Vol. 188, No. 2, 1992 October 30, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
905-911
SEQUENCEAND WETHYIATION IN THEp/A4 REGIONOF THE RABBITAMYLOID PRECURSOR PROTEIN GENE* Jeffety S. Davidson, Roger L. West, Padma Kotikalapudi SOUTHERN ILLINOIS
UNIVERSITY
and Leonard E. Maroun
SCHOOL OF MEDICINE
DEPARTMENT OF MEDICAL MICROBIOLOGY/IMMUNOLOGY P.O. Box 19230, SPRINGFIELD, IL 62794-9230
Received
September
21,
1992
Alzhefmer's disease is characterized by the accumulation of the B/A4 fragment of the amyloid precursor protein in the hippocampal regions of the brain. We report here the isolation of genomic clones carrying exons 15, 16 and 17 of the B/A4 coding region of the rabbit amyloid precursor protein gene. The complete sequence of these exons predicts that all three peptides are identical to their human counterparts. An unexpectedly high concentration of CpG dinucleotides seen in exon 15 were conserved and continued into the intron 15 region. MspI/HpaII southern blot analysis revealed the presence of a number of methylated CpG dinucleotides in the cloned region of the gene. These data suggest that the rabbit amyloid precursor protein gene could provide a new and useful model for the study of this important gene. 0 1992 Academx Press, 1~.
The
trisomy
cause
of mental
(AD)
is
the
pathological
of
chromosome
retardation most
frequent
a peptide
amyloid.
share fragment
The APP gene
chromosome
chromosome
13
400,000
base
(bp)
responsible mutation has
pairs for
has
Using
16 (4).
rabbit
[FAD])
cause
conditions
containing
(5).
intensified
in
of
syndrome
been in
situ The
mental the to
human
and contains
dominant
form
to human chromosome
effort
to produce
plaques
mapped to
this
gene
be more
(6,7).
study
to than
The gene disease
of a specific
FAD pedigrees the
B/A4
and mouse
Alzheimer's
different for
These
termed
21 (3),
The discovery
model
disease
neuronal
18 exons
of AD (familial 21 (8).
frequent
(2).
(APP)
estimated
at least
an animal
*Sequence data from this article have been MBL/GenBank Data Libraries under Accession
of
chromosome
been
most
aged
protein we have
has
the
the
accumulation
hybridization, APP gene
is
and Alzheimer's
in
precursor
17 of the human APP gene in nine the
(1)
deficit
amyloid
mapped
[DS])
population
common
of the
in length
an autosomal
has been mapped in exon
21 (Downs
in the newborn
(9,lO)
of this
gene.
deposited with the Nos. M83558 and
M83657.
0006-291X/92 905
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188,
No.
We report 15.5
2, 1992
here
the
kilobases)
BIOCHEMICAL
isolation
that
AND
BIOPHYSICAL
and analysis
carry
of
the @/A4 amyloid MATERIALS
RESEARCH
COMMUNICATIONS
two overlapping coding
exons
X clones of the
(14.5
rabbit
and
APP gene.
AND METHODS
Cloning grown filter
An adult New Zealand on a lawn of E. d lifts were probed
rabbit liver EMBL X library (Clontech (strain NM538) in 12" x 12" plates. essentially according to Maniatis (11).
#TL 1003d) Nitrocellulose
was
The probe used for screening was a 1056 bp EcoRI fragment of the human APP cDNA kindly provided by Kang (12). The probe was radiolabeled by nick translation using 32P ATP (specific activity 3000 Ci/mmol, NEN). Inserts from two positive X clones were digested with either EcoRI, EcoRI and HindIII, orXba1 and sublconed into Bluescript sequencing plasmids (Stratagene, LaJolla, CA). Seauencing The exons were sequenced using exonuclease III/mung bean nuclease nested deletions of the Bluescript plasmids and 35S dideoxynucleotide sequencing procedures (Sequenase, USB) usingBluescriptsequencingprimers. Electrophoresis was carried out on a 38 cm x 80 cm Biorad apparatus in 5% Long Ranger gel containing 7 M urea. The sequencing project management and genbank analysis were performedwiththe online Intelligenetics suite of DNA sequence analysis programs (Intelligenetics, Mountain View, CA). Southern
Blot
Analysis
MspI and HpaII digestions were according to manufacturers recommendations (BRL) with the exception that 8 units of enzymes were used for each pg of DNA and the reaction was extended to 12 hrs. Lambda DNA was added to some samples as a test for complete digestion. Digested DNA (8 pg per lane) was electrophoresed in 0.8% agarose gels at 9 v/cm pulsed with a programmable power invertor (MJ Research, Watertown, MA). Twenty centimeter by 5 cm gels were run with buffer recirculation and coolant in a Hoefer super sub-gel apparatus (Hoefer Scientific Instruments, San Francisco, CA). Capillary blotting was for 16 hrs according to Southern (13) with 0.6N NaOH, 0.5 M NaCl buffer. The DNA was fixed to the nylon membranes (Zetaprobe, Biorad, Inc., Richmond, CA) by UV exposure (Stratalinker, Stratagene Inc.) and baking at 80°C for 2 hrs. Hybridization was at 42°C overnight in 20% formamide (Hybrisol, Oncor Inc., Gaithersburg, MD). Filters were washed at 50°C in 0.5 x SSC, 0.5% SDS and wrapped in Saran wrap. RP-Xomat x-ray film (Kodak) was exposed with Cronex lighting plus screens for 7 days at 70°C. RESULTS AND DISCUSSION The probe human fragment
contains
according Xl
[14.5
of
the
double
for
screening
kb insert] rabbit
of
of
et
al.
