WO1997046678A9 - Nucleic acids and polypeptides related to presenilin - Google Patents
Nucleic acids and polypeptides related to presenilinInfo
- Publication number
- WO1997046678A9 WO1997046678A9 PCT/US1997/009272 US9709272W WO9746678A9 WO 1997046678 A9 WO1997046678 A9 WO 1997046678A9 US 9709272 W US9709272 W US 9709272W WO 9746678 A9 WO9746678 A9 WO 9746678A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- seq
- polypeptide
- nucleotide sequence
- sequence
- Prior art date
Links
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Definitions
- AD cases are late in onset (> 60 yr. of age), and
- a nucleic acid in accordance with the present disclosure is a nucleic acid in accordance with the present disclosure.
- a nucleic acid according to the present invention also comprises an
- PSl/429 A nucleic acid capable of hybridizing to such sequence, prefer ⁇
- strand or act as a template for one in the presence of a polymerase (i.e., an
- nucleic acid synthesizing enzyme which has a corresponding amount of nucleotide identity or similarity to Fig. 1 (SEQ ED NO:l).
- nucleic acid a nucleic acid
- nucleotide sequence comprising a nucleotide sequence hybridizing to the nucleotide sequence of
- Fig. 1 (SEQ ED NO: 1) also represents a nucleic acid which possesses at least
- nucleic acid or polypeptide can be any nucleic acid or polypeptide. According to the present invention, a nucleic acid or polypeptide.
- a nucleic acid according to the invention can comprise nucleotides
- nucleic acid will hybridize to a nucleotide sequence as set
- nucleic acid or polypeptide of the present invention can be any nucleic acid or polypeptide of the present invention.
- Homologs from mammalian and non-mammalian organisms can be any mammalian and non-mammalian organisms.
- oligonucleo ⁇ de (see below) selective for PSl/429 can be employed to select
- Modifications to a PSl/429 sequence e.g., mutations, can also be
- a mutated PSl/429 gene can comprise conserved amino acids
- sequence can comprise conserved residues from any number of homologous
- Mutations can be made in specific regions of nucleic acid coding for
- PSl/429 polypeptide e.g., in the membrane-spanning domain
- amino acid sequence e.g., different codons for the same amino acid
- conservative amino acids e.g., (based on the size of the side chain and degree of polarization) small nonpolar: cysteine, proline, alanine, threonine;
- small polar serine, glycine, aspartate, asparagine
- large polar glutamate
- a nucleic acid can therefore com ⁇
- tion 44 is leucine
- 77 is histidine
- 101 is threonine
- 105 is threonine
- 108 is
- valine 108 is leucine, 125 is tyrosine, 125 is arginine, 193 is threonine, 208
- glutamine is glutamine
- 222 is valine
- 225 is arginine
- 226 is leucine
- 229 is serine
- 242 is alanine
- 242 is glycine
- 247 is valine
- 248 is valine
- 346 is alanine
- 354 is valine, 372 is tyrosine, or a combination thereof; a nucleotide
- DNA DNA, RNA, synthetic nucleic acid, peptide nucleic acid, modified nucleo-
- nucleic acid comprising a nucleic acid can be joined via various kncwn linkages, e.g.,
- nucleic acid probes Such oligonucleotides or nucleic acid probes can be
- Detection can be desirable for a variety of different purposes,
- PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g., PCR e.g
- Fig. 1 (SEQ ED NO:l). Accordingly, a preferred antisense oligonucleotide
- a preferred sense oligonucleotide comprises GTTACT and a preferred sense oligonucleotide comprises
- More preferred oligonucleotides comprise about 1-4 nucleotides
- annealing at about 50°C, preferably 53.4°C.
- Another aspect of the present invention is a nucleotide sequence
- PSl/429 which is unique to PSl/429.
- a unique sequence to PSl/429 it is meant a
- nucleotide sequence which occurs in PSl/429, e.g., in the nucleotide
- nucleic acids especially not in an animal nucleic acid, preferably mammal, such as human, rat, mouse, etc, more preferably not in PS 1/467.
- sequence of PSl/429 can be used as a hybridization probe to identify the
- hybridization probe to detect PSl/429 in mRNA samples on a Northern blot.
- a unique PSl/429 nucleotide sequence can also be fused in-frame, at
- Hybridization can be performed under different conditions, depending on
- tide can be hybridized to a target nucleic acid under conditions in which the oligonucleotide only hybridizes to PSl/429, e.g., where the oligonucleotide
- complementarity at least about, e.g., 99%, 97%, 95%, 90%, 70%, 67%
- the present invention can be used diagnosticalry.
- a patient For example, a patient
- Alzheimer's disease having symptoms of Alzheimer's disease such as age-dependent,
- the present invention relates to
- a method of diagnosing Alzheimer's disease comprising contacting a sample
- PSl/429 preferably a unique sequence of PSl/429;
- sequence can be determined according to various methods, including isolat- ing the target nucleic acid, or a cDNA thereof, and determining its sequence
- Oligonucleotides according to the present invention can be of any length
- oligonucleotides in length, or more.
- oligonucleotides can have non-naturally-occurring nucleotides, e.g., inosine.
- the oligonucleotide can comprise a
- kit where the kit includes a desired buffer (e.g., phosphate, tris, etc.),
- a desired buffer e.g., phosphate, tris, etc.
- the oligonucleotide can be labeled or
- Anti-sense nucleic acid can also be prepared from a nucleic acid
- Fig. 1 SEQ ED NO:l
- Fig. 4 more preferably an anti-sense to the
- Antisense nucleic acid can be used in various ways, such as to
- the nucleic acid can be labeled using any desired method.
- the nucleic acid can be labeled using any desired method.
- radioactive tracers such as ⁇ P, 35 S, 125 I, 3 H, or 14 C, to mention only the most
- radioactive labelling can be carried out
- a non-radioactive labeling can also be used, combining
- a nucleic acid according to the present invention including oligo ⁇
- nucleotides can be used to detect expression of
- PSl or PS2 particularly, PSl/429.
- levels of PSl/429 can be determined alone or in combination with other
- the amount (e.g., its
- RGFVF amino acid 7-11 of SEQ ED NO:2
- FCETV amino acid
- a polypeptide of the invention e.g., having a polypeptide sequence
- amino acid sequence can also have a conservative nucleotide or amino acid
- compared sequences can be aligned and calculated according to any desired
- a PSl/429 polypeptide according to the present invention can also be any PSl/429 polypeptide according to the present invention.
- polypeptide according to the present invention includes polypeptides
- amino acid amino acid substitution
- amino acid deletion amino acid addition
- a mutation to amino acids 1-19 can be selected to have a biological
- amino acids 1-19 as set forth in Fig. 1 SEQ ED NO:2
- Fig. 1 SEQ ED NO:2
- Fig. 2 amino acids 1-19 as set forth in Fig. 1 (SEQ ED NO:2) or Fig.
- SEQ ED NO:7 can contain a cellular trafficking function, preferably for
- mitochondria such as mitochondria, cytoskeleton, nuclear, cell membrane, calveolae, etc.
- amino acids 1-19 can act as a signal peptide and be cleaved
- fusion gene can be introduced into a cell line and the transformed cell line
- a polypeptide coding for a polypeptide of the present invention is a polypeptide coding for a polypeptide of the present invention.
- amino acids 1-19 or a PSl/429 unique nucleotide sequence can
- chimeric polypeptide can be prepared according to various methods, includ-
- a chimeric nucleic acid coding for a chimeric polypeptide can contain the
- reading frame e.g., containing introns, splice sites, enhancers, etc.
- the chimeric nucleic acid can be produced according to various methods.
- a domain or desired polypeptide can possess any desired property, includ ⁇
- a biological function such as catalytic, signalling, growth promoting,
- nucleic acid coding for an amino acid sequence according to the
- present invention can be fused in frame to a desired coding sequence and act
- fusion encodes a cleavage site.
- a polypeptide according to the present invention can be produced in
- an expression system e.g., in vivo, in vitro, cell-free, recombinant, cell
- polypeptide imparted by such system include, glycosylation, amino acid
- polypeptide processing such as
- lines can remove the terminal methionine from an expressed polypeptide.
- a polypeptide according to the present invention can be recovered
- transformed host cells obtained from natural sources, transformed host cells (culture medium or cells)
- a nucleic acid or polypeptide of the present invention can be used as a nucleic acid or polypeptide of the present invention.
- the present invention also relates to antibodies which specifically
- PSl/429 polypeptide recognizes a PSl/429 polypeptide, and/or preferably antibodies which bind or
- Antibodies e.g., polyclonal,
- polypeptide according to Fig. 1 (SEQ ID NO:2), preferably comprising
- a regulator compound, or mixture of compounds, can be synthe ⁇
- a regulator compound is preferably a regulator of PSl/429, e.g.,
- an anti-sense nucleic acid e.g., aribozyme
- an aptamer an aptamer, a synthetic compound, or a naturally-occurring compound.
- composition comprising a pharmaceutically acceptable carrier and other
- nucleic acids for other aspects of the nucleic acids, polypeptides, antibodies, etc.,
- S182 429 was amplified from a human cortical cDNA library using high-fidelity PCR and oligonucleotide primers 5'-TCA CAT CGG AAA CAA AAC AG and 5'-ACC TCG TCC CTC AAA TCT.
- the cDNA library used as template in the reaction was constructed from post-mortem adult human cortex using the ⁇ ZAP Ef vector (Stratagene). After a double- round of PCR amplification, electrophoretic separation and purification, the purified amplification product was ligated into the pCRK vector and used to transform E.coli (TA cloning kit, Invitrogen). Clones with inserts were sequenced on both strands using an automated DNA sequencing system (Applied Biosystems).
- AtGW-AcG ⁇ V52 ⁇ TM-2 lie H ⁇ Thr fs) AIT 6W * AcT t ⁇ fcff) TM-2 Met f Values) ,aTG 6»gTG ⁇ 73) TM-2 Met « ⁇ • Leutfetf) aTG ⁇ cTG «/73 TM-2
Abstract
The present invention relates to a PS1/429 nucleic acid, polypeptide, and derivatives thereof, comprising novel nucleotide and amino acid sequences. The invention also relates to methods of using such nucleic acids, polypeptides, or derivatives thereof, e.g., in therapeutics, diagnostics, and as research tools. Another aspect of the present invention involves antibodies to PS1/429, regulators of PS1/429 gene expression and activity, and methods of treating Alzheimer's disease. The invention especially relates to an isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO:1, with the proviso that the nucleic acid does not contain all or part of the coding sequence for a polypeptide having 467 amino acids as set forth in SEQ ID NO:4 or SEQ ID NO:8. In addition, the invention includes an isolated nucleic acid comprising a PS1/429 unique nucleotide sequence and comprising nucleotides 206 and 207 as set forth in SEQ ID NO:1.
