Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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MOLECULES FOR DISEASE DETECTION AND TREATMENT
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of molecules
for disease
detection and treatment and to the use of these sequences in the diagnosis,
treatment, and prevention
of cell proliferative, autoimriune/inflammatory, developmental, and
neurological disorders , and in
the assessment of the effects of exogenous compounds on the expression of
nucleic acid and amino
acid sequences of molecules for disease detection and treatment.
BACKGROUND OF THE INVENTION
It is estimated that only 2% of mammalian DNA encodes proteins, and only a
small fraction
of the genes that encode proteins are actually expressed in a particular cell
at any time. The various
types of cells in a multicellular organism differ dramatically both in
structure and function, and the
identity of a particular cell is conferred by its unique pattern of gene
expression. In addition,
different cell types express overlapping but distinctive sets of genes
throughout development. Cell
growth and proliferation, cell differentiation, the immune response,
apoptosis, and other processes
that contribute to organismal development and survival are governed by
regulation of gene
expression. Appropriate gene regulation also ensures that cells function
efficiently by expressing
only those genes whose functions are required at a given time. Factors that
influence gene
expression include extracellular signals that mediate cell-cell communication
and coordinate the
activities of different cell types. Gene expression is regulated at the level
of DNA and RNA
transcription, and at the level of mRNA translation.
Aberrant expression or mutations in genes and their products may cause, or
increase
susceptibility to, a variety of human diseases such as cancer and other cell
proliferative disorders.
The identification of these genes and their products is the basis of an ever-
expanding effort to find
markers for early detection of diseases and targets for their prevention and
treatment. For example,
cancer represents a type of cell proliferative disorder that affects nearly
every tissue in the body. The
development of cancer, or oncogenesis, is often correlated with the conversion
of a normal gene into
a cancer-causing gene, or oncogene, through abnormal expression or mutation.
Oncoproteins, the
products of oncogenes, include a variety of molecules that influence cell
proliferation, such as
growth factors, growth factor receptors, intracellular signal transducers,
nuclear transcription factors,
and cell-cycle control proteins. In contrast, tumor-suppressor genes are
involved in inhibiting cell
proliferation. Mutations which reduce or abrogate the function of tumor-
suppressor genes result in
aberrant cell proliferation and cancer. Thus a wide variety of genes and their
products have been
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found that are associated with cell proliferative disorders such as cancer,
but many more may exist
that are yet to be discovered.
DNA-based arrays can provide an efficient, high-throughput method to examine
gene
expression and genetic variability. For example, SNPs, or single nucleotide
polymorphisms, are the
most common type of human genetic variation. DNA-based arrays can dramatically
accelerate the
discovery of SNPs in hundreds and even thousands of genes. Likewise, such
arrays can be used for
SNP genotyping in which DNA samples from individuals or populations are
assayed for the presence
of selected SNPs. These approaches will ultimately lead to the systematic
identification of all
genetic variations in the human genome and the correlation of certain genetic
variations with disease
susceptibility, responsiveness to drug treatments, and other medically
relevant information. (See, for
example, Wang, D.G. et al. (1998) Science 280:1077-1082.)
DNA-based array technology is especially important for the rapid analysis of
global gene
expression patterns. For example, genetic predisposition, disease, or
therapeutic treatment may
directly or indirectly affect the expression of a large number of genes in a
given tissue. In this case,
it is useful to develop a profile, or transcript image, of all the genes that
are expressed and the levels
at which they are expressed in that particular tissue. A profile generated
from an individual or
population affected with a certain disease or undergoing a particular therapy
may be compared with a
profile generated from a control individual or population. Such analysis does
not require knowledge
of gene function, as the expression profiles can be subjected to mathematical
analyses which simply
treat each gene as a marker. Furthermore, gene expression profiles may help
dissect biological
pathways by identifying all the genes expressed, for example, at a certain
developmental stage, in a
particular tissue, or in response to disease or treatment. (See, for example,
Lander, E.S. et al. (1996)
Science 274:536-539.) '
Certain genes are known to be associated with diseases because of their
chromosomal
location, such as the genes in the myotonic dystrophy (DM) regions of mouse
and human. The
mutation underlying DM has been localized to a gene encoding the DM-kinase
protein, but another
active gene, DMR-N9, is in close proximity to the DM-kinase gene (Jansen, G.
et al. (1992) Nat.
Genet. 1:261-266). DMR-N9 encodes a 650 amino acid protein that contains WD
repeats, motifs
found in cell signaling proteins. DMR-N9 is expressed in all neural tissues
and in the testis,
suggesting a role for DMR-N9 in the manifestation of mental and testicular
symptoms in severe cases
of DM (Jansen, G. et al: (1995) Hum. Mol. Genet. 4:843-852).
Other genes are identified based upon their expression patterns or association
with disease
syndromes. For example, autoantibodies to subcellular organelles are found in
patients with systemic
rheumatic diseases. A recently identified protein, golgin-67, belongs to a
family of Golgi
2
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autoantigens having alpha-helical coiled-coil domains (Eystathioy, T. et al.
(2000) J. Autoimmun.
14:179-187). The Stac gene was identified as a brain specific, developmentally
regulated gene. The
Stac protein contains an SH3 domain, and is thought to be involved in neuron-
specific signal
transduction (Suzuki, H. et al. (1996) Biochem. Biophys. Res. Commun. 229:902-
909).
Calponin is an actin-binding protein that may participate in the function and
organization the
cytoskeleton (Takahashi, K. et al. (1986) Biochem. Biophys. Res. Commun.
141:20-26). The N-
terminus of calponin can interact with calcium-binding proteins and
tropomyosin. Also at located at
the N-terminus is the CH-domain (calponin homology domain) that is found
withimthe structure of
several additional actin-binding proteins (Gusev, N.B. (2001) Biochemistry
(Most) 66:1112-1121).
E~ression prof ling
Array technology can provide a simple way to explore the expression of a
single polymorphic
gene or the expression profile of a large number of related or unrelated
genes. When the expression
of a single gene is examined, arrays are employed to detect the expression of
a specific gene or its
variants. When an expression profile is examined, arrays provide a platform
for identifying genes
that are tissue specific, are affected by a substance being tested in a
toxicology assay, are part of a
signaling cascade, carry out housekeeping functions, or are specifically
related to a particular genetic
predisposition, condition, disease, or disorder.
The potential application of gene expression profiling is particularly
relevant to improving
diagnosis, prognosis, and treatment of disease. For example, both the levels
and sequences expressed
in tissues from subjects with prostate cancer may be compared with the levels
and sequences
expressed in normal tissue.
Prostate cancer is a common malignancy in men over the age of S0, and the
incidence
increases with age. In the US, there are approximately 132,000 newly diagnosed
cases of prostate
cancer and more than 33,000 deaths from the disorder each year.
Once cancer cells arise in the prostate, they are stimulated by testosterone
to a more rapid
growth. Thus, removal of the testes can indirectly reduce both rapid growth
and metastasis of the
cancer. Over 95 percent of prostatic cancers are adenocarcinomas which
originate in the prostatic
acini. The remaining 5 percent are divided between squamous cell and
transitional cell carcinomas,
both of which arise in the prostatic ducts or other parts of the prostate
gland.
As with most cancers, prostate cancer develops through a multistage
progression ultimately
resulting in an aggressive, metastatic phenotype. The initial step in tumor
progression involves the
hyperproliferation of normal luminal and/or basal epithelial cells that become
hyperplastic and evolve
into early-stage tumors. The early-stage tumors are localized in the prostate
but eventually may
metastasize, particularly to the bone, brain or lung. About 80% of these
tumors remain responsive to
androgen treatment, an important hormone controlling the growth of prostate
epithelial cells.
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However, in its most advanced state, cancer growth becomes androgen-
independent and there is
currently no known treatment for this condition.
A primary diagnostic marker for prostate cancer is prostate specific antigen
(PSA). PSA is a
tissue-specific serine protease almost exclusively produced by prostatic
epithelial cells. The quantity
of PSA correlates with the number and volume of the prostatic epithelial
cells, and consequently, the
levels of PSA are an excellent indicator of abnormal prostate growth. Men with
prostate cancer
exhibit an early linear increase in PSA levels followed by an exponential
increase prior to diagnosis.
However, since PSA levels are also influenced by factors such as inflammation,
androgen and other
growth factors, some scientists maintain that changes in PSA levels are not
useful in detecting
individual cases of prostate cancer.
Current areas of cancer research provide additional prospects for markers as
well as potential
therapeutic targets for prostate cancer. Several growth factors have been
shown to play a critical role
in tumor development, growth, and progression. The growth factors Epidermal
Growth Factor
(EGF), Fibroblast Growth Factor (FGF), and Tumor Growth Factor alpha (TGFa)
are important in
the growth of normal as well as hyperproliferative prostate epithelial cells,
particularly at early stages
of tumor development and progression, and affect signaling pathways in these
cells in various ways
(Lin J et al. (1999) Cancer Res. 59:2891-2897; Putz T et al. (1999) Cancer Res
59:227-233). The
TGF-(3 family of growth factors are generally expressed at increased levels in
human cancers and the
high expression levels in many cases correlates with advanced stages of
malignancy and poor
survival (Gold LI ( 1999) Crit Rev Oncog 10:303-360). Finally, there are huW
an cell lines
representing both the androgen-dependent stage of prostate cancer (LNCap) as
well as the androgen-
independent, hormone refractory stage of the disease (PC3 and DU-145) that
have proven useful in
studying gene expression patterns associated with the progression of prostate
cancer, and the effects
of cell treatments on these expressed genes (Chung TD ( 1999) Prostate 15:199-
207).
Tumor necrosis factor a is a pleiotropic cytokine that mediates immune
regulation and
inflammatory responses. TNF-a-related cytokines generate partially overlapping
cellular responses,
including differentiation, proliferation, nuclear factor-xB (NF-xB)
activation, and cell death, by
triggering the aggregation of receptor monomers (Smith, C.A. et al. (1994)
Cell 76:959-962). The
cellular responses triggered by TNF-a are initiated through its interaction
with distinct cell surface
receptors (TNFRs). NF-oB is a transcription factor with a pivotal role in
inducing genes involved in
physiological processes as well as in the response to injury and infection.
Activation of NF-xB
involves the phosphorylation and subsequent degradation of an inhibitory
protein, IKB, and many of
the proximal kinases and adaptor molecules involved in this process have been
elucidated.
Additionally, the NF-xB activation pathway from cell membrane to nucleus for
IL-1 and TNF-a is
now understood (Bowie, A. and L.A. O~leill (2000) Biochem. Pharmacol. 59:13-
23).
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Treatment of confluent cultures of vascular smooth muscle cells (SMCs) with
TNF-a
suppresses the incorporation of [3H]proline into both collagenase-digestible
proteins (CDP) and
noncollagenous proteins (NCP). Such suppression by TNF-a is not observed in
confluent bovine
aortic endothelial cells and human fibroblastic IMR-90 cells. TNF-a decreases
the relative
proportion of collagen types IV and V suggesting that TNF-a modulates collagen
synthesis by SMCs
depending on their cell density and therefore may modify formation of
atherosclerotic lesions
(Hiraga, S. et al. (2000) Life Sci. 66:235-244).
