MXPA99001134A - Bovine leptin protein, nucleic acid sequences coding therefor and uses thereof - Google Patents

Abstract

A bovine adipocyte-specific polypeptide, termed leptin, is expressed in the fat tissue of cattle. Expression may be altered in over fat cattle, or expression may be in the form of a protein of lesser biological activity relative to that of leaner cattle. The bovine adipocyte polypeptide, DNA and RNA molecules coding therefor, methods for its preparation, and antibodies specific for the polypeptide are disclosed. Methods for determining the susceptibility of cattle to fat deposition are based on measuring the levels of the bovine adipocyte polypeptide in a biological fluid or tissue extract or by measuring mRNA encoding the bovine adipocyte polypeptide in cells of the subject. Methods of evaluating an agent related to the deposition of fat in cattle comprise contacting the agent with an adipocyte in vitro and measuring the amount of the bovine adipocyte polypeptide or mRNA that is produced by the adipocyte. Methods of limiting fat deposition include administering leptin or leptin DNA, and methods of altering intake include administering leptin, leptin DNA, or an antibody directed against leptin.

Description

PROTEIN OF BOVINE LEPTINE, NUCLEIC ACID SEQUENCES THAT CODIFY FOR THE SAME AND USES OF THE SAME BACKGROUND OF THE INVENTION 1. Field of the Invention: This invention relates to the regulation of energy consumption and metabolism in developing, finished, lactating or non-lactating cattle, and in gestation. More specifically, it refers to a specific bovine polypeptide called leptin, which is secreted by adipocytes or other types of cells and which has an influence on the consumption of energy and metabolism, fat deposition and weight gain in cattle. Furthermore, this invention relates to the sequences encoding the bovine leptin polypeptide, the antibodies directed against the bovine leptin polypeptide and methods for determining the susceptibility to fat deposition, altering the energy consumption and minimizing the Excessive fat deposition in cattle. 2. Description of the Prior Art: The National Institutes of Health have stated that obesity is a public health hazard and has prompted the animal feed industry to seek methods to limit the deposition of fat in animals for food. In addition, the energy cost of having animals for food converts dietary energy into fat instead of the deficient tissue providing a considerable incentive to develop technology to facilitate the efficient production of poorer meat products and to exactly match the nutrient content of the diet with the animal's nutrient needs. To combat these problems of health and production, both prophylactic and therapeutic approaches are necessary. For prophylactic purposes, it may be necessary to predict and measure the propensity and susceptibility to excessive fat deposition. For therapeutic purposes, it may be of great benefit to improve current methods to minimize the deposition of food energy as fat in the adipocyte. Currently, none of these desired objectives has been fully achieved. Proteins from genes expressed only (or predominantly) in adipose tissue and for which the level of expression may be related to fat deposition serve as primary targets for approaches aimed at predicting the fat-building potential and control of fat deposition of fat. For example, a specific polypeptide in the mammalian adipocyte, designated p154, was reported in the U.S. patent. 5,268,295 of Serrero, which is incorporated herein by reference, as being expressed in high amounts in adipogenic cell lines after cell differentiation and is abundant in the fatty cushions of normal and genetically obese mice. However, until now, there have been no reports of specific proteins in adipocyte expressed at different levels in cattle fattening compared to normal controls. Leptin, the protein produced by the leptin (orb) gene, is possibly related to fat deposition in cattle, as research has shown that mutations in genetically obese (ob / ob) mice result in excessive deposition. of fat are associated with the altered expression of the leptin gene. In addition, 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 Nati Acad Sci 92: 6957, Leroy et al., 1996, J. Biol. Chem. 271 (5): 2365 ). In addition, leptin is a regulator of feed consumption (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), the bovine leptin cDNA or genomic sequence was not available before the initiation of this project. In this way, the insights obtained regarding bovine metabolism were not accessible to bovine systems. In addition, purified, biologically active bovine protein (ie, leptin) has not been obtained.

