Note: Descriptions are shown in the official language in which they were submitted.
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VARI~NT PRESENILIN-2 GENES
INTRODUCTION
This invention was made in the course of research sponsored by the National In.~titu~Ps of
5 Health. The U.S. Government may have certain rights in this invention.
BACKGROUND OF THE INVENTION
.AI,IIr; llr.1'5 Disease (AD) is a progressive neurod,,~ ,dli\re disorder cllcu;.. ~ t by
memory loss and dP.mPnti~ The major F ' ~'~ I feature of AD is the presence of ~-u-~ uus
10 ncu~url~illary tangles and senile plaques cu~ osed priniarily of the amyloid protein. These
plaques contain beta-amyloid, a peptide varying from 39 to 43 amino acids in length, derived from
a larger amyloid ~JI~;UI~UI protein (APP) (Goate et al. Nature 19gl, 349, 704-6; Masters et al.
PNAS 1985, 82, 42454249; Kang et al. Nature 1987, 325, 733-736). Studies have shown that
;l~s in the g~ dliull of the 42 amino acid peptide lead to AD in APP encoded disease.
AD is typically a disease of the elderly, S.rn;. ~;"g up to 6% of those aged 65 and up to
20% of 80 year olds. This type of AD is termed late-onset. In addition, a small number of
p~i,5.~s have been des~lilxd wherein the disease is inherited as an ~lt~ nm~l tlom~ mt with age
~r~ r~ ce. Most con~ -ly, the age of onset of the disease is below 60 years in these
families (presenile). Thus, this type of AD is termed early-onset. Genetic factors have been
20 ;".~ t~i in both early and late onset AD.
Several large families have been il1r"1;r~d in which ~ lliL AD s~ les as fully
losu~ dorninant trait. Linkage analysis studies in presenile AD farnilies have
j,lr..,~ir,~i four genes, on ~l.l.)ll~s-,"~-~ 1, 14, 19, and 21, that when mutated, cause presenile AD.
The first presenile AD gene i-lrl 11 ir.Pd maps to chromosorne 21 and codes for the beta A4-amyloid
protein ~,~;u,~c" (APP) (Goate et al. Nature 1991, 349, 704-706; Murrell et al. Science 1991,
254, 97-99; Chartier-Harlin et aL Nature 1991, 353, 844-846). Mutations in this gene account for
a~uAil-~l~ly 5% of the families. These disease causing ~ n~ have been modeled in~I,.".~r~ 1 or primary cultured cells and have been shown to lead to altered proteolytic ~luces~illg
of APP in a way that favors p~olu~;lion of its amylc~ and potentially n~;ulul~JAic Ab
fragments. Tld--s~,~ ov~.~A~,.~sioll of one mutant APP has resulted in the first mouse model of
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AD, in which age-linked cerebral deposition of the Ab fragrnent is 7~Ccr~n~ - ' by neuronal,
astrocytic, and ., ~,~,lial pathology (Garnes et al. Nature 1995, 373, 523-527).Genetic variability in the apolip.~l"ut~l E locus on chrornosorne 19 have also been shown
to be ill4)UI~ in the etiology of AD (St.i~ et al. PNAS 1993, 90, 1977-1981; Saunders et
S al. Neurology 1993, 43, 1467-1472). Inl,~liti~ce of the I4 (112 Cys-Arg) allele has been reported
to lower the age of onset in a dose ~ r~ r~-l rnanner. Conversely, il~lcliL~Iee of the
apoli~lot~l E I2 allele appears to confer a decreased risk of dcv~ g AD (Corder et al.
Science 1993, 261, 921-923; Corder et a1. Nature Genet. 1994, 7, 180-184).
Presenilin-1 (PS-1), located on cl.~ s~ 14 harbors an P,Stinn~ 70% of the disease
10 causing ".,-l~l;. l-s, rnaking it the rnajor gene for farnilial presenile AD (e~l ~' 10% of all AD
cases~ (Sl-r- 1 ill~ lll et al. Nature 1995, 375, 754-760; van Br~.u~ren et al. Nature Genet. 1992.
2, 335-339; St. George-Hyslop et al. Nature Genet. 1992, 2, 330-334; Srh~llPnhP~g et al. Science
1992, 258, 668-670). This gene, ~ S182 or PS-l, is rnade up of 10 coding exons
.~} 3 to 12). PS-1 is ~ d to be an integral ~ Ll~u~e protein with at least 7
15 Il,...s..r.,~ e dornains. At the tirne of its i.co~ ion, five different rnissense mnt~ti-~n.c were
it1PntifiPd in 8 chromosorne 14 lir~ced families (Sl-r~ I et al. Nature 1995, 375, 754-760). A
total of ~ different ",..l~ .c in 40 families of various ethnic origins has been i~iP.ntifiP~I (van
Broeckhoven et al. Nature Genet. 1995, 11, 23~233). Two drfferent ..,..~ nc were i-lPntifiPA at
each of the codons 139, 146, 163, and 280. However, rnost ... "; ~ .,.c are scattered over the
20 protein with mnt~tionc found in S of the 7 putative L~ e domains and in 3 of the 6
hydl~ ~ ' loops. Mutations have been found in 6 of the 10 coding exons, with exon 5 and 8
a ~ g for 65% of the l.~ The same mutation occurs in several AD families ofdifferent ethnic origin, su~P,cting there are ;...lryr~..1r..~l mutation events in the PS-l gene.
A third gene for presenile AD (PS-2) rnaps to ~ hllllnl).CI~IIP I in the Volga-German AD
families, a group of families in which AD is the result of a founder effect (Levy-Lahad et al.
Science 1995, 269, 970-973). This gene (STM-2 or E5-1) was irl~.ntifi~ as a direct result of its
high homology to PS-l. The same Ill ~Sr.ll!~e mutation was found in 7 Volga-German AD families
and more recently, a second l.hss~l.3e mutation has been found in an Italian AD family (Levy-
Lahad et al. Science 1995, 269,97~973; Rogaev et al. Nature 1995, 376, 775-778; Barinaga
Science 1995, 269,917-918).
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Both PS-I and PS-2 have applv~dllldlly 450 amino acids and share an overall homology
of 67% with the highest similarity observed in the TM dornains (Levy-Lahad et al. Science 1995,
269,973-977; Rogaev et al. Nature 1995, 376,775-778. This degree of homology is indicative of a
similar biological function. The putative seven ~ " ~l ,. dne domain structure of the presenilins
S is cull~ 1c with a function as a receptor l~ le, an ion channel or a ~ e structural
protein. It has been determined that ~..--~ "~ in PS-I and PS-2 cause an increase in the
;IdtiUII of Ab42 thus ' lg biological interaction with amyloid.
