The PrP-like protein Doppel gene in sheep and cattle:
cDNA sequence and expression
Michael A. Tranulis,
Department of Biochemistry, Physiology and Nutrition, Norwegian School of Veterinary Science, N-0033 Oslo, Norway
Department of Morphology, Genetics and Aquatic Biology, Norwegian School of Veterinary Science, N-0033 Oslo, Norway
Dipartimento de Genetica e Microbiologia, Universita` di Pavia, via Abbiategrasso 207, 27100 Pavia, Italy
Received: 22 August 2000 / Accepted: 12 January 2001
Abstract. cDNAs encoding the ovine and bovine prion protein-
like protein Doppel (Dpl) have been cloned. Sequencing revealed
cDNAs of 2.85 and 3.31 kb from ovine and bovine testicular
tissue, in accordance with observations of single transcripts of 3.2
and 3.6 kb on Northern blots. Sequence alignments showed a very
high degree of identity between the sheep and cattle Dpl cDNAs,
except for a 0.4-kb stretch in the bovine 3Ј untranslated region and
the terminal 3Ј end of the sequences. The expression pattern of the
Dpl gene (Prnd) in adult tissues from both species was compared
by Northern blot and RT-PCR analyses. The Prnd gene was ex-
pressed strongly in testicular tissue, while low levels of expression
were seen in other tissues. The open reading frame of the ovine and
bovine sequences encodes a 178-amino acid protein with 95%
sequence identity between the two species. Predicted structural
features are in close agreement with previous reports for mouse,
human, and rat Dpl.
Prion diseases such as Creutzfeldt-Jakob disease in human, scrapie
in sheep and goats, and bovine spongiform encephalopathy (BSE)
in cattle, are fatal, transmissible, neurodegenerative diseases.
These diseases are associated with an abnormal isoform of the host
prion protein (PrP), encoded by the Prnp gene (Basler et al. 1986).
The underlying pathogenic process appears to involve in situ con-
version of the normal cellular prion protein (PrP
) to the scrapie
. The structural conversion from PrP
thought to involve several cellular components, such as the pro-
posed “Protein X”, which could catalyze this energetically unfa-
vorable structural change in a chaperone-like manner (Prusiner
1998). However, attempts to identify proteins of relevance to PrP
physiology or pathology have been unsuccessful (Moore et al.
1999). Studies in yeast have recently identified a Ras-regulated
protein (Mks1) that seems to be required for de novo generation,
but not propagation, of the yeast “prion” URE3 (Edskes and Wick-
The discovery in mice of a Prnp-like gene, called Prnd, located
about 16 kb downstream of the Prnp gene, has intensified the
search for factors of relevance to PrP biology (Moore et al. 1999).
The Prnd protein product, Doppel (Dpl), shows structural simi-
larities to the C-terminal two-thirds of PrP that could indicate
similar cellular roles. However, Dpl lacks the putative Cu(II)-
binding region, present in the N-terminal part of PrP, and a pal-
indrome sequence of importance for the orientation of PrP in the
endoplasmic reticulum membrane (Hedge et al. 1998).
Interestingly, in two strains of transgenic mice with ablated PrP
), predisposed to late-onset cerebellar ataxia, the
Prnd gene is strongly up-regulated in the central nervous system,
but not in other Prnp
strains without ataxia, suggesting a role for
Dpl in the degenerative processes leading to loss of Purkinje cells
and ataxia (Moore et al. 1999). Furthermore, reintroduction of a
wild-type Prnp transgene in a Prnp
strain prone to develop
ataxia prevented disease, suggesting interaction between PrP and
Dpl (Nishida et al. 1999). It is also noteworthy that introduction
mice of transgenes encoding an N-terminally trun-
cated PrP molecule, resembling the Dpl protein, causes develop-
ment of ataxia and cerebellar granule cell degeneration (Shmerling
et al. 1998).
The Prnd gene has been detected in both humans and rats in
addition to mice (Moore et al. 1999). To facilitate further studies
of the putative role of Dpl in the development of scrapie in sheep
and BSE in cattle, we here present the cloning and characterization
of the ovine and bovine Prnd cDNAs as well as expression pat-
terns of Prnd in tissues from adult sheep and cattle.
Materials and methods
Amplification of a Prnd fragment from ovine and bovine genomic
Genomic fragments corresponding to part of the Prnd gene were
amplified from ovine and bovine DNA using degenerated primers (F1, R1,
Table 1). PCR cycling conditions were 94°C for 30 s, 55°C for 30 s, 72°C
for 60 s, and 35 cycles.
The nucleotide sequence data reported in this paper have been submitted to
the EMBL Data Library and assigned the accession numbers AJ 251331
and AJ 251332 for the ovine and bovine Prnd genomic sequences, and AJ
278010 and AJ 278011 for the ovine and bovine mRNA sequences, re-
Correspondence to: I. Harbitz; E-mail: Ingrid.Harbitz@veths.no
Table 1. Sequences of oligonucleotide primers.
F1 and R1 are primers based on the human and murine Prnd sequences.
Primers corresponding to ovine and bovine cDNA. 3ЈF1 is degenerated; 3ЈF3 has
one mismatch (G in position 15) in the bovine sequence.
Numbers flanking the sequences correspond to ovine cDNA.
Mammalian Genome 12, 376–379 (2001).
© Springer-Verlag New York Inc. 2001