COMPENDIUM OF THE INVENTION The present invention provides sequences of genes, peptides, antibodies and methods for using them where the prediction and modulation of fat deposition and regulation of feed intake (ie, appetite) in bovine species is allowed. In one aspect, this invention is directed to a bovine adipocyte polypeptide, the bovine leptin protein, substantially free of other bovine proteins, or a functional derivative thereof. The present invention includes a bovine adipocyte polypeptide consisting essentially of at least about 8 amino acids of the amino acid sequence illustrated in Figures 1 and 3-5 (SEQ ID NOS: 4, 7 and 8), or a functional derivative of the same. The present invention is also directed to a DNA molecule of single or double chain structure or to an RNA molecule consisting essentially of a nucleotide sequence encoding the above polypeptide or a functional derivative thereof, the DNA or RNA molecule being substantially free of other bovine DNA or RNA sequences. The DNA molecule is preferably a DNA molecule of single or double chain structure having a nucleotide sequence consisting essentially of at least 20 nucleotides of the nucleotide sequence illustrated in Figures 1 and 2 (SEQ ID NO: 3) ) or a sequence complementary to at least part of the nucleotide sequence illustrated in Figures 1 and 2 (SEQ ID NO: 4) or a functional equivalent thereof, substantially free of other bovine DNA sequences. The RNA molecule is preferably an mRNA molecule that encodes the above bovine adipocyte polypeptide, or a functional derivative 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 plasmid p vehicle. 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 method for preparing a bovine adipocyte polypeptide or a functional derivative, as described above, the method comprising the steps of: (a) culturing a host capable of expressing the polypeptide under culture conditions; (b) expressing the polypeptide; and (c) recovering the polypeptide from the culture. Also included in the present invention is a method for detecting the presence of a nucleic acid molecule by having the sequence of the DNA molecule described above, or a complementary sequence, in a sample containing nucleic acid, the method comprising: (a) contacting the sample with an oligonucleotide probe complementary to the sequence of interest under hybridization conditions; and (b) measuring the hybridization of the probe to the nucleic acid molecule, thereby preserving the presence of the nucleic acid molecule. The above method may further comprise before step (a): (c) selectively amplifying the copy number of the nucleic acid sequence. Another embodiment of this invention is an antibody specific for an epitope of the bovine adipocyte polypeptide, or functional derivative, either polyclonal or monoclonal. A method for detecting the presence or measuring the amount of bovine 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 is also presented. , or measure the amount of bound antibody. The present invention includes methods for determining the susceptibility of cattle to fat deposition, which comprises removing a biological sample from a subject and measuring therein the amount of polypeptide or mRNA encoding it., when the amount of the polypeptide or mRNA is related to susceptibility. The present invention also includes methods for determining a subject's susceptibility 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 through gel electrophoresis, and relating the number of bands (pattern in band form) generated by restriction enzyme digestion to fat deposition (ie, RFLP techniques). Herein another method is provided for evaluating the efficacy of a drug (or other agent) directed at the regulation of fat deposition and food consumption, which comprises contacting the drug being tested with an adipocyte culture. in vitro and measuring the amount of adipocyte polypeptide or mRNA that is produced, the efficacy of the drug being related to the change in the production of the polypeptide or mRNA.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the nucleotide sequence of bovine leptin cDNA (upper) (SEQ ID NO: 3) and the amino acid sequence predicted for the coding region minus the secretion signal. Figure 2A shows a comparison of the sequence (SEQ ID NO: 3) of nucleotide of bovine leptin cDNA with the human nucleotide sequence. Figure 2B shows a comparison of the sequence (SEQ ID NO: 3) of nucleotide of bovine leptin cDNA with the murine nucleotide sequence. Figure 3A shows a comparison of the sequence (SEQ ID NO: 4) of bovine leptin amino acid predicted with the amino acid sequence of human leptin. Figure 3B shows a comparison of the sequence (SEQ ID NO: 4) of bovine leptin amino acid predicted with the amino acid sequence of murine leptin. Figure 4 illustrates a portion of the amino acid sequence of real bovine leptin amino acid sequence (SEQ ID NO: 5), which is an N-terminal sequence comprising 30 amino acids. Figure 5A shows a comparison of the sequence (SEQ ID NO: 7) of the amino acid of real bovine leptin with the amino acid sequence of human leptin. Figure 5B shows a comparison of the sequence (SEQ ID NO: 8) of the amino acid of real bovine leptin with the amino acid sequence of murine leptin. Figure 6 shows that a band of 449 base pairs was obtained from a cDNA of individual bovine chain structure amplified by PCR. Figure 7 illustrates the Northern staining analysis of bovine leptin mRNA.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention is directed to DNA and RNA molecules that encode a bovine adipocyte polypeptide called "leptin", or a functional derivative thereof, and to the same bovine leptin protein, or a functional derivative thereof. Bovine leptin protein is useful for the regulation of feed intake, energy metabolism, and fat deposition in cattle. Such targets can be obtained by administering recombinant or purified leptin, altering the expression of the bovine leptin gene or administering an antibody directed against the bovine leptin protein to obtain neutralization, depending on the desired result. The DNA, RNA and protein of bovine leptin, or derivatives thereof, and antibodies specific for the protein are used in tests to predict the potential for fat deposition in cattle. These molecules can also be used in the development of commercially valuable technology to alter feed intake and regulate fat deposition in livestock, and to match the nutrient content of the diet with the nutrient needs of livestock. In its first aspect, the present invention provides a bovine adipocyte polypeptide called "leptin". The term "polypeptide", as used herein, is intended to include not only the bovine leptin protein, and its functional derivatives, but also amino acid sequences having additional components, for example, amino acid sequences that have components additional 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 3-5 (SEQ ID NOS: 4, 7 and 8). Also within the scope of the present invention is any peptide having at least 8 amino acids present in the aforementioned sequence. Sequences of this length are useful as antigens and for making immunogenic conjugates with carriers for the production of antibodies specific for several whole protein epitopes. Said peptides are also useful for classifying said antibodies and in the methods of the present invention for the detection of the leptin protein in biological samples. It is well known in the art that about 8 amino acids are useful in the generation of antibodies to larger proteins of biological interest. The polypeptide of this invention is large enough to comprise an antigenically distinct determinant, or epitope, which can be used as an immunogen to produce antibodies against leptin, or a functional derivative thereof, and to test said antibodies. The polypeptide of this invention can also exist covalently or non-covalently bound to another molecule. For example, it can be fused (i.e., a fusion protein) to one or more other polypeptides via one or more peptide bonds. One embodiment includes the polypeptide substantially free of other bovine peptides. The polypeptide of the present invention can be biochemically or immunochemically purified from cells, tissues or a biological fluid. Alternatively, the polypeptide can be produced through recombinant means in a prokaryotic or eukaryotic host cell. "Substantially free of other bovine polypeptide" reflects the fact that because the gene for the bovine adipocyte polypeptide of interest can be cloned, the polypeptide can be expressed in a prokaryotic or eukaryotic organism, if desired. Methods for the synthesis of polypeptides of a desired sequence onto solid phase supports and their subsequent separation from the support are also well known. Alternatively, the protein can be purified from bovine tissue or fluids where it can occur naturally, so that it is purified by at least 90% (on a weight basis), and still at least 99%, if desired, of other bovine polypeptides and, therefore, is substantially free of them. This can be achieved by subjecting the tissue or fluids to standard purification techniques such as immunoabsorbent columns carrying monoclonal antibodies reactive against the protein. Alternatively, the purification of said tissue or fluids can be achieved through a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, and ion exchange chromatography. As alternatives to a native or recombinant bovine adipocyte polypeptide molecule, functional derivatives of the bovine adipocyte polypeptide can be used. As used herein, the term "functional derivative" refers to any "fragment", "variant", "analogue", or "chemical derivative" of the bovine adipocyte polypeptide that retains at least a portion of the function of the bovine adipocyte polypeptide that allows its utility according to the present invention. A "fragment" of the bovine adipocyte polypeptide, as used herein, refers to a subgroup of the molecule, i.e., a shorter peptide. A "variant" of the bovine adipocyte polypeptide, as used herein, refers to a molecule substantially similar to either the entire peptide or a fragment thereof. Variant peptides can be conveniently prepared through 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 through mutations in the DNA encoding the synthesized peptide (again using methods well known in the art). Such variants include, for example, deletions of, or insertions or substitutions of, residues within the amino acid sequence. Any combination of elimination, insertion and substitution can also be made to reach the final construction, as long as the final construction possesses the desired activity. Obviously, the mutations that will be made in the DNA encoding the variant peptide should not alter the reading frame and preferably will not create complementary regions that can produce secondary mRNA structures.