approximately
16,
[6]).
and 18 of
The screen kb insert], map of
15,
kb of
fragment
by Kang the
yielded each
obtained
et
al.
human
from
(12). gene
a
This
(numbered
two overlapping
clones,
exons
16 and 17
carrying
these
in FIGURE 1.
exons 18.6
provided 17,
A restriction
and is shown
encompassing
kindly
exons
and X2 [15.5
APP gene.
digests
was a 1056 bp EcoRI
plasmid
regions
to Yoshikai
kb overlap total
used
APP cDNA-containing
clones
15,
was prepared
using
The clones were found to share an 11.4 16 and 17. The combined clones represent a
the
rabbit 906
APP gene.
Vol.
188,
No.
2, 1992
BIOCHEMICAL
RAM
15
AND
RAM
BIOPHYSICAL
RESEARCH
16
RAM
17
H
k CLONE
S
E
bl
Eu
H
EK
COMMUNICATIONS
EH
EN
E
S
’ ’
E
E
N
X
X EH
H
d
Exon 15
hCLONE62
Exon 17
Exon 16
Black bars show the regions that have FIGURE 1. Restriction map of X clones. The location of a GC-rich region, a simple been subcloned into plasmid vectors. sequence tandem repeat (TR), and the j3/A4 amyloid coding regions are also shown. the following sequence : The tandem repeat was found in GCACACACCATCGCTATACTGCTACTCTACTTCACCCACTACACACTCCCctctctctcctccACACACTCCCac tctctctctctTATTTCCTTCAAAGCCACTGCTTTGGCGCCACACACC~CACTGCTCTCTCCACATATGAGCCCCTCT CAAAC. E-EcoRI, H-HindIII, N-NcoI, B-BamHI, K-KpnI, S-SalI.
DNA fragments
of
vectors.
Bacterial
plasmid
cDNA fragment synthetic
from
regions.
a 1800
have
bp EcoRI
been
of seen and
translational 5'
destabilize sequences significant
2.
(FIGURE
the
either
exon
of rabbit
APP exons
differences sequence.
mouse (15)
All this are that
have
consensus
region
of the
mutations
of
hairpin
loop.
shown both
3'-
end
labeled
to exons
15 and
its
exon
bars).
15,
termed
16 and 17.
flanking
intron
16 and its
flanking
carrying
exon
The available
accession
17
sequence
numbers:
Ram 15
the
noted
in exon
that
FIGURE rabbit
of
This
reported
907
human
and extend
to that
2 (8,9,17,18). this
in
the exon
16 and 17 to
the
loop found
24 and
in the
is underlined
FAD families
mutated retain
exons
compared
region
in
using
a hairpin
17 similar
region
and the
rabbit
presence
The bases in
confirm
in FIGURE
and co-workers
the
mRNA (16).
this
data
The complete
the
sequence
mouse
These
from
ferritin
(italicized)
16 and 17 are presented
by Johnstone
RNA (14).
15 DNA base Hyman
1056 bp
subclones
fragment
Genbank
found
and total the
the
Three
carrying
1, black
following
with
16 specific through
sequencing
and RAM 17 (M83657).
in
control
untranslated
FIGURE
regions
16 and 17 sequences
a total Tanzi
to sequence fragment
sequence
PCR primers
differences
used
the human and mouse sequences.