Human aortic endothelial cells (HAECs) are primary cells derived from the
endothelium of a
human aorta. Human iliac artery endothelial cells (HIAECs) are primary cells
derived from the
endothelium of an iliac artery. Human umbilical vein endothelial cells
(HUVECs) are primary cells
derived from the endothelium of an umbilical vein. Primary human endothelial
cell lines have been
used as an experimental model for investigating in vitro the role of the
endothelium in human
vascular biology. Activation of the vascular endothelium is considered to be a
central event in a wide
range of both physiological and pathophysiological processes, such as vascular
tone regulation,
coagulation and thrombosis, atherosclerosis, and inflammation.
Thus, vascular tissue genes differentially expressed during treatment of HAEC,
HIAEC, and
HLJVEC cell cultures with TNFa may reasonably be expected to be markers of the
atherosclerotic
process.
Most normal eukaryotic cells, after a certain number of divisions, enter a
state of senescence
in which cells remain viable and metabolically active but no longer replicate.
A number of
phenotypic changes such as increased cell size and pH-dependent beta-
galactosidase activity, and
molecular changes such as the upregulation of particular genes, occur in
senescent cells (Shelton
(1999) Current Biology 9:939-945). When senescent cells are exposed to
mitogens, a number of
genes are upregulated, but the cells do not proliferate. Evidence indicates
that senescent cells
accumulate with age in vivo, contributing to the aging of an organism. In
addition, senescence
suppresses tumorigenesis, and many genes necessary for senescence also
function as tumor
suppressor genes, such as p53 and the retinoblastoma susceptibility gene. Most
tumors contain cells
that have surpassed their replicative limit, i.e. they are immortalized. Many
oncogenes immortalize
cells as a first step toward tumor formation.
A variety of challenges, such as oxidative stress, radiation, activated
oncoproteins, and cell
cycle inhibitors, induce a senescent phenotype, indicating that senescence is
influenced by a number
of proliferative and anti-proliferative signals (Shelton su ra). Senescence is
correlated with the
progressive shortening of telomeres that occurs with each cell division.
Expression of the catalytic
component of telomerase in cells prevents telomere shortening and immortalizes
cells such as
fibroblasts and epithelial cells, but not other types of cells, such as CD8+ T
cells (Migliaccio et al.
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(2000) J Immunol 165:4978-4984). Thus, senescence is controlled by telomere
shortening as well as
other mechanisms depending on the type of cell.
A number of genes that are differentially expressed between senescent and
presenescent cells
have been identified as part of ongoing studies to understand the role of
senescence in aging and
tumorigenesis. Most senescent cells are growth arrested in the Gl stage of the
cell cycle. While
expression of many cell cycle genes is similar in senescent and presenescent
cells (Cristofalo (1992)
Ann N Y Acad Sci 663:187-194), expression of others genes such as cyclin-
dependent kinases p21
and p16, which inhibit proliferation, and cyclins D1 and E is elevated in
senescent cells. Other genes
that are not directly involved in the cell cycle are also upregulated such as
extracellular matrix
proteins fibronectin, procollagen, and osteonectin; and proteases such as
collagenase, stromelysin,
and cathepsin B (Chen (2000) Ann NY Acad Sci 908:111-125). Genes
underexpressed in senescent
cells include those that encode heat shock proteins, c-fos, and cdc-2 (Chen
supra).
For example, Alzheimer's disease (AD) is a progressive dementia characterized
neuropathologically by the presence of amyloid b-peptide-containing plaques
and neurofibrillary
tangles in specific brain regions. In addition, neurons and synapses are lost
and inflammatory
responses are activated in microglia and astrocytes. Gene expression profiling
of mild, moderate, and
severe AD cases is useful in defining the molecular mechanisms responsible for
functional loss.
DNA-based array technology is also useful as a method of analyzing cell
signaling pathways.
For example, protein kinase C (PKC) is a family of serine/threonine kinases
which play a critical role
in many signal transduction pathways in the cell (Kanashiro, C.A. and Khalil,
R.A. (1998) Clin. Exp.
Pharmacol. Physiol. 25:974-985). Phorbol 12-myristate 13-acetate (PMA) is a
broad activator of the
protein kinase C-dependent pathways. PMA promotes tumors in cells by over
activating PKC
pathways. PKC is also affected by intracellular calcium levels. Internal
calcium flux has been shown
have many effects on cells, particular in the areas of cell activation and
proliferation (Cole, K, and
Kohn, E. (1994) Cancer Metastasis Rev. 13:31-44). Ionomycin is a calcium
ionophore that permits
the entry of calcium into the cell, hence increasing the cytosolic calcium
concentration. Thus the
combination of PMA and ionomycin activates two of the major signaling pathways
used by
mammalian cells to interact with their environment. In T cells, for example,
the combination of PMA
and ionomycin mimics the type of secondary signaling events elicited during
optimal T cell
activation, while in endothelial cells, PMA and ionomycin reproduce the effect
of inflammatory
mediatiors.
Other proteins associated with cell signaling pathways are the guanine
nucleotide binding
proteins (GTP-binding proteins), which are present in all eukaryotic cells and
function in processes
including metabolism, cellular growth, differentiation, signal transduction,
cytoskeletal organization,
and intracellular vesicle transport and secretion. In higher organisms they
are involved in signaling
6
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that regulates such processes as the immune response (Aussel, C. et al. (
1988) J. Immunol. 140:215-
220), apoptosis, differentiation, and cell proliferation including oncogenesis
(Dhanasekaran, N. et al.
(1998) Oncogene 17:1383-1394). Low molecular weight GTP-binding proteins play
critical roles in
cellular protein trafficking events, such as the translocation of proteins and
soluble complexes from
the cytosol to the membrane through an exchange of GDP for GTP (Ktistakis,
N.T. (1998) BioEssays
20:495-504). In vesicle transport, the interaction between vesicle- and target-
specific identifiers (v-
SNAREs and tSNAREs) docks the vesicle to the acceptor membrane. The budding
process is
regulated by GTPases such as the closely related ADP ribosylation factors
(ARFs) and SAR proteins
(Rothman, J.E. and F.T. Wieland (1996) Science 272:227-234). The cycling of
LMW GTP-binding
proteins between the GTP-bound active form and the GDP-bound inactive form is
regulated by
additional proteins. Guanosine nucleotide exchange factors (GEFs) increase the
rate of nucleotide
dissociation by several orders of magnitude, thus facilitating release of GDP
and loading with GTP.
(Geyer, M. and Wittinghofer, A. (1997) Curr. Opin. Struct. Biol. 7:786-792).
The synaptonemal complex (SC) is a protein structure that stabilizes the
pairing of
homologous chromosomes in prophase. Stromalin 3 (STAG3) is a recently
identified member of the
SC. STAG3 gene duplications appear to predispose to germline chromosomal
rearrangements in a
region associated with Williams-Beuren syndrome, a neurobehavioral disorder
(Pezzi, N. et al.
(2000) FASEB J. 14:581-592). A gene encoding another member of the SC,
synaptonemal complex
protein 3 (SCP3) is transcribed exclusively in the testis, and is related to X-
linked lymphocyte-
regulated genes (Lammers, J.H. et al. (1994) Mol. Cell. Biol. 14:1137-1146).
The discovery of new molecules for disease detection and treatment, and the
polynucleotides
encoding them, satisfies a need in the art by providing new compositions which
are useful in the
diagnosis, prevention, and treatment of cell proliferative,
autoimmune/inflammatory, developmental,
and neurological disorders, and in the assessment of the effects of exogenous
compounds on the
expression of nucleic acid and amino acid sequences of molecules for disease
detection and
treatment.
SUMMARY OF THE INVENTION
The invention features purified polypeptides, molecules for disease detection
and treatment,
referred to collectively as "MDDT" and individually as "MDDT-1," "MDDT-2,"
"MDDT-3,"
"MDDT-4," "MDDT-5," "MDDT-6," "MDDT-7," "MDDT-8," "MDDT-9," "MDDT-10," "MDDT-
11," "MDDT-12," "MDDT-13," "MDDT-14," "MDDT-15," "MDDT-16," "MDDT-17," "MDDT-
18," "MDDT-19," "MDDT-20," "MDDT-21," "MDDT-22," "MDDT-23," "MDDT-24," "MDDT-
25," "MDDT-26," "MDDT-27," "MDDT-28," "MDDT-29," "MDDT-30," "MDDT-31," "MDDT-
32," "MDDT-33," "MDDT-34," "MDDT-35," "MDDT-36," and "MDDT-37." In one aspect,
the
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invention provides an isolated polypeptide selected from the group consisting
of a) a polypeptide
comprising an amino acid sequence selected from the group consisting of SEQ )D
NO:1-37, b) a
polypeptide comprising a naturally occurring amino acid sequence at least 90%
identical to an amino
acid sequence selected from the group consisting of SEQ )D NO:1-37, c) a
biologically active
fragment of a polypeptide having an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-37, and d) an immunogenic fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ >D NO:1-37. In one alternative, the
invention provides an
isolated polypeptide comprising the amino acid sequence of SEQ >D NO:1-37.
The invention further provides an isolated polynucleotide encoding a
polypeptide selected
from the group consisting of a) a polypeptide comprising an amino acid
sequence selected from the
group consisting of SEQ >D NO:1-37, b) a polypeptide comprising a naturally
occurring amino acid
sequence at least 90% identical to an amino acid sequence selected from the
group consisting of SEQ
ID NO:1-37, c) a biologically active fragment of a polypeptide having an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-37, and d) an immunogenic
fragment of a
polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D NO: l-37.
In one alternative, the polynucleotide encodes a polypeptide selected from the
group consisting of
SEQ )D NO:1-37. In another alternative, the polynucleotide is selected from
the group consisting of
SEQ ID N0:38-74.
Additionally, the invention provides a recombinant polynucleotide comprising a
promoter
sequence operably linked to a polynucleotide encoding a polypeptide selected
from the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ >D NO:1-37, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ )D NO:1-37, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ LD NO:1-37, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ ID NO:1-37. In one
alternative, the
invention provides a cell transformed with the recombinant polynucleotide. In
another alternative,
the invention provides a transgenic organism comprising the recombinant
polynucleotide.
The invention also provides a method for producing a polypeptide selected from
the group
consisting of a) a polypeptide comprising an amino acid sequence selected from
the group consisting
of SEQ 11..7 N0:1-37, b) a polypeptide comprising a naturally occurring amino
acid sequence at least
90% identical to an amino acid sequence selected from the group consisting of
SEQ 1D NO:1-37, c) a
biologically active fragment of a polypeptide having an amino acid sequence
selected from the group
consisting of SEQ ID NO:1-37, and d) an immunogenic fragment of a polypeptide
having an amino
acid sequence selected from the group consisting of SEQ >D NO:1-37. The method
comprises a)
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culturing a cell under conditions suitable for expression of the polypeptide,
wherein said cell is
transformed with a recombinant polynucleotide comprising a promoter sequence
operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the polypeptide so
expressed.
Additionally, the invention provides an isolated antibody which specifically
binds to a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ )17 NO:1-37, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-37, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-37, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ID NO:1-37.
The invention further provides an isolated polynucleotide selected from the
group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:38-74, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ ID
N0:38-74, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
In one alternative, the
polynucleotide comprises at least 60 contiguous nucleotides.
