JP3790102B2 - Melanocortin-4 receptor gene and use as a marker of lipid content, weight gain and / or food consumption in animals - Google Patents

Melanocortin-4 receptor gene and use as a marker of lipid content, weight gain and / or food consumption in animals Download PDF

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JP3790102B2
JP3790102B2 JP2000562559A JP2000562559A JP3790102B2 JP 3790102 B2 JP3790102 B2 JP 3790102B2 JP 2000562559 A JP2000562559 A JP 2000562559A JP 2000562559 A JP2000562559 A JP 2000562559A JP 3790102 B2 JP3790102 B2 JP 3790102B2
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mc4r
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lipid content
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JP2002521068A (en
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スク キム,クワン
ジェイ. ラーソン,ニールス
マックス エフ. ロスチルド
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アイオワ ステイト ユニバーシティ リサーチ ファウンデーション,インコーポレイティド
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    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/156Polymorphic or mutational markers
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    • C12Q2600/00Oligonucleotides characterized by their use
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Description

[0001]
Field of Invention
The present invention relates to a method of genetically assessing animals by assaying for the presence of at least one genetic marker that is one or more indicators of lipid content, growth rate and food consumption characteristics. . Specifically, this method analyzes diversity within the melanocortin-4 receptor (MC4R) gene, which is an indicator of the above characteristics. More specifically, the method analyzes polymorphisms within the MC4R gene.
[0002]
Background of the Invention
Consumer demand for low-fat meat products is increasing. This demand is further facilitated by the growing evidence in the scientific literature that high consumption of animal fats, especially fats with a high proportion of saturated fatty acids, represents a serious health crisis including risks related to cardiovascular disease. Other health issues related to high fat meat include its high cholesterol content and the salt helps to extract myosin, a native water-binding binding component from meat, to improve binding properties. The amount of salt produced may be relatively high. In addition, an increasing number of consumers do not accept meat products that contain chemical additives such as phosphoric acid, emulsifying additives, and antioxidants.
[0003]
With consumers looking for healthier meat products, meat producers are under pressure to continue to supply cheaper and healthier products.
Of course, cheaper products result from reduced production costs. Producers are always interested in improving their livestock growth and food intake. Lower production costs result from reduced time to shipment and reduced animal feeding costs. Thereby, an increase in profit margin in the livestock industry and / or a low price to consumers can be realized.
If an animal having the above characteristics can be selected, the producer can procure animals having these desired characteristics.
[0004]
There are genetic differences between breeds developed by breeding techniques to obtain individual meat producing animals and animals with these desired characteristics. For example, Chinese species are known to enter puberty at an early age and have a large number of litters, whereas American species are known to grow faster and have less fat. That is, it would be desirable to combine the best characteristics of both types of breeds, thereby improving pig production.
[0005]
However, the heritability of the desired trait is often low, for example, heritability for the number of litter is about 10% to 15%. Standard breeding methods that select individuals based on phenotypic diversity do not fully account for existing genetic variability or complex gene interactions. Thus, there is no need for a method that targets selection for low fatness, growth rate and food consumption at the cellular or DNA level. This method is intended to genetically evaluate animals so that the breeder can more accurately select animals that not only express the desired characteristics phenotypically but also express favorable basic genetic criteria. Provide a way. Traditionally, this work has been accomplished largely using marker-assisted sorting.
[0006]
Several groups have studied porcine DNA using restriction fragment length polymorphism (RFLP) analysis. Theung. Appl. Genet, 77: 271-274 (1989), referenced and incorporated herein by Jung et al., Discloses the use of the RFLP method to show genetic variability between two pig breeds. Polymorphism has been shown for porcine leukocyte antigen (SLA) class I genes of these varieties. Hoganson et al., Also referred to and incorporated herein, Abstract for Annual Meeting of Midwestern Section of the American Society of Animal Science, March 26-28, 1990, also used porcine tumor histocompatibility complex (MHC) for Chinese species using RFLP. ) Reported polymorphism of the gene.
[0007]
Jung et al., Referenced and incorporated hereAnimal Genetics26: 79-91 (1989) report on RFLP analysis of SLA class I genes in specific boars. The authors state that the results suggest a correlation between porcine SLA / MHC class I genes and productivity and performance characteristics. They also state that the use of SLA class I restriction fragments as genetic markers may improve future pig growth performance.
[0008]
The ability to track specific preferred alleles involves a novel and lengthy process of identifying DNA molecular markers for the main effective genes. The marker may be associated with a single gene with a major effect or with multiple genes with additional effects. DNA markers have multiple significance points; separation is easy and obvious to measure, and DNA markers are co-dominant, ie, heterozygous and homozygous animals can be clearly identified. Once the marker system is established, the selection decision can be made very easily because the DNA marker can be assayed at any time if tissue or blood samples can be collected from individual infants.
[0009]
Utilizing genetic differences within receptor genes is becoming a valuable marker system for selection. For example, U.S. Pat.Nos. 5,550,024 and 5,374,526 issued to Rothschild et al., Whose disclosures are referenced and incorporated herein, are among the porcine estrogen receptor genes associated with larger litter numbers. Polymorphs are disclosed. US Application Serial No. 08 / 812,208 discloses polymorphic markers in the porcine prolactin receptor gene that are associated with larger litter numbers and overall reproductive efficiency.
WO-A-07 / 47316 describes the presence of mutations in the MC4R protein in very obese human patients and that the predisposition to weight abnormalities can be confirmed by testing for mutations in the MC4R gene. However, Gotoda et al., Diabetologia 40 (1997) 976 describes that there is no correlation between a specific no mutation in the MC4R gene (Val 103 Ile) and obesity in white males. Thus, there is nothing in the prior art that shows a correlation between the MC4R gene and a means for selection of animals with improved metabolic traits.
