Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1996-07-31
2001-10-02
Saoud, Christine J. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S252300, C435S320100, C435S325000, C530S350000, C536S063000
Reexamination Certificate
active
06297027
ABSTRACT:
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 bovine. More specifically, it relates to a specific bovine 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 bovine. In addition, this invention relates to the nucleotide sequences encoding the bovine leptin polypeptide, the antibodies directed against the bovine leptin polypeptide, and methods to determine susceptibility to fat deposition, alter energy intake, and minimize excessive fat deposition in bovine.
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 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 U.S. Pat. No. 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 are no known reports of adipocyte-specific proteins expressed at different levels in fat cattle as compared with normal controls.
Leptin, the protein produced by the leptin (ob) gene, is possibly related to fat deposition in bovine because research has shown that mutations in genetically (ob/ob) 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), the bovine leptin cDNA or genomic sequence was not available prior to initiation of this project. Thus, the insights obtained with respect to bovine metabolism were not accessible to bovine systems. Furthermore, the biologically active purified bovine protein (i.e., 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 (i.e. appetite), in the bovine species.
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 depicted in FIGS.
1
and
3
-
5
(SEQ ID NOS:4, 7 and 8), or a functional derivative 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 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 single or double stranded DNA molecule having a nucleotide sequence consisting essentially of at least about 20 nucleotides of the nucleotide sequence depicted in
FIGS. 1 and 2
(SEQ ID NOS:3) or a sequence complementary to at least part of the nucleotide sequence depicted in
FIGS. 1 and 2
(SEQ ID NOS:4), or a functional equivalent thereof, substantially free of other bovine DNA sequences. The RNA molecule is preferably an mRNA sequence encoding 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 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 bovine adipocyte polypeptide or functional derivative as described above, the process 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 having the sequence of the DNA molecule described above, or a complementary sequence, in a nucleic acid-containing sample, the method comprising: (a) contacting the sample with an oligonucleotide probe complementary to the sequence of interest under hybridizing conditions; and (b) 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 (a): (c) 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 bovine adipocyte polypeptide, or functional derivative, either polyclonal or monoclonal. Also intended is a method for detecting the presence or measuring the quantity of the 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, or measuring the quantity of antibody bound.
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 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 (i.e., 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 vitr
Purina Mills, Inc.
Saoud Christine J.
Whyte Hirschboeck Dudek SC
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