Abstract
Scrapie is a prion disease affecting sheep and goats. Susceptibility to this neurodegenerative disease shows polygenic variance. The involvement of the laminin receptor (LRP/LR) in the metabolism and propagation of prions has previously been demonstrated. In the present work, the ovine laminin receptor gene (RPSA) was isolated, characterized, and mapped to ovine chromosome OAR19q13. Real-time RT-PCR revealed a significant decrease in RPSA mRNA in cerebellum after scrapie infection. Conversely, no differences were detected in other brain regions such as diencephalon and medulla oblongata. Association analysis showed that a polymorphism reflecting the presence of a RPSA pseudogene was overrepresented in a group of sheep resistant to scrapie infection. No amino acid change in the LRP/LR protein was found in the 126 sheep analyzed. However, interesting amino acid positions (241, 272, and 290), which could participate in the species barrier to scrapie and maybe to other transmissible spongiform encephalopathies, were identified by comparing LRP/LR sequences from various mammals with variable levels of resistance to scrapie.
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Acknowledgments
The authors thank the CERSYRA-Valdepeñas and AGRAMA breeders association, CSIC-León, CITA-Aragón, Prion Research Centre of the University of Zaragoza, INIA-Madrid, and ETSIA-Polytechnique University of Madrid for kindly providing Manchega, Awassi, Assaf, Rasa Aragonesa, and rabbit samples. The authors are very grateful to Dr. C. Mansilla and Dr. F. Ponz for helping improve the RT-PCR, to Dr. K.G. Dodds for correcting the English of the manuscript, to Dr. M.E.F. Alves for her continuous help, and to Dr. E.P. Cribiu and Dr. P. Zaragoza for allowing us to perform the cytogenetic mapping and the expression analysis in their respective laboratories. This work was supported by the RTA2006–00104 INIA project and a Predoctoral Grant from the INIA.
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Appendix 3
Figure A1 is a representation of the PCRs that confirm the presence of a RPSA pseudogene coamplifying with the RPSA functional gene when performing the amplification reactions to test the polymorphisms located in exon 4 (represented in capital bold letters) and intron 4 (represented in lower-case) for the association analysis. Underlined and enclosed between square brackets is the snoRNA E2. Primers used are in gray. The polymorphisms found among this region are indicated in bold and underlined letters. Table A1 summarizes the primers used in the amplification and sequencing reactions as well as several animals whose genotypes for the polymorphisms at positions 198 in exon 4 and 27 and 29 in intron 4 allowed us to infer the presence of a RPSA pseudogene which was not present in all the animals analyzed. The code of each sequence is also indicated. Thus, animal A did not bear the pseudogene, so results from both PCRs are consistent. Conversely, animals B and C bore the pseudogene, which was inferred by the insertion/deletion of a G (highlighted in gray) when performing PCR1 but not PCR2. In addition, animal B was heterozygous for the SNP at position 29 in intron 4, while animal C was homozygous (TT) for the same position. However, in the last case, the TT genotype was not possible to determine from the 02P7U sequence, which included the sequence of both the gene and the pseudogene.
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Marcos-Carcavilla, A., Calvo, J.H., González, C. et al. Structural and functional analysis of the ovine laminin receptor gene (RPSA): Possible involvement of the LRP/LR protein in scrapie response. Mamm Genome 19, 92–105 (2008). https://doi.org/10.1007/s00335-007-9085-6
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DOI: https://doi.org/10.1007/s00335-007-9085-6