Description
NUCLEIC ACIDS AND POLYPEPTTDES RELATED TO PRESENILIN
BACKGROUND OF THE INVENTION
Alzheimer's Disease (AD) is a progressive, debilitating deadly disease
of unknown etiology, that incapacitates an estimated 12% of the mature
population, first cognitively, and finally physically, over the course bf
several years. Monetary costs attributed to this disease, for which there is
currently neither cure nor adequate treatment, are estimated in the range of
$100 billion per year in the US alone. The number of cases is expected to
rise with the increasing age of the population.
The majority of AD cases are late in onset (> 60 yr. of age), and
non-Mendelian, thus offering no simple genetic cause that can be traced
conventionally to a single pathogenic gene product. However, a significant
proportion (< 15%) of AD can be linked to certain genes in a classical
autosomal dominant fashion. Other than a characteristically earlier onset
(< 60 yr. of age), which forms the basis of their designation as Early Onset
AD (EOAD), and the presence of a classical Mendelian inheritance pattern,
these rarer cases are indistinguishable from Late Onset AD (LOAD). Auto¬
somal dominance insures that causation lies within the mutated gene product
in these EOAD cases, and these genes have been sought and studied as the
only known causes of AD.
The great majority of EOAD cases were linked to a site on chromo¬
some 14, and the concerted search for the causative gene at this site has
recently been successful (Sherrington et al., 1995). The growing number of
single point mutations (> 26) identified in different families, barely
outstrips the number of alternative names ("S182", "PS1", "AD3", "STM1",
"presenillin-1") for this gene which, when mutated, causes Alzheimer's
disease.
DESCRIPTION OF THE INVENTION
The present invention relates to a PS1/429 nucleic acid, polypeptide,
and derivatives thereof, comprising novel nucleotide and amino acid
sequences. The invention also relates to methods of using such nucleic
acids, polypeptides, or derivatives thereof, e.g., in therapeutics, diagnostics,
and as research tools. Another aspect of the present invention involves anti¬
bodies to PS1/429, regulators of PS1/429 gene expression and activity, and
methods of treating Alzheimer's disease.
Various other objects, features and attendant advantages of the present
invention will be more fully appreciated as the same becomes better
understood when considered in conjunction with the accompanying
drawings, in which like reference characters designate the same or similar
parts throughout the several views, and wherein:
FIG. 1 shows the complete nucleotide sequence (SEQ ID NO:l) and
deduced amino acid sequence (SEQ ID NO:2) for a 429 amino acid polypep¬
tide encoded for by a human PS1 gene. See, Sherrington et al., Nature,
375:754-760, 1995. See, Sherrington et al. Nature, 375:754-760, 1995.
FIG. 2 shows the complete nucleotide sequence (SEQ ID NO:3) and
deduced amino acid sequence (SEQ ID NO:4) for a 467 amino acid polypep¬
tide encoded for by a human PS1 gene. See, Sherrington et al. Nature,
375:754-760, 1995.
FIG. 3 shows the deduced amino acid sequence (SEQ ID NO:8) for a
467 amino acid polypeptide encoded for by a murine PS1 gene.
FIG. 4 shows a comparison between amino acids 1-19 of Fig. 1 (SEQ
ID NO:2) and COX3 Sauroleishmania (SEQ ID NO:5), NEC2 precursor
(SEQ ID NO:6), and murine 429/PS1 (SEQ ID NO:7). Amino acids are
represented by the standard one letter amino acid code. Boxed amino acids
indicates identical amino acids; underlined amino acids indicate similar
amino acids.
FIG. 5 shows 3' UTR of human PS1 (SEQ ID NO.:9)
In accordance with the present invention, a novel transcript of a PS1
gene, coding for a 429 amino acid polypeptide, has been identified and
isolated from polyadenylated mRNA. This transcript, corresponding nucleic
acids, and polypeptides encoded therein, are termed PSl/429. The polypep-
tide encoded by PSl/429 differs from the PS1/467 polypeptide previously
identified (See, e.g., Sherrington et al., Nature 375:754-760, 1995) in that
the N-terminal 57 amino acids of PS1/467 is replaced by 19 alternative
amino acids in PSl/429. A PS1 gene is generally defined as a gene on
chromosome 14q24.3 which, when mutant, results in Alzheimer's disease.
In accordance with the present invention, a nucleic acid can comprise,
e.g., the complete coding sequence for amino acid 1 to amino acid 429 as
set forth in Fig. 1 (SEQ ID NO:2), a coding sequence for amino acids 1-19
in Fig. 1 (SEQ ID NO:2), a coding sequence which possesses the same
function as the polypeptide of Fig. 1 (SEQ ID NO:2) and which shares
sequence similarity with it, and a unique PSl/429 nucleotide sequence
spanning nucleotides 206/207 of the sequence set forth in Fig. 1 (SEQ ID
NO:l), e.g., comprising nucleotides 198 to 215 as set forth in Fig. 1 (SEQ
ID NO: 1) or its antisense. A nucleic acid according to the present invention
also comprises the murine PSl/429 nucleic acid and derivatives thereof,
e.g., Fig. 4 (SEQ ED NO:7). The present invention also relates to an
isolated nucleic acid coding for a polypeptide having 410 amino acids
comprising: (a) a nucleotide sequence coding for amino acids 20-429 as set
forth in SEQ ID NO:2; (b) a nucleotide sequence coding without interruption
for amino acids 20-429 as set forth in SEQ ID NO:2; or (c) a nucleotide
sequence which hybridizes to the nucleotide sequence set forth in SEQ ID
NO:l under stringent conditions.
A nucleic acid, especially PSl/429, according to the present invention
can be obtained from a variety of different sources. It can be obtained from
DNA or RNA, such as polyadenylated mRNA, e.g., isolated from tissues,
cells, or whole organism. The nucleic acid can be obtained directly from
DNA or RNA, or from a cDNA library. The nucleic acid can be obtained
from a particular cell-type, stage of development, genotype, or phenotype
(e.g., having Alzheimer's disease). A nucleic acid in accordance with the
present invention can also be obtained from an organism of a desired age.
For example, a nucleic acid can be obtained from the brain of a mouse or
human of a particular age, e.g., using 5'-ACCTCGTCCCTCAAATCT; and
5'-TCACATCGGAAACAAAACAG.
A nucleic acid comprising a nucleotide sequence coding for a
polypeptide according to the present invention can include only coding
sequence of PSl/429; coding sequence of PSl/429 and additional coding
sequence (e.g., sequences coding for leader, secretory, targeting, enzymatic,
fluorescent or other diagnostic peptides), coding sequence of PSl/429 and
non-coding sequences, e.g., untranslated sequences at either a 5' or 3' end,
or dispersed in the coding sequence, e.g., introns. A nucleic acid
comprising a nucleotide sequence coding without interruption for a PSl/429
polypeptide means that the nucleotide sequence contains an amino acid
coding sequence for a PSl/429 polypeptide, with no non-coding nucleotides
interrupting or intervening in the coding sequence, e.g., absent intron(s) or
the noncoding sequence found in the S182 clone of Sherrington et al., 1995,
Nature, 375:754. Such a nucleotide sequence can also be described as
contiguous.
A nucleic acid according to the present invention also comprises an
expression control sequence operably linked to a nucleotide sequence coding
for a polypeptide comprising amino acids 1-19 as set forth in SEQ ID NO:
2, SEQ ED NO:7, or a PSl/429 specific amino acid sequence selected from
the sequence of amino acid 1 to 19 as set forth in SEQ ED NO:2 and/or SEQ
D NO:7. The phrase "expression control sequence" means a nucleic acid
sequence which regulates expression of a polypeptide coded for by a nucleic
acid to which it is operably linked. Expression can be regulated at the level
of the mRNA or polypeptide. Thus, the expression control sequence in¬
cludes mRNA-related elements and protein-related elements. Such elements
include promoters, enhancers (viral or cellular), ribosome binding
sequences, transcriptional terminators, etc. An expression control sequence
is operably linked to a nucleotide coding sequence when the expression con¬
trol sequence is positioned in such a manner to effect or achieve expression
of the coding sequence. For example, when a promoter is operably linked
5' to a coding sequence, expression of the coding sequence is driven by the
promoter.
A nucleic acid -in accordance with the present invention can be
selected on the basis of nucleic acid hybridization. The ability of two
single-stranded nucleic acid preparations to hybridize together is a measure
of their nucleotide sequence complementarity, e.g., base-pairing between
nucleotides, such as A-T, G-C, etc. The invention thus also relates to
nucleic acids which hybridize to a nucleic acid comprising a nucleotide
sequence as set forth in Fig. 1 (SEQ ED NO:l), but preferably, which do not
contain all or part of a coding sequence for a polypeptide having 467 amino
acids as set forth in Fig. 2 (SEQ ED NO:4) or murine PS1/467, or STM2 as
disclosed in Levy-Lahad et al., Science, 269:970-973; 973-977, 1995, more
preferably which do not contain nucleotide sequences which are prepared by
PCR polymerase chain reaction using the oligonucleotide sequences
disclosed in Sherrington et al., i.e., (a) 5'-
TCACATCGGAAACAAAACAG, 5'-GAGTCACAGGGACAAAGAGC; (b)
5'-CACCTGAGCAATACTGTACG, 5'-TCATCTTGCTCCACCACCTG;
(c) 5'-AGNAAGATGAGGAAGAAGATG, 5'-
CACACCATTGTTGAGGAGT; (d) 5'-TCACAGAAGATACCGAGACT,
5'-CCCAACCATAAGAAGAACAG; (e) 5'-
TCTGTACTTTTTAAGGGTTGT, 5'-ACTTCAGAGTAATTCATCANCA;
(f) 5'-ATCTCCGGCAGGCATATCT-3\ 5'-
TGAAATCACAGCCAAGATGAG-3'; (g) 5'-
CACCCATTTACAAGTTTAGC, 5'-GATGAGACAAGTNCCNTGAA; (h)
5'-TGGAGACTGGAACACAAC, 5'-GTGTGGCCAGGGTAGAGAACT;
5'-GATTTAGTGGCIOTTTTGTG, 5'-ACCTCGTCCCTCAAATCT; and
does not contain: (j) 5'-CCATAGCCTGTTTCGTAGC; (k) 5'-
CCATAGCCTATTTCGTAGC; and more preferably which do not contain
the 3' UTR set forth in SEQ ED NO:9.