exon
Bluescript
screened
an exon
a 1450 bp XbaI
assigned
with
specific
or,
into
(GTTCTGGGTTGACAAATAT). carrying
intron
The complete partial
were
fragment
RAM 16 (M83558),
2 aligned
lifts
cloned
and RAM 17 has a 1900 bp EcoRI-Hind111
flanking
(M97377),
were
plasmid probe
RAM 16 has regions
and its
show
Kang's
RAM 16 and RAM 17 were
RAM 15 has
data
X inserts colony
oligonucleotide
RAM 15,
intron
the
in
serve
to
some FAD exon
17
Thus, potential
it
may be loop
even
Rabbit
Hun
House
Exon
Rabbit
Hman
HOUSe
Exon
17
16
IIIIIII
IIIIIIIIIII
IIIIIIII
II IIIIIIIIIIIIIIIII
IIIIIIlllll
IIIII IIIIIII
1620
II IIIIIIII
IIIIIII
IIIIII IIIIIIIIllllIIIIII
IIIIIIll
I IIIIIIIII
!!L
Arg
II
I 1940
FZGURF, 2. Rabbit axons 16 and 17 sequence comparison with the mouse and human sequences. All 15 rabbit The amino acid changes predicted for the mouse sequence changes conserve the human peptide amino acid sequence. Arrows point to start and end of B/A4 amyloid coding region. Two peptide are shown above the mouse sequence. of the bases found mutated in the FAD human gene sequence are italicized (#1924 and #1925). Bases are numbered according to Kang and co-workers and the putative iron binding consensus sequence is underlined (solid) along with the bases which close the loop (dotted).
I 1900
I
1660
1960
I
__._..- ...-..-...--...--...-...----.--.--.IIIIIIIIIIIIIIIIIIIIIIIII IIIIIIII IIIIIIIII
CATCACCTTG6TGATGCTGMGMGMACAGTACACATCCATT6
II I I I I I I I I II II II I II I II II II I II II I II III II I I II II I I I I II I I I lllllll
II IIIIIIIIIII
6T6nCm6CA6M6ATGT66GTC~C~G6TGCMTCATTGGACTCATGGTGGGCG6TGTTGTCATAGCGACA6TGATCGn
II IIIIIIIII
IIIIIIII
IIIIIII
Phe t
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ _..._..___.--.-..-..-._.-._.-
1740
1760
IIIIIlll
IIIIIIIIIIIIIIIIIIIIIIII
GnCCGBATTGACMATATCMGAeAGMGAGATCTCCGMGTOM6
IIII
GTTCT666TTGACAMTATCMGAC66A66A6ATCTCTM1166
IIIIIIII
G~CTe6sCT~C~CATCM~C~MMTCTC~MGT~~T~MTGCA~~C~CAT~~CA~~~6TCC6CCATC-CTG
61~ t
.:
< 0 .-
Vol.
188,
No.
2, 1992
observed
with
against
base
this
identical
BIOCHEMICAL
changes
human
neurotoxic three
iron
The
rabbit,
binding
sites
(20).
(FIGURE are
whereas
predicted
to have
the
sequence
acid
have
also
argued
retains
the
(CAGTGA (161). plaques
is predicted
B/A4 peptide
amino
(19)
mouse,
in neural
peptide
rabbit
COMMUNICATIONS
and Konings
unlike
accumulates
the
RESEARCH
Brosius
consensus that
BIOPHYSICAL
The mouse B/A4
2),
have
may
to be different
and complete
sequences
direct
exon
completely
at 16 and
identical
to
human peptide. Complete
amino
Of particular
3). distribution.
acid
sequence
conservation
is
also
seen
interest
is
conservation
of
the
unusual
This
peptide
or,
to
unexpectedly
could
3).
This
observed/expected
Garden,
al.
Since
of
genes
coding
region
of the
and
insensitive
like rabbit
then
Note
of
indicating
that of
the
Although that
of the
model,
the
the
mouse,
the
may
a 161 as
bp
the intron
61%,
region.
proposed
in the
rabbit
by
first
exon
and
These Gardiner-
20 bp of the
sites
responsible
the
in
methylation
probed
the
HspI bands
4 shows with
intron in the
B/A4
sensitive
FIGURE
flanking
of
the
sequentially
kb MspI bands for
expression
methylation
16 and its
and 2.2
CCGG cut
for
endonucleases. tissues
3.0,
modulate
isoschizomeric,
various
3.6,
the
regions. HpaII
lanes
are methylated
studied.
our knowledge
including
over
we tested
4.2,
tissues
of
restriction
fragment,
absence
rabbit
regions
gene using
human cDNA EcoRI
that
the
0.61
in the
We found
107 bp into
a CG island
APP gene,
(MspI),
residues
(6).
