EP1015634A1 - Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme - Google Patents

Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme

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Publication number
EP1015634A1
EP1015634A1 EP98944340A EP98944340A EP1015634A1 EP 1015634 A1 EP1015634 A1 EP 1015634A1 EP 98944340 A EP98944340 A EP 98944340A EP 98944340 A EP98944340 A EP 98944340A EP 1015634 A1 EP1015634 A1 EP 1015634A1
Authority
EP
European Patent Office
Prior art keywords
alleles
pigs
fabp
pig
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98944340A
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German (de)
English (en)
Inventor
Frans Gerbens
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pig Genes Bv Io
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Pig Genes Bv Io
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Filing date
Publication date
Application filed by Pig Genes Bv Io filed Critical Pig Genes Bv Io
Priority to EP98944340A priority Critical patent/EP1015634A1/fr
Publication of EP1015634A1 publication Critical patent/EP1015634A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the field of molecular biology as well as to the field of breeding methods for farm animals, in particular pigs.
  • the invention relates to the use of diagnostic methods derived from the field of molecular biology to be applied in breeding programmes that select animals on production traits that improve their breeding value .
  • breeding programmes By selecting animals on their breeding value calculated mainly from phenotypic measurements of production traits, breeding has greatly improved the genotype for production traits of livestock animals.
  • breeding programmes have selected for phenotypic characteristics of animals.
  • more recently selection for genotypic characteristics that are associated with improved production traits have gained interest in the field. Selection for phenotypic characteristics entails mainly selection of the respective animal, offspring or siblings or other relatives of the animals to be selected whereas selection of specific genotypic characteristics allows for earlier and specific detection of animals of interest.
  • One of the former methods is a marker assisted selection wherein polymorphisms in markers identified in a random manner are associated with production traits. For instance, meat production is closely linked to embryonic muscle formation, and, consecutively, to the distribution of muscle cells and fat cells. Biologically, production is concentrated in defined tissues of the animal, e.g. muscle tissue for lean meat production. In breeding programmes for optimising porcine lean meat production, various levels of selection pressure have been applied to different tissues (i.e. muscle, fat and bone) . However, when selecting for lean meat, and thus the absence of fat, one may lose certain traits that are wanted after all, i.e. traits that are associated with taste and thus with the consumers perception of the final product.
  • Fatty acid binding proteins are small intracellular proteins involved in fatty acid transport from the membrane to the sites of ⁇ oxidation and/or triacylglycerol or phospholipid synthesis (Veerkamp and Maatman, 1995) . Furthermore, FABP ' s modulate the intracellular fatty acid concentration (Veerkamp et al.,1993). Fatty acid metabolism has historically been linked to insuline resistance (Randle, 1963) , and therefore mutations in FABP genes may be associated with changes in cellular insulin resistance or dependency, fatty acid oxydation and fatty acid binding.
  • FABP ' s are members of a family of intracellular lipid binding proteins comprising at least eight structurally distinct types and named after their first tissue from which they were isolated or identified: adipocytes, brain, epidermal cells, heart, intestinal cells, ileal cells, liver and myelin cells.
  • the present invention provides among others an isolated or recombinant pig A-FABP gene specific nucleic acid molecule or pig A-FABP gene specific fragments thereof comprising or hybridising to the nucleotide sequence as shown in figure 1, or its complementary sequence or the RNA equivalents thereof.
  • the locus of this gene is on porcine chromosome 4.
  • the pig A-FABP gene can be assigned to functions in the regulation of intramuscular fat, thereby changing the ratio of fat deposited within the muscle versus fat deposited outside the muscles, i.e. in backfat depots. Since production and deposition of fat is energy consuming and takes away energy for other purposes, such as muscle growth, the regulation of intramuscular fat is correlated to the regulation of growth, and thus body weight and average daily gain and feed efficiency. Since FABP's are involved in fatty acid transport they can influence fatty acid oxidation rates, the metabolism of fatty acid derivatives in the tissue and the fatty acid composition of cells and thus of meat. Furthermore, FABP's may regulate cellular insulin dependency.
  • A-FABP regulates functional differentiation of mammary epithelial cells it is involved in regulating the quantity and composition of the milk available, thus influencing the growth and survival of newborn animals.
  • the present invention further provides a method to generate via recombinant DNA techniques an animal , such as small laboratory animals or farm animals, i.e. a pig, with additional genetic material originating from the pig A-FABP gene. Such animals may than encode wanted alleles of this gene and constitutively or transiently express allelic proteins or fragments thereof that enhance the production or physiological characteristics of those animals.
  • the invention further provides methods to generate proteins or (poly) peptides comprising various allelic proteins or fragments thereof derived from the pig A-FABP gene. Such peptides, or antibodies specifically directed against such peptides, may be used to influence production traits in the live animal, but may also be used in cell- culture systems in vi tro . Such (poly) peptides or proteins, or antibodies specifically directed against these, may also be used in diagnostic test systems to select animals that express wanted forms of allelic proteins or fragments thereof encoded by the pig A-FABP gene.
  • the invention further provides methods localising, identifying or marking genes or alleles or quantitative trait loci, in particular those corresponding to the pig A-FABP gene, in samples, in particular biological samples, cells or tissues, such as but not limited to hair, skin or blood, of farm animals, in particular pigs, by allowing for specific amplification of genomic fragments of those genes or alleles or quantitative trait loci of pigs. Since marker assisted selection of animals is frequently based upon genetic variation that exists within functional genes that influence a production trait directly, i.e.
  • genes such as the pig A- FABP that regulates fatty acid binding one of the methods that the invention provides is a method that identifies or marks loci or genes and that can distinguish between characteristics of alleles of those genes which characteristics serve as markers in selection programmes for animals with specific versions of those genes that are directly linked with improved production traits.
  • the invention further provides a method wherein polymorphic restriction sites within functional genes and thus different alleles of those genes are identified by allowing for specific amplification of genomic fragments of those genes, in particular by allowing for specific amplification of fragments of the A-FABP gene.
  • Amplification methods are well known in the art, the best known being PCR. A short description of the PCR used herein is given in the experimental part. Other primers, enzymes and conditions can of course be applied.
  • a suitable method of identifying wanted alleles and polymorphic sites related thereto is a restriction endonuclease treatment.
  • Suitable restriction enzymes for identifying pig A-FABP alleles are for example found by amplifying and subsequent digesting A- FABP allele fragments of various breeds of pigs. By these methods large numbers of pigs can be rapidly genotyped for studies in which genotypic variation can be associated with growth characteristics and other production or performance traits of pigs. Such production traits are for example body weight (BW) , back fat thickness (BFT) , intramuscular fat (IMF) and drip loss (DRIP) .