An "analogue" of the bovine adipocyte polypeptide, as used herein, refers to an unnatural molecule substantially similar to the entire molecule or to a fragment thereof. A "chemical derivative" of the bovine adipocyte polypeptide or peptide, as used herein, contains additional chemical moieties not normally part of the polypeptide. Covalent modifications are included within the scope of this invention. Such modifications can be introduced into the molecule by reacting target 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 through a nucleic acid molecule, a chain structure having the nucleotide sequence shown in Figures 1 and 2 (SEQ ID NO: 3). The present invention is directed to a DNA sequence encoding the polypeptide, or a functional derivative thereof, substantially free of other bovine DNA sequences. Said DNA may be individual chain structure (ie, sense, antisense or cDNA sequence) or double chain structure. The DNA sequence preferably should have approximately 20 or more nucleotides to allow hybridization to another polynucleotide. In order to obtain a specific higher hybridization characteristic, characterized by the absence of hybridization to sequences other than those encoding the polynucleotide, 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 an mRNA sequence encoding the polypeptide of this invention, or a functional derivative thereof. The present invention is also directed to the above DNA molecules, which are functional in recombinant expression systems, being used as hosts transfected or transformed with the vehicles and capable of expressing the polypeptide. Such hosts can be prokaryotic or eukaryotic. DNA can be incorporated into the host organism through transformation, transduction, transfection or a related procedure known in the art. In addition to a DNA or RNA sequence encoding the bovine adipocyte polypeptide molecule, this invention provides methods for the expression of nucleic acid sequences. In addition, the genetic sequences and oligonucleotides of the invention allow the identification and cloning of additional, yet undiscovered, adipocyte polypeptides having sequence homology to the bovine adipocyte polypeptide of the present invention. The recombinant DNA molecules of the present invention can be produced through any of a variety of means, such as, for example, synthesis of DNA or RNA, or most preferably, through the application of recombinant DNA techniques. The techniques for synthesizing said molecules are described, for example, by Wu, R., et al., Prog. Nucí. Acid Res. Molec. Biol. 21: 101-141 (1978), which is incorporated herein by reference. The methods for constructing recombinant molecules according to the method described above are described by Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference. Oligonucleotides representing a portion of the bovine adipocyte polypeptide of this invention are useful for classifying the presence of genes encoding such proteins and the cloning of bovine adipocyte polypeptide genes. Techniques for synthesizing such oligonucleotide are described, for example, by, Wu, R., et al. Prog. Nucí. Acid Res. Molec. Biol. 21: 101-141 (1978). An oligonucleotide, or a group of oligonucleotides, which is capable of encoding a fragment of the bovine adipocyte polypeptide gene of this invention (or which is complementary to said oligonucleotide, or group of oligonucleotides) is identified, synthesized and hybridized through means well known in the art, against a DNA or, most preferably, a cDNA preparation derived from cells which are capable of expressing the adipocyte polypeptide gene of bovine. Oligonucleotide molecules of individual chain structure complementary to the "very likely" bovine adipocyte polypeptide encoding sequences can be synthesized using procedures that are well known to those skilled in the art (See, e.g., U.S. Patent 5,268,295 ). In addition, DNA synthesis can be achieved through the use of automatic synthesizers. Nucleic acid hybridization techniques are described by Sambrook et al. (supra). In an alternative form for cloning the bovine adipocyte polypeptide gene of this invention, a collection of expression vectors is prepared by cloning the DNA or, most preferably, a cDNA (from a cell capable of expressing the bovine adipocyte polypeptide) to an expression vector. The collection is then classified for members capable of expressing a protein which binds to the anti-bovine adipocyte polypeptide antibody, and which has a nucleotide sequence that is capable of encoding polypeptides having the same amino acid sequence as the bovine adipocyte polypeptide of this invention, or fragments thereof. In this embodiment, the DNA, or most preferably the cDNA, is extracted and purified from a cell that is capable of expressing the bovine adipocyte polypeptide protein. The cDNA is fragmented (by cutting, endonuclease digestion, etc.) to produce a combination of DNA or cDNA fragments. The DNA or cDNA fragments of this combination are then cloned into an expression vector in order to produce a collection of expression vectors, each member of which contains a single cloned DNA or cDNA fragment. An "expression vector" is a vector which (due to the presence of appropriate transcriptional and / or translation control sequences) is capable of expressing a DNA (or cDNA) molecule, which has been cloned into the vector and this way produces a polypeptide or protein. The expression vectors of the present invention can 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 bovine adipocyte polypeptide of the present invention, or its functional derivative, can be recombined with the vector DNA according to conventional techniques such as those described 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 link is a link wherein the regulatory DNA sequences and the DNA sequence searched for to be expressed are connected in such a manner as to allow expression of the gene. The precise nature of the regulatory regions necessary for gene expression may vary from organism to organism, but should generally include a promoter region, which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription). ) as well as DNA sequences, which, when transcribed to RNA, will signal the initiation of protein synthesis. A promoter is a DNA or RNA molecule of double-stranded structure that is capable of binding the RNA polymerase and promoting the transcription of the "operably linked" nucleic acid sequence. The promoter sequences of the present invention can be either prokaryotic, eukaryotic or viral. However, strong promoters are preferred. Suitable promoters are repressible, or very preferably, constitutive. Examples of suitable prokaryotic promoters include the tetracycline promoter (Tet A) for pASK75 and T7lac for pET21. Examples of suitable eukaryotic promoters include alpha-actin or beta-actin. Examples of suitable viral promoters include Rous sarcoma or cytomegala. The present invention is also directed to an antibody specific for an epitope of the bovine adipocyte polypeptide of the present invention, and the use of said antibody to detect the presence of, or measure the amount or concentration of the polypeptide, or a functional derivative of the same, 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 antibody that may also be recognized by that antibody. The epitopes or "antigenic determinants" usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have three dimensional structural characteristics as well as specific charge characteristics. An antibody is said to be "capable of binding" to a molecule if it is capable of specifically reacting with the molecule to bind the molecule to the antibody. The bovine adipocyte polypeptide of the present invention, or a functional derivative thereof, preferably having at least 10 amino acids, is used as an antigen for the 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 through an antibody, which is also capable of inducing a animal to produce an antibody capable of binding an epitope of that antigen. An antigen can have one, or more than one, epitope. The specific reaction referred to above means that it indicates that the antigen will react, in a highly selective manner, with its antibody and not with the multitude of other antibodies, which can be evoked by other antigens. The term "antibody" means that it includes polyclonal antibodies, monoclonal antibodies (mAbs), and chimeric antibodies. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from animals immunized with an antigen. Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigenic epitopes. The mAbs can be obtained through methods known to those skilled in the art. (See, for example, Kholer and Milstein, Nature 256: 495-497 (1975) and U.S. Patent No. 4,376,110; from St. Groth, S. F. et al. J. Immunol. Methods, 35: 1-21 (1980); and Hartlow, E. et al., Antibiodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988). Chimeric antibodies are portions of different molecules, which are derived from different animal species, such as those having a variable region of a bovine mAb and a constant region of murine immunoglobulin. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Nati, Acad. Sci. USA 81: 3273-3277 (1984), Morrison et al., Proc. Nati. 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. Nati. Acad. Sci. USA 84: 3439-3443 (1987); Better et al., Science 240: 1041-1043 (1988)). These references are incorporated herein by reference. The term "antibody" also includes both intact molecules and fragments thereof, such as, for example, Fab and F (ab ') 2, which are capable of binding the antigen. The Fab and F (ab ') 2 fragments lack the Fe fragment of the intact antibody, are cleared more quickly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucí. 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 F8ab 'fragments) 2). 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). Antibodies or antibody fragments, useful in the present invention can be used to quantitatively or qualitatively detect the presence of cells expressing the bovine adipocyte polypeptide protein. This can be achieved through immunofluorescence techniques employing a fluorescently labeled antibody coupled with microscopy, flow cytometric or fluorometric detection. Antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectric microscopy, for the in situ detection of the bovine adipocyte polypeptide (ie, leptin). In situ detection can be achieved by removing a histological specimen from a subject, and providing a labeled antibody of the present invention to said specimen. The antibody (or fragment) is preferably provided by applying or covering the labeled antibody (or fragment) to a biological sample. Through the use of said method, it is possible to determine not only the presence of the bovine adipocyte polypeptide of the present invention but also its distribution in the examined tissue. Using the present invention, those skilled in the art will readily perceive that any of a variety of histological methods (such as staining procedures) can be modified in order to achieve such detection in situ. Such assays for the bovine adipocyte polypeptide of the present invention 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 labeled antibody capable of of identifying the bovine adipocyte polypeptide, and detecting the antibody through any number of techniques well known in the art, such as enzyme immunoassays (EIA or ELISA) or radioimmunoassays (RIA). The antibody molecules of the present invention can also be adapted for use in an immunometric assay, known as a "double-site" or "sandwich" assay. In a typical immunometric assay, an amount of an unlabeled antibody (or antibody fragment) is attached to a solid support (i.e., any carrier capable of binding the antigen or antibodies) and an amount of the detectably labeled soluble antibody is added to allow the detection and / or quantification of the tertiary complex formed between the solid phase antibody, the antigen and the labeled antibody.

