Comparative sequence analysis and expression of bovine PrP gene in mouse L-929 cells

Abstract

A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and six amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences, like other animal PrP genes, and consisted of 256 amino acids; the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to the 165th amino acid from the N-terminus. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP. The recombinant PrP comigrated with authentic bovine PrP in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at the perinuclear region of the cells.

Characterization of the Bovine Prion Protein Gene: The Expression Requires Interaction between the Promoter and Intron.

“…1, bold line); Figure 2A shows the nucleotide sequence of 3407-bp of the bovine PrP non-coding region. Compared with the nucleotide sequence of the cDNA reported by Yoshimoto et al [40], we found that the 151-bp 5' non-coding region of the cDNA consisted of two exons separated by a 2445-bp intron ( Fig. 2A).…”

“…The nucleotide sequence of the 5' end of the cDNA that was amplified and cloned into the plasmid was exactly identical to the 5' terminus of the cDNA reported by Yoshimoto et al [40] (Fig. 2A, bent arrow, +1).…”

“…Appropriate size-fractionated DNA fragments were ligated into EcoRI-cut λEMBL4 or λgt10 phage vectors (Stratagene, Lajolla, CA, U.S.A.) and then packaged using packaging extracts (Gigapack II Gold; Stratagene). The bovine leukocyte genomic library in λEMBL4 was screened with 187 base pairs (bp) of the 5'-terminal region of bovine PrP cDNA [40] (probe 1), and the λgt10 genomic library was screened with a DNA fragment of 716-bp (from 6-bp upstream of the translation start site of the sheep PrP gene to 710-bp downstream of it, probe 2). Phage DNAs of positive clones were digested with EcoRI and the resulting insert fragments were subcloned into the plasmid vector pBluescript SK(+) (Stratagene).…”

Complete Genomic Sequence and Analysis of the Prion Protein Gene Region from Three Mammalian Species

“…These possibilities are compatible with the apparent ubiquity of exon 2 splicing in other mammals; exon 2-containing cDNAs have now been described in cattle (Yoshimoto et al 1992) and recent reanalysis of the Syrian hamster (SHa) PrP gene, for which exon 1 originally appeared directly spliced to exon 3 (Basler et al 1986). The reanalysis showed that whereas 90% of SHaPrP mRNAs in the brain exhibit exon 1-3 splicing, 10% include exon 2 sequences closely related to those of sheep and mouse (Li and Bolton 1997).…”

“…AB001468) contains the mariner element as well, although the original bovine cDNA entries in GenBank (accession nos. D10612 and D90545) (Yoshimoto et al 1992) were partial and did not extend to the mariner integration site.…”