GC-rich
the
15 (FIGURE
CpG dinucleotide
had a G + C percentage
two CpG dinucleotides
DNA from
in each
of
of
in exon
arginine
DNA level.
extends
of
rabbit
the
blots
CpG's
15 sequence
methylation
housekeeping Wpa) , southern
are
the
segment
definition
There
intron
count
of
the
(21).
human
at
GC rich
ratio meet
et
available
function
some
CpG dinucleotide
(FIGURE
characteristics
the
be due to the need to conserve
retain
high
15 region an
region.
fragment
effects
17 peptides the
in the
possibility.
The ,9/A4 peptide
AND
of the
the
rabbit
the
possibility
genetics
provides of
of the
rabbit
is limited
many useful
characteristics
constructing
transgenic
compared
as an animal animals
(22).
1738
1685 alGluProValAspAlaArgProAlaAlaAspArgGlyLeuThrThrArgProG TrGAGCCTG~GAEgC~CCCTGCTGC~C~AGGACTGACCACC~ACCAGgtat ctgccctgcctgccttccacctgctggcctcctg~cttgagcttgagcaga~ccat cccccttctcccttataagcctctctgaat~cacatactgcagaacagaattggaaa
The exon 15 bases are in capitals just below FIGURE 3. Exon 15 sequence data. CpG dinucleotides are in bold capitals in both the predicted peptide sequence. This GC rich segment had a G + C the exon and adjacent intron sequence. percentage of 618, and an observed/expected ratio of CpG's of 0.61 over a 161bp These characteristics meet the definition of a CG island as proposed by region. Gardiner-Garden and Frommer (21). 909
to
Vol.
188,
No.
2,
1992
BIOCHEMICAL
Liver MH
FIGURE 4. with
been
sizes
studies
better,
model
associated
with
the
(23). for for both
Liver MH
the studying
by
APP gene
of rabbit tissue DNA. (A) Southern This probe had of the human APP cDNA. The same filter stripped and reprobed
role
flanking
reference
to
to produce
Our observations possibility
Downs syndrome
COMMUNICATIONS
analysis
fragment (B) 18.
calculated
the
RESEARCH
Kidney spleen Brain MHMHMH
which has exon 16 and its
were
using
further
blot
1056 EcoRI 16, 17 and
shown
disappointing
examined
southern
the 15,
our RAM 16 subclone
fragment
Extensive
with exons
BIOPHYSICAL
Kidney spleen Brain MHMHMH
MspI/HpaII
blot probed regions of
AND
it
of
the
may
and Alzheimer's
mice
that
the
provide
APP gene
regions.
have
rabbit
a new, in
The
markers.
transgenic
suggest
that
intron
X Hind111
the
thus should
and perhaps
far be a
neuropathology
disease.
ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association (Illinois Affiliate), Illinois Department of Public Health (grants #go630388 and Center Core Grant #P3OAGO8014, and #91630380), NIH/NIA Alzheimer's Disease Southern Illinois University School of Medicine. We sincerely appreciate the typing of the manuscript by Ms. Kathy Fritz. REFERENCES 1. 2.
3. 4. 5. 6.
D.J. and Chandley, A.C. (1983) In: Oxford Bond, Monographs on Medical Genetics (Fraser-Roberts, J.A., C.O. Carter, and A.G. Motulsky (Eds.). No. 11, pp. 1-159. Oxford University Press. Iqbal, K., D.R.C. McLachlan, B. Winblad and H.M. Wisniewski (1991) Alzheimer's Disease: Basic Mechanisms, Diagnosis and Therapeutic Strategies, pp. l-5. John Wiley & Sons, New York. Goldgaber, D., M.I. Lerman, O.W. McBride, U. Saffiotti and D.C. Gajdusek (1987) Science 235, 877. Reeves, R.H., J.D. Gearhart and J.W. Littlefield (1986) Brain Res. Bull. 16, 803. Maroun, L.E., M.J. Kovach, R.B. Adams, J. Colliver, C.M. Garvin, S. Mathur, M.E. Mourey-Metcalf and X. Yao (1990) Clin. Biotech. 2(l), 33-36. Yoshikai, S-i., H. Sasaki, K. Doh-ura, H. Furuya and Y. Sakaki (1990) Gene 87,
7. 8.
257-263.
Lamb, B.T. and J.D. Gearhart (1992) J. Cell. Biochem., S310, pg. 212, Keystone Symposia (abst). St. George-Hyslop, P.H., J.L. Haines, L.A. Farrer, R. Broeckhoven, et al. (1990) Nature 347, 194-198.
910
supplement Polinsky,
16E, C. Van
Vol.
9. 10. 11. 12.
13. 14.
15. 16. 17. 18.
19. 20. 21. 22. 23.