The binding activity of a given batch of antibody to the bovine adipocyte polypeptide can be determined according to well-known methods. Those skilled in the art will be able to determine the optimal and operative conditions of the assay for each determination using routine experimentation. The antibodies can be used in an immunoaffinity column to purify the binding of the adipocyte polypeptide of the invention through a one-step procedure, using methods known in the art. In accordance with the present invention, cattle that are susceptible to fat deposition are treated with the bovine adipocyte polypeptide of the present invention to limit said fat deposition. This treatment can be performed together with other anti-adipogenic therapies. A typical regimen for treating cattle with a propensity for fat deposition comprises administering an effective amount of the bovine adipocyte polypeptide administered over a period. The bovine adipocyte polypeptide of the present invention can be administered through any means that achieves its intended purpose, preferably to alter the consumption of food or limit the deposition of fat in a subject. For example, administration can be through various parenteral routes including, but not limited to, subcutaneous, intravenous, intradermal, intramuscular and intraperitoneal routes. Alternatively or concurrently, administration may be through the oral route, which can be achieved through the use of genetically altered food products, in which the bovine leptin gene has been inserted and expressed. Parenteral administration can be through bolus injection or through gradual perfusion for a time such as an osmotic delivery device implant. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions, which may contain auxiliary agents or excipients that are well known in the art. Pharmaceutical compositions such as tablets and capsules can also be prepared according to routine methods. It is understood that the dose of bovine adipocyte polypeptide of the present invention administered may depend on the age, sex, health and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the desired effect. The highly preferred dose will be designed for the individual subject, as understood and determined by one skilled in the art. The total dose required for each treatment can be administered through multiple doses or an individual dose. The bovine adipocyte polypeptide of the present invention can be administered alone or together with other therapeutic agents directed to the regulation of food consumption and / or fat deposition.

