTOOGTOTTT

AAAATOTOMA

ATGGGAOTGT

MATGMOCTTG

GACAGOTOC

TTTTTATOAC

TTGOOOAGTT

OOGTMAGTTT

OATTOTTOTG

CAMATGTCCT

OTTOOCTOAC

GCOAGGACT

CAGOATOATO

TOOTGGTGAC

AGOOTTTTTT

GTTTTOTOTG

AGMTTOAGA

GOAOGTTMAG

TAOTGGGMAG

GOGMOCGOAC

AGOACAGACA

TTMAAGAOGG

TACCTATATG

TATGGTGOGT

OTOAOAGTOA

TTOMATAGTT

GGCCTTGACG COTOCCOCAC OGOACCCOC OACGTCOTTA AGAGGMAGOC OTMAAGGTOO CCCTOTGGCO TOCATCAOGT AGCTGMOCTC TGAOOOTGOG OOTATAOAAO CCTOTACATG GGGTOCGOAG GOOGAGACOT AGACGAGGTG TGACTOCTCA TOOCTOCGTG TOTGAOOCT ATGTTACOGG MTCOAGGGT AGGGAGCOGA GCMAGCAGMA OGGGGAGGGA AGAGTCCCCO GAGTAOOMAT GTGGGGTCOA GCGCOTTGTC CAGGAGGGAC OTGCTCTTOT CTGOOCTAGA MTGCATAGG TCAGGGGTTA MATCTTOOMA TAAGOMAGGO

CCCOMOAA

TOOTOOTTOT

TGTGTGOOTT

OTOOGGMATG

GACGOOOCTT

TTTGOAMOCT

TCAGCOTCTT

CCOTGOTGTT

TAGTTTCOCT

OTTGCOTCAT

GTOTTTATTA

OGOOCATTGT

GATATOOGTT

TOTGTAGAGT

OTTAGAGTCG

ATTCOACOT

OTGOTTGTOT

1215 1275 1335 1395 1455 1515 1575 1635 1695 1755 1815 1875 1935 1995 2055 2115 2175 R: I BZZ] 04286.doc: mrr TGATTAGTTT TTACAMATCT GTTOGCAOAO TAAOTGCCTG AAGGTTGTTT TGAGATTAAA OGACTGAGAA CCATGAGACA CTGCTGTGAG CTGTGGTGTA GGTGTAGGCC GGTGCAGAGA GGGGTACGC TAAAAAAAGA GTGACTCCTC CACCAGGAGA AAGTTTTCGG TTGTGGCTTT AGAGCAGTCT GGAGACTTCC GGAGCOTGGG AGOAGTCCGG GGGACTTTOA AGAGAAAAC CAGATOCAGO GTGTCCCAGC

TAAACCATGG

AATTATTAGT

TTAGATAGGA

GGTTCGATOO

GGTGOAGAG

GOTOGGATTA

OAAAAGATGG

GATATCOOTG

GATAGGAGGO

GCATCTCCTG

TGGGCAGAAG

COATTAATGA GGGAAGAGAT OTGCCTCAGG ACTATCTTAA GTTCCTGTOG AGGOGCGAOG OTGGCTTTGT CAGTGGGTTA GTGGCTCGGA TCCCGCGTTG GACCOTAGO OTGGGAAGCT AAAAAAAAAA GGTTAGATTA OAGAACCAGG AOAGAGGATG AGCCTTAAAA GGGAGGTTTT GGOACTGTGA GTTTCTAACA CAGGGACAGG TGAGAAOCAG CGMAGCCOAT 2235 TAGTOTTCGO 2295 GAAACAGATC 2355 GGATCTGGTG 2415 CTGTGGCTGT 2475 GOATGTGCCG 2535 GATAAAGCMA 2595 CCTTCTTGAA 2655 CAATCTGCCC 2715 GTGGCCTTGG 2775 ATAGAGTCTT 2835 OTGGGCATGA 2895 AGGGYGYGGG 2955 AGTGGGGAGA 3015 GGCCTGGGGA 3075 AAGAGGCATC 3135 GGCAGAAGAC 3195 OTAAGTOTCC TTCTTCOAGC OATGCAACAG GTGTGTGGTG OAGGGAGTGA YGCTGOGNNY GGAGCTGAGG AGCGAGGCGG GGCATGGNGG GGOTGOAGOC TOCATCOTA CTTCATGAAG AGCOTGAGOA GNAGGGAGGG GCATGTGTGG AGGACCTCAG AGGOTAGACO CAAOTATGTG AGAPACAGAG AGTCGTGGCT GGTTOTACAG TGGAGGCCAT TCGAATGCCC AAAGOTGTCT GGGTGAGGCA GGGCTTGGTA

