Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACIDS AND PROTEINS
R~LATED TO ~T 71:~F.Tl~IER'S DISEASE,
AND USE~ THEREFOR
Field of the Invention
The present invention relates generally to the field of neurological and
physiological dysfunctions associated with Alzheimer's Disease. More particularly,
the invention is concerned with the identification, isolation and cloning of genes
which are associated with Alzheimer's Disease, as well as their corresponding
transcripts and protein products. The present invention also relates to methods for
detecting and diagnosing carriers of normal and mutant alleles of these genes, to
methods for tietecting and diagnosing Alzheimer's Disease, to methods of identifying
other genes and proteins related to, or interacting with, the genes and proteins of the
invention, to methods of screening for potential therapeutics for Alzheimer's Disease,
to methods of treatment for Alzheimer's Disease, and to cell lines and animal models
useful in sclc~nil~g for and ev~ ting potentially useful therapies for Alzheimer's
Disease.
Back~round of the Invention
Alzheimer's Disease (AD) is a degenerative disorder of the human central
nervous system characterized by progressive memory;~p~;....ent and cognitive andintellectual decline during mid to late adult life (K~t7m~n, 1986). The disease is
accompanied by a constellation of neuro-pathologic Çe~Lul~s principal amongst which
are the presence of extracellular amyloid or senile plaques, and neurofibrillary tangles
in neurons. The etiology of this disease is complex, although in some families it
appears to be inherited as an autosomal dominant trait. Linkage studies have
identified three genes associated with the development of AD~ amyloid precursor
protein (APP) (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell et al., 1991;
Karlinsky et al., 1992; Mullan et al., 1992), prt?s~nil;n-l (PS-1) (Sherrington, 1995),
and presenilin-2 (PS-2) (Rogaev, 1995, and Levy-Lahad, 1995).
The presenilins are multi-sp~nning membrane proteins which were described
in substantial detail in PCT Publication W096/34099, the entire disclosure of which
is incorporated herein by reference. Although the functions of the presenilins are
unknown, a number of autosomal dolllhl~ll presenilin mutations have been identified
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which are strongly associated with the development of early-onset, a~ essive,
Familial Alzheimer's Disease (FAD).
The present disclosure describes the identification, isolation, sequencing and
characterization of several human genes which interact with the presenilins, mutations
5 in which may lead to FAD. These prçsP!nilin-interacting protein genes may be
involved in the pathways which, when affected by mutant presenilin~, lead to thedevelopment of ~17heim~r's Disease. In addition, mutations in the presP.nilin-
interacting protein genes, even in the absence of defects in the pres~nilin~, may be
causative of ~l~heimer's Disease.
Summarv of the Invention
The present invention is based, in part, upon the identification, isolation,
seql~çncing arld characterization of several hurnan genes, referred to herein ~
"presenilin~ ,LdcLillg protein genes" or "PS-interacting protein genes." The products
of these genes are believed to interact in vivo with the human pres~.nilin-l proteins
15 and, therefore, are implicated in the bioeh~mic~l pathways which are affected in
Al7h~im~r's Disease. Each of these genes, therefore, ples~llL~ a new therapeutic target
for the treatrnent of ~ heimer's Disease. In addition, PS-interacting protein nucleic
acids, PS-interacting proteins and peptides, antibodies to the PS-interacting proteins,
cells L~ o~ ed with PS-interacting protein nucleic acids, and transgenic ~nim~l~20 altered with PS-interacting protein nucleic acids, all possess various utilities, as
described herein, for the ~i~gnosi~ therapy and contimled investigation of ~l~hejmer's
Disease and related disorders.
Thus, it is one object of the invention to provide isolated nucleic acids
encoding at least a PS-interacting domain of a PS-interacting protein. These PS-
25 interacting proteins include m~mm~ n S5a subunits of the 26S proteasome, theGT24 protein, the pO07 1 protein, the Rab l l protein, the retinoid X receptor-~, the
cytoplasmic chaperonin, and several sequences identified herein as clones Y2H35,Y2H171, and Y2H41. Preferred nucleotide and amino acid sequences are provided
herein. It is another object of the invention to provide probes and primers for these
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PS-interacting protein genes, and to provide nucleic acids which encode small
antigenic det~-.-,i"~"l~ ofthese genes. Therefore, preferred embollim~nt~ include
sequences of at least 10, 15 or 20 consecutive nucleotides selected from the disclosed
se~uences.
Using the nucleic acid sequences and antibodies disclosed and enabled
herein, methods for identifying allelic variants or heterospecific homologues of a
human PS-interacting protein and gene are provided. The methods may be practicedusing nucleic acid hybridization or amplification techniques, immllnoch~mic:~l
techniques, or any other technique known in the art. The allelic variants may include
other nor~nal human alleles as well as mutant alleles of the PS-interacting protein
genes which may be causative of Alzheimer's Disease. The heterospecific
homologues may be from other m~mm~ n species, such as mice, rats, dogs, cats or
non-hurnan primates, or may be from invertebrate species, such as Drosophila or C.
ele~ans. Thus, it is another object of the invention to provide nucleic acids which
encode allelic or heterospecific variants of the disclosed sequences, as well as the
allelic or heterospecific proteins encoded by them.
The it another object of the invention to provide vectors, and particularly
cs:iion vectors, which include any of the above-described nucleic acids. ~t is afurther object of the invention to provide vectors in which PS-interacting protein
nucleic acid sequences are operably joined to exogenous regulatory regions to produce
altered patterns of ~ es~ion, or to exogenous coding regions to produce fusion
proteins. Conversely, it is another object to provide nucleic acids in which PS-interacting protein regulatory regions are operably joined to exogenous coding
regions, including standard marker genes, to produce constructs in which the
regulation of PS-interacting protein genes may be studied and used in assays fortherapeutics.
It is another object of the invention to provide host cells and transgenic
~nim~l~ which have been transformed with any of the above-described nucleic acids
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of the invention. The host cells may be prokaryotic or eukaryotic cells and, in
particular, may be garnetes, zygotes, fetal cells, or stem cells useful in producing
transgenic animal models. ~.
In particularly plbr~lled embodiments, the present invention provides a
non-human animal model for Alzheimer's Disease, in which the genome of the
animal, or an ancestor thereof, has been modified by at least one recombinant
construct which has introduced one of the following modifications: ( l ) insertion of
nucleotide sequences encoding at least a functional domain of a heterospecific normal
PS-interacting protein, (2) insertion of nucleotide sequences encoding at least a
1~ functional domain of a heterospecific mut~nt PS-interacting protein, (3) insertion of
nucleotide sequences encoding at least a functional domain of a conspecific
homologue of a heterospecific mutant PS-interacting protein, and (4) inactivation of
an endogenous PS-interacting protein gene. Preferred transgenic animal models are
rats, mice, h~m~ters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-
human prim~t~s, but in~ tes are also contemplated for certain utilities.
It is another object of the invention to provide methods for producing at
least a fi~nctional domain of a PS-interacting protein using the nucleic acids of the
invention. In addition, the present invention also provides subst~nti~lly pure
preparations of such proteins, including short peptide sequences for used as
2û immunogens. Thus, the invention provides peptides comprising at least 10 or l 5
consecutive amino acid residues from the disclosed and otherwise enabled sequences.
The invention filrther provides subst~nti~lly pure plcp~lions of peptides which
compnse at least a PS-interacting domain of a PS-interacting protein, as well assubstantially pure pr~al~ions of the entire proteins. .,
Using the substantially pure peptides and proteins enabled herein, the .
invention also provides methods for producing antibodies which selectively bind to a
PS-interacting protein, as well as cell lines which produce these antibodies.
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Another object of the present invention is to provide methods of
identifying compounds which may have utility in the treatment of Al7heimer's
Disease and related disorders. These methods include methods for identifying
compounds which can modulate the c~ e:,sion of a PS-interacting protein gene,
5 methods for identifying compounds which can selectively bind to a PS-interacting
protein, and methods of identifying compounds which can modulate activity of a PS-
interacting protein. These methods may be con~ cte(l in vitro or in vivo~ and may
employ the transformed cell lines and transgenic animal models of &e invention. The
methods also may be part of a clinical trial in which compounds identified by the
10 methods of the invention are further tested in human subjects.
It is another object of the invention to provide methods of ~ no~ing or
screening for inherited forrns of ~l~heimer's Disease by dcl~ if a subiect bears
a mutant PS-interacting protein gene. Mutant PS-interacting genes may be detectec~
by assays including direct nucleotide seq~ cin~, probe specific hybri(li7~tion~
15 restriction enzyme digest and mapping, PCR mapping, ligase-m.o~i~tç~l PCR
detection, RNase protection, electrophoretic mobility shif~ detection, or chemic~l
mi~m~trh cleavage. ~lt~rn~tively, mutant forms of a PS-interacting protein may be
detected by assays including imml-no~ays, protease assays, or electrophoretic
mobility assays.
It is also an obJect of the invention to provide pharmaceutical ~J~c~ Lions
which may be used in the tre~tn Pnt of Alzheimer's Disease and related disorderswhich result from aberration in biochemic~ lW~S involving the PS-interacting
proteins disclosed and enabled herein. Thus, the present invention also providespharmaceutical preparations co~ ing a ~ubsl~llially pure PS-i~ ;ldcli"g protein, an
expression vector operably encoding a PS -interacting protein, an expression vector
operably encoding a PS-interacting protein ~nticrn.~e sequence, an antibody which
selectively binds to a mutant PS-interacting protein, or an antigenic f let~ n~nt of a
mutant PS-interacting protein. These ph~ re~ltical preparations may be used to
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treat a patient bearing a mutant PS-interacting protein gene which is causative of ..
Alzheimer's Disease or related disorders.
These an other objects of the present invention are described more fully in
the following specification and appended claims.
Detailed DescriPtion of the Invention
I. Definitions
In order to facilitate review of the various embo~1imentc of the invention,
and an underst~n(1ing of the various elements and con.ctitl~pMt~ used in making and
using the invention, the following definitions are provided for particular terms used in
10 the description and appended claims:
Presenilin. As used without filrther modification herein, the terrns
"preserlilin" or 'presenilins" mean the pr~?s~onil;n-l (PS 1) and/or the presenilin-2 (PS2)
genes/proteins. In particular, the unmodified terms "presenilin" or "presenilin.c" refer
to the m~mm~ n PS 1 and/or PS2 genes/proteins and, ~ref~.dbly, the human PS l
15 and/or PS2 genes/proteins as described and disclosed in PCT Publication
W096/34099.
Norrnal. As used herein with respect to genes, the terrn "normal" refers to
a gene which encodes a no~nal protein. As used herein with respect to proteins, the
term "normal" means a protein which perforrns its usual or normal physiological role
20 and which is not associated with, or causative of, a pathogenic condition or state.
Therefore, as used herein, the terrn "normal" is e~ t~ y synonymous with the usual
meaning of the phrase "wild type." For any given gene, or corresponding protein, a
multiplicity of normal allelic variants may exist, none of which is associated with the
development of a pathogenic condition or state. Such norrnal allelic variants include,
25 but are not limited to, variants in which one or more nucleotide substitutions do not
result in a change in the encoded amino acid sequence.
Mutant. As used herein with respect to genes, the term "mutant" refers to
a gene which encodes a mutant protein. As used herein with respect to proteins, the
term "Illu~ " means a protein which does not perform its usual or norrnal
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physiological role and which is associated with, or causative of, a pathogerlic
condition or state. Therefore, as used herein, the term "mutant" is essentially
synonymous with the terms "dysfunctional," "pathogenic," "disease-ca11~ing," and"deleterious." With respect to the presenilin and pres~niiin-interacting protein genes
5 and proteins of the present invention, the terrn "mutant" refers to genes/proteins
bearing one or more nucleotide/amino acid substitutions, insertions and/or deletions
which typically lead to the development ofthe syrnptoms of ~17heimer's Disease
and/or other relevant inheritable phenotypes (e.g. cerebral hemorrhage, mental
retardation, schizophrenia, psychosis, and depression) when expressed in h11m~n~10 This definition is understood to include the various mutations that naturally exist,
including but not limited to those disclosed herein, as well as synthetic or recombinant
mutations produced by human illlt;- v~ tion. The term "rnutant," as applied to these
genes, is not int~n~ie-l to embrace sequence variants which, due to the degeneracy of
the genetic code, encode proteins icl~ntic:~l to the normal sequences disclosed or
15 otherwise enabled herein; nor is it int~n(led to embrace sequence variants which,
although they encode different proteins, encode proteins which are functionally
equivalent to normal proteins.
SubstantiallY Pure. As used herein with respect to proteins (including
antibodies) or other pl~dlions, the term "~j..l.s~ lly pure" means that the
20 preparation is ess~nti~lly free of other substances to an extent practical and
~r~iate for its int~nded use. In particular, a protein ~ d~ion is ~u1 ~ 1y
pure if it is sufficiently free from other biological constituents so as to be useful in, for
example, generating antibodies, sequencing, or producing pha~naceutical
pL~aldlions. By techniques well known in the art, ~ul~ lly pure proteins or
25 peptides may be produced in light of the nucleic acid and amino acid sequences
disclosed herein. In particular, in light of the nucleic acid and arnino acid sequences
disclosed herein, one of ol-;lh~a. y skill in the art may, by application or serial
application of well-known methods including HPLC or imm11nQ-affinity
chromatography or electrophoretic separation, obtain proteins or peptides of any30 generally feasible purity. Preferably, but not necessarily, "subst~nti~lly pure"
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dLions include at least 60% by weight (dry weight) the compound of interest.
More preferably the preparation is at least 75% or 90%, and most preferably at least
99%, by weight the compound of interest. Purity can be measured by any app~ iatemethod, e.g., column chromatography, gel electrophoresis, or HPLC analysis. With5 respect to proteins, including antibodies, if a L)le~dlalion includes two or more
different compounds of interest (e.g., two or more different antibodies, imrnunogens,
functional domains, or other polypeptides of the invention)~ a "~ub~ ly pure"
~ ;p~Lion is preferably one in which the total weight (dry weight) of all the
compounds of interest is at least 60% of the total dry weight. Similarly, for such
10 ~ preparations co.-lzt;.,i~-g two or more compounds of interest, it is preferred that the
total weight of the compounds of interest be at least 75%, more plerel~ly at least
90%, and most preferably at least 99%, of the total dry weight of the preparation.
Finally, in the event that the protein of interest is mixed with one or more other
proteins (e.g., serum albumin) or compounds ~e.g., flill1~nt~ excipients, salts,15 polys~c~-h~ri(1es~ sugars, lipids) for purposes of ~lmini~tration~ stability, storage, and
the like, such other proteins or compounds may be ignored in calculation of the purity
of the ~,lep~lion.
Isolated nucleic acid. As used herein, an "isolated nucleic acid" is a
ribonucleic acid, deoxyribonucleic acid, or nucleic acid analog comprising a
20 polynucleotide sequence that is isolated or separate from sequences that are
imme~ tely contiguous (one on the 5' end and one on the 3' end) in the naturallyoccurring genome of the organism from which it is derived. The te~n thel~,role
includes, for example, a recombinant nucleic acid which is incorporated into a vector,
into an autonomously replicating plasmid or virus, or into the genomic DNA of a
25 prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a
genomic DNA fragment produced by PCR or restriction endonuclease trç~tment)
independent of other sequences. It also includes a recombinant DNA which is part of
a hybrid gene encoding additional polypeptide sequences and/or including exogenous
re~gulatory elements.
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SubstantiallY identical sequence. As used herein, a "subst~nti~ly
identical" amino acid se~uence is an amino acid sequence which differs only by
constl ~aLive amino acid substitutions, for example, substitution of one amino acid for
another of the sa~ne class ~e.g., valine for glycine, a~ginine ~or lysine, etc.) or by one
5 or more non-conservative substitutions, deletions, or insertions located at positions of
the amino acid sequence which do not destroy the function of the protein (assayed,
e.g., as described herein). Preferably, such a sequence is at least 85%, more
preferably 90%, and most preferably 95% identical at the amino acid level to thesequence of the protein or peptide to which it is being compared. For nucleic acids,
10 the length of comparison sequences will generally be at least 50 nucleotides,preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most
preferably l 10 nucleotides. A "S11~St~nt~ Y identical" nucleic acid sequence codes
for a ~ub~ lly identical amino acid sequence as defined above.
Transformed cell. As used herein, a "l.~lsrO,ll-ed cell" is a cell into which
15 (or into an ~noçstor of which) has been introduced, by means of recombinant DNA
techniques, a nucleic acid molecule of interest. The nucleic acid of interest will
typically encode a peptide or protein. The transformed cell may express the sequence
of interest or may be used only to propagate the sequence. The term "transformed"
may be used herein to embrace any method of introducing exogenous nucleic acids
20 including, but not limited to, LLdl1~rOI ~ tion~ transfection, electroporation,
microinjection, viral-mediated transfection, and the like.
OperablY ioined. As used herein, a coding sequence and a regulatory region are
said to be "operably joined" when they are covalently linked in such a way as to place
the ~ s:jion or transcription of the coding sequence under the inflli~n~e or control
25 of the regulatory region. If it is desired that the coding sequences be translated into a
fimctional protein, two DNA sequences are said to be operably joined if induction of
promoter function results in the transcription of the coding se~uence and if the nature
of the linkage between the t~vo DNA sequences does not (l) result in the introduction
of a frame-shift mutation, (2) h~ r~- e with the ability of the regulatory region to
30 direct the ~ scliption of the coding sequences, or (3) interfere with the ability of the
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corresponding RNA transcript to be tr~n~l~te~l into a protein. Thus, a regulatory
region would be operably joined to a coding sequence if the regulatory region were
capable of effecting transcription of that DNA sequence such that the resulting
transcript might be tr~n~l~ted into the desired protein or polypeptide.
Strin~ent hYbridization conditions. Str-ngPnt hybridization conditions is a termof art understood by those of ordinary skill in the art. For any given nucleic acid
sequence, slringpnt hybridization conditions are those conditions of temperature,
chaotrophic acids, buffer, and ionic strength which will permit hybridization of that
nucleic acid sequence to its complementary sequence and not to subst~nti~lly different
10 sequences. The exact conditions which constitute "stringent" conditions, depend upon
the nature of the nucleic acid sequence, the length of the sequence, and the frequency
of occurrence of subsets of that sequence within other non-identical sequences. By
varying hybridization conditions from a level of stringency at which non-specific
hybridization occurs to a level at which only specif~c hybridization is observed, one of
15 ordinary skill in the art can, without undue ~AI,C. j..-~nt~tion, cletermine conditions
which will allow a given sequence to hybridize only with complementary sequences.
Suitable ranges of such stringency conditions are described in Krause and Aaronson
(1991). Hybridization conditions, depending upon the length and commonality of asequence, may include tempe~ s o~20~C-65~C and ionic strengths from Sx to O.lx
20 SSC. Highly stringent hybridization conditions may include temperatures as low as
40-42~C (when d~l~Lu~ such as r~ ,alllide are included) or up to 60-65~C in ionic
strengths as low as O.lx SSC. These ranges, however, are only illustrative arld,depending upon the nature of the target sequence, and possible future technological
developments, may be more stringent than nt?ce~s~ry. Less than stringent conditions
25 are employed to isolate nucleic acid sequences which are substarltially similar, allelic
or homologous to any given sequence.
Selectivelv binds. As used herein with respect to antibodies, an antibody
is said to "selectively bind" to a target if the antibody recognizes and binds the target
of interest but does not substantially recognize and bind other molecules in a sample,
30 e.g., a biological sample, which includes the target of interest. That is, the antibody
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must bind to its target with sufficient specificity so as to distingt1ish the target from
r essentially all of molecules which would reasonably be present in a biological sample
including the target.
II. The Presenilins and Pres~nilin-Interactin~ Proteins
The present invention is based, in part, upon the discovery of a family of
m~mm~ n genes which, when ml7t~te-1, are associated with the development of
Alzheimer's Disease. The discovery of these genes, designated presenilin- 1 (PS 1 ) and
presP.nilin-2 (PS2), as well as the chara~ tion of these genes, their protein
products, Illul~ v~lle~ldLehoInologues~ and possible functional roles, are
described in PCT Publication W096/34099. The present invention is further based, in
part, upon the discovery of a group of proteins which interact with the pres~nilin~
under physiological conditions and which, therefore, are believed to be involved in the
biochemical pathways which are altered in .Al7heimçr's Disease. These proteins are
referred to herein as presenilin-interacting (PS-interacting) proteins. Because
mutations in the presenilin~ are known to be causative of Alzheimer's Disease, each of
the PS-interacting genes and proteins disclosed and described herein presents a novel
tOEget for therapeutic intervention in ~l7heim~r's Disease. That is, modulation of the
hllela-;Lions of these proteins with the pre~s~nilin~, or modulation of the interactions of
at least the PS-interacting domains of these PS-int~r~.ting proteins with at least the
inter~ctin~ domains of the presenilins, provides a means of mod~ ting the activity
and/or availability of the prçs~nilin~, or of mofl~ ting the activity and/or availability
of the PS-interacting proteins. Furthennore, as aberrations in the interactions of
mutant pres~nilin~ with one or more of these PS-interacting proteins is causative of
Alzheimer's Disease, mutations in one or more of these PS-interacting proteins are
also likely to be causative of ~ hejrner's Disease. Therefore, each of the PS-
interacting genes and proteins disclosed and described herein presents a novel target
for diagnosis of forms of .f~mili~tl and/or sporadic Alzheimer's Disease with anetiology independent of mutations in the pres.o.nilin~. Finally, as described more fully
below, the PS-interacting genes and proteins described and disclosed herein provide
for new assays for compounds which affect the interactions of the presenilins and PS-
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interacting proteins, assays for other members of the biochemical pathways involved
in the etiology of Alzheimer's Disease, and new cell lines and transgenic animalmodels for use in such assays.
5 1. Presenilin Processin~
Employing the antibodies and protein-binding assays described and/or
enabled in PCT Publication W096/34099, the processing and protein-protein
interactions of both normal and mutant presenilins were investigated. It was found
that mutations in the presenilins appear to lead to changes in both their intracellular
10 processing (e.g, endoproteolytic cleavage, ubiquitination, and clearance) and their
intracellular interactions with other proteins expressed in human brain. As described
below, knowledge of presenilin proce~in~ and interactions, and particularly changes
in mutant pres~nilin processing and interactions, provides for new diagnostic and
therapeutic targets for ~l7h~imer's Disease and related disorders.
Western blot analysis s~ Pst~ that the normal prPs~nilin~ undergo
proteolytic cleavage to yield characteristic N- and C-tennin~l fr~ment~ As notedabove, the normal presenilin proteins have an expected molecular mass of 47-51 kDa
depending, in part, upon rnRNA splice variations, electrophoretic conditions, etc.
Analysis of Western blots SllE~geSt~, however, that the normal presPnilin proteins
undergo proteolytic cleavage to yield an approximately 35 kDa N-~Pnnin~l fragment
and an approximately 18 kDa C-tPnnin~l fr~nPnt In particular, Western blots
bearing lysates ~om wild-type native human fibroblasts, human neocortical brain
tissue from control subjects, and neocortical brain tissue from non-transgenic and PSl
transgenic mice using antibodies ("14.2") recognizing PS1-specific residues 1-25 at
~5 the N-terminu~ reveal the presence of a strong immlm-reactive band of approximately
35 kDa and, after longer exposures, a weaker band of approximately 45 kDa which
presumably represents the full-length PS1 protein. Antibodies ("520"~ directed at
residues 304-318 at the apex of the TM6~7 loop of PS 1, and antibodies ~"4627")
directed at residues 457-467 in the ~-t~ormin-~c of PS1, both recognize the same strong
band of approximately l 8 kDa. Antibodies 520 also recognize a weak band of 45 kDa
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coincident with the PSI band detected by 14.2. SeqllPncing ofthe major C-terminal
fragrnent from PS 1-kansfected human embryonic kidney cells (EIEK 293) showed
that the principal endoproteolytic cleavage occurs near M298 in the proximal portion
of the TM6~7 loop, possibly by enzymes other than the proteasome. These
5 observations suggest that an endoproteolytic cleavage event occurs near the junction
of exons 9 and 10 of PS 1. Full length PS 1 in these cells is quickly turned over (tl,
60 min.) by the proteasome.
To ~let~lmine whether mutations in the presenilin proteins result in
alterations of their proteolytic cleavage, Western blots co~ lysates of fibroblast
10 and neocortical brain homogenates from normal subjects and subjects carrying PS1
mutations were investigated with the PS1 specific antibody Ab 14.2. In fibroblasts,
there were no obvious di~ ces in the relative int~n.cities of the protein bands when
Iysates from heterozygous carriers of the PS 1 mutations were cu~ art;d with normal
homozygotes. In contrast, there appeared to be a difiference between PS 1 mutation
15 carriers and nnnn~lc in homo~enates of temporal neocortex from AD affected
heterozygous carriers of either the PSl A246E or C410Y mutations (which are located
in TM6 and TM7 respectively). In heterozygotes, a strongly i..,."l-"~reactive band of
approximately 45 kDa was detecte :1 which initially appeared to correspond to the full-
length PS 1 protein. Further analysis, however, revealed that this band ~cpres~ s an
20 ~lt~.rn~tively processed presenilin product. A similar band corresponding to this
mutant processed PS 1 was observed in neocortical homogenates from some sporadiclate-onset AD patients. These data suggest that (1 ) some pathogenic PS 1 mutations
associated with early-onset AD alter the way in which the presenilins are processed
through endoproteolytic and proteasome pathways and (2) the presenilin proteins, and
25 charlges in the processing of the presenilins in the brain, are also implicated in late-
onset and sporadic AD.
2. Presenilin-Interacting Proteins
In order to identif~ proteins which may bind to or otherwise interact with
30 the presenilins in vivo. a yeast two-hybrid system was used as described below
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(Example 1). In particular, because mutations in the TM6~7 loop domains are
known to be causative of AD, a yeast two-hybrid system was used to identify cellular
proteins which may interact with normal and mutant pres~nilin TM6~7 loop
domains. Yeast two-hybrid studies were also done with cDNAs corresponding to the5 C-te~rnin~l 18 kDa endoproteolytic cleavage fragment, and with cDNAs
corresponding to the TM1~2 intr~ min~l loop domain, which is also the site oftheFAD associated Y1 15H missense mutation. In brief, cDNA sequences encoding the
TM6~7 loop (i.e., residues 266 to 409 of PS1) were ligated in-frame to the GAL4
DNA-binding domain in the pAS2-1 yeast ~ c;ssion plasmid vector (Clontech).
10 This plasmid was then co-l~ ro~ ed into S. cerevisiae strain Y190 together with a
library of human brain cDNAs ligated into the pACT2 yeast e~ s ,ion vector bearing
the GAL4 activation domain (Clontech). After a~rupliate selection and re-screening,
a number of clones were recovered and sequenced bearing human brain cDNAs
encoding peptides which interacted with the normal pres~nilin TM6~7 dom~in To
15 ~et~rmine whether these pres~nilin interactions would be modified by AD related
mutations within the TM6~7 loop, the yeast two-hybrid system was again used withTM6~7 loop peptides collt;~ the L286V, the L392V, and the exon 10 splicing
When these mutant constructs were used as "bait" to re-screen the brain
cDNA:GAL4 activation domain library, some but not all of the brain cDNA
20 sequences which interacted with the normal prçs~-nilin were recovered. In addition,
several new clones were identified which interacted with the mutant but not the
normal pr~s~nilin~ The clones collc;~ln~llding to the PS-interacting proteins with the
highest presenilin affinity are described in Example 1 and below.
PS-interacting proteins, particularly those which interact selectively with
2~ either the normal or mutant presen;lin~, provide new targets for the identification of
useful pharmaceuticals, new targets for diagnostic tools in the identification of
individuals at risk, new se~uences for the production of transformed cell lines and
transgenic animaI models, and new bases for therapeutic intervention in Alzheimer's
Disease. In particular, the onset of AD may be associated with aberrant interactions
30 between mutant presenilin proteins and no~nal forms of PS-interacting proteins such
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as those identified using the methods described herein. These changes may increase
or decrease interactions present with normal PS l or may cause interaction with a
novel mutation-specific PS-interacting protein. In addition, however, aberrant
interactions may result from normal pres~onilin~ binding to mutant forms of the PS-
ir~Ateracting proteins and, therefore, mutations in the PS-interacting proteins may also
be causative of AD.
A. The S5a Subunit of the 26S Proteasome
Two overlapping clones have been identified as repres~nting a portion of
the human protein ~lt~rn~tively known as Antisecretory ~actor ("ASF") or the
10 Multiubiquitin chain-binding S5a subunit of the 26S proteasome ("S5a"). Theseclones, which together include residues 70-377 of S5a, were shown to interact with
the normal presenitin TM~7 loop domain but only weakly with two TM6~7 loop
domain mllt~n~ tested (L286V, L392V). The PS 1 :S5a interaction was confirmed byco-hllllluAloAv,eei~ tion studies, arld immlmocytochemical studies showed S5a and
15 PSl are c2rAAulcssed in contiguous intracell~ r compartments in brain cells typically
affected by AD.
The interaction between PS1 and the proteasome could be relevant to the
pathogenesis of AL7Jheimer's Disease (AD) through several possible mec.h~ni.cm~
First, most m~mm~ n cells seem to m~in~in very low levels of the PS l holoprotein.
20 A notable exception to this are cells c~AAvlc~ g the PS 1 Q290-3 l9 splicing mutation,
which results in a mutant PS 1 holoplotcill which is not endoproteolytically cleaved
and which is, therefore, readily cletect~hle. In the case of the ~290-319 splicing
mutation at least, the presence of the mutant PS 1 holoprotein, or the absence or
reduction in the 35 kDa N-termin~l and l 8 kDa C-t~nnin~l fr~ment.c, appears
25 sufficient to cause AD. It is possible, therefore, that even very subtle changes in the
turnover of the mutant PS 1 holoprotein might have significant pathophysiological
effects. I'hus, mutations in either the pres~nilin~ or S5a which perturb the PS 1 :S5a
interaction in the m~mm~ n CNS may cause the presenilin holoprotein to be
aberrantly processed and cause AD. Therefore, modulation of presenilin proteolytic
3û pathways might be applied therapeutically to enhance removal of mutant holoprotein.
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To assess a potential in vivo relationship between PS 1 and the S5a subunit
of the 26S proteasome, the effects of proteasome inhibitors on PS 1 metabolism were
investigated. Short term organotypic cultures of neonatal rat hippocampus and
carcinoma of colon (CaCo2) cells (which express high levels of both PSI and PS2)5 were ~lmini~tered either the specific, reversible proteasome inhibitor N-acetyl-
leucinyl-leucinyl-norleucinyl-H (LLnL) ~Rock et al., 19943, or the specific il~;velsible
proteasome inhibitor lactacystin (Fenteany et al., 1995). Both agents caused an
increase in the steady state levels of PS 1 holoprotein. Both agents also prolonged the
half-life of the PS 1 holoprotein in pulse chase experiments in hippoc~mr~l slices from
10 ~15 minlltes to ~35 minutes As noted above, the PSl holoprotein appears to berapidly turned over in normal cells. However, even after four hours of metaboliclabelling, neither of the proteasome inhibitors affected the level of the 35 kDa N-
tl-nninz~l PS 1 fr~ment or resulted in the apl,ea,~,ce of novel species. These studies
imply that the majority of the PS 1 holoprotein is catabolized directly via a rapid,
15 proteasome dependent pathway in a manner similar to several other integral
membrane proteins (e.g. Sec61 and CFTR). On the other hand, because the ~35 kDa
and ~ 18 kDa t.-rm;n~l fr~rnent~ are still produced in the presence of proteasome
inhibitors, this endoproteolytic cleavage of PS 1 is probably not mediated by the
proteasome ~dLhw~y. Tll~,ro~, it appears that at least two proteolytic pathways act
20 upon the PS1 holoprotein.
An ~ltPrn~te possibility is that mutant PS 1 :S5a interactions may modify
the function or the cellular regulation of S5a. To address this possibility, S5a levels
were examined by Western blotting of lysates from postmortem temporal neocortex
from non-AD neurologic controls (n - 8), sporadic AD (n = 8) and PS 1 -linked FAD
25 (n = 4). In the majority of non-AD brains, polyclonal anti-S5a antibodies specifically
detectecl an S5a species with Mr of~ 50 kDa, which could be abolished by
preabsorption of the antibody with recombinant His6-SSa or with extracts of myc-S5a
transfected cells. In a subset of these control cases an additional S5a reactive band
was observed at ~34 kDa. In contrast, in tissue from all subjects with sporadic late
30 onset AD, the predo~ l SSa reactive species was observed at ~ 40 kDa which was
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not seen in control tissue. The origin, and the functional significance of this altered
electrophoretic mobility is unclear but indicates that SSa processing is altered in AD
brains, irrespective of whether the AD is presenilin-linked or sporadic.