In a preferred embodiment, the concentration of the bovine adipocyte polypeptide or mRNA of this invention is measured in a cell preparation, tissue extract or biological fluid of a subject as a means to determine the susceptibility or propensity of the subject to the deposition. of fat. Susceptibility to fat deposition is related to the level of the bovine adipocyte polypeptide, or its mRNA. In addition, restriction fragment length polymorphisms in the bovine adipocyte gene will be used to predict the fat deposition potential. Another embodiment of the invention is to evaluate the efficacy of a drug, or other agent, directed to increase or reduce food consumption by measuring the ability of the drug or agent to stimulate or suppress the production of the bovine adipocyte polypeptide or mRNA of this invention. through a cell or cell line capable of producing said polypeptides or mRNAs. Preferred cells are cells of an adipogenic cell line. Antibodies, the cDNA or riboprobe probe of the present invention are useful in the method for evaluating these drugs or other agents since they can be used to determine the amount of the bovine adipocyte polypeptide or mRNA using one of the aforementioned immunoassays. A further embodiment of the present invention is directed to assays for measuring the susceptibility of cattle to fat deposition based on the measurement in a tissue or fluid of the subject of the amount of the mRNA sequences present encoding the adipocyte polypeptide of bovine, or a functional derivative thereof, preferably using an RNA or DNA hybridization assay. Susceptibility to fat deposition is related to the amount of such mRNA sequences present. For such assays, the source of the mRNA sequences is preferably the adipogenic cells of the cattle. The preferred technique for measuring the amount of mRNA is a hybridization assay using RNA (e.g., a ribonuclease protection assay) or DNA (e.g., Northern or Slot staining assays) of the complementary base sequence as probes. Nucleic acid detection assays, especially hybridization assays, can be predicted on any characteristic of the nucleic acid molecule, such as its size, sequence, susceptibility to digestion via restriction endonucleases, etc. The sensitivity of these tests can be increased by altering the way in which the detection is reported or signaled to the observer. In this way, for example, the sensitivity of the assay can be increased through the use of detectably labeled reagents. A wide variety of such labels has been used for this purpose. Kourilsky et al. (U.S. Patent No. 4,581,333) describe the use of enzyme labels to increase sensitivity in a detection assay. Radioisotope labels are described by Falkow et al. (U.S. Patent No. 4,358,535), and by Berninger (U.S. Patent No. 4,446,237). Fluorescent labels (Albarella et al., EP 144914), chemical labels (Sheldon III et al., U.S. Patent No. 4,582,789; Albarella et al., Patent of E.U.A. 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. One method for overcoming the limitation of the sensitivity of the nucleic acid concentration is to selectively amplify the nucleic acid, the detection of which is desired before carrying out the assay. Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have been widely recognized. Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplifications of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Patent No. 4,237,224), Maniatis, T., et al., etc. Recently, an in vitro enzymatic method has been described, which is capable of increasing the concentration of said 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 50,424, EP 84,796, EP 258,017, EP237,362, Mullis, K., EP 201,184, Mullis et al., US Patent No. 4,683,202, Erlich, H., US Patent No. 4,582,788, and Saiki, R. et al., US patent 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 an individual copy in a particular sample. The method can be used to amplify DNA either of single or double chain structure. The essence of the method involves the use of two oligonucleotide probes that serve as primers for template-dependent polymerase-mediated replication of a desired nucleic acid molecule. Having described the invention, it will be more readily understood by reference to the following examples, which are provided by way of example, and are not intended to limit the present invention, unless otherwise indicated.

EXAMPLE I Isolation and Identification of Bovine Leptin cDNA from Adipose Tissue A. Isolation of Bovine Leptin cDNA 1. RNA Extraction: Total RNA was extracted from bovine adipose tissue, using a standard RNA extraction protocol: extraction by guanidinium thiocyanate acid-phenol-chloroform (Chomczynski and Sacchi, 1987, Analytic Biochemistry 162: 156). Poly A + mRNA was then purified from total RNA using an oligo (dT) -cellulose mini-column (Stratagene Cloning Systems, La Jolla, CA). In order to make a template for the PCR amplification, the poly A + mRNA was then reverse transcribed to a single strand structure cDNA using a reverse transcriptase (Gibco BRL, Gaithersburg, MD). 2. PCR and Primer Information: The combination of individual chain structure bovine cDNA was used as a template to amplify the bovine leptin cDNA in a PCR reaction with synthetic DNA primers based on the leptin cDNA sequence of published mouse. Two pairs of oligonucleotide degeneration primers specific for the human leptin and murine gene were designed and synthesized (DNA International, Lake Oswego, OR). The primers were designed to amplify the coding region of the bovine leptin gene, excluding the secretion signal at the 5 'end of the coding region). The forward primer has a sequence of 5'-GGA TCC GGT CTC AGG CCG TGC CYA TCC ARA AAG TCC-3 '(contains a Bsal site), and the reverse primer has a sequence of 5'-GAA TTC AGC GCT GCA YYC AGG GCT RAS RTC-3 '(contains an Eco47III site), where R = (A, G), S = (C, G), Y = (C, T). The PCR was carried out using the following conditions: 1 X pH regulator of PCR, 1.5 mM of MgCl 2, 1 μM of primers, 0.2 mM of dNTPs and 5 units of Taq polymerase per 100 μl of reaction. A total of 32 cycles were performed with the following conditions: 94 ° C, 1 minute; 55 ° C, 1.5 minutes; and 72 ° C, 1.5 minutes. After running the PCR product on a 1% agarose gel, a band of 449 base pairs of the amplified bovine chain structure cDNA amplified by PCR was obtained as illustrated in Figure 6. Specifically, lane 1 of Figure 6 contains the 449 bp bovine leptin cDNA, lane 2 contains the vector DNA pASK75, and lane 3 contains a scale of 100 standard base pairs. The size of the PCR product was consistent with the predicted size of the coding region of the bovine leptin gene. This PCR product was verified in a secondary PCR procedure.

B. Subcloning of the PCR Products to the Expression Vector pASK75 The bovine leptin cDNA obtained through the above procedures was cloned into specific restriction endonuclease cleavage sites (Bsal and Eco47III) of the vector pASK75 (Biometra Ltd., Tampa, FL). This vector, originally derived from pASK60, carries the promoter / operator region of the TetA resistance gene, and allows the precise insertion of a gene and the direct expression of a structural gene with the OmpA signal sequence and a Strep-Tag polypeptide, which is designated for the affinity purification of the recombinant protein. Briefly, the 449 bp PCR product was gel purified, and then partially cut through Bsal and Eco47III to facilitate expression in frame of the inserted DNA. The vector plasmid pASK75 was cut with the same enzymes and then digested with bovine intestinal alkaline phosphatase (CIAP, Gibco BRL) to remove the 5'-phosphate group. This step avoids vector-vector ligations during the ligation reaction. After confirming through gel electrophoresis that the PCR product and the vector were properly digested, ligation was accomplished using T4 DNA ligase (Gibco BRL) with incubation at 14 ° C for 20 hours. The recombinant bovine leptin DNA product was then transformed into an E. coli strain (XL-1-Blue, Stratagene Clonin Systems, La Jolla, CA) using the protocol recommended by the supplier. E. coli was grown in culture, and the recombinant plasmid DNA induced expression of the bovine leptin gene by adding anhydrotetracycline to a concentration below that required for antibiotic activity. The bovine leptin protein was then purified either through SDS-PAGE or Strep-Tag affinity chromatography. The recombinant plasmid DNA was also purified using a plasmid miniprep kit (Promega). The purified plasmid containing the bovine leptin insert was subjected to National Bioscience, Inc., for DNA sequencing to verify that the clone was the bovine leptin homolog and to establish homology with human and murine leptin genes. A clone obtained using the procedure described above, particularly E. coli C1, was deposited at American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., 20852-1776, on June 27, 1996, and has been designated as ATCC No. 98087. This microorganism was deposited under the conditions of the Budapest Treaty in the International Recognition of Deposit of Microorganisms for the purpose of patent procedure. All restrictions on the availability to the public of the material thus deposited will be irrevocably removed after granting a patent. This deposit will be maintained for a period of 30 years from the date of deposit or 5 years after the last request for the material, whichever lasts longer.