From the above, it can be seen that there is a need for a method for screening animals with improved metabolic properties with respect to lipid content, growth rate and food consumption.
[0010]
Summary of invention
An object of the present invention is to provide genetic markers on or within the MC4R gene that are indicators of lipid content, growth rate and / or food consumption.
Another object of the invention is to provide a method for assessing animals that increases the accuracy of selection and breeding methods for the desired attributes.
Yet another object of the invention is to provide a PCR amplification test that will greatly facilitate the determination of the presence of a marker.
An additional object of the invention is to provide a certainty for evaluating animal DNA samples for specific genetic markers.
These and other objects, features and advantages will become apparent upon review of the following description of the invention and the claims.
[0011]
The present invention relates to the discovery of polymorphisms within the melanocortin-4 receptor (MC4R) gene that are associated with animal lipid content, growth rate and food consumption characteristics. According to the invention, a genetic marker relating to a specific breed (breed) or a genetic line (line) can be identified from the association between the MC4R polymorphism and the characteristic. The Taq1 restriction pattern that identifies the polymorphism is used to assay for the presence of markers associated with the desired metabolic characteristics.
[0012]
Variety-dependent marker genotypes (ie, markers in some varieties but not markers in other varieties) include polymorphisms in MC4R, a guanine to adenine change at position 678 of the PCR product (A missense mutation in which the aspartate codon (GAU) at the 298 amino acid site of the MC4R protein is changed to the asparagine codon (AAU)). The invention includes assays for detecting markers and sequences characteristic of polymorphisms, and includes novel sequences within the MC4R gene that are utilized in the design of amplification primers suitable for such assays. In addition, the invention includes methods relating to the use of the assay in a mating program suitable for animal selection, and kits for performing the assay.
[0013]
Definition
As used herein, “low lipid content” or “low fat” means a biologically significant reduction in body fat relative to the mean value of a particular population.
[0014]
Detailed Description of the Preferred Embodiment
Obesity is a disease related to energy balance. Control of energy metabolism is simple: excess energy is stored as lipids and energy is managed to avoid excess energy accumulation, ie obesity. Obesity involves multiple genes and signal transduction systems, but little is known about the relationship between energy homeostasis mechanisms and genetic polymorphisms. Melanocortin-4-receptor (MC4R) has been shown to be an important mediator of long-term body weight homeostasis. MC4R antagonists increase food intake and body weight when administered chronically. Skuladottir, G. V et al., "Long term orexigenic effect of a novel melanocortin 4 receptor selective antagonist", British J. of Pharm., 126 (1): 27-34 (1999).
[0015]
Lu et al. (1994) suggested that melanocortin receptors are involved in the control of food intake and energy balance through studies of antagonism against Agouti obesity syndrome. Huszar et al. (1997) found that inactivation of the melanocortin-4 receptor gene (MC4R) causes adult-onset obesity syndrome in mice, and the main role of MC4R protein in controlling energy balance is agouti obesity. It was shown to be related to the syndrome. In addition, the MC4R protein also transmits the action of leptin, an important signaling molecule for energy homeostasis (Seeley et al., 1997).
[0016]
According to the present invention, the displacement or polymorphism of the MC4R gene is localized and its genetic variability is associated with differential expression of metabolic characteristics of lipid content, growth rate and / or food consumption. .
In one embodiment of the invention, the assay is subjected to detection of the presence of the desired genotype. The assay involves amplifying genomic DNA purified from a suitable source of blood, tissue, semen, or other genetic material, using standard methods such as primers and polymerase chain reaction (PCR), followed by DNA amplification. Digestion with restriction enzymes (eg TaqI) to generate genetic fragments of various lengths, and separation of at least some fragments from others (eg, using electrophoresis).
[0017]
Genes whose fragments are hybridized to isolated fragments using nucleotide probes (eg, radiolabeled cDNA probes) containing all or at least a portion of the MC4R gene cDNA sequence, and the results of hybridization are known to have markers It is also detected by comparison with assay results for sequences known to have no sequence or marker. The fractionation and use of probes suitable for the detection of MC4R sequences based on known and disclosed MC4R sequences is generally known to those skilled in the art. The probe may be any sequence that hybridizes with the separated digestion product to allow detection.
[0018]
Another embodiment of the invention provides a kit for assaying the presence of the genetic marker MC4R gene sequence. Markers are indicative of the genetic nature of lipid content, growth rate, and / or food consumption. A kit of a preferred embodiment is a novel PCR primer comprising 4-30 contiguous bases at either end of the polymorph that provides an amplification system that allows detection of TaqI polymorphism by PCR and TaqI digestion of PCR products Including. Preferred primers are SEQ ID NO: 8 and SEQ ID NO: 9.
Another embodiment comprises a breeding method in which the above type of assay is performed on a plurality of gene sequences obtained from various animals or animal embryos selected based on the results, and a specific animal is selected or excluded from the mating program. To do.
[0019]
According to the present invention, a polymorphism of the MC4R gene identified by the TaqI restriction pattern is disclosed. As is known in the art, the restriction pattern is not an accurate determination of fragment size, but only an approximation. One homozygous genotype (allele 1) is due to three bands, 466, 225 and 76 base pairs (bp) resulting from TaqI digestion of the PCR product; another homozygous genotype (allele 2) is By the two bands 542 and 225 bp; and for the heterozygous genotype (542, 466, 225 and 76 bp), the polymorphism can be identified from the four bands. Markers for low lipid and low food intake can be identified by the 466/225/76 band except for Chinese pigs, whereas the low lipid marker for Chinese pigs is the 542/225 band. A marker for fast growth rate acquisition can be identified by the 542/225 band.
