Journal of General Virology (1994), 75, 2947 2953. Printedin Great Britain
2947
Transmissible mink encephalopathy species barrier effect between ferret
and mink: PrP gene and protein analysis
Jason C. Bartz, Debbie I. McKenzie, Richard A. Bessen, Richard F. Marsh and Judd M . Aiken*
Department of Animal Health and Biomedical Sciences, 1655 Linden Drive, University of Wisconsin, Madison,
Wisconsin 53706, U.S.A.
Experimental infection of transmissible mink encephalopathy (TME) in two closely related mustelids, black
ferret (Mustela putorius furo) and mink (Mustela visa),
revealed differences in their susceptibility to the TME
agent. When challenged with the Stetsonville TME
agent, a longer incubation period was observed in ferrets
(28 to 38 months) than mink (4 months). Western blot
analysis of ferret and mink prion proteins (PrP)
demonstrated no detectable differences between the
proteins. Northern blot analysis of ferret brain RNA
indicated that PrP mRNA abundance is similar in
infected and uninfected individuals. We amplified the
PrP coding region from ferret DNA using the polymerase
chain reaction and compared the deduced amino acid
sequence of the ferret PrP gene with the mink PrP gene.
This comparison revealed six silent base changes and
two amino acid changes between mink and ferret:
Phe --->Lys at codon 179 and Arg ---, Gln at codon 224,
respectively. These changes may indicate the region of
PrP that is responsible for the species barrier effect
between mink and ferret.
Introduction
(Kimberlin et al., 1989). Back passage of inoculum into
the original host species often results in an increase in
incubation period on first passage suggesting adaptation
of the agent to the new host (Kimberlin et al., 1987).
Host genotype has been shown to influence incubation
time (Hunter et al., 1987). The scrapie incubation gene
(Sinc) was identified by genetic studies as having a
significant effect on the incubation period of scrapieinfected mice (Dickinson et al., 1968). The Sine gene has
been found to influence incubation time with every
known strain of mouse scrapie agent (Dickinson &
Meikle, 1971). In mice, the PrP gene contains polymorphisms that are correlated with specific incubation
times suggesting that the PrP gene is identical or closely
linked to the Sinc gene (Westaway et al., 1987; Carlson
et al., 1988). Polymorphisms located in the coding region
of the PrP gene also correlate with the susceptibility of
sheep to scrapie (Hunter et al., 1991; Goldmann et al.,
1991a), while human PrP gene variability has been
associated with familial Creutzfeldt-Jakob disease and
Gerstmann-Str~iussler syndrome (Hsiao et at., 1989;
Goldfarb et al., 1990).
The most extensively characterized species barrier
occurs between mice and hamsters. First passage of
hamster-adapted scrapie produces a disease in mice after
an abnormally long incubation period (Kimberlin et al.,
1987). Transgenic mice containing the hamster PrP gene
(in addition to the endogenous mouse PrP gene) are
Transmissible spongiform encephalopathies (TSEs) are
slowly progressive neurologic disorders that affect sheep,
cattle, mink, mule deer, elk, cats and humans (for review
see Prusiner, 1991). The neuropathology of these diseases
includes spongiform degeneration, astrocytosis and PrP
amyloid formation in the central nervous system. The
causative agent of TSEs remains elusive although it has
been hypothesized that an isoform of a cellular protein,
the prion protein (PrP), may be the infectious agent
(Prusiner, 1982). PrP is a host-encoded protein (Oesch et
al., 1985) that, in uninfected animals, is sensitive to
proteinase K treatments (PrP-sen). During the disease
process, PrP is modified resulting in its resistance to a
limited proteinase K digestion (PrP-res; Prusiner, 1982).
Infectious preparations, when inoculated into a different host species, typically produce an abnormally long
incubation period, an observation referred to as the
species barrier effect (Pattison, 1966). Subsequent passages reduce incubation time in the new host, which
eventually stabilizes at a shorter incubation period
The nucleotidesequencedata reported in this paper will appear in
the EMBL, GenBank and DDBJ databases under the accession
number U08952.
0001-2596 © 1994SGM
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2948
J. C. Bartz and others
Table 1. M i n k and ferret susceptibility to the
Stetsonville source o f the T M E agent
Incubation period
Passage
First
Second
Third
Mink
4 months
(14/14)
-
Ferret
28-30 months
(7/8)
8-9 months
(15/15)
5 months
(12/12)
* Number affected/number inoculated in parentheses.
