Note: Descriptions are shown in the official language in which they were submitted.
21267~7
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TITLE OF THE lN VL-~ 1 lON:
AGENTS FOR THE PR~vL-.llON AND TREATMENT OF
~UMAN ALZHEIMER'S DISEASE
FIELD OF THE lN V ~ lON:
The invention relates to therapeutic agents for the
prevention and treatment of human Alzheimer's Disease.
More specifically, the invention relates to a
proteinase/esterase-like protein implicated in
Alzheimer's Disease, to analogues and derivatives of
this molecule, and to nucleic acid molecules encoding
such molecules, or influencing their expression. The
invention also relates to therapeutic methods for using
such agents.
CROSS-REFERENCE TO RELATED APPLICATIONS:
This application is a continuation-in-part of U.S.
patent applications serial nos. 08/029,401 (filed on
March 4, 1993) and 08/115,013 (filed on September 1,
1993).
RA~KG~OUND OF THE lNvL~ ON:
Alzheimer's disease ("AD") is a progressive disease
of the human central nervous system. It is manifested
by dementia in the elderly, by disorientation, loss of
memory, difficulty with language, calculation, or
visual-spacial skills, and by psychiatric
manifestations. It is associated with degenerating
neurons in several regions of the brain. Alzheimer's
disease is reviewed by Price, D.L. et al. (Clin.
Neuropharm. 14:S9-S14 (1991)); Pollwein, P. et al.
2126787
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(Nucl. Acids Res. 20:63-68 (1992)); Regland, B. et al.
(Med. Hypoth. 38:11-19 (1992)) and Johnson, S.A. (In:
Review of Biological Research in Aging, Vol. 4.,
Rothstein, M. (Ed.), Wiley-Liss, NY, 163-170 (1990)).
I. Amyloid Protein
Pathologically, the disease is recognized by the
presence of intracellular tangles, and extracellular
deposits or "plaques" of amyloid protein ("AP") in the
neuropil and in blood vessels. The principal component
of the amyloid protein plaques is a 40 amino acid
protein known as the ~/A4 amyloid protein (Price, D.L.
et al., Clin. Neuropharm. I4:S9-S14 (1991)). This
protein forms fibrils, which are concentrated in amyloid
deposits in the extracellular space of the brain
parenchyma and in the vascular elements of the brain and
the pia-arachnoid (Currie, J.R. et al., J. Neurosci.
Res. 30:687-689 (1991)). All cases of Alzheimer's
disease show such deposition of amyloid in brain
parenchyma.
The ~/A4 amyloid protein is a 4 kD protein fragment
whose sequence is contained within a family of larger
proteins, which are alternatively spliced transcripts of
a single gene known as Alzheimer ~-amyloid peptide
precursor (APP) (Podlisny, M.B. et al., Science 238:669-
671 (1987); Currie, J.R. et al., J. Neurosci. Res.
30:687-689 (1991)). This precursor is normally cleaved
to form the ~-amyloid protein ("AP"). The isolation of
cDNA encoding the ~/A4 protein has led to the recent
recognition that the protein is, in fact, synthesized as
part of APP, and that ~/A4 protein comprises an abnormal
form of AP (Zain, S.B. et al., Proc. Natl. Acad. Sci.
(U.S.A.) 85:929-933 (1988); Vitek, M.P. et al., Molec.
Brain Res. 4:121-131 (1988); Johnson, S.A. (In: Review
of Biological Research in Aging, Vol. 4., Rothstein, M.
(Ed.), Wiley-Liss, NY, 163-170 (1990)).
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The APP gene is located on chromosome 21 and is
preferentially expressed in the neuronal cells of the
central nervous system. In addition to alternate
splicing programs, the different isoforms of APP are
generated by proteolytic cleavage of the translational
products (Pollwein, P. et al. (Nucl. Acids Res. 20:63-68
(1992); Price, D.L. et al., Clin. Neuropharm. 14:S9-S14
( 19 9 1 ) ) .
Although the accumulation of ~/A4 protein in
Alzheimer's disease is believed to result from the
faulty processing of one or more of the APP isoforms
(Currie, J.R. et al., J. Neurosci. Res. 30:687-689
(1991)), the exact mechanism of ~/A4 protein formation
is not yet known (see, Johnson, S.A. (In: Review of
Biological Research in Aging, Vol. 4., Rothstein, M.
(Ed.), Wiley-Liss, NY, 163-170 (1990); Roch, J.M. et
al., J. Biol. Chem.267:2214-2221 (1992)).
II. Apolipoprotein E
The E4 allele of apoliprotein apoE4 has been associated
with late onset familial and sporadic Alzheimer's
disease (Saunders, A.M. et al., Neurology 43: 1467-1472
(1993)), (Namba, Y., et al. Brain Res. 514 163-166
(1991)). The apoE gene is located on chromosome 19 and
is widely experessed, but is highest concentrated in
brain tissue. Apolipoproteins are implicated in other
serious human diseases involving faulty lipid
metabolism, e.g., cardiovascular diseases.
Type V hyperlipoproteinemia (HLP) is characterized
clinically by hepatosplenomegaly, occasional eruptive
xanthomas and an increased incidence of pancreatitis
(Ghiselli, G., et al., Lancet 2:405-407 (1982)). These
patients have striking hypertriglyceridemia due to
increased plasma chylomicron and very low density
lipoprotein concentrations in the fasting state, without
212~7~7
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a deficiency of lipoprotein lipase or its activator
protein, apolipoprotein (apo) C-II.
Apolipoprotein E (apoE), a protein constituent of
triglyceride-rich lipoproteins, has been implicated in
the receptor-mediated hepatic uptake of these particles
((Ghiselli, G., et al., Lancet 2:405-407 (1982)). The
protein is important in modulating the catabolism of
remnants of triglyceride-rich lipoprotein particles
(Gregg, R.E. et al., J. Clin. Invest. 78:815-821
(1986)), in particular, by facilitating the plasma
transport of cholesterol (Lalazar, A. et al., J. Biol.
Chem. 263:3542-3545 (1988)). It is a polymorphic
protein with the three common alleles coding for apoE2,
apoE3, and apoE4 (Utermann, G. et al., J. Lipid Res.
25:378-382 (1984); Utermann, G., J. Inher. Metab. Dis.
11 :74_86 (1988); Ghiselli, G., et al., Lancet 2:405-407
(1982)). In addition to these major isoforms of apoE,
minor variant isoforms (apoE1, apoE5, and apoE7) have
been detected by isoelectric focusing (Maeda, H. et al.,
J. Biochem. (Tokyo) 105:51-54 (1989)).
ApoE3 is considered the normal isoform. The apoE2
variant possesses a Cys substituted for an Arg at
residue 158 in its amino acid sequence. The apoE4
variant possesses an Arg substituted for Cys at residue
112 in its amino acid sequence, and lacks cysteine
residues (Weisgraber. K.H., J. Lipid Res. 31:1503-1512
(1990); Utermann, G. et al., J. Lipid Res. 25:378-382
(1984)).
Apolipoprotein (apo) E polymorphism has a
significant effect on plasma cholesterol and low density
lipoprotein cholesterol concentrations (Wardell, M.R. et
al., J. Lipid. Res. 32:521-528 (1991)). The common
variants, apoE2 and apoE4 have a significant impact on
interindividual variation of lipid and lipoprotein
levels in normal subjects. The common variant apoE2 and
more than half a dozen rare variants are defective in
binding to the low-density lipoprotein (LDL) receptor,
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and all are causally associated with the lipid
disorder, type III hyperlipoproteinemia (HLP). The mode
of inheritance of the disorder can be either dominant or
recessive, depending on the particular mutation(s) in
apoE, although the mechanisms involved are not fully
understood (Rall, S.C. et al., J. Intern. Med. 231:653-
659 (1992)). The apoE4 isoform is associated both with
HPL and type V HPL (Gregg, R.E. et al., J. Clin. Invest.
78:815-821 (1986); Kuusi, T. et al., J. Lipid Res.
29293-298 (1988); Yanagi, H. et al., Clin. Genet.
38:264-269 (1990)). Diabetic patients with the ApoE4
allele appear to be more susceptible to HPL than
diabetic patients with other alleles (Eto., M. et al.,
Diabetes 361301-1306 (1987)). In contrast, diabetic
patients with apoE2 were characterized by increased
levels of plasma triglyceride, total cholesterol, very
low density lipoprotein (VLDL)-chol, and apoE and an
increased VLDL-chol/VDLD-triglyceride ratio, i.e. the
accumulation of remnants.
In addition, fasting plasma glucose and hemoglobin-
A1 levels were significantly higher in
hyperlipoproteinemic diabetic patients with apoE2 than
in normolipidemic diabetic patients with apoE2 (Eto, M.
et al., J. Clin. Endocrinol. Metab. 69:1207-1212
(1989)). Indeed, homozygosity for ApoE2 invariably gives
rise to dysbetalipoproteinemia, and when associated with
obesity or a gene for hyperlipidemia, results in type
III hyperlipoproteinemia (Wardell, M.R. et al., J. Lipid
Res. 31:535-544 (1990)). Thus, diabetes appears to
predispose individuals having the ApoE2 allele to
hyperlipoproteinemia (particularly type III) and may be
a factor linking diabetes with hyperlipoproteinemia and
cardiovascular disease (Eto, M. et al., J. Clin.
Endocrinol. Metab. 69:1207-1212 (1989)).
Gene sequences that encodes several of the ApoE
isoforms has been cloned (Gill, L.L. et al., Biochem.
Biophys. Res. Commun. 130:1261-1266 (1985); Maeda, H. et
212S~87
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al., J. Biochem. (Tokyo) 105:491-493 (1989); Horie, Y.
et al., J. Biol. Chem. 267:1962-1968 (1992)).
III c-fos
The majority of early onset Alzheimer cases, are related
to a genetic lesion (mutation) in the 14q.24 region of
chromosome # 14, (St. George-Hyslop, P, et al. Nature
Genet. 2: 330-334 (1992)). The gene encoding C-Fos
protooncogene is located in the "center" of this region,
and abnormal expression of C-Fos has been documented in
the hippocampus of Alzheimer's victims, (Zangh. P., et
al., Neuroscience 46: 9-21 (1992)). The C-Fos protein
has been shown to activate the APP gene promoter;
however, but C-Fos does not display evidence of allelic
association with familial Alzheiner's disease,(FAD) and,
although it has been concluded that the protein coding
region of the C-Fos gene is not the site of FAD mutation
on chromosome #14 the possibility exist that mutations
in non coding regions of the of the C-Fos gene, which
might effect tissue specific expression of C-Fos, could
be the FAD mutation site, (Rogarve E.I., et al.,
Neurology 43: 2275-2279 (1993)). This invention
additionally relates to the discovery of a gene "C-
Fos5L" which is 100~ homologous to the anti sense strandof a region of the 5' untranslated region of C-Fos
protooncogene from nl35-n792 inclusive. The protein
encoded by C-Fos5L, "C-Fos5Lp" is a neuropeptide
hormone-like molecule made up of 121 amino acids which
has all the required biological characteristic for
combining with pac and apoE4Lx2 to cause the full range
of neuropathological symptoms of human Alzheimer's
disease. The neuropeptide harbours two amidation sites
and a secretory signal which can be used to produce four
distinct, activated, unique neuropepides. In addition C-
Fos5Lp, by similarity, is a trans-acting transcriptional
activator which can mimic or modulate the activity of a
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number of transcription factors including; the SRF
factor, REV protein, ERB-1, nuclear factor NF-1, DNA
binding protein UL42, transforming proteig INF 3, and
the androgen receptor transcriptional transactviting
region; it can also modulate activity or mimic, the
growth arrest specific protein, sperm histone protamine,
the acetylcholine receptor protein, fibrillanin,
tenacsin, a cysteine protease, the serotonin receptor,
the paired amphipathic helix protein, and two
serine/threonine protein kinases. In addition amino
acids (aa) 91 - 118 in the c terminal region of C-Fos5Lp
has significant, unique homology to aa 152 - 166 and 231
- 243 in apoE4; aa 40-59 and aa 102-121 shares homology
with regions of apoeLx2.
Without some type of effective treatment,
Alzheimer's disease would probably affect one out of
every 10 humans alive today. To date there is no
treatment for Alzheimer's disease at any stage of its
development. Two therapeutic reagents, Cognex and
Menthane, appear to give slight relief to some victims
but do not alter the course of the disease.
In view of the importance of diagnosing,
predicting, and treating Alzheimer's disease and
hyperlipoproteinemia and cardiovascular disease, an
affective means for achieving these goals would be
highly desirable. The present invention supplies such
means.