The exonic structure and the ~ t~-~re of alternate splicing in the PS-1 gene have been
determined. The use of an alternate splice donor site at the 3' end of exon 3 results in clones with
and without a VRSQ ~tif at codons 26-29 (Al7heimPr's Disease Collaborative Group. Nature
Genet. 1995, 11, 219-222). Alternate splicing has also been found in both exon 8 in PS- 1 and the
exon 8 in PS-2.
A number of other variant, alternatively spliced PS-2 genes have now been i~lr.lli~lP~l
These genes are useful in the ~l;~ cic of early-onset AD and in evaluating agents which rnay be
useful for the ~ lll of this disease.
SUMMARY OF THE INVENTION
An object of the present invention is to provide novel, variant PS-2 s~l, Ir~ll C'S
Another object of the present invention is to provide a rnethod of ~ o~;~.g Alzheimer's
disease using these novel PS-2 .ce l ~ or the exonic or intronic .se~ rl~ of the PS-2 gene.
Yet another object of this invention is to provide a model system for Alzheirner's disease
COIl~l~lllg variant PS-2 genes.
BRIEF DESCRIPIION OF THE FIGURES
- Figure 1 provides a s~ ."~ co.. y ~ nn of the Ol~ of the PS-l and PS-2 gene.
25 Labeled arrows indicate sites of known m~lt~ti-n~ Unlabeled arrows indicate intron/exon
bul....l,.. ies Hatched areas in PS-2 indicate sites of alternate splicing. USF#15 contains exons 3,
4 and 8. W.U.#2 lacks exons 3, 4 and 8. W.U.#15 lacks only exon 8.
Figure 2 provides the gene s~ -e of the PS-2 gene (SEQ ID NO: 31).
30 DETAILED DESCR~ION OF THE INVENTION
Mutations in the APP gene on ~hl----~ ---~ 21, the ApoE gene on CI1IUIIIOS(JII~ 19, and
the S182 or PS-I gene on chromosome 14 account for the majority of j(l. .llirl~l cases of early
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onset ~17h~imPr's disease (StlitLIllaLl et al. PNAS 1993, 90, 1977-1981; Sh~llillclc,n et al. Nature
1995, 375, 75~760). However, in the Volga-Gerrnan kindreds, and in several other families in
which AD appears to be inherited as an ~ n~ ."~ trait, these loci have been r~r~The Volga-German families are a culturally distinct ~ul~ulalion in Russia, whose5 Iwll~ did not rnarry into the Russian population. The relative onset of AD in this group is
eYreptinn~lly early, ranging from 50 to 70 years of age. However, clinically and pathologically,
AD in these families is ;,.,~ ;"~ hle from typical AD. The a~ltosom~l d locus,
~ onsibh, for AD in the Volga-German kindreds has been loc~li7P~l to ChI~ C~ P~ lq31~2
(Levy-Lahad et al. Science 1995, 269, 970-973). (~nl' ' genes which rnap to this locus have
10 also been i~ ntifi~ Levy-Lahad et al. isolated STM2 whose p~ t~d amino acid se~l"r..,l~e is
l c to that of S 182 (PS-l). A point 1 " ~ l, in STM2, resulting in the ~ub~ uliol- of an
icr,~ cin~ for an asparagine (N~4~I), was itlPntifi~l in affected individuals. This N,4,I mnt~ti~m
occurs at an arnino acid residue that is conserved in hurnan S182 and at the mouse S182 hom~'~g
Rogaev et al. reported the cloning of E5-1 on chrornosorne 1 which is also l-~ cc ~c to S182
(Nature 1995, 376, 775-778). Analysis of the mlrl~ti~l~ se~ r~re of the open reading frarne of
E5-1 (STM2) led to the discovery of two missense 5.~1~5~ c at conserved amino acid residues
in affected II~ of the Volga-German (N,4,I) and Italian (M~39V) p~licl~s.
In order to better el~, ' the structure of the PS-2 gene and to d - possible sites of
alternative splicing, the gene was cloned and s~ ~d and PCR was used to d~t; l.lille alternate
20 splice products (variants) and exon/intron b~ s. F~ ir,l, of intron/exon boundary
.r..r~s revealed that PS-2 is encoded by 10 coding exons. The PS-2 gene s~ r~.re was
deterrnined by both se.ll.r"~ g the EST seq ~~nre T03796 and isolating PS-2 cDNA's using the
GeneTrapper kit (Gibco BRL, Gailht;~ul~" MD).
The intron/exon structure of the PS-2 gene is shown in Table 1. Positions of introns that
25 interrupt the PS-2 cDNA are shown. Exonic se~l~nre is ~ ; -L~d in upper case and intronic
s~ in lower case letter. Exons are ~-wl~~red from the 5' end of the cDNA s~ ~enre
TABLE 1
E;XONl
(to-195) . CTTTTcccAAGGTcGcccAGgt~rg~t~t~ rcslg
EXON2 ~ k-lk~ CGAGGACGTGGGACTTCTCA
EXON2
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(-194 to -21) GCGGCCCCAAGTGTTCGTGGgt5cg~ttrAgArtrtrt
EXON3 tc~ cl~ nGTGCTTCCAGAGGCAGGGCT
EXON3
(-20 tO 140) GAGAGAACACTGCCCAGTGGgt~ cc~JrA~A~rtg
EXON4 c~c~At~ c~c~Ay~GAAGCCAGGAGAACGAGGA
EXON4
(141 to 355) ACAGAGAAGAATGGACAGCT~t~
EXON5 ~A~A~AA~Al~rA~t~ATcTAcAcGAcATTcAcTG
EXON5
(356 to 497) ACAAGTACCGCTGCTACAA(~lg~ c~ c~ cc
15 EXON6 Ç~ A~ GTTcATccATGGcTGGTTGA
EXON6
(498 to 565) TCACCTATATCTACCTTGGGtAA~A~tA~g~.A~A.
EXON7 A~cAr~ grAGAAAGTGCTCAAGACCTACA
EXON7
(566 to 786) GGGCGCCATCTCTGTGTATC.~A~I~.-A~
EXON8 a~atgt~ ~lgl~ l -GATCTCGTGGCTGTGCTGTG
EXON8
(787 to 885) CCCTGCCCTGATATACTCATgtg~cccccE;I~c
EXON9 AA~A-III.~ ~C~CTGCCATGGTGTGGACGGTT
EXON9
(886to969) CCCCTACGACCCGGAGATGGgt~
EXON10 ~ Arr~rA ~AGAAGAcTccTATGAcAGT
EXON10
(970 to 1071) GCTGGAGGAAGAGGAGGAAA~A~ ccAt~trAcA
EXONll t~t~rt~ctrAr~AgGTCAAGGGGGCGTGAAGCTT
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EXON11
(1072to 1190) CTTCGTGGCCATCCTCATTGt~g~t~ g~,
EXON12
S (1191 to3'end) ct(~ k~ ct~gGGCTTGTGTCTGACCCTCCT
cDNA s~u~n~ing revealed multiple positions at which mltlli(ms in sequence were
observed; four of which occurred at exon boun~Lies. A surnmary of this splicing data is provided
in Table 2.