TME infected
Uninfected
28S - -
18S
Fig. 1. Northern blot analysis of total ferret RNA isolated from
uninfected and TME-infected ferret brain hybridized with a random
primed ferret PrP coding region probe. The positions of the 28S and
18S rRNA are indicated.
susceptible to both mouse- and hamster-adapted scrapie
agent (Scott et al., 1989). Transgenic mice homozygous
for an ablated PrP gene, when challenged with mouseadapted scrapie, do not develop spongiform encephalopathy (Bfieler et al., 1992, 1993). Introduction of hamster
PrP transgenes into PrP-ablated mice renders the mice
susceptible to hamster-adapted agent but not to mouseadapted agent (Bfieler et al., 1993). These data further
imply a strong involvement of PrP in pathogenesis
and/or agent replication as well as for transmission
between species.
In this study we have examined the transmissibility of
TME into ferrets and mink. The identification of a
species barrier between these two closely related mustelids prompted us to examine PrP mRNA, PrP-res protein
abundance, and the PrP gene coding region of TMEinfected mink and ferret. Identification of differences in
the PrP gene between ferret and mink may give further
insight as to the role of PrP in the species barrier effect.
Methods
Infectivity assays. Mink brains were obtained from an incident of
TME in Stetsonville, Wisconsin, U.S.A. (Marsh et al., 1991). Black
ferret and mink were inoculated, in the right cerebral hemisphere, with
100 lal of 10% (w/v) brain homogenate. Animals were housed at
Charmany Farms and handled and cared for according to NIH and
University of Wisconsin Research Animal Resource Center guidelines.
Animals were observed weekly for clinical signs of TME.
Northern blot analysis. Frozen ferret brain tissue (0.05 to 0.10 g) was
powdered with a mortar and pestle and homogenized in 0.5 ml of
extraction buffer (4 M-guanidine thiocyanate, 25 raM-sodium citrate
pH 7-0, 0.5% N-lauroylsarcosine, 0.1% 2-mercaptoethanol, 0"2Msodium acetate pH 4.0). The homogenate was extracted once with an
equal volume of acidic phenol (pH 5.5) and a one-tenth volume of
chloroform, precipitated with propan-2-ol, and the RNA pellet
resuspended in RNAase-free distilled water (Sambrook et al., 1989).
Total ferret RNA (10 lag) was size fractionated by formaldehyde
agarose gel electrophoresis and transferred to positively charged nylon
membrane (Boehringer Mannheim) by capillary transfer (Sambrook et
al., 1989). A random primed probe (Prime-a-Gene, Promega) was
prepared from purified PCR product of the ferret PrP gene (con 1 and
con 2 primers). The probe was hybridized to the membrane in an
aqueous hybridization solution (0.9 M-NaC1, 1 mM-EDTA, 10mMTris-HC1 pH 8"0, 1'0% SDS, 100 lag/ml salmon sperm DNA) at 68 °C
for 16 h (Sambrook et al., 1989). The membranes were washed three
times in 0.1 x SSC/1.0 % SDS at 68 °C for 20 min and exposed to X-ray
film at - 8 0 °C for 5 days.
Enrichment ofPrP-res. PrP-res was prepared following the protocol
of Bolton et al. (1987). TME-infected mink and ferret brains were
minced and homogenized to 10% (w/v) in TEND pH 8-3 (10 mMTri~HCI pH 8"3, 1 mM-EDTA, 133 mM-NaC1, 1 IaM-DTT) containing
10 % N-lauroylsarcosine. Homogenate (50 ml) was clarified by centrifugation for 15 min at 23 000 g and the supernatant centrifuged for 3 h
at 145000g. The resulting pellet was resuspended in TEND pH 7.4
(10 mM-Tri~HC1 pH 7.4, 5 mM-EDTA, 133 mM-NaC1, 1 laM-DTT)
containing 1% N-lauroylsarcosine and 10% NaC1, sonicated (Fisher
550 sonic dismembrator) and centrifuged for 2 h at 215 000 g. The pellet
was resuspended in nuclease digestion buffer (10 mM-Tris-HCl pH 7.4,
100 mM-NaC1, 5 mM-CaCI~, 5 mM-MgCI~), sonicated and incubated
overnight in the presence of 100 gg/ml RNAase A and 20 gg/ml
DNAase 1. Buffer conditions were adjusted to 10% NaC1, 18 mMEDTA and 1% N-lauroylsarcosine, and the samples were centrifuged
at 215000g for 3 h over a 1 M-sucrose pad (1 M-sucrose in 1% Nlauroylsarcosine, 10 % NaC1, 10 mM-Tris HC1 pH 7.4, 5 mM-EDTA).
