U.S. patent application number 09/922261 was filed with the patent office on 2002-08-15 for compositions and methods for diagnosing and treating conditions, disorders, or diseases involving cell death.
This patent application is currently assigned to COGENT NEUROSCIENCE, INC.. Invention is credited to Barney, Shawn, Katz, Lawrence C., Lo, Donald C., Portbury, Stuart D., Puranam, Kasturi, Thomas, Mary Beth.
Application Number | 20020111471 09/922261 |
Document ID | / |
Family ID | 23833591 |
Filed Date | 2002-08-15 |
United States Patent
Application |
20020111471 |
Kind Code |
A1 |
Lo, Donald C. ; et
al. |
August 15, 2002 |
Compositions and methods for diagnosing and treating conditions,
disorders, or diseases involving cell death
Abstract
The present invention relates to compositions and methods for
the treatment and diagnosis of conditions, disorders, or diseases
involving cell death. The invention encompasses protective nucleic
acids which, when introduced into a cell predisposed to undergo
cell death or in the process of undergoing cell death, prevent,
delay, or rescue the cell from death relative to a corresponding
cell into which no exogenous nucleic acids have been introduced.
The invention encompasses nucleic acids of the protective sequence,
host cell expression systems of the protective sequence, and hosts
that have been transformed by these expression systems, including
transgenic animals. The invention also encompasses novel protective
sequence products, including proteins, polypeptides and peptides
containing amino acid sequences of the proteins, fusion proteins of
proteins, polypeptides and peptides, and antibodies directed
against such gene products. The invention further relates to target
sequences, including upstream and downstream regulatory sequences
or complete gene sequences, antibodies, antisense molecules or
sequences, ribozyme molecules, and other inhibitors or modulators
directed against such protective sequences, protective sequence
products, genes, gene products, and/or their regulatory elements
involved in cell death. The present invention also relates to
methods and compositions for the diagnosis and treatment of
conditions, disorders, or diseases, involving cell death,
including, but not limited to, treatment of the types of
conditions, disorders, or diseases, which can be prevented, delayed
or rescued from cell death and include, but are not limited to,
those associated with the central nervous system, including
neurological and psychiatric conditions, disorders, or diseases,
and those of the peripheral nervous system. Further, the invention
relates to methods of using the protective sequence, protective
sequence products, and/or their regulatory elements for the
identification of compounds that modulate the expression of the
protective sequence and/or the activity of the protective sequence
product. Such compounds can be useful as therapeutic agents in the
treatment of various conditions, disorders, or diseases involving
cell death.
Inventors: |
Lo, Donald C.; (Chapel Hill,
NC) ; Barney, Shawn; (Apex, NC) ; Thomas, Mary
Beth; (Chapel Hill, NC) ; Portbury, Stuart D.;
(Durham, NC) ; Puranam, Kasturi; (Durham, NC)
; Katz, Lawrence C.; (Durham, NC) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1155 Avenue of the Americas
New York
NY
10036-2711
US
|
Assignee: |
COGENT NEUROSCIENCE, INC.
DURHAM
NC
27704
|
Family ID: |
23833591 |
Appl. No.: |
09/922261 |
Filed: |
August 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09922261 |
Aug 3, 2001 |
|
|
|
09461697 |
Dec 14, 1999 |
|
|
|
Current U.S.
Class: |
536/23.2 |
Current CPC
Class: |
C07K 14/4747
20130101 |
Class at
Publication: |
536/23.2 ;
514/44 |
International
Class: |
A61K 048/00; C07H
021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes a protective sequence product comprising: (a)
an amino acid sequence shown in FIGS. 4(A-L); (b) an amino acid
sequence shown in FIGS. 5(A-X); (c) an amino acid sequence shown in
FIGS. 6(A-AD); (d) an amino acid sequence shown in FIGS. 7(A-H);
(e) an amino acid sequence shown in FIGS. 8(A-O); (f) an amino acid
sequence shown in FIGS. 9(A-AL); (g) an amino acid sequence shown
in FIGS. 10(A-O); (h) an amino acid sequence shown in FIGS.
11(A-AG); (i) an amino acid sequence shown in FIGS. 12(A-AY); or
(j) the amino acid sequence shown in FIG. 13.
2. An isolated nucleic acid molecule comprising: (a) a nucleic acid
shown in FIGS. 4(A-L); (b) a nucleic acid shown in FIGS. 5(A-X);
(c) a nucleic acid shown in FIGS. 6(A-AD); (d) a nucleic acid shown
in FIGS. 7(A-H); (e) a nucleic acid shown in FIGS. 8(A-O); (f) a
nucleic acid shown in FIGS. 9(A-AL); (g) a nucleic acid shown in
FIGS. 10(A-O); (h) a nucleic acid shown in FIGS. 11(A-AG); (i) a
nucleic acid shown in FIGS. 12(A-AY); (j) a nucleic acid shown in
FIG. 13; or (k) a nucleic acid shown in FIGS. 1(A-J);
3. An isolated nucleic acid molecule comprising a complement of the
nucleic acid molecule of any one of claims 1-2.
4. An isolated nucleic acid molecule which hybridizes to the
complement of the nucleic acid molecule of any one of claims 1-2
under highly stringent conditions.
5. An isolated nucleic acid molecule which hybridizes to the
complement of the nucleic acid molecule of any one of claims 1-2
under moderately stringent conditions.
6. The isolated nucleic acid molecule of claim 4, wherein said
isolated nucleic acid molecule encodes a protective sequence
product.
7. The isolated nucleic acid molecule of claim 5, wherein said
isolated nucleic acid molecule encodes a protective sequence
product.
8. A vector comprising the nucleic acid of any one of claims
1-2.
9. The vector of claim 8, wherein said vector is a viral
vector.
10. An expression vector comprising the nucleic acid of any one of
claims 1-2 operatively associated with a regulatory nucleic acid
controlling the expression of the nucleic acid in a host cell.
11. A host cell genetically engineered to contain the nucleic acid
of any one of claims 1-2.
12. A host cell genetically engineered to express the nucleic acid
of any one of claims 1-2 operatively associated with a regulatory
nucleic acid controlling expression of the nucleic acid in said
host cell.
13. A transgenic, non-human animal which has been genetically
engineered to contain a transgene comprising the nucleic acid of
any one of claims 1-2.
14. The transgenic, non-human animal of claim 13, wherein the
transgene is expressed.
15. An isolated polypeptide comprising the amino acid sequence of a
protective sequence product comprising: (a) an amino acid sequence
shown in FIGS. 4(A-L); (b) an amino acid sequence shown in FIGS.
5(A-X); (c) an amino acid sequence shown in FIGS. 6(A-AD); (d) an
amino acid sequence shown in FIGS. 7(A-H); (e) an amino acid
sequence shown in FIGS. 8(A-O); (f) an amino acid sequence shown in
FIGS. 9(A-AL); (g) an amino acid sequence shown in FIGS. 10(A-O);
(h) an amino acid sequence shown in FIGS. 11(A-AG); (i) an amino
acid sequence shown in FIGS. 12(A-AY); or (j) the amino acid
sequence shown in FIG. 13.
16. An isolated polypeptide comprising an amino acid sequence
encoded by the isolated nucleic acid molecule of claim 4.
17. An isolated polypeptide comprising an amino acid sequence
encoded by the isolated nucleic acid molecule of claim 5.
18. An isolated fusion polypeptide comprising a fusion peptide and
an amino acid sequence of a protective sequence product comprising:
(a) an amino acid sequence shown in FIGS. 4(A-L); (b) an amino acid
sequence shown in FIGS. 5(A-X); (c) an amino acid sequence shown in
FIGS. 6(A-AD); (d) an amino acid sequence shown in FIGS. 7(A-H);
(e) an amino acid sequence shown in FIGS. 8(A-O); (f) an amino acid
sequence shown in FIGS. 9(A-AL); (g) an amino acid sequence shown
in FIGS. 10(A-O); (h) an amino acid sequence shown in FIGS.
11(A-AG); (i) an amino acid sequence shown in FIGS. 12(A-AY); or
(j) the amino acid sequence shown in FIG. 13.
19. An isolated fusion polypeptide comprising a fusion peptide and
an amino acid sequence encoded by the isolated nucleic acid
molecule of claim 4.
20. An isolated fusion polypeptide comprising a fusion peptide and
an amino acid sequence encoded by the isolated nucleic acid
molecules of claim 5.
21. An antibody which binds to the isolated polypeptide of claim
15.
22. A method for treating a protective sequence-mediated condition,
disorder or disease in an individual comprising administering to
the individual a compound which modulates the function, activity
and/or expression of a protective sequence in a cell, cells,
tissue, organ, organism or individual.
23. The method of claim 22, wherein the compound inhibits or
potentiates the function, activity and/or expression of a
protective sequence in a cell, cells, tissue, organ, organism or
individual.
24. The method of claim 22, wherein the compound enhances or
increases the function, activity and/or expression of a protective
sequence in a cell, cells, tissue, organ, organism or
individual.
25. The methods of any one of claims 22-24, wherein the compound is
selected from the group consisting of a small organic molecule, an
antibody, a ribozyme or an antisense molecule.
26. The methods of any one of claims 22-24, wherein the protective
sequence-mediated condition, disorder, or disease is a condition,
disorder, or disease of the central nervous system, including but
not limited to, neurological and psychiatric conditions, disorders,
or diseases.
27. The method of claim 26, wherein the neurological condition is
an ischemia-related condition.
28. The method of claim 27, wherein the neurological condition is a
stroke.
29. The method of claim 22, wherein the protective sequence encodes
a protective sequence product comprising: (a) an amino acid
sequence shown in FIGS. 4(A-L); (b) an amino acid sequence shown in
FIGS. 5(A-X); (c) an amino acid sequence shown in FIGS. 6(A-AD);
(d) an amino acid sequence shown in FIGS. 7(A-H); (e) an amino acid
sequence shown in FIGS. 8(A-O); (d) an amino acid sequence shown in
FIGS. 9(A-AL); (g) an amino acid sequence shown in FIGS. 10(A-O);
(h) an amino acid sequence shown in FIGS. 11(A-AG); (i) an amino
acid sequence shown in FIGS. 12(A-AY); or (j) the amino acid
sequence shown in FIG. 13.
30. The method of claim 22, wherein the individual is a mammal.
31. The method of claim 30, wherein the mammal is a human.
32. A method for treating a protective sequence-mediated condition,
disorder or disease in an individual comprising administering to
the individual a compound which modulates the expression or
activity of a protective sequence product and/or protective
sequence regulatory product in the individual.
33. The method of claim 32, wherein the compound inhibits or
potentiates the expression or activity of a protective sequence
product and/or protective sequence regulatory product in the
individual.
34. The method of claim 32, wherein the compound enhances or
increases the expression or activity of a protective sequence
product and/or protective sequence regulatory product in the
individual.
35. The method of claim 32, wherein the compound is selected from
the group consisting of a small organic molecule, an antibody, a
ribozyme.or an antisense molecule.
36. The method of claim 32, wherein the protective
sequence-mediated condition, disorder, or disease is a condition,
disorder, or disease of the central nervous system, including but
not limited to, neurological and psychiatric conditions, disorders,
or diseases.
37. The method of claim 36, wherein the neurological condition is
an ischemia-related condition.
38. The method of claim 37, wherein the neurological condition is a
stroke.
39. The method of claim 32, wherein the protective sequence product
comprises: (a) an amino acid sequence shown in FIGS. 4(A-L); (b) an
amino acid sequence shown in FIGS. 5(A-X); (c) an amino acid
sequence shown in FIGS. 6(A-AD); (d) an amino acid sequence shown
in FIGS. 7(A-H); (e) an amino acid sequence shown in FIGS. 8(A-O);
(f) an amino acid sequence shown in FIGS. 9(A-AL); (g) an amino
acid sequence shown in FIGS. 10(A-O); (h) an amino acid sequence
shown in FIGS. 11(A-AG); (i) an amino acid sequence shown in FIGS.
12(A-AY); or (j) the amino acid sequence shown in FIG. 13.
40. The method of claim 32, wherein the individual is a mammal.
41. The method of claim 40, wherein the mammal is a human.
42. A method for identifying a compound which modulates expression
of a protective sequence comprising: (a) contacting a test compound
to a cell that expresses a protective sequence; (b) measuring a
level of protective sequence expression in the cell; (c) comparing
the level of protective sequence expression in the cell in the
presence of the test compound to a level of protective sequence
expression in the cell in the absence of the test compound, wherein
if the level of protective sequence expression in the cell in the
presence of the test compound differs from the level of expression
of the protective sequence in the cell in the absence of the test
compound, a compound that modulates expression of a protective
sequence is identified.
43. The method of claim 42, wherein the protective sequence is
endogenously expressed within the cell.
44. The method of claim 42, wherein the protective sequence encodes
a protective sequence product comprising: (a) an amino acid
sequence shown in FIGS. 4(A-L); (b) an amino acid sequence shown in
FIGS. 5(A-X); (c) an amino acid sequence shown in FIGS. 6(A-AD);
(d) an amino acid sequence shown in FIGS. 7(A-H); (e) an amino acid
sequence shown in FIGS. 8(A-O); (f) an amino acid sequence shown in
FIGS. 9(A-AL); (g) an amino acid sequence shown in FIGS. 10(A-O);
(h) an amino acid sequence shown in FIGS. 11(A-AG); (i) an amino
acid sequence shown in FIGS. 12(A-AY); or (j) the amino acid
sequence shown in FIG. 13.
45. The method of claim 42, wherein the protective sequence
comprises: (a) a nucleic acid shown in FIGS. 4(A-L); (b) a nucleic
acid shown in FIGS. 5(A-X); (c) a nucleic acid shown in FIGS.
6(A-AD); (d) a nucleic acid shown in FIGS. 7(A-H); (e) a nucleic
acid shown in FIGS. 8(A-O); (f) a nucleic acid shown in FIGS.
9(A-AL); (g) a nucleic acid shown in FIGS. 10(A-O); (h) a nucleic
acid shown in FIGS. 11(A-AG); (i) a nucleic acid shown in FIGS.
12(A-AY); (j) a nucleic acid shown in FIG. 13; or (k) a nucleic
acid shown in FIGS. 1(A-J);
46. A method for identifying a compound which modulates expression,
function or activity of a protective sequence product or protective
sequence regulatory element comprising: (a) contacting a test
compound to a cell that expresses a protective sequence product or
protective sequence regulatory element; (b) measuring a level of
protective sequence product or protective sequence regulatory
element expression, function or activity in the cell; (c) comparing
the level of protective sequence product or protective sequence
regulatory element expression, function or activity in the cell in
the presence of the test compound to a level of protective sequence
product or protective sequence regulatory element expression or
activity in the cell in the absence of the test compound, wherein
if the level of protective sequence product or protective sequence
regulatory element expression, function or activity in the cell in
the presence of the test compound differs from the level of
protective sequence product or protective sequence regulatory
element expression, function or activity in the cell in the absence
of the test compound, a compound that modulates expression or
activity of a protective sequence product or protective sequence
regulatory element is identified.
47. The method of claim 46, wherein the protective sequence product
or protective sequence regulatory element comprises: (a) an amino
acid sequence shown in FIGS. 4(A-L); (b) an amino acid sequence
shown in FIGS. 5(A-X); (c) an amino acid sequence shown in FIGS.
6(A-AD); (d) an amino acid sequence shown in FIGS. 7(A-H); (e) an
amino acid sequence shown in FIGS. 8(A-O); (f) an amino acid
sequence shown in FIGS. 9(A-AL); (g) an amino acid sequence shown
in FIGS. 10(A-O); (h) an amino acid sequence shown in FIGS.
11(A-AG); (i) an amino acid sequence shown in FIGS. 12(A-AY); or
(j) the amino acid sequence shown in FIG. 13.
48. A method for transferring a protective sequence into a cell
comprising contacting the cell with a nucleic acid comprising a
protective sequence such that the protective sequence is
transferred into the cell.
49. The method of claim 48 wherein the protective sequence is
expressed in the cell.
50. The method of claim 48 wherein the protective sequence confers
protection to the cell from cell death.
51. A method for modulating the function, activity and/or
expression of a protective sequence in a cell comprising
administering to the cell a compound which modulates the function,
activity and/or expression of a protective sequence in the
cell.
52. The method of claim 51, wherein the compound inhibits or
potentiates the function, activity and/or expression of a
protective sequence in the cell.
53. The method of claim 51, wherein the compound enhances or
increases the function, activity and/or expression of a protective
sequence in the cell.
54. The methods of any one of claims 51-53, wherein the compound is
selected from the group consisting of a small organic molecule, an
antibody, a ribozyme.or an antisense molecule.
55. The method of claim 51, wherein the protective sequence encodes
a protective sequence product comprising: (a) an amino acid
sequence shown in FIGS. 4(A-L); (b) an amino acid sequence shown in
FIGS. 5(A-X); (c) an amino acid sequence shown in FIGS. 6(A-AD);
(d) an amino acid sequence shown in FIGS. 7(A-H); (e) an amino acid
sequence shown in FIGS. 8(A-O); (f) an amino acid sequence shown in
FIGS. 9(A-AL); (g) an amino acid sequence shown in FIGS. 10(A-O);
(h) an amino acid sequence shown in FIGS. 11(A-AG); (i) an amino
acid sequence shown in FIGS. 12(A-AY); or (j) the amino acid
sequence shown in FIG. 13.
Description
1 INTRODUCTION
[0001] The present invention relates to compositions and methods
for the treatment and diagnosis of conditions, disorders, or
diseases involving cell death, including, but not limited to,
neurological disorders such as stroke. Nucleic acids are described
herein which, when introduced into a cell either predisposed to
undergo cell death or in the process of undergoing cell death,
prevent, delay, or rescue the cell from death relative to a
corresponding cell into which no exogenous nucleic acids have been
introduced Such nucleic acids are referred to as "protective
sequences". Protective sequences or their products are identified
by their ability to prevent, delay, or rescue a cell, cells,
tissues, organs, or organisms from dying. Protective sequences or
their products are also identified via their ability to interact
with other genes or gene products involved in conditions or
disorders involving cell death.
[0002] The invention further includes recombinant DNA molecules and
cloning vectors comprising protective sequences, and host cells and
host organisms engineered to contain such DNA molecules and cloning
vectors. The present invention further relates to protective
sequence products and to antibodies directed against such
protective sequence products.
[0003] The protective sequences identified, their products, or
antibodies may be used diagnostically, prophylactically,
therapeutically or as targets for therapeutic intervention. In this
regard, the present invention provides methods for the
identification and prophylactic or therapeutic use of compounds in
the treatment and diagnosis of conditions, disorders, or diseases
involving cell death. Additionally, methods are provided for the
diagnostic monitoring of patients undergoing clinical evaluation
for the treatment of conditions or disorders involving cell death,
for monitoring the efficacy of compounds in clinical trials and for
identifying subjects who may be predisposed to such conditions,
disorders, or diseases involving cell death.
2 BACKGROUND OF THE INVENTION
[0004] 2.1 Mechanisms which Lead to Cell Death
[0005] It is widely recognized that at least two distinct cell
death mechanisms exist for mammalian cells. These two mechanisms
are necrosis and apoptosis, and are significant components of
numerous conditions, disorders and disease states.
[0006] Necrosis plays an important physiologic role in signaling
the presence of certain conditions. When cells die as a result of
necrosis, the dying cells release substances that activate the
body's immune response in a local, and in some cases widespread,
reaction to the necrosis-inducing condition. This response is
important in, for example, bacterial infection.
[0007] Experimental evidence in a wide variety of cells throughout
the body has revealed that every cell can initiate a program of
self-destruction, called apoptosis. This program can be initiated
by a wide variety of natural and unnatural events. There are at
least four distinct pathways for executing this program of cell
death, and it is virtually certain that dozens, if not hundreds, of
different intracellular biochemical cascades interact with each
pathway. It is equally likely that certain cell types, such as
cells in the heart or neurons, will utilize specialized signaling
pathways that are not generally represented elsewhere in the body.
However, since cell death is neither always necessary nor desired,
it would be desirable to manipulate the manner in which cells start
their death process. In some circumstances, preventing, delaying,
or rescuing cells from death would either alleviate the disease or
allow more time for definitive treatment to be administered to the
patient. An example of this situation is brain cell death caused by
ischemic stroke: preventing, delaying, or rescuing cells from death
until the blood supply to the brain could be restored would greatly
reduce, if not eliminate, the possibility of a person's death
and/or long-term disability from stroke (Lee J M, et al. Nature
1999, 399(supp): A7-A14; Tarkowski E, et al. Stroke 1999, 30(2):
321-7; Pulera M R, et al. Stroke 1998, 29(12): 2622-30). In still
other circumstances, the failure of cells to die may itself lead to
disease such as cancer (Hetts S W. JAMA 1998, 297(4): 300-7).
[0008] Cell death plays an important role in the normal function of
mammalian organisms. While it may seem counterintuitive for cells
to have death as a normal part of their life cycle, controlled and
physiologically appropriate cell death is important in regulating
both the absolute and relative numbers of cells of a specific type.
(Hetts S W. JAMA 1998, 297(4): 300-7; Garcia I, et al. Science
1992, 258(5080): 302-4). When the mechanism of apoptosis does not
function properly and normal cell death does not occur, the
resulting disease is characterized by unregulated cellular
proliferation, as occurs in a neoplastic disease or an autoimmune
disease (Hetts S W. JAMA 1998, 297(4): 300-7; Yachida M, et al.
Clin Exp Immunol 1999, 116(1): 140-5).
[0009] One method for regulating cell death involves manipulating
the threshold at which the process of cell death begins. This
threshold varies significantly by cell type, tissue type, the type
of injury or insult suffered by the cell, cellular maturity, and
the physiologic conditions in the cell's environment (Steller H.,
Science 1995, 267(5203): 1445-9). Although it is probable that
certain cellular injuries or insults irrevocably induce death,
lesser injuries or insults may begin the dying process without
inducing irreversible cell death. What constitutes a lesser injury
or insult may vary tremendously with changes in the factors
influencing that cell's death threshold. The ability to alter a
cell's threshold for responding to an injury or insult, that is, to
either promote or discourage cell death, would be a desirable goal
for the treatment of conditions involving cell death. The ability
to better control cell death, by either discouraging or promoting
the mechanisms of cell death, would be an important invention for
ameliorating disease (U.S. Pat. Nos. 5,925,640; 5,786,173;
5,858,715; 5,856,171).
[0010] Recent evidence suggests that the mechanisms of cellular
death may be more complex than the two discrete pathways of
apoptosis and necrosis. Examples of this evidence may be found in
the central nervous system (CNS). In the complex CNS cellular
environment, both necrosis and apoptosis are observed with commonly
studied conditions, disorders, or diseases such as focal ischemia,
global ischemia, toxic insults, prolonged seizures, excitotoxicity,
and traumatic brain injury. In some reports, both apoptosis and
necrosis have been described (Choi W S, et al. J Neurosci Res 1999,
57(1): 86-94; Li Y, et al. J Neurol Sci 1998, 156(2): 119-32; Lee
J- M, et al. Nature 1999, 399(supp): A8-A14; Baumgartner W A, et
al. Ann Thorac Surg 1999, 67(6): 1871-3; Fujikawa D G, et al. Eur J
Neurosci 1999, 11(5): 1605-14; Gwag B J, et al Neuroscience 1999,
90(4): 1339-48; Mitchell I J, et al. 1998, 84(2): 489-501;
Nakashima K, et al. J Neurotrauma 1999, 16(2): 143-51; Ginsburg, M
D Cerebrovascular Disease: Pathophysiology, Diagnosis, and
Management 1998 Ch 42; Rink A D, et al. Soc Neurosci Abstr 1994,
20:250(Abstract)). Similar observations also occurred with brain
tumor cells. (Maurer B J, et al. J Natl Cancer Inst 1999, 91(13):
1138-46) Other investigators found that neurons die by either
apoptosis or necrosis under different environmental conditions
(Taylor D L, et al. Brain Pathol 1999, 9(1): 93-117). There also
are reports of a unique type of neuronal cell death following
stroke. This new type of cell death has features common to both
necrosis and apoptosis (Fukuda T, et al. Neurosci Res 1999, 33(1):
49-55). Other investigators believe that neuronal cell death is
best represented by a continuum between apoptosis and necrosis,
possibly mediated by calcium levels (Lee J- M, et al.1999,
399(supp): A7-A14), or a combination of direct ischemic damage
followed by indirect damage from excitotoxicity and loss of
interneuronal connections (Martin L J, et al. Brian Res Bull 1998,
46(4): 281-309). Further complicating the picture of neuronal cell
death is the observation that the death of one or more neurons in
one region of the brain can induce the death of neurons in other
brain regions. This phenomenon has been observed with stroke as
described above (Martin L J, et al. Brain Res Bull 1998, 46(4):
281-309) as well as neuronal cell death induced by the withdrawal
of growth factors (Ryu B R, et al. J Neurobiol 1999, 39(4):
536-46). Given the complex nature of actions and interactions among
the many physiologic and molecular forces in brain tissue, and the
different abilities of many substances acting either alone or in
combination to induce cellular injury or death, it is difficult to
determine with any degree of certainty if a nerve cell death
process is due to apoptosis or necrosis (Graham D I, Greenfield's
Neuropathology Ch 3 1997).
[0011] Despite the challenges in classifying the mechanism of
cellular death, there is broad agreement that most, if not all,
cells share common features in their death mechanisms (see, e.g.,
Lee J. M., et al., Nature 1999, 399 (supp): A7-A14).
[0012] 2.2 Selected Factors and Conditions which Inhibit Cell Death
Mechanisms
[0013] Several factors have been reported to inhibit the cell death
pathway. One of the best-known factors is the gene product bcl-2
(Adams J M, et al. Science 1998, 281(5381): 1322-6; Vaux D L, et
al. Proc Natl Acad Sci 1993, 90(3): 786-9; U.S. Pat. No. 5,856,171
and references cited therein). Expression of bcl-2 is believed to
regulate apoptotic death in neurons, kidney, heart, liver, blood
and skin cells under experimental conditions. In addition to
regulating death by apoptosis, bcl-2 is believed to regulate death
caused by non-apoptotic mechanisms. Factors related to bcl-2 have
been shown to be over-expressed in cancer and autoimmune
conditions, disorders, or diseases (U.S. Pat. No. 5,856,171 and
references cited therein). Other related factors acting on the same
pathway as bcl-2 also delay or prevent cell death.
[0014] In the brain, several factors have been shown to influence
the cell death pathway. In excitotoxic injury to neurons, it was
shown that lithium or bcl-2 each individually protected neurons
against cell death (Nonaka S, et al. Proc Natl Acad Sci 1998,
95(5): 2642-7; Behl C, et al. Biochem Biophys Res Commun 1993,
197(2): 949-56). During ischemic injury to neurons, it was shown
that nerve growth factor (NGF) and bcl-2 individually offered
protection against neuronal death (Guegan C, et al. Neurobiol Dis
1999, 6(3): 180-9; Linnik M D, et al. Stroke 1995, 26(9):
1670-4).
[0015] Factors acting to prevent cell death do not act solely in
the brain. In the heart, increased tolerance to non-lethal ischemic
injury was associated with an increased expression of the bcl-2
gene, suggesting that bcl-2 was involved in protecting the cardiac
muscle cells against ischemic injury (Maulik N, et al. Ann NY Acad
Sci 1999, 874:401-11). This same study demonstrated that lower
levels of bcl-2 expression were associated with higher rates of
cardiac cell death. A similar result was found for mechanical
injury to heart papillary muscle cells.
[0016] Recently, it has been demonstrated that bcl-2 prevented cell
death in a brain ischemia model (Guegan C, et al. Neurobiol Dis
1999, 6(3): 180-9; Linnik M D, et al. Stroke 1995, 26(9): 1670-4).
It was shown that the activity of bcl-2 to prevent neuronal death
was consistently demonstrated across several different physiologic
insults. It also has been demonstrated that the distinction between
apoptotic death and necrotic death is open to question, so the
possibility exists that bcl-2 can prevent or delay the necrotic
cell death pathway, the apoptotic cell death pathway or perhaps an
as yet undemonstrated cell death pathway.
[0017] Preventing cell death is an important medical goal. Several
types of mammalian cells, most notably neurons and cardiac muscle
cells, have limited if any capacity to regenerate. Preventing the
death of these cells from conditions such as heart attack, stroke,
shock, infection, cancer, Alzheimer's disease or traumatic injury,
to name a few, would be an important medical advance as the heart
and brain cannot grow sufficient cells to replace those cells lost
to disease or infection.
[0018] In addition to preventing cell death, delaying and/or
rescuing cells from programmed cell death is also an important
medical goal. In many pathological conditions where there is an
expectation that the disease will be successfully treated, such as
many types of infection, hypoxia, ischemia or metabolic
disturbances, delaying cell death would allow the pathological
condition to be treated without permanent damage to the cells. In
other words, the cells may be put into a suspended state from which
they could successfully be rescued and emerge with their normal
function intact.
3 SUMMARY OF THE INVENTION
[0019] The present invention relates to the discovery,
identification and characterization of protective sequences and to
compositions and methods for the treatment and diagnosis of
conditions, disorders, or diseases involving cell death. Protective
sequences refer to nucleic acid molecules comprising nucleic acid
sequences which, when introduced into a cell either predisposed to
undergo cell death or in the process of undergoing cell death,
prevent, delay, or rescue the cell from death relative to a
corresponding cell into which no exogenous nucleic acids have been
introduced. For example, protective sequences may act to prevent,
delay, ameliorate, inhibit, reduce, or rescue neuronal cell death
(e.g. apoptosis, necrosis and related cellular events). The
invention further relates to the discovery, identification and
characterization of gene products encoded by such nucleic acid
molecules, or by degenerate, e.g., allelic or homologous, variants
thereof. Protective sequences also can be regulatory nucleic acids.
Protective sequences further can be both coding sequences and
regulatory sequences.
[0020] The invention further relates to target sequences. Target
sequences include, but are not limited to, upstream and downstream
regulatory sequences, upstream and downstream complete or partial
gene or gene product sequences, antibodies, antisense molecules or
sequences, ribozyme molecules, and other inhibitors or modulators
directed against such protective sequences and protective sequence
products.
[0021] Protective sequences and protective sequence products can be
utilized prophylactically and/or therapeutically to prevent, delay
ameliorate, inhibit, reduce, or rescue conditions of cell death or
symptoms of conditions, disorders, or diseases involving cell
death. The modulation of the expression of protective sequences,
e.g., endogenous protective sequences, and/or the activity of the
protective sequence products, e.g., endogenous protective sequence
products, can also be utilized prophylactically or therapeutically
to prevent, delay, ameliorate, inhibit, reduce, or rescue
conditions of cell death or symptoms of conditions, disorders, or
diseases involving cell death. Further, protective sequences and
protective sequence products can be used to diagnose individuals
exhibiting or predisposed to such conditions, disorders, or
diseases involving cell death.
[0022] The compositions of the present invention include, in
particular, nucleic acid molecules which comprise the following
sequences: (a) nucleic acids of protective sequences, as well as
allelic variants, homologs, mutants and fragments thereof; (b)
nucleic acids which encode protective sequence products; (c)
nucleic acids which encode protective sequence regulatory elements;
(d) nucleic acids which encode fusion proteins comprising
protective sequence products or one or more protective sequence
product domains fused to a heterologous polypeptide; (e) nucleic
acids which encode fusion proteins comprising protective sequence
regulatory elements fused to a heterologous polypeptide; (f)
nucleic acids which hybridize to the above described sequences
under highly stringent or moderately stringent conditions,
including, but not limited to, human homologs; and (g)
complementary (e.g., antisense) nucleic acids of the sequences
described in (a) through (f), above. The nucleic acid molecules of
the invention include, but are not limited to, cDNA, genomic DNA
and RNA sequences.
[0023] The present invention also encompasses expression gene
products of the protective sequences listed above; i.e., proteins
and/or polypeptides that are encoded by the above protective
sequences.
[0024] Mimics, agonists and antagonists of the protective
sequences, protective sequence products, genes, gene products, or
their regulatory elements are also included in the present
invention. Such mimics, agonists and antagonists will include, for
example, small molecules, large molecules (e.g., protective
sequence product fragments or protective sequence product ligands)
and antibodies directed against a protective sequence product.
Mimics, agonists and antagonists of the invention also include
nucleic acids, such as antisense and ribozyme molecules, and gene
or regulatory sequence replacement constructs, which can be used to
modulate, inhibit or enhance expression of a protective
sequence.
[0025] The present invention further encompasses cloning and
expression vectors, which may include, but are not limited to,
bacterial, fungal, insect, plant, and mammalian vectors, which
contain the protective nucleic acid sequences of the invention,
which can be used as probes or to express those protective nucleic
acid sequences, protective sequence products, genes and/or gene
products in host cells or organisms. The present invention also
relates to cells that have been transformed, transfected, or
infected with such vectors, and to cells engineered to contain or
express the protective nucleic acid sequences, protective sequence
products, genes, gene products, and/or regulatory elements of the
invention. Further, non-human host organisms which have been
transformed, transfected, or infected with these protective nucleic
acid sequences, or their regulatory elements, are also encompassed
in the present invention. Host organisms of the invention include
organisms transformed, transfected, or infected with the cloning
vectors described above, including, but not limited to, non-human
transgenic animals, and particularly transgenic non-human mammals
which have been engineered to express a protective sequence,
protective sequence product, gene, gene product, or regulatory
element of the invention, or "knock-outs" which have been
engineered to not express the protective sequence, protective
sequence product, gene, gene product, or regulatory element of the
invention.
[0026] The transgenic animals of the invention include animals
which express a mutant variant or polymorphism of a protective
sequence, protective sequence product, gene, gene product, or
regulatory element, particularly a mutant variant or polymorphism
of a protective sequence, protective sequence product, gene, gene
product, or regulatory element which is associated with a
condition, disorder, or disease involving cell death. The
transgenic animals of the invention further include those that
express a protective sequence transgene at higher or lower levels
than normal. The transgenic animals of the invention further
include those which express the protective sequence, protective
sequence product, gene, gene product, or regulatory element in all
their cells, "mosaic" animals which express the protective
sequence, protective sequence product, gene, gene product, or
regulatory element in only some of their cells, and those in which
the protective sequence, protective sequence product, gene, gene
product, or regulatory element is selectively introduced into and
expressed in a specific cell type(s). The transgenic animals of the
invention also include "knock-out" animals. Knock-out animals
comprise animals that have been engineered to no longer express the
protective sequence, protective sequence product, gene, gene
product, or regulatory element.
[0027] The present invention also relates to methods and
compositions for the diagnosis of conditions, disorders, or
diseases involving cell death, as well as for the identification of
subjects susceptible to such conditions, disorders, or diseases.
Such methods comprise, for example, measuring expression of the
protective sequence, protective sequence product, gene, gene
product, or regulatory element in a patient sample, or detecting a
mutation in the protective sequence, protective sequence product,
gene, gene product, or regulatory element in the genome of a
mammal, including a human, suspected of exhibiting such a
condition, disorder, or disease. The protective nucleic acid
molecules of the invention can be used also as diagnostic
hybridization probes, or as primers for diagnostic PCR analysis to
identify protective sequences, protective sequence products, genes,
gene products, or regulatory element mutations, allelic variations
or regulatory defects, such as defects in the expression of the
protective sequence, protective sequence product, gene, gene
product, or regulatory element. Such diagnostic PCR analyses can be
used to diagnose individuals with a condition, disorder, or disease
involving cell death associated with a particular protective
sequence, protective sequence product, gene, gene product, or
regulatory element mutation, allelic variation or regulatory
defect. Such diagnostic PCR analyses can be used also to identify
individuals susceptible to such conditions, disorders, or diseases
involving cell death.
[0028] Methods and compositions, including pharmaceutical
compositions, for the treatment of conditions, disorders, or
diseases involving cell death also are included in the invention.
Such methods and compositions can increase, decrease or otherwise
modulate the level of protective sequences, protective sequence
products, genes, gene products, or their regulatory elements in a
patient in need of such treatment. Such methods and compositions
can also modulate the level of protective sequence expression
(e.g., endogenous protective sequence expression) and/or the level
of activity of a protective sequence product, (e.g., endogenous
protective sequence product). Further, since the protective
sequence or protective sequence product need not normally be
involved in such conditions, disorders, or diseases, such methods
include, for example, modulating the expression of the protective
sequence and/or the activity of the protective sequence product for
the treatment of conditions, disorders, or diseases involving cell
death which are normally mediated by some other gene.
[0029] In one embodiment, such methods and compositions are
utilized for the treatment of the types of conditions, disorders,
or diseases, which can be prevented, delayed or rescued from cell
death and include, but are not limited to, those associated with
the central nervous system including neurological and psychiatric
conditions, disorders, or diseases; those of the peripheral nervous
system; conditions, disorders, or diseases caused by physical
injury; conditions, disorders, or diseases of the blood vessels or
heart; conditions, disorders, or diseases of the respiratory
system; neoplastic conditions, disorders, or diseases; conditions,
disorders, or diseases of blood cells; conditions, disorders, or
diseases of the gastrointestinal tract; conditions, disorders, or
diseases of the liver; conditions, disorders, or diseases of the
pancreas; conditions, disorders, or diseases of the kidney;
conditions, disorders, or diseases of the ureters, urethra or
bladder; conditions, disorders, or diseases of the male genital
system; conditions, disorders, or diseases of the female genital
tract; conditions, disorders, or diseases of the breast;
conditions, disorders, or diseases of the endocrine system;
conditions, disorders, or diseases of the thymus or pineal gland;
conditions, disorders, or diseases of the skin or mucosa;
conditions, disorders, or diseases of the musculoskeletal system;
conditions, disorders, or diseases causing a fluid or hemodynamic
derangement; inherited conditions, disorders, or diseases;
conditions, disorders, or diseases of the immune system or spleen;
conditions, disorders, or diseases caused by a nutritional disease;
and conditions, disorders, or diseases typically occurring in
infancy or childhood, as described in Section 5.4.1.1. below.
[0030] In yet another embodiment, the methods and compositions of
the invention are utilized for the prevention, or delay, of cell
death in the event of one or more infections which may be caused by
bacteria; viruses; members of the family rickettsiae or chlamydia;
fungi, yeast, hyphae or pseudohyphae; prions; protozoans; or
metazoans.
[0031] In a further embodiment, the compounds and methods of the
invention can be used to treat infections or conditions, disorders,
or diseases which cause cell death in organ systems including, but
not limited to, blood vessels, heart, red blood cells, white blood
cells, lymph nodes, spleen, respiratory system, oral cavity,
gastrointestinal tract, liver and biliary tract, pancreas, kidney,
lower urinary tract, upper urinary tract and bladder, male sexual
organs and genitalia, female sexual organs and genitalia, breast,
thyroid gland, adrenal gland, parathyroid gland, skin,
musculoskeletal system, bone marrow or bones.
[0032] In another embodiment, the compounds and methods of the
invention can be used to treat further physiological impacts on
organs caused by the infections which induce cell death including,
but not limited to, fever equal to or greater than 101.5 degrees
Fahrenheit, a decrease or increase in pulse rate by more than 20
beats per minute, a decrease or increase in supine systolic blood
pressure by more than 30 millimeters of mercury, an increase or
decrease in respiratory rate by more than 8 breaths per minute, an
increase or decrease in blood pH by more than 0.10 pH units, an
increase or decrease in one or more serum electrolytes outside of
the clinical laboratory's usual reference range, an increase or
decrease in the partial pressure of arterial oxygen or carbon
dioxide outside of the clinical laboratory's usual reference range,
an increase or decrease in white or red blood cells outside of the
laboratory's usual reference range, an acute confusional state such
as delirium where delirium is defined by the American Psychiatric
Association's DSM-IV Manual or a diminished level of consciousness
or attention.
[0033] In another embodiment, the compounds and methods of the
invention can be used to promote cell death. These compounds could
be useful for treating and/or ameliorating conditions caused by,
for example, cancer and autoimmune diseases, both of which are
manifested by an uncontrolled growth of cells.
[0034] The invention still further relates to methods for
identifying compounds which modulate the expression of a protective
sequence and/or the synthesis or activity of a protective sequence
product. Such compounds include therapeutic compounds which can be
used as pharmaceutical compositions to reduce or eliminate the
symptoms of conditions, disorders, or diseases involving cell
death. Cellular and non-cellular assays are described which can be
used to identify compounds which interact with a protective
sequence, protective sequence product, gene, gene product, and/or
regulatory element, e.g., modulate the activity of a protective
sequence and/or bind to a protective sequence product. Such
cell-based assays of the invention utilize cells, cell lines, or
engineered cells or cell lines that express the protective
sequence, protective sequence product, gene, gene product, and/or
regulatory element.
[0035] In one embodiment, such methods comprise contacting a
compound to a cell which expresses a protective sequence,
protective sequence product, gene, gene product, and/or regulatory
element, measuring the level of protective sequence expression,
gene product expression or gene product activity, and comparing
this level to the level of protective sequence expression, gene
product expression or gene product activity produced by the cell in
the absence of the compound, such that if the level obtained in the
presence of the compound differs from that obtained in its absence,
a compound which modulates the expression of the protective
sequence and/or the synthesis or activity of protective sequence
products has been identified.
[0036] In an alternative embodiment, such methods comprise
administering a compound to a host, e.g., a transgenic animal which
expresses a protective sequence transgene or a mutant protective
sequence transgene, and measuring the level of protective sequence
expression, gene product expression or gene product activity. The
measured level is compared to the level of protective sequence
expression, gene product expression or gene product activity in a
host which is not exposed to the compound, such that if the level
obtained when the host is exposed to the compound differs from that
obtained when the host is not exposed to the compound, a compound
which modulates the expression of the protective sequence and/or
the synthesis or activity of protective sequence products, and/or
the symptoms of conditions, disorders, or diseases involving cell
death, has been identified.
[0037] 3.1 Definitions
[0038] "Protective sequence", as used herein, refers to nucleic
acid molecules comprising nucleic acid sequences which, when
introduced into a cell predisposed to either undergo cell death or
in the process of undergoing cell death, prevent, delay, or rescue
the cell from death relative to a corresponding cell into which no
exogenous protective nucleic acids have been introduced. In one
embodiment, a protective sequence encodes a protective sequence
product. In another embodiment, protective sequences are any
transcriptional products of the sequences disclosed herein. In
another embodiment, protective sequences comprise regulatory
elements of the sequences disclosed herein which modulate the
expression of a nucleic acid within a cell. For example, protective
sequences, their products, or their regulatory elements may act to
prevent, delay, or rescue a cell, cells, tissues, organs, or
organisms from dying. Compounds which modulate protective sequence
expression or activity of the protective sequence product can be
used in the treatment of conditions, disorders or diseases
associated with cell death processes. It is to be understood that
the protective sequences described above can act to ameliorate or
delay symptoms related to cell death. Although the protective
sequences may be involved directly in such cell death related
conditions or disorders, in certain cases, the protective sequences
will not normally be involved in such conditions or disorders, but
will be effective for the treatment and/or prevention of such
disorders. In these cases, modulation of the expression of the
protective sequence and/or the activity of the protective sequence
product will be useful for the treatment of conditions, disorders,
or diseases involving cell death which are normally mediated by
some other gene.
[0039] "Cell death", as used herein, refers to any mechanism and/or
pathway whereby a cell undergoes a series of events which
ultimately would lead to the death of the cell. For example, cell
death may be caused by various processes including, but not limited
to, apoptosis or programmed cell death, necrosis, or an as yet
unidentified cell death pathway. Cell death may be induced in
individual cells as a consequence of numerous internal and external
stimuli including, but not limited to, genetic predisposition,
toxic chemicals or processes, heat, cold, rapid environmental
changes, radiation, viruses, prions, bacteria, disruption of
nutrient balance, or exposure to bi-products and signaling from
other cells undergoing cell death. The protective sequences
disclosed herein, when introduced into a cell (e.g. a neuronal
cell) which has undergone an event that would ultimately lead to
cell death (e.g. ischemia), are capable of rescuing the cell from
cell death. Moreover, when a protective sequence, in combination
with a reporter gene (e.g. green fluorescent protein), is
introduced into a cell which has undergone an event that would
ultimately lead to cell death, expression of the reporter gene is
an indication that the protective sequence is capable of rescuing
the cell from cell death.
4 BRIEF DESCRIPTION OF THE FIGURES
[0040] FIGS. 1(A-J). Protective nucleic acids. See Table 1 for the
identity, the sequence identifier number, the length in base pairs
and the Accession Number for each of the sequences shown in these
figures.
[0041] FIG. 2. Restriction map and diagram of plasmid
pCMV.cndot.SPORT2. This plasmid was used as the cloning vector for
the protective sequences. Each clone was ligated into the SalI-NotI
restriction sites of the plasmid.
[0042] FIGS. 3(A-N). Protected Cortical Neurons Visualized by
Detection of EGFP Expressing Cells. FIGS. 3A and 3B represent
non-stroked, positive control samples. FIG. 3C represents a
positive control, stroked sample using Bcl-2. FIG. 3D represents a
stroked, negative control sample. FIG. 3E represents a stroked
sample protected by CNI-00711. FIG. 3F represents a stroked sample
protected by CNI-00712. FIG. 3G represents a stroked sample
protected by CNI-00714. FIG. 3H represents a stroked sample
protected by CNI-00715. FIG. 3I represents a stroked sample
protected by CNI-00716. FIG. 3J represents a stroked sample
protected by CNI-00717. FIG. 3K represents a stroked sample
protected by CNI-00720. FIG. 3L represents a stroked sample
protected by CNI-00721. FIG. 3M represents a stroked sample
protected by CNI-00723. FIG. 3N represents a stroked sample
protected by CNI-00724.
[0043] FIGS. 4(A-L). Open Reading Frames for CNI-00711. This Figure
depicts the twelve (12) potential ORFs for CNI-00711. Also shown
are the nucleotide sequences which encode the ORFs.
[0044] FIGS. 5(A-X). Open Reading Frames for CNI-00712. This Figure
depicts the 24 potential ORFs for CNI-00712. Also shown are the
nucleotide sequences which encode the ORFs.
[0045] FIGS. 6(A-AD). Open Reading Frames for CNI-00714. This
Figure depicts the 30 potential ORFs for CNI-00714. Also shown are
the nucleotide sequences which encode the ORFs.
[0046] FIGS. 7(A-H). Open Reading Frames for CNI-00715. This Figure
depicts the eight (8) potential ORFs for CNI-00715. Also shown are
the nucleotide sequences which encode the ORFs.
[0047] FIGS. 8(A-O). Open Reading Frames for CNI-00716. This Figure
depicts the fifteen (15) potential ORFs for CNI-00716. Also shown
are the nucleotide sequences which encode the ORFs.
[0048] FIGS. 9(A-AL). Open Reading Frames for CNI-00717. This
Figure depicts the 38 potential ORFs for CNI-00717. Also shown are
the nucleotide sequences which encode the ORFs.
[0049] FIGS. 10(A-O). Open Reading Frames for CNI-00720. This
Figure depicts the fifteen (15) potential ORFs for CNI-00720. Also
shown are the nucleotide sequences which encode the ORFs.
[0050] FIGS. 11(A-AG). Open Reading Frames for CNI-00721. This
Figure depicts the 33 potential ORFs for CNI-00721. Also shown are
the nucleotide sequences which encode the ORFs.
[0051] FIGS. 12(A-AY). Open Reading Frames for CNI-00723. This
Figure depicts the 51 potential ORFs for CNI-00723. Also shown are
the nucleotide sequences which encode the ORFs.
[0052] FIG. 13. Open Reading Frame for CNI-00724. This Figure
depicts the single potential ORF for CNI-00724. Also shown is the
nucleotide sequence which encodes the ORF.
5 DETAILED DESCRIPTION OF THE INVENTION
[0053] Protective sequences of the invention are described herein.
Also described are recombinant, cloned and degenerate variants,
homologs, orthologs, mutants and fragments thereof. The
compositions of the invention further include protective sequence
products (e.g. proteins or RNA) which are encoded or produced by
the nucleic acid molecules of the invention, and the modulation of
protective sequence expression and/or gene product activity in the
treatment of conditions, disorders, or diseases involving cell
death. Further, antibodies directed against the protective sequence
products, or conserved variants or fragments thereof, and viral-,
cell-, plant-, and animal-based models by which the protective
sequences may be further characterized and utilized are also
discussed in this section.
[0054] 5.1 The Protective Sequences
[0055] The protective sequences of the invention are described in
this section. Specifically, these protective sequences have been
shown to prevent, delay, or rescue cell death in a cell predisposed
for undergoing cell death, whether the pathway that leads to the
cell death involves apoptosis, necrosis or an as yet undefined
pathway. The protective sequences, their SEQ ID NOS and additional
information related to the protective sequences are listed below,
in Table 1.
[0056] The protective sequences listed in Table 1 may be obtained
using cloning methods well known to those skilled in the art,
including but not limited to the use of appropriate probes to
detect the protective sequences within an appropriate cDNA or gDNA
(genomic DNA) library. (See, for example, Sambrook et al., 1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, which is incorporated by reference herein in its
entirety). Probes for the novel sequences reported herein may be
obtained directly from CNI-NPP1-CP10, which represents a composite
deposit containing the isolated clones, which was deposited with
the NRRL as Accession No. B-30231. Alternatively, oligonucleotide
probes for the novel protective sequences may be synthesized based
on the DNA sequences disclosed herein.
1TABLE 1 PROTECTIVE SEQUENCES Length (bp) (NotI-SalI Protective
sequence SEQ ID NO: Figure No. fragment) CNI-00711 1 1A 852
CNI-00712 26 1B 1096 CNI-00714 75 1C 1825 CNI-00715 136 1D 542
CNI-00716 153 1E 771 CNI-00717 184 1F 1669 CNI-00720 261 1G 1182
CNI-00721 292 1H 1965 CNI-00723 359 1I 2702 CNI-00724 462 1J
979
[0057] The isolated protective nucleic acid molecules of the
invention include, in particular, nucleic acid molecules which
comprise the following sequences: (a) nucleic acids of protective
sequences, as well as allelic variants, homologs, mutants and
fragments thereof; (b) nucleic acids which encode protective
sequence products and/or their regulatory elements, or fragments
thereof; (c) nucleic acids which encode fusion proteins comprising
protective sequence products and/or their regulatory elements, or
one or more protective sequence product domains and/or their
regulatory elements fused to a heterologous polypeptide; (d)
nucleic acids which hybridize to the above described sequences
under highly stringent or moderately stringent conditions,
including, but not limited to human homologs; and (e) complementary
(e.g., antisense) nucleic acids of the sequences described in (a)
through (d), above. The nucleic acid molecules of the invention
include, but are not limited to, cDNA, genomic DNA and RNA
sequences.
[0058] The nucleic acids of the invention also include nucleic
acids which have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or
more nucleic acid identity to the protective nucleic acids of
(a)-(d) above. The nucleic acids of the invention further include
nucleic acids which encode polypeptides having at least 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98% or higher amino acid sequence identity
to the polypeptides encoded by the protective nucleic acids of
(a)-(d).
[0059] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical overlapping positions/total # of positions.times.100). In
one embodiment, the two sequences are the same length.
[0060] The determination of percent identity between two sequences
also can be accomplished using a mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268,
modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST
and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.
215:403-410. BLAST nucleotide searches can be performed with the
NBLAST program, score=100, wordlength=12 to obtain nucleic acids
homologous to a nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
a protein molecules of the invention. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
that detects distant relationships between molecules (Id.). When
utilizing BLAST, Gapped BLAST and PSI-Blast programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used (see http://www.ncbi.nlm.nih.gov). Another preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of sequences is the algorithm of Myers and Miller,
(1988) CABIOS 4:11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4 can be used.
[0061] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0062] The nucleic acids of the invention further include: (a) any
nucleic acid which hybridizes to a nucleic acid molecule of the
invention under moderately stringent conditions, e.g.,
hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C. followed by
one or more washes in 0.2.times.SSC/0.1% SDS at about 50-65.degree.
C., or (b) under highly stringent conditions, e.g., hybridization
to filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68.degree. C., or under other hybridization conditions which are
apparent to those of skill in the art (see, for example, Ausubel F.
M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol.
I, Green Publishing Associates, Inc., and John Wiley & sons,
Inc., New York, at pp. 6.3.1-6.3.6 and 2.10.3). Preferably the
nucleic acid molecule that hybridizes to the nucleic acid of (a)
and (b), above, is one which comprises the complement of a nucleic
acid molecule which encodes a protective sequence product. In a
preferred embodiment, nucleic acid molecules comprising the nucleic
acids of (a) and (b), above, encode protective sequence
products.
[0063] Functionally equivalent protective sequence products include
naturally occurring protective sequence products present in the
same or different species. Functionally equivalent protective
sequence products also include gene products which retain at least
one of the biological activities of the protective sequence
products, and/or which are recognized by and bind to antibodies
(polyclonal or monoclonal) directed against the protective sequence
products.
[0064] Among the nucleic acid molecules of the invention are
deoxyoligonucleotides ("oligos") which hybridize under highly
stringent or moderately stringent conditions to the nucleic acid
molecules described above. In general, for probes between 14 and 70
nucleotides in length the melting temperature (TM) is calculated
using the formula: Tm (.degree. C.)=81.5+16.6(log[monovalent
cations (molar)])+0.41 (% G+C)-(500/N) where N is the length of the
probe. If the hybridization is carried out in a solution containing
formamide, the melting temperature is calculated using the equation
Tm (.degree. C.)=81.5+16.6(log[monovalen- t cations
(molar)])+0.41(% G+C)-(0.61% formamide)-(500/N) where N is the
length of the probe. In general, hybridization is carried out at
about 20-25 degrees below Tm (for DNA-DNA hybrids) or 10-15 degrees
below Tm (for RNA-DNA hybrids).