Hybridization conditions can be chosen to select nucleic acids which
have a desired amount of nucleotide complementarity with the nucleotide
sequence set forth in Fig. 1 (SEQ ED NO:l) or the corresponding murine
PSl/429. A nucleic acid capable of hybridizing to such sequence, prefer¬
ably, possesses 50%, more preferably, 70% complementarity, between the
sequences. The present invention particularly relates to DNA sequences
which hybridize to the nucleotide sequence set forth in Fig. 1 (SEQ ED
NO:l) or a DNA coding for murine PSl/429 polypeptide as set forth in Fig.
4 (SEQ ED NO:7) under stringent conditions. As used here, "stringent
conditions" means any conditions in which hybridization will occur where
there is at least about 95%, preferably 97%, nucleotide complementarity
between the nucleic acids. A nucleotide sequence hybridizing to the
sequence of Fig. 1 (SEQ ED NO: 1) will have a complementary nucleic acid
strand, or act as a template for one in the presence of a polymerase (i.e., an
appropriate nucleic acid synthesizing enzyme), which has a corresponding
amount of nucleotide identity or similarity to Fig. 1 (SEQ ED NO:l). The
present invention includes both strands of nucleic acid, e.g., a sense strand
and an anti-sense strand. Thus, it is understood that a nucleic acid
comprising a nucleotide sequence hybridizing to the nucleotide sequence of
Fig. 1 (SEQ ED NO: 1) also represents a nucleic acid which possesses at least
about 95%, preferably 97% nucleotide sequence identity.
According to the present invention, a nucleic acid or polypeptide can
comprise one or more differences in the nucleotide or amino acid sequence
set forth in Fig. 1 (SEQ ED NO:l and SEQ ED NO:2) or murine PSl/429.
Changes or modifications to the nucleotide and/or amino acid sequence can
be accomplished by any method available, including directed or random
mutagenesis.
A nucleic acid according to the invention can comprise nucleotides
which occur in a naturally-occurring PS1 gene e.g., naturally-occurring
polymorphisms, normal or mutant alleles (nucleotide or amino acid), muta¬
tions which are discovered in a natural population of mammals, such as
humans, monkeys, pigs, mice, rats, or rabbits. By the term naturally-
occurring, it is meant that the nucleic acid is obtained from a natural source,
e.g., animal tissue and cells, body fluids, tissue culture cells, forensic
samples. Numerous naturally-occurring mutations have been identified in
human presenilin genes, disclosed, e.g., in Table 1. A nucleic acid accord¬
ing to the present invention can contain such mutations at a corresponding
position of the PSl/429 polypeptide. Table 1 shows examples of 22 muta¬
tions in the PSl gene. These mutations can be incorporated into the nucleic
acid or polypeptide at a corresponding position as indicated in Table 1. A
nucleic acid of the present invention can comprise one or more of such
mutations. Such a nucleic acid will hybridize to a nucleotide sequence as set
forth in Fig. 1 (SEQ ED NO:l). Other modifications to the sequence can
comprise mutations found in familial or genetic cases of disease, preferably
Alzheimer's disease. Nucleotide variations and genetic polymorphisms pre¬
sent in nucleic acid can be detected in accordance with various methods,
e.g., U.S. Pat. 5,468,613; Conner et al., Proc. NatL Acad. Sci. 80, 78
(1983); Angelini et al., Proc. NatL Acad., 83, 4489 (1986); Myers et al.,
Science 230, 1242 (1985). A nucleic acid, as described above, can also be
selected by its ability to hybridize to a sequence as set forth in Fig. 1 (SEQ
ED NO:l) or a murine PSl/429, where hybridization is more preferably
under stringent conditions, however less stringent conditions can also be
employed.
A nucleotide sequence coding for a polypeptide of the invention can
contain codons found in a naturally-occurring gene, transcript, or cDNA, for
example, e.g., as set forth in Fig. 1 (SEQ ED NO:l), or it can contain
degenerate codons coding for the same amino acid sequences of SEQ ED
NO:l or SEQ D NO:7.
In addition, a nucleic acid or polypeptide of the present invention can
be obtained from non-mammalian organisms, including C. elegans (Levitan
and Greenwald, Nature, 377:351, 1995). Mutations to a PSl/429 gene can
be based on a normal homolog or a mutated homolog of a non-mammalian
organism.
Homologs from mammalian and non-mammalian organisms can be
obtained according to various methods. For example, hybridization with an
oligonucleoύde (see below) selective for PSl/429 can be employed to select
such homologs, e.g., as described in Sambrook et al., Molecular Cloning,
1989, Chapter 11. Such homologs have varying amounts of nucleotide and
amino acid sequence identity and similarity to PSl/429. Non-mammalian
organisms include, e.g., vertebrates, invertebrates, zebra fish, chicken,
Drosophila, yeasts, C. elegans, roundworms, prokaryotes, plants,
Arabidopsis, viruses, etc.
Modifications to a PSl/429 sequence, e.g., mutations, can also be
prepared based on homology searching from gene data banks, e.g.,
Genbank, EMBL. Sequence homology searching can be accomplished using
various methods, including algorithms described in the BLAST family of
computer programs, the Smith-Waterman algorithm, etc. For example,
conserved amino acids can be identified between various sequences, PSl,
PS2, etc.. A mutation(s) can then be introduced into a PSl/429 sequence by
identifying conserved amino acids and modifying an amino acid in a
conserved position. A mutated PSl/429 gene can comprise conserved amino
acids, e.g., between corresponding regions of homologous nucleic acids,
especially amino acids 1-19 of Fig. 1 (SEQ ED NO:2), the transmembrane
(see below), and loop domain (see below), e.g., between PSl and PS2; PSl
and SEL-12 (Levitan and Greenwald, Nature:377 ,351 1995); or PSl and
SPE-4, etc. Amino acids conserved between multiple sequences (e.g., PS2,
SEL-12, SPE-4, E5-1, PSl, ALG-1 to -6, etc.) can also be selected as the
basis for introducing mutations into PSl/429. For example, a mutated
sequence can comprise conserved residues from any number of homologous
sequences as mentioned-above and/or determined from an appropriate
searching algorithm.
Mutations can be made in specific regions of nucleic acid coding for
the PSl/429 polypeptide, e.g., in the membrane-spanning domain (see
below), such as replacing it, changing amino acid sequences within it, etc.,
to analyze a function of the polypeptide coded for by the nucleic acid.
A nucleic acid and corresponding polypeptide of the present invention
include sequences which differ from the nucleotide sequence of Fig. 1 (SEQ
ED NO:l) or murine PSl/429 but which are phenotypically silent. These
sequence modifications include, e.g., nucleotide substitution which do not
affect the amino acid sequence (e.g., different codons for the same amino
acid), replacing naturally-occurring amino acids with homologous or
conservative amino acids, e.g., (based on the size of the side chain and
degree of polarization) small nonpolar: cysteine, proline, alanine, threonine;
small polar: serine, glycine, aspartate, asparagine; large polar: glutamate,
glutamine, lysine, arginine; intermediate polarity: tyrosine, histidine,
tryptophan; large nonpolar: phenylalanine, methionine, leucine, isoleucine,
valine. Such conservative substitutions also include those described by
Dayhoff in the Atlas of Protein Sequ nce and Structure 5 (1978), and by
Argos in EMBO J.. &, 779-785 (1989). A nucleic acid can therefore com¬
prise a nucleotide sequence coding for a polypeptide having an amino acid
sequence as set forth in SEQ ED NO:2, except where the amino acid at posi-
tion 44 is leucine, 77 is histidine, 101 is threonine, 105 is threonine, 108 is
valine, 108 is leucine, 125 is tyrosine, 125 is arginine, 193 is threonine, 208
is glutamine, 222 is valine, 225 is arginine, 226 is leucine, 229 is serine,
242 is alanine, 242 is glycine, 247 is valine, 248 is valine, 346 is alanine,
354 is valine, 372 is tyrosine, or a combination thereof; a nucleotide
sequence coding for a polypeptide having an amino acid sequence as set
forth in SEQ ED NO:2, except where one or more positions are substituted
by conservative amino acids, and/or is a biologically-active polypeptide; or a
nucleotide sequence coding for a polypeptide having an amino acid sequence
as set forth in SEQ ED NO:2, except having 5, 10, 15, or 20 substitutions,
e.g., wherein the substitutions are conservative amino acids. The invention
also relates to polypeptides coded for by such nucleic acids. In addition, it
may be desired to change the codons in the sequence to optimize the
sequence for expression in a desired host.
A nucleic acid according to the present invention can comprise, e.g.,
DNA, RNA, synthetic nucleic acid, peptide nucleic acid, modified nucleo-
tides, or mixtures. A DNA can be double- or single-stranded. Nucleotides
comprising a nucleic acid can be joined via various kncwn linkages, e.g.,
ester, sulfamate, sulfamide, phosphorothioate, phosphoramidate, methylphos-
phonate, carbamate, etc., depending on the desired purpose, e.g., resistance
to nucleases, such as RNase H, improved in vivo stability, etc. Various
modifications can be made to the nucleic acids, such as attaching detectable
markers (avidin, biotin, radioactive elements), moieties which improve
hybridization, detection, or stability. The nucleic acids can also be attached
to solid supports, e.g., nitrocellulose, nylon, agarose, diazotized cellulose,
latex solid microspheres, polyacrylamides, etc., according to a desired
method. See, e.g., U.S. Pat. Nos. 5,470,967, 5,476,925, 5,478,893.