[0020]
Furthermore, the polymorphism associated with this pattern has also been identified at the nucleotide level. Polymorphic TaqI sites have been sequenced including a common peripheral region. See SEQ ID NO: 1. The sequence around the polymorphic site is a PCR test that uses a primer of about 4-30 contiguous bases derived from the sequence immediately adjacent to the polymorphic site, together with the polymerase chain reaction, to greatly amplify the region before treatment with TaqI restriction enzyme. Promote. Primers need not be exactly complementary; essentially equivalent sequences are acceptable.
[0021]
From the sequence data, it was observed that in allelic gene 2, guanine at position 678 of the PCR product was replaced with adenine, or aspartate codon (GAU) was changed to asparagine codon (AAU) at amino acid 298 of MC4R protein. It was. PCR tests for polymorphisms were performed using 5′-TGG CAA TAG CCA AGA ACA AG-3 ′ (SEQ ID NO: 6) forward primer and 5′-CAG GGG ATA GCA ACA GAT GA-3 ′ (SEQ ID NO: 7) reverse Directional primers were utilized. The pig-specific primers used were 5'-TTA AGT GGA GGA AGA AGG-3 '(SEQ ID NO: 8) forward primer and 5'-CAT TAT GAC AGT TAA GCG G-3' (SEQ ID NO: 9) reverse Directional primer. The resulting amplification product of approximately 750 bp yields 466,225 and 76 bp (allele I) or 542 and 225 bp (allele 2) allelic fragments when digested with TqaI.
[0022]
Markers can be any known to those skilled in the art that identify the presence or absence of markers including, for example, single-stranded conformational polymorphism analysis (SSCP), RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, and temperature gradient electrophoresis of the MC4R gene. These methods, ligase chain reaction, or direct sequencing of the MC4R gene will be identified by verification with respect to the TaqIRFLP recognition pattern.
One or more additional restriction enzymes and / or probes and / or primers can be utilized. Additional enzymes, constructed probes and primers can be determined by routine experimentation by those skilled in the art.
[0023]
Other techniques include non-gel systems such as TaqMan ™ (Perkin Elmer). In this system, oligonucleotide PCR primers are designed to touch the displacement side of interest and allow PCR amplification of that region. A third oligonucleotide probe is then designed to hybridize to the region containing the base that has undergone changes between other alleles of the gene. The probe is labeled with a fluorescent dye at both the 5 ′ and 3 ′ ends.
[0024]
These dyes are selected such that when they are in close proximity to each other, one fluorescence quenches the other and consequently cannot be detected. Extension from the PCR primer present at the 5 'position on the template relative to the probe with Taq DNA polymerase, through the 5' nuclease activity of the Taq DNA polymerase, cleaves the dye bound to the 5 'end of the annealed probe. This eliminates the quenching action and allows the fluorescence from the dye at the 3 ′ end of the probe to be detected.
[0025]
The difference between different DNA sequences is distinguished by the fact that dye cleavage does not occur if probe hybridization to the template molecule is incomplete, i.e., some form of mismatch exists. That is, the quenching action is removed only if the nucleotide sequence of the oligonucleotide probe is completely complementary to the template molecule to which it binds. The reaction mixture can contain two types of probe sequences that are designed for each different allele that exists, ie, the reaction allows detection of both alleles.
[0026]
Although the use of RFLP is one method for detecting polymorphisms, other methods known to those skilled in the art may be used. Such methods include methods that detect polymorphisms by analyzing polymorphic gene products and detecting differences that occur in the gene products.
Although the preferred method for separating restriction fragments is gel electrophoresis, other alternative methods known to those skilled in the art may be used for separation and sizing of restriction fragments.
It is also possible to jointly select polymorphisms using other DNA markers. Linkage between alleles of a DNA marker that is known to be related to a specific allele of another DNA and an MC4R gene that has already been shown to be associated with a particular attribute can be established. Examples of markers on the public PiGMaP chromosome map that are associated with the MC4R gene are S0331, BHT0433, and S0313.
[0027]
Suitable reagents for application of the method of the invention will be packaged in a conventional kit. The kit provides the necessary materials packaged in suitable containers. At a minimum, the kit contains reagents that identify polymorphisms in the MC4R gene that are associated with attributes of interest, lipid content, growth rate and food consumption. Preferably, the reagent that specifies the polymorphism is a PCR set (primer, DNA polymerase, and four types of nucleoside triphosphates) that hybridizes with the MC4R gene or a fragment thereof.
[0028]
Preferably, the kit includes a PCR set and a restriction enzyme that cleaves the MC4R gene in at least one place. Preferably, the kit further comprises additional means such as reagents for detecting or measuring the detectable component or providing a control. Other reagents utilized for hybridization, prehybridization, DNA extension, visualization, and similar purposes may also be included.
[0029]
The genetic markers, methods and kits of the invention are useful in mating programs to improve lipid content, growth rate and food consumption characteristics within a breed, strain, or animal population. Sequentially selecting and mating animals that are at least heterozygous and preferably homozygous for the desired polymorphism associated with a particular trait leads to a breed, strain, or population with these desired traits Will be able to. That is, the marker is a selection tool.
[0030]
The following examples are for illustrative purposes and do not limit the invention.
Example 1. Melanocortin 4 receptor PCR-RFLP Exam- MC4R Genetic TaqI Polymorphism and genetic linkage map
Primer:
Primers were designed from homologous regions of human and rat MC4R sequences (gene bank accession numbers s77415 and u67863, respectively). Using these primers, the 750 bp sequence of the porcine MC4R gene was amplified.