TABLE 2
Splicing Event PCR cDNA Codons
exons 3 and 4 + W.U. #2, W.U. #15 1-119
exon 8 + W.U. #15 263-296
3 bp deletion - IB913 324
6 bp deletion - IB913 357-359
Splicing events in Table 2 are listed with respective cDNAs in which they were i-l~ntifi~l
as well as with cc)llr~ ldi-g arnino acid residues. Splicing events detected by RT-PCR are
denoted by a "+" in the PCR colurnn. All events are relative to the full length cDNA c10ne,
referred to as USF#15. Sirnplified ~ cLu,~ of the four cDNA clones are shown in Figure 1.
Three of the v~i~Liu"~ i-lr.-lir~l involve alternate splicing events. These include splicing
out of exon 3 and 4, and splicing in or out of exon 8 in the PS-2 gene. Amplifir~tinn by PCR over
the relevant b~u~lJ~ ies showed that these clones occurred in cDNA from all of the tested tissues.
No other alternate splicing was detected these tissues. The other two variations involved small
changes from the selr-r .~ bli~hed by Levy-Lahad et al. Science 1995, 269,973-977. These
changes include a deletion of a ~ residue at codon 324 and insertion of six base pairs
which change the encoded amino acid sr~ e from ERGV to ESQGG at codons 357-359.
In contrast, little evidence has been found for naturally oc.. i.. g alternate splicing in the
PS-l gene. The only alternate splicing previously reported for PS-I has been in exon 8 and at the
25 3' end of exon 3 resulting in clones with and without a VRSQ motif. While these splicing events
were also observed in PS-2, additional alternative splicing events were observed in PS-2 which
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were without c~,uivalence in PS-I. Some of these events lead to significant alterations in the
structure of the protein.
The rnost striking alternate ~ sclipt~ are those which lack exon 3 and 4. Variants
lacking exons 3 and 4 lose the normal start rnPJh: ~ - and if translated would be ~,.. ' : ' to begin
S at the methionine at codon 145 . This start site occurs after the proposed l~ c~ ~ ~r~ l ll ,l dile domain I
in the middle of L,;....~n,r...ll"d,le dom.ain 2. This protein is different than the Volga German AD
mutant N,4~I.
The i-lPntific-~tic n of these variants ir.dicates that certain ~ c in the PS-2 gene are
Ac.cori~tc~1 with the "Volga German" type of AD. Therefore, these variants may be useful in the
10 rl;~;"f~;c of AD or in the dc~. lO~IIr.l~l of models of AD.
The ic~ntifirAti. n of the intronic ~ u ~ c is also useful in the early dPJc-cti. n of variant
forms of the PS-2 gene. The genomic anal.ysis of the PS-2 gene (see Figure 1) has led to the
dc~el.~lllelll of a rnethod for iclr.,lir,.,.li..., of intronic poly"ul~ "~ which are predictive of
disease. F.lllrirlAtir)n, d~tr~tir~n, and .~ c of ....~ iu..c in both intronic se~l..r..rPi ~csori~
15 with splice variation and in the open reading frames proximal to these intron-exon buullJa.ies of
the PS-2 gene can be p~r~ ~d through use of intronic s~lllrl~rr.~. Tllr.llir~r~ n and analysis of
mutants or variants arising from ...--l ~ -.c in splice donor or acceptor sites are enabled by
hlv..l~dge of these intronic se~ e-~ Fullh~ , a . '~~ analysis of the intron-exon
buu-l~i~ makes possible ~,r<l~r-.-; ~ primers that would allow accurate s~lllr..-. e
of the first or last 10 to 20 1 ll ~ l ;llr~c of coding exons especially near cDNA termini.
At present there is no known effective therapy for the various forms of AD. However,
there are several other forms of dr~n~.ntiA for which ll~llllelll is available and which give rise to
plugl~si~e ll~hlAl delellulation closely l~sellll,lillg the dr~n~ntiA AC~; ''~ with Alzheimer's
disease. A rliagnl-ctir test for AD would therefore provide a useful tool in the rliaE;noci.C and
25 ll~lll~ll of these other c~ ;r~.C, by way of being able to exclude early onset Alzheirner's
&seace. It will also be of value when a suitable therapy for AD is available. There are several
mth~Ylr l~gies available from leCOIl~ lalll DNA t~hn~lccy which may be used for dele~iillg and
identifying genetic ....~l~ ti~ IèS~ ''~ for Al;~h~l~ disease. These include, but are not
limited to, direct probing, ligase chain reaction (LCR) and polymerase chain reaction (PCR)
30 mPth~ I~cY
Detection of variants or mutants using direct probing involves the use of olignn~lc!~ti~
probes which may be prepared synth.-Ji~Ally or by nick trAnCl~~inn. In a plerell~J embodiment, the
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probes are CO~ nt:~ry to at least a portion of the variant PS-2 genes i~lPnfifiP~ herein. The
DNA probes may be suitably labelled using, for exarnple, a ~ hPl enzyrne label, fluorescent
label, or biotin-avidin label, for slll.s~ vic~l~li7~ti~n in for exarnple a Southern blot
hybridization ~ll~;t:dUI~. The labelled probe is reacted with a sarnple of DNA from a patients
S ~u~;t~d of having AD bound to nitroce]llllr~s~P or Nylon 66 substrate. The areas that carry DNA
se l~r~ ec comple~ u y to the labeled DNA probe become labelled themselves as a c..~lg~lur~re
of the re~nnP~ling reaction. The areas of the filter that exhibit such labeling rnay then be
vic~l~li7~, for example, by autoradiography.
Alternative probe l: ' qllPC, such as ligace chain reaction (LCR) involve the use of a
10 1":~"~ h probe, i.e., probes which have full c ~ ~ ' ~ .d;.. ;Iy with the target except at the point of
the mutation or variation. The target sP~IIlP-nrP is then allowed to hybridize both with the
nlignnllrl~tirlPc having full c~ l...llr.l~dly, i.e., cti, ~ r~ collq,L.,~ ,y to the PS-2
variants of the present invention, and olignmlrl~oti-lPs containing a ~ "L~ , under cnn~ ionc
which will .li n;,~";cl, between the two. By u, ll~ting the reaction conditions, it is possible to
15 obtain hylL, ;.li".~ n only where there is full c~lllq~l~ .llr~ l ;Iy. If a ",:i"L~ h is present, then there
is significantly reduced hyl.. ;. li ,.-~ ;""
The polyrnerase chain reaction (PCR) is a 1~' qnP that aln~ s specific DNA
ce~ r~rP$ Repeated cycles of ~LIldluldli~ll, primer ~nnP~ling and ~ n~;on carried out with a heat
stable enzyme Taq polyrnerase leads to ~ontllLidl il-~as~ in the ~;~m~lltldli()ll of desired DNA
20 5~P~ "- PS
Given the knowledge of mlrlPotirlP seqllPnrpc encoding the PS-2 gene, it is possible to
prepare synthetic r~ omlcl~otit1Pc co ,l ~~t~ry to the selnr..,rPc which flank the DNA of
interest. Each oliE;."-~ lP is c~ ~ ' y to one of the two strands. The DNA is then
d~ -dul~d at high l~il~ldulcs (e.g., 95~C) and then rP~nnP~h~l in the presence of a large rnolar
25 excess of o!i~o.,.l.~ The oligcn lr!~ti~lec, oriented with their 3' ends pointing towards each
other, hybridize to opposite strands of the target sP~lPnrp and prirne enzymatic e;~lr~ I~ ,n along the
nucleic acid template in the presence of the four deoxyribnnllr!~tirl~P. l.i"h p~ The end
product is then d~..dulcd again for another cycle. After this thre~step cycle has been repeat
several times, an~l;r~=nl;on of a DNA segment by rnore than one rnillion fold can be achieved.