Another aspect of the present invention relates to oligonucleotides and
nucleic acid probes. Such oligonucleotides or nucleic acid probes can be
used, e.g., to detect, quantitate, or isolate a PSl/429 nucleic acid in a test
sample. Detection can be desirable for a variety of different purposes,
including research, diagnostic, and forensic. For diagnostic purposes, it
may be desirable to identify the presence or quantity of a PSl/429 nucleic
acid sequence in a sample, where the sample is obtained from tissue, cells,
body fluids, etc. In a preferred method, the present invention relates to a
method of detecting a PSl/429 nucleic acid comprising, contacting a target
nucleic acid in a test Sample with an oligonucleotide under conditions effec¬
tive to achieve hybridization between the target and oligonucleotide; and
detecting hybridization. An oligonucleotide in accordance with the invention
can also be used in synthetic nucleic acid amplification such as PCR, e.g.,
Saiki et al., 1988, Science, 241:53; U.S. Pat. No. 4,683,202. Such an
oligonucleotide preferably contains nucleotides 206-207, or its antisense, of
Fig. 1 (SEQ ED NO:l). Accordingly, a preferred antisense oligonucleotide
comprises GTTACT and a preferred sense oligonucleotide comprises
ATACAG. More preferred oligonucleotides comprise about 1-4 nucleotides
from one side of the 206/207 junction and about 10-20 nucleotides from the
other side, e.g., GTATTTCTATACAGT and TGCCGGGAGTTACTG. To
amplify a coding segment of PSl/429 containing all or part of amino acids
1-19 by polymerase chain reaction, a pair of oligonucleotides 5'
TCACATCGGAAACAAAACAG and 5' ACCTGCCGGGAGTTACTGTAT
are preferred, annealing at about 50°C, preferably 53.4°C.
Another aspect of the present invention is a nucleotide sequence
which is unique to PSl/429. By a unique sequence to PSl/429, it is meant a
defined order of nucleotides which occurs in PSl/429, e.g., in the nucleotide
sequence of Fig. 1 (SEQ ED NO:l), but rarely or infrequently in other
nucleic acids, especially not in an animal nucleic acid, preferably mammal,
such as human, rat, mouse, etc, more preferably not in PS 1/467. Both
sense and antisense nucleotide sequences are included. A unique nucleic
acid according to the present invention comprises nucleotides 206-207 of
SEQ ED NO:l and its antisense. A nucleic acid comprising a unique
sequence of PSl/429 can be used as a hybridization probe to identify the
presence of PSl/429 in a sample comprising a mixture of nucleic acids. For
example, the antisense of nucleotides 198-215 as set forth in Fig. 1 (SEQ ED
NO:l), can be labeled according to various methods and used as a
hybridization probe to detect PSl/429 in mRNA samples on a Northern blot.
Other hybridization probes can comprise nucleotides 198-215, or can be 1-4
nucleotides shorter at either or both ends, or can contain 1-4 additional
nucleotides at either or both ends. Hybridization can be performed under
stringent conditions to select nucleic acids having at least 95% identity (i.e.,
complementarity) to the probe, but less stringent conditions can also be
used. A unique PSl/429 nucleotide sequence can also be fused in-frame, at
either its 5' or 3' end, to various nucleotide sequences as mentioned
throughout the patent, including coding sequences for other parts of
PSl/429, enzymes, GFP, etc, expression control sequences, etc.
Hybridization can be performed under different conditions, depending
on the desired selectivity, e.g., as described in Sambrook et al., Molecular
Cloning, 1989. For example, to specifically detect PSl/429, an oligonucleo¬
tide can be hybridized to a target nucleic acid under conditions in which the
oligonucleotide only hybridizes to PSl/429, e.g., where the oligonucleotide
is 100% complementary to the target. Different conditions can be used if it
is desired to select target nucleic acids which have less than 100% nucleotide
complementarity, at least about, e.g., 99%, 97%, 95%, 90%, 70%, 67%
(See, e.g., Levy-Hahad et al.), etc. Since a mutation in a PSl gene can
cause diseases, e.g., Alzheimer's disease, an oligonucleotide according to
the present invention can be used diagnosticalry. For example, a patient
having symptoms of Alzheimer's disease such as age-dependent,
progressive, memory disorder, and other dysfunctions, can be diagnosed
with the disease by using an oligonucleotide according to the present
invention, in polymerase chain reaction followed by DNA sequencing to
identify whether the sequence is normal, in combination with other PSl
oligonucleotides, etc. In a preferred method, the present invention relates to
a method of diagnosing Alzheimer's disease comprising contacting a sample
comprising a target nucleic acid with an oligonucleotide under conditions
effective to permit hybridization between the target and oligonucleotide;
detecting hybrization, wherein the oligonucleotide comprises a sequence of
PSl/429, preferably a unique sequence of PSl/429; and determining the
nucleotide sequence of the target nucleic acid to which the oligonucleotide is
hybridized. See, e.g., Sorbi et al., Tanahashi et al., Sherrington et al. The
sequence can be determined according to various methods, including isolat-
ing the target nucleic acid, or a cDNA thereof, and determining its sequence
according to a desired method.
Oligonucleotides according to the present invention can be of any
desired size, preferably 14-16 oligonucleotides in length, or more. Such
oligonucleotides can have non-naturally-occurring nucleotides, e.g., inosine.
In accordance with the present invention, the oligonucleotide can comprise a
kit, where the kit includes a desired buffer (e.g., phosphate, tris, etc.),
detection compositions, etc. The oligonucleotide can be labeled or
unlabeled, with radioactive or non-radioactive labels as known in the art.
Anti-sense nucleic acid can also be prepared from a nucleic acid
according to the present, preferably an anti-sense to a coding sequence of
Fig. 1 (SEQ ED NO:l) or Fig. 4, more preferably an anti-sense to the
sequence comprising nucleotides 198-215 of Fig. 1 (SEQ ED NO:l or SEQ
ED NO:7). Antisense nucleic acid can be used in various ways, such as to
regulate expression of PSl, e.g., inhibit it, to detect its expression, or for in
situ hybridization.
The nucleic acid according to the present invention can be labelled
according to any desired method. The nucleic acid can be labeled using
radioactive tracers such as ^P, 35S, 125I, 3H, or 14C, to mention only the most
commonly used tracers. The radioactive labelling can be carried out
according to any method such as, for example, terminal labeling at the 3' or
5' end using a radiolabeled nucleotide, polynucleotide kinase (with or
without dephosphorylation with a phosphatase) or a ligase (depending on the
end to be labelled). A non-radioactive labeling can also be used, combining
a nucleic acid of the "present invention with residues having immunological
properties (antigens, haptens), a specific affinity for certain reagents
(ligands), properties enabling detectable enzyme reactions to be completed
(enzymes or coenzymes, enzyme substrates, or other substances involved in
an enzymatic reaction), or characteristic physical properties, such as
fluorescence or the emission or absorption of light at a desired wavelength,
etc.
A nucleic acid according to the present invention, including oligo¬
nucleotides, anti-sense nucleic acid, etc., can be used to detect expression of
PSl/429 in whole organs, tissues, cells, etc., by various techniques, in¬
cluding Northern blot, PCR, in situ hybridization, etc. Such nucleic acids
can be particularly useful to detect disturbed expression, e.g., cell-specific
and/or subcellular alterations, of PSl or PS2, particularly, PSl/429. The
levels of PSl/429 can be determined alone or in combination with other
genes products, transcripts, etc. In particular, the amount (e.g., its
expression level) of PSl/429 can be compared (e.g., as a ratio) to the
amounts of other transcripts in the same or different sample, e.g, transcripts
of PSl or PS2 such as transcript corresponding to the 467 amino acid
polypeptide.
A nucleic acid according to the present invention can be expressed in
a variety of different systems, n vitro and in vivo, according to the desired
purpose. For example, a nucleic acid can be inserted into an expression
vector, introduced into a desired host, and cultured under conditions effec-
tive to achieve expression of a polypeptide coded for the nucleic acid.
Effective conditions includes any culture conditions which are suitable for
achieving production of the polypeptide by the host cell, including effective
temperatures, pH, medias, additives to the media in which the host cell is
cultured (e.g., additives which amplify or induce expression such as
butyrate, or methotrexate if the coding nucleic acid is adjacent to a dhfr
gene), cyclohexamide, cell densities, culture dishes, etc. A nucleic acid can
be introduced into the cell by any effective method including, e.g., calcium
phosphate precipitation, electroporation, injection, DEAE-Dextran mediated
transfection, fusion with liposomes, and viral transfection. A cell into which
a nucleic acid of the present invention has been introduced is a transformed
host cell. The nucleic acid can be extrachromosomal or integrated into a
chromosome(s) of the host cell. It can be stable or transient. An expression
vector is selected for its compatibility with the host cell. Host cells include,
mammalian cells, e.g., COS-7, CHO, HeLa, LTK, insect cells, such as Sf9
and Drosophila, bacteria, such as E. coli, Streptococcus, bacillus, yeast,
fungal cells, plants, embryonic stem cells (e.g., mammalian, such as mouse
or human), neuronal cells (primary or immortalized), e.g., NT-2, NT-2N,
PC-12, SY-5Y, neuroblastoma. See, also Methods in Enz mology, volume
185, ed., D.V. Goeddel. Expression control sequences are similarly
selected for host compatibility and a desired purpose, e.g., high copy
number, high amounts, induction, amplification, controlled expression.
Other sequences which can be employed include enhancers such as from
SV40, CMV, inducible promoters, neuronal specific elements, or sequences
which allow selective or specific cell expression, such as in neuronal cells,
glial cells, etc.
In addition to a PSl/429 nucleic acid, another gene of interest can be
introduced into the same host for purposes of, e.g., modulating expression
PSl/429, elucidating PSl/429 function or that of the gene of interest. Genes
of interest include APP, E5-1, apoliprotein E, apoptosis genes such as ALG-
1 to -6 (Vito et al., 1995, Science, 271: 521), Bcl-2/Bax gene family, etc.
Such genes can be the normal gene, or a variation, e.g., a mutation,
chimera, polymorphism, etc.
A polypeptide according to the present invention preferably comprises
amino acid sequences 1-19, 1-429, or a PSl/429 specific amino acid
sequence selected from amino acids 1-19 as set forth in Fig. 1 (SEQ ED
NO:2), or Fig. 4 (SEQ ED NO:7). Polypeptides of the present invention
include polypeptides isolated from natural sources such as those discussed
above, and products of nucleic acid sequences as described above and
below. The invention also relates to an isolated polypeptide having 410
polypeptides and comprising amino acids 20-429 as set forth in SEQ ED
NO:2. The latter can occur as cleavage products of the polypeptide having
429 amino acids, e.g.-, by removal of the 19 amino acid sequence at the N-
terminus of a protein set forth in SEQ ED NO:2, a peptide formed by speci-
fie cleavage at the amino 19/20 junction or anywhere specifically within the
19 amino acid 5' to this junction. Such a protein can be the biologically-
active component of the 429 polypeptide precursor protein, e.g., when its
activity is mutated, Alzheimer's Disease occurs.