BRIEF DESCRIPTION OF THE FIGURES:
Figure lA shows the protein coding sequence of
"Pac" cDNA and the deduced sequence of the protein is
shown in Figure lB. "Tpac" cDNA is reconstructed from
negative strands of exons and from introns in the
amyloid precursor related human APRP gene. The protein
212S7~7
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coding sequence of the cDNA is shown in Figure lC and
the deduced sequence of the protein in the embodiment of
Figure lD.
Figure 2 shows the sequence of cDNA molecules. The
protein coding sequence of "hsaN" cDNA is shown in (A)
and the deduced sequence of the protein is shown in (B).
Figure 3A shows the nucleotide sequence of cDNA
encoding the apoE4L protein. Figure 3B shows the amino
acid sequence of the protein. In Figure 3B, raised "p"
indicates kinase c phosphorylation sites; "pck"
identifies casein kinase phosphorylation sites; a down
arrow indicates the location of a cleavage/secretory
site. Double underscoring is used to denote sequences
with significant identity and homology to apoE4.
Figure 4A shows the nucleotide sequence of cDNA
encoding the apoE4L1 protein. Figure 4B shows the amino
acid sequence of the protein. In Figure 4B, the raised
"p" indicates kinase c phosphorylation sites; a down
arrow indicates the location of a cleavage/secretory
site.
Figure 5 shows the upstream (5') regions that are
responsible for the transcription of the ApoE4L/Ll/Lx2
proteins.
Figure 6A shows the nucleotide sequence of cDNA
encoding the apoE4Lx2 protein. Figure 6B shows the
amino acid sequence of the protein. In Figure 6B, the
raised "p" indicates kinase c phosphorylation sites; a
down arrow indicates the location of a
cleavage/secretory site. The amino acids that join the
E4L protein to the E4L1 protein are underlined. Double
underscoring is used to denote sequences with
significant identity and homology to apoE4.
Figure 7 shows the regulatory region (Pac/reg) in
the 5 'upstream region of pac. This sequence contain
five promoter elements (three individual and two tandem)
correlated with "cap" sites. These promoters program the
2126787
g
transcription of the Pac family of mRNA's, probably in a
tissue specific manner.
Figure 8A shows the nucleotide sequence of the cDNA
encoding C-Fos5Lp; figure 5B shows the sequence of the
C-Fos5Lp neuropeptide, glycine residues (G) at which
amidation/activation can occur have a NH2 above, the
downwards pointing arrow indicates the secretory signal.
Figure 8C shows the 5' upstream regulatory region of C-
Fos5L, "C-Fos5Lreg" the "TATA box" promoter element is
underlined, the correlated "cap site" is overlined.
SUMMARY OF THE lNvL-.llON:
The invention concerns agents and methods for the
diagnosis and therapy of Alzheimer's disease and related
conditions, as well as hyperlipoproteinemia and
associated cardiovascular disease. Such agents include
a proteinase/esterase-like protein implicated in
Alzheimer's Disease, as well as analogues and
derivatives of this molecule, and nucleic acid molecules
encoding such molecules, or influencing their
expression.
In detail, the invention provides a nucleic acid
molecule, substantially free of natural cont~m;n~nts,
that encodes a protein selected from the group
consisting of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2,
hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-Fos5L. In
particular, the invention provides the above-described
nucleic acid molecule wherein the sequence is SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:12 or SEQ ID NO:22
The invention also provides a protein,
substantially free of natural cont~m;n~nts, selected
from the group consisting of Pac, Pac-1, Pac-2, Pace,
Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-
Fos5Lp. In particular, the invention provides the
above-described protein having a sequence of SEQ ID
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NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:13 os SEQ ID NO:23.
The invention also provides a reagent capable of
diagnosing the presence of a molecule selected from the
group consisting of Pac, a Pac-encoding nucleic acid
molecule, Pac-1, a Pac-1-encoding nucleic acid molecule,
Pac-2, a Pac-2-encoding nucleic acid molecule, Pace, a
Pace-encoding nucleic acid molecule, Tpac, a Tpac-
encoding nucleic acid molecule, Tpac-2, a Tpac-encoding
nucleic acid molecule, hsaP, an hsaP-encoding nucleic
acid, ApoE4L, an ApoE4L-encoding molecule, ApoE4L1, an
ApoE4L1-encoding molecule, ApoE4Lx2, an ApoE4Lx2-
encoding molecule and an C-Foc5Lp encoding molecule.
The invention particularly concerns the embodiments
wherein the reagent is a nucleic acid molecule,
(especially a ribozyme produced from nucleic acid
molecules having a sequence of SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12.or
SEQ ID NO:22, or a nucleic acid molecule obtainable by
mutating a nucleic acid molecule having a sequence of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:12, SEQ ID NO:21, SEQ ID NO: 22 or
SEQ ID NO:24) or a protein (especially an antibody, or a
fragment of an antibody, which is capable of binding to
Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L,
ApoE4L1, ApoE4Lx2 and C-Fos5Lp).
The invention also provides a method of treating
Alzheimer's disease, Down's Syndrome, Parkinson's
Disease Schizophrenia, hyperlipoproteinemia, or other
cardiovascular disease which comprises providing to an
individual, in need of such treatment, an effective
amount of an inhibitor of Pac, Pac-1, Pac-2, Pace, Tpac,
Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp.
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DESCRIPTION OF THE PREFERRED EMBODIMENTS:
Although it has been recognized that Alzheimer's
disease reflects the abnormal processing of APP to
produce ~/A4 protein, the exact mechanism of this
processing has not been elucidated. Studies have
demonstrated that at least 3 different forms of APP are
produced in neuronal cells (Roch, J.M. et al., J. Biol.
Chem. 267:2214-2221 (1992)). Two of these forms (having
751 and 770 amino acids) contain a domain that has
substantial homology to Kunitz-type protease inhibitors.
Normally, APP is processed to form AP throughout
life, however, in Alzheimer's disease, an abnormal AP --
~/A4 protein -- which is longer at the C-terminal by 2
or 3 amino acids is expressed. The deposition of this
abnormal protein in plaques coupled with a constant
destruction of acetylcholine in neurons cause the
dementia and other degenerative symptoms that
characterize the advanced stages of the disease.
Researchers have proposed that APP is a cell
surface receptor or a transmembrane protein, in which
the ~/A4 domain is partly embedded in the cell membrane.
The secretion of the ~/A4 protein thus reflects the
cleavage of the domain from the precursor molecule (see,
Roch, J.M. et al., J. Biol. Chem. 267:2214-2221 (1992)).
Under normal physiological conditions, the proteolysis
of APP into AP is believed to prevent the formation of
~/A4 protein. In sum, the consensus of all experimental
evidence suggests that the ~/A4 variant of AP protein
plays a major role in the expression of Alzheimer's
disease, and that the ~/A4 protein is produced via
proteolysis. The identity of the protease responsible
for ~/A4 formation has hitherto been unknown.
The present invention derives, in part, from the
discovery of a protein with proteinase and
acetylcholinesterase domains that catalyzes the
formation of the ~/A4 protein. The protease is encoded
2126 7~7
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by a copy of the antisense strand of the APP gene. The
present invention also derives in part from the
discovery of a truncated, modified version of the
protease that is encoded on the antisense strand of a
gene -- the human APRP gene -- that is structurally and
functionally related to APP. The present invention
provides the sequence of these molecules, as well as
that of the cDNA that encodes them. These molecules may
be used in the diagnosis, prediction and treatment of
Alzheimer's disease.
The present invention also derives, in part, from
the recognition of the role of the ApoE gene in human
Alzheimer's disease, and in cardiovascular disease, such
as hyperlipoproteinemia. In particular, the invention
relates to the discovery two tandem open reading frames
("orfs") on the antisense strand of ApoE4 gene. The
first orf, designated herein as "ApoE4L" or "E4L,"
commences 82 base pairs (bp) upstream from the ApoE4
stop translation signal (nucleotide 4403 on the coding
strand) and terminates with a TAG codon at nucleotide
3817. The second orf, designated herein as "ApoE4Ll" or
"E4Ll," is located within an exon, and encodes a
putative 198 amino acid protein. Because the two orfs
are in the same reading frame, a mutation that
obliterates the termination codon of E4L results in the
creation of a fusion protein, designated herein as
"ApoE4Lx2" or "E4Lx2."
Alternate splicing occurs in many eukaryotic genes,
and since different spliced varieties of mRNAs are found
in different cell types, it is believed that splicing is
tissue specific and may be a method of local
environmental control over the function of some gene(s).
The present invention further concerns gene sequences
produced via such alternate splicing mechanisms, and the
proteins that such gene sequences encode.
The common factor in all human Alzheimer's disease
is the formation of ~ amyloid containing plaques which
212~ 787
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indicated that all mechanisms causing Alzheimer's must
involv the abnormal formation of ~ amyloid protein and
hence the APP gene. The family of pac proteins have all
the biological properties required for the full
phenotype of Alzheimer's. Hence it is logical to
conclude that the disease is caused by the overt
expression of these proteins which are expressed in
Alzhemier's victims but not at significant or detectable
levels in healthy humans. ApoE4Lx2 has the potential to
influence the expression of pac and pac-isoforms and C-
Fos5Lp can influence the expression of apoE4Lx2 and also
mimic the protamine, acetylcholine inhibition (pac by
destruction of acetylcholine, C-Fos5Lp by blocking
interaction with acetylcholine receptor), and the
lysosomal protease activity of pac. Silent mutations
like the T-C mutation in exon 2 of C-Fos gene, although
insignificant to C-Fos expression can activate
expression of the anti-sense gene which, from what we
know of other systems we have discovered, is sometimes
under subtile repression by the sense gene. Therefore,
although mutations have not been found, or looked for,
in region of C-Fos gene which encodes C-Fos5L, it
appears that interaction between C-Fos5Lp, apoE4Lx2 gene
and APP gene, as well as specific mutation in the APP
gene can account for all aspects of human Alzheimer's
disease.
I. The Molecules of the Present Invention
The molecules of the present invention comprise
Pac, Tpac, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 and C-FosL5p,
as well as the analogs of these proteins, and gene
sequences that encode them. The molecules of the
invention also include the novel set of gene sequences
and encoded proteins (Pac-1, Pac-2, Pace, Tpac-1) that
result from the alternative splicing of the mRNA
antisense to the amyloid forming APP gene. These
2126 7~7
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molecules of the present invention are described below
in greater detail.
A. The Pac Protease
The negative strand of exons and introns in the
human APP gene was evaluated in a search for the
putative Alzheimer's disease protease. This evaluation
led to the recognition that the APP antisense strand
encoded a protein -- designated herein as "Pac." The
protein-encoding portion of the Pac cDNA sequence is
shown in Figure lA (SEQ ID NO:l) and the deduced
sequence of the protein in Figure lB (SEQ ID NO:2). The
cDNA encoding "Pac" is 100% homologous to a region of
the negative strand of the human APP mRNA, starting at
amino acid 7 within the AD protein. The molecular
weight of the Pac proteinase/esterase was found to be
12,040 kd, and to comprise 104 amino acid residues. The
protein is predicted to be unstable.
B. The Tpac Protease
In a similar manner to that described above, the
introns and exons of the antisense strand of the amyloid
precursor-related gene ("APRP") was evaluated, and found
to encode a protein designated herein as "Tpac." The
protein-encoding portion of the Tpac cDNA sequence is
shown in Figure lC (SEQ ID NO:3) and the deduced amino
acid sequence of the protein is shown in Figure lD (SEQ
ID NO:4). The mRNA encoding ~Tpac~ is 100% homologous
with a region of the negative strand of APRP mRNA which
traverses a portion of the AD protein, the protease
inhibitor domain and an intron of APRP protein.
C. The Acidic Activator Protein, hsaP
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A number of patients from families with inherited
Alzheimer disease exhibit mutations on chromosome #14 in
the region of the major heat shock 70 kb protein
(hsp70). We have constructed a cDNA (hsaN) shown in (A)
of Figure 2 which is 100~ homologous to nucleotides 313
to 471 inclusive on the antisense strand of the human
APP gene (SEQ ID NO:5). The cDNA encodes a 5.45 kD, 51
amino acid, intracellular, acidic activator protein
(hsaP), shown in (B) of Figure 2 (SEQ ID NO:6). hsaP
shares ~75~ homology with a 45 amino acid domain of
hsp70 in which 16 of 18 possible hydrophobic amino acid
residues match, strongly indicating that hsaP can
interact specifically with hsp70.