C. DNA and Protein Sequencing The insert DNA sequence (both sense and antisense strand structures) was performed through a commercial laboratory (National Bioscience, Inc.) using the Sanger dideoxy-nucleotide method. Briefly, the PCR product containing the 449 bp band was separated on a 1% low melting agarose gel. The 449 bp band was cut from the gel, further purified using a Genecleaning kit (Bio101, Inc., Vista, CA), and subjected to sequencing. The sequences were then compared with the Genbank and other databases using the GCG software. The sequence data confirm that the 449 bp product of two independent clones share approximately 87.6% homology with the human leptin cDNA (Figure 2A) (SEQ ID NO: 3) and 84.9% with the mouse leptin cDNA (Figure 2B) (SEQ ID NO: 3). The predicted amino acid sequence also shares approximately 87% homology with the human leptin protein (Figure 3A) (SEQ ID NO: 4) and approximately 86.3% homology to the murine leptin protein (Figure 3B) (SEC ID NO: 4). In addition, a portion of the predicted amino acid sequence was confirmed through amino terminal sequencing. Specifically, 30 amino acids comprising the N-terminal sequence have been obtained (Figure 4) (SEQ ID NO: 4). The actual amino acid sequence (ie, the N-terminal sequence (Figure 4) (SEQ ID NO: 4)) shares approximately 100% homology with the human leptin protein (Figure 5A) (SEQ ID NO: 7) , and approximately 100% homology with the murine leptin protein (Figure 5B) (SEQ ID NO: 8).

EXAMPLE II Isolation of the mRNA Corresponding to the Bovine Leptin cDNA The bovine leptin cDNA was used as a probe for the detection of full-length mRNA in a Northern stain containing bovine adipose tissue poly A + mRNA and total adipose RNA. ob / ob mouse (Figure 7). The RNA samples were separated on a 1% formaldehyde agarose gel and then transferred to a nylon membrane (Zeta-probe, Biorad) through the capillary transfer method in 10 X SSC (NaCl 1.5 M, citrate sodium 0.15 M, pH 7.0). The staining was hydridized with bovine leptin cDNA labeled with alpha- [32 P] dCTP in a hybridization solution (Gibco BRL, 0.9 M NaCl, 0.09 M sodium citrate (pH 7.0), 0.01 M EDTA (pH 8.0), 5X Denhart solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA) 0.5% SDS, 100 μg / ml denatured salmon sperm DNA, divided) at 55 ° C for 20 hours. The stain was washed with a final severity of 0.1 X SSC (0.015 M NaCl, 0.0015 M sodium citrate (pH 7), 0.1% SDS at 10 60 ° F and exposed to an X-ray film. Leptin mRNA of bovine (approximately 3,090 bp) was clearly evident in the bovine adipose tissue and a leptin mRNA of approximately 3,240 bp was detected in mouse ob / ob adipose tissue As shown in Figure 7, lanes 6-8 contain the total adipose RNA of mouse ob / ob and lane 10 contains bovine poly A + adipose mRNA The abundance of bovine leptin mRNA was low, therefore, a more sensitive RNAse protection assay was established (RPA) for quantifying bovine leptin mRNA in the adipose tissue In summary, a T7 promoter DNA sequence was added to the antisense leptin primer or via PCR with the sense primer as described in FIG. Example I. This modified antisense initiator produced a fragment of 478 bp containing the T7 promoter. A radiolabelled radiosonde 25 was then generated through in vitro transcription with alpha- [32 P] -UTP and the 478 bp PCR fragment. The RPA was performed using commercially available equipment (RPA II, Ambion, Inc.). Hybridization was performed with 50,000 cpm of bovine leptin radiosonde and 10 μg of total fat RNA for 20 hours at 42-45 ° C. The RNA of individual chain structure was then digested by a 1:50 dilution of T1 RNAse for 30 minutes at 37 ° C. After precipitation with ethanol, the protected fragment was separated in 5% of a polyacrylamide gel with 8M urea. The gel was then dried and exposed to an X-ray film and a single 449 bp fragment was protected. Beta-actin was used as an internal control for the standardization of RPA results.

EXAMPLE III Isolation of the Genomic DNA Clone Corresponding to Bovine Leptin The bovine leptin cDNA was also used to classify a collection of bovine genomic DNA. Specifically, a genomic collection of bovine (Holstein dairy cow) was purchased from a commercial source (Stratagene, Inc.). The collection, containing 2 X 106 plaque formation units (pfu) before amplification, was constructed in a FIX II lambda vector with insert sizes of 9-23 kb. The procedures for the classification of the genomic collection were those recommended by the provider. Approximately 1.2 X 106 pfu were classified on the primary classification plates. Specifically, two groups of replica nylon filters were lifted from the plates and pre-hybridized for 3 hours at 40-42 ° C in 0.8 M NaCl, 0.02 M tubes (pH 6.5), 50% or formamide, 0.5% of SDS and 100 μg / ml of salmon sperm DNA with sound application, denatured. The filters were hybridized overnight with a bovine leptin cDNA probe labeled with [alpha-32P] dCTP in hybridization buffer with the same composition as the pre-hybridization solution for 21 hours. The filters were subsequently washed with a final severity of 0.1 X SSC, 0.1% SDS at 60 ° C for 30 minutes. After exposure to the X-ray film, the positive clones that showed signals on both replica filters were recovered from the agar plates, re-titrated and tested in secondary and tertiary classification using the same protocol. After three qualifying rounds, four individual positive clones were identified for additional use.

EXAMPLE IV Purification of the Bovine Leptin Gene Product The polypeptide sequence encoded by the bovine leptin cDNA was purified through SDS-polyacrylamide gel electrophoresis and then the recombinant protein band was electroeluted from the gel. The purified protein will be used for the production of antibodies and the development of ELISA and other assay methodologies.