MC4R1: 5'TGG CAA TAG CCA AGA ACA AG3 '(SEQ ID NO: 6)
MC4R4: 5'CAG GGG ATA GCA ACA GAT GA3 '(SEQ ID NO: 7)
[0031]
[0032]
10 μL of Mixture 1 and DNA were combined into one in the reaction tube, and then mineral oil was overlaid. The following PCR program was run; 94 ° C. for 2 minutes; 94 ° C. for 30 seconds; 58 ° C. for 1 minute and 72 ° C. for 1 minute and 30 seconds for 35 cycles; followed by a final extension at 72 ° C. for 15 minutes.
5 μl of PCR product was examined on a standard 1% agarose gel to confirm successful amplification and clean negative controls. The product size is about 750 base pairs. Digestion was performed by the following method.
[0033]
TaqI Digestive reaction                            Ten μ L Reaction liquid
PCR product 5.0 μL
10 x TaqINE buffer 1.0 μL
BSA (10mg / ml) 0.1μL
TaqI enzyme (20U / μL) 0.5μL
dd sterile water 3.4μL
[0034]
A cocktail of buffer, enzyme, BSA and water was made. 5 μL was added to each reaction tube containing DNA. The mixture was then incubated at 65 ° C. for at least 4 hours to overnight. The loading dye was mixed with the digestion reaction solution and the whole amount was loaded on a 3% agarose gel. The major bands for allele I are approximately 466, 225, and 76 bp. Allele 2 genotype bands are 542 and 255 bp. The heterozygous genotype has both allele I and allele 2.
[0035]
result
The amplified PCR product is approximately 750 bp. The PCR product sequence confirmed that the MC4R gene had 97.6% and 92.2% identity with the corresponding human sequence at the amino acid level and DNA level, respectively (see FIGS. 2 and 3).
TaqI digestion of PCR products yielded 466, 225 and 76 bp (allele 1) or 542 and 255 bp (allele 2) allelic fragments. Heterozygous genotypes have both allelic types. Mendel's inheritance was observed for the three international generations of the three standard lines used for linkage analysis of this gene.
[0036]
The polymorphism between allele 1 and allele 2 that caused a change from G to A at position 678 in the PCR product is an aspartate codon (GAU) to asparagine codon (GAU) at amino acid 298 of the MC4R protein. A missense displacement to (AAU) was represented (see FIG. 1, SEQ ID NO: 1).
[0037]
Allele frequencies were determined by examining the genotypes of DNA samples obtained from a small number of animals of various breeds (Table 1). Allele 1 was observed with a frequency of 1 in Meishan, but was not observed in Hampshire and Yorkshire, or very infrequently. The frequency of allele 1 in Landrace and Chester White was 0.5 respectively.
Figures 2 and 3 illustrate the differences between the DNA and amino acid sequences of human and porcine MC4R genes (SEQ ID NOs: 2-5).
[0038]
[Table 1]
[0039]
Linkage analysis
Two-point and multipoint linkage analyzes were performed on the genotypes of international standard strains. See Figures 4a-4c. Data was analyzed using the CR1-MAP program. MC4R was significantly linked to multiple markers on porcine chromosome (SSC) 1. The markers most strongly linked by two-point linkage analysis (recombinant fractions and LOD scores in parentheses) were SO331 (0.02, 21.97), BHT0433 (0.02, 21.32) and SO313 (0.00, 17.76). As a result of multipoint linkage analysis, optimal map layout of markers and MC4R (indicated by Losambi cM distance): KGF-5, 8-CAPN3-2, 5-MEF2A-6.1, 1-MC4R-5, 6-SO313 were obtained. .
[0040]
MC4R was assigned to the cytogenetic region using porcine and rodent somatic cell hybrid panels. PCR products with pig specific primers were amplified in clones 7, 8, 16, 18, and 19. MC4R was located at SSC1q22-27.
[0041]
Example 2 Using pig-specific primers MC4R Receptor PCR-RFLP Test and pig MC4R Gene linkage map of genes
Pig specific primer sequence
Forward primer: 5'-TTA AGT GGA GGA AGA AGG-3 '(SEQ ID NO: 8)
Reverse primer: 5'-CAT TAT GAC AGT TAA GCG G-3 '(SEQ ID NO: 9)
[0042]
Detection method
PCR reaction in 10 μL final volume,
Pig genomic DNA 12.5ng
1x PCR buffer
MgCl2                            1.5mM
dNTP 0.124mM
Forward primer 0.3μM
Reverse primer 0.3μM
TaqDNA polymerase (5U / μL) 0.35U
Was carried out.
[0043]
PCR profile was 94 ° C for 2 minutes on a Robocycler (Statagene, La Jolla, CA); 94 ° C for 30 seconds; 56 ° C for 1 minute, 72 ° C for 1 minute and 30 seconds for 35 cycles; followed by 72 ° C for 15 minutes Included. 5 μl of PCR product was digested overnight at 65 ° C. in a total volume of 10 μl containing 10 U TaqI. The digested product was electrophoresed on a 3% agarose gel.
[0044]
Description of polymorphism
TaqI digestion of the PCR product yielded 466, 225 and 76 bp fragments for allele 1 and 542 and 225 bp fragments for allele 2. The heterozygous genotype had both allele 1 and 2 fragments.
[0045]
Genetic pattern
Mendelian inherited autosomal segregation was observed in three and three generations of European PiGMaP lines (Archibald et al., 1995).
Allele frequency
Allele frequencies were determined by determining the genotype of two generations of pre-animals for unrelated animals from the European PiGMaP line and the ISU standard line. The observed frequency of allele 1 is as follows.