Thus, the presenilin-proteasome interaction appears significant in several
5 respects. First, the facts that the normal presenilin TM6~7 loop domain interacts
with the S5a protein, that the mutant prçs~nilin TM6~7 loop domains fail to interact
(or interact very weakly) with the SSa protein, that pres~.nilin~ bearing mutations in
the TM6~7 loop domain appear to be ~lirrelGlltly cleaved and multiubiquitinated, that
proteasomes are known to be involved in the cleavage and clearance of a variety of
10 . proteins (particularly multiubiqnitin~te(i proteins), that inhibition of proteasome
activity inhibits cleavage of the presenilin holoproteins, and that S5a processing is
altered in AD brains, all suggest (l) that the S5a subunit and the 26S proteasome are
involved in the no~mal processing of the presP-nilin~c and that mutations which disrupt
this normal interaction may be ,~ ,nsible for the abnormal processing observed in
15 TM6~7 loop domain mllt~nt~; or (2) that the pr~ct~Milin-proteasome interaction may
modulate the activity of PS 1, SSa, or both, with or without involving proteasome-
mediated pr~s~nilin processing; or (3) that modulation of the norrnal quality control
function of proteasome-mediated degradation of misfolded or mutant membrane
proteins tr~ffickin~ through the ER and Golgi (such as APP, Notch, or Prion proteins),
20 and of misfolded, mutant, or ubiqllitin~tecl cytoplasmic proteins (including structural
proteins such as tau, and short lived, proteasome processed ~i n~lin~ molecules such
as NFkB). Thus, defective proteasome function might selectively cause these proteins
(especially ~APP, tau, Prion) to be aberrantly metabolized. The latter would lead to
the accumulation of neurotoxic, amyloidogenic protease-resistant derivatives such as
25 A,B and PrPsc, the accumulation of neurofibrilla~y tangles, and defective intracellular
sign~ling functions. In support of these hypotheses, it should be noted that failure to
clear hyperubiqlTitin~ted phosphorylated tau and other microtubule associated proteins
is a prominent feature of Alzheimer's Disease (Kosik and Greenberg, l 994),
suggesting a possible link between TM6~7 loop domain ~ x pres~nilin-
30 proteasome interactions, tau-proteasome interactions, and the neurofibrillary tangles
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of tau protein in AD brains. Finally, proteasomes are known to be capable of
degrading APP and of binding the A,B peptides which are associated with Alzheimer's
Disease, suggesting a possible link between TM6~7 loop domain mllt~ntc,
prçsenilin-proteasome interactions, APP-proteasome interactions, and the ~nyloid5 plaques characteristic of AD brains. Furthermore, ~lmini~tr~tion of proteasomeinhibitors such as LLnL and Lactacystin cause severe disturbances in ~APP
metabolism with increases in intracellular innm~tl]re N-glycosylated ,BAPP, and the
secretion of much larger amounts of A,~42 isoforms into the media (Klafki, et al.,
1996).
Therefore, pr~ nilin processing and the prçs~-ni~in-proteasome interaction
are clear targets for the diagnosis as well as therapeutic intervention in AD. Thus~ as
described below, assays may now be provided for drugs which affect the proteasome-
mediated cleavage ofthe pre~P~ilin.~, which affect the ~lt~ tive endoproteolyticcleavage and ubiquitination of the mutant pr~senilinc, or which otherwise affect the
15 proce~.~inp and trafficking of the presenilin~ or the S5a subunit of the proteasome. In
addition, as mutations in the 26S proteasome which disrupt the normal processing of
the presenilin~ are likely to be causative of Alzheimer's Disease, additional diagnostic
assays are provided for 11etectin~ mutations in the SSa or other subunits of theproteasome. Finally, additional transformed cell lines and transgenic models may20 now be provided which have been altered by the introduction of a normal or mutant
sequence encoding at least a functional domain of the proteasome. The appearance of
abnormal electrophoretic forms of S5a (and/or other proteasome subunits) in biologic
tissues and fluids can be used as a clinical test for diagnosis and monitoring of disease
activity in subjects with sporadic forms of AD.
2~ B. GT24: A Protein with "~ rlillo" RePeats
Another PS-interacting protein, designated GT24, was identified from
several over-lapping clones obtained using a PS l26ti409 domain as bait in the yeast two-
hybrid system arld a human adult brain cDNA library. Six longer GT24 clones of
~3.8 kb in size were subsequently obtained by screening of conventional cDNA
30 libraries. The o~en reading frame within the longest GT24 clone obtained to date
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(Accession number U81004~ suggests that GT24 is a protein of at least 1040 aminoacids with a uni~ue N-t~rminu~, and considerable homology to several ~
(~ repeat proteins at its C-t~nim~ Thus, for example, residues 440-862 of GT24
(numbering from Accession number U81004) have 32-56% identity (p=1.2e-l33) to
residues 440-854 of murine p 120 protein (Accession number Z17804), and residues367-815 of GT24 have 26-42% identity (p=0.0017) to residues 245-465 of the D.
melano aster ~rm~lillo segment polarity protein (Accession number P18824). The
(~T24 gene maps to chromosome ~pl5 near the anonymous microsatellite marker
D5S748 and the Cri-du-Chat syndrome locus.
~Iybridization of unique 5' sequences of GT24 to Northern blots reveals
that the GT24 gene is expressed as a range of L.~lS~iliplS varying in size between ~3.9
and 5.0 kb in several regions of human brain, and in several non-neurologic tissues
such as heart. In addition, in situ hybridization studies using a 289 bp single copy
fragment from the 5' end of GT24 in four month old murine brain reveal GT24
15 ll~lsc~il,tion closely parallels that of PS 1, with robust t;~ ssion in dentate and
hippocampal neurons, in scattered neocortical neurons, and in cerebellar Purkinje
cells. In day E13 murine embryos, GT24 is widely ~ essed at low levels, but is
expressed at somewhat higher levels in somites and in the neural tube. A
physiological in vivo interaction between GT24 and PSl is ~u~po.led by co-
20 immunop.~ci~ ion studies in HEK293 cells transiently transfected with a wild typehuman PS 1 cDNA, a c-mvc-tagged cDNA encoding residues 484- 1040 of GT24
(inciu-ling the C-t~rmin~l arm repeats~, or both cDNAs. Cell lysates were
immunopl~ci~itated with anti-PS 1 antibodies and then investip;~te~1 for the presence of
the mvc-GT24 protein by immuno-blotting. In PSl/mvc-GT~4 double transfected
25 cells, the imm~lnnprecipitates contained a robust anti-mvc reactive band of Mr ~60
kDa, which co-migrated with a mvc-GT24 control. In cells transfected with mYc-
GT24 only, a very weak band was detectecl after long exposures, presumably
reflecting interaction of the mvc-GT24 with low levels of endogenous PS 1. No mvc-
reactive bands were detected in cells transfected with PS 1 alone, or in any of the
30 transfected cells imrnunoprecipitated with pre-immune serum. Taken together, these
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observations strongly suggest that the observed PS 1 :GT24 interaction is
physiologically relevant.
To explore whether mutations in the TM6-TM7 loop of PS 1 might
influence the PS 1 :GT24 interaction, we employed q~l~ntit~tive liquid 13-galactosidase
5 assays to directly compare the yeast-two-hybrid interaction of the C-t~-rmin~l residues
499-1040 of GT24 with wildtype and mutant PS12664og~ These studies revealed thatthe interaction of GT24499,o40 with a L286V mutant PS 1 domain was not significantly
different from the interaction with the corresponding wild type PS 1 domain. In
contrast, there was a sigIuficant reduction in the GT24499 lo40 interaction with the
L392V mutant PS 1 construct. The absence of an effect of the L286V mutation, andthe presence of an effect with the L3g2V mutation, may suggest that some mutations
may effect PS 1 :GT24 binding, while others may modulate the PS 1 response to GT24
binding.
The PS 1 :GT24 interaction could support several functions. The arm repeat
15 motif of GT24 has been detected in several proteins with diverse functions including
,B-catenin and its invertebrate homologue :~nn~(1illo plakoglobin, pl20, the
adenomatous polyposis coli (APC) gene, ~u~ ssor of RNA polymerase 1 in yeast
(SRP1), and smGDS. For example"B-c~t~nin, pl20 and plakoglobin play an essentialrole in intercellular adhesion. ~-catenin/~nn~1;llo is involved in transduction of
20 win,~less/Wnt signals during cell fate specification, and ,B-catenin and pl20 may play
a role in other receptor mediated signal tr~nC~ f tion events including responses to
trophic factors such as PDGF, EGF, CSF-l and NGF.
If the PS 1 :GT24 interaction is part of intercellular ~ign~lin~ pathways for
trophic factors, or is involved in cell-cell adherence, disruption of the interaction may
25 be involved in the neurodegenerative processes in PS-linked ~AD brains, and in the
increased sensitivity of PS 1 or PS2 transfected cells to apoptosis ~Wolozin et al.,
1996). It is of note that at least one arm protein, smGDS, stim~ tes GDP/GTP
exchange on intracellular G-proteins (Kikuchi et al. 1992; Borguski et al., 1993), and
that mutant forms of both ~APP and PS2 are thought to activate programmed cell
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death pathways through me~h~ni~m~ involving heterotrimeric GTP/GDP proteins
(Wolozin et al, 1996; Okamoto, et al., 1995; Yamatsuji, et al, 1996).
The interaction between PS 1 and GT24 may also be involved in some of
the development~l phenotypes associated with homozygous PS1 knockouts in mice
such as failed somitogenesis of the caudal embryo, short tail, and fatal cerebral
hemorrhage at around day E13.5 (Wong et al., 1996). The resemblance ofthese
skeletal phenotypes to those associated with null mutations in PAX1 and Notch, and
the a~paLcllt suppressor effect of mutations in sell2 on Notch/linl2 mediated
sign~ling in C. ele~ans suggest that the PS proteins fimction in the Notch sign~linf~
10 pathway. In addition, mice homozygous for a knockout of the Wnt-3a gene (Takada
et al., 1994), and murine homozygotes for a spontaneous mutation, "vestigial tail" or
y~, in the Wnt-3a gene ~Greco et al., 1996), have skeletal phenotypes of defective
caudal somite and tail bud formation. The Wnt-3a knockouts are embryonic lethal by
day 12.5. These phenotypes are sirnilar to those of homozygous knockouts of the
murine PSl gene (Wong et al., 1996). The observation that GT24 binds to PS1, is
expressed in embryonic somites, and contains the ~ lo repeat motif of other
proteins used in the d~wll~Llc~ll signaling in the Win~less/Wnt pathway suggests that
PS 1 is a dov~ can~ element in the GT24-Win~less/Wnt pathway. This can be
exploited to create a bioassay for drugs affecting the GT24-PS 1 interaction directly, or
20 affecting u~sllc~ll or downstream components of that interaction pathway, and can
therefore be used to monitor the effects of pres~nilin mutations. For example, cells
transfected with normal or mutant presenilins may be exposed to soluble Wnt-3a
protein (or other Wnt proteins such as Wnt-1) and assayed for changes which are
specific to the Win~less/Wnt sign~ling pathway, or for any of the other changes
25 described herein for cell assays (e.g., intracellular ion levels, A,B processing,
-
apoptosis, etc.).
Thus, the GT24 protein also presents new targets for diagnosis as well as
therapeutic intervention in AD. For exarnple, as mutations in the GT24 protein may
also be causative of Alzheimer's Disease, additional diagnostic assays are provided for
30 detecting mutations in these sequences. Similarly, additional transformed cell lines
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and transgenic models may now ~e provided which have been altered by introduction
of a normal or mutant nucleic acid encoding at least a functional domain of the GT24
protein, and particularly the functional domains (e.g., residues 70-377) which interact
with the pre~t~.nil;n.~ Such transformed cells and transgenics will have utility in assays
5 for compounds which modulate the presenilin-GT24 interactions.
C. pO071: A Protein with "~ lillo" Repeats
Another independent clone isolated in the initial screening with the wild
type PSI26,j409 "bait" also encodes a peptide with C-te~min~l a~rn repeats (clone
Y2H25, Accession number U81005). A longer cDNA sequence corresponding to the
Y2H25 clone has been deposited with GenBank as human protein pO071 (Accession
number X81889) and is reproduced herein as SEQ ID NO: 5. Clone Y2H25
co,le~onds ~cc~nti~lly to nucleotide positions 1682- 1994 of SEQ ID NO: 5.
Comparison of the predicted sequence of the Y2H25/pO071 ORF with that of GT24
confirms that they are related proteins with 47% overall amino acid sequence identity,
and with 70% identity between residues 346-862 of GT24 and residues 509-1022 of
pO071. This suggests that PS 1 interacts with à novel class of arm repeat cc l It~
proteins. The broad ~4.5 kb hybridization signal obtained on Northern blots with the
unique S' end of GT24 could reflect either ~ltP.rn~tive splicing/polyadenylation of
GT24 or, less likely, the exi.~ten~e of additional members of this family with higher
2û degrees of N-t~ l homology to GT24 than pO071. Cells l~ r,l.l.ed with thesesequences, or transgenic ~nim~l~ including these sequences, will have additionalutility as animal models of AD and for use in screening for compounds which
modulate the action of normal and mutant pres~onilin~
D. Rab 11
One clone (Y2H9), disclosed herein as SEQ ID NO: 5, was identified as
interacting with the normal PS I TM6~7 loop domain and appears to correspond to a
known gene, Rabl 1, available through Accession numbers X56740 and X53143.
Rabl 1 is believed to be involved in protein/vesicle trafficking in the ER/Golgi. Note
the possible relationship to processing of membrane ploteills such as 13APP and Notch
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with resultant overproduction of toxic A~ peptides (especially neurotoxic Al3l42(43
isoforms) (Scheuner, et al, 1995).
E. Retinoid X Receptor-,B
One clone (Y2H23b), disclosed herein as SEQ ID NO: 6, was identified as
interacting with the no~nal PS 1 TM6~i loop domain and appears to correspond to a
known gene, known variously as the retinoid X receptor-,B, nuclear receptor co-
regulator, or MHC Class I regulatory element, and is available through Accessionnumbers M84820, X63522 and M81766. This gene is believed to be involved in
intercellular si~n~lin~, suggesting a possible relationship to the intercellular signaling
fimction mediated by C. ele~ans sell2 and Notch/lin-12 (transcription activator).
F. Cvtoplasmic Chaperonin
One clone (Y2H27), disclosed herein as SEQ ID NO: 8, was identified as
interacting with the normal PS 1 TM6~7 loop domain and appears to correspond to a
known gene, a cytoplasmic chaperonin colllnilli~-g TCP-l, available through
Accession numbers U17104 and X74801.
G. Clone Y2H35
One clone (Y2H35), disclosed herein as SEQ ID NO: 7, was identified as
interacting with the normal PS 1 TM6~7 loop domain and appears to co~Tespond to a
sequence that codes for a protein of unknown function, available through Accession
number R12984, but which displays evolutionary conservation in yeast sequences.
H. Clone Y2H171
One clone (Y2H171), disclosed herein as SEQ ID NO: 9, was identified as
interacting with the normal PS l TM6~7 loop domain and appears to cc,~ olld to aknown expressed repeat sequence available through Accession number D55326.
I. Clone Y2H41
One clone (Y2H41) was identified which reacts strongly with the TM6~7
loop domains of both PS 1 and PS2 as well as the mutant loop domains of PS 1. The
sequence, disclosed as SEQ ID NO: 10, shows strong homology to an EST of
unknown function (~ccession number T64843).
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III. Preferred Embodiments
Based, in part, upon the discoveries disclosed and described herein, the
following ~lert;.led embodiments of the present invention are provided.
l. Isolated Nucleic Acids
In one series of embo~iment.c, the present invention provides isolated
nucleic acids corresponding to, or relating to, the nucleic acid sequences disclosed
herein, which encode at least the PS-interacting domain of a PS-interacting protein.
10 As described more fillly below, the disclosed and enabled sequences include normal
sequences from humans and other m~mm~ n species, mutant sequences from
hl~m~n~ and other m~mm~ n species, homologous sequences from non-m~mm~ n
species such as Drosophila and C. ele~ans. subsets of these sequences useful as probes
and PCR primers, subsets of these sequences encoding fr~ nt~ of the PS-interacting
15 proteins or corresponding to particular structural domains or polymorphic regions,
complementary or :~nti~.o.n~e sequences corresponding to fragments of the PS-
interacting protein genes, sequences in which the PS-interacting protein coding
regions have been operably joined to exogenous regulatory regions, and sequencesencoding fusion proteins in which portions of the PS-interacting proteins are fused to
20 other proteins useful as m~rker.~ of ~ ion, as "tags" for pl-rific~tion, or in screens
and assays for other proteins which interact with the PS-interacting proteins.
Thus, in a first series of embof~iment.c, isolated nucleic acid sequences are
provided which encode at least a PS-interacting domain of a normal or mutant version
of a PS-interacting protein. Examples of such nucleic acid sequences are disclosed
25 herein as SEQ ID NOs: l, 3, and 5-lO. In addition, given the sequences ofthe PS-
interacting domains of the PS-interacting proteins disclosed herein, one of ordinary
skill in the art is clearly enabled to obtain the entire genomic or cDNA sequence
encoding the entire PS-interacting proteins. Thus, for example, based upon the initial
clone ofthe GT24 protein obtained using the yeast two-hybrid system (Example l),30 the larger GT24 clone disclosed as SEQ ID NO: 3 was obtained by standard methods
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known in the art. Complete cDNA or genomic clones of each of the genes encoding
the disclosed sequences may be similarly obtained by one of ordinary skill in the art.
Therefore, the present invention provides complete genomic sequences as well as
cDNA sequences corresponding to the PS-interacting protein genes of the invention.
5 Alternatively, the nucleic acids of the invention may comprise recombinant genes or
1'minigenes" in which all or some introns of the PS-interacting protein genes have
been removed, or in which various combinations of introns and exons and local CiS-
acting regulatory elements have been engineered in propagation or ~ ression
constructs or vectors. ~or purposes of reducing the size of a recombinant PS-
10 interacting protein gene, a cDNA gene may be employed, or various combinations ofintrons and untr~n~l~te~l exons may be removed from a DNA construct. These and
many variations on these embo-liment~ are now enabled by the identification and
description of the PS-interacting proteins provided herein.
In addition to the disclosed PS-interacting protein and gene sequences, one
15 of oldill~ y skill in the art is now enabled to identify and isolate nucleic acids
representing PS-interacting genes or cDNAs which are allelic to the disclosed
sequences or which are heterospecific homologues. Thus, the present invention
provides isolated nucleic acids corresponding to these alleles and homologues, as well
as the various above-described recombinant constructs derived from these sequences,
20 by means which are well known in the art. Briefly, one of ordinary skill in the art
may now screen p-c~,~dlions of genomic or cDNA, including samples prepared firomindividual org;~ni~m~ (e.g., human AD patients or their family members) as well as
bacterial, viral, yeast or other libraries of genomic or cDNA, using probes or PCR
primers to identify allelic or homologous sequences. Because it is desirable to
25 identify mutations in the PS-interacting proteins which may contribute to thedevelopment of AD or other disorders, because it is desirable to identify
polymorphisms in the PS-interacting proteins which are not pathogenic, and because
it is also desirable to create a variety of animal models which may be used to study
AD and screen for potential therapeutics, it is particularly co~ l~lated that additional
30 PS-interacting protein sequences will be isolated from other plc~dlions or libraries
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of human nuc}eic acids and from ~ lions or libraries from ~nim~lc including rats,
mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human
primates. Furtherrnore, PS-interacting protein homologues from yeast or invertebrate
species, including C. ele,~ans and other nematodes, as well as Drosophila and other
5 insects, may have particular utility for drug screening.
Standard hybridization screening or PCR techniques may be employed (as
used, for example, in the identification of the mPS l gene disclosed in PCT
Publication W096/34099) to identify and/or isolate such allelic and homologous
sequences using relatively short PS-interacting protein gene sequences. The
10 sequences may include 8 or fewer nucleotides depending upon the nature of the target
sequences, the method employed, and the specificity required. Future technological
developments may allow the advantageous use of even shorter sequences. With
current technology, sequences of 9-50 nucleotides, and preferably about 18-24 are
~lc;f~ d. These sequences may be chosen from those disclosed herein, or may be
15 derived from other allelic or heterospecific homologues enabled herein. When
probing mRNA or screening cDNA libraries, probes and primers from coding
sequences (rather than introns) are l)refcldbly employed, and sequences which are
omitted in ~lt~ tive splice variants typically are avoided unless it is speci~lcally
desired to identify those variants. Allelic variants of the PS-interacting protein genes
20 may be expected to hybridize to the disclosed sequences under stringent hybridization
conditions, as defined herein, whereas lower stringency may be employed to identify
heterospecific homologues.
In another series of embo(liments, the present invention provides for
isolated nucleic acids which include subsets of the PS-interacting protein sequences or
25 their complements. As noted above, such sequences will have utility as probes and
PCR primers in the identification and isolation of allelic and homologous variants of
the PS-interacting protein genes. Subsequences corresponding to polyrnorphic
regions of the PS-interacting proteins, will also have particular utility in screening
and/or genotyping individuals ~or diagnostic purposes, as described below. In
30 addition, and also as described below, such subsets will have utility for encoding (l)
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fr~gment~ of the PS-interacting proteins for inclusion in fusion proteins, (2) fragm~nt~
which comprise functional domains of the PS-interacting proteins for use in binding
studies, (3) fragments of the PS-interacting proteins which may be used as
imml~nogens to raise antibodies against the PS-interacting proteins, and (4) fragments
5 of the PS-interacting proteins which may act as competitive inhibitors or as mimetics
of the PS-interacting proteins to inhibit or mimic their physiological functions.
Final~y, such subsets may encode or lel)res~lll complement~T~ or ~nti~n~e sequences
which can hybridize to the PS-interacting protein genes or PS-interacting protein
mRNA transcripts under physiological conditions to inhibit the transcription or
10 translation of those sequences. Therefore, depending upon the int~nllecl use, the
present invention provides nucleic acid subsequences of the PS-interacting protein
genes which may have lengths varying from 8-lO nucleotides (e.g., for use as PCRprimers) to nearly the full size of the PS-interacting protein genomic or cDNAs.Thus, the present invention provides isolated nucleic acids comprising sequencesco~ ding to at least 8-10, preferably l5, and more preferably at least 20
consecutive nucleotides of the PS-interacting protein genes, as disclosed or otherwise
enabled herein, or to their complements. As noted above, however, shorter sequences
may be useful with different technologies.
In another series of embodiments, the present invention provides nucleic
20 acids in which the coding sequences for the PS-interacting proteins, with or without
introns or recombinantly engineered as described above, are operably joined to
endogenous or exogenous 5' and/or 3' regulatory regions. Using the present disclosure
and standard genetic techniques (e.g., PCR extensions, targeting gene waLIcing), one of
ordinary skill in the art is now enabled to clone the 5' and/or 3' endogenous regulatory
25 regions of any of the disclosed PS-interacting protein genes. Similarly, allelic
variants of these endogenous regulatory regions, as well as endogenous regulatory
regions from other m~mm~ n homologues, are similarly enabled without undue
eXperim~nt~tion. Alternatively, exogenous regulatory regions (i.e., regulatory regions
from a different conspecific gene or a heterospecific regulatory region) may be
30 operably joined to the PS-interacting protein coding sequences in order to drive
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e~ ion. Appropriate 5' regulatory regions will include promoter elements and
may also include additional elements such as operator or enhancer sequences,
ribosome binding sequences, RN~ capping sequences, and the like. The regulatory
region may be selected from sequences that control the e~ ,s~ion of genes of
5 prokaryotic or eukaryotic cells, their viruses, and combinations thereof. Suchregulatory regions include, but are not limited to, the lac system, the trp system, the
tac system, and the trc system; major operator and promoter regions of phage ~; the
control region of the fd coat protein; early and late promoters of SV40; promoters
derived from polyoma, adenovirus, retrovirus, baculovirus, and simian virus; 3-
10 phosphoglycerate kinase promoter; yeast acid phosphatase promoters; yeast alpha-
mating factors; promoter elements of other eukaryotic genes expressed in neurons or
other cell types; and colllbillations thereof. In particular, regulatory elements may be
chosen which are inducible or l~ressible (e.g., the ~-g~ to~ se promoter) to allow
for controlled and/or manipulable ~pLcs~ion of the PS-interacting protein genes in
15 cells transformed with these nucleic acids. ~ltPrn~tively, the PS-interacting protein
coding regions may be operably joined with regulatory elements which provide fortissue specific t;~lession in multicellular or~ni~m~. Such coll~LIucLs are particularly
useful for the production of transgenic or~ni.cm~ to cause ~ es~ion of the PS-
interacting protein genes only in al~plopl;ate tissues. The choice of ~lu~iate
20 regulatory regions is within the ability and discretion of one ûf or~ ~y skill in the art
and the recombinant use of many such regulatory regions is now established in the art.
In another series of embo-1inn~nt~, the present invention provides for
isolated nucleic acids encoding all or a portion of the PS-interacting proteins in the
form of a fusion protein. In these embodiments, a nucleic acid regulatory region25 (endogenous or exogenous) is operably joined to a first coding region which is
covalently joined in-frame to a second coding region. The second coding region
optionally may be covalently joined to one or more additional coding regions and the
last coding region is joined to a t~rmin~tion codon and, optionally, appropriate 3'
regulatory regions (e.g., polyadenylation signals). The PS-interacting protein
30 sequences of the fusion protein may represent the first, second, or any additional
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coding regions. The PS-inter~cting protein sequences may be conserved or non-
conserved domains and can be placed in any coding region of the fusion. The non-PS-interacting protein sequences of the fusion may be chosen according to the needs
and discretion of the practitioner and are not limited by the present invention. Useful
5 non-PS-interacting protein sequences include, for example, short sequence "tags" such
as antigenic ~let~- ., - it ~ or poly-His tags which may be used to aid in the
i(lentifi~tion or purification of the resultant filsion protein. ~lt~rn~tively, the non-
PS-interacting protein coding region may encode a large protein or protein fr~ment,
such as an enzyme or binding protein which also may assist in the identifi~ tion and
10 purification of the protein, or which may be useful in an assay such as those described
below. Particularly contemplated fusion proteins include poly-His and GST
~gl~lt~thione S-transferase) fusions which are useful in isolating and purifying the
presenilins-interacting proteins, and the yeast two hybrid hlsions, described below,
which are useful in assays to identify other proteins which bind to or interact with the
15 PS-interacting proteins.
In another series of embotlimentc the present invention provides isolated
nucleic acids in the forrn of recombinant DNA constructs in which a marker or
reporter gene (e.g., ,B-g;ll~rtocidase~ luciferase) is operably joined to the 5' regulatory
region of a PS-interacting protein gene such that t;~lt;ssion of the marker gene is
20 under the control of those regulato~y sequences. Using the PS-interacting protein
regulatory regions enabled herein, including regulatory regions from hurnan and other
m~mm~ n species, one of ordinary skill in the art is now enabled to produce suchconstructs. As discussed more fillly below, such isolated nucleic acids may be used to
produce cells, cell lines or transgenic ~nim~l~ which are useful in the identification of
25 compounds which can, directly or indirectly, differentially affect the expression of the
PS-interacting proteins.
Finally, the isolated nucleic acids of the present invention include any of
~he above described se~uences when inc~ c1 in vectors. Appropriate vectors include
cloning vectors and expression vectors of all types, including plasmids, phagemids,
30 cosmids, episomes, and the like, as well as integration vectors. The vectors may also
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include various marker genes (e.g., antibiotic resi~t~nce or susceptibility genes) which
are useful in identifying cells successfully transformed therewith. In addition, the
vectors may include regulatory sequences to which the nucleic acids of the invention
are operably joined, and/or may also include coding regions such that the nucleic
5 acids of the invention, when a~lu~.iateiy ligated into the vector, are expressed as
fusion proteins. Such vectors may also include vectors for use in yeast "two hybrid,"
baculovirus, and phage-display systems. The vectors may be chosen to be useful for
prokaryotic, eukaryotic or viral t;~L,ression, as needed or desired for the particular
application. For example, vaccinia virus vectors or simian virus vectors with the
10 SV40 promoter (e.g., pSV2), or Herpes simplex virus or adeno-associated virus may
be useful for l,~lsre-;Lion of m~mm~ n cells int~ in~ neurons in culture or in vivo,
and the baculovirus vectors may be used in ~ re.;Lhlg insect cells (e.g., buLL~Iny
cells). A great variety of di~.t;llL vectors are now commercially available and
otherwise known in the art, and the choice of an ~lu~liate vector is within the
15 ability and discretion of one of o,dina~y skill in the art.
2. Substantiallv Pure Proteins
The present invention provides for ~ub~l ;". I i~lly pure preparations of the
PS-interacting proteins, fragrnt?nt.c of the PS-inleld~Ling proteins, and fusion proteins
20 including the PS-interacting proteins or fragments thereof. The proteins, fr~gmentc
and fusions have utility, as described herein, in the generation of antibodies to normal
and mutant PS-interacting proteins, in the identification of proteins (aside from the
presçnilin.c) which bind to the PS-interacting proteins, and in diagnostic and
therapeutic methods. Therefore, depending upon the intended use, the present
25 invention provides substantially pure proteins or peptides comprising amino acid
sequences which are subsequences of the complete PS-interacting proteins and which
may have lengths varying from 4-lO amino acids (e.g., for use as immunogens), or 10-
lO0 amino acids (e.g., for use in binding assays~, to the complete PS-interacting
proteins. Thus, the present invention provides subst~nti~lly pure proteins or peptides
30 comprising sequences corresponding to at least 4-5, preferably 6-lO, and more
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preferably at least 50 or 100 consecutive amino acids of the PS-interacting proteins, as
disclosed or otherwise enabled herein.
The proteins or peptides of the invention may be isolated and purified by
any of a variety of methods selected on the basis of the properties revealed by their
5 protein sequences. For example, the PS-interacting proteins may be isolated fiom
cells in which the PS-interacting protein is normally highly expressed. ~ltPm~tively
the PS-interacting protein, fusion protein, or fragment thereof, may be purified from
cells transformed or transfected with w~ ssion vectors (e.g., baculovirus systems
such as the pPbac and pMbac vectors (Str~t~gPne La Jolla, CA); yeast ~ es~,ion
10 systems such as the pYESHIS Xpress vectors (Invitrogen, San Diego, CA); eukaryotic
~Y.pl~sion systems such as pcDNA3 (Invitrogen, San Diego, CA) which has constantconstitutive ~ ylc;s~ion, or LacSwitch (str~t~gen~ La Jolla, CA) which is inducible;
or prokaryotic ~rcs~,ion vectors such as pKK233-3 (Clontech, Palo Alto, CA). ~n
the event that the protein or ~agment integrates into the endoplasmic reticulum or
1~ plasma membrane of the recombinant cells (e.g., eukaryotic cells), the protein may be
purified from the membrane fraction. ~Alt~ tively, if the protein aggregates in
inclusion bodies within the recombinant cells (e.g., prokaryotic cells), the protein may
be purified from whole Iysed cells or from solubilized inclusion bodies.
Purification can be achieved using standard protein purification procedures
20 including, but not limited to, gel-filtration chromatography, ion-e~ch~nge
chromatography, high-performance liquid chromatography (RP-HPLC, ion-~ch~nge
HPLC, size-exclusion HPLC, high-p~;.ro~ allce chromatofocusing chromatography,
hydrophobic interactionchromatography, immllnf.~ ;ipilalion, orimmnnoafifinity
pllrific~tion. Gel electrophoresis (e.g., PAGE, SDS-PAGE) can also be used to isolate
25 a protein or peptide based on its molecular weight, charge properties and
hydrophobicity.
A PS-interacting protein, or a fragment thereof, may also be conveniently
purified by creating a fusion protein including the desired PS-interacting protein
sequence fused to another peptide such as an antigenic detc7~ t or poly-His tag
3Q ~e.g., QIAexpress vectors, QIAGEN Corp., Chal~wo~ , CA), or a larger protein (e.g.,
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GST using the pGEX-27 vector (Amrad, USA~ or green fluorescent protein using theGreen Lantern vector (GIBCO/BRL. Gaithersburg, MD). The fusion protein may be
expressed and recovered from prokaryotic or eukaryotic cells and purified by anystandard method based upon the fusion vector se~uence. For example, the fusion
5 protein may be purified by immlm~affinity or irnmunoprecipitation with an antibody
to the non-PS-interacting protein portion of the fusion or, in the case of a poly-His tag,
by affinity binding to a niclcel column. The desired PS-interacting protein or ICragment
may then be further purified from the fusion protein by enzymatic cleavage of the
fusion protein. Methods for preparing and using such fusion constructs for the
10 purification of proteins are well known in the art and several kits are commercially
available for this purpose. In light of the present disclosure, one is now enabled to
employ such fusion constructs with the PS-interacting proteins.
3. Antibodies to the PS-interacting Proteins
The present invention also provides antibodies, and methods of m~k;ng
antibodies, which selectively bind to the PS-interacting proteins or fragm~-nt.c thereof.
Of particular importance, by identifying the PS-interacting domains of the PS-
interacting proteins, and methods of identifying mutant forms of the PS-interacting
proteins associated with ~l~h~imer's Disease, the present invention provides
2~ antibodies, and methods of m~king antibodies, which will selectively bind to and,
thereby, identify and/or distinguish norrnal and mutant (i.e., pathogenic) forrns of the
PS-interacting proteins. The antibodies of the invention have utility as laboratory
reagents for, inter alia. immllnnaffinity purification of the PS-interacting proteins,
Western blotting to identify cells or tissues ~ ,S::iillg the PS-interacting proteins, and
25 immunocytoch~ y or imrnunofluorescence techniques to establish the subcellular
location of the proteins. In addition, as described below, the antibodies of theinvention may be used as diagnostics tools to identify carriers of AD-related PS-
interacting protein alleles, or as thc,~ulic tools to selectively bind and inhibit
pathogenic forrns of the PS-interacting proteins in vivo.
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The antibodies of the invention may be generated using the entire PS-
interacting proteins of the invention, or using any PS-interacting protein epitope
which is characteristic of that protein and which suhst~nti~lly distin~ hes it from
other host proteins. Any method of choosing ~ntig~nic ~1etermin~nt.c known in the art
5 may, of course, be employed. Such epitopes may be identified by comr~ring
sequences of, for example, 4-10 amino acid residues from a PS-interacting protein
sequence to co,n~uLe. (l~t~b~es of protein sequences from the relevant host. In
addition, larger fi~pment~ (e.g., 8-20 or, ~ re~bly, 9-15 residues) including one or
more potential epitopes may also be employed. Antibodies to the PS-interacting
10 domains (identified by the yeast two-hybrid assays described below) are expected to
have the greatest utility both diagnostically and therapeutically. On the other hand,
antibodies against highly cons~ ed domains are expected to have the greatest utility
for purification or iclenti fic~t;on of PS-interacting proteins.