A PSl/429 specific amino acid sequence selected from the sequence
of amino acid 1 to 19 as set forth in SEQ ED NO:2 or SEQ ED NO:7 means
a defined amino acid sequence which is found in the recited PSl sequence
but not in another gene amino acid sequence. A specific amino acid
sequence can be found routinely, e.g., by searching a gene database using
the BLAST set of computer programs. Such specific sequences include
(using one letter amino acid code): KERTSRG (amino acid 2-8 of SEQ ED
NO:2), RGFVF (amino acid 7-11 of SEQ ED NO:2), FCETV (amino acid
11-15 of SEQ ED NO:2), VFLYS (amino acid 15-19 of SEQ ED NO:2), etc.
A nucleotide sequence coding for a PSl/429 specific amino acid sequence
can be useful to produce peptides as antigens to generate an immune
response specific for PSl/429. Antibodies obtained by such immunization
can then be used a specific probe for the PSl/429 protein.
A polypeptide of the invention, e.g., having a polypeptide sequence
as shown in Fig. 1 (SEQ ED NO:2), can by analyzed by available methods
to identify structural and/or functional domains in the polypeptide. For
example, when the polypeptide coding sequence set forth in Fig. 1 (SEQ ED
NO:2) is analyzed by hydrophobicity algorithms, a continuous coding
sequence comprising the following domains is identified: a 19-amino acid
peptide domain from amino acids 1-19; seven (7) membrane-spanning
domain at positions (82 to 100; 133 to 154; 164 to 183; 195 to 213; 221 to
238; 244 to 262; 408 to 428); and a loop domain at position 263-407. See,
Sherrington et al., Nature, 375:754-760. Various programs can be
employed to analyze structure of the polypeptide, including, EMBL Protein
Predict; Rost and Sander, Proteins, 19:55-72, 1994; Kyte and Doolittle, J.
Mol. Bio.: 157:105, 1982.
A polypeptide of the present invention can also have 100% or less
amino acid sequence identity to the amino acid sequence set forth in SEQ ED
NO: 1. For the purposes of the following discussion: Sequence identity
means that the same nucleotide or amino acid which is found in the sequence
set forth in Fig 1. (SEQ ED NO:l and SEQ ED NO:2) is found at the corre¬
sponding position of the compared sequence(s). However, the compared
amino acid sequence can also have a conservative nucleotide or amino acid
substituted at the corresponding position. See below for examples of
conservative amino acid substitution. The sum of the identical and con-
served residues divided by the total number of residues in the sequence over
which the PSl/429 polypeptide is compared is equal to the percent sequence
similarity. For purposes of calculating sequence identity and similarity, the
compared sequences can be aligned and calculated according to any desired
method, algorithm, computer program, etc., including, e.g., FASTA,
BLASTA.
A PSl/429 polypeptide according to the present invention can also
comprise amino acid sequence similarity and/or identity, when compared to
the amino acid sequence of Fig. 1 (SEQ ED NO:2), e.g., about 20%, 30%,
37%, 40%, preferably, 50, 53, 60, 65, more preferably, 67, 70, 78, 80, 90,
92, 96, 99, etc. For example, when compared to amino acids 1-19 of Fig.
1 (SEQ NO:l): a 17-amino acid (1-17) Sauroleishmania COX3 sequence is
about 41 % similar; a 10-amino acid Sauroleishmania COX3 (1-10) is about
70% similar; a 18-amino acid (1-18) of a murine NEC2 precursor is about
56% similar; a 15-amino acid (1-15) of a murine NEC2 precursor is about
67% similar; and a 19-amino acid domain of mouse PSl is about 78% simi¬
lar (15/19 amino acids) or 74% identical (14/19 amino acids). See, Fig. 4.
Thus, a polypeptide according to the present invention includes polypeptides
having the above-mentioned similarity. Also included are polypeptides
which have a consensus sequence(s), e.g., preferably between one or more
of COX3, NEC2, or PSl (murine, rat, etc.) as mentioned above.
All or part of amino acids 1-19 in Fig. 1 (SEQ ED NO:2) and Fig. 4
(SEQ ED NO:7) and the corresponding murine sequence can be present in a
polypeptide according to the present invention. Such a polypeptide can also
comprise a modified 19 amino acid sequence, where such modifications in-
elude glycosylation, covalent modifications (e.g., of an R-group of an amino
acid), amino acid substitution, amino acid deletion, or amino acid addition.
Modifications to the 19 amino acid sequence can be accomplished according
to various methods, including recombinant, synthetic, chemical, etc.
A mutation to amino acids 1-19 can be selected to have a biological
function of the human, murine, etc. naturally-occurring amino acids 1-19.
For example, amino acids 1-19 as set forth in Fig. 1 (SEQ ED NO:2) or Fig.
4 (SEQ ED NO:7) can contain a cellular trafficking function, preferably for
targeting to or from, or, retention in, an intra- or subcellular compartment,
such as mitochondria, cytoskeleton, nuclear, cell membrane, calveolae, etc.
For example, amino acids 1-19 can act as a signal peptide and be cleaved
during maturation. Amino acids 1-19 or a PSl/429 unique nucleotide
sequence, can therefore be modified and tested for retention of signal peptide
function. For example, a nucleotide sequence coding for amino acids 1-19
can be modified and joined to a coding sequence for a detectable marker
(e.g., an enzyme, a peptide recognized by an antibody, GFP, etc.). The
fusion gene can be introduced into a cell line and the transformed cell line
then examined for the cellular distribution of the fusion gene product to
determine whether its function as a signal peptide has been eliminated or
retained. Thus, a nucleic acid coding for a derivative of amino acids 1-19
means a nucleic acidΛvhich codes for a polypeptide which codes for the
biological activity, e.g., a signal sequence.
A polypeptide coding for a polypeptide of the present invention,
preferably amino acids 1-19, or a PSl/429 unique nucleotide sequence can
be combined with one or more structural domains, functional domains,
detectable domains, antigenic domains, and/or a desired polypeptides of
interest, in an arrangement which does not occur in nature, i.e., not
naturally-occurring, e.g., as in a normal PSl gene, a genomic fragment
prepared from the genome of a living organism, e.g., an animal, preferably
a mammal, such as human, mouse, or cell lines thereof. A polypeptide
comprising such features is a chimeric or fusion polypeptide. Such a
chimeric polypeptide can be prepared according to various methods, includ-
ing, chemical, synthetic, quasi-synthetic, and/or recombinant methods.
A chimeric nucleic acid coding for a chimeric polypeptide can contain the
various domains or desired polypeptides in a continuous or interrupted open
reading frame, e.g., containing introns, splice sites, enhancers, etc.
The chimeric nucleic acid can be produced according to various methods.
A domain or desired polypeptide can possess any desired property, includ¬
ing, a biological function such as catalytic, signalling, growth promoting,
cellular targeting, etc., a structural function such as hydrophobic, hydro-
philic, membrane-spanning, etc., receptor-ligand functions, and/or detectable
functions, e.g., combined with enzyme, fluorescent polypeptide, green
fluorescent protein GFP (Chalfie et al., 1994, Science, 263:802; Cheng
et al., 1996, Nature Biotechnology, 14:606; Levy et al., 1996, Nature
Biotechnology, 14:610, etc. In addition, a PSl/429 nucleic acid, or a part
of it, can be used as selectable marker when introduced into a host cell. For
example, a nucleic acid coding for an amino acid sequence according to the
present invention can be fused in frame to a desired coding sequence and act
as a tag for purification, selection, or marking purposes. The region of
fusion encodes a cleavage site.
A polypeptide according to the present invention can be produced in
an expression system, e.g., in vivo, in vitro, cell-free, recombinant, cell
fusion, etc., according to the present invention. Modifications to the
polypeptide imparted by such system include, glycosylation, amino acid
substitution (e.g., by differing codon usage), polypeptide processing such as
digestion, cleavage, endopeptidase or exopeptidase activity, attachment of
chemical moieties, including lipids, phosphates, etc. For example, some cell
lines can remove the terminal methionine from an expressed polypeptide.
A polypeptide according to the present invention can be recovered
from natural sources, transformed host cells (culture medium or cells)
according to the usual methods, including, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
hydroxyapatite chromatography and lectin chromatography. It is preferred
to have low concentrations (approximately 0.1-5 mM) of calcium ion present
during purification (Price, et al., J. Biol. Chem. , 244:917 (1969)). Protein
refolding steps can be used, as necessary, in completing configuration of the
mature protein. Finally, high performance liquid chromatography (HPLC)
can be employed for final purification steps.
A nucleic acid or polypeptide of the present invention can be used as
a size marker in nucleic acid or protein electrophoresis, chromatography,
etc. For example, a Hael restriction digest of a cDNA coding for PSl/429
yields 0.133 kb and 1.570 kb molecular weight band since the restriction site
is at nucleotide 134 of PSl/429. Other defined restriction fragments can be
determined by scanning the sequence for restriction sites, calculating the
size, and performing the corresponding restriction digest.
The present invention also relates to antibodies which specifically
recognize a PSl/429 polypeptide, and/or preferably antibodies which bind or
attach to amino acids 1-19 as set forth in Fig 1. (SEQ ED NO:2) or Fig. 4
(SEQ ED NO:7), or a derivative thereof. Antibodies, e.g., polyclonal,
monoclonal, recombinant, chimeric, can be prepared according to any
desired method. For example, for the production of monoclonal antibodies, a
polypeptide according to Fig. 1 (SEQ ID NO:2), preferably comprising
amino acids 1-19, can be administered to mice or rabbit subcutaneously
and/or intraperitoneally, with or without adjuvant, in an amount effective to
elicit an immune response. The antibodies can also be single chain or FAb.
A derivative or modified mentioned amino acid sequence of Fig. 1
(SEQ ED NO:2) or Fig. 4 (SEQ ED NO:7) can be selected for its ability to
elicit an in vivo or in vitro immunological response, e.g., cellular or
humoral. The immune response can be similar (in antibodies and their
affinity), differ in antibody type, or produce antibodies with higher affinity
for amino acids 1-19.