[0046]
[Table 2]
[0047]
Chromosomal localization
For the three strains of PiGMaP genotypes, two-point and multipoint linkage analysis was performed using the CRI-MAP program (Green et al., 1990). MC4R was significantly linked to multiple markers on porcine chromosome 1 (SSC1). According to two-point linkage analysis, the most strongly linked markers (recombinant fraction and LOD score in parentheses) are SO331 (0.02, 21.97), BHT0433 (0.02, 21.32) and SO313 (0.00, 17.76). It was. The optimal map arrangement of MC4R for other linkage markers obtained by multipoint linkage analysis was as follows (indicated by Losambi cM distance): KGF-5, 8-CAPN3-2, 5-MEF2A-6.1, 1- MC4R-5, 6-SO313.
[0048]
comment
Melanocortin-4 receptor is a G protein-coupled seven-transmembrane receptor expressed in the brain. Huszar et al. (1997) found that inactivation of the MC4R gene induces adult-onset obesity syndrome in mice and showed a major role in controlling the energy balance of the MC4R protein. The MC4R gene has been mapped to human chromosome 18q21.3 (Gantz et al., 1993).
[0049]
The localization of the MC4R gene in SSC1 is consistent with previous chromosomal painting data (Goureau et al., 1996) that showed the same staining between this chromosome and HSA18 and 15. However, the gene arrangement of multiple genes that have been mapped to SSC1 based on HSA18 and 15 including CAPN3, KGF and MEF2A is not conserved in MC4R. Thus, mapping of MC4R to SSC1 may identify an evolutionary breakpoint between HSA 18 and 15 associated with SSC1.
[0050]
Example 3 Association between enhanced metabolic properties and markers
In preliminary experiments to determine which alleles are associated with traits and are present in breeds, the number of days that the genotype of multiple strains of animals reaches 110 kg body weight, back fat mass, test day gain, and average daily food Correlated with intake. The pig used for the test used the strain | stump | stock obtained from Pig Improvement Company (PIC) (PIC).
Data was collected using the PCR test described above for alleles 1 and 2 of the MC4R gene. The collected data is summarized in Table 3-8 below.
[0051]
The results showed that allele 1 was a significant low lipid allele in all strains except for Chinese pigs, which are lipid-rich alleles (see P2 back lipid value). Allele 2 was associated with the acquisition of a markedly fast growth rate (test for daily gain) in the commercial strains tested. Overall allele 1 is associated with lower food intake
[0052]
[Table 3]
[0053]
[Table 4]
[0054]
[Table 5]
[0055]
[Table 6]
[0056]
[Table 7]
[0057]
[Table 8]
[0058]
Example 4 Porcine melanocortin-4 receptor ( MC4R ) Gene missense displacement is related to fat percentage, growth, and food intake characteristics
To examine whether there is an association between MC4R polymorphism and phenotypic displacement, a number of animal solids from multiple pig strains were tested for mutants. Analysis of growth and performance test records indicated that dorsal fat mass, growth rate and food intake were significantly associated with MC4R genotype in multiple strains. Displacement amino acid residues of MC4R mutants are thought to cause major changes in MC4R function. These results support the functional significance of the porcine MC4R missense mutation, while suggesting that genome comparison based on model species is as important for livestock applications as it is for human drugs.
[0059]
The identification of mutations in leptin and the leptin receptor provides information on genetic elements involved in the control of energy balance (Zhang et al., 1994; Tartaglia et al., 1995). Genetic studies using animal models have facilitated identification of the major genetic causes of obesity (Andersson 1996; Pomp 1997; Giridharan 1998). In addition, several other genes involved in energy homeostatic nerve signaling pathways have been identified (Filer and Maratons-Filer 1998; Schwartz et al., 1999). Of particular interest among the candidate signaling molecules involved in the regulation of energy homeostasis is the melanocortin-4 receptor (MC4R). MC4R, which responds to leptin signals, is a communication between food intake and body weight (Seeley et al., 1997; Marsh et al., 1999).
[0060]
Neuropeptide Y (NPY) signaling in the central nervous system is also transmitted by the MC4R protein (Kask et al., 1998). Multiple displacements in MC4R, including frameshift and nonsense displacement, are associated with dominant inherited obesity in humans (Vaisse et al., 1998; Yeo et al., 1998). Other MC4R missense displacements in humans have also been identified (Gotoda et al., 1997; Hinney et al., 1999), but the functional significance of these displacements has not been specified.
[0061]
Selection based on growth characteristics is extremely important for the pig farming industry due to the costs associated with feeding and consumer preference for low-fat meat. Efficient genetic improvement of these quantitative attributes will be augmented through the use of marker-assisted sorting (MAS) using high-density genetic maps (Dekkers and van Arendonk 1998; Rothschild and Plastow 1999). A well-developed comparative mapping using human and mouse genetic maps is an important tool in this process, which identifies the relevant gene regions or major genes that control growth and performance attributes in pigs. Support.
[0062]
Biological understanding of complex attributes in humans or model species provides another way to identify genes responsible for economically beneficial attributes in livestock. With respect to obesity and growth traits, several quantitative trait loci (QTL) linkages have been successfully implemented using analysis of phenotypically displaced varieties and candidate genes (Yu et al., 1995; Casas-Carrillo et al. 1997; Knott et al., 1997; Knott et al., 1998; Rohrer et al., 1998; Wang et al., 1998; Paszek et al., 1999), but individual genes that have a major impact on growth and performance are still established for commercial species Absent. The role of MC4R in food intake and obesity suggests that it is an important genetic marker for growth-related attributes in pigs.