30 The resulting DNA may then be directly sr~ P~ ed in order to locate any genetic alterations.
Alternatively, the i-lPntifiP~ PS-2 variants of the present invention rnake it possible to prepare
oli~omlr!~tirlps that will only bind to altered DNA, so that PCR ~11 only result in the
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multiplication of the DNA if the mutation is present. Following PCR, allele-specific
t~lig~ml~lel~ti~l~ hybridization may be used to detect the AD point mllt~tirn
Alternatively, an ~ pt~ti-~n of PCR called amplification of specific alleles (PASA) can be
employed; this method uses dirrelcll~ial ~mrlifi~tirln for rapid and reliable ~ tin~tjon between
S alleles that differ at a single base pair. Newton et al. Nucleic Acid Res. 1989, 17, 2503; Nichols et
al. Genomics 1989, 5, 535; Okayama et al. J. Lab. Clin. Med. 1989, 1214, 105; Sarkar et al.
Anal. Biochem. 1990, 186:64; Somrner et al. Mayo Clin. Proc. 1989, 64, 1361; Wu Proc. Nat'l
Acad. Sci. USA 1989, 86, 2757; and Dutton et al. Biotechniques 1991, 11, 700. PASA involves
A."~,];rl~ n with two cli~ m~ oti~ primers such that one is allele specific. The desired allele is
~fr~ tly ~ lirled, while the other allele(s) is poorly ~- .q)l; r.~ because it 1~ .c with a base
at or near the 3' end of the allele specific primer. Thus, PASA or the related rnethod PAMSA can
be used to specifically amplify one or more mutant PS-2 alleles. Where such AmrlifirAtion is
pelrulllr3d on genetic material obtained from a patient, it can serve as a method of detecting the
presence of one or ~re mutant PS-2 alleles in a patient. PCR-induced mutation restriction
analysis, often referred to as IMR~ can also be used in the ~ t~tinn of mutants.Also ill4/UI~ t iS thedc~ l of r~pf~ models of Al~ disease. Such
models can be used to screen for agents that alter the de~,cllel~lh/e course of AD. Having i-l~.ntifi~
specific ~ n~ in the PS-2 gene as a cause of early onset familial Alzheimer's disease, it is
possible using genetic manipulation, to develop transgenic model systerns and/or whole cell
systerns cf-.~ g a mutated PS-2 gene or a portion thereof. The model systems can be used for
~,lcelfillg drugs and evaluating the efficacy of drugs in treating Al~ disease. In addition,
these rnodel systems provide a tool for defining the underlying l,~ "~ of PS-2 and its
1~ IA1;~ ;I' to AD thereby providing a basis for rational drug design.
- One type of cell system which can be used in the present invention can be naturally
derived. For this, blood samples from an affected individual are obtained and pclll~nt;lltly
rulll~ into a l~llyllul~' ~1 cell line using, for example, Epstein-Barr virus. Once
established, such cell lines can be grown c....l;...~oucly in ~ cultures and can be used in a
variety of invitroeApelllllel-ls to study PS-2 cAl lessiOll and processing. Another cell line used in
these studies c~nl4~ es skin rlbl~l~l~ derived from patients.
Since the FAD mllt~ti--n is dominant, an all~llldlivè rnethod for CUII~IILIelillg a cell line is
to gen~ti~Ally engineer a PS-2 mutated gene, or portion thereof, as dcsclibcd herein, into an
e~L~hed cell line of choice. Such methods are well known in the art as exemplified by Sisodia
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Science 1990, 248, 492 and Oltersdork et al. J. Biol. C~en~ 1990, 265, 4492, wherein an amyloid
precursor peptide gene was l~a~l~r~;~ed into n~rnm~ n cells.
Baculovirus ~,~yl~ ivll systems have also been found to be useful for high levelC;A~ Ssiull of h~.~l~gous genes in eukaryotic cells.
The mutated gene can also be excised for use in the creation of llall~O~llic anirnals
c.",l~;";~g the mutated gene. For example, a PS-2 gene of the present invention can be cloned and
placed in a cloning vector. Examples of cloning vectors which can be used include, but are not
~imited to, ICharon35, cosmid, or yeast artificial cl..v"~s(Jll,r The variant PS-2 gene can then be
llal~ cd to a host ~ IL~ ~ animal such as a mouse. As a result of the transfer, the resultant
10 transgenic n~l"."~" anirnal will preferably express one or more of the variant PS-2 polypeptides.
Alternatively, minigenes e,~ ;"g variant PS-2 polypeptides can be rlP.ci~nPA Such
minigenes may contain a cDNA se~r.r.l,~ e ~ -colil-g a variant PS-2 polypeptide, preferably full-
length, a combination of PS-2 exons, or a co.,l, .~inn thereof, linked to a do~ll~ a
polyadenylation signal s~u~nce and an u~ lulllvl~l (and preferably ~ hanc~l). Such a
rninigene cu~ u~;l will, when illllu~luc~d into an a~>l).u~ le transgenic host, such as a rnouse or
rat, express a variant PS-2 polypeptide.
One a~lua~,l- to creating Llal O - animals is to target a m-lt~tinn to the desired gene by
hl logollc l~ b laLiull in an embryonic stem (ES) cell in vitro followed by vlnje~lion of
the mQAifi~l ES cell line into a host blastocyst and ~b~ellllrlll in~nb~ti-~n in a foster rnother.
Frohrnan and Martin Cell 1989, 56, 145. Alternatively, the ~ e of microinjection of the
mutated gene, or portion thereof, into a one-cell embryo followed by ;"~ im~ in a foster mother
can be used. .A~itinnql methods for ~-v lu~ g llal~L ~ animals are well known in the art.