[0065] Exemplary highly stringent conditions may refer, e.g., to
washing in 6.times.SSC/0.05% sodium pyrophosphate at 37.degree. C.
(for about 14-base oligos), 48.degree. C. (for about 17-base
oligos), 55.degree. C. (for about 20-base oligos) and 60.degree. C.
(for about 23-base oligos).
[0066] Fragments of the nucleic acid molecules can be at least 10
nucleotides in length. Fragments of the nucleic acid molecules can
refer also to exons or introns, and, further, can refer to portions
of coding regions that encode domains of protective sequence
products.
[0067] The invention also encompasses (a) DNA vectors which contain
any of the foregoing coding sequences and/or their complements
(i.e., antisense); (b) DNA expression vectors which contain any of
the foregoing coding sequences operatively associated with a
regulatory element which directs the expression of the coding
sequences; and (c) genetically engineered host cells which contain
such vectors or have been engineered to contain and/or express a
nucleic acid sequence of the invention, e.g., any of the foregoing
coding sequences operatively associated with a regulatory element
which directs the expression of the coding sequences in the host
cell. As used herein, regulatory elements include but are not
limited to inducible and non-inducible promoters, enhancers,
operators and other elements known to those skilled in the art
which drive and regulate expression. The invention further includes
fragments of any of the DNA sequences disclosed herein.
[0068] The nucleic acid molecules may encode or act as antisense
molecules, useful, for example, in protective sequence regulation,
and/or as hybridization probes and/or as primers in amplification
reactions of protective nucleic acid sequences. Further, such
sequences may be used as part of ribozyme and/or triple helix
sequences, also useful for protective sequence regulation. Still
further, such molecules may be used as components of diagnostic
methods whereby, for example, the presence of a particular allele
involved in a condition, disorder, or disease involving cell death
may be detected.
[0069] The protective nucleic acids of the invention can be readily
obtained, for example, by standard sequencing and the sequences
provided herein.
[0070] As will be appreciated by those skilled in the art, DNA
sequence polymorphisms of a protective sequence will exist within a
population of individual organisms (e.g., within a human
population). Such polymorphisms may exist, for example, among
individuals within a population due to natural allelic variation.
Such polymorphisms include ones that lead to changes in amino acid
sequence. An allele is one of a group of alternative forms of a
gene that occur at a given genetic locus.
[0071] As used herein, the phrase "allelic variant" refers to a
nucleic acid that occurs at a given locus or to a gene product
encoded by that nucleic acid. Such natural allelic variations can
typically result in 1-5% variance in the nucleic acid of a given
gene. Sequencing the gene of interest in a number of different
individuals can identify alternative alleles. Using hybridization
probes to identify the same genetic locus in a variety of
individuals can readily carry this out.
[0072] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising any of up to six open
reading frames which may or may not encode a polypeptide of the
invention. For example, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules encoding any of the open reading
frames shown in FIGS. 4-13, and described in Tables 2-11,
respectively. The term can further include nucleic acid molecules
comprising upstream and/or exon/intron sequences and
structures.
2TABLE 2 OPEN READING FRAMES FOR CNI-00711 OPEN READING FRAME
SEQUENCE ID. NUMBER LENGTH LOCATION NO. 1 30 Nucleotide 48-77 of 2
9 Amino Acid Seq. Id. No. 1 3 2 60 Nucleotide 78-137 of 4 19 Amino
Acid Seq. Id. No. 1 5 3 12 Nucleotide 131-142 of 6 3 Amino Acids
Seq. Id. No.1 7 4 33 Nucleotide 342-374 of 8 10 Amino Acids Seq.
Id. No. 1 9 5 15 Nucleotide 436-450 of 10 4 Amino Acids eq. Id. No.
1 11 6 42 Nucleotide 447-488 of 12 13 Amino Acids Seq. Id. No. 1 13
7 42 Nucleotide 647-688 of 14 13 Amino Acids Seq. Id. No. 1 15 8 63
Nucleotide 688-750 of 16 20 Amino Acids Seq. Id. No. 1 17 9 45
Nucleotide 706-750 of 18 14 Amino Acids Seq. Id. No. 1 19 10 33
Nucleotide 718-750 of 20 10 Amino Acids Seq. Id. No. 1 21 11 24
Nucleotide 727-750 of 22 7 Amino Acids Seq. Id. No. 1 23 12 106
Nucleotide 747-842 of 24 35 Amino Acids Seq. Id. No. 1 25
[0073]
3TABLE 3 OPEN READING FRAMES FOR CNI-00712 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 54 Nucleotide 20-73 of 27
17 Amino Acid Seq. Id. No. 26 28 2 57 Nucleotide 86-142 of 29 18
Amino Acid Seq. Id. No. 26 30 3 12 Nucleotide 228-239 of 31 3 Amino
Acids Seq. Id. No. 26 32 4 93 Nucleotide 249-341 of 33 30 Amino
Acids Seq. Id. No. 26 34 5 30 Nucleotide 304-333 of 35 9 Amino
Acids Seq. Id. No. 26 36 6 309 Nucleotide 338-646 of 37 102 Amino
Acids Seq. Id. No. 26 38 7 93 Nucleotide 360-452 of 39 30 Amino
Acids Seq. Id. No. 26 40 8 261 Nucleotide 386-646 of 41 86 Amino
Acids Seq. Id. No. 26 42 9 57 Nucleotide 396-452 of 43 18 Amino
Acids Seq. Id. No. 26 44 10 195 Nucleotide 452-646 of 45 64 Amino
Acids Seq. Id. No. 26 46 11 480 Nucleotide 456-935 of 47 159 Amino
Acid Seq. Id. No. 26 48 12 141 Nucleotide 506-646 of 49 46 Amino
Acids Seq. Id. No. 26 50 13 420 Nucleotide 516-935 of 51 139 Amino
Acids Seq. Id. No. 26 52 14 399 Nucleotide 537-935 of 53 132 Amino
Acids Seq. Id. No. 26 54 15 81 Nucleotide 566-646 of 55 26 Amino
Acids Seq. Id. No. 26 56 16 348 Nucleotide 588-935 of 57 115 Amino
Acids Seq. Id. No. 26 58 17 27 Nucleotide 620-646 of 59 8 Amino
Acids Seq. Id. No. 26 60 18 303 Nucleotide 633-935 of 61 100 Amino
Acids Seq. Id. No. 26 62 19 84 Nucleotide 796-879 of 63 27 Amino
Acids Seq. Id. No. 26 64 20 36 Nucleotide 900-935 of 65 11 Amino
Acids Seq. Id. No. 26 66 21 24 Nucleotide 991-1014 of 67 7 Amino
Acids Seq. Id. No. 26 68 22 18 Nucleotide 997-1014 of 69 5 Amino
Acids Seq. Id. No. 26 70 23 33 Nucleotide 1007-1039 of 71 10 Amino
Acids Seq. Id. No. 26 72 24 15 Nucleotide 1041-1055 of 73 4 Amino
Acids Seq. Id. No. 26 74
[0074]
4TABLE 4 OPEN READING FRAMES FOR CNI-00714 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 1239 Nucleotide 29-1267
of 76 412 Amino Acid Seq. Id. No. 75 77 2 105 Nucleotide 126-230 of
78 34 Amino Acid Seq. Id. No. 75 79 3 1092 Nucleotide 76-1267 of 80
363 Amino Acids Seq. Id. No. 75 81 4 18 Nucleotide 360-377 of 82 5
Amino Acids Seq. Id. No. 75 83 5 69 Nucleotide 393-461 of 84 22
Amino Acids Seq. Id. No. 75 85 6 24 Nucleotide 546-569 of 86 7
Amino Acids Seq. Id. No. 75 87 7 96 Nucleotide 573-668 of 88 31
Amino Acids Seq. Id. No. 75 89 8 87 Nucleotide 582-668 of 90 28
Amino Acids Seq. Id. No. 75 91 9 600 Nucleotide 668-1267 of 92 199
Amino Acids Seq. Id. No. 75 93 10 159 Nucleotide 684-842 of 94 52
Amino Acids Seq. Id. No. 75 95 11 510 Nucleotide 758-1267 of 96 169
Amino Acids Seq. Id. No. 75 97 12 51 Nucleotide 792-842 of 98 16
Amino Acids Seq. Id. No. 75 99 13 336 Nucleotide 932-1267 of 100
111 Amino Acids Seq. Id. No. 75 101 14 33 Nucleotide 1017-1049 of
102 10 Amino Acids Seq. Id. No. 75 103 15 216 Nucleotide 1052-1267
of 104 71 Amino Acids Seq. Id. No. 75 105 16 60 Nucleotide
1080-1139 of 106 19 Amino Acids Seq. Id. No. 75 107 17 48
Nucleotide 1092-1139 of 108 15 Amino Acids Seq. Id. No.75 109 18 30
Nucleotide 1110-1139 of 110 9 Amino Acids Seq. Id. No. 75 111 19
141 Nucleotide 1127-1267 of 112 46 Amino Acids Seq. Id. No.75 113
20 132 Nucleotide 1136-1267 of 114 43 Amino Acids Seq. Id. No.75
115 21 90 Nucleotide 1167-1256 of 116 29 Amino Acids Seq. Id. No.75
117 22 72 Nucleotide 1185-1256 of 118 23 Amino Acids Seq. Id. No.
75 119 23 57 Nucleotide 1211-1267 of 120 18 Amino Acids Seq. Id.
No.75 121 24 15 Nucleotide 1253-1267 of 122 4 Amino Acids Seq. Id.
No. 75 123 25 45 Nucleotide 1283-1327 of 124 14 Amino Acids Seq.
Id. No. 75 125 26 132 Nucleotide 1411-1542 of 126 43 Amino Acids
Seq. Id. No.75 127 27 105 Nucleotide 1438-1542 of 128 34 Amino
Acids Seq. Id. No. 75 129 28 75 Nucleotide 1493-1567 of 130 24
Amino Acids Seq. Id. No. 75 131 29 39 Nucleotide 1573-1611 of 132
12 Amino Acids Seq. Id. No. 75 133 30 33 Nucleotide 1528-1660 of
134 10 Amino Acids Seq. Id. No.75 135
[0075]
5TABLE 5 OPEN READING FRAMES FOR CNI-00715 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 39 Nucleotide 34-72 of
137 12 Amino Acid Seq. Id. No. 136 138 2 24 Nucleotide 38-61 of 139
7 Amino Acid Seq. Id. No. 136 140 3 138 Nucleotide 93-230 of 141 45
Amino Acids Seq. Id. No. 136 142 4 93 Nucleotide 138-230 of 143 30
Amino Acids Seq. Id. No. 136 144 5 72 Nucleotide 145-216 of 145 23
Amino Acids Seq. Id. No. 136 146 6 57 Nucleotide 160-216 of 147 18
Amino Acids Seq. Id. No. 136 148 7 30 Nucleotide 352-381 of 149 9
Amino Acids Seq. Id. No. 136 150 8 75 Nucleotide 399-473 of 151 24
Amino Acids Seq. Id. No. 136 152
[0076]
6TABLE 6 OPEN READING FRAMES FOR CNI-00716 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 108 Nucleotide 53-160 of
154 35 Amino Acid Seq. Id. No. 153 155 2 99 Nucleotide 62-160 of
156 32 Amino Acid Seq. Id. No. 153 157 3 21 Nucleotide 205-225 of
158 6 Amino Acids Seq. Id. No. 153 159 4 75 Nucleotide 226-300 of
160 24 Amino Acids Seq. Id. No. 153 161 5 48 Nucleotide 253-300 of
162 15 Amino Acids Seq. Id. No. 153 163 6 42 Nucleotide 259-300 of
164 13 Amino Acids Seq. Id. No. 153 165 7 99 Nucleotide 358-456 of
166 32 Amino Acids Seq. Id. No. 153 167 8 63 Nucleotide 394-456 of
168 20 Amino Acids Seq. Id. No. 153 169 9 39 Nucleotide 418-456 of
170 12 Amino Acids Seq. Id. No. 153 171 10 177 Nucleotide 459-635
of 172 58 Amino Acids Seq. Id. No. 153 173 11 27 Nucleotide 574-600
of 174 8 Amino Acids Seq. Id. No. 153 175 12 75 Nucleotide 604-678
of 176 24 Amino Acids Seq. Id. No. 153 177 13 33 Nucleotide 693-725
of 178 10 Amino Acids Seq. Id. No. 153 179 14 30 Nucleotide 696-725
of 180 9 Amino Acids Seq. Id. No. 153 181 15 42 Nucleotide 730-771
of 182 14 Amino Acids Seq. Id. No. 153 183
[0077]
7TABLE 7 OPEN READING FRAMES FOR CNI-00717 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 819 Nucleotide 80-898 of
185 272 Amino Acid Seq. Id. No. 184 186 2 774 Nucleotide 125-898 of
187 257 Amino Acid Seq. Id. No. 184 188 3 717 Nucleotide 182-898 of
189 238 Amino Acids Seq. Id. No. 184 190 4 699 Nucleotide 200-898
of 191 232 Amino Acids Seq. Id. No. 184 192 5 696 Nucleotide
203-898 of 193 231 Amino Acids Seq. Id. No. 184 194 6 72 Nucleotide
279-350 of 195 23 Amino Acids Seq. Id. No. 184 196 7 66 Nucleotide
285-350 of 197 21 Amino Acids Seq. Id. No. 184 198 8 57 Nucleotide
294-350 of 199 18 Amino Acids Seq. Id. No. 184 200 9 51 Nucleotide
369-419 of 201 16 Amino Acids Seq. Id. No. 184 202 10 306
Nucleotide 423-728 of 203 101 Amino Acids Seq. Id. No. 184 204 11
282 Nucleotide 447-728 of 205 93 Amino Acids Seq. Id. No. 184 206
12 231 Nucleotide 498-728 of 207 76 Amino Acids Seq. Id. No. 184
208 13 213 Nucleotide 516-728 of 209 70 Amino Acids Seq. Id. No.
184 210 14 195 Nucleotide 534-728 of 211 64 Amino Acids Seq. Id.
No. 184 212 15 189 Nucleotide 540-728 of 213 62 Amino Acids Seq.
Id. No. 184 214 16 174 Nucleotide 555-728 of 215 57 Amino Acids
Seq. Id. No. 184 216 17 156 Nucleotide 573-728 of 217 51 Amino
Acids Seq. Id. No. 184 218 18 126 Nucleotide 603-728 of 219 41
Amino Acids Seq. Id. No. 184 220 19 117 Nucleotide 612-728 of 221
38 Amino Acids Seq. Id. No. 184 222 20 96 Nucleotide 633-728 of 223
31 Amino Acids Seq. Id. No. 184 224 21 48 Nucleotide 681-728 of 225
15 Amino Acids Seq. Id. No. 184 226 22 42 Nucleotide 687-728 of 227
13 Amino Acids Seq. Id. No. 184 228 23 78 Nucleotide 741-818 of 229
25 Amino Acids Seq. Id. No. 184 230 24 60 Nucleotide 759-818 of 231
19 Amino Acids Seq. Id. No. 184 232 25 48 Nucleotide 771-818 of 233
15 Amino Acids Seq. Id. No. 184 234 26 36 Nucleotide 783-818 of 235
11 Amino Acids Seq. Id. No. 184 236 27 84 Nucleotide 846-929 of 237
27 Amino Acids Seq. Id. No. 184 238 28 69 Nucleotide 861-929 of 239
22 Amino Acids Seq. Id. No. 184 240 29 66 Nucleotide 864-929 of 241
21 Amino Acids Seq. Id. No. 184 242 30 75 Nucleotide 931-1005 of
243 24 Amino Acids Seq. Id. No. 184 244 31 75 Nucleotide 1062-1136
of 245 24 Amino Acids Seq. Id. No. 184 246 32 18 Nucleotide
1119-1136 of 247 5 Amino Acids Seq. Id. No. 184 248 33 15
Nucleotide 1162-1176 of 249 4 Amino Acids Seq. Id. No. 184 250 34
81 Nucleotide 1304-1384 of 251 26 Amino Acids Seq. Id. No. 184 252
35 24 Nucleotide 1361-1384 of 253 7 Amino Acids Seq. Id. No. 184
254 36 27 Nucleotide 1396-1422 of 255 8 Amino Acids Seq. Id. No.
184 256 37 90 Nucleotide 1478-1567 of 257 29 Amino Acids Seq. Id.
No.184 258 38 24 Nucleotide 1554-1577 of 259 7 Amino Acids Seq. Id.
No. 184 260
[0078]
8TABLE 8 OPEN READING FRAMES FOR CNI-00720 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 24 Nucleotide 62-85 of
262 7 Amino Acid Seq. Id. No. 261 263 2 228 Nucleotide 88-315 of
264 75 Amino Acid Seq. Id. No. 261 265 3 195 Nucleotide 121-315 of
266 64 Amino Acids Seq. Id. No. 261 267 4 69 Nucleotide 247-315 of
268 22 Amino Acids Seq. Id. No. 261 269 5 87 Nucleotide 321-407 of
270 28 Amino Acids Seq. Id. No. 261 271 6 270 Nucleotide 376-645 of
272 89 Amino Acids Seq. Id. No. 261 273 7 21 Nucleotide 593-559 of
274 6 Amino Acids Seq. Id. No. 261 275 8 42 Nucleotide 604-645 of
276 13 Amino Acids Seq. Id. No. 261 277 9 18 Nucleotide 623-640 of
278 5 Amino Acids Seq. Id. No. 261 279 10 99 Nucleotide 651-749 of
280 32 Amino Acids Seq. Id. No. 261 281 11 33 Nucleotide 661-693 of
282 10 Amino Acids Seq. Id. No. 261 283 12 54 Nucleotide 742-795 of
284 17 Amino Acids Seq. Id. No. 261 285 13 15 Nucleotide 1020-1034
of 286 4 Amino Acids Seq. Id. No. 261 287 14 48 Nucleotide
1034-1081 of 288 15 Amino Acids Seq. Id. No. 261 289 15 12
Nucleotide 1126-1137 of 290 3 Amino Acids Seq. Id. No. 261 291
[0079]
9TABLE 9 OPEN READING FRAMES FOR CNI-00721 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 207 Nucleotide 112-318 of
293 68 Amino Acids Seq. Id. No. 292 294 2 147 Nucleotide 172-318 of
295 48 Amino Acids Seq. Id. No. 292 296 3 24 Nucleotide 236-259 of
297 7 Amino Acids Seq. Id. No. 292 298 4 18 Nucleotide 345-362 of
299 5 Amino Acids Seq. Id. No. 292 300 5 51 Nucleotide 352-402 of
301 16 Amino Acids Seq. Id. No. 292 302 6 132 Nucleotide 362-493 of
303 43 Amino Acids Seq. Id. No. 292 304 7 33 Nucleotide 370-402 of
305 10 Amino Acids Seq. Id. No. 292 306 8 21 Nucleotide 382-402 of
307 6 Amino Acids Seq. Id. No. 292 308 9 12 Nucleotide 426-437 of
309 3 Amino Acids Seq. Id. No. 292 310 10 201 Nucleotide 589-789 of
311 66 Amino Acids Seq. Id. No. 292 312 11 93 Nucleotide 738-830 of
313 30 Amino Acids Seq. Id. No. 292 314 12 21 Nucleotide 776-796 of
315 6 Amino Acids Seq. Id. No. 292 316 13 42 Nucleotide 789-830 of
317 13 Amino Acids Seq. Id. No. 292 318 14 27 Nucleotide 840-866 of
319 8 Amino Acids Seq. Id. No. 292 320 15 324 Nucleotide 866-1189
of 321 107 Amino Acids Seq. Id. No. 292 322 16 78 Nucleotide
870-947 of 323 25 Amino Acids Seq. Id. No. 292 324 17 54 Nucleotide
894-947 of 325 17 Amino Acids Seq. Id. No. 292 326 18 30 Nucleotide
918-947 of 327 9 Amino Acids Seq. Id. No. 292 328 19 24 Nucleotide
976-999 of 329 7 Amino Acids Seq. Id. No. 292 330 20 66 Nucleotide
1057-1122 of 331 21 Amino Acids Seq. Id. No. 292 332 21 15
Nucleotide 1108-1122 of 333 4 Amino Acids Seq. Id. No. 292 334 22
69 Nucleotide 1346-1414 of 335 22 Amino Acids Seq. Id. No. 292 336
23 63 Nucleotide 1352-1414 of 337 20 Amino Acids Seq. Id. No. 292
338 24 15 Nucleotide 1400-1414 of 339 4 Amino Acids Seq. Id. No.
292 340 25 18 Nucleotide 1491-1508 of 341 5 Amino Acids Seq. Id.
No. 292 342 26 42 Nucleotide 1523-1564 of 343 13 Amino Acids Seq.
Id. No. 292 344 27 15 Nucleotide 1528-1542 of 345 4 Amino Acids
Seq. Id. No. 292 346 28 111 Nucleotide 1647-1757 of 347 36 Amino
Acids Seq. Id. No. 292 348 29 87 Nucleotide 1654-1740 of 349 28
Amino Acids Seq. Id. No. 292 350 30 24 Nucleotide 1826-1849 of 351
7 Amino Acids Seq. Id. No. 292 352 31 12 Nucleotide 1859-1870 of
353 3 Amino Acids Seq. Id. No. 292 354 32 51 Nucleotide 1867-1917
of 355 16 Amino Acids Seq. Id. No. 292 356 33 57 Nucleotide
1881-1937 of 357 18 Amino Acids Seq. Id. No. 292 358
[0080]
10TABLE 10 OPEN READING FRAMES FOR CNI-00723 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 36 Nucleotide 217-252 of
360 11 Amino Acids Seq. Id. No. 359 361 2 48 Nucleotide 288-335 of
362 15 Amino Acids Seq. Id. No. 359 363 3 18 Nucleotide 332-349 of
364 5 Amino Acids Seq. Id. No. 359 365 4 63 Nucleotide 393-455 of
366 20 Amino Acids Seq. Id. No. 359 367 5 24 Nucleotide 412-435 of
368 7 Amino Acids Seq. Id. No. 359 369 6 51 Nucleotide 439-489 of
370 16 Amino Acids Seq. Id. No. 359 371 7 1473 Nucleotide 489-1961
of 372 490 Amino Acids Seq. Id. No. 359 373 8 1467 Nucleotide
495-1961 of 374 488 Amino Acids Seq. Id. No. 359 375 9 90
Nucleotide 544-633 of 376 29 Amino Acids Seq. Id. No. 359 377 10 78
Nucleotide 556-633 of 378 25 Amino Acids Seq. Id. No. 359 379 11 63
Nucleotide 571-633 of 380 20 Amino Acids Seq. Id. No. 359 381 12 33
Nucleotide 614-646 of 382 10 Amino Acids Seq. Id. No. 359 383 13 12
Nucleotide 622-633 of 384 3 Amino Acids Seq. Id. No. 359 385 14 42
Nucleotide 634-675 of 386 13 Amino Acids Seq. Id. No. 359 387 15
1260 Nucleotide 702-1961 of 388 419 Amino Acids Seq. Id. No. 359
389 16 45 Nucleotide 736-780 of 390 14 Amino Acids Seq. Id. No. 359
391 17 108 Nucleotide 740-847 of 392 35 Amino Acids Seq. Id. No.
359 393 18 1128 Nucleotide 834-1961 of 394 375 Amino Acids Seq. Id.
No. 359 395 19 1017 Nucleotide 945-1961 of 396 338 Amino Acids Seq.
Id. No. 359 397 20 15 Nucleotide 986-1000 of 398 4 Amino Acids Seq.
Id. No. 359 399 21 30 Nucleotide 1000-1029 of 400 9 Amino Acids
Seq. Id. No. 359 401 22 936 Nucleotide 1026-1961 of 402 311 Amino
Acids Seq. Id. No. 359 403 23 12 Nucleotide 1061-1072 of 404 3
Amino Acids Seq. Id. No. 359 405 24 39 Nucleotide 1069-1107 of 406
12 Amino Acids Seq. Id. No. 359 407 25 33 Nucleotide 1075-1107 of
408 10 Amino Acids Seq. Id. No. 359 409 26 870 Nucleotide 1092-1961
of 410 289 Amino Acids Seq. Id. No. 359 411 27 54 Nucleotide
1258-1311 of 412 17 Amino Acids Seq. Id. No. 359 413 28 678
Nucleotide 1284-1961 of 414 225 Amino Acids Seq. Id. No. 359 415 29
21 Nucleotide 1342-1362 of 416 6 Amino Acids Seq. Id. No. 359 417
30 24 Nucleotide 1379-1402 of 418 7 Amino Acids Seq. Id. No. 359
419 31 15 Nucleotide 1429-1443 of 420 4 Amino Acids Seq. Id. No.
359 421 32 486 Nucleotide 1476-1961 of 422 161 Amino Acids Seq. Id.
No. 359 423 33 54 Nucleotide 1489-1542 of 424 17 Amino Acids Seq.
Id. No. 359 425 34 15 Nucleotide 1528-1542 of 426 4 Amino Acids
Seq. Id. No. 359 427 35 57 Nucleotide 1543-1599 of 428 18 Amino
Acids Seq. Id. No. 359 429 36 24 Nucleotide 1576-1599 of 430 7
Amino Acids Seq. Id. No. 359 431 37 240 Nucleotide 1722-1961 of 432
79 Amino Acids Seq. Id. No. 359 433 38 90 Nucleotide 1872-1961 of
434 29 Amino Acids Seq. Id. No. 359 435 39 54 Nucleotide 1915-1968
of 436 17 Amino Acids Seq. Id. No. 359 437 40 39 Nucleotide
1993-2031 of 438 12 Amino Acids Seq. Id. No. 359 439 41 309
Nucleotide 2004-2312 of 440 102 Amino Acids Seq. Id. No. 359 441 42
21 Nucleotide 2011-2031 of 442 6 Amino Acids Seq. Id. No. 359 443
43 204 Nucleotide 2109-2312 of 444 67 Amino Acids Seq. Id. No. 359
445 44 198 Nucleotide 2115-2312 of 446 65 Amino Acids Seq. Id. No.
359 447 45 57 Nucleotide 2198-2254 of 448 18 Amino Acids Seq. Id.
No. 359 449 46 231 Nucleotide 2269-2499 of 450 76 Amino Acids Seq.
Id. No. 359 451 47 216 Nucleotide 2284-2499 of 452 71 Amino Acids
Seq. Id. No. 359 453 48 153 Nucleotide 2300-2454 of 454 50 Amino
Acids Seq. Id. No. 359 455 49 30 Nucleotide 2423-2452 of 456 9
Amino Acids Seq. Id. No. 359 457 50 48 Nucleotide 2452-2499 of 458
15 Amino Acids Seq. Id. No. 359 459 51 15 Nucleotide 2522-2536 of
460 4 Amino Acids Seq. Id. No. 359 461
[0081]
11TABLE 11 OPEN READING FRAME FOR CNI-00724 OPEN READING FRAME
SEQUENCE NUMBER LENGTH LOCATION ID. NO. 1 243 Nucleotide 567-809 of
Seq. Id. 463 80 Amino Acids No. 462 464
[0082] In a specific embodiment, the nucleic acid molecules
comprise nucleic acids that encode an open reading frame of at
least 3 contiguous amino acid residues from a full-length protein.
In alternate embodiments, the nucleic acid molecules comprise an
open reading frame which encodes at least about 5, 8, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or
more contiguous amino acid residues of a protein.
[0083] The sequence obtained from clones containing partial coding
sequences or non-coding sequences can be used to obtain the entire
coding region by using the RACE method, for example (Chenchik, et
al., 1995, CLONTECHniques (X) 1: 5-8; Barnes, 1994, Proc. Natl.
Acad. Sci. USA 91: 2216-2220; and Cheng et al., Proc. Natl. Acad.
Sci. USA 91: 5695-5699). Oligonucleotides can be designed based on
the sequence obtained from the partial clone that can amplify a
reverse transcribed mRNA encoding the entire coding sequence.
Alternatively, probes can be used to screen cDNA libraries prepared
from an appropriate cell or cell line in which the protective
sequence is transcribed.
[0084] With respect to allelic variants of protective sequences
associated with a condition, disorder, or disease involving cell
death, any and all such nucleotide variations and resulting amino
acid polymorphisms or variations which are the result of natural
allelic variation of the protective sequence are intended to be
within the scope of the present invention. Such allelic variants
include, but are not limited to, ones that do not alter the
functional activity of the protective sequence product.
[0085] With respect to the cloning of additional allelic variants
of the isolated protective sequence and homologues and orthologs
from other species (e.g., guinea pig, cow, mouse), the isolated
protective sequences disclosed herein may be labeled and used to
screen a cDNA library constructed from mRNA obtained from
appropriate cells or tissues (e.g., brain) derived from the
organism (e.g., guinea pig, cow and mouse) of interest. The
hybridization conditions used generally should be of a lower
stringency when the cDNA library is derived from an organism
different from the type of organism from which the labeled sequence
was derived, and can routinely be determined based on, e.g.,
relative relatedness of the target and reference organisms.
[0086] Alternatively, the labeled fragment may be used to screen a
genomic library derived from the organism of interest, again, using
appropriately stringent conditions. Appropriate stringency
conditions are well known to those of skill in the art as discussed
above, and will vary predictably depending on the specific
organisms from which the library and the labeled sequences are
derived. For guidance regarding such conditions, see, for example,
Sambrook, et al., 1989, Molecular Cloning, A Laboratory Manual,
Second Edition, Cold Spring Harbor Press, N.Y.; and Ausubel, et
al., 1989-1999, Current Protocols in Molecular Biology, Green
Publishing Associates and Wiley lnterscience, N.Y., both of which
are incorporated herein by reference in their entirety.
[0087] Additionally, the cloning of homologs and orthologs of the
isolated protective sequence from other species (e.g. mouse) could
also occur using the knowledge of syntenic regions and/or genes.
Syntenic genes are genes which are believed to be located on the
same chromosome because they are lost along with a marker gene
which is known to be located on that chromosome. There are
well-established genetic maps of specific chromosome regions that
show syntenic regions between chromosomes of humans and other
species that can be utilized, by one skilled in the art, for this
purpose.
[0088] Further, a protective sequence allelic variant may be
isolated from, for example, human nucleic acid, by performing PCR
using two degenerate oligonucleotide primer pools designed on the
basis of amino acid sequences within the protective sequence
product of interest. The template for the reaction may be cDNA
obtained by reverse transcription of mRNA prepared from, for
example, human or non-human cell lines or tissue known or suspected
to express a wild type or mutant protective sequence allele. In one
embodiment, the allelic variant is isolated from an individual who
has a condition, disorder, or disease involving cell death. Such
variants are described in the examples below.
[0089] The PCR product may be subcloned and sequenced to ensure
that the amplified sequences represent the sequences of a
protective nucleic acid sequence. The PCR fragment may then be used
to isolate a full-length cDNA clone by a variety of methods. For
example, the amplified fragment may be labeled and used to screen a
bacteriophage cDNA library. Alternatively, the labeled fragment may
be used to isolate genomic clones via the screening of a genomic
library.
[0090] PCR technology also may be utilized to isolate full-length
cDNA sequences. For example, RNA may be isolated, following
standard procedures, from an appropriate cellular or tissue source.
A reverse transcription reaction may be performed on the RNA using
an oligonucleotide primer specific for the most 5' end of the
amplified fragment for the priming of first strand synthesis. The
resulting RNA/DNA hybrid may then be "tailed" with guanines using a
standard terminal transferase reaction. The hybrid may be digested
with RNAase H and second strand synthesis may then be primed with a
poly-C primer. Thus, cDNA sequences upstream of the amplified
fragment may easily be isolated. For a review of cloning strategies
which may be used, see e.g., Sambrook et al., 1989, supra, or
Ausubel et al., supra.
[0091] In cases where the isolated protective sequence is the
normal, or wild type gene, this gene may be used to isolate mutant
alleles of the protective sequence. Such an isolation is preferable
in processes and disorders that are known or suspected to have a
genetic basis. Mutant alleles may be isolated from individuals
either known or suspected to have a genotype which contributes to
symptoms of conditions, disorders, or diseases involving cell
death. Mutant alleles and mutant allele products may then be
utilized in the therapeutic and diagnostic assay systems described
below.
[0092] A cDNA of the mutant protective sequence may be isolated,
for example, by using PCR, a technique well known to those of skill
in the art. In this case, the first cDNA strand may be synthesized
by hybridizing an oligo-dT oligonucleotide to mRNA isolated from
tissue known or suspected to be expressed in an individual
putatively carrying the mutant allele, and by extending the new
strand with reverse transcriptase. The second strand of the cDNA is
then synthesized using an oligonucleotide that hybridizes
specifically to the 5' end of the normal protective sequence. Using
these two primers, the product is then amplified via PCR, cloned
into a suitable vector and subjected to DNA sequence analysis
through methods well known to those of skill in the art. By
comparing the DNA sequence of the mutant protective sequence to
that of the normal protective sequence, the mutation(s) responsible
for the loss or alteration of function of the mutant gene product
can be ascertained.
[0093] Alternatively, a genomic or cDNA library can be constructed
and screened using DNA or RNA, respectively, from a tissue known to
or suspected of expressing the protective sequence of interest in
an individual suspected of or known to carry the mutant allele. The
normal protective sequence or any suitable fragment thereof may
then be labeled and used as a probed to identify the corresponding
mutant allele in the library. The clone containing this protective
sequence may then be purified through methods routinely practiced
in the art, and subjected to sequence analysis as described above
in this Section.
[0094] Additionally, an expression library can be constructed
utilizing DNA isolated from or cDNA synthesized from a tissue known
to or suspected of expressing the protective sequence of interest
in an individual suspected of or known to carry the mutant allele.
In this manner, protective sequence products made by the tissue
containing the putative mutant alleles may be expressed and
screened using standard antibody screening techniques in
conjunction with antibodies raised against the normal protective
sequence product, as described, below, in Section 5.3 (For
screening techniques, see, for example, Harlow, E. and Lane, eds.,
1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press,
Cold Spring Harbor.) In cases where the mutation results in an
expressed protective sequence product with altered function (e.g.,
as a result of a missense mutation), a polyclonal set of antibodies
are likely to cross-react with the mutant protective sequence
product. Library clones detected via their reaction with such
labeled antibodies can be purified and subjected to sequence
analysis as described in this Section, above.
[0095] The invention also includes nucleic acid molecules,
preferably DNA molecules that are the complements of the nucleic
acids of the preceding paragraphs.
[0096] In certain embodiments, the protective nucleic acid
molecules of the invention are present as part of protective
nucleic acid molecules comprising nucleic acid sequences which do
not contain heterologous (e.g., cloning vector or expression
vector) sequences. In other embodiments, the protective nucleic
acid molecules of the invention further comprise vector sequences,
e.g., cloning vectors or expression vectors.
[0097] 5.2 Protein Products of the Protective Sequences
[0098] Protective sequence products or fragments thereof of the
invention can be prepared for a variety of uses, including but not
limited to, prophylactic or therapeutic modulators of protective
sequence product function, for the generation of antibodies,
diagnostic assays, or for the identification of other cellular or
extracellular protective sequence products involved in the
regulation of conditions, disorders, or diseases involving cell
death.
[0099] The protective sequence products of the invention include,
but are not limited to, human protective sequence products and
non-human protective sequence products, e.g., mammalian (such as
bovine or guinea pig), protective sequence products.
[0100] Protective sequence products of the invention, sometimes
referred to herein as a "protective sequence protein" or
"protective sequence polypeptide," includes those gene products
encoded by any of up to six translational reading frames of the
protective sequence sequences depicted in Table 1, as well as gene
products encoded by other human allelic variants and non-human
variants of protective sequence products which can be identified by
the methods herein described. Among such protective sequence
product variants are protective sequence products comprising amino
acid residues encoded by polymorphisms of such protective sequence
products.
[0101] In addition, protective sequence products of the invention
may include proteins that represent functionally equivalent gene
products. Functionally equivalent protective sequence products may
include, for example, protective sequence products encoded by one
of the nucleic acid molecules described in Section 5.1, above. In
preferred embodiments, such functionally equivalent protective
sequence products are naturally occurring gene products.
Functionally equivalent protective sequence products also include
gene products which retain at least one of the biological
activities of the protective sequence products described above,
and/or which are recognized by and bind to antibodies (polyclonal
or monoclonal) directed against protective sequence products of the
invention.
[0102] Equivalent protective sequence products may contain
deletions, including internal deletions, additions, including
additions yielding fusion proteins, or substitutions of amino acid
residues within and/or adjacent to the amino acid sequence encoded
by the protective sequence sequences described, above, in Section
5.1. Generally, deletions will be deletions of single amino acid
residues, or deletions of no more than about 2, 3, 4, 5, 10 or 20
amino acid residues, either contiguous or non-contiguous.
Generally, additions or substitutions, other than additions which
yield fusion proteins, will be additions or substitutions of single
amino acid residues, or additions or substitutions of no more than
about 2, 3, 4, 5, 10 or 20 amino acid residues, either contiguous
or non-contiguous. Preferably, these modifications result in a
"silent" change, in that the change produces a protective sequence
product with the same activity as the original protective sequence
product. However, nucleic acid changes resulting in amino acid
additions or substitutions may also be made for the purpose of
modifying the protective sequence product in order to generally
enhance their use as therapeutic agents or components for assays,
such modifications to include, but not be limited to, stabilizing
the product against degradation, enhancing pharmacokinetic
properties, modifying site tropisms at the level of cells, tissues,
organs, or organisms.
[0103] Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity and/or the amphipathic nature of the residues
involved. For example, nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan and methionine; polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine and
glutamine; positively charged (basic) amino acids include arginine,
lysine and histidine; and negatively charged (acidic) amino acids
include aspartic acid and glutamic acid. Additionally, non-natural
amino acids, including, but not limited to, D-amino acids may be
used.
[0104] Alternatively, where alteration of function is desired,
addition(s), deletion(s) or non-conservative alterations can
produce altered, including reduced-activity, protective sequence
products. Such alterations can, for example, alter one or more of
the biological functions of the protective sequence product.
Further, such alterations can be selected so as to generate
protective sequence products which include, but are not limited to,
products which are better suited for expression, scale up, etc. in
the host cells chosen. For example, cysteine residues can be
deleted or substituted with another amino acid residue in order to
eliminate disulfide bridges.
[0105] Protein fragments and/or peptides of the invention may
comprise at least as many contiguous amino acid residues as
necessary to represent an epitope fragment (that is to be
recognized by an antibody directed to the protein). Examples of
such protein fragments and/or peptides of the invention are shown
by the open reading frames of the protective sequences shown in
FIGS. 4-13, and described in Tables 2-11, respectively. In one
nonlimiting embodiment of the invention, such protein fragments or
peptides comprise at least about 3 contiguous amino acid residues
from a full-length protein. In alternate embodiments, the protein
fragments and peptides of the invention can comprise about 5, 8,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 450 or more contiguous amino acid residues of a protein.
[0106] Peptides and/or proteins corresponding to one or more
domains of the protein as well as fusion proteins in which a
protein, or a portion of a protein such as a truncated protein or
peptide or a protein domain, is fused to an unrelated protein are
also within the scope of this invention. Such proteins and peptides
can be designed on the basis of the nucleic acids disclosed in
Section 5.1, above. Fusion proteins include, but are not limited
to, IgFc fusions which stabilize the protein or peptide and prolong
half-life in vivo; or fusions to any amino acid sequence which
allows the fusion protein to be anchored to the cell membrane; or
fusions to an enzyme, fluorescent protein, luminescent protein or a
epitope tagged protein or peptide which provides a marker
function.
[0107] The protein sequences described above can include a domain,
which comprises a protein transduction domain which targets the
protective sequence product for delivery to various tissues and
more particularly across the brain blood barrier, using, for
example, the protein transduction domain of human immunodeficiency
virus TAT protein (Schwarze et al., 1999, Science 285:
1569-72).
[0108] The protein sequences described above can include a domain,
which comprises a signal sequence that targets the gene product for
secretion. As used herein, a signal sequence includes a peptide of
at least about 15 or 20 amino acid residues in length which occurs
at the N-terminus of secretory and membrane-bound proteins and
which contains at least about 70% hydrophobic amino acid residues
such as alanine, leucine, isoleucine, phenylalanine, proline,
tyrosine, tryptophan or valine. In a preferred embodiment, a signal
sequence contains at least about 10 to 40 amino acid residues,
preferably about 19-34 amino acid residues and has at least about
60-80%, more preferably 65-75% and more preferably at least about
70% hydrophobic residues. A signal sequence serves to direct a
protein containing such a sequence to a lipid bilayer.
[0109] A signal sequence of a polypeptide of the invention can be
used to facilitate secretion and isolation of the secreted protein
or other proteins of interest. Signal sequences are typically
characterized by a core of hydrophobic amino acids, which are
generally cleaved from the mature protein during secretion in one
or more cleavage events. Such signal peptides contain processing
sites that allow cleavage of the signal sequence from the mature
proteins as they pass through the secretory pathway. Thus, the
invention pertains to the described polypeptides having a signal
sequence (that is, "immature" polypeptides), as well as to the
signal sequences themselves and to the polypeptides in the absence
of a signal sequence (i.e., the "mature" cleavage products). It is
to be understood that polypeptides of the invention can further
comprise polypeptides comprising any signal sequence having
characteristics as described above and a mature polypeptide
sequence.
[0110] In one embodiment, a nucleic acid sequence encoding a signal
sequence of the invention can be operably linked in an expression
vector to a protein of interest, such as a protein which is
ordinarily not secreted or is otherwise difficult to isolate. The
signal sequence directs secretion of the protein, such as from a
eukaryotic host into which the expression vector is transformed,
and the signal sequence is subsequently or concurrently cleaved.
The protein can then be readily purified from the extracellular
medium by art recognized methods. Alternatively, the signal
sequence can be linked to the protein of interest using a sequence
which facilitates purification, such as with a GST domain.
[0111] Finally, the proteins of the invention also include protein
sequences wherein domains encoded by any transcriptional or
post-transcriptional, and/or translational or post-translational
modifications, or fragments thereof, have been deleted. The
polypeptides of the invention can further comprise
posttranslational modifications, including, but not limited to
glycosylations, acetylations and myrisalations.
[0112] The protective sequence products, peptide fragments thereof
and fusion proteins thereof may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods
for preparing the protective sequence products, polypeptides,
peptides, fusion peptide and fusion polypeptides of the invention
by expressing nucleic acid containing protective sequence sequences
are described herein. Methods that are well known to those skilled
in the art can be used to construct expression vectors containing
protective sequence product coding sequences and appropriate
transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques and in vivo genetic recombination. See, for
example, the techniques described in Sambrook, et al., 1989, supra,
and Ausubel, et al., 1989, supra. Alternatively, RNA capable of
encoding protective sequence product sequences may be chemically
synthesized using, for example, synthesizers. See, for example, the
techniques described in "Oligonucleotide Synthesis", 1984, Gait,
ed., IRL Press, Oxford.
[0113] A variety of host-expression vector systems may be utilized
to express the protective sequence product coding sequences of the
invention. Such host-expression systems represent vehicles by which
the coding sequences of interest may be produced and subsequently
purified, but also represent cells which may, when transformed or
transfected with the appropriate nucleotide coding sequences,
exhibit the protective sequence product of the invention in situ.
These include, but are not limited to, microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing protective sequence product coding sequences; yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast
expression vectors containing the protective sequence product
coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the
protective sequence product coding sequences; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing protective sequence product coding sequences;
or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionine promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter).
[0114] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
protective sequence product being expressed. For example, when a
large quantity of such a protein is to be produced, for the
generation of pharmaceutical compositions of protective sequence
product or for raising antibodies to protective sequence product,
for example, vectors which direct the expression of high levels of
fusion protein products which are readily purified may be
desirable. Such vectors include, but are not limited to, the E.
coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which the protective sequence product coding sequence
may be ligated individually into the vector in frame with the lacZ
coding region so that a fusion protein is produced; pIN vectors
(Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van
Heeke and Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the
like. pGEX vectors may also be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In
general, such fusion proteins are soluble and can easily be
purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned protective sequence product can
be released from the GST moiety.
[0115] In an insect system, Autographa californica, nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
protective sequence product coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter
(for example the polyhedrin promoter). Successful insertion of
protective sequence product coding sequence will result in
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedrin gene). These recombinant viruses are then
used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed. (e.g., see Smith, et al., 1983, J. Virol.
46:584; Smith, U.S. Pat. No. 4,215,051).
[0116] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the protective sequence product coding sequence
of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing protective sequence
products in infected hosts. (See, e.g., Logan and Shenk, 1984,
Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiation
signals may also be required for efficient translation of inserted
protective sequence product coding sequences. These signals include
the ATG initiation codon and adjacent sequences. In cases where an
entire protective sequence, including its own initiation codon and
adjacent sequences, is inserted into the appropriate expression
vector, no additional translational control signals may be needed.
However, in cases where only a portion of the protective sequence
coding sequence is inserted, exogenous translational control
signals, including, perhaps, the ATG initiation codon, must be
provided. Furthermore, the initiation codon must be in phase with
the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational
control signals and initiation codons can be of a variety of
origins, both natural and synthetic. The efficiency of expression
may be enhanced by the inclusion of appropriate transcription
enhancer elements, transcription terminators, etc. (see Bittner, et
al., 1987, Methods in Enzymol. 153:516-544).
[0117] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells that possess the cellular machinery for
proper processing of the primary transcript, glycosylation and
phosphorylation of the gene product may be used. Such mammalian
host cells include, but are not limited to, CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3 and WI38.
[0118] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express the protective sequence product may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express the protective sequence product. Such engineered
cell lines may be particularly useful in screening and evaluation
of compounds that affect the endogenous activity of the protective
sequence product.
[0119] A number of selection systems may be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigler,
et al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for the following genes: dhfr, which confers
resistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad.
Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA
78:1527); gpt, which confers resistance to mycophenolic acid
(Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro,
which confers resistance to hygromycin (Santerre, et al., 1984,
Gene 30:147).
[0120] Alternatively, the expression characteristics of an
endogenous protective sequence within a cell line or microorganism
may be modified by inserting a heterologous DNA regulatory element
into the genome of a stable cell line or cloned microorganism such
that the inserted regulatory element is operatively linked with the
endogenous protective sequence. For example, an endogenous
protective sequence which is normally "transcriptionally silent",
i.e., a protective sequence which is normally not expressed, or is
expressed only at very low levels in a cell line or microorganism,
may be activated by inserting a regulatory element which is capable
of promoting the expression of a normally expressed protective
sequence product in that cell line or microorganism. Alternatively,
a transcriptionally silent, endogenous protective sequence may be
activated by insertion of a promiscuous regulatory element which
works across cell types.
[0121] Methods, which are well known to those skilled in the art,
can be used to construct vectors containing the protective sequence
operatively associated with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, and synthetic
techniques. See, for example, the techniques described in Sambrook,
et al., 1992, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory, N.Y. and Ausubel et al., 1989, Current Protocols
in Molecular Biology, Greene Publishing Associates & Wiley
Interscience, N.Y.
[0122] The protective sequences may be associated operatively with
a variety of different promoter/enhancer elements. The expression
elements of these vectors may vary in their strength and
specificities. Depending on the host/vector system utilized, any
one of a number of suitable transcription and translation elements
may be used. The promoter may be in the form of the promoter that
is associated naturally with the gene of interest. Alternatively,
the DNA may be positioned under the control of a recombinant or
heterologous promoter, i. e., a promoter that is not associated
normally with that gene. For example, tissue specific
promoter/enhancer elements may be used to regulate the expression
of the transferred DNA in specific cell types. Examples of
transcriptional control regions which exhibit tissue specificity
which have been described and could be used, include, but are not
limited to: choline acetyltransferase (ChAT) gene control region
which is active in cholinergic cells in the brain (Lonnerberg et
al., 1996, JBC 271:33358-65; Lonnerberg et al., 1995, PNAS 92:
4046-50; Ibenez and Perrson, 1991 Eur. J. Neurosci. 3: 1309-15),
mouse Thy-1.2 gene control region which is active in adult neurons
including hippocampus, thalamus, cerebellum, cortex, RGC, DRG, and
MN in the brain (Caroni, 1997, J Neurosci. Meth. 71: 3-9; Vidal et
al., 1990, EMBO J 9: 833-40), neuron specific enolase (NSE) gene
control region which is active in pan-neuronal, neuron specific,
deep layers of cerebral and neocortex (not in white matter) areas
of the brain (Hannas-Djebbara et al., 1997, Brain Res. Mol. Brain
Res. 46: 91-9; Peel et al., 1997, Gene Therapy 4: 16-24; Twyman et
al., 1997, J Mol Neurosci 8: 63-73; Forss-Petter et al., 1990,
Neuron 5:187-97), elastase I gene control region which is active in
pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646;
Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122); immunoglobulin gene control region which
is active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-658; Adams et al., 1985, Nature 318:533-538; Alexander et
al., 1987, Mol. Cell. Biol. 7:1436-1444); albumin gene control
region which is active in liver (Pinkert et al., 1987, Genes and
Devel. 1:268-276); alpha-fetoprotein gene control region which is
active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58);
alpha-1-antitrypsin gene control region which is active in liver
(Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene
control region which is active in myeloid cells (Magram et al.,
1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94);
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell
48:703-712); myosin light chain-2 gene control region which is
active in skeletal muscle (Shani, 1985, Nature 314:283-286) and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
Promoters isolated from the genome of viruses which grow in
mammalian cells (e.g., CMV, RSV, vaccinia virus 7.5K, SV40, HSV,
adenoviruses MLP, and MMTV LTR promoters) may be used, as well as
promoters produced by recombinant DNA or synthetic techniques.
Further, promoters specifically activated within bone, i.e., the
osteocalcin promoter, which is specifically activated within cells
of osteoblastic lineage, may be used to target expression of
nucleic acids within bone cells.
[0123] A heterologous regulatory element may be inserted into a
stable cell line or cloned microorganism, such that it is
operatively linked with an endogenous protective sequence, using
techniques, such as targeted homologous recombination, which are
well known to those of skill in the art, and described e.g., in
Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667,
published May 16, 1991.
[0124] Alternatively, utilizing an antibody specific for the fusion
protein being expressed may readily purify any fusion protein. For
example, a system described by Janknecht, et al. allows for the
ready purification of non-denatured fusion proteins expressed in
human cell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci.
USA 88:8972-8976). In this system, the gene of interest is
subcloned into a vaccinia recombination plasmid such that the
gene's open reading frame is translationally fused to an
amino-terminal tag consisting of six histidine residues. Extracts
from cells infected with recombinant vaccinia virus are loaded onto
Ni.sup.2+-nitriloacetic acid-agarose columns and histidine-tagged
proteins are selectively eluted with imidazole-containing
buffers.
[0125] The protective sequence products can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs,
goats, sheep, cows and non-human primates, e.g., baboons, monkeys
and chimpanzees may be used to generate transgenic animals. The
term "transgenic," as used herein, refers to animals expressing
protective sequences from a different species (e.g., mice
expressing human protective sequences), as well as animals which
have been genetically engineered to overexpress endogenous (i.e.,
same species) sequences or animals which have been genetically
engineered to no longer express endogenous protective sequences
(i.e., "knock-out" animals), and their progeny.
[0126] Any technique known in the art may be used to introduce a
protective sequence transgene into animals to produce the founder
lines of transgenic animals. Such techniques include, but are not
limited to, pronuclear microinjection (Hoppe and Wagner, 1989, U.S.
Pat. No. 4,873,191); retrovirus mediated gene transfer into germ
lines (Van der Putten, et al., 1985, Proc. Natl. Acad. Sci., USA
82:6148-6152); gene targeting in embryonic stem cells (Thompson, et
al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983,
Mol. Cell. Biol. 3:1803-1814); and sperm-mediated gene transfer
(Lavitrano et al., 1989, Cell 57:717-723) (For a review of such
techniques, see Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol.
115, 171-229).
[0127] Any technique known in the art may be used to produce
transgenic animal clones containing a protective sequence
transgene, for example, nuclear transfer into enucleated oocytes of
nuclei from cultured embryonic, fetal or adult cells induced to
quiescence (Campbell, et al., 1996, Nature 380:64-66; Wilmut, et
al., Nature 385:810-813).
[0128] The present invention provides for transgenic animals which
carry a protective sequence transgene in all their cells, as well
as animals which carry the transgene in some, but not all their
cells, i.e., mosaic animals. The transgene may be integrated as a
single transgene or in concatamers, e.g., head-to-head tandems or
head-to-tail tandems. The transgene also may be selectively
introduced into and activated in a particular cell type by
following, for example, the teaching of Lasko et al. (Lasko, et
al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236). The regulatory
sequences required for such a cell-type specific activation will
depend on the particular cell type of interest, and will be
apparent to those of skill in the art. When it is desired that the
cerebral transgene be integrated into the chromosomal site of the
endogenous protective sequence, gene targeting is preferred.
Briefly, when such a technique is to be utilized, vectors
containing some nucleic acids homologous to the endogenous
protective sequence are designed for the purpose of integrating,
via homologous recombination with chromosomal sequences, into and
disrupting the function of the nucleic acid of the endogenous
protective sequence. The transgene also maybe selectively
introduced into a particular cell type, thus inactivating the
endogenous protective sequence in only that cell type, by
following, for example, the teaching of Gu, et al. (Gu, et al.,
1994, Science 265, 103-106). The regulatory sequences required for
such a cell-type specific inactivation will depend on the
particular cell type of interest, and will be apparent to those of
skill in the art.
