Note: Descriptions are shown in the official language in which they were submitted.
CA 02456777 2004-03-O1
~~.F OF THF INVEN''1'IDN:
METHODS FOR THE DIAGNOSIS, PROGNOSIS AND TREATMENT OF
GLAUCOMA AND RELATED DISORDERS
The present invention is in the fields of diagnostics, prognosis, and
treatment,
and concerns methods and reagents for diagnosing and treating glaucoma and
,~.
related disorders.
"Glaucomas" are a group of debilitating eye diseases that are the leading
cause of preventable blindness in the United States and other developed
nations.
Priaaary Open Angle Glaucoma ("POAG'~ is the most eomrnon form of glaucoma.
The disease is characterized by the alteration of the trabecular meshwork,
leading to
obstruction of the normal ability of aqueous humor to leave the eye without
closure
of the spare (e:g., the "angle") between the iris and cornea (see, Vaughan, D.
et al.,
In: General Ophthalmology, Appleton & Lange, Norwalk, CT, pp. 213-230 (1992)).
A
characteristic of such obstruction in this disease is an increased intraocular
pressure
("IOP"), resulting in progressive visual Ioss and blindness if not treated
appropriately and in a timely fashion.
The disease is estimated to affect between 0.4% and 3.3% of all adults over 40
years old (Leske, M.C. et aL, Amer. J. Epidemiol. l I3:1843-1846 (1986);
Bengtsson, B.,
Br. J. OphthamoI: 73:483-487 (1989); Strong, N:P., Ophthul. Physiol. Opt. 12:3-
7 (1992)).
Moreover, the prevalence of the disease rises with age to over 6% of those 75
years
- 30 or older (Strong, N.P., Ophthal. Physiol. Opt. T2:3-7 (1992)).
CA 02456777 2004-03-O1
A link between the IOP response of patients to glucocorticoids and
the
disease of POAG has long been suspected. While only 5% of the normal
population
shown a high IOP increase {16 mm Hg) to topical glucocorticoid testing,
greater than
' 40-50% of patients with POAG show this response: In addition; an
Open Angle
glaucoma may be induced by exposure to glucocorticoids. This observation
has
suggested that an increased or abnormal glucocorticoid response
in trabecular cells
may be involved in POAG {than, G.L. et al., Exper. dye Res. 54:211-218
(1992); Yun,
A.j. et al., Invest. Ophtharnol. Vis. Sci. 30:2012-2022 (1989);
Clark, A.F., Exper. Eye Res.
55:265 (1992); Klemetti, A., Acts Ophthamol. 68:29 33 {1990); Knepper,
P.A.; U.S.
Patent No. 4,617,299). The ability of glucocorticoids to induce
a glaucoma-Iike
condition has led to efforts to identify genes or gene products
that would be
induced by the cells of the trabecular meshwork in response to glucocorticoids
{Polansky, J.R et al., in: Glaucoma Lipdate IV, Springer-Verlag,
Berlin, pp. 20-29
{1991)). Initial efforts using short-term exposure to dexamethasone
revealed only
changes in specific protein synthesis. Extended exposure to relatively
high-levels of
dexamethasone was, however, found to induce the expression of related
66 kD and
55 kD proteins that could be visualized by gel electrophoresis (Polansky,
j:It et al.;
in: Glaucoma i.Ipdate IV, Springer-Verlag, Berlin, pp. 20-29 (1991)).
The induction
kinetics of these proteins as well as their dose response characteristics
were similar
to the kinetics that were required for steroid-induced IOP eie~ration
in human
subjects (Polansky, J.R et al.; In: Glaucoma i,ipdate IV, Springer-Verlag,
Berlin; pp. 20-
29 (1991)). Problems of aggregation and apparent instability or
loss of protein in the
purification process were obstacles in obtaining a direct protein
sequence.
Because increased IOP is a readily measurable characteristic of
glaucoma, the
diagnosis of the disease is largely screened for by measuring intraocular
pressure
{tonometry) (Strong, N.P., Ophthal. Physiol. Opt. 22:3-7 (1992);
Greve, M. et al.; Can. J.
Ophthamol. 28:201-206 (1993)). Unfortunately, because glaucomatous
and normal
pressure ranges overlap, such methods are of limited value unless
multiple readings
are obtained (Hitchings, RA:, Br. j. Ophfhamol. 7T:326 (1993); Tuck,
M.W: et al.;
Ophthal. PhysioL Opt: 23:227-232 (1993); Vaughan, D. et al., In:
General Ophthamology,
Appleton & Lange, Norwalk, C"T, pp. 213-230 (1992); Vernon, S.A.,
Eye 7:134-137
(1993)). For this reason, additional methods; such as direct examination
of the optic
disk and determination of the extent of a patient's visual field
loss are soften
conducted to improve the accuracy of diagnosis (Greve, M. et aL,
Can. ]: Ophthamol.
28:201- 206 (1993)). Moreover, these techniques are of limited prognostic
value.
CA 02456777 2004-03-O1
Nguyen et al., U.S. Patent Number 5,789,169,
disclosed a novel protein sequence highly induced by
glucocorticoids in the endothelial lining cells of the human
trabecular meshwork. Nguyen et al., U.S. Patent Number
5,789,169 also disclosed the cDNA sequence for that
protein, the protein itself, molecules that bind to it, and nucleic acid
molecules that
encode it, and provided improved methods and reagents for diagnosing glaucoma
and related disorder, as well as for diagnosing other diseases or conditions,
such as
cardiovascular, immunological, or other diseases or conditions that affect the
expression or activity of the protein.
The present invention provides improved diagnostic agents, prognostic
agents.; therapeutic agents and methods.
S.IIMMARY OF THE L~.VE~(,O~j:
An object of the invention is to provide a method for diagnosing glaucoma in
a patient which comprises the steps: (A) incubating under conditions
permitting
nucleic acid hybridization: a marker nucleic acid molecule, said marker
nucleic and
molecule comprising a nueleotide sequence of a polynucleotide that
specifically
hxbridizes to a polxnucleotide that is linked to a TIER promoter, and a
complementary nucleic acid molecule obtained from a cell or a bodily fluid of
said
patient, wherein nucleic acid hybridization between said marker nucleic acid
molecule, and said complementary nucleic acid molecule obtained from said
patient
permits the detection of a polymorphism whose presence is predictive of a
mutation
affecting TIGR response in said patient; (B) permitting hybridization between
said
marker nucleic acid molecule and said complementary nucleic acid molecule
obtained- from said patient; and (C) detecting the presence of said
polymorphism,
wherein the detection of the polymorphism is cliagnostie of glaucoma.
Another object of the invention is to provide a method for , prognosing
glaucoma in a patient which comprises the steps: (A) incubating under
conditions
~0 permitting nucleic acid hybridization: a marker nucleic acid molecule, said
marker
nucleic acid molecule comprising a nucleotide sequence of a polynucleotide
that
specifically hxbridizes to a polynudeotide that is linked to a TIGR promoter,
and a
complernentar~,.r nucteic acid molecule obtained from a cell or a bodily fluid
of said
patient, wherein nucleic acid hybridization between said marker nucleic acid
3
CA 02456777 2004-03-O1
molecule, and said complementary nucleic add molecule obtained from said
patient
permits the detection of a poly~orpi~ism whose presence js predicgve of a
mutation
affecting TIGR response in said patient; {B) permitting hybridization between
said
marker nucleic acid molecule and said complementary nucleic acid molecule
obtained from said patient; and (C) detecting the presence of said
polymorphism,
wherein the detection of the polymorphism is prognostic of glaucoma. -
Another object of the invention is to provide marker nucleic acid molecules
capable of specifically detecting TIGRmtI, TlGRmt2, TIGRmt3, TIGRmt4, TIGRmtS
and TIGRsvI .
Another object of the invention is to provide a method for diagnosing steroid
sensitivity in a patient which comprises the steps: (A) incubating under
conditions
permitting nucleic acid hybridization: a marker nucleic and molecule, the
marker
nucleic acid molecule comprising a nucleotide sequence of a polynudeotide that
is
linked to a TIGR promoter, and a complementary nucleic acid molecule obtained
from a cell or a bodily fluid of the patient, wherein nucleic add
hybridization
between the marker nucleic add molecule, and the complementary nucleic add
molecule obtained from the patient permits the detection of a polymorphism
whose
presence is predictive of a mutation affecting TIGR response in the patient;
{B)
permitting hybridization between said TIER-encoding marker nucleic acid
molecule
and the complementary nucleic acid molecule obtained from the patient; and
(C).
detecting the presence of the polymorphism, wherein the detection of the
polymorphism is diagnostic of steroid sensitivity.
Further objects of the invention provide a nucleic acid molecule that
comprises the sequence of SEQ ID NO: 1, recombinant DNA molecules containing a
polynudeotide that specifically hybridizes to SEQ ID NO: 1 and substantially
purified molecules that specifically bind to a nueleic acid molecule that
comprises
the sequence of SEQ ID NO:1.
Further objects of the invention provide a nucleic acid molecule that
comprises the sequence of SEQ ID NO: 3, recombinant DNA molecules containing a
polynucleotide that specifically hybridizes to SEQ ID NO: 3 and substantially -
-- .-.
purified molecules that specifically bind to a nucleic add molecule that
comprises
the sequence of SEQ ID NO: 3.
Additional objects of the invention provide a nucleic acid molecule that
comprises the sequence of SEQ ID NO: 4, recombinant DNA molecules containing a
polynucleotide that specifically hybridizes to SEQ ID NO: 4 and substantially
4
CA 02456777 2004-03-O1
z
purified molecules that specifically bind to a nucleic acid
molecule that comprises the sequence of SEQ ID NO: 4.
Additional objects of. the invention provide a
nucleic acid molecule that comprises the sequence of
SEQ ID NO: 5, recombinant DNA molecules containing a
polynucleotide that specifically hybridizes to SEQ ID N0: 5
and substantially purified molecules that specifically bind
to a nucleic acid molecule that comprises the sequence of
SEQ ID N0: 5.
An additional object of the present invention is
to provide a method of treating glaucoma which comprises
administering to a glaucomatous patient an effective amount
of an agent that inhibits the synthesis of a TIGR protein.
Indeed, the molecules of the present invention may
be used to diagnose diseases or conditions which are
characterized by alterations in the expression of
extracellular proteins.
According to one aspect of the present invention,
there is provided a method for diagnosing glaucoma in a
sample obtained from a cell or a bodily fluid by detecting
mutants in the promoter region of the TIGR gene, comprising
the steps of: (A) incubating under conditions permitting
nucleic acid hybridization, a marker nucleic acid molecule,
said marker nucleic acid molecule having a nucleic acid
sequence that specifically hybridizes to a nucleic acid
molecule having the sequence of SEQ ID NO: 1, SEQ ID N0: 2,
or their complements, and a complementary nucleic acid
molecule obtained from a sample, wherein nucleic acid
hybridization between said marker nucleic acid molecule and
said complementary nucleic acid molecule permits the
detection of a polymorphism; (B) permitting hybridization
5
CA 02456777 2004-03-O1
between said marker nucleic acid molecule and said
complementary nucleic acid molecule; and (C) detecting the
presence of said polymorphism, wherein the detection of said
polymorphism is diagnostic or prognostic of glaucoma:
According to another aspect of the present
invention, there is provided a method for diagnosing steroid
sensitivity in a sample obtained from a cell or a bodily
fluid by detecting mutants in the promoter region of the
TIGR gene, comprising the steps of: (A) incubating under
conditions permitting nucleic acid hybridization, a marker
nucleic acid molecule, said marker nucleic acid molecule
having a nucleic acid sequence that specifically hybridizes
to a nucleic acid molecule having the sequence of
SEQ ID NO: 1; SEQ ID NO: 2, or their complements, and a
complementary nucleic acid molecule obtained from a sample,
wherein nucleic acid hybridization between said marker
nucleic acid molecule and said complementary nucleic acid
molecule permits the detection of a polymorphism;
(B) permitting hybridization between said marker nucleic
acid molecule and said complementary nucleic acid molecule;
and (C) detecting the presence of said polymorphism; wherein
the detection of said polymorphism is diagnostic of steroid
sensitivity.
According to still another aspect of the present
invention, there is provided a method for prognosing
glaucoma in a sample obtained from a cell or a bodily fluid
by detecting mutants in the promoter region of the TIGR
gene, comprising the steps of: (A) incubating under
conditions permitting nucleic acid hybridization, a marker
nucleic acid molecule, said marker nucleic acid molecule
having a nucleic acid sequence that specifically hybridizes
to a nucleic acid molecule having the sequence of
5a
CA 02456777 2004-03-O1
SEQ ID NO: 1, SEQ ID NO: 2, or their complements, and a
complementary nucleic acid molecule obtained from a sample,
wherein nucleic acid hybridization between said marker
nucleic acid molecule and said complementary nucleic acid
molecule permits the detection of a polymorphism;
(B) permitting hybridization between said marker nucleic
acid molecule and said complementary nucleic acid molecule;
and (C) detecting the presence of said polymorphism, wherein
the detection of said polymorphism is diagnostic or
prognostic of glaucoma.
According to yet another aspect of the present
invention, there is provided a use of an agent capable of
binding a cis element located within SEQ ID NO: 1 to treat
glaucoma.
According to yet another aspect of the present
invention, there is provided an oligonucleotide comprising a
nucleic acid fragment from about 15 to about 250 nucleotides
having a sequence of SEQ ID NO: 1, SEQ ID NO: 2, or
complements thereof.
According to a further aspect of the present
invention, there is provided an oligonucleotide that binds
to an about 15 to about 250 nucleotide fragment of
SEQ ID NO: l, SEQ LD NO: 2 or their complements, wherein
said oligonucleotide may be used for detecting the presence
of the TIGRmtl, TIGRmt2, TIGRmt3, TIGRmt4, TIGRmt5, or
TIGRsvI mutations.
According to yet a further aspect of the present
invention, there is provided a method for detecting the
specific binding of a molecule to a nucleic acid comprising
the steps of providing the nucleic acid as described herein,
contacting the nucleic acid with a sample containing the
5b
CA 02456777 2004-03-O1
molecule to be tested, and identifying binding of the
molecule to the nucleic acid.
According to still a further aspect of the present
invention, there is provided a method of prognosticating an
increased susceptibility to glaucoma, a progressive ocular
hypertensive disorder resulting in loss of visual field, or
the presence of steroid sensitivity in a patient by
determining the genotype of an individual with regard to the
presence of the TIGRmtl, TIGRmt2, TIGRmt3, TIGRmt4, TIGRm t5,
or TIGRsv2 mutations, said method comprising the steps of:
(a) providing a nucleic acid as described herein,
(b) contacting the nucleic acid with a sample containing the
molecule to be tested, and (c) identifying binding of the
molecule to the nucleic acid:
BRIEF DESCRIPTION OF THE FIGURES:
Figures 1A, 1B, 1C, 1D and 1E provide the nucleic
acid sequence of a TIGR promoter region (SEQ ID NO: 1) from
an individual without glaucoma.
Figures 2A, 2B, 2C and 2D provide the location and
sequence changes highlighted.in bold and underlined that are
associated with the TIGRm t1, TIGRm t2, TIGRmt3; TIGRmt4,
TIGRmtS, and TIGRsvI mutations in SEQ ID NO: 2.
Figures 3A, 3B, 3C, 3D, 3E, 3F, and 3G provide
nucleic acid sequences of a TIGR promoter, and TIGR exons,
TIGR introns and TIGR downstream sequences (SEQ ID NO: 3,
SEQ ID NO: 4, and SEQ ID NO: 5).
Figure 4 provides a diagrammatic representation of
the location of primers on the TIGR gene promoter for Single
Strand Conformational Polymorphism (SSCP) analysis.
5c
CA 02456777 2004-03-O1
Figure 5 provides a diagrammatic representation of
the TIGR exons and the arrangement of SSCP primers.
Figure 6 provides a homology analysis of TIER
homology with olfactomedin and olfactomedin-related
proteins.
Figure 7 shows the nucleotide sequence of TIGR
(SEQ ID N0: 26) .
Figure 8 shows the amino acid sequence of TIGR
(SEQ ID NO: 32) .
DETAILED DESCRIPTION OF TFiE INVENTION:
I. Agents of the Invention
5d
CA 02456777 2004-03-O1
As used herein, the term "glaucoma" has its art recognized meaning, and
includes both primary glaucomas, secondary glaucomas, juvenile glaucomas,
congenital glaucomas, and familial glaucomas, including, without limitation,
pigmentary glaucoma, high tension glaucoma and low tension glaucoma and their
related diseases. The methods of the present invention are particularly
relevant to
the diagnosis of POAG, OAG, juvenile glaucoma, and inherited glaucomas. The -
methods of the present invention are also particularly relevant to the
prognosis of
POAG, OAG, juvenile glaucoma, and inherited glaucomas. A disease or condition
is said to be related to glaucoma if it possesses or exh~its a symptom of
glaucoma,
20 for example, an increased intro-ocular pressure resulting from aqueous
outflow
resistance (see, Vaughan, D. et aL; in: General Ophtha'nology, Appleton &
Lange,
Norwalk, CT, pp. 2T3-230 (1992)). The preferred agents of the present
invention are
discussed in detail below.
The agents of the present invention are capable of being used to diagnose the
presence or severity of glaucoma and its related diseases in a patient
suffering from
glaucoma (a "glaucomatous patient'. The agents of the present invention are
also
capable of being used to progwose the presence or severity of glaucoma and its
related diseases in a person not yet suffering from any clinical
manifestations of
glaucoma. Such agents may be either naturally occurring or non-naturally
occurring. As used herein, a naturally occurring molecule may be
"substantially
purified," if desired, such that one,or more molecules that is or may be
present in a
naturally occurring preparation containing that molecule will have been
removed or
will be present at a lower concentration than that at which it would normally
be
found.
The agents of the present invention will preferably be "biologically active"
with respect to either a structural attribute, such as the capacity of a
nucleic acid to
hybridize to another nucleic acid molecule, or the ability of a protein to be
bound by
antibody (or to compete with another molecule for such binding).
Alternatively,
such an attribute may be catalytic, and thus involve the capacity of the agent
to
mediate a chemical reaction or response.
As used herein, the term '">3GR protein" refers to a protein having the amino
acid sequence of SEQ ID NO: 32. As used herein, the agents of the present
invention comprise nucleic acid molecules, proteins, and orgar<ic molecules.
As indicated above, the trabecular meshwork has been proposed to play an
important role in the normal flow of the aqueous, and has been presumed to be
the
6
CA 02456777 2004-03-O1
major site of outflow resistance in glaucomatous eyes. Human trabecutar
meshwork
(F~TA~I) cells are endothelial Like cells which line the outflow channels by
which
aqueous humor e~cits the eye; altered synthetic function of the cells may be
involved
in the pathogenesis of steroid glaucoma and other types of glaucoma. Sustained
S steroid treatment of these cells are interesting because it showed that a
major
difference was observed when compared to 1-2 day glucocorticoid (GC) exposure.
This difference appears relevant to the conical onset of steroid glaucoma (1-6
weeks).
Although trabecular meshwork cells had been found to induce specific
10~ proteins in response to glucocorticoids (see, Polansky, J.R.; In: "Basic
Aspects of
Glaucoma Restarch III", Schattauer, New York 307-318 (1993)), efforts to
purify the
expressed protein were encumbered by insolubility and other problems. Nguyen,
T.D: et aL, (In: "Basic Aspects of Glaucoma Research III", Schattauer, Nem
York, 331-343
(199-3y) used a molecular cloning approach to ' -
iS isolate a highly induced mRNA species from glueocorticoid-induced human
trabecular cells. . The mRNA exhibited a time course of induction that was
similar to
the glucocorticoid-induced proteins. The clone was designated "IL2" (ATCC No:
9994, American Type Culture Colle~ion,, Rocl~v~.le Maryland):
Nguyea et al., U.S. Pateat Number 5,171,788,
20 isolated a IL2 clone which enrnded a novel secretory protein that is
induced in cells
of the trabecular meshwork upon exp~ure to glucocorticoids. - It has been
proposed
that this protein may become deposited in the extracellular spaces of the
trabecular
meshwork and bind to the surface of the endothelial cells that line the
trabecular
meshwork, thus causing a decrease in aqueous #low. 6~uantitative dot blot
analysis
2S. and PCR evaluations have shown that the mRNA exhibits a progressive
induction
yvith time whereas other known GC=inductions from other systems and found in
HTM cells (metallothionein, alpha-1 acid glycoprotein and alpha-1
antichymotrypsin) reached maximum level at one day or earlier. Cf particular
interest, the induction level of this clone was very high (4-6% total cellular
mRNA)
30 with control levels undetectable without PCR method: Based on studies of
3SS
methionine cell labeling; the clone has the characteristics recently
discovered for the
major GC-induced extracellular glyaoprotein in these cells, which is a
sialenated, N-
glycosylated molecule with a putative inositol phosphate anchor: The induction
of
mRNA agproa~ched 4% of the total cellular mRNA. The mRNA increased
3S progressively over 10 days of dexamethasone treatment. The IL2 clone is 2:0
Kb
7
CA 02456777 2004-03-O1
whereas the Northern blotting shows a band of .2.~5 Kb. Although not including
a
poly A tail; the 3' end ~of the clone contains two consensus polyadenylation
signals.
A genomic clone was isolated and designated PITIGR clone (A?'CC No:
97570, American Type Culture Collection, Rockville; Maryland). In-situ
hybridization using the P1TIGR clone shows a TfGR gene and/or a sequence or
sequences that specifically hybridize to the TIGR gene located at chromosome
1,
q21-27, and more preferably to the T'1GR gene located at chromosome 1, q~2-26,
and
most preferably to the TIGR gene located at duomosome 1, q24. Clone PiTIGR
comprises human genomic sequences that specifically hybridize to the TIGR gene
cloned into the BamHI site of vector pCYPAC (ioaru~ou ef al., fVature
Gene~fics, fi:84-89
(1994) ) .
.As used herein, the term "TIGR gex~p refers to the region of DNA involved
in producing a TiGR protein; it includes, without limitation, iaegions
pr~eceeding and
following the coding region as well as intervening sequences between
individual
coding regions.
As used herein, the term "TIGR axon" refers to any interrupted region of the
TIGR gene that serves as a template for a mature 3'IGR mRNA molecule. As used
herein, the term "TIGR intron" refers to a region of the TIGR gene which is
non-
coding and serves as a template for a TIGR mRNA molecule.
Localization stuclies using a Stanford G3 radiation hybrid panel mapped the
TIGR gene near the D1S2536 marker with a LOD score of 6:0 (Richard e# aL,
American Journal of Human Genetics 53:5: 915-921 (i993),r
Frazer et al., Genomics 14.3: 574 578 (1992) ~ .
Research Genetics, Huntsville, Alabama): Other markers in this region
include: D1S210; D1S1552; DIS2536; DIS2790; SHC~-12820; and D18.
Sequences located upstream of the ~'IGR coding region are isolated and
sequenced in a non-glaucomic individual. The upstream sequence is set forth in
SEQ ID. No: 1. Sequence comparisons of the upstream .region of a non
:.glaucoma
individual and individuals with glaucoma identify a number of anutations in
individuals with glaucoma. These mutations are illustrated in'~'rigure 2. Five
mutations are identified. TIGRmf1 is the result of a replacement of a cytosine
with a
guanine at position 4337 (SEQ ID NO: 1, SEQ ID NO: 2, and SEQ TD iyl0: 3).
TIGRrnt2 is the result of a replacement of a cytosine with a thvfr~~,~e afi
position 4950
(SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3)» TIGRmf3 is the result of an
addition in the following order of a guanine, a thymine, a guanine, and a
thymine
8
CA 02456777 2004-03-O1
(GTGT) at position 4998 {SEQ ID NO: I, SEQ ID NO: 2, and SEQ ID NO: 3):
TIGRmt4 is the result of a replacement of an adenine with a guanine at
position 4256
{SEQ ID NO: 1, SEQ ID NO: 2; and SEQ ID NO: 3). TIGRmtS is the result of a
replacement of a guanine with an adenine at position 4262 {SEQ ID NO: 1, SEQ
ID
NO: 2 and SEQ iD NO: 3). One or more of TIGRmt I, TIGRmf2, TIGRrnt3, TTGRmt4,
and TIGRmfS canbe homozygous or heterozygous.
Sequence comparisons of the upstream region of a non-glaucoma individual
and individuals with glaucoma identify at least one sequence variation in
individuals with glaucoma. One such sequence variant is illustrated in Figure
2.