PS-interacting protein immllnogen L)lcl)al~ions may be produced from
15 crude extracts (e.g., Iysates or membrane fractions of cells highly t;~LIJlt;SSillg the
proteins), from proteins or peptides ~ub~lalllially purified from cells which naturally
or recombinantly express them or, for short immlmogens, by chemical peptide
synthesis. The immllnogens may also be in the form of a fusion protein in which the
non-PS-interacting protein region is chosen ~or its adjuvant properties. As used20 herein, a PS-interacting protein immunogen shall be defined as a ~ Lion
including a peptide comprising at least 4-8, and preferably at least 9-15 consecutive
arnino acid residues of a PS-interacting proteins, as disclosed or otherwise enabled
herein. Sequences of fewer residues may, of course, also have utility depending upon
the intended use and future technological development~ Therefore, any PS-
25 interacting protein derived sequences which are employed to generate antibodies tothe PS-interacting proteins should be regarded as PS-interacting protein imml]nogens.
The antibodies of the invention may be polyclonal or monoclonal, or may
be antibody fr~grnPntc, including Fab fr~grnPntc F(ab')2, and single chain antibody
fragments. In addition, after identifying useful antibodies by the method of the30 invention, recornbinant antibodies may be generated, including any of the antibody
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fragments listed above, as well as hllm~ni7ecl antibodies based upon non-human
antibodies to the PS-interacting proteins. In light of the present disclosure, as well as
the characterization of other PS-interacting proteins enabled herein, one of ordinary
skill in the art may produce the above-described antibodies by any of a variety of
standard means well known in the art. For an overview of antibody techniques, see
Antibodv En~ineerin~: A Practical Guide. Borrebaek, ed., W.H. Freeman &
Company, NY (l 992~, or Antibody En~ineerin~. 2nd Ed., Borrebaek, ed., Oxford
Ulliv~ y Press, Oxford (1995).
As a general matter, polyclonal antibodies may be generated by first
10 jmmllni7in~ a mouse, rabbit, goat or other suitable animal with the PS-interactin~
protein immunogen in a suitable carrier. To increase the immllnllgenicity of thepreparation, the immunogen may be coupled to a carrier protein or mixed with an
adjuvant (e.g., Freund's adjuvant). Booster injections, although not necessary are
recommen(1~ After an ~p.o~liate period to allow for the development of a humoral15 response, preferably several weeks, the ~nim~lc may be bled and the sera may be
purified to isolate the immunoglobulin c(~ )ol,e~lt.
.~imil~rly, as a general matter, monoclonal anti-PS-interacting protein
antibodies may be produced by first injecting a mouse, rabbit, goat or other suitable
anirnal with a PS-i,lLel~clillg protein irnmunogen in a suitable carrier. As above,
20 carrier proteins or adjuv~l~s may be utilized and booster injections (e.g., bi- or tri-
weekly over 8-l0 weeks) are lc;co"""~n~ 1 After allowing for development of a
humoral response, the ~nim~l.c are sacrificed and their spleens are removed and
resuspended in, for example, phosphate buffered saline {PBS). The spleen cells serve
as a source of Iyrnphocytes, some of which are producing antibody of the a~propliate
25 specificity. These cells are then fused with an immortalized cell line (e.g., myeloma~,
and the products of the fusion are plated into a number of tissue culture wells in the
presence of a selective agent such as HAT. The wells are serially screened and
replated, each time selecting cells making useful antibody. Typically, several
screening and replating procedures are carried out until over 90% of the wells contain
30 single clones which are positive for antibody production. Monoclonal antibodies
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produced by such clones may be purified by standard methods such as affinity
chromatography using Protein A Sepharose, by ion-exchange chromatography, or by
variations and combinations of these techniques.
The antibodies of the invention may be labelled or conjugated with other
compounds or materials for diagnostic and/or therapeutic uses. For example, theymay be coupled to radionuclides, fluorescent compounds, or enzymes for im~ging or
therapy, or to liposomes for the targeting of compounds contained in the liposomes to
a specific tissue location.
10 4. Tlallsrollned Cell Lines
The present invention also provides for cells or cell lines, both prokaryotic
and eukaryotic, which have been tr~n.~formed or transfected with the nucleic acids of
the present invention so as to cause clonal propagation of those nucleic acids and/or
e~r~s~ion of the ~oteil,s or peptides encoded thereby. Such cells or cell lines will
15 have utility both in the propagation and production of the nucleic acids and proteins of
the present invention but also, as further described herein, as model systems for
diagnostic and thc;l~eu~ic assays. In particular, it is expected that cells co-
transformed with PS-interacting protein sequences as well as pres~nilin sequences will
have improved utility as models of the biochemical pathways which may be affected
20 in AD. For exarnple, cells co-l~ srolll~ed with the interacting domains of PS-
interacting sequences and pres~Dnilini in yeast two-hybrid fusion constructs, will have
utility in s~ .illg for compounds which either enh~n~e or inhibit interactions
between these domains. Similarly, for cells transformed with a heterospecific
pres~n;lin, co-~ldl~r,llllation with a similarly heterospecific PS-interacting protein, or
25 co-transformation and homologous recombination to introduce a similarly
heterospecific PS-interacting domain of a PS-interacting protein (e.g., "hlTm~ni7ing" a
non-human endogenous PS-interacting protein), will result in a better model system
for studying the interactions of the preseni~in~ and the PS-interacting proteins. Cells
transformed with only PS-interacting sequences will, of course, have utility of their
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own for studying the role of these proteins in the etiology of AD, and also as
precursors for presenilin co-transformed cells.
As used herein, the te~ ro~ ed cell" is intende~l to embrace any
cell, or the descendant of any cell, into which has been introduced any of the nucleic
5 acids of the invention, whether by l.~l~ro,l..ation, transfection, infection, or other
means. Methods of producing ~ iate vectors, transforming cells with those
vectors, and identifying transformants are well known in the art and are only briefly
reviewed here ~see, for example, Sambrook et al. (1989) Molecular Clonin~: A
Laboratory ManuaL 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring
10 Harbor, New York~.
Prokaryotic cells useful for producing the transformed cells of the
invention include members of the b~cteri~l genera Escherichia (e.g., E. coli),
Psell-lom-~nas (e.g., P. aerll~inos~), and Bacillus (e.g., B. subtillus. B.
stearothermol~hilus), as well as many others well known and frequently used in the
15 art. Prokaryotic cells are particularly useful for the production of large quantities of
the proteins or peptides of the invention (e.g., norrnal or mutant PS-interacting
proteins, fr~ment~ of the PS-interacting proteins, fusion proteins of the PS-
interacting proteins). Bacterial cells ~e.g., E. coli) may be used with a variety of
ssion vector systems including, for example, plasmids with the T7 RNA
20 polymerase/promoter system, bacteriophage ~ regulatory sequences, or M13 Phage
mGPI-2. B~ctt~n~l hosts may also be l~ r~l-ned with fusion protein vectors whichcreate, for example, lacZ, t~pE, maltose-binding protein, poly-His tags, or ~hlt~t1lione-
S-transferase filsion proteins. All of these, as well as many other prokaryotic
lci,sion systems, are well known in the art and widely available commercially
25 (e.g., pGEX-27 (Amrad, USA) for GST fusions).
Eukaryotic cells and cell lines useful for producing the l~ r~ ed cells of
the invention include m~mm~ n cells and cell lines (e.g., PC12, COS, CHO,
fibroblasts, myelomas, neurobl~tQm~s~ hybridomas, human embryonic kidney 293,
oocytes, embryonic stem cells), insect cells lines (e.g., using baculovirus vectors such
30 as pPbac or pMbac (Stratagene, La Jolla, CA~), yeast (e.g., using yeast expression
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vectors such as pYESHIS (Invitrogen, CA)), and fungi. Eukaryotic cells are
palticularly useful for embortimentc in which it is necessary that the PS-interacting
proteins, or functional fragments thereof, pclro~ the functions and/or undergo the
intracellular interactions associated with either the normal or mutant proteins. Thus,
5 for example, transfo~ned eukaryotic cells are preferred for use as models of PS-
interacting protein function or interaction, and assays for screening candidate
thefa~ lics preferably employ transformed eukaryotic cells.
To accomplish e~3les~ion in eukaryotic cells, a wide variety of vectors
have been developed and are commercially available which allow inducible (e.g.,
i0 LacSwitch ~ ssion vectors, Stratagene, La Jolla, CA) or cognate ~e.g., pcDNA3vectors, Invitrogen, Chatsworlh, CA) e~les~ion of PS-interacting protein nucleotide
sequences under the regulation of an artificial promoter element. Such promoter
elements are often derived from CMV or SV40 viral genes, although other strong
promoter etern~o-nt~ which are active in eukaryotic cells can also be employed to induce
15 transcription of PS-interacting protein nucleotide sequences. Typically, these vectors
also contain an artificial polyadenylation sequence and 3' UTR which can also bederived from exogenous viral gene sequences or from other eukaryotic genes.
Furthermore, in some constructs, artificial, non-coding, spliceable introns and exons
are included in the vector to enhance ~ les~ion of the nucleotide sequence of interest.
20 These e,~res~ion systems are commonly available from commercial sources and are
typified by vectors such as pcDNA3 and pZeoSV (Invitrogen, San Diego, CA).
Innumerable commercially-available as well as custom-designed t;~)re~sion vectors
are available from commercial sources to allow ~lGs~ion of any desired PS-
interacting protein l~ s~;l;pt in more or less any desired cell type, either constitutively
2~ or after exposure to a certain exogenous stim~ (e.g., withdrawal of tetracycline or
exposure to IPTG).
Vectors may be introduced into the recipient or "host" cells by various
methods well known in the art including, but not limited to, calcium phosphate
transfection, :jLlUlllilllll phosphate L~ r~ ion, DE~E dextran kansfection,
30 electroporation, lipofection (e.g., Dosper Liposomal transfection rea~ent, Boehringer
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Mannheim, Germany), microinjection, ballistic insertion on micro-beads, protoplast
fusion or, for viral or phage vectors, by infection with the recombinant virus or phage.
5. Trans~enic Animal Models
The present invention also provides for the production of transgenic non-
human animal models in which mutant or wild type PS-interacting protein se~uences
are expressed, or in which the PS-interacting protein genes have been inactivated
(e.g., "knock-out" deletions), for the study of Alzheimer's Disease, for the screening of
candidate ph~rm~e~ltical compounds, for the creation of explanted m~mm~ n CNS
10 cell cultures (e.g., neuronal, glial, organotypic or mixed cell cultures), and for the
evaluation of potential therapeutic h~ ions. Prior to the present invention, a
partial animal model for Alzheimer's Disease existed via the insertion and over-es~ion of a mutant form of the human amyloid precursor protein gene as a
minigene under the regulation of the platelet-derived growth factor ~ receptor
15 promoter element (Games et al., l995). This mutant (~APP7~7 Val~Ile) causes the
appearance of synaptic pathology and amyloid ,B peptide deposition in the brain of
transgenic ~nim~l~ bearing this transgene in high copy number. These changes in the
brain of the transgenic animal are very similar to that seen in human AD (Games et
al., l 995). It is, however, as yet unclear whether these ~nim~l~ become ~lementef1 but
there is general consensus that it is now possible to recreate at least some aspects of
AD in mice. In addition, transgenic animal models in which the presPnilin genes are
gt~nçtic~lly ~ngineered are disclosed in PCT Publication W096/34099. These
transgenic animal models have been shown to have altered A~ production and altered
hippocampus-dependent memory function.
Animal species suitable for use in the animal models of the present
invention include, but are not limited to, rats, mice, h~m~t~r.~, guinea pigs, rabbits,
dogs, cats, goats, sheep, pigs, and non-human primates (e.g., Rhesus monkeys,
cl~illlpa,~es). For initial studies, transgenic rodents ~e.g., mice) may be ~lc;rt~ ,d due
to their relative ease of ,~ rll~nce and shorter life spans. E~owever, transgenic yeast
or invertebrates ~e.g., nematodes, insects) may be preferred for some studies because
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they will allow for even more rapid and inexpensive screening. For example,
in~ lebl~tes bearing mutant PS-interacting protein homologues (or m~mm~ n PS-
interacting protein transgenes) which cause a rapidly occurring and easily scored
phenotype (e.g., abnormal vulva or eye development after several days) can be used as
5 screens fior drugs which block the effect of the mutant gene. Such invertebrates may
prove far more rapid and efficient for mass screenings than larger vertebrate ~nim~
Once lead compounds are found through such screens, they may be tested in higher~nim~ such a rodents. Ultim~t~ly, transgenic non-hurnan primates may be preferred
for longer term studies due to their greater similarity to hl~m~n~ and their higher
10 cognitive abilities.
Using the nucleic acids disclosed and otherwise enabled herein, there are
now several available approaches for the creation of a transgenic animal model for
Alzheimer's Disease. Thus, the enabled animal models include: (I) Animals in which
sequences encoding at least a functional domain of a noImal hurnan PS-interacting
15 protein gene have been recolllbi~ llly introduced into the ~enome of the animal as an
additional gene, under the regulation of either an exogenous or an endogenous
promoter element, and as either a m;nigene or a large genomic fr~gment; in whichsequences encoding at least a functional domain of a normal human PS-interactingprotein gene have been recombinantly substituted for one or both copies of the
20 animal's homologous PS-inl~ lg protein gene by homologous recombination or
gene targeting; and/or in which one or both copies of one of the animalls homologous
PS-interacting protein genes have been recombinantly 'Ihllm~ni7~ by the partial
substitution of sequences encoding the human homologue by homologous
recombination or gene l~gelillg. These animals are useful for evaluating the effects
25 of the transgenic procedures, and the effects of the introduction or substitution of a
human or hllm~ni7~1 PS-interacting protein gene. (2) Animals in which sequences
encoding at least a functional domain of a mutant (i.e., pathogenic~ human PS-
interacting protein gene have been recombinantly introduced into the genome of the
animal as an additional gene, under the regulation of either an exogenous or an
30 endogenous promoter element, and as either a minigene or a large genomic fragment;
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in which sequences encoding at least a fimctional domain of a mutant human PS-
interacting protein gene have been recombinantly substituted for one or both copies of
the animal's homologous PS-interacting protein gene by homologous recombination
or gene targeting, and/or in which one or both copies of one of the animal's
5 homologous PS-interacting protein genes have been recombinantly "hllnn~ni7ed" by
the partial ~ul)s~i~u~ion of sequences encoding a mutant human homologue by
homologous recombination or gene targeting. These ~nim~l.s are useful as models
which will display some or all o~the characteristics, whether at the biochemical,
physiological and/or behavioral level, of hllm~n~ carrying one or more alleles which
10 are pathogenic of ~17.heimer's Disease or other ~ e~es associated with mutations in
the PS-interacting protein genes. (3) Animals in which sequences encoding at least a
functional domain of a mutant version of one of that animal's PS-interacting protein
genes (bearing, for exarnple, a specific mutation corresponding to, or similar to, one
of the pathogenic mutations of the human PS-interacting proteins) have been
15 recombinantly introduced into the genome of the animal as an additional gene, under
the regulation of either an exogenous or an endogenous promoter element, and as
either a minigene or a large genomic fragrnent and/or in which sequences encoding at
least a functional domain of a mutant version of one of that animal's PS-interacting
protein genes (bearing, for example, a specific mutation corresponding to, or similar
20 to, one of the pathogenic mutations of the human PS-inter~t~tin~ proteins) have been
recombinantly substituted for one or both copies of the animal's homologous PS-
interacting protein gene by homologous recombination or gene targeting. These
~nim~ are also useful as models which will display some or all of the characteristics,
whether at the biochemical, physiological and/or behavioral level, of hl-m~n~ carrying
2~ one or more alleles which are pathogenic of Alzheimer's Disease. (4) "Knock-out"
~nim~ in which one or both copies of one of the animal's PS-interacting protein
genes have been partially or completely deleted by homologous recombination or
gene targeting, or have been ina.;liv~ted by the insertion or substitution by
homologous recombination or gene targeting of exogenous sequences (e.g., stop
30 codons, lox p sites). Such ~n;m~l~ are useful models to study the effects which loss of
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PS-interacting protein gene ~yles~ion may have, to evaluate whether loss of function
is preferable to continued expression of mutant forms, and to ex~min~ whether other
genes can be recruited to replace a mutant PS-interacting protein or to intervene with
the effects of other genes (e.g., PS1, PS2, APP or ApoE) causing AD as a tre~t~nent
5 for AD or other disorders. For example, a normal PS-interacting protein gene may be
necçc.c~ry for the action of mutant pres~on;lin or APP genes to actually be expressed as
AD and, therefore, transgenic PS-interacting protein animal models may be of use in
elucidating such multigenic interactions.
In addition to transgenic animal models in which the ~ yl~s~ion of one or
10 more of the PS-interacting proteins is altered, the present invention also provides for
the production of transgenic animal models in which the ~ ession of one or more of
the presenilinc, APP, or ApoE is altered. The nucleic acids encoding the presçnilinc7
APP, and ApoE are known in the art, a methods for producing transgenic ~nim~lc with
these sequences are also known (see, e.g., PCT Publication W096/34099, Games et
15 al., 1995). Indeed, because non-human ~nim~lc may differ from hllm~nc not only in
their PS-interacting protein sequences, but also in the sequences of their pres~nilin,
APP and/or ApoE homologues, it is particularly collL~ plated that transgenics may be
produced which bear recombinant normal or mutant human sequences for at least one
pres~nilin, APP and/or ApoE gene in addition to recombinant sequences for one or20 more PS-interacting proteins. Such co-transformed animal models would possessmore elements of the human molecular biology and, therefore, are expected to be
better models of human disorders. Thus, in accordance with the present invention,
transgenic animal models may be produced bearing norrnal or mutant sequences forone or more PS-interacting proteins, or interacting domains of these proteins. These
25 .~nim~lc will have utility in that they can be crossed with animals bearing a variety of
mormal or mutant presenilin, APP or ApoE sequences to produce co-transformed
animal models. Furthermore, as detailed below, it is expected that mutations in the
PS-interacting genes, like mutations in the presenilinc themselves, may be causative
of Alzheimer's Disease and/or other disorders as well (e.g., other cognitive,
30 intellectual, neurological or psychological disorders such as cere~ral hemorrhage,
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schizophrenia, depression, mental retardation and epilepsy). Therefore, transgenic
animal models bearing normal or mutant sequences corresponding to the PS-
interacting proteins, absent transformation with any presenilin, APP or ApoE
sequences, will have utility of their own in the study of such disorders.
As detailed below, p,efel,~d choices for transgenic animal models
transformed with PS-interacting proteins, or domains of PS-interacting proteins,include those transformed with nor~nal or mutant sequences corresponding to the
clones identified and described in Example 1 and disclosed in SEQ ID NOs: 1-12.
These clones, which interact with normal or mutant PS 1 TM6~7 loop domains, were10 identified according to the methods described in Example 1, below, and PCT
Publication W096/34099. These clones, longer nucleic acid sequences comprising
these clones, and other clones i~l~ntifie~l according to this and other methods of the
invention (e.g., allelic and splice variants or heterospecific homologues of these
clones) may all be employed in accordance with the present invention to produce
15 animal models which, with or without co-l-;-~r ll " ,~tion with presenilin, APP arld/or
ApoE sequences, will have utility in the study of Alzheimer's Disease and/or other
cognitive, intellectual, neurological or psychological disorders.
Thus, using the nucleic acids disclosed and otherwise enabled herein, one
of oldilla, y skill in the art may now produce any of the following types of transgenic
20 animal models with altered PS-interacting protein ~.cs,ion~ Animals in which
sequences encoding at least a functional domain of a normal human PS-interactingprotein gene have been recombinantly introduced into the genome of the animal as an
additional gene, under the regulation of either an exogenous or an endogenous
promoter element, and as either a minigene or a large genomic fr~gment in which
25 sequences encoding at least a functional domain of a normal human PS-interacting
protein gene have been recombinantly substituted for one or both copies of the
animal's homologous PS-interacting protein gene by homologous recombination or
gene l~ ling; and/or in which one or both copies of one of the animal's homologous
PS-interacting protein genes have been recombinantly "hl-m~ni7ed" by the partial30 substitution of sequences encoding the human homologue by homologous
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recombination or gene ~g~ g. These animals are particularly useful for providingtransgenic models which express human PS-interacting proteins as well as human
presenilin proteins. They are also useful in evaluating the effects of the transgenic
procedures, and the effects of the introduction or substitution of a human or
5 hl-m~ni7~ PS-interacting protein gene. (2) Animals in which sequences encoding at
least a functional domain of a mutant (i.e., pathogenic) human PS-interacting protein
gene have been recombinantly introduced into the genome of the animal as an
additional gene, under the regulation of either an exogenous or an endogenous
promoter element, and as either a minigene or a large genomic fr~grn~nt in which10 sequences encoding at least a functional domain of a mutant human PS-interacting
protein gene have been recombinantly ~ub~LiLuLed for one or both copies of the
animal's homologous PS-interacting protein gene by homologous recombination or
gene targeting; and/or in which one or both copies of one of the animal's homologous
PS-interacting protein genes have been lecol~ ina.~Lly "hllm~ni7~-1" by the partial
15 substitution of sequences encoding a mutant human homologue by homologous
recombination or gene tal~,eti,lg. These animals are useful as models which willdisplay some or all of the characteristics, whether at the biochemic~l, physiological
and/or behavioral level, of hnm~n~ carrying one or more alleles which are pathogenic
of Alzheimer's Disease or other ~ e~c associated with mutations in these PS-
20 interacting genes. (3) Animals in which sequences encoding at least a functionaldomain of a mutant version of one of that animal's PS-interacting protein genes
(bearing, for example, a specific mutation corresponding to, or similar to, one of the
pathogenic mutations of the human PS-interacting proteins) have been recombinantly
introduced into the genome of the animal as an additional gene, under the regulation
25 of either an exogenous or an endogenous promoter element, and as either a minigene
or a large genomic fr~nent; and/or in which sequences encoding at least a functional
domain of a mutant version of one of that animal's PS-interacting protein genes
(bearing, for example, a specific mutation corresponding to, or similar to, one of the
pathogenic mutations of the hllm~n~ PS-interacting proteins) have been recombinantly
3Q substituted ror one or both copies of the animal's homologous PS-interacting protein
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gene by homologous recombination or gene targeting. These ~mim~l.c are also useful
as models which will display some or all of the characteristics, whether at the
biochemical, physiological and/or behavioral level, of hlln~n~ car~ying one or more
alleles which are pathogenic of Alzheimer's Disease. (4) "Knock-out" ~nim~l.c in5 which one or both copies of one of the animal's PS-interacting protein genes have
been partially or completely deleted by homologous recombination or gene targeting,
or have been inactivated by the insertion or substitution by homologous
recombination or gene targeting of exogenous sequences (e.g., stop codons, lox psites). Such ~nim~l~ are useful models to study the effects which loss of PS-
10 interacting protein gene c;~le~ion may have, to evaluate whether loss of function ispreferable to continue~ res~ion, and to ex~mine whether other genes can be
recruited to replace a mutant PS-interacting protein or to i~ l V~llC with the effects of
other genes (e.g., APP or ApoE) causing AD as a tre~trn~nt for AD or other disorders.
For example, a normal PS-interacting protein may be necessary for the action of
1~ mutant PS1, PS2 or APP genes to actually be expressed as AD and, therefore,
transgenic PS-interacting protein animal models may be of use in elucidating such
multigenic hlteld-;~ions.
In some ~re~led embodiments, transgenic animal models are produced in
which just the PS-interacting domains of the PS-interacting proteins are introduced
20 into the genome of the animal by homologous recombination. Thus, for example,preferred embodiments include transgenic animals in which the PS~ te.~ g
domains of PS-interacting proteins are "hl-m~ni7tod" by homologous recombinationwith sequences f~om human PS-interacting proteins. These ~nim~l~ may then be bred
with transgenics in which normal or mutant prec~nilin sequences have been
25 introduced. The progeny of these ~nim~ls, having both human pr~sçnilin and human
PS-interacting protein sequences, will provide improved animal models for
Alzheimer's Disease.
To create an animal model (e.g., a tr~n~g~nic mouse), a normal or mutant
PS-interacting gene ~e.g., normal or mutant S5a, GT24, pO071, Rabl 1, etc.), or a
30 normal or mutant version of a recombinant nucleic acid encoding at least a functional
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,
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domain of a PS-interacting gene (e.g., the PS-interacting domains obtained in the
yeast two-hybrid system), can be inserted into a germ line or stem cell using standard
techniques of oocyte microin~ection, or transfection or microinjection into embryonic
stem cells. Animals produced by these or similar processes are referred to as
transgenic. Similarly, if it is desired to inactivate or replace an endogenous pres~n;l;n
or PS-interacting protein gene, homologous recombination using embryonic stem
cells may be employed. Animals produced by these or similar processes are referred
to as "knock-out" (inactivation) or "knock-in" (replacement) models.
For oocyte injection, one or more copies of the recombinant DNA
10 constructs of the present invention may be inserted into the pronucleus of a just-
fertilized oocyte. This oocyte is then reimplanted into a pseudo-~le~ foster
mother. The liveborn ~n;m~l~ are screened for integrants using analysis of DNA (e.g.,
from the tail veins of offspring mice) for the presence of the inserted recombinant
transgene sequences. The transgene may be either a complete genomic sequence
15 injected as a YAC, BAC, PAC or other chromosome DNA fr~mçnt a cDNA with
either the natural promoter or a heterologous promoter, or a minigene Co.~ g all of
the coding region and other elements found to be necess~ y for ol)limu~ c;ssion~Retroviral infection of early embryos can also be done to insert the
recombinant DNA constructs of the invention. In this method, the transgene (e.g., a
20 normal or mutant S5a, GT24, pO071, Rab l l, etc., sequence~ is inserted into a
lvvil~l vector which is used to infect embryos (e.g., mouse or non-human primateembryos) directly during the early stages of development to generate chimeras, some
of which will lead to germline tr~ncmic~ion~
Homologous recombination using stem cells allows for the screening of
25 gene transfer cells to identify the rare homologous recombination events. Once
identified, these can be used to generate chimeras by injection of blastocysts, and a
l~r~3~(,lLion of the res~llting ~nim~lc will show germline tr~n.cmiccion from the
recombinant line. This methodology is especially useful if inactivation of a gene is
desired. For example, inactivation of the S5a gene in mice may be accomplished by
30 rlesigning a DNA fragment which contains sequences from an S5a coding region
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fl~nking a selectable marker. Homologous recombination leads to the insertion of the
marker sequences in the middle of the coding region, c~lleing inactivation of the SSa
gene and/or deletion of intP!rn~l sequences. DNA analysis of individual clones can
then be used to recognize the homologous recombination events.
The techniques of generating transgenic ~nim~c, as well ac the techniques
for homologous recombination or gene targeting, are now widely accepted and
practiced. A laboratory manual on the manipulation of the mouse embryo, for
example, is available ~l.ot~iling standard laboratory techniques for the production of
transgenic mice (Hogan et al., 1986). To create a ~ sg~l.e, the target sequence of
10 interest (e.g., norrnal or mutant presenilin sequences, normal or mutant PS-interacting
protein sequences) are typically ligated into a cloning site located dowllsl~c~ll of
some promoter element which will regulate the ~ ession of RNA from the
sequence. Duwll~ ll of the coding sequence, there is typically an artificial
polyadenylation sequence. ~n the trar~cgenic models that have been used to
15 successfully create ~nim~le which mimic aspects of inherited human
neurodegenerative tliceslees~ the most sllccescfil1 promoter elements have been the
platelet-derived growth factor rec~Lor~B gene subunit promoter and the h~mct~r prion
protein gene promoter, although other promoter elements which direct e,~lession in
central nervous system cells would also be usefill. An ~It~rn~te approach to creating a
20 tr~ncgto,ne is to use an endogenous pres~nilin or PS-interacting protein gene promoter
and regulatory sequences to drive t;~les~ion of the tr~n~gene Finally, it is possible
to create tr~n~g~n~ss using large genomic DNA fra~rnent.c such as YACs which
contain the entire desired gene as well as its ap~ropliate regulatory sequences. Such
consLIu~;ls have been s~lcces~fi~lly used to drive human APP ~ res~ion in kansgenic
25 mice (Lamb et al., 1993).
Animal models can also be created by targeting the endogenous presenilin
or PS-interacting protein gene in order to alter the endogenous sequence by
homologous recombination. These targeting events can have the effect of removingendogenous sequence ~knock-out) or altering the endogenous sequence to create an30 amino acid change associated with human disease or an otherwise abnormal sequence
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(e.g., a sequence which is more like the human sequence than the original anirnal
sequence) (knock-in animal models). A large nurnber of vectors are available to
accomplish this and a~.~ro~iate sources of genomic DNA for mouse and other animal
genomes to be targeted are comrnercially available from companies such as
5 GenomeSystems Inc. ~St. Louis, Missouri, USA). The typical feature of these
~geLillg vector constructs is that 2 to 4 kb of genomic DNA is ligated 5' to a
selectable marker (e.g., a bacterial neomycin re~i~t~n~e gene under its own promoter
element termed a "neomycin cassette"). A second DNA fragrnent from the gene of
interest is then ligated do~llsllc~n of the neomycin c~sette but ~ sll~ll of a second
10 selectable marker ~e.g., thymidine kinase). The DNA fr~grnent~ are chosen such that
mutant sequences can be introduced into the germ line of the targeted animal by
homologous replacement of the endogenous sequences by either one of the sequences
included in the vector. ~ltern~tively, the sequences can be chosen to cause deletion of
sequences that would normally reside between the left and right arms of the vector
15 surrounding the neomycin c~ette The former is known as a knock-in, the latter is
known as a knock-out. Again, innumerable model systems have been created,
particularly for targeted knock-outs of genes including those relevant to
neurodegenerative ~li.ce~.~es (e.g., targeted deletions of the murine APP gene by Zheng
et al., 1995; targeted deletion of the murine prion gene associated with adult onset
20 human CNS degeneration by Bueler et al., 1996).
Finally, equivalents of transgenic ~nim~l~, including ~nim~l~ with m~lt~ted
or inactivated pres~nilin genes, or mllt~ted or inactivated PS-interacting protein genes,
may be produced using chemic~l or X-ray mllt~ nesis of g~mt~t~s, followed by
fertilization. Using the isolated nucleic acids disclosed or otherwise enabled herein,
25 one of ordinary skill may more rapidly screen the r~s--lting offspring by, for exarnple,
direct sequencing RFLP, PCR, or hybridization analysis to detect mllt~nt~, or
Southern blotting to demonstrate loss of one allele by dosage.
6. Assa~s for Dru~s Which Affect PS-Interactin~ Protein Expression
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In another series of embodiments, the present invention provides assays for
identifying small molecules or other compounds which are capable of inducing or
inhibiting the eAIlre;~ion of the PS-interacting genes and proteins (e.g., S5a or GT24).
The assays may be pcLr~ ed in vitro using non-transformed cells, immortalized cell
5 lines, or recombinant cell lines, or in vivo using the transgenic animal models enabled
herein.
In particular, the assays may detect the presence of increased or decreased
e;AI~eS~iOn of S5a, GT24, pO071, Rab 11, or other PS-interacting genes or proteins on
the basis of increased or decreased mRNA ~ es:~ion (using, e.g., the nucleic acid
10 probes disclosed and enabled herein), increased or decreased levels of PS-interacting
proteins (using, e.g., the anti-PS-hllt; dclillg protein antibodies disclosed and enabled
herein), or increased or decreased levels of ~A~le3~ion of a marker gene (e.g., ~-
galactosidase or luciferase) operably joined to a PS-hl~ g protein 5' regulatory
region in a recombinant construct.
Thus, for example, one may culture cells known to express a particular PS-
interacting protein and add to the culture medium one or more test compounds. After
allowing a sufficient period of time (e.g., 0-72 hours) for the compound to induce or
inhibit the ~A~ ion of the PS-interacting protein, any change in levels of t;A~les~ion
from an established baseline may be detected using any of the techniques described
20 above and well known in the art. In particularly plcr~ d embo~iment~, the cells are
from an immortalized cell line such as a human neuroblastoma, glioblastoma or a
hybridoma cell line. Using the nucleic acid probes and /or antibodies disclosed and
enabled herein, detection of changes in the ~AI ,c;s~ion of a PS-interacting protein, and
thus identifie~tion of the compound as an inducer or repressor of PS-interacting25 protein ~;A~lc;ssion, requires only routine experiment~tion.
In particularly preferred embodiments, a recombinant assay is employed in
which a reporter gene such a ,B-galactosidase, green fluorescent protein, ~lk~ine
phosphatase, or luciferase is operably joined to the S' regulatory regions of a PS-
interacting protein gene. Preferred vectors include the Green T .~nteItl 1 vector
30 (GIBCO/BRL, Gaithersburg, MD) and the Great EScAPe pSEAP vector (Clontech,
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Palo Alto). The PS-interacting protein regulatory regions may be easily isolated and
cloned by one of on~ ,y skill in the art in light of the present disclosure of coding
regions from these genes. The reporter gene and regulatory regions are joined in-
frame ~or in each of the three possible reading frames~ so that transcription and
5 translation of the reporter gene may proceed under the control of the PS-interacting
protein regulatory elements. The recombinant construct may then be introduced into
any a~plu~.,iate cell type, although m~rnm~ n cells are l~lere-l~d, and human cells
are most pref~ ed. The transformed cells may be grown in culture and, after
establishing the baseline level of ~I)res~ion of the reporter gene, test compounds may
10 be added to the medium. The ease of detection of the e~les~ion of the reporter gene
provides for a rapid, high through-put assay for the i-l~ntification of inducers and
r~esso~ o~the PS-interacting protein gene.
