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Continued Characterization of the Callipyge Mutation in Sheep

Continued Characterization of the Callipyge Mutation in Sheep. Noelle E. Cockett Utah State University Logan, Utah USA. Carpenter et al. (1995). MULGE5. DLK1. GTL2. OY3. (Berghmans et al. 2000). Positional Cloning of the Callipyge Mutation. OAR 18. CLPG. (Cockett et al. 1994).

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Continued Characterization of the Callipyge Mutation in Sheep

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  1. Continued Characterization of the Callipyge Mutation in Sheep Noelle E. Cockett Utah State University Logan, Utah USA

  2. Carpenter et al. (1995)

  3. MULGE5 DLK1 GTL2 OY3 (Berghmans et al. 2000) Positional Cloning of the Callipyge Mutation OAR18 CLPG (Cockett et al. 1994)

  4. NPNM CPNM NPCM CPCM  Polar overdominance (Cockett et al. 1996) Inheritance of the Callipyge Trait

  5. Freking et al. (1998) F values* for linkage on Chromosome 18 under various genetic models Loin Rump Leg Lean Additive .5 2.1 1.4 .2 Maternal dominance 2.0 1.6 .9 2.7 Polar overdominance 164.4 110.6 371.8 145.3 *Maximum F threshold value (3 df) = 8.05

  6. Genomic Imprinting: • Allele-specific reversible epigenetic • modification dependent on parental • origin of the allele • Input by maternal and paternal • alleles is different

  7. Mechanism of Genomic Imprinting: • Methylation of cytosine in CpG • islands • Imprinting signals erased • during migration of primordial germ • cells into the genital ridge • Imprinting signals reestablished • in late gametes

  8. Imprinted Genes (Mouse): • 34+ genes on 10 chromosomes • Found in clusters • Code for proteins (Igf2, Igf2r) • Code for untranslated RNA (H19) • Species differ in imprinting

  9. Other Examples of Polar Overdominance Hybrid dysgenesis (Drosophila) Polar lethality • (Mouse)

  10. Male parent CC CNNC NN CC All norm All norm All norm All norm CN All norm 50%:50% 25%:75% 25%:75% Female parent NC All norm 50%:50% 25%:75% 25%:75% NN All callip All norm 50%:50% 50%:50%

  11. 211A17-B 293G8-T 288D6-T/S 288D6-T/S 359E3-T 277K17-S 918E6-S 271D12-S 265F11-T 602C10-S 229G11-T 355B7-S 359E3-S 363D1-T 497C1-T 218E10-T 65H10-T 120M3-D 145B8-T 918E6-T 557E1-T 355B7-T 271D12-T 48J17-S 573F8-S 239G7-T 386H8-T 293G8-S 56A24-D 6G20-A 120M3-T 201P6-S 497C1-S 84J15-S 6G20-T 56A24-A 19H2-S 9G7-T DLK1 WARS 84J15-T Gtl2 97 30 363D1 88 18 355B7 73 48 724D11 557E1 20 30 80 130 275H4 15 20 63 120 602C10 573F8 65 140 15 73 145B8 229G11 63 48 343D2 60 110 90 239G7 265F11 30 73 126 271D12 359E3 30 97 90 15 293G8 19H2 112 82 23 278C11 386H8 110 20 55 24 218E10 918E6 15 73 20 497C1 10 8 OY3 6G20C 239G7A 497C1A BM1561 497C1B 218E10A IDVGA30 724D11A BULGE33 265F11A Contig (900 Kb) Fine mapping (450 Kb) Sequence (250 Kb)

  12. % similarity: 94% < 86% < < 94% 78% < < 86% 70% < < 78% 60% < < 70% 60% < < 60% MEG8 PEG11 GTL2 Ovine sequence DAT DLK1 Human sequence

  13. Marker Brain Heart Adipose Kidney Liver Lung L. node Muscle Pancreas Intestine Spleen Thymus Marker MEG8

  14. DLK1 DAT P + M P + M Genomic DNA Muscle cDNA P P GTL2 MEG8 Genomic DNA P + M P + M Muscle cDNA M M

  15. 8-Week Muscles LD GM SP +/+ +/CmCp/+C/C +/+ +/CmCp/+ C/C +/+ +/CmCp/+C/C PEG11 AntiPEG11 DLK1 MEG8 GTL2 18S RNA

  16. Maternally Expressed Genes in the CLPG Region Paternal Expression Maternal C C N N C N C N +++ + +++ +

  17. Paternally Expressed Genes in the CLPG Region Paternal Expression Maternal C C N N C N C N +++ +++ + +

  18.  The CLPG mutation enhances gene expression: “C” maternal DLK1 GTL2 “C” paternal DLK1 GTL2 (Charlier et al. 2001) Gene Expression in the CLPG Domain  Genes in the CLPG region exhibit imprinting: “N” maternal DLK1 GTL2 “N” paternal DLK1 GTL2

  19. DLK1 immunohistochemistry CLPG/+ DLK1 is only detected in skeletal muscleof callipyge(CLPG/+) individuals

  20. In vivo overexpression: DLK1ORF ATG BGH polyA MLC-3F Promoter 3’ enhancer pBS Amp R 2 3 DLK1/+

  21. DLK1 GTL2 A to G transition = SNPCLPG Identification of SNPCLPG (Freking et al. 2002)

  22. Cross-Species Comparative Sequencing SNPCLPG CONSENSUS CC..GG.C..GG.CCACCTGTC....CC.T.C SHEEP (N) --GA--G-TG--A---------AGAT--T-T- SHEEP (C) --GA--G-TG--G---------AGAT--T-T- AOUDAD --GA--G-TG--A---------AGAT--T-T- BHARAL --GA--G-TG--A---------AGAT--T-T- CATTLE --GA--G-TG--A---------AGAT--T-T- HORSE --CA--G-TG--A---------AGAT--C- - PIG --AA--G-CG--A---------AGAT--T- - MOUSE --AG--G-CA--A---------C AC--T- - RAT --AG--G-CA--A---------C AC--T- - VOLE --AG--G-CA--A---------C AC--T- - RABBIT --AG--G-CA--A---------C AC--T- - SQUIRREL --AG--T-TG--A---------GGAT--T- - DOG --AG--G-CG--A---------AGAG--T- - HUMAN --AG--G-TG--A---------AGGT--T- -

  23. Solid Gold – Founder of Callipyge

  24. Analysis of SNPCLPG in “Solid Gold” Solid Gold NN CC CN “C” allele N > C in “Solid Gold” “N” allele (Smit et al. 2003)

  25. “Solid Gold” is Mosaic at SNPCLPG mutation event “Solid Gold” germline somatic tissues

  26. DNA “probe”: Nuclear extract: “+” allele: mutant: Identification of DNA-binding Proteins Technique:Electrophoretic Mobility Shift Assay (EMSA)

  27. EMSA Results N + NE C + NE N C protein + DNA complex SNPCLPG mutation reduces protein binding in vitro free probe

  28. NE Binding Across the 12-bp motif SNPCLPG G G A C C A C C T G T C Normal ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Mutated++ ++ + ++ -- -- -- + -- -- + +

  29. DLK1 DLK1 GTL2 GTL2 pat mat N pat mat C Current Model for the Function of SNPCLPG

  30. Acknowledgements University of Liege, Belgium Michel Georges, Carole Charlier, Karin Sergers, Erica Davis Purdue University Chris Bidwell USDA/ARS Gary Snowder Utah State University Tracy Shay, Maria Smit

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