Tlansg - anirnals are used in the ~C.~ lnr~l of new Illel~;ulic cull4,o~ilivl,s and in
cal~ ..ngr.l ~ y testing, as ~r~ rl~d by U.S. Patent 5,223,610. These animals are also used in
25 the dcv~lopl~ of predictive anirnal models for human disease states, as exe~rlifi~d in U.S.
Patent5,221,778. Tl. sO ~animalshavenowbeendcvelo~dfor ~ r-~ g ~17h~imPr's&sease(U.S. Patent 7,769,626), multi-drug l~i~ ce to a~;r~ r, agents (U.S. Patent 7,260,827), and
carcino" ~ rec (U.S. Patent 4,736,866). Therefore, the PS-2 genes of the present
invention which are believed to cause early onset .Al7hPi~r's &sease in Cl~ . so"~ 14-linked
30 pe.li~ provide a useful means for developing ll ,, ~ anirnals to assess this disease.
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WO 97/38133 PCT/US97/04683
Site directed m l~gPnP.ci~ and/or gene conversion can also be used to a mutate a non
human PS-2 gene allele, either e,~ ge"~ 1y or via tlall~r~licJll, such that the mutated gene
encodes a polypeptide with an altered amino acid as described in the present invention.
In addition, ~ il)c ' to the PS-2 gene and variants thereof can be raised for use in the
5 examination of the function of the truncated llans~ of the PS-2 gene. These antibodies can be,
for P~ !P polyclonal or nnon~lon~l antibodies. The present invention also includes chirneric,
single chain, and 1l~ antibodies, as well as Fab fragrnents, or the product of an Fab
~sion library. Various l)r~lul~s known in the art may be used for the production of such
o~ and fragnnents.
Antibodies ge~ t~i against the PS-2 genes of the present invention can be obtained by
direct injection into an anirnal or by A~' ' ~ ~Pring the gene to an animal, pl~rtlably a nc,-l,.,.l~-,.
The antibody so obtained will then bind the PS-2 gene or itself. In this manner, even a fragment of
the gene can be used to generate thee antibodies.
For ~ iUII of lli~ l all~Odi~s, any l~', which provides antibodies
produced by c~ l;."l~ cell line cultures can be used. Examples include the hybridorna ~ lle
(Kohler and Milstein, Nature 1975, 256, 495497), the trioma t~rl-n;ql-P the hurnan B-cell
hybridorna technique (Kozbor et al., Immlmology Today 1983, 4, 72), and the EBV-hybridoma
' l to produce human l-K,~ Al ~tibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., 1985, pp.77-96).
T~ 3IIP~ llr~ for the production of single chain antibodies (U.S. Patent
4,946,778) can be adapted to produce single chain alltil,r ' to the PS-2 genes of this invention.
Also, t, ~ mice rnay be used to express l.~ ~ antibodies to the PS-2 genes of this
The following l - " py~ are provided to further illustrate the present invention.
EXAMPLES
Example 1: Cloning ~e PS-2 gene
PS-2 cDNA's were isolated using the Gene Trapper kit (Gibco BRL) acc~.d;..g to the
m~m-f~rtllrer's directions. A human SU~ " brain library (Gibco BRL, Gaithersburg, MD) in
30 pCMV.SPORT was probcd with the primer 5'-CATTCACTGAGGACACACCC-3' (SEQ ID
NO: I) (derived from the EST selllP.nl~e T03796). However, use of this sequence consi~ ly
(three times), resulted in the isolation of different clones which c--nt~inPd the 5' and 3' untranslated
- 11 -
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W O 97/38133 PCTAUS97/04683
gene s~ll~nrf-c but were missing the region of the gene around the putative start codon. The
library was ~ ~l,ed using the prirner 5'-CAAATACGGAGCGAAGACAG-3' (SEQ ID NO: 2),derived from the region omitted in the previous clones, again using the Gene Trapper kit. This
enabled the isolation of a clone containing the missing region.
Example 2: cDNA preparation
Human RNA from brain, he~t, liver, lung, placenta, and skeletal muscle was obtained
from Clontech (Palo Alto, CA). First strand cDNA was Sy~ ~ following the S~
Preamplifir~tinn System for First Strand cDNA Synthesis (Gibco BRL). Two mg of total RNA
was cull~ ed with 1 mg of r~lig~ cl~oti-~P (dT),2 ,8 prirner, 100 ng of random hexamer primer
and DEPC treated water in a 0.5 ml tube. Samples were ;..~ l~1 at 70~C for 10 rninutes and
placed on ice. Kit coll~llull~ were added in acc~.dance with the rn~mlf~rhlrer's protocol, and
samples were i ~ ~'ul ~. ~t~d at 37~C for 2 minutes. Su~~ t lI Reverse Tl~ls~l i~se (RT; 200 U)
was added and sarnples were inruh~t~d at 37~C for 1 hour. Samples were then i l.;LL ' at 70~C
for 15 minutes, chilled on ice, and 2 U of RNAse E~ was added. All sarnples were stored at -20~C.
For ~ll.se~ PCR, 1 ml of final product was used for each reaction.
Example 3: PCR to deternnne alternate splice products
Primers were designed throughout the cDNA s~ll--nr~ to allow the ~~ rc.~ r-ll of the
20 alternate splicing in a variety of tissues. Exonic pr~mers, along with the c~ ''t r,n,c for their use are
provided in Table 3. Intronic primers, along with the conditions for their use are provided in Table
4.
TABLE 3
Primer Name SEQ Se~ - (5'~3') 1 or~ff ~ ~ g
ID Temp.
NO (~C)
LP313F ~ 5 AGCCTGCTGAGAAGAAGAAACCA EXON 3 50
LPSOlF 6 AGGCAGGGCCCAGAGGATGGAGA EXON 4 50
GA
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Primer Name SEQ Sequence (5'-3') l.o~ Qn ~r~
ID Temp.
NO (~C)
LP676R 7 AGAGTGACAGGCACAAACAGCAT EXON5 50
LP71lF 8 ACCATCAAGTCTGTGCGCTTCTAC EXON5 50
LP861R 9 AGATGGTCATAACCACGATGACG EXON6 50
LP835F 10 TCAGCGTCATCGTGGTTATGACC EXON6 50
LP960R 11 AGGTAGATATAGGTGAAGAGGAA EXON7 50
LP921F 12 TCTTCACTGATGCTGCTGTTCC EXON7 59
LP1012R 13 AAGAGGGTGGGGTAGTCCATGGC EXON8 50
LP1081F 14 AGCAGGCCTACCTCATCATGATC EXON8 50
LP1254R 15 TCATTTCTCTCCTGGGCAGTTTC EXON9 50
LP1231F 16 TAGAAACTGCCCAGGAGAGAAAT EXON9 50
LP1290F 17 TGGTGTGGACGGTTGGCATGGCG EXON10 48
LP1334R 18 GGTCCAGCTTCGCCATGCCAACC EXON9 59
LP1460R 19 TCTTCCTCCAGCTCCTCCCCTGG EXONII 50
LP1380F 20 ATGACAGTTTTGGGGAGCCTTCA EXONII 50
LP1535R 21 TGGCAGCCGCCTTGCCCACCAGC EXON12 48
LP1820R 22 ACTAGAGTGTAAAACTATACAA EXON12 50
5;UTR 23 GCTTCTGTCTCAGGTTCCTTC 5'UTR 54
- FORWARD
5'UTR 24 CGGTGTTTGGCTG m TATCA 5'UTR 54
REVERSE
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PnmerN~ne SEQ Sequence(5'-3') T.or~ g
nD Temp.