TlGRsvI is the result of a replacement of an adenine with a guanine at
position 4406
(SEQ ID NO: I, SEQ ID NO: 2 and SEQ ID NO: 3).
Molecules comprising sequences upstream of the TIGR coding region
provide useful markers for polymorphic studies. Such molecules include primers
suitable far single strand conformational polymorphic studies; examples of
which
are as follows: forward primer "Sk-la": 5'-TGA GGC TTC CTC TGG AAA C-3' (SEQ
ID NO: 6); reverse primer "cat": 5'-TGA AAT CAG CAC ACC AGT AG3' (SEQ ID
NO: 7); forward primer "CA2": 5'-GCA CCC ATA CCC CAA TAA TAGS' (SEQ ID
NO: 8); reverse primer "Pr+1 ": 5'-AGA GTT CCC CAG ATT TCA CC-3' (SEQ ID
NO: 9); forward primer NPr-1": 5' ATC TGG GGA ACT CTT CTC AG ;3' (SEQ ID
NO: 10); reverse primer "Pr+2(4A2)": 5'-TAC AGT TGT TGC AGA TAC G3' (SEQ
ID NO: 11); forward primer °'Pr-2{4A)": 5'-ACA ACG TAT CTG CAA CAA
CTG3'
(SEQ ID NO: 12); reverse primer "Pr+3(4A)": 5'-TCA GGC TTA ACT GCA GAA
CC-3' (SEQ ID NO: I3); forward primer "Pr-3(4A)": 5'-TTG GTT CTG CAG TTA
AGC C-3' (SEQ ID NO: I4); reverse primer "Pr+2(4A1)": 5'-AGC AGC ACA AGG
GCA ATC C-3' (SEQ ID NO: 15); reverse primer "Pr+1(4A)": 5'-ACA GGG CTA
TAT TGT GGG3' (SEQ ID NO: I6).
In addition, molecules conlprisiztg sequences within TIGR exons provide
useful markers for polymorphic studies. Such molecules include primers
suitable
for single strand conformational polymorphic studies, examples of which are as
follows: forward primer "KSIX": 5'-CCT GAG ATG CCA GCT GTC C=3' (SEQ ID
NO: I7); reverse primer "SK1XX": 5'-CTG AAG CAT TAG AAG CCA AC 3' (SEQ
ID NO: 18); forward primer "KS2a1": 5'-ACC TTG GAC CAG GCT GCC AG3'
(SEQ ID N0:19); reverse primer "SK3" 5' AGG TTT GTT CGA GTT CCA G3' (SEQ
ID NO: 20); forward primer "KS4": 5'-ACA ATT ACT GGC AAG TAT GG3' (SEQ
ID NO: 21 ); reverse primer "SK6A": 5'-CCT TCT CAG CCT TGC TAC C-3' (SEQ ID
9
CA 02456777 2004-03-O1
NO: 22); forward primer "KS5": 5'-ACA CCT CAG CAG ATG CTA CC-3' (SEQ ID
NO: 23); reverse primer "SK8": 5'-ATG GAT GAC TGA CAT GGC C 3' (SEQ ID NO:
24); forward primer "KS6": 5'-AAG GAT GAA CAT GGT CAC C-3' (SEQ ID NO:
The locations of primers: Sk-1 a, cat, CA2, Pr+1, Pr-1, Pr+2(4A2), Pr-2(4A);
Pr+3(4A), Pr-3(4A), Pr-3(4A), Pr+2(4AI), and Pr+1(4A) are diagramatically set
forth
in Figure 4. The location of primers: KSIX, SKIXX, Ks2al, SK3, KS4, SK6A, KSS,
SKB, and KS6 are diagramatically set forth in Figure 5.
The primary structure of the TIGR coding region initiates from an ATG
IO initiation site (SEQ iD N0:3, residues 5337-5339) and includes a 20 amino
acid
consensus signal sequence a second ATG (SEQ ID NO: 3, residues 5379-5381),
indicating that the protein is a secretory protein. The nucleotide sequence
for the
TIGR coding region is depicted in Figure 7 (SEQ ID NO: 26). The protein
contains
an N-linked glycosylation site located in the most hydrophilic region of the
molecule. The amino terminal portion of the protein is highly polarized and
adopts
alpha helical structure as shown by its hydropathy profile and the Gamier-
Robison
structure analysis. In contrast, the protein contains a 25 amino acid
hydrophobic
region near its carboxy terminus. This region may comprise a glucocorticoid
induced protein (GIP) anchoring sequence. The amino acid sequence of TIGR is
depicted in Figure 8 (SEQ ID NO: 33).
Study of cyclohexamide treatment in the absence and presence of GC suggest
that the induction of TIGR may involve factors in addition to the GC receptor.
The
TIGR gene may be involved in the cellular stress response since it is also
indueed by
stimulants such as H202, 12-0-tetradecanolyphorbol-I3-acetate (TPA), and high
glucose; this fact may relate to glaucoma pathogenesis and treatment:
Sequence comparison of the upstream region identify a number of DNA
motifs (cis elements). These DNA motifs or cis elements are shown in Figure 1:
These motifs include, without limitation, glucocorticoid response motif(s),
shear
stress response motif(s), NFxB recognition motif(s), and API motif(s). The
locations
of these and other motifs axe diagramaticaily set forth in Figure 1. As used
herein,
the term "cis elements capable of binding" refers to the ability of one or
more of the
described cis elements to specifically bind an agent: Such binding may be by
any
chemical, physical or biological interaction between the cis element ar,d the
agent,
including, but not limited, to any covalent, steric, agostic, electronic and
ionic
interaction between the cis element and the agent. As used herein, the term
IO
CA 02456777 2004-03-O1
"specifically binds" refers to the ability of the agent to bind to a specified
cis element
but not to wholly unrelated nucleic acid sequences.
A preferred loss of agents comprises TIGR nucleic acid molecules (NTIGR
molecules'. Such molecules may be either DNA or RNA. A second preferred class
5._. of agents ('"ITGR molecules") comprises the TIGR protein, its peptide
fragments,
fusion proteins, and analogs.
Expression of the rat PRL gene is highly restricted to pituitary lackotroph
cells
and is. induced by the CAMP-dependent protein lcinase A pathway. At least one
of
the redundant pituitary specific elements (PRL-FP11I) of the proximal rat PRL
promotor is required for this protein kinase A effect (Rajnarayan et aL,
Molecular
Endochrvnoiogy 4: 502-512 (1995), herein incorporated by reference). A
sequence
corresponding to an upstream motif or cis element characteristic of PRL-Fell l
is set
forth in Figure 1 at residues 370-388 and 4491-4502, respectively. In
accordance
with the embodiments of the present invention, transcription of TIGR molecules
can
be effected bx agents capable of altering the biochemical properties or
concentration
of molecules that bind the PRL-FPlll upstream motif or cis element: Such
agents
can be used in the study of glaucoma pathogenesis. In another embodiment, such
agents can also be used in the study of glaucoma prognosis. In another
embodiment
uch agents can be used in the treatment of glaucoma.
A consensus sequence (GR/PR), recognized by both the glucocorticoid
receptor of rat liver and the progesterone receptor from rabbit uterus, has
been
reported to be involved in glucocorticoid and progesterone-dependent gene
expression (Von der Ahe ef aL, Nature 3t3: 706-7~09~ (1985'). __
A. sequence corresponding to a GC/PR upstream .motif or cis element is
set forth in Figure 1 at residues 433-44,5. In accordance with the embodiments
of the
present invention; transcription of TIER molecules can be effected by agents
capable
of altering the biochemical properties or concentration of glucocorticoid or
progesterone or their homologues, including, but not limited to, the
concentration of
glucocorticoid or progesterone or their homologues bound to an GC/PR upstream
34 motif or cis element. Such agents can be used in the study of glaucoma
pathogenesis: In another embodiment, such agents can also be used in the study
of
glaucoma prognosis. In another . embodiment such agents can be used in the
treatment of glaucoma.
Shear stress motif (SSRE) or cis element has been identified in a number of ;.
genes including platelet-derived growth factor B chain, tissue plasaiinogen
activator
11
CA 02456777 2004-03-O1
(tPA), ICAM-1 and TGF-~1 (Resnick et aL, Proc. NatL Acad. Sci. (LISA) 80: 4591-
4595) .
3'ranscription of these genes has been
assotiated with humoral stimuli such as cytolcines and bacterial products as
well as
hemodynamic stress forces. Sequences corresponding to a upstream shear stress
motif or cis element are set forth in Figure 1 at residues 44G-4a1, 128&1293,
3597-
3602, 4~1-477b, and 5240-5245, respectively. In accordance with the
:embodiments .
of the present invention, transcription of TdGR molecules can be effected by
agents
capable of altering the biochemical properties or concentration of
~olecules~capable
of binding the shear stress motif. Such agents can be used in the study of.
glaucoma
pathogenesis. in another embodiment, such agents can also be used in the study
of
glaucoma prognosis. In another embodiment such agents can be used in the
treatment of glaucoma.
A consensus sequence for a glucocorticoid response upstream motif (GRE) or
cis element has been characterized (Beato, CeII 56: 335-344 (1989); Beclcer et
al:,
Nature 324: 686-688 (1986), herein incorporated by reference; Salcai et aL,
Genes acrd
DeveIoprnent Z: II44-1154 (I988), herein incorporated by reference). Genes
containing this upstream motif or cis element are regulated by
glucocorticoids,
progesterone, androgens and mineral corticoids (Beato, Cel156: 335-344
(1989)). .
Sequences corresponding to glucocorticoid response upstream 'motif or cis
element
are set forth in Figure 1 at residues 574-600,1042-1055, 2444-2468, 2442-?369,
3536-
3563, 4574-4593, 4595-4b14, 4851-4865; 4844-4864, 5079 X084; and 5083-5111,
respectively. In accordance with the embodiments of the present invention,
transcription of TIER molecules can be effected by agents capable of al#ering
the
biochemical properties or concentration of molecules capable of binding a
glucocorticoid response upstream motif or cis element. ~5uch agents can be
used in
the study of glaucoma pathogenesis. In another embodiment, such agents can
also
be used in the study of glaucoma prognosis. In another embodiment such ag~nfs
can be used in the treatment of glaucoma.
A sequence specific binding site (CBE) for the wild type nuclear
phosphoprotein, p53, has been identified and appears to be ~ associated with
replication origins (Kern et aI. Science 252: 1708-1711 (1991~))~
A sequence rnrresponding to an C8E upstream motif or cis element is
set forth in Figure 1 at residues 735-74b. In accordance with the embadunents
of the
present invention, transcription of TIGR molecules can be effected by agents
capable
of altering the biochemical properties or eoncentration of p53 or its
homologues,
12
CA 02456777 2004-03-O1
including, but not limited to, the concentration of p53. or its homologues
bound to
an CBE upstream motif or cis element. Such agents can be used in the study of
glaucoma pathogene..~is. in another embodiment, such agents can also be used
in
the studg of glaucoma prognosis. In another embodiment such agents can be used
in the treatment of glaurnma.
Nuclear factor ets-like (NFE); a transcriptional activator that facilitates
p50
and c-Rel-dependent IgH 3' enhancer activit~r has been shown to bind to an NFE
site
in the Rel-dependent IgH 3' er~han~r (Linderson et aL; European j. lrnmunoiogy
27:
468-475 (199~.j . A sequence corresponding to an
NF'E upstream motif or cis element is set forth in Figure, l at residues 774-
795. In
accordance with the embodi~atents of the present invention, transcription of
TIGR
molecules. can be effected by agents capable of altering the biochemical
properties or
concentration of nuclear factors or their homologues, including, but not
limited to,
the concentration of nuclear factors or their homologues bound to an NFE
upstream
motif or cis element. Such agents can be used in the study of glaucoma
pathogenesis. In another embodiment, such agents can also be used in the study
of
glaucoma prognosis. In another embodiment such agents can be used in the
treatment of glaucoma.
An upstream motif or cis element (K'TF.1-CS) for a control element 3' to the
human keratin 1 gene that regulates cell type and differentiation-specific
expression
has been identified (Huff et al., J. Biological Chemistry 268: 377 3$4 (1993)
) .
A sequence corresponding to an upstream motif or cis
element characteristic of KTF.I-CS is set forth in Figure 1 at residues 843-
854: In
accordance with the embodiments of the present invention, transcription of
TIGR
molecules can be effected bx agents capable of altering the biochemical
prorperties or
concentration of KTF.1-CS or its homologues, including, but not limited to,
the
concentration of K'TF.I-CS. or its homologues bound to a KTF.1-CS upstream
motif
or cis element Such agents can be used in the study of glaucoma pathogenesis.
In
another embodiment, such agents can also be used ~in the study of glaucoma
prognosis. In another embodiment such agents can be used in the treatment of
glaucoma.
A progesterone responsive element (PRE) that maps to the far upstream
steroid dependent DNase hypersensitive site of chicken lysozyme chromatin has
been characterized (Hecht et aL, EMBO J. 7:2063-2073 (1988).:) .
The element confers hormonal regulation to a heterologous promoter
I3
CA 02456777 2004-03-O1
and is composed of a cluster of progesterone receptor binding sites. A
sequence
corresponding to an ups~irearn motif or cis element characteristic of P~ is
set forth
in Figure 1 at residues 987-1026. In accordance with the embodiments of the
present invention, transcription of TiGR molecules can be effected by agent
capable
of altering the biochemical properties or concentrati~ri of molecules capable
of
binding a progesterone responsive PRE upstream motif or cis.element. Such
agents
may be useful in the study of glaucoma pathogenesis: In another e~bodia~ent,
such
agents can also be used in the study of giaurnma prognosis. In another
eriibodiment
such agents cain be used in the treatment of glaucoma.
A sequence (ETP-EGFR) has been characterized which serves as a motif ~'ror a
traps-active transcription factor that regulates expression of the epidermal
growth
factor receptor (Regec et al., Blood 85:2711-2719 (199'a~):
A sequence corresponding to an ETF-EGFR upstream motif or cis
element is set forth in Figure 1 at residues 1373-1388. In accordance v~ith
the
embodiments of the present invention, transcription of 'T1GR molecules can be
effected by agents capable of altering the biochemical properties or
concentration of
nuclear factors or their homologues, including, but not limited to, the
concentration
of nuclear factors or their homologues bound to an ETF-EGFR upstream motif
or~cis
element. Such agents can be used in the study of glaucoma pathogenesis. In
another embodiment, such agents can also be used in the study of glaucoma
prognosis. In another embodiment such agents can be used in the treatment of
glaucoma.
A common traps-acting factor (SRE-cFos) has been shown to regulate skeletal
and cardiac alpha-Actin gene transcription in muscle (A~Iuscat ef al.;
Molecular and
Cellular Biology 10: 4120-4133 (1988)) - A sequence
corresponding to an SRE-cFos upstream motif or cis element is set forth in
figure 1
at residues 1447-1456. In accordance with the embodiments of the present
invention, transcription of TiGR molecules can be effected by agents capable
of
altering the biochemical properties or concentration of nuclear factors or
their
homologues, including, but not limited to, the concentration of nuclear
'factors or
their homologues bound to an SRE-cFos upstream motif or cis element. Such
agents
can be used in the study of glaucoma pathogenesis. In another embodiment, such
agents can also be used in the study of glaucoma prQ~osis. In
another~embodiment
such agents can be used in the treatment of ~giaumma.
14
CA 02456777 2004-03-O1
Alu repetitive elements are unique to primates and are interspersed within
the human genome with an average spacing of 4Kb. While some Alu sequences are
actively transcribed bx polymerase III; normal transcripts may also contain
Alu
derived sequences in 5' or 3' untranslated regions (Jurka and Mikahanljaia, J:
MoI.
Evolution 32: 105-121 (1991j, Claveria and
Makalowski, Nature 3~I: 251-752 (1994)?, A
sequence corresponding to an Alu upstream motif or Gis element is set forth in
Figure 1 at residues 1331-1550. In accordance with the embodiments of the
present
invention, transcription of TIER molecules can be effected by agents capable
of
altering the biochemical properties or concentration of nuclear factors or
their
homologues, including, but not limited to, the concentration of nuclear
factors or
their homologues. bound to an Alu upstream motif or cis element. Such agents
can
be used in the study of glaueoma pathogenesis. In another embodiment, such
agents can also be used in the study of glaucoma prognosis. In another
embodiment
such agents can be used in the treatment of glaucoma.
A consensus sequence for a vitellogenin gene-binding protein (VBP)
upstream motif or cis element has been characterized (Iyer et aL, Molecular
and
Cellular Biology T 1: 4863-48?5 (1991)x. Expression
of the VBP gene commences early in liver ontogeny and is not subject to
circadian
control. A sequence corresponding to an upstream motaf or cIS element capable
of
binding VBP is set forth in Figure 1 at residues 1?86-1797. In accordance with
the
embodiments of the present invention, transcription of TIER molecules can be
effected by agents capable of altering the biochemical properties or
concentration of
VBP or its homologues, including, but not limited to, the concentration of VBP
or its
homologues bound to an VBP upstream motif or cis element Such agents can be
used in the study of glaucoma pathogenesis. In another embodiment, such agents
can also be used in the study of glaucoma prognosis. In another embodiment
such
agents can be used in the treatment of glaucoma.
A structural motif (Malt-CS) or cis element involved in the -activation of all
promoters of the maltose operons in Escherichia toll and KlebsieIIa pneunwniae
has
been characterized (Vidal-Ingigliardi et aL, J. Mol: Biol. 2I8: 323-334
(1991),x,
A sequence corresponding to a upstream Malt-CS motif
or crs element is set forth in Figure 1 at residues 1832-1841. In accordance
with the
embodiments of the present invention, transcription of TIGR molecules can be
effected by agents capable of altering the biochemical properties or
concentration of
CA 02456777 2004-03-O1
molecules capable of binding the upstream Malt-CS motif or cis element. Such
agents can be used in the study of glaucoma pathogenesis. in another
embooiment,
such agents can also be used in the study of glaucoma prognosis. In another
embodiment such.agents can be used in the treatment of glaurnma.
A consensus sequence for an estrogen receptor upstream motif or cis ele;nent
has been characterized (ERE) (Forman ef al., MoI. ~ndocri»olagy 4: 1293-2301
(199U) ;
de Verneuii et aL, Nucleic Acid Res. 38: 4489-4497
(1990), herein incorporated by refs; Gaub ef aL, Cell'63:1~67 176 (19900:
A sequence corresponding to half an upstream motif or
cis element capable of binding estrogen receptor is set forth in Figure 1 at
residues
2166-2195, 3413-3429, and 3892-3896, respectively. In accordance with the
embodiments of the present invention, transcription of TTGR molecules can be
effected b~ agents capable of altering the biochemical properties or
concentration, of
the estrogen receptor or its homologues bound to an upstream motif or cis
element.
Such agents can be used in the study of glaucoma pathogenesis. In another
embodiment; such agents can also be used in the study of .glaucoma prognosis.
In
another embodiment such agents can be used in the treatment of glaucoma.
Certain proteiirbinding sites (NF mutagen) in Ig gene enhancers which
determine transcriptional activity and induc~ility have been shown to interact
with
nuclear factors (Lenardo et aL, Science X236:1573-1'577 (198Tj ) .
A sequence corresponding to an NF mutagen upstream motif or cis
element is set forth in Figure 1 at residues 2329-2338. In accordance with the
embodiments of the present invention, transcription of TTGR molecules van be
effected by agents capable of altering the biochemical propertses or
concentration of
nuclear factors or their homologues, including, but not limited to, the
concentration
of nuclear factors or their homologues bound to an NF-mutagen ups#ream motif
or
cis element. Such agents can be used lm the study of glaucoma pathogenesis. In
another embodiment, such agents can also be used in the study of glaucoma
prognosis. In another embodiment such agents carp be used in the trea#~nent of
glaucoma.
A consensus sequence for a transcriptional repressor of c-myc ~~(myc-PRF)
upstream motif or cis element has been identified (ICalckis.et al., Nature
339: 718-719
(3989)) , Myc-PRF interacts wig another widely ' v
distributed protein, myc-CFl (comalon factor 1), which binds nearby and this
3'~ association may be important in myc-PRF repression A sequence
corresponding to
w
1,6
CA 02456777 2004-03-O1
an upstream motif or cis element capable of binding myc-PRF is set forth in
Figure 1
at residues 2403-241b. In accordance with the embodiments of the present
invention, transcription of TIGR molecules can be effected by agents capable
of
altering the biochemical properties or concentration of myc-PRF or its
homologues,
including, but not limited to, the concentration of myc-PRF or its homologues
bound
to an mxc-PRF upsfream motif or cis element Such agents can be used in the
study
of glaucoma pathogenesis. . In another embodiment, such agents can also be
used in
the study of glaucoma prognosis. In another embodiment such agents can'be used
in the treatment of glaucoma.
Human transcription factor activator protein 2 (AP2) is a transcription factor
that has been shown to bind to Spl, nuclear factor 1 (NFl) and simian virus 40
transplantation (SV40 T) antigen binding sites. It is developmentally
regulated
(Williams and Tijan, Gene Dev. 5: 670-682 (1991);
Mitchell et aL, Genes Dea. 5: 105-7:19 (1991); Coubois
ef al., Nucleic Acid Researcls I8: 57-64 (1990); Comb
et al., NucI'eic Acid Research I8: 39?5 3982 (1990);
Winings et aL, Nucleic Acid Research I9: 3709 3714 (1991) ) .
Sequences ~rresponding to an upstream motif or cis element capable
of binding AP2 are set forth in Figure 1 at residues 2520-2535, and '5170-
5187,
respectively. In accordance with the embodiments of the present invention,
transcription of TIGR molecules can be effecfied by agents capable of
altering.the
biochemical properties or concentration of AP2 or its homologues, including,
but
not limited to, the concentration of AP2 or its homologues bound to an
upstream
motif or cis element. Such agents may be useful in the study of glaucoma
pathogenesis. In another embodiment, such agents can also be used in the study
of
glaucoma prognosis. In another embodiment such agents can be used in the
treatment of glaucoma.
Drosophila RNA polymerase II heat shock transcription factor (I~STF) is a
transcription factor that has been shown to be required for active
transcription of an
hsp 70 gene (Parker and Topoi, CeII 37:. 273-283 (1984) ) .
vequences corresponding to an upstream motif or cis element capable of
binding HSTF .are set forth in Figure 1 at residues 2622-2635, and 5105-SI32.
In
accordance with the embodiments of the present invention, transcription of
TIGR
f
molecules can be effected by agents capable of altering the biochemical
properties or
concentration of HSTF or its homologues, including, but not limited to, the
17
CA 02456777 2004-03-O1
concentration of I~ or its homologues bound to an ~STF upstream motif or cis
element. Such agents can be used in the study of glaucoma pathogenesis. In
another embodiment, such agents can also be used in the study of . glaucoma
prognosis. In another embodiment such agents can be used in the treatment of
glaucoma. ~ w
A sequence corresponding to an upstream motif or cis element characteristic
of SBF is set forth in Figure 1 at residues 2733-2743 (Shore et al., I:MBO j.
t;: 461-467
(1987)). In accordance with the embodiments of
the present invention, transcription of TIGR molecules can be effected by
agents
capable of altering the biocheaucal properties or concentration of molecules
that
bind the SBF upstream motif or cis element. Such agents can be used in the
study of
glaucoma pathogenesis. In another embodiment, such agents can also be used in
the study of glaucoma prognosis. In another embodiment such agents can be used
in the treatment of glaucoma.
IS An NFl motif or cis element has been identified which recognizes a family
of
at least six proteins (Courtois, ef aL, Nucleic Acid Res. 18: 57-64 tI990);
Mul et aL, j. Virol. b4: X510=6518 (1990);
Rossi et al., Cell 52: 405-414 (1988) ;
Gounari et al., EMBO j. L0: 559-5b6 (1990);
Goyal et aL, MoI. Cell BioL 10: 1041-1048 (1990); .
Mermond et aL, Nature 332: 557-561 (1988);
Gronosta~ski ef al:; Molecular and Cellular Biology 5: 9b4-971 (1985) ;
Hennighausen ef aL, EMBO j. 5: 1367-1371 (198~~. ;
Chodosh et aL, CeII a3: 11-24 (I988)> .