Under normal physiological conditions, the sense/
antisense relationship between APP and hsaP will not
allow the expression of hsaP in healthy humans; however,
acquired mutations in regions of the APP gene involved
in antisense repression of hsaP will allow hsaP to be
expressed. This free, intracellular, hsaP is
"neutralized" through interaction with wild type hsp70,
as is the case with some other undesirable intracellular
proteins.
Mutations in any of several dozen genes which share
the hsp70 region in chromosome #1 can be the cause of
inherited Alzheimer's disease. The role of hsp70 in the
disease may be caused by inherited mutations that reside
in the region of the gene and that are critical for
interaction with expressed hsaP. Thus, the pathophysio-
logical symptoms of Alzheimer's disease in these
families may be the result of the inherited mutation on
chromosome #14 and an acquired mutation on APP.
Alternatively, either hsa or pac may be an Alzheimer's
locus on human chromosome #14.
Nevertheless, regardless of the mechanisms of
intracellular interaction between hsaP and hsp70,
humanized antibodies prepared against hsaP, or ribozymes
prepared from selected regions of the APP gene against
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hsaN, will safely and efficiently prevent and cure
Alzheimer's disease in such families.
D. Comparison between Pac and Tpac
It was observed, from similarities, that the
deduced protein encoded by Pac and Tpac are highly
related, and that both are related to lysosomal membrane
proteins and have distinct domains. Three domains
~Pase" (endo-proteinase); ~Acase" (acetylcholinesterase)
and "Ease" (esterase) are indicated in Figure lB. A
domain "Btox" for botulism toxin (a zinc metalloprotease
which blocks neurotransmitter release form neurons) was
found to partially overlap the "Ease" domain.
The deduced protein encoded by "Tpac" is a
truncated form of "Pac." The protein was found to be
truncated at amino acid #23 and four new amino acids --
CITS --- were found to have been added to the truncated
c-terminal end of the protein. It comprises only the
proteinase domain of "Pac." This domain has significant
identity and homology with domains in, viper Russelli
proteinase, human cathepsin A, human complement c3
convertase, RNA 1 polyprotein protease cofactor, and a
species of yeast killer toxin with carboxypeptidase-like
activity. In addition "Tpac" has strong similarity and
homology to the powerful, 50 amino acid, testes DNA
binding "protamine Z1" protein. The latter protein
forms highly condensed chromatin packets consisting of
membranes and chromosomal DNA in sperm heads.
E. Mechanisms of Pac and Tpac Action
The factors responsible for inducing the expression
of "Pac" and "Tpac" have not been fully identified, but
such information is unnecessary for the purposes of the
present invention. Presumably, these proteins are not
normally expressed in healthy humans because the mRNA
2126 787
encoding them is in an antisense relationship with
APP/APRP mRNA. Without limitation, there are several
ways in which ~'Pac" may be involved as the causative
factor in Alzheimer's disease. The 12.4 kd native Pac
protein or two or more similar subunits, acting
together, may actively cut out the abnormal ~/A4 protein
from APP and actively destroy acetylcholine.
Alternatively, the 12.4 kd native Pac protein may
associate with other dissimilar protein subunits to form
a polyprotein which can mediate such enzymatic
activities. In another alternative, the 12.4 kd protein
acting may act as an activator of inactive proteinase
and acetylcholinesterase zymogens to form active enzyme
molecules which can carry out the enzymatic activity.
There are several ways in which "Tpac" may be
involved as a causative agent in Alzheimer's disease.
The 27 amino acid protein which lacks an
acetylcholinesterase domain, can function. Tpac may act
as a transducer of protease activity in an inactive
proteinase. In analogy to the action of its homologue,
~protamine Z1," Tpac may bind to chromosomal DNA and
influence the expression of critical genes. Tpac may
alternatively compact the relatively insoluble abnormal
AD protein, together with neural cell chromatin and
dendrites, into the ~'intracellular neurofibrillary
tangles" found in advanced stages of Alzheimer's
disease.
Irrespective of the manner of the involvement of
the "Pac" and "Tpac" in Alzheimer's disease any
therapeutic method which blocks the expression of these
proteins or blocks the proteins will significantly
contribute towards the control of the disease. The
diagnostic and/or therapeutic reagents of the invention
additionally include fusion proteins, antipeptide
reagents, etc., made from the deduced amino acid
sequences described in the embodiment of Figures lB and
lD. The invention also includes any cellular proteinase
~12G 787
-18-
or acetylcholinesterase activated by the proteins or
domains of the proteins described in the embodiment of
Figures lB and lD.
F. The ApoE4L Protein
The first orf identified in the ApoE antisense
sequence encodes the apoE4L protein. The nucleotide
sequence of ApoE4L cDNA is shown in Figure 3A (SEQ ID
NO:7). The apoE4L protein sequence is shown in Figure
3B (SEQ ID NO:8). Of particular interest is the
sequence 89-104 of SEQ ID NO:8, which possesses
significant identity and homology to apoE4 sequences.
Kinase c phosphorylation sites are present at positions
3, 42, 43, 54, 87, 112, 122, and 129 of SEQ ID NO:8.
Casein kinase phosphorylation sites are present at 176-
177 and 193-194 of SEQ ID NO:8. Cleavage/secretory
sites occur at positions 43-44 and 182-183.
G. The ApoE4Ll Protein
The second orf identified in the ApoE antisense
sequence encodes the apoE4L1 protein. The second orf
commences 16 bp downstream from E4L stop codon,
nucleotide 3801, in the same reading frame, and
terminates within the adjoining intron at nucleotide
3558. This second orf encodes a putative 107 amino acid
long protein designated herein as "E4L1". Only 21 bp of
exon sequence is included in the E4L1 sequence. The
nucleotide sequence of cDNA encoding the apoE4L1 protein
is shown in Figure 4A (SEQ ID NO:9).
The apoE4L1 protein sequence is shown in Figure 4B
(SEQ ID NO:10). A kinase c phosphorylation site is
present at position 54 of SEQ ID NO:10. A
cleavage/secretory site occurs at positions 46-47.
212G 787
_
-19 -
H. Transcriptional Regulation of the ApoE4L and
ApoE4L1 Gene Sequences
Both the Apo4L and Apo4L1 orf's can be transcribed
from promoter elements and coordinated GC box and cap
sites that are located 300-500 bp upstream from the
E4L/Ll orf. The sequence of this upstream regulatory
region is SEQ ID NO: 11 (Figure 5). Promoter elements
are present at positions 12-26 and 322-335 in SEQ ID
NO:11. A GC box is present at position 206-219. Cap
sites are present at positions 55 and 338 in SEQ ID
NO:11. A ribosome binding site is present 5 nucleotides
from the end of the sequence. The sequence "TATAAA"
located 100 bp downstream of the E4L1 stop codon might
be used as a transcription termination signal, however,
there is a perfect consensus termination signal at
nucleotide 2495 which may alternatively signal the end
of the putative antisense prime transcript.
I. The ApoE4Lx2 Protein
Despite the allelic differences of ApoE2, ApoE3 and
ApoE4, the antisense molecules encoded by each of these
alleles still encodes the E4L and E4L1 proteins; however
ApoE4 comprises two alleles. In one form the stop codon
(TAG) which closes E4L is replaced by GCT. As a result
of this mutation, the E4L and E4L1 combine with the
addition of six new codons to form a hybrid orf,
designated herein as "E4Lx2" which has coding
information for 311 amino acids. The nucleotide
sequence that encodes E4Lx2 is presented in Figure 6A
(SEQ ID NO:12); the six new codons are underlined.
The amino acid sequence of E4Lx2 (SEQ ID NO: 13) is
shown in Figure 6B. Of particular interest is the
sequence 89-104 of SEQ ID NO:13, which possesses
significant identity and homology to apoE4 sequences.
Kinase c phosphorylation sites are present at positions
2126787
-20-
3, 42, 43, 54, 87, 122, 129, 152 and 303 of SEQ ID
NO:13. Casein kinase phosphorylation sites are present
at 176-177 and 193-194 of SEQ ID NO:13.
Cleavage/secretory sites occur at positions 35-36, 43-44
and 182-183 and 295-296. A glycosylation site is
present at position 202.
The E4L protein is extremely basic (pI = 12.57,
charge at pH 7.0 = 38.34, MW= 22 kD, 9 potential
phosphorylation sites). It appears to be a DNA binding,
trans-activating transcriptional activator-type protein
with the structural characteristics of powerful
protamines. There are three secretory sites which could
provide four different biologically "active"
polypeptides. The E4L1 protein is a basic protein (pI
11.09, MW = 11.8 kD, one potential phosphorylation
site); it appears to be a DNA binding protein and may
also act as a coupled G-receptor signal transducer. The
E4Lx2 protein has the combined primary characteristics
of the E4L protein as well as those of the E4L1 protein,
plus a potential glycosylation site provided by the
codons linking the two proteins.
The E4Lx2 protein has several additional
potentially functional characteristics including
significant identity and homology with the herpes
associated "infected cell protein," and potential
protease activity. The primary structure of E4Lx2
appears to make this protein more likely than apo4E to
be responsible for the role in growth and repair of the
nervous system during development and injury ascribed to
apoE4; and, also, for the tight binding to the ~-amyloid
protein -- which, as indicated above is the central
player in the pathophysiology of human Alzheimer's
disease.
In view of the involvement of apolipoprotein in
Alzheimer's disease, it is therefore likely that E4Lx2,
rather than mutations in the ApoE4 protein, is
implicated in causing or contributing to late-onset
21~6787
-21-
familial Alzheimer's disease. Moreover, E4L and E4L1
appear to be involved in other diseases associated with
ApoE2, ApoE3, and ApoE4 -- such as hyperlipoproteinemia
and related cardiovascular conditions.
II. The C-Fos5Lp protein
The C-Fos5Lp protein is a trans acting transcriptional
activator which can function as a family of activated
neuro peptides with a very wide range of potential
biological activites, many of which are highly relevant
to the neuropathological symptoms of Alzheimer's
disease. In addition, like pac, C-Fos5Lp can function in
a tissue specific manner depending on wheather none, one
or both of the potential amidation sites are amidated,
and on wheather the secretory signal is used or not used
in a specific tissue type. Most significant, however is
the unique structural relationship in the c-terminal
domain of C-Fos5Lp with domains in apoE4Lx2, and apoE4,
which suggest a very specific interaction between these
proteins which is highly relevant to the role they play
in the etiology of human Alzheimer's disease.
J. Molecules resulting from
Alternative/Differential Splicing
As indicated above, alternate splicing occurs in
many eukaryotic genes, and since different spliced
varieties of mRNAs are found in different cell types, it
is believed that splicing is tissue specific and may be
a method of local environmental control over the
function of some gene(s). The present invention further
concerns gene sequences produced via such alternate
splicing mechanisms, and the proteins that such gene
sequences encode.
212~787
-22-
Pac/reg contains five promoter systems each
consisting of a "TATA box" (underlined in figure #7)
and correlated "cap site"(overlined in figure #7). These
systems are located between -455/-418, -420/-369, -389/-
343(two tandem promoters sharing a single correlated cap
site) , and -329/-286 and program the transcription of
Pac mRNA. Each promoter might power transcription of an
alternate spliced mRNA in a tissue specific manner. Pac
mRNA also harbours three potential transcription
termination sites in the 3' downstream untranslated
region located at + 337, + 372 and + 637 relative to the
Pac "orf" stop translation codon. Differentially mRNA's
might use different transcription termination signals.
The spliced DNA and encoded proteins described
here are probably involved in the neuropathology of
Alzheimer's disease. Alternate splicing of the mRNA
antisense to the amyloid forming APP gene, gives rise to
a set of novel gene sequences and encoded proteins (Pac-
1, Pac-2, Pace, Tpac-1) whose sequences are presented
below.
The above-described Tpac molecule plays a central
role in the formation of the amyloid plaques that are
characteristic of Alzheimer's disease. In Pac-1 the
separate domains for protease/protamine and for
acetylcholinesterase associated activity is unchanged
and the protein's changes in amino acid composition
occur only in the "center" domain of the protein which
has not been associated with any known activity in pac.
In pac-2, Tpac is unchanged and can be independently
expressed, a different protein ("Pace") is in the
acetylcholinesterase domain, and a neuropeptide-type
protein, Tpac-2, is encoded in the central domain. Both
Pac-1 and Pac-2 have potential mitochondrial transit
sequences.