NO (~C)
EXON 4 25 AGCCTCGAGGAGCAGTCAG S' EXON 4 49
INRTONIC
EXON4 26 GCAGACGGAGAGAAGGGT 3'EXON 4 49
INTRONIC
EXON7 27 GGGCAGGCTCTTCTTCAGGG 5'EXON7 57
INTRONIC
EXON 7 28 GAAAGCCACGGCCAGGAAG 3'EXON 7 57
INTRONIC
R05822F 29 TCACGGACAGGAAGCACAGC 5' EXON 12 56
R05822R 30 GTAACAAGAACAGGACTCAG 3' EXON 12 56
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TABLE 4
Primer SE Se~ (5'-3') Size Annealing
Name Q (bp) Temp.
ID (~C)
NO
53PS2X2 32 GAC TTG TGT CCA AGT CTC
33PS2X2 33 CTG TAA GGT ACA GTA GCC G 325
52PS2X3 34 AAA AAT CCG TGC ATT ACA T 50~C/30"
3PS2X3 35 GCT GGT GTG GAG CTG CAG GTA CAG 405
TG
SPS2X4 36 AGC CTC GAG GAG CAG TCA G 53~C/30"
3PS2X4 37 GCA GAC GGA GAG AAG GGT 240
SPS2X5 38 GGT ATC AGT CTC AGG ATC ATG GG 60~C/30"
3PS2X5 39 TGG GGA AGA CTG GAG CTC GAT G 263
52PS2X6 40 GTA AAG AGG GCC AGG TTG GG 53~C/30"
3PS2X6 41 GTG CAG CAC TGG GGA CGA TTT 360
52PS2X7 42 GGG CAG GCT CTT CTT CAG GG 60~C/30"
3PS2X7 43 GAA AGC CAC GGC CAG GAA G 255
SPS2X8 44 TTA GCA CCG CCT GAG ACG T 48~C/30"
3PS2X8 45 AGC TGG TCA GAG TGT TAC 510
32PS2X8 46 CCC CTC CTG AAC TCA TGC CT
SPS2X9 47 CTC TGA CCA GCT GTT GTT TC 57~C/30"
3PS2X9 48 AGC CTC CAC CCT CTG TCT 249
SPS2X10 49 TTC CAT TCT GTG CAC GCC TC 56~C/30"
3PS2X10 50 ACC TGC CCC CAC CAC AAT G 244
SPS2X11 51 ACA GCT CCT GTC CAC ACC A 53~C/30"
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W O 97/38133 PCT~US97/04683
Primer SE Sc4- e (5'-3') Si~ ~ '
Name Q (bp) Temp.
ID (~C)
NO
3PS2xl 1 52 ACT AGA GTG TAA AAC TAT ACA A 292
Aliquots of tissue cDNAs were amplified in a Perkin Elmer DNA Thermal Cycler 480(Perkin Elrner, Norwalk, CT). Each PCR reaction c~ .Pd I ml of final cDNA product, 25 pmol
of each primer (forward and reverse), 12.5 nmol of dNTP (Pl"~ Columbus, OH), 1.25 U of
S Taq polymerase (Promega, Madison, WI) for a total reaction volume of 50 ml overlaid with 60 ml
mineral oil (Fisher, Pi~ , PA). Primers used were designed to span at least two putative
intron/exon bul~ s of the PS-2 gene. For example, LP313F (forward, exon 2) and LP676R
(reverse, exon 4) were used to span exon 2/3 and exon 3/4 intron exon buundalies. For a given
reaction, sarnples were d~ tll~ at 94~C for S minutes. Samples then underwent 35 cycles of 0.5
10 minutes at 94~C, 0.5 minutes at the relevant ~nn~lin~ ll~;lalult; for the given primer pair, and
0.75 minutes at 72~C. This was followed by a final t;AL~Ilsiu" for 10 minutes at 72~C. Products
were visualiz_d on a 2% agarose gel (Promega) using ethidium brûmide staining. Product bands
were excised and purified using the Wizard PCR Preps DNA Purification System (Prornega). Five
rnicroliters of the final 50 ml purified product was used for 5e~ -g
Example 4: Sequencing for deternnnation of alternate splice p.~
PCR product was treated with EYnnn~lP~ce 1 and Shrimp Alkaline Ph-~rh~t~cP (PCR
lg kit-USB, Cleveland, OH). Five ""~ of PCR product was ;... ~ ~1 with I U ofEx.. ~ ~ 1 for 15 minutes at 37~C. The sample was then held at 80~C for 15 n~nutes after
20 which 2 U of Shrinnp Alkaline Pl.. !~l)h;.~P. was added. As before, the sarnple was held at 37~C
for 15 minutes and then at 80~C for 15 minutes. This final 7 rnl product was used in the
se~uencing protocol described in the Se~ r..~e PCR product S~lllr....-;..g Kit. The forward prirner
used in the original PCR reaction was used for manual s~ g
25 Example 5: PAC isolation
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P1 derived artificial cl..u.l,oscll~s (PACs) were isolated by s~lcc~ lg a gridded library
(Genome Systems, Inc.) with PCR products ~--yl~led with primers R05822F and R05822R.
Three PACs cn~ g the PS-2 gene were digested with Notl and sized using pulse field gel
d~:~lu~llulc~ia (PFGE). PAC DNA was run at 200 V for 21 hours at 14~C with switch times
S varying from 5-20 seconds. Sizes ranged from 90 kb to 110 kb. Primers used in the detection of
the 5' untranslated s~ .ce was 5'-GCTT(~'l'~'l'(l'l'CAG(i'l'l l'(~ l-l'C-3'(SEQ ID NO: 3) and 5'-
CG(~'l'~i'l'l'l'GG( :'l'(~'l'l'l'l'ATCA-3' (SEQ ID NO: 4). Int ronic prirners were used to detect
presence of exons 4 and 7. Prirners R05822F and R05822R were used to detect exon 12. PCR
reaction c.~".l;~in~-~ were as follows: 5 rninutes d~" alu dliull followed by 35 cycles of 94~C for 0.5
rninutcs, the respective ~nnP~l g lal~.dlulc for 0.5 minutes, and 72~C for 0.5 rninutes. A 10
rninute L.AlCllSiUII at 72~C c- ~ d~ each reaction. ~nnP~l;ng lr- ~ q~ e for each prirner pair are
provided in Table 2.