The IVFl protein will bind to an NFl motif or cis element either as a
dimer (if the motif is palindromic) or as an single molecule (if the motif is
not
palindromic). The IVF1 protein is induced by TGF~ (Faisst and Meyer, Nucleic
Acid
~ Z0: 3-26 (1992). Sequences correespotiding
to an upstream motif or cis element capable of binding ~TFI are set forth in
Figure 1
at residues 2923-2938, 4143-4167, and 4886-4900, respectively. in accordance
with
the embodiments of the present invention, transcription of ~TGR molecules can
be
effected by agents capable of altering the biochemical properties or
concentration of
NFl or its homologues, including; but not limited to, the roncentr anon of ~3
or i~
homologues bound to an upstream motif or cis element. 'Such agents can be used
in
the study of glaucoma pathogenesis. In another embodiment, such agents can
also
18
CA 02456777 2004-03-O1
be used in the study of glaucoma prognosis. In another embodiment such agents
can be used in the teeatment of glaucoma.
Conserved regulatory sequences (NF-MHCIIA/B} of a rabbit major
histocompatability complex (MHC) class II gene are responsible for binding two
5. ~distlnct nuclear factors NF-MI~iCIIA and NF-MfICIIB and are believed to be
- involved in the regulation of coordinate expression of the class II genes -
eg. MHC
class II gene in B lymphocytes (Sittisombut Molecular and Cellular Biology 5:
2034
2041 (1988) ) . A sequence corresponding to an NF
MHCIIA/B upstream motif or cis element is set forth in Figure 1 at residues
2936
10~ 2944. In accordance with the embodiments of the present invention,
.transcription of
TIER molecules can be effected by agents capable of altering the biochemical
properties or concentration of NF-MHCIIA or NF-MHCITiB or their homologues,
including, but not limited to, the concentration of NF-MHCIIA or NF-MHCIIB or
'their homologues bound to an NF-MI-ICIIA/B upstreaan motif or cis element.
Such
15 agents can be used in the study of glaucoma pathogenesis. 1n another
embodiment;
such agents can also be used in the study of glaucoma prognosis. In another
embodiment such agents can be used in the treatment 'of glaucoma.
PEA 1 binding motifs or cis elements have been identified (Piette and Yaniv,
EMBO j. 5:1331-1337 (198, herein incorporated by reference). The PEAL protein
is
20 a transcription factor that is reported to bind to both the polyoma virus
and c~'os
enhancers A sequence corresponding to an upstream motif or cis element capable
of binding PEAL is set forth in Figure 1 at residues 3285-3298. In accordance
with
the embodiments of the present invention, transcription of TIGR molecules can
be
effected by agents capable of altering the biochemical properties or
concentration of
25. PEAL or its homologues, including, but not limited to, the concentration
of PEAL or
its. homologues bound to an upstream motif or cis element. Such agents can be
used
in the study of glaucoma pathogenesis. In another embodiment, such agents can
also be used in the study of glaucoma prognosis. In another embodiment such
agents can be used in the treatnnent of glaucoma.
30 A conserved cis-acting regulatory element (ICS} has been shown to bind
traps-acting constituitive nuclear factors present in lymphocytes and
fibroblasts
which are involved in the interferon ((IFNNj-mediated transcriptional
enhancement of
MF3C lass I and other genes (Shirayoshi et aL, Proc. Nafl. Acad. Sci. (LISA)
85: 5884-
5888 (1988}) .. A sequence corresponding to an ICS '
3S upstream motif or cis element is set forth in Figure 1 at residues 3688-
3699. In
19
CA 02456777 2004-03-O1
accordance with the embodiments of the present invention, transcription .of
TIGIt
molecules can be effected by agents capab2e of altering the biochemical
properties or
concentration of nuclear factors or their homologues, including, but not
limited to,
the concentration of nuclear factors or their homologues bound to an ICS
upstream
motif or cis element. Such agents can be used in the study of glaucoma
pathogenesis. In another embodiment, such agents can also be used in he study
of
glaucoma prognosis. In another embodiment such agents can be used in the
treatment of glaucoma.
A consensus sequence for an ISGF2 upstream motif or cis element has been
characterized (Iman ef aL, Nucleic Acids Res.18: 6573~r~80 X1990);
Harada ef aL, CeII 63: 303-312 (1990);
Yu-Lee et aL, Ivlol. CeII Biol. 10: 3087-3094 (1990),
Pine et al., MoI. Cell Biol. I0: 32448-2457 (1990) )
ISGF2 is induced by interferon a and 7, prolactin and virus infections. A
sequence
corresponding to an upstream motif or cis element capable of binding ~2 is set
forth in Figure 1 at residues 4170-4179. In accordance with the embodiments of
the
present invention, transcription of TIGR molecules can be effected by
agents.~capable
of altering the biochemical properties or concentration of ISGF2 or its
homologues,
including, but not limited to, the concentration of i9GF2 or its homologues
bound to
an upstream motif or cis element Such agents can be used in the study of
glaucoma
pathogenesis. In another embodiment, such agents can also be used in the s#udy
of
glaucoma prognosis. In another embodiment such agents can be used in the
treatment of glaucoma.
A sequence corresponding to an upstream motif or cis element capable of
binding zinc is set forth in Figure 1 at residues 4285-492. In accordance with
the
embodiments of the present invention, transcription of TIGR molecules can be
effected by agents capable of altering the biochemical properties or
concentration of
zinc: Such agents can be used in the study of glaucoma pathogenesis. In
another
embodiment, such agents can also be used in the study of glaucoma prognosis.
In
another embodiment such agents can be used in the treatment of glaurnma.
A sequence corresponding to an upstream motif or cis element characteristic
of C:AP/CRP-gal0 is set forth in Figure 1 at residues 4379-4404 (Taniguchi et
al:,
Proc. Natd. Acad. Sci (LISA) ?6: 5090-5094 (1979 7 - In
accordance with the embodiments of the present invention, transcription of ~iG
molecules can be effected by agents capable of altering the biochemical
properties or
CA 02456777 2004-03-O1
concentration of molecules that bind the CAP/CRP-gal0 upstream motif or cis
element. Such agents can be used in the study of glaucoma pathogenesis. In
another embodiment, such agents can also be used in the study of glaucoma
prognosis. In another embodiment such agents can be used in the treatment of
giaucroma.
Human transcription factor activator protein 1 (API) is a transcription factor
that has been shown to regulate genes, which are highly expressed in
transformed
cells such as stromelysin, c 'os; al-anti-trypsin and collagenase (Gutman and
4llasxixk, EMBD j. ~:7: 2241-2246 (1990}, herein incorporated by reference;
Martin et
al:, Proc. Natl. Acrid. Sri. LISA 85: 5839-5843 (1988), herein incorporated by
reference;
Jones et al:, Genes and Development 2: 267-281 (1988)
Faissx and Meyer, Nucleic Acid Research 20: 3-26 (1992);
-Kim et aL, Molecular and Cellular Biology I0: 1492-1497
(1990) Baumhueter et al., ~EMBO j. 7: 2485-2493
(19$x8)). The APl transcription factor has been
associated with genes that are activated by 12-O-tetradecanolyphorbol-13-
acetate
(TPA) (Gutman and Wasylyk, EMBO j.7: 2241-2246 (1990)). Sequences
corresponding to an upstream motif or cis element capable of binding APl are
set
forth in Figure 1 at residues 4428-4434 and 4627-4639, respectively. In
accordance
with the embodiments of the present invention, transcription of TIGR molecules
can
be effected by agents capable of altering the biochemical properties or
concentration
of AP1 or its homologues, including; but not limited to, the concentration of
APl or
its homologues bound to an upstream motif or cis element. Such agents can be
used
in the study of glaucoma pathogenesis. In another eraabodiment, such agents
can
also be used in the study of glaucoma prognosis. In another embodiment such
agents can be used in the treatment of glaucoma.
The sex-determining region of the Y chromosome gene, sry, is expressed in
the fetal mouse for a brief period, just prior to testis differentiation SRY
is a DNA
binding protein known to bind to a CACA-rich region in the sry gene (Vriz et
aL;
Biochemistry and Molecular Biology International 37: 1137-1146 (1995) ) .
A sequence corresponding~to an upstream motif or cis
element capable of binding SRY is set forth in Figure 1 at residues 4625-4634.
In
accordance with the embodiments of the present invention, transcription of
TIGR
molecules can be effected by agents capable of altering the biochemical
properties or ro
concentration of SRY or its homologues, including, but not limited to, the
21
CA 02456777 2004-03-O1
concentration of SRY or its homologues bound to an upstream moti# or cis
element.
Such agents may be useful in the study of glaucoma pathogenesis. in another
embodiment, such agents can also be used in the study of .glaucoma prognosis.
In
another embodiment such agents can be used in the treatment of glaucomae
A sequence rnrresponding to arr upstream motif or cis element charaMe~#ic '
of GC2-GH is set forth in Figure 1 at residues 4689-4711 (West ef al.,
Molecular and
Cellular Biology 7: 1193-1197 (1987): ~
accordance with the embodiments of the present invention, transcription of
~'IGR
molecules can be effected by agents capable of altering the biochemical
properties or
concentration of GC2-GH or its homologues, including; but not limited to, the
concentration of GC2-GH or its homologues bound to an upstream motif or cis
element. Such agents can be used in the study of glaucoma pathogenesis. In
another embodiment, such agents can also be used in the study of ;glaucoma
prognosis. In another embodiment such agents can be used in thetreatment of
glaucoma.
PEA 3 binding motifs or cis elements have been identified (Martin et aL, Prnc.
NatI. Acad. Sci. ltISA) 85: 5839-5843 (1988);
Gutman and Wasylyk, EMBD J. 7: 2241-2246 (1990) ) .
The PEAS protein is a transcription factor that is xeported to interact
with APl like proteins (Martin et al., Proc. Nail. Acad. Sci. (11SA) 85: 5839-
ai843 (1988);
herein incorporated by reference): Sequences corresponding to an upstreann
motif
or cis element capable of binding PEAS is set forth in Figure 1 at residues
476'x-4769.
In accordance with the embodiments of the present invention, transcription of
TIGR
molecules can be effected by agents capable of altering the biochemical
properties or
concentration of PEA3 or its homologues, including, but not limited to, the
concentration of PEA3 or its homologues bound to an upstream motif or cis
element.
Such agents can be used in the study of glaucoma pathogenesis. In another
embodiment, such agents can also be used in the study of glaucoma prognosis.
in
another embodiment such agents can be used in the treatment of glaucoma.
Mammalian interspersed repetitive (MIR) is an element involved in the
coding and processing sequences of mammalian genes. The MIR element is at
least
260 by in length and numbers about 105 copies within the mammalian gervome
(Murnane ef al., Nucleic Aciris Researck 15: ?837-2839 (1995) ) .
A sequence corresponding to an MIR upstream motif or cis element is
set forth in Figure 1 at residues 4759-4954. In accordance with the
embodiments of
22
CA 02456777 2004-03-O1
the present invention, transcription of TIGR molecules can be effected by
agents
capable of altering the biochemical properties or concentration of nuclear
factors or
their homologues, including, but not limited to, the concentration of nuclear
factors
or their homologues bound to an Ivi~t upstream motif ~r cis element. Such
agents
can be used in the study of glaucoma pathogenesis. In another embodiment, such
agents can also be used in the study of glaucoma prognosis. In another
embodiment
such agents can be used in the treatment of glaucoma.
Normal liver and differentiated hepatoma cell lines contain a hepatocyte
specific nuclear factor (H~iF-1) which binds cis-acting element equences
within the
~.0 promoters of the alpha and beta chains of fibrinogen and alpha 1-
antitrypsin
(Baumhueter et al.; EMBO j. 8: 2485-2493,- A
sequence corresponding to an HNF-1 upstream motif or cis element is set forth
in
Figure 1 at residues 4923-4941. In accordance with the embodiments of the
present
invention, transcription of TIGR molecules can be effected by agents capable
of
altering the biochemical properties or concentration of HNF-1 or its
homologues,
including, but not limited to, the concentration of HNF-1 or its homologues
bound
to an HNF-1 upstream motif or cis element. Such agents can be used in the
study of
glaucoma pathogenesis. In another embodiment, such agents can also be used in
the study of glaucoma prognosis. In another embodiment such agents can be used
in the treatment of glaucoma.
A number of cis elements or upstream motifs halve been associated with gene
regulation by steroid and thyroid hormones {e.g: glucocorticoid and
estrogen}(Beato; Cedl 56: 335-344 (1989)' Brent et ut.,
Molecular Endocrinology 89:1996-2000 (1989}; Glass
ef al., Cell 54: 313-323 (1988); Evans, Science 240:
889-895 {1988) ) .
A consensus sequence for a thyroid receptor upstream motif or cis element
(TRE} has been characterized (B.eato, CeII 56: 335-344 (1989}, herein
incorporated by
reference}, A sequence corresponding to a -thyroid receptor upstream motif or
cis
element is set forth in Figure 1 at residues 5151-5156: Thyroid hornnones are
capable of regulating genes containing a thyroid receptor upstream motif or
cis
element (Glass et al., CedI 54: 313-323 (1988) ) .
Thyroid hormones can negatively regulate TIGR. In accordance with the
emiiodiments of the present invention; transcription of TIGR molecules can be
effected by agents capable of altering the biochemical properties or
concentration of
CA 02456777 2004-03-O1
molecules capable of binding a thyroid receptor upstream motif or cis element.
Such agents can be used in the-study of :giaucocna. pathogenesis. In another
embodiment, such agents can also be used in the study of glaucoma prognosis:
In
another embodiment such agents can be used in the treatment of glaucoma:
NFxB is a transcription factor that is reportedly also~ated with a number of
biological processes including T-cell activation and cytokine regulation
{Lenardo et
aL, CeId 58: 227 229 (1989) ). A consensus upstream
motif or cis element capable of binding NFxB has been reported (Lenardo et aL,
Neil
58: 227-229 (1989)). Sequences corresponding to an upstream motif or cis
element
capable of binding NFxB are set forth in Figure 1 at residues ~516b-5175: In
accordance with the embodiments of the present invention; transcription of
TIER
molecules can be effected by agents capable of altering the biochemical
properties or
concentration of NFxB or its homologues, including, but not limited to, the
concentration of NFxB or its homologues bound to an upstream motif or cis
element.
Such agents can be used in the study of glaucoma pathogenesis. In another
embodiment, such agents can also be used in the study of glaucoma prognosis.
In
another embodiment such agents can be used in the treatment of glaucoma.
Where one or more of the agents is a nucleic acid molecule, such nucleic acid
molecule may be sense, antisense or triplex oligonucleotides corresponding to
any
part of the TIGR promoter, TIGR cDNA, TIGR intron, TIGR axon or TIGR gene.
The TIGR promoter, or fragment thereof; of the present invention may be
cloned into a suitable vector and utilized to promote the expression of a
marker
gene (e.g. firefly luciferase {de Wet, MoI. Cell Baol. 7: ~2'S-737 (1987).
or GUS 9efferson ef aL, EMBO J. a6: 3901-3907 (1987; ~.
25In another embodiment of the present
invention, a TIGR promoter may be cloned into a suitable vector and utilized
~o
promote the expression of a TIGR gene in a suitable eukaryotic or prokaryotic
host
cell (e.g. human trabecular cell, Chinese hamster cell, ~. coli). In another
embodiment of the present invention, a TIGR promoter may be cloned into a
suitable vector and utilized to promote the expression of a homologous or
heterologous gene in a suitable eukaryotic or prokaryotic host cells (e.g.
human
trabecular cell lines, Chinese hamster cells, ~: coh~.
Practitioners are familiar with the standard resource mater3ais which des~be
specific conaitions and procedures for the construction, manipulation and
isolation
of macromolecules (e.g., DNA molecules, plasmids, Etc.), generation of
recombinant
24
CA 02456777 2004-03-O1
organs and the screening and isolating of c>aa~es, (see for example, Sambrook
et
al., In MoI~Clo~in~: A Ldboratory Marsual f Cold Sprirvg Harbor Press (1989.;
.Old .and .Primios~e, In Principles of Cane Ivlaxupulation: An lntrflductiQn
'To Ceitetic
Engineering, Blackwell (19~94~) .
The ~CxR promoter or any portion thereof of the present invention may be
used-in a gel-retardation or band shift assay (Old and Primrose, In Principles
of
Gene Manipulation: An Introduction To Genetic Engineering, Blackwell (1994)).
Any of the cis elements identified in the -pinvention may be used in a gel-
retardation or band shift assay to isolate proteins capable of binding the cis
element..
. Suitable D1~A fragments or molecules comprise' or consist ofvone or more of
the
following: sequences corresponding to ~ an upstream motif or cis element
characteristic of PRL-FPlll as set forth in: Figure 1 at residues 370-388, and
4491
4502, respectively, a sequence corresponding to an upstream motif or cis
element
capable of binding GR/PR as set forth in Figured at residues 433-445,
sequences
corresponding to an upstream shear stress motif or cis element as set forth in
~.gure
1 at residues 44b-45I, 1288-2293, 3597 X602, 4771-4776, and 5240-5245,
respectively,
sequences corresponding to glucocorticoid response upstream motif or cis
element
as set forth in Figure 1 at residues 574-600; 1042-1056, 2444-2468, 2442 2269,
3536-
2a 3563, 45~~4-4593, 4595-4614, 4851-4865, 4844 4864, 5079-5084, 5083-5111,
respectively,
a sequence 'corresponding to ~an upstream motif or cis element capable of
binding
CBE as set forth in Figure 1 at residues 735-746, a sequence corresponding to
an
. , ~.
upstream motif or cis element capable of binding NFE as set forth in Figure 1
at
residues Tf4-795, a sequence corresponding to an upstream motif or cis element
capable of binding KTF:1-CS ~s set forth in Figure 1 at residues 843-854, . a
, sequence
corresponding to an upstream motif or cis element capable of binding PRE is
set
forth in Figure 1, at . residues 987 1026, a sequence corresponding to an
upstream
motif or cis element capable of binding fiTF-EGFR as set forth in Figure 1 at
residues 1373-1388, a sequence corresponding to an upstream motif or cis
element
capable of binding SRE-cFos as set forth in Figure 1 at residues 1447-1456, a
sequence odrresponding to an upstream motif or cis element capable of binding
Alu
as set forth in. Figure 1 at residues 1331-1550, a sequence corresponding to
an
upstream motif or cis element capable of binding VEP as set forth in Figure,1
at
residues 1786-1797', a sequence corresponding to an upstream motif or cis
element
capable of binding Malt-CS as set forth in Figure 1 at residues 1832-1841,
sequences
CA 02456777 2004-03-O1
corresponding to an upstream motif or cis element capable of,binding ERE as
set
forth in Figure 1 at resiaues 2167-2195, 3413-3429, and 3892-3896,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
1VF-
mutagen as set forth in Figure l at residues 2329-2338, a sequence
corresponding to
an upstream motif or cis element capable of binding myc-PRF as set forth in
Figure
1 at residues 2403-2416, sequences corresponding to an upstream motif or cis
element capable of binding AP2 as set forth in Figure 1 at residues 2520-2535
and
5170-5187, respectively, sequences corresponding to an upstream motif or cis
element capable of binding HST'F as set forth in Figure 1 at residues 2622-
2635; and
5105-5132, respectively, a sequence corresponding to an upstream motif or cis
element characteristic of SBF as set forth in Figure 1 at residues 2733-2743,
sequences
corresponding to an upstream motif or cis element capable of binding NF-1 as
set
forth in Figure 1 at residues 2923-2938, 4144-4157, and 4887-4900,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
NF-
IviHCIIA/B as set forth in Figure 1 at residues 2936-2944, a sequence
corresponding
to an upstream motif or cis element capable of binding PEAL as set forth in
Figure 1
at residues 3285-3298, a sequence corresponding to an upstream motif or cis
element capable of binding ICS as set forth in Figure 1 at residues 3688-3699,
a
sequence corresponding to an upstream motif or cis element capable of binding
ISGF2 as set forth in Figure 1 at residues 4170-4179, a sequence corresponding
to an
upstream motif or cis element capable of binding zinc as set forth in Figure 1
at
residues 4285-4293, a sequence corresponding to an upstream motif or cis
element
characteristic of CAP/CRP-gal0 as set forth in Figure 1 at residues 4379-4404,
sequences corresponding to an upstream motif or cis element capable of binding
APl as set forth in Figure 1 at residues 4428-4434; and 4627-4639,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
SRY
as set forth in Figure 1 at residues 4625-4634, a sequence corresponding to an
upstream motif or cis element characteristic of GC2 as set forth in Figure 1
at
residues 4678-4711, a sequenee corresponding to an upstream motif or cis
element
capable of binding PEAS as set forth in Figure 1 at residues 4765-4769, a
sequence
corresponding to an upstream motif or cis element capable of MIR as set forth
in
Figure 1 at residues 4759-4954, a sequence corresponding to an upstream motif
or cis
element capable of binding NF-HNF-1 as set forth in Figure 1 at residues 4923-
4941,
a sequence corresponding to a thyroid receptor upstream motif or cis element
as set
forth in Figure 1 at residues 5151-5156, and a sequence corresponding to an
26
CA 02456777 2004-03-O1
upstream motif or cis element capable of binding NFxB as set forth in Figure 1
at
residues 5166-5275:
A preferred class of agents of the present invention comprises nucleic acid
molecules wiU encode all or a fragment of "TTGR promoter" or flanking gene
sequences. As used herein, the terms "'TIER promoter" or "promoter" is used in
an
expansive sense to refer to the regulatory sequences) that control mRNA
production: Such sequences include RNA polymerise binding sites,
glucocorticoid
response elements, enhancers, etc. All such TTGR molecules may be used to
diagnose the presence of glaucoma and severity of glaucoma. Such molecules may
'
be either DNA or RNA.
Fragment nucleic acid molecules may encode significant portions) of, or
indeed most of, SEQ ID N0:1 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5.
Alternatively, the fragments may comprise smaller oligonucleotides (having
from
about 15 to about 250 nucleotide residues, and more preferably, about 15 to
about 30
nucleotide residues.). Such oligonucleotides include SEQ ID N0: 6; SEQ ID NO:
7,
SEQ ID NO: 8; SEQ ID NO: 9, SEQ ID N0:10, SEQ ID NO:11, SEQ ID N0:12, SEQ
ID NO: 13, SEQ TD NO: 14; SEQ ID NO: 15, SEQ iD NO: 16, SEQ ID N0:17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22; SEQ ID
NO: 23; SEQ ID NO: 24, SEQ ID NO: 25.