AGAA

TAGA

TCTC

CGCT

ACC

Thr

MAG

Lys

OCT

Pro 90

GAO

Asp

AGG

Arg

OTG

Leu

CAG

Gin ~GGOOGT GAGACCAGOT TG' TAGGAT TGTGTGGMAG GG CACGCC TGCAGGAAGG CC~ OTCCCC TTCOTOOTGC AC GGT TTG GAO TTO ATO Gly Leu Asp Phe Ilie ATG GAO GAG ACO OTG Met Asp Gin Thr Leu 75 TOO AGA MAT GTG ATO Ser Arg Asn Val Ilie 95 OTT OTO GAO GTG OTG Leu Leu His Leu Leu 110 GOG GTG GAG AGO TTG Ala Leu Glu Thr Leu 125 TAG TOG AGG GAG GTG Tyr Ser Thr Glu Val 140 GOT GGG OTO OAT OCT GTO Pro Gly Leu His Pro Val 65 GOG ATO TAO GAA GAG ATO Ala Ilie Tyr Gln GIn Ilie 80 GMA ATA TOG MAT GAO GTG GIn Ilie Ser Asn Asp Leu 100 GOG TOO TOG AAG AGO TGO Ala Ser Ser Lys Ser Gys 115 GAG AGO CTG GGG GGO GTO Glu Ser Leu Gly Gly Val 130 GTG GOC GTG AGO AGG GTG Val Ala Leu Ser Arg Leu OTG AGT TTG Leu Ser Leu OTO AGO AGT Leu Thr Ser GAG MAC OTO Glu Asn Leu 000 TTG 000 Pro Leu Pro 120 OTG GMA GOG Leu Glu Ala 135 GAG GGG GOT GIn Gly Ala 150 TOO 3474 Ser GTG 3522 Leu OGG 3570 Arg 105 AGO 3618 Ser GAGGOTTG GOAGCOAOGO OAGGCMAGG AGTTOGGGGO 3255 GMAGAGGO AGOCGGAGGT GGGGGGTGGG GGTGGAOGGG 3315 AGGGGGTG CAGAGCCGMO ATCTOTCTCG OTGAGOGTGT 3375 AG CAG TOT GTO TGC TOO MAA GAG AGG GTG 3426 GIn Ser Val Ser Ser Lys GIn Arg Val