  • BW body weight
  • BFT back fat thickness
  • IMF intramuscular fat
  • DRIP drip loss
  • Yet another method for identifying wanted alleles is the detection of polymorphic sites such as microsatellites or CA-repeats found in the A- FABP gene.
  • Various alleles, such as the Al to A9 alleles can in this way be detected.
  • the methods according to the invention can be developed into diagnostic assays or kits by which selection of pigs with alleles of interest can be performed in routine screening protocols employed in breeding programmes. With such protocols better results of selection can be expected when genes responsible for regulation of commercially interesting body tissues can be rapidly identified and controlled.
  • such testing protocols can be used to identify, select and breed farm animals, such as pigs, which have better production traits, such as IMF% or backfat thickness or average daily weight gain or feed efficiency, than the average animal in the population.
  • Better production traits such as BW or daily weight gain will increase the production per year expressed as amount of meat per animal raised.
  • a population of animals with a higher and less variable IMF% will result in a more homogenous product (meat) which is also better appreciated by putative customers because of a better tenderness or taste.
  • selection for higher IMF% may be possible while at the same time selection against fat deposition in other depots, such as backfat, can be performed.
  • One objective in pig breeding programs is the reduction of fat in the carcass to meet the consumers ' demand for lean meat. This reduction is accomplished by selection for reduced back fat thickness. As a result of this selection the intramuscular fat depot, which is positively correlated with taste and meat acceptance (Wood et al . , 1988) may be reduced. However both fat depots are not or only moderately correlated (Hovenier et al . , 1992) indicating that IMF can be treated independently from BFT at least partially.
  • the respective gene was identified, sequenced and chromosomally localised. Moreover, genetic variation within this gene was identified. To establish the role of A-FABP in porcine IMF accretion this genetic variation was studied in a Duroc pig population.
  • a porcine genomic DNA EMBL3/SP6/T7 lambda library (Clontech Laboratories Inc. Palo Alto, CA) was screened by plaque hybridization (Sambrook et al . 1989) to mouse A-FABP (ALBP) cDNA (Bernlohr et al . , 1984) in the pGEM vector labeled with [ ⁇ - 32 P]dCTP by nick translation (Sambrook et al . 1989).
  • 500,000 plaques were transferred to replica nitrocellulose filters and incubated in denaturation buffer (1.5 M NaCl/0.5 M NaOH) for 2 min, neutralisation buffer (1.5 M NaCl/0.5 M Tris-HCl pH 8.0) for 5 min and fixation buffer (0.2 M Tris-HCl pH 7.5/2X SSC(0.3 M NaCl , 0.03 M sodium citrate)) for 30 s.
  • denaturation buffer 1.5 M NaCl/0.5 M NaOH
  • neutralisation buffer 1.5 M NaCl/0.5 M Tris-HCl pH 8.0
  • fixation buffer 0.2 M Tris-HCl pH 7.5/2X SSC(0.3 M NaCl , 0.03 M sodium citrate)
  • the filters were prehybridized (6 X SSC/0.5% (w/v) SDS/5 X Denhardt ' s and 100 mg/ml NaOH-treated salmon sperm DNA) for two h at 67°C and hybridized at 67°C overnight in the same buffer containing the radioactive probe.
  • the filters were washed four times with 2 X SSC, 0.1% (w/v) SDS for 30 min at room temperature.
  • a single plaque that showed positive signals on replicate filters was purified by two additional rounds of low density plaque hybridization. Phage DNA was isolated using the plate lysate method (Sambrook et al. 1989) .
  • DNA sequence analysis DNA from a positive phage clone was used to subclone the A- FABP gene. Therefore, the Ba ⁇ HI , HindiII and SacI restriction digestion fragments of the phage DNA were subcloned in pBS .
  • Recombinant plasmid DNA from A-FABP clones was purified with the Wizard Maxiprep kit (Promega, Madison, WI , U.S.A.) . The nucleotide sequence was determined by cycle sequencing
  • PCR amplifications were performed on 1 ⁇ l of a 1:1000 dilution of phage DNA preparations or 50 ng of genomic DNA in
  • primers (example of forward primer GGTACTTTCTGATCTAATGGTG and reverse: GGGAACTCTTGAAGTCTTTCTC) were designed to amplify the corresponding region. Annealing was performed at 56°C.
  • the forward primer was fluorescently labelled and the PCR product was analysed on a denaturing polyacrylamide gel on a ABI 373. The length of the PCR product was estimated according to standard marker fragments (Perkin Elmer) using the GENESCAN software package (Perkin
  • a pig/rodent somatic cell hybrid panel (Rettenberger et al . 1996) was used to assign the A-FABP gene to a specific chromosome by PCR.
  • DNA 100 ng
  • porcine chromosomes in various combinations was used in a PCR reaction which unambiguously amplified porcine A-FABP gene exon 3 through exon 4.
  • heritability estimates for each trait were taken from Hovenier et al . (1992) . Prior to statistical analysis, BFT was adjusted to a weight of 110 kg and BW was adjusted to a weight at 180 days of age, for each animal .
  • test station*test year*test month was replaced by slaughter date.
  • FIG 1 the complete DNA sequence of the porcine A-FABP gene is shown.
  • the A-FABP gene exhibits the four-exon/ three- intron stucture common to all known FABP genes.
  • the size of the introns is 2629 bp, 840 bp and 471 bp, respectively.
  • 2370 bp of the 5' upstream region and 1435 bp of the 3 ' downstream region are present .
  • the coding region of the porcine A-FABP gene (Fig 2) shows 90% and 83% similarity with the human and mouse A-FABP coding regions (Baxa et al.,1989; Bernlohr et al.,1984).
  • porcine A-FABP gene was assigned to chromosome 4. This assignment is consistent with the human and mouse A-FABP gene localisation on chromosome 8q21 (Prinsen et al . , 1997) and 3 (Heuckeroth et al.,1987), respectively. Heterologous chromosome painting demonstrated that the short arm and centromeric end of the long arm of porcine chromosome 4 have conserved synteny with human chromosome 8 (Rettenberger et al . , 1995).
  • the 3' poly A tract of SINEs could be a source of informative markers (Ellegren et al . , 1993) .
  • A-FABP gene two complete copies of porcine SINEs, including the poly A tract, were present in the 5' upstream region and in intron 2. The observed single adenine stretches display genetic polymorphism.
  • the porcine A-FABP gene microsatellite was tested as a potential marker for IMF content, BW, BFT and DRIP in a Duroc pig population previously used for a similar analysis with the porcine H-FABP gene.
  • This Duroc population three alleles were present Al , A2 and A3 resulting in 6 different genotype classes.
  • Table 2 shows the distribution of the genotype classes for each trait analysed. Because the frequency of the A3 allele is very low in this population, the genotype classes A2A3 and A3A3 can not be included in the analysis with respect to IMF and DRIP.
  • genotype classes with respect to the microsatellite in the porcine A-FABP gene, show a considerable and significant difference in IMF content. Moreover, this effect is independent from the genetic variation in the H-FABP gene which was previously demonstrated to affect IMF content . With respect to BW significant contrasts were detected between the A1A2 , A2A3 and A3A3 genotype classes and the A1A1 genotype class.
  • Table 1 The frequency of A-FABP microsatellite alleles in several pig breeds represented by unrelated animals.
  • Table 2 The number of animals for each A-FABP genotype class for each trait as used in the association analysis.
  • Table 3 The genotype class mean values and respective standard variation for each trait
  • Table 4 Contrasts between A-FABP genotype classes for IMF and IMF adjusted for BFT (IMF/BFT) .
  • A2A2 -A1A1 2.32 1.77 0.10 0.29 -0.75 1.55
  • Table 6 Contrasts between the A-FABP genotype classes for IMF when corrected for each H-FABP RFLP genotype classes.
  • Figure 1 The porcine A-FABP nucleic acid sequence.
  • Figure 2 The porcine A-FABP nucleic acid sequence encoding the A-FABP protein.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
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Abstract