Alternatively such oligonucleotides may derive from either the TIGR
promoter, TIGR introns, TIGR exons, TIGR cDNA and TIGR downstream sequences
comprise or consist of one or more of the following: sequences corresponding
to an
upstream motif or cis element characteristic of PRL-FPI11 as set forth.in
Figure 1 at
residues 370-388, and 4491-4502, respectively, a sequence corresponding to an
upstream motif or cis element capable of binding GR/PR as set forth in Figure
1 at
residues 433-445, sequences corresponding to an upstream shear stress motif or
cis
element as set forth in Figure l at residues 446-451,1288-1293, 3597 3602,
4771-4776,
and 5240-5245, respectively, sequences corresponding to glucocorticoid
response
upstream motif or cis element as set forth in Figure 1 at residues 574-
600,1042-1056,
2444-2468, 2442-2269, 3536-3563; 4574-4593, 4595-4614, 4851-4865, 4844-4864,
5079-
5084, 5083-5111, respectively, a sequence rnrresponding to an upstream motif
or cis
element capable of binding CBE as set forth in Figure 1 at residues 735-746, a
seauence corresponding to an upstream motif or cis element cacable of binding
NFE as set forth in Figure 1 at residues 774-795, a sequence corresponding to
an
upstream motif or cis element capable of binding KTF.1-CS as set forth in
Figure 1
27
CA 02456777 2004-03-O1
at residues 843-854, a sequence corresponding to an upstream motif or cis
element
capable of binding FRE is set forth in Figure 1 at residues 987-1026, a
sequence
corresponding ~ an upstream motif or cis element capable of binding ETF EGFR
as
set forth in Figure 1 at residues 1373-1388, a sequence corresponding to an
upstream
motaf or cis element capable of binding SRE-cFos as set forth in Figure 1 at
residues
1447-1456, a sequence corresponding to an uvstream motif or cis element
capable of
binding Alu as set forth in Figure 1 at residues 1331-1550, a sequence
corresponding
to an upstream motif or cis element capable of binding VBP as set forth in
Figure 1
at residues 1786-1797, a sequence corresponding to an upstream motif or cis
element capable of binding Malt-CS as set forth in Figure 1 at residues
1832=1841,
sequences corresponding to an upstream motif or cis element capable of binding
ERE as set forth in Figure 1 at residues 2167-2195, 3413-3429, and 3892-3896,
respectively, a sequence corresponding to an upstream motif or cis element
capable
of binding NF-mutagen as set forth in Figure 1 at residues 2329-2338, a
sequence
corresponding to an upstream motif or cis element capable of binding myo-PRF
as
set forth in Figure Z at residues 2403-2416, sequences corresponding to an
upstream
motif or cis element capable of binding AP2 as set forth in Figure 1 at
residues 2520-
2535 and 5170-5187; respectively, sequences corresponding to an upstream motif
or
cis element capable of binding HSTF as set forth in Figure 1 at residues 2622
2635,
and 5105-5132, respectively, a sequence corresponding to an upstream motif or
cis
element characteristic of SBF as set forth in Figure 1 at residues 2733-2743,
sequences
corresponding to an upstream motif or cis element capable of binding NF-1 as
set
forth in Figure 1 at residues 2923-2938, 4144-4157, and 4887-4900,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
NF-
MHCBA/B as set forth in Figure 1 at residues 2936-2944, a sequence
corresponding
to an upstream motif or cis element capable of binding PEAL as set forth in
Figure 1
at residues 3285-3298, a sequence corresponding to an upstream motif or cis
element capable of binding ICS as set forth in Figure 1 at residues 3688-3699,
a
sequence corresponding to an upstream motif or cis element capable of binding
ISGF2 as set forth in Figure 1 at residues 4170-4179, a sequence corresponding
to an
upstream motif or cis element capable of binding zinc as set forth in Figure 1
at
residues 4285-4293, a sequence corresponding to an upstream motif or cis
element
characteristic of CAP/CRP-gal0 as set forth in Figure 1 at residues 4379-4404,
sequences corresponding to an upstream motif or cis element capable of binding
APl as set forth in Figure 1 at residues 4428-4434, and 4627-4639,
respectively, a
28
CA 02456777 2004-03-O1
sequence corresponding to an upstream motif or cis element capable of binding
SRY
as set forth in Figure 1 at residues 465-4634, a sequence corresponding to an
upstream motif or _cis element characteristic of GC2 as set forth in Figure 1
at
residues 468-4?ll, a sequence coaesponding to an upstream motif or cis element
capable of binding PEA3 as set forth in Figure 1 at residues 4765-4769, a
sequence
corresponding to an upstream motif or cis element capable of MIIt as set forth
in
Figure 1 at residues 4T59-4954, a sequence corresponding to an upstream motif
or cis
element capable of binding NF-HNF 1 as set forth in Figure 1 at residues 49?3-
4941,
a sequence corresponding to a thyroid receptor upstream motif or cis element
as set
forth iw Figure 1 at residues 5151-5156, and a sequence corresponding to an
upstream motif or cis element capable of binding NFxB as set forth in Figure 1
at
residues SI66-5175. For such purpose, the oligonucleorides must be capable of
specifically hybridizing to a nucleic acid molecule genetically or physically
linked to
the TTGR gene. As used herein, the term "linked" refers to genetically,
physically or
. ogerablg linked:
As used herein, two nucleic acid molecules are said to be capable of
specifically hybridizing to one another if the two molecules are capable of
forming
an anti-parallel, double-stranded nucleic arid structure, whereas they are
unable to
form a double-stranded structure when incubated with a non TIGR nucleic and
molecule. Awucleic acid molecule is said to be the "complement" of another
nucleic
acid molecule if they exhibit complete complementarity. .As used herein,
molecules
are said to exhibit "complete complementarity" when every nucleotide of one of
the
molecules is complementary to a nucleotide of the other. Two molecules are
said to
be "minimally completrientary" if they can hybridize to one another with
sufficient
stability to permit them to remain annealed to one another under. at. least
conventional "Low-stringency" conditions. Similarly, the molecules are said to
be
"complementary" if they can hybridize to one another with sufficient stability
to
permit them to remain annealed to one another. under conventional "high-
stringency" conditions. Conventional. stringency., conditions are described by
Sambrook, j., et al., (In: Molecular Cloning, a Laboratory Manual, 2nd
Edition, Cold
Spring Harbor Press, Cold Spring.Harbor, New York (1989)), and by Haymes,
B:D., et
al. (In: Nucleic Aciai Hybridization, A Practical Alrproach,1RL Press;
Washington, DC
(1985)) . , Departures from complete
complementarity are therefore permissible, as long as such departures do not
35- completely preclude the capacity of the molecules to form a double-
stranded
29
CA 02456777 2004-03-O1
structure. Thus, in order for an oligonucleotide to serve as a primer it need
only be
sufficiently complennentary in sequence to be able to foraz a stable double-
stxar~ded
struchue under the particular solvent and salt concentrations employed.
Apart from their diagnostic or prognostic uses, such oligonucleotides may be
employed to obtain other TIGR nucleic acid molecules. Such molecules include
the
TIGR-encoding nucleic acid molecule of non-human animals (particularly, cats,
monkeys, rodents and dogs), fragments thereof, as well as their promoters and
flanking sequences. Such molecules can be readily obtained by using the above
described primers to screen cDNA or genomic libraries obtained from non-human
species. Methods for forming such libraries are well known in the art. Such
analogs
may differ in their nucleotide sequences from that of SEQ ID NO: 1, SEQ ID NO:
2,
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID
NO: 8, SEQ II7 NO: 9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID NO:
13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID NO:17, SEQ ID NO: 18,
SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23,
SEQ ID NO: 24, SEQ ID NO: 25, or from molecules consisting of sequences
corresponding to an upstream motif or cis element characteristic of PRL-Fl'I11
as
set forth in Figure 1 at residues 370-388, and 4491-4502, respectively, a
sequence
corresponding to an upstream motif or cis element capable of binding GR/PR as
set
forth in Figure 1 at residues 433-445, sequences corresponding to an upstream
shear
stress motif or cis element as set forth in Figure 1 at residues 446-451, 1288-
1293,
3597-3602, 4771-4776, and 5240-5245, respectively; sequences corresponding to
glucocorticoid response upstream motif or cis element as set forth in Figure 1
at
residues 574-600, 1042-1056, 2444-2468, 2442-2269; 3536-3563; 4574-4593, 4595-
4614,
4851-4865, 4844-4864, 5079-5084, 5083-5111, respectively, a sequence
corresponding
to an upstream motif or cas element capable of binding CBE as set forth in
Figure 1
at residues 735-746, a sequence corresponding to an upstream motif or cis
element
capable of binding NFE as set forth in Figure 1 at residues 774-795, a
sequence
corresponding to an upstream motif or cis element capable of binding KTF.1-CS
as
set forth in Figure i at residues 843-854, a sequence corresponding o an
upstream
motif or cis element capable of binding PRE is set forth in Figure 1 at
residues 987-
1026, a sequence corresponding to an upstream motif or cis element capable of
binding E~'F-EGFE, as set fobth in Figure 1 at residues 1373-1388, a sequence
corresponding to an upstream motif or cis element capable of binding SRE-cFos
as
set forth in Figure 1 at residues 1447-1456, a sequence corresponding to an
upstream
CA 02456777 2004-03-O1
motif or cis element capable of binding .Alu as set forth in Figure 1 at
residues 1331-
1550, a sequence corresponding to an upstream motif or cis element capable of
binding VBP as set forth in Figure 1 at residues 1786-1797, a sequence
corresponding
to an upstream motif or cis element capable of binding Malt-CS as set forth in
Figure 1 at residues 1832-1841, sequences corresponding to an upstream motif
or cis
element capable of binding ERE as set forth in Figure 1 at residues 2167-2195,
3413-
3429, and 3892-3896, respectively, a sequence corresponding to an upstream
motif or
cis element capable of binding NF-mutagen as set forth in Figure 1 at residues
2329-
2338, a sequence corresponding to an upstream motif or cis element capable of
binding myc-PRF as set forth in Figure 1 at residues 2403-2416, sequences
corresponding to an upstream motif or cis element capable of binding AP2 as
set
forth in Figure 1 at residues 2520-2535 and 5170-5187, respectively; sequences
corresponding to an upstream motif or cis element capable of binding HSTF as
set
forth in Figure 1 at residues 2622-2635, and 5105-5132, respectively; a
sequence
corresponding to an upstream motif or cis element characteristic of SBF as set
forth
in Figure 1 at residues 2733-2743, sequences corresponding to an upstream
motif or
cis element capable of binding NF-1 as set forth in Figure 1 at residues 2923-
2938,
4144-4157, and 4887-4900, respectively, a sequence corresponding to an
upstream
motif or cis element capable of binding NF-MHCIIA/B as set forth in Figure 1
at
residues 2936-2944, a sequence corresponding to an upstream motif or cis
element
capable of binding PEA1 as set forth in Figure 1 at residues 3285 3298, a
sequence
corresponding to an upstream motif or cis element capable of binding ICS as
set
forth in Figure 1 at residues 3688-3699, a sequence corresponding to an
upstream
motif or cis element capable of binding ISGF2 as set forth in Figure 1 at
residues
4170-4179, a sequence corresponding to an upstream motif or cis element
capable of
binding zinc as set forth in Figure 1 at xesidues 4285-4293, a sequence
corresponding
to an upstream motif or cis element characteristic of CAP/CRP-gal0 as set
forth in
Figure 1 at residues 4379-4404, sequences corresponding to an upstream motif
or
cis element capable of binding APl as set forth in Figure 1 at residues 4428-
4434,
and 4627-4639, respectively, a sequence corresponding to an upstream motif or
cis
element capable of binding SIZY as set forth in Figure 1 at residues 4625-
4634, a
sequence corresponding to an upstream motif or cis element characteristic of
GC2
as set forth in Figure 1 at residues 4678-4711, a sequence corresponding to an
upstream motif or cis element capable of binding PEAS as set forth in Figure 1
at
residues 4765-4769, a sequence corresponding to an upstream motif or cis
element
31
CA 02456777 2004-03-O1
capable of MIR as set forth in Figure 1 at residues 4759-4954, a sequence
correspoa~ding town upstream motif or cis element capable of binding NF-HIV~i
as
set forth in Figure 1 at residues 4923-4941; a sequence corresponding to a
thyroid
receptor upstream motif or cis element as set forth in Figure 1 at residues
5151-5156,
and a seguence, corresponding to an upstream motif or cis element ca~sable of
binding NFKB as set forth in Figure 1 at residues 5166-5175 because complete
complementarit~ is not needed for stable hybridization. The TIGR nucleic acid
molecules of the present invention therefore also include molecules that,
although
capable of specifically hybridizing with TIGR nucleic acid molecules may lack
.,complete complementarity."
Any of a variety of methods may be used to obtain the above-described
nucleic acid molecules (Ellen, Methods in Molecular Medicine: Molecular
Diagnosis
of Genetic Diseases.; ~ Humana Press (1996) ). SEQ
ID NO:1, SEø. ID N~ 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: S, SEQ ID NO: 6,
SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NC7: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ
ID N4.12, SEQ ID NO: I3, SEQ ID N0:14, SEQ ID N0:15, SEQ ID NO:16, SEQ ID
NO: I7~; SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, SEQ ID NO24, SEQ ID NO: 25, sequences corresponding to
an upstream motif or cis element characteristic of PRL FP1II as set forth in
Figure 1
at residues 370-388, and 4491-4502, respectively, a sequence corresponding to
an
upstream motif or cis element capable of binding GR/PR as set forth in Figure
1 at
residues 433-445, sequences corresponding to an upstream shear stress. motif
or .cis ,
element as set forth in Figure 1 at residues 446-451,128&1293, 3597-3602, 4771-
4776,
arid 5240-5245, respectively, sequences corresponding to glucocorticoid
response
25. upstream motif or cis element as set forth in Figure 1-at residues 574
600,1042-1056,
2444-2468, 2442-2263; 3536-3563, 45T4-4593, 4595-4614, 4851-4865, 4844-4864;
5079-
5084, 5083-5111, respectively, a sequence corresponding to an upstream motif
or cis
element capable of binding CBE as set forth in Figure 1 at residues 735-746, a
sequence corresponding to an upstream motif or cis element capable of binding
NFE as set forth in Figure 1 at residues ?74-795, a sequence corresponding to
an
upstream motif or cis element capable of binding ICTF.1-CS as set forth in
Figure 1
at residues 843-854, a sequence corresponding to an upstream motif or cis
element
capable of binding PRE is set forth in Figu>re 1 at residues 987-1026, a
sequence
corresponding to an upstream motif or cis element capable of binding ETF-EGFR
as
35~ set forth in Figure 1 at residues 1373-1388; a sequence corresponding to
an upstream
32
CA 02456777 2004-03-O1
motif or cis element capable of binding SRE-cFos as set forth in Figure 1 at
residues
1447-I45b, a sequence corresponding to an upstream motif or cis element
capable of
binding Alu as set forth in Figure 1 at residues I33I-1550, a sequence
corresponding
to an upstream motif or cis element capable of binding VBP as set forth in
Figure 1
at residues 1786-1797, a sequence corresponding to an upstream motif or cis
element capable of binding Malt-CS as set forth in Figure 1 at residues 1832-
1841,
sequences corresponding to an upstream motif or cis element capable of binding
ERE as set forth in Figure 1 at residues 2167-2195, 3413-3429, and 38923896;
respectively, a sequence corresponding to an upstream motif or cis element
capable
of binding NF-mutagen as set forth in Figure 1 at residues 2329-2338, a
sequence
corresponding to an upstream motif or cis element capable of binding myc-PRF
as
set forth in Figure I at residues 2403-2416, sequences corresponding to an
upstream
motif or cis element capable of binding AP2 as set forth in Figure 1 at
residues 2520-
2535 and 5170-5287, respectively, sequences corresponding to an upstream motif
or
I5 cis element capable of binding I3STF as set forth in Figure 1 at residues
2b22-2635,
and 5105-5132, respectively, a sequence corresponding to an upstream motif or
cis
element characteristic of SBF as set forth in Figure 1 at residues, 2733-2743,
sequences
corresponding to an upstream motif or cis element capable of binding NF-I as
set
forth in Figure 1 at residues 2923-2938, 4144-4157, and 4887-4900,
respectively, a
sequence corresponding to an upstream motif ar cis element capable of binding
NF-
MHCIIA/B as set forth in Figure 1 at residues 2936-2944; a sequence
corresponding
to an upstream motif or cis element capable of binding PEAL as set forth in
Figure 1
at residues 3285-3298, a sequence corresponding to an upstream.. motif or cas
element capable of binding ICS as set forth in Figure I at residues 3688-3699,
a
sequence corresponding to an upstream motif or cis element capable of binding
ISGF2 as set forth in Figure 1 at residues 41?0-4179, a sequence corresponding
to an
upstream motif or cis element capable -of binding zinc as set forth in Figure
1 at
residues 4285-4293, a sequence corresponding to an upstream motif or cis
element
characteristic of CAP/CRP-gal0 as set forth in Figure 1 at residues. 4379-
4404,
sequences corresponding to an upstream motif or cis element capable of binding
APl as set forth in Figure 1 at residues 4428-4434, and 4627-4639,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
SRY
as set forth in Figure 1 at residues 4b25-4634, a sequence corresponding to an
upstream motif or ca element characteristic of GC2 as set forth in Figure I at
residues 4678-4711, a sequence corresponding to an upstream motif or cis
element
33
CA 02456777 2004-03-O1
capable of binding PEA3 as set forth in Figure 1 at residues 4765-4769, a
sequence
corresponding to an upstream motif or cis~ element= capable of -MIR as set
forth in
Figure 1 at residues 4759-='_ 954, a sequence corresponding to an upstream
motif or cis
element capable of binding NF-HNF-1 as set forth in Figure 1 at residues 4923-
4941,
a sequence con~espanding t~ a thyroid receptor upstream motif or cis element
as set
forth in Figure 1 at residues 5151-5156, and a sequence corresponding to an
upstream motif or cis element capable of binding NF~cB as set forth in Figure
1 at
residues 5166-5175 may be used to synthesize all or any portion of the TIGR
promoter or anx of the TIGR upstream motifs or portions the TIGR cDNA
(Zameclvk et al., Pros. Naft. Acad. Sci. (LLS.A.) 83:4143 (1986); Goodchild et
aL, Proc.
NatI. Acad. Sci. (LI:S.A.) 85:55t?~ (1988); Wickstrom et aL; Proc. Natl: Acad.
Sa. (ILS.A.)
85:1028; Holt, j.T. et at., Motec. CeII. Biol. 8:963 (1988); Gervvirtz, A..M.
et al., Science
242:1303 (1988}; Anfossi, G:, et aL, Proc: Natl. Acad. Sci. (LLS:A:) 86:3379
(1989);
Becker, D., et aL, EMBC? J. 8:367'9 (1989)..) .
Automated nucleic and synthesizers may be employed for this purpose. In
lieu of such synthesis, the disclosed SEQ ID NO:1, SEQ ID NO: 2; SEQ ID NO: 3,
SEQ
ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO:
9, SEQ ID NO: 10r SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14,
SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 1?, SEQ ID NO: 18, SEQ ID NO: 19,
SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
SEQ ID . NO: 25, sequences corresponding to an upstream motif or cis element
characteristic of PRL-FP111 as set forth in Figure 1 at residues 370-388; and
4491-
4502; respectively, a sequence corresponding to an upstream motif or cis
element
capable of binding GR/PR as set forth in Figure 1 at residues 433-445,
sequences
corresponding to an upstream shear stress motif or cis element as set forth in
Figure
1 at residues 446-451,128&1293; 3597-3602, 4T~1=4T76, and 5240-5245;
respectively,,
sequences corresponding to glucocorticoid response upstream motif or cis
element
as set forth in Figure 1 at residues 5?4-600, 1042-105.6, 2444-2468, 2442-
2269, 3536-
3563, 457'4-45,93, 4595-4614, 4851-4865, 4844-4864, 5079-5084, 5~3-5111,
respectively,
a sequence corresponding to an upstream motif or cis element capable of
binding
CBE as, set forth in Figure 1 at residues 735-746, a sequence corresponding to
_ an
upstream motif or cis element capable of binding NFE as set forth in Figure 1
at
residues 7?4 795, a sequence corresponding to an upstream motif or cis element
capable of binding KTF.1-CS as set forth in Figure 1 at residues 843-854, a
sequence
34
CA 02456777 2004-03-O1
corresponding to an upstream motif or cis element capable of binding PRE is
set
forth in Figure 1 at residues 9871026, a sequence corresponding to an upstream
motif or cis element capable of binding E3'F-fiGFR as set forth in Figure 1 at
residues 1373-1388, a sequence corresponding to an upstream motif or cis
element
capable of binding SRE-cFos as set forth in Figure 1 at residues 144?-1456, a
sequence corresponding to an upstream motif or cis element capable of binding
Alu
as set forth in Figure 1 at residues 1331-1550, a sequence corresponding to an
upstream motif or cis element capable of binding VBP as set forth in Figure 1
at
residues 1786-1797, a sequence corresponding to an upstream motif or cis
element
capable of binding Malt-CS as set forth in Figure 1 at residues 1832-1841,
sequences
corresponding to an upstream motif or cis element capable of binding ERE as
set
forth in Figure l at residues 2167-2195, 3413-3429, and 3892-3896,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
NF-
mutagen as set forth in Figure 1 at residues 2329-2338, a sequence
corresponding to
an upstream motif or cfs element capable of binding myc-PRF as set forth in
Figure
1 at residues 2403-2416, sequences corresponding to an upstream motif or cis
element capable of binding AP2 as set forth in Figure I at residues 2520-2535
and
5170-5187, respectively, sequences corresponding to an upstream motif or cis
element capable of binding HSTF as set forth in Figure 1 at residues 2622
2635; and
ZO 5105-5132, respectively, a sequence corresponding to an upstream motif or
cis
element characteristic of SBF as set forth in Figure 1 at residues 2733-2743,
sequences
corresponding to an upstream motif or cis element capable of binding NF-1 as
set
forth in Figure 1 at residues 2923-2938; 4144-4157, and 4887-4900,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
NF-
MHCIIA/B as set forth in Figure 1 at residues 2936-2944, a sequence
corresponding
to an upstream motif or cis element capable of binding PEAL as set forth in
Figure 1
at residues 3285-3298, a sequence corresponding to an upstream motif or cis
element capable of binding ICS as set forth in Figure 1 at residues 3688-3699,
a
sequence corresponding to an upstream motif or cis element capable of binding -
ISGF2 as set forth in Figure 1 at residues 4170-4179, a sequence corresponding
to an
upstream motif or cis element capable of binding zinc as set forth in Figure 1
at
residues 4285-4293, a sequence corresponding to an upstream motif or cis
element
characteristic of CAP/CRP-gal0 as set forth in Figure 1 at residues 4379-4404;
sequences corresponding to an upstream motif or cis element capable of binaing
A.Pl as set forth in Figure 1 at residues 4428-4434, and 4627-4639,
respectively, a
CA 02456777 2004-03-O1
sequence corresponding to an upstream motif or crts element capable of binding
SRY
as set ffTrttl in Figure 1 at residues 4625-4634, a sequence corresponding to
an
upstream motif or cis element characteristic of GC2 as set forth in Figure 1
at
residues 4678-4711, a sequence corresponding to an upstream motif or cis
element
5- capable of binding PF.A3 as set forth in Figure ~ 1 at residues 4765-4769,
a sequence
corresponding to an upstream motif or cis element capable of MIR as set forth
in
Figure 1 at residues.4759-4954, a sequence corresponding to an upstream motif
or cis
element capable of binding NF-HNF-1 as set forth in Figure 1 at residues 4923-
4941,
a sequence corresponding to a thyroid receptor upstream motif or cis element
as set
forth in Figure 1 at residues 5151-5156, and a sequence corresponding to an
upstream motif or cis element capable of binding NFxB as set forth in Figure 1
at
residues 5166-5175 may be used to define a pair of primers that can be used
with the
polymerase chain reaction (Mullis, K. et al.; Cold Spring Harbor Symp. Quant.
Biol.
5I:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP
237,362;
Mullis, K., EP 201,184; Mullis K. ef al., US 4,683,202; Erlich, H., US
4,582,788; and
Saiki, R et aly US 4,683,194)) to amplify and obtain any desired TIGR gene DNA
molecule or fragment.
The TIGR promoter sequences} and TIGR flanking sequences can also be
obtained by incubating oligonucleotide probes of TIGR oligonucleotides with
2t~ members of genomic human libraries and recovering clones that hybridize to
the
probes. In a second embodiment, methods of "chromosome walking," or 3' or
5°
RACE may be used (Frohman, M.A. et aL, Proc. Natl. Acad. Sci. (I,LS.A.)
85:8998-9002
(1988}; ~ Ohara, O: ef al., Proc. Natl. Acad. Sci.
(LLS.A.) 86:5673-5677 (1989)) to obtain such
sequences.
IL Uses of the Molecules of the Invention in the Diagnosis and Prognosis of
Glaucoma and Related Diseases
A particularly desired use of the present invention relates to the diagnosis
of
glaucoma, POAG, pigmentary glaucoma, high tension glaucoma and low tension
glaucoma and their related diseases. Another particularly desired use of the
present
invention relates to the prognosis of glaucoma, POAG, pigmentary glaucoma,
high
tension glaucoma and low tension glaucoma and their related diseases. As used
Herein the term "glaucoma" includes both primary glaucomas, secondary
glaucomas; juvenile glaucomas, congenital glaucomas, and familial glaucomas,
CA 02456777 2004-03-O1
including, without limitation, pigmentary glaucoma, high tension glaucoma and
low tension glaucoma and their related diseases: As indicated above, methods
for
diagnosing or prognosing glaucoma suffer from inaccuracy, or require multiple
examinations. The molecules of the present invention may be used to define
superior assays for glaucoma. Quite apart from such usage, the molecules of
the
present invention may be used to dia~wsis or predict an individual's
sensitivity to
elevated intraocular pressure upon administration of steroids such as
glucornrtimids or corticosteroids, or anti-inflammatory steroids):
Dexamethasone;
cortisol and prednisolone are preferred steroids for this purpose. Medical
conditions such as inflammatory and allergic disorders, as deli as organ
~a~plantation recipients, benefit from treatment with glucocorticoids. Certain
individuals exhibit an increased sensitivity to such steroids (i.e., "steroid
sensitivity"), which is manifested by an undesired increase in intraocular
pressure.