Additionally, the invention provides a method for detecting a target
polynucleotide in a
sample, said target polynucleotide having a sequence of a polynucleotide
selected from the group
consisting of a) a polynucleotide comprising a polynucleotide sequence
selected from the group
consisting of SEQ ID N0:38-74, b) a polynucleotide comprising a naturally
occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence selected from the
group consisting of
SEQ ID N0:38-74, c) a polynucleotide complementary to the polynucleotide of
a), d) a
polynucleotide complementary to the polynucleotide of b), and e) an RNA
equivalent of a)-d). The
method comprises a) hybridizing the sample with a probe comprising at least 20
contiguous
nucleotides comprising a sequence complementary to said target polynucleotide
in the sample, and
which probe specifically hybridizes to said target polynucleotide, under
conditions whereby a
hybridization complex is formed between said probe and said target
polynucleotide or fragments
thereof, and b) detecting the presence or absence of said hybridization
complex, and optionally, if
present, the amount thereof. In one alternative, the probe comprises at least
60 contiguous
nucleotides.
The invention further provides a method for detecting a target polynucleotide
in a sample,
said target polynucleotide having a sequence of a polynucleotide selected from
the group consisting
of a) a polynucleotide comprising a polynucleotide sequence selected from the
group consisting of
9
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SEQ ll~ N0:38-74, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at
least 90% identical to a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:38-74, c) a polynucleotide complementary to the polynucleotide of a), d) a
polynucleotide
complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
The method
comprises a) amplifying said target polynucleotide or fragment thereof using
polymerase chain
reaction amplification, and b) detecting the presence or absence of said
amplified target
polynucleotide or fragment thereof, and, optionally, if present, the amount
thereof.
The invention further provides a composition comprising an effective amount of
a
polypeptide selected from the group consisting of a) a polypeptide comprising
an amino acid
sequence selected from the group consisting of SEQ ll~ NO:1-37, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-37, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-37, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ )Z7 NO:1-37, and a pharmaceutically acceptable excipient. In
one embodiment, the
composition comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:1-
37. The invention additionally provides a method of treating a disease or
condition associated with
decreased expression of functional MDDT, comprising administering to a patient
in need of such
treatment the composition.
The invention also provides a method for screening a compound for
effectiveness as an
agonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ )D NO:1-37, b) a
polypeptide comprising a
naturally occurnng amino acid sequence at least 90% identical to an amino acid
sequence selected
from the group consisting of SEQ >D NO:1-37, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ 117
NO:1-37, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ >D NO:1-37. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting agonist activity in the sample. In
one alternative, the
invention provides a composition comprising an agonist compound identified by
the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with decreased expression of
functional MDDT, comprising
administering to a patient in need of such treatment the composition.
Additionally, the invention provides a method for screening a compound for
effectiveness as
an antagonist of a polypeptide selected from the group consisting of a) a
polypeptide comprising an
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amino acid sequence selected from the group consisting of SEQ )D NO:1-37, b) a
polypeptide
comprising a naturally occurnng amino acid sequence at least 90% identical to
an amino acid
sequence selected from the group consisting of SEQ )D NO:1-37, c) a
biologically active fragment of
a polypeptide having an amino acid sequence selected from the group consisting
of SEQ )D NO:1-37,
and d) an immunogenic fragment of a polypeptide having an amino acid sequence
selected from the
group consisting of SEQ >D NO:1-37. The method comprises a) exposing a sample
comprising the
polypeptide to a compound, and b) detecting antagonist activity in the sample.
In one alternative, the
invention provides a composition comprising an antagonist compound identified
by the method and a
pharmaceutically acceptable excipient. In another alternative, the invention
provides a method of
treating a disease or condition associated with overexpression of functional
MDDT, comprising
administering to a patient in need of such treatment the composition.
The invention further provides a method of screening for a compound that
specifically binds
to a polypeptide selected from the group consisting of a) a polypeptide
comprising an amino acid
sequence selected from the group consisting of SEQ )D NO:1-37, b) a
polypeptide comprising a
.naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ B7 NO:1-37, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ ID
NO:1-37, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ ll7 NO:1-37. The method comprises a) combining the
polypeptide with at least
one test compound under suitable conditions, and b) detecting binding of the
polypeptide to the test
compound, thereby identifying a compound that specifically binds to the
polypeptide.
The invention further provides a method of screening for a compound that
modulates the
activity of a polypeptide selected from the group consisting of a) a
polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ m NO:1-37, b) a
polypeptide comprising a
naturally occurring amino acid sequence at least 90% identical to an amino
acid sequence selected
from the group consisting of SEQ ID NO:1-37, c) a biologically active fragment
of a polypeptide
having an amino acid sequence selected from the group consisting of SEQ m NO:1-
37, and d) an
immunogenic fragment of a polypeptide having an amino acid sequence selected
from the group
consisting of SEQ 1D NO:1-37. The method comprises a) combining the
polypeptide with at least
one test compound under conditions permissive for the activity of the
polypeptide, b) assessing the
activity of the polypeptide in the presence of the test compound, and c)
comparing the activity of the
polypeptide in the presence of the test compound with the activity of the
polypeptide in the absence
of the test compound, wherein a change in the activity of the polypeptide in
the presence of the test
compound is indicative of a compound that modulates the activity of the
polypeptide.
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The invention further provides a method for screening a compound for
effectiveness in
altering expression of a target polynucleotide, wherein said target
polynucleotide comprises a
polynucleotide sequence selected from the group consisting of SEQ ID N0:38-74,
the method
comprising a) exposing a sample comprising the target polynucleotide to a
compound, b) detecting
altered expression of the target polynucleotide, and c) comparing the
expression of the target
polynucleotide in the presence of varying amounts of the compound and in the
absence of the
compound.
The invention further provides a method for assessing toxicity of a test
compound, said
method comprising a) treating a biological sample containing nucleic acids
with the test compound;
b) hybridizing the nucleic acids of the treated biological sample with a probe
comprising at least 20
contiguous nucleotides of a polynucleotide selected from the group consisting
of i) a polynucleotide
comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:38-74, ii) a
polynucleotide comprising a naturally occurnng polynucleotide sequence at
least 90% identical to a
polynucleotide sequence selected from the group consisting of SEQ ID N0:38-74,
iii) a
polynucleotide having a sequence complementary to i), iv) a polynucleotide
complementary to the
polynucleotide of ii), and v) an RNA equivalent of i)-iv). Hybridization
occurs under conditions
whereby a specific hybridization complex is formed between said probe and a
target polynucleotide
in the biological sample, said target polynucleotide selected from the group
consisting of i) a
polynucleotide comprising a polynucleotide sequence selected from the group
consisting of SEQ )D
N0:38-74, ii) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least
90% identical to a polynucleotide sequence selected from the group consisting
of SEQ ID N0:38-74,
iii) a polynucleotide complementary to the polynucleotide of i), iv) a
polynucleotide complementary
to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
Alternatively, the target
polynucleotide comprises a fragment of a polynucleotide sequence selected from
the group
consisting of i)-v) above; c) quantifying the amount of hybridization complex;
and d) comparing the
amount of hybridization complex in the treated biological sample with the
amount of hybridization
complex in an untreated biological sample, wherein a difference in the amount
of hybridization
complex in the treated biological sample is indicative of toxicity of the test
compound.
BRIEF DESCRIPTION OF THE TABLES
Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the present invention.
Table 2 shows the GenBank identification number and annotation of the nearest
GenBank
homolog, and the PROTEOME database identification numbers and annotations of
PROTEOME
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database homologs, for polypeptides of the invention. The probability scores
for the matches
between each polypeptide and its homolog(s) are also shown.
Table 3 shows structural features of polypeptide sequences of the invention,
including
predicted motifs and domains, along with the methods, algorithms, and
searchable databases used for
analysis of the polypeptides.
Table 4 lists the cDNA and/or genomic DNA fragments which were used to
assemble
polynucleotide sequences of the invention, along with selected fragments of
the polynucleotide
sequences.
Table 5 shows the representative cDNA library for polynucleotides of the
invention.
Table 6 provides an appendix which describes the tissues and vectors used for
construction of
the cDNA libraries shown in Table 5.
Table 7 shows the tools, programs, and algorithms used to analyze the
polynucleotides and
polypeptides of the invention, along with applicable descriptions, references,
and threshold
parameters.
Table 8 shows single nucleotide polymorphisms found in polynucleotide
sequences of the
invention, along with allele frequencies in different human populations.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular
forms "a," "an,"
and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for example, a
reference to "a host cell" includes a plurality of such host cells, and a
reference to "an antibody" is a
reference to one or more antibodies and equivalents thereof known to those
skilled in the art, and so
forth.
Unless defined otherwise, all technical and scientific terms used herein have
the same
meanings as commonly understood by one of ordinary skill in the art to which
this invention belongs.
Although any machines, materials, and methods similar or equivalent to those
described herein can be
used to practice or test the present invention, the preferred machines,
materials and methods are now
described. All publications mentioned herein are cited for the purpose of
describing and disclosing
the cell lines, protocols, reagents and vectors which are reported in the
publications and which might
be used in connection with the invention. Nothing herein is to be construed as
an admission that the
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invention is not entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
"MDDT" refers to the amino acid sequences of substantially purified MDDT
obtained from
any species, particularly a mammalian species, including bovine, ovine,
porcine, murine, equine, and
human, and from any source, whether natural, synthetic, semi-synthetic, or
recombinant.
The term "agonist" refers to a molecule which intensifies or mimics the
biological activity of
MDDT. Agonists may include proteins, nucleic acids, carbohydrates, small
molecules, or any other
compound or composition which modulates the activity of MDDT either by
directly interacting with
MDDT or by acting on components of the biological pathway in which MDDT
participates.
An "allelic variant" is an alternative form of the gene encoding MDDT. Allelic
variants may
result from at least one mutation in the nucleic acid sequence and may result
in altered mRNAs or in
polypeptides whose structure or function may or may not be altered. A gene may
have none, one, or
many allelic variants of its naturally occurring form. Common mutational
changes which give rise to
allelic variants are generally ascribed to natural deletions, additions, or
substitutions of nucleotides.
Each of these types of changes may occur alone, or in combination with the
others, one or more times
in a given sequence.
"Altered" nucleic acid sequences encoding MDDT include those sequences with
deletions,
insertions, or substitutions of different nucleotides, resulting in a
polypeptide the same as MDDT or a
polypeptide with at least one functional characteristic of MDDT. Included
within this definition are
polymorphisms which may or may not be readily detectable using a particular
oligonucleotide probe
of the polynucleotide encoding MDDT, and improper or unexpected hybridization
to allelic variants,
with a locus other than the normal chromosomal locus for the polynucleotide
sequence encoding
MDDT. The encoded protein may also be "altered," and may contain deletions,
insertions, or
substitutions of amino acid residues which produce a silent change and result
in a functionally
equivalent MDDT. Deliberate 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, as long as the biological or immunological activity of MDDT is
retained. For example,
negatively charged amino acids may include aspartic acid and glutamic acid,
and positively charged
amino acids may include lysine and arginine. Amino acids with uncharged polar
side chains having
similar hydrophilicity values may include: asparagine and glutamine; and
serine and threonine.
Amino acids with uncharged side chains having similar hydrophilicity values
may include: leucine,
isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence, or a fragment of any of these, and to
naturally occurring or
synthetic molecules. Where "amino acid sequence" is recited to refer to a
sequence of a naturally
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occurring protein molecule, "amino acid sequence" and like terms are not meant
to limit the amino
acid sequence to the complete native amino acid sequence associated with the
recited protein
molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR)
technologies well
known in the art.
The term "antagonist" refers to a molecule which inhibits or attenuates the
biological activity
of MDDT. Antagonists may include proteins such as antibodies, nucleic acids,
carbohydrates, small
molecules, or any other compound or composition which modulates the activity
of MDDT either by
directly interacting with MDDT or by acting on components of the biological
pathway in which
MDDT participates.