The pellet was resuspended in nuclease digestion buffer containing
100 lag/ml RNAase A and 20 lag/ml DNAase 1, sonicated, and
incubated for 2 h at room temperature. Buffer conditions were adjusted
to 500 mM-NaC1 and 1% N-lauroylsarcosine and the samples were
centrifuged at 16000 g for 30 min. The final PrP-enriched pellets were
resuspended in water and the protein concentration was determined
using the Pierce Micro BCA protein assay. PrP samples were treated
with proteinase K as described by Bessen & Marsh (1992).
Total brain homogenate. Total brain homogenates were prepared
according to a modification of Serban et al. (1990). Mink or ferret
brains were minced and homogenized to 20 % (w/v) in TEN pH 8"3
(5 mM-Tris-HC1 pH 8.3, l mM-EDTA, 133 mM-NaC1) containing 0-5 %
Nonidet P40. Debris was removed by centrifugation at 500 g for 5 min.
Western blot analysis. Electrophoretic separation of proteins by
SDS-PAGE (Hoeffer Scientific) was performed on 4 % stacking, 12 %
resolving gels using a mini-slab gel apparatus (Laemmli, 1970). Proteins
were electrophoretically transferred to Immobilon-P membrane (Millipore) with a Hoeffer semi-dry transfer apparatus. The membrane was
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PrP gene and protein analysis
Mink
1.25
0.25
0.05
GT 3'); (ii) mink 5 (5' ATGGTGAAAAGCCACATAGGCAG 3')
and mink 3 (5' TCCCACTATCAGGAGAATGAGCA 3'); (iii) anticon 1 (5' GTTGCCTCCAGGRCTKCCCT 3') and anticon 2 (5' ACCACCAAGGGGGAGAACTTCA 3').
Ferret
0.01
0.01
0.05
0.25
2949
1.25 mg
1-25 mg
PCR amplifications. Genomic DNA template (250 to 750 ng) was
incubated in the presence of 10 gl 10 x Taq buffer (500 mM-KCI,
100 mM-Tris-HCl pH 8-8, 15 mM-MgCI2, 1% Triton X-100; Promega),
10/al dNTP solution (1 mM each of dGTP, dATP, dTTP and dCTP;
Pharmacia), 10 gl each of 1 laM-oligonucleotide primer and 2-5 units
Taq polymerase in a final volume of 100 ~tl. The PCR profile consisted
of a 5 min denaturation at 93 °C prior to 30 cycles of 1 min at 95 °C,
1 rain at 55 °C and 2 min at 72 °C, with a final incubation of 5 min at
72 °C in a Coy thermocycler. A no-template control reaction was
performed with every set of amplifications.
Fig. 2. Western blot analysis with a PrP antibody of protein equivalents
(mg) of mink and ferret total brain homogenates. (a) Uninfected mink
and ferret; (b) TME-infected mink and ferret. The position of a
molecular mass marker is indicated.
DNA sequencing. The PCR amplification products were ligated into
a plasmid vector (pGEM-3Z, Promega) prior to sequence analysis. The
amplified fragments were prepared for ligation by treatment with T4
DNA polymerase and T4 DNA kinase (Starr & Quaranta, 1992). The
insert was then ligated into Sinai-digested pGEM-3Z vector by
standard techniques (Sambrook et al., 1989). Recombinant plasmid
DNA was isolated by alkaline lysis minipreps and sequenced using the
dideoxy chain termination method with Sequenase enzyme (US
Biochemical). The sequence data obtained were the result of a minimum
of two separate amplification reactions from two animals. DNA
sequence data were analysed by Genetics Computer Group (Madison,
Wis., U.S.A.) software for amino acid translation and comparison with
other PrP sequences.