[0063]
Materials and methods
animal
Pigs were bred under normal production conditions at key farms in the US and Europe under the care of PIC employees. The pigs were subjected to performance tests at about 70 days of age and were removed from the study after 13 weeks. At the end of the test, the amount of back lipid was measured in real time (B mode) for the 10th rib located 2 cm from the center line using ultrasound. The average daily weight (growth) gain was calculated by dividing the resulting weight gain by the number of days tested. The number of days until the market weight of 110 kg was calculated using the usual method, and food intake was measured using a separate electronic measuring device.
[0064]
pig MC4R Gene fragment PCR amplification
Primers were designed from the homologous regions of human and rat MC4R (gene bank accession numbers s77415 and u67863, respectively). Primers are: forward primer: 5′-TGG CAA TAG CCA AGA ACA AG-3 ′ (SEQ ID NO: 6) and reverse primer: 5′-CAG GGG ATA GCA ACA GAT GA-3 ′ (SEQ ID NO: 7). PCR reaction is 12.5 mg porcine genomic DNA, 1 × PCR buffer, 1.5 mM MgCl2, 0.125 mM dNTPs, 0.2 mM of each primer, and 0.35 U Taq DNA polymerase (Promega) was performed in a final volume of 10 mL. PCR conditions are as follows: 35 cycles of 94 ° C for 2 minutes; 94 ° C for 30 seconds; 56 ° C for 1 minute, 92 ° C for 1 minute and 30 seconds on a Robocycler (Stratagene, La Jolla, CA) Followed by 72 ° C for 15 minutes.
[0065]
Sequencing and mutation detection
PCR products obtained from multiple porcine solids of various varieties were sequenced and nucleotide changes were detected by comparing the sequences. Sequencing was performed with ABI Sequencer 377 (Applied Biosystems). The porcine MC4R sequence was submitted to the gene bank and received accession number AF087937. Sequence analysis showed a single nucleotide substitution present within the TaqI restriction enzyme recognition site (Kim et al., 1999). Next, in order to identify polymorphic sites and facilitate the PCR-RFLP test, a primer set was designed to obtain an MC4R gene fragment containing only one informative TaqI restriction enzyme site. These primers were: forward 5′-TAC CCT GAC CAT CTT GAT TG-3 ′ (SEQ ID NO: 10) and reverse 5′-ATA GCA ACA GAT GAT CTC TTT G-3 ′ (SEQ ID NO: 11).
[0066]
Statistical analysis
Analysis of the variance procedure was utilized with a mixed model for farm, study duration, animal gender, MC4R genotype, and site (random) fixation effects. All animals of the US / European line (strains AD) were pooled as a global analysis and added into this analytical line of origin. Average effects were calculated for each genotype and are shown in Tables 9-15. The significance level was determined using the F test throughout.
[0067]
result
pig MC4R Identification of missense displacement of genes
The MC4R gene consists of an approximately 1 kb coding sequence contained within a single exon. An approximately 750p porcine MC4R gene fragment was generated by PCR (Kim et al., 1999). The PCR product sequence confirmed that the PCR product was an MC4R gene with 92.2% and 97.6% identity to the human MCR4R sequence at the nucleotide and amino acid levels, respectively. As a result of aligning multiple sequences obtained from multiple strains of animals, a single nucleotide substitution was identified (G to A; FIG. 5).
[0068]
The polymorphism represented an ISU sense displacement in which aspartic acid (GAU) at the same position as amino acid 298 of human MC4R protein was replaced by asparagine (AAU). In order to confirm the change in this salt k, we designed a pig-specific primer close to the polymorphic site and analyzed the polymorphism as a TaqIPCR-RFLP gel (FIG. 6). FIG. 6 shows TaqI digestion of PCR products analyzed by agarose gel electrophoresis. Allele 1 produced 156 and 170 bp fragments and allele 2 produced 226 bp fragments as PCR-RFLP. The heterozygote had both allele 1 and 2 fragments. The molecular weight marker (M) and MC4R genotype were shown at the top of each lane.
[0069]
MC4R Missense mutations are caused by melanocortin receptor ( MCR ) In a highly conserved area between.
MCR is a subfamily of GPCRs that contain certain conserved structural elements common to many other G protein coupled receptors (GPCRs), but the overall amino acid identity between MCRs and other GPCRs is low ( Tatro 1996). When the deduced amino acid sequence of porcine MC4R was juxtaposed with MC4R protein derived from other species, other MCR proteins, or representative GPCRs, aspartic acid at the 298th position of the seventh transmembrane domain was extremely highly conserved in MCR protein. (FIG. 7).
[0070]
But in other GPCRs, this position was occupied by asparagine. MCR proteins show 40-80% amino acid identity to each other (Tatro 1996), but the second intracellular domain loop and the seventh transmembrane domain are highly conserved among MCR proteins (Gantz et al., 1993). Some associations between MCR structure and function have been discovered by studies of neutral and experimental displacement in humans and mice (Robbins et al., 1993; Valverde et al., 1995; Frandberg et al., 1998).
[0071]
These studies have shown that several displacements within highly conserved regions result in structural changes and change receptor function. Asp298Asn displacement displacement will affect the function of the receptor. However, although this requires further validation, it is known that changes in homologous residues in MC1R (Asp294His) are associated with white skin and red hair in humans (Valverde et al., 1995). )
[0072]
MC4R Missense displacement is associated with obesity-related attributes.
To study the effects of this missense displacement, the effects on variability in growth rate, back fat, and food consumption of 1,800 animals from multiple commercial pig strains obtained from PIC, the international pig breeding company The correlation between action and MC4R genotype was analyzed. Animals were obtained from private commercial lines of European / US varieties (lines AD) and from lines generated by crossing between European and Chinese varieties (line E).