[0129] Once transgenic animals have been generated, the expression
of the recombinant protective sequence may be assayed utilizing
standard techniques. Initial screening may be accomplished by
Southern blot analysis or PCR techniques to analyze animal tissues
to assay whether integration of the transgene has taken place. The
level of mRNA expression of the transgene in the tissues of the
transgenic animals may also be assessed using techniques which
include, but are not limited to, Northern blot analysis of tissue
samples obtained from the animal, in situ hybridization analysis
and RT-PCR (reverse transcriptase PCR). Samples of protective
sequence-expressing tissue also may be evaluated
immunocytochemically using antibodies specific for the transgene
product.
[0130] Protective proteins can be used, e.g., to treat cell
death-related conditions, disorders, or diseases. Such protective
sequence products include, but are not limited to, soluble
derivatives such as peptides or polypeptides corresponding to one
or more domains of the protective sequence product which are
modified such that they are deleted for one or more hydrophobic
domains. Alternatively, antibodies to the protein or anti-idiotypic
antibodies which mimic the protective sequence product (including
Fab fragments), modulators, antagonists or agonists can be used to
treat cell death-related conditions, disorders, or diseases
involving the protective sequence product. In yet another approach,
nucleotide constructs encoding such protective sequence products
can be used to genetically engineer host cells to express such
protective sequence products in vivo; these genetically engineered
cells can function as "bioreactors" in the body delivering a
continuous supply of protective sequence product, peptides and
soluble polypeptides.
[0131] 5.3 Antibodies to the Protective Sequence Products
[0132] Described herein are methods for the production of
antibodies capable of specifically recognizing one or more
protective sequence product epitopes or epitopes of conserved
variants or peptide fragments of the protective sequence products
of the invention. Further, antibodies that specifically recognize
mutant forms of the protective sequence products of the invention
are encompassed by the invention. The terms "specifically bind" and
"specifically recognize" refer to antibodies which bind to
protective sequence product epitopes involved in conditions,
disorders, or diseases involving cell death at a higher affinity
than they bind to protective sequence product epitopes not involved
in such conditions, disorders, or diseases (e.g., random
epitopes).
[0133] Such antibodies may include, but are not limited to,
polyclonal antibodies, monoclonal antibodies (mAbs), humanized or
chimeric antibodies, single chain antibodies, Fab fragments,
F(ab').sub.2 fragments, fragments produced by a Fab expression
library, anti-idiotypic (anti-Id) antibodies and epitope-binding
fragments of any of the above. Such antibodies may be used, for
example, in the detection of a protective sequence product in a
biological sample and may, therefore, be utilized as part of a
diagnostic or prognostic technique whereby patients may be tested
for abnormal levels of protective sequence products, and/or for the
presence of abnormal forms of such protective sequence products.
Such antibodies also may be utilized in conjunction with, for
example, compound screening schemes, as described, below, in
Section 5.4.2, for the evaluation of the effect of test compounds
on protective sequence product levels and/or activity.
Additionally, such antibodies can be used in conjunction with the
gene therapy techniques described below, in Section 5.4.1.3., to
evaluate, for example, the normal and/or engineered cells prior to
their introduction into the patient.
[0134] Antibodies derived from the protective sequence or
protective sequence product, including, but not limited to,
antibodies and anti-idiotypic antibodies that mimic activity or
function additionally may be used in methods for inhibiting
abnormal protective sequence product activity. Thus, such
antibodies may, therefore, be utilized as part of treatment methods
for protective sequence product-mediated conditions, disorders, or
diseases.
[0135] For the production of antibodies against a protective
sequence, various host animals may be immunized with a protective
sequence or protective sequence product, or a portion thereof. Such
host animals may include, but are not limited to, rabbits, mice and
rats, to name but a few. Various adjuvants may be used to increase
the immunological response, depending on the host species,
including, but not limited to, Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially
useful human adjuvants such as BCG (bacille Calmette-Guerin) and
Corynebacterium parvum.
[0136] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen, such as protective sequence product, or an antigenic
functional derivative thereof. For the production of polyclonal
antibodies, host animals such as those described above, may be
immunized with protective sequence product supplemented with
adjuvants as also described above.
[0137] Monoclonal antibodies, which are homogeneous populations of
antibodies to a particular antigen, may be obtained by any
technique that provides for the production of antibody molecules by
continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique of Kohler and Milstein, (1975,
Nature 256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell
hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72;
Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing the
mAb of this invention may be cultivated in vitro or in vivo.
Production of high titers of mAbs in vivo makes this the presently
preferred method of production.
[0138] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison, et al., 1984, Proc. Natl. Acad.
Sci., 81:6851-6855; Neuberger, et al., 1984, Nature 312:604-608;
Takeda, et al., 1985, Nature, 314:452-454) by splicing the genes
from a mouse antibody molecule of appropriate antigen specificity
together with genes from a human antibody molecule of appropriate
biological activity can be used. A chimeric antibody is a molecule
in which different portions are derived from different animal
species, such as those having a variable region derived from a
murine mAb and a human immunoglobulin constant region. (See, e.g.,
Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat.
No. 4,816397, which are incorporated herein by reference in their
entirety.)
[0139] In addition, techniques have been developed for the
production of humanized antibodies. (See, e.g., Queen, U.S. Pat.
No. 5,585,089, which is incorporated herein by reference in its
entirety.) An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, referred to as complementarily determining regions (CDRs).
The extent of the framework region and CDRs have been precisely
defined (see, "Sequences of Proteins of Immunological Interest",
Kabat, E. et al., U.S. Department of Health and Human Services
(1983) ). Briefly, humanized antibodies are antibody molecules from
non-human species having one or more CDRs from the non-human
species and a framework region from a human immunoglobulin
molecule.
[0140] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988,
Science 242:423-426; Huston, et al., 1988, Proc. Natl. Acad. Sci.
USA 85:5879-5883; and Ward, et al., 1989, Nature 334:544-546) can
be adapted to produce single chain antibodies against protective
sequence products. Single chain antibodies are formed by linking
the heavy and light chain fragments of the Fv region via an amino
acid bridge, resulting in a single chain polypeptide.
[0141] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, such fragments include,
but are not limited to: the F(ab').sub.2 fragments, which can be
produced by pepsin digestion of the antibody molecule and the Fab
fragments, which can be generated by reducing the disulfide bridges
of the F(ab').sub.2 fragments. Alternatively, Fab expression
libraries may be constructed (Huse, et al., 1989, Science
246:1275-1281) to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity.
[0142] 5.4 Uses of the Protective Sequences, Protective Sequence
Products and Antibodies
[0143] Described herein are various uses and applications of
protective sequences, protective sequence products, including
peptide fragments and fusion proteins thereof and of antibodies and
anti-idiotypic antibodies derived from the protective sequence
products and peptide fragments thereof. The application relates to
compositions and methods for the treatment of conditions,
disorders, or diseases involving cell death. Such applications
include, but are not limited to, the prophylactic or therapeutic
use of protective sequences which, when introduced into a cell
predisposed to undergo cell death or in the process of dying, to
prevent, delay, or rescue a cell, cells, tissue, organs, or
organisms from dying, as described below in Section 5.4.1.
[0144] Additionally, such applications include methods for the
treatment of conditions, disorders, or diseases involving cell
death, including, but not limited to, those associated with the
central nervous system including neurological and psychiatric
conditions, disorders, or diseases, and others as described below,
in Section 5.4.1.1, and for the identification of compounds which
modulate the expression of the protective sequence and/or the
synthesis or activity of the protective sequence product, as
described below, in Section 5.4.1. Such compounds can include, for
example, other cellular products that are involved in such
processes as the regulation of cell death. These compounds can be
used, for example, in the amelioration of conditions, disorders, or
diseases involving cell death.
[0145] One example of the type of injury that can cause cell death
in neuronal cells is stroke, which often is the result of ischemic
injury. A relatively broad time window (8 hours to perhaps several
days or longer) exists between the onset of ischemic injury (i.e.
cessation or marked reduction in blood flow) before most neural
cells actually die. There are many complex pathways and perhaps
hundreds of different signaling molecules which are likely to be
involved, leaving many different intervention points each with the
potential to prevent, delay, arrest and reverse the cell death
program. These delayed biochemical intervention points represent
ideal clinical intervention points as they correspond to the time
period during which most stroke patients present for medical
treatment.
[0146] Many current medications for the treatment of stroke affect
the physical and biochemical events that are acutely related to the
initial onset of stroke, and, thus, must be administered soon after
the biochemical cascades begin. These approaches all suffer from
the necessity of administering the drugs within a very brief time
window following a stroke. However, many stroke patients do not
even realize that they have suffered from a stroke until a time
point at which many of the current treatments are ineffective. This
is because many stroke patients often do not present at the
emergency room prior to the passing of at least 13 hours from the
onset of the stroke. The methods and compounds of the present
invention, however, can be administered during the broader time
window between stroke and the onset of the pathways leading to cell
death.
[0147] In addition to stroke, a variety of other conditions,
disorders, and diseases lead to the activation of the same
biochemical cascades which lead to neuronal cell death in stroke.
There is growing evidence that numerous other disease states that
induce cell death programs are related to those induced by stroke.
Cell death programs have been increasingly implicated in
Alzheimer's disease, a well-known neurodegenerative condition which
leads to substantial loss of specific neuronal populations in the
neocortex and hippocampus. Vascular dementia (multi-infarct
dementia) is another disorder in which stroke-like cell death
pathways are active. In vascular dementia, a repetitive process of
small blood vessel diseases induces regional brain cell death,
leading to a progressive loss of cognitive abilities. A partial
list of other brain diseases which activate brain cell death
pathways similar to those observed in stroke include, but are not
limited to, Parkinson's disease, traumatic injury, Down's syndrome,
Huntington's disease, HIV infection and intracranial
infections.
[0148] One notable example from the preceding list is physical
trauma to the nervous system. Although such trauma can be caused by
a multitude of different physical insults to the head, neck, spine
and other parts of the nervous system, all result in focal damage
to, and death of, neural tissue and its component cells. Focally
damaged areas behave similarly to stroke-induced infarcts in that a
wider area of neural damage and death, a penumbra, is induced via
biochemical and cellular mechanisms which are similar or identical
to those occurring in stroke.
[0149] While, for clarity, the uses described in this section are
primarily uses related to conditions, disorders, or diseases
involving cell death, it is to be noted that each of the diagnostic
and therapeutic treatments described herein can be additionally
utilized in connection with other defects associated with the
protective sequences of the invention.
[0150] Additionally, described herein are various applications of
protective sequences, protective sequence products, genes, gene
products, and/or their regulatory elements, including, but not
limited to, prognostic and diagnostic evaluation of conditions,
disorders, or diseases as described below in Section 5.4.1.1.
[0151] A variety of methods can be employed for the diagnostic and
prognostic evaluation of conditions, disorders, or diseases
involving cell death and for the identification of subjects having
a predisposition to such conditions, disorders, or diseases.
[0152] Since protective sequences or protective sequence products
need not normally be involved in all conditions, disorders, or
diseases involving cell death, methods of the invention include,
for example, modulating the expression of the protective sequence
and/or the activity of the protective sequence product for the
treatment of conditions, disorders, or diseases involving cell
death which are normally mediated by some other gene.
[0153] For cell death related conditions, disorders, or diseases in
which the protective sequences or protective sequence products are
involved normally, such diagnostic and prognostic methods may, for
example, utilize reagents such as the protective nucleic acids
described in Section 5.1, and antibodies directed against
protective sequence products, including peptide fragments thereof,
as described, above, in Section 5.3.
[0154] Specifically, such reagents may be used, for example,
for:
[0155] (1) the detection of the presence of protective sequence
mutations, or the detection of either over- or under-expression of
the protective sequence relative to wild-type levels of
expression;
[0156] (2) the detection of over- or under-abundance of protective
sequence products relative to wild-type abundance of the protective
sequence product; and
[0157] (3) the detection of an aberrant level of protective
sequence product activity relative to wild-type protective sequence
product activity levels.
[0158] Protective nucleic acids can, for example, be used to
diagnose a condition, disorder, or disease involving cell death
using, for example, the techniques for mutation/polymorphism
detection described above in Section 5.1.
[0159] Mutations at a number of different genetic loci may lead to
phenotypes related to conditions, disorders, or diseases involving
cell death. Ideally, the treatment of patients suffering from such
conditions, disorders, or diseases will be designed to target the
particular genetic loci containing the mutation mediating the
condition, disorder, or disease. Genetic polymorphisms have been
linked to differences in drug effectiveness. Thus, identification
of alterations in protective sequence, protein or gene flanking
regions can be utilized in pharmacogenetic methods to optimize
therapeutic drug treatments.
[0160] In one embodiment of the present invention, therefore,
alterations, i.e., polymorphisms, in the protective sequence or
protein encoded by genes comprising such polymorphisms, are
associated with a drug or drugs' efficacy, tolerance or toxicity,
and may be used in pharmacogenomic methods to optimize therapeutic
drug treatments, including therapeutic drug treatments for one of
the conditions, disorders, or diseases described herein contained
in Section 5.4.1.1, e.g., central nervous system conditions,
disorders, or diseases. Such polymorphisms can be used, for
example, to refine the design of drugs by decreasing the incidence
of adverse events in drug tolerance studies, e.g., by identifying
patient subpopulations of individuals who respond or do not respond
to a particular drug therapy in efficacy studies, wherein the
subpopulations have a polymorphism associated with drug
responsiveness or unresponsiveness. The pharmacogenomic methods of
the present invention also can provide tools to identify new drug
targets for designing drugs and to optimize the use of already
existing drugs, e.g., to increase the response rate to a drug
and/or to identify and exclude non-responders from certain drug
treatments (e.g., individuals having a particular polymorphism
associated with unresponsiveness or inferior responsiveness to the
drug treatment) or to decrease the undesirable side effects of
certain drug treatments and/or to identify and exclude individuals
with marked susceptibility to such side effects (e.g., individuals
having a particular polymorphism associated with an undesirable
side effect to the drug treatment).
[0161] In an embodiment of the present invention, polymorphisms in
the protective sequence or flanking this sequence, or variations in
protective sequence expression, or activity, e.g., variations due
to altered methylation, differential splicing or post-translational
modification of the protective sequence product, may be utilized to
identify an individual having a disease or condition resulting from
a disorder involving cell death and thus define the most effective
and safest drug treatment. Assays such as those described herein
may be used to identify such polymorphisms or variations in
protective sequence expression or activity. Once a polymorphism in
the protective sequence or in a flanking sequence in linkage
disequilibrium with a disorder-causing allelle, or a variation in
protective sequence expression has been identified in an
individual, an appropriate drug treatment can be prescribed to the
individual.
[0162] For the detection of protective sequence mutations or
polymorphisms, any nucleated cell can be used as a starting source
for genomic nucleic acid. For the detection of protective sequence
expression or protective sequence products, any cell type or tissue
in which the protective sequence is expressed may be utilized.
[0163] Nucleic acid-based detection techniques are described,
below, in Section 5.4.1.4. Peptide detection techniques are
described, below, in Section 5.4.1.5.
[0164] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits. The invention therefore
also encompasses kits for detecting the presence of a polypeptide
or nucleic acid of the invention in a biological sample (i.e., a
test sample). Such kits can be used, e.g., to determine if a
subject is suffering from or is at increased risk of developing a
condition, disorder, or disease associated with a disorder-causing
allele, or aberrant expression or activity of a polypeptide of the
invention. For example, the kit can comprise a labeled compound or
agent capable of detecting the polypeptide or mRNA or DNA or
protective sequence sequences, e.g., encoding the polypeptide in a
biological sample. The kit can comprise further a means for
determining the amount of the polypeptide or mRNA in the sample
(e.g., an antibody that binds the polypeptide or an oligonucleotide
probe that binds to DNA or mRNA encoding the polypeptide). Kits can
also include instructions for observing that the tested subject is
suffering from, or is at risk of developing, a condition, disorder,
or disease associated with aberrant expression of the polypeptide
if the amount of the polypeptide or mRNA encoding the polypeptide
is above or below a normal level, or if the DNA correlates with
presence of an allele which causes a condition, disorder, or
disease.
[0165] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide of the invention; and, optionally, (2) a
second, different antibody which binds to either the polypeptide or
to the first antibody and is conjugated to a detectable agent.
[0166] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide (e.g., a detectably labeled
oligonucleotide) which hybridizes to a nucleic acid sequence
encoding a polypeptide of the invention, or (2) a pair of primers
useful for amplifying a nucleic acid molecule encoding a
polypeptide of the invention.
[0167] The kit also can comprise, for example, one or more
buffering agents, preservatives or protein stabilizing agents. The
kit also can comprise components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The kit can
contain also a control sample or a series of control samples that
can be assayed and compared to the test sample. Each component of
the kit usually is enclosed within an individual container and all
of the various containers are within a single package along with
instructions for observing whether the tested subject is suffering
from or is at risk of developing a condition, disorder, or disease
associated with polymorphisms which correlate with alleles which
cause conditions, disorders, or diseases involving cell death,
and/or aberrant levels of mRNA, polypeptides or activity.
[0168] Additionally, the application relates to the compositions
and methods for the development of screening assays for the
identification of compounds, described in Section 5.4.2 below,
which interact with or modulate protective sequences, protective
sequence products, genes, gene products, and/or their regulatory
elements.
[0169] 5.4.1 Composition and Methods for the Treatment of
Conditions, Disorders, or Diseases Involving Cell Death
[0170] This application relates to compositions and methods for the
treatment of conditions, disorders, or diseases involving cell
death. Such applications include, but are not limited to, the
prophylactic or therapeutic use of protective sequences, protective
sequence products, genes, gene products, or the regulatory
elements, target sequences, or variants of any of the
aforementioned sequences or products, which, when introduced into a
cell predisposed to undergo cell death or in the process of dying,
prevent, delay, or rescue a cell, cells, tissue, organs, or
organisms from dying. The application further relates to the
methods and compositions whereby a condition, disorder, or disease
involving cell death, including but not limited to, the conditions,
disorders, or diseases mentioned in Section 5.4.1.1, may be treated
wherein such methods can comprise administering antibodies,
antisense molecules or sequences, ribozyme molecules, or other
inhibitors or modulators directed against such protective
sequences, protective sequence products, genes, gene products, or
the regulatory elements, target sequences, or variants of any of
the aforementioned sequences or products.
[0171] The application relates to compositions and methods for
those instances whereby the condition, disorder, or disease
involving cell death results from protective sequence mutations,
such methods can comprise supplying the subject with a nucleic acid
molecule encoding an unimpaired protective sequence product such
that an unimpaired protective sequence product is expressed and the
cell, cells, tissue, organ, organism displaying symptoms of the
condition, disorder, or disease is prevented, delayed, or rescued
from death.
[0172] In another embodiment of methods for the treatment of
conditions, disorders, or diseases involving cell death resulting
from protective sequence mutations, such methods can comprise
supplying the subject with a cell comprising a nucleic acid
molecule which encodes an unimpaired protective sequence product
such that the cell expresses the unimpaired protective sequence
product and the cell, cells, tissue, organ, or organism displaying
symptoms of the condition, disorder, or disease is prevented,
delayed, or rescued from death.
[0173] In cases in which a loss of normal protective sequence
product function results in the development of a condition,
disorder, or disease involving cell death, an increase in
protective sequence product activity would facilitate progress
towards an asymptomatic state in individuals exhibiting a deficient
level of protective sequence expression and/or gene product
activity. Methods for enhancing the expression or synthesis of
protective sequence product can include, for example, methods such
as those described below, in Section 5.4.1.3.
[0174] Alternatively, symptoms of a condition, disorder, or disease
involving cell death may be prevented, delayed, or rescued by
administering a compound which decreases the level of protective
sequence expression and/or gene product activity. Methods for
inhibiting or reducing the level of protective sequence product
synthesis or expression can include, for example, methods such as
those described in Section 5.4.1.2.
[0175] In cases where the development of a condition, disorder, or
disease involving cell death is due to a sequence or gene other
than a protective sequence, modulating, including but not limited
to, mimicking, agonizing, or antagonizing the expression of a
protective sequence and/or the activity of a protective sequence
product, or their regulatory elements, can be used for the
treatment of the condition, disorder, or disease involving cell
death. This is because protective sequences are nucleic acid
molecules comprising nucleic acid sequences which, when introduced
into a cell predisposed to undergo cell death, prevent, delay, or
rescue such cell death relative to a corresponding cell into which
no exogenous protective sequence has been introduced.
[0176] The proteins and peptides which may be used in the methods
of the invention include synthetic (e.g., recombinant or chemically
synthesized) proteins and peptides, as well as naturally occurring
proteins and peptides. The proteins and peptides may have both
naturally occurring and non-naturally occurring amino acid residues
(e.g., D-amino acid residues) and/or one or more non-peptide bonds
(e.g., imino, ester, hydrazide, semicarbazide, and azo bonds). The
proteins or peptides may also contain additional chemical groups
(i.e., functional groups) present at the amino and/or carboxy
termini, such that, for example, the stability, bioavailability,
and/or inhibitory activity of the peptide is enhanced. Exemplary
functional groups include hydrophobic groups (e.g. carbobenzoxyl,
dansyl, and t-butyloxycarbonyl, groups), an acetyl group, a
9-fluorenylmethoxy-carbonyl group and macromolecular carrier groups
(e.g., lipid-fatty acid conjugates, polyethylene glycol, or
carbohydrates) including peptide groups. Additional proteins and
peptides which may be used in the methods of the invention include
those described in WO 99/59615, which is herein incorporated by
reference in its entirety.
[0177] 5.4.1.1 Examples of Conditions, Disorders, or Diseases
Involving Cell Death
[0178] The types of conditions, disorders, or diseases which can be
prevented, delayed, or rescued by the compounds and methods of the
present invention include, but are not limited to, those associated
with the central nervous system including neurological and
psychiatric conditions, disorders, or diseases; those of the
peripheral nervous system; conditions, disorders, or diseases
caused by physical injury; conditions, disorders, or diseases of
the blood vessels or heart; conditions, disorders, or diseases of
the respiratory system; neoplastic conditions, disorders, or
diseases; conditions, disorders, or diseases of blood cells;
conditions, disorders, or diseases of the gastrointestinal tract;
conditions, disorders, or diseases of the liver; conditions,
disorders, or diseases of the pancreas; conditions, disorders, or
diseases of the kidney; conditions, disorders, or diseases of the
ureters, urethra or bladder; conditions, disorders, or diseases of
the male genital system; conditions, disorders, or diseases of the
female genital tract; conditions, disorders, or diseases of the
breast; conditions, disorders, or diseases of the endocrine system;
conditions, disorders, or diseases of the thymus or pineal gland;
conditions, disorders, or diseases of the skin or mucosa;
conditions, disorders, or diseases of the musculoskeletal system;
conditions, disorders, or diseases causing a fluid or hemodynamic
derangement; inherited conditions, disorders, or diseases;
conditions, disorders, or diseases of the immune system or spleen;
conditions, disorders, or diseases caused by a nutritional disease;
and conditions, disorders, or diseases typically occurring in
infancy or childhood.
[0179] Conditions, disorders, or diseases involving the central
nervous system include, but are not limited to, common
pathophysiologic complications such as increased intracraneal
pressure and cerebral herniation, septic embolism, cerebral edema,
suppurative endovasculitis and hydrocephalus; infections such as
meningitis, acute meningitis, acute lymphocytic meningitis, chronic
meningitis, purulent meningitis, syphilitic gumma, encephalitis,
cerebral abscess, epidural abscess, subdural abscess, brain
abscess, viral encephalitis, acute viral encephalitis,
encephalomeningitis, aseptic meningitis, post-infectious
encephalitis, subacute encephalitis, chronic encephalitis, chronic
meningitis, chronic encephalomeningitis, slow virus diseases and
unconventional agent encephalopathies; protozoal infections such as
malaria, toxoplasmosis, amebiasis and trypanosomiasis; rickettsial
infections such as typhus and Rocky Mountain spotted fever;
metazoal infections such as echinococcosis and cysticercosis;
vascular diseases such as ischemic encephalopathy, cerebral
infarction, intracranial hemorrhage, intraparenchymal hemorrhage,
subarachnoid hemorrhage, mixed intraparenchymal and subarachnoid
hemorrhage; conditions involving the eye such as macular
degeneration, glaucoma, retinopathy of prematurity, retinitis
pigmentosa, diabetic retinopathy, or other traumatic injuries to
the retina or optic nerve; trauma such as epidural hematoma,
subdural hematoma, parenchymal injuries; tumors such as primary
intrachranial tumors, astrocytoma, oligodendroglioma, ependymoma,
medulloblastoma and meningioma; degenerative diseases such as
Altzheimer's disease, Huntington's disease, Parkinsonism,
idiopathic Parkinson's disease and motor neuron disease;
demyelinating diseases such as multiple sclerosis; nutritional,
environmental and metabolic conditions, disorders, or diseases.
[0180] Conditions, disorders, or diseases of the peripheral nervous
system include, but are not limited to, peripheral neuropathy,
acute idiopathic polyneuropathy, diabetic neuropathy and peripheral
nerve tumors.
[0181] Conditions, disorders, or diseases caused by physical injury
include, but are not limited to, the direct, indirect, immediate,
or delayed effects of: changes in temperature such as frostbite and
thermal burns; an increase in atmospheric pressure such as air
blast or immersion blast caused by an explosion; a decrease in
atmospheric pressure such as caisson disease or high-altitude
hypoxia; mechanical violence from penetrating or non-penetrating
traumatic injury; electromechanical energy such as radiation injury
from either charged particles or electromagnetic waves;
electrocution or non-ionizing radiation such as radio waves,
microwaves, laser light or ultrasound.
[0182] Conditions, disorders, or diseases of the blood vessels or
heart include, but are not limited to, hypertension (high blood
pressure), heart failure; ischemic or atherosclerotic heart
disease; myocardial infarction; cardiac arrest; hypertensive heart
disease; cor pulmonale; valvular heart disease such as that caused
by rheumatic fever, aortic valve stenosis, mitral annulus
calcification, carcinoid heart disease, nonbacterial thrombotic
endocarditis, or nonbacterial verrucous endocarditis; infectious
endocarditis caused by organisms including, but not limited to,
Streptococcus species, Staphylococcus species, enterococci,
pneumococci, gram-negative rods, Candida species, Aspergillus
species, or culture-negative endocarditis; congenital heart disease
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosis, coarctation of the aorta, Tetralogy of Fallot,
tricuspid atresia, pulmonary stenosis or atresia, aortic stenosis
or atresia, bicuspid aortic valve, or hypoplastic left heart
syndrome; cardiomyopathy; pericarditis; pericardial effusion;
rheumatoid heart disease; congenital anomalies of the blood
vessels; arteriosclerosis including, but not limited to
atherosclerosis, Monckeberg's medial calcific stenosis, hyaline
arteriosclerosis, or hyperplastic arteriosclerosis; one or more of
the vasculidities including, but not limited to, polyarteritis
nodosa, hypersensitivity angiitis, Wegener's granulomatosis, giant
cell (temporal) arteritis, Takayasu's arteritis, Kawasaki's
disease, thromboangiitis obliterans, infectious vasculitis,
Raynaud's disease; arteriosclerotic aortic aneurysm; syphilitic
aortic aneurysm; dissecting aortic aneurysm; varicose veins;
thrombophlebitis; lymphangitis; lymphedema; telangiectases; or
arteriovenous malformations (AVM).
[0183] Conditions, disorders, or diseases of the respiratory system
include, but are not limited to, pulmonary congestion; heart
failure; embolism; infarction; pulmonary hypertension; adult
respiratory distress syndrome (ARDS); obstructive lung disease;
restrictive lung disease; chronic obstructive pulmonary disease;
asthma; sarcoidosis; diffuse interstitial or infiltrative lung
diseases including, but not limited to, idiopathic pulmonary
fibrosis, pneumoconiosis, hypersensitivity pneumonitis,
Goodpasture's syndrome, idiopathic pulmonary hemosiderosis,
collagen-vascular diseases, or pulmonary eosinophilia;
serofibrinous pleuritis; suppurative pleuritis; hemorrhagic
pleuritis; pleural effusions; pneumothorax; hemothorax or
pneumohemothorax.
[0184] Neoplastic conditions, disorders, or diseases include, but
are not limited to, benign tumors composed of one parenchymal cell
type such as fibromas, myxomas, lipomas, hemangiomas, meningiomas,
leiomyomas, adenomas, nevi, moles, or papillomas; benign mixed
tumors derived from one germ layer such as a mixed tumor of
salivary gland origin; benign mixed tumors derived from more than
one germ layer such as a teratoma; primary malignant tumors or
metastases of malignant tumors composed of one parenchymal cell
type such as sarcomas, Ewing's tumor, leukemia, myeloma,
histiocytosis X, Hodgkin's disease, lymphomas, carcinomas,
melanomas, bronchial adenoma, small cell lung cancer, or seminoma;
primary malignant tumors or metastases of mixed malignant tumors
derived from one germ layer such as Wilms' tumor or malignant mixed
salivary gland tumor; primary malignant tumor or metastases of
mixed malignant tumors derived from one germ layer such as
malignant teratoma or teratocarcinoma; undifferentiated benign
tumor or undifferentiated malignant tumor.
[0185] Conditions, disorders, or diseases of blood cells include,
but are not limited to, anemia due to one or more of the following
conditions: acute blood loss, chronic blood loss, hemolytic anemia,
sickle cell disease, thalassemia syndromes, autoimmune hemolytic
anemia, traumatic anemia, or diminished erythropoesis from
megaloblastic anemia, iron deficiency, aplastic anemia, idiopathic
bone marrow failure; polycythemia; hemorrhagic diatheses related to
increased vascular fragility; hemorrhagic diatheses related to a
reduction in platelets; idiopathic or thrombotic thrombocytopenic
purpura; hemorrhagic diatheses related to defective platelet
function; hemorrhagic diatheses related to abnormalities in
clotting factor(s); disseminated intravascular coagulation (DIC);
neutropenia; agranulocytosis; leukocytosis; plasma cell dyscrasias
such as myeloma, Waldenstrom's macroglobulinemia, or heavy-chain
disease; or histiocytosis.
[0186] Conditions, disorders, or diseases of the gastrointestinal
tract include, but are not limited to, congenital anomalies such as
atresia, fistulas, or stenosis; periodontal disease; periapical
disease; xerostomia; necrotizing sialometaplasia; esophageal rings
or webs; hernia; Mallory-Weiss syndrome; esophagitis;
diverticulosis; diverticulitis; scleroderma; esophageal varices;
acute or chronic gastritis; peptic ulcer; gastric erosion or
ulceration; ischemic bowel disease; infarction; embolism; Crohn's
disease; obstruction from foreign bodies, hernia, adhesion,
intussusception, or volvulus; ileus; megacolon; angoidysplasia;
ulcerative colitis; psuedomembranous colitis; or polyps.
[0187] Conditions, disorders, or diseases of the liver include, but
are not limited to, acute hepatic failure due to one of more of
metabolic, circulatory, toxic, microbial, or neoplastic causes;
chronic hepatic failure due to one or more of metabolic,
circulatory, toxic, microbial, or neoplastic causes; hereditary
hyperbilirubinemias; infarct; embolism; hepatic circulation
thrombosis or obstruction; fulminant hepatic necrosis; portal
hypertension; alcoholic liver disease; post-necrotic cirrhosis;
biliary cirrhosis; cirrhosis associated with alpha-1-antitrypsin
deficiency; Wilson's disease; or Reye's syndrome.
[0188] Conditions, disorders, or diseases of the pancreas include,
but are not limited to, congenital aberrant pancreas, congenital
anomalies of pancreatic ducts, stromal fatty infiltration,
pancreatic atrophy, acute hemorrhagic pancreatitis, chronic
pancreatitis, chronic calcifying pancreatitis, chronic obstructive
pancreatitis, pancreatic psuedocyst, diabetes mellitus, or
gestational diabetes.
[0189] Conditions, disorders, or diseases of the kidney include,
but are not limited to, congenital anomalies; polycystic renal
disease; dialysis-associated cystic disease; glomerular disease,
including, but not limited to, acute glomerulonephritis, acute
proliferative glomerulonephritis, rapidly progressive
glomerulonephritis, postinfectious rapidly progressive
glomerulonephritis, Goodpasture's syndrome, idiopathic rapidly
progressive glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis, lipoid nephrosis, focal segmental
glomerulosclerosis, membranoproliferative glomerulonephritis, focal
proliferative glomerulonephritis, chronic glomerulonephritis, or
hereditary nephritis; acute tubular necrosis; acute renal failure;
tubulointerstitial diseases including, but not limited to,
pyelonephritis, drug-induced interstitial nephritis, analgesic
nephritis, urate nephropathy, hypercalcemia and nephrocalcinosis,
hypokalemic nephropathy, myeloma-induced tubulointerstitial
disease, radiation nephritis, immunologically medicated
tubulointerstitial disease; hypertension; malignant hypertension;
renal artery stenosis; renal diseases secondary to microangiopathic
hemolytic anemia; atheroembolic renal disease; sickle cell disease
nephropathy; diffuse cortical necrosis; renal infarcts; obstructive
uropathy; or urolithiasis.
[0190] Conditions, disorders, or diseases of the ureters, urethra
or bladder include, but are not limited to, congenital anomalies;
inflammatory diseases; physical obstruction by causes including,
but not limited to calculi, strictures, neoplasia, blood clot, or
pregnancy; sclerosing retroperitonitis; acute cystitis; chronic
cystitis; interstitial cystitis; emphysematous cystitis;
eosinophilic cystitis; encrusted cystitis; fistula; or neurogenic
bladder.
[0191] Conditions, disorders, or diseases of the male genital
system include, but are not limited to, congenital anomalies;
balanoposthitis; condyloma; phimosis; paraphimosis; dysplastic
epithelial lesions; nonspecific epididymitis or orchitis;
granulomatous orchitis; torsion of the testis or its vascular
supply; granulomatous prostatitis; acute or chronic prostatitis; or
benign prostatic hyperplasia.
[0192] Conditions, disorders, or diseases of the female genital
tract include, but are not limited to, congenital anomalies, lichen
scleroses, acute cervicitis, chronic cervicitis, cervical polyps;
acute endometritis; chronic endometritis; endometriosis;
dysfunctional uterine bleeding; endometrial hyperplasia; senile
cystic endometrial atrophy; salpingitis; polycystic ovary disease;
pre-eclampsia or eclampsia (toxemia of pregnancy); placentitis;
threatened abortion; or ectopic pregnancy.
[0193] Conditions, disorders, or diseases of the breast include,
but are not limited to, congenital anomalies, acute mastitis,
chronic mastitis, galactocele, granulomas, traumatic fat necrosis,
mammary duct ectasia, fibrocystic disease, sclerosing adenitis,
epithelial hyperplasia, hypertrophy, or gynecomastia.
[0194] Conditions, disorders, or diseases of the endocrine system
include, but are not limited to, congenital anomalies; Sheehan's
pituitary necrosis; empty sella syndrome; hyperthyroidism
(thyrotoxicosis) from causes including, but not limited to, Graves'
disease, toxic multinodular goiter, toxic adenoma, acute or
subacute thyroiditis, TSH-secreting tumor, neonatal thyrotoxicosis,
iatrogenic thyrotoxicosis; Hashimoto's thyroiditis; hypothyroidism
(cretinism or myxedema) from causes including, but not limited to,
surgical or radioactive ablation, primary idiopathic myxedema,
iodine deficiency, goitrogenic agents, hypopituitarism,
hypothalamic lesions, TSH resistance, subacute thyroiditis, or
chronic thyroiditis; diffuse nontoxic simple or multinodular
goiter; multiple endocrine neoplasia syndromes; primary or
secondary hyperparathyroidism; chief cell hyperplasia; clear cell
hyperplasia; hypoparathyroidism; pseudo- and
pseudopseudohypoparathyrodis- m; Addison's disease;
Waterhouse-Friderichsen syndrome; secondary adrenocortical
insufficiency; Cushing's syndrome; Conn's syndrome; or congenital
adrenal hyperplasia.
[0195] Conditions, disorders, or diseases of the skin or mucosa
include, but are not limited to, melanocytic proliferative
disorders; inflammatory dermatoses including, but not limited to,
eczematous dermatitis, urticaria, erythema multiforme, cutaneous
necrotizing vasculitis, cutaneous lupus erythematosus,
graft-versus-host disease, panniculitis, acne vulgaris, rosacea,
lichen planus, lichen sclerosus et atrophicus, pityriasis,
psoriasis, or parapsoriasis; blistering diseases including, but not
limited to, pemphigus, bullous pemphigoid, dermatitis
herpetiformis, or porphyria.
[0196] Conditions, disorders, or diseases of the musculoskeletal
system include, but are not limited to, muscular atrophy; segmental
necrosis; myositis; muscular dystrophy, including, but not limited
to, Duchenne type, Becker type, Fascioscapulohumeral, Limb-Girdle,
myotonic dystrophy, or ocular myopathy; congenital myopathies;
myasthenia gravis; traumatic myositis ossificans; nodular
fasciitis; desmoid tumors; palmar fibromatosis; congenital bone
disorders including, but not limited to, osteogenesis imperfecta,
achondroplasia, osteopetrosis, osteochondromatosis,
endochondromatosis; osteomyelitis; fractures; osteoporosis;
osteomalacia; bony changes secondary to hyperparathyroidism;
Paget's disease; hypertrophic osteoarthropathy; fibrous dysplasia;
or nonossifying fibroma.
[0197] Conditions, disorders, or diseases causing a fluid or
hemodynamic derangement include, but are not limited to, systemic
edema; anasarca; edema from increased hydrostatic pressure
including, but not limited to congestive heart failure, cirrhosis
of the liver, constrictive pericarditis, venous obstruction; edema
from reduced oncotic pressure including, but not limited to,
cirrhosis of the liver, malnutrition, protein-losing renal disease,
protein-losing gastroenteropathy, protein loss through increased
vascular permeability; edema from lymphatic obstruction including,
but not limited to, cancer, inflammatory injury, surgical injury,
traumatic injury, or radiation injury; edema from increased osmotic
tension in the interstitial fluid including, but not limited to,
sodium retention from excessive salt intake or increased renal
sodium retention, reduced renal perfusion, acute or chronic renal
failure, acute or chronic renal insufficiency; edema from increased
endothelial permeability including, but not limited to,
inflammation, shock, burns, trauma, allergic reaction, immunologic
reaction, or adult respiratory distress syndrome; ascites;
pericardial effusion; hydrothorax; hyperemia; hemorrhage; mural
thrombus or occlusive thrombus diminishing or obstructing vascular
flow; phlebothrombosis; blood clot; embolism; thromboembolism;
disseminated intravascular coagulation (DIC); amniotic fluid
infusion; amniotic fluid embolism; systemic embolism disease;
septic embolism; fat embolism; pulmonary embolism; air gas embolism
(caisson disease or decompression sickness); anemic (white)
infarction; hemorrhagic (red) infarction; cerebral infarction;
septic infarction; ischemia; cardiogenic shock from conditions
including, but not limited to, myocardial infarction, cardiac
arrest, cardiac rupture, cardiac tamponade, pulmonary embolism,
cardiac valvular obstruction, or cardiac arrhythmias; hypovolemic
shock from conditions including, but not limited to, hemorrhage,
vomiting, diarrhea, diaphoresis, extensive injury to bone or soft
tissues, burns, or accumulation of intraperitoneal fluid; shock due
to peripheral blood pooling from conditions including, but not
limited to, spinal cord injury, general anesthesia, regional
anesthesia, local anesthesia, drug-induced ganglionic or adrenergic
blockade, gram-negative septicemia, or gram-positive septicemia;
anaphylaxis, or disseminated intravascular coagulation (DIC).
[0198] Inherited conditions, disorders, or diseases include, but
are not limited to, Down's syndrome, Edwards' syndrome, Patau's
syndrome, other trisomies, Cri du Chat syndrome, Klinefelter's
syndrome, XYY syndrome, Turner's syndrome, Multi-X female syndrome,
hermaphrodism or pseudohermaphrodism, Marfan's syndrome,
neurofibromatosis, vonHippel-Lindau disease, familial
hypercholesterolemia, albinism, alkaptonuria, Fabry's disease,
Fragile-X syndrome, Ehlers-Danlos syndromes, inherited neoplastic
syndromes, inherited autosomal dominant conditions, Huntington's
disease, Alport's disease, sickle-cell disease, thalessemia,
tuberous sclerosis, vonWillebrand's disease, polycystic kidney
disease, Pompe's disease, GM1-gangliosidosis; Tay-Sachs disease,
Sandhoff-Jatzkewitz disease, metachromatic leukodystrophy, multiple
sufatase deficiency, Krabbe's disease, Gaucher's disease,
Niemann-Pick disease, all types of mucopolysaccharidoses, I-cell
disease, Hurler's polydystrophy, fucosidosis, mannosidosis,
aspartylglycosaminuria, Wolman's disease, or acid phosphatase
deficiency, inherited autosomal recessive conditions, inherited
sex-linked conditions.
[0199] Conditions, disorders, or diseases of the immune system or
spleen include, but are not limited to, Type I hypersensitivity
conditions (anaphylaxis and other basophil or mast cell mediated
conditions), Type II hypersensitivity conditions (cytotoxic
conditions involving phagocytosis or lysis of target cell), Type
III hypersensitivity conditions (immune complex conditions
involving antigen-antibody complexes), Type IV hypersensitivity
conditions (cell-mediated conditions), transplant rejection,
systemic lupus erythematosus, Sjogren's syndrome, CREST,
scleroderma, polymyositis-dermatomyositis, mixed connective tissue
disease, polyarteritis nodosa, amyloidosis, X-linked
agammaglobulinemia, common variable immunodeficiency, isolated IgA
deficiency, DiGeorge's syndrome, severe combined immunodeficiency,
Wiscott-Aldrich syndrome, infection with HIV virus, acquired immune
deficiency syndrome (AIDS), congenital anomalies of the immune
system, hypersplenism, splenomegaly, congenital anomalies of the
spleen, congestive splenomegaly, infarcts, or splenic rupture.
[0200] Conditions, disorders, or diseases caused by a nutritional
disease include, but are not limited to, marasmus, kwashiorkor,
fat-soluble vitamin deficiency or toxicity (Vitamins A, D, E, or
K), water-soluble vitamin deficiency or toxicity (thiamine,
riboflavin, niacin, pyridoxine, folate, cobalamin, Vitamin C),
mineral deficiency or toxicity (iron, calcium, magnesium, sodium,
potassium, chloride, zinc, copper, iodine, cobalt, chromium,
selenium, nickel, vanadium, manganese, molybdenum, rickets,
osteomalacia, beriberi, hypoprothrombinemia, pellagra,
megaloblastic anemia, scurvy, pernicious anemia, lack of gastric
intrinsic factor, removal or pathophysiological functioning in the
terminal ileum, microcytic anemia, or obesity.
[0201] Conditions, disorders, or diseases typically occurring in
infancy or childhood include, but are not limited to, preterm
birth, congenital malformations from genetic causes, congenital
malformations from infectious causes, congenital malformations from
toxic or teratogenic causes, congenital malformations from
radiation, congenital malformations from idiopathic causes, small
for gestational age infants, perinatal trauma, perinatal asphyxia,
perinatal ischemia or hypoxia, birth injury, intracranial
hemorrhage, deformations, respiratory distress syndrome of the
newborn, atelectasis, hemolytic disease of the newborn,
kernicterus, hydrops fetalis, congenital anemia of the newborn,
icterus gravis, phenylketonuria, galactosemia, cystic fibrosis,
hamartoma, or choristoma.
[0202] In another embodiment, the compounds and methods of the
invention can be used to treat infections that cause cell death.
The infections may be caused by bacteria; viruses; members of the
family rickettsiae or chlamydia; fungi, yeast, hyphae or
pseudohyphae; prions; protozoas; or metazoas.
[0203] Examples of aerobic or anaerobic bacteria which may cause
such infections include, but are not limited to, gram-positive
cocci, gram-positive bacilli (gram-positive rods), gram-negative
cocci, gram-negative bacilli (gram-negative rods), Mycoplasma
species, Ureaplasma species, Treponema species, Leptospira species,
Borrelia species, Vibrio species, Mycobacteria species, members of
Actinomycetes or L-forms (cell-wall deficient forms).
[0204] Examples of DNA, RNA or both DNA and RNA viruses which may
cause such infections include, but are not limited to, members of
the families adenoviridae, parvoviridae, papovaviridae,
herpesviridae, poxviridae, picornaviridae, orthomyxoviridae,
paramyxoviridae, rhabdoviridae, bunyaviridae, arenaviridae,
coronaviridae, retroviridae, reoviridae, togaviridae and
caliciviridae.
[0205] Examples of members of the families rickettsiae or
chlamydiae which may cause such infections include, but are not
limited to, Rickettsia species, Rochalimaea species, Coxiella
species or Chlamydia species.
[0206] Examples of fungi, yeast, hyphae or pseudohyphae which may
cause such infections include, but are not limited to, members of
Ascomycota, Basidiomycota, Zygomycota, or Deutoeromycota (Fungi
Imperfecti); Candida species, Cryptococcus species, Torulopsis
species, Rhodotorula species, Sporothrix species, Phialophora
species, Cladosporium species, Xylohypha species, Blastomyces
species, Histoplasma species, Coccidioides species,
Paracoccidioides species, Geotrichum species, Aspergillus species,
Rhizopus species, Mucor species, Pseudoallescheria species or
Absidia species.
[0207] Examples of prions which may cause such infections include,
but are not limited to, the causative agent of Creutzfeldt-Jakob
Disease, the causative agent of Gerstmann-Straussler-Scheinker
Disease, the causative agent of fatal familial insomnia, the
causative agent of kuru, and the causative agent of bovine
spongiform encephalopathy.
[0208] Examples of protozoa at any point in their life cycle which
may cause such infections include, but are not limited to,
Entamoeba species, Naegleria species, Acanthamoeba species,
Pneumocystis species, Balantidium species, members of order
Leptomyxida, Plasmodium species, Toxoplasma species, Leishmania
species and Trypanosoma species.
[0209] Examples of metazoa at any point in their life cycle which
may cause such infections include, but are not limited to, members
of Platyhelminthes such as the organisms in Cestoda (tapeworms) or
Trematoda (flukes); or members of Aschelminthes such as the
organisms in Acanthocephala, Chaetognatha, Cycliophora,
Gastrotricha, Nematoda or Rotifera.
[0210] In a further embodiment, the compounds and methods of the
invention can be used to treat infections or disorders which cause
cell death in organ systems including, but not limited to, blood
vessels, heart, red blood cells, white blood cells, lymph nodes,
spleen, respiratory system, oral cavity, gastrointestinal tract,
liver and biliary tract, pancreas, kidney, lower urinary tract,
upper urinary tract and bladder, male sexual organs and genitalia,
female sexual organs and genitalia, breast, thyroid gland, adrenal
gland, parathyroid gland, skin, musculoskeletal system, bone marrow
or bones.
[0211] In a further embodiment, the compounds and methods of the
invention can be used to treat further physiological impacts on
organs caused by the infections which induce cell death including,
but not limited to, fever equal to or greater than 101.5 degrees
Fahrenheit, a decrease or increase in pulse rate by more than 20
beats per minute, a decrease or increase in supine systolic blood
pressure by more than 30 millimeters of mercury, an increase or
decrease in respiratory rate by more than 8 breaths per minute, an
increase or decrease in blood pH by more than 0.10 pH units, an
increase or decrease in one or more serum electrolytes outside of
the clinical laboratory's usual reference range, an increase or
decrease in the partial pressure of arterial oxygen or carbon
dioxide outside of the clinical laboratory's usual reference range,
an increase or decrease in white or red blood cells outside of the
laboratory's usual reference range, an acute confusional state such
as delirium where delirium is defined by the American Psychiatric
Association's DSM-IV Manual or a diminished level of consciousness
or attention.
[0212] 5.4.1.2 Modulatory Antisense, Ribozyme and Triple Helix
Approaches
[0213] In another embodiment, the types of conditions, disorders,
or diseases involving cell death which may be prevented, delayed,
or rescued by modulating protective sequence expression, protective
sequence product activity, or their regulatory elements by using
protective sequences in conjunction with well-known antisense, gene
"knock-out," ribozyme and/or triple helix methods, are described.
Among the compounds which may exhibit the ability to modulate the
activity, expression or synthesis of the protective sequence, the
protective sequence product, or its regulatory elements, including
the ability to prevent, delay, or rescue a cell, cells, tissue,
organ, or organism from the symptoms of a condition, disorder, or
disease involving cell death are antisense, ribozyme and triple
helix molecules. Such molecules may be designed to modulate, reduce
or inhibit either unimpaired, or if appropriate, mutant protective
sequence activity. Techniques for the production and use of such
molecules are well known to those of skill in the art.
[0214] Antisense RNA and DNA molecules act to directly block the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense approaches involve the design of
oligonucleotides which are complementary to a protective sequence
mRNA. The antisense oligonucleotides will bind to the complementary
protective sequence mRNA transcripts and prevent translation.
Absolute complementarity, although preferred, is not required.
[0215] A sequence "complementary" to a portion of an RNA, as
referred to herein, means a sequence having sufficient
complementarity to be able to hybridize with the RNA, forming a
stable duplex; in the case of double-stranded antisense nucleic
acids, a single strand of the duplex DNA may thus be tested, or
triplex formation may be assayed. The ability to hybridize will
depend on both the degree of complementarity and the length of the
antisense nucleic acid. Generally, the longer the hybridizing
nucleic acid, the more base mismatches with an RNA it may contain
and still form a stable duplex (or triplex, as the case may be).
One skilled in the art can ascertain a tolerable degree of mismatch
by use of standard procedures to determine the melting point of the
hybridized complex.
[0216] In one embodiment, oligonucleotides complementary to
non-coding regions of the protective sequence of interest could be
used in an antisense approach to inhibit translation of endogenous
mRNA. Antisense nucleic acids should be at least six nucleotides in
length, and are preferably oligonucleotides ranging from 6 to about
50 nucleotides in length. In specific aspects, the oligonucleotide
is at least 10 nucleotides, at least 17 nucleotides, at least 25
nucleotides or at least 50 nucleotides.
[0217] Regardless of the choice of target sequence, it is preferred
that in vitro studies are first performed to quantitate the ability
of the antisense oligonucleotide to inhibit protective sequence
expression. It is preferred that these studies utilize controls
that distinguish between antisense gene inhibition and nonspecific
biological effects of oligonucleotides. It is also preferred that
these studies compare levels of the cerebral RNA or protein with
that of an internal control RNA or protein. Additionally, it is
envisioned that results obtained using the antisense
oligonucleotide are compared with those obtained using a control
oligonucleotide. It is preferred that the control oligonucleotide
is of approximately the same length as the test oligonucleotide and
that the nucleic acid of the oligonucleotide differs from the
antisense sequence no more than is necessary to prevent specific
hybridization to the target sequence.
[0218] The oligonucleotides can be DNA or RNA or chimeric mixtures
or derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, hybridization, etc. The
oligonucleotide may include other appended groups such as peptides
(e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger, et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
86:6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. U.S.A.
84:648-652; PCT Publication No. WO88/09810, published Dec. 15,
1988) or the blood-brain barrier (see, e.g., PCT Publication No.
WO89/10134, published Apr. 25, 1988), hybridization-triggered
cleavage agents (see, e.g., Krol et al., 1988, BioTechniques
6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm.
Res. 5:539-549). To this end, the oligonucleotide may be conjugated
to another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0219] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including but
not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0220] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including but not
limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0221] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group consisting of a phosphorothioate, a pbosphorodithioate, a
phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a
methylphosphonate, an alkyl phosphotriester, and a formacetal or
analog thereof.
[0222] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier, et al., 1987, Nucl.
Acids Res. 15:6625-6641). The oligonucleotide is a
2'-0-methylribonucleotide (Inoue, et al., 1987, Nucl. Acids Res.
15:6131-6148), or a chimeric RNA-DNA analogue (Inoue, et al, 1987,
FEBS Lett. 215:327-330).
[0223] Oligonucleotides of the invention may be synthesized by
standard methods known in the art, e.g., by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein, et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin, et al., 1988, Proc. Natl. Acad. Sci.
U.S.A. 85:7448-745 1), etc.
[0224] While antisense nucleotides complementary to the protective
sequence coding region sequence could be used, those complementary
to the transcribed, untranslated region are most preferred.
[0225] Antisense molecules should be delivered to cells that
express the protective sequence in vivo. A number of methods have
been developed for delivering antisense DNA or RNA to cells; e.g.,
antisense molecules can be injected directly into the tissue site,
or modified antisense molecules, designed to target the desired
cells (e.g., antisense linked to peptides or antibodies which
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systemically.
[0226] A preferred approach to achieve intracellular concentrations
of the antisense sufficient to suppress translation of endogenous
mRNAs utilizes a recombinant DNA construct in which the antisense
oligonucleotide is placed under the control of a strong pol III or
pol II promoter. The use of such a construct to transfect target
cells in the patient will result in the transcription of sufficient
amounts of single stranded RNAs which will form complementary base
pairs with the endogenous protective sequence transcripts and
thereby prevent translation of the protective sequence mRNA. For
example, a vector can be introduced e.g., such that it is taken up
by a cell and directs the transcription of an antisense RNA. Such a
vector can remain episomal or become chromosomally integrated, as
long as it can be transcribed to produce the desired antisense RNA.
Such vectors can be constructed by recombinant DNA technology
methods standard in the art. Vectors can be plasmid, viral, or
others known in the art, used for replication and expression in
mammalian cells. Expression of the sequence encoding the antisense
RNA can be by any promoter known in the art to act in mammalian,
preferably human cells. Such promoters can be inducible or
constitutive. Such promoters include but are not limited to: the
SV40 early promoter region (Bemoist and Chambon, 1981, Nature
290:304-310), the promoter contained in the 3'-long terminal repeat
of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),
the herpes thymidine kinase promoter (Wagner, et al., 1981, Proc.
Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of
the metallothionein gene (Brinster, et al., 1982, Nature
296:39-42), etc. Any type of plasmid, cosmid, YAC or viral vector
can be used to prepare the recombinant DNA construct that can be
introduced directly into the tissue site. Alternatively, viral
vectors can be used that selectively infect the desired tissue, in
which case administration may be accomplished by another route
(e.g., systemically).
[0227] Ribozyme molecules designed to catalytically cleave target
gene mRNA transcripts can also be used to prevent translation of
target gene mRNA and, therefore, expression of target gene product.
(See, e.g., PCT International Publication WO90/11364, published
Oct. 4, 1990; Sarver, et al., 1990, Science 247, 1222-1225).
[0228] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. (For a review, see Rossi, 1994,
Current Biology 4:469-471). The mechanism of ribozyme action
involves sequence specific hybridization of the ribozyme molecule
to complementary target RNA, followed by an endonucleolytic
cleavage event. The composition of ribozyme molecules must include
one or more sequences complementary to the target gene mRNA, and
must include the well known catalytic sequence responsible for mRNA
cleavage. For this sequence, see, e.g., U.S. Pat. No. 5,093,246,
which is incorporated herein by reference in its entirety.
[0229] While ribozymes that cleave mRNA at site specific
recognition sequences can be used to destroy target gene mRNAs, the
use of hammerhead ribozymes is preferred. Hammerhead ribozymes
cleave mRNAs at locations dictated by flanking regions which form
complementary base pairs with the target mRNA. The sole requirement
is that the target mRNA have the following sequence of two bases:
5'-UG-3'. The construction and production of hammerhead ribozymes
is well known in the art and is described more fully in Myers,
1995, Molecular Biology and Biotechnology: A Comprehensive Desk
Reference, VCH Publishers, New York, (see especially FIG. 4, page
833) and in Haseloff and Gerlach, 1988, Nature, 334:585-591, which
is incorporated herein by reference in its entirety.
[0230] Preferably the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the target gene
mRNA, i.e., to increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts.
[0231] The ribozymes of the present invention also include RNA
endoribonucleases (hereinafter "Cech-type ribozymes") such as the
one which occurs naturally in Tetrahymena thermophila (known as the
IVS, or L-1 9IVS RNA) and which has been extensively described by
Thomas Cech and collaborators (Zaug, et al., 1984, Science,
224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et
al., 1986, Nature, 324:429-433; published International patent
application No. WO 88/04300 by University Patents Inc.; Been and
Cech, 1986, Cell, 47:207-216). The Cech-type ribozymes have an
eight base pair active site that hybridizes to a target RNA
sequence whereafter cleavage of the target RNA takes place. The
invention encompasses those Cech-type ribozymes that target eight
base-pair active site sequences that are present in the target
gene.
[0232] As in the antisense approach, the ribozymes can be composed
of modified oligonucleotides (e.g., for improved stability,
targeting, etc.) and should be delivered to cells that express the
target gene in vivo. A preferred method of delivery involves using
a DNA construct "encoding" the ribozyme under the control of a
strong constitutive pol III or pol II promoter, so that transfected
cells will produce sufficient quantities of the ribozyme to destroy
endogenous target gene messages and inhibit translation. Because
ribozymes, unlike antisense molecules, are catalytic, a lower
intracellular concentration is required for efficiency.
[0233] Endogenous target gene expression can also be reduced by
inactivating or "knocking out" the target gene or its promoter
using targeted homologous recombination (e.g., see Smithies, et
al., 1985, Nature 317:230-234; Thomas and Capecchi, 1987, Cell
51:503-512; Thompson, et al., 1989, Cell 5:313-321; each of which
is incorporated by reference herein in its entirety). For example,
a mutant, non-functional target gene (or a completely unrelated DNA
sequence) flanked by DNA homologous to the endogenous target gene
(either the coding regions or regulatory regions of the target
gene) can be used, with or without a selectable marker and/or a
negative selectable marker, to transfect cells which express the
target gene in vivo. Insertion of the DNA construct, via targeted
homologous recombination, results in inactivation of the target
gene. Such approaches are particularly suited in the agricultural
field where modifications to ES (embryonic stem) cells can be used
to generate animal offspring with an inactive target gene (e.g.,
see Thomas and Capecchi, 1987 and Thompson, 1989, supra). However
this approach can be adapted for use in humans provided the
recombinant DNA constructs are directly administered or targeted to
the required site in vivo using appropriate viral vectors.
[0234] Alternatively, endogenous target gene expression can be
reduced by targeting deoxyribonucleotide sequences complementary to
the regulatory region of the target gene (i.e., the target gene
promoter and/or enhancers) to form triple helical structures which
prevent transcription of the target gene in target cells in the
body. (See generally, Helene, 1991, Anticancer Drug Des.,
6(6):569-584; Helene, et al., 1992, Ann. N.Y. Acad. Sci.,
660:27-36; and Maher, 1992, Bioassays 14(12):807-815).
[0235] Nucleic acid molecules to be used in triple helix formation
for the inhibition of transcription should be single stranded and
composed of deoxynucleotides. The base composition of these
oligonucleotides must be designed to promote triple helix formation
via Hoogsteen base pairing rules, which generally require sizable
stretches of either purines or pyrimidines to be present on one
strand of a duplex. Nucleic acids may be pyrimidine-based, which
will result in TAT and CGC.sup.+ triplets across the three
associated strands of the resulting triple helix. The
pyrimidine-rich molecules provide base complementarity to a
purine-rich region of a single strand of the duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may
be chosen which are purine-rich, for example, contain a stretch of
G residues. These molecules will form a triple helix with a DNA
duplex that is rich in GC pairs, in which the majority of the
purine residues are located on a single strand of the targeted
duplex, resulting in GGC triplets across the three strands in the
triplex.
[0236] Alternatively, the potential sequences that can be targeted
for triple helix formation may be increased by creating a so-called
"switchback" nucleic acid molecule. Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they
base pair with first one strand of a duplex and then the other,
eliminating the necessity for a sizable stretch of either purines
or pyrimidines to be present on one strand of a duplex.
[0237] In instances wherein the antisense, ribozyme, and/or triple
helix molecules described herein are utilized to inhibit mutant
gene expression, it is possible that the technique may so
efficiently reduce or inhibit the transcription (triple helix)
and/or translation (antisense, ribozyme) of mRNA produced by normal
target gene alleles which the possibility may arise wherein the
concentration of normal target gene product present may be lower
than is necessary for a normal phenotype. In such cases, to ensure
that substantially normal levels of target gene activity are
maintained, therefore, nucleic acid molecules which encode and
express target gene polypeptides exhibiting normal target gene
activity may, be introduced into cells via gene therapy methods
such as those described, below, in Section 5.4.1.3 which do not
contain sequences susceptible to whatever antisense, ribozyme, or
triple helix treatments are being utilized. Alternatively, in
instances whereby the target gene encodes an extracellular protein,
it may be preferable to co-administer normal target gene protein in
order to maintain the requisite level of target gene activity.
[0238] Anti-sense RNA and DNA, ribozyme, and triple helix molecules
of the invention may be prepared by any method known in the art for
the synthesis of DNA and RNA molecules, as discussed above. These
include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in
the art such as for example solid-phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules may be generated by in
vitro and in vivo transcription of DNA sequences encoding the
antisense RNA molecule. Such DNA sequences may be incorporated into
a wide variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
[0239] 5.4.1.3 Gene Replacement Therapy
[0240] Protective nucleic acid sequences, described above in
Section 5.1, can be utilized for transferring recombinant
protective nucleic acid sequences to cells and expressing said
sequences in recipient cells. Such techniques can be used, for
example, in marking cells or for the treatment of a condition,
disorder, or disease involving cell death. Such treatment can be in
the form of gene replacement therapy. Specifically, one or more
copies of a normal protective sequence or a portion of the
protective sequence which directs the production of a protective
sequence product exhibiting normal protective sequence function,
may be inserted into the appropriate cells within a patient, using
vectors which include, but are not limited to adenovirus,
adeno-associated virus and retrovirus vectors, in addition to other
particles which introduce DNA into cells, such as liposomes.
[0241] Because the protective sequence of the invention may be
expressed in the brain, such gene replacement therapy techniques
should be capable of delivering protective sequences to these cell
types within patients. Thus, in one embodiment, techniques which
are well known to those of skill in the art (see, e.g., PCT
Publication No. WO89/10134, published Apr. 25, 1988) can be used to
enable protective sequences to cross the blood-brain barrier
readily and to deliver the sequences to cells in the brain. With
respect to delivery which is capable of crossing the blood-brain
barrier, viral vectors such as, for example, those described above,
are preferable.
[0242] In another embodiment, techniques for delivery involve
direct administration, e.g., by stereotactic delivery of such
protective sequences to the site of the cells in which the
protective sequences are to be expressed.
[0243] Methods for introducing genes for expression in mammalian
cells are well known in the field. Generally, for such gene therapy
methods, the nucleic acid is directly administered in vivo into a
target cell or a transgenic mouse that expresses SP-10 promoter
operably linked to a reporter gene. This can be accomplished by any
methods known in the art, e.g., by constructing it as part of an
appropriate nucleic acid expression vector and administering it so
that it becomes intracellular, e.g., by infection using a defective
or attenuated retroviral or other viral vector (see U.S. Pat. No.
4,980,286), by direct injection of naked DNA, by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), by
coating with lipids or cell-surface receptors or transfecting
agents, by encapsulation in liposomes, microparticles, or
microcapsules, by administering it in linkage to a peptide which is
known to enter the nucleus, or by administering it in linkage to a
ligand subject to receptor-mediated endocytosis (see e.g., Wu and
Wu, 1987, J. Biol. Chem. 262:4429-4432), which can be used to
target cell types specifically expressing the receptors. In another
embodiment, a nucleic acid-ligand complex can be formed in which
the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated Apr. 16, 1992; WO 92/22635 dated Dec. 23, 1992;
WO92/20316 dated Nov. 26, 1992; WO93/14188 dated Jul. 22, 1993; WO
93/20221 dated Oct. 14, 1993).
[0244] Additional methods which may be utilized to increase the
overall level of protective sequence expression and/or gene product
activity include using targeted homologous recombination methods,
discussed in Section 5.2, above, to modify the expression
characteristics of an endogenous protective sequence in a cell or
microorganism by inserting a heterologous DNA regulatory element
such that the inserted regulatory element is operatively linked
with the endogenous protective sequence in question. Targeted
homologous recombination can thus be used to activate transcription
of an endogenous protective sequence which is "transcriptionally
silent", i.e., is not normally expressed or is normally expressed
at very low levels, or to enhance the expression of an endogenous
protective sequence which is normally expressed.
[0245] Further, the overall level of protective sequence expression
and/or gene product activity may be increased by the introduction
of appropriate protective sequence-expressing cells, preferably
autologous cells, into a patient at positions and in numbers which
are sufficient to ameliorate the symptoms of a condition, disorder,
or disease involving cell death. Such cells may be either
recombinant or non-recombinant.
[0246] Among the cells that can be administered to increase the
overall level of protective sequence expression in a patient are
normal cells, preferably brain cells, which express the protective
sequence. Alternatively, cells, preferably autologous cells, can be
engineered to express protective sequences, and may then be
introduced into a patient in positions appropriate for the
amelioration of the symptoms of a condition, disorder, or disease
involving cell death. Alternately, cells which express an
unimpaired protective sequence and which are from a MHC matched
individual can be utilized, and may include, for example, brain
cells. The expression of the protective sequences is controlled by
the appropriate gene regulatory sequences to allow such expression
in the necessary cell types. Such gene regulatory sequences are
well known to the skilled artisan. Such cell-based gene therapy
techniques are well known to those skilled in the art, see, e.g.,
Anderson, U.S. Pat. No. 5,399,349.
[0247] When the cells to be administered are non-autologous cells,
they can be administered using well-known techniques that prevent a
host immune response against the introduced cells from developing.
For example, the cells may be introduced in an encapsulated form
that, while allowing for an exchange of components with the
immediate extracellular environment, does not allow the introduced
cells to be recognized by the host immune system.
[0248] Additionally, compounds, such as those identified via
techniques such as those described, in Section 5.4.2, which are
capable of modulating protective sequences, protective sequence
product activity, or their regulatory sequences can be administered
using standard techniques which are well known to those of skill in
the art. In instances in which the compounds to be administered are
to involve an interaction with brain cells, the administration
techniques should include well known methods that allow for a
crossing of the blood-brain barrier.
[0249] 5.4.1.4 Detection of Protective Nucleic Acid Molecules
[0250] A variety of methods can be employed to screen for the
presence of protective sequence-specific mutations or polymorphisms
(including polymorphisms flanking protective sequences) and to
detect and/or assay levels of protective nucleic acid
sequences.
[0251] Mutations or polymorphisms within or flanking the protective
sequences can be detected by utilizing a number of techniques.
Nucleic acid from any nucleated cell can be used as the starting
point for such assay techniques, and may be isolated according to
standard nucleic acid preparation procedures that are well known to
those of skill in the art.
[0252] Protective nucleic acid sequences may be used in
hybridization or amplification assays of biological samples to
detect abnormalities involving protective sequence structure,
including point mutations, insertions, deletions, inversions,
translocations and chromosomal rearrangements. Such assays may
include, but are not limited to, Southern analyses, single-stranded
confornational polymorphism analyses (SSCP) and PCR analyses.
[0253] Diagnostic methods for the detection of protective
sequence-specific mutations or polymorphisms can involve for
example, contacting and incubating nucleic acids obtained from a
sample, e.g., derived from a patient sample or other appropriate
cellular source with one or more labeled nucleic acid reagents
including recombinant DNA molecules, cloned genes or degenerate
variants thereof, such as described in Section 5.1, above, under
conditions favorable for the specific annealing of these reagents
to their complementary sequences within or flanking the protective
sequence. The diagnostic methods of the present invention further
encompass contacting and incubating nucleic acids for the detection
of single nucleotide mutations or polymorphisms of the protective
sequence. Preferably, these nucleic acid reagent sequences within
the protective sequence are 15 to 30 nucleotides in length.
[0254] After incubation, all non-annealed nucleic acids are removed
from the reaction. The presence of nucleic acids that have
hybridized, if any such molecules exist, is then detected. Using
such a detection scheme, the nucleic acid from the cell type or
tissue of interest can be immobilized, for example, to a solid
support such as a membrane, or a plastic surface such as that on a
microtiter plate or polystyrene beads. In this case, after
incubation, non-annealed, labeled nucleic acid reagents of the type
described in Section 5.1 are easily removed. Detection of the
remaining, annealed, labeled nucleic acid reagents is accomplished
using standard techniques well known to those skilled in the art.
The protective sequences of the invention to which the nucleic acid
reagents have annealed can be compared to the annealing pattern
expected from a normal protective sequence of the invention in
order to determine whether a protective sequence mutation is
present.
[0255] In a preferred embodiment, protective sequence mutations or
polymorphisms can be detected by using a microassay of nucleic acid
sequences of the invention immobilized to a substrate or "gene
chip" (see, e.g. Cronin, et al., 1996, Human Mutation 7:244-255).
Alternative diagnostic methods for the detection of protective
sequence-specific nucleic acid molecules (or flanking sequences),
in patient samples or other appropriate cell sources, may involve
their amplification, e.g., by PCR (the experimental embodiment set
forth in Mullis, 1987, U.S. Pat. No. 4,683,202), followed by the
analysis of the amplified molecules using techniques well known to
those of skill in the art, such as, for example, those listed
above. The resulting amplified sequences can be compared to those
which would be expected if the nucleic acid being amplified
contained only normal copies of the protective sequence in order to
determine whether a protective sequence mutation or polymorphism in
linkage disequilibrium with a disease-causing allele exists.
[0256] Among those nucleic acid sequences that are preferred for
such amplification-related diagnostic screening analyses are
oligonucleotide primers that amplify exon sequences. The sequences
of such oligonucleotide primers are, therefore, preferably derived
from cerebral intron sequences so that the entire exon, or coding
region, can be analyzed as discussed below. Primer pairs useful for
amplification of cerebral exons are preferably derived from
adjacent introns. Appropriate primer pairs can be chosen such that
each of the cerebral exons present within the gene will be
amplified. Primers for the amplification of exons can be routinely
designed by one of ordinary skill.
[0257] Additional nucleic acid sequences which are preferred for
such amplification-related analyses are those which will detect the
presence of a polymorphism which differs from the sequence depicted
in the Figures. Such polymorphisms include ones that represent
mutations associated with a condition, disorder, or disease
involving cell death.
[0258] Amplification techniques are well known to those of skill in
the art and can routinely be utilized in connection with primers
such as those described above. In general, hybridization conditions
can be as follows: In general, for probes between 14 and 70
nucleotides in length, the melting temperature TM is calculated
using the formula: Tm(.degree. C.)=81.5+16.6(log[monovalent
cations])+0.41(% G+C)-(500/N) where N is the length of the probe.
If the hybridization is carried out in a solution containing
formamide, the melting temperature is calculated using the equation
Tm(.degree. C.)=81.5+16.6(log[monovalent cations])+0.41(%
G+C)-(0.61% formamide)-(500/N) where N is the length of the probe.
Additionally, well-known genotyping techniques can be performed to
identify individuals carrying protective sequence mutations. Such
techniques include, for example, the use of restriction fragment
length polymorphisms (RFLPs), which involve sequence variations in
one of the recognition sites for the specific restriction enzyme
used.
[0259] Further, improved methods for analyzing DNA polymorphisms,
which can be utilized for the identification of protective
sequence-specific mutations, have been described which capitalize
on the presence of variable numbers of short, tandemly repeated DNA
sequences between the restriction enzyme sites. For example, Weber
(U.S. Pat. No. 5,075,217) describes a DNA marker based on length
polymorphisms in blocks of (dC-dA)n-(dG-dT)n short tandem repeats.
The average separation of (dC-dA)n-(dG-dT)n blocks is estimated to
be 30,000-60,000 bp. Markers which are so closely spaced exhibit a
high frequency of co-inheritance, and are extremely useful in the
identification of genetic mutations, such as, for example,
mutations within the protective sequence of the invention, and the
diagnosis of diseases and disorders related to mutations of the
protective sequences of the invention.
[0260] Also, Caskey et al. (U.S. Pat. No. 5,364,759) describe a DNA
profiling assay for detecting short tri- and tetra nucleotide
repeat sequences. The process includes extracting the DNA of
interest, amplifying the extracted DNA and labeling the repeat
sequences to form a genotypic map of the individual's DNA.
[0261] Other methods well known in the art may be used to identify
single nucleotide polymorphisms (SNPs), including biallelic SNPs or
biallelic markers which have two alleles, both of which are present
at a fairly high frequency in a population. Conventional techniques
for detecting SNPs include, e.g., conventional dot blot analysis,
single stranded conformational polymorphism (SSCP) analysis (see,
e.g., Orita et al., 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770),
denaturing gradient gel electrophoresis (DGGE), heteroduplex
analysis, mismatch cleavage detection and other routine techniques
well known in the art (see, e.g., Sheffield et al., 1989, Proc.
Natl. Acad. Sci. 86:5855-5892; Grompe, 1993, Nature Genetics
5:111-117). Alternative, preferred methods of detecting and mapping
SNPs involve microsequencing techniques wherein an SNP site in a
target DNA is detecting by a single nucleotide primer extension
reaction (see, e.g., Goelet et al., PCT Publication No. WO92/15712;
Mundy, U.S. Pat. No. 4,656,127; Vary and Diamond, U.S. Pat. No.
4,851,331; Cohen et al., PCT Publication No. WO91/02087; Chee et
al., PCT Publication No. WO95/11995; Landegren et al., 1988,
Science 241:1077-1080; Nicerson et al., 1990, Proc. Natl. Acad.
Sci. U.S.A. 87:8923-8927; Pastinen et al., 1997, Genome Res.
7:606-614; Pastinen et al., 1996, Clin. Chem. 42:1391-1397; Jalanko
et al., 1992, Clin. Chem. 38:39-43; Shumaker et al., 1996, Hum.
Mutation 7:346-354; Caskey et al., PCT Publication No. WO
95/00669).
[0262] The level of protective sequence expression also can be
assayed. For example, RNA from a cell type or tissue known, or
suspected, to express the protective sequence, such as brain, may
be isolated and tested utilizing hybridization or PCR techniques
such as are described, above. The isolated cells can be derived
from cell culture or from a patient. The analysis of cells taken
from culture may be a necessary step in the assessment of cells to
be used as part of a cell-based gene therapy technique or,
alternatively, to test the effect of compounds on the expression of
the protective sequence. Such analyses may reveal both quantitative
and qualitative aspects of the expression pattern of the protective
sequence, including activation or inactivation of protective
sequence expression.
[0263] In one embodiment of such a detection scheme, a cDNA
molecule is synthesized from an RNA molecule of interest (e.g., by
reverse transcription of the RNA molecule into cDNA). A sequence
within the cDNA is then used as the template for a nucleic acid
amplification reaction, such as a PCR amplification reaction, or
the like. The nucleic acid reagents used as synthesis initiation
reagents (e.g., primers) in the reverse transcription and nucleic
acid amplification steps of this method are chosen from among the
protective sequence nucleic acid reagents described in Section 5.1.
The preferred lengths of such nucleic acid reagents are at least
9-30 nucleotides. For detection of the amplified product, the
nucleic acid amplification may be performed using radioactively or
non-radioactively labeled nucleotides. Alternatively, enough
amplified product may be made such that the product may be
visualized by standard ethidium bromide staining or by utilizing
any other suitable nucleic acid staining method.
[0264] Additionally, it is possible to perform such protective
sequence expression assays "in situ", i.e., directly upon tissue
sections (fixed and/or frozen) of patient tissue obtained from
biopsies or resections, such that no nucleic acid purification is
necessary. Nucleic acid reagents such as those described in Section
5.1 may be used as probes and/or primers for such in situ
procedures (see, for example, Nuovo, G. J., 1992, "PCR In Situ
Hybridization: Protocols And Applications", Raven Press, NY).
[0265] Alternatively, if a sufficient quantity of the appropriate
cells can be obtained, standard Northern blot analysis can be
performed to determine the level of mRNA expression of the
protective sequence.
[0266] 5.4.1.5 Detection of Protective Sequence Products
[0267] Protective sequence products of the invention, including
both wild-type and mutant protective sequence products, conserved
variants and polypeptide fragments thereof, which are discussed,
above, in Section 5.2, may be detected using antibodies which are
directed against such gene products. Such antibodies, which are
discussed in Section 5.3, above, may thereby be used as diagnostics
and prognostics for a condition, disorder, or disease involving
cell death. Such methods may be used to detect abnormalities in the
level of protective sequence expression or of protective sequence
product synthesis, or abnormalities in the structure, temporal
expression and/or physical location of protective sequence product.
The antibodies and immunoassay methods described herein have, for
example, important in vitro applications in assessing the efficacy
of treatments for conditions, disorders, or diseases involving cell
death. Antibodies, or fragments of antibodies, such as those
described below, may be used to screen potentially therapeutic
compounds in vitro to determine their effects on protective
sequence expression and protective sequence product production. The
compounds which have beneficial effects on conditions, disorders,
or diseases involving cell death can thereby be identified, and a
therapeutically effective dose determined.
[0268] In vitro immunoassays may also be used, for example, to
assess the efficacy of cell-based gene therapy for a condition,
disorder, or disease involving cell death. Antibodies directed
against protective sequence products may be used in vitro to
determine, for example, the level of protective sequence expression
achieved in cells genetically engineered to produce the protective
sequence product. In the case of intracellular protective sequence
products, such an assessment is done, preferably, using cell
lysates or extracts. Such analysis will allow for a determination
of the number of transformed cells necessary to achieve therapeutic
efficacy in vivo, as well as optimization of the gene replacement
protocol.
[0269] The tissue or cell type to be analyzed generally will
include those that are known, or suspected, to express the
protective sequence. The protein isolation methods employed herein
may, for example, be such as those described in Harlow and Lane
(1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). The isolated cells can
be derived from cell culture or from a patient. The analysis of
cells taken from culture may be a necessary step in the assessment
of cells to be used as part of a cell-based gene therapy technique
or, alternatively, to test the effect of compounds on the
expression of the protective sequence.
[0270] Preferred diagnostic methods for the detection of protective
sequence products, conserved variants or peptide fragments thereof,
may involve, for example, immunoassays wherein the protective
sequence products or conserved variants or peptide fragments are
detected by their interaction with an anti-protective sequence
product-specific antibody.
[0271] For example, antibodies, or fragments of antibodies, such as
those described, above, in Section 5.3, may be used to,
quantitatively or qualitatively, detect the presence of protective
sequence products or conserved variants or peptide fragments
thereof. This can be accomplished, for example, by
immunofluorescence techniques employing a fluorescently labeled
antibody (see below, this Section) coupled with light microscopic,
flow cytometric or fluorimetric detection. Such techniques are
especially preferred for protective sequence products that are
expressed on the cell surface.
[0272] The antibodies (or fragments thereof) useful in the present
invention may, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of protective sequence products, conserved variants or
peptide fragments thereof. in situ detection may be accomplished by
removing a histological specimen from a patient, and applying
thereto a labeled antibody which binds to a protective sequence
polypeptide. The antibody (or fragment) is preferably applied by
overlaying the labeled antibody (or fragment) onto a biological
sample. Through the use of such a procedure, it is possible to
determine not only the presence of the protective sequence product,
conserved variants or peptide fragments, but also its distribution
in the examined tissue. Using the present invention, those of
ordinary skill will readily recognize that any of a wide variety of
histological methods (such as staining procedures) can be modified
in order to achieve in situ detection of a protective sequence
product.
[0273] Immunoassays for protective sequence products, conserved
variants or peptide fragments thereof will typically comprise
incubating a sample, such as a biological fluid, a tissue extract,
freshly harvested cells or lysates of cells in the presence of a
detectably labeled antibody capable of identifying the protective
sequence product, conserved variants or peptide fragments thereof,
and detecting the bound antibody by any of a number of techniques
well-known in the art.
[0274] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier, such as
nitrocellulose, which is capable of immobilizing cells, cell
particles or soluble proteins. The support may then be washed with
suitable buffers followed by treatment with the detectably labeled
protective sequence product specific antibody. The solid phase
support may then be washed with the buffer a second time to remove
unbound antibody. The amount of bound label on the solid support
may then be detected by conventional means.
[0275] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0276] One of the ways in which the protective sequence
product-specific antibody can be detectably labeled is by linking
the same to an enzyme, such as for use in an enzyme immunoassay
(EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)",
1978, Diagnostic Horizons 2:1-7, Microbiological Associates
Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978,
J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.
73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press,
Boca Raton, Fla.,; Ishikawa, E. et al., (eds.), 1981, Enzyme
Immunoassay, Kgaku Shoin, Tokyo). The enzyme that is bound to the
antibody will react with an appropriate substrate, preferably a
chromogenic substrate, in such a manner as to produce a chemical
moiety which can be detected, for example, by spectrophotometric,
fluorimetric or by visual means. Enzymes which can be used to
detectably label the antibody include, but are not limited to,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, .alpha.-glycerophosphate,
dehydrogenase, triose phosphate isomerase, horseradish peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase,
.beta.-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and
acetylcholinesterase. The detection can be accomplished by
colorimetric methods that employ a chromogenic substrate for the
enzyme. Detection also may be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards.
[0277] Detection may be accomplished also using any of a variety of
other immunoassays. For example, by radioactively labeling the
antibodies or antibody fragments, it is possible to detect
protective sequence products through the use of a radioimmunoassay
(RIA) (see, for example, Weintraub, B., Principles of
Radioimmunoassays, Seventh Training Course on Radioligand Assay
Techniques, The Endocrine Society, March, 1986). The radioactive
isotope can be detected by such means as the use of a gamma counter
or a scintillation counter or by autoradiography.
[0278] It is also possible to label the antibody with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wavelength, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine.
[0279] The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0280] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester.
[0281] Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic
protein increases the efficiency of the chemiluminescent reaction.
The presence of a bioluminescent protein is determined by detecting
the presence of luminescence. Important bioluminescent compounds
for purposes of labeling are luciferin, luciferase and
aequorin.
[0282] 5.4.2 Screening Assays for Compounds which Interact with
Protective Sequence Products or Modulate Protective Sequence
Activity
[0283] The following assays are designed to identify compounds
which bind to a protective sequence product, compounds which bind
to proteins, or portions of proteins which interact with a
protective sequence product, compounds which modulate, e.g.,
interfere with, the interaction of a protective sequence product
with proteins and compounds which modulate the activity of the
protective sequence (i.e., modulate the level of protective
sequence expression and/or modulate the level of protective
sequence product activity). Assays may additionally be utilized
which identify compounds which bind to protective sequence
regulatory sequences (e.g., promoter sequences; see e.g., Platt,
1994, J. Biol. Chem. 269, 28558-28562), and which can modulate the
level of protective sequence expression. Such compounds may
include, but are not limited to, small organic molecules, such as
ones which are able to cross the blood-brain barrier, gain to
and/or entry into an appropriate cell and affect expression of the
protective sequence or some other gene involved in a protective
sequence regulatory pathway.
[0284] Methods for the identification of such proteins are
described, below, in Section 5.4.2.2. Such proteins may be involved
in the control and/or regulation of functions related to cell
death. Further, among these compounds are compounds which affect
the level of protective sequence expression and/or protective
sequence product activity and which can be used in the therapeutic
treatment of conditions, disorders, or diseases involving cell
death as described, below, in Section 5.4.2.3.
[0285] Compounds may include, but are not limited to, peptides such
as, for example, soluble peptides, including but not limited to,
Ig-tailed fusion peptides, and members of random peptide libraries;
(see, e.g., Lam, et al., 1991, Nature 354:82-84; Houghten, et al.,
1991, Nature 354:84-86), and combinatorial chemistry-derived
molecular library made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to members of random or
partially degenerate, directed phosphopeptide libraries; see, e.g.,
Songyang, et al., 1993, Cell 72:767-778), antibodies (including,
but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression library fragments, and
epitope-binding fragments thereof), and small organic or inorganic
molecules.
[0286] Such compounds may further comprise compounds, in particular
drugs or members of classes or families of drugs, known to
ameliorate the symptoms of a condition, disorder, or disease
involving cell death.
[0287] Such compounds include families of antidepressants such as
lithium salts, carbamazepine, valproic acid, lysergic acid
diethylamide (LSD), p-chlorophenylalanine, p-propyldopacetamide
dithiocarbamate derivatives e.g., FLA 63; anti-anxiety drugs, e.g.,
diazepam; monoamine oxidase (MAO) inhibitors, e.g., iproniazid,
clorgyline, phenelzine and isocarboxazid; biogenic amine uptake
blockers, e.g., tricyclic antidepressants such as desipramine,
imipramine and amitriptyline; serotonin reuptake inhibitors e.g.,
fluoxetine; antipsychotic drugs such as phenothiazine derivatives
(e.g., chlorpromazine (thorazine) and trifluopromazine)),
butyrophenones (e.g., haloperidol (Haldol)), thioxanthene
derivatives (e.g., chlorprothixene), and dibenzodiazepines (e.g.,
clozapine); benzodiazepines; dopaminergic agonists and antagonists
e.g., L-DOPA, cocaine, amphetamine, .alpha.-methyl-tyrosine,
reserpine, tetrabenazine, benzotropine, pargyline; noradrenergic
agonists and antagonists e.g., clonidine, phenoxybenzamine,
phentolamine, tropolone.
[0288] Compounds identified via assays such as those described
herein may be useful, for example, in elaborating the biological
function of protective sequence products and for ameliorating
conditions, disorders, or diseases involving cell death. Assays for
testing the effectiveness of compounds identified by, for example,
techniques such as those described in Sections 5.4.2.1-5.4.2.3, are
discussed, below, in Section 5.4.2.4.
[0289] 5.4.2.1 In Vitro Screening Assays for Compounds which Bind
to Protective Sequence Products
[0290] In vitro systems may be designed to identify compounds
capable of binding the protective sequence products of the
invention. Compounds identified may be useful, for example, in
modulating the activity of unimpaired and/or mutant protective
sequence products, may be useful in elaborating the biological
function of the protective sequence product, may be utilized in
screens for identifying compounds which disrupt normal protective
sequence product interactions or may in themselves disrupt such
interactions.
[0291] The principle of the assays used to identify compounds which
bind to the protective sequence product involves preparing a
reaction mixture of the protective sequence product and the test
compound under conditions and for a time sufficient to allow the
two components to interact and bind, thus forming a complex which
can be removed and/or detected in the reaction mixture. These
assays can be conducted in a variety of ways. For example, one
method to conduct such an assay involves anchoring a protective
sequence product or a test substance onto a solid support and
detecting protective sequence product/test compound complexes
formed on the solid support at the end of the reaction. In one
embodiment of such a method, the protective sequence product may be
anchored onto a solid support, and the test compound, which is not
anchored, may be labeled, either directly or indirectly.
[0292] In practice, microtiter plates are conveniently utilized as
the solid support. The anchored component may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished by simply coating the solid surface with a solution
of the protein and drying. Alternatively, an immobilized antibody,
preferably a monoclonal antibody, specific for the protein to be
immobilized may be used to anchor the protein to the solid surface.
The surfaces may be prepared in advance and stored.
[0293] In order to conduct the assay, the non-immobilized component
is added to the coated surface containing the anchored component.
After the reaction is complete, unreacted components are removed
(e.g., by washing) under conditions such that any complexes formed
will remain immobilized on the solid surface. The detection of
complexes anchored on the solid surface can be accomplished in a
number of ways. Where the previously non-immobilized component is
pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the previously
non-immobilized component is not pre-labeled, an indirect label can
be used to detect complexes anchored on the surface; e.g., using a
labeled antibody specific for the previously non-immobilized
component (the antibody, in turn, may be directly labeled or
indirectly labeled with a labeled anti-Ig antibody).
[0294] Alternatively, a reaction can be conducted in a liquid
phase, the reaction products separated from unreacted components,
and complexes detected; e.g. using an immobilized antibody specific
for the protective sequence product or the test compound to anchor
any complexes formed in solution, and a labeled antibody specific
for the other component of the possible complex to detect anchored
complexes.
[0295] 5.4.2.2 Assays for Proteins which Interact with Protective
Sequence Products
[0296] Any method suitable for detecting protein-protein
interactions may be employed for identifying protective sequence
product-protein interactions.
[0297] Among the traditional methods that may be employed are
co-immunoprecipitation, cross-linking and co-purification through
gradients or chromatographic columns. Utilizing procedures such as
these allows for the identification of proteins, including
intracellular proteins, which interact with protective sequence
products. Once isolated, such a protein can be identified and can
be used in conjunction with standard techniques, to identify
proteins it interacts with. For example, at least a portion of the
amino acid sequence of a protein which interacts with the
protective sequence product can be ascertained using techniques
well known to those of skill in the art, such as via the Edman
degradation technique (see, e.g., Creighton, 1983, "Proteins:
Structures and Molecular Principles," W. H. Freeman & Co.,
N.Y., pp.34-49). The amino acid sequence obtained may be used as a
guide for the generation of oligonucleotide mixtures that can be
used to screen for gene sequences encoding such proteins. Screening
may be accomplished, for example, by standard hybridization or PCR
techniques. Techniques for the generation of oligonucleotide
mixtures and the screening are well known. (See, e.g., Ausubel,
supra, and 1990, "PCR Protocols: A Guide to Methods and
Applications," Innis, et al., eds. Academic Press, Inc., New
York).
[0298] Additionally, methods may be employed which result in the
simultaneous identification of genes that encode a protein that
interacts with a protective sequence product. These methods
include, for example, probing expression libraries with labeled
protective sequence product, using the protective sequence product
in a manner similar to the well-known technique of antibody probing
of .lambda.gt11 libraries.
[0299] One method that detects protein interactions in vivo, the
two-hybrid system, is described in detail for illustration only and
not by way of limitation. One version of this system has been
described (Chien, et al., 1991, Proc. Natl. Acad. Sci. USA,
88:9578-9582) and is commercially available from Clontech (Palo
Alto, Calif.).
[0300] Briefly, utilizing such a system, plasmids are constructed
which encode two hybrid proteins: one consists of the DNA-binding
domain of a transcription activator protein fused to the protective
sequence product and the other consists of the transcription
activator protein's activation domain fused to an unknown protein
which is encoded by a cDNA which has been recombined into this
plasmid as part of a cDNA library. The DNA-binding domain fusion
plasmid and the cDNA library are transformed into a strain of the
yeast Saccharomyces cerevisiae that contains a reporter gene (e.g.,
HBS or lacZ) whose regulatory region contains the transcription
activator's binding site. Either hybrid protein alone cannot
activate transcription of the reporter gene: the DNA-binding domain
hybrid cannot because it does not provide activation function and
the activation domain hybrid cannot because it cannot localize to
the activator's binding sites. Interaction of the two hybrid
proteins reconstitutes the functional activator protein and results
in expression of the reporter gene, which is detected by an assay
for the reporter gene product.
[0301] The two-hybrid system or related methodologies may be used
to screen activation domain libraries for proteins that interact
with the "bait" gene product. By way of example, and not by way of
limitation, protective sequence products of the invention may be
used as the bait gene product. Total genomic or cDNA sequences are
fused to the DNA encoding an activation domain. This library and a
plasmid encoding a hybrid of a bait protective sequence product
fused to the DNA-binding domain are co-transformed into a yeast
reporter strain, and the resulting transformants are screened for
those which express the reporter gene. For example, a bait
protective sequence, such as the open reading frame of the gene,
can be cloned into a vector such that it is translationally fused
to the DNA encoding the DNA-binding domain of the GAL4 protein.
These colonies are purified and the library plasmids responsible
for reporter gene expression are isolated. DNA sequencing is then
used to identify the proteins encoded by the library plasmids.
[0302] A cDNA library of the cell line, from which proteins which
interact with bait protective sequence products are to be detected,
can be made using methods routinely practiced in the art. According
to the particular system described herein, for example, the cDNA
fragments can be inserted into a vector such that they are
translationally fused to the transcriptional activation domain of
GAL4. Such a library can be co-transformed along with the bait
protective sequence-GAL4 fusion plasmid into a yeast strain that
contains a lacZ gene driven by a promoter that contains GAL4
activation sequence. A cDNA encoded protein, fused to a GAL4
transcriptional activation domain that interacts with bait
protective sequence product will reconstitute an active GAL4
protein and thereby drive expression of the HIS3 gene. Colonies
that express HIS3 can be detected by their growth on petri dishes
containing semi-solid agar based media lacking histidine. The cDNA
can then be purified from these strains, and used to produce and
isolate the bait protective sequence product-interacting protein
using techniques routinely practiced in the art.
[0303] 5.4.2.3 Assays for Compounds which Interfere with or
Potentiate Protective Sequence Products Macromolecule
Interaction
[0304] The protective sequence products may, in vivo, interact with
one or more macromolecules, including intracellular macromolecules,
such as proteins. Such macromolecules may include, but are not
limited to, nucleic acid molecules and those proteins identified
via methods such as those described, above, in Sections
5.4.2.1-5.4.2.2. For purposes of this discussion, the
macromolecules are referred to herein as "binding partners".
Compounds that disrupt protective sequence product binding to a
binding partner may be useful in regulating the activity of the
protective sequence product, especially mutant protective sequence
products. Such compounds may include, but are not limited to
molecules such as peptides, and the like, as described, for
example, in Section 5.4.2.1 above.
[0305] The basic principle of an assay system used to identify
compounds which interfere with or potentiate the interaction
between the protective sequence product and a binding partner or
partners involves preparing a reaction mixture containing the
protective sequence product and the binding partner under
conditions and for a time sufficient to allow the two to interact
and bind, thus forming a complex. In order to test a compound for
inhibitory activity, the reaction mixture is prepared in the
presence and absence of the test compound. The test compound may be
initially included in the reaction mixture, or may be added at a
time subsequent to the addition of protective sequence product and
its binding partner. Control reaction mixtures are incubated
without the test compound or with a compound that is known not to
block complex formation. The formation of any complexes between the
protective sequence product and the binding partner is then
detected. The formation of a complex in the control reaction, but
not in the reaction mixture containing the test compound, indicates
that the compound interferes with the interaction of the protective
sequence product and the binding partner. Additionally, complex
formation within reaction mixtures containing the test compound and
normal protective sequence product also may be compared to complex
formation within reaction mixtures containing the test compound and
a mutant protective sequence product. This comparison may be
important in those cases wherein it is desirable to identify
compounds that disrupt interactions of mutant but not normal
protective sequence product.
[0306] In order to test a compound for potentiating activity, the
reaction mixture is prepared in the presence and absence of the
test compound. The test compound may be initially included in the
reaction mixture, or may be added at a time subsequent to the
addition of protective sequence product and its binding partner.
Control reaction mixtures are incubated without the test compound
or with a compound that is known not to block complex formation.
The formation of any complexes between the protective sequence
product and the binding partner is then detected. Increased
formation of a complex in the reaction mixture containing the test
compound, but not in the control reaction, indicates that the
compound enhances and therefore potentiates the interaction of the
protective sequence product and the binding partner. Additionally,
complex formation within reaction mixtures containing the test
compound and normal protective sequence product may also be
compared to complex formation within reaction mixtures containing
the test compound and a mutant protective sequence product. This
comparison may be important in those cases wherein it is desirable
to identify compounds that enhance interactions of mutant but not
normal protective sequence product.
[0307] In alternative embodiments, the above assays may be
performed using a reaction mixture containing the protective
sequence product, a binding partner and a third compound which
disrupts or enhances protective sequence product binding to the
binding partner. The reaction mixture is prepared and incubated in
the presence and absence of the test compound, as described above,
and the formation of any complexes between the protective sequence
product and the binding partner is detected. In this embodiment,
the formation of a complex in the reaction mixture containing the
test compound, but not in the control reaction, indicates that the
test compound interferes with the ability of the second compound to
disrupt protective sequence product binding to its binding
partner.
[0308] The assays for compounds that interfere with or potentiate
the interaction of the protective sequence products and binding
partners can be conducted in a heterogeneous or homogeneous format.
Heterogeneous assays involve anchoring either the protective
sequence product or the binding partner onto a solid support and
detecting complexes formed on the solid support at the end of the
reaction. In homogeneous assays, the entire reaction is carried out
in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested. For example, test compounds which interfere
with or potentiate the interaction between the protective sequence
products and the binding partners, e.g., by competition, can be
identified by conducting the reaction in the presence of the test
substance; i.e., by adding the test substance to the reaction
mixture prior to or simultaneously with the protective sequence
product and interactive intracellular binding partner.
Alternatively, test compounds which disrupt preformed complexes,
e.g., compounds with higher binding constants which displace one of
the components from the complex, can be tested by adding the test
compound to the reaction mixture after complexes have been formed.
The various formats are described briefly below.
[0309] In a heterogeneous assay system, either the protective
sequence product or the interactive binding partner, is anchored
onto a solid surface, while the non-anchored species is labeled,
either directly or indirectly. In practice, microtiter plates are
conveniently utilized. The anchored species may be immobilized by
non-covalent or covalent attachments. Non-covalent attachment may
be accomplished simply by coating the solid surface with a solution
of the protective sequence product or binding partner and drying.
Alternatively, an immobilized antibody specific for the species to
be anchored may be used to anchor the species to the solid surface.
The surfaces may be prepared in advance and stored.
[0310] In order to conduct the assay, the partner of the
immobilized species is exposed to the coated surface with or
without the test compound. After the reaction is complete,
unreacted components are removed (e.g., by washing) and any
complexes formed will remain immobilized on the solid surface. The
detection of complexes anchored on the solid surface can be
accomplished in a number of ways. Where the non-immobilized species
is pre-labeled, the detection of label immobilized on the surface
indicates that complexes were formed. Where the non-immobilized
species is not pre-labeled, an indirect label can be used to detect
complexes anchored on the surface; e.g., using a labeled antibody
specific for the initially non-immobilized species (the antibody,
in turn, may be directly labeled or indirectly labeled with a
labeled anti-Ig antibody). Depending upon the order of addition of
reaction components, test compounds which inhibit complex formation
or which disrupt preformed complexes can be detected.
[0311] Alternatively, the reaction can be conducted in a liquid
phase in the presence or absence of the test compound, the reaction
products separated from unreacted components, and complexes
detected; e.g., using an immobilized antibody specific for one of
the binding components to anchor any complexes formed in solution,
and a labeled antibody specific for the other partner to detect
anchored complexes. Again, depending upon the order of addition of
reactants to the liquid phase, test compounds which inhibit complex
formation or which disrupt preformed complexes can be
identified.
[0312] In an alternate embodiment of the invention, a homogeneous
assay can be used. In this approach, a preformed complex of the
protective sequence product and the interactive binding partner is
prepared in which either the protective sequence product or its
binding partners is labeled, but the signal generated by the label
is quenched due to complex formation (see, e.g., U.S. Pat. No.
4,109,496 by Rubenstein which utilizes this approach for
immunoassays). The addition of a test substance that competes with
and displaces one of the species from the preformed complex will
result in the generation of a signal above background. In this way,
test substances that disrupt protective sequence product/binding
partner interaction can be identified.
[0313] In another embodiment of the invention, these same
techniques can be employed using peptide fragments which correspond
to the binding domains of the protective sequence product and/or
the binding partner (in cases where the binding partner is a
protein), in place of one or both of the full length proteins. Any
number of methods routinely practiced in the art can be used to
identify and isolate the binding sites. These methods include, but
are not limited to, mutagenesis of the gene encoding one of the
proteins and screening for disruption of binding in a
co-immunoprecipitation assay. Compensating mutations in the gene
encoding the second species in the complex can then be selected.
Sequence analysis of the genes encoding the respective proteins
will reveal the mutations that correspond to the region of the
protein involved in interactive binding. Alternatively, one protein
can be anchored to a solid surface using methods described in this
Section above, and allowed to interact with and bind to its labeled
binding partner, which has been treated with a proteolytic enzyme,
such as trypsin. After washing, a short, labeled peptide comprising
the binding domain may remain associated with the solid material,
which can be isolated and identified by amino acid sequencing.
Also, once the gene coding for the segments is engineered to
express peptide fragments of the protein, it can then be tested for
binding activity and purified or synthesized.
[0314] For example, and not by way of limitation, a protective
sequence product can be anchored to a solid material as described,
above, in this Section by making a GST-1 fusion protein and
allowing it to bind to glutathione agarose beads. The binding
partner can be labeled with a radioactive isotope, such as
.sup.35S, and cleaved with a proteolytic enzyme such as trypsin.
Cleavage products can then be added to the anchored GST-1 fusion
protein and allowed to bind. After washing away unbound peptides,
labeled bound material, representing the binding partner binding
domain, can be eluted, purified and analyzed for amino acid
sequence by well-known methods. Peptides so identified can be
produced synthetically or produced using recombinant DNA
technology.
[0315] 5.4.2.4 Assays for the Identification of Compounds which
Modulate Conditions, Disorders, or Diseases Involving Cell
Death
[0316] Compounds, including, but not limited to, binding compounds
identified via assay techniques such as those described, above, in
Sections 5.4.2.1-5.4.2.3, can be tested for the ability to
ameliorate symptoms of a condition, disorder, or disease involving
cell death.
[0317] It should be noted that the assays described herein can be
used to identify compounds which affect activity by either
affecting protective sequence expression or by affecting the level
of protective sequence product activity. For example, compounds may
be identified which are involved in another step in the pathway in
which the protective sequence and/or protective sequence product is
involved, such as, for example, a step which is either "upstream"
or "downstream" of the step in the pathway mediated by the
protective sequence. Such compounds may, by affecting this same
pathway, modulate the effect on the development of conditions,
disorders, or diseases involving cell death. Such compounds can be
used as part of a therapeutic method for the treatment of the
condition, disorder, or disease.
[0318] Described below are cell-based and animal model-based assays
for the identification of compounds exhibiting such an ability to
ameliorate symptoms of a condition, disorder, or disease involving
cell death.
[0319] First, cell-based systems can be used to identify compounds
which may act to ameliorate symptoms of a condition, disorder, or
disease, including, but not limited to, those described in Section
5.4.1.1. Such cell systems can include, for example, recombinant or
non-recombinant cell, such as cell lines, which express the
protective sequence of interest.
[0320] In utilizing such cell systems, cells which express the
protective sequence of interest may be exposed to a compound
suspected of exhibiting an ability to ameliorate symptoms of a
condition, disorder, or disease involving cell death at a
sufficient concentration and for a sufficient time to elicit such
an amelioration of such symptoms in the exposed cells. After
exposure, the cells can be assayed to measure alterations in the
expression of the protective sequence, e.g., by assaying cell
lysates for cerebral mRNA transcripts (e.g., by Northern analysis)
or for protective sequence products expressed by the cell;
compounds which modulate expression of the protective sequence are
good candidates as therapeutics.
[0321] In addition, animal-based systems or models for a condition,
disorder, or disease involving cell death, for example, transgenic
mice containing a human or altered form of a protective sequence,
may be used to identify compounds capable of ameliorating symptoms
of the condition, disorder, or disease. Such animal models may be
used as test substrates for the identification of drugs,
pharmaceuticals, therapies and interventions. For example, animal
models may be exposed to a compound suspected of exhibiting an
ability to ameliorate symptoms, at a sufficient concentration and
for a sufficient time to elicit such an amelioration of symptoms of
a condition, disorder, or disease involving cell death. The
response of the animals to the exposure may be monitored by
assessing the reversal of the symptoms of the condition, disorder,
or disease.
[0322] With regard to intervention, any treatments that reverse any
aspect of symptoms of a condition, disorder, or disease involving
cell death, should be considered as candidates for human
therapeutic intervention in such conditions, disorders, or
diseases. Dosages of test agents may be determined by deriving
dose-response curves, as discussed in Section 5.5.1, below.
[0323] 5.4.3 Additional Uses for the Protective Sequences,
Protective Sequence Products, or Their Regulatory Elements
[0324] In addition to the uses described above, the polynucleotides
of the present invention can be used for various other purposes.
For example, they can be used to express recombinant protein for
analysis, characterization or therapeutic use; as molecular weight
markers on gels; as chromosome markers or tags (when labeled) to
identify chromosomes or to map related gene positions; to compare
with endogenous DNA sequences in patients to identify potential
genetic conditions, disorders, or diseases; as probes to hybridize
and thus discover novel, related DNA sequences; as a source of
information to derive PCR primers for genetic fingerprinting; as a
probe to "subtract-out" known sequences in the process of
discovering other novel polynucleotides; to raise anti-protein
antibodies using DNA immunization techniques; and as an antigen to
raise anti-DNA antibodies or elicit another immune response.
[0325] The proteins provided by the present invention can similarly
be used to raise antibodies or to elicit another immune response;
as a reagent (including the labeled reagent) in assays designed to
quantitatively determine levels of the protein (or its receptor) in
biological fluids; as markers for tissues in which the
corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation
or development or in a disease state); and, of course, to isolate
correlative receptors or ligands. Where the protein binds or
potentially binds to another protein (such as, for example, in a
receptor-ligand interaction), the protein can be used to identify
the other protein with which binding occurs or to identify
inhibitors of the binding interaction. Proteins involved in these
binding interactions can also be used to screen for peptide or
small molecule inhibitors or agonists of the binding
interaction.
[0326] Methods for performing the uses listed above are well known
to those skilled in the art. References disclosing such methods
include without limitation "Molecular Cloning: A Laboratory
Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in
Enzymology: Guide to Molecular Cloning Techniques", Academic Press,
Berger, S. L. and A. R. Kimmel eds., 1987.
[0327] 5.5 Pharmaceutical Preparations and Methods of
Administration
[0328] The compounds which are determined to affect protective
sequence expression or gene product activity can be administered to
a patient at therapeutically effective doses to treat or ameliorate
a condition, disorder, or disease involving cell death or modulate
a cell death-related process described herein. A therapeutically
effective dose refers to that amount of the compound sufficient to
result in amelioration of symptoms of such a condition, disorder,
or disease.
[0329] 5.5.1 Effective Dose
[0330] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
that exhibit large therapeutic indices are preferred. While
compounds which exhibit toxic side effects may be used, care should
be taken to design a delivery system which targets such compounds
to the site of affected tissue in order to minimize potential
damage to uninfected cells and, thereby, reduce side effects.
[0331] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
which includes the lC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0332] As defined herein, a therapeutically effective amount of
antibody, protein, or polypeptide (i.e., an effective dosage)
ranges from about 0.001 to 30 mg/kg body weight, preferably about
0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg
body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9
mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0333] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
condition, disorder, or disease, previous treatments, the general
health and/or age of the subject, and other diseases present.
Moreover, treatment of a subject with a therapeutically effective
amount of a protein, polypeptide, or antibody can include a single
treatment or, preferably, can include a series of treatments. In a
preferred example, a subject is treated with antibody, protein, or
polypeptide in the range of between about 0.1 to 20 mg/kg body
weight, one time per week for between about 1 to 10 weeks,
preferably between 2 to 8 weeks, more preferably between about 3 to
7 weeks, and even more preferably for about 4, 5, or 6 weeks. It
will also be appreciated that the effective dosage of antibody,
protein, or polypeptide used for treatment may increase or decrease
over the course of a particular treatment. Changes in dosage may
result and become apparent from the results of diagnostic assays as
described herein.
[0334] 5.5.2 Formulations and Use
[0335] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
[0336] Thus, the compounds and their physiologically acceptable
salts and solvates may be formulated for administration by
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral rectal or topical administration.
[0337] For oral administration, the pharmaceutical compositions may
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g. magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0338] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0339] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0340] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0341] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0342] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0343] In certain embodiments, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment. This may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0344] For topical application, the compounds may be combined with
a carrier so that an effective dosage is delivered, based on the
desired activity.