Compounds identified by ~is method will have potential utility in
modifying the ~ s~ion of the PS-interacting protein genes in vivo. These
15 compounds may be filrther tested in the animal models disclosed and enabled herein
to identify those compounds having the most potent in vivo effects. In addition, as
described herein with respect to small molecules having binding activity for PS-interacting proteins, these molecules may serve as "lead compounds" for the further
development of ph~ f ell~icals by, for example, subjecting the compounds to
20 sequential modifications, molecular modeling, and other routine procedures employed
in rational drug design.
7. I~l~r~t;fic~tion of Compounds with PS-Interactin~ Protein Bindin~ CapacitY
In light of the present disclosure, one of ordinary skill in the art is enabled
25 to practice new screening methodologies which will be useiill in the identification of
proteins and other compounds which bind to, or otherwise directly interact with, the
PS-interacting pl()leills. The proteins and compounds will include endogenous
cellular components, aside from the pres~nilin~, which interact with the PS-interacting
proteins in vivo and which, therefore, provide new targets for phalmaceutical and
30 therapeutic interventions, as well as recombinant, synthetic and otherwise exogenous
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compounds which may have PS-interacting protein binding capacity and, therefore,may be candidates for ph~ ce ~tical agents. Thus, in one series of embo-limerlt~,
cell Iysates or tissue homogenates (e.g., human brain homogenates, Iymphocyte
Iysates) may be screened for proteins or other compounds which bind to one of the
normal or mutant PS-interacting proteins. ~lt~.rn~tively, any of a variety of
exogenous compounds, both naturally occurring and/or synthetic (e.g., libraries of
small molecules or peptides), may be screened for PS-interacting protein bindingcapacity. Small molecules are particular plc;f~,~f~;d in this context because they are
more readily absorbed after oral ~1mini~tration, have fewer potential antigenic
~let~rmin~ntc, and/or are more likely to cross the blood brain barrier than larger
molecules such as nucleic acids or proteins. The methods of the present invention are
particularly useful in that they may be used to identify molecules which selectively or
preferentially bind to a mutant form of a PS-interacting protein (rather than a normal
form) and, therefore, may have particular utility in treating cases of AD which arise
1~ from mutations in the PS-interacting ~ L~ins.
Once ide~tified by the methods described above, the c~n~ te compounds
may then be produced in quantities sufficient for ph~rm~c elltical ~lmini~tration or
testing (e.g."ug or mg or greater quantities), and form~ ted in a ph~,rm~ceutically
acceptable carrier (see, e.g., Re~in~son's Pha~naceutical Sciences. Gennaro, A., ed.,
Mack Pub., 1990). These c~n~ 5e compounds may then be ~lmini~ctered to the
olllled cells of the invention, to the transgenic animal models of the invention, to
cell lines derived from the animal models or from human patients, or to Alzheimer's
patients. The animal models described and enabled herein are of particular utility in
further testing candidate compounds which bind to normal or mutant PS-interacting
proteins for their thel~peuLic efficacy.
In addition, once identified by the methods described above, the candidate
compounds may also serve as ~'lead compounds" in the design and development of
new pharrnaceuticals. For example, as in well known in the art, se~uential
modification of small molecules (e.g., amino acid residue replacement with peptides;
functional group replacement with peptide or non-peptide compounds) is a standard
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approach in the pharmaceutical industry for the development of new ph~rm~eeuticals.
Such development generally proceeds from a "lead compound" which is shown to
have at least some of the activity (e.g., PS-interacting protein binding or blocking
ability) of the desired ph~ ce~l~ical. In particular, when one or more compoundshaving at least some activity of interest (e.g., modulation of PS-interacting protein
activity) are identified, structural coml)~uison of the molecules can greatly inform the
skilled practitioner by suggesting portions of the lead compounds which should be
conserved and portions which may be varied in the design of new candidate
compounds. Thus, the present invention also provides a means of identifying lead10 compounds which may be sequentially modified to produce new candidate
compounds for use in the tre~ nt of Alzheimer's Disease. These new compounds
then may be tested both for binding to PS-interacting proteins and/or blocking PS-
interacting protein activity, and for therapeutic efficacy (e.g., in the animal models
described herein). This procedure may be iterated until compounds having the desired
15 therapeutic activity and/or efficacy are identified.
In each of the present series of embo~lim~nt~, an assay is con~-cte-i to
detect binding between a "PS-interacting protein component" and some other moiety.
Of particular utility will be sequential assays in which compounds are tested for the
ability to bind to only normal or only mutant forms of the PS-interacting clom~in~ of
20 PS-illL~ld.;~ g proteins in the binding assays. Such compounds are expected to have
the greatest therapeutic utilities, as described more fully below. The "PS-interacting
protein component" in these assays may be a complete normal or mutant form of a
PS-interacting protein (e.g., SSa, GT24, pO071, Rab l l, etc.) but need not be. Rather,
particular functional domains of the PS-interacting proteins, particularly the PS-
25 interacting domains as described above, may be employed either as separate
molecules or as part of a fusion protein. For example, to isolate proteins or
compounds that interact with these functional domains, s~l~ellillg may be carried out
using fusion constructs and/or synthetic peptides corresponding to these regions.
Thus, for S5a, GST-fusion peptides may be made including sequences corresponding30 ~p~ ,ately to amino acids 70-377 of SEQ ID NO: 2 (included in clones Y2H29
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and Y2H3 1, see Exarnple I ), approximately to amino acids 206-377 of SEQ ID NO: 2
(which includes protein-protein interaction motifs, see Ferrell et al., 1996), or to any
other SSa domain of interest. Similarly, for GT24, GST- or other fusion peptides may
be produced including sequences corresponding approximately to amino acids 440-
815 of SEQ ID NO: 4 (including part of the ~rrn~-lillo repeat segrnent). Obviously,
various combinations of fusion proteins and PS-interacting protein functional ~lom~in~
are possible and these are merely examples. In addition, the functional domains may
be altered so as to aid in the assay by, for example, introducing into the functional
domain a reactive group or amino acid residue (e.g., cysteine) which will facilitate
I0 immobilization of the domain on a substrate (e.g., using sulfhydryl reactions). Thus,
for example, the PS-interacting domain of S5a may be synthe~i7e~1 CO~ l;llg an
additional C-t~rrnin~l cysteine residue to facilitate immobilization of the ~10m~in
Such peptides may be used to create an affinity substrate for affinity chromatography
(Sulfo-link; Pierce~ to isolate binding proteins for microsequencing. Sirnilarly, other
15 functional domain or antigenic fragments may be created with modified residues (see,
e.g., Example 4).
The proteins or other compounds identified by these methods may be
purified and characterized by any of the standard methods known in the art. Proteins
may, for example, be purified and separated using electrophoretic (e.g., SDS-PAGE,
20 2D PAGE) or chromatographic (e.g., HPLC) techniques and may then be
microsequenced. For proteins with a blocked N-t~ .,.i~.l.~, cleavage (e.g., by CNBr
and/or trypsin) of the particular binding protein is used to release peptide fragments.
Further purification/characterization by HPLC and microseq~lenring and/or mass
spectrometry by convellLional methods provides intPrn~l sequence data on such
25 blocked proteins. For non-protein compounds, standard organic chemical analysis ,.
techniques (e.g., IR, NMR and mass spectrometry; functional group analysis; X-ray
crystallography) may be employed to ~let~nine their structure and identity.
Methods for screening cellular lysates, tissue homogenates, or small
molecule libraries for candidate PS-interaction protein-binding molecules are well
30 known in the art and, in light of the present disclosure, may now be employed to
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identify compounds which bind to normal or mutant PS-interacting protein
components or which modulate PS-interacting protein activity as defined by non-
specific measures (e.g., changes in intracellular Ca2+, GTP/GDP ratio) or by specific
measures (e.g., changes in A~ peptide production or ch~n~es in the ~ es~ion of
5 other do~ genes which can be monitored by differential display, 2D gel
electrophoresis, differential hybridization, or SAGE methods). The pl~r~ d methods
involve variations on the following techni~ues: (1) direct extraction by affinity
chromatography; (2) co-isolation of PS-interacting protein components and bound
proteins or other compounds by immllnoprecipitation; (3) the Biomolecular
10 Interaction Assay (BIAcore), and (4) the yeast two-hybrid systems. These and others
are discussed separately below.
A. Affinitv Chromato~raphY
In light of the present disclosure, a variety of affinity binding techniques
well known in the art may be employed to isolate proteins or other compounds which
15 bind to the PS-h~ d ;ling protein ~ ck~se-1 or otherwise enabled herein. In general, a
PS-interacting protein component may be immobilized on a substrate (e.g., a column
or filter) and a solution including the test compound(s) is contacted with the PS-
interacting protein, fusion or fragment under conditions which are permissive for
binding. The sub~llale is then washed with a solution to remove unbound or weakly
20 bound molecules. A second wash may then elute those compounds which strongly
bound to the immobilized normal or mutant PS-interacting protein component.
~Itt~rn~ively, the test compounds may be imrnobilized and a solution cc,~ ,;.,g one
or more PS-interacting protein components may be contacted with the column, filter
or other substrate. The ability of the PS-interacting protein colllpollent to bind to the
25 test compouIlds may be det~rmined as above or a labeled form of the PS-interacting
protein component (e.g., a radio-labeled or chemiluminescent functional domain) may
be used to more rapidly assess binding to the substrate-immobilized compound(s). B. Co-Tmmnn~-pleci~,il;lLion
Another well characterized technique for the isolation of PS-interacting
30 protein components and their associated proteins or other compounds is direct
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imm~ precipitation with antibodies. This procedure has been surcecsfill~y used, for
example, to isolate many of the synaptic vesicle associated proteins (Phizicky and
Fields, 1994). Thus, either normal or mutant, free or membrane-bound PS-interacting
protein components may be mixed in a solution with the candidate compound(s)
5 under conditions which are permissive for binding, and the PS-interacting protein
component may be immuno~leci~ ed. Proteins or other compounds which co-
immun~ e with the PS-interacting protein component may then be identified
by standard techniques as described above. General techniques for
immnn~-precipitation may be found in, for exarnple, Harlow and Lane, (1988)
10 Antibodies: A LaboratorY ManuaL Cold Spring Harbor Press, Cold Spring Harbor, NY.
The antibodies employed in this assay, as described and enabled herein,
may be polyclonal or monoclonal, and include the various antibody fragments (e.g.,
Fab, F(ab')2,) as well as single chain antibodies, and the like.
C. The Biomolecular Interaction Assay
Another useful method for the detection and isolation of binding proteins
is the Biomolecular Interaction Assay or "BLAcore" system developed by PharmaciaBiosensor and described in the m~nllf~ctllrer's protocol (LKB Ph~rm~ri~, Sweden). In
light of the present disclosure, one of c,l-lh~ y skill in the art is now enabled to
20 employ this system, or a ~ub~ lial equivalent, to identify proteins or other
compounds having PS-interacting protein binding capacity. The BIAcore system uses
an affinity purified anti-GST antibody to irnmobilize GST-fusion proteins onto asensor chip. Obviously, other fusion proteins and corresponding antibodies may be
substit~lte-l The sensor utilizes surface plasmon resonance which is an optical
25 phenomenon that detects changes in refractive indices. A homogenate of a tissue of
interest is passed over the immobilized fusion protein and protein-protein interactions
are registered as changes in the refractive index. This system can be used to
det~nnine the kinetics of binding and to assess whether any observed binding is of
physiological relevance.
.. .
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D. The Yeast Two-Hvbrid SYstem
The yeast "two-hybrid" system takes advantage of transcriptional factors
that are composed of two physically separable, functional domains (Phizicky and
Fields, l 994). The most commonly used is the yeast GAL4 transcriptional activator
5 consi~ting of a DNA binding domain and a transcriptional activation domain. Two
different cloning vectors are used to generate separate fusions of the GAL4 domains
to genes encoding potential binding proteins. The fusion proteins are co-expressed,
targeted to the nucleus and, if h~L~laclions occur, activation of a reporter gene (e.g.,
lacZ) produces a detect~le phenotype. For example, the Clontech ~tchm~k~r
10 System-2 may be used with the Clontech brain cDNA GAL4 activation domain fusion
library with PS-interacting protein-GAL4 binding domain fusion clones (Clontech,Palo Alto, CA). In light of the disclosures herein, one of o~ ~ y skill in the art is
now enabled to produce a variety of PS-interacting protein fusions, including fusions
including either normal or mutant functional domains of the PS-interacting proteins,
15 and to screen such fuslon libraries in order to identify PS-interacting protein binding
proteins.
E. Other Methods
The nucleotide sequences and protein products, including both mutant and
norrnal forms of these nucleic acids and their corresponding proteins, can be used with
2û the above techniques to isolate other interacting proteins, and to identify other genes
whose ~ ssion is altered by the over-~Lpl~c;ssion of norrnal PS-interacting protein
sequences, by the under-e~ sion of nonnal PS-interacting protein sequences, or by
the ~ ion of mutant PS-interacting protein sequences. Identification of these
other interacting proteins, as well as the if l~ntific~tion of other genes whose
25 e~ lcs~ion levels are altered in AD will identify other gene targets which have direct
relevance to the pathogenesis of this disease in its clinical or pathological forms.
Specifically, other genes will be identified which may themselves be the site of other
mutations causing Alzheimer's Disease, or which can themselves be targeted
therapeutically (e.g., to reduce their ~r~s~ion levels to normal, or to
30 pharmacologically block the effects of their over-~ s~ion) as a potential treatment
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for this disease. Specifically, these techniques rely on PCR-based and/or
hybridization-based methods to identify genes which are differentially expressedbetween two conditions (a cell line e,~plessillg normal PS-interacting proteins
colllp~hed to the same cell type ~Lc~illg a mutant PS-interacting protein). These
5 techniques include differential display, serial analysis of gene ~ c s~ion (SAGE), and
mass-spectrometry of protein 2D-gels and subtractive hybridization (reviewed in
Nowak, 1995 and Kahn, 1995).
As will be obvious to one of ol~dilldl y skill in the art, there are numerous
other methods of screening individual proteins or other compounds, as well as large
10 libraries of proteins or other compounds (e.g., phage display libraries and'cloning
systems from Stratagene, La Jolla, CA) to identify molecules which bind to normal or
mutant PS-interacting protein components. All of these methods comprise the step of
mixing a normal or mutant PS-interacting protein, filsion, or fragment with testcompounds, allowing for binding (if any), and assaying for bound complexes. All
15 such methods are now enabled by the present disclosure of substantially pure PS-
hll~ .cting proteins, substantially pure Ps-int~r~rtin~ functional domain fragments,
PS-interacting protein fusion proteins, PS~ f-- ~n. 1;1 Ig protein antibodies, and methods
of m~king and using the same.
20 8. Disru~tin~ PS-Interactin~ Protein Interactions
The ability to disrupt specific interactions of the PS-interacting proteins
with the prec~nii;n.~, or with other ylvl~hls~ is potentially of great therapeutic value,
and will be important in underst~n-ling the etiology of AD and in identif~ing
additional targets for therapy. The methods used to identify compounds which disrupt
25 PS-interacting protein interactions may be applied equally well to interactions
involving either normal or mutant PS-interacting proteins.
Assays for compounds which can disrupt PS-interacting protein
interactions may be performed by any of a variety of methods well known in the art.
In essence, such assays will parallel those assays for identifying proteins and
30 compounds with binding activity toward the PS-interacting proteins. Thus, once a
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compound with binding activity for a PS-interacting protein is identified by anymethod, that method or an equivalent method may be performed in the presence of
c~n~ te compounds to identify compounds which disrupt the interaction. Thus, forexample, the assay may employ methods including (1) affinity chromatography; (2)imrnunoprecipitation; (3) theBiomolecularInteractionAssay (BI~core); or (4) me
yeast two-hybrid systems. Such assays can be developed using either normal or
mutant purified PS-interacting proteins, and/or either normal or mutant purifiedbinding proteins (e.g., normal or mutant pr~çnilin~).
For affinity methods, either the PS-interacting protein or its binding
10 partner may be affixed to a matrix, for example in a column, and the counterpart
protein (e.g., the PS-interacting protein if pres~nilin or another binding partner is
affixed to the matrix; or a pr~enilin or other binding partner if the PS-interacting
protein is affixed to the matrix) is then exposed to the affixed protein/compound either
before or after adding the candidate compound(s~. In the absence of a disruptive15 effect by the c~n~ te compoumd(s), the interaction between the PS-interactingprotein and its binding par~er will cause the counterpart protein to bind to the affixed
protein. Any compound which disrupts the in~r~-~tion will cause release of the
counterpart protein from the matrix. Release of the counterpart protein from thematrix can be measured using methods lmown in the art.
For PS-interacting protein interactions which are detectable by yeast two-
hybrid systems, these assays may also be employed to identify compounds which
disrupt the interaction. Briefly, a PS-interacting protein and its binding partner (or
al~pl~liate structural domains of each) are employed in the fusion proteins of the
system, and the cells are exposed to candidate compounds to det~otmin~ their effect
25 uponthe~ es~ionofthel~o.Lel gene. By~p~ liatechoiceofareportergene,
such a system can be readily adapted for high through-put screening of large libraries
of compounds by, for example, using a reporter gene which confers rçs;~t~nce to an
antibiotic which is present in the medium, or which rescues an auxotrophic strain
grown m mmlm~l medlum.
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These assays may be used to screen many different types of compounds for
their disruptive effect on the interactions of the PS-interacting proteins. For example,
the compounds may belong to a library of synthetic molecules, or be specificallydesigned to disrupt the interaction. The compounds may also be peptides
5 corresponding to the interacting domain of either protein. This type of assay can be
used to identify compounds that disrupt a specific interaction between a given PS-
interacting protein variant and a given binding partner. In addition, compounds that
disrupt all interactions with PS-interacting proteins may be identified. For exarnple, a
compound that specifically disrupts the folding of PS-interacting proteins would be
10 expected to disrupt all interactions between PS-interacting proteins and other proteins.
,~lt~ tively, this type of disruption assay can be used to identify compounds which
disrupt only a range of dir~clclll PS-interacting protein interactions, or only a single
PS-interacting protein interaction.
15 9. Methods of IdentifYin~ Compounds Mo~ tin~ PS-Interactin~e Protein ActivitvIn another series of embo-lim~ntc, the present invention provides for
methods of identifying compounds with the ability to modulate the activity of normal
and mutant PS-interacting proteins. As used with respect to this series of
embodiments, the term "activity" broadly includes gene and protein c~ c~,~,iOn, PS-
20 interacting protein post-translation procçC~cin~, trafficking and loe~li7~tion~ and any
fimctional activity (e.g., enzyrnatic, receptor-effector, binding, channel), as well as
dowll ,llealn affects of any of these. It is known that Alzheimer's Disease is associated
with increased production of the long form of A~ peptides, the appearance of amyloid
plaques and neurofibrillary tangles, decreases in cognitive function, and apoptotic cell
25 death. Therefore, using the Ll~l~olllled cells and transgenic animal models ofthe
present invention, cells obtained from subjects bearing norrnal or mutant PS-
interacting protein genes, or ~nim~l~ or human subjects bearing naturally occurring
normal or mutaIlt PS-interacting proteins, it is now possible to screen candidate
~h~ cellticals and treatments for their therapeutic effects by detecting changes in
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one or more of these functional characteristics or phenotypic manifestations of normal
or mutant PS-interacting protein e~le~iOn.
Thus, the present invention provides methods for screening or assaying for
proteins, small molecules or other compounds which modulate PS-interacting protein
5 activity by contacting a cell in vivo or in vitro with a c:ln(li~l~te compound and
assaying for a change in a marker associated with normal or mutant PS-interacting
protein activity. The marker associated with PS-interacting protein activity may be
any measurable biochemical, physiological, histological and/or behavioral
characteristic associated with PS-interacting protein e~r~ion. In particular, useful
10 markers will include any measurable biochemical, physiological, histological and/or
behavioral characteristic which distinguishes cells, tissues, ~nim~ or individuals
bearing at least one mutant preePnilin or PS-interacting protein gene from their normal
counterparts. In addition, the marker may be any specific or non-specific measure of
pres.-nilin or PS-interacting protein activity. PS-interacting protein specific measures
15 include measures of PS-interacting protein ~ e~SiOn (e.g., PS-interacting protein
rnRNA or protein levels) which may employ the nucleic acid probes or antibodies of
the present invention. Non-specific measures include changes in cell physiology such
as pH, intracellular calcium, cyclic AMP levels, GTP/GDP ratios,
phosphatidylinositol activity, protein phosphorylation, etc., which can be monitored
20 on devices such as the cytosensor microphysiometer (Molecular Devices Inc., United
States). The activation or inhibition of PS-interacting protein activity in its mutant or
normal form can also be monitored by e~mining ch~n~es in the e~l~s~ion of other
genes (e.g., the prese~ilin~) which are specific to the PS-interacting protein pathway
leading to ~l7heimer's Disease. These can be assayed by such techniques as
25 dirr~ .Lial display, differential hybridization, and SAGE ~sequential analysis of gene
expression), as well as by two (1imtqncional gel electrophoresis of cellular lysates. In
each case, the dirrclelltially-~ essed genes can be ascertained by inspection ofi~enti~zll studies before and after application of the candidate compound.
Furthermore, as noted elsewhere, the particular genes whose ~ sion is modulated
30 by the a-lmini~tration of the candidate compound can be asc~ illed by cloning,
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nucleotide sequencing, amino acid sequencing, or mass spectrometry (reviewed in
Nowak, 1995).
In general, a cell may be contacted with a candidate compound and, a~er
an ~l-lupliate period (e.g., 0-72 hours for most biochemical measures of cultured
5 cells), the marker of pr~s~nilin or PS-interacting protein activity may be assayed and
compared to a baseline measurement. The baseline measurement may be made prior
to contacting the cell with the candidate compound or may be an external baseline
established by other ~c~ lents or known in the art. The cell may be a ll~l~rollned
cell of the present invention or an explant from an animal or individual. ln particular,
10 the cell may be an explant from a carrier of a presenil;n or PS-interacting protein
mutation (e.g., a hurnan subject with ~17heimer's Disease~ or an animal model of the
invention (e.g., a ll~lSg~lliC nematode or mouse bearing a mutant pres~nilin or PS-
interacting protein gene). To augment the effect of pre~nilin or PS-interacting
protein mutations on the A13 ~dlllw~, transgenic cells or :~nim~1~ may be employed
15 which have increased A,3 pro(1~lction Preferred cells include those from neurological
tissues such as neuronal, glial or mixed cell cultures; and cultured fibroblasts, liver,
kidney, spleen, or bone marrow. The cells may be contacted with the candidate
compounds in a culture in vitro or may be ?~rimini.ct~,red in vivo to a live animal or
human subject. For live ~nim~l.c or human subjects, the test compound may be
20 ~1mini~t~ored orally or by any parenteral route suitable to the compound. For clinical
trials of human subjects, measul~ ents may be conducted periodically (e.g., daily,
weekly or monthly) for several months or years.
Because most individuals bearing a mutation in a particular gene are
heterozygous at that locus (i.e., bearing one normal and one mutant allele),
25 compounds may be tested for their ability to modulate normal as well as mutant
pres~nilin or PS-interacting protein activity. Thus, for example, compounds which
enhance the function of normal prçcç~ilin~ or PS-interacting proteins may have utility
in treating Alzheimer's Disease or related disorders. ~lt.om~tively, because
~u~ es~ion of the activity of both normal and mutant copies of a gene in a
30 heterozygous individual may have less severe clinical consequences than progression
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of the associated fli~e~ce it may be desired to identify compound which inactivate or
~u~rt;SS all fo~ns of the presenilins, the PS-interacting proteins, or their interactions.
Preferably, however, compounds are identified which selectively or specifically
inactivate or ~u~leS~ the activity of mutant presenilin or PS-interacting proteins
5 without disrupting the function of their normal counterparts.
In light of the identifir~tion~ characterization, and disclosure herein of a
novel group of PS-interacting genes and proteins, the PS-interacting protein nucleic
acid probes and antibodies, and the PS-interacting protein transformed cells andtr~ncg( nic ~nim~lc of the invention, one of ordinary skill in the art is now enabled by
10 perform a great variety of assays which will detect the modulation of presenilin and/or
PS-interacting protein activity by c~n~ te compounds. Particularly ~lcr~l~d and
contemplated embo-limentc are ~1iccllc~ed in some detail below.
A. PS-Interactin~ Protein Ex~ression
In one series of embo-lim~nt.c, specific measures of PS-interacting protein
15 ~ c~ion are employed to screen candidate compounds for their ability to affect
pres~nilin activity. Thus, using the PS-inter~çting protein nucleic acids and antibodies
disclosed and otherwise enabled herein, one may use mRNA levels or protein levels
as a marker for the ability of a candidate compound to modulate PS-interacting
protein activity. The use of such probes and antibodies to measure gene and protein
20 t;2~ression is well known in the art and ~iccl-cce(l elsewhere herein. C~f particular
interest may be the icl~ntification of compounds which can alter the relative levels of
dirf~lelll variants (e.g., mutant and norrnal) of the PS-interacting proteins.
B. Intracellular Localization
In another series of embo~limerltc~ compounds may be screened for their
25 ability to modulate the activity of the PS-interacting proteins based upon their effects
on the kafficking and inkacellular localization of the PS-interacting proteins. The
presenilins and some of the PS-interacting proteins (e.g., S5a) have been seen
immnn~ cyto~ hemic~lly to be localized in membrane structures associated with the
endoplasmic reticulum and Golgi a~pal~lus. Differences in localization of mutant and
30 normal pr~,c~nilin.c or PS-interacting proteins may, therefore, contribute to the etiology
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of Al7heTmer's Disease and related disorders. C~ompounds which can affect the
localization of these proteins may, therefore, be identified as potential therapeutics.
Standard techniques known in the art may be employed to detect the localization of
the pres~nilin~ and PS-interacting proteins. Generally, these techniques will employ
5 the antibodies of the present invention, and in particular antibodies which selectively
bind to one or more mutant PS-interacting proteins but not to normal proteins. As is
well known in the art, such antibodies may be labeled by any'of a variety of
techniques (e.g., fluorescent or radioactive tags, labeled secondary antibodies, avidin-
biotin, etc.) to aid in vi~ li7ing the intr~ell~ r location of these proteins. The PS-
10 interacting proteins may be co-localized to particular structures, as in known in the
art, using antibodies to markers of those structures (e.g., TGN38 for the Golgi,kansferrin receptor for post-Golgi transport vesicles, LAMP2 for lysosomes).
Western blots of purified fractions from cell lysates enriched for different intracellular
membrane bound organelles (e.g., lysosomes, synaptosomes, Golgi) may also be
15 employed.
B. Ion ~egulation/Metabolism
In another series of embodiments, compounds may be screened for their
ability to modulate the activity of the prPsenilin~ or PS-interacting proteins based
upon measures in intracellular Ca2+, Na~ or K~ levels or metabolism. As noted above,
20 the presenilin~ are membrane associated proteins which may serve as, or interact with,
ion lec~l~ or ion channels. Thus, compounds may be screened for their ability tomodulate pres~nilin and PS-interacting protein-related metabolism of calcium or other
ions either in vivo or in vitro by, for example, measurements of ion channel fluxes
and/or tr~n~m~mhrane voltage and/or current fluxes, using patch clamps, voltage
25 clamps or fluorescent dyes sensitive to intracellular ion levels or tr~n~memhrane
voltage. Ion channel or receptor function can also be assayed by measurements ofactivation of second messengers such as cyclic AMP, cGMP tyrosine kin~ces,
phosphates, increases in intracellular Ca2~ levels, etc. Recombinantly made proteins
may also be reconstructed in artificial membrane systems to study ion channel
30 con~uctz~nce and, therefore, the "cell" employed in such assays may comprise an
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artificial membrane or cell. Assays for changes in ion regulation or metabolism can
be performed on cultured cells ~ es~ g endogenous normal or mutant presenilins
and PS-interacting proteins. Such studies also can be performed on cells transfected
with vectors capable of ~lCS~illg one of the presenilinc or PS~ L~l~cLi~lg proteins, or
functional domains of one of the pres~nilin~ or PS-interacting proteins, in normal or
mutant form. In addition, to enhance the signal measured in such assays, cells may be
co-transfected with genes encoding ion channel proteins. For example, Xenopus
oocytes or rat kidney (HEK293) cells may be co-transfected with sequences encoding
rat brain Na+ ~1 subunits, rabbit skeletal muscle Ca2+ ~ ul)U~ , or rat heart K+ ,B 1
10 subunits. Changes in prç~enilin or PS-interacting protein-mediated ion channel
activity can be measured by, for example, two-microelectrode voltage-clamp
recordings in oocytes, by whole-cell patch-clamp recordings in HEK293 cells. or by
equivalent means.
C. Apoptosis or Cell Death
In another series of embo-lim~?nt~, compounds may be screened for their
ability to modulate the activity of the pres~nilin~ or PS-interacting proteins based
upon their effects on presen;lin or PS-hlLel~ ;lillg protein-related apoptosis or cell
death. Thus, for example, baseline rates of apoptosis or cell death may be established
for cells in culture, or the baseline degree of neuronal loss at a particular age may be
established post-mortem for animal models or human subjects, and the ability of a
candidate compound to ~U~ S~ or inhibit apoptosis or cell death may be measured.Cell death may be measured by standard microscopic techniques (e.g., light
microscopy) or apoptosis may be measured more specifically by characteristic nuclear
morphologies or DNA fr~nent~tion patterns which create nucleosomal ladders (see,e.g., Gavrieli et al., 1992; Jacobson et al., 1993; Vito et al., 1996). TUNEL may also
be employed to evaluate cell death in brain (see, e.g., T ~m~nn et al., 1995). In
preferred embo~iment~, compounds are screened for their ability to ~LIppleSS or inhibit
neuronal loss in the transgenic animal models of the invention. Transgenic mice
bearing, for example, a mutant human, mutant mouse, or hllm~ni7e-1 mutant presenilin
or PS-interactin~ protein gene may be employed to identify or evaluate compounds
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which may delay or arrest the neurodegeneration associated with Alzheimer's
Disease. A similar transgenic mouse model, bearing a mutant APP gene, has recently
been reported by Games et al. (1995).
D. A,B Peptide Production
In another series of embodiments, compounds may be screened for their
ability to modulate pres~nilin or PS-interacting protein-related ch~n~es in APP
processing. The A,13 peptide is produced in several isoforms rçsulting from differences
in APP processing. The A,B peptide is a 39 to 43 amino acid derivative of ,BAPP
which is progressively deposited in diffuse and senile plaques and in blood vessels of
subjects with AD. In human brain, A~ peptides are heterogeneous at both the N- and
C-termini. Several observations, however, suggest that both the full length and N-
t~ min~l trlm~ ~te~l forms of the long-tailed A~ peptides ending at residue 42 or 43
(i.e., A~ 1-42/43 and A~x-42/43) have a more important role in AD than do peptides
ending at residue 40. Thus, A~ 42/43 and A,Bx-42/43 are an early and prominent
feature of both senile plaques and diffuse plaques, while peptides ending at residue 40
(i.e., A,B 1 -40 and A,Bx-40) are predoll~illalllly associated with a subset of mature
plaques and with amyloidotic blood vessels (see, e.g., Iwatsubo et al., 1995; Gravina
et al., 1995; Tamaoka et al., 1995; Podlisny et al. 1995). Furthermore, the long-tailed
isoforms have a greater ~lupellsil~ to fibril for nation, and are thought to be more
neurotoxic than A,B1-40 peptides (Pike et al., 1993; Hilbich et al., 1991). Finally,
mics.-nce mutations at codon 717 of the ,BAPP gene are associated with early onset
FAD, and result in overproduction of long-tailed A,B in the brain of affected mutation
carriers, in peripheral cells and plasma of both affected and presymptomatic carners,
and in cell lines tr~ncfecterl with l3APP717 mutant cDNAs (T~ k~ et al., 1994;
26 Suzuki et al., 1994).
Thus, in one series of embo-limentc, the present invention provides
methods for screening candidate compounds for their abil;ty to block or inhibit the
increased production of long isoforms of the A,B peptides in cells or transgenic~nim~lc ~.fessi.lg a normal or mutant presenilin gene and/or a normal or mutant PS-
interacting protein gene. In particular, the present invention provides such methods in
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which cultured m~mm~ n cells, such as brain cells or fibroblasts, have been
transformed according to the methods disclosed herein, or in which transgenic
~nlm~l~, such as rodents or non-human primates, have been produced by the methods
disclosed herein, to express relatively high levels of a normal or mutant pres~nilin or
5 PS-interacting protein. Optionally, such cells or tr~n~g~nic ~nim~l~ may also be
rc,lll,ed so as to express a normal or mutant form of the ,BAPP protein at
relatively high levels.