An antibody specific for such sequence means that an antibody recog-
nizes a defined sequence of amino acids within or including the amino acid
sequence of Fig. 1 (SEQ ED NO:2) or Fig. 4 (SEQ ED NO:7). Thus, a spe¬
cific antibody will bind with higher affinity to an amino acid sequence, i.e.,
an epitope, found in Fig 1 (SEQ ED NO:2) than to a different epitope(s),
e.g., as detected and/or measured by an immunoblot assay. Thus, an anti-
body which is specific for an epitope having a peptide sequence within and
including Fig. 1 (SEQ ED NO:2) is useful to detect the presence of the
epitope in a sample, e.g., a sample of tissue containing PSl gene product,
distinguishing it from samples in which the epitope is absent.
In addition, ligands which bind to a polypeptide according to the
present invention, preferably amino acids 1-19, or a derivative thereof, can
also be prepared, e.g., using synthetic peptide libraries or aptamers (e.g.,
Pitrung et al., U.S. Pat. No. 5,143,854; Geysen et al., 1987, J. Immunol.
7/46678
- 30 - Methods, 102:259-274; Scott et al., 1990, Science, 249:386; Blackweli et
al., 1990, Science, 250:1104; Tuerk et al., 1990, Science, 249: 505).
Antibodies and other ligands which bind PSl/429 can be used in
various ways, including as therapeutic, diagnostic, and commercial research
tools, e.g, to quantitate the levels of PSl/429 polypeptide in animals,
tissues, cells, etc., to identify the cellular localization and/or distribution of
PSl/429, to purify PSl/429 or a polypeptide comprising a part of ?Sl/429,
such as amino acids 1-19. Antibodies to PSl/429, or a derivative thereof,
can be used in Western blots, ELIZA, immunoprecipitation, RIA, etc. The
present invention relates to such assays, compositions and kits for
performing them, etc.
An antibody according to the present invention can be used to detect
PSl/429 polypeptide or fragments thereof in various samples, including
tissue, body fluid, blood, urine, cerebrospinal fluid. A method of the
present invention comprises contacting an antibody which binds to a peptide
of SEQ ED NO:l under conditions effective, as known in the art, to achieve
binding, detecting specific binding between the antibody and target, such as
polypeptide, aptamer, etc. By specific binding, it is meant that the antibody
attaches to a defined sequence of amino acids, e.g., within or including the
amino acid sequence of SEQ ED NO:2 or SEQ ED NO:7 or derivatives
thereof. The antibodies or derivatives thereof can also be used to inhibit
expression of PSl/429 or a fragment thereof. The levels of PSl/429 poly-
peptide can be determined alone or in combination with other gene products.
In particular, the amount (e.g., its expression level) of PSl/429 polypeptide
can be compared (e.g., as a ratio) to the amounts of other polypeptides in
the same or different sample, e.g., polypeptides of a PSl gene, such
PS 1/467 polypeptide, or polypeptides coded for by PS2.
An antibody can be used in combination with other antibodies, e.g.,
antibodies that recognize pathological markers of Alzheimer's Disease,
including paired helical filaments, micro-tubule associated protein 2
(MAP-2), tau, and ubiquitin (e.g., U.S. Pat. Nos 5,385,915; 5,397,712; and
methods therein) or in combination with other diagnostics, e.g, an amyloid
peptide such as in U.S. Pat. No 5,434,050, haptoglobin in U.S. Pat. No.
5,429,947, etc. In general, reagents which are specific for PSl/429 can be
used in diagnostic and/or forensic studies according to any desired method,
e.g., as U.S. Pat. Nos. 5,397,712; 5,434,050; 5,429,947.
The present invention also relates to a labelled PSl/429 polypeptide,
prepared according to a desired method, e.g., as disclosed in U.S. Pat. No.
5,434,050. A labelled polypeptide can be used, e.g., in binding assays,
such as to identify substances that bind or attach to PSl/429, to track the
movement of PSl/429 in a cell, in an in vitro, in vivo, or in situ system,
etc.
A nucleic acid, polypeptide, antibody, etc., according to the present
invention can be isolated. The term "isolated" means that the material is in
a form in which it is not found in its original environment, e.g., more con¬
centrated, more purified, separated from component, etc. An isolated
nucleic acid includes, je.g., a nucleic acid having the sequence of PSl/429
separated from the chromosomal DNA found in a living animal. This
nucleic acid can be part of a vector or other desired nucleic acid and still be
isolated in that it is not in a form which it is found in its natural
environment.
The present invention also relates to a non-human transgenic animal,
preferably a mammal, more preferably a mouse, comprising a PSl/429
nucleic acid, especially a mutation (e.g., those in Table 1) which has been
introduced into the animal. Transgenic animals can be prepared according
to known methods, including, e.g., by pronuclear injection of recombinant
genes into pronuclei of 1-cell embryos, incorporating an artificial yeast
chromosome into embryonic stem cells, gene targeting methods, embryonic
stem cell methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191;
4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385;
5,221,778; Gordon et al., Proc. NatL Acad. Sc , 77:7380-7384 (1980);
Palmiter et al., Cell, 41:343-345 (1985); Palmiter et al., Ann. Rev. Genet. ,
20:465-499 (1986); Askew et al., Mol. Cell. Bio. , 13:4115-4124, 1993;
Games et al. Nature, 373:523-527, 1995; Valancius and Smithies, Mol.
Cell. Bio. , 11:1402-1408, 1991 ; Stacey et al., Mol. Cell. Bio. , 14: 1009-
1016, 1994; Hasty et al., Nature, 350:243-246, 1995; Rubinstein et al.,
Nucl. Acid Res. , 21:2613-2617,1993. A nucleic acid according to the
present invention can be introduced into any non-human mammal, including
a mouse (Hogan et aL, 1986, in Manipulating the Mouse Embryo: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York), pig (Hammer et al., Nature, 315:343-345, 1985), sheep
(Hammer et al., Nature, 315:343-345, 1985), cattle, rat, or primate. See
also, e.g., Church, 1987, Trends in Biotech. 5:13-19; Clark et al., 1987,
Trends in Biotech. 5:20-24; and DePamphilis et al., 1988, BioTechniques,
6:662-680. In addition, e.g., custom transgenic rat and mouse production is
commercially available. These transgenic animals are useful as an AD
model, e.g., to test drugs, or as food for a snake.
The present invention also relates to the treatment and prevention of
diseases, e.g., Alzheimer's disease, diseases associated with mitochondria!
cytopathology, apoptosis, or neurodegeneration. For example, the invention
relates to a method of treating Alzheimer's disease comprising administer¬
ing, to a subject in need of treatment, an amount of a compound effective to
treat the disease, where the compound is a regulator of PSl/429 gene or
polypeptide expression, effecting, e.g., apoptosis, neuronal degeneration,
and/or mitochondrial degeneration. Treating the disease can mean, delaying
its onset, delaying the progression of the disease, improving or delaying
clinical and pathological signs of disease, e.g., memory, and/or other
complex deficits, neurodegeneration, and other symptoms observed in
O 97/46678
- 34 - patients. A regulator compound, or mixture of compounds, can be synthe¬
tic, naturally-occurring, or a combination. A regulator compound can
comprise amino acids, nucleotides, hydrocarbons, lipids, polysaccharides,
etc. A regulator compound is preferably a regulator of PSl/429, e.g.,
inhibiting or increasing its mRNA, protein expression, or processing, e.g.,
cleavage, at the junction between amino acids 19 and 20. Expression can be
regulated using different agents, e.g., an anti-sense nucleic acid, aribozyme,
an aptamer, a synthetic compound, or a naturally-occurring compound.
Additionally, a polypeptide comprising PSl/429 can be employed to target
an agent to a desired location in the cell (e.g., using amino acids 1-19) or
cells can be supplemented with PSl/429, or derivatives thereof. To treat the
disease, the compound, or mixture, can be formulated into pharmaceutical
composition comprising a pharmaceutically acceptable carrier and other
excipients as apparent to the skilled worker. See, e.g., Remington's
Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company,
1990. Such composition can additionally contain effective amounts of other
compounds, especially for treatment of Alzheimer's disease, including,
agents disclosed in U.S. Pat. 5,385,915.
Generally, the nucleic acids, polypeptides, antibodies, etc. of the
present invention can be used as described in, U.S. Pat. Nos. 5,501,969;
5,506, 133; or 5,441,870.
7/46678
- 35 -
For other aspects of the nucleic acids, polypeptides, antibodies, etc.,
reference is made to standard textbooks of molecular biology, protein
science, and immunology. See, e.g., Davis et al. (1986), Basic Methods in
Molecular Biology, Elsevir Sciences Publishing, Inc., New York; Hames et
al. (1985), Nucleic Acid Hybridization, EL Press, Molecular Cloning,
Sambrook et al.; Current Protocols in Molecular Biology, Edited by F.M.
Ausubel et al., John Wiley & Sons, Inc; Current Protocols in Human
Genetics, Edited by Nicholas C. Dracopoli et al., John Wiley & Sons, Inc.;
Current Protocols in Protein Science; Edited by John E. Coligan et al., John
Wiley & Sons, Inc.; Current Protocols in Immunology; Edited by John E.
Coligan et al., John Wiley & Sons, Inc.
EXAMPLE
S182429 was amplified from a human cortical cDNA library using high-fidelity PCR and oligonucleotide primers 5'-TCA CAT CGG AAA CAA AAC AG and 5'-ACC TCG TCC CTC AAA TCT. The cDNA library used as template in the reaction was constructed from post-mortem adult human cortex using the λ ZAP Ef vector (Stratagene). After a double- round of PCR amplification, electrophoretic separation and purification, the purified amplification product was ligated into the pCRK vector and used to transform E.coli (TA cloning kit, Invitrogen). Clones with inserts were sequenced on both strands using an automated DNA sequencing system (Applied Biosystems).
High fidelity PCR was used (Expand PCR kit; Boehringer Mannheim): 2 μl of cDNA template (titer = 1 x 1010/ml), 30 pmol of each primer mentioned above, 0.75 μl of TAQ/PWO polymerase mix, 350 μM
97/46678
- 36 - dNTPs in a 50 μl reaction volume for 10 cycles of 94°C for 10s, 53 °C for 30s, and 68 °C for 90s followed by 20 more cycles of increasing duration in 20s increments at the 68 °C extension step on a Gene Amp 9600 thermocycler (Periόn* Elmer).