The sequences of cDNA molecules Pac-1 and Pac-2 are
as follows:
2126787
-23-
Pac-1 (SEQ ID NO:14):
ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
TATTTGTCAACCCAGAACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCA
GGGCGGGCATCAACAGGCTCAACTGGGCACAGGAAGCAAGGGACACACAA
AGCAAACAAGACAAATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGAC
TCTTACCTTCGTTTTCTGTGTTGGCTGGCACAGAGTCAGCCCCAAAAGAA
TGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCATTCACGGGAAGGAG
CTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCATGAGAGCATCGTTTC
CGTAACTGATGGTTGGTTCACTAATCATGTTGGCCAAGACGTCATCTGA
Pac-2 (SEQ ID NO:15):
ATGTCGGAATTCTGCATCCATCTTCACTTCAGAGATCTCCTCCGTCTTGA
TATTTGTCAACCCAGAACCTGTATTACATCATAATTAAAGGGTCACTTCA
AATTCTACTCTGCAGTAAGATCAATTGAGAGAGGCTTAAAATGCAGAAAG
GAGACAACGTCTGCTCGAGCTTAGGCCCAAGATGCGGAGAGGCAGAAGTC
AAGCGGTTGTGATACCTGGTCGAGTGGTCAGTCCTCGGTCGGCAGCAGGG
CGGGCATCAAGATGGAGAACGCCCTTGCTGGCTCAGGGGACTCTTACCTT
CGTTTTCTGTGTTGGCTGGCACGAGTCAGCCCCAAAAGAATGCCACGAGT
CAGCCCCAAAAGAATGCCACGGCTGGAGATCGTCCAGGCTGAACTCTCCA
TTCACGGGAAGGAGCTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGCAT
GAGAGCATCGTTTCCGTAACTGATGGTTGGTTCACTAATCATGTTGGCCA
AGACGTCATCTGA
The cDNA sequence of Pace is as follows
(SEQ ID NO:16):
ATGCGGAGAGGCACAAGTCAAGCGGTTCTGATACCTGGTCGAGTGGTCAG
TCCTCGGTCGGCAGCAGGGCGGGCATCAACAGGCTCAAGTGGGCACAGGA
AGCAAGGGACACAGAAAGCAAACAAGACAAATCAAGATGGAGAACGCCCT
TGCTGGCTCAGGGGACTCTTACCTTCGAAAT~l~l~llGGCTGGCACAGA
GTCAGCCCCAAAAGAATGCCACGGCTGGAGATCGTCCAGGCTGAACTCTC
CATTCACGGGAAGGAGCTCCACGGTGGTTTTCGTTTCGGTCAAAGATGGC
ATGAGAGCATCGTTTCCGTAA
The cDNA sequence of Tpac-1 is as follows
(SEQ ID NO:17):
ATGCAGAAAGCAGACAACGTCTGCTCGAGCTTAGGCCCAAGATGCGGAGA
GGCACAAGTCAAGCGGTTCTGA
The deduced amino acid sequences of the proteins
expressed by antisense mRNAs, Pac-1 and Pac-2, produced
by the alternative/differential splicing in the pac
antisense gene is as follows:
_ 2126787
-24-
Pac-1 (SEQ ID NO:18):
MSEFCIHLHFRDLLRLDICQPRTWSSGQSSVGSRAGINRLKWAQEARDTE
SKQDKSRWRTPLLAQGTLTFEICVGWHRVSPKRMPRLEIVQAELSIHGKE
PKRMPRLEIVQAELSIHGKELHGGFRFGQRWHESIVSVTDGWFTNHVGQD
VI
Tpac-2 (SEQ ID NO:19): MQKGDNVCSSLGPRCGEAQVKRF
Pace (SEQ ID NO:20):
MRRGTSQAVLIPGRWSPRSAAGRASTGSSGHRKQGTQKANKTNQDGERP
CWLRGLLPSKSVLAGTESAPKECHGWRSSRLNSPFTGRSST W FVSVKDG
MRASFP
Pac/reg (SEQ ID NO: 21)
CTAAATGCAATATAATTTACAATTTATAAACGCAATTAGAAGAATTTCAT
TTCTTAAATGCAGGGGACATTTGGATGAGGTTATATAAAAAGTTTCAGTA
TATTCTCTGCCCAACTGGTTGGTCAAATATTTGATGGTTACTTTAAAAAA
AAAAAAAAAATTTAGTAGAGATGGGGTTTCACCTAATTGGCCAGGCTGGT
CTCGAATTTCTGACCTCGTGATCCTCCCGCCTTGGCCTCCCAAAGTGCTG
C-Fos5L (SEQ ID NO: 22)
ATGAGGGGTTTCGGGGATGGCTCCCCCCAGGGCTACAGGGAAAGGCCGTG
GAAACCTGCTGACGCAGATGTCCTAATATGGACATCCTGTGTAAGGGGGG
AGGGATTGACGGGAACTGCTCGCGGGCTGCAGCCAACACCGAGGGTGCAG
TGCGGGGGGAGGCGGGGGCCGCGGCTGGGGGAGGGGAGGCGGGAACGGCG
CAGAATGAGAGAGAACATTCGCACCTGGTTCAATGCGGACCCTTGTTCCC
GAGGTCGGGGGGGATGGGGCAGAGAGCGC~ ACCCTTGTACGGAAA
CTGAAGACAGTTCTGAGGCTCAGAGATAGGAGAAACGGCATCGAGTACAG
GACCCCGAGGACTTAA
C-Fos5Lp
MRGFGDGSPQGYRERPWKPADADVLIWTSCVRGEGLTGTARGLQPTPRVQ
CGGRRGPRLGEGRRERRRMRENIRTWFNADPCSRGRGGWGRERLFTLVRK
LKTVLRLRDRRNGIEYRTPRT
C-Fos5Lreg
TTCGCTCGCCGCGGCCGCCGGCTCAGTCTTGGCTTCTCAGTTGCTCGCTG
CAGATGCGGTTGGAGTACGAGGCGCCGCAGCCACTG~'l"l"l"l'ATAACAAGC
(:111lATGAATGAGTGTAAAcGTcAcGGGcAcAAccAcGGTGGcGccAGA
GGGGTGGCGCGCGGGCCTGGGCGCTTCTGCGGCCGCCCGGCTGCGTCCCC
AGCGCTCGCAGCTCCCTGCCCCCGCCTTGGCGCGTGTCCTAATCTCGTGA
GCAATTCGCAGTTCCTGTCTCAGAGGTCTCGTGGGCCCCCCAAG
II. The Uses of the Molecules of the Present Invention.
2126 ~ 87
-25-
The elucidation of the significance of Pac, Pac-l,
Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll,
ApoE4Lx2 and C-Fos5Lp in the etiology of Alzheimer's
disease provides improved means for diagnosing the
presence and clinical grade of the disease. Moreover,
it provides an improved means for predicting whether an
asymptomatic individual is predisposed to the disease.
It further provides a means for treating the disease.
In addition, because of the pathological similarity
between Alzheimer's disease and Down's Syndrome,
Parkinson's Disease and Schizophrenia, the molecules and
methods of the present invention can be used in the
diagnosis and treatment of these diseases and
conditions. In particular, any of the proteins
described herein, or mutants thereof may be used in the
treatment of or in the development of reagents for the
treatment of these diseases and conditions.
A. Diagnostic Uses
Since neither Pac, Tpac, hsaP nor the other
antisense sequences described herein are expressed by
normal cells, the detection of these molecules in a
tissue or fluid sample -- such as a biopsy sample, or a
blood or spinal fluid sample -- is indicative of the
presence of Alzheimer's disease in a patient.
Similarly, detection of ApoE4L, ApoE4Ll, and, in
particular, ApoE4Lx2 or C-Fos5Lp in such a sample is
indicative of Alzheimer's disease, as well as
cardiovascular disorders (such as hyperlipoproteinemia,
etc.).
The detection of these molecules may be done by any
of a variety of methods. In one embodiment, antibodies
are employed that are capable of binding to the Pac,
Pac-l, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4Ll
ApoE4Lx2 or C-Fos5Lp molecules, and the presence of such
molecules is determined via and immunoassay. A large
2126~87
-26-
number of suitable immunoassay formats have been
described (Yolken, R.H., Rev. Infect. Dis. 4:35 (1982);
Collins, W.P., In: Alternative Immunoassays, John Wiley
& Sons, NY (1985); Ngo, T.T. et al., In: Enzyme Mediated
Immunoassay, Plenum Press, NY (1985); incorporated by
reference herein.
Suitable antibodies can be either polyclonal or
monoclonal, of either a species homologous to or
heterologous to the species from which the sample was
derived. In lieu of such antibodies, equivalent binding
molecules, such as antibody fragments (F(ab'), F(ab') 21
single chain antibodies, etc.), recombinant antibodies,
chimeric antibodies, etc. may be employed. Such
antibodies can be obtained using conventional methods
with Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP,
ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp as an antigen.
Such molecules are preferably obtained through the
expression of the gene sequences described herein.
The simplest immunoassay involves merely incubating
an anti-Pac, anti-Tpac, anti-hsaP, anti-ApoE4L, anti-
ApoE4L1, anti-ApoE4Lx2, anti-C-Fos5Lp etc. antibody with
a sample suspected to contain the target molecule --
Pac, Tpac, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2, C-Fos5Lp
etc. The presence of the target molecule is determined
by the presence, and proportional to the concentration,
of any antibody bound to the target molecule. In order
to facilitate the separation of target-bound antibody
from the unbound antibody initially present, a solid
phase is typically employed. Thus, for example the
sample can be passively bound to a solid support, and,
after incubation with the antibody, the support can be
washed to remove any unbound antibody.
In more sophisticated immunoassays, the
concentration of the target molecule is determined by
binding the antibody to a support, and then permitting
the support to be in contact with a sample suspected to
contain the target molecule. Target molecules that have
2126737
-27-
become bound to the immobilized antibody can be detected
in any of a variety of ways. For example, the support
can be incubated in the presence of a labelled, second
antibody that is capable of binding to a second epitope
of the target molecule. Immobilization of the labelled
antibody on the support thus requires the presence of
the target, and is proportional to the concentration of
the target in the sample. In an alternative assay, the
target is incubated with the sample and with a known
amount of labelled target. The presence of any target
molecules in the sample competes with the labelled
target molecules for antibody binding sites. Thus, the
amount of labelled target molecules that are able to
bind the antibody is inversely proportional to the
concentration of target molecule in the sample.
As indicated above, immunoassay formats may employ
labelled antibodies to facilitate detection.
Radioisotopic immunoassays ("RIAs") have the advantages
of simplicity, sensitivity, and ease of use.
Radioactive labels are of relatively small atomic
dimension, and do not normally affect reaction kinetics.
Such assays suffer, however, from the disadvantages
that, due to radioisotopic decay, the reagents have a
short shelf-life, require special handling and disposal,
and entail the use of complex and expensive analytical
equipment. RIAs are described in Laboratory Techniques
and Biochemistry in Molecular Biology, by Work, T.S., et
al., North Holland Publishing Company, NY (1978), with
particular reference to the chapter entitled "An
Introduction to Radioimmune Assay and Related
Techniques~ by Chard, T., incorporated by reference
herein.
Enzyme-based immunoassay formats (ELISAs) have the
advantage that they can be conducted using inexpensive
equipment, and with a myriad of different enzymes, such
that a large number of detection strategies --
colorimetric, pH, gas evolution, etc. -- can be used to
2126787
-28-
quantitate the assay. In addition, the enzyme reagents
have relatively long shelf-lives, and lack the risk of
radiation contamination that attends to RIA use. ELISAs
are described in ELISA and Other Solid Phase
Immunoassays (Kemeny, D.M. et al., Eds.), John Wiley &
Sons, NY (1988), incorporated by reference herein. For
these reasons, enzyme labels are particularly preferred.
No single enzyme is ideal for use as a label in
every conceivable immunometric assay. Instead, one must
determine which enzyme is suitable for a particular
assay system. Criteria important for the choice of
enzymes are turnover number of the pure enzyme (the
number of substrate molecules converted to product per
enzyme site per unit of time), purity of the enzyme
preparation, sensitivity of detection of its product,
ease and speed of detection of the enzyme reaction,
absence of interfering factors or of enzyme-like
activity in the test fluid, stability of the enzyme and
its conjugate, availability and cost of the enzyme and
its conjugate, and the like. Examples of suitable
enzymes include peroxidase, acetylcholine esterase,
alpha-glycerol phosphate dehydrogenase, alkaline
phosphatase, asparaginase, ~-galactosidase, catalase,
delta-5-steroid isomerase, glucose oxidase, glucose-6-
phosphate dehydrogenase, glucoamylase, glycoamylase,luciferase, malate dehydrogenase, peroxidase,
ribonuclease, staphylococcal nuclease, triose phosphate
isomerase, urease, yeast-alcohol dehydrogenase, etc.
Peroxidase and urease are among the more preferred
enzyme labels, particularly because of chromogenic pH
indicators which make its activity readily visible to
the naked eye.