TGO

Ser

GTG

Leu 3666 3714 3763

GAG

Asp 155 ATG CTG OGG GAG Met Leu Arg GIn 145 CTG GAO Leu Asp 160 CTO AGO OCT GGC TGC TGMAGOGTTG Leu Ser Pro Gly Cys 165 MAGGOCTOTG TGOGGAGAGT OGGGGGMAGA MGOCTGAGOT TGGAGGAGTO GAGAGGGTGT GOGGAGOTGO TGTOTGGAGG TGTGGGGACG ATTTCTOTGT AGOTGOTOTT OGAMAGGOAG MAAGCTOOM GGCAOGAOAC OAMAGAOAGA TOOGOGGOCA OOGGMAAGGG GGOGOGGTOO AGCOAAOGGT GGAOTAGATT CAOGMGCGTO TTOOTTGOTG TTGCATOTGO AGCTGACGG GTGOTTCAGO GGATTTCAGA GOOTTTGGAG GATGMGOCAG GGTTOCATGT GAGMATTOOG TGMAGGOTAO AGGOAGAGAG AGOTGGATGO TGCCACGGOM GAOMGTTGG TTATTTATTA TGOGGTGTAT TOTGGTTGGA TTTGAAGOAA MAAGGCC GTOTGGGGTO AGCCGGGGOT AGGGGGAGGO TCOOGAGGTG GTGTTTGOAG GGGGCTGGTG AGGOGMGGGC ATTTTGAGTG ACTTGAGGGG TOTOMAGGTG GAGTGGOTTT GTTTGTAOTG TGAGTGAOTT TAMAACTGOA GCGTGTGOAO TGOTGGAGMA 3823 OGGTOOGGTA 3883 MAGGCCTGGT 3943 TCGGATTTTG 4003 GTGAOGGGGG 4063 GGGAGOAOGG 4123 MAGGATTTCT 4183 TTTGCAGGGT 4243 TAGOATOGAT 4303 GTTGTGTAGA 4363 TGGGATOGGO 4423 [R:\LIBZZ]04286.doc:mrr TGCGCGGATC TCGMAGGGCC AGGTTCTCTT GTGTACGCGG AGACAGGAAG TGTGTTGGTG TTGTGTGATG GACATTTGTG TGAGGGGCTC TCGTGGTTTT CATGAGAGAA GGAGATGATT CGCCCGTGCA GGAGTGGGGA AGGGGCTGAA AGAPAGAAGA AGATGAACTT TGTCAGGGGT GGCGGGGCAT GGATCCAGAA TGTGTATTTC TCTGGACAGG GTGAGGTCAT TGTCTCATCT CCTTCACGGG GGTCGTGGGG TTTTGCCAGC GCCGAAGACC GTTGGGGGCC GTGGTGAGCT TTCTCATCAG GGAGTGAGGG TCTCGCGTTG CCGTGGCCCT CTGAATGGTC TGGGTGATCC GGTTTGGGAT TTGTATGCTC ACCCAAAGCA ATTTTTTCTC CAGGGGGAGG GTGAAAGCTC 4483 4543 4603 4663 4723 4783 4843 4903 4963

CTGCCTTCTC

GAGACAGTGA

CACACTGATG

AGGGCCTGCT

TGGGAGGTCT

TGACCGCCTC

GCATGGGTTT

GTCCAAGGAA

CCCCAACTGG

CCTTCAGGAC

TCATTTCACC

CTGAGCTGAG

TTTTACTGTT

ATAGCTGAGA

GATCCAGTGT

CCTGGGAACT

AAAAAAAAAC

CTGACCCCTA

TTGGGCAGGG

CAGCTGCTAG AGGCTGGTCT TCCCCAGGGC GGGATCCTTG TCATAACAGG GAAGTGCCCT TCCCATCCAT TTTGGGAAGG GTGGGCTTAC GAGATGGTCC GAGGAGGCOC CTTCTCCCTA CCACCATCCT GCCGCTGTGA ATTTGAATCT CAAGCAATCA TGCTATGGGC TCAGAGAAGC CTTGCATTCC AGATGATAAC ATGGCAACCG AGCAGCTGAA GAGGGCAGTG CCCGGGCCCA CGGGGCGCAT CAGCATCTAT CTATAAAAAT AAGAAAATAC TGTTGCTGCA GCAGCTTGGG TTCACATGTT GCAGGCAAGG AAGACCATAA CAGCAGACTG GAGTCAGTGT CCCCTGAGCC AGTTCAGGAA GTGTTTGCTG