24K
Mink
1.25
0.25
0.05
Ferret
0.01
0.01
0.05
0.25
24K -
incubated for 3 h at 37 °C in blocking buffer (1% nonfat dried milk,
2% newborn calf serum, 0.9% NaCI, 10mM-Tris-HCl, pH 8.8)
followed by incubation at room temperature for 18 h with a 1:5000
dilution of C3 antibody (kindly provided by Richard Rubenstein) in
Buffer A (50 mM-Tris-HC1 pH 7.4), 150 mM-NaCI, 1 mM-EDTA,
0.05 % Tween 20, 0.25 % gelatin). The filters were then washed four
times for 15 rain each in wash buffer (50 mM-Tris-HCl pH 7.4, 150 mMNaCI, 1 mM-EDTA, 0.005 % Tween 20) and incubated with alkaline
phosphatase conjugated to anti-hamster IgG (Kirkegaard & Perry
Laboratories). Washes were repeated as described above followed by
one additional rinse in Develop buffer (50 mM-Tris-HCl pH 9-8, 3 mMMgCl~). Bands were visualized by development in Buffer C (50 mMTris, 3 mM-MgC12) containing 0.05 mg/ml BCIP and 0.01% NBT as
described by Blake et al. (1984).
Results
Inoculation of the Stetsonville source of TME into mink
produced the expected 4 months incubation period
(Marsh et al., 1991). The first passage of the Stetsonville
source of TME into ferrets, however, resulted in an
unexpectedly long incubation period of 28 to 38 months
(Table 1). The second serial passage of ferret-adapted
TME had an incubation period of 8 to 9 months while
third serial passage reduced the incubation period to 5
months. Further passage does not significantly shorten
incubation period (data not shown).
Northern blot analysis indicated that ferret PrP
mRNA is approximately 2.3 kb (Fig. 1) in length and
thus similar in size to the mink PrP transcript
PCR amplification primers. Three sets of primers were used for the
amplification of the ferret PrP gene: (i) con 1 (5' AGGGMAGYCCTGGAGGCAAC 3') and con 2 (5" TGAAGTTCTCCCCCTTGGTG-
(a)
R---~ Q
F --~ L
Signal
Octapeptide repeat
Hydrophobic region
N-Glycosylation Hydrophobic region
sites
(b)
Mink 5
0
Con 1
Anticon
2
>
100
200
300
400
500
600
700
1
I
I
I
I
I
k
•
Anticon 1
Con 2
800
I
<
Mink 3
Fig. 3. (a) Diagram of ferret PrP based on the
deduced amino acid sequence. The location of the
amino acid differences at positions 179 (F ~ L)
and 224 ( R ~ Q ) between ferret and mink,
respectively, are illustrated. (b) Location and
orientation of PCR primers used.
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2950
J. C. Bartz and others
Mink
Ferret
Mink
Ferret
1 ATGGTGAAAAGCCACATAGGCAGCTGGCTCCTGGTTCTCTTTGTGGCCACATGGAGTGAC
.
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60
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S
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D
Mink
Ferret
Mink
Ferret
61
Mink
Ferret
Mink
Ferret
121
Mink
Ferret
Mink
Ferret
181
GGCCAGCCCCACGGGGGTGGCTGGGGACAGCCCCACGGGGGTGGCTGGGGTCAGCCCCAC
..................................................
A .........
G
Q
P
H
G
G
G
W
G
Q
P
H
G
G
G
W
G
Q
P
H
.
.
.
.
240
Mink
Ferret
Mink
Ferret
241
GGGGGTGGCTGGGGACAGCCGCATGGTGGCGGTGGCTGGGGTCAAGGTGGTGGGAGCCAC
............................................................
G
G
G
W
G
Q
P
H
G
G
G
G
W
G
Q
G
G
G
-
300
Mink
Ferret
Mink
Ferret
301
GGTCAGTGGGGCAAGCCCAGTAAGCCCAAAACCAACATGAAGCATGTGGCGGGAGCCGCA
..........................
T ..........................
T ......
G
Q
W
G
K
P
S
K
P
K
T
N
M
K
H
V
A
G
A
A
.
.
.
.
.
360
Mink
Ferret
Mink
Ferret
361
GCAGCCGGGGCGGTCGTGGGGGGCCTGGGCGGCTACATGCTGGGGAGCGCCATGAGCAGG
.............. T .............................................
A
A
G
A
V
V
G
G
L
G
G
Y
M
L
G
S
A
M
S
-
420
Mink
Ferret
Mink
Ferret
421
Mink
Ferret
Mink
Ferret
481
Mink
Ferret
Mink
Ferret
541
Mink
Ferret
Mink
Ferret
601
Mink
Ferret
Mink
Ferret
661
Mink
Ferret
Mink
Ferret
721
ATTGGCTTCTGCAAGAAGCGGCCAAAGCCTGGAGGAGGCTGGAACACTGGGGGGAGCCGA
.