In lieu of such enzyme labels, radioisotopic,
chemiluminescent or fluorescent labels may be employed.
Examples of suitable radioisotopic labels include 3H,
In, 2sI, l3lI, 32p, 35S, 14C slCr 57To 58Co 59F 75S
Eu, 90Y, 67CU, 2l7ci, 2llAt 2l2Pb 47Sc 109Pd et
212S787
-29-
Examples of suitable chemiluminescent labels include a
l-lmi n~l label, an isoluminal label, an aromatic
acridinium ester label, an imidazole label, an
acridinium salt label, an oxalate ester label, a
luciferin label, an aequorin label, etc. Examples of
suitable fluorescent labels include a fluorescein label,
an isothiocyanate label, a rhodamine label, a
phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, a
fluorescamine label, etc.
As an alternative to such immunoassay formats, the
presence of Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP,
ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp in a cell can be
determined by any means capable of detecting mRNA
encoding these proteins. Thus, molecules comprising
nucleic acid probes capable of hybridizing to such
molecules may be used in the diagnosis of Alzheimer's
disease. As used herein, a "probe" is a detectably
labelled nucleic acid molecule that is capable of
hybridizing to a defined site of a target molecule. Any
of the nucleotide sequences disclosed herein can be used
as a probe; the general requirement for such use being
merely that the nucleic acid molecule be sufficiently
long (generally 10 or more nucleotides in length) that
it possesses the capacity to form stable hybridization
products with the target molecule. Any of a wide
variety of labels (see above) may be used to label
nucleic acids: enzyme labels (Kourilsky et al., U.S.
Patent 4,581,333), radioisotopic labels (Falkow et al.,
U.S. Patent 4,358,535; Berninger, U.S. Patent
4,446,237), fluorescent labels (Albarella et al., EP
144914), chemical labels (Sheldon III et al., U.S.
Patent 4,582,789; Albarella et al., U.S. Patent
4,563,417), modified bases (Miyoshi et al., EP 119448),
etc.
Such nucleic acid based assays may use either DNA
or RNA to detect the Pac, Pac-1, Pac-2, Pace, Tpac,
212G787
-30-
Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L mRNA.
In one embodiment, the assays may be performed on RNA
that has been extracted from neuronal cells.
Alternatively, and more preferably, the assays may be
done in situ on biopsied tissue.
Where the concentration of such mRNA in a sample is
too low to be detected, such mRNA may be specifically
amplified through the use of any of a variety of
amplification protocols, such as PCR (Mullis, K.B., Cold
Spring Harbor Symp. Ouant. Biol. 51:263-273 (1986);
Saiki, R.K., et al., Bio/Technology 3:1008-1012 (1985);
Mullis K. et al., U.S. Patent 4,683,202; Erlich, H.,
U.S. Patent 4,582,788; Saiki, R. et al., US 4,683,194
and Mullis, K.B., et al., Met. Enzymol. 155:335-350
(1987), transcription-based amplification systems (Kwoh
D et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173
(1989); Gingeras TR et al., PCT appl. WO 88/10315
(priority: US Patent applications serial nos. 064,141
and 202,978); Davey, C. et al. (European Patent
Application Publication no. 329,822), etc.
In yet another embodiment, the diagnosis of Pac,
Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1,
ApoE4Lx2 or C-Fos5L expression is performed using a
ribozyme produced from nucleic acid molecules having a
sequence of such molecules.
B. Prognostic Uses
The present invention additionally provides a
capacity to predict whether an individual is at risk for
Alzheimer's disease. Thus, any of the above-described
assays may be performed on an asymptomatic individual in
order to assess that individual's predisposition to
Alzheimer's disease.
21267~7
-
-31-
C. Therapeutic Uses
Significantly, the present invention provides a
means for treating Alzheimer's disease. Such treatment
may be either "prophylactic" or "therapeutic." A
prophylactic treatment is one that is provided in
advance of any symptom of Alzheimer's disease in order
to prevent or attenuate any subsequent onset of the
disease. A therapeutic treatment is one that is
provided in response to the onset of a symptom of
Alzheimer's disease, and serves to attenuate an actual
symptom of the disease.
In one embodiment, such treatment is provided by
administering to a patient in need of such treatment an
effective amount of an antibody, or an antibody fragment
(F(ab'), F(ab' )21 single chain antibodies, etc.) that is
capable of binding to Pac, Pac-1, Pac-2, Pace, Tpac,
Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5Lp. As
used herein, an effective amount is an amount sufficient
to mediate a clinically significant change in the
severity of a symptom, or a clinically significant delay
in the onset of a symptom.
As will be appreciated, for acute administration,
polyclonal or monoclonal antibodies (or fragments of
either) may be administered. More preferably, and
especially for chronic administration, the use of non-
immunogenic antibodies is preferred. Such molecules can
be pseudo-homologous (i.e. produced by a non-human
species, but altered to a form that is immunologically
indistinct from human antibodies). Examples of such
pseudo-homologous molecules include ~humanized" (i.e.
non-immunogenic in a human) prepared by recombinant or
other technology. Such antibodies are the equivalents
of the monoclonal and polyclonal antibodies, but are
less immunogenic, and are better tolerated by the
patient.
2125787
-32-
Humanized antibodies may be produced, for example
by replacing an immunogenic portion of an antibody with
a corresponding, but non-immunogenic portion (i.e.
chimeric antibodies) (Robinson, R.R. et al.,
International Patent Publication PCT/US86/02269; Akira,
K. et al., European Patent Application 184,187;
Taniguchi, M., European Patent Application 171,496;
Morrison, S.L. et al., European Patent Application
173,494; Neuberger, M.S. et al., PCT Application WO
86/01533; Cabilly, S. et al., European Patent
Application 125,023; Better, M. et al., Science
240:1041-1043 (1988); Liu, A.Y. et al., Proc. Natl.
Acad. Sci. USA 84:3439-3443 (1987); Liu, A.Y. et al., J.
Immunol. 139:3521_3526 (1987); Sun, L.K. et al., Proc.
Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y.
et al., Canc. Res. 47:999-1005 (1987); Wood, C.R.
et al., Nature 314:446-449 (1985)); Shaw et al., J.
Natl.Cancer Inst. 80:1553-1559 (1988); all of which
references are incorporated herein by reference).
General reviews of "humanized" chimeric antibodies are
provided by Morrison, S.L. (Science, 229:1202-1207
(1985)) and by Oi, V.T. et al., BioTechniques 4:214
(1986); which references are incorporated herein by
reference).
Suitable "humanized" antibodies can alternatively
be produced by CDR or CEA substitution (Jones, P.T.
et al., Nature 321:552-525 (1986); Verhoeyan et al.,
Science 239:1534 (1988); Beidler, C.B. et al., J.
Immunol. 141:4053-4060 (1988); all of which references
are incorporated herein by reference).
In another embodiment, the nucleic acid molecules
of the present invention may be mutated and expressed in
order to identify Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2,
hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos mutant
molecules that can complex with and significantly
inactivate Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP,
Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2 or C-FosL5 molecules
~ 2126787
present in a cell. In one sub-embodiment, such mutated
protein molecules may be administered to a patient.
Alternatively, nucleic acid expressing such molecules
may be administered.
In yet another embodiment, "antisense" or ~triplex"
nucleic acid molecules may be used to provide the
desired therapy. As used herein, an "antisense
oligonucleotide" is a nucleic acid (either DNA or RNA)
whose sequence is complementary to the sequence of at
least part of the Pac-, Pac-1-, Pac-2-, Pace-, Tpac-,
Tpac-2-, hsaP-, Pac/reg, ApoE4L-, ApoE4L1-, ApoE4Lx2 or-
C-Fos5L -encoding sequences described herein, such that
it is capable of binding to, or hybridizing with, an
endogenous Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP,
ApoE4L, ApoE4L1, ApoE4Lx2 or fosC5L mRNA molecule, and
thereby impair (i.e. attenuate or prevent) its the
translation into Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2,
hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L. A "triplex"
molecule is a nucleic acid molecule that is capable of
binding to double-stranded DNA in a manner sufficient to
impair its transcription.
To act as a triplex oligonucleotide, the nucleic
acid molecule must be capable of binding to the region
of the double-stranded DNA genome that encodes Pac, Pac-
1, Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L,
ApoE4L1, ApoE4Lx2, fosC5L or C-Fos5Lreg in a manner
sufficient to impair the transcription of either gene.
Triplex oligonucleotides are disclosed by Hogan, U.S.
Patent 5,176,996 and by Varma et al., U.S. Patent
5,175,266. To act as an antisense oligonucleotide, the
nucleic acid molecule must be capable of binding to or
hybridizing with that portion of the Pac, Pac-1, Pac-2,
Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1,
ApoE4Lx2 or C-Fos5L mRNA molecule which mediates the
translation of the target mRNA. Antisense
oligonucleotides are disclosed in European Patent
Application Publication Nos. 263,740; 335,451; and
2126787
-34-
329,882, and in PCT Publication No. WO90/00624, all of
which references are incorporated herein by reference.
Such a molecule can be of any length that is effective
for this purpose. Preferably, the antisense
oligonucleotide will be about 10-30 nucleotides in
length, most preferably, about 15-24 nucleotides in
length.
Thus, in one embodiment of this invention, an
antisense oligonucleotide that is designed to
specifically block transcription or translation of a
Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP, ApoE4L,
ApoE4L1, ApoE4Lx2 or C-Fos5L mRNA transcript can be used
to impair the expression of Pac, Pac-1, Pac-2, Pace,
Tpac, Tpac-2, hsaP, ApoE4L, ApoE4L1, ApoE4Lx2 or C-Fos5L
in a cell, and thereby provide a treatment for
Alzheimer's disease.
In general, the antisense oligomer is prepared in
accordance with the nucleotide sequence of Pac, Pac-1,
Pac-2, Pace, Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L,
ApoE4L1, ApoE4Lx2, C-Fos5L or Fos5Lreg as reported
herein.
The sequence of the antisense oligonucleotide may
contain one or more insertions, substitutions, or
deletions of one or more nucleotides provided that the
resulting oligonucleotide is capable of binding to or
hybridizing with the above-described translation locus
of either Pac, Pac-1, Pac-2, Pace, Tpac, Tpac-2, hsaP,
ApoE4L, ApoE4L1, ApoE4Lx2 or Fos5L mRNA.
Any means known in the art to synthesize the
antisense oligonucleotides of the present invention may
be used (Zamechik et al., Proc. Natl. Acad. Sci.
(U.S.A.) 83:4143 (1986); Goodchild et al., Proc. Natl.
Acad. Sci. (U.S.A.) 85:5507 (1988); Wickstrom et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 85:1028; Holt, J.T. et
al., Mol. Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et
al., Science 242:1303 (1988); Anfossi, G., et al., Proc.
Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D., et
2126787
-
-35-
al., EMBO J. 8:3679 (1989); all of which references are
incorporated herein by reference). Automated nucleic
acid synthesizers may be employed for this purpose. In
addition, desired nucleotides of any sequence can be
obtained from any commercial supplier of such custom
molecules.
Most preferably, the antisense oligonucleotides of
the present invention may be prepared using solid phase
"phosphoramidite synthesis." The synthesis is performed
with the growing nucleotide chain attached to a solid
support derivatized with the nucleotide which will be
the 3'-hydroxyl end of the oligonucleotide. The method
involves the cyclical synthesis of DNA using monomer
units whose 5'-hydroxyl group is blocked (preferably
with a 5'-DMT (dimethoxytrityl) group), and whose amino
groups are blocked with either a benzoyl group (for the
amino groups of cytosine and adenosine) or an isobutyryl
group (to protect guanosine). Methods for producing
such derivatives are well known in the art.
III. Administration of the Molecules of the Present
Invention
The above-described therapeutic agents of the
present invention can be formulated according to known
methods used to prepare pharmaceutically useful
compositions, whereby these materials, or their
functional derivatives, are combined in admixture with a
pharmaceutically acceptable carrier vehicle. Suitable
vehicles and their formulation, inclusive of other human
proteins, e.g., human serum albumin, are described, for
example, in Remington's Pharmaceutical Sciences (16th
ed., Osol, A., Ed., Mack, Easton PA (1980)). In order
to form a pharmaceutically acceptable composition
suitable for effective administration, such compositions
will contain an effective amount of such agents,
together with a suitable amount of carrier vehicle.