La présente invention porte sur une nouvelle séquence du gène A-FABP du porc, ainsi que sur des procédés d'utilisation de ce gène et de ses produits. L'invention porte notamment sur des procédés de détection de différents allèles du gène A-FABP du porc qui sont associés à des différences de caractères génotypiques et/ou phénotypiques des porcs possédant ces allèles. L'invention porte encore sur des procédés permettant de faire une distinction entre des allèles issus de différents phénotypes, notamment à l'aide de techniques consistant à réaliser une amplification sélective de matériaux dérivés du gène A-FABP du porc. Ces techniques sont particulièrement adaptées pour sélectionner des animaux destinés à être utilisés dans des programmes d'amélioration génétique. L'invention porte également sur des programmes d'amélioration génétique utilisant ces techniques.
EP98944340A 1997-09-18 1998-09-18 Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme Withdrawn EP1015634A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP98944340A EP1015634A1 (fr) 1997-09-18 1998-09-18 Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP97202857 1997-09-18
EP97202857 1997-09-18
PCT/NL1998/000541 WO1999014365A1 (fr) 1997-09-18 1998-09-18 Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme
EP98944340A EP1015634A1 (fr) 1997-09-18 1998-09-18 Gene codant la proteine se liant a l'acide gras des adipocytes du porc, et procedes de localisation, d'identification ou marquage des genes ou alleles ou loci de caractere quantitatif des animaux de ferme