AAGTCCTGGG

CAGGTCATGT

TGCAGCCATC

CTTTCAAGAC

TCATCAAATA

ACOTOCCOCA

ACACAGTGCG

CAGGCTAACC

TACTGAGAAG

CAGAGTTCCC

TCACGGCTGT

CCAAAAAAAA

GTGGCAAACC

AGCTAGTGTT

GAAGAGCGGA

TCAGTGAGTC

TCAATAGGTC

GCACTACAGG

TGAGCATCTA

AATATTAAAA

CACCCCGTCT

GTCCTCAGCA

CTGCTTGCAC

CCGCATCCCT

TTGTGGCACA

GGCAAGGGTT

ATAAATAAAT

AGGACTAGAA

CTCTGGGGAC

GTTTCCAGGC

CCGGGACTCG 5023 AAACAAGGAG 5083 AGGTAGATCT 5143 TTGTGCTCAG 5203 TCCAGTCCTG 5263 GCAGAGGCTG 5323 GGTGGAAAGG 5383 TTGGTAGCAT 5443 TTGAAGCAGG 5503 GAGGGCTMAG 5563 CGATCCCTGG 5623 AAAAATAAAC 5683 CCTGGGTCCT 5743 GGGAACAGGG 5803 TGATTTTGCA 5863 GGAGGTGAGG GAAGTGGATT GCCTGGAGGG AGGAGGCTGT TTTGTTTGAA GCTT INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 166 amino acids TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 5917 Arg Cys Gly Pro Tyr Val Glu Ala 20 Leu Cys Arg Phe Leu 10 Val Pro Ilie Trp Arg Leu Ala Leu Ala Tyr Val Gin Asp Asp Thr Thr Leu Ilie Gin Ser Val 50 Lys Thr Ilie Val Ser Ser Lys Gin 55 Pro Val Leu Ser Thr 40 Ser Asp Ilie Ser His Met Gly Leu His Arg Val Thr Gly Leu Leu Ser Lys Met Asp 75 Ser Leu Pro Ser Arai Asp Phe Ilie Tyr GIn GIn Ilie Leu GIn Thr Leu Ala Asn Val Ilie Gin Thr Ilie Ser Asn Asp 100 Glu Asn Leu Arg 105 Pro Leu Pro Ser Ser Ser Lys 115 Ser Cys Asp Leu Leu His Leu 110 Arg Ala Leu Glu Thr 125 Leu Tyr Ser Thr Giu Leu 120 Leu Glu Val Val Ser Ala Leu Gly Gly Val Leu Glu 130 135 Leu Ser Arg Leu Gin Gly 150 Leu Ser Pro Gly Cys 165 Ala Ser 140 Ala Leu GIn Asp Met 155 Leu Arg Gin Leu 160 INFORMATION FOR SEQ ID NO:3: R\ I B ZZ] 042 86 doc: mrr SEQUENCE CHARACTERISTICS: LENGTH: 435 base pairs TYPE: nucleic acid (C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..435 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GTG CCC ATC TGG AGA GTC CAG GAT GAC ACC AAA ACC CTC ATC AAG ACG 48 Val Pro lie Trp Arg Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr 1 5 10 ATT GTC ACC AGG ATC AGT GAC ATT TCA CAC ATG CAG TCT GTC TCC TCC 96 lie Val Thr Arg lie Ser Asp Ile Ser His Met Gin Ser Val Ser Ser 25 AAA CAG AGG GTC ACC GGT TTG GAC TTC ATC CCT GGG CTC CAT CCT GTC 144 Lys Gin Arg Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro Val 40 CTG AGT TTG TCC AAG ATG GAC CAG ACC CTG GCG ATC TAC CAA CAG ATC 192 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala Ie Tyr GIn Gin lIe 55 CTC ACC AGT CTG CCT TCC AGA AAT GTG ATC CAA ATA TCG AAT GAC CTG 240 Leu Thr Ser Leu Pro Ser Arg Asn Val lie GIn lie Ser Asn Asp Leu 70 75 GAG AAC CTC CGG GAC CTT CTC CAC CTG CTG GCC TCC TCC AAG AGC TGC 288 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser Lys Ser Cys 90 CCC TTG CCC AGC AGG GCC CTG GAG ACC TTG GAG AGC CTG GGC GGC GTC 336 Pro Leu Pro Ser Arg Ala Leu Glu Thr Leu Glu Ser Leu Gly Gly Val 100 105 110 CTG GAA GCC TCC CTC TAC TCC ACG GAG GTG GTG GCC CTG AGC AGG CTG 384 Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu 115 120 125 CAG GGG GCT CTG CAG GAC ATG CTG CGG CAC GTG GAC CTC AGC CCT GGC 432 Gin Gly Ala Leu Gin Asp Met Leu Arg His Val Asp Leu Ser Pro Gly 130 135 140 TGC 435 a a a a a INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 145 amino acids TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Val Pro lie Trp Arg Val Gin Asp Asp Thr Lys Thr Leu lie Lys 1 5 10 Ile Val Thr Arg Ile Ser Asp lie Ser His Met Gin Ser Val Ser 25 Lys Gin Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro 40 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala lie Tyr Gin Gin 55 Leu Thr Ser Leu Pro Ser Arg Asn Val lie Gin lie Ser Asn Asp 70 75 Thr Ser Val Ile Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Ser Ser 90 Pro Leu Pro Ser Arg Ala Leu Glu Thr Leu Glu Ser Leu 100 105 Lys Ser Cys Gly Gly Val [R:\LIBZZ1042 86.Ac mrr Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu 115 120 125 GIn Gly Ala Leu Gin Asp Met Leu Arg His Val Asp Leu Ser Pro Gly 130 135 140 Cys 145 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 504 base pairs TYPE: nucleic acid (C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID ATGCATTGGG GAACCCTGTG CGGATTCTTG TGGCTTTGGC CCTATCTTTT CTATGTCCAA GCTGTGCCCA TCCAAAAAGT CCAAGATGAC ACCAAAACCC TCATCAAGAC AATTGTCACC 120 AGGATCAATG ACATTTCACA CACGCAGTCA GTCTCCTCCA AACAGAAAGT CACCGGTTTG 180 GACTTCATTC CTGGGCTCCA CCCCATCCTG ACCTTATCCA AGATGGACCA GACACTGGCA 240 GTCTACCAAC AGATCCTCAC CAGTATGCCT TCCAGAAACG TGATCCAAAT ATCCAACGAC 300 CTGGAGAACC TCCGGGATCT TCTTCACGTG CTGGCCTTCT CTAAGAGCTG CCACTTGCCC 360 TGGGCCAGTG GCCTGGAGAC CTTGGACAGC CTGGGGGGTG TCCTGGAAGC TTCAGGCTAC 420 TCCACAGAGG TGGTGGCCCT GAGCAGGCTG CAGGGGTCTC TGCAGGACAT GCTGTGGCAG 480 CTGGACCTCA GCCCTGGGTG CTGA 504 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 504 base pairs TYPE: nucleic acid (C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGTGCTGGA GACCCCTGTG TCGGTTCCTG TGGCTTTGGT CCTATCTGTC TTATGTTCAA GCAGTGCCTA TCCAGAAAGT CCAGGATGAC ACCAAAACCC TCATCAAGAC CATTGTCACC 120 AGGATCAATG ACATTTCACA CACGCAGTCG GTATCCGCCA AGCAGAGGGT CACTGGCTTG 180 GACTTCATTC CTGGGCTTCA CCCCATTCTG AGTTTGTCCA AGATGGACCA GACTCTGGCA 240 GTCTATCCAC AGGTCCTCAC CAGCCTGCCT TCCCAAAATG TGCTGCAGAT AGCCAATGAC 300 CTGGAGAATC TCCGAGACCT CCTCCATCTG CTGGCCTTCT CCAAGAGCTG CTCCCTGCCT 360 CAGACCAGTG GCCTGCAGAA GCCAGAGAGC CTGGATGGCG TCCTGGAAGC CTCACTCTAC 420 TCCACAGAGG TGGTGGCTTT GAGCAGGCTG CAGGGCTCTC TGCAGGACAT TCTTCAACAG 480 TTGGATGTTA GCCCTGAATG CTGA 504 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid (C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGATCCGGTC TCAGGCCGTG CCYATCCARA AAGTCC 36 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid (C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GAATTCAGCG CTGCAYYCAG GGCTRASRTC C. 9 [R:\LIBZZ]04286.doc:mrr