EXAMPLE V Antibodies to Bovine Leptin and its Use to Detect Bovine Leptin in Adipogenic Cells Polyclonal and / or monoclonal antibodies were produced with the recombinant bovine leptin protein. The techniques used to produce, classify, detect and / or quantify antibodies or leptin are discussed at length in "Antibodies: a Laboratory • Manual "(Harlow et al., 1988, Cold Spring Harbor Laboratory) All media or components of media, mouse or cell strains (eg, BALB / C mouse, sp2 / 0 myeloma cells, JA744A macrophages. 1, etc.) are commercially available 15 A. Animal Immunization • 1. Rabbits: Purified bovine leptin protein was injected into rabbits for the production of polyclonal antibodies Specifically, each rabbit received repeated subcutaneous injections with antigen in auxiliary Complete of Freund followed by at least one booster injection of approximately 200 μg to 1 mg When the serum titration of the immunized rabbits was sufficiently high when tested using bovine leptin as the antigen, the rabbit serum was harvested as the polyclonal antiserum for bovine leptin. 2. BALB / C mice (4 weeks old) Purified bovine leptin protein was injected into BALB / C mice for the production of monoclonal antibodies. Specifically, each mouse was injected with approximately 50 μg of bovine leptin protein with Ribi S-TDCM helpers (RIBI ImmunoChem Research, Inc., Hamilton, Montana). The number of injections depends on the titration of the antibody in the serum of immunized mice as determined through ELISA using bovine leptin as the antigen. In the course of the production of monoclonal antibodies against bovine leptin protein, the spleens of the immunized mice were used to prepare spleen loops. Hybridoma cells were made by fusing the spleen oxytes with sp2 / 0 myeloma cells (treated with a medium containing 8-Azaguanine) in the presence of 50% PEG-1500. Hybridoma cells were incubated in a HAT medium (hypoxanthine), aminopterin and thymidine) of selection). The subsequent classification for positive clones uses recombinant bovine leptin as the antigen in ELISA or Western staining methodology. Positive clones that produce a strong anti-bovine leptin antibody are characterized by specific character, subtype, affinity, binding sites, etc. When large amounts of purified antibody are required, positive clones are cultured on a large scale and the antibody purified from the culture supernatant, or injected into the intraperitoneal cavity of BALB / C mice for the production of ascites. The latter procedure requires approximately 1-2x10 6 hybridoma cells per mouse, and usually takes approximately 7-14 days. Large amounts of antibody are purified from the ascites through techniques such as ammonium sulfate precipitation and ion exchange chromatography (e.g., DEAE-Tricacryl M). Having already fully described this invention, it will be appreciated by those skilled in the art that it can be carried out within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. Although this invention has been described in relation to its specific embodiments, it will be understood that it is capable of other modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention and including said outputs of the present description as within known or customary practice within the art to which the invention pertains and in that it can be applied to the essential aspects set forth above as follows in the scope of the appended claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) ADDRESS: Purina Mili, Inc. (B) STREET: 1401 South Hanley (C) CITY: St. Louis (D) STATE: Missouri (E) COUNTRY: E.U.A. (F) POSTAL CODE: 63144 (ii) TITLE OF THE INVENTION: BOVINE LEPTINE PROTEIN, NUCLEIC ACID SEQUENCES CODING FOR THE SAME AND USES THEREOF (iii) NUMBER OF SEQUENCES: 8 (iv) COMPUTER LEGIBLE FORM : (A) TYPE OF MEDIUM: flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTAWARE: Patentln Relay # 1.0, Version # 1.30 (v) REQUEST DATA CURRENT: (A) APPLICATION NUMBER: PCT / US97 / 12532 (B) SUBMISSION DATE: July 17, 1997 (C) CLASSIFICATION: (vi) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 688,908 (B) DATE OF SUBMISSION: July 31, 1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "initiator" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 GGATCCGGTC TCAGGCCGTG CCYATCCARA AAGTCC 36 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "initiator" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GAATTCAGCG CTGCAYYCAG GGCTRASRTC 30 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (x) ASPECT: (A) NAME / KEY: CDS (B) LOCATION: 6 .. 443 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3 AGGCC GTG CCT ATC CAG AAA GTC CAG GAT GAC ACC AAA ACC CTC ATC 47 Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He 1 5 10 AAG ACA ATT GTC ACC AGG ATC AAT GAC ATC TCA CAC ACG CAG TCC GTC 95 Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val 15 20 25 30 TCC TCC AAA CAG AGG GTC ACT GGT TTG GAC TTC ATC CCT GGG CTC CAC 143 Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His 35 40 45 CCT CTC CTG AGT TTG TCC AAG ATG GAC CAG ACA TTG GCG ATC TAC CAA 191 Pro Leu Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Wing He Tyr Gln 50 55 60 CAG ATC CTC ACC AGT CTG CCT TCC AGA AAT GTG GTC CA T ATA TCC AAT 239 Gln He Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln He Ser Asn 65 70 75 GAC CTG GAG AAC CTC CGG GAC CTT CTC CTG CTG GCC GCC TCC AAG 287 Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Wing Ser Lys 80 85 90 AGC TGC CCC TTG CCG CAG GTC AGG GCC CTG GAG AGC TTG GAG AGC TTG 335 Ser Cys Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu 95 100 105 110 GGT GTC GTC CTG GAA GCC TCC CTC TAC TCC ACC GAG GTG GTG GCC CTG 383 Gly Val Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu 115 120 125 AGC CGG CTG CAG GGG CA CTA CAG GAC ATG TTG CGG CAG C u GAC CTC 431 Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu 130 135 140 AGC CCT GAA TGC AGCGCT 449 Ser Pro Glu Cys 145 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 base pairs (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Val Pro He Gln Lys Val Gln Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15 He Val Thr Arg He Asn Asp He Ser His Thr Gln Ser Val Ser Ser 20 25 30 Lys Gln Arg Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Leu 35 40 45 Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Wing He Tyr Gln Gln He 50 55 60 Leu Thr Ser Leu Pro Ser Arg Asn Val Val Gln He Ser Asn Asp Leu 65 70 75 80 Glu Asn Leu Arg Asp Leu Leu His Leu Leu Wing Ala Ser Lys Ser Cys 85 90 95 Pro Leu Pro Gln Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val 100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gln Gly Ser Leu Gln Asp Met Leu Arg Gln Leu Asp Leu Ser Pro 130 135 140 Glu Cys 145 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 445 base pairs (B) TYPE: nucleic acid (C) STRING STRUCTURE: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AAGCTGTGCC CATCCAAAAA GTCCAAGATG ACACCAAAAC CCTCATCAAG ACAATTGTCA 60 CCAGGATCAA TGACATTTCA CACACGCAGT CAGTCTCCTC CAAACAGAAA GTCACCGGTT 120 TGGACTTCAT TCCTGGGCTC CACCCCATCC TGACCTTATC CAAGATGGAC CAGACACTGG 180 CAGTCTACCA ACAGATCCTC ACCAGTATGC CTTCCAGAAA CGTGATCCAA ATATCCAACG 240 ACCTGGAGAA CCTCCGGGAT CTTCTTCACG TGCTGGCCTT CTCTAAGAGC TGCCACTTGC 300 CCTGGGCCAG TGGCCTGGAG ACCTTGGACA GCCTGGGGGG TGTCCTGGAA GCTTCAGGCT 360 ACTCCACAGA GGTGGTGGCC CTGAGCAGGC TGCAGGGGTC TCTGCAGGAC ATGCTGTGGC 420 AGCTGGACCT CAGCCCTGGG TGCTG 445 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 445 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: cDNA (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAGCAGTGCC TATCCAGAAA GTCCAGGATG ACACCAAAAC CCTCATCAAG ACCATTGTCA 60 CCAGGATCAA TGACATTTCA CACACGCAGT CGGTATCCGC CAAGCAGAGG GTCACTGGCT 120 TGGACTTCAT TCCTGGGCTT CACCCCATTC TGAGTTTGTC CAAGATGGAC CAGACTCTGG 180 CAGTCTATCA ACAGGTCCTC ACCAGCCTGC CTTCCCAAAA TGTGCTGCAG ATAGCCAATG 240 ACCTGGAGAA TCTCCGAGAC CTCCTCCATC TGCTGGCCTT CTCCAAGAGC TGCTCCCTGC 300 CTCAGACCAG TGGCCTGCAG AAGCCAGAGA GCCTGGATGG CGTCCTGGAA GCCTCACTCT 360 ACTCCACAGA GGTGGTGGCT TTGAGCAGGC TGCAGGGCTC TCTGCAGGAC ATTCTTCAAC 420 AGTTGGATGT TAGCCCTGAA TGCTG 445 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 167 base pairs (B) TYPE: amino acid (C) CHAIN STRUCTURE: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp Pro Tyr Leu 1 5 10 15 Phe Tyr Val Gln Ala Val Pro He Gln Lys Val Gln Asp Asp Thr Lys 20 25 30 Thr Leu He Lys Thr He Val Thr Arg He " Asn Asp He Ser His Thr 35 40 45 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Wing 65 70 75 80 Val Tyr Gln Gln He Leu Thr Ser Met Pro Ser Arg Asn Val He Gln 85 90 95 He Be Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Wing 100 105 110 Phe Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu Thr Leu 115 120 125 • Asp Ser Leu Gly Gly Val Leu Glu Wing Ser Gly Tyr Ser Thr Glu Val 130 135 140 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 145 150 155 160 Leu Asp Leu Ser Pro Gly Cys 165 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: • 5 (A) LENGTH: 4167 base pairs (B) TYPE: amino acid (C) CHAIN STRUCTURE: (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: protein 10 (ix) SEQUENCE DESCRIPTION: SEQ ID NO: 8: • Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp Ser Tyr Leu 1 5 10 15 Ser Tyr Val Gln Ala Val Pro He Gln Lys Val Gln Asp Asp Thr Lys 20 25 30 Thr Leu He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr 35 40 45 Gln Ser Val Be Wing Lys Gln Arg Val "Thr Gly Leu Asp Phe He Pro 50 55 60 Gly Leu His Pro He Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Wing 65 70 75 80 Val Tyr Gln Gln Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln 85 90 95 He Wing Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Wing 100 105 110 Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro 115 120 125 Glu Ser Leu Asp Gly Val Leu Glu Wing Ser Leu Tyr Ser Thr Glu Val 130 135 140 Val Wing Leu Being Arg Leu Gln Gly Being Leu Gln Asp He Leu Gln Gln 145 150 155 160 Leu Asp Val Ser Pro Glu Cys 165