188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Goate, A., M-C. Chartier-Harlin, M. Mullan, J. Brown, F. Crawford, et al. (1991) Nature 349, 704-706. Goate, A.M., M.J. Mullan, M.-C. Chartier-Harlin, J. Brown, F. Crawford, et al. (1992) J. Cell. Biochem., supplement 16E, S105, pg.204, Keystone Symposia (abst). Maniatis, T., E.F. Fritsch and J. Sambrook (1992) A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. A. Unterbeck, J.M. Salbaum, C.L. Masters, et al. Kang, J., H-G. Lemaire, (1987) Nature 325, 733-736. Southern, E. (1975) J. Mol. Biol. 98, 503. Johnstone, E.M., M.O. Chaney, F.H. Norris, R. Pascual and S.P. Little (1991) Mol. Brain Res. 10, 299-305. Tanzi R.E. and B.T. Hyman (1991) Nature 350, 564. Aziz, N. and Hamish N. Munro (1987) Proc. Natl. Acad. Sci. 84, 8478-8482. Murrell, J., M. Farlow, B. Ghetti and M. Benson (1991) Science 254, 97-99. Chartier-Harlin, M-C., H.H. Crawford, A. Warren, D. Hughes, L. Fidani, et al. (1991) Nature 353, 844. Brosius, J. and D. Konings (1992) Aging 13, 449-451. Kowall, N.W., M.F. Beal, J. Busciglio, L.K. Duffy and B.A. Yankner (1991) Proc. Natl. Acad. Sci. 88, 7247-7251. Gardiner-Garden, M. and M. Frommer (1992) J. Mol. Biol. 196, 261-282. Hammer, R.E., V.G. Pursel, C.E. Rexroad, Jr., R.J. Wall, D.J. Bolt, et al. (1985) Nature 315, 680-683. Marx, J. (1992) Science 255(5049), 1200-1202.
911
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages
905-911
SEQUENCEAND WETHYIATION IN THEp/A4 REGIONOF THE RABBITAMYLOID PRECURSOR PROTEIN GENE* Jeffety S. Davidson, Roger L. West, Padma Kotikalapudi SOUTHERN ILLINOIS
UNIVERSITY
and Leonard E. Maroun
SCHOOL OF MEDICINE
DEPARTMENT OF MEDICAL MICROBIOLOGY/IMMUNOLOGY P.O. Box 19230, SPRINGFIELD, IL 62794-9230
Received
September
21,
1992
Alzhefmer's disease is characterized by the accumulation of the B/A4 fragment of the amyloid precursor protein in the hippocampal regions of the brain. We report here the isolation of genomic clones carrying exons 15, 16 and 17 of the B/A4 coding region of the rabbit amyloid precursor protein gene. The complete sequence of these exons predicts that all three peptides are identical to their human counterparts. An unexpectedly high concentration of CpG dinucleotides seen in exon 15 were conserved and continued into the intron 15 region. MspI/HpaII southern blot analysis revealed the presence of a number of methylated CpG dinucleotides in the cloned region of the gene. These data suggest that the rabbit amyloid precursor protein gene could provide a new and useful model for the study of this important gene. 0 1992 Academx Press, 1~.
The
trisomy
cause
of mental
(AD)
is
the
pathological
of
chromosome
retardation most
frequent
a peptide
amyloid.
share fragment
The APP gene
chromosome
chromosome
13
400,000
base
(bp)
responsible mutation has
pairs for
has
Using
16 (4).
rabbit
[FAD])
cause
conditions
containing
(5).
intensified
in
of
syndrome
been in
situ The
mental the to
human
and contains
dominant
form
to human chromosome
effort
to produce
plaques
mapped to
this
gene
be more
(6,7).
study
to than
The gene disease
of a specific
FAD pedigrees the
B/A4
and mouse
Alzheimer's
different for
These
termed
21 (3),
The discovery
model
disease
neuronal
18 exons
of AD (familial 21 (8).
frequent
(2).
(APP)
estimated
at least
an animal
*Sequence data from this article have been MBL/GenBank Data Libraries under Accession
of
chromosome
been
most
aged
protein we have
has
the
the
accumulation
hybridization, APP gene
is
and Alzheimer's
in
precursor
17 of the human APP gene in nine the
(1)
deficit
amyloid
mapped
[DS])
population
common
of the
in length
an autosomal
has been mapped in exon
21 (Downs
in the newborn
(9,lO)
of this
gene.
deposited with the Nos. M83558 and
M83657.
0006-291X/92 905
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
188,
No.
We report 15.5
2, 1992
here
the
kilobases)
BIOCHEMICAL
isolation
that
AND
BIOPHYSICAL
and analysis
carry
of
the @/A4 amyloid MATERIALS
RESEARCH
COMMUNICATIONS
two overlapping coding
exons
X clones of the
(14.5
rabbit
and
APP gene.