The present invention may be employed to diagnosis or predict such
sensitivity, as
well as glaucoma and related diseases.
In a first embodiment, the TIGR molecules of the present invention are used
to determine whether an individual has a mutation affecting the level (i.e.,
the
concentration of TIGR mRNA or protein in a sample, etc.) or pattern (i.e., the
kinetics of expression; rate of decomposition, stability profile, etc.) of the
TIGR
expression (collectively, the °"TIGR response" of a cell or bodily
fluid) (for example;
a mutation in the TIGR gene, or in a regulatory regions) or other genes) that
control or affect the expression of TIGR), and being predictive of individuals
who
would be predisposed to glaucoma (prognosis), related diseases, or steroid
sensitivity. As used herein, the TIGR response manifested by a cell or bodily
fluid is
said to be "altered" if it differs from the TIER response of cells or of
bodily fluids of
normal individuals. Such alteration may be manifested by either abnormally
increased or abnormally diminished TIGR response. To determine whether a TIER
response is altered, the TIGR response manifested by the cell or bodily fluid
of .the
patient is compared with that of a similar cell sample (or bodily fluid
sample) of
normal individuals. As will be appreciated, it is not necessary to re-
determine the
TIGR response of the cell sample (or bodily fluid sample) of normal
individuals each
time such a comparison is made; rather, the TIGR response of a particular
individual
may be compared with previously obtained values of normal individuals.
In one sub-embodiment, such an analysis is conducted by determining the
presence and/or identity of polymorphism(s) in the TIGR gene or its flanking
37
CA 02456777 2004-03-O1
regions which are associated with glaucoma, or a predisposition (prognosis) to
glaucoma, related diseases, or steroid sensitivity. As used herein, the term
"TIER
flanking regions" refers to those regions which are located either upstream or
downstream of the TIGR coding region.
Any of a variety of molecules can be used to identify such polymorphism(s).
In one embodiment, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ
ID
NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,
SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,
SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ II7 NO: 24, SEQ ID NO: 25,
sequences corresponding to an upstream motif or cis element characteristic of
PRL-
FPlli as set forth in Figure 1 at residues 370-388, and 4491-4502,
respectively, a
sequence corresponding to an upstream motif or cis element capable of binding
GR/PR as set forth in Figure 1 at residues 433-X45, sequences corresponding to
an
upstream spar stress motif or cis element as set forth in Figure Z at residues
446-
451, 1288-1293, 3597-3602, 4771-4776, and 5240-5245, respectively, sequences
rnrresponding to glucocorticoid response upstream motif or cis element as set
forth
in Figure l at residues 574-600, 1042-1056, 2444-2468, 2442 2269, 3536-3563,
4574-
4593, 4595:4614, 4851-4865, 4844-4864; 5079-5084, 5083-S1I1; respectively, a
sequence
corresponding to an upstream motif or cis element capable of binding CBE as
set
forth in Figure I at residues 735-746, a sequence corresponding to an upstream
motif
or cis element capable of binding NFE as set forth in Figure 1 at residues 774-
795, a
sequence corresponding to an upstream motif or cis element capable of binding
ICTF.1-CS as set forth in Figure 1 at residues 843-854, a sequence
corresponding to an
upstream motif or cis element capable of binding PRE is set forth in Figure I
at
residues 987-1026, a sequence corresponding to an upstream motif or cis
element
capable of binding ETF-EGFR as set forth in Figure 1 at residues 1373-1388, a
sequence corresponding to an upstream motif or cis element capable of binding
SRE-cFos as set forth in Figure I at residues 1447-1456, a sequence
coaesponding to
an upstream motif or cis element capable of binding Alu as set forth in Figure
1 at
residues 1331-I550, a sequence corresponding to an upstream motif or cis
element
capable of binding VBP as set forth in Figure 1 at residues 1786-1797, a
sequence
corresponding to an upstream motif or cis element capable of binding Malt-CS
as
set forth in Figure 1 at residues 1832-1841, sequences corresponding to an
upstream
motif or cis element capable of binding ERE as set forth in Figure 1 at
residues 2167-
38
CA 02456777 2004-03-O1
2195, 3413-3429; and 3892-3896, respectively, a sequence corresponding to an
upstream motif or cis element capable of binding NF-mutagen as set forth in
Figure
1 at residues 2329-2338, a sequence corresponding to an upstream motif or cis
element capable of binding myc-PRF as set forth in Figure 1 at residues 2403-
2416,
sequences corresponding to an upstream motif or cis element capable of binding
AP2 as set forth in Figure 1 at residues 2520-2535 and 5170-5187,
respectively,
sequences corresponding to an upstream motif or cis element capable of binding
.
HSTF as set forth in Figure 1 at residues 2622-2635, and 5105-5132,
respectively, a
sequence corresponding to an upstream motif or cis element characteristic of
SBF as
set forth in Figure 1 at residues 2733-2743, sequences corresponding to an
upstream
motif or cis element capable of binding NF-1 as set forth fn Figure 1 at
residues
2923-2938, 4144-4157, and 4887-4900, respectively, a sequence corresponding to
an
upstream motif or cis element capable of binding IeTF-MHCIIA/B as set forth in
Figure 1 at residues 2936-2944, a sequence corresponding to an upstream motif
or cis
element capable of binding PEAI as set forth in Figure 1 at residues 3285-
3298, a
sequence corresponding to an upstream motif or cis element capable of binding
ICS
as set forth in Figure 1 at residues 3688-3699, a sequence corresponding to an
upstream motif or cis element capable of binding ISGF2 as set forth in Figure
1 at
residues 4170-4179, a sequence corresponding to an upstream motif or cis
element
capable of binding zinc as set forth in Figure 1 at residues 4285-4293, a
sequence
corresponding to an upstream motif or cis element characteristic of
CAl'/CRl'=gal0
as set forth in Figure 1 at residues 4379-4404, sequences corresponding to an
upstream motif or cis element capable of binding APl as set forth in Figure 1
at
residues 4428-4434, and 4627-4639, respectively, a sequence corresponding to
an
upstream motif or cis element capable of binding SRY as set forth in Figure 1
at
residues 4625-4634, a sequence corresponding to an upstream motif or cis
element
characteristic of GC2 as set forth in Figure 1 at residues 4678-4711, a
sequence
corresponding to an upstream motif or cis element capable of binding PEA3 as
set
forth in Figure 1 at residues 4765-4769, a sequenee corresponding to an
upstream
motif or cis element capable of MtR as set forth in Figure 1 at residues.4759-
4954, a
sequence corresponding to an upstream anotif or cis element capable of binding
NF-
HNF-1 as set forth in Figure 1 at residues 4923-4941, a sequence corresponding
to a
thyroid receptor upstream motif or cis element as set forth in Figure I at
residues
5151-5156, and a sequence corresponaing to an upstream motif or cis element
capable of binding NFxB as set forth in Figure 1 at residues 5166-5175 (or a
sub-
39
CA 02456777 2004-03-O1
sequence thereof) may be employed as a marker nucleic acid molecule to
identify
such polymorphism(s).
Alternatively; such polymorphisms can be detected through the use of a
marker nucleic acid molecule or a marker protein that is genetically linked to
(i.e.; a
polynucleotide that co-segregates with) such polymorphism(s). As stated above,
the
~'iGR gene and/or a sequence or sequences that specifically hybridize to the
TIGR
gene have been mapped to chromosome iq, 21-32, and more preferably to the TIER
gene located at chromosome 1, q21-27, and more preferably to this TIER gene
located at chromosome 1, q22-26, and most preferably to the TIGR gene located
at
chromosome I, q24. In a preferred aspect of this embodiment, such marker
nucleic
acid molecules will have the nucleotide sequence of a polynucleotide that is
closely
genetically linked to such polymorphism(s) (e.g., markers located at
chromosome 1,
q19-25 (and more pxeferably chromosome 1, q23-25, and most preferably
chromosome 1; q24.
Localization studies using a Stanford G3 radiation hybrid panel mapped the
TIGR gene with the D1S2536 marker nucleic acid molecules at the D1S2536 locus
with a LOD score of 6Ø Other marker nucleic acid molecules in this region
include:
D1S210; D1S1552; D1S2536; D1S2790; SI3GC-12820; and D1S2558. Other
polynucleotide markers that map to such locations are known and can be
employed
to identify such polymorphism(s).
The genomes of animals and plants naturally undergo spontaneous mutation
in the course of their continuing evolution (Gusella, J.F., Ann. Rev. Biochem.
55:831-
854 (1986)). A "polymorphism" in the TIGR gene or its flanking regions is a
variation or difference in the sequence of the TIGR gene or its flanking
regions that
arises in some of the members of a species. The variant sequence and the
"original"
sequence co-exist in the species' population In some instances, such co-
existence is
in stable or quasi-stable equilibrium.
A polymorphism is thus said to be "allelic," in that, due to the existence of
the polymorphism, some members of a species may have the original sequence
~(i.e.
the original '°alleie") whereas other members may have the variant
sequence (i.e. the
variant "allele"). In the simplest case, only one variant sequence may exist,
and the
polymorphism is thus said to be di-allelic. In other cases, the species'
population
may contain multiple alleles, and the polurnorphism is termed tri-allelic,
etc. A
single gene may have multiple different unrelated polymorphisms. For example,
it
CA 02456777 2004-03-O1
may have a di-allelic pollnnorphism at one site, and a mufti-allelic
polymorphism at
another site.
The variation that defines the polymorphism may range from a single
nucleotide variation to the insertion or deletion of extended regions within a
gene.
5~ In sonne cases, the DNA sequence variations are in regions of -the genoine
that are
characterized bx short tandem repeats (STRs) that include tandem dl- or tri-
nucleotide repeated motifs of nucleotides. Polymorphisms characterized by such
tandem repeats are referred to as "variable number tandem repeat" ("VIvTTR")
poly~no~phisms. VNTRs have been used in identity and paternity analysis
(Weber,:
J.L., >;1:S. Patent 5,0T5,21?; Armour, J.A:L. d al., FEBS Left. 307:113-Ii5
(1992); Jones; .
L. et al., Eur. j..Haernafol. 39:144-I4?. (1987); Horn, G.T. et al., PCT'
Application
W09~1/14003; Jeffreys, A.J., European Patent Application 3?0,719; Jeffreys,
A.J., U.S.
Patent 5~,1?5;~~ jeffreys. A.J. et al., Amen. ): Hum. Genet. 39:11-24 (1986);
Jeffieys.
A.J. et al., Nature 3I'6:?6-?9 (1985); Gray, LC. et al.; PrQC. R: Acad. Soc:
Land: 243:241-
253 (1991); Moore, S.S. et al., Genomies 10:654-660 (1991); Jeffreys, A.J. et
al.,Anim.
Genet. T 8:I-15 (198; I~llel, J. et aL, Anim. Genet. 20:145-155 (1989); ~llel,
J. et al.,
Genet. I24:?83-7°81 (1990)?
In an alternative embodiment, such polymorphisms can be detected through
the use of a marker nucleic acid molecule that is physically linked to ,such
24 polxatorphism(s). For this purpose, marker nucleic acid molecules
comprising a
nucleotide sequence of a polynudeotide located within 1 mb of the .
polynnorphism(s}, and more preferably within 100 kb .of the polymprphism(s),
and
most preferably within IO kb of the golymo=phism(s) can be employed. EXamples
of
such marker nucleic ands are set out in SE(~ ID NC1: 1, SEQ ID NO: 2, SEQ 1D
NO: 3,
SEQ .ID NO: 4, SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: ?, SEQ ID NO: 8, SEQ ID
N0: ,9; SEQ ID NO:10, SEQ ID NO: II, SEQ ID N0:12, SEQ ID NO: I3, SEQ ID NO:
I4, SEQ ID NO: I5, SEQ ID N0:16, SEQ ID NO:1?, SEQ Ip NO:18, SEQ ID N0:19;
SEQ ID~ NO: 20, SEQ ID N4: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,
SEQ ID NO: 25.
In another embodiment a marker nucleic acid will be used that is capable of
specifically deteding~TIGRrntI, TIGRmt2, TIGRmt3, TIGRmt4, TIGRmtS, TTGRsaI,or
a combinatiori of these mutations. Methods to detect bases) substitutions;
base(s),
deletions and bases) additions are known in the art (i,e. methods to genotype
an
indivitduai). For example, the ~'~;enetic Bit Analysis ("GBA") method '
disclosed by.
Goelet, P. ef aL, WO 32/157'12, gay ~ u~d for
41
CA 02456777 2004-03-O1
detecting the single nucleotide polymorphisms of the present invention. GBA is
a
method of polymorph~c site iraerrogation id which the nucleotide sequence
infonriation surrounding the site of variation in a target DNA sec~uente is
used to
design an oiigonudeotide primer that is complementary to the region
immediately
adjacent to, but not including, the variable nucleotide in the target DNA. The
target
DNA template is selected from the biological sample ar<d hybridized to the
interrogating primer. This primer is extended by a single labeled
dideoxynucleotide
using DNA polymerise in the presence of two, and preferably all four chain
termiinatitig nucleoside triphosphate precursors. Cohere, D. et al:, (PCT
Application
IO W091/02087) descn'bes a related method of genotyping.
Other primer-guided nucleotide incorporation procedures for assaying
polymorphic sites in DNA have been described (Komher, J. S. ef al., Nucl.
Acids. lies.
17:7779-7784 {1989)~:'Sokolov; B. P., hTucl. Acids R~es. '
T 8:3671 (1990) ; Syvanen, A.-C:, et al., Genamics
8:654 - 692 {1990), r Kuppuswamy, M.N. et ii., Proc..
NafI. Acid. Sci. (II:S.A.) 88:1143-1147 (1991).; '
Prezant, T.R. et al., Hum.-Mufaf. 1:159-164 (1992),.
Ugozzoli, L. ef al., GATA 9:107-112 (1992); ,; Nyr6n,
P. et aL, Anal. Biochem. 208:171-175 {1993) ) .
The detection of polymorphic sites in a. sample of DNA may be facilitated
through the use of nucleic acid amplification methods. Such methods
specifically
increase the concentration of polynucleotides th~s.t span the polymorphic
site, or
include that site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis or other
means.
Another preferred method of achieving such amplification employs the
polymerise chain reaction ("PCR") (Mullis, K. et aL, Cold Spring Harbor Symp.
Quartt.
JBiol. 51:263-273 (1986); .'a MK. ,ef uL. US.1'a~nt hio.
4,683,202; Eriich, H., U.S. Patent No. 482,788; and Saiki, R ef al., U.S.
Patent No.
4,683,194); using primer pairs that are capable of hybridizing to the proximal
sequences that define a polymorphism in its double-stranded form.
In lieu of PCR, a3ternat;ve methods, such as the "Ligasp wChain &eaon"
("LCR") may be used (Barany, F., Proc. Nafl. Acid. yci. (LLS.A.> 88:189-193
(1991)) .
LCR uses two pairs of oligonucleotide probes to exponentially amplify a
specific
42
CA 02456777 2004-03-O1
target. The sequences of each pair of oligonucleotides is selected to permit
the pair
to hybridize to abutting sequences of the same strand of the target. Such
hybridization forms a substrate for a template-dependent ligase. As with PCR,
the
resulting products thus sense as a template in subsequent cycles and an
exponential
S amplification of the desired sequence is obtained.
LCR can be performed with oligonucleotides having the proximal and distal
sequences of the same strand of a polymorphic site: In one embodiment, either
oligonudeotide will be designed to include the actual poiymorphic site of the
polymorphism. In such an embodiment, the reaction conditions are selected such
1i? that the oligonucleotides can be ligated together only if the target
molecule either
contains or lacks the specific nucleotide that is complementary to the
polymorphic
site present on the oligonucleotide. Alternatively, the oligonucleotides may
be
selected such that they do not include the polymorphic site (see, Segev, D.,
PCT
Application WO 90/01069).
15 The "Oligonucleotide Ligation Assay" ("OLA") may alternatively be
employed (Landegren, U. et aL, Science 24I:10?7:1080 (1988)). The OLA protocol
uses two oligonucleotides which are designed to be capable of hybridizing to
abutting sequences of a single strand of a target. OLA, like LCR, is
particularly
suited for the detection of point mutations. Unlike LCR, however, OLA results
in
ZO "linear" rather than exponential amplification of the target sequence.
Nickerson, D.A. et al., have described a nucleie acid detection assay that
cofnbines- attributes of PCR and OLA (Nickerson, D.A. et aL, Proc. Nufl. Acad:
Sci.
(LLS.A.) 8t:89~3-892 (1990)). In this method, PCR is used to achieve the
exponential
amplification of target DNA, which is then detected using OLA. In addition to
25 requiring multiple, and separate, processing steps, one problem associated
with
such combinations is that they inherit all of the problems associated with PCR
and
Schemes based on Iigation of two (or more) oligonucleotides in the presence
of nucleic acid having the sequence of the resulting "di-oligonucleotide",
thereby
30 amplifying the di-oligonucleotide, are also known (Wu, I~.Y. ef aL,
Genomics 4:560
(1989)), and may be readily adapted to the purposes of the present invention
Other known nucleic acid amplification procedures, such as allele-specific
aligomers, branched DNA technology, transcription~ased amplification systems,
or
isothermal amplification methods may also be used to amplify and analyze such
35- polymorphisms (Malek, L.T: et al., U.S. Patent 5,130,238r
43
CA 02456777 2004-03-O1
Schuster ~t ~k., ~~.f.S, Pat~xrat 5,1~69f7~6l~v~~oh; W: et~ ui., Pro; . ~Va#1.
Acad. aci. ~~,i.S.A.)
f~:ali~ (1989); Walker, G.T. et al., Pros: Nail. Acad. , Sci. (U.S.A.) 89:392-
396
(1992)). ~l the foregoing nucleic aeid amplification methods could be used to
predict or diagnose glaucoma.
The identification of a polymorphism in the TIGR gene can be determined iar
a variety of ways. By correlating the presence or absence of glaucoma in an
individual with the presence or absence of a polymorphism in the TIER gene or
its
flanking regions, it is possible to diagnose the predisposition (prognosis) of
an
asymptomatic patient to glaucoma; related diseases, or steroid sensitivity: If
a
polymorphism creates or destroys a restriction endonuclease cleavage site, or
if it
results in the loss or insertion of DNA (e.g., a ~1VTR polymor~hism)~ it will
alter the
size or profile of the DNA fragments that are generated by digestion with that
restriction endonuclease. As such, individuals that possess a variant sequence
can
be distinguished from those having the original sequence by restriction
fragment
analysis. Polymorphisms that can be identified in this manner are termed
"restriction fragment length polymorphisms" ("RFhPs"). RPLP's have been widely
used in human and animal genetic analyses ( Skolnick, M:H. et al., Cytogen.
Cell
Genet. 32:'58-67 (1982); liotstein, D: ef al., Ate. j. ~ Hum. Genet. 32:314-
331 (7t91~0) ,
zo
The role of TIvR in glaucoma pathogenesis indicates that the presence of
genetic
alterations (e.g., DNA polymorphisms) that affect the TIGR response can be
employed to predict glaucoma
A preferred method of achieving such identification employs the single-
strand rnnformational polymorphism (SSCP) approach. The SSCP technique is a
method capable of identifying most sequence variations in a single strand of
DNA,
typically between 150 and 250 nucleotides in length (Ellen, Methods in
Molecular
Medicine: Molecular Diagnosis of Genetic .Diseases, Humans Press (1996).;
prita et aL, Ger~omics 5: 874-879 X1989) ) .
Under denaturing conditions a single strand of i~IVA
will adopt a conformation that is uniquely dependent on its sequence
conformation.
This conformation usually will be dL~terent, even if only a single base is
~chauged.
Most conformations have been reported to alter the physical configuration or
siie
sufficiently to be detectable by electrophoresis. A number of protocols have
been
44
CA 02456777 2004-03-O1
described for SSCP including, but not limited to Lee et al., Arxal. Bioche~xx.
205: 289-
' 293 (1992); Suzuki et al., Anal. Bioclze>7x.192: 82-84 (1991); Lo et al.,
Nucleic Acids
S Research 20:1005-1009 (1992); Sarkar et al., Gerxomics 13: 441-443 (1992).
In accordance with this embodiment of the invention, a sample DNA is
obtained from a patient's cells. In a preferred embodiment, the DNA sample is
obtained from the patient's blood. However, any source of DNA may be used. The
DNA is subjected to restriction endonuclease digestion. TIGR is used as a
probe in
IO accordance with the above-described RFLP methods. By comparing the RFLP
pattern of the TIGR gene obtained from normal and glaucomatous patients, one
can
determine a patient's predisposition (prognosis) to glaucoma. The polymorphism
obtained in this approach can then be-cloned to identify the mutation at the
coding
region which alters the protein's structure or regulatory region of the gene
which
15 affects its. expression level. Changes involving promoter interactions with
other
regulatory proteins can be identified by, for example, gel shift assays using
I-TTM
cell extracts; fluid from the anterior chamber of the eye, serum, etc.
Interactions of
TIER protein in glaucomatous cell extracts, fluid from the anterior chamber of
the
. eye, serum, etc. can be compared to control samples to thereby identify
changes in
20 those properties of TTGR that relate to the pathogenesis of glaucoma:
Similarly such
extracts and fluids. as well as others (blood, etc) can be used to diagnosis
or predict
steroid sensitivity.
- Several different classes of polymorphisms may be identified through such
methods. Examples of such classes include: {1) polymorphisms present in the
TIGR
25. cDNA of different individuals; {2) polymorphisms in non-translated TIGR
gene
sequences, including the promoter or other regulatory regions of the T'IGR
gene; (3)
polvmorphisms in genes whose products interact with TIGR regulatory sequences;
{4) polvmorphisms in gene sequences whose products interact with the TIGR
protein, or to which the TIGR protein binds.
~a In an alternate sub-embodiment, the evaluation is conducted using
oligonucleotide "probes" whose sequence is complementary to that of a portion
of
~EQ ID NO: 1, SEQ ID NO: 2 SEQ ID NO: 3; SEQ ID NO: 4, or SEQ ID NO: 5. Such
molecules are then incubated with cell extracts of a patient under conditions
sufficient to permit nucleic acid hybridization.
CA 02456777 2004-03-O1
In one sub-embodiment of this aspect of the present invention, one can
diagnose or predict glaucoma, related diseases and steroid sensitivity by
ascertaining the TIGR response in a biopsy (or a macrophage or other blood
cell
sample), or other cell sample, or more preferably, in a sample of bodily fluid
(especially, blood, serum, plasma, tears, buccal cavity, etc.). Since the TIGR
gene is
induced in response to the presence of glucocorticoids, a highly preferred
embodiment of this method comprises ascertaining such TIGR response prior to,
during and/or subsequent to, the administration of a giucocorticoid. Thus, by
way
of illustration, glaucoma could be diagnosed or predicted by determining
whether
the administration of a glucocorticoid (administered topically, intraocularly,
intramuscularly, systemically, or otherwise) alters the TIGR response of a
particular
individual, relative to that of normal individuals. Most preferably, for this
purpose,
at least a "TIGR gene-inducing amount" of the glucocorticoid will be provided.
As
used herein, a TIER gene-inducing amount of a glucocorticoid is an amount of
glucocorticoid sufficient to cause a detectable induction of TIGR expression
in cells
of glaucomatous or non-glaucomatous individuals.
III. Methods of Administration
The agents of the present invention can be formulated according to known
methods to prepare pharmacologically acceptable compositions, whereby these '.
materials, or their functional derivatives, having the desired degree of
purity are
combined in admixture with a physiologically acceptable carrier, excipient, or
stabilizer. Such materials are non-toxic to recipients at the. dosages and
concentrations employed. The active component of such compositions may be
agents analogs or mimetics of such molecules. Where nucleic and molecules are
employed, such molecules may be sense, antisense or triplex oligonucleotides
of the
TIGR promoter, TIGR cDNA, TIGR intron, TIGR exon or TIGR gene.
A composition is said to be "pharmacologically acceptable" if its
administration can be tolerated by a recipient patient. An agent is
physiologically
significant if its presence results in a detectable change in the physiology
of a
recipient patient.
Suitable vehicles and their formulation, inclusive of other human proteins,
e.g., human serum albumin, are described, for example, in Remington's
Pha~wnaceuncal jciences 116th ed.; Osol, A., Rd., Mack, ~aston PA (19&0 j j.