[0073]
In line AD, a significant correlation with the MC4R genotype was observed for all performance attributes. Homozygous animals for allele 1 averaged back fat (p <.001), daily gain (P <.001) and food intake (P <0.000) compared to homozygous 22 genotype animals. .01) was significantly lower (Tables 11, 13, and 15). Overall, 11 genotype pigs had about 9% less back fat than 22 genotype pigs (Table 11), while 22 genotype pigs grew significantly faster than 11 genotype pigs. (37g / day) (Table 13).
[0074]
These results may represent an appetite relationship for 22 genotype animals to eat more (Table 15). The association between the missense displacement of the MC4R gene and the associated performance attributes is clearly established in European / US varieties. Although fewer animals were tested, no such results were observed in the more obese Chinese breeding line (strain E). Interestingly, for back fat, line E showed a trend opposite to that observed for other lines (Table 11).
[0075]
Consideration
This study clearly showed that porcine MC4R missense mutations were significantly associated with multiple performance attributes in pigs. Allele 1 representing Asp298, an amino acid well conserved in other MCR subtypes and other species MC4R, is associated with low back fat, slow growth rate and low food intake, and allele 2 which is Asn298 is Related to obesity, high food intake and fast animal growth. Because highly conserved residues in the melanocortin receptor protein play important roles in ligand binding or intracellular signaling (Tatro 1996), MC4R displacement is functionally distinct in the control of food intake and body weight. Will demonstrate their abilities. Further validation of this hypothesis will provide important trials on the structural basis of MCR function and molecular targets for the treatment of human obesity.
[0076]
Allele 1 in line E was associated with the most obese animal derived from a cross between a large white variety from China and a line from Meishan. This is surprising that the displacement results in significant amino acid changes within the well conserved region. The result will depend on sampling. However, if more results are added, some explanations are possible if the results are equally significant. One possibility may be due to differences in background gene effects (epistasis). Since growth and obesity are complex polygenic attributes, hundreds of years of selection have allowed Chinese varieties to have different allelic interactions, and these predicted interactions can be crossed to a wide range of strains. There could be a diversity of polygenic traits (Frankel and Schork 1996).
[0077]
A QTL study on obesity and growth characteristics using branched lines was performed (Cases-Carrill et al., 1997; Knott et al., 1998; Rohrer et al., 1998l Wang et al., 1998; Paszek et al., 1999) on chromosome 1. No QTL has been reported for the vicinity of the C4R locus, which is mapped approximately 80 cm on the linkage map (data not shown). The epithelial effect of the MC4R allele proposed for line E makes it difficult to observe the MC4R locus in most QTL experiments, including crosses between Chinese and European / US lines. The effects of some alleles may vary with background and may be difficult to detect in QTL events involving genetically divergent varieties.
[0078]
The effect of the MC4R mutation is probably explained by future studies on the biological effects caused by this mutation in other pig breeds and strains. However, if MC4R mutations are strongly correlated with low lipidity, growth and food intake, this displacement can be used to help select producers who develop pig lines that meet specific consumer requirements. Could be done (Meuwissen and Goddard 1996). For example, in some strains where appetite generally decreases after delivery, food intake may be improved by selecting for the biallelic MC4R.
[0079]
In addition, selection for allele 1 was available for some lines considered to be excessive obesity, and selection for allele 2 was also available for lines considered to be overly slow. Thus, genotyping for MC4R displacement in porcine mating lines will improve the efficiency of selection of productivity attributes associated with food, including growth and low fat. Candidate gene methods have also been used to study the role of the porcine leptin gene (Jiang and Gibson 1999). However, in the case of leptin, there is evidence for an association between leptin polymorphism and back fat thickness in crosses between commercial varieties and unmodified lines, but there is a clear association for another commercial line tested. There was no sex (Jiang and Gibson 1999).
[0080]
Therefore, you should not think that you have found a gene that can be presumed to have a correlation. In contrast, with regard to MC4R, we have determined that displacement within this candidate gene can account for significant changes in back fat, growth rate and food intake in commercial pig strains. These MC4R results illustrate the potential value of comparative genetic analysis using candidate genes in livestock genetics.
Effects of MC4R genotypes on multiple reproductive characteristics in pigs
[0081]
[Table 9]
[0082]
[Table 10]
[0083]
[Table 11]
[0084]
[Table 12]
[0085]
[Table 13]
[0086]
[Table 14]
[0087]
[Table 15]
[0088]
While the invention has been described with reference to specific compositions, theory of effectiveness, etc., it is not meant that the invention be limited by these illustrated embodiments or mechanisms, and is defined by the appended claims. It will be apparent to those skilled in the art that modifications can be made without departing from the scope or spirit of the invention as described. The claims are meant to encompass any claim components and steps in any order effective to meet their intended purpose, unless the context indicates a specific contradiction.
[0089]
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[Brief description of the drawings]
FIG. 1 is the sequence (SEQ ID NO: 1) listed for porcine MC4R. “X” indicates the position of the polymorph.
FIG. 2 represents a DNA sequence comparison between MC4R genes of human (SEQ ID NO: 2) and pig (SEQ ID NO: 3).
FIG. 3 represents a comparison of amino acid sequences between human (SEQ ID NO: 4) and porcine (SEQ ID NO: 5) MC4R genes.
FIG. 4a represents a linkage report for MC4R by CR1-MAP.
FIG. 4b represents a linkage report for MC4R by CR1-MAP.
FIG. 4c represents a linkage report for MC4R by CR1-MAP.
FIG. 5 depicts a partial nucleotide and amino acid sequence (SEQ ID NO: 12) of the porcine MC4R gene.
FIG. 6 is an electrophoresis gel of TaqI digests of PCR products. The molecular weight marker (M) and MC4R genotype are shown at the top of each lane.