AND METHODS
Cloning grown filter
An adult New Zealand on a lawn of E. d lifts were probed
rabbit liver EMBL X library (Clontech (strain NM538) in 12" x 12" plates. essentially according to Maniatis (11).
#TL 1003d) Nitrocellulose
was
The probe used for screening was a 1056 bp EcoRI fragment of the human APP cDNA kindly provided by Kang (12). The probe was radiolabeled by nick translation using 32P ATP (specific activity 3000 Ci/mmol, NEN). Inserts from two positive X clones were digested with either EcoRI, EcoRI and HindIII, orXba1 and sublconed into Bluescript sequencing plasmids (Stratagene, LaJolla, CA). Seauencing The exons were sequenced using exonuclease III/mung bean nuclease nested deletions of the Bluescript plasmids and 35S dideoxynucleotide sequencing procedures (Sequenase, USB) usingBluescriptsequencingprimers. Electrophoresis was carried out on a 38 cm x 80 cm Biorad apparatus in 5% Long Ranger gel containing 7 M urea. The sequencing project management and genbank analysis were performedwiththe online Intelligenetics suite of DNA sequence analysis programs (Intelligenetics, Mountain View, CA). Southern
Blot
Analysis
MspI and HpaII digestions were according to manufacturers recommendations (BRL) with the exception that 8 units of enzymes were used for each pg of DNA and the reaction was extended to 12 hrs. Lambda DNA was added to some samples as a test for complete digestion. Digested DNA (8 pg per lane) was electrophoresed in 0.8% agarose gels at 9 v/cm pulsed with a programmable power invertor (MJ Research, Watertown, MA). Twenty centimeter by 5 cm gels were run with buffer recirculation and coolant in a Hoefer super sub-gel apparatus (Hoefer Scientific Instruments, San Francisco, CA). Capillary blotting was for 16 hrs according to Southern (13) with 0.6N NaOH, 0.5 M NaCl buffer. The DNA was fixed to the nylon membranes (Zetaprobe, Biorad, Inc., Richmond, CA) by UV exposure (Stratalinker, Stratagene Inc.) and baking at 80°C for 2 hrs. Hybridization was at 42°C overnight in 20% formamide (Hybrisol, Oncor Inc., Gaithersburg, MD). Filters were washed at 50°C in 0.5 x SSC, 0.5% SDS and wrapped in Saran wrap. RP-Xomat x-ray film (Kodak) was exposed with Cronex lighting plus screens for 7 days at 70°C. RESULTS AND DISCUSSION The probe human fragment
contains
according Xl
[14.5
of
the
double
for
screening
kb insert] rabbit
of
of
et
al.
approximately
16,
[6]).
and 18 of
The screen kb insert], map of
15,
kb of
fragment
by Kang the
yielded each
obtained
et
al.
human
from
(12). gene
a
This
(numbered
two overlapping
clones,
exons
16 and 17
carrying
these
in FIGURE 1.
exons 18.6
provided 17,
A restriction
and is shown
encompassing
kindly
exons
and X2 [15.5
APP gene.
digests
was a 1056 bp EcoRI
plasmid
regions
to Yoshikai
kb overlap total
used
APP cDNA-containing
clones
15,
was prepared
using
The clones were found to share an 11.4 16 and 17. The combined clones represent a
the
rabbit 906
APP gene.
Vol.
188,
No.
2, 1992
BIOCHEMICAL
RAM
15
AND
RAM
BIOPHYSICAL
RESEARCH
16
RAM
17
H
k CLONE
S
E
bl
Eu
H
EK
COMMUNICATIONS
EH
EN
E
S
’ ’
E
E
N
X
X EH
H
d
Exon 15
hCLONE62
Exon 17
Exon 16
Black bars show the regions that have FIGURE 1. Restriction map of X clones. The location of a GC-rich region, a simple been subcloned into plasmid vectors. sequence tandem repeat (TR), and the j3/A4 amyloid coding regions are also shown. the following sequence : The tandem repeat was found in GCACACACCATCGCTATACTGCTACTCTACTTCACCCACTACACACTCCCctctctctcctccACACACTCCCac tctctctctctTATTTCCTTCAAAGCCACTGCTTTGGCGCCACACACC~CACTGCTCTCTCCACATATGAGCCCCTCT CAAAC. E-EcoRI, H-HindIII, N-NcoI, B-BamHI, K-KpnI, S-SalI.
DNA fragments
of
vectors.
Bacterial
plasmid
cDNA fragment synthetic
from
regions.
a 1800
have
bp EcoRI
been
of seen and
translational 5'
destabilize sequences significant
2.