The term "antibody" refers to intact immunoglobulin molecules as well as to
fragments
thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding
an epitopic
determinant. Antibodies that bind MDDT polypeptides can be prepared using
intact polypeptides or
using fragments containing small peptides of interest as the immunizing
antigen. The polypeptide or
oligopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit)
can be derived from the
translation of RNA, or synthesized chemically, and can be conjugated to a
carrier protein if desired.
Commonly used carriers that are chemically coupled to peptides include bovine
serum albumin,
thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is
then used to
immunize the animal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an
epitope) that
makes contact with a particular antibody. When a protein or a fragment of a
protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (particular regions or three-
dimensional structures
on the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
The term "aptamer" refers to a nucleic acid or oligonucleotide molecule that
binds to a
specific molecular target. Aptamers are derived from an in vitro evolutionary
process (e.g., SELEX
(Systematic Evolution of Ligands by EXponential Enrichment), described in U.S.
Patent No.
5,270,163), which selects for target-specific aptamer sequences from large
combinatorial libraries.
Aptamer compositions may be double-stranded or single-stranded, and may
include
deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other
nucleotide-like molecules.
The nucleotide components of an aptamer may have modified sugar groups (e.g.,
the 2'-OH group of
a ribonucleotide may be replaced by 2'-F or 2'-NHZ), which may improve a
desired property, e.g.,
resistance to nucleases or longer lifetime in blood. Aptamers may be
conjugated to other molecules,
CA 02445366 2003-10-23
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e.g., a high molecular weight carrier to slow clearance of the aptamer from
the circulatory system.
Aptamers may be specifically cross-linked to their cognate ligands, e.g., by
photo-activation of a
cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-
13.)
The term "intramer" refers to an aptamer which is expressed in vivo. For
example, a vaccinia
virus-based RNA expression system has been used to express specific RNA
aptamers at high levels in
the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA
96:3606-3610).
The term "spiegelmer" refers to an aptamer which includes L-DNA, L-RNA, or
other left-
handed nucleotide derivatives or nucleotide-like molecules. Aptamers
containing left-handed
nucleotides are resistant to degradation by naturally occurring enzymes, which
normally act on
substrates containing right-handed nucleotides.
The term "antisense" refers to any composition capable of base-pairing with
the "sense"
(coding) strand of a specific nucleic acid sequence. Antisense compositions
may include DNA;
RNA; peptide nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as
phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides
having modified
sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having
modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-
deoxyguanosine. Antisense
molecules may be produced by any method including chemical synthesis or
transcription. Once
introduced into a cell, the complementary antisense molecule base-pairs with a
naturally occurring
nucleic acid sequence produced by the cell to form duplexes which block either
transcription or
translation. The designation "negative" or "minus" can refer to the antisense
strand, and the
designation "positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
The term "biologically active" refers to a protein having structural,
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active" or
"immunogenic" refers to the capability of the natural, recombinant, or
synthetic MDDT, or of any
oligopeptide thereof, to induce a specific immune response in appropriate
animals or cells and to bind
with specific antibodies.
"Complementary" describes the relationship between two single-stranded nucleic
acid
sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement,
3'-TCA-5'.
A "composition comprising a given polynucleotide sequence" and a "composition
comprising
a given amino acid sequence" refer broadly to any composition containing the
given polynucleotide
or amino acid sequence. The composition may comprise a dry formulation or an
aqueous solution.
Compositions comprising polynucleotide sequences encoding MDDT or fragments of
MDDT may be
employed as hybridization probes. The probes may be stored in freeze-dried
form and may be
associated with a stabilizing agent such as a carbohydrate. In hybridizations,
the probe may be
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deployed in an aqueous solution containing salts (e.g., NaCI), detergents
(e.g., sodium dodecyl
sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
subjected to repeated
DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit
(Applied
Biosystems, Foster City CA) in the 5' and/or the 3' direction, and
resequenced, or which has been
assembled from one or more overlapping cDNA, EST, or genomic DNA fragments
using a computer
program for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison
WI) or Phrap (University of Washington, Seattle WA). Some sequences have been
both extended
and assembled to produce the consensus sequence.
"Conservative amino acid substitutions" are those substitutions that are
predicted to least
interfere with the properties of the original protein, i.e., the structure and
especially the function of
the protein is conserved and not significantly changed by such substitutions.
The table below shows
amino acids which may be substituted for an original amino acid in a protein
and which are regarded
as conservative amino acid substitutions.
Original Residue Conservative Substitution
Ala Gly, Ser
Arg His, Lys
Asn Asp, Gln, His
Asp Asn, Glu
Cys Ala, Ser
Gln Asn, Glu, His
Glu Asp, Gln, His
Gly Ala
His Asn, Arg, Gln, Glu
Ile Leu, Val
Leu Ile, Val
Lys Arg, Gln, Glu
Met Leu, Ile
Phe His, Met, Leu, Trp, Tyr
Ser Cys, Thr
Thr Ser, Val
Trp Phe, Tyr
Tyr His, Phe, Trp
Val Ile, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of
the polypeptide
backbone in the area of the substitution, for example, as a beta sheet or
alpha helical conformation,
(b) the charge or hydrophobicity of the molecule at the site of the
substitution, and/or (c) the bulk of
the side chain.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
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The term "derivative" refers to a chemically modified polynucleotide or
polypeptide.
Chemical modifications of a polynucleotide can include, for example,
replacement of hydrogen by an
alkyl, acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a
polypeptide which
retains at least one biological or immunological function of the natural
molecule. A derivative
polypeptide is one modified by glycosylation, pegylation, or any similar
process that retains at least
one biological or immunological function of the polypeptide from which it was
derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable
of generating a
measurable signal and is covalently or noncovalently joined to a
polynucleotide or polypeptide.
"Differential expression" refers to increased or upregulated; or decreased,
downregulated, or
absent gene or protein expression, determined by comparing at least two
different samples. Such
comparisons may be carried out between, for example, a treated and an
untreated sample, or a
diseased and a normal sample.
"Exon shuffling" refers to the recombination of different coding regions
(exons). Since an
exon may represent a structural or functional domain of the encoded protein,
new proteins may be
assembled through the novel reassortment of stable substructures, thus
allowing acceleration of the
evolution of new protein functions.
A "fragment" is a unique portion of MDDT or the polynucleotide encoding MDDT
which is
identical in sequence to but shorter in length than the parent sequence. A
fragment may comprise up
to the entire length of the defined sequence, minus one nucleotide/amino acid
residue. For example,
a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid
residues. A fragment
used as a probe, primer, antigen, therapeutic molecule, or for other purposes,
may be at least 5, 10,
15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous
nucleotides or amino acid
residues in length. Fragments may be preferentially selected from certain
regions of a molecule. For
example, a polypeptide fragment may comprise a certain length of contiguous
amino acids selected
from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide
as shown in a certain
defined sequence. Clearly these lengths are exemplary, and any length that is
supported by the
specification, including the Sequence Listing, tables, and figures, may be
encompassed by the present
embodiments.
A fragment of SEQ >D N0:38-74 comprises a region of unique polynucleotide
sequence that
specifically identifies SEQ )D N0:38-74, for example, as distinct from any
other sequence in the
genome from which the fragment was obtained. A fragment of SEQ m N0:38-74 is
useful, for
example, in hybridization and amplification technologies and in analogous
methods that distinguish
SEQ m N0:38-74 from related polynucleotide sequences. The precise length of a
fragment of SEQ
)D N0:38-74 and the region of SEQ )D N0:38-74 to which the fragment
corresponds are routinely
determinable by one of ordinary skill in the art based on the intended purpose
for the fragment.
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A fragment of SEQ ID NO:1-37 is encoded by a fragment of SEQ ID N0:38-74. A
fragment
of SEQ 1D NO:1-37 comprises a region of unzque amino acid sequence that
specifically identifies
SEQ >D NO:1-37. For example, a fragment of SEQ ID NO:1-37 is useful as an
immunogenic peptide
for the development of antibodies that specifically recognize SEQ )D NO:1-37.
The precise length of
a fragment of SEQ ID NO:1-37 and the region of SEQ )D NO:1-37 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment.
A "full length" polynucleotide sequence is one containing at least a
translation initiation
codon (e.g., methionine) followed by an open reading frame and a translation
termination codon. A
"full length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
"Homology" refers to sequence similarity or, interchangeably, sequence
identity, between
two or more polynucleotide sequences or two or more polypeptide sequences.
The terms "percent identity" and "% identity," as applied to polynucleotide
sequences, refer
to the percentage of residue matches between at least two polynucleotide
sequences aligned using a
standardized algorithm. Such an algorithm may insert, in a standardized and
reproducible way, gaps
in the sequences being compared in order to optimize alignment between two
sequences, and
therefore achieve a more meaningful comparison of the two sequences.
Percent identity between polynucleotide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program. This program is part of the LASERGENE software
package, a suite of
molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is
described in
Higgins, D.G. and P.M. Sharp (1989) CABIOS 5:151-153 and in Higgins, D.G. et
al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the default
parameters are set as
follows: Ktuple=2, gap penalty=5, window=4, and "diagonals saved"=4. The
"weighted" residue
weight table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent
similarity" between aligned polynucleotide sequences.
Alternatively, a suite of commonly used and freely available sequence
comparison algorithms
is provided by the National Center for Biotechnology Information (NCBI) Basic
Local Alignment
Search Tool (BLAST) (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410),
which is available
from several sources, including the NCBI, Bethesda, MD, and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite includes various
sequence
analysis programs including "blastn," that is used to align a known
polynucleotide sequence with
other polynucleotide sequences from a variety of databases. Also available is
a tool called "BLAST
2 Sequences" that is used for direct pairwise comparison of two nucleotide
sequences. "BLAST 2
Sequences" can be accessed and used interactively at
http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
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The "BLAST 2 Sequences" tool can be used for both blastn and blastp (discussed
below). BLAST
programs are commonly used with gap and other parameters set to default
settings. For example, to
compare two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version
2Ø12 (April-21-2000) set at default parameters. Such default parameters may
be, for example:
Matrix: BLOSUM62
Reward for match: 1
Penalty for mismatch: -2
Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off.' S0
Expect: l0
Word Size: 11
Filter: on
Percent identity may be measured over the length of an entire defined
sequence, for example,
as defined by a particular SEQ ID number, or may be measured over a shorter
length, for example,
over the length of a fragment taken from a larger, defined sequence, for
instance, a fragment of at
least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or
at least 200 contiguous
nucleotides. Such lengths are exemplary only, and it is understood that any
fragment length
supported by the sequences shown herein, in the tables, figures, or Sequence
Listing, may be used to
describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree of identity may
nevertheless encode
similar amino acid sequences due to the degeneracy of the genetic code. It is
understood that changes
in a nucleic acid sequence can be made using this degeneracy to produce
multiple nucleic acid
sequences that all encode substantially the same protein.
The phrases "percent identity" and "% identity," as applied to polypeptide
sequences, refer to
the percentage of residue matches between at least two polypeptide sequences
aligned using a
standardized algorithm. Methods of polypeptide sequence alignment are well-
known. Some
alignment methods take into account conservative amino acid substitutions.
Such conservative
substitutions, explained in more detail above, generally preserve the charge
and hydrophobicity at the
site of substitution, thus preserving the structure (and therefore function)
of the polypeptide.