Claims (45)

1. A single or double-stranded DNA molecule consisting essentially of a nucleotide sequence encoding a porcine adipocyte polypeptide, leptin.

2. The DNA molecule according to Claim 1, which is substantially free of other porcine DNA sequences.

3. The DNA molecule according to Claim 1 having a nucleotide sequence consisting essentially of at least about 20 nucleotides of the nucleotide sequence depicted in Figure 1 or a sequence complementary to at least about 20 nucleotides of the nucleotide sequence depicted in Figure 1, substantially free of other porcine DNA sequences.

4. The DNA molecule according to Claim 3 consisting essentially of the nucleotide sequence depicted in Figure 1 or a sequence complementary to sequence of Figure 1.

5. The DNA molecule according to Claim 3 consisting essentially of the nucleotide sequence depicted in Figure 2 or a sequence complementary to sequence of 20 Figure 2.

6. The DNA molecule according to Claim 3 consisting essentially of the nucleotide sequence depicted in Figure 3 or a sequence complementary to sequence of Figure 3.

7. The DNA molecule according to Claim 1 which is an expression vector.

8. The DNA molecule according to Claim 7 wherein said vector is a plasmid.

9. A host cell transformed or transfected with the DNA molecule according to Claim 8. An RNA molecule substantially free of other RNA sequences, consisting essentially of an mRNA sequence encoding a porcine adipocyte polypeptide, leptin.

11. The RNA molecule according to Claim 10, consisting essentially of an mRNA S" sequence encoding a porcine adipocyte polypeptide of at least about 10 amino acids encoded by the nucleotide sequence depicted in Figure 1.

12. The RNA molecule according to Claim 11, consisting essentially of an mRNA sequence encoding a porcine adipocyte polypeptide of at least about 10 amino acids encoded by the nucleotide sequence depicted in Figure 2.

13. The RNA molecule according to Claim 11, consisting essentially of an mRNA 20 sequence encoding a porcine adipocyte polypeptide of at least about 10 amino acids encoded by the nucleotide sequence depicted in Figure 3.

14. A DNA sequence encoding antisense RNA which is complementary to the gene encoding a porcine adipocyte polypeptide, said antisense RNA capable of regulating expression of said gene.

15. A porcine adipocyte polypeptide encoded by a nucleic acid molecule consisting essentially of the nucleotide sequence depicted in Figure 1 or a sequence complementary to the sequence depicted in Figure 1.

16. The porcine adipocyte polypeptide according to Claim 15, wherein the nucleic acid molecule consists essentially of the nucleotide sequence depicted in Figure 2, or a sequence complementary to the sequence depicted in Figure 2. o•

17. The porcine adipocyte polypeptide according to Claim 16, wherein the nucleic acid molecule consists essentially of the nucleotide sequence depicted in Figure 3, or a sequence complementary to the sequence depicted in Figure 3.

18. An antibody directed against the porcine adipocyte polypeptide of Claim 1. An isolated single or double-stranded DNA molecule consisting of a nucleotide sequence which encodes a porcine adipocyte polypeptide leptin having the amino acid sequence of SEQ ID NO:4, or the complement to the DNA molecule. 2. The DNA molecule of claim 1 consisting of the nucleotide sequence of SEQ S ID NO:3. 3. An isolated single or double-stranded DNA molecule consisting of a nucleotide sequence which encodes a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the examples. 4. An expression vector comprising the DNA molecule of any one of claims 1 to 3. The vector of claim 4 in which the vector is plasmid. 6. An expression vector comprising a nucleotide sequence encoding a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples. 7. A host cell transformed or transfected with the plasmid according to any one of claims 4 to 6. 8. A host cell transformed or transfected with a DNA molecule according to any one of claims 1 to 3. 9. A method for transforming a host cell to express a porcine adipocyte 20 polypeptide leptin, said method comprising contacting said cell with a DNA molecule according to any one of claims 1 to 3 encoding a porcine adipocyte polypeptide leptin, or an expression vector according to any one of claims 4 to 6. A method for transforming a host cell to express a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples. 11. A host cell transformed by a method according to claim 9 or claim 12. A host cell transformed with a DNA molecule consisting essentially of a nucleotide sequence encoding a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples. 13. A process for preparing a porcine adipocyte polypeptide leptin, or functional derivative thereof, the process comprising the steps of: culturing a host cell according to any one of claims 7, 8, 11 or 12; expressing the polypeptide; and recovering the polypeptide from the culture. [I:\DayLib\LIBZZ]03938.doc:mrr 36 14. A process for preparing a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples. A porcine adipocyte polypeptide prepared by a process according to claim 13 or 14. 16. An isolated porcine adipocyte polypeptide leptin encoded by a nucleic acid molecule consisting essentially of the nucleotide sequence SEQ ID NO:3, or a sequence complementary to sequence SEQ ID NO:3. 17. An isolated porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples. 18. A method for the regulation of feed intake, energy metabolism or fat deposition in swine, said method comprising administering to said swine an effective amount of a polypeptide according to any one of claims 15 to 17.