46
CA 02456777 2004-03-O1
If the composition is to be water soluble, it may be formulated in a buffer
such as phosphate or other organic acid salt preferably at.a pH of about 7 to
8. If the
composition is only partially soluble in water, it may be , prepared as a
microemulsion by formulating it with a nonionic surfactant such as Tweeri,.
Pluronics, or PEG, eg., Tween 80; in an amount af, for example, 0.04-0.05%
(w/v), bn
incfease its solubility. The term "water soluble" as applied to the
polysaccharides
and polyethylene glycols is meant to include colloidal solutions and
dispersions. In
general, the solubility of the cellulose derivatives is determined by the
degree of
substitution of ether groups, and the stabilizing derivatives useful herein
should
have a sufficient quantity of such ether groups per anhydroglucose unit in
.the
celluiose chain to render the derivatives water soluble. A degree of ether
substitution of at least 0.35 ether groups per anhydroglucose unit is
generally
sufficient. Additionally, the cellulose derivatives may be in the form of
alkali metal
salts, for example, the Li, Na, K or Cs salts.
Optionally other ingredients may be added such as antioxidants, e.g.,
ascorbic acid; low molecular weight (less than about ten residues)
poiypeptides, e.g.;
polyargirune or tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino
acids, such as glycine, glutamic acid, aspartic and, or arginine;
monosaccharides,
disaccl~aridesr and other carbohydrates including cellulose or its
derivatives,
glucose, mannose, or dextrins; chelating agents such as EDTA; and sugar
alcohols
such as mannitol or sorbibol.
Additional pharmaceutical methods may be employed to control the
duration of action. Controlled or sustained release preparations may be
achieved
through the use of polymers to complex or absorb the TIGR molecule{s) of the
composition The controlled delivery may be exercised by selecting appropriate
macromolecules (for example polyesters; polyamino acids, polyvinyl
pyrrolidone,
ethylenevinylacetate; methylcellulase, carboxymethylcellulose, or protamine
sulfate)
and the concentration of macromolecules as well as the methods of
incorporation in
order to control release.
Sustained release formulations may also be prepared, and include the
formation of microcapsular particles and implantable articles. For preparing
sustained-release compositions, the TIGR molecule{s) of the composition is
preferably incorporated into a biodegradable matrix or microcapsule. A
suitable
material for this purpose is a polylactide, although other polymers of poly-{a-
*Trade-mark
4?
CA 02456777 2004-03-O1
hydroxycarboxylic acids), such as poly-D-{-)-3-hydroxybutyric acid, ~P
133,988A),
can be used. Other biodegradable polymers iniclude poly~{lafito~es);
poly(orthoesters), polyamino acids, hydrogels, or poly~(orthocarbona~es)
poly(acetais). The polymeric material may also comprise polyesters,
poly(la~etic
acid) or ethylene vinylacetate copolymers. For examples of sustained ~~elease
compositions, see U.S. Patent No. 3;773;919, EP ~58,481A, U.S. Patent Ne.
3,887;°99, .
EP 158,277A, Canadian Patent No. 1176565, Sidman, U. et aL, Biopoiymers 22:547
(1983), and Larger, R et al., Chtm. Teelr:12:98 (I982).
Alternatively, instead of incorporating the TIER molecules) of the
composition into polymeric particles, it is possible to entrap these materials
in
microcapsules prepared, for example, by ooacervatian techniques or by
interfacial
polymerization, for example; hydroxymethylcellulose or gelatine-mi~ocapsules
and
poly(methylmethacylate) micxocapsules, respectively, or in colloidal drug
delivery
systems, for example, liposomes, albuaun microspheres, microemulsions;
nanoparticles, and nanocapsules or in macroemulsions: Such techniques are
disclosed in Remington's Pharmaceutical Sciences (19811).
In an alternative embodiment, liposome formulations and methods that
permif intracellular uptake of the molecule will be emuloved. Suitable methods
are
known in the art, see, for example,
..._y~~h, D.B. (US.
Patent No: 5,190,762), Callahan, M.V. ef at. (U.S. Patent No. ~5,270,OS2) and
Gonzalezro, R.J. (PCT Application 91/0577i).
Having now generaDy described the invention, the same will be more readily
understood through reference to the following examples which are provided by
?5 way of illustration, and are not intended to be limiting of the present
invention,
unless specified.
Single strand conformational polymorphism ,(SSCP) screening is~caned out
according to the procedure of Hue et al., The Journal of Invesfigative
Ophtlralrnolo~y
105.4: 529-632 (1995)_. SSCP primers are
constructed corresponding to sequences found within the TIGR promoter and two
of exons of TIGR The following primers are canst~ucted: forwa~3 primer "Sk-
1a":
5' TGA GGC TTC CTC TGG AAA C 3' (SEQ ID NO: 6); reverse primer "1a2":
°'S'-
TGA AAT CAG CAC ACC AGT AG3' (SEQ ID NO: 7); forward primer "CA2": ~S'-
48
CA 02456777 2004-03-O1
~ CCC ATA CCC CAA TAA TAG3' (SEQ ID NO: 8); reverse primer "Pr+1": 5'-
AGA GTT CCC CAG ATT.TCA CC-3'' (SEQ ID NO: 9); forward primer "Pr-I": 5'-
ATC TGG GGA ACT CTT CTC AG3' (SEQ ID NO:10); reverse primer "Pr+2(4A2)":
5'-'FAC AGT TGT TGC AGA TAC G3' (SEQ ID NO: 11); forward primer "Pr-
2(4A)": 5' ACA ACG TAT CTG CAA CAA CTG3' (SEQ ID N0:12); reverse primer
"Pr+3(4A)": 5' TCA GGC TTA ACT GCA GAA CC-3' (SEQ ID NO: I3); forward
primer "Pr-3(4A)": 5' TTG GTT CTG CAG TTA AGC C-3° (SEQ ID NO: 14);
reverse
primer "Pr+2(4A1)": 5'-AGC AGC ACA AGG GCA ATC C-3' (SEQ ID NO: 15);
reverse primer "1'r+1(4A}": 5'-ACA GGG CTA TAT TGT GGG-3' (5EQ ID NO: 16);
forward primer "KSIX": 5'fiCT GAG ATG CCA GCT GTC C-3' (SEQ ID NO: 1~;
reverse primer "SK1XX": 5'-CTG AAG CAT TAG AAG CCA AC 3' (SEQ ID NO:
18); forward primer "KS2aI": 5'-ACC TTG GAC CAG GCT GCC AG3' (SEQ ID
NO:1~.}; reverse primer "SK3" 5'-AGG TTT GTT CGA GTT CCA G3' (SEQ ID NO:
20}forward primer "KS4": 5'-ACA ATT ACT GGC AAG TAT GG-3' (SEQ ID NO:
21); reverse primer "SK6A": 5'-CCT TCT CAG CCT TGC TAC C-3' (SEQ ID NO: 22);
forward primer "ICSS": 5'-ACA CCT CAG CAG ATG CTA CC3' (SEQ ID NO: 23);
reverse primer "SK8": 5'-ATG GAT GAC TGA CAT GGC C-3' (SEQ ID NO: 24);
forward primer "KS6": 5' AAG GAT GAA CAT GGT CAC C 3' (SEQ ID NO: 25).
The locations of primers: Sk-la, cat; CA2, Pr+I, Pr-1, Pr+2(4A2), Pr-2(4A),
Pr+3(4A}, Pr-3 (4A), Pr-3(4A}, Pr+2(4A1), and Pr+1(4A) are diagramatically set
forth
in Figure 4. . The location of primers: KS1X, SKIXX, Ks2al, SK3, KS4, SK6A,
KSS,
SK8, and KS6 are diagramatically set forth in F'igu=e 5.
Families with a history of POAG in Klamath Falls, Oregon, are screened by
SSCP according to the method of Hue et aL, The journal o~' Investigative
O~htluiImoIogy
25r 2U5.4; 529-G32 (1995). SSCP primers SK-Ia; ca2,
CA2, Pr+1, Pr-2(4A), Pr+3(4A), SK1XX, and KS6 detect single strand
conformational r
polymorphisms in .this population. An SSCP is detected using SSCP primers
Pr+3(4A) and Pr-2(4A). 70 family. members of the Klamath Fall, Oregon are
screened with these primers and the results are set forth in Table 1.
49
CA 02456777 2004-03-O1
TABLE 1
~'e~tai SSCP+
Glaucoma ,positive individuals) 12 12 0
Glaucoma negative individuals 13 0 13
Spouses (glaurnma negative) 16 2 T4 -'
Others2 29 6 23
1= glaucoma positive individuals as determined by IOP of greater than 25 mmHg
2 = unidentified glaucoma due to the age of the individual.
A second SSCP is detected using SSCP primers Pr+1 and CA2. 14 #amily
members of the Kiamath Fall, Oregon are screened with these primers. A
characteristic polymorphism is found in the 6 affected family, members but
absent in
the,8 unaffected members. A third SSCP is detected using SSCP primers cat and
sk-
la. The same 14 family members of the Klamath Fall, Oregon that are screened
with
Pr+1 and CA2 are screened with cat and sk-la primers. A characteristic
polymorphism is found in the 6 affected family members but absent in the 8
unaffected members. A fourth SSCP is detected using SSCP primers KS6 and
SK1XX. 22 family members of the Klamath Fall, Oregon and 10 members of a
Portland; Oregon pedigree are screened with these primers. A polymorphism is
found in exon 3. The results are as set forth in Table 2.
_ TABLE 2
Total SSCP+ SSCP-
IQamath Fall, Oregon
Glaucoma positive individuals)3 3 0
Glaucoma negative individuals6 0 6
Others2 13 6 7
Portland, Oregon
Glaucoma positive individuals)6 6 0
Glaucoma negative individuals4 0 4
O.thers2 0 0 0
1= glaucoma positive individuals d by IOP of greater than 25 mmHg
as determine
2 = unidentified glaucoma individual.
due to the age of the
CA 02456777 2004-03-O1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
(ii) TITLE OF INVENTION: METHODS FOR THE DIAGNOSIS, PROGNOSTSAND
TREATMENT OF GLAUCOMA AND RELATED DISORDERS
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SMART & BIGGAR
(B) STREET: P.O. BOX 2999, STATION D
(C) CITY: OTTAWA
(D) STATE: ONT
(E) COUNTRY: CANADA
(F) ZIP: K1P 5Y6
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (text)
(vi) CURRENT APPLICATTON DATA:
(A) APPLICATION NUMBER: Division of CA 2,278,782
(B) FILING DATE: 09-JAN-1998
(C) CLASSIFICATTON:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/791,154
(B) FILING DATE: 28-JAN-1997
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/938,669
(B) FILING DATE: 26-SEP-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SMART & BIGGAR
(B) REGISTRATION NUMBER:
(C) REFERENCE/DOCKET NUMBER: 73185-12D
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613)-232-2486
(B) TELEFAX: (613)-232-8440
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
ATCTTTGTTC AGTTTACCTC AGGGCTATTA TGAAATGAAA TGAGATAACC 60
AATGTGAAAG
TCCTATAAAC TGTATAGCCT CCATTCGGAT GTATGTCTTT GGCAGGATGA 120
TAAAGAATCA
GGAAGAAGGA GTATCCACGT TAGCCAAGTG TCCAGGCTGT GTCTGCTCTT 180
ATTTTAGTGA
CAGATGTTGC TCCTGACAGA AGCTATTCTT CAGGAAACAT CACATCCAAT 240
ATGGTAAATC
CATCAAACAG GAGCTAAGAA ACAGGAATGA GATGGGCACT TGCCCAAGGA 300
AAAATGCCAG
GAGAGCAAAT AATGATGAAA AATAAACTTT TCCCTTTGTT TTTAATTTCA 360
GGAAAAAATG
ATGAGGACCA AAATCAATGA ATAAGGAAAA CAGCTCAGAA AAAAGATGTT 420
TCCAAATTGG
TAATTAAGTA TTTGTTCCTT GGGAAGAGAC CTCCATGTGA GCTTGATGGG 480
AAAATGGGAA
AAACGTCAAA AGCATGATCT GATCAGATCC CAAAGTGGAT TATTATTTTA 540
AAAACCAGAT
GGCATCACTC TGGGGAGGCA AGTTCAGGAA GGTCATGTTA GCAAAGGACA 600
TAACAATAAC
AGCAAAATCA AAATTCCGCA AATGCAGGAG GAAAATGGGG ACTGGGAAAG 660
CTTTCATAAC
AGTGATTAGG CAGTTGACCA TGTTCGCAAC ACCTCCCCGT CTATACCAGG 720
GAACACAAAA
ATTGACTGGG CTAAGCCTGG ACTTTCAAGG GAAATATGAA AAACTGAGAG 7$0
CAAAACAAAA
GACATGGTTA AAAGGCAACC AGAACATTGT GAGCCTTCAA AGCAGCAGTG 840
CCCCTCAGCA
GGGACCCTGA GGCATTTGCC TTTAGGAAGG CCAGTTTTCT TAAGGAATCT 900
TAAGAAACTC
TTGAAAGATC ATGAATTTTA ACCATTTTAA GTATAAAACA AATATGCGAT 960
GCATAATCAG
TTTAGACATG GGTCCCAATT TTATAAAGTC AGGCATACAA GGATAACGTG 1020
TCCCAGCTCC
GGATAGGTCA GAAATCATTA GAAATCACTG TGTCCCCATC CTAACTTTTT 10$0
CAGAATGATC
51
CA 02456777 2004-03-O1
TGTCATAGCC CTCACACACAGGCCCGATGTGTCTGACCTACAACCACATCTACAACCCAA1140
GTGCCTCAAC CATTGTTAACGTGTCATCTCAGTAGGTCCCATTACAAATGCCACCTCCCC1200
TGTGCAGCCC ATCCCGCTCCACAGGAAGTCTCCCCACTCTAGACTTCTGCATCACGATGT1260
TACAGCCAGA AGCTCCGTGAGGGTGAGGGTCTGTGTCTTACACCTACCTGTATGCTCTAC1320
ACCTGAGCTC ACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGTCTCAGCCTCC1380
CGCGTAGCTG GGACTACAGGCGCACGCCCGGCTAATTTTTGTATTGTTAGTAGAGATGGG1440
GTTTCACCAT ATTAGCCCGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCACCCACCTC1500
AGCCTCCTAA AGTGCTGGGATTACAGGCATGAGTCACCGCGCCCGGCCAAGGGTCAGTGT1560
TTAATAAGGA ATAACTTGAATGGTTTACTAAACCAACAGGGAAACAGACAAAAGCTGTGA1620
TAATTTCAGG GATTCTTGGGATGGGGAATGGTGCCATGAGCTGCCTGCCTAGTCCCAGAC1680
CACTGGTCCT CATCACTTTCTTCCCTCATCCTCATTTTCAGGCTAAGTTACCATTTTATT1740
CACCATGCTT TTGTGGTAAGCCTCCACATCGTTACTGAAATAAGAGTATACATAAACTAG1800
TTCCATTTGG GGCCATCTGTGTGTGTGTATAGGGGAGGAGGGCATACCCCAGAGACTCCT1860
TGAAGCCCCC GGCAGAGGTTTCCTCTCCAGCTGGGGGAGCCCTGCAAGCACCCGGGGTCC1920
TGGGTGTCCT GAGCAACCTGCCAGCCCGTGCCACTGGTTGTTTTGTTATCACTCTCTAGG1980
GACCTGTTGC TTTCTATTTCTGTGTGACTCGTTCATTCATCCAGGCATTCATTGACAATT2040
TATTGAGTAC TTATATCTGCCAGACACCAGAGACAAAATGGTGAGCAAAGCAGTCACTGC2100
CCTACCTTCG TGGAGGTGACAGTTTCTCATGGAAGACGTGCAGAAGAAAATTAATAGCCA2160
GCCAACTTAA ACCCAGTGCTGAAAGAAAGGAAATAAACACCATCTTGAAGAATTGTGCGC2220
AGCATCCCTT AACAAGGCCACCTCCCTAGCGCCCCCTGCTGCCTCCATCGTGCCCGGAGG2280
CCCCCAAGCC CGAGTCTTCCAAGCCTCCTCCTCCATCAGTCACAGCGCTGCAGCTGGCCT2340
GCCTCGCTTC CCGTGAATCGTCCTGGTGCATCTGAGCTGGAGACTCCTTGGCTCCAGGCT2400
CCAGAAAGGA AATGGAGAGGGAAACTAGTCTAACGGAGAATCTGGAGGGGACAGTGTTTC2460
CTCAGAGGGA AAGGGGCCTCCACGTCCAGGAGAATTCCAGGAGGTGGGGACTGCAGGGAG2520
TGGGGACGCT GGGGCTGAGCGGGTGCTGAAAGGCAGGAAGGTGAAAAGGGCAAGGCTGAA2580
GCTGCCCAGA TGTTCAGTGTTGTTCACGGGGCTGGGAGTTTTCCGTTGCTTCCTGTGAGC2640
CTTTTTATCT TTTCTCTGCTTGGAGGAGAAGAAGTCTATTTCATGAAGGGATGCAGTTTC2700
ATAAAGTCAG CTGTTAAAATTCCAGGGTGTGCATGGGTTTTCCTTCACGAAGGCCTTTAT2760
TTAATGGGAA TATAGGAAGCGAGCTCATTTCCTAGGCCGTTAATTCACGGAAGAAGTGAC2820
TGGAGTCTTT TCTTTCATGTCTTCTGGGCAACTACTCAGCCCTGTGGTGGACTTGGCTTA2880
TGCAAGACGG TCGAAAACCTTGGAATCAGGAGACTCGGTTTTCTTTCTGGTTCTGCCATT2940
GGTTGGCTGT GCGACCGTGGGCAAGTGTCTCTCCTTCCCTGGGCCATAGTCTTCTCTGCT3000
ATAAAGACCC TTGCAGCTCTCGTGTTCTGTGAACACTTCCCTGTGATTCTCTGTGAGGGG3060
GGATGTTGAG AGGGGAAGGAGGCAGAGCTGGAGCAGCTGAGCCACAGGGGAGGTGGAGGG3120
GGACAGGAAG GCAGGCAGAAGCTGGGTGCTCCATCAGTCCTCACTGATCACGTCAGACTC3180
CAGGACCGAG AGCCACAATGCTTCAGGAAAGCTCAATGAACCCAACAGCCACATTTTCCT3240
TCCCTAAGCA TAGACAATGGCATTTGCCAATAACCAAAAAGAATGCAGAGACTAACTGGT3300
GGTAGCTTTT GCCTGGCATTCAAAAACTGGGCCAGAGCAAGTGGAAAATGCCAGAGATTG3360
TTAAACTTTT CACCCTGACCAGCACCCCACGCAGCTCAGCAGTGACTGCTGACAGCACGG3420
AGTGACCTGC AGCGCAGGGGAGGAGAAGAAAAAGAGAGGGATAGTGTATGAGCAAGAAAG3480
ACAGATTCAT TCAAGGGCAGTGGGAATTGACCACAGGGATTATAGTCCACGTGATCCTGG3540
GTTCTAGGAG GCAGGGCTATATTGTGGGGGGAAAAAATCAGTTCAAGGGAAGTCGGGAGA3600
CCTGATTTCT AATACTATATTTTTCCTTTACAAGCTGAGTAATTCTGAGCAAGTCACAAG3660
GTAGTAACTG AGGCTGTAAGATTACTTAGTTTCTCCTTATfiAGGAACTCTTTTTCTCTGT3720
GGAGTTAGCA GCACAAGGGCAATCCCGTTTCTTTTAACAGGAAGAAAACATTCCTAAGAG3780
TAAAGCCAAA CAGATTCAAGCCTAGGTCTTGCTGACTATATGATTGGTTTTTTGAAAAAT3840
CATTTCAGCG ATGTTTACTATCTGATTCAGAAAATGAGACTAGTACCCTTTGGTCAGCTG3900
TAAACAAACA CCCATTTGTAAATGTCTCAAGTTCAGGCTTAACTGCAGAACCAATCAAAT3960
AAGAATAGAA TCTTTAGAGCAAACTGTGTTTCTCCACTCTGGAGGTGAGTCTGCCAGGGC4020
AGTTTGGAAA TATTTACTTCACAAGTATTGACACTGTTGTTGGTATTAACAACATAAAGT4080
TGCTCAAAGG CAATCATTATTTCAAGTGGCTTAAAGTTACTTCTGACAGTTTTGGTATAT4140
TTATTGGCTA TTGCCATTTGCTTTTTGTTTTTTCTCTTTGGGTTTATTAATGTAAAGCAG4200