Percent identity between polypeptide sequences may be determined using the
default
parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN
version 3.12e
sequence alignment program (described and referenced above). For pairwise
alignments of
polypeptide sequences using CLUSTAL V, the default parameters are set as
follows: Ktuple=1, gap
penalty=3, window=5, and "diagonals saved"=5. The PAM250 matrix is selected as
the default
residue weight table. As with polynucleotide alignments, the percent identity
is reported by
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CLUSTAL V as the "percent similarity" between aligned polypeptide sequence
pairs.
Alternatively the NCBI BLAST software suite may be used. For example, for a
pairwise
comparison of two polypeptide sequences, one may use the "BLAST 2 Sequences"
tool Version
2Ø12 (April-21-2000) with blastp set at default parameters. Such default
parameters may be, for
example:
Matrix: BLOSUM62
Open Gap: l l and Extension Gap: 1 penalties
Gap x drop-off.' S0
Expect: 10
Word Size: 3
Filter: on
Percent identity may be measured over the length of an entire defined
polypeptide sequence,
for example, as defined by a particular SEQ ID number, or may be measured over
a shorter length,
for example, over the length of a fragment taken from a larger, defined
polypeptide sequence, for
instance, a fragment of at least 15, at least 20, at least 30, at least 40, at
least 50, at least 70 or at least
150 contiguous residues. Such lengths are exemplary only, and it is understood
that any fragment
length supported by the sequences shown herein, in the tables, figures or
Sequence Listing, may be
used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain
DNA sequences of about 6 kb to 10 Mb in size and which contain all of the
elements required for
chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the
amino acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to the process by which a polynucleotide strand anneals
with a
complementary strand through base pairing under defined hybridization
conditions. Specific
hybridization is an indication that two nucleic acid sequences share a high
degree of
complementarity. Specific hybridization complexes form under permissive
annealing conditions and
remain hybridized after the "washing" step(s). The washing steps) is
particularly important in
determining the stringency of the hybridization process, with more stringent
conditions allowing less
non-specific binding, i.e., binding between pairs of nucleic acid strands that
are not perfectly
matched. Permissive conditions for annealing of nucleic acid sequences are
routinely determinable
by one of ordinary skill in the art and may be consistent among hybridization
experiments, whereas
wash conditions may be varied among experiments to achieve the desired
stringency, and therefore
hybridization specificity. Permissive annealing conditions occur, for example,
at 68°C in the
21
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 p.g/ml sheared,
denatured salmon
sperm DNA.
Generally, stringency of hybridization is expressed, in part, with reference
to the temperature
under which the wash step is carried out. Such wash temperatures are typically
selected to be about
5°C to 20°C lower than the thermal melting point (Tin) for the
specific sequence at a defined ionic
strength and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe. An equation for
calculating Tm and
conditions for nucleic acid hybridization are well known and can be found in
Sambrook, J. et al.
( 1989) Molecular Cloning: A Laboratory Manual, 2°d ed., vol. 1-3, Cold
Spring Harbor Press,
Plainview NY; specifically see volume 2, chapter 9.
High stringency conditions for hybridization between polynucleotides of the
present
invention include wash conditions of 68°C in the presence of about 0.2
x SSC and about 0.1% SDS,
for 1 hour. Alternatively, temperatures of about 65°C, 60°C,
55°C, or 42°C may be used. SSC
concentration may be varied from about 0.1 to 2 x SSC, with SDS being present
at about 0.1%.
Typically, blocking reagents are used to block non-specific hybridization.
Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at about 100-200
p,g/ml. Organic
solvent, such as formamide at a concentration of about 35-50% v/v, may also be
used under particular
circumstances, such as for RNA:DNA hybridizations. Useful variations on these
wash conditions
will be readily apparent to those of ordinary skill in the art. Hybridization,
particularly under high
stringency conditions, may be suggestive of evolutionary similarity between
the nucleotides. Such
similarity is strongly indicative of a similar role for the nucleotides and
their encoded polypeptides.
The term "hybridization complex" refers to a complex formed between two
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a solid
support (e.g., paper, membranes, filters, chips, pins or glass slides, or any
other appropriate substrate
to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively.
"Immune response" can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by expression
of various factors, e.g., cytokines, chemokines, and other signaling
molecules, which may affect
cellular and systemic defense systems.
An "immunogenic fragment" is a polypeptide or oligopeptide fragment of MDDT
which is
capable of eliciting an immune response when introduced into a living
organism, for example, a
22
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
mammal. The term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment
of MDDT which is useful in any of the antibody production methods disclosed
herein or known in
the art.
The term "microarray" refers to an arrangement of a plurality of
polynucleotides,
polypeptides, or other chemical compounds on a substrate.
The terms "element" and "array element" refer to a polynucleotide,
polypeptide, or other
chemical compound having a unique and defined position on a microarray.
The term "modulate" refers to a change in the activity of MDDT. For example,
modulation
may cause an increase or a decrease in protein activity, binding
characteristics, or any other
biological, functional, or immunological properties of MDDT.
The phrases "nucleic acid" and "nucleic acid sequence" refer to a nucleotide,
oligonucleotide, polynucleotide, or any fragment thereof. These phrases also
refer to DNA or RNA
of genomic or synthetic origin which may be single-stranded or double-stranded
and may represent
the sense or the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like
material.
"Operably linked" refers to the situation in which a first nucleic acid
sequence is placed in a
functional relationship with a second nucleic acid sequence. For instance, a
promoter is operably
linked to a coding sequence if the promoter affects the transcription or
expression of the coding
sequence. Operably linked DNA sequences may be in close proximity or
contiguous and, where
necessary to join two protein coding regions, in the same reading frame.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone of
amino acid residues ending in lysine. The terminal lysine confers solubility
to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation, and may be pegylated to extend their lifespan in the cell.
"Post-translational modification" of an MDDT may involve lipidation,
glycosylation,
phosphorylation, acetylation, racemization, proteolytic cleavage, and other
modifications known in
the art. These processes may occur synthetically or biochemically. Biochemical
modifications will
vary by cell type depending on the enzymatic milieu of MDDT.
"Probe" refers to nucleic acid sequences encoding MDDT, their complements, or
fragments
thereof, which are used to detect identical, allelic or related nucleic acid
sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a detectable label or
reporter molecule.
Typical labels include radioactive isotopes, ligands, chemiluminescent agents,
and enzymes.
"Primers" are short nucleic acids, usually DNA oligonucleotides, which may be
annealed to a target
polynucleotide by complementary base-pairing. The primer may then be extended
along the target
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WO 02/092759 PCT/US02/13392
DNA strand by a DNA polymerase enzyme. Primer pairs can be used for
amplification (and
identification) of a nucleic acid sequence, e.g., by the polymerase chain
reaction (PCR).
Probes and primers as used in the present invention typically comprise at
least 15 contiguous
nucleotides of a known sequence. In order to enhance specificity, longer
probes and primers may
also be employed, such as probes and primers that comprise at least 20, 25,
30, 40, 50, 60, 70, 80, 90,
100, or at least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and
primers may be considerably longer than these examples, and it is understood
that any length
supported by the specification, including the tables, figures, and Sequence
Listing, may be used.
Methods for preparing and using probes and primers are described in the
references, for
example Sambrook, J. et al. (1989) Molecular Clonin~A Laboratory Manual,
2°d ed., vol. 1-3, Cold
Spring Harbor Press, Plainview NY; Ausubel, F.M. et al. (1987) Current
Protocols in Molecular
Bio_ IoQV, Greene Publ. Assoc. & Wiley-Intersciences, New York NY; Innis, M.
et al. (1990) PCR
Protocols, A Guide to Methods and Applications, Academic Press, San Diego CA.
PCR primer pairs
can be derived from a known sequence, for example, by using computer programs
intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical
Research, Cambridge
MA).
Oligonucleotides for use as primers are selected using software known in the
art for such
purpose. For example, OLIGO 4.06 software is useful for the selection of PCR
primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and larger
polynucleotides of up to
5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases.
Similar primer
selection programs have incorporated additional features for expanded
capabilities. For example, the
PrimOU primer selection program (available to the public from the Genome
Center at University of
Texas South West Medical Center, Dallas TX) is capable of choosing specific
primers from
megabase sequences and is thus useful for designing primers on a genome-wide
scope. The Primer3
primer selection program (available to the public from the Whitehead
Institute/MTT Center for
Genome Research, Cambridge MA) allows the user to input a "mispriming
library," in which
sequences to avoid as primer binding sites are user-specified. Primer3 is
useful, in particular, for the
selection of oligonucleotides for microarrays. (The source code for the latter
two primer selection
programs may also be obtained from their respective sources and modified to
meet the user's specific
needs.) The PrimeGen program (available to the public from the UK Human Genome
Mapping
Project Resource Centre, Cambridge UK) designs primers based on multiple
sequence alignments,
thereby allowing selection of primers that hybridize to either the most
conserved or least conserved
regions of aligned nucleic acid sequences. Hence, this program is useful for
identification of both
unique and conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and
polynucleotide fragments identified by any of the above selection methods are
useful in hybridization
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WO 02/092759 PCT/US02/13392
technologies, for example, as PCR or sequencing primers, microarray elements,
or specific probes to
identify fully or partially complementary polynucleotides in a sample of
nucleic acids. Methods of
oligonucleotide selection are not limited to those described above.
A "recombinant nucleic acid" is a sequence that is not naturally occurring or
has a sequence
that is made by an artificial combination of two or more otherwise separated
segments of sequence.
This artificial combination is often accomplished by chemical synthesis or,
more commonly, by the
artificial manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques
such as those described in Sambrook, supra. The term recombinant includes
nucleic acids that have
been altered solely by addition, substitution, or deletion of a portion of the
nucleic acid. Frequently,
a recombinant nucleic acid may include a nucleic acid sequence operably linked
to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector that is
used, for example, to
transform a cell.
Alternatively, such recombinant nucleic acids may be part of a viral vector,
e.g., based on a
vaccinia virus, that could be use to vaccinate a mammal wherein the
recombinant nucleic acid is
expressed, inducing a protective immunological response in the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from
untranslated
regions of a gene and includes enhancers, promoters, introns, and 5' and 3'
untranslated regions
(IJTRs). Regulatory elements interact with host or viral proteins which
control transcription,
translation, or RNA stability.
"Reporter molecules" are chemical or biochemical moieties used for labeling a
nucleic acid,
amino acid, or antibody. Reporter molecules include radionuclides; enzymes;
fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors;
magnetic particles; and
other moieties known in the art.
An "RNA equivalent," in reference to a DNA sequence, is composed of the same
linear
sequence of nucleotides as the reference DNA sequence with the exception that
all occurrences of the
nitrogenous base thymine are replaced with uracil, and the sugar backbone is
composed of ribose
instead of deoxyribose.
The term "sample" is used in its broadest sense. A sample suspected of
containing MDDT,
nucleic acids encoding MDDT, or fragments thereof may comprise a bodily fluid;
an extract from a
cell, chromosome, organelle, or membrane isolated from a cell; a cell; genomic
DNA, RNA, or
cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that
interaction between a
protein or peptide and an agonist, an antibody, an antagonist, a small
molecule, or any natural or
synthetic binding composition. The interaction is dependent upon the presence
of a particular
structure of the protein, e.g., the antigenic determinant or epitope,
recognized by the binding
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
molecule. For example, if an antibody is specific for epitope "A," the
presence of a polypeptide
comprising the epitope A, or the presence of free unlabeled A, in a reaction
containing free labeled A
and the antibody will reduce the amount of labeled A that binds to the
antibody.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least 60% free,
preferably at least 75% free, and most preferably at least 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acid residues
or nucleotides
by different amino acid residues or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
A "transcript image" or "expression profile" refers to the collective pattern
of gene
expression by a particular cell type or tissue under given conditions at a
given time.