In this series of embo(liment~, the candidate compound is ~1mini~t~red to
the cell line or transgenic ~nim~l~ (e.g., by addition to the media of cells in culture; or
10 by oral or p~llLt;lal ~1mini~tration to an animal) and, after an ap~l~,pl;ate period
(e.g., 0-72 hours for cells in culture, days or months for animal models), a biological
sample is collected (e.g., cell culture supe~t~nt or cell lysate from cells in culture;
tissue homogenate or plasma from an animal) and tested for the level of the longisoforms of the A,B peptides. The levels of the peptides may be ~let~rmine-l in an
15 absolute sense (e.g., nMol/ml) or in a relative sense (e.g., ratio of long to short A,B
isoforms). The A,~ isoforms may be detecte~l by any means known in the art (e.g.,
electrophoretic separation and se~uencing) but, preferably, antibodies which arespecific to the long isoform are employed to ~1et~nnine the absolute or relative levels
of the A~ l -42/43 or A,Bx-42/43 peptides. Candidate ph~rrn~cellticals or therapies
20 which reduce the absolute or relative levels of these long A~ isofolms, particularly in
the transgenic animal models of the invention, are likely to have ~t;l~eutic utility in
the treatment of Alzheimer's Disease, or other disorders caused by mutations in the
presenilins or PS-interacting proteins, or by other aberrations in APP metabolism.
E. PhosPhorvlation of Microtubule Associated Proteins
In another series of embodiments, c~n(lil1~tç compounds may be screened
for their ability to modulate prest-nilin or PS-interacting protein activity by ~çs~ing
~ the effect of the compound on levels of phosphorylation of microtubule associated
proteins (MAPs) such as tau. The abnormal phosphorylation of tau and other MAPs
in the brains of victims of Alzheimer's Disease is well known in the art. Thus,
3~ compounds which prevent or inhibit the abnormal phosphorylation of MAPs may
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have utility in treating presenilin or PS-interacting protein-associated diseases such as
AD. As above, cells from normal or mutant ~nim~l~ or subjects, or the transformed
cell lines and animal models of the invention may be employed. Preferred assays will
employ cell lines or animal models transformed with a mutant human or hllm~ni7f--1
5 mutant presenilin or PS-interacting protein gene. The baseline phosphorylation state
of MAPs in these cells may be established and then ç~n~ te compounds may be
tested for their ability to prevent, inhibit or counteract the hyperphosphorylation
associated with 111~ The phosphorylation state ofthe MAPs may be determined
by any standard method known in the art but, preferably, antibodies which bind
10 selectively to phosphorylated or unphosphorylated epitopes are employed. Suchantibodies to phosphorylation epitopes of the tau protein are known in the art (e.g.,
ALZ50).
10. Screening and Dia~nostics for Alzheimer's Disease
A. General Dia~nostic Methods
The PS-i-lt~dclil-g genes and gene products, as well as the PS-interacting
protein derived probes, primers and antibodies, disclosed or otherwise enabled herein,
are useful in the screening for carriers of alleles associated with Alzheimer's Disease,
for (1 j~osi-c of victims of ~l7h-oimer's Disease, and for the screening and ~ no~ic of
20 related presenile and senile cl~m~nti~, psychiatric ~ e~cçs such as schizophrenia and
depression, and neurologic ~ ez~es such as stroke and cerebral hemorrhage, all of
which are seen to a greater or lesser extent in symptomatic human subjects bearing
mutations in the PS 1 or PS2 genes or in the APP gene. Individuals at risk for
Alzheimer's Disease, such as those with AD present in the family pedigree, or
25 individuals not previously known to be at risk, may be routinely screened using
probes to detect the presence of a mutant PS-interacting protein gene or protein by a
variety of techniques. Diagnosis of inherited cases of these diseases can be
accomplished by methods based upon the nucleic acids (including genomic and
mRNA/cDNA sequences), proteins, and/or antibodies disclosed and enabled herein,
30 including functional assays designed to detect failure or ~ nent~tion of the normal
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presenilin or PS-interacting protein activity and/or the presence of specific new
activities conferred by mutant PS-interacting proteins. Preferably, the methods and
products are based upon the human nucleic acids, proteins or antibodies, as disclosed
or otherwise enabled herein. As will be obvious to one of ordinary skill in the art,
5 however, the significant evolutionary co~ v~lion of large portions of nucleotide and
amino acid sequences, even in species as diverse as hnm~n~, mice, C. ele~ans, and
Drosophila, allow the skilled artisan to make use of non-human homologues of thePS-interacting proteins to produce useful nucleic acids, proteins and antibodies, even
for applications directed toward human or other animal subjects. Thus, for brevitv of
10 exposition, but without limiting the scope of the invention, the following description
will focus upon uses of the human homologues of PS-interacting proteins and genes.
It will be un(1erstQod~ however, that homologous sequences from other species will be
equivalent for many purposes.
As will be appreciated by one of ordinary skill in the art, the choice of
15 diagnostic methods of the present invention will be jnfll~enfe-l by the nature of the
available biological samples to be tested and the nature of the inforrnation required.
~l~heimer's Disease is, of course, primarily a disease of the brain, but brain biopsies
are illV~lSiV~ and expensive procedures, particularly for routine screening. Other
tissues which express the presPnilin~ or PS-interacting proteins at ~i nific~nt levels
20 may, therefore, be plere.,~d as sources for samples.
B. Protein Based Screens and Dia~nostics
When a diagnostic assay is to be based upon PS-interacting proteins, a variety
of approaches are possible. For example, diagnosis can be achieved by monitoringdifferences in the electrophoretic mobility of normal and mutant proteins. Such an
25 approach will be particularly useful in identifying mllt~nt~ in which charge
substitutions are present, or in which insertions, deletions or substitutions have
resulted in a significant change in the electrophoretic migration of the res--lt~nt
protein. AltPrn~tively~ diagnosis may be based upon differences in the proteolytic
cleavage pattems of normal and mutant proteins, differences in molar ratios of the
-
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various amino acid residues, or by functional assays demonstrating altered function of
the gene products.
In ~lef~ d embodiments, protein-based diagnostics will employ differences
in the ability of antibodies to bind to normal and mutant PS-interacting proteins. Such
~ nostic tests may employ antibodies which bind to the normal proteins but not to
mutant proteins, or vice versa. ~n particular, an assay in which a plurality of
monoclonal antibodies, each capable of binding to a mutant epitope, may be
employed. The levels of anti-mutant antibody binding in a sample obtained from atest subject (vi~ ed by, ~or example, radiolabelling, ELISA or chetn~ min~ccence)
10 may be compared to the levels of binding to a control sample. ~lt~tn~t;vely,
antibodies which bind to normal but not mutant proteins may be employed, and
decreases in the level of antibody binding may be used to distin~li~h homozygousnormal individuals from mutant heterozygotes or homozygotes. Such antibody
diagnostics may be used for in situ immllnohi~toch~rni~try using biopsy samples of
15 CNS tissues obtained antemortem or l)o~ llem, including neuropathological
structures associated with these diseases such as neurofibrillary tangles and amyloid
plaques, or may be used with fluid samples such a cwe6l~ al fluid or with
peripheral tissues such as white blood cells.
C. Nucleic Acid Based Screens and Di~nostics
When the diagnostic assay is to be based upon nucleic acids from a sample,
the assay may be based upon ml?NA, cDNA or genomic DNA. When mRNA is used
from a sample, there are considerations with respect to source tissues and the
possibility of alternative splicing. I'hat is, there may be little or no ~les~ion of
transcripts unless ~lv~iate tissue sources are chosen or available, and alternative
~5 splicing may result in the loss of some inforrn~tion or difficulty in i~ yl~Lation~
Whether mRNA, cDNA or genomic DNA is assayed, standard methods well known in
the art may be used to detect the presence of a particular sequence either in situ or in
vitro (see, e.g., Sambrook et al., (1989) Molecular Clonin~: A Laboratorv Manual,
2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY). As a general matter,30 however, any tissue with nucleated cells may be ex~mined.
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Genomic DNA used for the diagnosis may be obtained from body cells, such
as those present in the blood, tissue biopsy, surgical specimen, or autopsy material.
The DNA may be isolated and used directly for detection of a specific sequence or
may be amplified by the polymerase chain reaction (PCR) pnor to analysis.
5 Similarly, RNA or cDNA may also be used, with or without PCR amplification. Todetect a specific nucleic acid sequence, direct nucleotide sequencing, hybridization
using specific oligonucleotides, restriction enzyme digest and mapping, PCR
mapping, RNase protection, chemical mi~m~t~h cleavage, ligase-me~ ted detection,and various other methods may be employed. Oligonucleotides specific to particular
lû sequences can be chemically syn1hesi7~1 and labeled ~adioactively or non-
radioactively (e.g., biotin tags, ethidium bromide), and hybridized to individual
samples immobilized on membranes or other solid-supports (e.g., by dot-blot or
transfer from gels after electrophoresis), or in solution. The presence or absence of
the target sequences may then be vicn~li7~1 using methods such ~ autoradiography,
15 fluorometry, or colorimetry. These procedures can be automated using rc(ll-ntl~nt,
short oligonucleotides of known sequence fixed in high density to silicon chips.~I) Appropriate Probes and Primers
Whether for hybridization, RNase protection, ligase-me~ te-1 detection, PCR
amplification or any other standards methods described herein and well known in the
20 art, a variety of subsequences of the PS-interacting protein sequences disclosed or
otherwise enabled herein will be useful as probes and/or primers. These sequences or
subsequences will include both normal sequences and deleterious mutant sequences.
In general, useful sequences will include at least 8-9, more preferably l 0-50, and most
pleL~lably 18-24 consecutive nucleotides from introns, exons or intron/exon
25 boundaries. Depending upon the target sequence, the specificity required, and future
technological developments, shorter sequences may also have utility. Therefore, any
PS-interacting protein derived sequence which is employed to isolate, clone, amplify,
identify or otherwise manipulate a PS-interacting protein sequence may be regarded as
an ~ Liate probe or primer. Particularly contemplated as useful will be sequences
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including nucleotide positions from the PS-interacting protein genes in which disease-
causing mutations are lcnown to be present, or sequences which flank these positions.
(2) Hvbridization Screenin~
For in situ detection of a normal or mutant PS-interacting protein-related
nucleic acid sequence, a sample of tissue may be prepared by standard techniques and
then contacted with one or more of the above-described probes, preferably one which
is labeled to facilitate detection, and an assay for nucleic acid hybridization is
conducted under stringent conditions which perrnit hybridization only between the
probe and highly or perfectly complementary sequences. Because many mutations
10 consist of a single nucleotide substitution, high stringency hybridization conditions
may be required to ~ tingui~h normal sequences from most mutant sequences. When
the PS-interacting protein genotypes of the subject's parents are known, probes may
be chosen accordingly. Alternatively, probes to a variety of ,~ may be
employed sequentially or in combination. Because most individuals carrying
15 mutations in the PS-interacting proteins will be heterozygous, probes to normal
sequences also may be employed and homozygous normal individuals may be
~ictin~li~hed from mutant heterozygotes by the amount of binding (e.g., by intensity
of radioactive signal). In another variation, competitive binding assays may be
employed in which both normal and mutant probes are used but only one is labeled.
~3) RestrictionMappin~
Sequence alterations may also create or destroy fortuitous restriction enzyme
recognition sites which are revealed by the use of a~l~l;ate enzyme digestion
followed by gel-blot hybridization. DNA fr~ nt~ carrying the site (normal or
mutant) are detected by their increase or reduction in size, or by the increase or
25 decrease of corresponding restriction fragment nurnbers. Such restriction fragment
length polyrnorphism analysis (RF~P), or restriction mapping, may be employed with
genomic DNA, mRNA or cDNA. The PS-interacting protein sequences may be
amplified by PCR using the above-described primers prior to restriction, in which
case the lengths of the PCR products may indicate the presence or absence of
30 particular restriction sites~ and/or may be subjected to restriction after amplification.
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The restriction fragments may be vi.~ ed by any convenient means (e.g., under W
light in the presence of ethidium bromide).
(4) PCR Mappin~
In another series of embodiment.s, a single base sTIhstitlltion mutation may be
detected based on differential PCR product length or production in PCR. Thus,
primers which span mutant sites or which, preferably, have 3' termini at mutation
sites, may be employed to amplify a sample of genomic DNA, mRNA or cDNA ~om
a subject. A mi~m~tch at a mutational site may be expected to alter the ability of the
no~nal or mutant primers to promote the polymerase reaction and, thereby, result in
product profiles which differ between normal subjects and heterozygous ~d/or
homozygous ~ ; The PCR products of the normal and mutant gene may be
differentially separated and detected by standard techniques, such as polyacrylamide
or agarose gel electrophoresis and vi.~u~ tion with labeled probes, ethidium
bromide or the like. Because of possible non-specific priming or readthrough of
mutation sites, as well as the fact that most carriers of mutant alleles will beheterozygous, the power of this technique may be low.
(5) Electrophoretic Mobilitv
Genetic testing based on DNA sequence dirr~ ces also may be achieved by
detection of alterations in electrophoretic mobility of DNA, mRNA or cDNA
fragments in gels. Small sequence deletions and insertions, for example, can be
visualized by high resolution gel ele~ ul~hore~is of single or double stranded DNA, ûr
as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel
electrophoresis. Mutations or polyrnorphisms in the PS-interacting protein genes may
also be rletectefT by methods which exploit mobility shifts due to single-skanded
conformational polymorphisms (SSCP) associated with mRNA or single-stranded
DNA secondary structures.
~6) ChemicalCleava eofMi~m~tches
Mutations in the PS-interacting protein genes may also be detected by
employing the chemical cleavage of mi~m~tch (CCM) method (see, e.g., Saleeba andCûtton~ 1993, and references therein). In this technique, probes (up to ~ 1 kb) may be
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mixed with a sample of genomic DNA, cDNA or mRNA obtained from a subject.
The sample and probes are mixed and subjected to conditions which allow for
heteroduplex formation (if any). Preferably, both the probe and sample nucleic acids
are double-stranded, or the probe and sample may be PCR amplified together, to
ensure creation of all possi~le micm~t~h heteroduplexes. Micm~tched T residues are
reactive to osmium tetroxide and micm~tr.hed C residues are reactive to
hydroxylamine. Because each micm~tched A will be accompanied by a micm~t-~hed
T, and each mi.cm~tcheri G will be accompanied by a micm~tched C, any nucleotidedifferences bet~,veen the probe and sample (including small insertions or deletions~
I0 will lead to the formation of at least one reactive heteroduplex. After treatment with
osmium tetroxide and/or hydroxylamine to modify any micm~tc h sites, the ~ ule is
subjected to chemical cleavage at any modified micm~tch sites by, for example,
reaction with piperidine. The l~ Lw e may then be analyzed by standard techniques
such as gel electrophoresis to detect cleavage products which would in~lic~t~
mi.cm~tches between the probe and sample.
(7) Other Methods
Various other methods of detecting PS-interacting protein mutations, based
upon the sequences disclosed and othe~wise enabled herein, will be a~n~ to thoseof ordinary skill in the art. Any of these may be employed in accordance with the
present invention. These in- IIT(ie, but are not limited to, nuclease protection assays
(S l or ligase-me~ tçd), ligated PCR, cl~ . gradient gel electrophoresis (DGGE;
see, e.g., Fischer and T f nn~n, 1983), restriction ~n~lonllclease fin~ g
combined with SSCP (REF-SSCP; see, e.g., Liu and Sommer, 1995~, and the like.
D. Other Screens and Dia~nostics
In inherited cases, as the primary event, and in non-inherited cases as a
secondary event due to the disease state, abnormal processing of the presenilinc, PS-
interacting proteins, APP, or proteins reacting with the presenilins, PS-interacting
proteins, or APP may occur. This can be detected as abnorrnal phosphorylation,
glycosylation, glycation amidation or proteolytic c}eavage products in body tissues or
fluids (e.g., CSF or blood).
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Diagnosis also can be made by observation of alterations in transcription,
translation, and post-translational modification and processing, as well as alterations
in the intracellular and extracellular trafficking of gene products in the brain and
peripheral cells. Such changes will include alterations in the amount of messenger
S RNA and/or protein, alteration in phosphorylation state, abnormal intracellular
location/distribution, abnormal ext~acellular distribution, etc. Such assays will
include: Northern Blots (e.g., with PS-interacting protein-specific and non-specific
nucleotide probes), Westem blots and enzyme-linked immunosorbent assays (ELISA)
(e.g., with antibodies raised specifically to a PS-interacting protein or PS-interacting
10 functional domain, including various post-translational modification states including
glycosylated and phosphorylated isoforms). These assays can be performed on
peripheral tissues (e.g., blood cells, plasma, cultured or other fibroblast tissues, etc.)
as well as on biopsies of CNS tissues obtained ~nt~mf~rtem or postmortem, and upon
cerebrospinal fluid. Such assays might also include in situ hybricli7~tion and
15 immlmohistochemistry (to localize mP~Pnger RNA and protein to specific subcellular
cun~ ~ ents and/or within neuropathological structures associated with these
diseases such as neurofibrillary tangles and amyloid plaques).
. Screenin~ and Diagnostic Kits
In accordance with the present invention, (ii~gnos~ic kits are also provided
20 which will include the reagents necessary for the above-described diagnostic screens.
For example, kits may be provided which include antibodies or sets of antibodieswhich are specific to one or more mutant epitopes. These antibodies may, in
particular, be labeled by any of the standard means which facilitate vi~u~li7~tion of
binding. ~ltern~tively~ kits may be provided in which oligonucleotide probes or PCR
25 lprimers, as described above, are present for the detection and/or amplification of
normal or mutant pr~enilin and/or PS-interacting protein nucleotide sequences.
Again, such probes may be labeled for easier detection of specific hybridization. As
a~lo~liate to the various diagnostic embodiments described above, the
oligonucleotide probes or antibodies in such kits may be immobilized to substrates
30 and ~p.opliate controls may be provided.
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1 l . Methods of Treatment
The present invention now provides a basis for therapeutic intervention in
e~cec which are caused, or which may be caused, by mutations in the PS-
5 interacting proteins. As noted above, mutations in the hPS 1 and hPS2 genes havebeen associated with the development of early onset forms of Al7heimer's Disease
and, therefore, the present invention is particularly directed to the trP~tment of
subjects ~i~gnosed with, or at risk of developing, ~l7heimer's Disease.
Without being bound to any particular theory of the invention, the effect of
10 the ~ heimer's Disease related mutations in the precPnilinc appears to be a gain of a
novel function, or an acceleration of a normal function, which directly or indirectly
causes aberrant processing of the Amyloid Precursor Protein (APP) into A,B peptide,
abnormal phosphorylation homeost~cic, and/or abnormal apoptosis in the brain. Such
a gain of function or acceleration of function model would be conci.ct~nt with the adult
15 onset of the sy,~ lol~s and the dominant inh~rit~nce of Alzheimer's Disease.
Nonetheless, the lllecl~ m by which mutations in the prPsenilin~ may cause theseeffects remains unknown.
The present invention, by identifying a set of PS-interacting proteins,
provides new therapeutic targets for ill~ ing in the etiology of precenilin-related
20 AD. In addition, as mutations in the pres~-nilinc may cause AD, it is likely that
mutations in the PS-interacting proteins may also cause AD. The fact that the PS-
interacting protein SSa is alternately processed in the brains of victims of sporadic
AD, as well as in the brains of victims of presenilin-linked AD, suggests that, at the
very least, this PS-interacting protein is involved in the etiology of AD independent of
25 mutations in the presPnilinc It is likely that the other PS-interacting proteins also
may be involved in non-presenilin-linked AD.
Therapies to treat PS-interacting protein-associated ~ cP~5 such as AD
may be based upon (1) ~dminictration of normal PS-interacting proteins, (2~ genetherapy with normal PS-interacting protein genes to compensate for or replace the
30 mutant genes, (3~ gene therapy based upon antisense sequences to mutant PS-
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interacting protein genes or which "knock out" the mutant genes, (4) gene therapy
based upon sequences which encode a protein which blocks or corrects the deleterious
effects of PS-interacting protein mutants, (5) irnrnunotherapy based upon antibodies to
normal and/or mutant PS-interacting proteins, or (6) small molecules ~drugs) which
5 alter P~-interacting protein ~ ,s~ion, alter interactions between PS-interacting
proteins and other proteins or ligands, or which otherwise block the aberrant function
of mutant pres~nilin or PS-interacting proteins by ~lt~ring the structure ofthe mutant
proteins, by enhancing their metabolic clearance, or by inhibiting their function.
A. Protein Thera~y
Treatment of .AI7h~imer's Disease, or other disorders res-llting from PS-
interacting protein mutations, may be ~rulllled by replacing the mutant protein with
normal protein, by modlll~tin~ the function of the mutant protein, or by providing an
excess of normal protein to reduce the effect of any aberrant function of the mutant
proteins.
To accomplish this, it is n~c~ ry to obtain, as described and enabled
herein, large amounts of substantially pure PS-interacting protein from cultured cell
systems which can express the protein. Delivery of the protein to the affected brain
areas or other tissues can then be accomplished using a~ru~l;ate p~ck~ing or
~lmini.ctration systems including, for example, liposome mediated protein delivery to
2û the target cells.
B. Gene Therapv
Tn one series of embodiments, gene therapy may be employed in which
normal copies of a PS-interacting protein gene are introduced into patients to code
sl~cces~llly for normal protein in one or more different affected cell types. The gene
25 must be delivered to those cells in a form in which it can be taken up and code for
sufficient protein to provide effective function. Thus, it is plcr~ l~c;d that the
recombinant gene be operably joined to a strong promoter so as to provide a highlevel of e~l!L~ssion which will compensate for, or out-compete, the mutant proteins.
As noted above, the recombinant construct may contain endogenous or exogenous
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regulatory elements, inducible or repressible regulatory elements, or tissue-specific
regulatory elements.
In another series of embodiments, gene therapy may be employed to
replace the mutant gene by homologous recombination with a recombinant construct.
5 The recombinant construct may contain a normal copy of the targeted PS-interacting
protein gene, in which case the defect is coll~.;led in situ, or may contain a "knock-
out" construct which introduces a stop codon, mi~sPnce mutation, or deletion which
abolished function of the mutant gene. It should be noted in this respect that such a
construct may knock-out both the normal and mutant copies of the targeted gene in a
10 heterozygous individual, but the total loss of gene function may be less deleterious to
the individual than contimle-l progression of the disease state.
In another series of embo~lim~ntc, :mti~cpnce gene therapy may be
employed. The ~nticence therapy is based on the fact that sequence-specific
~uyp~es~ion of gene ~ ,res~ion can be achieved by intracell~ r hybridization between
15 mRNA or DNA and a complement~ ntic~n~ce species. The formation of a hybrid
duplex may then interfere with the Ll~ls~ ,Lion of the gene and/or the pr~c~ccin~,
transport, translation and/or stability of the target rnRNA. Anticen.ce strategies may
use a variety of approaches including the ~flminictration of ~nti.e-once oligonucleotides
or antisense oligonucleotide analogs (e.g., analogs with phosphorothioate backbones)
20 or l~ reclion with ~ntictonce RNA ~yles~ion vectors. Again, such vectors may
include exogenous or endogenous regulatory regions, inducible or ~ ible
regulatory elements, or tissue-specific regulatory elements.
II1 another series of embodiments, gene therapy may be used to introduce a
recombinant construct encoding a protein or peptide which blocks or otherwise
25 corrects the aberrant fimction caused by a mutant presenilin or PS-interacting protein
gene. In one embodiment, the recombinant gene may encode a peptide which
corresponds to a domain of a PS-interacting which has been found to abnormally
interact with another cell protein or other cell ligand (e.g., a mutant prçsenilin). Thus,
for example, if a mutant PS l TM6~7 domain is found to interact with a PS-
30 interacting protein but the corresponding normal TM6~7 domain does not undergo
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this interaction, gene therapy may be employed to provide an excess of the mutant
TM6~7 domain which may compete with the mutant prçsloni1in protein and inhibit or
b}ock the aberrant interaction. ~1t~ tively, the PS-interacting domain of a PS-
interacting protein which interacts with a mutant, but not a normal, presenilin may be
5 encoded and expressed by a recombinant construct in order to compete with, and thereby inhibit or block, the aberrant interaction.
Retroviral vectors can be used for somatic cell gene therapy especially
because of their high efficiency of infection and stable integration and ~;~pl~ssion. A
full length PS-interacting protein gene, subsequences encoding fimctional domains of
10 ~ these proteins, or any of the other therapeutic peptides described above, can be cloned
into a retroviral vector and ~ es~ion may be driven fiom its endogenous promoter,
from the retroviral long tt-rmin~1 repeat, or from a promoter specific for the target cell
type of interest (e.g., neurons). Other viral vectors which can be used include adeno-
associated virus, vaccinia virus, bovine papilloma virus, or a herpes virus such as
15 Epstein-Barr virus.
C. Tmmlmothera~y
Irnmunotherapy is also possible for ~17h~imer's Disease. Antibodies may
be raised to a norrnal or mutant PS-interacting protein (or a portion thereof) arrd are
2~-1min;etered to the patient to bind or block an aberrant interaction ~e.g., with a mutant
20 pr~eenilin) and y~ rellL its deleterious effects. Simn1t~n~ously, ~ ssion of the
normal protein product could be encouraged. ~1t~rn~tively, antibodies may be raised
to specific complexes between mutant or wild-type PS-interacting proteins and their
interaction partners.
A further approach is to stim~ te endogenous antibody production to the
25 desired arltigen. A-lminietration could be in the forrn of a one time imm1mngenic
r~l~lion or vaccine immunization. The PS-interacting protein or other antigen may
be mixed with ph~rm~ceutically acceptable caIriers or excipients compatible with the
protein. The immtmogenic composition and vaccine may fi~rther contain auxiliary
substances such as emulsifying agents or adiuvants to enhance effectiveness.
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Tmmllnngenic compositions and vaccines may be ~lmini.~tered palel,LeLdlly by
injection subcutaneously or i~ uscularly.
D. Small Molecule Thera~eutics
As described and enabled herein, the present invention provides for a
5 number of methods of identifying small molecules or other compounds which may be
useful in the treatment of Alzheimer's Disease or other disorders caused by mutations
in the presPnilin~ or PS-interacting proteins. Thus, for example, the present invention
provides for methods of identifying proteins which bind to normal or mutant PS-
interacting proteins (aside from the presenilins). The invention also provides for
10 . methods of identifying small molecules which can be used to disrupt aberrant
interactions between mutant presenilin~ and/or Ps-hll~ lg proteins and such other
binding proteins or other cell components.
Exam~les
Example 1. Isolation of PS-interacting ~roteins bv t~,vo-hybrid Yeast system.
To identify proteins interacting with the pres~nilin proteins, a
commercially available yeast two-hybrid kit ("M~t~hm~kPr System 2" from Clontech,
Palo Alto, CA) was employed to screen a brain cDNA library for clones which
interact with fùnctional domains of the pres~nilinc. In view of the likelihood that the
TM6~7 loop domains of the pres~nilin~ are important functional domains, partial
cDNA sequences encoding either residues 266-409 of the normal PS l protein or
residues 272-390 of the normal PS2 protein were ligated in-frame into the EcoRI and
BamHI sites of the pAS2- I fusion-protein expression vector (Clontech). The reSlllt~nt
fusion proteins contain the GAL4 DNA binding domain coupled in-frame either to the
TM6~7 loop of the PS 1 protein or to the TM6~7 loop of the PS2 protein. These
ion plasmids were co-transformed into S. cerevisiae skain Yl90 together with
a library of human brain cDNAs ligated into the pACT2 yeast fusion-protein
~ression vector (Clontech) bearing the GAL4 activation domain using modified
lithium acetate protocols of the "M~t-~hm~ker System 2" yeast two-hybrid kit
(Clontech, Palo Alto, CA). Yeast clones bearing human brain cDNAs which interact
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with the TM6~7 loop domain were selected for His- rc~ict~nce by plating on SD
minim~l medium lacking hi~ti(lin~ a~d for ,Bgal+ activation by color selection. The
His+ ,Bgal~ clones were then purged of the pAS2-1 "bait" construct by culture inlO~lg/ml cyclohexamide and the unknown "trapped" inserts of the human brain
5 cDNAs encoding PS-interacting proteins were isolated by PCR and sequenced. Of 6
million initial transformants, 200 positive clones were obtained af[er His- selection,
and 42 after ,Bgal+ color selection, carried out in accordance with the m~nnf~rturer's
protocol for selection of positive colonies. Of these 42 clones there were several
independent clones represen~ing the same genes.
To address the likelihood that mutations in the pr~c~nilin~ cause ~D
through the acquisition of a novel but toxic function (i.e., dc~ gain of function
mutation) which is meAi~teA by a novel interaction between the mutant proteins and
one or more other cellular proteins, the human brain cDNA libra~y cloned into the
pACT2 ~ s~ion vector (Clontech) was re-screened using mutant TM6~7 loop
domain sequences as described above and according to m~nllf~-~tllrer's protocols. In
particular, mutant pre~e~ilin sequences corresponding to residues 260-409 of PS l
TM6~7 loop domains bearing mutations L286V, L392V and ~290-3 l 9 were ligated
in-frame into the GAL4 DNA-binding domain of the pAS2- l vector (Clontech3 and
used to screen the human brain cDNA:GAL4 activation domain library of pACT
vectors (Clontech). Yeast were co-transformed, positive colonies were selected, and
"trapped" sequences were recovered and sequenced as described above. In addition to
some of the same sequences recovered with the normal TM6~7 loop domains,
several new sequences were obtained which reflect aberrant interactions of the mutant
pr~sPnitin~ with normal cellular proteins.
The recovered and sequenced clones corresponding to these PS-interacting
proteins were colllp~ed to the public sequence databases using the BL:ASTN
algoli~ll.,l via the NCBI e-mail server. Descriptions of several of these clones follow:
Antisecretorv Factor/ Proteasome SSa Subunit. Two overlapping clones
(Y2H29 and Y2H3 1) were identified which correspond to a C-t--~min~l fragment of a
protein ~lt~rn~tively identified as Antisecretory Factor ("ASF") or the Multiubiquitin
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chain binding S5a subunit of the 26S proteasome ("SSa") (Johansson et al. 1995;
Ferrell et al., 1996). The complete nucleotide and amino acid sequences of the S5a
subunit are avaiIable through the public databases under Accession number U51007and are reproduced here as SEQ ID NO: 1 and SEQ ID NO: 2. The nucleotide
5 sequences of the Y2H29 and Y2H31 clones include nucleotides 351-1330 of SEQ IDNO: 1 and amino acid residues 70-377 of SEQ ID NO: 2. Thus, residues 70-377 of
the full SSa subunit include the PS-interacting domain ofthis protein. l~e~ es 206-
377 of S5a contain certain motifs that are important for protein-protein interactions
(Ferrell et al., 1996).
The PS l-S5a subunit h~ a iLion was directly re-tested for both wild type
and mutant PS1 TM6~7 loop (residues 260-409) by tlallsrollllillg Y187 yeast cells
with the ay~lopliate wild type or mutant (L286V, L392V or ~290-319~ cDNA ligatedin-frarne to the GAL4-DNA binding domain of pACT2. The ~290-319 mutant fusion
construct displayed autonomous ,Bgal activation in the absence of any S5a ''target
sequence" and, therefore, could not be filrther analyzed. In contrast, both the L286V
and L392V mutant constructs interacted specifically with the S5a construct.
Q~ ntit~tive assays, however, showed that these interactions were weaker than those
involving the wild type PS 1 260409 sequence and that the degree of interaction was
crudely correlated with the age of onset of FAD. The difference in ,Bgal activation
was not attributable to instability of the mutant PS 126a4O9 construct mRNAs or fusion
proteins because Western blots of Iysates of transformed yeast showed equivalentquantities of mutant or wild-type fusion proteins.
Because one of the putative functions of S5a is to bind multi-ubiq l;tin~t~-l
proteins, the PS 1 :S5a interaction observed in S. cerevisiae could arise either through
yeast-dependent ubiquitination ofthe PS1260409 construct, or by direct interaction. The
former would reflect a degradative pathway, a functional and perhaps reciprocal
interaction between PS1 and S5a, or both. A direct interaction is favored by the fact
that the PS 1 :SSa interaction is decreased rather than increased by the presence of the
L286V and L392V mutations, and by the fact that neither of these mutations affect
ubiquitin conjugation sites in the PS 1260409 loop (i.e., K265, K311, K314 or K395).
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To further examine this possibility, we investigated the direct interaction of
recombinant His-tagged fusion proteins corresponding to full length SSa and the
PSl260"09 loop. Partially purified recombinant His-tagged PSl260409100p and His-tagged SSa proteins and ~~ iate controls were mixed in phosphate buffered saline.
5 The ~ Ul~ was then subjected to size exclusion cl~ul~ ography, and eluates were
e~-nine~1 by SDS-PAGE and Western blotting using anti-His-tag monoclonal
antibodies ~Quiagen). In the crude PSl260409100p ~ Lalion alone, the PSl26040~ loop
eluted from the size exclusion colurnn as a broad peak at 35 l~i....(es In the crude SSa
l~l~al~lion alone, S5a eluted at 25 mlnllt.os However, when the crude PS126o~,og lOop
10 and S5a pl~udlions were mixed~ there was a significant shift in the elution of PS 126o
409 toward a higher molecular weight complex. Co-elution of S5a and PS126o~og in the
same fraction was confinned by SDS-PAGE and Western blotting of fractions using
the anti-~Iis-tag antibody. These results are con.~i~tent with a ubiquitin-independent
and, therefore, possibly functional il~ eLion.