19. A polypeptide according to any one of claims 15 to 17, when used for the regulation of feed intake, energy metabolism or fat deposition in swine. s 20. Use of a polypeptide according to any one of claims 15 to 17 for the manufacture of a composition for the regulation of feed intake, energy metabolism or fat deposition in swine.

21. An isolated mRNA molecule encoding a porcine adipocyte polypeptide leptin, the mRNA molecule encoded by the nucleotide sequence of SEQ ID NO:3. 20 22. An isolated mRNA molecule consisting of a nucleotide sequence that encodes n* a porcine adipocyte polypeptide leptin having the amino acid sequence of SEQ ID NO:4.

23. An isolated mRNA molecule encoding a porcine adipocyte polypeptide leptin, substantially as hereinbefore described with reference to any one of the Examples.

24. An isolated RNA molecule substantially free of other RNA sequences, consisting essentially of a sequence complementary to an RNA molecule according to any one of claims 21 to 23. A method for producing an antibody capable of binding porcine leptin polypeptide, the method substantially as hereinbefore described with reference to any one of the examples.

26. An antibody capable of binding the porcine adipocyte polypeptide leptin according to any one of claims 15 to 17.

27. An antibody capable of binding a porcine adipocyte polypeptide leptin, the antibody produced by the method of claim

28. An antibody capable of binding a porcine adipocyte polypeptide leptin, s3 substantially as hereinbefore described with reference to any one of the examples. [I:\DayLib\LI BZZ]03938.doc:mrr 37

29. A method for detecting the presence of a nucleic acid molecule encoding a porcine adipocyte polypeptide leptin, or a complementary sequence, in a nucleic acid- containing sample, the method comprising: contacting the sample under hybridising conditions with a probe selected from: a DNA molecule according to any one of claims 1 Sto 3 or a fragment thereof; or an RNA molecule according to any one of claims 21 to 24 or a fragment thereof; and measuring the hybridisation of the probe to the nucleic acid molecule, thereby detecting the presence of the nucleic acid molecule. A method according to claim 29, wherein the nucleic acid molecule has been selectively amplified before contact with the probe.

31. A method for detecting the presence of a nucleic acid molecule encoding a porcine adipocyte polypeptide leptin, or a complementary sequence, in a nucleic acid- containing sample, substantially as hereinbefore described with reference to any one of the Examples.

32. A nucleic acid molecule encoding a porcine adipocyte polypeptide leptin 15 detected by a method according to any one of claims 29 to 31.

33. A DNA molecule according to any one of claims 1 to 3 or an RNA molecule according to any one of claims 21 to 24, when used for detecting the presence of a nucleic acid molecule encoding a porcine adipocyte polypeptide leptin.

34. A method for detecting the presence of a porcine adipocyte polypeptide leptin 20 in a biological sample, the method comprising contacting the sample with an antibody according to any one of claims 26 to 28 and detecting the binding of the antibody to an antigen in the sample, or measuring the quantity of the antibody bound.

35. A method for detecting the presence of a porcine adipocyte polypeptide leptin in a biological sample, substantially as hereinbefore described.

36. A porcine adipocyte polypeptide leptin detected by a method according to :claim 34 or claim

37. An antibody according to any one of claims 26 to 28, when used for detecting the presence of a porcine adipocyte polypeptide leptin in a biological sample.