2126737
-
-36-
Additional pharmaceutical methods may be employed
to control the duration of action. Control release
preparations may be achieved through the use of polymers
to complex or absorb the agents. The controlled
delivery may be exercised by selecting appropriate
macromolecules (for example polyesters, polyamino acids,
polyvinyl, pyrrolidone, ethylenevinylacetate, methyl-
cellulose, carboxymethylcellulose, or protamine,
sulfate) and the concentration of macromolecules as well
as the methods of incorporation in order to control
release. Another possible method to control the
duration of action by controlled release preparations is
to incorporate the agents into particles of a polymeric
material such as polyesters, polyamino acids, hydrogels,
poly(lactic acid) or ethylene vinylacetate copolymers.
Alternatively, instead of incorporating these agents
into polymeric particles, it is possible to entrap these
materials in microcapsules prepared, for example, by
coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatine-microcapsules and poly(methylmethacylate)
microcapsules, respectively, or in colloidal drug
delivery systems, for example, liposomes, albumin
microspheres, microemulsions, nanoparticles, and
nanocapsules or in macroemulsions. Such techniques are
disclosed in Remington's Pharmaceutical Sciences (1980).
In one embodiment of the present invention, nucleic
acid molecule(s) comprising antisense or triplex
molecules, or encoding mutated Pac, Pac-1, Pac-2, Pace,
Tpac, Tpac-2, hsaP, Pac/reg, ApoE4L, ApoE4L1, ApoE4Lx2
or C-Fos5L molecules may be administered using viral or
retroviral vectors in accordance with the methods of
"gene therapy".
The principles of gene therapy are disclosed by
Oldham, R.K. (In: Principles of Biotherapy, Raven Press,
NY, 1987), and similar texts. Disclosures of the
methods and uses for gene therapy are provided by Boggs,
21~7~7
-37-
S.S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E.M.
(Biol. Reprod. 42:39-49 (1990)); Ledley, F.D., In:
Biotechnology, A Comprehensive Treatise, volume 7B, Gene
Technology, VCH Publishers, Inc. NY, pp 399-458 (1989));
all of which references are incorporated herein by
reference.
Although, as indicated above, such gene therapy can
be provided to a recipient in order to treat (i.e.
suppress, or attenuate) an existing condition, the
principles of the present invention can be used to
provide a prophylactic gene therapy to individuals who,
due to inherited genetic mutations, or somatic cell
mutation, are predisposed to Alzheimer's disease.
Having now generally described the invention, the
same will be more readily understood through reference
to the following examples which are provided by way of
illustration, and are not intended to be limiting of the
present invention, unless specified.
Example 1
Cloning and Expression of ~'Pac" and "Tpac" cDNA
Oligonucleotides corresponding to "Pac" and "Tpac"
cDNA were prepared by oligonucleotide synthesis on an
Applied Biosystems synthesizer using the column method
according to the recommendations of the manufacturer,
and gel purified.
For "Pac" the positive and negative strands were
synthesized as three fragments, which were ligated to
form a continuous strand, and gel purified. For "Tpac"
full-length strands were synthesized and gel purified.
Appropriate strands were mixed together in
equimolar amounts and processed in the EXPRESS system
(Invitrogen corporation pTrcHis Xpress-Prokaryotic
Expression and Purification system) according to the
recommen~tions of the manufacturer.
2125787
-38-
The sense-antisense relationship between the cDNAs
encoding these proteins and the cDNA encoding the
amyloid forming APP gene, and the similarities between
the amino acid sequences deduced for the protein, and
amino acid sequences in proteinases and esterases which
are involved in pathogenic processes, make the proteins
perfect candidates for factors that cause and maintain
human Alzheimer's disease. Since the mRNAs encoding
these proteins in healthy humans are repressed by mRNA
encoding the normal APP gene and the gene encoding the
amyloid related protein APRP363, they are potentially
excellent targets for a safe, effective, immunological
approach aimed at preventing, perhaps even stopping and
reversing, the course of this, and related diseases.
While the invention has been described in
connection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any
variations, uses, or adaptations of the invention
following, in general, the principles of the invention
and including such departures from the present
disclosure as come within known or customary practice
within the art to which the invention pertains and as
may be applied to the essential features hereinbefore
set forth and as follows in the scope of the appended
claims.
2~26787
-39 -
SBQUBNC8 LISTING
(1) GBNBRAL INPORMATION:
(i) APPLICANT: PRBDDIB, RICK
BBRGMANN, ~OHANNA
(ii) TITLB OF INVBNTION: AGBNTS FOR THB PRBVBNTION AND TRRATMR~lT
OF HUMAN AT.~RTMRu DISBASB
(iii) NUMBBR OF I _ : 24
(iv) ~u~.~k~ ADDRBSS:
1 0 IAI ~nRR.csRR: RICK PRBDDIB
:B.I STRBBT: 4855 COTB ST LUC RD, Suite# 201
C CITY: MONTRBAL
D STATB: QUBBBC
l~BI COUNTRY: CANADA
:FI ZIP: H3W 2H5
(v) COM?UTBR RBADABLB FORM:
~A MBDIUM TYPB: Floppy disk
:BI COMPUTBR: IBM PC compatible
ICJ OPBRATING SYSTBM: PC-DOS/MS-DOS
ID:I SOFTWARB: PatentIn Release #1.0, Version #1.25
(vi) CURRBNT APPLICATION DATA:
(A) APPLICATION NUMBBR:CAN#
(B) FILING DATB:
(C) CLASSIFICATION:
2 5 (viii) ATTORNBY/AGBNT INFORMATION:
(A) NAMB: NONB
(B) RBGISTRATION NUMBBR:
(C) RBFBRBNCB/DOCKBT NUMBBR:
(ix) TRn- CATION INFORMATION:
3 0 (A) TBLBPHONB: (0014940) 86 25 53
(B) T LBFAX: (0014940) 86 01 76
(2) INFORMATION FOR SBQ ID NO:1:
(i) SBQUBNCB CHARACTBRISTICS:
~A) LBNGTH: 315 hase pairs
I:B) TYPB: nucleic acid
~:C) STRA : single
l:D) TOPOLOGY: linear
(ii) MOLBCULB TYPB: cDNA
(iii) ~Y~u~ CAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: PAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:1:
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGCTCTCC l~l-ll~A l~Ill~l~AA 60
~rrA~AA~T GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA wG~T CAACAGGCTC 120
AACTTCGTTT I~l~l~ll~G CTGGCACAGA GTCAGCCCCA AAAGAATGCC ACGGCTGGAG 180
ATCGTCCAGG CTGAACTCTC CATTCACGGG AAGGAGCTCC A~l~lll l~lll~l 240
CAAAGATGGC ATGAGAGCAT ~lll~ A ACTGATCCTT GGTTCACTAA TCAAGTTGGC 300
CAAGACGTCA TGTGA 315
(2) INFORMATION FOR SBQ ID NO:2:
(i) SBQUBNCB CHARACTLRISTICS:
(A) LBNGTH: 104 amino acids
(B) TYPB: amino acid
(D) TOPOLOGY: linear
(ii) MOLBCULB TYPB: protein
(iii) ~Y~ul~llCAL: NO
(vi) ORIGINAL SOURCB:
6 0 (A) ORGANISM: HOMO SAPIBNS
~26 7~7
-40 -
(vii) TMMRnTATR SOURCE:
(B) CLONB: PAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:2:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu
1 s lo lS
A3p Ile Cys Gln Pro Arg Thr Trp Ser Ser Gly Gln Ser Ser Val Gly
Ser Arg Ala Gly Ile Asn Arg Leu Asn Phe Val Phe Cy8 Val Gly Trp
1 0 His Arg Val Ser Pro Lys Arg Met Pro Arg Leu Glu Ile Val Gln Ala
50 55 60
Glu Leu Ser Ile His Gly Lys Glu Leu His Gly Gly Phe Arg Phe Gly
65 70 75 80
Gln Arg Trp His Glu Ser Ile Val Ser Val Thr Asp Asp Trp Phe Thr
Asn His Val Gly Gln Asp Val Ile
100
(2) INFORMATION POR SBQ ID NO:3:
(i) SBQUBNCB rHARArTRRT5TIcs
2 0 (A LBNGTH: 84 baoe pairs
(Bl TYPB: nucleic acid
(Cl sTRAt~RnNRq~s: single
(Dl TOPOLOGY: linear
(ii) MOLBCULB TYPB: cDNA
(iii) H~Vl~llCAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) TMMRnTATB SOURCB:
3 0 (B) CLONB: TPAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:3:
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC l`~ ~A TATTTGTCAA 60
rrrAr.AArrT GTATTACATC ATAA 84
(2) INPORMATION POR SBQ ID NO:4:
3 5 (i) SBQUBNCB rRARArTRRT.5TICS:
~A LBNGTH: 27 amino acids
B I TYPB: amino acid
C:I sTRpt~RnNRe~s: single
Dl TOPOLOGY: linear
4 0 ( i i ) MOLBCULB TYPB: cDNA
(iii) HY~Old~llCAL: NO
(iv) ANTI-SBNSB: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: TPAC
(xi) SBQUBNCB DBSCRIPTION: SBQ ID NO:4:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu
5 10 15
5 0 Asp Ile Cys Gln Pro Arg Thr Cys Ile Thr Ser
20 25
(2) INPORMATION POR SEQ ID NO:S:
(i) SBQUBNCB rRARArTRRT~eTIcs
(A~ LBNGTH: 159 base pairs
5 5 (B:l TYPB: nucleic acid
(C, STRP ~~: single
(Dl TOPOLOGY: linear
2~26 737
-
-41 -
(ii) MOLBCULB TYPB cDNA
(iii) ~Y~uln~ CAL NO