Claims (12)

1. - A DNA molecule of single or double chain structure consisting essentially of a nucleotide sequence encoding a bovine adipocyte polypeptide leptin.

2. The DNA molecule according to claim 1, characterized in that it is substantially free of other bovine DNA sequences.

3. The DNA molecule according to claim 1, characterized in that it has a nucleotide sequence consisting essentially of at least 20 nucleotides of the nucleotide sequence SEQ ID NO: 3, or a sequence complementary to at least about 20 nucleotides of the nucleotide sequence SEQ ID NO: 3, substantially free of other bovine DNA sequences.

4. The DNA molecule according to claim 3, characterized in that it consists essentially of the nucleotide sequence SEQ ID NO: 3 or a sequence complementary to the sequence SEQ ID NO: 3.

5. The DNA molecule according to claim 1, characterized in that it is an expression vector.

6. The DNA molecule according to claim 5, wherein said vector is a plasmid.

7. A host cell transformed or transfected with a DNA molecule according to claim 6.

8. - An RNA molecule substantially free of other RNA sequences, consisting essentially of a mRNA sequence encoding a bovine adipocyte polypeptide leptin.

9. - The RNA molecule according to claim 8, characterized in that it consists essentially of a mRNA sequence encoding a bovine adipocyte polypeptide of at least about 10 amino acids encoded by the nucleotide sequence SEQ ID NO: 3.

10. A DNA sequence encoding an antisense RNA, characterized in that it is complementary to the gene encoding a bovine adipocyte polypeptide, said antisense RNA capable of regulating the expression of said gene.

11. A bovine adipocyte polypeptide encoded through a nucleic acid molecule consisting essentially of the nucleotide sequence SEQ ID NO: 3, or a sequence complementary to the sequence SEQ ID NO: 3.

12. An antibody directed against the bovine adipocyte polypeptide of claim 11.