Exsnnple 6: T ' ~ i of ~ t,. boundalies
Exon/intron buull~ics were obtained by PCR/ligation lri l.";~lllP~ Purified PAC DNA
was digested with a variety of blunt-cutting enzyrnes and ligated to a .c~ifir:~lly designed linker.
S~uPn~Ps were then specifically amplified by PCR using a linker-derived prirner and a PS-2
derived prirner for boundary sPqlPn~ine S~-Pn~';n~ of products was pu roll~d directly in low-
rnelting point agarose by using a rnodified dideox~""~ lr. sP~ Pnrillg rnethod with a 32p end
labelled prirner and Taq DNA polyrnerase t~ll~ldtUl~ cycled reactions.
Example 7: Del~c~on of small sequence varia~o~
The Ph~ ALF rlaE;~ rnanager was used to detect srnall se~-lr~ e changes not
dt~ hlP by standard agarose gellethidium brornide vi.~ li7~inn Relevant cDNA sefl~lr..l~P.
25 were a-l4~1illed by PCR using a s'-nuol~c~l,l tagged primer.
CA 022228l3 l997-ll-28
WO g7/38133 PCT/US97/04683
SEQUENCE LISTING
~1) GENERAL INFORMATION:
(i) APPLICANTS:University of South Florida, Washington University and
The Institute of Genomic Research
(ii) TITLE OF INVENTION: Variant Presenilin-2 Genes
(iii) NUMBER OF SEQUENCES: 52
(iv) CORRESPONDENCE ADDRESS:
~A) ADDRESSEE: SmithKline Beecham Corporation
(B) STREET: 709 Swedeland Road
(C) CITY: King of Prussia
(D) STATE: PA
(E) COUNTRY USA
(F) ZIP: 19406
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE:
(B) COMPUTER:
(C) OPERATING SYSTEM:
~D) SOFTWARE:
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: not yet assigned
(B) FILING DATE: Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:60/014,860
(B) FILING DATE: March 4, 1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: William T. Han
(B) REGISTRATION NUMBER: 34,344
(C) REFERENCE/DOCKET NUMBER: ATG50001
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (610) 270-5219
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W O97/38133 PCT~US97/04683
(B) TELEFAX: (610) 270-5090
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20
(B~ TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
! ~D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CATTCACTGA GGACACACCC 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
~iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CAAATACGGA GCGAAGACAG 20
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
G~ CAGGTTTCTT C 21
~2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: Nucleic Acld
~C) STRANDEDNESS: Single
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(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CGGTGTTTGG c~ ATC A 2l
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AGCCTGCTGA GAAGAAGAAA CCA 23
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
AGGCAGGGCC CAGAGGATGG AGAGA 25
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 24
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
AGAGTGACAG GCACAAACAG CATG 24
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 24
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
ACCATCAAGT CTGTGCGCTT CTAC 24
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
AGATGGTCAT AACCACGATG ACG 23
(2) INFORMATION FOR SEQ ID NO: l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
TCAGCGTCAT C~lG~llATG ACC 23
(2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:
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WO 97/38133 PCTrUS97/04683
AGGTAGATAT AGGTGAAGAG GAA 23
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
TCTTCACTGA TGCTGCTGTT CC 22
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
AAGAGGGTGG GGTAGTCCAT GGC 23
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
~B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
AGCAGGCCTA CCTCATCATG ATC 23
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
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(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
TCATTTCTCT CCTGGGCAGT TTC 23
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENG?H: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
TAGAAACTGC CCAGGAGAGA AAT 23
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TGGTGTGGAC GGTTGGCATG GCG 23
(2) INFORMATION FOR SEQ ID NO: l8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
TGGTGTGGAC GGTTGGCATG GCG 23
(2) INFORMATION FOR SEQ ID NO: l9:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv~ ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l9:
TCTTCCTCCA GCTCCTCCCC TGG 23
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
ATGACAGTTT TGGGGAGCCT TCA 23
(2) INFORMATION FOR SEQ ID NO: 2l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:
TGGCAGCCGC CTTGCCCACC AGC 23
(2) INFORMATION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
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~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
ACTAGAGTGT AAAACTATAC AA 22
~2) INFORMATION FOR SEQ ID NO: 23:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 2l
~B) TYPE: Nucleic Acid
~C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
G~ ~l~l CAGGTTCCTT C 2l
(2) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 2l
~B) TYPE: Nucleic Acid
~C) sTR~Nn~nN~s: Single
~D) TOPOLOGY: Linear
~iv) ANTI-SENSE: NO
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:
CGG~ llGG ~ ATC A 2l
(2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
~D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
AGCCTCGAGG AGCAGTCAG l9
~2) INFORMATION FOR SEQ ID NO: 26:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 18
(B) TYPE: Nucleic Acid
-25-
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W O97/38133 PCTAUS97/04683
~C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:
GCAGACGGAG AGAAGGGT l8
(2) INFORMATION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:
GGGCAGGCTC TTCTTCAGGG 20
(2) INFORMATION FOR SEQ ID NO: 28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
GAAAGCCACG GCCAGGAAG l9
(2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
TCACGGACAG GAAGCACAGC 20
(2) INFORMATION FOR SEQ ID NO: 30:
-~6-
CA 02222813 1997-11-28
W O97/38133 PCT~US97/04683