(iv) ANTI-SENSB NO
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(B) STRAIN hsaP
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 5
CACTGCTTGC ~ ,l GCACCAGTTC TGGATGGTCA ~ll~l- 60
GGCTTCTACC TCATTGGTGA TCTGCAGTTC ArrrTArArT TCTTGGCAAT ACTGCAGGAT 120
10 ~iu~.~C~.. `~ GTATCAATGC A~.. ~. CCCTGATGG 159
(2) INFORMATION FOR SBQ ID NO 6
(i) SBQUBNCB CHARACTBRISTICS
(A) LBNGTH 51 amino acid~3
(B) TYPB amino acid
(D) TOPOLOGY linear
(ii) MOLBCULB TYPB protein
(iii) IIY~O~ llCAL NO
(iv) ANTI-SBNSB NO
(vi) ORIGINAL SOURCB
2 0 (A) ORGANISM HOMO SAPIBNS
(B) STRAIN huaP
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 6
Met Gly Leu Ala Leu Leu Ala Ala Pro Leu Ala Pro Val Leu AE~p Gly
5 10 15
2 5 Hi3 Trp Leu Val Gly Phe Tyr Hi~ Ile Gly Asp Leu Gln Phe Arg Val
20 25 30
Aop Phe Leu Ala Ile Leu Gln A~p Ala Phe Leu Gly Ile A~n Ala Gly
35 40 45
3 o Phe Gly Pro
(2) INFORMATION FOR SBQ ID NO 7
(i) SEQUBNCB CHARACTBRISTICS
~A) LBNGTH 597 ba3e pair~
IB) TYPB nucleic acid
3 5 , c) STRANDBDNBSS E~ingle
lD~ TOPOLOGY linear
(ii) MOLBCULB TYPB cDNA
(iii) tlY~ CAL NO
(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4L
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 7
4 5 A~ ll. A rrAr-r~r-GrTc rAArrArrTc TTGAGGCGGG CCTGGAAGGC ~lC~C~l~,C 60
A~,.~T~,l G~.~l~, CTCCTCCAGC ll~ --ACCTCCGCCAC Cl~lC~llc 120
AC~il~ lC A W~ li ~lCl~ i C~IClC~l CCATCCGCGC GCGCAGCCGC 180
TCGCCCCAGG ~ CTCCTGTAGC GG~ ~ rr~ CC CACAGTGGCG 240
GCCCGCACGC b~ rArr~r-r~r~r-c CCCAGGCGCT CGCGGATGGC GCTGAGGCCG 300
5 0 ~/~.c~,c -~ ~c cc~ w~ TACACTGCCA ~,~ l~ CAGGTCATCG 360
GCATCGCGGA GGAGCCGCTT ACGCAGCTTG CGCAGGTGGG AGGCGAGGCG CACCCGCAGC 420
i.~l.~l~C I.l~iGC~iAG CATwC~l,c A - ~;--~-' GGTACTGCAC rArr,r,rrrrr 480
-- ~12~ ~ ~7
-42 -
CGCACGTCCT CCATGTCCGC GCCCAGCCGG GC~17c~b CCTGCAGCTC CTTGGACAGC 540
~71~7~ 7~Ll~7~ rAc~ Tc A~711,11~1 CCAGTTCCGA TTTGTAG 597
(2) INPORMATION POR SBQ ID NO 8
(i) SBQUBNCB ChABACTBRISTICS
(A) LBNGTH 198 amino acid~
(B) TYPB amino acid
(D) TOPOLOGY linear
(ii) MOLBCULB TYPB protein
(iii) nr~ulnhllCAL NO
1 0 (vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4L
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 8
Met Ser Ser Thr Arg Gly Ser Ann Gln Leu Leu Arg Arg Ala Trp Ly~
1 5 10 15
Ala Ser Ala Cy3 Arg Arg Ile Cy~ Trp Ala cy8 Ser Ser Ser Leu Ala
20 25 30
Arg Thr Ser Ala Thr Cyo Ser Phe Thr Ser Ser Arg Arg Ser Arg Val
2 0 35 40 45
Arg Leu Pro Ile Ser Ser Ile Arg Ala Arg Ser Arg Ser Pro Gln Ala
Trp Ala Arg Ser Cy~ Ser Gly Trp Pro Ala Arg Glu Pro Thr Val Ala
2 5 Ala Arg Thr Arg Pro Cy3 Ser Thr Arg Gly Pro Arg Arg Ser Arg Met
85 90 95
Ala Leu Arg Pro Arg Ser Ala Pro Ser Arg Ala Pro Ala Trp Tyr Thr
100 105 110
Ala Arg Arg Phe Cy~ Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg
3 0 115 120 125
Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu
130 135 140
Trp Pro Ser Met Ala Cy~ Thr Ser Pro Arg Tyr Cy~ Thr Arg Ala Pro
145 150 155 160
3 5 Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala Cy9 Ala Ala Cys Ser
165 170 175
Ser Leu A~p Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cy~
180 185 190
195
(2) INFORMATION POR SBQ ID NO 9
(i) SEQUBNCB ~ARArTRRTqTIcs
lAI LBNGTH 324 ba~e pairo
IBI TYPB nucleic acid
~c sTRANnRr ~.c ~ingle
Dl TOPOLOGY linear
(ii) MOLBCULB TYPB cDNA
(iii) ~r~ln~llCAL NO
(iv) ANTI-SBNSB YBS
5 0 (vi ) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4Ll
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 9
A'~71~ ,1 CCATCTGCGC ~ ~GC~ ArAr~,cr,r~, A~,l~,lCG~ ACAGTGGGGA 60
~- AAGGGCTGGG A~ ~7 CGAGATG~3G ATGAGCCAGA ~rrAA~ 120
A~A~ArAA ~AAr~C AGAGGCCGAG A~AA~A~A~ A~A~A~A~AT G~ ~A 180
2i2~787
-
-43 -
GATGCAGAGG r.rAr.Arr.rA Ar.Arr.AArAA GGAGCTAGGA G~ w AA GGTGCTCATG 240
CCTCTAATCC CAGCACTTTG rrArGrrrAr Grr-r-r-Ar~r-AT CGCTTGAGCC CAGAAGTTCA 300
AGACCAGCCT GGGCAACACA GTGA 324
(2) INFORMATION POR SBQ ID NO 10
(i) SBQUBNCu CHARACTERISTICS
(A) LENGTH 107 amino acids
(B) TYPB amino acid
(D) TOPOLOGY linear
(ii) MOLBCULE TYPB protein
1 0 (iii) dY~u-dhllCAL NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONE APOB4Ll
(xi) SBQUBNCE DBSCRIPTION SBQ ID NO 10
Met Val Ser Ser Ile Cys Ala Val Arg Pro Arg Gly Arg Glu Gly Val
1 5 10 15
Ala Gln Trp Gly Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp
2 0 Gly Asp Glu Pro Glu Arg Pro Lyo Arg Glu Arg Arg Asn Gly Ala Glu
35 40 45
Ala Glu Arg Arg Arg Gln Arg Gln Met Gln Arg Ala Asp Ala Glu Gly
50 55 60
2 5 Arg Gly Arg Asp Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met
Pro Leu Ile Pro Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu
85 90 95
Pro Arg Ser Ser Arg Pro Ala Trp Ala Thr Gln
100 105
3 0 (2) INFORMATION POR SBQ ID NO ll
(i) SEQUBNCB CHARACTBRISTICS
l,A,I LBNGTH 532 base pairs
I B I TYPB nucleic acid
'Cl sTRr ~ single
3 5 ~D TOPOLOGY linear
(ii) MOLBCULB TYPB DNA (genomic)
(iii) dY~uldhLlcAL NO
(iv) ANTI-suNsB NO
(vi) ORIGINAL SOURCB
4 0 (A) ORGANISM HOMO SAPIBNS
(xi) SBQUENCB DBSCRIPTION SBQ ID NO ll
CTCTACTAAA AATArAAAAA TTAGCCGGGT ~l~ GAGCCTGTAA TCCCAGCTAC 60
TGAGGCAGCA GAATCGCTTG AArrrAArAr GCAGAGGTTG CAGTGAGCCA AGATCGTGCC 120
ACTGCACTCT A~C~ ~l~ ACAGAGCCAG A~ lA AAAAr~rr~r~r~r~ AArr.~rrArAr. 180
4 5 ArAAArATAr ArA~ArArAr AGATGGAGAG rDrr,r,rrrTG AGAATTGTGT GGCAGTATGT 240
GGGCAGAAAG AGAAACTGAG G~1~GW~11 ArArrAAATc ArArrrrrAr GCCAGCAGAT 300
GCGTGAAACT TGGTGAATCT TTATTA-DACT AGGGTCCACC rrArr.ArrDr ~l~GW~ 360
r,rrArArGr~T ~ VC AWL-1G~L~ rArr.rArr.A~. GCACGGwTG G~l~l~ 420
Gu~l~G~l~C AGGCTTCGGC GTTCAGTGAT I~l.~l~ w rArDr~r~r~r~rG G~l~l~ 480
5 0 rrArGGrAr~c CTGCACCTTC TcrArrAr~rr rr~rrrArTG GCGCTGCATG TC 532
(2) INPORMATION POR SBQ ID NO 12:
(i) SBQUBNCB r~ARArTRRT~qTICS
(A LPNGTH 936 base pairs
(B I TYPB nucleic acid
5 5 (c sTRANnun~uq~q~ single
(D TOPOLOGY linear
- 2126787
-44 -
(ii) MOLBCULB TYPB cDNA
(iii) ~Y~uld~llCAL NO
(iv) ANTI-SBNSE YBS
(vi) ûRIGINAL SOURCB:
5(A) ORGANISM HOMû SAPIENS
(vii) IMMBDIATB SOURCB
(B) CLONB APOB4LX2
(xi) SBQUBNCB DBSCRIPTION SBQ ID Nû 12
Arl~l.ll~A CCA wGGCTC GAACCAGCTC TTGAwCwG CCTGGAAGGC ~l~W~l~C 60
1 0 AGGCGTATCT G~l~ WC~l~ CTCCTCCAGC ll~G~ a C~l~C~AC ~l~lC~llc 120
A~lC~l~a W~b~l~C~ ~blC~W~l~ CCCATCTCCT CCATCCGCGC GCGCAGCCGC 180
TCGCCCCAGG ~l~ WC~ CTCCTGTAGC W~ W rrArrrArrr CACAGTGGCG 240
GCCCGCACGC W~ ArrAr-r~r-r rrrAr~r,rCrT CGCwATGGC GCTGAGGCCG 300
~ W~C ~l~W~C CCCwCCT w TACACTGCCA W~llCl~ CA wTCATCG 360
GCATCGCwA wAGCCGCTT ACGCAGCTTG CGCAwT ww ArrrrArrrr CACCCGCAGC 420
TC~l~bbl~C TCT wCCGAG CA'l~b~.~C A~ ~C wTACTGCAC rArrr.rrrrr 480
CGCACGTCCT CCATGTCCGC GrrrAr~crw GC~ ~C-~ CCTGCAGCTC CTT wACAGC 540
~ ~ T.~C~.C~C rArrr~GcrTc A~l.~..~. CCAGTTCCGA lll~G~l~'C 600
TTCAACTCCT CCAT wTCTC ~l.~I.l~ GC~l~wC CGAGAwwCG wAwwTGTC 660
2 0 GCACAGT ww r~Ar~rr~r~r~r~r- ArAArrrrTG wAT w wCG wCGAGAT w wATGAGCCA 720
aAr-Ar-ArrrA A~Arr~ArAr AAr~rAArGrr GCAGAGGCCG ArArAArrAr ArArArArAr 780
ATGCAGAGAG CAGATGCAGA r~r-rAr~Ar~r-c ArArArrAAr AArrArrTAr rArrrrr,r~C 840
AAGGTGCTCA TGCCTCTAAT rrrAr-rArTT TGc~r-Ar~r/rrr- Arr,rrrrArr. AT~ll~AG 900
CCCAGAAGTT rAArArrArC CTGwCAACA CAGTGA 936
(2) INFORMATION POR SEQ ID NO 13
(i) SBQUENCB ruARArTR~TcTIcs
(A) LBNGTH 311 amino acids
(B) TYPB amino acid
(D) TOPOLOGY linear
3 0 (ii) MOLBCULB TYPB protein
(iii) ~Y~Ol~hllCAL NO
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) TMMRnTATR SOURCB
3 5 (B) CLONB APOB4LX2
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 13
Met Ser Ser Thr Arg Gly Ser A3n Gln Leu Leu Arg Arg Ala Trp Ly~
1 5 10 15
Ala Ser Ala Cy~ Arg Arg Ile Cy9 Trp Ala Cyo Ser Ser Ser Leu Ala
4 0 20 25 30
Arg Thr Ser Ala Thr C,v8 Ser Phe Thr Ser Ser Arg Arg Ser Arg Val
Arg Leu Pro Ile Ser Ser Ile Arg Ala Arg Ser Arg Ser Pro Gln Ala
4 5 Trp Ala Arg 5er Cys Ser Gly Trp Pro Ala Arg Glu Pro Thr val Ala
65 70 75 80
Ala Arg Thr Arg Pro Cy~ Ser Thr Arg Gly Pro Arg Arg Ser Arg Met
85 90 95
Ala Leu Arg Pro Arg Ser Ala Pro Ser arg Ala Pro Ala Trp Tyr Thr
5 0 loo 105 110
Ala Arg Arg Phe Cy~ Arg Ser Ser Ala Ser Arg Arg Ser Arg Leu Arg
115 120 125
Ser Leu Arg Arg Trp Glu Ala Arg Arg Thr Arg Ser Ser Ser Val Leu
212~ 787
-45 -
130 135 140
Trp Pro Ser Met Ala Cyo Thr Ser Pro Arg Tyr Cys Thr Arg Ala Pro
145 150 155 160
Arg Thr Ser Ser Met Ser Ala Pro Ala Arg Ala CYB Ala Ala Cyn Ser
165 170 175
Ser Leu Asp Ser Arg Ala Arg Val Ser Ser Ala Thr Gly Val Ser Cys
180 185 190
Ser Ser Ser Ser Asp Leu Ala Ala Phe Asn Ser Ser Met Val Ser Ser
195 200 205
1 0 Ile Cy9 Ala Val Arg Pro Arg Gly Arg Glu Gly Val Ala Gln Trp Gly