FIG. 7 shows multiple parallel arrangements of a putative seventh transmembrane domain of porcine MC4R with other MCRs and GPCRs. "*" Indicates the predicted sequence position of porcine MC4R. Other amino acid sequences were obtained from the gene bank database (registration numbers P32245, P70596, P41983, P56451, P34974, P41968, P33033, Q01718, Q01726, Q28031, AF011466, P21554, P18089, P30680, P47211). In the missense displacement body of porcine MC4R, amino acid N at the position indicated by the arrow was replaced with D. Asp (D) residues are well conserved among MCRs, and Asn (N) residues are well conserved in most other GPCRs.

Claims (20)

  1. In a method for identifying pigs having genotypes that exhibit metabolic traits of lipid content, growth rate and food consumption,
    a) obtaining a nucleic acid sample from a pig, and b) identifying a polymorphism at nucleotide position 678 in the MC4R gene of said sample, wherein said polymorphism comprises lipid content, growth rate and food consumption Associated with one or more metabolic traits selected from:
    A method comprising that.
  2. The method of claim 1 , wherein the polymorphism at nucleotide position 678 is associated with lipid content.
  3. The method of claim 1 , wherein the guanine at nucleotide position 678 is associated with low food intake.
  4.   The method of claim 1, wherein the adenine at nucleotide position 678 is associated with a faster rate of weight gain.
  5.   The method according to claim 1, wherein the step of identifying the polymorphism is a method using an allele-specific oligonucleotide.
  6.   The step of identifying the polymorphism includes restriction fragment length polymorphism (RFLP) analysis, heteroduplex analysis, single-strand conformation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis ( 2. The method of claim 1 selected from the group consisting of TGGE), and the use of linked genetic markers.
  7.   The method of claim 6, wherein identifying the polymorphism comprises RFLP analysis.
  8.   The method of claim 1, further comprising amplifying the MC4R gene sequence.
  9.   9. The method of claim 8, further comprising digesting the amplified region with the restriction endonuclease TaqI.
  10.   The method according to claim 8 or 9, wherein the primer used for amplification is selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.
  11. A single-chain oligonucleotide primers useful for detecting PCR product nucleotide position 678 of the nucleotide porcine MC4R gene, the oligonucleotide primers SEQ ID NO: nucleotide sequence selected from the group consisting of 6 to 10 of 30 or fewer nucleotides that comprise become and continuously along the sequence of the porcine NC4R gene, oligonucleotide primers are those which amplify the region containing the nucleotide position 678 of porcine MC4R gene.
  12. In a method for identifying a pig having a genotype representing any one of lipid content, growth rate and metabolic traits of food intake,
    a) obtaining a sample of genomic DNA from a pig;
    b) digesting the sample with TaqI to obtain a fragment;
    c) separating the resulting fragment from the digest; and d) identifying the presence or absence of a TaqI site at nucleotide position 678 of the PCR product of the porcine MC4R gene;
    A method comprising that.
  13. The method of claim 12 stages further comprising a for selecting a pig for having the genotypes for breeding.
  14.   When using TaqI as a restriction enzyme that cleaves the same recognition site, it can be identified by 466, 225 and 76 bp fragments when guanine is present at base 678, and 542 and 225 bp fragments when adenine is present The method according to claim 12, which can be identified by
  15. 15. The method of claim 14 , wherein the identifying step comprises detecting a TaqI site by amplification.
  16. Consists more than 30 contiguous nucleotides of the sequence selected from the group consisting of SEQ ID NO: 6-10 along and porcine MC4R gene consists Nde including, for amplifying a region of pig MCR4 gene comprising a nucleotide nucleotide position 678 Primers for assaying for the presence of polymorphic TaqI sites within the MC4R gene.
  17. In a method of screening pigs for any traits of lower lipid content, faster growth rate and lower food consumption,
    a) obtaining a nucleic acid sample from a pig;
    b) identifying the polymorphism characterized by nucleotide position 678 in the PCR product of the porcine MC4R gene; and c) selecting pigs;
    The method of claim 1, wherein the polymorphism is associated with one or more selected from the group consisting of low lipid content, high growth rate and low food consumption.
  18. In a method for identifying pigs with any genotype of lipid content, growth rate and metabolic traits of food consumption: a) obtaining a pig sample from the strain or breed of interest at nucleotide position 678 in the MC4R gene Determining the association of the MC4R genotype with one or more metabolic traits selected from the group consisting of lipid content, growth rate and food consumption;
    b) preparing genomic DNA in samples from each pig;
    c) determining the genotype of the MC4R gene at nucleotide position 678 in the MC4R gene; and d) calculating the association between the genotype of the MC4R gene at nucleotide position 678 in the MC4R gene and the trait;
    A method comprising that.
  19. In a method for screening pigs having an MC4R genotype of any of the metabolic traits of lipid content, growth rate and food consumption,
    a) obtaining a nucleic acid sample from a pig;
    b) identifying the genotype of the MC4R gene at nucleotide position 678 of the pig; and c) a pig having a genotype associated with one or more metabolic traits selected from the group consisting of lipid content, growth rate and food consumption Sorting out;
    A method comprising that.
  20. In a method for identifying a pig having any genotype of lipid content, growth rate and metabolic traits of food consumption,
    a) obtaining a nucleic acid sample from a pig; and b) an Asp (GAU) madone at amino acid position 298 of the MC4R protein is assayed for the presence of a polymorph with a change to the Asn (AAU) codon, wherein the polymorphism Is associated with one or more metabolic traits selected from the group consisting of lipid content, growth rate and food consumption;
    A method comprising that.
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