GT24 and related ~enes with homology to pl20/Plako~lobin familY. Five
over-lapping clones (Y2H6, Y2HlOb, Y2H17h2, Y2H24, and Y2H25) were obtained
which interact with the normal PS l TM6~7 loop domain and which appear to
represent at least one novel gene. The Y2H24 clone was also found to interact with
the mutant PS l TM6~7 loop domains. Note that it appears that more than one
member of the gene family was isolated, suggesting a family of genes interactingdifferentially with different presenilins. The most complete availsble cDNA
corresponding to these clones was d~ei~l~t~-l GT24 and is disclosed herein as SEQ ID
NO: 3 and has been deposited with GenBank as Accession number U8 l O04. The
open reading frame suggests that GT24 is a protein of at least 1040 amino acids with a
unique N-Le. .-,;",~ and considerable homology to several ~ lillo (~) repeat
proteins at its C-tl-,nnimls The predicted amino acid sequence of GT24 is disclosed
herein as SEQ ID NO: 4. Thus, for example, residues 440-862 of GT24 have 32-56%
identity (p=1.2e-l33) to residues 440-854 of murine pl20 protein (Accession number
Z17804), and residues 367-815 of GT24 have 26-42% identity (p=0.00 l 7~ to residues
245-465 of the D. melano~aster ~ tiillo segment pol~rity protein (Accession
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number P18824). The C~T24 gene maps to chromosome SplS near the anonymous
microsatellite marker DSS748 and the Cri-du-Chat syndrome locus. This sequence is
also nearly identical to portions of two human ESTs of unknown function (i.e.,
nucleotides 2701 -3018 of Accession number F08730 and nucleotides 2974-3348 of
5 Accession number T18858). These clones also show lower degrees of homology with
other partial cDNA and gDNA sequences (e.g., H17245, T06654, T77214, H24294,
M62015, T87427 and G04019).
pO071 ~ene. An additional His-, ~gal~ clone isolated in the initial screening
with wild type PS 1266409 "bait" had a similar nucleotide sequence to GT24 (target
clone Y2H25; Accession nurnber U81005), and would also be predicted to encode a
peptide with C~-t~rrnin~l arm repeats. A longer cDNA sequence closely corresponding
to the Y2H25 clone has been deposited in GenBank as human protein pO071
(Accession number X81889). The nucleotide and collc;spollding amino acid
sequences of pO071 are reproduced herein as SEQ ID NOs: 5 and 6. Comparison of
the predicted se(luence of the pO071 ORF with that of GT24 confirms that they are
related proteins with 47% overall amino acid sequence identity, and with 70% identity
between residues 346-862 of GT24, and residues 509-1022 of pO071 (which includesresidues encoded by the Y2H25 cDNA). The latter result strongly suggests that PS 1
interacts with a novel class of arm repeat co~ proteins. The broad ~ 4 kb
hybridization signal obtained on Northern blots with the unique 5' end of GT24 could
reflect either alternate splicing/polyadenylation of GT24, or the ~ ten~e of
additional members of this family with higher degrees of N-t~nnin~l homology to
GT24 than pO071.
Rabl 1 ~ene. This clone (Y2H9), disclosed herein as SEQ ID NO: 7, was
identified as interacting with the normal PS 1 TM6~7 loop domain and appears to
correspond to a known gene, Rab l l, available through Accession numbers X56740
and X53143. Rabl 1 is believed to be involved in protein/vesicle traffickin~ in the
ER/Golgi. Note the possible relationship to processing of membrane proteins such as
BAPP and Notch with r~slllt~nt overproduction of toxic A13 peptides (especially
neurotoxic A~ 2(43) isoforms) (Scheuner, et al, 1995).
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Retinoid X receptor-B ~ene. This clone (Y2H23b~, disclosed herein as
SEQ ID NO: 8, was identified as interacting with the normal PSl TM6~7 loop
domain and appears to correspond to a known gene, known variously as the retinoid X
receptor-,B, nuclear receptor co-regulator or MHC Class I regulatory element, and
available through Accession numbers M84820, X63522 and M81766. This gene is
believed to be involved in intercellular ~ 1ing, suggesting a possible relationship to
the intercellular sign~ling function metii~tec~ by C. ele~ans sell2 and Notchllin-12
(transcription activator).
CYtoplasmic chal~ero.lill ~ene. This clone (Y2H27), disclosed herein as
10 SEQ ID NO: 9, was identified as interacting with the nor nal PSl T~6~7 loop
domain and appears to correspond to a known gene, a cytoplasmic chaperonin
col-t~...io~ TCP-1, available through Accession numbers U17104 and X74801.
Unknown gene (~2H35). This clone (Y2H35), disclosed herein as SEQ
ID NO: 10, was identified as interacting with the normal PS 1 TM6~7 loop domain
and appears to correspond to a known gene of unknown function, available throughAccession number R12984, which shows conservation down through yeast.
Unknown gene ~Y2H171). This clone (Y2H171), disclosed herein as SEQ
ID NO: 11, was identified as interacting with the normal PSl TM6~7 loop domain
and appears to correspond to a known expressed repeat sequence available throughAccession ~ b-,l D55326.
Unknown gene (Y2H41). This clone (Y2H41) was identified which reacts
strongly with the TM6~7 loop domains of both PSl and PS2 as well as the mutant
loop domains of PSl. The sequence, disclosed as SEQ ID NO: 12, shows strong
homology to an EST of unknown function (Accession number T64843).
Exarnple 2. Isolation of pres~nilin bindin~ proteins by affinitY chromato~raphy.To identify the proteins which may be involved in the biochemical
- function of the pres~nilin~, PS-interacting proteins were isolated using affinity
chromatography. A GST-fusion protein co..~ .g the PSl TM6~7 loop, prepared
as described in Example 3, was used to probe human brain extracts, prepared by
30 homogenizing brain tissue by Polytron in physiological salt solution. Non-specific
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binding was elimin~tç(l by pre-clearing the brain homogenates of endogenous GST-binding components by incubation with glutathione-Sepharose beads. These GST-
free homogenates were then incubated with the GST-PS fusion proteins to produce the
desired complexes with functional binding proteins. These complexes were then
5 recovered using the affinity glutathione-Sepharose beads. Af'~er extensive washing
with phosphate buffered saline, the isolated collection of proteins was separated by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Tris-tricine gradient gel 4-
20%). Two major bands were observed at ~14 and 20 kD in addition to several
weaker bands ranging from 50 to 60 kD.
The same approach may now be used to identi~y proteins which have
binding activity for the PS-interacting proteins and, thereby, to further elucidate the
etiology of AD and to identify additional thc.dl)~;ulics targets for intervention in AD
and related disorders.
Example 3. Eukarvotic and prokarvotic ~ ession vector svstems.
Constructs suitable for use in eukaryotic and prokaryotic t;~ ;s~ion
systems have been generated using different classes of PS 1 nucleotide cDNA
sequence inserts. In the first class, termed full-length constructs, the entire PS 1 cDNA
sequence is inserted into the ~ es~ion plasmid in the correct orientation, and
includes both the natural 5' UTR and 3' UTR sequences as well as the entire openreading frame. The open reading frames bear a nucleotide se~uence c~sette which
allows either the wild type open reading frame to be in~ ded in the ~ t;s~ion system
or altc;lllalively, single or a combination of double mutations can be inserted into the
open reading frame. This was accomplished by removing a restriction fragment from
the wild type open reading frame using the enzymes NarI and PflmI and replacing it
with a similar fragment generated by reverse L~ scli~l~se PCR and bearing the
nucleotide sequence encoding either the M146L mutation or the H163R mutation. A
second restriction fragment was removed from the wild type normal nucleotide
sequence for the open reading frame by cleavage with the enzyrnes PflmI and NcoIand replaced with a restriction fragment beanng the nucleotide sequence encoding the
A246E mutation, the A260V mutation, the A285V mutation, the L2~6V mutation, the
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L392V mutation or the C410Y mutation. A third variant, bearing a combination of
either the M146L or H163R mutation in tandem with one ofthe re~ i"i"g mutations,was made by linlcing a NarI-PflmI fragment bearing one of the former mutations and a
PflmI-NcoI fragment bearing one of the latter mutations.
The second class of cDN~ inserts, termed truncated constructs, was
constructed by removing the 5' UTR and part of the 3' I~R sequences from full
length wild type or mutant cDNA sequences. The 5' UTR sequence was replaced witha synthetic oligonucleotide cu"t~,"i--g a KpnI restriction site (GGTAC/C) and a small
sequence (GCCACC) to create a Kozak initiation site around the ATG at the
lQ beginning of the PS 1 ORF. The 3' UTl? was replaced with an oligonucleotide with an
artificial EcoRI site at the 5' end. Mutant vanants of this construct were then made by
inserting the mutant sequences described above at the NarI-PflmI and PsImI-NcoI
sites as described above.
For eukaryotic ~ ion, these various cDNA constructs bearing wild
15 type and mutant sequences, as described above, were cloned into the ~ ,ssion
vector pZeoSV in which the SV60 promoter cassette had been removed by restriction
digestion and replaced with the CMV promoter element of pcDNA3 (Invitrogen). Forprokaryotic ~ s~,ion, constructs have been made using the glutathione S-l~ 7r~ldse
(GST) fusion vector pGEX-kg. The inserts which have been ~tt~chefl to the GST
20 fusion nucleotide sequence are the same nucleotide sequences described above
bearing either the normal open reading frame nucleotide sequence, or bearing a
combination of single and double mutations as described above. These GST fusion
constructs allow ~ ssion of the partial or full-length protein in prokaryotic cell
systems as mutant or wild type GST fusion proteins, thus allowing purification of the
25 ffill-length protein followed by removal of the GST fusion product by thrombin
digestion. A filrther cDNA construct was made with the GST fusion vector, to allow
the production of the amino acid sequence corresponding to the hydrophilic acidic
loop domain between TM6 and TM7 of the full-length protein, either as a wild type
nucleotide sequence or as a mutant sequence bearing either the A285V mutation, the
30 L286V mutation or the L392V mutation. This was accomplished by recovering wild
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type or mutant sequence from a~plopliate sources of RNA using a ~' oligonucleotide
primer with a 5' BamHI restriction site (G/GATCC), and a 3' primer with a 5' EcoRI
restriction site (G/AATTC). This allowed cloning of the ~plopl;ate mutant or wild
type nucleotide sequence corresponding to the hydrophilic acidic loop domain at the
BamHI and the EcoRI sites within the pGEX-KG vector.
The PS-interacting protein genes may be similarly manipulated by
recombinant means for ~ ssion in prokaryotic or eukaryotic hosts. In particular,GST or other fusion proteins may be produced which will be useful in assays (e.g.,
yeast two-hybrid studies) for therapeutics.
10 Exam~le 4. AntibodY Production.
Peptide antigens corresponding to portions of the PS l protein were
synth~i7e-1 by solid-phase techniques and purified by reverse phase high P1~;7~;L11e
liquid chro~ ography. Peptides were covalently linked to keyhole limpet
hemocyanin (KLH) via di~lllfide linkages that were made possible by the addition of a
15 cysteine residue at the peptide C-t~rmimls of the pr~s~nilin fr~nent This additional
residue does not appear nonn~lly in the protein sequence and was included only to
f~f~ilit~te linkage to the KLH molecule.
A total ofthree New ~e~nd white rabbits were i.l~ ed with peptide-
KLH complexes for each peptide antigen in combination with Freund's adjuvant and20 were subsequently given booster injections at seven day intervals. Antisera were
collected for each peptide and pooled and IgG ~ ci~ ted with ammonium sulfate.
Antibodies were then affinity purified with Sulfo-link agarose (Pierce) coupled with
the a~plo~liate peptide. This final pllrifi~fion is required to remove non-specific
interactions of other antibodies present in either the pre- or post-immllne serum.
The specificity of each antibody was confirrned by three tests. First, each
detected single predominant bands of the approxirnate size predicted for pres~nilin-l
on Western blots of brain homogenate. Second, each cross-reacted with recombinant
fusion proteins bearing the ayplol~liate sequence. Third each could be specifically
blocked by pre-absorption with recombinant PS l or the i ~ i7ing peptide.
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Antibodies to peptides derived from the PS-interacting proteins may be
produced by similar means.
Example 5. Trans~enic mice.
A series of wild type and mutant PS 1 and PS2 genes were constlucted for
use in the ~ lion of l,~lsgenic mice. Mutant versions of PS 1 and PS2 were
generated by site-directed mutagenesis of the cloned cDNAs using standard
techniques.
The cDNAs and their mutant versions were used to prepare two classes of
mutant and wild type PS 1 and PS2 cDNAs, as described in Example 3. The first
10 class, referred to as "full-length" cDNAs, were prepared by removing approximately
200 bp of the 3' untr~ncl~ted region imm~ t~ly before the polyA site by digestion
with EcoRI (PS1) or PvuII (PS2). The second class, referred to as "I~ n~efl"
cDNAs, were prepared by replacing the 5' untr~n~l~te(l region with a ribosome
binding site (Kozak COll5ell~US sequence) placed immediately 5' of the ATG start15 codon.
Various full length and t~mr~te~i wild type and mutant PSl and PS2
cDNAs, p.c;par~d as described above, were introduced into one or more of the
following vectors and the rçslllting constructs were used as a source of gene for the
production of transgenic mice.
The cos.TET ~ s~ion vector: This vector was derived from a cosmid
clone co~ ;..;..g the Syrian h~m~tPr PrP gene. It has been described in detail by Scott
et aL (1992) and Hsiao et al. (1995). PSl and PS2 cDNAs (full length or trlm-,~te~)
were inserted into this vector at its SalI site. The final constructs contain 20 kb of 5'
sequence fl~nk;ng the inserted cDNA. This 5' fl~nkin~ sequence includes the PrP
25 gene promoter, 50 bp of a PrP gene 5' untr~n~ te~l region exon, a splice donor site, a I
kb intron, and a splice acceptor site located immediately adjacent to the SalI site into
- which the PS 1 or PS2 cDNA was inserted. The 3' sequence fl~nking the inserted
cDNA includes an approximately 8 kb segment of PrP 3' untr~n~l~te-l region
including a polyadenylation signal. Digestion of this construct with NotI (PS 1 ) or
30 FseI (PS2) released a fir~rnent C~ i 1 ~g a mutant or wild type PS gene under the
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control ofthe PrP promoter. The released fragment was gel purified and injected into
the pronuclei of fertilized mouse eggs using the method of Hsiao et al. (1995).
Platelet-derived ~rowth factor receptor ~-subunit constructs: PS cDNAs
were also introduced between the SalI (full length PS 1 cDNAs) or HindIII (trllnf~tefl
5 PS1 cDNAs, full length PS2 cDNAs, and tr ~neate~l PS2 cDNAs) at the 3' end of the
human platelet derived growth factor receptor ~-subunit promoter and the EcoRI site
at the'5' end of the SV40 polyA sequence and the entire r.~csette was cloned into the
pZeoSV vector (Invitrogen, San Diego, CA.). Fr~m~nt~ rele~ed by ScaI/BamHI
digestion were gel purified and injected into the pronuclei of fertilized mouse eggs
10 using the method of Hsiao et al. (1995).
Human R-actin constructs: PS 1 and PS2 cDNAs were inserted into the SalI
site of pBAcGH. The construct produced by this insertion includes 3.4 kb of the
human ~ actin 5' fl~nking sequence (the human ,B actin promoter, a spliced 78 bphuman ~ actin 5' untr~n~l~te~l exon and intron3 and the PS 1 or PS2 insert folIowed by
15 2.2 kb of human growth horrnone ~enl)mic sequence co~ .g several introns and
exons as well as a polyadenylation signal. SffI was used to release a PS-cul.l;1;.-i..g
fragment which was gel purified and injected into the pronuclei of fertilized mouse
eggs using the method of Hsiao et al. (1995).
Phospho~lycerate lcinase constructs: PS1 and PS2 cDNAs were introduced
20 into the pL~90 vector. The cDNAs were inserted between the KpnI site downstream
of the human phosphoglycerate kinase promoter and the XbaI site u~sl1ealll of the 3'
untr~n~l~terl region of the hurnan phosphoglycerate kinase gene. PvuII/~in~lTTT (PS 1
cDNAs) or PvuII (PS2 cDNAs) digestion was used to release a PS-co~
fragment which was then gel purified and injected into the pronuclei of fertilized
25 mouse eggs as described above.
Analvsis of A~ in trans~enic murine hippocampus: To analyze the effect
of a mutant human PS 1 transgene in mice, a PS 1 mutation observed in conjunction
with a particularly severe fonn of early-onset PSl-linked Alzheimer's disease was
used, namely the M146L mis~n~e mutation (!~helTington et al., 1995). The ~nim~
30 which were het ~erozygous ~or the PS 1 mutant transgene on a mixed F~B-C57BL/6
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strain background, were cross-bred with similar mice bearing the human wild-type,~APP69s cDNA under the same Syrian h~m~t-~.r PrP promoter similar to those ~nim~l~
recently described by Hsiao et al., l995. These cross breedings were done because it
is thought that human A13 is more susceptible to the forrnation of aggregates than are
5 murine A,B peptides.
The progeny ofthese PS1MI46L x ,BAPPWT cross-breedings were then
genotyped to identify ~nim~le that contained both the human wild-type ,BAPP69s
transgene and also the mutant human PS1MI46L transgene. These mice were aged until
two to three months of age and then sacrificed, with the hippocatnpus and neocortex
10 being ~ secte~1 rapidly from the brain and frozen. Litter mates of these mice, which
contained only the wild-type human ~APP695 transgene were also sacrificed, and their
hippocampi and neocortices were ~ secte~l and rapidly frozen as well.
The concentration of both total A~ peptides (A,BX 4" and A~X 42 (43)) as well
as the subset of A,B peptides ending on residues 42 or 43 (long-tailed A~42 peptides)
15 were then measured using a two-sandwich ELISA as described previously (Tamaoka
et al., l 994; Suzuki et al., l 994). These results convincingly showed a small increase
in total A,B peptides in the double ll~lsgel~ic ~nim~l~ bearing wild-type human
~APP69s and mutant human PSlM,46L transgenes compared to the wild-type human
~APP695 controls. More hllpl~s~ ely, these mea~ul~lllents also showed that there was
20 an increase in the amount of long-tailed A~ peptides ending on residues 42 or 43
(A~42). In contrast, litter mates bearing only the wild-type human ~APP69s transgene
had A,B42 long-tailed peptide values which were below the limit of qll~ntit~tion("BLQ").
These observations therefore confirrn that the construction of transgenic
25 ~nim~1~ can lecapiLulate some of the bioehemie~1 features of human ~17heimer's
disease (namely the overproduction of A,B peptide and, in particular, overproduction
- of long-tailed isoforms of A~ peptide). These observations thus prove that the
transgenic models are in fact useful in exploring therapeutic targets relevant to the
tre~tment and pr~ ion of Alzheimer's disease.
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Analvsis of hiPPocampus dependent memorv fimctions in PS1 trans~enic
mice: Fourteen transgenic C57BL/6 x FVB mice bearing the human PS1MI46V mutant
transgene under the PrP promoter (as described) above and 12 wild type litter mates
aged 2.5-3 months of age (both groups were balanced for age, weight, and sex) were
5 investigated for behavioral differences attributable to the mutant transgene. Also the
qualitative observation of murine behavior in their home cages did not indicate
bimodal distribution of behaviors in the sample of ~nim~l~
Exneriment 1. To test for subtle differences in exploratory
behavior (e.g. locomotion, sç~nnin~ of the ellvilvlllllent through rearing, and patterns
10 of investigation of l-nf~n~ r environment), both PS 1M~46V and wild type litter mates
were tested in the open-field (Janus, et al. 1995). The results of the test revealed no
.cignific~nt differences bt;lwet;ll ll ,.. .~g5~., ic ~ and controls in exploration of a new
environment measured by mice locomotor behaviors (walking, p~ ing, wall le~ning,rearing, grooming), (F(1,24~ = .98, NS). Thus, dirrel~llces any in behavior on the
15 Morris water maze test (see below) cannot be attributed to dirr~ ces in locomotor
abilities, etc.
Experiment 2. One week after the open-field test, the PS1MI46V
mutant transgenic mice and their litter mates were trained in the Morris water maze.
In this test, a mouse has to swim in a pool in order to find a submerged escape
20 platform. The animal solves that test through learning the location of the platform
using the available extra-maze spatial cues (Morris, 1990). This test was chosenbecause there is strong evidence that the hippocampal -formation is involved in this
form of le~rning The hippocampus is also a major site of AD neuropathology in
hl-m~n.c and defects in spatial learning (geographic disorientation, losing objects,
25 wandering, etc.) are prominent early features of human AD. As a result the test is
likely to detect early changes equivalent to those seen in human AD. The Morris test
is conducted in three phases. In the first phase (the learning acquisition phase), the
mouse has to learn the spatial position of the platform. In the second phase (the probe
trial), the platform is removed from the pool and the mouse's search for the platform
30 is recorded. In the final phase (the learning transfer phase), the platform is replaced in
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a new position in the pool, and the mouse has to learn that new spatial position of the
platform.
Transgenic and wild type mice did not differ in their latencies to find the
platform during learning acquisition (F(1,24) = 0.81, NS), and both groups showed
5 rapid learning across trials (F(10,15) - 11.57, p < 0.001). During the probe trial
phase, mice from both groups searched the quadrant of the pool which originally
contained the platform si~nific~ntly longer than other areas of the pool which had not
contained the platfolm (F(3,22) = 28.9, p C 0.001). However, the wild type controls
showed a trend which was not ~uite st~ti~tiC~llly significant (t(24) = 1.21, p = 0.24) for
an increased nurnber of crossings of the exact previous position of the platform. In
the learning transfer test, both groups showed the same latency of finding the new
position of the platform in the initial block of trials (t(24) = 1.11, NS). Such long
latency to find the new spatial position is expected because the mice spent most of
their time searching for the platform in the old spatial position. However, in later
trials in the learning I~ l phase, the wild type mice showed shorter swim latencies
to the new position of the platform cO~ d to the PS 1M~46V mutant transgenics
(F~1,24) 2.36, p = 0.14). The results indicate that PS1MI46V mutant ll~llSg~lliC mice
were less flexible in transferring learned information to a new situation and tended to
persevere in their search for the platform in the old location.
Thus, although no differences were found in the spontaneous exploration
of a new enviromnent and in the acquisition of new spatial information between the
wild type and the PS1MI46V mutant transgenic mice, the PS1MI46V mutant transgenic
mice were hllpail ed in switching and/or adapting this knowledge in later situations.
Electro~hysiolo~ical Recordin~es in the hippocam~us of mutant trans~enic
mice: Five to six months old litter mate control and human PS1MI46V mutant
transgenic mice on the same C57BL/6 x FVB strain backgrounds as above were used
~ to study long term potentiation (LTP) as an electrophysiologic correlate of learning
and memory in the hippocampus. Recordings were carried out on 400 ,um thick
hippocampal slices according to conventional techni~ues. Briefly, brains were
removed and transverse sections cont~ining hippocampi were obtained within 1 min.
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after mice were decapitated under halothane anesthesia. Slices were kept at roomtemperature in oxygenated artificial cerebrospinal fluid for one hour prior to
recording. One slice at a time was transferred to the recording chamber, where they
were m~int~ined at 32 ~C in an interface between oxyg~n~ted artificial cerebrospinal
5 fluid and humidified air. Slices were then allowed to equilibrate in the recording
chamber for another hour.
Extracellular field recordings were carried out in the CAl subfield of the
hippocampus at the Schaeffer collateral-pyramidal cell synapse. Synaptic responses
were induced by the stim~ tir n of Schaeffer collaterals at a frequency of 0.03 Hz and
10 an intensity of 30-50 % of m~im~l response. Tetani to evoke long-term potentiation
consisted of ~ trains of l 00 Hz stimulation lasting for 200 ms at an illLc~ llail~ interval
of 10 seconds. Field potentials were recorded using an Axopatch 200B amplifier
(Axon Instrument). Glass pipettes were fa~ricated ~om borosilicate glass with anouter diameter of l.S mm, and pulled with a two step Narishige puller. Data were15 acquired on a 486-IBM compatible co~ ,ul~. using PCLAMP6 software (Axon
Instrument).
To test for any abnormality in presynaptic function, we investigated the
dirr~ lces in paired-pulse f~rilit~tion, which is an example of use-dependent increase
in synaptic efficacy and is considered to be presynaptic in origin. In hippoc~ll~us,
20 when two stimuli are delivered to the Schaeffer collaterals in rapid sllccçssion, paired-
pulse facilitation manifests itself as an enh~nce~ dendritic response to the second
stiTnlllu~ as the interstimulus interval gets shorter. In three pairs of wild-
type/transgenic mice, we did not observe any difference in the paired-pulse facilitation
over an illLt;l~lilllulus interval range of 20 ms to l sec. These data suggest that in
25 PSIMI46V mutant tr~n~enic mice, the excitability of Sch~ef~Pr collateral fibers and
neuro~ er release are likely to be normal.
Tetanic stim~ tion induced a long-lasting increase in the synaptic strength
in both control (n = 3~ and PS1MI46V mutant transgenic mice (n = 2). In slices obtained
from the PS1MI46V mutant transgenic mice, long-lasting increase in the synaptic
30 strength was 30 % more than that obtained from control mice.
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Although preferred embot1iment~ of the invention have been described
herein in detail, it will be understood by those sl~illed in the art that variations may be
made thereto without departing from the spirit of the invention or the scope of the
5 appended claims.
SUBSTITUTE SHEET (RULE 26
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Rcfel~ces
Borguski et al. (1993) Nature 366:643-654.
Bueler et al. (1996) Nature 356:577-582.
Chartier-Harlin et al. (1991) Nature 353:844-846.
Ferrell et al. (1996) FEBS Lett. 381:143-148.
Fenteany et al. (1995) Science 268:726-731.
Fischer and T .~rm~n (1983) Proc. Natl. Acad. Sci. ~USA) 80: 1579-1583.
Garnes et al. (1995) Nature 373:523-527.
Gavrieli et al. (1992) J. Cell Biol. 119:493-501.
10 Goate et al. (1991) Nature 349:704-706.
Gravina et al. (1995) J. Biol. Chem. 270:7013-7016.
Greco et al. (1996) Genes & Dev. 10:313-324.
Hilbich et al. (1991) J. Mol. Biol. 218:149-163.
Hogan et al. (1986) Manipulatin~ the Mouse Embrvo, Cold Spring Harbor Laboratory15 Press,
Cold Spring Harbor, New York.
Hsiao et al. (1995) Neuron. (in press).
Iwatsubo et al. (1995) ~nn. Neurol. 37:294-299.
Jacobson et al. (1993) Nature 361 :365.
20 Janus et al. (1995). Neurobiolo~y o~Lea~nin~ and MemorY~ 64:58-67.
Johansson et al. (1995) J.Biol.Chem. 270:20615-20620.
Kahn (1995) Science 270:369-370.
K~rlin~ky et al. (1992) Neurologv 42:1445-1453.
T~t7.tn~n (1986) N. Eng. J. Med. 314:964-973.
2~ Kikuchi et al. (1992) Onco~ene 7:289-293.
Klafki et al. (1996) J. Biol. Chem. 271:28655-28659.
Kosik and Greenberg (1994) Alzheimer Disease. New York, Raven Press. 335-344.
Krause and Aaronson (1991) Methods in Enz~nolo~v. 200:546-S56.
Lamb et al. (1993) Nature Genetics 5:22-29
30 T ~.sm~nn et al. (1995) Acta Neuropathol. 89:35.
Levy-Lehad et al. (1995) Science 269:970-973.
SlJtsS ~ JTE SHEET (RULE 26)
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-95-
Liu and Sommer (1995) Biotechniques 18:470-477.
Morris (1990) Cold Sprine~ Harbor SvmPosia on Quantitative Biolo~, 55:161-173.
Mullan et al. (1992) Nature Genetics 1:345-347.
Murrell et al. (1991) Science 254:97-99.
Nowak (1995) Science 270:368-371.
Okamoto, et al. (1995) J. Biol. Chem. 270:4205-4208.
Phizicky aIld Fields (1994) Microbiol. Reviews 59:94-123.
Pike et al. (1993) J. Neurosci. 13:1676-1678.
Podlisny et al. (1995) J. Biol. Chem. 270:9564-9570.
Rock et al. (1994) Cell 78:761-771.
Rogaev et al. (19953 Nature 376:775-778.
Saleeba and Cotton (1993) Methods in Enzvmology 217:286-295.
Scheuner et al. (1995) Soc. Neurosci. Abstr. 21:1500.
Scott et al. (1992) Protein Sci. 1:986-997.
Sherrington et al. ~1995) Nature 375:754-760.
Suzulci et al. (1994) Science 264:1336-1340.
Takada et al. (1994) Genes & Dev. 8:174-189.
Tamaoka et al. (1994) J. Biol. Chem. 269:32721-32724.
Tamaoka et al. (1995) Brain Res. 679:151-156.
2û Vito et al. (1996) Science 271:521-525.
Wolozin et al. (1996) Science 274:1710-1713.
Wong et al. (1996) Neuroscience 22:728.
y~m~ ji et al. (1996) Science 272:1349-1352.
Zheng et al. (1995) Cell 81:525-531
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i~ APPLICANT: ST. GEORGE-HYSLOP, PETER H.
ROMMENS, JOHANNA M.
FRASER, PAUL E.
(ii) TITLE OF INVENTION: NUCLEIC ACIDS AND PROTEINS RELATED TO
ALZHEIMER'S DISEASE, AND USES THEREFOR
(iii~ NUMBER OF SEQUENCES: 12
(iv) CO~E;PnNDENCE ADDRESS:
.', ..D)hESSEE: Sim & McBurney
T~FET: 330 University Avenue, 6TH Floor
' I ~.IT': Toronto
~ ~T..TE: Ontario
., ro NTRY: Canada
,'l :I': M5G lR7
(v3 COMPUT';l -EADABLE FORM:
(.'.~ M~ TYP-.: Floppy disk
(_ CI~P'rER: 'BM PC compatible
(~. O~ R.TING YSTEM: PC-DOS/MS-DOS
(l SO-T ~RE: .~atentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 27-JAN-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A~ APPLICATION NUMBER: US 08/592,591
(B) FILING DATE: Z6-JAN-1996
(vii) PRIOR APPLICATION DATA:
(A~ APPLICATION NUMBER: US 60/021,673
(B) FILING DATE: 05-JUL-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/021,700
(B) FILING DATE: 12-JUL-1996
(vii) PRIOR APP1ICATION DATA:
(A) APPLICATION NUMBER: US 60/029,895
(B) FILING DATE: 08-NOV-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: DOCKET# CAN-006PR(d)
(B) FILING DATE: 02-JAN-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: RAE, Patricia A.