210 215 220
Gly Gly Gly Glu Gly Leu Gly Trp Gly Gly Arg Asp Gly Asp Glu Pro
22s 230 235 240
Glu Arg Pro Ly~ Arg Glu Arg Arg Asn Gly Ala Glu Ala Glu Arg Arg
245 250 255
Arg Gln Arg Gln Met Gln Arg Ala Asp Ala GlU Gly Arg Gly Arg Asp
260 265 270
Glu Glu Gly Ala Arg Arg Pro Gly Lys Val Leu Met Pro Leu Ile Pro
275 280 285
2 0 Ala Leu Trp Glu Ala Glu Ala Gly Gly Ser Leu Glu Pro Arg Ser Ser
290 295 300
Arg Pro Ala Trp Ala Thr Gln
305 310
(2) INFORMATION FOR SBQ ID NO:14:
(i) SBQUBNCB r~A~ArTR~T~TIcs:
(A,l L8NGTH: 399 base pairs
(Bl TYPB: nucleic acid
(Cl STRANDBDNESS: single
(D:l TOPOLOGY: linear
(ii) MOLBCULB TYPB: cDNA
(iii) ~Y~l~llCAL: NO
(iv) ANTI-SBNSB: YBS
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: Pac-1
(xi) SEQU8NCB DBSCRIPTION: SBQ ID NO:14:
ATGTCGGAAT TCTGCATCCA TCTT QCTTC AGAGATCTCC T~ iA TATTTGTCAA 60
rrrArAArrT GGTCGAGTGG TCAGTCCTCG GTCGGCAGCA ~ T CAACAGGCTC 120
4 0 AACTGGGCAC AGGAAGCAAG cc~ArArAA ArrAAArAA~ ACAAATCAAG ATGGAGAACG 180
~-llG~l~ CTCAGGGGAC TCTTACCTTC ~llll~l~li ll~i~l~i~A CAGAGTCAGC 240
rrrAAAArAA TGCCACGGCT GGAGATCGTC CAGGCTGAAC TCTCCATTCA rr,r~ARr~r 300
CTCCACGGTG ~llll~illl CGGTCAAAGA TGGCATGAGA GCATCGTTTC CGTAACTGAT 360
~ill~ll~A CTAATCATGT TGGCCAAGAC GTCATCTGA 399
(2) INFORMATION FOR SBQ ID NO:15:
(i) SBQUBNCB r~A~ArTR~T.CTICS:
(A) LBNGTH: 513 base pairs
(B) TYPB: nucleic acid
(C) STR~.J~uN~SS: single
(D) TOPOLOGY: linear
(ii) MOLBCULB TYPB: cDNA
(iii) ~Y~ul~llCAL: NO
(iv) ANTI-SBNSB: YBS
(vi) ORIGINAL SOURCB:
5 5 (A) ORGANISM: HOMO SAPIBNS
(vii) IMMBDIATB SOURCB:
(B) CLONB: Pac-2
2125~7
-46 -
(xi) SEQUBNCB DBSCRIPTION SBQ ID NO 15
ATGTCGGAAT TCTGCATCCA TCTTCACTTC AGAGATCTCC TC~--ll~A TATTTGTCAA 60
rrrArAArrT GTATTACATC ATAATTAAAG GGTCACTTCA AATTCTACTC Tr-rAr-TAAr-A 120
TCAATTGAGA GAGGCTTAAA ATGCAGAAAG rArArAArrT CTGCTCGAGC TTArrrrrAA 180
GATGCGGAGA rGrArAArTc AAGCGGTTGT GATACCTGGT CGAGTGGTCA ~l~ ~lC 240
rrrArrArrr CGGGCATCAA GATGGAGAAC GCC~ ~ GCTCAGGGGA CTCTTACCTT 300
~llll~l~l ~ll~lvG~ ACGAGTCAGC rrrAAAArAA TGCCACGAGT CAGCCCCAAA 360
AGAATGCCAC GGCTGGAGAT CGTCCAGGCT GAACTCTCCA TTCACGGGAA GGAGCTCCAC 420
~l~llllC ~lll~l A AAGATGGCAT GAGAGCATCG TTTCCGTAAC TGATGGTTGG 480
1 0 TTCACTAATC Ai~ll~A AGACGTCATC TGA513
(2) INPORMATION FOR SBQ ID NO 16
(i) SBQUBNCB CHARACTBRISTICS
~A) LBNGTH 321 base pairs
B) TYPB nucleic acid
c) STRr-~RT)NRqq: Eiingle
l~D) TOPOLOGY linear
(ii) MOLBCULB TYPB cDNA
(iii) dY~ld~llCAL NO
(iv) ANTI-SENSB YES
2 0 (vi ) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB Pace
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 16
ATGCGGAGAG G QCAAGTCA AG~ll.l~ ATACCTGGTC GAGTGGTCAG l~l-~l~ 60
rr~rrArrrC GGGCATCAAC AGGCTCAAGT Grr,rArArrA Arrr~ ArA~AAAr~rA 120
AArAArArAA ATCAAGATGG AGAACGCCCT l~ A GGGGACTCTT ACCTTCGAAA 180
I.l~l~ll~ CTGwCACAGA GTCAGCCCCA AAAGAATGCC ACwCTGwAG ATCGTCCAGG 240
CTGAACTCTC CATTCACGw AAGGAGCTCC A~wl~ll~ 1~111 - Wl CAAAGATGwC 300
3 0 ATGAGAGCAT ~lll.~lA A 321
(2) INFORMATION FOR SBQ ID NO 17
(i) SBQUBNCB rHARArTRRTqTICS
(A) LBNGTH 72 base pairo
(B) TYPB nucleic acid
(c) STR~'~)Rr'--"9: oingle
(D) TOPOLOGY linear
(ii) MOLBCULB TYPB cDNA
(iii) HY~VlHhllCAL NO
(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIBNS
(vii) IMMBDIATB SOURCB
(B) CLONB Tpac-1
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 17
ATGCAGAAAG rArArAA~rT CTGCTCGAGC TTAr-GrrrAA GATGCGwAGA wCACAAGTC 60
AAGCGGTTCT GA 72
(2) INFORMATION FOR SBQ ID NO 18
(i) SBQUBNCB r~ARArTRRT~qTIcs
(A) LBNGTH 152 amino acido
5 0 (B) TYPB amino acid
(D) TOPOLOGY linear
(ii) MOLBCULB TYPB protein
212 ~ r~ ~ r~
-47 -
(iii) ~IYt~ bLlCAL: NO
(vi) ORIGINAL SOURCB:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMBDIATE SOURCE:
(B) CLONE: Pac-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Ser Glu Phe Cys Ile His Leu His Phe Arg Asp Leu Leu Arg Leu
Asp Ile Cys Gln Pro Arg Thr Trp Ser Ser Gly Gln Ser Ser Val Gly
Ser Arg Ala Gly Ile Asn Arg Leu Lys Trp Ala Gln Glu Ala Arg Asp
Thr Glu Ser Lys Gln Asp Lys Ser Arg Trp Arg Thr Pro Leu Leu Ala
Gln Gly Thr Leu Thr Phe Glu Ile Cys Val Gly Trp His Arg Val Ser
65 70 75 80
Pro Lys Arg Met Pro Arg Leu Glu Ile Val Gln Ala Glu Leu Ser Ile
85 90 95
2 o His Gly Lys Glu Lys His Gly Gly Phe Arg Phe Gly Gln Arg Trp His
100 105 110
Glu Ser Ile Val Ser Val Thr Asp Gly Trp Phe Thr Asn His Val Gly
115 120 125
Gln Asp Val Ile
(2) INFORMATION FOR SEQ ID NO:19:
(i) S--QUENCE rH~R Z~rTRR T .qTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
3 0 (ii) MOLECULE TYPE: protein
(iii) ~IY~UldbLlCAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Tpac-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Gln Lys Gly Asp Asn Val Cys Ser Ser Leu Gly Pro Arg Cys Gly
Glu Ala Gln Val Ly~3 Arg Phe
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE rul~R~rTRRT~qTIcs:
(A) LENGTH: 106 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) ~IY~ULrlbLlcAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: Pace
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Arg Arg Gly Thr Ser Gln Ala Val Leu Ile Pro Gly Arg Val Val
5 5 Ser Pro Arg Ser Ala Ala Gly Arg Ala Ser Thr Gly Ser Ser Gly His
2126787
-48--
Arg Lys Gln Gly Thr Gln Lys Ala Asn Lys Thr Asn Gln Asp Gly Glu
Arg Pro Cys Trp Leu Arg Gly Leu Leu Pro Ser Lys Ser Val Leu Ala
Gly Thr Glu Ser Ala Pro Lys Glu Cys His Gly Trp Arg Ser Ser Arg
6s 70 75 80
Leu Asn Ser Pro Phe Thr Gly Arg Ser Ser Thr Val Val Phe Val Ser
85 90 95
Val Lys Asp Gly Met Arg Ala Ser Phe Pro
100 105
(2) INFORMATION POR SEQ ID NO 21
(i) SEQUENC_ CHARACTERISTICS
(A) LENGTH 250 base pairs
(B) TYPE nucleic acid
(c) STR~ ~q single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE cDNA
(iii) ~Y~ulrl~llCAL NO
(iv) ANTI-SBNSE YES
(vi) ORIGINAL SOURCE
(A) ORGANISM HOMO SAPIENS
(vii) IMMEDIATE SOURCR
(B) CLONE Pac/reg
(xi) SEQUENCE DESCRIPTION SEQ ID NO 21
CTAAATr-r3ATATAATTTAcAATTTATAAACGc~AATTArAAr-AA~llcAlll.l.~AATG 60
rArrrrArATTTGGATGAGGTTATATAAAAArTTTCAGTATA~ l~CC AACTGGTT 120
GGTcAAATATTTGATGcTTAcTTTA7~AAAAAAAAAAAA7\7\TTTAGTAGAGA~ 180
ACCATATTGGCCA~G~l~l.l.iAATTTCTGACCTCGTGA~C~l.~iC~ ~l.C 240
CAAAGTGCTG 300
3 0 (2) INFORMATION FOR SEQ ID NO 22
(i) SEQUENCE CHARACT8RISTICS
(A) LENGTH 366 base pairs
(B) TYPE nucleic acid
(C) STRANnRnNRcc single
3 5 (D) TOPOLOGY linear
(ii) MOLECULE TYPE cDNA
(iii) 11Y~Ul ~ CAL NO
(iv) ANTI-SENSE YES
(vi) ORIGINAL SOURC_
4 0 (A) ORGANISM HOMO SAPIENS
(vii) IMMEDIATE SûURCE
(B) CLONE C-FosSL
212~787
-49 -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
ATGA~lll.~-''-'''''Arrr.rTA~rrr~AA~"~l~AAACCTGCT 60
GAcGcAGATGTccTAATATGGAcA~lccl~~ Ar~r,r~rArcrATTr-Arrrr.AArTGcT 120
~ r~rrAArArrr-Ar~r~r~TGcA~l~-l~x:~xx/~ l~G~ l~bb~ 180
r~rr~r~rrrrraAArGr~rr~cAGAATr~r~r~r~ArATTcGcAc~-l~bll~AATGcGGAc 240
~A~lll~ ~X-~l~rArArAl-~ IllllACCCTTGTACGGAAA 300
cTr~AAr~ArAr~TTcTGAGGcTrAr~Ar~ATAr~r~Ar~AAArr-r~rATcGAGTArAr~r~Arrrr~r~Ar~r~ 360ACTTAA 420
(2) INFORMATION FOR SEQ ID NO:23:
1 0 (i) SBQUENCE rHARArTR~T~cTIcs:
(A) LENGTH: 121 amino acido
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) ~Y~ul~llCAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HOMO SAPIENS
(vii) IMMEDIATE SOURCE:
(B) CLONE: C-Fos5Lp
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Arg Gly Phe Gly Asp Gly Ser Pro Gln Gly Tyr Arg Glu Arg Pro
1 5 10 15
Trp Lys Pro Ala Asp Ala A~p Val Leu Ile Trp Thr Ser Cy5 Val Arg
20 25 30
2 5 Gly Glu Gly Leu Thr Gly Thr Ala Arg Gly Leu Gln Pro Thr Pro Arg
35 40 45
Val Gln Cy~ Gly Gly Arg Arg Gly Pro Arg Leu Gly Glu Gly Arg Arg
50 55 60
Glu Arg Arg Arg Met Arg Glu Asn Ile Arg Thr Trp Phe Asn Ala Asp
3 0 65 70 75 80
Pro Cys Ser Arg Gly Arg Gly Gly Trp Gly Arg Glu Arg Leu Phe Thr
85 90 95
Leu Val Arg Lyo Leu Ly~ Thr Val Leu Arg Leu Arg Asp Arg Arg A~n
100 105 110
3 5 Gly Ile Glu Tyr Arg Thr Pro Arg Thr
115 120
(2) INFORMATION EOR SEQ ID NO:24:
(i) SEQUENCE ChARACTERISTICS:
(A) LENGTH: 294 ba~e pairs
(B) TYPE: nucleic acid
(C) STRP ~: ~ingle
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
(iii) ~Y~ul~llCAL: NO
2 126787
-50 -
(iv) ANTI-SBNSB YBS
(vi) ORIGINAL SOURCB
(A) ORGANISM HOMO SAPIENS
(vii) TMMRnTATR SOURCB
(B) CLONB Fo~SLreg
(xi) SBQUBNCB DBSCRIPTION SBQ ID NO 24
TTcG~l~(ic~w~ ~l~AGTcTT wcTTcTcA~ ~AGATGcwT 60
TGr-Ar-TArr-Ar~r-cGccGcAGccA~ TAArAAr-cGTTTTATGAATGAGTGTAAA 120
cGTcAcwwrArAArrA~ l~r-Ar~-l~i((-((-((~-(-(l(iW~i~ll.l~C i80
0 l~AG-l~ lvG~ 240
AAI~l~l~AGcATTTcGcA~ll~ AGAh~l~ l~ wl~ Ar~ 300