[0345] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0346] The compositions may, if desired, be presented in a pack or
dispenser device that may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
6 EXAMPLE
[0347] Sequence and Characterization of Protective Sequences
[0348] In the example presented herein, the sequence and
characterization of the protective sequences are provided.
[0349] 6.1 Materials and Methods
[0350] 6.1.1 Preparation of DNA
[0351] A human fetal brain cDNA library (Gibco), in which
individual clones were inserted into the NotI-SalI site of the
pCMV.cndot.SPORT2 vector, was diluted 200,000 fold in LB broth
(DIFCO Laboratories) containing 0.2 mg/ml ampicillin (Sigma). The
diluted library (100-140 .mu.l) was then plated and grown on LB
agar (DIFCO Laboratories) bioassay plates with 0.2 mg/ml
ampicillin. Plates were incubated at 37.degree. C. for 24 hours.
Single colonies were then used to inoculate deep-well blocks
containing 1.5 ml LB broth containing 0.2 mg/ml ampicillin.
Inoculated cultures were incubated at 37.degree. C. with agitation
at 150-200 rpm for 18-24 hours. Replicate plates were created from
the cultures by adding 20 .mu.l of culture to 80 .mu.l of LB broth
containing 18% glycerol and 0.2 mg/ml ampicillin and stored at
-80.degree. C. Remaining bacterial cells were centrifuged at
1000.times.g for 6 minutes to collect the cells at the bottom.
Following centrifugation, the broth was decanted off of the
bacterial pellet and the pellet resuspended and then stored in 100
.mu.l of Cell Resuspension Solution (Promega) at 4.degree. C. for
up to one week.
[0352] Plasmid DNA was extracted using Promega MagneSil kits with a
modified protocol. The pelleted bacteria were re-suspended and 50
.mu.l was transferred into a round bottom plate that rests on a
magnet. Cell Lysis Solution (50 .mu.l) was added and the plate was
incubated at room temperature without agitation for 30 seconds.
Following lysis, 70 .mu.l of a Neutralization Solution/MagneSil
Paramagnetic Particles mixture (pre-mixed at a ratio of 6:1) was
added. The reaction was mixed by pipetting and incubated at room
temperature without agitation for 5 minutes to allow the magnetic
particles to be drawn to the magnet. The supernatant containing
plasmid DNA was then transferred to a new plate and stored at
-20.degree. C.
[0353] Individual clones were chosen for their ability to delay or
prevent cell death when introduced into a cell predisposed to
undergoing cell death, relative to a corresponding cell into which
no exogenous protective sequence had been introduced.
[0354] 6.1.2 Sequence Characterization of the DNA
[0355] The cDNA inserts of the clonally pure plasmids which are
selected for their ability to protect cells from cell death when
introduced into cells predisposed to undergo cell death are
sequenced using the ABI Big Dye terminator Cycle Sequencing Ready
Reaction Kit and subsequently analyzed on the ABI310 capillary
sequencing machine (PE Biosystems, Foster City, Calif.).
[0356] Briefly, 0.5 .mu.g of plasmid DNA is mixed with 3.2 pmole of
either the M13 forward (5'-TGTAAAACGACGGCCAGT-3'; SEQ ID NO:465) or
the M13 reverse (5'-CAGGAAACAGCTATGACC-3'; SEQ ID NO:466)
sequencing primer and 8 .mu.l of the terminator ready reaction mix
in a total volume of 20 .mu.l. The cycle sequencing reaction is
carried out in a thermocycler (PCR machine) using standard methods
known by those skilled in the art. The extension products from the
sequencing reaction are purified by precipitation using
isopropanol. 80 .mu.l of 75% isopropanol is added to the sample and
after thorough mixing, the sample is incubated at room temperature
(25.degree. C.) for 20 minutes. The sample is then centrifuged at
12,000.times.g for 20 minutes at room temperature. The supernatant
is removed and the pellet is rinsed once by addition of 250 .mu.l
of 75% isopropanol followed by centrifugation as above for 5
minutes. The supernatant is removed and the sample air-dried for 10
minutes. The sample is then resuspended in 20 .mu.l of TSR
(template suppression reagent) and denatured by heating at
94.degree. C. for 2 minutes and rapidly cooling on ice. The
subsequent electrophoresis and analysis is carried out on the
ABI310 sequencer according to the manufacturer's protocol. The
entire cDNA clone is similarly sequenced by the use of sequence
specific internal primers as required.
[0357] 6.1.3 Sequence Comparison
[0358] The sequence data for the protective cDNA clones is compared
using the BLAST 2.0 algorithm (Altschul, S F et al., 1997, Nuc.
Acids Res. 25:3389) against known sequences in the GeneBank
sequence database maintained by NCBI (National Center for
Biotechnology Information). This program uses the two-hit method to
find homology within the database. The BLAST nucleotide searches
are performed with the "BLAST N" program (wordlength=11) to obtain
nucleic acids homologous to nucleic acid molecules of the
invention. BLAST protein searches of potential ORFs are performed
with the "BLAST P" program (wordlength=3) to obtain amino acid
sequences homologous to the ORFs of the invention.
[0359] 6.1.4 Immuno-cytochemistry Protocol for the Characterization
of Protected Cells
[0360] Transfected tissue is immersed in freshly prepared 2.5%
paraformaldehyde (PFA) in phosphate buffered saline (PBS) for two
hours to fix the tissue. PFA is removed by aspiration and the fixed
tissue washed consecutively four times in PBS for 15 minutes,
changing the PBS solution between each wash. Upon removal of the
final PBS wash, the tissue is immersed in a blocking solution
consisting of 10% goat serum, 2% bovine serum albumin (BSA), and
0.25% Triton X-100 for a duration of two hours.
[0361] After removal of the blocking solution, the tissue is
immersed in a primary antibody solution, freshly prepared by adding
rabbit anti-GFP polyclonal (1:2000 ul) into blocking solution, for
an incubation period of twelve hours at 4.degree. C.
[0362] After removal of the primary antibody solution, the tissue
is washed consecutively four times in PBS for 10 minutes, changing
the PBS solution between each wash. An anti-rabbit, flourescently
conjugated secondary antibody, diluted in PBS at a concentration of
1:500, is then added to the tissue and allowed to incubate at room
temperature for four hours. The secondary antibody solution is
removed by aspiration and the tissue washed consecutively four
times in PBS for 15 minutes, changing the PBS solution between each
wash. After the final wash is removed, the tissue is mounted on
glass slides and dried at 37.degree. C. for thirty minutes. A
three-minute xylene incubation is performed before the addition of
coverslips to preserve the slices.
[0363] 6.2 Results
[0364] The following protective sequences, which were obtained
using the methods described in Section 6.1, were chosen based on
their ability to prevent, delay, or rescue cells predisposed to
undergo cell death, relative to a corresponding cell into which no
exogenous protective sequence had been introduced.
[0365] 6.2.1 Protective Sequence CNI-00711
[0366] Protective sequence CNI-00711 (SEQ ID NO:1) comprises 852
nucleotides. Twelve (12) potential open reading frames ("ORFs")
have been identified within the protective sequence and are
depicted in Table 2. BLAST sequence comparison analysis of
CNI-00711 against known sequences in the GenBank sequence database
reveals 89% homology, at the nucleotide level, with the UV1 exon,
containing part of the envelope region of a human endogenous
retrovirus (HERV) type C (ACC. No. AF058907). The homologous clone
was initially described as a germ-line insertion of HERV into the
human pleiotrophin gene. The insertion occurred between the 5'
untranslated region (UTR) and the coding region. The homology
between CNI-00711 and HERV exists for 366 base pairs out of the 412
bases found within the UV1 exon of the HERV insertion.
[0367] 6.2.2 Protective Sequence CNI-00712
[0368] Protective sequence CNI-00712 (SEQ.ID NO:26) is a completely
novel sequence which comprises 1096 nucleotides. Twenty-four (24)
potential ORFs have been identified within the protective sequence
and are depicted in Table 3. The longest ORF of the clone is 160
amino acids. BLAST sequence comparison analysis of CI-00712 against
known nucleotide and protein sequences in the GenBank database
reveals no significant homology at either the nucleotide or amino
acid level.
[0369] 6.2.3 Protective Sequence CNI-00714
[0370] Protective sequence CNI-00714 (SEQ. ID NO:75) comprises 1825
nucleotides. Thirty (30) potential ORFs have been identified within
the protective sequence and are depicted in Table 4. The longest
ORF of the cDNA encodes 412 amino acids. BLAST sequence comparison
analysis of CNI-00714 against known nucleic acids in the GenBank
database reveals homology with the sequence encoding the human
KIAA0764 gene (ACC. No. AB018307). At the nucleotide level, the
overall percent homology between CNI-00714 and KIAA0764 is 76%. At
the amino acid level, the CNI-00714 and KIAA0764 proteins are
identical except for a 2 amino acid deletion near the N-terminus.
KIAA0764 is an unidentified brain cDNA that shows high level
expression in lung and brain.
[0371] 6.2.4 Protective Sequence CNI-00715
[0372] Protective sequence CNI-00715 (SEQ. ID NO:136) comprises 542
nucleotides. Eight (8) potential ORFs have been identified within
the protective sequence and are depicted in Table 5. BLAST sequence
comparison analysis of CNI-00715 against known nucleic acids in the
GenBank database reveals a 97% identity (503/520 bases) with a
human DNA sequence, clone 425C14, which is from chromosome 6Q22.
This clone contains the heat shock factor 2 gene (HSF2) and an
unknown gene which is similar to the gene which encodes the
placental protein, DIFF33 (ACC. No. HS425C14). Additionally, a
relatively high homology--74% identity (64/87 bases)--is also
observed with a short region within the coding region of the
bestrophin gene. Bestrophin is the gene responsible for Best
macular dystrophy (ACC.No. AF057170).
[0373] 6.2.5 Protective Sequence CNI-00716
[0374] Protective sequence CNI-00716 (SEQ. ID NO:153) is a
completely novel sequence which comprises 771 nucleotides. Fifteen
(15) potential ORFs have been identified within the protective
sequence and are depicted in Table 6. The longest ORF is 58 amino
acids. BLAST sequence comparison analysis of CNI-00716 against
known nucleotide and protein sequences in the GenBank database
reveals no significant homology at either the nucleotide or the
amino acid level.
[0375] 6.2.6 Protective Sequence CNI-00717
[0376] Protective sequence CNI-00717 (SEQ ID NO:184) comprises 1669
nucleotides. Thirty eight (38) potential ORFs have been identified
within the protective sequence and are depicted in Table 7. BLAST
sequence comparison analysis of CNI-00717 against known nucleic
acids in the GenBank database reveals 61% (573/935 bases) identity
within the coding region of the mouse GARP34 mRNA (ACC No.
AB018374). The ORF of CNI-00717 in this region of homology is 272
amino acids in length. When this amino acid sequence is compared to
the amino acid sequence of GARP34, there is 50% identity (132/265
amino acids).
[0377] 6.2.7 Protective Sequence CNI-00720
[0378] Protective sequence CNI-00720 (SEQ. ID NO:261) comprises
1182 nucleotides. Fifteen (15) potential ORFs have been identified
within the protective sequence and are depicted in Table 8. Two
relatively long ORFs of 75 and 89 amino acids were documented.
Neither ORF was homologous to any known sequences in the Genbank
database. BLAST sequence comparison analysis of CNI-00720 against
known nucleic acids in the GenBank database reveals homology with
the 3'UTR of two human genes--neuroendocrine-specif- ic
protein-like protein 1(NSPL1) (ACC.No. 119297) and reticulon 3
(RTN3) (ACC. No. RTN3). NSPL1 and RTN 3 are identical
neuron-specific genes that belong to the reticulon gene family.
There is 99% identity between the nucleic acid of CNI-00720 and the
3' UTRs of NSPL1 and RTN3. Additionally, there is 97% identity
within the 5' UTR of the human protein tyrosine kinase,
t-Ror-1(Ror1) mRNA (ACC. No.HSU38894).
[0379] 6.2.8 Protective Sequence CNI-00721
[0380] Protective sequence CNI-00721 (SEQ. ID NO:292) comprises
1965 nucleotides. Thirty-three (33) potential ORFs have been
identified within the protective sequence and are depicted in Table
9. BLAST sequence comparison analysis of CNI-00721 against known
nucleic acids in the GenBank database revealed strong homology with
the human p311 mRNA (ACC. No.HSU36189). There is 99% identity
(465/470 bases) between the CNI-00721 cDNA and the p311 mRNA. The
homology is 100% within the coding sequence region.
[0381] 6.2.9 Protective Sequence CNI-00723
[0382] Protective sequence CNI-00723 (SEQ. ID NO:359) comprises
2702 nucleotides. Fifty-one (51) potential ORFs have been
identified within the protective sequence and are depicted in Table
10. BLAST sequence comparison analysis of CNI-00723 against known
nucleic acids in the GenBank database reveals a short stretch of
homology with the Drosophila asteroid mRNA (ACC.No AF047010). The
CNI-00723 cDNA is 35% identical (73/209 bases) to the asteroid mRNA
with the homology occurring within the coding region of the mRNA.
The longest ORF of CNI-00723 is 490 amino acids. This ORF is 37%
identical (55/144 amino acids) to the Drosophila asteroid
protein.
[0383] 6.2.10 Protective Sequence CNI-00724
[0384] Protective sequence CNI-00724 (SEQ. ID NO:462) is a
completely novel sequence which comprises 979 nucleotides. Only a
single potential ORF has been identified within the protective
sequence and it is depicted in Table 11. This ORF, which is 80
amino acids in length, revealed no homology with any known amino
acid sequence in the GenBank database. BLAST sequence comparison
analysis of CNI-00724 against known nucleic acids in the GenBank
database reveals that homology exists with a human VGF nerve growth
factor inducible mRNA (ACC. No. NM.sub.--003378). There is 94%
identity (821/872 bases) between the CNI-00724 insert and the VGF
nerve growth factory inducible mRNA. This homology is observed in
the 3' portion of the coding sequence and in the 3' UTR of the VGF
nerve growth factory inducible mRNA.
[0385] 7 Deposit of Microorganisms
[0386] The following microorganisms were deposited with the
Agricultural Research Service (NRRL), U. S. Department of
Agriculture, 1815 N. University Street, Peoria, Ill., 61604, under
the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure, and comply with the criteria set forth in 37
C.F.R. .sctn.1.801-1.809 regarding availability and permanency of
deposits. The deposits were made on the date indicated and assigned
the indicated accession number:
12 Microorganism Deposit NRRL Deposit No. Date of Deposit
Escherichia coli B-30231 November 3, 1999 CNI-NPP1-CP10
[0387] CNI-NPP1-CP10 represents a composite deposit of a mixture of
ten (10) strains. To distinguish and isolate each of the individual
strains, an aliquot of the mixture can be streaked out to single
colonies on nutrient media (e.g., LB plates) supplemented with 100
.mu.g/ml ampicillin, single colonies grown and then DNA can be
extracted using standard procedures.
[0388] Next, a sample of the DNA preparation can be digested with
Not I and Sal I, and the resulting products can be separated by
standard gel electrophoresis techniques using a 1% agarose gel in
TAE buffer. Liberated inserts are of the following approximate
sizes:
13 1: CNI-00711 852 bp 2: CNI-00712 1096 bp 3: CNI-00714 1825 bp 4:
CNI-00715 542 bp 5: CNI-00716 771 bp 6: CNI-00717 1669 bp 7:
CNI-00720 1182 bp 8: CNI-00721 1965 bp 9: CNI-00723 2702 bp 10:
CNI-00724 979 bp
[0389] 8 References Cited
[0390] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings.
[0391] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
466 1 852 DNA Homo sapiens 1 tcgacccacg cgtccgggaa catatctcaa
aataataata actatttatg acaaacccac 60 agtcaatatc atactgaatg
ggcaaaagct ggaagcattc taaataccaa aggacatcat 120 tagttaacaa
atgctagact aactagatac caaagcttgc tctgtgaaaa atccccacat 180
aaccattgaa gtttacaaca ccctaaaccc tgccaccttg ctcccagtat cagagagccc
240 agttaaacat aactatgtag aggtattaga ctcagtttat tctagtaggc
ccaacctcca 300 agaccatcgt tgaacatcag tagactggga gctgtacgtg
gatgggagca gctttgccaa 360 cccctgcaaa gtgactcttg aagaagacca
caaaccctgc tccagtcaac atctggaagc 420 ttgactagtc cacgcatggc
tgaagcatga ggaaactcat cacaggactc attttcctta 480 aaatttagac
ttgtacagta aagacttcaa cttgaccttc ctcagactga gggctgttcc 540
cagagtatac atcaagtcac tgaggtagga caaaaggttg ctacagtcct attattttac
600 agttattata agtgtactgg aactctaaaa agaacttgtt tttataatgt
tattctatac 660 aattatttat aatacaatat acaaataatg tatttagccc
aggaaatgac caacctgatg 720 tgtgttatga cccatctgag cctcccatga
ccacagtttt taaaataaga ttaagaactg 780 aagactggtg ggggctcata
aacaatatga gtaaagtgtt agccaaaata aaacaaaaaa 840 aaaagggcgg cc 852 2
30 DNA Homo sapiens 2 atgacaaacc cacagtcaat atcatactga 30 3 9 PRT
Homo sapiens 3 Met Thr Asn Pro Gln Ser Ile Ser Tyr 1 5 4 60 DNA
Homo sapiens 4 atgggcaaaa gctggaagca ttctaaatac caaaggacat
cattagttaa caaatgctag 60 5 19 PRT Homo sapiens 5 Met Gly Lys Ser
Trp Lys His Ser Lys Tyr Gln Arg Thr Ser Leu Val 1 5 10 15 Asn Lys
Cys 6 12 DNA Homo sapiens 6 atgctagact aa 12 7 3 PRT Homo sapiens 7
Met Leu Asp 1 8 33 DNA Homo sapiens 8 atgggagcag ctttgccaac
ccctgcaaag tga 33 9 10 PRT Homo sapiens 9 Met Gly Ala Ala Leu Pro
Thr Pro Ala Lys 1 5 10 10 15 DNA Homo sapiens 10 atggctgaag catga
15 11 4 PRT Homo sapiens 11 Met Ala Glu Ala 1 12 42 DNA Homo
sapiens 12 atgaggaaac tcatcacagg actcattttc cttaaaattt ag 42 13 13
PRT Homo sapiens 13 Met Arg Lys Leu Ile Thr Gly Leu Ile Phe Leu Lys
Ile 1 5 10 14 42 DNA Homo sapiens 14 atgttattct atacaattat
ttataataca atatacaaat aa 42 15 13 PRT Homo sapiens 15 Met Leu Phe
Tyr Thr Ile Ile Tyr Asn Thr Ile Tyr Lys 1 5 10 16 63 DNA Homo
sapiens 16 atgtatttag cccaggaaat gaccaacctg atgtgtgtta tgacccatct
gagcctccca 60 tga 63 17 20 PRT Homo sapiens 17 Met Tyr Leu Ala Gln
Glu Met Thr Asn Leu Met Cys Val Met Thr His 1 5 10 15 Leu Ser Leu
Pro 20 18 45 DNA Homo sapiens 18 atgaccaacc tgatgtgtgt tatgacccat
ctgagcctcc catga 45 19 14 PRT Homo sapiens 19 Met Thr Asn Leu Met
Cys Val Met Thr His Leu Ser Leu Pro 1 5 10 20 33 DNA Homo sapiens
20 atgtgtgtta tgacccatct gagcctccca tga 33 21 10 PRT Homo sapiens
21 Met Cys Val Met Thr His Leu Ser Leu Pro 1 5 10 22 24 DNA Homo
sapiens 22 atgacccatc tgagcctccc atga 24 23 7 PRT Homo sapiens 23
Met Thr His Leu Ser Leu Pro 1 5 24 106 DNA Homo sapiens 24
atgaccacag tttttaaaat aagattaaga actgaagact ggtgggggct cataaacaat
60 atgagtaaag tgttagccaa aataaaacaa aaaaaaaagg gcggcc 106 25 35 PRT
Homo sapiens 25 Met Thr Thr Val Phe Lys Ile Arg Leu Arg Thr Glu Asp
Trp Trp Gly 1 5 10 15 Leu Ile Asn Asn Met Ser Lys Val Leu Ala Lys
Ile Lys Gln Lys Lys 20 25 30 Lys Gly Gly 35 26 1096 DNA Homo
sapiens 26 tcgacccacg cgtccgggca tggccaggcc ggctgggctg cagagcgccg
gcacgggtcc 60 acgcctcggg tgacgggctt ccaggatgtt cgggcgcggg
gcggcccatc cgcatccccc 120 aacaccccca cctccggcct gagcctccca
gcgccggggg aaccacctcc tgtccgctgt 180 tgctggcccg catcctagca
gcggcctgac gccctcccca ccctggcatg cccccttgac 240 ctgggacgat
gagcatacga ctggggagcc cagtggaggc gccctcccga agcgccactg 300
cccatgctga ccacccagcc ctccggctgc tgatgtcatg agtaacacca ctgtgcccaa
360 tgccccccag gccaacagcg actccatggt gggctatgtg ttggggccct
tcttcctcat 420 caccctggtc ggggtggtgg tggctgtggt aatgtatgta
cagaagaaaa agcgggtgga 480 ccggctgcgc catcacctgc tccccatgta
cagctatgac ccagctgagg aactgcatga 540 ggctgagcag gagctgctct
ctgacatggg agaccccaag gtggtacatg gctggcagag 600 tggctaccag
cacaagcgga tgccactgct ggatgtcaag acgtgacctg acccccttgc 660
cccacccttc agagcctggg gtcctggact gcctggggcc ctgccatctg cttcccctgc
720 tgtcacctgg ctccccctgc tgggtgctgg gtctccattt ctccctccac
ccaccctcag 780 cagcatctgc ttcccatgcc ctcaccatca cctcactgcc
cccaggcctt ctgccctttg 840 tgggtgttga gctcaccgcc cacccacagg
cactcatagg aagaggcttt ccttctggga 900 tggcggcggc tggtagacac
ctttgctttc tctagccctc ctgggctggg cttgggccca 960 aatccccagg
caggctttgg agttgtttcc atggtgatgg ggccagatgt atagtattca 1020
gtatatattt tgtaaataaa atgttttgtg gctaaaaaaa aaaaaaaaaa aaaaaaaaaa
1080 aaaaaaaagg gcggcc 1096 27 54 DNA Homo sapiens 27 atggccaggc
cggctgggct gcagagcgcc ggcacgggtc cacgcctcgg gtga 54 28 17 PRT Homo
sapiens 28 Met Ala Arg Pro Ala Gly Leu Gln Ser Ala Gly Thr Gly Pro
Arg Leu 1 5 10 15 Gly 29 57 DNA Homo sapiens 29 atgttcgggc
gcggggcggc ccatccgcat cccccaacac ccccacctcc ggcctga 57 30 18 PRT
Homo sapiens 30 Met Phe Gly Arg Gly Ala Ala His Pro His Pro Pro Thr
Pro Pro Pro 1 5 10 15 Pro Ala 31 12 DNA Homo sapiens 31 atgccccctt
ga 12 32 3 PRT Homo sapiens 32 Met Pro Pro 1 33 93 DNA Homo sapiens
33 atgagcatac gactggggag cccagtggag gcgccctccc gaagcgccac
tgcccatgct 60 gaccacccag ccctccggct gctgatgtca tga 93 34 30 PRT
Homo sapiens 34 Met Ser Ile Arg Leu Gly Ser Pro Val Glu Ala Pro Ser
Arg Ser Ala 1 5 10 15 Thr Ala His Ala Asp His Pro Ala Leu Arg Leu
Leu Met Ser 20 25 30 35 30 DNA Homo sapiens 35 atgctgacca
cccagccctc cggctgctga 30 36 9 PRT Homo sapiens 36 Met Leu Thr Thr
Gln Pro Ser Gly Cys 1 5 37 309 DNA Homo sapiens 37 atgagtaaca
ccactgtgcc caatgccccc caggccaaca gcgactccat ggtgggctat 60
gtgttggggc ccttcttcct catcaccctg gtcggggtgg tggtggctgt ggtaatgtat
120 gtacagaaga aaaagcgggt ggaccggctg cgccatcacc tgctccccat
gtacagctat 180 gacccagctg aggaactgca tgaggctgag caggagctgc
tctctgacat gggagacccc 240 aaggtggtac atggctggca gagtggctac
cagcacaagc ggatgccact gctggatgtc 300 aagacgtga 309 38 102 PRT Homo
sapiens 38 Met Ser Asn Thr Thr Val Pro Asn Ala Pro Gln Ala Asn Ser
Asp Ser 1 5 10 15 Met Val Gly Tyr Val Leu Gly Pro Phe Phe Leu Ile
Thr Leu Val Gly 20 25 30 Val Val Val Ala Val Val Met Tyr Val Gln
Lys Lys Lys Arg Val Asp 35 40 45 Arg Leu Arg His His Leu Leu Pro
Met Tyr Ser Tyr Asp Pro Ala Glu 50 55 60 Glu Leu His Glu Ala Glu
Gln Glu Leu Leu Ser Asp Met Gly Asp Pro 65 70 75 80 Lys Val Val His
Gly Trp Gln Ser Gly Tyr Gln His Lys Arg Met Pro 85 90 95 Leu Leu
Asp Val Lys Thr 100 39 93 DNA Homo sapiens 39 atgcccccca ggccaacagc
gactccatgg tgggctatgt gttggggccc ttcttcctca 60 tcaccctggt
cggggtggtg gtggctgtgg taa 93 40 30 PRT Homo sapiens 40 Met Pro Pro
Arg Pro Thr Ala Thr Pro Trp Trp Ala Met Cys Trp Gly 1 5 10 15 Pro
Ser Ser Ser Ser Pro Trp Ser Gly Trp Trp Trp Leu Trp 20 25 30 41 261
DNA Homo sapiens 41 atggtgggct atgtgttggg gcccttcttc ctcatcaccc
tggtcggggt ggtggtggct 60 gtggtaatgt atgtacagaa gaaaaagcgg
gtggaccggc tgcgccatca cctgctcccc 120 atgtacagct atgacccagc
tgaggaactg catgaggctg agcaggagct gctctctgac 180 atgggagacc
ccaaggtggt acatggctgg cagagtggct accagcacaa gcggatgcca 240
ctgctggatg tcaagacgtg a 261 42 72 PRT Homo sapiens 42 Met Val Gly
Tyr Val Leu Gly Pro Phe Phe Leu Ile Thr Leu Val Gly 1 5 10 15 Val
Val Val Ala Val Val Met Tyr Val Gln Lys Lys Lys Arg Val Asp 20 25
30 Arg Leu Arg His His Leu Leu Pro Met Tyr Ser Tyr Asp Pro Ala Glu
35 40 45 Glu Leu His Glu Ala Glu Gln Glu Leu Leu Ser Asp Met Gly
Asp Pro 50 55 60 Lys Val Val His Gly Trp Gln Ser 65 70 43 57 DNA
Homo sapiens 43 atgtgttggg gcccttcttc ctcatcaccc tggtcggggt
ggtggtggct gtggtaa 57 44 18 PRT Homo sapiens 44 Met Cys Trp Gly Pro
Ser Ser Ser Ser Pro Trp Ser Gly Trp Trp Trp 1 5 10 15 Leu Trp 45
195 DNA Homo sapiens 45 atgtatgtac agaagaaaaa gcgggtggac cggctgcgcc
atcacctgct ccccatgtac 60 agctatgacc cagctgagga actgcatgag
gctgagcagg agctgctctc tgacatggga 120 gaccccaagg tggtacatgg
ctggcagagt ggctaccagc acaagcggat gccactgctg 180 gatgtcaaga cgtga
195 46 64 PRT Homo sapiens 46 Met Tyr Val Gln Lys Lys Lys Arg Val
Asp Arg Leu Arg His His Leu 1 5 10 15 Leu Pro Met Tyr Ser Tyr Asp
Pro Ala Glu Glu Leu His Glu Ala Glu 20 25 30 Gln Glu Leu Leu Ser
Asp Met Gly Asp Pro Lys Val Val His Gly Trp 35 40 45 Gln Ser Gly
Tyr Gln His Lys Arg Met Pro Leu Leu Asp Val Lys Thr 50 55 60 47 480
DNA Homo sapiens 47 atgtacagaa gaaaaagcgg gtggaccggc tgcgccatca
cctgctcccc atgtacagct 60 atgacccagc tgaggaactg catgaggctg
agcaggagct gctctctgac atgggagacc 120 ccaaggtggt acatggctgg
cagagtggct accagcacaa gcggatgcca ctgctggatg 180 tcaagacgtg
acctgacccc cttgccccac ccttcagagc ctggggtcct ggactgcctg 240
gggccctgcc atctgcttcc cctgctgtca cctggctccc cctgctgggt gctgggtctc
300 catttctccc tccacccacc ctcagcagca tctgcttccc atgccctcac
catcacctca 360 ctgcccccag gccttctgcc ctttgtgggt gttgagctca
ccgcccaccc acaggcactc 420 ataggaagag gctttccttc tgggatggcg
gcggctggta gacacctttg ctttctctag 480 48 159 PRT Homo sapiens 48 Met
Tyr Arg Arg Lys Ser Gly Trp Thr Gly Cys Ala Ile Thr Cys Ser 1 5 10
15 Pro Cys Thr Ala Met Thr Gln Leu Arg Asn Cys Met Arg Leu Ser Arg
20 25 30 Ser Cys Ser Leu Thr Trp Glu Thr Pro Arg Trp Tyr Met Ala
Gly Arg 35 40 45 Val Ala Thr Ser Thr Ser Gly Cys His Cys Trp Met
Ser Arg Arg Asp 50 55 60 Leu Thr Pro Leu Pro His Pro Ser Glu Pro
Gly Val Leu Asp Cys Leu 65 70 75 80 Gly Pro Cys His Leu Leu Pro Leu
Leu Ser Pro Gly Ser Pro Cys Trp 85 90 95 Val Leu Gly Leu His Phe
Ser Leu His Pro Pro Ser Ala Ala Ser Ala 100 105 110 Ser His Ala Leu
Thr Ile Thr Ser Leu Pro Pro Gly Leu Leu Pro Phe 115 120 125 Val Gly
Val Glu Leu Thr Ala His Pro Gln Ala Leu Ile Gly Arg Gly 130 135 140
Phe Pro Ser Gly Met Ala Ala Ala Gly Arg His Leu Cys Phe Leu 145 150
155 49 141 DNA Homo sapiens 49 atgtacagct atgacccagc tgaggaactg
catgaggctg agcaggagct gctctctgac 60 atgggagacc ccaaggtggt
acatggctgg cagagtggct accagcacaa gcggatgcca 120 ctgctggatg
tcaagacgtg a 141 50 46 PRT Homo sapiens 50 Met Tyr Ser Tyr Asp Pro
Ala Glu Glu Leu His Glu Ala Glu Gln Glu 1 5 10 15 Leu Leu Ser Asp
Met Gly Asp Pro Lys Val Val His Gly Trp Gln Ser 20 25 30 Gly Tyr
Gln His Lys Arg Met Pro Leu Leu Asp Val Lys Thr 35 40 45 51 420 DNA
Homo sapiens 51 atgacccagc tgaggaactg catgaggctg agcaggagct
gctctctgac atgggagacc 60 ccaaggtggt acatggctgg cagagtggct
accagcacaa gcggatgcca ctgctggatg 120 tcaagacgtg acctgacccc
cttgccccac ccttcagagc ctggggtcct ggactgcctg 180 gggccctgcc
atctgcttcc cctgctgtca cctggctccc cctgctgggt gctgggtctc 240
catttctccc tccacccacc ctcagcagca tctgcttccc atgccctcac catcacctca
300 ctgcccccag gccttctgcc ctttgtgggt gttgagctca ccgcccaccc
acaggcactc 360 ataggaagag gctttccttc tgggatggcg gcggctggta
gacacctttg ctttctctag 420 52 139 PRT Homo sapiens 52 Met Thr Gln
Leu Arg Asn Cys Met Arg Leu Ser Arg Ser Cys Ser Leu 1 5 10 15 Thr
Trp Glu Thr Pro Arg Trp Tyr Met Ala Gly Arg Val Ala Thr Ser 20 25
30 Thr Ser Gly Cys His Cys Trp Met Ser Arg Arg Asp Leu Thr Pro Leu
35 40 45 Pro His Pro Ser Glu Pro Gly Val Leu Asp Cys Leu Gly Pro
Cys His 50 55 60 Leu Leu Pro Leu Leu Ser Pro Gly Ser Pro Cys Trp
Val Leu Gly Leu 65 70 75 80 His Phe Ser Leu His Pro Pro Ser Ala Ala
Ser Ala Ser His Ala Leu 85 90 95 Thr Ile Thr Ser Leu Pro Pro Gly
Leu Leu Pro Phe Val Gly Val Glu 100 105 110 Leu Thr Ala His Pro Gln
Ala Leu Ile Gly Arg Gly Phe Pro Ser Gly 115 120 125 Met Ala Ala Ala
Gly Arg His Leu Cys Phe Leu 130 135 53 399 DNA Homo sapiens 53
atgaggctga gcaggagctg ctctctgaca tgggagaccc caaggtggta catggctggc
60 agagtggcta ccagcacaag cggatgccac tgctggatgt caagacgtga
cctgaccccc 120 ttgccccacc cttcagagcc tggggtcctg gactgcctgg
ggccctgcca tctgcttccc 180 ctgctgtcac ctggctcccc ctgctgggtg
ctgggtctcc atttctccct ccacccaccc 240 tcagcagcat ctgcttccca
tgccctcacc atcacctcac tgcccccagg ccttctgccc 300 tttgtgggtg
ttgagctcac cgcccaccca caggcactca taggaagagg ctttccttct 360
gggatggcgg cggctggtag acacctttgc tttctctag 399 54 132 PRT Homo
sapiens 54 Met Arg Leu Ser Arg Ser Cys Ser Leu Thr Trp Glu Thr Pro
Arg Trp 1 5 10 15 Tyr Met Ala Gly Arg Val Ala Thr Ser Thr Ser Gly
Cys His Cys Trp 20 25 30 Met Ser Arg Arg Asp Leu Thr Pro Leu Pro
His Pro Ser Glu Pro Gly 35 40 45 Val Leu Asp Cys Leu Gly Pro Cys
His Leu Leu Pro Leu Leu Ser Pro 50 55 60 Gly Ser Pro Cys Trp Val
Leu Gly Leu His Phe Ser Leu His Pro Pro 65 70 75 80 Ser Ala Ala Ser
Ala Ser His Ala Leu Thr Ile Thr Ser Leu Pro Pro 85 90 95 Gly Leu
Leu Pro Phe Val Gly Val Glu Leu Thr Ala His Pro Gln Ala 100 105 110
Leu Ile Gly Arg Gly Phe Pro Ser Gly Met Ala Ala Ala Gly Arg His 115
120 125 Leu Cys Phe Leu 130 55 81 DNA Homo sapiens 55 atgggagacc
ccaaggtggt acatggctgg cagagtggct accagcacaa gcggatgcca 60
ctgctggatg tcaagacgtg a 81 56 26 PRT Homo sapiens 56 Met Gly Asp
Pro Lys Val Val His Gly Trp Gln Ser Gly Tyr Gln His 1 5 10 15 Lys
Arg Met Pro Leu Leu Asp Val Lys Thr 20 25 57 348 DNA Homo sapiens
57 atggctggca gagtggctac cagcacaagc ggatgccact gctggatgtc
aagacgtgac 60 ctgaccccct tgccccaccc ttcagagcct ggggtcctgg
actgcctggg gccctgccat 120 ctgcttcccc tgctgtcacc tggctccccc
tgctgggtgc tgggtctcca tttctccctc 180 cacccaccct cagcagcatc
tgcttcccat gccctcacca tcacctcact gcccccaggc 240 cttctgccct
ttgtgggtgt tgagctcacc gcccacccac aggcactcat aggaagaggc 300
tttccttctg ggatggcggc ggctggtaga cacctttgct ttctctag 348 58 115 PRT
Homo sapiens 58 Met Ala Gly Arg Val Ala Thr Ser Thr Ser Gly Cys His
Cys Trp Met 1 5 10 15 Ser Arg Arg Asp Leu Thr Pro Leu Pro His Pro
Ser Glu Pro Gly Val 20 25 30 Leu Asp Cys Leu Gly Pro Cys His Leu
Leu Pro Leu Leu Ser Pro Gly 35 40 45 Ser Pro Cys Trp Val Leu Gly
Leu His Phe Ser Leu His Pro Pro Ser 50 55 60 Ala Ala Ser Ala Ser
His Ala Leu Thr Ile Thr Ser Leu Pro Pro Gly 65 70 75 80 Leu Leu Pro
Phe Val Gly Val Glu Leu Thr Ala His Pro Gln Ala Leu 85 90 95 Ile
Gly Arg Gly Phe Pro Ser Gly Met Ala Ala Ala Gly Arg His Leu 100 105
110 Cys Phe Leu 115 59 27 DNA Homo sapiens 59 atgccactgc tggatgtcaa
gacgtga 27 60 8 PRT Homo sapiens 60 Met Pro Leu Leu Asp Val Lys Thr
1 5 61 303 DNA Homo sapiens 61 atgtcaagac gtgacctgac ccccttgccc
cacccttcag agcctggggt cctggactgc 60 ctggggccct gccatctgct
tcccctgctg tcacctggct ccccctgctg ggtgctgggt 120 ctccatttct
ccctccaccc accctcagca gcatctgctt cccatgccct caccatcacc 180
tcactgcccc caggccttct gccctttgtg ggtgttgagc tcaccgccca cccacaggca
240 ctcataggaa gaggctttcc ttctgggatg gcggcggctg gtagacacct
ttgctttctc 300
tag 303 62 100 PRT Homo sapiens 62 Met Ser Arg Arg Asp Leu Thr Pro
Leu Pro His Pro Ser Glu Pro Gly 1 5 10 15 Val Leu Asp Cys Leu Gly
Pro Cys His Leu Leu Pro Leu Leu Ser Pro 20 25 30 Gly Ser Pro Cys
Trp Val Leu Gly Leu His Phe Ser Leu His Pro Pro 35 40 45 Ser Ala
Ala Ser Ala Ser His Ala Leu Thr Ile Thr Ser Leu Pro Pro 50 55 60
Gly Leu Leu Pro Phe Val Gly Val Glu Leu Thr Ala His Pro Gln Ala 65
70 75 80 Leu Ile Gly Arg Gly Phe Pro Ser Gly Met Ala Ala Ala Gly
Arg His 85 90 95 Leu Cys Phe Leu 100 63 84 DNA Homo sapiens 63
atgccctcac catcacctca ctgcccccag gccttctgcc ctttgtgggt gttgagctca
60 ccgcccaccc acaggcactc atag 84 64 27 PRT Homo sapiens 64 Met Pro
Ser Pro Ser Pro His Cys Pro Gln Ala Phe Cys Pro Leu Trp 1 5 10 15
Val Leu Ser Ser Pro Pro Thr His Arg His Ser 20 25 65 36 DNA Homo
sapiens 65 atggcggcgg ctggtagaca cctttgcttt ctctag 36 66 11 PRT
Homo sapiens 66 Met Ala Ala Ala Gly Arg His Leu Cys Phe Leu 1 5 10
67 24 DNA Homo sapiens 67 atggtgatgg ggccagatgt atag 24 68 7 PRT
Homo sapiens 68 Met Val Met Gly Pro Asp Val 1 5 69 18 DNA Homo
sapiens 69 atggggccag atgtatag 18 70 5 PRT Homo sapiens 70 Met Gly
Pro Asp Val 1 5 71 33 DNA Homo sapiens 71 atgtatagta ttcagtatat
attttgtaaa taa 33 72 10 PRT Homo sapiens 72 Met Tyr Ser Ile Gln Tyr
Ile Phe Cys Lys 1 5 10 73 15 DNA Homo sapiens 73 atgttttgtg gctaa
15 74 4 PRT Homo sapiens 74 Met Phe Cys Gly 1 75 1825 DNA Homo
sapiens 75 tcgacccacg cgtccgtctt attccaaaat gttgagatac tggggagaga
taccaatatc 60 atcaagccag accaacagaa gttccttcga tttgctccca
cgggagttcc gtctggtgga 120 agtccatgac ccacccctgc accaaccctc
agccaacaag ccgaagcccc ccactatgct 180 ggacatcccc tcagagccat
gtagtctcac catccatacg attcagttga ttcagcacaa 240 ccgacgtctt
cgcaacctta ttgccacagc tcaggcccag aatcagcagc agacagaagg 300
tgtaaaaact gaagagagtg aacctcttcc ctcgtgccct gggtcacctc ctctccctga
360 tgacctcctg cctttagatt gtaagaatcc caatgcacca ttccagatcc
ggcacagtga 420 cccagagagt gacttttatc gtgggaaagg ggaacctgtg
actgaactca gctggcactc 480 ctgtcggcag ctcctctacc aggcagtggc
cacaatcctg gcccacgcgg gctttgactg 540 tgctaatgag agtgtcctgg
agaccctaac tgatgtggca catgagtatt gccttaagtt 600 taccaagttg
ctgcgttttg ctgtggaccg ggaggcccgg ctgggacaga ctccttttcc 660
tgatgtgatg gagcaggtat tccatgaagt gggtattggc agtgtgctct ccctccagaa
720 gttctggcag caccgcatca aggactatca cagttacatg ctacagatta
gtaagcaact 780 ctctgaagaa tatgaaagga ttgtcaatcc tgagaaggcc
acagaggacg ctaaacctgt 840 gaagatcaag gaggaacctg tgagcgacat
cacttttcct gtcagtgagg agctggaggc 900 tgaccttgct tctggagacc
agtcactgcc tatgggagtg cttggggctc agagcgaacg 960 cttcccatct
aacctggagg ttgaagcttc accacaggct tcaagtgcag aggtaaatgc 1020
ttctcctctt tggaatctgg cccatgtgaa aatggagcct caagaaagtg aagaaggcaa
1080 tgtctctggg catggtgtgc tgggcagtga tgtcttcgag gagcctatgt
caggcatgag 1140 tgaagctggg attcctcaga gccctgatga ctcagatagc
agctatggtt cccactccac 1200 tgacagcctc atggggtcct cccctgtttt
caaccagcgc tgcaagaaga ggatgaggaa 1260 aatataaaag gaaaagaggg
agatgttttg tccagaccta ctagacccaa cagaaaaggt 1320 tagctgacta
cagcagaccc tttgcagcag tagttttaac attgacttca catattcaga 1380
agtgattcta aaggactgtg gcacatagaa atgtattttg ctgagctgta caacaggatg
1440 gcacaaaatc ctgctgatag aaataagtgt aaccggccag gcacagtggc
tcatgcctgt 1500 aatcccagca ttttgggagg cccaggtggg tggatcatct
gaggtcagga gttcgagacc 1560 agcctgacca acatggaaaa aaccccatct
ctactaaaaa tacaaaatta gccgggtgtg 1620 gtggcacatg cctgtaatcc
cagctactca ggaaggctga ggcaggagaa ctgcttgaac 1680 ctgggaggtg
gaggttgtgg tgagccgaga ctccagcctg ggcaacaaga gtgaaactcc 1740
gtctcaaaaa taaataaata aataaaagaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1800 aaaaaaaaaa aaaaaaaggg cggcc 1825 76 1239 DNA Homo sapiens 76
atgttgagat actggggaga gataccaata tcatcaagcc agaccaacag aagttccttc
60 gatttgctcc cacgggagtt ccgtctggtg gaagtccatg acccacccct
gcaccaaccc 120 tcagccaaca agccgaagcc ccccactatg ctggacatcc
cctcagagcc atgtagtctc 180 accatccata cgattcagtt gattcagcac
aaccgacgtc ttcgcaacct tattgccaca 240 gctcaggccc agaatcagca
gcagacagaa ggtgtaaaaa ctgaagagag tgaacctctt 300 ccctcgtgcc
ctgggtcacc tcctctccct gatgacctcc tgcctttaga ttgtaagaat 360
cccaatgcac cattccagat ccggcacagt gacccagaga gtgactttta tcgtgggaaa
420 ggggaacctg tgactgaact cagctggcac tcctgtcggc agctcctcta
ccaggcagtg 480 gccacaatcc tggcccacgc gggctttgac tgtgctaatg
agagtgtcct ggagacccta 540 actgatgtgg cacatgagta ttgccttaag
tttaccaagt tgctgcgttt tgctgtggac 600 cgggaggccc ggctgggaca
gactcctttt cctgatgtga tggagcaggt attccatgaa 660 gtgggtattg
gcagtgtgct ctccctccag aagttctggc agcaccgcat caaggactat 720
cacagttaca tgctacagat tagtaagcaa ctctctgaag aatatgaaag gattgtcaat
780 cctgagaagg ccacagagga cgctaaacct gtgaagatca aggaggaacc
tgtgagcgac 840 atcacttttc ctgtcagtga ggagctggag gctgaccttg
cttctggaga ccagtcactg 900 cctatgggag tgcttggggc tcagagcgaa
cgcttcccat ctaacctgga ggttgaagct 960 tcaccacagg cttcaagtgc
agaggtaaat gcttctcctc tttggaatct ggcccatgtg 1020 aaaatggagc