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The entire disclosure of all applications, patents and publications, cited above and in the figures are hereby incorporated by reference.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Tαbk -1 - 37 -
PS-1 Mutations Mutation . Domain
Val 82; Lβu fy ) g G ^cTG (t%t) TM-1
ΫV *τι
Tyr r His (IT) tAT eg cAT f fa") - H L«1 Met «£»■ Val IfO aTG6βygTG - TM-2
<o<
Met ffi Thr#*/) AtGW-AcG ^V52θ TM-2 lie HφThr fs) AIT 6W*AcT tøfcff) TM-2 Met f Values) ,aTG 6»gTG^ 73) TM-2 Met «§• Leutfetf) aTG ^cTG «/73 TM-2
«* JΛttj
8. His *>? Tyr^S") cAT *» tAT y) HL-2
9. His *ξ Arg «r) CaT WL-QgT ( i HL-2
10. Ala jw- Trw//?9) gCC ∞ aCC f7j.2 TM-5
11. Ala » » Glu £*Λ GOG « GaG V TM-6
12. Ala «£ Val &zz) GcT 3uσ GfT «fc) TM-6
13. Cys β r Arg(Zzs) tGT «^ cGT føf) HL-6 i
1 . Pro «4- Leu zzt) CcG wa^CtG ^?zΛHL-6
15. Pro «?- Ser zz cCA V*r tCA fø ) HL-6
16. Glu ∞f-AIa (ztz) GaA J^GcA ^7( HL-6
17. GluΛθθ- Gly/zvz) GaA «β£ GgA « fr HL-6
18. Ala » Val 2V7) Get «« GtT _W) HL-6
19. Leu £ge-Vat zw) cTC JJ« gTC ^«3 HL-6
20. Gly ** Ala/3V6) GgA4∞9 GcA &*i HL-6
21. Leu sea- val^sv) cTG «j gTG #»/"> HL-6
References:
(1.) Campion, O. era/. Mutations of the PraseniGπ-1 gene in families with eariy-onset Alzheimer's disease. Hum. malec Genet 4, 2373-2377 (1995) (2.) Clarfc, R.F. etal. The structure of the presenilin 1 (S182) gene and identification of six novel mutations In early-onset AD families. Nature Genet. 11, 219-222 (1995). (3.) Cruts, M. et al. Molecular genetic analysis of familial early-onset Alzheimer's disease linked to chromosome 14q24.3. Hum. molβo. Genet 4, 2363-2371 (1995) (4.) Sherrington, R. et al. Cloning of a gene bearing mis-sense mutations in early-onset familial Alzheimer's disease. Nature 375, 754-760 (1995). (5.) Sorbi, S. er at Missense mutation of S182 gene In ItaOan families with early-onset Alzheimer's disease. Lancet 346, 439-440 (1995). (6.) Tanahashl, H. ef al. Missense mutation of S182 gene in Japanese familial Alzheimer's disease. Lancet 346.440 (1995). (7.) Rogaev, E.I. ef al. Familial Alzheimer's disease In kindreds with missense mutations In a gene on chromosome 1 related to trio Alzheimer's disease type 3 gene. Nature 376, 77G-77Θ (1995). (8.) Wasco, W. ef al Familial Alzhoimor's chromosome 14 mutations. Nature Mβd.1, 848 (1995).
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: DAVIS, JOHN N.
CHISHOLM, JANE C. DRACHE, BETTINA
(ii) TITLE OF INVENTION: Novel Nucleic Acids and Polypeptides Related to Presenilin
(iii) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Bayer Corporation
(B) STREET: 400 Morgan Lane
(C) CITY: West Haven
(D) STATE: Connecticut
(E) COUNTRY: US
(F) ZIP: 06516-4175
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/659,296
(B) FILING DATE: 18-JULY-1996
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/683,315
(B) FILING DATE: 18-JULY-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Jones, Huw R.
(B) REGISTRATION NUMBER: 33,916
(C) REFERENCE/DOCKET NUMBER: WH 501 OP 1 -PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (203) 812-2317
(B) TELEFAX: (203) 812-5492
SUBSππilE SHEET (RULE 26)
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
ACCTCGTCCC TCAATCT 17
(2) INFORMATION FOR SEQ ID NO:l 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11 :
TCACATCGGA AACAAAACAG 20
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
GAGTCACAGG GACAAAGAGC 20
SOBSΠTDIE SHEET (RULE 26)
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
CACCTGAGCA ATACTGTACG 20
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
TCACAGAAGA TACCGAGACT 20
srosππjTE SBEET (R LE 2β>
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
AGNAAGATGA GGAAGAAG AT G 21
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
CACACCATTG TTGAGGAGT 19
SUBS UIE SHEET PLE26)
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TCTGTACTTT TTAAGGGTTG T 21
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
CCCAACCATA AGAAGAACAG 20
(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
TCTGTACTTT TTAAGGGTTG T 21
SBBSITTUTE SHEET (ROLE 26)
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
ACTTCAGAGT AATTCATCAN CA 22
(2) INFORMATION FOR SEQ ID NO:21 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
ATCTCCGGCA GGCATATCT 19
SUBSπnrrE SHEET RULE 26)
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TGAAATCACA GCCAAGATGA G 21
SUBSTTTUTE SHEET RULE 26
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
CACCCATTTA CAAGTTTAGC 20
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GATGAGACAA GTNCCNTGAA 20
sβrørruTE SHEET (msn)
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
TGGAGACTGG AACACAAC 18
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
GTGTGGCCAG GGTAGAGAAC T 21
S∞STΠWTE SHEET (JMIIE26
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GATTTAGTGG CTGTTTTGTG 20
SUBSmUTE SHEET (RULE 26)
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CCATAGCCTG TTTCGTAGC 19
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CCATAGCCTA TTTCGTAGC 19
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
CCATAGCCTA TTTCGTAGC 19
(2) INFORMATION FOR SEQ ID NO:31 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31 :
TGCCGGGAGT TACTG 15
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TCACATCGGA AACAAAACAG 20
SUBSΠΠJTE SHEET (RBLE 26)
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
ACCTGCCGGG AGTTACTGTA T 21
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Lys Glu Arg Thr Ser Arg Gly 1 5
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Arg Gly Phe Val Phe 1 5
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Phe Cys Glu Thr Val 1 5
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Val Phe Leu Tyr Ser
1 5
SMSπrUIΪ SHEET (R0i£26)
Claims
1. An isolated nucleic acid comprising a nucleotide sequence coding without interruption for a PSl/429 polypeptide comprising amino acid 1 to amino acid 429 as set forth in SEQ ID NO:2.
2. An isolated nucleic acid comprising a nucleotide sequence coding for a PSl/429 polypeptide comprising amino acid 1 to amino acid 429 as set forth in SEQ ID NO: 2 and having 429 amino acids.
3. An isolated nucleic acid of claim 1, wherein the nucleic acid comprises a naturally-occurring nucleotide sequence.
4. An isolated nucleic acid of claim 2, wherein the nucleic acid comprises a naturally-occurring nucleotide sequence.
5. An isolated nucleic acid of claim 1, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1.
6. An isolated nucleic acid of claim 2, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 1.
7. An isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: l, with the proviso that the nucleic acid does not contain all or part of the coding sequence for a polypeptide having 467 amino acids as set forth in SEQ ID NO:4 or SEQ ID NO:8).
8. An isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to and which contains at least 95 % nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: l, with the proviso that the nucleic acid does not contain all or part of the coding sequence for a polypeptide having 467 amino acids as set forth in SEQ ID NO:4 or SEQ ID NO:8).
9. An isolated nucleic acid comprising an expression control sequence operably linked to a nucleotide sequence coding for a polypeptide comprising amino acid 1 to amino acid 19 as set forth in SEQ ID NO:2.
10. An isolated nucleic acid comprising an expression control sequence operably linked to a nucleotide sequence coding for a PSl/429 specific amino acid sequence selected from the sequence of amino acid 1 to 19 as set forth in SEQ ID NO:2.
11. An isolated nucleic acid comprising a PSl/429 unique nucleotide sequence and comprising nucleotides 206 and 207 as set forth in SEQ ID NO:l.
SUBSTfflm SHEET (IULE 26)
12. An isolated nucleic acid of claim 11, wherein the nucleotide sequence is operably linked to an expression control sequence.
13. An isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of claim 11.
14. An isolated nucleic acid comprising a nucleotide sequence which hybridizes to under stringent conditions and which contains at least 95 % nucleotide sequence identity to the nucleotide sequence of claim 11.
15. An isolated nucleic acid of claim 13, wherein the nucleic acid is operably linked to an expression control sequence.
16. An isolated nucleic acid coding for a polypeptide having 410 amino acids comprising:
(a) a nucleotide sequence coding for amino acids 20-429 as set forth in SEQ ID NO:2;
(b) a nucleotide sequence coding without interruption for amino acids 20-429 as set forth in SEQ ID NO:2; or
(c) a nucleotide sequence which hybridizes to a nucleotide sequence coding for the amino sequence set forth in SEQ ID NO: 2 under stringent conditions.
17. A method of expressing, in transformed host cells, a PSl/429 polypeptide coded for by a nucleic acid, comprising culturing transformed host cells containing a nucleic acid according to claim 1 under conditions effective to express the polypeptide.
18. A method of expressing, in transformed host cells, a PSl/429 polypeptide coded for by a nucleic acid, comprising transforming host cells with a nucleic acid according to claim 1 , and culturing the transformed host cells containing the nucleic acid under conditions effective to express the polypeptide.
19. A method of expressing, in transformed host cells, a polypeptide coded for by a nucleic acid, comprising transforming host cells with a nucleic acid according to claim 7, and culturing the transformed host cells containing the nucleic acid under conditions effective to express the polypeptide.
20. A method of expressing, in transformed host cells, a polypeptide coded for by a nucleic acid, comprising transforming host cells with a nucleic acid according to claim 12, and culturing the transformed host cells containing the nucleic acid under conditions effective to express the polypeptide.
21. A method of expressing, in transformed host cells, a polypeptide coded for by a nucleic acid, comprising transforming host cells with a nucleic acid according to claim 14, and culturing the transformed host cells containing the nucleic acid under conditions effective to express the polypeptide.
22. A polypeptide produced by a method of claim 19.
23. A polypeptide produced by a method of claim 21.
24. An isolated polypeptide comprising,
(a) amino acid 1 to amino acid 429 as set forth in SEQ ID NO:2;
(b) amino acid 1 to 19 as set forth in SEQ ID NO:2; (c) amino acid 1 to amino acid 19 as set forth in SEQ ID NO:7; or
(d) a PSl/429 specific amino acid sequence selected from the sequence of amino acid 1 to 19 as set forth in SEQ ID NO: 2 or SEQ ID NO:7.