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 20
(B) TYPE Nucleic Acld
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO 30:
GTAACAAGAA CAGGACTCAG 20
(2) INFORMATION FOR SEQ ID NO: 3l
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 2277
(B) TYPE Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION SEQ ID NO 31
GAATTCCCGG GTCGACCCAC GCGTCCGCGA GCGGCGGCGG AGCAGGCATT 50
TCCAGCAGTG AGGAGACAGC CAGAAGCAAG CTATTGGAGC TGAAGGAACC l00
TGAGA~.AA GCTAGTCCCC CCTCTGAATT TTACTGATGA AGAAACTGAG 150
GCCACAGAGC TAAAGTGACT TTTCCCAAGG TCGCCCAGCG AGGACGTGGG 200
ACTTCTCAGA CGTCAGGAGA GTGATGTGAG GGAGCTGTGT GACCATAGAA 250
AGTGACGTGT TAAAAACCAG CGCTGCCCTC TTTGAAAGCC AGGGAGCATC 300
ATTCATTTAG CCTGCTGAGA AGAAGAAACC AAGTGTCCGG GATTCAGACC 350
TCTCTGCGGC CCCAAGTGTT CGTGGTGCTT CCAGAGGCAG GGCTATGCTC 400
ACATTCATGG CCTCTGACAG CGAGGAAGAA ~l~l~lGATG AGCGGACGTC 450
CCTAATGTCG GCCGAGAGCC CCACGCCGCG ~lC~lGCCAG GAGGGCAGGC 500
AGGGCCCAGA GGATGGAGAG AACACTGCCC AGTGGAGAAG CCAGGAGAAC 550
GAGGAGGACG GTGAGGAGGA CCCTGACCGC TATGTCTGTA GTGGGGTTCC 600
CGGGCGGCCG CCAGGCCTGG AGGAAGAGCT GACCCTCAAA TACGGAGCGA 650
AGCACGTGAT CATGCTGTTT GTGCCTGTCA ~ GCAT GATCGTGGTG 700
GTAGCCACCA TCAAGTCTGT GCGCTTCTAC ACAGAGAAGA ATGGACAGCT 750
CATCTACACG ACATTCACTG AGr~rArArc CTCGGTGGGC CAGCGCCTCC 800
TCAACTCCGT GCTGAACACC CTCATCATGA TCAGCGTCAT CGTGGTTATG 850
-27-
CA 022228l3 l997-ll-28
W O97/38133 PCT~US97/04683
ACCATCTTCT TGGTGGTGCT CTACAAGTAC CGCTGCTACA AGTTCATCCA 900
TGGCTGGTTG ATCATGTCTT CACTGATGCT GCTGTTCCTC TTCACCTATA 950
TCTACCTTGG GGAAGTGCTC AAGACCTACA ATGTGGCCAT GGACTACCCC 1000
ACCCTCTTGC TGACTGTCTG GAACTTCGGG GCAGTGGGCA TGGTGTGCAT 1050
CCACTGGAAG GGCCCTCTGG TGCTGCAGCA GGCCTACCTC ATCATGATCA 1100
GTGCGCTCAT GGCCCTAGTG TTCATCAAGT ACCTCCCAGA GTGGTCCGCG 1150
TGGGTCATCC TGGGCGCCAT CTCTGTGTAT GATCTCGTGG CTGTGCTGTG 1200
TCCCAAAGGG CCTCTGAGAA TGCTGGTAGA AACTGCCCAG GAGAGAAATG 1250
AGCCCATATT CCCTGCCCTG ATATACTCAT CTGCCATGGT GTGGACGGTT 1300
GGCATGGCGA AGCTGGACCC CTCCTCTCAG GGTGCCCTCC AGCTCCCCTA 1350
CGACCCGGAG ATGGAAGAAG ACTCCTATGA CAGTTTTGGG GAGCCTTCAT 1400
ACCCCGAAGT CTTTGAGCCT CCCTTGACTG GCTACCCAGG GGAGGAGCTG 1450
GAGGAA&AGG AGGAAAGGGG CGTGAAGCTT GGCCTCGGGG ACTTCATCTT 1500
CTACAGTGTG CTGGTGGGCA AGGCGGCTGC CACGGGCAGC GGGGACTGGA 1550
ATACCACGCT GGCCTGCTTC GTGGCCATCC TCATTGGCTT ~~ ~l~ACC 1600
CTCCTGCTGC TTGCTGTGTT CAAGAAGGCG CTGCCCGCCC TCCCCATCTC 1650
CATCACGTTC GGGCTCATCT TTTACTTCTC CACGGACAAC CTGGTGCGGC 1700
CGTTCATGGA CACCCTGGCC TCCCATCAGC TCTACATCTG AGGGACATGG 1750
TGTGCCACAG GCTGCAAGCT GCAGGGAATT TTCATTGGAT GCAGTTGTAT 1800
AGTTTTACAC TCTAGTGCCA TATATTTTTA AGA~"l"l"l"l'~"l' TTCCTTAAAA 1850
AATAAAGTAC GTGTTTACTT GGTGAGGAGG AGGCAGAACC AGCTCTTTGG 1900
TGCCAGCTGT TTCATCACCA GACTTTGGCT CCCGCTTTGG GGAGCGCCTC 1950
GCTTCACGGA CAGGAAGCAC AGCAGGTTTA TCCAGATGAA CTGAGAAGGT 2000
CAGATTAGGG CG~JGr-~AAG AGCATCCGGC ATGAGGGCTG AGATGCGCAA 2050
AGAGTGTGCT CGGGAGTGGC CCCTGGCACC TGGGTGCTCT GGCTGGAGAG 2100
GAAAAGCCAG TTCCCTACGA GGAGTGTTCC CAATGCTTTG TCCATGATGT 2~50
CCTTGTTATT TTATTGCCTT TAGAAACTGA GTC~ TGTTACGGCA 2200
GTCACACTGC TGGGAAGTGG CTTAATAGTA ATATCAATAA ATAGATGAGT 2250
CCTGTTAGAA AAAGCGGCCG CTCTAGA 2277
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: Nucleic Acid
-28-
CA 02222813 1997-11-28
W O97138133 PCTAUS97/04683
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
GACTTGTGTC CAAGTCTC l~
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
CTGTAAGGTA CAGTAGCCG l9
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
AAAAATCCGT GCATTACAT l9
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
GCTGGTGTGG AGCTGCAGGT ACAGTG 26
(2) INFORMATION FOR SEQ ID NO: 36:
-29-
CA 022228l3 l997-ll-28
WO 97138133 I'CTIUS97104683
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
AGCCTCGAGG AGCAGTCAG 19
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18
~B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GCAGACGGAG AGAAGGGT 18
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GGTATCAGTC TCAGGATCAT GGG 23
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
- 30 -
CA 022228l3 l997-ll-28
WO 97138133 PCT/US97/04683
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3g:
TGGGGAAGAC TGGAGCTCGA TG 22
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GTAAAGAGGG CCAGGTTGGG 20
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: Nuclelc Acid
(C) STRANDEDNESS: Single
(D) TOPO~OGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
GTGCAGCACT GGGGACGATT T 21
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GGGCAGGCTC TTCTTCAGGG 20
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: Nucleic Acid
- 31 -
CA 02222813 1997-11-28
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~C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
GAAAGCCACG GCCAGGAAG l9
~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TTAGCACCGC CTGAGACGT l9
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l8
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: ~inear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
AGCTGGTCAG AGTGTTAC l8
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
CCCCTCCTGA ACTCATGCCT 20
(2) INFORMATION FOR SEQ ID NO: 47:
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CA 02222813 1997-11-28
WO 97/38133 PCT/US97/04683
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
- (D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
CTCTGACCAG ~ "l'C 20
(2) INFORMATION FOR SEQ ID NO: 48:
UU~N~ CHARACTERISTICS:
(A) LENGTH: 18
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
AGCCTCCACC CTCTGTCT 18
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
TTCCATTCTG TGCACGCCTC 20
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
-33-
CA 022228l3 l997-ll-28
W O 97/38133 PCTrUS97/04683
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
ACCTGCCCCC ACCACAATG 19
(2) INFORMATION FOR SEQ ID NO: 51:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19
~B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
ACAGCTCCTG TCCACACCA 19
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
ACTAGAGTGT AAAACTATAC AA 22
- 34-
