NR AUMM
AU Eckert,J.
TI Analysis of new variants in the bovine and ovine PRNP
QU Inaugural-Dissertation zur Erlangung des Grades einer Doktorin der Veterinärmedizin (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover, vorgelegt von Julia Eckert aus Böblingen, 28.5.2004
PT Dissertation
AB It is of great interest to clarify the role of the PRNP plays in TSE pathogenesis. Especially for sheep, an association of ovine PRNP variants with scrapie susceptibility has been attested, and variants at three ORF positions are proposed to show the highest effect. However, they do not explain all the observed variability, and it is necessary to identify additional markers in the PRNP in order to fine map the effects. The bovine PRNP is still being analysed for similar associations. By creating a dense set of readily accessible markers in the PRNP region, futurre association studies, as well as genotyping and breeding for TSE resistance will be facilitated. GELDERMANN et al. (2003) identified microsatellite sites in the bovine and ovine PRNP region on hand of software assisted screening of published sequences. In the present study these sites were amplified, cloned, sequenced and analysed. Eight polymorphic bovine microsatellite sites and four polymorphic ovine microsatellite sites for which sequence information was available at the Genbank database were subjected to analysis. In addition seven other ovine sites upstream of the Genbank database sequence were amplified using primers defined for bovine PRNP sites, three of which displayed polymorphisms after fragment length analysis on the A.L.F. sequencer. PRC was performed for individuals of eight cattle breeds and eleven sheep bredds to identify as many polymorphisms as possible. Two to eight different samples were cloned for the bovine sites and led to the identification of altogether 28 different sequences. For the eleven ovine sites one to seven samples per site were cloned and also led to the identification of 28 different sequences. All sequences gained for bovine polymorphic PRNP microsatellite loci could be attributed to the respective PRNP sequences. In sheep for four of the six polymorphic microsatellite sites all variants were attributed to the respective published sequences as well, while the origin of some variants found for sites S05 and S09 could not be identified. Though repeat variation of the microsatellite motif was the main cause of length polymorphisms, insertions and deletions were involved at eight sites. At seven of the polymorphic sites SNPs were also found and in one bovine and one ovine site alleles wer identified that differed in sequences, but showed the same fragment length. The results of this study show the merit of software assisted screening of DNA sequences for the identification of polymorphic microsatellite sites. Through the application of hetereologous primers it was also possible to access previously unsequenced areas of the ovine PRNP region. The sequence origin and the nature of polymorphisms was successfully analysed by cloning and sequencing, and most sites can now be used for the fine mapping of the bovine and ovine PRNP region in genotyping studies.
SP englisch
PO Deutschland