ctcaagaaag tgaagaaggc aatgtctctg ggcatggtgt gctgggcagt 1080
gatgtcttcg aggagcctat gtcaggcatg agtgaagctg ggattcctca gagccctgat
1140 gactcagata gcagctatgg ttcccactcc actgacagcc tcatggggtc
ctcccctgtt 1200 ttcaaccagc gctgcaagaa gaggatgagg aaaatataa 1239 77
412 PRT Homo sapiens 77 Met Leu Arg Tyr Trp Gly Glu Ile Pro Ile Ser
Ser Ser Gln Thr Asn 1 5 10 15 Arg Ser Ser Phe Asp Leu Leu Pro Arg
Glu Phe Arg Leu Val Glu Val 20 25 30 His Asp Pro Pro Leu His Gln
Pro Ser Ala Asn Lys Pro Lys Pro Pro 35 40 45 Thr Met Leu Asp Ile
Pro Ser Glu Pro Cys Ser Leu Thr Ile His Thr 50 55 60 Ile Gln Leu
Ile Gln His Asn Arg Arg Leu Arg Asn Leu Ile Ala Thr 65 70 75 80 Ala
Gln Ala Gln Asn Gln Gln Gln Thr Glu Gly Val Lys Thr Glu Glu 85 90
95 Ser Glu Pro Leu Pro Ser Cys Pro Gly Ser Pro Pro Leu Pro Asp Asp
100 105 110 Leu Leu Pro Leu Asp Cys Lys Asn Pro Asn Ala Pro Phe Gln
Ile Arg 115 120 125 His Ser Asp Pro Glu Ser Asp Phe Tyr Arg Gly Lys
Gly Glu Pro Val 130 135 140 Thr Glu Leu Ser Trp His Ser Cys Arg Gln
Leu Leu Tyr Gln Ala Val 145 150 155 160 Ala Thr Ile Leu Ala His Ala
Gly Phe Asp Cys Ala Asn Glu Ser Val 165 170 175 Leu Glu Thr Leu Thr
Asp Val Ala His Glu Tyr Cys Leu Lys Phe Thr 180 185 190 Lys Leu Leu
Arg Phe Ala Val Asp Arg Glu Ala Arg Leu Gly Gln Thr 195 200 205 Pro
Phe Pro Asp Val Met Glu Gln Val Phe His Glu Val Gly Ile Gly 210 215
220 Ser Val Leu Ser Leu Gln Lys Phe Trp Gln His Arg Ile Lys Asp Tyr
225 230 235 240 His Ser Tyr Met Leu Gln Ile Ser Lys Gln Leu Ser Glu
Glu Tyr Glu 245 250 255 Arg Ile Val Asn Pro Glu Lys Ala Thr Glu Asp
Ala Lys Pro Val Lys 260 265 270 Ile Lys Glu Glu Pro Val Ser Asp Ile
Thr Phe Pro Val Ser Glu Glu 275 280 285 Leu Glu Ala Asp Leu Ala Ser
Gly Asp Gln Ser Leu Pro Met Gly Val 290 295 300 Leu Gly Ala Gln Ser
Glu Arg Phe Pro Ser Asn Leu Glu Val Glu Ala 305 310 315 320 Ser Pro
Gln Ala Ser Ser Ala Glu Val Asn Ala Ser Pro Leu Trp Asn 325 330 335
Leu Ala His Val Lys Met Glu Pro Gln Glu Ser Glu Glu Gly Asn Val 340
345 350 Ser Gly His Gly Val Leu Gly Ser Asp Val Phe Glu Glu Pro Met
Ser 355 360 365 Gly Met Ser Glu Ala Gly Ile Pro Gln Ser Pro Asp Asp
Ser Asp Ser 370 375 380 Ser Tyr Gly Ser His Ser Thr Asp Ser Leu Met
Gly Ser Ser Pro Val 385 390 395 400 Phe Asn Gln Arg Cys Lys Lys Arg
Met Arg Lys Ile 405 410 78 105 DNA Homo sapiens 78 atgacccacc
cctgcaccaa ccctcagcca acaagccgaa gccccccact atgctggaca 60
tcccctcaga gccatgtagt ctcaccatcc atacgattca gttga 105 79 34 PRT
Homo sapiens 79 Met Thr His Pro Cys Thr Asn Pro Gln Pro Thr Ser Arg
Ser Pro Pro 1 5 10 15 Leu Cys Trp Thr Ser Pro Gln Ser His Val Val
Ser Pro Ser Ile Arg 20 25 30 Phe Ser 80 1092 DNA Homo sapiens 80
atgctggaca tcccctcaga gccatgtagt ctcaccatcc atacgattca gttgattcag
60 cacaaccgac gtcttcgcaa ccttattgcc acagctcagg cccagaatca
gcagcagaca 120 gaaggtgtaa aaactgaaga gagtgaacct cttccctcgt
gccctgggtc acctcctctc 180 cctgatgacc tcctgccttt agattgtaag
aatcccaatg caccattcca gatccggcac 240 agtgacccag agagtgactt
ttatcgtggg aaaggggaac ctgtgactga actcagctgg 300 cactcctgtc
ggcagctcct ctaccaggca gtggccacaa tcctggccca cgcgggcttt 360
gactgtgcta atgagagtgt cctggagacc ctaactgatg tggcacatga gtattgcctt
420 aagtttacca agttgctgcg ttttgctgtg gaccgggagg cccggctggg
acagactcct 480 tttcctgatg tgatggagca ggtattccat gaagtgggta
ttggcagtgt gctctccctc 540 cagaagttct ggcagcaccg catcaaggac
tatcacagtt acatgctaca gattagtaag 600 caactctctg aagaatatga
aaggattgtc aatcctgaga aggccacaga ggacgctaaa 660 cctgtgaaga
tcaaggagga acctgtgagc gacatcactt ttcctgtcag tgaggagctg 720
gaggctgacc ttgcttctgg agaccagtca ctgcctatgg gagtgcttgg ggctcagagc
780 gaacgcttcc catctaacct ggaggttgaa gcttcaccac aggcttcaag
tgcagaggta 840 aatgcttctc ctctttggaa tctggcccat gtgaaaatgg
agcctcaaga aagtgaagaa 900 ggcaatgtct ctgggcatgg tgtgctgggc
agtgatgtct tcgaggagcc tatgtcaggc 960 atgagtgaag ctgggattcc
tcagagccct gatgactcag atagcagcta tggttcccac 1020 tccactgaca
gcctcatggg gtcctcccct gttttcaacc agcgctgcaa gaagaggatg 1080
aggaaaatat aa 1092 81 363 PRT Homo sapiens 81 Met Leu Asp Ile Pro
Ser Glu Pro Cys Ser Leu Thr Ile His Thr Ile 1 5 10 15 Gln Leu Ile
Gln His Asn Arg Arg Leu Arg Asn Leu Ile Ala Thr Ala 20 25 30 Gln
Ala Gln Asn Gln Gln Gln Thr Glu Gly Val Lys Thr Glu Glu Ser 35 40
45 Glu Pro Leu Pro Ser Cys Pro Gly Ser Pro Pro Leu Pro Asp Asp Leu
50 55 60 Leu Pro Leu Asp Cys Lys Asn Pro Asn Ala Pro Phe Gln Ile
Arg His 65 70 75 80 Ser Asp Pro Glu Ser Asp Phe Tyr Arg Gly Lys Gly
Glu Pro Val Thr 85 90 95 Glu Leu Ser Trp His Ser Cys Arg Gln Leu
Leu Tyr Gln Ala Val Ala 100 105 110 Thr Ile Leu Ala His Ala Gly Phe
Asp Cys Ala Asn Glu Ser Val Leu 115 120 125 Glu Thr Leu Thr Asp Val
Ala His Glu Tyr Cys Leu Lys Phe Thr Lys 130 135 140 Leu Leu Arg Phe
Ala Val Asp Arg Glu Ala Arg Leu Gly Gln Thr Pro 145 150 155 160 Phe
Pro Asp Val Met Glu Gln Val Phe His Glu Val Gly Ile Gly Ser 165 170
175 Val Leu Ser Leu Gln Lys Phe Trp Gln His Arg Ile Lys Asp Tyr His
180 185 190 Ser Tyr Met Leu Gln Ile Ser Lys Gln Leu Ser Glu Glu Tyr
Glu Arg 195 200 205 Ile Val Asn Pro Glu Lys Ala Thr Glu Asp Ala Lys
Pro Val Lys Ile 210 215 220 Lys Glu Glu Pro Val Ser Asp Ile Thr Phe
Pro Val Ser Glu Glu Leu 225 230 235 240 Glu Ala Asp Leu Ala Ser Gly
Asp Gln Ser Leu Pro Met Gly Val Leu 245 250 255 Gly Ala Gln Ser Glu
Arg Phe Pro Ser Asn Leu Glu Val Glu Ala Ser 260 265 270 Pro Gln Ala
Ser Ser Ala Glu Val Asn Ala Ser Pro Leu Trp Asn Leu 275 280 285 Ala
His Val Lys Met Glu Pro Gln Glu Ser Glu Glu Gly Asn Val Ser 290 295
300 Gly His Gly Val Leu Gly Ser Asp Val Phe Glu Glu Pro Met Ser Gly
305 310 315 320 Met Ser Glu Ala Gly Ile Pro Gln Ser Pro Asp Asp Ser
Asp Ser Ser 325 330 335 Tyr Gly Ser His Ser Thr Asp Ser Leu Met Gly
Ser Ser Pro Val Phe 340 345 350 Asn Gln Arg Cys Lys Lys Arg Met Arg
Lys Ile 355 360 82 18 DNA Homo sapiens 82 atgacctcct gcctttag 18 83
5 PRT Homo sapiens 83 Met Thr Ser Cys Leu 1 5 84 69 DNA Homo
sapiens 84 atgcaccatt ccagatccgg cacagtgacc cagagagtga cttttatcgt
gggaaagggg 60 aacctgtga 69 85 22 PRT Homo sapiens 85 Met His His
Ser Arg Ser Gly Thr Val Thr Gln Arg Val Thr Phe Ile 1 5 10 15 Val
Gly Lys Gly Asn Leu 20 86 24 DNA Homo sapiens 86 atgagagtgt
cctggagacc ctaa 24 87 7 PRT Homo sapiens 87 Met Arg Val Ser Trp Arg
Pro 1 5 88 96 DNA Homo sapiens 88 atgtggcaca tgagtattgc cttaagttta
ccaagttgct gcgttttgct gtggaccggg 60 aggcccggct gggacagact
ccttttcctg atgtga 96 89 31 PRT Homo sapiens 89 Met Trp His Met Ser
Ile Ala Leu Ser Leu Pro Ser Cys Cys Val Leu 1 5 10 15 Leu Trp Thr
Gly Arg Pro Gly Trp Asp Arg Leu Leu Phe Leu Met 20 25 30 90 87 DNA
Homo sapiens 90 atgagtattg ccttaagttt accaagttgc tgcgttttgc
tgtggaccgg gaggcccggc 60 tgggacagac tccttttcct gatgtga 87 91 28 PRT
Homo sapiens 91 Met Ser Ile Ala Leu Ser Leu Pro Ser Cys Cys Val Leu
Leu Trp Thr 1 5 10 15 Gly Arg Pro Gly Trp Asp Arg Leu Leu Phe Leu
Met 20 25 92 600 DNA Homo sapiens 92 atggagcagg tattccatga
agtgggtatt ggcagtgtgc tctccctcca gaagttctgg 60 cagcaccgca
tcaaggacta tcacagttac atgctacaga ttagtaagca actctctgaa 120
gaatatgaaa ggattgtcaa tcctgagaag gccacagagg acgctaaacc tgtgaagatc
180 aaggaggaac ctgtgagcga catcactttt cctgtcagtg aggagctgga
ggctgacctt 240 gcttctggag accagtcact gcctatggga gtgcttgggg
ctcagagcga acgcttccca 300 tctaacctgg aggttgaagc ttcaccacag
gcttcaagtg cagaggtaaa tgcttctcct 360 ctttggaatc tggcccatgt
gaaaatggag cctcaagaaa gtgaagaagg caatgtctct 420 gggcatggtg
tgctgggcag tgatgtcttc gaggagccta tgtcaggcat gagtgaagct 480
gggattcctc agagccctga tgactcagat agcagctatg gttcccactc cactgacagc
540 ctcatggggt cctcccctgt tttcaaccag cgctgcaaga agaggatgag
gaaaatataa 600 93 199 PRT Homo sapiens 93 Met Glu Gln Val Phe His
Glu Val Gly Ile Gly Ser Val Leu Ser Leu 1 5 10 15 Gln Lys Phe Trp
Gln His Arg Ile Lys Asp Tyr His Ser Tyr Met Leu 20 25 30 Gln Ile
Ser Lys Gln Leu Ser Glu Glu Tyr Glu Arg Ile Val Asn Pro 35 40 45
Glu Lys Ala Thr Glu Asp Ala Lys Pro Val Lys Ile Lys Glu Glu Pro 50
55 60 Val Ser Asp Ile Thr Phe Pro Val Ser Glu Glu Leu Glu Ala Asp
Leu 65 70 75 80 Ala Ser Gly Asp Gln Ser Leu Pro Met Gly Val Leu Gly
Ala Gln Ser 85 90 95 Glu Arg Phe Pro Ser Asn Leu Glu Val Glu Ala
Ser Pro Gln Ala Ser 100 105 110 Ser Ala Glu Val Asn Ala Ser Pro Leu
Trp Asn Leu Ala His Val Lys 115 120 125 Met Glu Pro Gln Glu Ser Glu
Glu Gly Asn Val Ser Gly His Gly Val 130 135 140 Leu Gly Ser Asp Val
Phe Glu Glu Pro Met Ser Gly Met Ser Glu Ala 145 150 155 160 Gly Ile
Pro Gln Ser Pro Asp Asp Ser Asp Ser Ser Tyr Gly Ser His 165 170 175
Ser Thr Asp Ser Leu Met Gly Ser Ser Pro Val Phe Asn Gln Arg Cys 180
185 190 Lys Lys Arg Met Arg Lys Ile 195 94 159 DNA Homo sapiens 94
atgaagtggg tattggcagt gtgctctccc tccagaagtt ctggcagcac cgcatcaagg
60 actatcacag ttacatgcta cagattagta agcaactctc tgaagaatat
gaaaggattg 120 tcaatcctga gaaggccaca gaggacgcta aacctgtga 159 95 52
PRT Homo sapiens 95 Met Lys Trp Val Leu Ala Val Cys Ser Pro Ser Arg
Ser Ser Gly Ser 1 5 10 15 Thr Ala Ser Arg Thr Ile Thr Val Thr Cys
Tyr Arg Leu Val Ser Asn 20 25 30 Ser Leu Lys Asn Met Lys Gly Leu
Ser Ile Leu Arg Arg Pro Gln Arg 35 40 45 Thr Leu Asn Leu 50 96 510
DNA Homo sapiens 96 atgctacaga ttagtaagca actctctgaa gaatatgaaa
ggattgtcaa tcctgagaag 60
gccacagagg acgctaaacc tgtgaagatc aaggaggaac ctgtgagcga catcactttt
120 cctgtcagtg aggagctgga ggctgacctt gcttctggag accagtcact
gcctatggga 180 gtgcttgggg ctcagagcga acgcttccca tctaacctgg
aggttgaagc ttcaccacag 240 gcttcaagtg cagaggtaaa tgcttctcct
ctttggaatc tggcccatgt gaaaatggag 300 cctcaagaaa gtgaagaagg
caatgtctct gggcatggtg tgctgggcag tgatgtcttc 360 gaggagccta
tgtcaggcat gagtgaagct gggattcctc agagccctga tgactcagat 420
agcagctatg gttcccactc cactgacagc ctcatggggt cctcccctgt tttcaaccag
480 cgctgcaaga agaggatgag gaaaatataa 510 97 169 PRT Homo sapiens 97
Met Leu Gln Ile Ser Lys Gln Leu Ser Glu Glu Tyr Glu Arg Ile Val 1 5
10 15 Asn Pro Glu Lys Ala Thr Glu Asp Ala Lys Pro Val Lys Ile Lys
Glu 20 25 30 Glu Pro Val Ser Asp Ile Thr Phe Pro Val Ser Glu Glu
Leu Glu Ala 35 40 45 Asp Leu Ala Ser Gly Asp Gln Ser Leu Pro Met
Gly Val Leu Gly Ala 50 55 60 Gln Ser Glu Arg Phe Pro Ser Asn Leu
Glu Val Glu Ala Ser Pro Gln 65 70 75 80 Ala Ser Ser Ala Glu Val Asn
Ala Ser Pro Leu Trp Asn Leu Ala His 85 90 95 Val Lys Met Glu Pro
Gln Glu Ser Glu Glu Gly Asn Val Ser Gly His 100 105 110 Gly Val Leu
Gly Ser Asp Val Phe Glu Glu Pro Met Ser Gly Met Ser 115 120 125 Glu
Ala Gly Ile Pro Gln Ser Pro Asp Asp Ser Asp Ser Ser Tyr Gly 130 135
140 Ser His Ser Thr Asp Ser Leu Met Gly Ser Ser Pro Val Phe Asn Gln
145 150 155 160 Arg Cys Lys Lys Arg Met Arg Lys Ile 165 98 51 DNA
Homo sapiens 98 atgaaaggat tgtcaatcct gagaaggcca cagaggacgc
taaacctgtg a 51 99 16 PRT Homo sapiens 99 Met Lys Gly Leu Ser Ile
Leu Arg Arg Pro Gln Arg Thr Leu Asn Leu 1 5 10 15 100 336 DNA Homo
sapiens 100 atgggagtgc ttggggctca gagcgaacgc ttcccatcta acctggaggt
tgaagcttca 60 ccacaggctt caagtgcaga ggtaaatgct tctcctcttt
ggaatctggc ccatgtgaaa 120 atggagcctc aagaaagtga agaaggcaat
gtctctgggc atggtgtgct gggcagtgat 180 gtcttcgagg agcctatgtc
aggcatgagt gaagctggga ttcctcagag ccctgatgac 240 tcagatagca
gctatggttc ccactccact gacagcctca tggggtcctc ccctgttttc 300
aaccagcgct gcaagaagag gatgaggaaa atataa 336 101 111 PRT Homo
sapiens 101 Met Gly Val Leu Gly Ala Gln Ser Glu Arg Phe Pro Ser Asn
Leu Glu 1 5 10 15 Val Glu Ala Ser Pro Gln Ala Ser Ser Ala Glu Val
Asn Ala Ser Pro 20 25 30 Leu Trp Asn Leu Ala His Val Lys Met Glu
Pro Gln Glu Ser Glu Glu 35 40 45 Gly Asn Val Ser Gly His Gly Val
Leu Gly Ser Asp Val Phe Glu Glu 50 55 60 Pro Met Ser Gly Met Ser
Glu Ala Gly Ile Pro Gln Ser Pro Asp Asp 65 70 75 80 Ser Asp Ser Ser
Tyr Gly Ser His Ser Thr Asp Ser Leu Met Gly Ser 85 90 95 Ser Pro
Val Phe Asn Gln Arg Cys Lys Lys Arg Met Arg Lys Ile 100 105 110 102
33 DNA Homo sapiens 102 atgcttctcc tctttggaat ctggcccatg tga 33 103
10 PRT Homo sapiens 103 Met Leu Leu Leu Phe Gly Ile Trp Pro Met 1 5
10 104 216 DNA Homo sapiens 104 atggagcctc aagaaagtga agaaggcaat
gtctctgggc atggtgtgct gggcagtgat 60 gtcttcgagg agcctatgtc
aggcatgagt gaagctggga ttcctcagag ccctgatgac 120 tcagatagca
gctatggttc ccactccact gacagcctca tggggtcctc ccctgttttc 180
aaccagcgct gcaagaagag gatgaggaaa atataa 216 105 71 PRT Homo sapiens
105 Met Glu Pro Gln Glu Ser Glu Glu Gly Asn Val Ser Gly His Gly Val
1 5 10 15 Leu Gly Ser Asp Val Phe Glu Glu Pro Met Ser Gly Met Ser
Glu Ala 20 25 30 Gly Ile Pro Gln Ser Pro Asp Asp Ser Asp Ser Ser
Tyr Gly Ser His 35 40 45 Ser Thr Asp Ser Leu Met Gly Ser Ser Pro
Val Phe Asn Gln Arg Cys 50 55 60 Lys Lys Arg Met Arg Lys Ile 65 70
106 60 DNA Homo sapiens 106 atgtctctgg gcatggtgtg ctgggcagtg
atgtcttcga ggagcctatg tcaggcatga 60 107 19 PRT Homo sapiens 107 Met
Ser Leu Gly Met Val Cys Trp Ala Val Met Ser Ser Arg Ser Leu 1 5 10
15 Cys Gln Ala 108 48 DNA Homo sapiens 108 atggtgtgct gggcagtgat
gtcttcgagg agcctatgtc aggcatga 48 109 15 PRT Homo sapiens 109 Met
Val Cys Trp Ala Val Met Ser Ser Arg Ser Leu Cys Gln Ala 1 5 10 15
110 30 DNA Homo sapiens 110 atgtcttcga ggagcctatg tcaggcatga 30 111
9 PRT Homo sapiens 111 Met Ser Ser Arg Ser Leu Cys Gln Ala 1 5 112
141 DNA Homo sapiens 112 atgtcaggca tgagtgaagc tgggattcct
cagagccctg atgactcaga tagcagctat 60 ggttcccact ccactgacag
cctcatgggg tcctcccctg ttttcaacca gcgctgcaag 120 aagaggatga
ggaaaatata a 141 113 46 PRT Homo sapiens 113 Met Ser Gly Met Ser
Glu Ala Gly Ile Pro Gln Ser Pro Asp Asp Ser 1 5 10 15 Asp Ser Ser
Tyr Gly Ser His Ser Thr Asp Ser Leu Met Gly Ser Ser 20 25 30 Pro
Val Phe Asn Gln Arg Cys Lys Lys Arg Met Arg Lys Ile 35 40 45 114
132 DNA Homo sapiens 114 atgagtgaag ctgggattcc tcagagccct
gatgactcag atagcagcta tggttcccac 60 tccactgaca gcctcatggg
gtcctcccct gttttcaacc agcgctgcaa gaagaggatg 120 aggaaaatat aa 132
115 43 PRT Homo sapiens 115 Met Ser Glu Ala Gly Ile Pro Gln Ser Pro
Asp Asp Ser Asp Ser Ser 1 5 10 15 Tyr Gly Ser His Ser Thr Asp Ser
Leu Met Gly Ser Ser Pro Val Phe 20 25 30 Asn Gln Arg Cys Lys Lys
Arg Met Arg Lys Ile 35 40 116 90 DNA Homo sapiens 116 atgactcaga
tagcagctat ggttcccact ccactgacag cctcatgggg tcctcccctg 60
ttttcaacca gcgctgcaag aagaggatga 90 117 29 PRT Homo sapiens 117 Met
Thr Gln Ile Ala Ala Met Val Pro Thr Pro Leu Thr Ala Ser Trp 1 5 10
15 Gly Pro Pro Leu Phe Ser Thr Ser Ala Ala Arg Arg Gly 20 25 118 72
DNA Homo sapiens 118 atggttccca ctccactgac agcctcatgg ggtcctcccc
tgttttcaac cagcgctgca 60 agaagaggat ga 72 119 23 PRT Homo sapiens
119 Met Val Pro Thr Pro Leu Thr Ala Ser Trp Gly Pro Pro Leu Phe Ser
1 5 10 15 Thr Ser Ala Ala Arg Arg Gly 20 120 57 DNA Homo sapiens
120 atggggtcct cccctgtttt caaccagcgc tgcaagaaga ggatgaggaa aatataa
57 121 18 PRT Homo sapiens 121 Met Gly Ser Ser Pro Val Phe Asn Gln
Arg Cys Lys Lys Arg Met Arg 1 5 10 15 Lys Ile 122 15 DNA Homo
sapiens 122 atgaggaaaa tataa 15 123 4 PRT Homo sapiens 123 Met Arg
Lys Ile 1 124 45 DNA Homo sapiens 124 atgttttgtc cagacctact
agacccaaca gaaaaggtta gctga 45 125 14 PRT Homo sapiens 125 Met Phe
Cys Pro Asp Leu Leu Asp Pro Thr Glu Lys Val Ser 1 5 10 126 132 DNA
Homo sapiens 126 atgtattttg ctgagctgta caacaggatg gcacaaaatc
ctgctgatag aaataagtgt 60 aaccggccag gcacagtggc tcatgcctgt
aatcccagca ttttgggagg cccaggtggg 120 tggatcatct ga 132 127 43 PRT
Homo sapiens 127 Met Tyr Phe Ala Glu Leu Tyr Asn Arg Met Ala Gln
Asn Pro Ala Asp 1 5 10 15 Arg Asn Lys Cys Asn Arg Pro Gly Thr Val
Ala His Ala Cys Asn Pro 20 25 30 Ser Ile Leu Gly Gly Pro Gly Gly
Trp Ile Ile 35 40 128 105 DNA Homo sapiens 128 atggcacaaa
atcctgctga tagaaataag tgtaaccggc caggcacagt ggctcatgcc 60
tgtaatccca gcattttggg aggcccaggt gggtggatca tctga 105 129 34 PRT
Homo sapiens 129 Met Ala Gln Asn Pro Ala Asp Arg Asn Lys Cys Asn
Arg Pro Gly Thr 1 5 10 15 Val Ala His Ala Cys Asn Pro Ser Ile Leu
Gly Gly Pro Gly Gly Trp 20 25 30 Ile Ile 130 75 DNA Homo sapiens
130 atgcctgtaa tcccagcatt ttgggaggcc caggtgggtg gatcatctga
ggtcaggagt 60 tcgagaccag cctga 75 131 24 PRT Homo sapiens 131 Met
Pro Val Ile Pro Ala Phe Trp Glu Ala Gln Val Gly Gly Ser Ser 1 5 10
15 Glu Val Arg Ser Ser Arg Pro Ala 20 132 39 DNA Homo sapiens 132
atggaaaaaa ccccatctct actaaaaata caaaattag 39 133 12 PRT Homo
sapiens 133 Met Glu Lys Thr Pro Ser Leu Leu Lys Ile Gln Asn 1 5 10
134 33 DNA Homo sapiens 134 atgcctgtaa tcccagctac tcaggaaggc tga 33
135 10 PRT Homo sapiens 135 Met Pro Val Ile Pro Ala Thr Gln Glu Gly
1 5 10 136 542 DNA Homo sapiens 136 tcgacccacg cgtccgggac
aatagtgtag gttatggatg gaggtgtcgg tactaaattg 60 aataacgagt
aaataatctt acttgggtag agatggcctt tgccaacaaa gtgaactgtt 120
ttggttgttt taaactcatg aagtatgggt tcagtggaaa tgtttggaac tctgaaggat
180 ttagacaagg ttttgaaaag gataatcatg ggttagaagg aagtgtttga
aagtcacttt 240 gaaagttagt tttgggccag cacggtagct cacccttgta
atcccagcac tttgggaggc 300 tgaggtgggt agattacttg agcccaggaa
ttcaagacca gcctgggcaa catggtgaaa 360 ccctgtttct ataaaaaata
atctgggctt tgtagcatat gcctgtggtc ccagctactg 420 aggaggctga
ggtgggagga ttgcttgagc ccaggaggca gaggttgcag tgagccaagg 480
tcacgtcact gcactctagc ctgggcaaca gagtaagaca aaaaaaaaaa aaaagggcgg
540 cc 542 137 39 DNA Homo sapiens 137 atggatggag gtgtcggtac
taaattgaat aacgagtaa 39 138 12 PRT Homo sapiens 138 Met Asp Gly Gly
Val Gly Thr Lys Leu Asn Asn Glu 1 5 10 139 24 DNA Homo sapiens 139
atggaggtgt cggtactaaa ttga 24 140 7 PRT Homo sapiens 140 Met Glu
Val Ser Val Leu Asn 1 5 141 138 DNA Homo sapiens 141 atggcctttg
ccaacaaagt gaactgtttt ggttgtttta aactcatgaa gtatgggttc 60
agtggaaatg tttggaactc tgaaggattt agacaaggtt ttgaaaagga taatcatggg
120 ttagaaggaa gtgtttga 138 142 45 PRT Homo sapiens 142 Met Ala Phe
Ala Asn Lys Val Asn Cys Phe Gly Cys Phe Lys Leu Met 1 5 10 15 Lys
Tyr Gly Phe Ser Gly Asn Val Trp Asn Ser Glu Gly Phe Arg Gln 20 25
30 Gly Phe Glu Lys Asp Asn His Gly Leu Glu Gly Ser Val 35 40 45 143
93 DNA Homo sapiens 143 atgaagtatg ggttcagtgg aaatgtttgg aactctgaag
gatttagaca aggttttgaa 60 aaggataatc atgggttaga aggaagtgtt tga 93
144 30 PRT Homo sapiens 144 Met Lys Tyr Gly Phe Ser Gly Asn Val Trp
Asn Ser Glu Gly Phe Arg 1 5 10 15 Gln Gly Phe Glu Lys Asp Asn His
Gly Leu Glu Gly Ser Val 20 25 30 145 72 DNA Homo sapiens 145
atgggttcag tggaaatgtt tggaactctg aaggatttag acaaggtttt gaaaaggata
60 atcatgggtt ag 72 146 23 PRT Homo sapiens 146 Met Gly Ser Val Glu
Met Phe Gly Thr Leu Lys Asp Leu Asp Lys Val 1 5 10 15 Leu Lys Arg
Ile Ile Met Gly 20 147 57 DNA Homo sapiens 147 atgtttggaa
ctctgaagga tttagacaag gttttgaaaa ggataatcat gggttag 57 148 18 PRT
Homo sapiens 148 Met Phe Gly Thr Leu Lys Asp Leu Asp Lys Val Leu
Lys Arg Ile Ile 1 5 10 15 Met Gly 149 30 DNA Homo sapiens 149
atggtgaaac cctgtttcta taaaaaataa 30 150 9 PRT Homo sapiens 150 Met
Val Lys Pro Cys Phe Tyr Lys Lys 1 5 151 75 DNA Homo sapiens 151
atgcctgtgg tcccagctac tgaggaggct gaggtgggag gattgcttga gcccaggagg
60 cagaggttgc agtga 75 152 24 PRT Homo sapiens 152 Met Pro Val Val
Pro Ala Thr Glu Glu Ala Glu Val Gly Gly Leu Leu 1 5 10 15 Glu Pro
Arg Arg Gln Arg Leu Gln 20 153 771 DNA Homo sapiens 153 tcgacccacg
cgtccgcaaa acctaaatag aagttgttgt taccgtgtgc caatgtgtcc 60
catgtgggtt gtgccaggta gagaaacagg aagtcaatca tctgtgacag tctctattct
120 gtcgttttgc tccttggtat ttgatttgca ctatatttag ttgaagcctg
ttcactgttt 180 aaaaccggag gtatcttcaa aggcatggag acctggttcc
agtaaatgtc ccaccagtgg 240 ggtatagaaa gcatgctcat gaccctgccg
tgtcgtctga ggtacccgtt cttatcctag 300 tggttcagga agagaaaacg
cagtttgcac tttcaagaca gcttctctaa ggctggcatg 360 ttatctcctt
gctttgcttt ttgccgtttt aaaatgtgta attgttccag cattccaatg 420
gtcttgtgca tagcagggga ctgtaaccaa aaataaacat gtatttgtgt aattggtttg
480 aagaagtctt gaatagctct ttactgtctt acttggggtt gataagattt
gagtgtttgc 540 aattttttac taaatgtagc tccaaagtct taaatggctt
gtttgttctt aaactgttaa 600 ttgatgaaac tgtgcataag tttacaatgt
actaacttat tttgcttatt atatatagtg 660 ttttattgga aattgtaacc
acacacttca gcatgatgaa aataaagatt agtgtttcca 720 tttaaataaa
tgttttatcc tcccataaaa aaaaaaaaaa aaagggcggc c 771 154 108 DNA Homo
sapiens 154 atgtgtccca tgtgggttgt gccaggtaga gaaacaggaa gtcaatcatc
tgtgacagtc 60 tctattctgt cgttttgctc cttggtattt gatttgcact atatttag
108 155 35 PRT Homo sapiens 155 Met Cys Pro Met Trp Val Val Pro Gly
Arg Glu Thr Gly Ser Gln Ser 1 5 10 15 Ser Val Thr Val Ser Ile Leu
Ser Phe Cys Ser Leu Val Phe Asp Leu 20 25 30 His Tyr Ile 35 156 99
DNA Homo sapiens 156 atgtgggttg tgccaggtag agaaacagga agtcaatcat
ctgtgacagt ctctattctg 60 tcgttttgct ccttggtatt tgatttgcac tatatttag
99 157 32 PRT Homo sapiens 157 Met Trp Val Val Pro Gly Arg Glu Thr
Gly Ser Gln Ser Ser Val Thr 1 5 10 15 Val Ser Ile Leu Ser Phe Cys
Ser Leu Val Phe Asp Leu His Tyr Ile 20 25 30 158 21 DNA Homo
sapiens 158 atggagacct ggttccagta a 21 159 6 PRT Homo sapiens 159
Met Glu Thr Trp Phe Gln 1 5 160 75 DNA Homo sapiens 160 atgtcccacc
agtggggtat agaaagcatg ctcatgaccc tgccgtgtcg tctgaggtac 60
ccgttcttat cctag 75 161 24 PRT Homo sapiens 161 Met Ser His Gln Trp
Gly Ile Glu Ser Met Leu Met Thr Leu Pro Cys 1 5 10 15 Arg Leu Arg
Tyr Pro Phe Leu Ser 20 162 48 DNA Homo sapiens 162 atgctcatga
ccctgccgtg tcgtctgagg tacccgttct tatcctag 48 163 15 PRT Homo
sapiens 163 Met Leu Met Thr Leu Pro Cys Arg Leu Arg Tyr Pro Phe Leu
Ser 1 5 10 15 164 42 DNA Homo sapiens 164 atgaccctgc cgtgtcgtct
gaggtacccg ttcttatcct ag 42 165 13 PRT Homo sapiens 165 Met Thr Leu
Pro Cys Arg Leu Arg Tyr Pro Phe Leu Ser 1 5 10 166 99 DNA Homo
sapiens 166 atgttatctc cttgctttgc tttttgccgt tttaaaatgt gtaattgttc
cagcattcca 60 atggtcttgt gcatagcagg ggactgtaac caaaaataa 99 167 32
PRT Homo sapiens 167 Met Leu Ser Pro Cys Phe Ala Phe Cys Arg Phe
Lys Met Cys Asn Cys 1 5 10 15 Ser Ser Ile Pro Met Val Leu Cys Ile
Ala Gly Asp Cys Asn Gln Lys 20 25 30 168 63 DNA Homo sapiens 168
atgtgtaatt gttccagcat tccaatggtc ttgtgcatag caggggactg taaccaaaaa
60 taa 63 169 20 PRT Homo sapiens 169 Met Cys Asn Cys Ser Ser Ile
Pro Met Val Leu Cys Ile Ala Gly Asp 1 5 10 15 Cys Asn Gln Lys 20
170 39 DNA Homo sapiens 170 atggtcttgt gcatagcagg ggactgtaac
caaaaataa 39 171 12 PRT Homo sapiens 171 Met Val Leu Cys Ile Ala
Gly Asp Cys Asn Gln Lys 1 5 10 172 177 DNA Homo sapiens 172
atgtatttgt gtaattggtt tgaagaagtc ttgaatagct ctttactgtc ttacttgggg
60 ttgataagat ttgagtgttt gcaatttttt actaaatgta gctccaaagt
cttaaatggc 120 ttgtttgttc ttaaactgtt aattgatgaa actgtgcata
agtttacaat gtactaa 177 173 58 PRT Homo sapiens 173 Met Tyr Leu Cys
Asn Trp Phe Glu Glu Val Leu Asn Ser Ser Leu Leu 1 5 10 15 Ser Tyr
Leu Gly Leu Ile Arg Phe Glu Cys Leu Gln Phe Phe Thr Lys 20 25 30
Cys Ser Ser Lys Val Leu Asn Gly Leu Phe Val Leu Lys Leu Leu Ile 35
40 45 Asp Glu Thr Val His Lys Phe Thr Met Tyr 50 55 174 27 DNA Homo
sapiens 174 atggcttgtt tgttcttaaa ctgttaa 27 175 8 PRT Homo sapiens
175 Met Ala Cys Leu Phe Leu Asn Cys 1 5 176 75 DNA Homo sapiens 176
atgaaactgt gcataagttt acaatgtact aacttatttt gcttattata tatagtgttt
60 tattggaaat tgtaa 75
177 24 PRT Homo sapiens 177 Met Lys Leu Cys Ile Ser Leu Gln Cys Thr
Asn Leu Phe Cys Leu Leu 1 5 10 15 Tyr Ile Val Phe Tyr Trp Lys Leu
20 178 33 DNA Homo sapiens 178 atgatgaaaa taaagattag tgtttccatt taa
33 179 10 PRT Homo sapiens 179 Met Met Lys Ile Lys Ile Ser Val Ser
Ile 1 5 10 180 30 DNA Homo sapiens 180 atgaaaataa agattagtgt
ttccatttaa 30 181 9 PRT Homo sapiens 181 Met Lys Ile Lys Ile Ser
Val Ser Ile 1 5 182 42 DNA Homo sapiens 182 atgttttatc ctcccataaa
aaaaaaaaaa aaaagggcgg cc 42 183 14 PRT Homo sapiens 183 Met Phe Tyr
Pro Pro Ile Lys Lys Lys Lys Lys Arg Ala Ala 1 5 10 184 1669 DNA
Homo sapiens 184 tcgacccacg cgtccgcagg cagtgactgc cttcggcttt
ttttctgctg actaagatct 60 cctatagaga gctacaacaa tgcccaaaag
aaagccaaag agaagatctg ccaggttgtc 120 tgctatgctt gtgccagtta
caccagaggt gaagcctaaa agaacatcaa gttcaaggaa 180 aatgaagaca
aaaagtgata tgatggaaga aaacatagat acaagtgccc aagcagttgc 240
tgaaaccaag caagaagcag ttgttgaaga agactacaat gaaaatgcta aaaatggaga
300 agccaaaatt acagaggcac cagcttctga aaaagaaatt gtggaagtaa
aagaagaaaa 360 tattgaagat gccacagaaa agggaggaga aaagaaagaa
gcagtggcag cagaagtaaa 420 aaatgaagaa gaagatcaga aagaagatga
agaagatcaa aacgaagaga aaggggaagc 480 tggaaaagaa gacaaagatg
aaaaagggga agaagatgga aaagaggata aaaatggaaa 540 tgagaaagga
gaagatgcaa aagagaaaga agatggaaaa aaaggtgaag acggaaaagg 600
aaatggagaa gatggaaaag agaaaggaga agatgaaaaa gaggaagaag acagaaaaga
660 aacaggagtt ggaaaagaga atgaagatgg aaaagagaag ggagataaaa
aagaggggaa 720 agatgtaaaa gtcaaagaag atgaaaaaga gagagaagat
ggaaaagaag atgaaggtgg 780 aaatgaggaa gaagctggaa aagagaaaga
agatttaaaa gaagaggaag aaggaaaaga 840 ggaagatgag atcaaagaag
atgatggaaa aaaagaggag ccacagagta ttgtttaaaa 900 ctgccctatg
tagtttcata atttggtaac atgtaccttc atgttgtaaa gttaatagag 960
ataaatattt ttatcaaaaa ttttataaac acagcctttc tttagcattg atttaatttc
1020 agaacatctt catattgatt attagccata aagtttctaa catgaaacat
ttatctataa 1080 attttgtgat tatagtagtg gaatacatag aaaaaaatat
gctttcaact ttgtgagtga 1140 atttcgtgtt gtgtaagtta tatgtcaaat
ctttgaattt taattttact ccttttatac 1200 atgtgataat ttcataaagt
gagggatccc aaaaaaagag tttcatccca acattcttgt 1260 tctgcaggtt
gcttttataa agaaggtgaa ctattttcat gtaatgttaa gagttaaact 1320
tatctttccc aaatataact ttattattag cttgggaaaa atgaaattgt attcccattt
1380 ttaaaataaa tacaaatgtt tatttcagaa gggcagtttt gattatatgt
gaatacacaa 1440 attttactgg atttatctta ataaaaagac tctgacgatg
attgtgtttt gttatatctt 1500 caaaaatata gctagtgaaa tattgtgctt
aatttttttc tattgtgtta ttcatgaaaa 1560 tatttaatat tcactgacat
aaaattaata taaagtaaaa ttcaccattt taattataat 1620 aaaaataaag
tatataattc aaaaaaaaaa aaaaaaaaaa agggcggcc 1669 185 819 DNA Homo
sapiens 185 atgcccaaaa gaaagccaaa gagaagatct gccaggttgt ctgctatgct
tgtgccagtt 60 acaccagagg tgaagcctaa aagaacatca agttcaagga
aaatgaagac aaaaagtgat 120 atgatggaag aaaacataga tacaagtgcc
caagcagttg ctgaaaccaa gcaagaagca 180 gttgttgaag aagactacaa
tgaaaatgct aaaaatggag aagccaaaat tacagaggca 240 ccagcttctg
aaaaagaaat tgtggaagta aaagaagaaa atattgaaga tgccacagaa 300
aagggaggag aaaagaaaga agcagtggca gcagaagtaa aaaatgaaga agaagatcag
360 aaagaagatg aagaagatca aaacgaagag aaaggggaag ctggaaaaga
agacaaagat 420 gaaaaagggg aagaagatgg aaaagaggat aaaaatggaa
atgagaaagg agaagatgca 480 aaagagaaag aagatggaaa aaaaggtgaa
gacggaaaag gaaatggaga agatggaaaa 540 gagaaaggag aagatgaaaa
agaggaagaa gacagaaaag aaacaggagt tggaaaagag 600 aatgaagatg
gaaaagagaa gggagataaa aaagagggga aagatgtaaa agtcaaagaa 660
gatgaaaaag agagagaaga tggaaaagaa gatgaaggtg gaaatgagga agaagctgga
720 aaagagaaag aagatttaaa agaagaggaa gaaggaaaag aggaagatga
gatcaaagaa 780 gatgatggaa aaaaagagga gccacagagt attgtttaa 819 186
272 PRT Homo sapiens 186 Met Pro Lys Arg Lys Pro Lys Arg Arg Ser
Ala Arg Leu Ser Ala Met 1 5 10 15 Leu Val Pro Val Thr Pro Glu Val
Lys Pro Lys Arg Thr Ser Ser Ser 20 25 30 Arg Lys Met Lys Thr Lys
Ser Asp Met Met Glu Glu Asn Ile Asp Thr 35 40 45 Ser Ala Gln Ala
Val Ala Glu Thr Lys Gln Glu Ala Val Val Glu Glu 50 55 60 Asp Tyr
Asn Glu Asn Ala Lys Asn Gly Glu Ala Lys Ile Thr Glu Ala 65 70 75 80
Pro Ala Ser Glu Lys Glu Ile Val Glu Val Lys Glu Glu Asn Ile Glu 85
90 95 Asp Ala Thr Glu Lys Gly Gly Glu Lys Lys Glu Ala Val Ala Ala
Glu 100 105 110 Val Lys Asn Glu Glu Glu Asp Gln Lys Glu Asp Glu Glu
Asp Gln Asn 115 120 125 Glu Glu Lys Gly Glu Ala Gly Lys Glu Asp Lys
Asp Glu Lys Gly Glu 130 135 140 Glu Asp Gly Lys Glu Asp Lys Asn Gly
Asn Glu Lys Gly Glu Asp Ala 145 150 155 160 Lys Glu Lys Glu Asp Gly
Lys Lys Gly Glu Asp Gly Lys Gly Asn Gly 165 170 175 Glu Asp Gly Lys
Glu Lys Gly Glu Asp Glu Lys Glu Glu Glu Asp Arg 180 185 190 Lys Glu
Thr Gly Val Gly Lys Glu Asn Glu Asp Gly Lys Glu Lys Gly 195 200 205
Asp Lys Lys Glu Gly Lys Asp Val Lys Val Lys Glu Asp Glu Lys Glu 210
215 220 Arg Glu Asp Gly Lys Glu Asp Glu Gly Gly Asn Glu Glu Glu Ala
Gly 225 230 235 240 Lys Glu Lys Glu Asp Leu Lys Glu Glu Glu Glu Gly
Lys Glu Glu Asp 245 250 255 Glu Ile Lys Glu Asp Asp Gly Lys Lys Glu
Glu Pro Gln Ser Ile Val 260 265 270 187 774 DNA Homo sapiens 187
atgcttgtgc cagttacacc agaggtgaag cctaaaagaa catcaagttc aaggaaaatg
60 aagacaaaaa gtgatatgat ggaagaaaac atagatacaa gtgcccaagc
agttgctgaa 120 accaagcaag aagcagttgt tgaagaagac tacaatgaaa
atgctaaaaa tggagaagcc 180 aaaattacag aggcaccagc ttctgaaaaa
gaaattgtgg aagtaaaaga agaaaatatt 240 gaagatgcca cagaaaaggg
aggagaaaag aaagaagcag tggcagcaga agtaaaaaat 300 gaagaagaag
atcagaaaga agatgaagaa gatcaaaacg aagagaaagg ggaagctgga 360
aaagaagaca aagatgaaaa aggggaagaa gatggaaaag aggataaaaa tggaaatgag
420 aaaggagaag atgcaaaaga gaaagaagat ggaaaaaaag gtgaagacgg
aaaaggaaat 480 ggagaagatg gaaaagagaa aggagaagat gaaaaagagg
aagaagacag aaaagaaaca 540 ggagttggaa aagagaatga agatggaaaa
gagaagggag ataaaaaaga ggggaaagat 600 gtaaaagtca aagaagatga
aaaagagaga gaagatggaa aagaagatga aggtggaaat 660 gaggaagaag
ctggaaaaga gaaagaagat ttaaaagaag aggaagaagg aaaagaggaa 720
gatgagatca aagaagatga tggaaaaaaa gaggagccac agagtattgt ttaa 774 188
257 PRT Homo sapiens 188 Met Leu Val Pro Val Thr Pro Glu Val Lys
Pro Lys Arg Thr Ser Ser 1 5 10 15 Ser Arg Lys Met Lys Thr Lys Ser
Asp Met Met Glu Glu Asn Ile Asp 20 25 30 Thr Ser Ala Gln Ala Val
Ala Glu Thr Lys Gln Glu Ala Val Val Glu 35 40 45 Glu Asp Tyr Asn
Glu Asn Ala Lys Asn Gly Glu Ala Lys Ile Thr Glu 50 55 60 Ala Pro
Ala Ser Glu Lys Glu Ile Val Glu Val Lys Glu Glu Asn Ile 65 70 75 80
Glu Asp Ala Thr Glu Lys Gly Gly Glu Lys Lys Glu Ala Val Ala Ala 85
90 95 Glu Val Lys Asn Glu Glu Glu Asp Gln Lys Glu Asp Glu Glu Asp
Gln 100 105 110 Asn Glu Glu Lys Gly Glu Ala Gly Lys Glu Asp Lys Asp
Glu Lys Gly 115 120 125 Glu Glu Asp Gly Lys Glu Asp Lys Asn Gly Asn
Glu Lys Gly Glu Asp 130 135 140 Ala Lys Glu Lys Glu Asp Gly Lys Lys
Gly Glu Asp Gly Lys Gly Asn 145 150 155 160 Gly Glu Asp Gly Lys Glu
Lys Gly Glu Asp Glu Lys Glu Glu Glu Asp 165 170 175 Arg Lys Glu Thr
Gly Val Gly Lys Glu Asn Glu Asp Gly Lys Glu Lys 180 185 190 Gly Asp
Lys Lys Glu Gly Lys Asp Val Lys Val Lys Glu Asp Glu Lys 195 200 205
Glu Arg Glu Asp Gly Lys Glu Asp Glu Gly Gly Asn Glu Glu Glu Ala 210
215 220 Gly Lys Glu Lys Glu Asp Leu Lys Glu Glu Glu Glu Gly Lys Glu
Glu 225 230 235 240 Asp Glu Ile Lys Glu Asp Asp Gly Lys Lys Glu Glu
Pro Gln Ser Ile 245 250 255 Val 189 717 DNA Homo sapiens 189
atgaagacaa aaagtgatat gatggaagaa aacatagata caagtgccca agcagttgct
60 gaaaccaagc aagaagcagt tgttgaagaa gactacaatg aaaatgctaa
aaatggagaa 120 gccaaaatta cagaggcacc agcttctgaa aaagaaattg
tggaagtaaa agaagaaaat 180 attgaagatg ccacagaaaa gggaggagaa
aagaaagaag cagtggcagc agaagtaaaa 240 aatgaagaag aagatcagaa
agaagatgaa gaagatcaaa acgaagagaa aggggaagct 300 ggaaaagaag
acaaagatga aaaaggggaa gaagatggaa aagaggataa aaatggaaat 360
gagaaaggag aagatgcaaa agagaaagaa gatggaaaaa aaggtgaaga cggaaaagga
420 aatggagaag atggaaaaga gaaaggagaa gatgaaaaag aggaagaaga
cagaaaagaa 480 acaggagttg gaaaagagaa tgaagatgga aaagagaagg
gagataaaaa agaggggaaa 540 gatgtaaaag tcaaagaaga tgaaaaagag
agagaagatg gaaaagaaga tgaaggtgga 600 aatgaggaag aagctggaaa
agagaaagaa gatttaaaag aagaggaaga aggaaaagag 660 gaagatgaga
tcaaagaaga tgatggaaaa aaagaggagc cacagagtat tgtttaa 717 190 238 PRT
Homo sapiens 190 Met Lys Thr Lys Ser Asp Met Met Glu Glu Asn Ile
Asp Thr Ser Ala 1 5 10 15 Gln Ala Val Ala Glu Thr Lys Gln Glu Ala
Val Val Glu Glu Asp Tyr 20 25 30 Asn Glu Asn Ala Lys Asn Gly Glu
Ala Lys Ile Thr Glu Ala Pro Ala 35 40 45 Ser Glu Lys Glu Ile Val
Glu Val Lys Glu Glu Asn Ile Glu Asp Ala 50 55 60 Thr Glu Lys Gly
Gly Glu Lys Lys Glu Ala Val Ala Ala Glu Val Lys 65 70 75 80 Asn Glu
Glu Glu Asp Gln Lys Glu Asp Glu Glu Asp Gln Asn Glu Glu 85 90 95
Lys Gly Glu Ala Gly Lys Glu Asp Lys Asp Glu Lys Gly Glu Glu Asp 100
105 110 Gly Lys Glu Asp Lys Asn Gly Asn Glu Lys Gly Glu Asp Ala Lys
Glu 115 120 125 Lys Glu Asp Gly Lys Lys Gly Glu Asp Gly Lys Gly Asn
Gly Glu Asp 130 135 140 Gly Lys Glu Lys Gly Glu Asp Glu Lys Glu Glu
Glu Asp Arg Lys Glu 145 150 155 160 Thr Gly Val Gly Lys Glu Asn Glu
Asp Gly Lys Glu Lys Gly Asp Lys 165 170 175 Lys Glu Gly Lys Asp Val
Lys Val Lys Glu Asp Glu Lys Glu Arg Glu 180 185 190 Asp Gly Lys Glu
Asp Glu Gly Gly Asn Glu Glu Glu Ala Gly Lys Glu 195 200 205 Lys Glu
Asp Leu Lys Glu Glu Glu Glu Gly Lys Glu Glu Asp Glu Ile 210 215 220
Lys Glu Asp Asp Gly Lys Lys Glu Glu Pro Gln Ser Ile Val 225 230 235
191 699 DNA Homo sapiens 191 atgatggaag aaaacataga tacaagtgcc
caagcagttg ctgaaaccaa gcaagaagca 60 gttgttgaag aagactacaa
tgaaaatgct aaaaatggag aagccaaaat tacagaggca 120 ccagcttctg
aaaaagaaat tgtggaagta aaagaagaaa atattgaaga tgccacagaa 180
aagggaggag aaaagaaaga agcagtggca gcagaagtaa aaaatgaaga agaagatcag
240 aaagaagatg aagaagatca aaacgaagag aaaggggaag ctggaaaaga
agacaaagat 300 gaaaaagggg aagaagatgg aaaagaggat aaaaatggaa
atgagaaagg agaagatgca 360 aaagagaaag aagatggaaa aaaaggtgaa
gacggaaaag gaaatggaga agatggaaaa 420 gagaaaggag aagatgaaaa
agaggaagaa gacagaaaag aaacaggagt tggaaaagag 480 aatgaagatg
gaaaagagaa gggagataaa aaagagggga aagatgtaaa agtcaaagaa 540
gatgaaaaag agagagaaga tggaaaagaa gatgaaggtg gaaatgagga agaagctgga
600 aaagagaaag aagatttaaa agaagaggaa gaaggaaaag aggaagatga
gatcaaagaa 660 gatgatggaa aaaaagagga gccacagagt attgtttaa 699 192
232 PRT Homo sapiens 192 Met Met Glu Glu Asn Ile Asp Thr Ser Ala
Gln Ala Val Ala Glu Thr 1 5 10 15 Lys Gln Glu Ala Val Val Glu Glu
Asp Tyr Asn Glu Asn Ala Lys Asn 20 25 30 Gly Glu Ala Lys Ile Thr
Glu Ala Pro Ala Ser Glu Lys Glu Ile Val 35 40 45 Glu Val Lys Glu
Glu Asn Ile Glu Asp Ala Thr Glu Lys Gly Gly Glu 50 55 60 Lys Lys
Glu Ala Val Ala Ala Glu Val Lys Asn Glu Glu Glu Asp Gln 65 70 75 80
Lys Glu Asp Glu Glu Asp Gln Asn Glu Glu Lys Gly Glu Ala Gly Lys 85
90 95 Glu Asp Lys Asp Glu Lys Gly Glu Glu Asp Gly Lys Glu Asp Lys
Asn 100 105 110 Gly Asn Glu Lys Gly Glu Asp Ala Lys Glu Lys Glu Asp
Gly Lys Lys 115 120 125 Gly Glu Asp Gly Lys Gly Asn Gly Glu Asp Gly
Lys Glu Lys Gly Glu 130 135 140 Asp Glu Lys Glu Glu Glu Asp Arg Lys
Glu Thr Gly Val Gly Lys Glu 145 150 155 160 Asn Glu Asp Gly Lys Glu
Lys Gly Asp Lys Lys Glu Gly Lys Asp Val 165 170 175 Lys Val Lys Glu
Asp Glu Lys Glu Arg Glu Asp Gly Lys Glu Asp Glu 180 185 190 Gly Gly
Asn Glu Glu Glu Ala Gly Lys Glu Lys Glu Asp Leu Lys Glu 195 200 205
Glu Glu Glu Gly Lys Glu Glu Asp Glu Ile Lys Glu Asp Asp Gly Lys 210
215 220 Lys Glu Glu Pro Gln Ser Ile Val 225 230 193 696 DNA Homo
sapiens 193 atggaagaaa acatagatac aagtgcccaa gcagttgctg aaaccaagca
agaagcagtt 60 gttgaagaag actacaatga aaatgctaaa aatggagaag
ccaaaattac agaggcacca 120 gcttctgaaa aagaaattgt ggaagtaaaa
gaagaaaata ttgaagatgc cacagaaaag 180 ggaggagaaa agaaagaagc
agtggcagca gaagtaaaaa atgaagaaga agatcagaaa 240 gaagatgaag
aagatcaaaa cgaagagaaa ggggaagctg gaaaagaaga caaagatgaa 300
aaaggggaag aagatggaaa agaggataaa aatggaaatg agaaaggaga agatgcaaaa
360 gagaaagaag atggaaaaaa aggtgaagac ggaaaaggaa atggagaaga
tggaaaagag 420 aaaggagaag atgaaaaaga ggaagaagac agaaaagaaa
caggagttgg aaaagagaat 480 gaagatggaa aagagaaggg agataaaaaa
gaggggaaag atgtaaaagt caaagaagat 540 gaaaaagaga gagaagatgg
aaaagaagat gaaggtggaa atgaggaaga agctggaaaa 600 gagaaagaag
atttaaaaga agaggaagaa ggaaaagagg aagatgagat caaagaagat 660
gatggaaaaa aagaggagcc acagagtatt gtttaa 696 194 231 PRT Homo
sapiens 194 Met Glu Glu Asn Ile Asp Thr Ser Ala Gln Ala Val Ala Glu
Thr Lys 1 5 10 15 Gln Glu Ala Val Val Glu Glu Asp Tyr Asn Glu Asn
Ala Lys Asn Gly 20 25 30 Glu Ala Lys Ile Thr Glu Ala Pro Ala Ser
Glu Lys Glu Ile Val Glu 35 40 45 Val Lys Glu Glu Asn Ile Glu Asp