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EP1015634A1 true EP1015634A1 (fr) 2000-07-05

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EP (1) EP1015634A1 (fr)
AU (1) AU9189998A (fr)
CA (1) CA2304349A1 (fr)
WO (1) WO1999014365A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1111043A1 (fr) * 1999-12-21 2001-06-27 Institute for Pig Genetics B.V. Nouveaux QTL sur les chromosomes X, 2, 6 et 7 de cochons
WO2007109514A2 (fr) * 2006-03-16 2007-09-27 Wisconsin Alumni Research Foundation Détection du gène de la létalité pour une meilleure fertilité chez les mammaliens
CN101935706B (zh) * 2010-09-02 2012-05-30 中国农业科学院北京畜牧兽医研究所 一种检测猪肉品质性状的方法及专用引物对

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Publication number Priority date Publication date Assignee Title
WO1992006104A1 (fr) * 1990-09-28 1992-04-16 The Dana-Farber Cancer Institute Sequences d'adn specifiques aux adipocytes et leur utilisation dans la production d'animaux transgeniques presentant un metabolisme des graisses modifie
DE4338817A1 (de) * 1993-11-13 1995-06-14 Max Delbrueck Centrum Gensequenzen von Cellular X Binding Proteinen
AU725544B2 (en) * 1996-03-28 2000-10-12 Pig Genes B.V.I.O. The porcine heart fatty acid-binding protein encoding gene and methods to identify polymorphisms associated with body weight

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Title
See references of WO9914365A1 *

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AU9189998A (en) 1999-04-05
CA2304349A1 (fr) 1999-03-25
WO1999014365A1 (fr) 1999-03-25

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