Abstract
The susceptibility of humans to the variant Creutzfeldt-Jakob disease is greatly influenced by polymorphisms within the human prion protein gene (PRNP). Similar genetic differences exist in sheep, in which PRNP polymorphisms modify the susceptibility to scrapie. However, the known coding polymorphisms within the bovine PRNP gene have little or no effect on bovine spongiform encephalopathy (BSE) susceptibility in cattle. We have recently found a tentative association between PRNP promoter polymorphisms and BSE susceptibility in German cattle (Sander, P., Hamann, H., Pfeiffer, I., Wemheuer, W., Brenig, B., Groschup, M., Ziegler, U., Distl, O., and Leeb, T. (2004) Neurogenetics 5, 19-25). A plausible hypothesis explaining this observation could be that the bovine PRNP promoter polymorphisms cause changes in PRNP expression that might be responsible for differences in BSE incubation time and/or BSE susceptibility. To test this hypothesis, we performed a functional promoter analysis of the different bovine PRNP promoter alleles by reporter gene assays in vitro and by measuring PRNP mRNA levels in calves with different PRNP genotypes in vivo. Two variable sites, a 23-bp insertion/deletion (indel) polymorphism containing a RP58-binding site and a 12-bp indel polymorphism containing an SP1-binding site, were investigated. Band shift assays indicated differences in transcription factor binding to the different alleles at the two polymorphisms. Reporter gene assays demonstrated an interaction between the two postulated transcription factors and lower expression levels of the ins/ins allele compared with the del/del allele. The in vivo data revealed substantial individual variation of PRNP expression in different tissues. In intestinal lymph nodes, expression levels differed between the different PRNP genotypes.
Highlights
Wasting disease in elk and deer, feline spongiform encephalopathy in cats, and transmissible mink encephalopathy
Putative Transcription Factor-binding Sites—The bovine prion protein gene (PRNP) promoter region from positions Ϫ2676 to ϩ2564 was screened in silico for transcription factor-binding sites
We hypothesized that mutations affecting the PRNP expression level might have an influence on bovine spongiform encephalopathy (BSE) incubation time and/or BSE susceptibility
Summary
In Silico Analysis of Transcription Factor-binding Sites—The computer software MatInspector was used to scan the bovine PRNP promoter region for possible transcription factor-binding sites [16]. The region spanned from positions 46754 to 51993 according to the bovine PRNP genomic sequence (GenBankTM accession number AJ298878), including the putative promoter [17]. The oligonucleotides with the sequence surrounding the polymorphism ϩ571A3 G were used for EMSA with the gel shift assay system (Promega, Mannheim, Germany) according to the manufacturer’s instructions. A PCR product including 2676 bp of the 5Ј-flanking sequence, exon 1, intron 1, and the first part of exon 2 of the bovine PRNP gene was cloned as an MluI-BglII fragment into the promoterless reporter vector pGL3-Basic (Promega). The RP58-expressing vector pRP58 was generated by cloning a PCR product with the cDNA from the human RP58 gene into the pGL3Control vector (Promega), replacing its luciferase box. The restriction sites HindIII and XbaI were used for cloning this PCR product into the pGL3-Control vector. The XbaI fragment from plasmid pSP1-778C [21]
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