GGATTATTAA CCTACAGTCCAGAAAGCCTGTGAATTTGAATGAGGAAAAAATTACATTTT4260
TGTTTTTACC ACCTTCTAACTAAATTTAACATTTTATTCCATTGCGAATAGAGCCATAAA4320
CTCAAAGTGG TAATAACAGTACCTGTGATTTTGTCATTACCAATAGAAATCACAGACATT4380
TTATACTATA TTACAGTTGTTGCAGATACGTTGTAAGTGAAATATTTATACTCAAAA.CTA4440
CTTTGAAATT AGACCTCCTGCTGGATCTTGTTTTTAACATATTAATAAAACATGTTTAAA4500
ATTTTGATAT TTTGATAATCATATTTCATTATCATTTGTTTCCTTTGTAATCTATATTTT4560
ATATATTTGA AAACATCTTTCTGAGAAGAGTTCCCCAGATTTCACCAATGAGGTTCTTGG4620
CATGCACACA CACAGAGTAAGAACTGATTTAGAGGCTAACATTGACATTGGTGCCTGAGA4680
TGCAAGACTG AAATTAGAAAGTTCTCCCAAAGATACACAGTTGTTTTAAAGCTAGGGGTG4740
AGGGGGGAAA TCTGCCGCTTCTATAGGAAfiGCTCTCCCTGGAGCCTGGTAGGGTGCTGTC4800
CTTGTGTTCT GGCTGGCTGTTATTTTTCTCTGTCCCTGCTACGTCTTAAAGGACTTGTTT4860
GGATCTCCAG TTCCTAGCATAGTGCCTGGCACAGTGCAGGTTCTCAATGAGTTTGCAGAG4920
52
CA 02456777 2004-03-O1
TGAATGGAAA AAATATATCC TTGTTGAAATCAGCACACCA GTAGTCCTGG4980
TATAAACTAG
TGTAAGTGTGTGTACGTGTGTGTGTGTGTG TGTGTGTGTGTGTAAAACCA GGTGGAGATA5040
TAGGAACTATTATTGGGGTATGGGTGCATA AATTGGGATGTTCTTTTTAA AAAGAAACTC5100
CAAACAGACTTCTGGAAGGTTATTTTCTAA GAATCTTGCTGGCAGCGTGA AGGCAACCCC5160
CCTGTGCACAGCCCCACCCAGCCTCACGTG GCCACCTCTGTCTTCCCCCA TGAAGGGCTG5220
GCTCCCCAGTATATATAAACCTCTCTGGAG CTCGGGCATGAGCCAGCAAG GCCACCCATC5280
CAGGCACCTCTCAGCACAGC 5300
(2) INFORMATTON ID N0:2:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:
5304
base
pairs
(B) TYPE:
nucleic
acid
(C) STRANDEDNESS:
single
(D) TOPOLOGY:linear
(xi) SEQUENCE SEQ ID
DESCRIPTION: N0:2:
ATCTTTGTTC AGTTTACCTCAGGGCTATTATGAAATGAAATGAGATAACCAATGTGAAAG60
TCCTATAAAC TGTATAGCCTCCATTCGGATGTATGTCTTTGGCAGGATGATAAAGAATCA120
GGAAGAAGGA GTATCCACGTTAGCCAAGTGTCCAGGCTGTGTCTGCTCTTATTTTAGTGA180
CAGATGTTGC TCCTGACAGAAGCTATTCTTCAGGAAACATCACATCCAATATGGTAAATC240
CATCAAACAG GAGCTAAGAAACAGGAATGAGATGGGCACTTGCCCAAGGAAAAATGCCAG300
GAGAGCAAAT AATGATGAAAAATAAACTTTTCCCTTTGTTTTTAATTTCAGGAAAAAATG360
ATGAGGACCA AAATCAATGAATAAGGAAAACAGCTCAGAAAAAAGATGTTTCCAAATTGG420
TAATTAAGTA TTTGTTCCTTGGGAAGAGACCTCCATGTGAGCTTGATGGGAAAATGGGAA480
AAACGTCAAA AGCATGATCTGATCAGATCCCAAAGTGGATTATTATTTTAAAAACCAGAT540
GGCATCACTC TGGGGAGGCAAGTTCAGGAAGGTCATGTTAGCAAAGGACATAACAATAAC600
AGCAAAATCA AAATTCCGCAAATGCAGGAGGAAAATGGGGACTGGGAAAGCTTTCATAAC660
AGTGATTAGG CAGTTGACCATGTTCGCAACACCTCCCCGTCTATACCAGGGAACACAAAA720
ATTGACTGGG CTAAGCCTGGACTTTCAAGGGAAATATGAAAAACTGAGAGCAAAACAAAA780
GACATGGTTA AAAGGCAACCAGAACATTGTGAGCCTTCAAAGCAGCAGTGCCCCTCAGCA840
GGGACCCTGA GGCATTTGCCTTTAGGAAGGCCAGTTTTCTTAAGGAATCTTAAGAAACTC900
TTGAAAGATC ATGAATTTTAACCATTTTAAGTATAAAACAAATATGCGATGCATAATCAG960
TTTAGACATG GGTCCCAATTTTATAAAGTCAGGCATACAAGGATAACGTGTCCCAGCTCC1020
GGATAGGTCA GAAATCATTAGAAATCACTGTGTCCCCATCCTAACTTTTTCAGAATGATC1080
TGTCATAGCC CTCACACACAGGCCCGATGTGTCTGACCTACAACCACATCTACAACCCAA1140
GTGCCTCAAC CATTGTTAACGTGTCATCTCAGTAGGTCCCATTACAAATGCCACCTCCCC1200
TGTGCAGCCC ATCCCGCTCCACAGGAAGTCTCCCCACTCTAGACTTCTGCATCACGATGT1260
TACAGCCAGA AGCTCCGTGAGGGTGAGGGTCTGTGTCTTACACCTACCTGTATGCTCTAC1320
ACCTGAGCTC ACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGTCTCAGCCTCC1380
CGCGTAGCTG GGACTACAGGCGCACGCCCGGCTAATTTTTGTATTGTTAGTAGAGATGGG1440
GTTTCACCAT ATTAGCCCGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCACCCACCTC1500
AGCCTCCTAA AGTGCTGGGATTACAGGCATGAGTCACCGCGCCCGGCCAAGGGTCAGTGT1560
TTAATAAGGA ATAACTTGAATGGTTTACTAAACCAACAGGGAAACAGACAAAAGCTGTGA1620
TAATTTCAGG GATTCTTGGGATGGGGAATGGTGCCATGAGCTGCCTGCCTAGTCCCAGAC1680
CACTGGTCCT CATCACTTTCTTCCCTCATCCTCATTTTCAGGCTAAGTTACCATTTTATT1740
CACCATGCTT TTGTGGTAAGCCTCCACATCGTTACTGAAATAAGAGTATACATAAACTAG1800
TTCCATTTGG GGCCATCTGTGTGTGTGTATAGGGGAGGAGGGCATACCCCAGAGACTCCT1860
TGAAGCCCCC GGCAGAGGTTTCCTCTCCAGCTGGGGGAGCCCTGCAAGCACCCGGGGTCC1920
TGGGTGTCCT GAGCAACCTGCCAGCCCGTGCCACTGGTTGTTTTGTTATCACTCTCTAGG1980
GACCTGTTGC TTTCTATTTCTGTGTGACTCGTTCATTCATCCAGGCATTCATTGACAATT2040
TATTGAGTAC TTATATCTGCCAGACACCAGAGACAAAATGGTGAGCAAAGCAGTCACTGC2100
CCTACCTTCG TGGAGGTGACAGTTTCTCATGGAAGACGTGCAGAAGAAAATTAATAGCCA2160
GCCAACTTAA ACCCAGTGCTGAAAGAAAGGAAATAAACACCATCTTGAAGAATTGTGCGC2220
AGCATCCCTT AACAAGGCCACCTCCCTAGCGCCCCCTGCTGCCTCCATCGTGCCCGGAGG2280
CCCCCAAGCC CGAGTCTTCCAAGCCTCCTCCTCCATCAGTCACAGCGCTGCAGCTGGCCT2340
GCCTCGCTTC CCGTGAATCGTCCTGGTGCATCTGAGCTGGAGACTCCTTGGCTCCAGGCT2400
CCAGAAAGGA AATGGAGAGGGAAACTAGTCTAACGGAGAATCTGGAGGGGACAGTGTTTC2460
CTCAGAGGGA AAGGGGCCTCCACGTCCAGGAGAATTCCAGGAGGTGGGGACTGCAGGGAG2520
TGGGGACGCT GGGGCTGAGCGGGTGCTGAAAGGCAGGAAGGTGAAAAGGGCAAGGCTGAA2580
GCTGCCCAGA TGTTCAGTGTTGTTCACGGGGCTGGGAGTTTTCCGTTGCTTCCTGTGAGC2640
CTTTTTATCT TTTCTCTGCTTGGAGGAGAAGAAGTCTATTTCATGAAGGGATGCAGTTTC2700
ATAAAGTCAG CTGTTAAAATTCCAGGGTGTGCATGGGTTTTCCTTCACGAAGGCCTTTAT2760
TTAATGGGAA TATAGGAAGCGAGCTCATTTCCTAGGCCGTTAATTCACGGAAGAAGTGAC2820
TGGAGTCTTT TCTTTCATGTCTTCTGGGCAACTACTCAGCCCTGTGGTGGACTTGGCTTA2880
53
CA 02456777 2004-03-O1
TGCAAGACGG TCGAAAACCTTGGAATCAGGAGACTCGGTTTTCTTTCTGGTTCTGCCATT2940
GGTTGGCTGT GCGACCGTGGGCAAGTGTCTCTCCTTCCCTGGGCCATAGTCTTCTCTGCT3000
ATAAAGACCC TTGCAGCTCTCGTGTTCTGTGAACACTTCCCTGTGATTCTCTGTGAGGGG3060
GGATGTTGAG AGGGGAAGGAGGCAGAGCTGGAGCAGCTGAGCCACAGGGGAGGTGGAGGG3120
GGACAGGAAG GCAGGCAGAAGCTGGGTGCTCCATCAGTCCTCACTGATCACGTCAGACTC3180
CAGGACCGAG AGCCACAATGCTTCAGGAAAGCTCAATGAACCCAACAGCCACATTTTCCT3240
TCCCTAAGCA TAGACAATGGCATTTGCCAATAACCAAAAAGAATGCAGAGACTAACTGGT3300
GGTAGCTTTT GCCTGGCATTCAAAAACTGGGCCAGAGCAAGTGGAAAATGCCAGAGATTG3360
TTAAACTTTT CACCCTGACCAGCACCCCACGCAGCTCAGCAGTGACTGCTGACAGCACGG3420
AGTGACCTGC AGCGCAGGGGAGGAGAAGAAAAAGAGAGGGATAGTGTATGAGCAAGAAAG3480
ACAGATTCAT TCAAGGGCAGTGGGAATTGACCACAGGGATTATAGTCCACGTGATCCTGG3540
GTTCTAGGAG GCAGGGCTATATTGTGGGGGGAAAAAATCAGTTCAAGGGAAGTCGGGAGA3600
CCTGATTTCT AATACTATATTTTTCCTTTACAAGCTGAGTAATTCTGAGCAAGTCACAAG3660
GTAGTAACTG AGGCTGTAAGATTACTTAGTTTCTCCTTATTAGGAACTCTTTTTCTCTGT3720
GGAGTTAGCA GCACAAGGGCAATCCCGTTTCTTTTAACAGGAAGAAAACATTCCTAAGAG378Q
TAAAGCCAAA CAGATTCAAGCCTAGGTCTTGCTGACTATATGATTGGTTTTTTGAAAAAT3840
CATTTCAGCG ATGTTTACTATCTGATTCAGAAAATGAGACTAGTACCCTTTGGTCAGCTG3900
TAAACAAACA CCCATTTGTAAATGTCTCAAGTTCAGGCTTAACTGCAGAACCAATCAAAT3960
AAGAATAGAA TCTTTAGAGCAAACTGTGTTTCTCCACTCTGGAGGTGAGTCTGCCAGGGC4020
AGTTTGGAAA TATTTACTTCACAAGTATTGACACTGTTGTTGGTATTAACAACATAAAGT4080
TGCTCAAAGG CAATCATTATTTCAAGTGGCTTAAAGTTACTTCTGACAGTTTTGGTATAT4140
TTATTGGCTA TTGCCATTTGCTTTTTGTTTTTTCTCTTTGGGTTTATTAATGTAAAGCAG4200
GGATTATTAA CCTACAGTCCAGAAAGCCTGTGAATTTGAATGAGGAAAAAATTACGTTTT4260
TATTTTTACC ACCTTCTAACTAAATTTAACATTTTATTCCATTGCGAATAGAGCCATAAA4320
CTCAAAGTGG TAATAAGAGTACCTGTGATTTTGTCATTACCAATAGAAATCACAGACATT4380
TTATACTATA TTACAGTTGTTGCAGGTACGTTGTAAGTGAAATATTTATACTCAAAACTA4440
CTTTGAAATT AGACCTCCTGCTGGATCTTGTTTTTAACATATTAATAAAACATGTTTAAA4500
ATTTTGATAT TTTGATAATCATATTTCATTATCATTTGTTTCCTTTGTAATCTATATTTT4560
ATATATTTGA AAACATCTTTCTGAGAAGAGTTCCCCAGATTTCACCAATGAGGTTCTTGG4620
CATGCACACA CACAGAGTAAGAACTGATTTAGAGGCTAACATTGACATTGGTGCCTGAGA4680
TGCAAGACTG AAATTAGAAAGTTCTCCCAAAGATACACAGTTGTTTTAAAGCTAGGGGTG4740
AGGGGGGAAA TCTGCCGCTTCTATAGGAATGCTCTCCCTGGAGCCTGGTAGGGTGCTGTC4800
CTTGTGTTCT GGCTGGCTGTTATTTTTCTCTGTCCCTGCTACGTCTTAAAGGACTTGTTT4860
GGATCTCCAG TTCCTAGCATAGTGCCTGGCACAGTGCAGGTTCTCAATGAGTTTGCAGAG4920
TGAATGGAAA TATAAACTAGAAATATATCTTTGTTGAAATCAGCACACCAGTAGTCCTGG4980
TGTAAGTGTG TGTACGTGTGTGTGTGTGTGTGTGTCTGTGTGTGTGTAAAACCAGGTGGA5040
GATATAGGAA CTATTATTGGGGTATGGGTGCATAAATTGGGATGTTCTTTTTAAAAAGAA5100
ACTCCAAACA GACTTCTGGAAGGTTATTTTCTAAGAATCTTGCTGGCAGCGTGAAGGCAA5160
CCCCCCTGTG CACAGCCCCACCCAGCCTCACGTGGCCACCTCTGTCTTCCCCCATGAAGG5220
GCTGGCTCCC CAGTATATATAAACCTCTCTGGAGCTCGGGCATGAGCCAGCAAGGCCACC5280
CATCCAGGCA CCTCTCAGCACAGC 5304
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6169 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:3:
ATCTTTGTTC AGTTTACCTC AGGGCTATTATGAAATGAAATGAGATAACCAATGTGAAAG60
TCCTATAAAC TGTATAGCCT CCATTCGGATGTATGTCTTTGGCAGGATGATAAAGAATCA120
GGAAGAAGGA GTATCCACGT TAGCCAAGTGTCCAGGCTGTGTCTGCTCTTATTTTAGTGA180
CAGATGTTGC TCCTGACAGA AGCTATTCTTCAGGAAACATCACATCCAATATGGTAAATC240
CATCAAACAG GAGCTAAGAA ACAGGAATGAGATGGGCACTTGCCCAAGGAAAAATGCCAG300
GAGAGCAAAT AATGATGAAA AATAAACTTTTCCCTTTGTTTTTAATTTCAGGAAAAAATG360
ATGAGGACCA AAATCAATGA ATAAGGAAAACAGCTCAGAAAAAAGATGTTTCCAAATTGG420
TAATTAAGTA TTTGTTCCTT GGGAAGAGACCTCCATGTGAGCTTGATGGGAAAATGGGAA480
AAACGTCAAA AGCATGATCT GATCAGATCCCAAAGTGGATTATTATTTTAAAAACCAGAT540
GGCATCACTC TGGGGAGGCA AGTTCAGGAAGGTCATGTTAGCAAAGGACATAACAATAAC600
AGCAAAATCA AAATTCCGCA AATGCAGGAGGAAAATGGGGACTGGGAAAGCTTTCATAAC660
AGTGATTAGG CAGTTGACCA TGTTCGCAACACCTCCCCGTCTATACCAGGGAACACAAAA720
ATTGACTGGG CTAAGCCTGG ACTTTCAAGGGAAATATGAAAAACTGAGAGCAAAACAAAA780
GACATGGTTA AAAGGCAACC AGAACATTGTGAGCCTTCAAAGCAGCAGTGCCCCTCAGCA840
54
CA 02456777 2004-03-O1
GGGACCCTGAGGCATTTGCCTTTAGGAAGGCCAGTTTTCTTAAGGAATCTTAAGAAACTC900
TTGAAAGATCATGAATTTTAACCATTTTAAGTATAAAACAAATATGCGATGCATAATCAG960
TTTAGACATGGGTCCCAATTTTATAAAGTCAGGCATACAAGGATAACGTGTCCCAGCTCC1020
GGATAGGTCAGAAATCATTAGAAATCACTGTGTCCCCATCCTAACTTTTTCAGAATGATC1080
TGTCATAGCCCTCACACACAGGCCCGATGTGTCTGACCTACAACCACATCTACAACCCAA1140
GTGCCTCAACCATTGTTAACGTGTCATCTCAGTAGGTCCCATTACAAATGCCACCTCCCC1200
TGTGCAGCCCATCCCGCTCCACAGGAAGTCTCCCCACTCTAGACTTCTGCATCACGATGT1260
TACAGCCAGAAGCTCCGTGAGGGTGAGGGTCTGTGTCTTACACCTACCTGTATGCTCTAC1320
ACCTGAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGTCTCAGCCTCC1380
CGCGTAGCTGGGACTACAGGCGCACGCCCGGCTAATTTTTGTATTGTTAGTAGAGATGGG1440
GTTTCACCATATTAGCCCGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCACCCACCTC1500
AGCCTCCTAAAGTGCTGGGATTACAGGCATGAGTCACCGCGCCCGGCCAAGGGTCAGTGT1560
TTAATAAGGAATAACTTGAATGGTTTACTAAACCAACAGGGAAACAGACAAAAGCTGTGA1620
TAATTTCAGGGATTCTTGGGATGGGGAATGGTGCCATGAGCTGCCTGCCTAGTCCCAGAC1680
CACTGGTCCTCATCACTTTCTTCCCTCATCCTCATTTTCAGGCTAAGTTACCATTTTATT1740
CACCATGCTTTTGTGGTAAGCCTCCACATCGTTACTGAAATAAGAGTATACATAAACTAG1800
TTCCATTTGGGGCCATCTGTGTGTGTGTATAGGGGAGGAGGGCATACCCCAGAGACTCCT1860
TGAAGCCCCCGGCAGAGGTTTCCTCTCCAGCTGGGGGAGCCCTGCAAGCACCCGGGGTCC1920
TGGGTGTCCTGAGCAACCTGCCAGCCCGTGCCACTGGTTGTTTTGTTATCACTCTCTAGG1980
GACCTGTTGCTTTCTATTTCTGTGTGACTCGTTCATTCATCCAGGCATTCATTGACAATT2040
TATTGAGTACTTATATCTGCCAGACACCAGAGACAAAATGGTGAGCAAAGCAGTCACTGC2100
CCTACCTTCGTGGAGGTGACAGTTTCTCATGGAAGACGTGCAGAAGAAAATTAATAGCCA2160
GCCAACTTAAACCCAGTGCTGAAAGAAAGGAAATAAACACCATCTTGAAGAATTGTGCGC2220
AGCATCCCTTAACAAGGCCACCTCCCTAGCGCCCCCTGCTGCCTCCATCGTGCCCGGAGG2280
CCCCCAAGCCCGAGTCTTCCAAGCCTCCTCCTCCATCAGTCACAGCGCTGCAGCTGGCCT2340
GCCTCGCTTCCCGTGAATCGTCCTGGTGCATCTGAGCTGGAGACTCCTTGGCTCCAGGCT2400
CCAGAAAGGAAATGGAGAGGGAAACTAGTCTAACGGAGAATCTGGAGGGGACAGTGTTTC2460
CTCAGAGGGAAAGGGGCCTCCACGTCCAGGAGAATTCCAGGAGGTGGGGACTGCAGGGAG2520
TGGGGACGCTGGGGCTGAGCGGGTGCTGAAAGGCAGGAAGGTGAAAAGGGCAAGGCTGAA2580
GCTGCCCAGATGTTCAGTGTTGTTCACGGGGCTGGGAGTTTTCCGTTGCTTCCTGTGAGC2640
CTTTTTATCTTTTCTCTGCTTGGAGGAGAAGAAGTCTATTTCATGAAGGGATGCAGTTTC2700
ATAAAGTCAGCTGTTAAAATTCCAGGGTGTGCATGGGTTTTCCTTCACGAAGGCCTTTAT2760
TTAATGGGAATATAGGAAGCGAGCTCATTTCCTAGGCCGTTAATTCACGGAAGAAGTGAC2820
TGGAGTCTTTTCTTTCATGTCTTCTGGGCAACTACTCAGCCCTGTGGTGGACTTGGCTTA2880
TGCAAGACGGTCGAAAACCTTGGAATCAGGAGACTCGGTTTTCTTTCTGGTTCTGCCATT2940
GGTTGGCTGTGCGACCGTGGGCAAGTGTCTCTCCTTCCCTGGGCCATAGTCTTCTCTGCT3000
ATAAAGACCCTTGCAGCTCTCGTGTTCTGTGAACACTTCCCTGTGATTCTCTGTGAGGGG3060
GGATGTTGAGAGGGGAAGGAGGCAGAGCTGGAGCAGCTGAGCCACAGGGGAGGTGGAGGG3120
GGACAGGAAGGCAGGCAGAAGCTGGGTGCTCCATCAGTCCTCACTGATCACGTCAGACTC3180
CAGGACCGAGAGCCACAATGCTTCAGGAAAGCTCAATGAACCCAACAGCCACATTTTCCT3240
TCCCTAAGCATAGACAATGGCATTTGCCAATAACCAAAAAGAATGCAGAGACTAACTGGT3300
GGTAGCTTTTGCCTGGCATTCAAAAACTGGGCCAGAGCAAGTGGAAAATGCCAGAGATTG3360
TTAAACTTTTCACCCTGACCAGCACCCCACGCAGCTCAGCAGTGACTGCTGACAGCACGG3420
AGTGACCTGCAGCGCAGGGGAGGAGAAGAAAAAGAGAGGGATAGTGTATGAGCAAGAAAG3480
ACAGATTCATTCAAGGGCAGTGGGAATTGACCACAGGGATTATAGTCCACGTGATCCTGG3540
GTTCTAGGAGGCAGGGCTATATTGTGGGGGGAAAAAATCAGTTCAAGGGAAGTCGGGAGA3600
CCTGATTTCTAATACTATATTTTTCCTTTACAAGCTGAGTAATTCTGAGCAAGTCACAAG3660
GTAGTAACTGAGGCTGTAAGATTACTTAGTTTCTCCTTATTAGGAACTCTTTTTCTCTGT3720
GGAGTTAGCAGCACAAGGGCAATCCCGTTTCTTTTAACAGGAAGAAAACATTCCTAAGAG3780
TAAAGCCAAACAGATTCAAGCCTAGGTCTTGCTGACTATATGATTGGTTTTTTGAAAAAT3840
CATTTCAGCGATGTTTACTATCTGATTCAGAAAATGAGACTAGTACCCTTTGGTCAGCTG3900
TAAACAAACACCCAGTTGTAAATGTCTCAAGTTCAGGCTTAACTGCAGAACCAATCAAAA3960
AGAATAGAATCTTTAGAGCAAACTGTGTTTCTCCACATCTGGAGGTGAGTCTGCCAGGGC4020
AGTTTGGAAATATTTACTTCACAAGTATTGACACTGTTGTTGGTATTAACAACATAAAGT4080
TGCTCAAAGGCAATCATTATTTCAAGTGGCTTAAAGTTACTTCTGACAGTTTTGGTATAT4140
TTATTGGCTATTGCCATTTGCTTTTTGTTTTTTCTCTTTGGGTTTATTAATGTAAAGCAG4200
GGATTATTAACCTACAGTCCAGAAAGCCTGTGAATTTGAATGAGGAAAAAATTACATTTT4260
TGTTTTTACCACCTTCTAACTAAATTTAACATTTTATTCCATTGCGAATAGAGCCATAAA4320
CTCAAAGTGGTAATAACAGTACCTGTGATTTTGTCATTACCAATAGAAATCACAGACATT4380
TTATACTATATTACAGTTGTTGCAGATACGTTGTAAGTGAAATATTTATACTCAAAACTA4440
CTTTGAAATTAGACCTCCTGCTGGATCTTGTTTTTAACATATTAATAAAACATGTTTAAA4500
ATTTTGATATTTTGATAATCATATTTCATTATCATTTGTTTCCTTTGTAATCTATATTTT4560
ATATATTTGAAAACATCTTTCTGAGAAGAGTTCCCCAGATTTCACCAATGAGGTTCTTGG4620
CATGCACACACACAGAGTAAGAACTGATTTAGAGGCTAACATTGACATTGGTGCCTGAGA4680
CA 02456777 2004-03-O1
TGCAAGACTG TTGTTTTAAA 4740
AAATTAGAAA GCTAGGGGTG
GTTCTCCCAA
AGATACACAG
AGGGGGGAAA TCTGCCGCTTCTATAGGAATGCTCTCCCTGGAGCCTGGTAGGGTGCTGTC4800
CTTGTGTTCT GGCTGGCTGTTATTTTTCTCTGTCCCTGCTACGTCTTAAAGGACTTGTTT4860
GGATCTCCAG TTCCTAGCATAGTGCCTGGCACAGTGCAGGTTCTCAATGAGTTTGCAGAG4920
TGAATGGAAA TATAAACTAGAAATATATCCTTGTTGAAATCAGCACACCAGTAGTCCTGG4980
TGTAAGTGTG TGTACGTGTGTGTGTGTGTGTGTGTGTGTGTGTAAAACCAGGTGGAGATA5040
TAGGAACTAT TATTGGGGTATGGGTGCATAAATTGGGATGTTCTTTTTAAAAAGAAACTC5100
CAAACAGACT TCTGGAAGGTTATTTTCTAAGAATCTTGCTGGCAGCGTGAAGGCAACCCC5160
CCTGTGCACA GCCCCACCCAGCCTCACGTGGCCACCTCTGTCTTCCCCCATGAAGGGCTG5220
GCTCCCCAGT ATATATAAACCTCTCTGGAGCTCGGGCATGAGCCAGCAAGGCCACCCATC5280
CAGGCACCTC TCAGCACAGCAGAGCTTTCCAGAGGAAGCCTCACCAAGCCTCTGCAATGA5340
GGTTCTTCTG TGCACGTTGCTGCAGCTTTGGGCCTGAGATGCCAGCTGTCCAGCTGCTGC5400
TTCTGGCCTG CCTGGTGTGGGATGTGGGGGCCAGGACAGCTCAGCTCAGGAAGGCCAATG5460
ACCAGAGTGG CCGATGCCAGTATACCTTCAGTGTGGCCAGTCCCAATGAATCCAGCTGCC5520
CAGAGCAGAG CCAGGCCATGTCAGTCATCCATAACTTACAGAGAGACAGCAGCACCCAAC5580
GCTTAGACCT GGAGGCCACCAAAGCTCGACTCAGCTCCCTGGAGAGCCTCCTCCACCAAT5640
TGACCTTGGA CCAGGCTGCCAGGCCCCAGGAGACCCAGGAGGGGCTGCAGAGGGAGCTGG5700
GCACCCTGAG GCGGGAGCGGGACCAGCTGGAAACCCAAACCAGAGAGTTGGAGACTGCCT5760
ACAGCAACCT CCTCCGAGACAAGTCAGTTCTGGAGGAAGAGAAGAAGCGACTAAGGCAAG5820
AAAATGAGAA TCTGGCCAGGAGGTTGGAAAGCAGCAGCCAGGAGGTAGCAAGGCTGAGAA5880
GGGGCCAGTG TCCCCAGACCCGAGACACTGCTCGGGCTGTGCCACCAGGCTCCAGAGAAG5940
GTAAGAATGC AGAGTGGGGGGACTCTGAGTTCAGCAGGTGATATGGCTCGTAGTGACCTG6000
CTACAGGCGC TCCAGGCCTCCCTGCCCTTTCTCCTAGAGACTGCACAGCTAGCACAAGAC6060
AGATGAATTA AGGAAAGCACACGATCACCTTCAAGTATTACTAGTAATTTAGCTCCTGAG6120
AGCTTCATTT AGATTAGTGGTTCAGAGTTCTTGTGCCCCTCCATGTCAG 6169