38. A method for determining the susceptibility of swine to fat deposition, the method comprising removing a biological sample from a subject, extracting the DNA therefrom, digesting the DNA with restriction endonucleases, contacting the sample under hybridising conditions with a DNA molecule according to any one of claims 1 to 3 or a fragment thereof and separating the restriction fragments by electrophoresis and relating the number or pattern of hybridised bands to fat deposition. [I:\DayLib\LI BZZ]03938.doc:mrr 38

39. A method for determining the susceptibility of swine to fat deposition, the method comprising removing a biological sample from a subject and measuring the amount of mRNA coding for a porcine adipocyte polypeptide leptin by contacting the sample under hybridising conditions with a probe comprising a DNA molecule according to any one of claims 1 to 3 or a fragment thereof, and measuring the hybridisation of the probe to mRNA, wherein the amount of mRNA is related to susceptibility. A method for determining the susceptibility of swine to fat deposition, the method comprising removing a biological sample from a subject and measuring the amount of mRNA coding for a porcine adipocyte polypeptide leptin by contacting the 0t sample under hybridising conditions with a probe comprising an RNA molecule according to any one of claims 21 to 24 or a fragment thereof, and measuring the hybridisation of the probe to mRNA, wherein the amount of mRNA is related to susceptibility.

41. A method for determining the susceptibility of swine to fat deposition, the i5 method comprising removing a biological sample from a subject and measuring therein the amount of porcine adipocyte polypeptide leptin by contacting the sample under hybridising conditions with an antibody according to any one of claim 26 to 28 and detecting the binding of the antibody to an antigen in the sample, or measuring the quantity of the antibody bound, wherein the amount of polypeptide is related to susceptibility.

42. A method for determining the susceptibility of swine to fat deposition, substantially as hereinbefore described. )43. A DNA molecule according to any one of claims 1 to 3, when used for determining the susceptibility of swine to fat deposition.

44. An RNA molecule according to any one of claims 21 to 24, when used for determining the susceptibility of swine to fat deposition. An antibody according to any one of claims 26 or claim 28, when used for determining the susceptibility of swine to fat deposition.

46. A method for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake, the method comprising contacting the agent being tested with an adipocyte culture in vitro, removing a sample from the culture and measuring therein the amount of mRNA coding for a porcine adipocyte polypeptide leptin by contacting the sample under hybridising conditions with a probe comprising a DNA molecule according to any one of claims 1 to 3 or a fragment thereof, and measuring the []:\DayLib\LIBZZ03938.doc:nrr 39 hybridisation of the probe to mRNA, wherein the efficacy of the agent is related to its ability to change the production of the mRNA.

47. A method for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake, the method comprising contacting the agent being tested with an adipocyte culture in vitro, removing a sample from the culture and measuring therein the amount of mRNA coding for a porcine adipocyte polypeptide leptin by contacting the sample under hybridising conditions with a probe comprising an RNA molecule according to any one of claims 21 to 24 or a fragment thereof, and measuring the hybridisation of the RNA molecule to the mRNA, wherein the efficacy of the agent is tI related to its ability to change the production of the mRNA.

48. A method for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake, the method comprising contacting the agent being tested with an adipocyte culture in vitro, removing a sample from the culture and measuring therein the amount of porcine adipocyte polypeptide leptin by contacting the sample i5 under hybridising conditions with an antibody according to any one of claim 26 to 28 and detecting the binding of the antibody to an antigen in the sample, or measuring the quantity of the antibody bound, wherein the efficacy of the agent is related to its ability to change the production of the polypeptide.

49. A method for evaluating the efficacy of an agent directed to the regulation of 20 fat deposition and feed intake, substantially as hereinbefore described.

50. A DNA molecule according to any one of claims 1 to 3, when used for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake.

51. An RNA molecule according to any one of claims 21 to 24, when used for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake.

52. An antibody according to any one of claims 26 to 28, when used for evaluating the efficacy of an agent directed to the regulation of fat deposition and feed intake.

53. A composition manufactured according to claim Dated 20 September, 2001 Purina Mills, Inc. and Purdue Research Foundation Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [I:\DayLib\1.1BZZ]0393 8.doc:mrr