(iX) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 595-1155
(B) TELEFAX: (416) 595-1163 - -
(2) INFORMATION FOR SEQ ID NO:1:
(i) SE~UE~C. CHARACTERISTICS:
(..) _E ~GTH: 1330 base pairs
( ) TY E: nucleic acid
(.) 'T~ANDEDNESS: single
~ O_'OLOGY: linear
(ix)-FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 145..1275
(D) OTHER INFORMATION: /product= "S5a"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AATTCCCAAA TGACCTTTTA TTTCATACAG AGATACAAAG GCAACTATGT GCAGCAACAA 60
TCTGATGGGC AGTCCAAACT CTTGGGAGGA AGTAAATTCA TGGTAAATGT CATGATGGCG 120
GTCGGGAGGG AGGAAGGTGG CAAG ATG-GTG TTG GAA AGC ACT ATG GTG TGT 171
Met Val Leu Glu Ser Thr Met Val Cys
1 5
GTG GAC AAC AGT GAG TAT ATG CGG AAT GGA GAC TTC TTA CCC ACC AGG 219
Val Asp Asn Ser Glu Tly5 Met Arg Asn Gly A2po Phe Leu Pro Thr Ar2
CTG CAG GCC CAG CAG GAT GCT GTC AAC ATA GTT TGT CAT TCA AAG ACC 26
Leu Gln Ala Gln Gln Asp Ala Val Asn Ile Val Cys His Ser Lys Thr
30 35 40
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g er Asn Pro Glu Asn Asn Val GGlGyC LTT AT C AcA CTG GCT AAT GAC 315
TGT GAA GTG CTG ACC ACA CTC ACC CCA GAC ACT GGC CGT ATC CTG TCC 363
Cys Glu Val Leu Thr Thr Leu Thr Pro Asp Thr Gly Ar~ Ile Leu Ser
AAG CTA CAT ACT GTC CAA CCC AAG GGC AAG ATC ACC TTC TGC ACG GGC 411
Lys Le5u His Thr Val Gln Pro Lys Gly Lys Ile T~r Phe Cys Thr Gly
ATC CGC GTG GCC CAT CTG GCT CTG AAG CAC CGA CAA GGC AAG AAT CAC 459
I e Arg Val Ala His Leu Ala Leu Lys His ArOg Gln Gly Lys Asn Hloi5
AAG ATG CGC ATC ATT GCC TTT GTG GGA AGC CCA GTG GAG GAC AAT GAG 507
Lys Met Arg Ile le Ala Phe Val Gly Sle5r Pro Val Glu Asp Alz0n Glu
AAG GAT CTG GTG AAA CTG GCT AAA CGC CTC AAG AAG GAG AAA GTA AAT 555
Lys Asp Leu V1215 Lys Leu Ala Lys Arg Leu Lys Lys Glu Lys Val Asn
GTT GAC ATT ATC AAT TTT GGG GAA GAG GAG GTG AAC ACA GAA AAG CTG 603
Val Asp Ile Ile Asn Phe Gly Glu Glu Glu Val Asn Thr Glu Lys Leu
ACA GCC TTT GTA AAC ACG TTG AAT GGC AAA GAT GGA ACC GGT TCT CAT 651
Thr Ala Phe Val Asn Thr Leu Asn Gly Lys Asp G y Thr Gly Ser His
CTG GTG ACA GTG CCT CCT GGG CCC AGT TTG GCT GAT GCT CTC ATC AGT 699
L17e0u Val Thr Val Pro lP7r5o Gly Pro Ser Leu A a Asp Ala Leu Ile Ser
TCT CCG ATT TTG GCT GGT GAA GGT GGT GCC ATG CTG GGT CTT GGT GCC 747
Ser Yro Ile ~eu A a Gly Glu Gly Gly Ala Met Leu Gly Leu Gly Ala
AGT GAC TTT GAA TTT GGA GTA GAT CCC AGT GCT GAT CCT GAG CTG GCC 795
Ser Asp Phe G u Phe Gly Val Asp Pro Ser Ala Asp Pro Glu Leu Ala
TTG GCC CTT CGT GTA TCT ATG GAA GAG CAG CGG CAG CGG CAG GAG GAG 843
Leu Ala Leu Arg Val Ser Met Glu Glu Gln Arg Gln 2A3r0g Gln Glu Glu
GAG GCC CGG CGG GCA GCT GCA GCT TCT GCT GCT GAG GCC GGG ATT GCT 891
Glu 2A13a5 Arg Arg Ala Ala Ala Ala Ser Ala Ala Glu Ala Gly Ile Ala
ACG ACT GGG ACT GAA GAC TCA GAC GAT GCC CTG CTG AAG ATG ACC ATC 939
25h0r Thr Gly Thr Glu 2As55p Ser Asp Asp Ala 2Le60u Leu Lys Met Thr 26e5
AGC CAG CAA GAG TTT GGC CGC ACT GGG CTT CCT GAC CTA AGC AGT ATG 987
Ser Gln Gln Glu Phe Gly Arg Thr Gly Leu Pro Asp Leu Ser Ser Met
ACT GAG GAA GAG CAG ATT GCT TAT GCC ATG CAG ATG TCC CTG CAG GGA 1035
Thr Glu Glu Glu Gln Ile Ala Tyr Ala Met Gln Met Ser Leu Gln Gly
GCA GAG TTT GGC CAG GCG GAA TCA GCA GAC ATT GAT GCC AGC TCA GCT 1083
Ala Glu 3Poho Gly Gln Ala Glu Ser Ala Asp Ile Asp A}a Ser Ser Ala
ATG GAC ACA TCC GAG CCA GCC AAG GAG GAG GAT GAT TAC GAC GTG ATG 1131
Met 3Aslp Thr Ser Glu Pro 3A2a0 Lys Glu Glu Asp 3A25p Tyr Asp Val Met
CAG GAC CCC GAG TTC CTT CAG AGT GTC CTA GAG AAC CTC CCA GGT GTG 1179
3G310n Asp Pro Glu Phe 3Leu Gln Ser Val Leu Glu Asn Leu Pro Gly Va
GAT CCC AAC AAT GAA GCC ATT CGA AAT GCT ATG GGC TCC CTG GCC TCC 1227
Asp Pro Asn Asn G u Ala Ile Arg Asn A a Met Gly Ser Leu A a Ser
CAG GCC ACC AAG GAC GGC AAG AAG GAC AAG AAG GAG GAA GAC AAG AAG 1275
Gln Ala Thr 3L6y5s Asp Gly Lys Lys 3A70p Lys Lys Glu Glu 3A75p Lys Lys
TGAGACTGGA GGGAAAGGGT AGCTGAGTCT GCTTAGGGAC TGCATGGGGG AATTC 1330
~2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 377 amino a~ids
IB) TYPE: amino acid
(D~ TOPOLOGY: linear
(ii) MOLECU~E TYPE: prote1n
Sl~ 111 UTE SHEET (RULE 26)
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(xi) SEQUENCE DESCP~IPTION: SEQ ID NO:2:
Met Val Leu Glu Ser Thr Met Val Cys Val Asp Asn Ser Glu Tyr Met
Arg Asn Gly Asp Phe Leu Pro Thr Arg Leu Gln Ala Gln Gln Asp Ala
Val Asn Ile Val Cys His Ser Lys Thr Arg Ser Asn Pro Glu Asn Asn
Val Gly Leu Ile Thr Leu Ala Asn Asp Cys Glu Val Leu Thr Thr Leu
Thr Pro Asp Thr Gly Arg Ile Leu Ser Lys Leu Bis Thr Val Gln Pro
Lys Gly Lys Ile Thr Phe Cys Thr Gly Ile Arg Val Ala His Leu Ala
Leu Lys His lAr00g Gln Gly Lys Asn His Lys Met Arg Ile le Ala Phe
115 120 125
Lys lA3r0g Leu Lys Lys Glu Lys Val Asn Val Asp Ile Ile Asn Phe Gly
G u Glu Glu Val Asn Thr Glu Lys Leu Thr Ala Phe Val Asn Thr Leu
Asn Gly Lys Asp Gly Thr Gly Ser His Leu Val Thr Val Pro lP7r5o Gly
Pro Ser Leu Ala Asp Ala Leu Ile Ser Ser Pro Ile Leu lAla0 Gly Glu
Gly Gly A a Met Leu Gly Leu Gly Ala Ser Asp Phe Glu Phe Gly Val
Asp Pro Ser Ala Asp Pro Glu Leu Ala Leu Ala 2Le20u Arg Val Ser Met
Glu Glu Gln Arg Gln Arg Gln Glu Glu Glu A15a Arg Arg Ala Ala 2A10a
Ala Ser Ala Ala G u Ala Gly Ile Ala Thr Thr Gly Thr Glu A5s5p Ser
Asp Asp Ala Leu Leu Lys Met Thr Ile Ser Gln Gln Glu Phe Gly Arg
Thr Gly 2L7eu5 Pro Asp Leu Ser Ser Met Thr Glu Glu Glu Gln Ile Ala
Tyr Ala Met Gln Met Ser Leu Gln Gly Ala Glu Phe Gly Gln Ala Glu
Ser Ala Asp Ile Asp Ala Ser Ser Ala Met Asp Thr Ser Glu Pro Ala
Lys Glu Glu Asp 3A25p Tyr Asp Val Met Gln Asp Pro Glu Phe Leu Gln
340 345 350
Arg Asn A355a Met Gly Ser Leu 3A160a Ser Gln Ala Thr Ly65 Asp Gly Lys
Lys 3A7s0p Lys Lys Glu Glu 3A75p Lys Lys
~2) INFORMATION FOR SEQ ID N3:3:
~i) SEQUE~:-. CHARACT-.RISTICS
GTH: 384_ base pairs
PE: nucle.c acid
) ~TRANDEDNES : single
~ O'OLOGY: l_near
(ix) FEATURE-
~A) NAMEtREY: CDS
~B) LOCATION: 2..3121
~ix) FEATURE-
~A) NAME/KEY: misc ~eature
~B) LOCATION: 1..3~41
~D) OTHER INFORMATION: /note= ~GT24"
SU~;~ JTE SHEET (RULE 26~
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
T TCA CAG CTC CCG GCC CGA GGC ACA CAA GCC CGA GST ACG GGC CAG 46
Ser Gln Leu Pro Ala Arg Gly Thr Gln A a Arg Xaa Thr Gly G n
AGC TTC AGC CAG GGC ACG ACC AGC CGC GCC GGC CAC CTG GCG GGG CCC 94
Ser Phe Ser Gln G y Thr Thr Ser Arg Ala Gly His Leu Ala G31y Pro
GAG CCC GCG CCG CCG CCG CCG CCG CCR CCG CGG GAG CCG TTC GCG CCC 142
Glu Pro Ala Pro Pro Pro Pro Pro Xaa Pro Arg Glu Pro Phe Ala Pro
AGC CTG GGC AGC GCC TTC CAC CTG CCC GAC GCG CCG CCC GCC GCC GCC 190
Ser Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala
GCC GCC GCG CTC TAC TAC TCC A~C TCC ACG CTG CCC GCG CCG CCG CGC 238
Ala Ala Ala Leu Tyr Tyr Ser Xaa Ser Thr Leu Pro Ala Pro Pro Arg
GGG GGC TCC CCG CTG GCC GCG CCC CAG GGC GGT TCG CCC ACC AAG CTG 286
G80y Gly Ser Pro Leu A a Ala Pro Gln Gly G y Ser Pro Thr Lys Leu
CAG CGC GGC GGC TCG GCC CCC GAG GGC GCC ACC TAC GCC GCG CCG CGC 334
Gln Arg Gly Gly Ser Ala Pro Glu Gly A a Thr Tyr Ala Ala Pro Arg
GGC TCC TCG CCC AAG CAG TCG CCC AGC CGC CTG GCC AAG TCC TAC AGC 382
Gly Ser Ser Pro Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser
ACC AGC TCG CCC ATC AAC ATC GTC GTG TCC TCG GCC GGC CTG TCC CCG 430
Thr Ser Ser Pro Ile Asn Ile Val Val Ser Ser Ala Gly Leu Ser Pro
ATC CGC GTG ACC TCG CCC CCC ACC GTG CAG TCC ACC ATC TCC TCC TCG 478
Ile Arg Val Thr Ser Pro Pro Thr Val Gln Ser Thr Ile Ser Ser Ser
CCC ATC CAC CAG CTG AGC TCC ACC ATC GGC ACG TAC GCC ACC CTG TCG 526
Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser
CCC ACC AAG CGC CTG GTC CAC GCG TCC GAG CAG TAC AGC AAG CAC TCG 574
Pro Thr Lys Arg Leu Val Hls Ala Ser G85u Gln Tyr Ser Lys 190 ser
CAG GAG CTG TAT GCC ACG GCC ACC CTC CAG AGG CCG GGC AGC CTG GCA 622
Gln Glu Leu lTgyr5 Ala Thr Ala Thr Leu Gln Arg Pro Gly Ser Leu Ala
GCT GGT TCC CGA GCC TCA TAC AGC AGC CAG CAT GGG CAC CTG GGC CCA 670
210 215 220
GAG TTG CGG GCC CTG CAG TCC CCA GAA CAC CAC ATA GAT CCC ATC TAT 718
Glu Leu Arg Ala Leu Gln Ser Pro Glu His His 235e Asp Pro Ile Tyr
GAA GAC CGC GTC TAT CAG AAG CCC CCT ATG AGG AGT CTC AGC CAG AGC 766
Glu Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser
CAG GGG GAC CCT CTG CCG CCA GCA CAC ACC GGC ACC TAC CGC ACG AGC 814
Gln Gly Asp Pro 2Le60u Pro Pro Ala His Th65r Gly Thr Tyr Arg 2Th0r Ser
ACA GCC CCA TCT TCC CCT GGT GTC GAC TCC GTC CCC TTG CAG CGC ACA 862
275 280 285
GGC AGC CAG CAC GGC CCA CAG AAT GCC GCC GCG GCC ACC TTC CAG AGG 910
Gly Ser G n His Gly Pro Gln Asn Ala Ala Ala Ala Thr Phe Gln Arg
GCC AGC TAT GCC GCC GGC CCA GCC TCC AAT TAC GCG GAC CCC TAC CGA 958
Ala Ser Tyr Ala Ala Gly Pro Ala Ser Asn Tyr 3A15a Asp Pro Tyr Arg
CAG CTG CAG TAT TGT CCC TCT GTT GAG TCT CCA TAC AGC AAA TCC GGC 1006
Gln Leu Gln Tyr Cys Pro Ser Val Glu Ser Pro Tyr Ser Lys Ser Gly
CCT GCT CTC CCG CCT GAA GGC ACC TTG GCC AGG TCC CCG TCC ATT GAT 1054
Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg Ser Pro Ser le Asp
AGC ATT CAG AAA GAT CCC AGA GAA TTT GGA TGG AGA GAC CCG GAA CTG 1102
Ser Ile Gln 3L5y5s Asp Pro Arg Glu Phe Gly Trp Arg Asp Pro Glu Leu
CCG GAA GTG ATT C-AG ATG TTG CAG CAC CAG TTT CCC TCG GTC CAG TCT 1150
Sl~ 111 UTE SHEFT (RULE 26)
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Pro Glu Val Ile Gln Met Leu GLn His Gln Phe Pro Ser Val Gln Ser
AAC GCG GCA GCC TAC TTG GAnA CHiASC Leu Cys Phe Gly Asp Asn Ly 1198
AAA GCC GAG ATA AGG AGA CAA GGA GGC ATC CAG CTC CTG GTG GAC CTG 1246
4LoyOs Ala Glu Ile Ary Arg Gln Gly Gly Ile Gln Leu Leu Val Asp Leu
TTG GAT CAT CGG ATG ACC GAA GTC CAC CGT AGT GCC TGT GGA GCT CTG 1294
Leu Asp Hls Arg Met Thr Glu Val His A2rg Ser Ala Cys Gly 4A30a Leu
AGA AAC CTG GTG TAT GGG AAG GCC AAC GAT GAT AAC AAA ATT GCC CTG 1342
Arg Asn Leu Val Tyr Gly Lys Ala Asn Asp Asp Asn Lys I e Ala Leu
AAA AAC TGT GGT GGC ATC CCA GCA CTG GTG AGG TTA CTC CGC AAG ACG 1390
Lys Asn 4c5yO Gly Gly Ile Pro A a Leu Val Arg Leu Leu Arg Lys Thr
ACT GAC CTG GAG ATC CGG GAG CTG GTC ACA GGA GTC CTT TGG AAC CTC 1438
Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Va Leu Trp Asn Leu
TCC TCA TGC GAT GCA CTC AAA ATG CCA ATC ATC CAG GAT GCC CTA GCA 1486
4S8eOr Ser Cys Asp Ala L8e5u Lys Met Pro Ile Ile Gln Asp Ala Leu Ala
GTA CTG ACC AAC GCG GTG ATT ATC CCC CAC TCA GGC TGG GAA AAT TCG 1534
Val Leu Thr Asn Ala Val Ile Ile Pro H s Ser Gly Trp Glu Asn Ser
CCT CTT CAG GAT GAT CGG AAA ATA CAG CTG CAT TCA TCA CAG GTG CTG 1582
Pro Leu Gln 5Als5p Asp Arg Lys Ile Gln Leu His Ser Ser Gln Val Leu
CGT AAC GCC ACC GGG TGC CTA AGG AAT GTT AGT TCG GCC GGA GAG GAG 1630
Arg Asn 5Ala Thr Gly Cys Leu Arg Asn Val Ser Ser A a Gly Glu Glu
GCC CGC AGA AGG ATG AGA GAG TGT GAT GGG CTT ACG GAT GCC TTG CTG 1678
Ala 5A4r5y Arg Arg Met Arg 5G150u Cys Asp Gly Leu 5T5h5r Asp Ala Leu Leu
TAC GTG ATC CAG TCT GCG CTG GGG AGC AGT GAG ATC GAT AGC AAG ACC 1726
Tyr Val Ile Gln Ser Ala Leu Gly Ser Ser G710u Ile Asp Ser Lys 5T7h5r
GTT GAA AAC TGT GTG TGC ATT TTA AGG AAC CTC TCG TAC CGG CTG GCG 1774
Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu Ser Tyr Arg 5LgeOu Ala
GCA GAA ACG TCT CAG GGA CAG CAC ATG GGC ACG GAC GAG CTG GAC GGG 1822
Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr Asp Glu L6eOu5 Asp Gly
CTA CTC TGT GGC GAG GCC AAT GGC AAG GAT GCT GAG AGC TCT GGG TGC 1870
Leu Leu C~s Gly Glu Ala Asn G y Lys Asp Ala Glu S6er Ser Gly Cys
TGG GGC AAG AAG AAG AAG AAA AAG AAA TCC CAA GAT CAG TGG GAT GGA 1918
Trp G625y Lys Lys Lys Lys L3yo Lys Lys Ser Gln As~ Gln Trp Asp Gly
GTA GGA CCT CTT CCA GAC TGT GCT GAA CCA CCA AAA GGG ATC CAG ATG 1966
Va Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln ~et
CTG TGG C-AC CCA TCA ATA GTC AAA CCC TAC CTC ACA CTG CTC TCT GAG 2014
Leu Trp His Pro Ser Ile Val Lys Pro Tyr Leu Thr Leu Leu Ser Glu
TGC TCA AAT CCA GAC ACG CTG GAA GGG GCG GCA GGC GCC CTG CAG AAC 2062
Cys Ser Asn P67r5o Asp Thr Leu Glu Gly Ala Ala Gly Ala Leu Gln Asn
TTG GCT GCA GGG AGC TGG AAG TGG TCA GTA TAT ATC CGA GCC GCT GTC 2110
Leu Ala Ala Gly Ser Trp Lys rp Ser Val Tyr Ile Arg Ala Ala Val
CGA AAA GAG AAA GGC CTG CCC ATC CTC GTG GAG CTG CTC CGA ATA GAC 2158
Arg L70ys5 Glu Lys Gly Leu P7rl0o Ile Leu Val Glu Leu Leu Arg Ile Asp
AAT GAC CGT GTG GTG TGC GCG GTG GCC ACT GCG CTG CGG AAC ATG GCC 2206
7A20n Asp Arg Val Val Cys Ala Val Ala Thr A a Leu Arg Asn Met Ala
TTG GAC GTC AGA AAT AAG GAG CTC ATC GGC AAA TAC GCC ATG CGA GAC 2254
Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp
SUBSTITUTE SHEET (RULE 26)
CA 02244412 1998-07-27
W 097~7296 PCT/CA97/00~51
-1 0 1 -
CTA GTC CAC AGG CTT CCA GGA GGG AAC ~AC AGC AAC AAC ACT GCA AGC 2302
Leu Val His Arq Leu Pro Gly Gly Asn Asn Ser Asn Asn Thr Ala Ser
755 760 765
AAG GCC ATG TCG GAT GAC ACA GTG ACA GCT GTC TGC TGC ACA CTG CAC 2350
Lys Ala Met Ser Asp ASp Thr Val Thr Ala Val Cys Cys Thr Leu His
770 775 730
~ GAA GTG ATT ACC AAG AAC ATG GAG AAC GCC AAG GCC TTA CGG GAT GCC 2398 Glu Va Ile Thr Lys Asn et Glu Asn Ala Lys Ala Leu Arg Asp Ala
GGT GGC ATC GAG AAG TTG GTC GGC ATC TCC AAA AGC AAA GGA GAT AAA 2446
Glooy Gly Ile Glu Lys Le5u Val Gly Ile Ser Lys Ser Lys Gly Asp Lys
CAC TCT CCA AAA GTG GTC AAG GCT GCA TCT CAG GTC CTC AAC AGC ATG 2494
His Ser Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met
820 825 830
TGG CAG TAC CGA GAT CTG AGG AGT CTC TAC AAA AAG GAT GGA TGG TCA 2542
Trp Gln Tyr A83rg5 Asp Leu Arg Ser 8Le4uO Tyr Lys Lys Asp 3G45y Trp Ser
CAA TAC CAC TTT GTA GCC TCG TCT TCA ACC ATC GAG AGG GAC CGG CAA 2590
Gln Tyr His Phe Val Ala Ser Ser Ser Thr Ile Glu Arc Asp Arg Gln
850 855 860
AGG CCC TAC TCC TCC TCC CGC ACG CCC TCC ATC TCC CCT GTG CGC GTG 2638
Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val
TCT CCC AAC AAC CGC TCA GCA AGT GCC CCA GCT TCA CCT CGG GAA ATG 2686
Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro Ala Ser Pro Arg Glu Met
880 885 890 895
ATC AGC CTC AAA GAA AGG AAA ACA GAC TAC GAG TGC ACC GGC AGC AAC 2734
Ile Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Cys Thr Gly Ser Asn
900 905 910
GCC ACC TAC CAC GGA GGT AAA GGC GAA CAC ACT TCC AGG AAA GAT GCC 2782
Ala Thr Tyr His Gly Gly Lys Gly Glu His Thr Ser Arg Lys Asp Ala
ATG ACA GCT CAA AAC ACT GGA ATT TCA ACT TTG TAT AGG AAT TCT TAT 2830
Met Thr Ala Gln Asn Thr Gly I e Ser Thr Leu Tyr Arg Asn Ser Tyr
GGT GCG CCC GCT GAA GAC ATC AAA CAC AAC CAG GTT TCA GCA CAG CCA 2878
Gly Ala Pro Ala Glu Asp Ile Lys His Asn Gln Val Ser Ala Gln Pro
945 950 955
GTC CCA CAG GAG CCC AGC AGA AAA GAT TAC GAG ACC TAC CAG CCA TTT 2926
Val Pro Gln Glu Pro Ser Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe
960 965 970 975
CAG AAT TCC ACA AGA AAT TAC GAT GAG TCC TTC TTC GAG GAC CAG GTC 2974
Gln Asn Ser Thr Arg Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val
~ 980 985 99o
CAC CAT CGC CCT CCC GCC AGC GAG TAC ACC ATG CAC CTG GGT CTC AAG 3022
His His Arg Pro Pro Ala Ser Glu Tyr Thr Met His Leu Gly Leu Lys
995 1000 1005
TCC ACC GGC AAC TAC GTT GAC TTC TAC TCA GCT GCC CGT CCC TAC AGT 3070
Ser Thr lG01yOAsn Tyr Val Asp lPhe Tyr Ser Ala Ala Arg Pro Tyr Ser
GAA CTG AAC TAT GAA ACG AGC CAC TAC CCG GCC TCC CCC GAC TCC TGG 3118
Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp
1025 1030 1035
gTG TGAGGAGCAG GGCACAGGCG CTCCGGGAAA CAGTGCATGT GCATGCATAC 3171
1040
CACAAGACAT TTCTTTCTGT TTTGGTTTTT TTCTCCTGCA AATTTAGTTT GTTAAAGCCT 3231
GTTCCATAGG AAGGCTGTGA TAACCAGTAA GGGAAATATT AAGAGCTATT TTAGAAAGCT 3291
~ AAATGAATCG CAAGTTAACT TGGAAATCAG TAGAAAGCTA AAGTGATCCT AAATATGACA 3351
GTGGGCAGCA CCTTTCCTAG CGTGTTNTGT TAGGAGTAAC GAGAAGTGCT TTATACTGAA 3411
CGTGGGTTGN TTGGTAGGGT GGAGNCGAGG CATTCGGGCC GGTGGGGCGT AAGGGTTATC 3471
GTTAAGCACA AGACACAGAA TAGTTTACAC ACTGTGTGGG GGACGGCTTC TCACGCTTTG 3531
TTTACTCTCT TCATCCGTTG TGACTCTAGG CTTCAGGTTG CATTGGGGTT CCTCTGTACA 3591
GCAAGATGTT TCTTGCCTTT TGTTAATGCA TTGTTGTAAA GTATTTGATG TACATTACAG 3651
ATTAAAGAAG NAAAGCGCGT TGTGTATATT ACACCAATNC CGCCGTGTTT CCTCATCTAT 3711
GGTTCTAAAT ATTGCTTCAA TTTCNAACTT TTGAAAGATG TATGGATTTC CAGTTTTTCT 3771
SUBSTITUTESHEET(RULE26)
CA 02244412 1998-07-27
W O 97/27296 PCT/CA97/00051
-102-
TTACTTTCTC CCAGTATGTT TTAACCNMMN AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 3831
AAAACTCGAG
3841
(2~ INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 1040 amino acids
(B) TYPE: amino acld
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: protein
(xi) SEQVENCE DESCRIPTION: SEQ ID NO:4:
Ser Gln Leu Pro Ala Arg Gly Thr Gln Ala Arg Xaa Thr Gly Gln Ser
Phe Ser Gln G y Thr Thr Ser Arg Ala Gly His Leu Ala Gly Pro Glu
Pro Ala Pro Pro Pro Pro Pro Xaa Pro Arg Glu Pro Phe Ala Pro Ser
Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala Ala
Ala Ala Leu Tyr Tyr Ser Xaa Ser Thr Leu Pro Ala Pro Pro Arg G y
Gly Ser Pro Leu Ala Ala Pro Gln Gly Gly Ser Pro Thr Lys Leu Gln
Arg Gly Gly Ser Ala Pro Glu Gly Ala Thr Tyr Ala Ala lPlr0o Arg Gly
Ser Ser lPlr5o Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser Thr
Ser Ser Pro Ile Asn Ile Val Val Ser Ser Ala Gl~ Leu Ser Pro Ile
lA4rg5 Val Thr Ser Pro PrOo Thr Val Gln Ser Thr Ile Ser Ser Ser Pro
Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser Pro
Thr Lys Arg Leu Val His Ala Ser Glu Gln Tyr Ser Lys His Ser Gln
Glu Leu Tyr Ala Thr Ala Thr Leu Gln Arg Pro Gly 2S0e5r Leu Ala Ala
Gly Ser Arg Ala Ser Tyr Ser Ser Gln His Gly His Leu Gly Pro Glu
Leu Arg Ala Leu Gln Ser Pro Glu His His 215e Asp Pro Ile Tyr 2Glou
Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser Gln
Gly Asp Pro Leu Pro Pro Ala His Thr Gly Thr Tyr Arg Thr Ser Thr
Ala Pro Ser Ser Pro Gly Val Asp Ser Val Pro Leu Gln Arg Thr Gly
Ser Gln His Gly Pro Gl~ Asn Ala Ala Ala Ala Thr Phe Gln Arg Ala
S3e0r5 Tyr Ala Ala Gly 3Plro Ala Ser Asn Tyr Ala Asp Pro Tyr Arq Gln
Leu Gln Tyr Cys 3P2ro5 Ser Val Glu Ser Pro Tyr Ser Lys Ser Gly Pro
Ala Leu Pro Pro Glu Gly Thr Leu A31a5 Arg Ser Pro Ser 350e Asp Ser
Ile Gln 3L5y5 Asp Pro Arg Glu Ph-e Gly Trp Arg Asp Pro Glu Leu Pro
Glu Va Ile Gln Met Leu Gln His Gln Phe Pro Ser Val Gln Ser Asn
Ala Ala Ala Tyr Leu Gln His Leu Cys Phe G 5y Asp Asn Lys Ile L~s
Ala Glu Ile Arg 4A0r5g Gln Gly Gly Ile G n Leu Leu Val Asp Leu Leu
S~ JTE !iHEET (RULE 26~
-
CA 02244412 1998-07-27
W O 97~27~96 PCT/CA97100051
Asp His Arg Met Thr Glu Val His Arg Ser Ala Cys Gly Ala Leu Arg
Asn Leu Va Tyr Gly Ly3 Ala Asn Asp Asp Asn Lys Ile Ala Leu Lys
Asn Cys Gly Gly Ile Pro A a Leu Val Arg Leu Leu Arg Lys Thr Thr
Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Val Leu Trp Asn Leu Ser
Ser Cys Asp Ala Leu Lys Met Pro Ile e Gln Asp Ala Leu A a Val
Leu Thr Asn Ala Val Ile Ile Pro H s Ser Gly Trp Glu Asn Ser Pro
Leu Gln 5A~5p Asp Arg Lys Ile G n Leu His Ser Ser Gln Val Leu Arg
Asn 5A30a Thr Gly Cys Leu 5A3g Asn Val Ser Ser A a Gly Glu Glu Ala
5A4r5g Arg Arg Met Arg 5Glou Cys Asp Gly Leu 5T5h5r Asp Ala Leu Leu 5Ty60r
Val Ile Gln Ser A a Leu Gly Ser Ser Glu Ile Asp Ser Lys Thr Val
Glu Asn Cys 58aO Cys Ile Leu Arg Asn Leu Ser Tyr Arg Leu Ala Ala
Glu Thr Ser Gln Gly Gln His M8t Gly Thr Asp Glu 6Lg5u Asp Gly Leu
Leu C61yO Gly Glu Ala Asn G615y Lys Asp Ala Glu 6S2eOr Ser Gly Cys Trp
6 3 0 Y S e r G l n 6A 3 5p G l n T r p A s p G l y V a
Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln Met Leu
Trp E~is Pro S6e60 Ile Val Lys Pro T66Y5r Leu Thr Leu Leu S67eOr Glu Cys
Ser Asn 6P7r5o Asp Thr Leu Glu G61y Ala Ala Gly Ala Leu Gln Asn Leu
Ala A690a Gly Ser Trp Lys T69r5p Ser Val Tyr Ile Ar~ Ala Ala Val Arg
Lys Glu Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Ile Asp Asn
Asp Arg Val Val 7C2ys5 Ala Val Ala Thr Ala Leu Arg Asn Met Ala Leu
Asp Val Arg Asn Lys Glu Leu Ile G y Lys Tyr Ala Met Arg ASp Leu
Val His 7A5r5g Leu Pro Gly Gly 7A6n Asn Ser Asn Asn Thr Ala Ser Lys
Ala M77eOt Ser Asp Asp Thr Val Thr Ala Val Cys Cys Thr Leu His Glu
Va Ile Thr Lys Asn Met Glu Asn Ala Lys A a Leu Arg Asp Ala G y
Gly Ile Glu Lys Lgu Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His
Ser Pro Lys Va Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp
835 840 345
Tyr Hi5s Phe Val Ala Ser Ser Ser Thr Ile Glu Arg Asp Arg Gln Arg
Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser
Pro Asn Asn Arg Ser Ala Ser Ala Pro A a Ser Pro Arg Glu Met Ile
Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Cys Thr Gly Ser Asn Ala
Thr Tyr s Gly Gly Lys Gly G u His Thr Ser Arg Lys Asp Ala Met
SUcs~ JTE SHEET (RULE 26)
CA 02244412 1998-07-27
WO 97127296 PCT/CA97/00051
- 1 04-
Thr A a Gln Asn Thr Gly I e Ser Thr Leu Tyr-Arg Asn Ser Tyr Gly
Ala Pro Ala Glu Asp Ile Lys His Asn Gln Val Ser Ala Gln Pro Val
Pro Gln Glu Pro 9e65r Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Gln
970 975
Asn Ser Thr A93r0g Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val ~is
His Arg gPgr5o Pro Ala Ser Glu lT0y0r0Thr Met His Leu lG100y5Leu Lys Ser
Thr Gly Asn Tyr Val Asp Phe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu
Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val
1025 1030 1035 1040
~2) INFORMATION FOR SEQ ID NO:5:
(i) SE~'UE;iC- CHARACT:RISTICS:
~) LE GTH: 390 base pairs
) TY E: nucle c acid
() 'TXANDEDNES : single
.n) ~-O~OLOGY: l_near
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 142..3777
(D) OTHER INFORMATION: /note= "pO071"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTACTGTTGT TTTTGAGGGG CGGGCAGCCG CGCCGCCGCG GCACTTTTTT AATTTTTTCG 60
GGTGCCGCAG CAGCGACCCC TCGGCGCCGA TGTCCCTGAT CCCTGGAGCG ACGACGGCCG 120
CTGCCTAAGC TGGGAAGAGG A ATG CCA GCT CCT GAG CAG GCC TCA TTG GTG 171
Met Pro Ala Pro Glu Gln Ala Ser Leu Val
1 5 10