(2) INFORMATION ID N0:4:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 926 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:SEQ ID
N0:4:
AAGGTAGGCACATTGCCCTG CAATTTATAATTTATGAGGTGTTCAATTAT GGAATTGTCA60
AATATTAACAAAAGTAGAGA GACTACAATGAACTCCAATGTAGCCATAAC TCAGGCCCAA120
CTGTTATCAGCACAGTCCAA TCATGTTTTATCTTTCCTTCTCTGACCCCC AACCCATCCC180
CAGTCCTTATCTAAAATCAA ATATCAAACACCATACTCTTTGGGAGCCTA TTTATTTAGT240
TAGTTAGTTTTCAGACAGAG TTTCTTTCTTGTTCCCAAGCTGGAGTACAA TAGTGTAGTC300
TCGGCTAACAGCAATCTCCC CCTCCTTGGTTCAAGCAATTCTCCTGCCTC AGTCTCCCAA360
GAAGCTGGGATTATAGACAC CTGCCACCACATCCAGCTAATTTTTTTGTG TTTTAGAAAA420
GACAGGGTTTCACCATGTTG GCCAGGCTGGTTTCGAACTCCTGACCTCAG GTGATCCGCC480
TGCCTCGGCCTCCCAAAGTG CTGGGATTACAGGCATGAGCCACCACGCCT GGCCGGCAGC540
CTATTTAAATGTCATCCTCA ACATAGTCAATCCTTGGGCCATTTTTTCTT ACAGTAAAAT600
TTTGTCTCTTTCTTTTAATC AGTTTCTACGTGGAATTTGGACACTTTGGC CTTCCAGGAA660
CTGAAGTCCGAGCTAACTGA AGTTCCTGCTTCCCGAATTTTGAAGGAGAG CCCATCTGGC720
TATCTCAGGAGTGGAGAGGG AGACACCGGTATGAAGTTAAGTTTCTTCCC TTTTGTGCCC780
ACGTGGTCTTTATTCATGTC TAGTGCTGTGTTCAGAGAATCAGTATAGGG TAAATGCCCA840
CCCAAGGGGGAAATTAACTT CCCTGGGAGCAGAGGGAGGGGAGGAGAAGA GGAACAGAAC900
TCTCTCTCTCTCTCTGTTAC CCTTGT 926
(2) INFORMATION ID N0:5:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 2099 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:SEQ ID
N0:5:
TGGCTCTGCCAAGCTTCCGC ATGATCATTGTCTGTGTTTGGAAGATTATG GATTAAGTGG60
TGCTTCGTTTTCTTTCTGAA TTTACCAGGATGTGGAGAACTAGTTTGGGT AGGAGAGCCT120
CTCACGCTGAGAACAGCAGA AACAATTACTGGCAAGTATGGTGTGTGGAT GCGAGACCCC180
AAGCCCACCTACCCCTACAC CCAGGAGACCACGTGGAGAATCGACACAGT TGGCACGGAT240
56
CA 02456777 2004-03-O1
GTCCGCCAGG TTTTTGAGTATGACCTCATCAGCCAGTTTATGCAGGGCTA CCCTTCTAAG300
GTTCACATAC TGCCTAGGCCACTGGAAAGCACGGGTGCTGTGGTGTACTC GGGGAGCCTC360
TATTTCCAGG GCGCTGAGTCCAGAACTGTCATAAGATATGAGCTGAATAC CGAGACAGTG420
AAGGCTGAGA AGGAAATCCCTGGAGCTGGCTACCACGGACAGTTCCCGTA TTCTTGGGGT480
GGCTACACGG ACATTGACTTGGCTGTGGATGAAGCAGGCCTCTGGGTCAT TTACAGCACC540
GATGAGGCCA AAGGTGCCATTGTCCTCTCCAAACTGAACCCAGAGAATCT GGAACTCGAA600
CAAACCTGGG AGACAAACATCCGTAAGCAGTCAGTCGCCAATGCCTTCAT CATCTGTGGC660
ACCTTGTACA CCGTCAGCAGCTACACCTCAGCAGATGCTACCGTCAACTT TGCTTATGAC720
ACAGGCACAG GTATCAGCAAGACCCTGACCATCCCATTCAAGAACCGCTA TAAGTACAGC780
AGCATGATTG ACTACAACCCCCTGGAGAAGAAGCTCTTTGCCTGGGACAA CTTGAACATG840
GTCACTTATG ACATCAAGCTCTCCAAGATGTGAAAAGCCTCCAAGCTGTA CAGGCAATGG900
CAGAAGGAGA TGCTCAGGGCTCCTGGGGGGAGCAGGCTGAAGGGAGAGCC AGCCAGCCAG960
GGCCCAGGCA GCTTTGACTGCTTTCCAAGTTTTCATTAATCCAGAAGGAT GAACATGGTC1020
ACCATCTAAC TATTCAGGAATTGTAGTCTGAGGGCGTAGACAATTTCATA TAATAAATAT1080
CCTTTATCTT CTGTCAGCATTTATGGGATGTTTAATGACATAGTTCAAGT TTTCTTGTGA1140
TTTGGGGCAA AAGCTGTAAGGCATAATAGTCTTTTCCTGAAAACCATTGC TCTTGCATGT1200
TACATGGTTA CCACAAGCCACAATAAAAAGCATAACTTCTAAAGGAAGCA GAATAGCTCC1260
TCTGGCCAGC ATCGAATATAAGTAAGATGCATTTACTACAGTTGGCTTCT AATGCTTCAG1320
ATAGAATACA GTTGGGTCTCACATAACCCTTACATTGTGAAATAAAATTT TCTTACCCAA1380
CGTTCTCTTC CTTGAACTTTGTGGGAATCTTTGCTTAAGAGAAGGATATA GATTCCAACC1440
ATCAGGTAAT TCCTTCAGGTTGGGAGATGTGATTGCAGGATGTTAAAGGT GTGTGTGTGT1500
GTGTGTGTGT GTGTGTAACTGAGAGGCTTGTGCCTGGTTTTGAGGTGCTG CCCAGGATGA1560
CGCCAAGCAA ATAGCGCATCCACACTTTCCCACCTCCATCTCCTGGTGCT CTCGGCACTA1620
CCGGAGCAAT CTTTCCATCTCTCCCCTGAACCCACCCTCTATTCACCCTA ACTCCACTTC1680
AGTTTGCTTT TGATTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGGGT CTCGCTCTGT1740
CACCCAGGCT GGAGTGCAGTGGCACGATCTCGGCTCACTGCAAGTTCCGC CTCCCAGGTT1800
CACACCATTC TCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACC TGCCACCACG1860
CCTGGCTAAT TTTTTTTTTTTCCAGTGAAGATGGGTTTCACCATGTTAGC CAGGATGGTC1920
TCGATCTCCT GACCTTGTCATCCACCCACCTTGGCCTCCCAAAGTGCTGG GATTACAGGC1980
GTGAGCCACC ACGCCCAGCCCCTCCACTTCAGTTTTTATCTGTCATCAGG GGTATGAATT2040
TTATAAGCCA CACCTCAGGTGGAGAAAGCTTGATGCATAGCTTGAGTATT CTATACTGT2099
(2) INFORMATION ID N0:6:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH:
19 base
pairs
(B) TYPE: eic acid
nucl
(C) STRANDEDNESS:
single
(D) TOPOLOGY:linear
(xi) SEQUENCE SEQ ID
DESCRTPTION: N0:6:
TGAGGCTTCC TCTGGAAAC 19
(2) INFORMATION ID N0:7:
FOR SEQ
(i) SEQUENCE
CHARACTERTSTICS:
(A) LENGTH: base pairs
20
(B) TYPE: eic acid
nucl
(C) STR.ANDEDNESS:
single
(D) TOPOLOGY:linear
(xi) SEQUENCE SEQ ID :
DESCRIPTION: N0:7
TGAAATCAGC ACACCAGTAG 20
(2 ) INFORMATION FOR ID N0:8:
SEQ
(i) S EQUENCE S:
CHARACTERISTIC
(A) LENGTH: base pairs
21
(B) TYPE: eic acid
nucl
(C) STRANDEDNESS: single
(D) TOPOLOGY:linear
(xi) SEQUENCE SCRIPTION:SEQ ID :
DE N0:8
GCACCCATAC CCCAATAATAG 2I
57
CA 02456777 2004-03-O1
20
40
SO
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
AGAGTTCCCC AGATTTCACC 20
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
ATCTGGGGAA CTCTTCTCAG 20
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
TACAGTTGTT GCAGATACG 19
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
ACAACGTATC TGCAACAACT G 21
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
TCAGGCTTAA CTGCAGAACC 20
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
TTGGTTCTGC AGTTAAGCC 19
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
58
CA 02456777 2004-03-O1
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
AGCAGCACAA GGGCAATCC 19
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
ACAGGGCTAT ATTGTGGG 18
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
CCTGAGATGC CAGCTGTCC 19
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
CTGAAGCATT AGAAGCCAAC 20
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
ACCTTGGACC AGGCTGCCAG 20
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
AGGTTTGTTC GAGTTCCAG 19
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
ACAATTACTG GCAAGTATGG 20
59
CA 02456777 2004-03-O1
20
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
CCTTCTCAGC CTTGCTACC 19
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
ACACCTCAGC AGATGCTACC 20
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
ATGGATGACT GACATGGCC 19
(2) INFORMATION ID N0:25:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 19 base
pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION:SEQ ID
N0:25:
AAGGATGAAC ATGGTCACC 19
(2) INFORMATION ID N0:26:
FOR SEQ
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 1548 base rs
pai
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
cDNA
(xi) SEQUENCE DESCRIPTION:SEQ ID
N0:26:
AGAGCTTTCC AGAGGAAGCC TCACCAAGCCTCTGCAATGAGGTTCTTCTG TGCACGTTGC60
TGCAGCTTTG GGCCTGAGAT GCCAGCTGTCCAGCTGCTGCTTCTGGCCTG CCTGGTGTGG120
GATGTGGGGG CCAGGACAGC TCAGCTCAGGAAGGCCAATGACCAGAGTGG CCGATGCCAG180
TATACCTTCA GTGTGGCCAG TCCCAATGAATCCAGCTGCCCAGAGCAGAG CCAGGCCATG240
TCAGTCATCC ATAACTTACA GAGAGACAGCAGCACCCAACGCTTAGACCT GGAGGCCACC300
AAAGCTCGAC TCAGCTCCCT GGAGAGCCTCCTCCACCAATTGACCTTGGA CCAGGCTGCC360
AGGCCCCAGG AGACCCAGGA GGGGCTGCAGAGGGAGCTGGGCACCCTGAG GCGGGAGCGG420
GACCAGCTGG AAACCCAAAC CAGAGAGTTGGAGACTGCCTACAGCAACCT CCTCCGAGAC480
AAGTCAGTTC TGGAGGAAGA GAAGAAGCGACTAAGGCAAGAAAATGAGAA TCTGGCCAGG540
AGGTTGGAAA GCAGCAGCCA GGAGGTAGCAAGGCTGAGAAGGGGCCAGTG TCCCCAGACC600
CGAGACACTG CTCGGGCTGT GCCACCAGGCTCCAGAGAAGTTTCTACGTG GAATTTGGAC660
ACTTTGGCCT TCCAGGAACT GAAGTCCGAGCTAACTGAAGTTCCTGCTTC CCGAATTTTG720
AAGGAGAGCC CATCTGGCTA TCTCAGGAGTGGAGAGGGAGACACCGGATG TGGAGAACTA780
GTTTGGGTAG GAGAGCCTCT CACGCTGAGAACAGCAGAAACAATTACTGG CAAGTATGGT840
GTGTGGATGC GAGACCCCAA GCCCACCTACCCCTACACCCAGGAGACCAC GTGGAGAATC900
GACACAGTTG GCACGGATGT CCGCCAGGTTTTTGAGTATGACCTCATCAG CCAGTTTATG960
CA 02456777 2004-03-O1
CAGGGCTACC CTTCTAAGGTTCACATACTGCCTAGGCCACTGGAAAGCACGGGTGCTGTG1020
GTGTACTCGG GGAGCCTCTATTTCCAGGGCGCTGAGTCCAGAACTGTCATAAGATATGAG1080
CTGAATACCG AGACAGTGAAGGCTGAGAAGGAAATCCCTGGAGCTGGCTACCACGGACAG1140
TTCCCGTATT CTTGGGGTGGCTACACGGACATTGACTTGGCTGTGGATGAAGCAGGCCTC1200
TGGGTCATTT ACAGCACCGATGAGGCCAAAGGTGCCATTGTCCTCTCCAAACTGAACCCA1260
GAGAATCTGG AACTCGAACAAACCTGGGAGACAAACATCCGTAAGCAGTCAGTCGCCAAT1320
GCCTTCATCA TCTGTGGCACCTTGTACACCGTCAGCAGCTACACCTCAGCAGATGCTACC1380
GTCAACTTTG CTTATGACACAGGCACAGGTATCAGCAAGACCCTGACCATCCCATTCAAG1440
AACCGCTATA AGTACAGCAGCATGATTGACTACAACCCCCTGGAGAAGAAGCTCTTTGCC1500
TGGGACAACT TGAACATGGTCACTTATGACATCAAGCTCTCCAAGATG 1548
(2) INFORMATION ID N0:27:
FOR SEQ
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Thr Gly Ala val Val Tyr Ser Gly Ser Leu Tyr Phe Gln Gly Ala Glu
1 5 10 15
Ser Arg Thr Val Ile Arg Tyr Glu Leu Asn Thr Glu Thr Val Lys Ala
20 25 30
Glu Lys Glu Ile Pro Gly Ala Gly Tyr His Gly Gln Phe Pro Tyr Ser
35 40 45
Trp Gly Gly Tyr Thr Asp Ile Asp Leu Ala Val Asp Glu Ala Gly Leu
50 55 60
Trp Val Ile Tyr Ser Thr Asp Glu Ala Lys Gly Ala Ile Val Leu Ser
65 70 75 80
Lys Leu Asn Pro Glu Asn Leu Glu Leu Glu Gln Thr Trp Glu Thr Asn
85 90 95
Ile Arg Lys Gln Ser Val Ala Asn Ala Phe Ile Ile Cys Gly Thr Leu
100 105 110
Tyr Thr Val Ser Ser Tyr Thr Ser Ala Asp Ala Thr Val Asn Phe Ala
115 120 125
Tyr Asp Thr Gly Thr Gly Ile Ser Lys Thr Leu Thr Ile Pro Phe Lys
130 135 140
Asn Arg Tyr Lys Tyr Ser Ser Met Ile Asp Tyr Asn Pro Leu Glu Lys
145 150 155 160
Lys Leu Phe Ala Trp Asp Asn Leu Asn Met Val Thr Tyr Asp Ile Lys
165 170 175
Leu Ser
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 131 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Arg Phe Asp Leu Lys Thr Glu Thr Ile Leu Lys Thr Arg Ser Leu Asp
1 5 10 15
Tyr Ala Gly Tyr Asn Asn Met Tyr His Tyr Ala Trp Gly Gly His Ser
20 25 30
Asp Ile Asp Leu Met Val Asp Glu Ser Gly Leu Trp Ala Val Tyr Ala
35 40 45
Thr Asn Gln Asn Ala Gly Asn Ile Val Val Ser Arg Leu Asp Pro Val
50 55 60
Ser Leu Gln Thr Leu Gln Thr Trp Asn Thr Ser Tyr Pro Lys Arg Xaa
70 75 80
61
CA 02456777 2004-03-O1
Pro Gly Xaa Ala Phe Ile Ile Cys Gly Thr Cys Tyr Val Thr Asn Gly
85 90 95
Tyr Ser Gly Gly Thr Lys Val His Tyr Ala Tyr Gln Thr Asn Ala Ser
100 105 110
Thr Tyr Glu Tyr Ile Asp Ile Pro Phe Gln Asn Lys Leu Xaa Pro His
115 120 125
Phe Pro Cys
130
(2) INFORMATION ID N0:29:
FOR
SEQ
(i) CS:
SEQUENCE
CHARACTERISTI
(A) LENGTH:178amino ids
ac
(B) TYPE: acid
amino
(C) STRANDEDNESS:
single
(D) TOPOLOGY:
linear
(ii) TYPE:
MOLECULE None
(xi) DESCRIPTION: SEQID N0:29:
SEQUENCE
Gly ThrGly Gln ValTyr Asn GlySerIleTyr PheAsn LysPhe
Val
1 5 10 15
Gln SerHis Ile IleArg Phe AspLeuLysThr GluThr IleLeu
Ile
20 25 30
Lys ThrArg Ser AspTyr Ala GlyTyrAsnAsn MetTyr HisTyr
Leu
35 40 45
Ala TrpGly Gly SerAsp Ile AspLeuMetVal AspGlu AsnGly
His
50 55 60
Leu TrpAla Val AlaThr Asn GlnAsnAlaGly AsnIle ValIle
Tyr
65 70 75 80
Ser LysLeu Asp ValSer Leu GlnIleLeuGln ThrTrp AsnThr
Pro
85 90 95
Ser TyrPro Lys SerAla Gly GluAlaPheIle IleCys GlyThr
Arg
100 105 110
Leu TyrVal Thr GlyTyr Ser GlyGlyThrLys ValHis TyrAla
Asn
115 120 125
Tyr GlnThr Asn SerThr Tyr GluTyrIleAsp IlePro PheGln
Ala
130 135 140
Asn LysTyr Ser IleSer Met LeuAspTyrAsn ProLys AspArg
His
145 150 155 160
Ala LeuTyr Ala AsnAsn Gly HisGlnThrLeu TyrAsn ValThr
Trp
165 170 175
Leu Phe
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 177 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Gly Ala Gly Val Val Val His Asn Asn Asn Leu Tyr Tyr Asn Cys Phe
1 5 10 15
Asn Ser His Asp Met Cys Arg Ala Ser Leu Thr Ser Gly Val Tyr Gln
20 25 30
Lys Lys Pro Leu Leu Asn Ala Leu Phe Asn Asn Arg Phe Ser Tyr Ala
35 40 45
Gly Thr Met Phe Gln Asp Met Asp Phe Ser Ser Asp Glu Lys Gly Leu
50 55 60
Trp Val Ile Phe Thr Thr Glu Lys Ser Ala Gly Lys Ile Val Val Gly
70 75 80
Lys Val Asn Val Ala Thr Phe Thr Val Asp Asn Ile Trp Ile Thr Thr
85 90 95
62
CA 02456777 2004-03-O1
Gln Asn Lys Ser Asp Ala Ser Asn Ala Phe Met Tle Cys Gly Val Leu
100 105 110
Tyr Val Thr Arg Ser Leu Gly Pro Lys Met Glu Glu Val Phe Tyr Met
115 120 125
Phe Asp Thr Lys Thr Gly Lys Glu Gly His Leu Ser Ile Met Met Glu
130 135 140
Lys Met Ala Glu Lys Val His Ser Leu Ser Tyr Asn Ser Asn Asp Arg
145 150 155 160
Lys Leu Tyr Met Phe Ser Glu Gly Tyr Leu Leu His Tyr Asp Ile Ala
165 170 175
Leu
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS; single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: None
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Gly Val Val Tyr Ser Arg Leu Thr Glu Thr Leu Ala Gly Tyr Asn Asn
1 5 10 15
Tyr Ala Trp Gly Gly Asp Ile Asp Leu Val Asp Glu Gly Leu Trp Tyr
20 25 30
Thr Ala Gly Ile Val Ser Lys Leu Pro Leu Gln Thr Trp Thr Lys Ala
35 40 45
Phe Ile Ile Cys Gly Thr Leu Tyr Val Thr Tyr Val Tyr Ala Tyr Thr
50 55 60
Ile Tyr Asp Tyr Asn Pro Lys Leu Tyr Leu
65 70
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 504 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Met Arg Phe Phe Cys Ala Arg Cys Cys Ser Phe Gly Pro Glu Met Pro
1 5 10 15
Ala Val Gln Leu Leu Leu Leu Ala Cys Leu Val Trp Asp Val Gly Ala
20 25 30
Arg Thr Ala Gln Leu Arg Lys Ala Asn Asp Gln Ser Gly Arg Cys G.ln
35 40 45
Tyr Thr Phe Ser Val Ala Ser Pro Asn Glu Ser Ser Cys Pro Glu Gln
50 55 60
Ser Gln Ala Met Ser Val Ile His Asn Leu Gln Arg Asp Ser Ser Thr
65 70 75 80
Gln Arg Leu Asp Leu Glu Ala Thr Lys Ala Arg Leu Ser Ser Leu Glu
85 90 95
Ser Leu Leu His Gln Leu Thr Leu Asp Gln Ala Ala Arg Pro Gln Glu
100 105 110
Thr Gln Glu Gly Leu Gln Arg Glu Leu Gly Thr Leu Arg Arg Glu Arg
115 120 125
Asp Gln Leu Glu Thr Gln Thr Arg Glu Leu Glu Thr Ala Tyr Ser Asn
130 135 140
Leu Leu Arg Asp Lys Ser Val Leu Glu Glu Glu Lys Lys Arg Leu Arg
145 150 155 160
Gln Glu Asn Glu Asn Leu Ala Arg Arg Leu Glu Ser Ser Ser Gln Glu
165 170 175
63
CA 02456777 2004-03-O1
Val Ala Leu ArgArgGly GlnCys ProGlnThr ArgAspThr Ala
Arg
180 185 190
Arg AlaValPro FroGlySer ArgGlu ValSerThr TrpAsnLeu Asp
195 200 205
Thr LeuAlaPhe GlnGluLeu LysSer GluLeuThr GluValPro Ala
210 215 220
Ser ArgIleLeu LysGluSer ProSer GlyTyrLeu ArgSerGly Glu
225 230 235 240
Gly AspThrGly CysGlyGlu LeuVal TrpValGly GluProLeu Thr
245 250 255
Leu ArgThrAla GluThrIle ThrGly LysTyrGly ValTrpMet Arg
260 265 270
Asp ProLysPro ThrTyrPro TyrThr GlnGluThr ThrTrpArg Ile
275 280 285
Asp ThrValGly ThrAspVal ArgGln ValPheGlu TyrAspLeu Ile
290 295 300
Ser GlnPheMet GlnGlyTyr ProSer LysValHis IleLeuPro Arg
305 310 315 320
Pro LeuGluSer ThrGlyAla ValVal TyrSerGly SerLeuTyr Phe
325 330 335
Gln GlyAlaGlu SerArgThr ValIle ArgTyrGlu LeuAsnThr Glu
340 345 350
Thr ValLysAla GluLysGlu IlePro GlyAlaGly TyrHisGly Gln
355 360 365
Phe ProTyrSer TrpGlyGly TyrThr AspIleAsp LeuAlaVal Asp
370 375 380
Glu AlaGlyLeu TrpValIle TyrSer ThrAspGlu AlaLysGly Ala
385 390 395 400
Ile ValLeuSer LysLeuAsn ProGlu AsnLeuGlu LeuGluGln Thr
405 410 415
Trp GluThrAsn IleArgLys GlnSer ValAlaAsn AlaPheIle Ile
420 425 430
Cys GlyThrLeu TyrThrVal SerSer TyrThrSer AlaAspAla Thr
435 440 445
Val AsnPheAla TyrAspThr GlyThr GlyIleSer LysThrLeu Thr
450 455 460
Ile ProPheLys AsnArgTyr LysTyr SerSerMet IleAspTyr Asn
465 470 475 480
Pro LeuGluLys LysLeuPhe AlaTrp AspAsnLeu AsnMetVal Thr
485 490 495
Tyr AspIleLys LeuSerLys Met
500
64