(FIGURE
the
either
exon
of rabbit
APP exons
differences sequence.
mouse (15)
All this are that
have
consensus
region
of the
mutations
of
hairpin
loop.
shown both
3'-
end
labeled
to exons
15 and
its
exon
bars).
15,
termed
16 and 17.
flanking
intron
16 and its
flanking
carrying
exon
The available
accession
17
sequence
numbers:
Ram 15
the
noted
in exon
that
FIGURE rabbit
of
This
reported
907
human
and extend
to that
2 (8,9,17,18). this
in
the exon
16 and 17 to
the
loop found
24 and
in the
is underlined
FAD families
mutated retain
exons
compared
region
in
using
a hairpin
17 similar
region
and the
rabbit
presence
The bases in
confirm
in FIGURE
and co-workers
the
mRNA (16).
this
data
The complete
the
sequence
mouse
These
from
ferritin
(italicized)
16 and 17 are presented
by Johnstone
RNA (14).
15 DNA base Hyman
1056 bp
subclones
fragment
Genbank
found
and total the
the
Three
carrying
1, black
following
with
16 specific through
sequencing
and RAM 17 (M83657).
in
control
untranslated
FIGURE
regions
16 and 17 sequences
a total Tanzi
to sequence fragment
sequence
PCR primers
differences
used
the human and mouse sequences.
exon
Bluescript
screened
an exon
a 1450 bp XbaI
assigned
with
specific
or,
into
(GTTCTGGGTTGACAAATAT). carrying
intron
The complete partial
were
fragment
RAM 16 (M83558),
2 aligned
lifts
cloned
and RAM 17 has a 1900 bp EcoRI-Hind111
flanking
(M97377),
were
plasmid probe
RAM 16 has regions
and its
show
Kang's
RAM 16 and RAM 17 were
RAM 15 has
data
X inserts colony
oligonucleotide
RAM 15,
intron
the
in
serve
to
some FAD exon
17
Thus, potential
it
may be loop
even
Rabbit
Hun
House
Exon
Rabbit
Hman
HOUSe
Exon
17
16
IIIIIII
IIIIIIIIIII
IIIIIIII
II IIIIIIIIIIIIIIIII
IIIIIIlllll
IIIII IIIIIII
1620
II IIIIIIII
IIIIIII
IIIIII IIIIIIIIllllIIIIII
IIIIIIll
I IIIIIIIII
!!L
Arg
II
I 1940
FZGURF, 2. Rabbit axons 16 and 17 sequence comparison with the mouse and human sequences. All 15 rabbit The amino acid changes predicted for the mouse sequence changes conserve the human peptide amino acid sequence. Arrows point to start and end of B/A4 amyloid coding region. Two peptide are shown above the mouse sequence. of the bases found mutated in the FAD human gene sequence are italicized (#1924 and #1925). Bases are numbered according to Kang and co-workers and the putative iron binding consensus sequence is underlined (solid) along with the bases which close the loop (dotted).
I 1900
I
1660
1960
I
__._..- ...-..-...--...--...-...----.--.--.IIIIIIIIIIIIIIIIIIIIIIIII IIIIIIII IIIIIIIII
CATCACCTTG6TGATGCTGMGMGMACAGTACACATCCATT6
II I I I I I I I I II II II I II I II II II I II II I II III II I I II II I I I I II I I I lllllll
II IIIIIIIIIII
6T6nCm6CA6M6ATGT66GTC~C~G6TGCMTCATTGGACTCATGGTGGGCG6TGTTGTCATAGCGACA6TGATCGn
II IIIIIIIII
IIIIIIII
IIIIIII
Phe t
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ _..._..___.--.-..-..-._.-._.-
1740
1760
IIIIIlll
IIIIIIIIIIIIIIIIIIIIIIII
GnCCGBATTGACMATATCMGAeAGMGAGATCTCCGMGTOM6
IIII
GTTCT666TTGACAMTATCMGAC66A66A6ATCTCTM1166
IIIIIIII
G~CTe6sCT~C~CATCM~C~MMTCTC~MGT~~T~MTGCA~~C~CAT~~CA~~~6TCC6CCATC-CTG
61~ t
.:
< 0 .-
Vol.
188,
No.
2, 1992
observed
with
against
base
this
identical
BIOCHEMICAL
changes
human
neurotoxic three
iron
The
rabbit,
binding
sites
(20).