"Transformation" describes a process by which exogenous DNA is introduced into
a
recipient cell. Transformation may occur under natural or artificial
conditions according to various
methods well known in the art, and may rely on any known method for the
insertion of foreign
nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is
selected based on the type of host cell being transformed and may include, but
is not limited to,
bacteriophage or viral infection, electroporation, heat shock, lipofection,
and particle bombardment.
The term "transformed cells" includes stably transformed cells in which the
inserted DNA is capable
of replication either as an autonomously replicating plasmid or as part of the
host chromosome, as
well as transiently transformed cells which express the inserted DNA or RNA
for limited periods of
time.
A "transgenic organism," as used herein, is any organism, including but not
limited to
animals and plants, in which one or more of the cells of the organism contains
heterologous nucleic
acid introduced by way of human intervention, such as by transge~nic
techniques well known in the
art. The nucleic acid is introduced into the cell, directly or indirectly by
introduction into a precursor
of the cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with
a recombinant virus. In one alternative, the nucleic acid can be introduced by
infection with a
recombinant viral vector, such as a lentiviral vector (Loin, C. et al. (2002)
Science 295:868-872).
The term genetic manipulation does not include classical cross-breeding, or in
vitro fertilization, but
rather is directed to the introduction of a recombinant DNA molecule. The
transgenic organisms
contemplated in accordance with the present invention include bacteria,
cyanobacteria, fungi, plants
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WO 02/092759 PCT/US02/13392
and animals. The isolated DNA of the present invention can be introduced into
the host by methods
known in the art, for example infection, transfection, transformation or
transconjugation. Techniques
for transferring the DNA of the present invention into such organisms are
widely known and
provided in references such as Sambrook et al. (1989), supra.
A "variant" of a particular nucleic acid sequence is defined as a nucleic acid
sequence having
at least 40% sequence identity to the particular nucleic acid sequence over a
certain length of one of
the nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of nucleic acids may show, for
example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99% or greater
sequence identity over a certain defined length. A variant may be described
as, for example, an
"allelic" (as defined above), "splice," "species," or "polymorphic" variant. A
splice variant may have
significant identity to a reference molecule, but will generally have a
greater or lesser number of
polynucleotides due to alternate splicing of exons during mRNA processing. The
corresponding
polypeptide may possess additional functional domains or lack domains that are
present in the
reference molecule. Species variants are polynucleotide sequences that vary
from one species to
another. The resulting polypeptides will generally have significant amino acid
identity relative to
each other. A polymorphic variant is a variation in the polynucleotide
sequence of a particular gene
between individuals of a given species. Polymorphic variants also may
encompass "single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one
nucleotide base. The
presence of SNPs may be indicative of, for example, a certain population, a
disease state, or a
propensity for a disease state.
A "variant" of a particular polypeptide sequence is defined as a polypeptide
sequence having
at least 40% sequence identity to the particular polypeptide sequence over a
certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool
Version 2Ø9 (May-07-
1999) set at default parameters. Such a pair of polypeptides may show, for
example, at. least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least
92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
or greater sequence
identity over a certain defined length of one of the polypeptides.
THE INVENTION
The invention is based on the discovery of new human molecules for disease
detection and
treatment (MDDT), the polynucleotides encoding MDDT, and the use of these
compositions for the
diagnosis, treatment, or prevention of cell proliferative,
autoimmune/inflammatory, developmental,
and neurological disorders.
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Table 1 summarizes the nomenclature for the full length polynucleotide and
polypeptide
sequences of the invention. Each polynucleotide and its corresponding
polypeptide are correlated to
a single Incyte project identification number (Incyte Project ID). Each
polypeptide sequence is
denoted by both a polypeptide sequence identification number (Polypeptide SEQ
ID NO:) and an
Incyte polypeptide sequence number (Incyte Polypeptide )D) as shown. Each
polynucleotide
sequence is denoted by both a polynucleotide sequence identification number
(Polynucleotide SEQ
ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte
Polynucleotide ID) as
shown.
Table 2 shows sequences with homology to the polypeptides of the invention as
identified by
BLAST analysis against the GenBank protein (genpept) database and the PROTEOME
database.
Columns 1 and 2 show the polypeptide sequence identification number
(Polypeptide SEQ >D NO:)
and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide
>D) for polypeptides
of the invention. Column 3 shows the GenBank identification number (GenBank >D
NO:) of the
nearest GenBank homolog and the PROTEOME database identification numbers
(PROTEOME >D
NO:) of the nearest PROTEOME database homologs. Column 4 shows the probability
scores for the
matches between each polypeptide and its homolog(s). Column S shows the
annotation of the
GenBank and PROTEOME database homolog(s) along with relevant citations where
applicable, all
of which are expressly incorporated by reference herein.
Table 3 shows various structural features of the polypeptides of the
invention. Columns 1
and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the
corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention.
Column 3 shows the number of amino acid residues in each polypeptide. Column 4
shows potential
phosphorylation sites, and column 5 shows potential glycosylation sites, as
determined by the
MOTIFS program of the GCG sequence analysis software package (Genetics
Computer Group,
Madison WI). Column 6 shows amino acid residues comprising signature
sequences, domains, and
motifs. Column 7 shows analytical methods for protein structure/function
analysis and in some
cases, searchable databases to which the analytical methods were applied.
Together, Tables 2 and 3 summarize the properties of polypeptides of the
invention, and
these properties establish that the claimed polypeptides are molecules for
disease detection and
treatment. For example, SEQ )D NO:1 is 98% identical, from residue M1 to
residue V2061, to
human myoferlin (GenBank ll~ 86731235) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ ID NO:l
also contains
multiple C2 domains as determined by searching for statistically significant
matches in the hidden
Markov model (HMM)-based PFAM database of conserved protein family domains.
(See Table 3.)
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Data from additional BLAST analyses provide further corroborative evidence
that SEQ ID NO:1 is a
ferlin family protein. In an alternative example, SEQ ID N0:9 is 92%
identical, from residue M1 to
residue K127, to Mus musculus testis-specific histone H2B (GenBank ID
g1518338) as determined
by the Basic Local Alignment Search Tool (BLAST). (See Table 2.) The BLAST
probability score
is S.Oe-56, which indicates the probability of obtaining the observed
polypeptide sequence alignment
by chance. SEQ ID N0:9 also contains a core histone H2A/H2B/H3/H4 domain as
determined by
searching for statistically significant matches in the hidden Markov model
(HMM)-based PFAM
database of conserved protein family domains. (See Table 3.) Data from BLIMPS,
MOTIFS, and
PROFILESCAN analyses provide further corroborative evidence that SEQ ID N0:9
is a histone. In
an alternative example, SEQ >D N0:17 is 93% identical, from residue M1 to
residue V1288, to rat 5-
oxo-L-prolinase (GenBank >D g1732065) as determined by the Basic Local
Alignment Search Tool
(BLAST). (See Table 2.) The BLAST probability score is 0.0, which indicates
the probability of
obtaining the observed polypeptide sequence alignment by chance. SEQ ID N0:17
also contains
hydantoinase/oxoprolinase domains as determined by searching for statistically
significant matches in
the hidden Markov model (HMM)-based PFAM database of conserved protein family
domains. In an
alternative example, SEQ ID N0:18 is 53% identical, from residue M96 to
residue 8748, to human
zinc finger protein 304 (GenBank ID g9968290) as determined by BLAST analysis.
(See Table 2.)
The BLAST probability score is 4.1e-180. SEQ ID N0:18 also contains KRAB box
and zinc forger
domains as determined by searching for statistically significant matches in
the hidden Markov model
(HMM)-based PFAM database of conserved protein family domains. (See Table 3.)
Data from
MOTIFS analysis provides further corroborative evidence that SEQ 117 N0:18 is
a zinc-forger
protein. In an alternative example, SEQ )D N0:34 is 99% identical, from
residue M515 to residue
T1047, to human hepatocellular carcinoma-associated antigen 67 (GenBank >17
g7645038) as
determined by the Basic Local Alignment Search Tool (BLAST). (See Table 2.)
The BLAST
probability score is 1.2e-287, which indicates the probability of obtaining
the observed polypeptide
sequence alignment by chance. SEQ D7 N0:34 also contains a pleckstrin homology
(PH) domain and
a Sec7 GTP exchange factor domain as determined by searching for statistically
significant matches
in the hidden Markov model (HMM)-based PFAM database of conserved protein
family domains.
(See Table 3.) Data from BLIMPS and additional BLAST analyses provide further
corroborative
evidence that SEQ ID N0:34 is a disease associated protein. In an alternative
example, SEQ LD
N0:36 is 52% identical, from residue A59 to residue D444, to leucine-rich
neuronal protein
(GenBank )D g3135309) as determined by the Basic Local Alignment Search Tool
(BLAST). (See
Table 2.) The BLAST probability score is 1.9e-126, which indicates the
probability of obtaining the
observed polypeptide sequence alignment by chance. SEQ >D N0:36 also has
homology to the
human leucine-rich neuronal protein which has leucine-rich repeats and a
potential membrane anchor,
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WO 02/092759 PCT/US02/13392
as determined by BLAST analysis using the PROTEOME database. SEQ ID N0:36 also
contains a
calponin homology domain, and a leucine-rich repeat domain as determined by
searching for
statistically significant matches in the hidden Markov model (HMM)-based PFAM
database of
conserved protein family domains. (See Table 3.) Data from BLIMPS, MOTIFS, and
additional
BLAST analyses provide further corroborative evidence that SEQ ID N0:36 is a
leucine-rich
protein. SEQ )D N0:2-8, SEQ ID NO:10-16, SEQ ID N0:19-33, SEQ )D N0:35, and
SEQ >D N0:37
were analyzed and annotated in a similar manner. The algorithms and parameters
for the analysis of
SEQ ID NO:1-37 are described in Table 7.
As shown in Table 4, the full length polynucleotide sequences of the present
invention were
assembled using cDNA sequences or coding (exon) sequences derived from.genomic
DNA, or any
combination of these two types of sequences. Column 1 lists the polynucleotide
sequence
identification number (Polynucleotide SEQ 117 NO:), the corresponding Incyte
polynucleotide
consensus sequence number (Incyte ID) for each polynucleotide of the
invention, and the length of
each polynucleotide sequence in basepairs. Column 2 shows the nucleotide start
(5') and stop (3')
positions of the cDNA and/or genomic sequences used to assemble the full
length polynucleotide
sequences of the invention, and of fragments of the polynucleotide sequences
which are useful, for
example, in hybridization or amplification technologies that identify SEQ ID
N0:38-74 or that
distinguish between SEQ ID N0:38-74 and related polynucleotide sequences.