25. An isolated polypeptide having 410 amino acids as set forth as amino acids 20-429 of SEQ ID NO:2.
26. An isolated polypeptide of claim 25, further comprising an amino acid 1 to 19 as set forth in SEQ ID NO:2.
27. An isolated peptide of claim 24 which comprises amino acid sequence RGFVF (SEQ ID NO: 2) or VFLYS (SEQ ID NO: 2).
28. A transformed host cell containing a nucleic acid of claim 1.
29. A transformed host cell containing a nucleic acid of claim 7.
30. A transformed host cell containing a nucleic acid of claim 9.
31. A transformed host cell containing a nucleic acid of claim 14.
32. A vector comprising a nucleic acid of claim 1.
33. A vector comprising a nucleic acid of claim 9.
34. An isolated antibody which is specific for an amino acid sequence of amino acid 1 to amino acid 19 as set forth in SEQ ID NO: 2 or SEQ ID NO:7.
35. A method of diagnosing Alzheimer's disease comprising, hybridizing an oligonucleotide comprising a nucleotide sequence according to claim 11 to a target nucleic acid contained in a test sample, under conditions effective to achieve hybridization, detecting the target nucleic acid in the test sample hybridized to the oligonucleotide, and determining the nucleotide sequence of the target nucleic acid.
36. A method of diagnosing Alzheimer's disease comprising, contacting a polypeptide in a test sample with an antibody of claim 34, under conditions effective for specific binding between said antibody and said polypeptide; and detecting binding between said antibody and said polypeptide.
37. A transgenic non-human mammal comprising a nucleic acid of claim 1.
38. An isolated nucleic acid of claim 1, wherein the nucleic acid is DNA or RNA.
39. An isolated nucleic acid of claim 7, wherein the amino acid at position 44 is leucine, 77 is histidine, 101 is threonine, 105 is threonine, 108 is valine, 108 is leucine, 125 is tyrosine, 125 is arginine, 193 is threonine, 208 is glutamine, 222 is valine, 225 is arginine, 226 is leucine, 229 is serine, 242 is alanine, 242 is glycine, 247 is valine, 248 is valine, 346 is alanine, 354 is valine, 372 is tyrosine, or a combination thereof.
40. An isolated nucleic acid of claim 11, wherein the amino acid at position 44 is leucine, 77 is histidine, 101 is threonine, 105 is threonine, 108 is valine, 108 is leucine, 125 is tyrosine, 125 is arginine, 193 is threonine, 208 is glutamine, 222 is valine, 225 is arginine, 226 is leucine, 229 is serine, 242 is alanine, 242 is glycine, 247 is valine, 248 is valine, 346 is alanine, 354 is valine, 372 is tyrosine, or a combination thereof.
41. An isolated nucleic acid of claim 7, wherein the nucleic acid further comprises a detectable label.
42. A method of claim 21, further comprising modulating expression of the polypeptide.
43. A method of claim 21, further comprising isolating the polypeptide.
44. A process for amplifying a PSl/429 nucleic acid contained in a nucleic acid or a mixture of nucleic acids, which process comprises:
(a) treating the nucleic acid with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
(b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and
(c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; wherein the oligonucleotide primers are
5' TCACATCGGAAACAAAACAG and 5' ACCTGCCGGGAGTTACTGTAT.
45. An isolated nucleic acid of claim 7, which comprises a nucleotide sequence as set forth in SEQ ID NO: 1.
46. An isolated nucleic acid of claim 7, which comprises a nucleotide sequence having an amino acid sequence as set forth in SEQ ID NO:2.
47. An isolated nucleic acid of claim 7, which comprises a nucleotide sequence coding for a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, except where the amino acid at position 44 is leucine, 77 is histidine, 101 is threonine, 105 is threonine, 108 is valine, 108 is leucine, 125 is tyrosine, 125 is arginine, 193 is threonine, 208 is glutamine, 222 is valine, 225 is arginine, 226 is leucine, 229 is serine, 242 is alanine, 242 is glycine, 247 is valine, 248 is valine, 346 is alanine, 354 is valine, 372 is tyrosine, or a combination thereof.
48. An isolated nucleic acid of claim 7, which comprises a nucleotide sequence coding for a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, except where one or more positions are substituted by conservative amino acids.
49. An isolated nucleic acid of claim 7, which comprises a nucleotide sequence coding for a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, except where one or more positions are substituted by conservative amino acids and the polypeptide coded for by the nucleic acid is biologically-active .
50. An isolating nucleic acid of claim 7, which comprises a nucleotide sequence having an amino acid sequence as set forth in SEQ ID NO:2, except having 5, 10, 15, or 20 amino acid substitutions.
51. An isolating nucleic acid of claim 50, wherein the substitutions are by conservative amino acids.
52. An isolated nucleic acid comprising a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: l , with the proviso that the nucleic acid does not contain nucleotide sequence which is amplified by polymerase chain reaction using the following nucleotides:
(a) 5 ' -TCACATCGGAAACAAAACAG, 5 ' -GAGTC AC AGGGAC AAAG AGC ;
(b) 5 ' -CACCTGAGC AATACTGTACG, 5'-TCATCTTGCTCCACCACCTG;
(c) 5'-AGNAAGATGAGGAAGAAGATG, 5 '-CACACCATTGTTGAGGAGT;
(d) 5*-TCACAGAAGATACCGAGACT, 5 '-CCCAACCATAAGAAGAACAG;
(e) 5'-TCTGTACTTTTTAAGGGTTGT,
5 ' -ACTTCAGAGTAATTC ATC ANC A;
(f) 5 ' -ATCTCCGGC AGGCATATCT-3 ' ,
5 '-TGAAATCACAGCCAAGATGAG-3 ' ; (g) 5 ' -C ACCC ATTTAC AAGTTTAGC ,
5 '-GATGAGACAAGTNCCNTGAA; (h) 5'-TGGAGACTGGAACACAAC,
5 ' -GTGTGGCCAGGGTAGAGAACT; (i) 5'-GATTTAGTGGCTGTTTTGTG,
5'-ACCTCGTCCCTCAAATCT; and does not contain: 0) 5'-CCATAGCCTGTTTCGTAGC; (k) 5'-CCATAGCCTATTTCGTAGC; (1) 3 ' UTR as set forth in SEQ ID NO : 9.
SOBSimiTE SHEET (RULE 26)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU33720/97A AU3372097A (en) | 1996-06-06 | 1997-06-03 | Nucleic acids and polypeptides related to presenilin |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US65929696A | 1996-06-06 | 1996-06-06 | |
US08/659,296 | 1996-06-06 | ||
US68331596A | 1996-07-18 | 1996-07-18 | |
US08/683,315 | 1996-07-18 |
Publications (2)
Publication Number | Publication Date |
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WO1997046678A1 WO1997046678A1 (en) | 1997-12-11 |
WO1997046678A9 true WO1997046678A9 (en) | 1998-04-16 |
Family
ID=27097802
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1997/009272 WO1997046678A1 (en) | 1996-06-06 | 1997-06-03 | Nucleic acids and polypeptides related to presenilin |
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WO (1) | WO1997046678A1 (en) |
Families Citing this family (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997027296A1 (en) * | 1996-01-26 | 1997-07-31 | Hsc Research And Development Limited Partnership | Nucleic acids and proteins related to alzheimer's disease, and uses therefor |
AU3251997A (en) * | 1996-07-05 | 1998-02-02 | Governing Council Of The University Of Toronto, The | Genetic sequences and proteins related to Alzheimer's disease, and uses herefor |
FR2768346B1 (en) * | 1997-09-15 | 2002-04-19 | Fond Jean Dausset Ceph | COMPOUND FOR INHIBITION OF PRESENILINE 1 FOR THE PREPARATION OF A MEDICAMENT AND DIAGNOSTIC AGENT |
AR020329A1 (en) * | 1998-07-09 | 2002-05-08 | Boehringer Ingelheim Pharma | A SUBSTANCE, IN PARTICULAR A RIBOZIMA, IS ABLE TO INHIBIT THE PRESENILINE EXPRESSION 2, A RECOMBINATING MOLECULA OF DNA THAT CODIFY SUCH RIBOZIMA, A RECOMBINATING UNVECTOR THAT UNDERSTANDS THE CORRECTING CELL TO A RIBO BELLING ONE. |
DE19916417A1 (en) * | 1999-04-01 | 2000-10-19 | Schering Ag | New amyloid-specific aptamer, useful for diagnosis and/or treatment of e.g. Alzheimer's disease, is stabilized against nucleases |
WO2001066745A2 (en) * | 2000-03-03 | 2001-09-13 | The Salk Institute For Biological Studies | Presenilin/crk binding polypeptides (pcbp) and methods of use thereof |
DE10303974A1 (en) | 2003-01-31 | 2004-08-05 | Abbott Gmbh & Co. Kg | Amyloid β (1-42) oligomers, process for their preparation and their use |
RU2432362C2 (en) | 2005-11-30 | 2011-10-27 | Эбботт Лэборетриз | Monoclonal antibodies and applications thereof |
PL2289909T3 (en) | 2005-11-30 | 2015-04-30 | Abbvie Inc | Screening method, process for purifying of non-diffusible a-beta oligomers, selective antibodies against said non-diffusible a-beta oligomers and a process for manufacturing of said antibodies |
US8455626B2 (en) | 2006-11-30 | 2013-06-04 | Abbott Laboratories | Aβ conformer selective anti-aβ globulomer monoclonal antibodies |
EP2486928A1 (en) | 2007-02-27 | 2012-08-15 | Abbott GmbH & Co. KG | Method for the treatment of amyloidoses |
EP2558494B1 (en) | 2010-04-15 | 2018-05-23 | AbbVie Inc. | Amyloid-beta binding proteins |
CN103298833B (en) | 2010-08-14 | 2015-12-16 | Abbvie公司 | Amyloid beta associated proteins |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US5986054A (en) * | 1995-04-28 | 1999-11-16 | The Hospital For Sick Children, Hsc Research And Development Limited Partnership | Genetic sequences and proteins related to alzheimer's disease |
US5973133A (en) * | 1995-07-18 | 1999-10-26 | Washington University | Mutant S182 genes |
US6248555B1 (en) * | 1995-08-31 | 2001-06-19 | The General Hospital Corporation | Genetic alterations related to familial alzheimer's disease |
JPH10327897A (en) * | 1996-01-19 | 1998-12-15 | Washington Univ | Diagnosis and prognostic diagnosis of disease with sequence of presenilin-1 gene |
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