GAG GAG GGG CAA CCA CAG ACC CGC CAG GAA GCT GCC TCC ACT GGC CCA 219
Glu Glu Gly Gln Pro Gln Thr Arg Gln Glu Ala Ala Ser Thr Gly Pro
GGC ATG GAA CCC GAG ACC ACA GCC ACC ACT ATT CTA GCA TCC GTG AAG 267
Gly Met Glu Pro Glu Thr Thr Ala Thr Thr Ile Leu Ala Ser Val Lys
30 35 40
GAG CAG GAG CTT CAG TTT CAG CGA CTC ACC CGA GAA CTG GAA GTG GAA 315
Glu Gln Glu Leu Gln Phe Gln Arg Leu Thr Arg Glu Leu Glu Val Glu
AGG CAG ATT GTT GCC AGT CAG CTA GAA AGA TGT AGG CTT GGA GCA GAA 363
Arg G n Ile Val Ala Ser G n Leu Glu Arg Cys Arg Leu Gly Ala Glu
TCA CCA AGC ATC GCC AGC ACC AGC TCA ACT GAG AAG TCA TTT CCT TGG 411
Ser Pro Ser Ile Ala Ser Thr Ser Ser Thr Glu Lys Ser Phe Pro Trp
AGA TCA ACA GAC GTG CCA AAT ACT GGT GTA AGC AAA CCT AGA GTT TCT 459
Arg Ser Thr Asp Val Pro Asn Thr Gly Val Ser Lys Pro Arg V10al5 Ser
GAC GCT GTC CAG CCC AAC AAC TAT CTC ATC AGG ACA GAG CCA GAA CAA 507
Asp Ala Val lGln0 Pro Asn Asn Tyr Leu Ile Arg Thr Glu Pro Glu Gln
GGA ACC CTC TAT TCA CCA GAA CAG ACA TCT CTC CAT GAA AGT GAG GGA 555
Gly Thr lL2e5u Tyr Ser Pro Glu G30n Thr Ser Leu His G u Ser Glu Gly
TCA TTG GGT AAC TCA AGA AGT TCA ACA CAA ATG AAT TCT TAT TCC GAC 603
Ser Leu Gly Asn Ser Arg Ser Ser Thr Gln Met Asn Ser Tyr Ser Asp
AGT GGA TAC CAG GAA GCA GGG AGT TTC CAC AAC AGC CAG AAC GTG AGC 651
Ser Gly Tyr Gln Glu A a Gly Ser Phe His Asn Ser Gln Asn Val Ser
AAG GCA GAC AAC AGA CAG CAG CAT TCA TTC ATA GGA TCA ACT AAC AAC 699
Lys Ala Asp Asn A7r5g Gln Gln His Ser Phe Ile Gly Ser Thr Asn Asn
CAT GTG GTG AGG AAT TCA AGA GCT GAA GGA CAA ACA CTG GTT CAG CCA 747
Hls Val Val Arg A-sn Ser Arg Ala Glu Gly Gln Thr Leu Val Gln Pro
S~ 111 UTE SHEET (RULE 26~
CA 02244412 1998-07-27
W 097/27296 PCT/CA97/00051
-105-
190 195 200
TCA GTA GCC AAT CGG GCC ATG AGA AGA GTT AGT TCA GTT CCA TCT AGA 795
Ser Val Ala Asn Arg Ala Met Arg Arg Val Ser Ser Val Pro Ser Arg
GCA CAG TCT CCT TCT TAT GTT ATC AGC ACA GGC GTG TCT CCT TCA AGG 843
Ala Gln Ser Pro Ser Tyr Val Ile Ser Thr Gly 2V3aO Ser Pro Ser Arg
GGG TCT CTG AGA ACT TCT CTG GGT AGT GGA TTT GGC TCT CCG TCA GTG 891
2G3$y Ser Leu Arg Thr 2S4eOr Leu Gly Ser Gly 2P4h5e Gly Ser Pro Ser 25aO
ACC GAC CCC CGA CCT CTG AAC CCC AGT GCA TAT TCC TCC ACC ACA TTA 939
Thr Asp Pro Arg Pro Leu Asn Pro Ser A a Tyr Ser Ser Thr Thr Leu
CCT GCT GCA CGG GCA GCC TCT CCG TAC TCA CAG AGA CCC GCC TCC CCA 987
Pro Ala Ala 2A7rOg Ala Ala Ser Pro Tyr Ser Gln Arg Pro Ala Ser Pro
ACA GCT ATA CGG CGG ATT GGG TCA GTC ACC TCC CGG CAG ACC TCC AAT 1035
Thr Ala I e Arg Arg Ile Gly Ser Val Thr Ser Arg Gln Thr Ser Asn
CCC AAC GGA CCA ACC CCT CAA TAC CAA ACC ACC GCC AGA GTG GGG TCC 1083
Pro 3AOOn Gly Pro Thr Pro 3GlOn Tyr Gln Thr Thr Ala Arg Val Gly Ser
CCA CTG ACC CTG ACG GAT GCA CAG ACT CGA GTA GCT TCC CCA TCC CAA 1131
Pro Leu Thr Leu Thr Asp Ala Gln Thr Arg Val Ala Ser Pro Ser G n
GGC CAG GTG GGG TCG TCG TCC CCC AAA CGC TCA GGG ATG ACC GCC GTA 1179
Gly Gln Val Gly 3S3e5r Ser Ser Pro Lys 3ArOg Ser Gly Met Thr A15a Val
CCA CAG CAT CTG GGA CCT TCA CTG CAA AGG ACT GTT CAT GAC ATG GAG 1227
Pro Gln His 350u Gly Pro Ser Leu Gln Arg Thr Val His ASp Met Glu
CAA TTC GGA CAG CAG CAG TAT GAC ATT TAT GAG AGG ATG GTT CCA CCC 1275
Gln Phe 3G6y Gln Gln Gln Tyr Asp Ile Tyr Glu Arg Met Val Pro Pro
AGG CCA GAC AGC CTG ACA GGC TTA CGG AGT TCC TAT GCT AGT CAG CAT 1323
Arg Pro Asp Ser Leu Thr Gly Leu Arg Ser ser 3T9yOr Ala Ser Gln His
AGT CAG CTT GGG CAA GAC CTT CGT TCT GCC GTG TCT CCC GAC TTG CAC 1371
Ser Gln Leu Gly Gln Asp Leu Arg Ser Ala Val Ser Pro Asp Leu 4H1sO
ATT ACT CCT ATA TAT GAG GGG AGG ACC TAT TAC AGC CCA GTG TAC CGC 1419
Ile Thr Pro Ile 4Tly5r Glu Gly Arg Thr 4Tyr Tyr Ser Pro Val Tyr Arg
AGC CCA AAC CAT GGA ACT GTG GAG CTC CAA GGA TCG CAG ACG GCG TTG 1467
Ser Pro Asn 43s0 Gly Thr Val Glu Leu Gln Gly Ser Gln Thr Ala Leu
TAT CGC ACA GGT GTA TCA GGT ATT GGA AAT CTA CAA AGG ACA TCC AGC 1515
Tyr Arg 4T4hSr Gly Val Ser Gly 5e Gly Asn Leu Gln Arg Thr Ser Ser
CAA CGA AGT ACC CTT ACA TAC CAA AGA AAT AAT TAT GCT CTG AAC ACA 1563
Gln Arg Ser Thr Leu Thr T6y5r Gln Arg Asn Asn T47yOr Ala Leu Asn Thr
ACA GCT ACC TAC GCG GAG CCC TAC AGG CCT ATA CAA TAC CGA GTG CAA 1611
4T7h5r Ala Thr Tyr Ala 4G81uo Pro Tyr Arg Pro I18e5 Gln Tyr Arg Val 4GlgOn
GAG TGC AAT TAT AAC AGG CTT CAG CAT GCA GTG CCG GCT GAT GAT GGC 1659
Glu Cys Asn Tyr A95n Arg Leu Gln HiS AlOa Val Pro Ala Asp 5ASop5 Gly
ACC ACA AGA TCC CCA TCA IAlTeA AsAp SAer Ile Gln Lys Asp 5p2rOo A g 1707
CC TGG CGT GAT CCT GlAG LTTG CprcOT Glu gal Ile 53i5 Met Le 1755
510n Phe Pro Ser Val G n AlAa AAA5Tn AlCA GCG GCC TAC CTG CAG CAC 1803
CTG TGC TTT GGT GAC AAC AAA GTG AAG ATG GAG GTG TGT AGG TTA GGG 1851
GGA ATC AAG CAT CTG GTT GAC CTT CTG GAC CAC AGA GTT TTG GAA GTT 1899
SU~ JTE SHEET (RULE 26)
CA 02244412 1998-07-27
W O 97/27296 PCT/CA97/000~1
-106-
Gly Ile Lys His Leu Val Asp Leu Leu Asp His Arg Val Leu Glu Val
575 580 - 585
CAG AAG AAT GCT TGT GGT GCC CTT CGA AAC CTC GTT TTT GGC AAG TCT 1947
Gln Lys Asn Ala Cys Gly Ala Leu Arg Asn Leu Val Phe Gly Lys Ser
59 60
ACA GAT GAA AAT AAA ATA GCA ATG AAG AAT GTT GGT GGG ATA CCT GCC 1995
Thr Asp Glu Asn Lys Ile Ala Met Lys Asn Val Gly Gly Ile Pro Ala
610 61
TTG TTG CGA CTG TTG AGA AAA TCT ATT GAT GCA GAA GTA AGG GAG CTT 2043
Leu L62eOu Arg Leu Leu Arg Lys Ser Ile Asp Ala Glu Val Arg Glu Leu
5 630
GTT ACA GGA GTT CTT TGG AAT TTA TCC TCA TGT GAT GCT GTA AAA ATG 2091
Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Val Lys Met
635 640 645 650
ACA ATC ATT CGA GAT GCT CTC TCA ACC TTA ACA AAC ACT GTG ATT GTT 2139
Thr Ile Ile Arg Asp Ala Leu Ser Thr Leu Thr Asn Thr Val Ile Val
655 660 665
CCA CAT TCT GGA TGG AAT AAC TCT TCT TTT GAT GAT GAT CAT AAA ATT 2187
Pro His Ser Gly Trp Asn Asn Ser Ser Phe Asp Asp Asp His Lys Ile
675 680
AAA TTT CAG ACT TCA CTA GTT CTG CGT AAC ACG ACA GGT TGC CTA AGG 2235
Lys Phe Gln Thr Ser Leu Val Leu Arg Asn Thr Thr Gly Cys Leu Arg
690 69
AAC CTC ACG TCC GCG GGG GAA GAA GCT CGG AAG CAA ATG CGG TCC TGC 2283
Asn Leu Thr Ser Ala Gly G u Glu Ala Arg Lys Gln Met Arg Ser Cys
5 710
GAG GGG CTG GTA GAC TCA CTG TTG TAT GTG ATC CAC ACG TGT GTG AAC 2331
7Glu5 Gly Leu Val Asp 52r Leu Leu Tyr Val Ile ~is Thr Cys Val Asn
725 730
ACA TCC GAT TAC GAC AGC AAG ACG GTG GAG AAC TGC GTG TGC ACC CTG 2379
Thr Ser Asp Tyr Asp Ser Lys Thr Val Glu Asn Cys Val Cys Thr Leu
735 740 745
AGG AAC CTG TCC TAT CGG CTG GAG CTG GAG GTG CCC CAG GCC CGG TTA 2427
Arg Asn Leu Ser Tyr Arg Leu Glu Leu Glu Val Pro Gln Ala Arg Leu
750 755 760
CTG GGA CTG AAC GAA TTG GAT GAC TTA CTA GGA AAA GAG TCT CCC AGC 2475
Leu Gly Leu Asn Glu Leu Asp Asp Leu Leu Gly Lys 7G75u Ser Pro Ser
AAA GAC TCT GAG CCA AGT TGC TGG GGG AAG AAG AAG AAA AAG AAA AAG 2S23
Lys 7As3pO Ser Glu Pro ser 7C8ys Trp Gly Lys Lys 7LgyO Lys Lys Lys Lys
AGG ACT CCG CAA GAA GAT CAA TGG GAT GGA GTT GGT CCT ATC CCA GGA 2571
Ar3 Thr Pro Gln Glu Asp Gln Trp Asp Gly Val Gly Pro Ile Pro Gly
79 80v 805 810
CTG TCG AAG TCC CCC AAA GGG GTT GAG ATG CTG TGG CAC CCA TCG GTG 26}9
Leu Ser Lys Ser Pro Lys Gly Val Glu Met Leu Trp His Pro Ser Val
815 820 825
GTA AAA CCA TAT CTG ACT CTT CTA GCA GAA AGT TCC AAC CCA GCC ACC 2667
Val Lys Pro Tyr Leu Thr Leu Leu Ala Glu Ser Ser Asn Pro Ala Thr
830 835 840
TTG GAA GGC TCT GCA GGG TCT CTC CAG AAC CTC TCT GCT AGC AAC TGG 2715
Leu Glu Gly Ser Ala Gly Ser Leu Gln Asn Leu Ser Ala Ser Asn Trp
845 850 855
AAG TTT GCA GCA TAT ATC CGG GGC GGC CGT CCG AAA AGA AAA GGG CTC 2763
Lys Phe Ala Ala Tyr Ile Arg Gly Gly Arg Pro 8L7yO Arg Lys Gly Leu
CCC ATC CTT GTG GAG CTT CTG AGA ATG GAT AAC GAT AGA GTT GTT TCT 2811
Pro Ile Leu Val Glu Leu Leu Arg Met Asp Asn Asp Arg Val Val Ser
875 880 885 890
TCC GGT GCA ACA GCC TTG AGG AAT ATG GCA CTA GAT GTT CGC AAC AAG 2859
Ser Gly Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys
900 905
GAG CTC ATA GGC AAA TAC GCC ATG CGA GAC CTG GTC AAC CGG CTC CCC 2907
Glu Leu Ile G y Lys Tyr Ala Met Arg Asp Leu Val Asn Arg Leu Pro
92
GGC GGC AAT GGC CCC AGT GTC TTG TCT GAT GAG ACC ATG GCA GCC ATC 2955
Gly Gly Asn Gly Pro Ser Val Leu Ser Asp Glu Thr Met Ala Ala Ile
TGC TGT GCT CTG CAC GAG GTC ACC AGC AAA AAC ATG GAG AAC GCA AAA 3003
Cys Cys Ala Leu His Glu Val Thr Ser Lys Asn Met Glu Asn Ala Lys
9 0 945 950
SUBSTITUTE SHEET (RULE 26)
CA 02244412 1998-07-27
W ~ 97127296 PCT/CA97/00051
-107-
GCC CTG GCC GAC TCA GGA GGC ATA GAG AAG CTG GTG AAC ATA ACC AAA 3051
A a Leu Ala Asp Ser G y Gly Ile Glu Lys Leu Val Asn Ile Thr Lys
965 9 0
GGC AGG GGC GAC AGA TCA TCT CTG AAA GTG GTG AAG GCA GCA GCC CAG 3099
Gly ~rg Gly Asp Ar~ Ser Ser Leu Lys Val Val Lys Ala Ala Ala Gln
97~ 980 985
~ GTC TTG AAT ACA TTA TGG CAA TAT CGG GAC CTC CGG AGC ATT TAT AAA 3147Val Leu Asn Thr Leu Trp Gln Tyr Ar~ Asp Leu Arg Ser Ile Tyr Lys
AAG GAT GGG TGG AAT CAG AAC CAT TTT ATT ACA CCT GTG TCG ACA TTG 3195
Lys Asp Gloy Trp Asn Gln Asn His Phe Ile Thr,Pro Va Ser Thr Leu
GAG CGA GAC CGA TTC AAA TCA CAT CCT TCC TTG TCT ACC ACC AAC CAA 3243
Glu Arg Asp Arg Phe Lys Ser His Pro Ser Leu Ser Thr Thr Asn Gln
CAG ATG TCA CCC ATC ATT CAG TCA GTC GGC AGC ACC TCT TCC TCA CCA 3291
Gln Met Ser Pro Ile Ile Gln Ser Val Gly Ser Thr Ser Ser Ser Pro
1035 1040 104S 1050
GCA CTG TTA GGA ATC AGA GAC CCT CGC TCT GAA TAC GAT AGG ACC CAG 3339
Ala Leu Leu Gly Ile Arg Asp Pro Arg Ser Glu Tyr Asp Arg Thr Gln
1055 1060 1065
CCA CCT ATG CAG TAT TAC AAT AGC CAA GGG GAT GCC ACA CAT AAA GGC 3387
Pro Pro Met lG17n0Tyr Tyr Asn Ser Gln Gly Asp Ala Thr HLS LYS Gly
CTG TAC CCT GGC TCC AGC AAA CCT TCA CCA ATT TAC ATC AGT TCC TAT 3435
Le,u Tyr Pro Gly Ser Ser Lys lP0ro0Ser Pro Ile Tyr 10e Ser Ser Tyr
TCC TCA CCA GCA AGA GAA CAA AAT AGA CGG CTA CAG CAT CAA CAG CTG 3483
Ser Ser Pro Ala Arg Glu Gln Asn Arg Arg Leu Gln His Gln Gln Leu
1100 1105 1110
TAT TAT AGT CAA GAT GAC TCC AAC AGA AAG AAC TTT GAT GCA TAC AGA 3531
Tllylr5Tyr Ser Gln Asp lA1~0Ser Asn Arg Lys lAls2n5Phe Asp Ala Tyr Allr3g0
TTG TAT TTG CAG TCT CCT CAT AGC TAT GAA GAT CCT TAT TTT GAT GAC 3579
Leu Tyr Leu Gln Ser Pro His Ser Tyr Glu Asp Pro Tyr Phe Asp Asp
1135 1140 1145
CGA GTT CAC TTT CCA GCT TCT ACT GAT TAC TCA ACA CAG TAT GGA CTG 3627
Arg Val Hia lPlh5e0Pro Ala Ser Thr lA15p5Tyr Ser Thr Gln lTlyr60Gly Leu
AAA TCG ACC ACA AAT TAT GTA GAC TTT TAT TCC ACT AAA CGA CCT TCT 3675
1165 1170 1175
TAT AGA GCA GAA CAG TAC CCA GGG TCC CCA GAC TCA TGG GTG TAC GAT 3723
Tyr lAlr~0Ala Glu Gln Tyr lPlr8o5Gly Ser Pro Asp lSlegr0Trp Val Tyr Asp
CAA GAT GCC CAA CAG AGG AAC TCT TTC TTT CTA ACC TTG TTC AGA TTG 3771
Gln Asp Ala Gln Gln Arg Asn Ser Phe Phe Leu Thr Leu Phe Arg Leu
1195 1200 1205 1210
AGG TGA AAAGTCCATC TTGCTGATTT CATGATTGAA ATGTGAAAGT GAAGTGGAAG 3827
GAATGAATGA AGTGTGTTTT TTTTTCCTTT TTGAGGAATT ATCAGGGGAA TTCGATATCA 3887
AGCTTATCGA TACCGTCGAC 3907
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 1212 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: protein
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Pro Ala Pro Glu Gln Ala Ser Leu Val Glu Glu Gly Gln Pro Gln
1 5 10 15
Thr Arg Gln Glu Ala Ala Ser Thr Gly Pro Gly Met Glu Pro Glu Thr
2 30
Thr Ala Thr Thr Ile Leu Ala Ser Val Lys Glu Gln Glu Leu Gln Phe
Gln Arg Leu Thr Arg Glu Leu Glu Val Glu Arg Gln Ile Val Ala Ser
SU~ 111 ~JTE SHEET (RULE 26~
CA 02244412 1998-07-27
W O 97/27296 PCT/CA97/00051
-108-
Gln Leu Glu Arg Cys Arg Leu Gly Ala Glu Ser Pro Ser Ile Ala Ser
Thr Ser Ser Thr Glu Lys Ser Phe Pro Trp Arg Ser Thr Asp Val Pro
Asn Thr Gly Va Ser Lys Pro Arg Va Ser Asp Ala Val Glr, Pro Asn
Asn Tyr Lle5u Ile Arg Thr Glu PrOo Glu Gln Gly Thr Leu Tyr Ser Pro
Glu Gln Thr Ser Leu His Glu Ser Glu Gly Ser LeU Gly Asn Ser Arg
Ser Ser Thr Gln Met Asn Ser Tyr Ser Asp Ser Gly Tyr Gln Glu Ala
Gly Ser Phe His lAs65n Ser Gln Asn Val lS7erO Lys Ala Asp Asn A17r5g Gln
Gln His Ser Phe Ile Gly Ser Thr Asn Asn His Val Val Arg Asn Ser
Arg Ala G u Gly Gln Thr Leu Va Gln Pro Ser Val A a Asn Arg Ala
~et Arg Arg Val Ser Ser Val Pro Ser Arg Ala Gln Ser Pro Ser Tyr
Val Ile Ser Thr Gly Val Ser Pro Ser Arg 2G3y Ser Leu Arg Thr 2S4eOr
Leu Gly Ser Gly Phe Gly Ser Pro Ser Val Thr Asp Pro Arg Pro Leu
Asn Pro Ser Ala Tyr Ser Ser Thr Th5r Leu Pro Ala Ala 2A7rOg Ala Ala
Ser Pro Tyr Ser Gln Arg Pro Ala Ser Pro Thr Ala le Arg Arg Ile
Gly Ser Val Thr Ser Arg Gln Thr Ser Asn Pro Asn Gly Pro Thr Pro
3Gln5 Tyr Gln Thr Thr AlOa Arg Val Gly Ser Pro Leu Thr Leu Thr Asp
Ala Gln Thr Arg Va Ala Ser Pro Ser G n Gly Gln Val Gly Ser Ser
Ser Pro Lys 3A4rOg Ser Gly Met Thr Ala Val Yro Gln His Leu Gly Pro
Ser Leu G355n Arg Thr Val His Asp Met Glu Gln Phe Gly Gln Gln Gln
Tyr 3A750p Ile Tyr Glu Arg 3Me75 Val Pro Pro Arg P3rOo Asp Ser Leu Thr
Gly Leu Arg Ser Ser Tyr Ala Ser Gln His Ser Gln Leu Gly Gln Asp
Leu Arg Ser Ala 4VOal5 Ser Pro Asp Leu 4Hilo Ile Thr Pro Ile 4Tlyr Glu
Gly Arg Thr 4T2yO Tyr Ser Pro Val Tyr Arg Ser Pro Asn His Gly Thr
Val Glu 4Le3u5 Gln Gly Ser Gln Thr Ala Leu Tyr Arg Thr Gly Val Ser
Gly I50 Gly Asn Leu Gln A4r5g5 Thr Ser Ser Gln A4r60g Ser Thr Leu Thr
T46y5r Gln Arg Asn Asn 4T7yOr Ala Leu Asn Thr 4T7h5r Ala Thr Tyr Ala GloU
Pro Tyr Arg Pro le Gln Tyr Arg Val Gln Glu Cys Asn Tyr Asn Arg
Leu Gln His AOa Val Pro Ala Asp 5AOsp5 Gly Thr Thr Arg 51erO Pro Ser
515 520 525
Glu Leu Pro Glu Val Ile His ~et Leu Glu His Gln Phe Pro Ser Val
Gln Ala Asn Ala Ala A a Tyr Leu Gln His 5L55u Cys Phe Gly Asp 5As50n
Lys Val Lys Met G~u Val Cys Arg Leu Gly Gly Ile Lys His Leu Val
SUBSTITUTE SHEET (RULE 26)
CA 02244412 1998-07-27
W~97127296 PCT/CA97/00051
56S 570 575
Asp Leu Leu Asp His Arg Val Leu G u Val Gln Lys Asn 5AgOa Cys Gly
Ala Leu Arg Asn Leu Val Phe Gly Lys Ser Thr Asp Glu Asn Lys Ile
~ Ala Met Lys Asn Val Gly G y Ile Pro Ala Leu L20u Arg Leu Leu Arg
6L2ys Ser Ile Asp Ala Glu Val Arg Glu Leu Va Thr Gly Val Leu Trp
Asn Leu Ser Ser Cys Asp Ala Val Lys ~et Thr Ile Ile Arg Asp Ala
Leu Ser Thr L u Thr Asn Thr Val e Val Pro His Ser Gly Trp Asn
Asn Ser Ser Phe Asp Asp Asp His Lys Ile Lys Phe G68Sn Thr Ser Leu
Val Leu Arg Asn Thr Thr Gly Cys Leu Arg Asn Leu Thr Ser Ala Gly
Glu Glu Ala Arg Lys G n Met Arg Ser Cys G u Gly Leu Val Asp 72erO
Leu Leu Tyr Val Ile His Thr Cys Val Asn Thr Ser Asp Tyr Asp Ser
Lys Thr Val Glu Asn Cys Val Cys Thr Leu Arg Asn Leu S5eOr Tyr Arg
Leu Glu Leu Glu Val Pro Gln A a Arg Leu Leu Gly Leu Asn Glu Leu
Asp Asp Leu Leu Gly Lys Glu Ser Pro Ser Lys A78sOp Ser Glu Pro Ser
C78y5s Trp Gly Lys Lys 7Lys Lys Lys Lys Lys Arg Thr Pro Gln Glu Asp
Gln Trp Asp Gly Val Gly Pro Ile Pro Gly Leu Ser Lys Ser 81rSo Lys
Gly Val Glu 82eO Leu Trp His Pro Ser Val Val Lys Pro Tyr Leu Thr
8 3 5 8 4 0 8 4 5
Ser Leu Gln Asn Leu Ser Ala Ser Asn Trp Lys Phe Ala Ala Tyr Ile
Ar65g Gly Gly Arg Pro 8L7yO Arg Lys Gly Leu 8P7ro5 Ile Leu Val Glu 8L8eOu
Leu Arg Met Asp Asn Asp Arg Val Val Ser Ser Gly Ala Thr A a Leu
Arg Asn Met A a Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr
Ala Met Arg Asp Leu Val Asn Arg Leu Pro Gly Gly Asn Gly Pro Ser
Val Leu Ser Asp Glu Thr Met Ala Ala Ile Cys Cys Ala Leu His Glu
Val Thr Ser Lys Asn Met Glu Asn Ala Lys Ala Leu Ala Asp Ser Gly
Gly Ile Glu Lys Leu Val Asn Ile Thr Lys Gly Arg Gly Asp Arg Ser
Ser Leu Lys Val Val Lys Ala Ala Ala Gln Val Leu Asn Thr Leu Trp
Gln Tyr Aggr5g Asp Leu Arg Ser le Tyr Lys Lys Asp Gly Trp Asn Gln
Asn His Phe Ile Thr Pro Val Ser Thr Leu Glu lAOr2gOAsp Arg Phe Lys
Ser His Pro Ser Leu Ser Thr Thr Asn Gln Gln Met Ser 2ro Ile e
Gln Ser Val Gly Ser Thr Ser Ser Ser Pro Ala Leu Leu Gly Ile Arg
Asp Pro Arg lSeO6rOGlu Tyr Asp Arg TlOhr65Gln Pro Pro Me~ GlOn70TYr Tyr
SU~ 11 UTE SHEET (RULE 26)
CA 02244412 1998-07-27
W O 97/27296 PCT/CA97/00051
-110-
Asn Ser lG07n5Gly Asp Ala Thr His8 Lys Gly Leu Tyr Pro Gly Ser Ser
Lys Pro Ser Pro Ile Tyr I e Ser Ser Tyr Ser Ser Pro Ala Arg Glu
lGlOn5Asn Arg Arg Leu G n His Gln Gln Leu Tyr Tyr Ser Gln Asp Asp
Ser Asn Arg Lys Asn Phe Asp Ala Tyr Arg Leu Tyr Leu Gln Ser Pro
1125 11 0 1135
His Ser Tyr lG14uoAsp Pro Tyr Phe lAslp45Asp Arg Val His lPhe5 Pro Ala
Ser Thr lA1~5Tyr Ser Thr Gln Tyr Gly Leu Lys Ser Thr Thr Asn Tyr
Val Asp Phe Tyr Ser Thr Lys Arg Pro Ser Tyr Arg Ala Glu Gln Tyr
Pro Gly Ser Pro Asp Ser Trp Val Tyr Asp Gln Asp Ala Gln Gln Ar
1185 1190 1195 1200
Asn Ser Phe Phe Leu Thr Leu Phe Arg Leu Arg
1205 1210
(2) INFORMATION FOR SEQ ID NO:7:
(i) S~QUENC3 CHARACTERISTICS:
..l _E~GTH: 370 base pairs
'T'~ANDnEDNCESS single
:\ l'O'OLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..970
(D) OTHER INFORMATION: /note~ "Y2H9"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCCCAC AGATACCACT GCTGCTCCCG CCCTTTCGCT CCTCGGCCGC GCAATGGGCA 60
CCCGCGACGA CGAGTACGAC TACCTCTTTA AAGTTGTCCT TATTGGAGAT TCTGGTGTTG 120
GAAAGAGTAA TCTCCTGTCT CGATTTACTC GAAATGAGTT TAATCTGGAA AGCAAGAGCA 180
CCATTGGAGT AGAGTTTGCA ACAAGAAGCA TCCAGGTTGA TGGAAAAACA ATAAAGGCAC 240
AGATATGGGA CACAGCAGGG CAAGAGCGAT ATCGAGCTAT AACATCAGCA TATTATCGTG 300
GAGCTGTAGG TGCCTTATTG GTTTATGACA TTGCTAAACA TCTCACATAT GAAAATGTAG 360
AGCGATGGCT GAAAGAACTG AGAGATCATG CTGATAGTAA CATTGTTATC ATGCTTGTGG 420
GCAATAAGAG TGATCTACGT CATCTCAGGG CAGTTCCTAC AGATGAAGCA AGAGCTTTTG 480
CAGAAAAGAA TGGTTTGTCA TTCATTGAAA CTTCGGCCCT AGACTCTACA AATGTAGAAG 540
CTGCTTTTCA GACAATTTTA ACAGAGATTT ACCGCATTGT TTCTCAGAAG CAAATGTCAG 600
ACAGACGCGA AAATGACATG TCTCCAAGCA ACAATGTGGT TCCTATTCAT GTTCCACCAA 660
CCACTGAAAA CAAGCCAAAG GTGCAGTGCT GTCAGAACAT CTAAGGCATT TCTCTTCTCC 720
CCTAGAAGGC TGTGTATAGT CCATTTCCCA GGTCTSASAT TTAAATATAW TTGTAATTCT 780
TGTGTCAC-TT TTGTGTTTTA TTACTTCATA CTTATGAATT TTTCCATGTC CTAAGTCTTT 840
TGATTTTGMT TTATAAAATC ATCCACTTGT NCCGAATGNC TGCAGCTTTT TTTCATGCTA 900
TGGCTTCACT AGCCTTAGTT TNATAAACTG AATGTTTGGA TTCCTCCCCC CAAAAAAAAA 960
AAAACTCGAG 970
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUE~C CHARACTERISTICS:
..) _E GTH: 264 base pairs
) ~Y E: nucleic acid
) T~ANDEDNESS: single
D) ~O?OLOGY: linear
(ix) FEAT~RE:
(A) NAME/KEY: misc feature
(B~ LOCATION: 1..2~4
~D) OTHER INFORMATION: /note- "Y2H23b"
S~ JTE SHEET (RULE 26~
CA 02244412 1998-07-27
W O 97/27296 PCT/CA97/00051
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
GAATTCGCGG CCGNGTCGAC CCCCCACCCC CGATGCCACC ACCCCCANTG GGNTCTCCCN 60
NCCCAGTCAT CAGTTCTTCC ATGGNGTNCC CTGGTCTGCC CCCTCCAGCT CCCCCAGGCN 120
TTCTCCGGGT CTGNCAGCAG CCNCCAGATT AACTCAACAG TGTCACTCCC TGGGGGTGGG 180
TCTGGNCCCC CTGANGATGT GAAGCCACCA GTCTNAGNGG TCCGGGGTCT GTACTGTCCA 240
CCCCCTCCAG GTGGACCTGG CGCT 264
(2) INFOR~ATION FOR SEQ ID NO:9:
(i) SEQUENC CHARACTERISTICS:
(..) LE GTH: 340 base pairs
(.) TY E: nucleic acid
(C) STRANDEDNESS: single
(~) TO,'OLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: misc ~eature
(B) LOCATION: 1..3~0
(D) OTHER INFORMATION: /note= "Y2H27"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GAATTCGCGG CCGCGTCGAC CGCGGTCGCG TCGACCTGTT GCCCAGGCCC TAGAGGTCAT 60
TCCTCGTACC CTGATCCAGA ACTGTGGGGC CAGCACCATC CGTCTACTTA CCTCCCTTCG 120
GGCCAAGCAC ACCCAGGAGA ACTGTGAGAC CTGGGGTGTA AATGGTGAGA CGGGTACTTT 180
GGTGGACATG AAGGAACTGG GCATATGGGA GCCATTGGCT GTGAAGCTGC AGACTTATAA 240
GACAGCAGTG GAGACGGCAG TTCTGCTACT GCGAATTGAT GACATCGTTT CAGGCCACAA 300
AAAGAAAGGC GATGACCAGA GCCGGCAAGG CGGNGCTCCT 340
(2) INFORMATION FOR SEQ ID NO:10:
(i) S_QUENC]- CHARACTERISTICS:
E GTH: 404 base pairs
. TY'E: nucleic acid
~T~ANDEDNESS: single
~ 'O?OLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: misc ~eature
(B) LOCATION: 1..4~4
(D) OTHER INFORMATION: /note= "Y2H35"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GAATTCGCGG TCGCGTCGAC GGTTAGTCCC ACTGGNCGCA TCGAGGGNTT CACCAACGTC 60
ATGGAGCTGT ATGGCANGAT CGCCGAGGTC TTCCNCCTGC CAACTGCCGA GGTGATGTTC 120
TGCACCCTGA NCACCCACAA AGTGGACATN GACAAGCTCC TGGGGGGCCA GATCGGGCTG 180
GAGGACTTCA TCTTCGCCCA CGTGAAGGGG YAGCGCAAGG AGGTGGAGGT GTTCAWGTCG 240
GAGGATGYAC TCGGRCTCAC CATCACGGAC AACGGGGCTG GCTACGCTTC CATCAAGCGC 300
ATCAAGGAGG GCAGCGTGAT CGACCACATC CACCTCATCA GCGTGGGCGA CATGATCGAG 360
GCCATTAACG GGCAGAGCTT CCTGGGCTGC CGGCATTACG AGGT 404
(2) INFORMATION FOR SEQ ID NO:11:
(i) SE~-~UENCE CHARACTERISTICS:
E GTH: 350 base pairs
~) TY E: nucleic acid
TRANDEDNESS: single
'O?OLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: misc ~eature
(B) LOCATION: 1..3~0
(D) OTHER INFORMATION: /note- "Y2H171"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GAATTCGCGG CCGCGTCGAC AAAAAAAGTA AAAGGAACTC GGCAAATCTT ACCCCGCCTG 60
TTTACCAAAA ACATCACCTC TAGCATCACC AGTATTAGAG GCACCGCCTG CCCAGTGACA 120
SUBSTITUTE SHEET (RULI~ 26)
CA 02244412 1998-07-27
W 097/27296 PCT/CA97100051
-112-
CATGTTTAAC GGCCGCGGTA CCCTAACCGT GCAAAGGTAG CATAATCACT TGTTCCTTAA 180
GTAGGGACCT GTATGAATGG CTCCACGAGG GTTCAGCTGT CTCTTACTTT TAACCARTGA 240
AATTGACCTG CCCG~GAAGA GGCGGGCATG ACACAGCAAG ACGAGAAGAC CCTATGGAGC 300
TTTAATTTAT TAATGCAAAC AGTACCTAAC AAACCCACAG GGTCCTAAAC 350
(2~ INFORMATION FOR SEQ ID NO:12:
(i) S:~UE~C- CHARACTERISTICS:
E GTH: 3S0 base pairs
,) TY'E: nucleic acid
r~ TRANDEDNESS: single
D) _O'OLOGY: linear
(ix) FEATURE:
(A) NA~E/KEY: misc feature
(B) LOCATION: 1..3~0
(D) OTHER INFOR~ATION: /note= "Y2H41"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GAATTCGCGG NCGCGTCGAC AGATAATGAA AAAACCAGAG GTTCCCTTCT TTGGTCCCCT 60
NNNNGATGGT GCTATTGTGA ATGGAAAGGT TCTACCCATT ATGGTTAGAG CAACAGCTAT 120
AAATGCAAGC CGTGCTCTGA AATCTCTGAT TCCATTGTAT CAAAACTTCT ATGAGGAGAG 180
AGCACGATAC CTGCAAACAA TTGTCCAGCA CCACTTAGAA CCAACAACAT TTGAAGATTT 240
TGNAGCACAG GTTTTTTCTC CAGCTCCCTA CCACCATTTA CCATCTGATG CCGTTGGCTC 300
CTACCCAGAG ATTCTACCCA GTGAAAACTC CCACAGCAAC GCAGGTAGGA 350
S~ ~S 111 IJTE SHEET (RULE 26)