Ala Thr Glu Lys Gly Gly Glu Lys 50 55 60 Lys Glu Ala Val Ala Ala
Glu Val Lys Asn Glu Glu Glu Asp Gln Lys 65 70 75 80 Glu Asp Glu Glu
Asp Gln Asn Glu Glu Lys Gly Glu Ala Gly Lys Glu 85 90 95 Asp Lys
Asp Glu Lys Gly Glu Glu Asp Gly Lys Glu Asp Lys Asn Gly 100 105 110
Asn Glu Lys Gly Glu Asp Ala Lys Glu Lys Glu Asp Gly Lys Lys Gly 115
120 125 Glu Asp Gly Lys Gly Asn Gly Glu Asp Gly Lys Glu Lys Gly Glu
Asp 130 135 140 Glu Lys Glu Glu Glu Asp Arg Lys Glu Thr Gly Val Gly
Lys Glu Asn 145 150 155 160 Glu Asp Gly Lys Glu Lys Gly Asp Lys Lys
Glu Gly Lys Asp Val Lys 165 170 175 Val Lys Glu Asp Glu Lys Glu Arg
Glu Asp Gly Lys Glu Asp Glu Gly 180 185 190 Gly Asn Glu Glu Glu Ala
Gly Lys Glu Lys Glu Asp Leu Lys Glu Glu 195 200 205 Glu Glu Gly Lys
Glu Glu Asp Glu Ile Lys Glu Asp Asp Gly Lys Lys 210 215 220 Glu Glu
Pro Gln Ser Ile Val 225 230 195 72 DNA Homo sapiens 195 atgaaaatgc
taaaaatgga gaagccaaaa ttacagaggc accagcttct gaaaaagaaa 60
ttgtggaagt aa 72 196 23 PRT Homo sapiens 196 Met Lys Met Leu Lys
Met Glu Lys Pro Lys Leu Gln Arg His Gln Leu 1 5 10 15 Leu Lys Lys
Lys Leu Trp Lys 20 197 66 DNA Homo sapiens 197 atgctaaaaa
tggagaagcc aaaattacag aggcaccagc ttctgaaaaa gaaattgtgg 60 aagtaa 66
198 21 PRT Homo sapiens 198 Met Leu Lys Met Glu Lys Pro Lys Leu Gln
Arg His Gln Leu Leu Lys 1 5 10 15 Lys Lys Leu Trp Lys 20 199 57 DNA
Homo sapiens 199 atggagaagc caaaattaca gaggcaccag cttctgaaaa
agaaattgtg gaagtaa 57 200 18 PRT Homo sapiens 200 Met Glu Lys Pro
Lys Leu Gln Arg His Gln Leu Leu Lys Lys Lys Leu 1 5 10 15 Trp Lys
201 51 DNA Homo sapiens 201 atgccacaga aaagggagga gaaaagaaag
aagcagtggc agcagaagta a 51 202 16 PRT Homo sapiens 202 Met Pro Gln
Lys Arg Glu Glu Lys Arg Lys Lys Gln Trp Gln Gln Lys 1 5 10 15 203
306 DNA Homo sapiens 203 atgaagaaga agatcagaaa gaagatgaag
aagatcaaaa
cgaagagaaa ggggaagctg 60 gaaaagaaga caaagatgaa aaaggggaag
aagatggaaa agaggataaa aatggaaatg 120 agaaaggaga agatgcaaaa
gagaaagaag atggaaaaaa aggtgaagac ggaaaaggaa 180 atggagaaga
tggaaaagag aaaggagaag atgaaaaaga ggaagaagac agaaaagaaa 240
caggagttgg aaaagagaat gaagatggaa aagagaaggg agataaaaaa gaggggaaag
300 atgtaa 306 204 101 PRT Homo sapiens 204 Met Lys Lys Lys Ile Arg
Lys Lys Met Lys Lys Ile Lys Thr Lys Arg 1 5 10 15 Lys Gly Lys Leu
Glu Lys Lys Thr Lys Met Lys Lys Gly Lys Lys Met 20 25 30 Glu Lys
Arg Ile Lys Met Glu Met Arg Lys Glu Lys Met Gln Lys Arg 35 40 45
Lys Lys Met Glu Lys Lys Val Lys Thr Glu Lys Glu Met Glu Lys Met 50
55 60 Glu Lys Arg Lys Glu Lys Met Lys Lys Arg Lys Lys Thr Glu Lys
Lys 65 70 75 80 Gln Glu Leu Glu Lys Arg Met Lys Met Glu Lys Arg Arg
Glu Ile Lys 85 90 95 Lys Arg Gly Lys Met 100 205 282 DNA Homo
sapiens 205 atgaagaaga tcaaaacgaa gagaaagggg aagctggaaa agaagacaaa
gatgaaaaag 60 gggaagaaga tggaaaagag gataaaaatg gaaatgagaa
aggagaagat gcaaaagaga 120 aagaagatgg aaaaaaaggt gaagacggaa
aaggaaatgg agaagatgga aaagagaaag 180 gagaagatga aaaagaggaa
gaagacagaa aagaaacagg agttggaaaa gagaatgaag 240 atggaaaaga
gaagggagat aaaaaagagg ggaaagatgt aa 282 206 93 PRT Homo sapiens 206
Met Lys Lys Ile Lys Thr Lys Arg Lys Gly Lys Leu Glu Lys Lys Thr 1 5
10 15 Lys Met Lys Lys Gly Lys Lys Met Glu Lys Arg Ile Lys Met Glu
Met 20 25 30 Arg Lys Glu Lys Met Gln Lys Arg Lys Lys Met Glu Lys
Lys Val Lys 35 40 45 Thr Glu Lys Glu Met Glu Lys Met Glu Lys Arg
Lys Glu Lys Met Lys 50 55 60 Lys Arg Lys Lys Thr Glu Lys Lys Gln
Glu Leu Glu Lys Arg Met Lys 65 70 75 80 Met Glu Lys Arg Arg Glu Ile
Lys Lys Arg Gly Lys Met 85 90 207 231 DNA Homo sapiens 207
atgaaaaagg ggaagaagat ggaaaagagg ataaaaatgg aaatgagaaa ggagaagatg
60 caaaagagaa agaagatgga aaaaaaggtg aagacggaaa aggaaatgga
gaagatggaa 120 aagagaaagg agaagatgaa aaagaggaag aagacagaaa
agaaacagga gttggaaaag 180 agaatgaaga tggaaaagag aagggagata
aaaaagaggg gaaagatgta a 231 208 76 PRT Homo sapiens 208 Met Lys Lys
Gly Lys Lys Met Glu Lys Arg Ile Lys Met Glu Met Arg 1 5 10 15 Lys
Glu Lys Met Gln Lys Arg Lys Lys Met Glu Lys Lys Val Lys Thr 20 25
30 Glu Lys Glu Met Glu Lys Met Glu Lys Arg Lys Glu Lys Met Lys Lys
35 40 45 Arg Lys Lys Thr Glu Lys Lys Gln Glu Leu Glu Lys Arg Met
Lys Met 50 55 60 Glu Lys Arg Arg Glu Ile Lys Lys Arg Gly Lys Met 65
70 75 209 213 DNA Homo sapiens 209 atggaaaaga ggataaaaat ggaaatgaga
aaggagaaga tgcaaaagag aaagaagatg 60 gaaaaaaagg tgaagacgga
aaaggaaatg gagaagatgg aaaagagaaa ggagaagatg 120 aaaaagagga
agaagacaga aaagaaacag gagttggaaa agagaatgaa gatggaaaag 180
agaagggaga taaaaaagag gggaaagatg taa 213 210 70 PRT Homo sapiens
210 Met Glu Lys Arg Ile Lys Met Glu Met Arg Lys Glu Lys Met Gln Lys
1 5 10 15 Arg Lys Lys Met Glu Lys Lys Val Lys Thr Glu Lys Glu Met
Glu Lys 20 25 30 Met Glu Lys Arg Lys Glu Lys Met Lys Lys Arg Lys
Lys Thr Glu Lys 35 40 45 Lys Gln Glu Leu Glu Lys Arg Met Lys Met
Glu Lys Arg Arg Glu Ile 50 55 60 Lys Lys Arg Gly Lys Met 65 70 211
195 DNA Homo sapiens 211 atggaaatga gaaaggagaa gatgcaaaag
agaaagaaga tggaaaaaaa ggtgaagacg 60 gaaaaggaaa tggagaagat
ggaaaagaga aaggagaaga tgaaaaagag gaagaagaca 120 gaaaagaaac
aggagttgga aaagagaatg aagatggaaa agagaaggga gataaaaaag 180
aggggaaaga tgtaa 195 212 64 PRT Homo sapiens 212 Met Glu Met Arg
Lys Glu Lys Met Gln Lys Arg Lys Lys Met Glu Lys 1 5 10 15 Lys Val
Lys Thr Glu Lys Glu Met Glu Lys Met Glu Lys Arg Lys Glu 20 25 30
Lys Met Lys Lys Arg Lys Lys Thr Glu Lys Lys Gln Glu Leu Glu Lys 35
40 45 Arg Met Lys Met Glu Lys Arg Arg Glu Ile Lys Lys Arg Gly Lys
Met 50 55 60 213 189 DNA Homo sapiens 213 atgagaaagg agaagatgca
aaagagaaag aagatggaaa aaaaggtgaa gacggaaaag 60 gaaatggaga
agatggaaaa gagaaaggag aagatgaaaa agaggaagaa gacagaaaag 120
aaacaggagt tggaaaagag aatgaagatg gaaaagagaa gggagataaa aaagagggga
180 aagatgtaa 189 214 62 PRT Homo sapiens 214 Met Arg Lys Glu Lys
Met Gln Lys Arg Lys Lys Met Glu Lys Lys Val 1 5 10 15 Lys Thr Glu
Lys Glu Met Glu Lys Met Glu Lys Arg Lys Glu Lys Met 20 25 30 Lys
Lys Arg Lys Lys Thr Glu Lys Lys Gln Glu Leu Glu Lys Arg Met 35 40
45 Lys Met Glu Lys Arg Arg Glu Ile Lys Lys Arg Gly Lys Met 50 55 60
215 174 DNA Homo sapiens 215 atgcaaaaga gaaagaagat ggaaaaaaag
gtgaagacgg aaaaggaaat ggagaagatg 60 gaaaagagaa aggagaagat
gaaaaagagg aagaagacag aaaagaaaca ggagttggaa 120 aagagaatga
agatggaaaa gagaagggag ataaaaaaga ggggaaagat gtaa 174 216 57 PRT
Homo sapiens 216 Met Gln Lys Arg Lys Lys Met Glu Lys Lys Val Lys
Thr Glu Lys Glu 1 5 10 15 Met Glu Lys Met Glu Lys Arg Lys Glu Lys
Met Lys Lys Arg Lys Lys 20 25 30 Thr Glu Lys Lys Gln Glu Leu Glu
Lys Arg Met Lys Met Glu Lys Arg 35 40 45 Arg Glu Ile Lys Lys Arg
Gly Lys Met 50 55 217 156 DNA Homo sapiens 217 atggaaaaaa
aggtgaagac ggaaaaggaa atggagaaga tggaaaagag aaaggagaag 60
atgaaaaaga ggaagaagac agaaaagaaa caggagttgg aaaagagaat gaagatggaa
120 aagagaaggg agataaaaaa gaggggaaag atgtaa 156 218 51 PRT Homo
sapiens 218 Met Glu Lys Lys Val Lys Thr Glu Lys Glu Met Glu Lys Met
Glu Lys 1 5 10 15 Arg Lys Glu Lys Met Lys Lys Arg Lys Lys Thr Glu
Lys Lys Gln Glu 20 25 30 Leu Glu Lys Arg Met Lys Met Glu Lys Arg
Arg Glu Ile Lys Lys Arg 35 40 45 Gly Lys Met 50 219 126 DNA Homo
sapiens 219 atggagaaga tggaaaagag aaaggagaag atgaaaaaga ggaagaagac
agaaaagaaa 60 caggagttgg aaaagagaat gaagatggaa aagagaaggg
agataaaaaa gaggggaaag 120 atgtaa 126 220 41 PRT Homo sapiens 220
Met Glu Lys Met Glu Lys Arg Lys Glu Lys Met Lys Lys Arg Lys Lys 1 5
10 15 Thr Glu Lys Lys Gln Glu Leu Glu Lys Arg Met Lys Met Glu Lys
Arg 20 25 30 Arg Glu Ile Lys Lys Arg Gly Lys Met 35 40 221 117 DNA
Homo sapiens 221 atggaaaaga gaaaggagaa gatgaaaaag aggaagaaga
cagaaaagaa acaggagttg 60 gaaaagagaa tgaagatgga aaagagaagg
gagataaaaa agaggggaaa gatgtaa 117 222 38 PRT Homo sapiens 222 Met
Glu Lys Arg Lys Glu Lys Met Lys Lys Arg Lys Lys Thr Glu Lys 1 5 10
15 Lys Gln Glu Leu Glu Lys Arg Met Lys Met Glu Lys Arg Arg Glu Ile
20 25 30 Lys Lys Arg Gly Lys Met 35 223 96 DNA Homo sapiens 223
atgaaaaaga ggaagaagac agaaaagaaa caggagttgg aaaagagaat gaagatggaa
60 aagagaaggg agataaaaaa gaggggaaag atgtaa 96 224 31 PRT Homo
sapiens 224 Met Lys Lys Arg Lys Lys Thr Glu Lys Lys Gln Glu Leu Glu
Lys Arg 1 5 10 15 Met Lys Met Glu Lys Arg Arg Glu Ile Lys Lys Arg
Gly Lys Met 20 25 30 225 48 DNA Homo sapiens 225 atgaagatgg
aaaagagaag ggagataaaa aagaggggaa agatgtaa 48 226 15 PRT Homo
sapiens 226 Met Lys Met Glu Lys Arg Arg Glu Ile Lys Lys Arg Gly Lys
Met 1 5 10 15 227 42 DNA Homo sapiens 227 atggaaaaga gaagggagat
aaaaaagagg ggaaagatgt aa 42 228 13 PRT Homo sapiens 228 Met Glu Lys
Arg Arg Glu Ile Lys Lys Arg Gly Lys Met 1 5 10 229 78 DNA Homo
sapiens 229 atgaaaaaga gagagaagat ggaaaagaag atgaaggtgg aaatgaggaa
gaagctggaa 60 aagagaaaga agatttaa 78 230 25 PRT Homo sapiens 230
Met Lys Lys Arg Glu Lys Met Glu Lys Lys Met Lys Val Glu Met Arg 1 5
10 15 Lys Lys Leu Glu Lys Arg Lys Lys Ile 20 25 231 60 DNA Homo
sapiens 231 atggaaaaga agatgaaggt ggaaatgagg aagaagctgg aaaagagaaa
gaagatttaa 60 232 19 PRT Homo sapiens 232 Met Glu Lys Lys Met Lys
Val Glu Met Arg Lys Lys Leu Glu Lys Arg 1 5 10 15 Lys Lys Ile 233
48 DNA Homo sapiens 233 atgaaggtgg aaatgaggaa gaagctggaa aagagaaaga
agatttaa 48 234 15 PRT Homo sapiens 234 Met Lys Val Glu Met Arg Lys
Lys Leu Glu Lys Arg Lys Lys Ile 1 5 10 15 235 36 DNA Homo sapiens
235 atgaggaaga agctggaaaa gagaaagaag atttaa 36 236 11 PRT Homo
sapiens 236 Met Arg Lys Lys Leu Glu Lys Arg Lys Lys Ile 1 5 10 237
84 DNA Homo sapiens 237 atgagatcaa agaagatgat ggaaaaaaag aggagccaca
gagtattgtt taaaactgcc 60 ctatgtagtt tcataatttg gtaa 84 238 27 PRT
Homo sapiens 238 Met Arg Ser Lys Lys Met Met Glu Lys Lys Arg Ser
His Arg Val Leu 1 5 10 15 Phe Lys Thr Ala Leu Cys Ser Phe Ile Ile
Trp 20 25 239 69 DNA Homo sapiens 239 atgatggaaa aaaagaggag
ccacagagta ttgtttaaaa ctgccctatg tagtttcata 60 atttggtaa 69 240 22
PRT Homo sapiens 240 Met Met Glu Lys Lys Arg Ser His Arg Val Leu
Phe Lys Thr Ala Leu 1 5 10 15 Cys Ser Phe Ile Ile Trp 20 241 66 DNA
Homo sapiens 241 atggaaaaaa agaggagcca cagagtattg tttaaaactg
ccctatgtag tttcataatt 60 tggtaa 66 242 21 PRT Homo sapiens 242 Met
Glu Lys Lys Arg Ser His Arg Val Leu Phe Lys Thr Ala Leu Cys 1 5 10
15 Ser Phe Ile Ile Trp 20 243 75 DNA Homo sapiens 243 atgtaccttc
atgttgtaaa gttaatagag ataaatattt ttatcaaaaa ttttataaac 60
acagcctttc tttag 75 244 24 PRT Homo sapiens 244 Met Tyr Leu His Val
Val Lys Leu Ile Glu Ile Asn Ile Phe Ile Lys 1 5 10 15 Asn Phe Ile
Asn Thr Ala Phe Leu 20 245 75 DNA Homo sapiens 245 atgaaacatt
tatctataaa ttttgtgatt atagtagtgg aatacataga aaaaaatatg 60
ctttcaactt tgtga 75 246 24 PRT Homo sapiens 246 Met Lys His Leu Ser
Ile Asn Phe Val Ile Ile Val Val Glu Tyr Ile 1 5 10 15 Glu Lys Asn
Met Leu Ser Thr Leu 20 247 18 DNA Homo sapiens 247 atgctttcaa
ctttgtga 18 248 5 PRT Homo sapiens 248 Met Leu Ser Thr Leu 1 5 249
15 DNA Homo sapiens 249 atgtcaaatc tttga 15 250 4 PRT Homo sapiens
250 Met Ser Asn Leu 1 251 81 DNA Homo sapiens 251 atgttaagag
ttaaacttat ctttcccaaa tataacttta ttattagctt gggaaaaatg 60
aaattgtatt cccattttta a 81 252 26 PRT Homo sapiens 252 Met Leu Arg
Val Lys Leu Ile Phe Pro Lys Tyr Asn Phe Ile Ile Ser 1 5 10 15 Leu
Gly Lys Met Lys Leu Tyr Ser His Phe 20 25 253 24 DNA Homo sapiens
253 atgaaattgt attcccattt ttaa 24 254 7 PRT Homo sapiens 254 Met
Lys Leu Tyr Ser His Phe 1 5 255 27 DNA Homo sapiens 255 atgtttattt
cagaagggca gttttga 27 256 8 PRT Homo sapiens 256 Met Phe Ile Ser
Glu Gly Gln Phe 1 5 257 90 DNA Homo sapiens 257 atgattgtgt
tttgttatat cttcaaaaat atagctagtg aaatattgtg cttaattttt 60
ttctattgtg ttattcatga aaatatttaa 90 258 29 PRT Homo sapiens 258 Met
Ile Val Phe Cys Tyr Ile Phe Lys Asn Ile Ala Ser Glu Ile Leu 1 5 10
15 Cys Leu Ile Phe Phe Tyr Cys Val Ile His Glu Asn Ile 20 25 259 24
DNA Homo sapiens 259 atgaaaatat ttaatattca ctga 24 260 7 PRT Homo
sapiens 260 Met Lys Ile Phe Asn Ile His 1 5 261 1182 DNA Homo
sapiens 261 tcgacccacg cgtccgtgat aaataactta taggtgatag tgataattcc
tgattccaag 60 aatgccatct gataaaaaag aatagaaatg gaaagtggga
ctgagaggga gtcagcaggc 120 atgctgcggt ggcggtcact ccctctgcca
ctatccccag ggaaggaaag gctccgccat 180 ttgggaaagt ggtttctacg
tcactggaca ccggttctga gcattagttt gagaactcgt 240 tcccgaatgt
gctttcctcc ctctcccctg cccacctcaa gtttaataaa taaggttgta 300
cttttcttac tataaaataa atgtctgtaa ctgctgtgca ctgctgtaaa cttgttagag
360 aaaaaaataa cctgcatgtg ggctcctcag ttattgagtt tttgtgatcc
tatctcagtc 420 tgggggggaa cattctcaag aggtgaaata caagaaagcc
tttttttctt ggatcttttc 480 ccgagattca aatctccgat ttcccatttg
ggggcaagtt tttttcttca ccttcaatat 540 gagaattcag cgaacttgaa
agaaaaatca tctgtgagtt ccttcaggtt ctcactcata 600 gtcatgatcc
ttcagaggga atatgcactg gcgagtttaa agtaagggct atgatatttg 660
atggtcccaa agtacggcag ctgcaaaaag tagtggaagg aaattgtcta cgtgtcttgg
720 aaaaattagt taggaatttg gatgggtaaa aggtaccctt gccttactcc
atcttatttt 780 cttagccccc tttgagtgtt ttaactggtt tcatgtccta
gtaggaagtg cattctccat 840 cctcatcctc tgccctccca ggaagtcagt
gattgtcttt ttgggcttcc cctccaaagg 900 accttctgca gtggaagtgc
cacatccagt tcttttcttt tgttgctgct gtgtttagat 960 aattgaagag
atctttgtgc cacacaggat tttttttttt ttttaagaaa aacctataga 1020
tgaaaaatta ctaatgaaac tgtgtgtacg tgtctgtgcg tgcaacataa aaatacagta
1080 gcacctaagg agcttgaatc ttggttcctg taaaatttca aattgatgtg
gtattaataa 1140 aaaaaaaaaa aacccaaaaa aaaaaaaaaa aaaagggcgg cc 1182
262 24 DNA Homo sapiens 262 atgccatctg ataaaaaaga atag 24 263 7 PRT
Homo sapiens 263 Met Pro Ser Asp Lys Lys Glu 1 5 264 228 DNA Homo
sapiens 264 atggaaagtg ggactgagag ggagtcagca ggcatgctgc ggtggcggtc
actccctctg 60 ccactatccc cagggaagga aaggctccgc catttgggaa
agtggtttct acgtcactgg 120 acaccggttc tgagcattag tttgagaact
cgttcccgaa tgtgctttcc tccctctccc 180 ctgcccacct caagtttaat
aaataaggtt gtacttttct tactataa 228 265 75 PRT Homo sapiens 265 Met
Glu Ser Gly Thr Glu Arg Glu Ser Ala Gly Met Leu Arg Trp Arg 1 5 10
15 Ser Leu Pro Leu Pro Leu Ser Pro Gly Lys Glu Arg Leu Arg His Leu
20 25 30 Gly Lys Trp Phe Leu Arg His Trp Thr Pro Val Leu Ser Ile
Ser Leu 35 40 45 Arg Thr Arg Ser Arg Met Cys Phe Pro Pro Ser Pro
Leu Pro Thr Ser 50 55 60 Ser Leu Ile Asn Lys Val Val Leu Phe Leu
Leu 65 70 75 266 195 DNA Homo sapiens 266 atgctgcggt ggcggtcact
ccctctgcca ctatccccag ggaaggaaag gctccgccat 60 ttgggaaagt
ggtttctacg tcactggaca ccggttctga gcattagttt gagaactcgt 120
tcccgaatgt gctttcctcc ctctcccctg cccacctcaa gtttaataaa taaggttgta
180 cttttcttac tataa 195 267 64 PRT Homo sapiens 267 Met Leu Arg
Trp Arg Ser Leu Pro Leu Pro Leu Ser Pro Gly Lys Glu 1 5 10 15 Arg
Leu Arg His Leu Gly Lys Trp Phe Leu Arg His Trp Thr Pro Val 20 25
30 Leu Ser Ile Ser Leu Arg Thr Arg Ser Arg Met Cys Phe Pro Pro Ser
35 40 45 Pro Leu Pro Thr Ser Ser Leu Ile Asn Lys Val Val Leu Phe
Leu Leu 50 55 60 268 69 DNA Homo sapiens 268 atgtgctttc ctccctctcc
cctgcccacc tcaagtttaa taaataaggt tgtacttttc 60 ttactataa 69 269 21
PRT Homo sapiens 269 Met Cys Phe Pro Pro Ser Pro Leu Pro Thr Ser
Ser Leu Ile Asn Lys 1 5 10 15 Val Val Leu Phe Leu 20 270 87 DNA
Homo sapiens 270 atgtctgtaa ctgctgtgca ctgctgtaaa cttgttagag
aaaaaaataa cctgcatgtg 60 ggctcctcag ttattgagtt tttgtga 87 271 28
PRT Homo sapiens 271 Met Ser Val Thr Ala Val His Cys Cys Lys Leu
Val Arg Glu Lys Asn 1 5 10 15 Asn Leu His Val Gly Ser Ser Val Ile
Glu Phe Leu 20 25 272 270 DNA Homo sapiens 272 atgtgggctc
ctcagttatt gagtttttgt gatcctatct cagtctgggg gggaacattc 60
tcaagaggtg aaatacaaga aagccttttt ttcttggatc ttttcccgag attcaaatct
120 ccgatttccc atttgggggc aagttttttt cttcaccttc aatatgagaa
ttcagcgaac 180 ttgaaagaaa aatcatctgt gagttccttc aggttctcac
tcatagtcat gatccttcag 240 agggaatatg
cactggcgag tttaaagtaa 270 273 89 PRT Homo sapiens 273 Met Trp Ala
Pro Gln Leu Leu Ser Phe Cys Asp Pro Ile Ser Val Trp 1 5 10 15 Gly
Gly Thr Phe Ser Arg Gly Glu Ile Gln Glu Ser Leu Phe Phe Leu 20 25
30 Asp Leu Phe Pro Arg Phe Lys Ser Pro Ile Ser His Leu Gly Ala Ser
35 40 45 Phe Phe Leu His Leu Gln Tyr Glu Asn Ser Ala Asn Leu Lys
Glu Lys 50 55 60 Ser Ser Val Ser Ser Phe Arg Phe Ser Leu Ile Val
Met Ile Leu Gln 65 70 75 80 Arg Glu Tyr Ala Leu Ala Ser Leu Lys 85
274 21 DNA Homo sapiens 274 atgagaattc agcgaacttg a 21 275 6 PRT
Homo sapiens 275 Met Arg Ile Gln Arg Thr 1 5 276 42 DNA Homo
sapiens 276 atgatccttc agagggaata tgcactggcg agtttaaagt aa 42 277
13 PRT Homo sapiens 277 Met Ile Leu Gln Arg Glu Tyr Ala Leu Ala Ser
Leu Lys 1 5 10 278 18 DNA Homo sapiens 278 atgcactggc gagtttaa 18
279 5 PRT Homo sapiens 279 Met His Trp Arg Val 1 5 280 99 DNA Homo
sapiens 280 atgatatttg atggtcccaa agtacggcag ctgcaaaaag tagtggaagg
aaattgtcta 60 cgtgtcttgg aaaaattagt taggaatttg gatgggtaa 99 281 32
PRT Homo sapiens 281 Met Ile Phe Asp Gly Pro Lys Val Arg Gln Leu
Gln Lys Val Val Glu 1 5 10 15 Gly Asn Cys Leu Arg Val Leu Glu Lys
Leu Val Arg Asn Leu Asp Gly 20 25 30 282 33 DNA Homo sapiens 282
atggtcccaa agtacggcag ctgcaaaaag tag 33 283 10 PRT Homo sapiens 283
Met Val Pro Lys Tyr Gly Ser Cys Lys Lys 1 5 10 284 54 DNA Homo
sapiens 284 atgggtaaaa ggtacccttg ccttactcca tcttattttc ttagccccct
ttga 54 285 17 PRT Homo sapiens 285 Met Gly Lys Arg Tyr Pro Cys Leu
Thr Pro Ser Tyr Phe Leu Ser Pro 1 5 10 15 Leu 286 15 DNA Homo
sapiens 286 atgaaaaatt actaa 15 287 4 PRT Homo sapiens 287 Met Lys
Asn Tyr 1 288 48 DNA Homo sapiens 288 atgaaactgt gtgtacgtgt
ctgtgcgtgc aacataaaaa tacagtag 48 289 15 PRT Homo sapiens 289 Met
Lys Leu Cys Val Arg Val Cys Ala Cys Asn Ile Lys Ile Gln 1 5 10 15
290 12 DNA Homo sapiens 290 atgtggtatt aa 12 291 3 PRT Homo sapiens
291 Met Trp Tyr 1 292 1965 DNA Homo sapiens 292 tcgacccacg
cgtccggagg agagagagtg aacagggagc ggggcttttg tctgttggtc 60
tccctggact gaagagaggg agaatagaag cccaagacta agattctcaa aatggtttat
120 tacccagaac tctttgtctg ggtcagtcaa gaaccatttc caaacaagga
catggaggga 180 aggcttccta agggaagact tcctgtccca aaggaagtga
accgcaagaa gaacgatgag 240 acaaacgctg cctccctgac tccactgggc
agcagtgaac tccgctcccc aagaatcagt 300 tacctccact ttttttaatc
gtaacacctc catttgtatt acatatggtg tatgggtatt 360 gatgaggtca
tggtatcata tatgggattt ttttctgtgt aaatcatcaa gtataagaag 420
aaactatggg actctgagcc ttgctttaga gaatttacag tggacaaata ggtgtcatca
480 aaccagtttt taatcattct gactcaagtg aaaacgctca gaatttcaca
ctgtgaatcc 540 cgtttacaac ccttacaggt gggccttcag gcctggttcg
ctacaacaat gtcttccaca 600 actcaaactc ccaccgcgct cacacaaccg
gtccactcct gccttttcac tcacacagct 660 cccgactgct tcttgcagag
gctgagagtc ccccccccac cttttttttc atttagatgt 720 aacaaaccta
gtagtttatg ttcatcaatt gtctgtatat ctctatattt tatccatgta 780
ctcttttgat gtatagaagt agtttgaaac tcattgtttc cttgtggtaa gtgaccgaga
840 tgctgccaca ggacctgaga cactgatgaa tggtgctatt ttggactttc
aacatgctcc 900 ttggcgaggt agctctgatg gagttatttt ttatttccat
gttctaagaa ggtgttggta 960 ctctgtttcc cttgaatgtt gttctctaga
ctggattgac ttgttttcct tgtgtcttca 1020 gtgtggcttt cttcctcagt
gttgtaggtt gagcgaatgc taccagagtg tgagagacca 1080 ttgtctcgtt
ggctggcgct cacggacatg cagtcacggt agcgggagca atcacaaaac 1140
tgtaatttac ttaccaaatc tcttcctttc cgtagcctcg cctgcctgac ttagagaaag
1200 aaaagcaata attttacagg cattttgagg tgtctctttg ggttctttct
gtttgaaagg 1260 atatttgtcg aaaaaaagag caaaaccgtt ttaaataaac
tccccctgga aaaaaaccca 1320 aaacactggc atactgagtg ggaatatgaa
aatgacacct tttccaaata ttaaattgga 1380 aaacaaggtc tacaaaatca
tgatactttt ttaaaaggca gagcattctt ttttcggcaa 1440 ttttgataag
caaggtgtag atttacattt ttgtccttgc tcccaacgaa atggataaac 1500
aaaaataaat taccatctac tcatggaatg ttgttgtgtt agccagtctg aaagcccacc
1560 ttaattttta tataactgtc tttagctctt cttttgacag ggcaggcctt
gttctgaact 1620 gtttcgcttc tgactgttaa acaccgatga cgcatgcact
gcacttcttc gttttcttct 1680 tgctccccca ttggcctgag tttcttgtgc
attactcctc tccctccttc gttagaatag 1740 gtgtatcagc tgtgtaaata
gagcaagaaa acagtattct gcatctgtgg catttatgta 1800 gagttgcagt
tgtgtactgc tgaaaatgca ggcttttgta acagtgtgat ctttactgat 1860
gcactcatga caagtaccca atgtatttta gctattttag tagtatttgt tcaataaata
1920 cgcaagctgt aaggtaaaaa aaaaaaaaaa aaaaaaaggg cggcc 1965 293 207
DNA Homo sapiens 293 atggtttatt acccagaact ctttgtctgg gtcagtcaag
aaccatttcc aaacaaggac 60 atggagggaa ggcttcctaa gggaagactt
cctgtcccaa aggaagtgaa ccgcaagaag 120 aacgatgaga caaacgctgc
ctccctgact ccactgggca gcagtgaact ccgctcccca 180 agaatcagtt
acctccactt tttttaa 207 294 68 PRT Homo sapiens 294 Met Val Tyr Tyr
Pro Glu Leu Phe Val Trp Val Ser Gln Glu Pro Phe 1 5 10 15 Pro Asn
Lys Asp Met Glu Gly Arg Leu Pro Lys Gly Arg Leu Pro Val 20 25 30
Pro Lys Glu Val Asn Arg Lys Lys Asn Asp Glu Thr Asn Ala Ala Ser 35
40 45 Leu Thr Pro Leu Gly Ser Ser Glu Leu Arg Ser Pro Arg Ile Ser
Tyr 50 55 60 Leu His Phe Phe 65 295 147 DNA Homo sapiens 295
atggagggaa ggcttcctaa gggaagactt cctgtcccaa aggaagtgaa ccgcaagaag
60 aacgatgaga caaacgctgc ctccctgact ccactgggca gcagtgaact
ccgctcccca 120 agaatcagtt acctccactt tttttaa 147 296 48 PRT Homo
sapiens 296 Met Glu Gly Arg Leu Pro Lys Gly Arg Leu Pro Val Pro Lys
Glu Val 1 5 10 15 Asn Arg Lys Lys Asn Asp Glu Thr Asn Ala Ala Ser
Leu Thr Pro Leu 20 25 30 Gly Ser Ser Glu Leu Arg Ser Pro Arg Ile
Ser Tyr Leu His Phe Phe 35 40 45 297 24 DNA Homo sapiens 297
atgagacaaa cgctgcctcc ctga 24 298 7 PRT Homo sapiens 298 Met Arg
Gln Thr Leu Pro Pro 1 5 299 18 DNA Homo sapiens 299 atggtgtatg
ggtattga 18 300 5 PRT Homo sapiens 300 Met Val Tyr Gly Tyr 1 5 301
51 DNA Homo sapiens 301 atgggtattg atgaggtcat ggtatcatat atgggatttt
tttctgtgta a 51 302 16 PRT Homo sapiens 302 Met Gly Ile Asp Glu Val
Met Val Ser Tyr Met Gly Phe Phe Ser Val 1 5 10 15 303 132 DNA Homo
sapiens 303 atgaggtcat ggtatcatat atgggatttt tttctgtgta aatcatcaag
tataagaaga 60 aactatggga ctctgagcct tgctttagag aatttacagt
ggacaaatag gtgtcatcaa 120 accagttttt aa 132 304 43 PRT Homo sapiens
304 Met Arg Ser Trp Tyr His Ile Trp Asp Phe Phe Leu Cys Lys Ser Ser
1 5 10 15 Ser Ile Arg Arg Asn Tyr Gly Thr Leu Ser Leu Ala Leu Glu
Asn Leu 20 25 30 Gln Trp Thr Asn Arg Cys His Gln Thr Ser Phe 35 40
305 33 DNA Homo sapiens 305 atggtatcat atatgggatt tttttctgtg taa 33
306 10 PRT Homo sapiens 306 Met Val Ser Tyr Met Gly Phe Phe Ser Val
1 5 10 307 21 DNA Homo sapiens 307 atgggatttt tttctgtgta a 21 308 6
PRT Homo sapiens 308 Met Gly Phe Phe Ser Val 1 5 309 12 DNA Homo
sapiens 309 atgggactct ga 12 310 3 PRT Homo sapiens 310 Met Gly Leu
1 311 201 DNA Homo sapiens 311 atgtcttcca caactcaaac tcccaccgcg
ctcacacaac cggtccactc ctgccttttc 60 actcacacag ctcccgactg
cttcttgcag aggctgagag tccccccccc accttttttt 120 tcatttagat
gtaacaaacc tagtagttta tgttcatcaa ttgtctgtat atctctatat 180
tttatccatg tactcttttg a 201 312 66 PRT Homo sapiens 312 Met Ser Ser
Thr Thr Gln Thr Pro Thr Ala Leu Thr Gln Pro Val His 1 5 10 15 Ser
Cys Leu Phe Thr His Thr Ala Pro Asp Cys Phe Leu Gln Arg Leu 20 25
30 Arg Val Pro Pro Pro Pro Phe Phe Ser Phe Arg Cys Asn Lys Pro Ser
35 40 45 Ser Leu Cys Ser Ser Ile Val Cys Ile Ser Leu Tyr Phe Ile
His Val 50 55 60 Leu Phe 65 313 93 DNA Homo sapiens 313 atgttcatca
attgtctgta tatctctata ttttatccat gtactctttt gatgtataga 60
agtagtttga aactcattgt ttccttgtgg taa 93 314 30 PRT Homo sapiens 314
Met Phe Ile Asn Cys Leu Tyr Ile Ser Ile Phe Tyr Pro Cys Thr Leu 1 5
10 15 Leu Met Tyr Arg Ser Ser Leu Lys Leu Ile Val Ser Leu Trp 20 25
30 315 21 DNA Homo sapiens 315 atgtactctt ttgatgtata g 21 316 6 PRT
Homo sapiens 316 Met Tyr Ser Phe Asp Val 1 5 317 42 DNA Homo
sapiens 317 atgtatagaa gtagtttgaa actcattgtt tccttgtggt aa 42 318
13 PRT Homo sapiens 318 Met Tyr Arg Ser Ser Leu Lys Leu Ile Val Ser
Leu Trp 1 5 10 319 27 DNA Homo sapiens 319 atgctgccac aggacctgag
acactga 27 320 8 PRT Homo sapiens 320 Met Leu Pro Gln Asp Leu Arg
His 1 5 321 324 DNA Homo sapiens 321 atgaatggtg ctattttgga
ctttcaacat gctccttggc gaggtagctc tgatggagtt 60 attttttatt
tccatgttct aagaaggtgt tggtactctg tttcccttga atgttgttct 120
ctagactgga ttgacttgtt ttccttgtgt cttcagtgtg gctttcttcc tcagtgttgt
180 aggttgagcg aatgctacca gagtgtgaga gaccattgtc tcgttggctg
gcgctcacgg 240 acatgcagtc acggtagcgg gagcaatcac aaaactgtaa
tttacttacc aaatctcttc 300 ctttccgtag cctcgcctgc ctga 324 322 107
PRT Homo sapiens 322 Met Asn Gly Ala Ile Leu Asp Phe Gln His Ala
Pro Trp Arg Gly Ser 1 5 10 15 Ser Asp Gly Val Ile Phe Tyr Phe His
Val Leu Arg Arg Cys Trp Tyr 20 25 30 Ser Val Ser Leu Glu Cys Cys
Ser Leu Asp Trp Ile Asp Leu Phe Ser 35 40 45 Leu Cys Leu Gln Cys
Gly Phe Leu Pro Gln Cys Cys Arg Leu Ser Glu 50 55 60 Cys Tyr Gln
Ser Val Arg Asp His Cys Leu Val Gly Trp Arg Ser Arg 65 70 75 80 Thr
Cys Ser His Gly Ser Gly Ser Asn His Lys Thr Val Ile Tyr Leu 85 90
95 Pro Asn Leu Phe Leu Ser Val Ala Ser Pro Ala 100 105 323 78 DNA
Homo sapiens 323 atggtgctat tttggacttt caacatgctc cttggcgagg
tagctctgat ggagttattt 60 tttatttcca tgttctaa 78 324 25 PRT Homo
sapiens 324 Met Val Leu Phe Trp Thr Phe Asn Met Leu Leu Gly Glu Val
Ala Leu 1 5 10 15 Met Glu Leu Phe Phe Ile Ser Met Phe 20 25 325 54
DNA Homo sapiens 325 atgctccttg gcgaggtagc tctgatggag ttatttttta
tttccatgtt ctaa 54 326 17 PRT Homo sapiens 326 Met Leu Leu Gly Glu
Val Ala Leu Met Glu Leu Phe Phe Ile Ser Met 1 5 10 15 Phe 327 30
DNA Homo sapiens 327 atggagttat tttttatttc catgttctaa 30 328 9 PRT
Homo sapiens 328 Met Glu Leu Phe Phe Ile Ser Met Phe 1 5 329 24 DNA
Homo sapiens 329 atgttgttct ctagactgga ttga 24 330 7 PRT Homo
sapiens 330 Met Leu Phe Ser Arg Leu Asp 1 5 331 66 DNA Homo sapiens
331 atgctaccag agtgtgagag accattgtct cgttggctgg cgctcacgga
catgcagtca 60 cggtag 66 332 21 PRT Homo sapiens 332 Met Leu Pro Glu
Cys Glu Arg Pro Leu Ser Arg Trp Leu Ala Leu Thr 1 5 10 15 Asp Met
Gln Ser Arg 20 333 15 DNA Homo sapiens 333 atgcagtcac ggtag 15 334
4 PRT Homo sapiens 334 Met Gln Ser Arg 1 335 69 DNA Homo sapiens
335 atgaaaatga caccttttcc aaatattaaa ttggaaaaca aggtctacaa
aatcatgata 60 cttttttaa 69 336 22 PRT Homo sapiens 336 Met Lys Met
Thr Pro Phe Pro Asn Ile Lys Leu Glu Asn Lys Val Tyr 1 5 10 15 Lys
Ile Met Ile Leu Phe 20 337 63 DNA Homo sapiens 337 atgacacctt
ttccaaatat taaattggaa aacaaggtct acaaaatcat gatacttttt 60 taa 63
338 20 PRT Homo sapiens 338 Met Thr Pro Phe Pro Asn Ile Lys Leu Glu
Asn Lys Val Tyr Lys Ile 1 5 10 15 Met Ile Leu Phe 20 339 15 DNA
Homo sapiens 339 atgatacttt tttaa 15 340 4 PRT Homo sapiens 340 Met
Ile Leu Phe 1 341 18 DNA Homo sapiens 341 atggataaac aaaaataa 18
342 5 PRT Homo sapiens 342 Met Asp Lys Gln Lys 1 5 343 42 DNA Homo
sapiens 343 atggaatgtt gttgtgttag ccagtctgaa agcccacctt aa 42 344
13 PRT Homo sapiens 344 Met Glu Cys Cys Cys Val Ser Gln Ser Glu Ser
Pro Pro 1 5 10 345 15 DNA Homo sapiens 345 atgttgttgt gttag 15 346
4 PRT Homo sapiens 346 Met Leu Leu Cys 1 347 111 DNA Homo sapiens
347 atgacgcatg cactgcactt cttcgttttc ttcttgctcc cccattggcc
tgagtttctt 60 gtgcattact cctctccctc cttcgttaga ataggtgtat
cagctgtgta a 111 348 36 PRT Homo sapiens 348 Met Thr His Ala Leu
His Phe Phe Val Phe Phe Leu Leu Pro His Trp 1 5 10 15 Pro Glu Phe
Leu Val His Tyr Ser Ser Pro Ser Phe Val Arg Ile Gly 20 25 30 Val
Ser Ala Val 35 349 87 DNA Homo sapiens 349 atgcactgca cttcttcgtt
ttcttcttgc tcccccattg gcctgagttt cttgtgcatt 60 actcctctcc
ctccttcgtt agaatag 87 350 28 PRT Homo sapiens 350 Met His Cys Thr
Ser Ser Phe Ser Ser Cys Ser Pro Ile Gly Leu Ser 1 5 10 15 Phe Leu
Cys Ile Thr Pro Leu Pro Pro Ser Leu Glu 20 25 351 24 DNA Homo
sapiens 351 atgcaggctt ttgtaacagt gtga 24 352 7 PRT Homo sapiens
352 Met Gln Ala Phe Val Thr Val 1 5 353 12 DNA Homo sapiens 353
atgcactcat ga 12 354 3 PRT Homo sapiens 354 Met His Ser 1 355 51
DNA Homo sapiens 355 atgacaagta cccaatgtat tttagctatt ttagtagtat
ttgttcaata a 51 356 16 PRT Homo sapiens 356 Met Thr Ser Thr Gln Cys
Ile Leu Ala Ile Leu Val Val Phe Val Gln 1 5 10 15 357 57 DNA Homo
sapiens 357 atgtatttta gctattttag tagtatttgt tcaataaata cgcaagctgt
aaggtaa 57 358 18 PRT Homo sapiens 358 Met Tyr Phe Ser Tyr Phe Ser
Ser Ile Cys Ser Ile Asn Thr Gln Ala 1 5 10 15 Val Arg 359 2702 DNA
Homo sapiens 359 tcgacccacg cgtccgggaa cgtacgtccc agccctcttt
agctacttag cgcctctggg 60 cccgagaaca cctgctcctt ggctcagtct
ggcgccaccg gcatcacgga actgtacttc 120 ccagagacgt cacaccggga
gacttccgat tcccgctctt gagattggac tctcacgtgc 180 aggagccagt
cctcgctggg ctctagcggg cttctgatgg aggagctact cctctgggag 240
gacagaaatt agcagcagcc tctgtcacca tccaaagatt acaacccatg aaaccattga
300 gtttgtgcct tgtatcagaa agcaaaggag aatgaaaaag cacagctaac
attgcttgag 360 gatctaggcg attaattctt tagactgtca tcatgggtat
cccgaggact aatgagtttt 420 gtgggaagat cataagtaat gaagttcttc
actgatttga agttgcgggg acacaaaaat 480 tgtcattgat ggttatgctc
ttttccaccg tctttgcttc agtttcaaac ttggatctcc 540 ggtatggagg
ggactatgat tcttttgcag atgttgtaca aaaattcttt gaatcactgt 600
ttgcttgtaa tatatgccca tatgttgtat tagatggagg atgtgacatt tcagataaaa
660 agcttacaac tttaaaggat agagctagag agaagatcca gatggcccat
tccctttctg 720 ttggtgggag tgggtatgta tgtcccttac tcatccggga
agtattcata caggttttga 780 tcaagctgcg ggtgtgtttt gtccagtgct
tttcagaagc agatcgggac attatgacac 840 ttgctaacca ttggaattgc
cctgtgttat catcagatag tgacttttgc atttttgacc 900 tgaaaactgg
gttttgccca ttgaatagct ttcagtggag aaatatgaac actattaagg 960
gcacacaaaa ctatatccct gccaaatgct tttcccttga tgcattctgc catcacttca
1020 gcaatatgaa taaagctcta ctacctctct ttgcggtgct atgtggaaat
gaccatgtta 1080 atctacccat catggagaca ttcttaagta aagcgcgtct
tcctcttgga gctaccagtt 1140 ctaaagggag gagacaccac cgaatcctgg
gacttctgaa ttggttgtct cattttgcca 1200 accctaccga agcactagat
aatgttctga aatacctccc aaaaaaggat cgagaaaatg 1260 ttaaggaact
tctctgctgt tccatggaag aataccaaca gtcccaggtg aagctacagg 1320
acttcttcca gtgtggtact tatgtctgtc cagatgcctt gaatcttggt ttaccagaat
1380 gggtattagt ggctttagct aaaggccagc tatctccttt catcagtgat
gctttggtcc 1440 taagacggac cattcttccc acacaggtgg aaaacatgca
gcaaccaaat gcccacagaa 1500 tatctcagcc catcaggcaa atcatctatg
ggcttctttt aaatgcctca ccacatctgg 1560 acaagacatc ctggaatgca
ttgcctcctc agcctctagc tttcagtgaa gtggaaagga 1620 ttaataaaaa
tatcagaacc
tcaatcattg atgcagtaga actggccaag gatcattctg 1680 acttaagcag
attgactgag ctctccttga ggaggcggca gatgcttctg ttagaaaccc 1740
tgaaggtgaa acagaccatt ctggagccaa tccctacttc actgaagttg cccattgctg
1800 tcagttgcta ctggttgcag cacaccgaga ccaaagcaaa gctacatcat
ctacaatcct 1860 tactgctcac aatgctagtg gggcccttga ttgccataat
caacagccct ggaaatgtgg 1920 accctgtacc caggcaggct cagtgtcttg
ctcctcgcta gttggtaaaa ggtaaggaag 1980 agctgcagga agatggtgct
aagatgttgt atgcagagtt ccaaagagtg aaggcgcaga 2040 cacggctggg
cacaagactg gacttagaca cagctcacat cttctgtcag tggcagtcct 2100
gtctccagat ggggatgtat ctcaaccagc tgctgtccac tcctctccca gagccagacc
2160 taactcgact gtacagtgga agcctggtgc acggactatg ccagcaactg
ctagcatcga 2220 cctctgtaga aagtctcctg agcatatgtc ctgaggctaa
gcaactttat gaatatctat 2280 tcaatgccca caaggtcata tgcccccgct
gaaatattcc taccaaaagg tagatcaaat 2340 tcaaaaaaaa aaaggcagaa
gaaacagaat accagctgtt ctaagaacag agggagaacc 2400 actgcacaca
ccaagtgttg gtatgaggga aacaaccggt ttgggttgtt aatggttgaa 2460
aacttagagg aacatagtga ggcctccaac attgaataaa actcagtttg catcaaacta
2520 gatgtattta atataatcct tacttaaaat tcttccgtta ccacccttga
aacaattagc 2580 tttttcttta ggactgacct gttaggggat aaacatcaca
ataatctgaa ttccaagtta 2640 ttttgtattt tgtttttaat aaatacaacc
tgatttaaga aaaaaaaaaa aaaagggcgg 2700 cc 2702 360 36 DNA Homo
sapiens 360 atggaggagc tactcctctg ggaggacaga aattag 36 361 11 PRT
Homo sapiens 361 Met Glu Glu Leu Leu Leu Trp Glu Asp Arg Asn 1 5 10
362 48 DNA Homo sapiens 362 atgaaaccat tgagtttgtg ccttgtatca
gaaagcaaag gagaatga 48 363 15 PRT Homo sapiens 363 Met Lys Pro Leu
Ser Leu Cys Leu Val Ser Glu Ser Lys Gly Glu 1 5 10 15 364 18 DNA
Homo sapiens 364 atgaaaaagc acagctaa 18 365 5 PRT Homo sapiens 365
Met Lys Lys His Ser 1 5 366 63 DNA Homo sapiens 366 atgggtatcc
cgaggactaa tgagttttgt gggaagatca taagtaatga agttcttcac 60 tga 63
367 20 PRT Homo sapiens 367 Met Gly Ile Pro Arg Thr Asn Glu Phe Cys
Gly Lys Ile Ile Ser Asn 1 5 10 15 Glu Val Leu His 20 368 24 DNA
Homo sapiens 368 atgagttttg tgggaagatc ataa 24 369 7 PRT Homo
sapiens 369 Met Ser Phe Val Gly Arg Ser 1 5 370 51 DNA Homo sapiens
370 atgaagttct tcactgattt gaagttgcgg ggacacaaaa attgtcattg a 51 371
16 PRT Homo sapiens 371 Met Lys Phe Phe Thr Asp Leu Lys Leu Arg Gly
His Lys Asn Cys His 1 5 10 15 372 1473 DNA Homo sapiens 372
atggttatgc tcttttccac cgtctttgct tcagtttcaa acttggatct ccggtatgga
60 ggggactatg attcttttgc agatgttgta caaaaattct ttgaatcact
gtttgcttgt 120 aatatatgcc catatgttgt attagatgga ggatgtgaca
tttcagataa aaagcttaca 180 actttaaagg atagagctag agagaagatc
cagatggccc attccctttc tgttggtggg 240 agtgggtatg tatgtccctt
actcatccgg gaagtattca tacaggtttt gatcaagctg 300 cgggtgtgtt
ttgtccagtg cttttcagaa gcagatcggg acattatgac acttgctaac 360
cattggaatt gccctgtgtt atcatcagat agtgactttt gcatttttga cctgaaaact
420 gggttttgcc cattgaatag ctttcagtgg agaaatatga acactattaa
gggcacacaa 480 aactatatcc ctgccaaatg cttttccctt gatgcattct
gccatcactt cagcaatatg 540 aataaagctc tactacctct ctttgcggtg
ctatgtggaa atgaccatgt taatctaccc 600 atcatggaga cattcttaag
taaagcgcgt cttcctcttg gagctaccag ttctaaaggg 660 aggagacacc
accgaatcct gggacttctg aattggttgt ctcattttgc caaccctacc 720
gaagcactag ataatgttct gaaatacctc ccaaaaaagg atcgagaaaa tgttaaggaa
780 cttctctgct gttccatgga agaataccaa cagtcccagg tgaagctaca
ggacttcttc 840 cagtgtggta cttatgtctg tccagatgcc ttgaatcttg
gtttaccaga atgggtatta 900 gtggctttag ctaaaggcca gctatctcct
ttcatcagtg atgctttggt cctaagacgg 960 accattcttc ccacacaggt
ggaaaacatg cagcaaccaa atgcccacag aatatctcag 1020 cccatcaggc
aaatcatcta tgggcttctt ttaaatgcct caccacatct ggacaagaca 1080
tcctggaatg cattgcctcc tcagcctcta gctttcagtg aagtggaaag gattaataaa
1140 aatatcagaa cctcaatcat tgatgcagta gaactggcca aggatcattc
tgacttaagc 1200 agattgactg agctctcctt gaggaggcgg cagatgcttc
tgttagaaac cctgaaggtg 1260 aaacagacca ttctggagcc aatccctact
tcactgaagt tgcccattgc tgtcagttgc 1320 tactggttgc agcacaccga
gaccaaagca aagctacatc atctacaatc cttactgctc 1380 acaatgctag
tggggccctt gattgccata atcaacagcc ctggaaatgt ggaccctgta 1440
cccaggcagg ctcagtgtct tgctcctcgc tag 1473 373 490 PRT Homo sapiens
373 Met Val Met Leu Phe Ser Thr Val Phe Ala Ser Val Ser Asn Leu Asp
1 5 10 15 Leu Arg Tyr Gly Gly Asp Tyr Asp Ser Phe Ala Asp Val Val
Gln Lys 20 25 30 Phe Phe Glu Ser Leu Phe Ala Cys Asn Ile Cys Pro
Tyr Val Val Leu 35 40 45 Asp Gly Gly Cys Asp Ile Ser Asp Lys Lys
Leu Thr Thr Leu Lys Asp 50 55 60 Arg Ala Arg Glu Lys Ile Gln Met
Ala His Ser Leu Ser Val Gly Gly 65 70 75 80 Ser Gly Tyr Val Cys Pro
Leu Leu Ile Arg Glu Val Phe Ile Gln Val 85 90 95 Leu Ile Lys Leu
Arg Val Cys Phe Val Gln Cys Phe Ser Glu Ala Asp 100 105 110 Arg Asp
Ile Met Thr Leu Ala Asn His Trp Asn Cys Pro Val Leu Ser 115 120 125
Ser Asp Ser Asp Phe Cys Ile Phe Asp Leu Lys Thr Gly Phe Cys Pro 130
135 140 Leu Asn Ser Phe Gln Trp Arg Asn Met Asn Thr Ile Lys Gly Thr
Gln 145 150 155 160 Asn Tyr Ile Pro Ala Lys Cys Phe Ser Leu Asp Ala
Phe Cys His His 165 170 175 Phe Ser Asn Met Asn Lys Ala Leu Leu Pro
Leu Phe Ala Val Leu Cys 180 185 190 Gly Asn Asp His Val Asn Leu Pro
Ile Met Glu Thr Phe Leu Ser Lys 195 200 205 Ala Arg Leu Pro Leu Gly
Ala Thr Ser Ser Lys Gly Arg Arg His His 210 215 220 Arg Ile Leu Gly
Leu Leu Asn Trp Leu Ser His Phe Ala Asn Pro Thr 225 230 235 240 Glu
Ala Leu Asp Asn Val Leu Lys Tyr Leu Pro Lys Lys Asp Arg Glu 245 250
255 Asn Val Lys Glu Leu Leu Cys Cys Ser Met Glu Glu Tyr Gln Gln Ser
260 265 270 Gln Val Lys Leu Gln Asp Phe Phe Gln Cys Gly Thr Tyr Val
Cys Pro 275 280 285 Asp Ala Leu Asn Leu Gly Leu Pro Glu Trp Val Leu