(FIGURE are
whereas
predicted
to have
the
sequence
acid
have
also
argued
retains
the
(CAGTGA (161). plaques
is predicted
B/A4 peptide
amino
(19)
mouse,
in neural
peptide
rabbit
COMMUNICATIONS
and Konings
unlike
accumulates
the
RESEARCH
Brosius
consensus that
BIOPHYSICAL
The mouse B/A4
2),
have
may
to be different
and complete
sequences
direct
exon
completely
at 16 and
identical
to
human peptide. Complete
amino
Of particular
3). distribution.
acid
sequence
conservation
is
also
seen
interest
is
conservation
of
the
unusual
This
peptide
or,
to
unexpectedly
could
3).
This
observed/expected
Garden,
al.
Since
of
genes
coding
region
of the
and
insensitive
like rabbit
then
Note
of
indicating
that of
the
Although that
of the
model,
the
the
mouse,
the
may
a 161 as
bp
the intron
61%,
region.
proposed
in the
rabbit
by
first
exon
and
These Gardiner-
20 bp of the
sites
responsible
the
in
methylation
probed
the
HspI bands
4 shows with
intron in the
B/A4
sensitive
FIGURE
flanking
of
the
sequentially
kb MspI bands for
expression
methylation
16 and its
and 2.2
CCGG cut
for
endonucleases. tissues
3.0,
modulate
isoschizomeric,
various
3.6,
the
regions. HpaII
lanes
are methylated
studied.
our knowledge
including
over
we tested
4.2,
tissues
of
restriction
fragment,
absence
rabbit
regions
gene using
human cDNA EcoRI
that
the
0.61
in the
We found
107 bp into
a CG island
APP gene,
(MspI),
residues
(6).
GC-rich
the
15 (FIGURE
CpG dinucleotide
had a G + C percentage
two CpG dinucleotides
DNA from
in each
of
of
in exon
arginine
DNA level.
extends
of
rabbit
the
blots
CpG's
15 sequence
methylation
housekeeping Wpa) , southern
are
the
segment
definition
There
intron
count
of
the
(21).
human
at
GC rich
ratio meet
et
available
function
some
CpG dinucleotide
(FIGURE
characteristics
the
be due to the need to conserve
retain
high
15 region an
region.
fragment
effects
17 peptides the
in the
possibility.
The ,9/A4 peptide
AND
of the
the
rabbit
the
possibility
genetics
provides of
of the
rabbit
is limited
many useful
characteristics
constructing
transgenic
compared
as an animal animals
(22).
1738
1685 alGluProValAspAlaArgProAlaAlaAspArgGlyLeuThrThrArgProG TrGAGCCTG~GAEgC~CCCTGCTGC~C~AGGACTGACCACC~ACCAGgtat ctgccctgcctgccttccacctgctggcctcctg~cttgagcttgagcaga~ccat cccccttctcccttataagcctctctgaat~cacatactgcagaacagaattggaaa
The exon 15 bases are in capitals just below FIGURE 3. Exon 15 sequence data. CpG dinucleotides are in bold capitals in both the predicted peptide sequence. This GC rich segment had a G + C the exon and adjacent intron sequence. percentage of 618, and an observed/expected ratio of CpG's of 0.61 over a 161bp These characteristics meet the definition of a CG island as proposed by region. Gardiner-Garden and Frommer (21). 909
to
Vol.
188,
No.
2,
1992
BIOCHEMICAL
Liver MH
FIGURE 4. with
been
sizes
studies
better,
model
associated
with
the
(23). for for both
Liver MH
the studying
by
APP gene
of rabbit tissue DNA. (A) Southern This probe had of the human APP cDNA. The same filter stripped and reprobed
role
flanking
reference
to
to produce
Our observations possibility
Downs syndrome
COMMUNICATIONS
analysis
fragment (B) 18.
calculated
the
RESEARCH
Kidney spleen Brain MHMHMH
which has exon 16 and its
were
using
further
blot
1056 EcoRI 16, 17 and
shown
disappointing
examined
southern
the 15,
our RAM 16 subclone
fragment
Extensive
with exons
BIOPHYSICAL
Kidney spleen Brain MHMHMH
MspI/HpaII
blot probed regions of
AND
it
of
the
may
and Alzheimer's
mice
that
the
provide
APP gene
regions.
have
rabbit
a new, in
The
markers.
transgenic
suggest
that
intron
X Hind111
the
thus should
and perhaps
far be a
neuropathology
disease.
ACKNOWLEDGMENTS This work was supported by grants from the American Heart Association (Illinois Affiliate), Illinois Department of Public Health (grants #go630388 and Center Core Grant #P3OAGO8014, and #91630380), NIH/NIA Alzheimer's Disease Southern Illinois University School of Medicine. We sincerely appreciate the typing of the manuscript by Ms. Kathy Fritz. REFERENCES 1. 2.
3. 4. 5. 6.
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188,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
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