The polynucleotide fragments described in Column 2 of Table 4 may refer
specifically, for
example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from
pooled cDNA
libraries. Alternatively, the polynucleotide fragments described in column 2
may refer to GenBank
cDNAs or ESTs which contributed to the assembly of the full length
polynucleotide sequences. In
addition, the polynucleotide fragments described in column 2 may identify
sequences derived from
the ENSEMBL (The Sanger Centre, Cambridge, UK) database (i.e., those sequences
including the
designation "ENST"). Alternatively, the polynucleotide fragments described in
column 2 may be
derived from the NCBI RefSeq Nucleotide Sequence Records Database (i.e., those
sequences
including the designation "NM" or "NT") or the NCBI RefSeq Protein Sequence
Records (i.e., those
sequences including the designation "NP"). Alternatively, the polynucleotide
fragments described in
column 2 may refer to assemblages of both cDNA and Genscan-predicted exons
brought together by
an "exon stitching" algorithm. For example, a polynucleotide sequence
identified as
FL XXXXXX_N, NZ YYYYY N3 NQ represents a "stitched" sequence in which XXXXXX
is the
identification number of the cluster of sequences to which the algorithm was
applied, and YYYYY is
the number of the prediction generated by the algorithm, and N,,z,j.._, if
present, represent specific
exons that may have been manually edited during analysis (See Example V).
Alternatively, the
polynucleotide fragments in column 2 may refer to assemblages of exons brought
together by an
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
"exon-stretching" algorithm. For example, a polynucleotide sequence identified
as
FLXXXXXX_gAAAAA~BBBBB_1 N is a "stretched" sequence, with XXXXXX being the
Incyte
project identification number, gAAAAA being the GenBank identification number
of the human
genomic sequence to which the "exon-stretching" algorithm was applied, gBBBBB
being the
GenBank identification number or NCBI RefSeq identification number of the
nearest GenBank
protein homolog, and N referring to specific exons (See Example V). In
instances where a RefSeq
sequence was used as a protein homolog for the "exon-stretching" algorithm, a
RefSeq identifier
(denoted by "NM," "NP," or "NT") may be used in place of the GenBank
identifier (i.e., gBBBBB).
Alternatively, a prefix identifies component sequences that were hand-edited,
predicted from
genomic DNA sequences, or derived from a combination of sequence analysis
methods. The
following Table lists examples of component sequence prefixes and
corresponding sequence analysis
methods associated with the prefixes (see Example IV and Example V).
Prefix Type of analysis and/or examples of programs
GNN, GFG,Exon prediction from genomic sequences using,
for example,
ENST GENSCAN (Stanford University, CA, USA) or
FGENES
(Computer Genomics Group, The Sanger Centre,
Cambridge, UK).
GBI Hand-edited analysis of genomic sequences.
FL Stitched or stretched genomic sequences
(see Example V).
INCY Full length transcript and exon prediction
from mapping of EST
sequences to the genome. Genomic location
and EST composition
data are combined to predict the exons and
resulting transcript.
In some cases, Incyte cDNA coverage redundant with the sequence coverage shown
in Table
4 was obtained to confirm the final consensus polynucleotide sequence, but the
relevant Incyte cDNA
identification numbers are not shown.
Table 5 shows the representative cDNA libraries for those full length
polynucleotide
sequences which were assembled using Incyte cDNA sequences. The representative
cDNA library is
the Incyte cDNA library which is most frequently represented by the Incyte
cDNA sequences which
were used to assemble and confirm the above polynucleotide sequences. The
tissues and vectors
which were used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide
sequences
of the invention, along with allele frequencies in different human
populations. Columns I and 2
show the polynucleotide sequence identification number (SEQ ID NO:) and the
corresponding Incyte
project identification number (P117) for polynucleotides of the invention.
Column 3 shows the Incyte
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identification number for the EST in which the SNP was detected (EST )D), and
column 4 shows the
identification number for the SNP (SNP >D). Column 5 shows the position within
the EST sequence
at which the SNP is located (EST SNP), and column 6 shows the position of the
SNP within the full
length polynucleotide sequence (CB 1 SNP). Column 7 shows the allele found in
the EST sequence.
Columns 8 and 9 show the two alleles found at the SNP site. Column 10 shows
the amino acid
encoded by the codon including the SNP site, based upon the allele found in
the EST. Columns 11-
14 show the frequency of allele 1 in four different human populations. An
entry of n/d (not
detected) indicates that the frequency of allele 1 in the population was too
low to be detected, while
n/a (not available) indicates that the allele frequency was not determined for
the population .
The invention also encompasses MDDT variants. A preferred MDDT variant is one
which
has at least about 80%, or alternatively at least about 90%, or even at least
about 95% amino acid
sequence identity to the MDDT amino acid sequence, and which contains at least
one functional or
structural characteristic of MDDT.
The invention also encompasses polynucleotides which encode MDDT. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence selected
from the group consisting of SEQ ID N0:38-74, which encodes MDDT. The
polynucleotide
sequences of SEQ >D N0:38-74, as presented in the Sequence Listing, embrace
the equivalent RNA
sequences, wherein occurrences of the nitrogenous base thymine are replaced
with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
The invention also encompasses a variant of a polynucleotide sequence encoding
MDDT. In
particular, such a variant polynucleotide sequence will have at least about
70%, or alternatively at
least about 85%, or even at least about 95% polynucleotide sequence identity
to the polynucleotide
sequence encoding MDDT. A particular aspect of the invention encompasses a
variant of a
polynucleotide sequence comprising a sequence selected from the group
consisting of SEQ ID
N0:38-74 which has at least about 70%, or alternatively at least about 85%, or
even at least about
95% polynucleotide sequence identity to a nucleic acid sequence selected from
the group consisting
of SEQ ID N0:38-74. Any one of the polynucleotide variants described above can
encode an amino
acid sequence which contains at least one functional or structural
characteristic of MDDT.
In addition, or in the alternative, a polynucleotide variant of the invention
is a splice variant
of a polynucleotide sequence encoding MDDT. A splice variant may have portions
which have
significant sequence identity to the polynucleotide sequence encoding MDDT,
but will generally
have a greater or lesser number of polynucleotides due to additions or
deletions of blocks of sequence
arising from alternate splicing of exons during mRNA processing. A splice
variant may have less
than about 70%, or alternatively less than about 60%, or alternatively less
than about 50%
polynucleotide sequence identity to the polynucleotide sequence encoding MDDT
over its entire
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WO 02/092759 PCT/US02/13392
length; however, portions of the splice variant will have at least about 70%,
or alternatively at least
about 85%, or alternatively at least about 95%, or alternatively 100%
polynucleotide sequence
identity to portions of the polynucleotide sequence encoding MDDT. For
example, a polynucleotide
comprising a sequence of SEQ ll~ N0:73 is a splice variant of a polynucleotide
comprising a
sequence of SEQ ID N0:39 and a polynucleotide comprising a sequence of SEQ ID
N0:74 is a splice
variant of a polynucleotide comprising a sequence of SEQ >D N0:54. Any one of
the splice variants
described above can encode an amino acid sequence which contains at least one
functional or
structural characteristic of MDDT.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding MDDT, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring MDDT, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode MDDT and its variants are generally
capable
of hybridizing to the nucleotide sequence of the naturally occurring MDDT
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences encoding
MDDT or its derivatives possessing a substantially different codon usage,
e.g., inclusion of non-
naturally occurring codons. Codons may be selected to increase the rate at
which expression of the
peptide occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with
which particular codons are utilized by the host. Other reasons for
substantially altering the
nucleotide sequence encoding MDDT and its derivatives without altering the
encoded amino acid
sequences include the production of RNA transcripts having more desirable
properties, such as a
greater half-life, than transcripts produced from the naturally occurring
sequence.
The invention also encompasses production of DNA sequences which encode MDDT
and
MDDT derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding MDDT or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular, to
those shown in SEQ )D
N0:38-74 and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987) Methods
Enzymol.
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152:507-511.) Hybridization conditions, including annealing and wash
conditions, are described in
"Definitions."
Methods for DNA sequencing are well known in the art and may be used to
practice any of
the embodiments of the invention. The methods may employ such enzymes as the
Klenow fragment
of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase
(Applied
Biosystems), thermostable T7 polymerase (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerases and proofreading exonucleases such as those found
in the ELONGASE
amplification system (Life Technologies, Gaithersburg MD). Preferably,
sequence preparation is
automated with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno NV),
PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal
cycler
(Applied Biosystems). Sequencing is then carried out using either the ABI 373
or 377 DNA
sequencing system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system
(Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The
resulting sequences
are analyzed using a variety of algorithms which are well known in the art.
(See, e.g., Ausubel, F.M.
(1997) Short Protocols in Molecular Biolo~y, John Wiley & Sons, New York NY,
unit 7.7; Meyers,
R.A. (1995) Molecular Biology and BiotechnoloQV, Wiley VCH, New York NY, pp.
856-853.)
The nucleic acid sequences encoding MDDT may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream sequences,
such as promoters and regulatory elements. For example, one method which may
be employed,
restriction-site PCR, uses universal and nested primers to amplify unknown
sequence from genomic
DNA within a cloning vector. (See, e.g., Sarkar, G. ( 1993) PCR Methods
Applic. 2:318-322.)
Another method, inverse PCR, uses primers that extend in divergent directions
to amplify unlrnown
sequence from a circularized template. The template is derived from
restriction fragments
comprising a known genomic locus and surrounding sequences. (See, e.g.,
Triglia, T. et al. (1988)
Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR
amplification of DNA
fragments adjacent to known sequences in human and yeast artificial chromosome
DNA. (See, e.g.,
Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:11 l-119.) In this method,
multiple restriction
enzyme digestions and ligations may be used to insert an engineered double-
stranded sequence into a
region of unknown sequence before performing PCR. Other methods which may be
used to retrieve
unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991)
Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries
(Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need
to screen libraries
and is useful in finding intron/exon junctions. For all PCR-based methods,
primers may be designed
using commercially available software, such as OLIGO 4.06 primer analysis
software (National
Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30
nucleotides in
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WO 02/092759 PCT/US02/13392
length, to have a GC content of about 50°10 or more, and to anneal to
the template at temperatures of
about 68°C to 72°C.
When screening for full length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to analyze
the size or confirm the nucleotide sequence of sequencing or PCR products. In
particular, capillary
sequencing may employ flowable polymers for electrophoretic separation, four
different nucleotide
specific, laser-stimulated fluorescent dyes, and a charge coupled device
camera for detection of the
emitted wavelengths. Outpudlight intensity may be converted to electrical
signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Applied Biosystems), and the
entire
process from loading of samples to computer analysis and electronic data
display may be computer
controlled. Capillary electrophoresis is especially preferable for sequencing
small DNA fragments
which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode MDDT may be cloned in recombinant DNA molecules that direct
expression of
MDDT, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent
degeneracy of the genetic code, other DNA sequences which encode substantially
the same or a
functionally equivalent amino acid sequence may be produced and used to
express MDDT.
The nucleotide sequences of the present invention can be engineered using
methods generally
known in the art in order to alter MDDT-encoding sequences for a variety of
purposes including, but
not limited to, modification of the cloning, processing, and/or expression of
the gene product. DNA
shuffling by random fragmentation and PCR reassembly of gene fragments and
synthetic
oligonucleotides may be used to engineer the nucleotide sequences. For
example, oligonucleotide-
mediated site-directed mutagenesis may be used to introduce mutations that
create new restriction
sites, alter glycosylation patterns, change codon preference, produce splice
variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling
techniques such
as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent
No.
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians,
F.C. et al. (1999) Nat.
