Dataset Information

Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3.

ABSTRACT: Amino acid replacements encoded by the prion protein gene (PRNP) have been associated with transmissible and hereditary spongiform encephalopathies in mammalian species. However, an association between bovine spongiform encephalopathy (BSE) and bovine PRNP exon 3 has not been detected. Moreover, little is currently known regarding the mechanisms of evolution influencing the bovine PRNP gene. Therefore, in this study we evaluated the patterns of nucleotide variation associated with PRNP exon 3 for 36 breeds of domestic cattle and representative samples for 10 additional species of Bovinae. The results of our study indicate that strong purifying selection has intensely constrained PRNP over the long-term evolutionary history of the subfamily Bovinae, especially in regions considered to be of functional, structural, and pathogenic importance in humans as well as other mammals. The driving force behind this intense level of purifying selection remains to be explained.

SUBMITTER: Seabury CM

PROVIDER: S-EPMC524052 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3.

Seabury Christopher M CM Honeycutt Rodney L RL Rooney Alejandro P AP Halbert Natalie D ND Derr James N JN

Proceedings of the National Academy of Sciences of the United States of America 20041011 42

Amino acid replacements encoded by the prion protein gene (PRNP) have been associated with transmissible and hereditary spongiform encephalopathies in mammalian species. However, an association between bovine spongiform encephalopathy (BSE) and bovine PRNP exon 3 has not been detected. Moreover, little is currently known regarding the mechanisms of evolution influencing the bovine PRNP gene. Therefore, in this study we evaluated the patterns of nucleotide variation associated with PRNP exon 3 fo ...[more]

PMID: 15477588

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Project description:Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNAVal promoter. Six target sites of bovine PRNP were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire bPRNP coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or Renilla luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a Bsp MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting.Four siRNA expression plasmid vectors, six target sites of bPRNP, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of bPRNP in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.

Dataset Information

Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3.

Publications

Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3.

Similar Datasets

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