.
.
I
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G
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F
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C
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K
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K
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.
.
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G
.
120
.
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G
.
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.
S
TACCCAGGGCAGGGCAGTCCTGGAGGCAACCGCTACCCACCCCAGGGTGGTGGCGGCTGG
.....................................................
Y
P
G
Q
G
S
P
G
G
N
R
Y
P
P
Q
G
G
G
.
.
.
.
.
.
.
R
180
G
W
S
CCCCTCATTCATTTTGGCAACGACTATGAGGACCGCTACTACCGTGAGAACATGTACCGC
............................................................
P
L
I
H
F
G
N
D
Y
E
D
R
Y
Y
R
E
N
M
-
H
R
480
TACCCCAACCAAGTGTACTACAAGCCGGTGGATCAGTACAGCAACCAGAACAACTTCGTG
........................................................
Y
P
N
Q
V
Y
Y
K
P
V
D
Q
Y
S
N
Q
N
N
-
Y
R
F
L
G--V
-
540
CATGACTGCGTCAACATCACGGTCAAGCAGCACACGGTGACCACCACCACCAAGGGCGAG
............................................................
H
D
C
V
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V
K
Q
H
T
V
T
T
T
T
K
.
.
.
.
G
E
AACTTCACGGAGACCGACATGAAGATCATGGAGCGCGTGGTGGAGCAGATGTGTGTCACC
.
N
.
.
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F
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.
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.
T
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660
.
.
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V
CAGTACCAGCGAGAGTCCGAGGCTTACTACCAGAGGGGGGCGAGCGCCATCCTCTTCTCG
.......... A .................................................
Q
Y
Q
R
E
S
E
A
Y
Y
Q
R
G
A
S
A
I
L
F
Q
CCCCCTCC~GTGATCCTCCTCATCTCACTGCTCATTCTCCTGATAGTGGGA
...........................
G ........................
P
P
P
V
I
L
L
I
S
L
L
I
L
L
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G
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600
771
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720
S
-
P r P gene and protein analysis
(Kretzschmar et al., 1992; J. Bartz, unpublished data).
Similar levels of PrP mRNA were present in both
uninfected and TME-infected ferret brain (Fig. 1).
PrP-res was purified from ferret and mink brains and
analysed by Western blot (Fig. 2). No differences in
molecular mass between the species were noted. Comparison of PrP abundance between species revealed that
mink PrP was present at slightly higher levels than ferret
PrP (Fig. 2). Levels of PrP were significantly higher in
TME-infected mink and ferret in comparison to normal
animals. Digestion of PrP-res with proteinase K revealed
similar digestion patterns in TME-infected mink and
ferret (data not shown).
Three sets of primers were used for the amplification
of the ferret PrP gene (Fig. 3). The initial amplification
used consensus primers (con 1 and con 2) based on
conserved regions of PrP in several species" sheep,
human, hamster, mouse, rat and bovine (Goldmann et
al., 1990; Kretzschmar et al., 1986; McKinley &
Prusiner, 1986; Locht et al., 1986; Liao et al., 1987;
Goldmann et al., 1991b). These primers are located
internal to the PrP open reading frame (ORF) translation
initiation and termination sites and.yield a 480 bp
amplification product. Primers flanking the 5' and 3f
ends of the PrP ORF were derived from the mink
sequence (Kretzschmar et al., 1992) and were used to
confirm the sequence of the internal primer sites. The
amplified products were ligated into plasmid vectors and
sequenced. The sequence data indicate that the ferret PrP
coding region is 771 nucleotides in length resulting in a
deduced peptide of 257 amino acids which contains a
signal peptide, five copies of an octapeptide repeat,
hydrophobic regions and two N-linked glycosylation
sites (Fig. 3). Sequence analysis of the ferret PrP gene
identified six nucleotide differences at positions 84
( C ~ A ) , 231 ( A N T ) , 327 ( T ~ C ) , 354 ( T ~ A ) , 375
(T ~ C), 537 (G--* C), 671 (A ~ G ) and 747 (G ~ A )
between ferret and mink respectively. The nucleotide
variability at position 537 and 671 resulted in amino acid
differences: (i) a phenylalanine (mink) to leucine (ferret)
at position 179 and (ii) an arginine (mink) to glutamine
(ferret) at position 224 (Figs 3 and 4).