Val Ala Leu Ala 290 295 300 Lys Gly Gln Leu Ser Pro Phe Ile Ser Asp
Ala Leu Val Leu Arg Arg 305 310 315 320 Thr Ile Leu Pro Thr Gln Val
Glu Asn Met Gln Gln Pro Asn Ala His 325 330 335 Arg Ile Ser Gln Pro
Ile Arg Gln Ile Ile Tyr Gly Leu Leu Leu Asn 340 345 350 Ala Ser Pro
His Leu Asp Lys Thr Ser Trp Asn Ala Leu Pro Pro Gln 355 360 365 Pro
Leu Ala Phe Ser Glu Val Glu Arg Ile Asn Lys Asn Ile Arg Thr 370 375
380 Ser Ile Ile Asp Ala Val Glu Leu Ala Lys Asp His Ser Asp Leu Ser
385 390 395 400 Arg Leu Thr Glu Leu Ser Leu Arg Arg Arg Gln Met Leu
Leu Leu Glu 405 410 415 Thr Leu Lys Val Lys Gln Thr Ile Leu Glu Pro
Ile Pro Thr Ser Leu 420 425 430 Lys Leu Pro Ile Ala Val Ser Cys Tyr
Trp Leu Gln His Thr Glu Thr 435 440 445 Lys Ala Lys Leu His His Leu
Gln Ser Leu Leu Leu Thr Met Leu Val 450 455 460 Gly Pro Leu Ile Ala
Ile Ile Asn Ser Pro Gly Asn Val Asp Pro Val 465 470 475 480 Pro Arg
Gln Ala Gln Cys Leu Ala Pro Arg 485 490 374 1467 DNA Homo sapiens
374 atgctctttt ccaccgtctt tgcttcagtt tcaaacttgg atctccggta
tggaggggac 60 tatgattctt ttgcagatgt tgtacaaaaa ttctttgaat
cactgtttgc ttgtaatata 120 tgcccatatg ttgtattaga tggaggatgt
gacatttcag ataaaaagct tacaacttta 180 aaggatagag ctagagagaa
gatccagatg gcccattccc tttctgttgg tgggagtggg 240 tatgtatgtc
ccttactcat ccgggaagta ttcatacagg ttttgatcaa gctgcgggtg 300
tgttttgtcc agtgcttttc agaagcagat cgggacatta tgacacttgc taaccattgg
360 aattgccctg tgttatcatc agatagtgac ttttgcattt ttgacctgaa
aactgggttt 420 tgcccattga atagctttca gtggagaaat atgaacacta
ttaagggcac acaaaactat 480 atccctgcca aatgcttttc ccttgatgca
ttctgccatc acttcagcaa tatgaataaa 540 gctctactac ctctctttgc
ggtgctatgt ggaaatgacc atgttaatct acccatcatg 600 gagacattct
taagtaaagc gcgtcttcct cttggagcta ccagttctaa agggaggaga 660
caccaccgaa tcctgggact tctgaattgg ttgtctcatt ttgccaaccc taccgaagca
720 ctagataatg ttctgaaata cctcccaaaa aaggatcgag aaaatgttaa
ggaacttctc 780 tgctgttcca tggaagaata ccaacagtcc caggtgaagc
tacaggactt cttccagtgt 840 ggtacttatg tctgtccaga tgccttgaat
cttggtttac cagaatgggt attagtggct 900 ttagctaaag gccagctatc
tcctttcatc agtgatgctt tggtcctaag acggaccatt 960 cttcccacac
aggtggaaaa catgcagcaa ccaaatgccc acagaatatc tcagcccatc 1020
aggcaaatca tctatgggct tcttttaaat gcctcaccac atctggacaa gacatcctgg
1080 aatgcattgc ctcctcagcc tctagctttc agtgaagtgg aaaggattaa
taaaaatatc 1140 agaacctcaa tcattgatgc agtagaactg gccaaggatc
attctgactt aagcagattg 1200 actgagctct ccttgaggag gcggcagatg
cttctgttag aaaccctgaa ggtgaaacag 1260 accattctgg agccaatccc
tacttcactg aagttgccca ttgctgtcag ttgctactgg 1320 ttgcagcaca
ccgagaccaa agcaaagcta catcatctac aatccttact gctcacaatg 1380
ctagtggggc ccttgattgc cataatcaac agccctggaa atgtggaccc tgtacccagg
1440 caggctcagt gtcttgctcc tcgctag 1467 375 488 PRT Homo sapiens
375 Met Leu Phe Ser Thr Val Phe Ala Ser Val Ser Asn Leu Asp Leu Arg
1 5 10 15 Tyr Gly Gly Asp Tyr Asp Ser Phe Ala Asp Val Val Gln Lys
Phe Phe 20 25 30 Glu Ser Leu Phe Ala Cys Asn Ile Cys Pro Tyr Val
Val Leu Asp Gly 35 40 45 Gly Cys Asp Ile Ser Asp Lys Lys Leu Thr
Thr Leu Lys Asp Arg Ala 50 55 60 Arg Glu Lys Ile Gln Met Ala His
Ser Leu Ser Val Gly Gly Ser Gly 65 70 75 80 Tyr Val Cys Pro Leu Leu
Ile Arg Glu Val Phe Ile Gln Val Leu Ile 85 90 95 Lys Leu Arg Val
Cys Phe Val Gln Cys Phe Ser Glu Ala Asp Arg Asp 100 105 110 Ile Met
Thr Leu Ala Asn His Trp Asn Cys Pro Val Leu Ser Ser Asp 115 120 125
Ser Asp Phe Cys Ile Phe Asp Leu Lys Thr Gly Phe Cys Pro Leu Asn 130
135 140 Ser Phe Gln Trp Arg Asn Met Asn Thr Ile Lys Gly Thr Gln Asn
Tyr 145 150 155 160 Ile Pro Ala Lys Cys Phe Ser Leu Asp Ala Phe Cys
His His Phe Ser 165 170 175 Asn Met Asn Lys Ala Leu Leu Pro Leu Phe
Ala Val Leu Cys Gly Asn 180 185 190 Asp His Val Asn Leu Pro Ile Met
Glu Thr Phe Leu Ser Lys Ala Arg 195 200 205 Leu Pro Leu Gly Ala Thr
Ser Ser Lys Gly Arg Arg His His Arg Ile 210 215 220 Leu Gly Leu Leu
Asn Trp Leu Ser His Phe Ala Asn Pro Thr Glu Ala 225 230 235 240 Leu
Asp Asn Val Leu Lys Tyr Leu Pro Lys Lys Asp Arg Glu Asn Val 245 250
255 Lys Glu Leu Leu Cys Cys Ser Met Glu Glu Tyr Gln Gln Ser Gln Val
260 265 270 Lys Leu Gln Asp Phe Phe Gln Cys Gly Thr Tyr Val Cys Pro
Asp Ala 275 280 285 Leu Asn Leu Gly Leu Pro Glu Trp Val Leu Val Ala
Leu Ala Lys Gly 290 295 300 Gln Leu Ser Pro Phe Ile Ser Asp Ala Leu
Val Leu Arg Arg Thr Ile 305 310 315 320 Leu Pro Thr Gln Val Glu Asn
Met Gln Gln Pro Asn Ala His Arg Ile 325 330 335 Ser Gln Pro Ile Arg
Gln Ile Ile Tyr Gly Leu Leu Leu Asn Ala Ser 340 345 350 Pro His Leu
Asp Lys Thr Ser Trp Asn Ala Leu Pro Pro Gln Pro Leu 355 360 365 Ala
Phe Ser Glu Val Glu Arg Ile Asn Lys Asn Ile Arg Thr Ser Ile 370 375
380 Ile Asp Ala Val Glu Leu Ala Lys Asp His Ser Asp Leu Ser Arg Leu
385 390 395 400 Thr Glu Leu Ser Leu Arg Arg Arg Gln Met Leu Leu Leu
Glu Thr Leu 405 410 415 Lys Val Lys Gln Thr Ile Leu Glu Pro Ile Pro
Thr Ser Leu Lys Leu 420 425 430 Pro Ile Ala Val Ser Cys Tyr Trp Leu
Gln His Thr Glu Thr Lys Ala 435 440 445 Lys Leu His His Leu Gln Ser
Leu Leu Leu Thr Met Leu Val Gly Pro 450 455 460 Leu Ile Ala Ile Ile
Asn Ser Pro Gly Asn Val Asp Pro Val Pro Arg 465 470 475 480 Gln Ala
Gln Cys Leu Ala Pro Arg 485 376 90 DNA Homo sapiens 376 atggagggga
ctatgattct tttgcagatg ttgtacaaaa attctttgaa tcactgtttg 60
cttgtaatat atgcccatat gttgtattag 90 377 29 PRT Homo sapiens 377 Met
Glu Gly Thr Met Ile Leu Leu Gln Met Leu Tyr Lys Asn Ser Leu 1 5 10
15 Asn His Cys Leu Leu Val Ile Tyr Ala His Met Leu Tyr 20 25 378 78
DNA Homo sapiens 378 atgattcttt tgcagatgtt gtacaaaaat tctttgaatc
actgtttgct tgtaatatat 60 gcccatatgt tgtattag 78 379 25 PRT Homo
sapiens 379 Met Ile Leu Leu Gln Met Leu Tyr Lys Asn Ser Leu Asn His
Cys Leu 1 5 10 15 Leu Val Ile Tyr Ala His Met Leu Tyr 20 25 380 63
DNA Homo sapiens 380 atgttgtaca aaaattcttt gaatcactgt ttgcttgtaa
tatatgccca tatgttgtat 60 tag 63 381 20 PRT Homo sapiens 381 Met Leu
Tyr Lys Asn Ser Leu Asn His Cys Leu Leu Val Ile Tyr Ala 1 5 10 15
His Met Leu Tyr 20 382 33 DNA Homo sapiens 382 atgcccatat
gttgtattag atggaggatg tga 33 383 10 PRT Homo sapiens 383 Met Pro
Ile Cys Cys Ile Arg Trp Arg Met 1 5 10 384 12 DNA Homo sapiens 384
atgttgtatt ag 12 385 3 PRT Homo sapiens 385 Met Leu Tyr 1 386 42
DNA Homo sapiens 386 atggaggatg tgacatttca gataaaaagc ttacaacttt aa
42 387 13 PRT Homo sapiens 387 Met Glu Asp Val Thr Phe Gln Ile Lys
Ser Leu Gln Leu 1 5 10 388 1260 DNA Homo sapiens 388 atggcccatt
ccctttctgt tggtgggagt gggtatgtat gtcccttact catccgggaa 60
gtattcatac aggttttgat caagctgcgg gtgtgttttg tccagtgctt ttcagaagca
120 gatcgggaca ttatgacact tgctaaccat tggaattgcc ctgtgttatc
atcagatagt 180 gacttttgca tttttgacct gaaaactggg ttttgcccat
tgaatagctt tcagtggaga 240 aatatgaaca ctattaaggg cacacaaaac
tatatccctg ccaaatgctt ttcccttgat 300 gcattctgcc atcacttcag
caatatgaat aaagctctac tacctctctt tgcggtgcta 360 tgtggaaatg
accatgttaa tctacccatc atggagacat tcttaagtaa agcgcgtctt 420
cctcttggag ctaccagttc taaagggagg agacaccacc gaatcctggg acttctgaat
480 tggttgtctc attttgccaa ccctaccgaa gcactagata atgttctgaa
atacctccca 540 aaaaaggatc gagaaaatgt taaggaactt ctctgctgtt
ccatggaaga ataccaacag 600 tcccaggtga agctacagga cttcttccag
tgtggtactt atgtctgtcc agatgccttg 660 aatcttggtt taccagaatg
ggtattagtg gctttagcta aaggccagct atctcctttc 720 atcagtgatg
ctttggtcct aagacggacc attcttccca cacaggtgga aaacatgcag 780
caaccaaatg cccacagaat atctcagccc atcaggcaaa tcatctatgg gcttctttta
840 aatgcctcac cacatctgga caagacatcc tggaatgcat tgcctcctca
gcctctagct 900 ttcagtgaag tggaaaggat taataaaaat atcagaacct
caatcattga tgcagtagaa 960 ctggccaagg atcattctga cttaagcaga
ttgactgagc tctccttgag gaggcggcag 1020 atgcttctgt tagaaaccct
gaaggtgaaa cagaccattc tggagccaat ccctacttca 1080 ctgaagttgc
ccattgctgt cagttgctac tggttgcagc acaccgagac caaagcaaag 1140
ctacatcatc tacaatcctt actgctcaca atgctagtgg ggcccttgat tgccataatc
1200 aacagccctg gaaatgtgga ccctgtaccc aggcaggctc agtgtcttgc
tcctcgctag 1260 389 419 PRT Homo sapiens 389 Met Ala His Ser Leu
Ser Val Gly Gly Ser Gly Tyr Val Cys Pro Leu 1 5 10 15 Leu Ile Arg
Glu Val Phe Ile Gln Val Leu Ile Lys Leu Arg Val Cys 20 25 30 Phe
Val Gln Cys Phe Ser Glu Ala Asp Arg Asp Ile Met Thr Leu Ala 35 40
45 Asn His Trp Asn Cys Pro Val Leu Ser Ser Asp Ser Asp Phe Cys Ile
50 55 60 Phe Asp Leu Lys Thr Gly Phe Cys Pro Leu Asn Ser Phe Gln
Trp Arg 65 70 75 80 Asn Met Asn Thr Ile Lys Gly Thr Gln Asn Tyr Ile
Pro Ala Lys Cys 85 90 95 Phe Ser Leu Asp Ala Phe Cys His His Phe
Ser Asn Met Asn Lys Ala 100 105 110 Leu Leu Pro Leu Phe Ala Val Leu
Cys Gly Asn Asp His Val Asn Leu 115 120 125 Pro Ile Met Glu Thr Phe
Leu Ser Lys Ala Arg Leu Pro Leu Gly Ala 130 135 140 Thr Ser Ser Lys
Gly Arg Arg His His Arg
Ile Leu Gly Leu Leu Asn 145 150 155 160 Trp Leu Ser His Phe Ala Asn
Pro Thr Glu Ala Leu Asp Asn Val Leu 165 170 175 Lys Tyr Leu Pro Lys
Lys Asp Arg Glu Asn Val Lys Glu Leu Leu Cys 180 185 190 Cys Ser Met
Glu Glu Tyr Gln Gln Ser Gln Val Lys Leu Gln Asp Phe 195 200 205 Phe
Gln Cys Gly Thr Tyr Val Cys Pro Asp Ala Leu Asn Leu Gly Leu 210 215
220 Pro Glu Trp Val Leu Val Ala Leu Ala Lys Gly Gln Leu Ser Pro Phe
225 230 235 240 Ile Ser Asp Ala Leu Val Leu Arg Arg Thr Ile Leu Pro
Thr Gln Val 245 250 255 Glu Asn Met Gln Gln Pro Asn Ala His Arg Ile
Ser Gln Pro Ile Arg 260 265 270 Gln Ile Ile Tyr Gly Leu Leu Leu Asn
Ala Ser Pro His Leu Asp Lys 275 280 285 Thr Ser Trp Asn Ala Leu Pro
Pro Gln Pro Leu Ala Phe Ser Glu Val 290 295 300 Glu Arg Ile Asn Lys
Asn Ile Arg Thr Ser Ile Ile Asp Ala Val Glu 305 310 315 320 Leu Ala
Lys Asp His Ser Asp Leu Ser Arg Leu Thr Glu Leu Ser Leu 325 330 335
Arg Arg Arg Gln Met Leu Leu Leu Glu Thr Leu Lys Val Lys Gln Thr 340
345 350 Ile Leu Glu Pro Ile Pro Thr Ser Leu Lys Leu Pro Ile Ala Val
Ser 355 360 365 Cys Tyr Trp Leu Gln His Thr Glu Thr Lys Ala Lys Leu
His His Leu 370 375 380 Gln Ser Leu Leu Leu Thr Met Leu Val Gly Pro
Leu Ile Ala Ile Ile 385 390 395 400 Asn Ser Pro Gly Asn Val Asp Pro
Val Pro Arg Gln Ala Gln Cys Leu 405 410 415 Ala Pro Arg 390 45 DNA
Homo sapiens 390 atgtatgtcc cttactcatc cgggaagtat tcatacaggt tttga
45 391 14 PRT Homo sapiens 391 Met Tyr Val Pro Tyr Ser Ser Gly Lys
Tyr Ser Tyr Arg Phe 1 5 10 392 108 DNA Homo sapiens 392 atgtccctta
ctcatccggg aagtattcat acaggttttg atcaagctgc gggtgtgttt 60
tgtccagtgc ttttcagaag cagatcggga cattatgaca cttgctaa 108 393 35 PRT
Homo sapiens 393 Met Ser Leu Thr His Pro Gly Ser Ile His Thr Gly
Phe Asp Gln Ala 1 5 10 15 Ala Gly Val Phe Cys Pro Val Leu Phe Arg
Ser Arg Ser Gly His Tyr 20 25 30 Asp Thr Cys 35 394 1128 DNA Homo
sapiens 394 atgacacttg ctaaccattg gaattgccct gtgttatcat cagatagtga
cttttgcatt 60 tttgacctga aaactgggtt ttgcccattg aatagctttc
agtggagaaa tatgaacact 120 attaagggca cacaaaacta tatccctgcc
aaatgctttt cccttgatgc attctgccat 180 cacttcagca atatgaataa
agctctacta cctctctttg cggtgctatg tggaaatgac 240 catgttaatc
tacccatcat ggagacattc ttaagtaaag cgcgtcttcc tcttggagct 300
accagttcta aagggaggag acaccaccga atcctgggac ttctgaattg gttgtctcat
360 tttgccaacc ctaccgaagc actagataat gttctgaaat acctcccaaa
aaaggatcga 420 gaaaatgtta aggaacttct ctgctgttcc atggaagaat
accaacagtc ccaggtgaag 480 ctacaggact tcttccagtg tggtacttat
gtctgtccag atgccttgaa tcttggttta 540 ccagaatggg tattagtggc
tttagctaaa ggccagctat ctcctttcat cagtgatgct 600 ttggtcctaa
gacggaccat tcttcccaca caggtggaaa acatgcagca accaaatgcc 660
cacagaatat ctcagcccat caggcaaatc atctatgggc ttcttttaaa tgcctcacca
720 catctggaca agacatcctg gaatgcattg cctcctcagc ctctagcttt
cagtgaagtg 780 gaaaggatta ataaaaatat cagaacctca atcattgatg
cagtagaact ggccaaggat 840 cattctgact taagcagatt gactgagctc
tccttgagga ggcggcagat gcttctgtta 900 gaaaccctga aggtgaaaca
gaccattctg gagccaatcc ctacttcact gaagttgccc 960 attgctgtca
gttgctactg gttgcagcac accgagacca aagcaaagct acatcatcta 1020
caatccttac tgctcacaat gctagtgggg cccttgattg ccataatcaa cagccctgga
1080 aatgtggacc ctgtacccag gcaggctcag tgtcttgctc ctcgctag 1128 395
375 PRT Homo sapiens 395 Met Thr Leu Ala Asn His Trp Asn Cys Pro
Val Leu Ser Ser Asp Ser 1 5 10 15 Asp Phe Cys Ile Phe Asp Leu Lys
Thr Gly Phe Cys Pro Leu Asn Ser 20 25 30 Phe Gln Trp Arg Asn Met
Asn Thr Ile Lys Gly Thr Gln Asn Tyr Ile 35 40 45 Pro Ala Lys Cys
Phe Ser Leu Asp Ala Phe Cys His His Phe Ser Asn 50 55 60 Met Asn
Lys Ala Leu Leu Pro Leu Phe Ala Val Leu Cys Gly Asn Asp 65 70 75 80
His Val Asn Leu Pro Ile Met Glu Thr Phe Leu Ser Lys Ala Arg Leu 85
90 95 Pro Leu Gly Ala Thr Ser Ser Lys Gly Arg Arg His His Arg Ile
Leu 100 105 110 Gly Leu Leu Asn Trp Leu Ser His Phe Ala Asn Pro Thr
Glu Ala Leu 115 120 125 Asp Asn Val Leu Lys Tyr Leu Pro Lys Lys Asp
Arg Glu Asn Val Lys 130 135 140 Glu Leu Leu Cys Cys Ser Met Glu Glu
Tyr Gln Gln Ser Gln Val Lys 145 150 155 160 Leu Gln Asp Phe Phe Gln
Cys Gly Thr Tyr Val Cys Pro Asp Ala Leu 165 170 175 Asn Leu Gly Leu
Pro Glu Trp Val Leu Val Ala Leu Ala Lys Gly Gln 180 185 190 Leu Ser
Pro Phe Ile Ser Asp Ala Leu Val Leu Arg Arg Thr Ile Leu 195 200 205
Pro Thr Gln Val Glu Asn Met Gln Gln Pro Asn Ala His Arg Ile Ser 210
215 220 Gln Pro Ile Arg Gln Ile Ile Tyr Gly Leu Leu Leu Asn Ala Ser
Pro 225 230 235 240 His Leu Asp Lys Thr Ser Trp Asn Ala Leu Pro Pro
Gln Pro Leu Ala 245 250 255 Phe Ser Glu Val Glu Arg Ile Asn Lys Asn
Ile Arg Thr Ser Ile Ile 260 265 270 Asp Ala Val Glu Leu Ala Lys Asp
His Ser Asp Leu Ser Arg Leu Thr 275 280 285 Glu Leu Ser Leu Arg Arg
Arg Gln Met Leu Leu Leu Glu Thr Leu Lys 290 295 300 Val Lys Gln Thr
Ile Leu Glu Pro Ile Pro Thr Ser Leu Lys Leu Pro 305 310 315 320 Ile
Ala Val Ser Cys Tyr Trp Leu Gln His Thr Glu Thr Lys Ala Lys 325 330
335 Leu His His Leu Gln Ser Leu Leu Leu Thr Met Leu Val Gly Pro Leu
340 345 350 Ile Ala Ile Ile Asn Ser Pro Gly Asn Val Asp Pro Val Pro
Arg Gln 355 360 365 Ala Gln Cys Leu Ala Pro Arg 370 375 396 1017
DNA Homo sapiens 396 atgaacacta ttaagggcac acaaaactat atccctgcca
aatgcttttc ccttgatgca 60 ttctgccatc acttcagcaa tatgaataaa
gctctactac ctctctttgc ggtgctatgt 120 ggaaatgacc atgttaatct
acccatcatg gagacattct taagtaaagc gcgtcttcct 180 cttggagcta
ccagttctaa agggaggaga caccaccgaa tcctgggact tctgaattgg 240
ttgtctcatt ttgccaaccc taccgaagca ctagataatg ttctgaaata cctcccaaaa
300 aaggatcgag aaaatgttaa ggaacttctc tgctgttcca tggaagaata
ccaacagtcc 360 caggtgaagc tacaggactt cttccagtgt ggtacttatg
tctgtccaga tgccttgaat 420 cttggtttac cagaatgggt attagtggct
ttagctaaag gccagctatc tcctttcatc 480 agtgatgctt tggtcctaag
acggaccatt cttcccacac aggtggaaaa catgcagcaa 540 ccaaatgccc
acagaatatc tcagcccatc aggcaaatca tctatgggct tcttttaaat 600
gcctcaccac atctggacaa gacatcctgg aatgcattgc ctcctcagcc tctagctttc
660 agtgaagtgg aaaggattaa taaaaatatc agaacctcaa tcattgatgc
agtagaactg 720 gccaaggatc attctgactt aagcagattg actgagctct
ccttgaggag gcggcagatg 780 cttctgttag aaaccctgaa ggtgaaacag
accattctgg agccaatccc tacttcactg 840 aagttgccca ttgctgtcag
ttgctactgg ttgcagcaca ccgagaccaa agcaaagcta 900 catcatctac
aatccttact gctcacaatg ctagtggggc ccttgattgc cataatcaac 960
agccctggaa atgtggaccc tgtacccagg caggctcagt gtcttgctcc tcgctag 1017
397 338 PRT Homo sapiens 397 Met Asn Thr Ile Lys Gly Thr Gln Asn
Tyr Ile Pro Ala Lys Cys Phe 1 5 10 15 Ser Leu Asp Ala Phe Cys His
His Phe Ser Asn Met Asn Lys Ala Leu 20 25 30 Leu Pro Leu Phe Ala
Val Leu Cys Gly Asn Asp His Val Asn Leu Pro 35 40 45 Ile Met Glu
Thr Phe Leu Ser Lys Ala Arg Leu Pro Leu Gly Ala Thr 50 55 60 Ser
Ser Lys Gly Arg Arg His His Arg Ile Leu Gly Leu Leu Asn Trp 65 70
75 80 Leu Ser His Phe Ala Asn Pro Thr Glu Ala Leu Asp Asn Val Leu
Lys 85 90 95 Tyr Leu Pro Lys Lys Asp Arg Glu Asn Val Lys Glu Leu
Leu Cys Cys 100 105 110 Ser Met Glu Glu Tyr Gln Gln Ser Gln Val Lys
Leu Gln Asp Phe Phe 115 120 125 Gln Cys Gly Thr Tyr Val Cys Pro Asp
Ala Leu Asn Leu Gly Leu Pro 130 135 140 Glu Trp Val Leu Val Ala Leu
Ala Lys Gly Gln Leu Ser Pro Phe Ile 145 150 155 160 Ser Asp Ala Leu
Val Leu Arg Arg Thr Ile Leu Pro Thr Gln Val Glu 165 170 175 Asn Met
Gln Gln Pro Asn Ala His Arg Ile Ser Gln Pro Ile Arg Gln 180 185 190
Ile Ile Tyr Gly Leu Leu Leu Asn Ala Ser Pro His Leu Asp Lys Thr 195
200 205 Ser Trp Asn Ala Leu Pro Pro Gln Pro Leu Ala Phe Ser Glu Val
Glu 210 215 220 Arg Ile Asn Lys Asn Ile Arg Thr Ser Ile Ile Asp Ala
Val Glu Leu 225 230 235 240 Ala Lys Asp His Ser Asp Leu Ser Arg Leu
Thr Glu Leu Ser Leu Arg 245 250 255 Arg Arg Gln Met Leu Leu Leu Glu
Thr Leu Lys Val Lys Gln Thr Ile 260 265 270 Leu Glu Pro Ile Pro Thr
Ser Leu Lys Leu Pro Ile Ala Val Ser Cys 275 280 285 Tyr Trp Leu Gln
His Thr Glu Thr Lys Ala Lys Leu His His Leu Gln 290 295 300 Ser Leu
Leu Leu Thr Met Leu Val Gly Pro Leu Ile Ala Ile Ile Asn 305 310 315
320 Ser Pro Gly Asn Val Asp Pro Val Pro Arg Gln Ala Gln Cys Leu Ala
325 330 335 Pro Arg 398 15 DNA Homo sapiens 398 atgcttttcc cttga 15
399 4 PRT Homo sapiens 399 Met Leu Phe Pro 1 400 30 DNA Homo
sapiens 400 atgcattctg ccatcacttc agcaatatga 30 401 9 PRT Homo
sapiens 401 Met His Ser Ala Ile Thr Ser Ala Ile 1 5 402 936 DNA
Homo sapiens 402 atgaataaag ctctactacc tctctttgcg gtgctatgtg
gaaatgacca tgttaatcta 60 cccatcatgg agacattctt aagtaaagcg
cgtcttcctc ttggagctac cagttctaaa 120 gggaggagac accaccgaat
cctgggactt ctgaattggt tgtctcattt tgccaaccct 180 accgaagcac
tagataatgt tctgaaatac ctcccaaaaa aggatcgaga aaatgttaag 240
gaacttctct gctgttccat ggaagaatac caacagtccc aggtgaagct acaggacttc
300 ttccagtgtg gtacttatgt ctgtccagat gccttgaatc ttggtttacc
agaatgggta 360 ttagtggctt tagctaaagg ccagctatct cctttcatca
gtgatgcttt ggtcctaaga 420 cggaccattc ttcccacaca ggtggaaaac
atgcagcaac caaatgccca cagaatatct 480 cagcccatca ggcaaatcat
ctatgggctt cttttaaatg cctcaccaca tctggacaag 540 acatcctgga
atgcattgcc tcctcagcct ctagctttca gtgaagtgga aaggattaat 600
aaaaatatca gaacctcaat cattgatgca gtagaactgg ccaaggatca ttctgactta
660 agcagattga ctgagctctc cttgaggagg cggcagatgc ttctgttaga
aaccctgaag 720 gtgaaacaga ccattctgga gccaatccct acttcactga
agttgcccat tgctgtcagt 780 tgctactggt tgcagcacac cgagaccaaa
gcaaagctac atcatctaca atccttactg 840 ctcacaatgc tagtggggcc
cttgattgcc ataatcaaca gccctggaaa tgtggaccct 900 gtacccaggc
aggctcagtg tcttgctcct cgctag 936 403 311 PRT Homo sapiens 403 Met
Asn Lys Ala Leu Leu Pro Leu Phe Ala Val Leu Cys Gly Asn Asp 1 5 10
15 His Val Asn Leu Pro Ile Met Glu Thr Phe Leu Ser Lys Ala Arg Leu
20 25 30 Pro Leu Gly Ala Thr Ser Ser Lys Gly Arg Arg His His Arg
Ile Leu 35 40 45 Gly Leu Leu Asn Trp Leu Ser His Phe Ala Asn Pro
Thr Glu Ala Leu 50 55 60 Asp Asn Val Leu Lys Tyr Leu Pro Lys Lys
Asp Arg Glu Asn Val Lys 65 70 75 80 Glu Leu Leu Cys Cys Ser Met Glu
Glu Tyr Gln Gln Ser Gln Val Lys 85 90 95 Leu Gln Asp Phe Phe Gln
Cys Gly Thr Tyr Val Cys Pro Asp Ala Leu 100 105 110 Asn Leu Gly Leu
Pro Glu Trp Val Leu Val Ala Leu Ala Lys Gly Gln 115 120 125 Leu Ser
Pro Phe Ile Ser Asp Ala Leu Val Leu Arg Arg Thr Ile Leu 130 135 140
Pro Thr Gln Val Glu Asn Met Gln Gln Pro Asn Ala His Arg Ile Ser 145
150 155 160 Gln Pro Ile Arg Gln Ile Ile Tyr Gly Leu Leu Leu Asn Ala
Ser Pro 165 170 175 His Leu Asp Lys Thr Ser Trp Asn Ala Leu Pro Pro
Gln Pro Leu Ala 180 185 190 Phe Ser Glu Val Glu Arg Ile Asn Lys Asn
Ile Arg Thr Ser Ile Ile 195 200 205 Asp Ala Val Glu Leu Ala Lys Asp
His Ser Asp Leu Ser Arg Leu Thr 210 215 220 Glu Leu Ser Leu Arg Arg
Arg Gln Met Leu Leu Leu Glu Thr Leu Lys 225 230 235 240 Val Lys Gln
Thr Ile Leu Glu Pro Ile Pro Thr Ser Leu Lys Leu Pro 245 250 255 Ile
Ala Val Ser Cys Tyr Trp Leu Gln His Thr Glu Thr Lys Ala Lys 260 265
270 Leu His His Leu Gln Ser Leu Leu Leu Thr Met Leu Val Gly Pro Leu
275 280 285 Ile Ala Ile Ile Asn Ser Pro Gly Asn Val Asp Pro Val Pro
Arg Gln 290 295 300 Ala Gln Cys Leu Ala Pro Arg 305 310 404 12 DNA
Homo sapiens 404 atgtggaaat ga 12 405 3 PRT Homo sapiens 405 Met
Trp Lys 1 406 39 DNA Homo sapiens 406 atgaccatgt taatctaccc
atcatggaga cattcttaa 39 407 12 PRT Homo sapiens 407 Met Thr Met Leu
Ile Tyr Pro Ser Trp Arg His Ser 1 5 10 408 33 DNA Homo sapiens 408
atgttaatct acccatcatg gagacattct taa 33 409 10 PRT Homo sapiens 409
Met Leu Ile Tyr Pro Ser Trp Arg His Ser 1 5 10 410 870 DNA Homo
sapiens 410 atggagacat tcttaagtaa agcgcgtctt cctcttggag ctaccagttc
taaagggagg 60 agacaccacc gaatcctggg acttctgaat tggttgtctc
attttgccaa ccctaccgaa 120 gcactagata atgttctgaa atacctccca
aaaaaggatc gagaaaatgt taaggaactt 180 ctctgctgtt ccatggaaga
ataccaacag tcccaggtga agctacagga cttcttccag 240 tgtggtactt
atgtctgtcc agatgccttg aatcttggtt taccagaatg ggtattagtg 300
gctttagcta aaggccagct atctcctttc atcagtgatg ctttggtcct aagacggacc
360 attcttccca cacaggtgga aaacatgcag caaccaaatg cccacagaat
atctcagccc 420 atcaggcaaa tcatctatgg gcttctttta aatgcctcac
cacatctgga caagacatcc 480 tggaatgcat tgcctcctca gcctctagct
ttcagtgaag tggaaaggat taataaaaat 540 atcagaacct caatcattga
tgcagtagaa ctggccaagg atcattctga cttaagcaga 600 ttgactgagc
tctccttgag gaggcggcag atgcttctgt tagaaaccct gaaggtgaaa 660
cagaccattc tggagccaat ccctacttca ctgaagttgc ccattgctgt cagttgctac
720 tggttgcagc acaccgagac caaagcaaag ctacatcatc tacaatcctt
actgctcaca 780 atgctagtgg ggcccttgat tgccataatc aacagccctg
gaaatgtgga ccctgtaccc 840 aggcaggctc agtgtcttgc tcctcgctag 870 411
289 PRT Homo sapiens 411 Met Glu Thr Phe Leu Ser Lys Ala Arg Leu
Pro Leu Gly Ala Thr Ser 1 5 10 15 Ser Lys Gly Arg Arg His His Arg
Ile Leu Gly Leu Leu Asn Trp Leu 20 25 30 Ser His Phe Ala Asn Pro
Thr Glu Ala Leu Asp Asn Val Leu Lys Tyr 35 40 45 Leu Pro Lys Lys
Asp Arg Glu Asn Val Lys Glu Leu Leu Cys Cys Ser 50 55 60 Met Glu
Glu Tyr Gln Gln Ser Gln Val Lys Leu Gln Asp Phe Phe Gln 65 70 75 80
Cys Gly Thr Tyr Val Cys Pro Asp Ala Leu Asn Leu Gly Leu Pro Glu 85
90 95 Trp Val Leu Val Ala Leu Ala Lys Gly Gln Leu Ser Pro Phe Ile
Ser 100 105 110 Asp Ala Leu Val Leu Arg Arg Thr Ile Leu Pro Thr Gln
Val Glu Asn 115 120 125 Met Gln Gln Pro Asn Ala His Arg Ile Ser Gln
Pro Ile Arg Gln Ile 130 135 140 Ile Tyr Gly Leu Leu Leu Asn Ala Ser
Pro His Leu Asp Lys Thr Ser 145 150 155 160 Trp Asn Ala Leu Pro Pro
Gln Pro Leu Ala Phe Ser Glu Val Glu Arg 165 170 175 Ile Asn Lys Asn
Ile Arg Thr Ser Ile Ile Asp Ala Val Glu Leu Ala 180 185 190 Lys Asp
His Ser Asp Leu Ser Arg Leu Thr Glu Leu Ser Leu Arg Arg 195 200 205
Arg Gln Met Leu Leu Leu Glu Thr Leu Lys Val Lys Gln Thr Ile Leu 210
215 220 Glu Pro Ile Pro Thr Ser Leu Lys Leu Pro Ile Ala Val Ser Cys
Tyr 225 230 235 240 Trp Leu Gln His Thr Glu Thr Lys Ala Lys Leu His
His Leu Gln Ser
245 250 255 Leu Leu Leu Thr Met Leu Val Gly Pro Leu Ile Ala Ile Ile
Asn Ser 260 265 270 Pro Gly Asn Val Asp Pro Val Pro Arg Gln Ala Gln
Cys Leu Ala Pro 275 280 285 Arg 412 54 DNA Homo sapiens 412
atgttaagga acttctctgc tgttccatgg aagaatacca acagtcccag gtga 54 413
17 PRT Homo sapiens 413 Met Leu Arg Asn Phe Ser Ala Val Pro Trp Lys
Asn Thr Asn Ser Pro 1 5 10 15 Arg 414 678 DNA Homo sapiens 414
atggaagaat accaacagtc ccaggtgaag ctacaggact tcttccagtg tggtacttat
60 gtctgtccag atgccttgaa tcttggttta ccagaatggg tattagtggc
tttagctaaa 120 ggccagctat ctcctttcat cagtgatgct ttggtcctaa
gacggaccat tcttcccaca 180 caggtggaaa acatgcagca accaaatgcc
cacagaatat ctcagcccat caggcaaatc 240 atctatgggc ttcttttaaa
tgcctcacca catctggaca agacatcctg gaatgcattg 300 cctcctcagc
ctctagcttt cagtgaagtg gaaaggatta ataaaaatat cagaacctca 360
atcattgatg cagtagaact ggccaaggat cattctgact taagcagatt gactgagctc
420 tccttgagga ggcggcagat gcttctgtta gaaaccctga aggtgaaaca
gaccattctg 480 gagccaatcc ctacttcact gaagttgccc attgctgtca
gttgctactg gttgcagcac 540 accgagacca aagcaaagct acatcatcta
caatccttac tgctcacaat gctagtgggg 600 cccttgattg ccataatcaa
cagccctgga aatgtggacc ctgtacccag gcaggctcag 660 tgtcttgctc ctcgctag
678 415 225 PRT Homo sapiens 415 Met Glu Glu Tyr Gln Gln Ser Gln
Val Lys Leu Gln Asp Phe Phe Gln 1 5 10 15 Cys Gly Thr Tyr Val Cys
Pro Asp Ala Leu Asn Leu Gly Leu Pro Glu 20 25 30 Trp Val Leu Val
Ala Leu Ala Lys Gly Gln Leu Ser Pro Phe Ile Ser 35 40 45 Asp Ala
Leu Val Leu Arg Arg Thr Ile Leu Pro Thr Gln Val Glu Asn 50 55 60
Met Gln Gln Pro Asn Ala His Arg Ile Ser Gln Pro Ile Arg Gln Ile 65
70 75 80 Ile Tyr Gly Leu Leu Leu Asn Ala Ser Pro His Leu Asp Lys
Thr Ser 85 90 95 Trp Asn Ala Leu Pro Pro Gln Pro Leu Ala Phe Ser
Glu Val Glu Arg 100 105 110 Ile Asn Lys Asn Ile Arg Thr Ser Ile Ile
Asp Ala Val Glu Leu Ala 115 120 125 Lys Asp His Ser Asp Leu Ser Arg
Leu Thr Glu Leu Ser Leu Arg Arg 130 135 140 Arg Gln Met Leu Leu Leu
Glu Thr Leu Lys Val Lys Gln Thr Ile Leu 145 150 155 160 Glu Pro Ile
Pro Thr Ser Leu Lys Leu Pro Ile Ala Val Ser Cys Tyr 165 170 175 Trp
Leu Gln His Thr Glu Thr Lys Ala Lys Leu His His Leu Gln Ser 180 185
190 Leu Leu Leu Thr Met Leu Val Gly Pro Leu Ile Ala Ile Ile Asn Ser
195 200 205 Pro Gly Asn Val Asp Pro Val Pro Arg Gln Ala Gln Cys Leu
Ala Pro 210 215 220 Arg 225 416 21 DNA Homo sapiens 416 atgtctgtcc
agatgccttg a 21 417 6 PRT Homo sapiens 417 Met Ser Val Gln Met Pro
1 5 418 24 DNA Homo sapiens 418 atgggtatta gtggctttag ctaa 24 419 7
PRT Homo sapiens 419 Met Gly Ile Ser Gly Phe Ser 1 5 420 15 DNA
Homo sapiens 420 atgctttggt cctaa 15 421 4 PRT Homo sapiens 421 Met
Leu Trp Ser 1 422 486 DNA Homo sapiens 422 atgcagcaac caaatgccca
cagaatatct cagcccatca ggcaaatcat ctatgggctt 60 cttttaaatg
cctcaccaca tctggacaag acatcctgga atgcattgcc tcctcagcct 120
ctagctttca gtgaagtgga aaggattaat aaaaatatca gaacctcaat cattgatgca
180 gtagaactgg ccaaggatca ttctgactta agcagattga ctgagctctc
cttgaggagg 240 cggcagatgc ttctgttaga aaccctgaag gtgaaacaga
ccattctgga gccaatccct 300 acttcactga agttgcccat tgctgtcagt
tgctactggt tgcagcacac cgagaccaaa 360 gcaaagctac atcatctaca
atccttactg ctcacaatgc tagtggggcc cttgattgcc 420 ataatcaaca
gccctggaaa tgtggaccct gtacccaggc aggctcagtg tcttgctcct 480 cgctag
486 423 161 PRT Homo sapiens 423 Met Gln Gln Pro Asn Ala His Arg
Ile Ser Gln Pro Ile Arg Gln Ile 1 5 10 15 Ile Tyr Gly Leu Leu Leu
Asn Ala Ser Pro His Leu Asp Lys Thr Ser 20 25 30 Trp Asn Ala Leu
Pro Pro Gln Pro Leu Ala Phe Ser Glu Val Glu Arg 35 40 45 Ile Asn
Lys Asn Ile Arg Thr Ser Ile Ile Asp Ala Val Glu Leu Ala 50 55 60
Lys Asp His Ser Asp Leu Ser Arg Leu Thr Glu Leu Ser Leu Arg Arg 65
70 75 80 Arg Gln Met Leu Leu Leu Glu Thr Leu Lys Val Lys Gln Thr
Ile Leu 85 90 95 Glu Pro Ile Pro Thr Ser Leu Lys Leu Pro Ile Ala
Val Ser Cys Tyr 100 105 110 Trp Leu Gln His Thr Glu Thr Lys Ala Lys
Leu His His Leu Gln Ser 115 120 125 Leu Leu Leu Thr Met Leu Val Gly
Pro Leu Ile Ala Ile Ile Asn Ser 130 135 140 Pro Gly Asn Val Asp Pro
Val Pro Arg Gln Ala Gln Cys Leu Ala Pro 145 150 155 160 Arg 424 54
DNA Homo sapiens 424 atgcccacag aatatctcag cccatcaggc aaatcatcta
tgggcttctt ttaa 54 425 17 PRT Homo sapiens 425 Met Pro Thr Glu Tyr
Leu Ser Pro Ser Gly Lys Ser Ser Met Gly Phe 1 5 10 15 Phe 426 15
DNA Homo sapiens 426 atgggcttct tttaa 15 427 4 PRT Homo sapiens 427
Met Gly Phe Phe 1 428 57 DNA Homo sapiens 428 atgcctcacc acatctggac
aagacatcct ggaatgcatt gcctcctcag cctctag 57 429 18 PRT Homo sapiens
429 Met Pro His His Ile Trp Thr Arg His Pro Gly Met His Cys Leu Leu
1 5 10 15 Ser Leu 430 24 DNA Homo sapiens 430 atgcattgcc tcctcagcct
ctag 24 431 7 PRT Homo sapiens 431 Met His Cys Leu Leu Ser Leu 1 5
432 240 DNA Homo sapiens 432 atgcttctgt tagaaaccct gaaggtgaaa
cagaccattc tggagccaat ccctacttca 60 ctgaagttgc ccattgctgt
cagttgctac tggttgcagc acaccgagac caaagcaaag 120 ctacatcatc
tacaatcctt actgctcaca atgctagtgg ggcccttgat tgccataatc 180
aacagccctg gaaatgtgga ccctgtaccc aggcaggctc agtgtcttgc tcctcgctag
240 433 79 PRT Homo sapiens 433 Met Leu Leu Leu Glu Thr Leu Lys Val
Lys Gln Thr Ile Leu Glu Pro 1 5 10 15 Ile Pro Thr Ser Leu Lys Leu
Pro Ile Ala Val Ser Cys Tyr Trp Leu 20 25 30 Gln His Thr Glu Thr
Lys Ala Lys Leu His His Leu Gln Ser Leu Leu 35 40 45 Leu Thr Met
Leu Val Gly Pro Leu Ile Ala Ile Ile Asn Ser Pro Gly 50 55 60 Asn
Val Asp Pro Val Pro Arg Gln Ala Gln Cys Leu Ala Pro Arg 65 70 75
434 90 DNA Homo sapiens 434 atgctagtgg ggcccttgat tgccataatc
aacagccctg gaaatgtgga ccctgtaccc 60 aggcaggctc agtgtcttgc
tcctcgctag 90 435 29 PRT Homo sapiens 435 Met Leu Val Gly Pro Leu
Ile Ala Ile Ile Asn Ser Pro Gly Asn Val 1 5 10 15 Asp Pro Val Pro
Arg Gln Ala Gln Cys Leu Ala Pro Arg 20 25 436 54 DNA Homo sapiens
436 atgtggaccc tgtacccagg caggctcagt gtcttgctcc tcgctagttg gtaa 54
437 17 PRT Homo sapiens 437 Met Trp Thr Leu Tyr Pro Gly Arg Leu Ser
Val Leu Leu Leu Ala Ser 1 5 10 15 Trp 438 39 DNA Homo sapiens 438
atggtgctaa gatgttgtat gcagagttcc aaagagtga 39 439 12 PRT Homo
sapiens 439 Met Val Leu Arg Cys Cys Met Gln Ser Ser Lys Glu 1 5 10
440 309 DNA Homo sapiens 440 atgttgtatg cagagttcca aagagtgaag
gcgcagacac ggctgggcac aagactggac 60 ttagacacag ctcacatctt
ctgtcagtgg cagtcctgtc tccagatggg gatgtatctc 120 aaccagctgc
tgtccactcc tctcccagag ccagacctaa ctcgactgta cagtggaagc 180
ctggtgcacg gactatgcca gcaactgcta gcatcgacct ctgtagaaag tctcctgagc
240 atatgtcctg aggctaagca actttatgaa tatctattca atgcccacaa
ggtcatatgc 300 ccccgctga 309 441 102 PRT Homo sapiens 441 Met Leu
Tyr Ala Glu Phe Gln Arg Val Lys Ala Gln Thr Arg Leu Gly 1 5 10 15
Thr Arg Leu Asp Leu Asp Thr Ala His Ile Phe Cys Gln Trp Gln Ser 20
25 30 Cys Leu Gln Met Gly Met Tyr Leu Asn Gln Leu Leu Ser Thr Pro
Leu 35 40 45 Pro Glu Pro Asp Leu Thr Arg Leu Tyr Ser Gly Ser Leu
Val His Gly 50 55 60 Leu Cys Gln Gln Leu Leu Ala Ser Thr Ser Val
Glu Ser Leu Leu Ser 65 70 75 80 Ile Cys Pro Glu Ala Lys Gln Leu Tyr
Glu Tyr Leu Phe Asn Ala His 85 90 95 Lys Val Ile Cys Pro Arg 100
442 21 DNA Homo sapiens 442 atgcagagtt ccaaagagtg a 21 443 6 PRT
Homo sapiens 443 Met Gln Ser Ser Lys Glu 1 5 444 204 DNA Homo
sapiens 444 atggggatgt atctcaacca gctgctgtcc actcctctcc cagagccaga
cctaactcga 60 ctgtacagtg gaagcctggt gcacggacta tgccagcaac
tgctagcatc gacctctgta 120 gaaagtctcc tgagcatatg tcctgaggct
aagcaacttt atgaatatct attcaatgcc 180 cacaaggtca tatgcccccg ctga 204
445 67 PRT Homo sapiens 445 Met Gly Met Tyr Leu Asn Gln Leu Leu Ser
Thr Pro Leu Pro Glu Pro 1 5 10 15 Asp Leu Thr Arg Leu Tyr Ser Gly
Ser Leu Val His Gly Leu Cys Gln 20 25 30 Gln Leu Leu Ala Ser Thr
Ser Val Glu Ser Leu Leu Ser Ile Cys Pro 35 40 45 Glu Ala Lys Gln
Leu Tyr Glu Tyr Leu Phe Asn Ala His Lys Val Ile 50 55 60 Cys Pro
Arg 65 446 198 DNA Homo sapiens 446 atgtatctca accagctgct
gtccactcct ctcccagagc cagacctaac tcgactgtac 60 agtggaagcc
tggtgcacgg actatgccag caactgctag catcgacctc tgtagaaagt 120
ctcctgagca tatgtcctga ggctaagcaa ctttatgaat atctattcaa tgcccacaag
180 gtcatatgcc cccgctga 198 447 65 PRT Homo sapiens 447 Met Tyr Leu
Asn Gln Leu Leu Ser Thr Pro Leu Pro Glu Pro Asp Leu 1 5 10 15 Thr
Arg Leu Tyr Ser Gly Ser Leu Val His Gly Leu Cys Gln Gln Leu 20 25
30 Leu Ala Ser Thr Ser Val Glu Ser Leu Leu Ser Ile Cys Pro Glu Ala
35 40 45 Lys Gln Leu Tyr Glu Tyr Leu Phe Asn Ala His Lys Val Ile
Cys Pro 50 55 60 Arg 65 448 57 DNA Homo sapiens 448 atgccagcaa
ctgctagcat cgacctctgt agaaagtctc ctgagcatat gtcctga 57 449 18 PRT
Homo sapiens 449 Met Pro Ala Thr Ala Ser Ile Asp Leu Cys Arg Lys
Ser Pro Glu His 1 5 10 15 Met Ser 450 231 DNA Homo sapiens 450
atgaatatct attcaatgcc cacaaggtca tatgcccccg ctgaaatatt cctaccaaaa
60 ggtagatcaa attcaaaaaa aaaaaggcag aagaaacaga ataccagctg
ttctaagaac 120 agagggagaa ccactgcaca caccaagtgt tggtatgagg
gaaacaaccg gtttgggttg 180 ttaatggttg aaaacttaga ggaacatagt
gaggcctcca acattgaata a 231 451 76 PRT Homo sapiens 451 Met Asn Ile
Tyr Ser Met Pro Thr Arg Ser Tyr Ala Pro Ala Glu Ile 1 5 10 15 Phe
Leu Pro Lys Gly Arg Ser Asn Ser Lys Lys Lys Arg Gln Lys Lys 20 25
30 Gln Asn Thr Ser Cys Ser Lys Asn Arg Gly Arg Thr Thr Ala His Thr
35 40 45 Lys Cys Trp Tyr Glu Gly Asn Asn Arg Phe Gly Leu Leu Met
Val Glu 50 55 60 Asn Leu Glu Glu His Ser Glu Ala Ser Asn Ile Glu 65
70 75 452 216 DNA Homo sapiens 452 atgcccacaa ggtcatatgc ccccgctgaa
atattcctac caaaaggtag atcaaattca 60 aaaaaaaaaa ggcagaagaa
acagaatacc agctgttcta agaacagagg gagaaccact 120 gcacacacca
agtgttggta tgagggaaac aaccggtttg ggttgttaat ggttgaaaac 180
ttagaggaac atagtgaggc ctccaacatt gaataa 216 453 71 PRT Homo sapiens
453 Met Pro Thr Arg Ser Tyr Ala Pro Ala Glu Ile Phe Leu Pro Lys Gly
1 5 10 15 Arg Ser Asn Ser Lys Lys Lys Arg Gln Lys Lys Gln Asn Thr
Ser Cys 20 25 30 Ser Lys Asn Arg Gly Arg Thr Thr Ala His Thr Lys
Cys Trp Tyr Glu 35 40 45 Gly Asn Asn Arg Phe Gly Leu Leu Met Val
Glu Asn Leu Glu Glu His 50 55 60 Ser Glu Ala Ser Asn Ile Glu 65 70
454 153 DNA Homo sapiens 454 atgcccccgc tgaaatattc ctaccaaaag
gtagatcaaa ttcaaaaaaa aaaaggcaga 60 agaaacagaa taccagctgt
tctaagaaca gagggagaac cactgcacac accaagtgtt 120 ggtatgaggg
aaacaaccgg tttgggttgt taa 153 455 50 PRT Homo sapiens 455 Met Pro
Pro Leu Lys Tyr Ser Tyr Gln Lys Val Asp Gln Ile Gln Lys 1 5 10 15
Lys Lys Gly Arg Arg Asn Arg Ile Pro Ala Val Leu Arg Thr Glu Gly 20
25 30 Glu Pro Leu His Thr Pro Ser Val Gly Met Arg Glu Thr Thr Gly
Leu 35 40 45 Gly Cys 50 456 30 DNA Homo sapiens 456 atgagggaaa
caaccggttt gggttgttaa 30 457 9 PRT Homo sapiens 457 Met Arg Glu Thr
Thr Gly Leu Gly Cys 1 5 458 48 DNA Homo sapiens 458 atggttgaaa
acttagagga acatagtgag gcctccaaca ttgaataa 48 459 15 PRT Homo
sapiens 459 Met Val Glu Asn Leu Glu Glu His Ser Glu Ala Ser Asn Ile
Glu 1 5 10 15 460 15 DNA Homo sapiens 460 atgtatttaa tataa 15 461 4
PRT Homo sapiens 461 Met Tyr Leu Ile 1 462 979 DNA Homo sapiens 462
tcgacccacg cgtccgcctg ccagcggacg acgtggtcag catcatcgag gaggtggagg
60 agaagcggaa gcggaagaag aacgcccctc ccgagcccgt gccgcccccc
cgtgccgccc 120 ccgcccccac ccacgtccgc tccccgcagc ccccgccccc
cgcccccgct cccgcacgag 180 acgagctgcc ggactggaac gaggtgctcc
cgccctggga tcgggaggag gacgaggtgt 240 acccgccagg gccgtaccac
cctttcccca actacatccg gccgcggaca ctgcagccgc 300 cctcggcctt
gcgccgccgc cactaccacc acgccttgcc gccttcgcgc cactatcccg 360
gccgggaggc ccaggcgcgg cgcgcgcagg aggaggcgga ggcggaggag cgccggctgc
420 aggagcagga ggagctggag aattacatcg agcacgtgct gctccggcgc
ccgtgactgc 480 ccttcccgta accgcccccg cgcgcccccg ccgcgcgcgc
gcgccggcgc ccccctccgt 540 gttgcccgct ccccctcggt gtttgcatgc
gccccggccc tgccccttgg ccctgcccct 600 gtccccgggc tgcgtcggga
cctgccagac ccccctcccg ggtcctgagc ccgaactccc 660 agagctcacc
cgcgggtgac cgggggccag cccaggaggg cgggtggttt gtgcgagttc 720
ccttgccacg cggggccccg gccccatcaa gtccctctgg ggacgtcccc gtcggaaacc
780 ggaaaaagca gttccagtta attgtgtgaa gtgtgtctgt ctccagccct
tcgggcctcc 840 cacgagcccc tccagcctct ccaagtcgct gtgaattgac
cccttctttc ctttctctgt 900 tgtaaatacc cctcacggag gaaatagttt
tgctaagaaa taaaagtgac tattttaaaa 960 aaaaaaaaaa agggcggcc 979 463
243 DNA Homo sapiens 463 atgcgccccg gccctgcccc ttggccctgc
ccctgtcccc gggctgcgtc gggacctgcc 60 agacccccct cccgggtcct
gagcccgaac tcccagagct cacccgcggg tgaccggggg 120 ccagcccagg
agggcgggtg gtttgtgcga gttcccttgc cacgcggggc cccggcccca 180
tcaagtccct ctggggacgt ccccgtcgga aaccggaaaa agcagttcca gttaattgtg
240 tga 243 464 80 PRT Homo sapiens 464 Met Arg Pro Gly Pro Ala Pro
Trp Pro Cys Pro Cys Pro Arg Ala Ala 1 5 10 15 Ser Gly Pro Ala Arg
Pro Pro Ser Arg Val Leu Ser Pro Asn Ser Gln 20 25 30 Ser Ser Pro
Ala Gly Asp Arg Gly Pro Ala Gln Glu Gly Gly Trp Phe 35 40 45 Val
Arg Val Pro Leu Pro Arg Gly Ala Pro Ala Pro Ser Ser Pro Ser 50 55
60 Gly Asp Val Pro Val Gly Asn Arg Lys Lys Gln Phe Gln Leu Ile Val
65 70 75 80 465 18 DNA Artificial Sequence Ologonucleotide 465
tgtaaaacga cggccagt 18 466 18 DNA Artificial Sequence
Oligonucleotide 466 caggaaacag ctatgacc 18
* * * * *
References