Genomic organization, comparative analysis, and genetic
polymorphisms of the bovine and ovine prion Doppel genes (PRND)
Maria G. Foti,
Michael A. Tranulis,
Giovanni Di Guardo,
John L. Williams,
Dipartimento di Genetica e Microbiologia, Universita` di Pavia, Pavia, Italy
Norwegian School of Veterinary Science, Oslo, Norway
Roslin Institute (Edinburgh), Roslin, Midlothian, UK
Istituto Zooprofilattico Sperimentale delle regioni Lazio e Toscana, Roma, Italy
Istituto Superiore della Sanita`, Laboratorio di Medicina Veterinaria, Roma, Italy
Received: 28 February 2001 / Accepted: 1 May 2001
Abstract. The doppel protein (Dpl) is a prion-like protein en-
coded by the gene PRND, which has been found downstream of
the prion gene, PRNP, in human and mouse. This paper describes
the isolation and structural organization of the bovine and ovine
PRND genes, which are composed of two exons compared with the
three of human and mouse. Intergenic distances between PRNP
and PRND were covered by means of long-range PCR and found
to be 16.8 and 20 kb, in cattle and sheep respectively. The 5Ј and
3Ј untranslated regions (UTR) were analyzed to identify transcrip-
tion regulatory sequences and compared with those from the
PRND and PRNP sequences published for other species. Three
polymorphisms (R50H, N110H, and R132Q) were revealed in the
cattle coding region; two synonymous substitutions (I12I, A26A)
were found in sheep. None of the polymorphisms was significantly
associated with either Bovine Spongiform Encephalopathy (BSE)
in cattle or scrapie in sheep.
Prion diseases comprise a group of fatal neurodegenerative pa-
thologies such as Creutzfeldt Jakobs disease (CJD) in humans,
BSE in cattle, and scrapie in sheep and goat. As these diseases
develop, an abnormal conformer of the host-encoded prion protein
(PrP) accumulates in the brain, accompanied by neurodegeneration
(Prusiner et al. 1998).
It has proved difficult to identify factors other than PrP that
could be directly involved in the pathogenesis of prion diseases.
However, PRNP has a paralog, PRND, localized about 12 and 21
kbp downstream of the murine and human prion genes, respec-
tively (Moore et al. 1999). PRND, referred to as Doppel, encodes
a protein (Dpl) that is similar to the C-terminal globular domain of
PrP. The murine PRND encodes a polypeptide of 179 amino acids
with 19% identity and 50% amino acid similarity to the C-terminal
globular domain of PrP. The human gene (PRND, OMIM 604263)
maps to chromosomal location 20p12-pter, like PRNP. Structural
predictions for Dpl suggest that it is anchored at the cell surface,
like PrP, by a glycosylphosphatidylinositol anchor (Silverman et
al. 2000). PRNP and PRND are transcribed separately, but chi-
meric transcripts are also generated by intergenic splicing (Moore
et al. 1999). Recently, the presence of the PRND gene in cattle and
sheep has been shown by cloning and sequencing of the cDNA
(Tranulis et al. 2001).
Here we describe the organization of PRND in cattle and
sheep, position them with respect to PRNP, and report on the
analysis of genetic polymorphisms in the ruminants’ coding re-
gions in terms of association with TSE.
Materials and methods
Amplification of introns and long-range PCR.
Intron sequences were
detected by using primers BP1/U (CCAGGAGTATGTGCAGAAGGT)
and M2/L (AAACTGCCAATAGTTGGCCTCATAGT), corresponding to
nt 15–35 and 355–330, and 1–21 and 341–316, of cattle and sheep cDNA
sequences respectively (AJ278011 and AJ278010). The amplified frag-
ments were cloned by using the TOPO TA Cloning Kit (Invitrogen) and
were sequenced on both strands.
Long-PCR fragments were produced from two YACs containing the
bovine and sheep prion loci with primers 104/U (TTCCCAGATGGTGC-
CATGCT at position 30315–30334 of the sheep PRNP region, acc.
U67922) and B1/L (ACCTTCTGCACATACTCCTGG), corresponding to
nt 35–15 and 21–1 of bovine and sheep cDNA sequences, respectively, by
using the Takara LA Long-PCR Kit (Takara). PCR products were cloned
into TOPO PCR Cloning XL Kit (Invitrogen).
Cattle and sheep chromosome spreads were prepared
and FISH localization of PRND was performed as described in Castiglioni
et al. (1998) with YAC clones containing the entire prion locus as probes.
Fresian cattle from the UK (45 BSE-affected
and 48 controls), Italian Sarda sheep (32 scrapie-affected and 24 controls),
and Norwegian Rygja (16 scrapie-affected and 34 controls) were studied.
The Dpl coding region of cattle and Sarda sheep was amplified from 50
ng genomic DNA with primers BP2/U (ATGAGGAAACATCTGGGTG-
GATG) and BP2/L (TTATTTCACAATAAACCAAATGAAAAC), corre-
sponding to nt 101–123 and 623–597, 87–109 and 623–597, respectively,
of cattle and sheep cDNA sequences. In the case of the Rygja sheep,
primers were GT1F (CGACACAATGAGGAAACATCTG) and GT2R
(TTGCCTGCAAGCTTATTTCACA), corresponding to nt 80–101 and
635–614 of the cDNA sequence.
DNA sequencing and analyses.
Reactions were performed by dye-
terminator cycle sequencing and were analyzed on an ABI310 (Applied
Biosystems) on both strands of either cloned PCR fragments or PCR prod-
ucts. Genome-wide repeats and low complexity regions were identified
with the program RepeatMasker (http://ftp.genome.washington.edu/RM/
RepeatMasker.html). Database searches were performed with BLAST pro-
grams (Altschul et al. 1990; http://www.ncbi.nlm.nih.gov/blast/), and
analyses of non-coding regions were performed with BLASTN Advantage
options (settings: -W7 -G1 -e 3.00 -q-1). Potential coding regions were
searched with GRAIL (Uberbacher and Mural 1991) and GENESCAN
Correspondence to: L. Ferretti Universita` degli Studi di Pavia, Diparti-
mento di Genetica e Microbiologia, via Ferrata 1, 27100 Pavia, Italy.
email: firstname.lastname@example.org; FAX (0039) 0382 528496.
Mammalian Genome 12, 729–733 (2001).
© Springer-Verlag New York Inc. 2001