Discussion
Spongiform encephalopathies can be readily transmitted
to experimental animals of most species on first passage.
Notable exceptions include: (i) the transmission of
mouse-adapted scrapie to hamsters (Kimberlin et al.,
2951
1987; Scott et al., t989) and (ii) attempts to transmit
TME to mice (Marsh & Hanson, 1969). Our transmission
studies with TME demonstrate that a species barrier
exists between ferret and mink. Since PrP has been
hypothesized to play an important role in delineating the
species barrier, we compared the PrP gene sequence as
well as the size and abundance of the PrP mRNA and
protein in these two closely related species.
In several species, PrP mRNA abundance has been
shown to be similar in infected and uninfected individuals
suggesting that PrP transcription is not involved in the
disease process (Chesebro et al., 1985; Oesch et al.,
1985). Not surprisingly, PrP mRNA abundance in TMEinfected and uninfected ferrets remained at equivalent
levels indicating that differences in mRNA levels are not
involved in the disease process.
Western blot analysis of PrP-res enriched preparations
from infected mink and ferret identified no observable
differences in the migration of the PrP protein indicating
no major differences in molecular mass or glycosylation
of PrP between the two species. Western blot analysis
did, however, reveal a 10-fold increase in the total
amount of PrP from TME-infected animals relative to
uninfected animals (Fig. 2). Although TME-infected
mink appear to have slightly more PrP than TMEinfected ferrets this difference is probably due to
variability in the clinical disease stage of the animals
being analysed.
Although involvement of PrP in spongiform encephalopathies has been well established, its exact function in
the disease process is not clearly understood (Chesebro,
1990; Prusiner, 1992; Rohwer, 1991 ; Weissmann, 1991).
Several lines of evidence suggest that PrP is involved in
the susceptibility of an individual to spongiform encephalopathies. For example, incubation periods in mice are
influenced by two alleles of the PrP gene that differ in
only two amino acid positions (Westaway et al., 1987).
Recent experiments with transgenic mice further demonstrate how minor differences in PrP can affect the
species barrier. Transgenic mice carrying two PrP amino
acid substitutions from Syrian hamster are susceptible
only to mouse-adapted agent while mice that carry five
amino acid substitutions are susceptible to both mouse
and hamster-adapted agent (Scott et al., 1993).
Comparison of the ferret PrP open reading frame with
other species using the GAP program (sequence analysis
software package version 7.0; Genetics Computer
Group) revealed 83 to 98 % nucleotide sequence identity
(Table 2). The ferret ORF shared the highest nucleotide
Fig. 4. Nucleotide and amino acid sequence comparison of ferret and mink PrP ORFs. (Ferret sequence, this paper, mink sequence,
Kretzschmar et al., 1992.) Locations of PCR primers are underlined. Letters in bold type mark the nucleotide and amino acid
differences between ferret and mink.
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J. C. Bartz and others
Table 2. Nucleotide and amino acid sequence
comparisons of ferret PrP with other PrP sequences
Nucleotide
identity
Species
(%)
Amino acid
similarity
Mink
Sheep
Bovine
Syrian hamster
Human
Mouse
98-9
86.9
86.6
85.8
85-5
83-0
99.6
97-7
96-9
94-6
96-1
94-0
(%)
sequence identity with mink (98.7%) and was least
similar to the mouse (82.9%) while the deduced ferret
PrP amino acid sequence shared the highest sequence
similarity with mink (99.6 %) and the least with mouse
(94.0 %).
Sequence analysis of the ferret PrP gene identified two
amino acid differences with mink at codons 179 and 224
(Fig. 4). The locations of the amino acid changes in
mink and ferret are not analogous to other amino acid
positions in PrP that influence incubation period. The
change at codon 179, however, is located within five
amino acids of a codon change that has been implicated
in the species barrier between mice and hamsters (Scott
et al., 1993).
The similarity of the PrP ORF between ferret and
mink in conjunction with the species barrier exhibited by
the two species makes them a useful model for the study
of the species barrier effect. The data presented here
suggest that if the PrP protein is the sole factor in
governing species barrier, amino acid differences in ferret
and mink PrP play an important role in the observed
species barrier effect.
The authors are grateful for the technical assistance of Jean
Kaczkowski and to Lynne Borchardt-Sendek for her helpful discussions. These studies were supported by grant 9203792 from the
United States Department of Agriculture to J.M.A.
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