Biotechnol. 17:259-264; and Crameri, A. et al. (1996) Nat. Biotechnol. 14:315-
319) to alter or
improve the biological properties of MDDT, such as its biological or enzymatic
activity or its ability
to bind to other molecules or compounds. DNA shuffling is a process by which a
library of gene
variants is produced using PCR-mediated recombination of gene fragments. The
library is then
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
subjected to selection or screening procedures that identify those gene
variants with the desired
properties. These preferred variants may then be pooled and further subjected
to recursive rounds of
DNA shuffling and selection/screening. Thus, genetic diversity is created
through "artificial"
breeding and rapid molecular evolution. For example, fragments of a single
gene containing random
point mutations may be recombined, screened, and then reshuffled until the
desired properties are
optimized. Alternatively, fragments of a given gene may be recombined with
fragments of
homologous genes in the same gene family, either from the same or different
species, thereby
maximizing the genetic diversity of multiple naturally occurring genes in a
directed and controllable
manner.
In another embodiment, sequences encoding MDDT may be synthesized, in whole or
in part,
using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et
al. (1980) Nucleic
Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic Acids Symp.
Ser. 7:225-232.)
Alternatively, MDDT itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solution-phase or
solid-phase techniques.
(See, e.g., Creighton, T. (1984) Proteins, Structures and Molecular
Properties, WH Freeman, New
York NY, pp. 55-60; and Roberge, J.Y. et al. (1995) Science 269:202-204.)
Automated synthesis
may be achieved using the ABI 431A peptide synthesizer (Applied Biosystems).
Additionally, the
amino acid sequence of MDDT, or any part thereof, may be altered during direct
synthesis and/or
combined with sequences from other proteins, or any part thereof, to produce a
variant polypeptide or
a polypeptide having a sequence of a naturally occurring polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g., Chiez, R.M. and F.Z. Regnier (1990) Methods
Enzymol. 182:392-421.)
The composition of the synthetic peptides may be confirmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, sera, pp. 28-53.)
In order to express a biologically active MDDT, the nucleotide sequences
encoding MDDT
or derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
sequence in a suitable host. These elements include regulatory sequences, such
as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding MDDT. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding MDDT. Such signals include the ATG initiation codon and
adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding MDDT and its
initiation codon and
upstream regulatory sequences are inserted into the appropriate expression
vector, no additional
transcriptional or translational control signals may be needed. However, in
cases where only coding
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CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
sequence, or a fragment thereof, is inserted, exogenous translational control
signals including an in-
frame ATG initiation codon should be provided by the vector. Exogenous
translational elements and
initiation codons may be of various origins, both natural and synthetic. The
efficiency of expression
may be enhanced by the inclusion of enhancers appropriate for the particular
host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to
construct expression
vectors containing sequences encoding MDDT and appropriate transcriptional and
translational
control elements. These methods include in vitro recombinant DNA techniques,
synthetic
techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al.
(1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4,
8, and 16-17;
Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolo~y, John Wiley
& Sons, New York
NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and
express sequences
encoding MDDT. These include, but are not limited to, microorganisms such as
bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors;
yeast transformed with
yeast expression vectors; insect cell systems infected with viral expression
vectors (e.g., baculovirus);
plant cell systems transformed with viral expression vectors (e.g.,
cauliflower mosaic virus, CaMV,
or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or
animal cell systems. (See, e.g., Sambrook, supra; Ausubel, supra; Van Heeke,
G. and S.M. Schuster
(1989) J. Biol. Chem. 264:5503-5509; Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu,
N. (1987) EMBO
J. 6:307-311; The McGraw Hill Yearbook of Science and TechnoloQV (1992) McGraw
Hill, New
York NY, pp. 191-196; Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659; and
Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors
derived from retroviruses,
adenoviruses, or herpes or vaccinia viruses, or from various bacterial
plasmids, may be used for
delivery of nucleotide sequences to the targeted organ, tissue, or cell
population. (See, e.g., Di
Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356; Yu, M. et al. (1993)
Proc. Natl. Acad. Sci.
USA 90(13):6340-6344; Buller, R.M. et al. (1985) Nature 317(6040):813-815;
McGregor, D.P. et al.
(1994) Mol. Immunol. 31(3):219-226; and Verma, LM. and N. Somia (1997) Nature
389:239-242.)
The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected depending
upon the use intended for polynucleotide sequences encoding MDDT. For example,
routine cloning,
subcloning, and propagation of polynucleotide sequences encoding MDDT can be
achieved using a
multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La Jolla CA)
or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding MDDT into the
vector's multiple
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WO 02/092759 PCT/US02/13392
cloning site disrupts the lacZ gene, allowing a colorimetric screening
procedure for identification of
transformed bacteria containing recombinant molecules. In addition, these
vectors may be useful for
in vitro transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M.
Schuster (1989) J. Biol.
Chem. 264:5503-5509.) When large quantities of MDDT are needed, e.g. for the
production of
antibodies, vectors which direct high level expression of MDDT may be used.
For example, vectors
containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
Yeast expression systems may be used for production of MDDT. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH
promoters, may be used in the yeast Saccharom~es cerevisiae or Pichia
pastoris. In addition, such
vectors direct either the secretion or intracellular retention of expressed
proteins and enable
integration of foreign sequences into the host genome for stable propagation.
(See, e.g., Ausubel,
1995, supra; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544; and
Scorer, C.A. et al. (1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of MDDT. Transcription of
sequences
encoding MDDT may be driven by viral promoters, e.g., the 355 and 195
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small subunit of
RUBISCO or heat shock
promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-
1680; Brogue, R. et al.
(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell
Differ. 17:85-105.)
These constructs can be introduced into plant cells by direct DNA
transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of
Science and Technolo~y
(1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells, a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding MDDT
may be ligated into
an adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader
sequence. Insertion in a non-essential E1 or E3 region of the viral genome may
be used to obtain
infective virus which expresses MDDT in host cells. (See, e.g., Logan, J. and
T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such
as the Rous sarcoma
virus (RSV) enhancer, may be used to increase expression in mammalian host
cells. 5V40 or EBV-
based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments of
DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb
to 10 Mb are
constructed and delivered via conventional delivery methods (liposomes,
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. (1997) Nat. Genet.
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15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression
of MDDT in cell lines is preferred. For example, sequences encoding MDDT can
be transformed
into cell lines using expression vectors which may contain viral origins of
replication and/or
endogenous expression elements and a selectable marker gene on the same or on
a separate vector.
Following the introduction of the vector, cells may be allowed to grow for
about 1 to 2 days in
enriched media before being switched to selective media. The purpose of the
selectable marker is to
confer resistance to a selective agent, and its presence allows growth and
recovery of cells which
successfully express the introduced sequences. Resistant clones of stably
transformed cells may be
propagated using tissue culture techniques appropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk- and apY cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell 11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic,
or herbicide resistance can be.used as the basis for selection. For example,
dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides neomycin and G-
418; and als and pat
confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively. (See, e.g.,
Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA 77:3567-3570; Colbere-
Garapin, F. et al. ( 1981 )
J. Mol. Biol. 150:1-14.) Additional selectable genes have been described,
e.g., trpB and hisD, which
alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and
R.C. Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins
(GFP; Clontech), f3 glucuronidase and its substrate 13-glucuronide, or
luciferase and its substrate
luciferin may be used. These markers can be used not only to identify
transformants, but also to
quantify the amount of transient or stable protein expression attributable to
a specific vector system.
(See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of interest is
also present, the presence and expression of the gene may need to be
confirmed. For example, if the
sequence encoding MDDT is inserted within a marker gene sequence, transformed
cells containing
sequences encoding MDDT can be identified by the absence of marker gene
function. Alternatively,
a marker gene can be placed in tandem with a sequence encoding MDDT under the
control of a single
promoter. Expression of the marker gene in response to induction or selection
usually indicates
expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding MDDT
and that express
MDDT may be identified by a variety of procedures known to those of skill in
the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations,
PCR
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amplification, and protein bioassay or immunoassay techniques which include
membrane, solution, or
chip based technologies for the detection and/or quantification of nucleic
acid or protein sequences.
Immunological methods for detecting and measuring the expression of MDDT using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on MDDT is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St. Paul MN,
Sect. N; Coligan, J.E. et al. (1997) Current Protocols in Immunolo~y, Greene
Pub. Associates and
Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical
Protocols, Humana
Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art and
may be used in various nucleic acid and amino acid assays. Means for producing
labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding MDDT
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled nucleotide.
Alternatively, the sequences encoding MDDT, or any fragments thereof, may be
cloned into a vector
for the production of an mRNA probe. Such vectors are known in the art, are
commercially
available, and may be used to synthesize RNA probes in vitro by addition of an
appropriate RNA
polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures
may be conducted
using a variety of commercially available kits, such as those provided by
Amersham Pharmacia
Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules
or labels which
may be used for ease of detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or
chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic
particles, and the like.
Host cells transformed with nucleotide sequences encoding MDDT may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell may be secreted or retained intracellularly
depending on the sequence
and/or the vector used. As will be understood by those of skill in the art,
expression vectors
containing polynucleotides which encode MDDT may be designed to contain signal
sequences which
direct secretion of MDDT through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications of
the polypeptide include, but are not limited to, acetylation, carboxylation,
glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro" or
"pro" form of the protein may also be used to specify protein targeting,
folding, and/or activity.
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Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are
available from the
American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the correct
modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant
nucleic acid
sequences encoding MDDT may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric MDDT protein
containing a heterologous moiety that can be recognized by a commercially
available antibody may
facilitate the screening of peptide libraries for inhibitors of MDDT activity.
Heterologous protein
and peptide moieties may also facilitate purification of fusion proteins using
commercially available
affinity matrices. Such moieties include, but are not limited to, glutathione
S-transferase (GST),
maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide
(CBP), 6-His, FLAG,
c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification of their
cognate fusion proteins on immobilized glutathione, maltose, phenylarsine
oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable
immunoaffmity
purification of fusion proteins using commercially available monoclonal and
polyclonal antibodies
that specifically recognize these epitope tags. A fusion protein may also be
engineered to contain a
proteolytic cleavage site located between the MDDT encoding sequence and the
heterologous protein
sequence, so that MDDT may be cleaved away from the heterologous moiety
following purification.
Methods for fusion protein expression and purification are discussed in
Ausubel ( 1995, supra, ch.
10). A variety of commercially available kits may also be used to facilitate
expression and
purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled MDDT may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system
(Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with the
T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, for example, 35S-methionine.
MDDT of the present invention or fragments thereof may be used to screen for
compounds
that specifically bind to MDDT. At least one and up to a plurality of test
compounds may be
screened for specific binding to MDDT. Examples of test compounds include
antibodies,
oligonucleotides, proteins (e.g., receptors), or small molecules.
In one embodiment, the compound thus identified is closely related to the
natural ligand of
MDDT, e.g., a ligand or fragment thereof, a natural substrate, a structural or
functional mimetic, or a
natural binding partner. (See, e.g., Coligan, J.E. et al. (1991) Current
Protocols in Immunoloey 1(2):
Chapter 5.) Similarly, the compound can be closely related to the natural
receptor to which MDDT
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binds, or to at least a fragment of the receptor, e.g., the ligand binding
site. In either case, the
compound can be rationally designed using known techniques. In one embodiment,
screening for
these compounds involves producing appropriate cells which express MDDT,
either as a secreted
protein or on the cell membrane. Preferred cells include cells from mammals,
yeast, Drosophila, or
E. coli. Cells expressing MDDT or cell membrane fractions which contain MDDT
are then contacted
with a test compound and binding, stimulation, or inhibition of activity of
either MDDT or the
compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide,
wherein binding is
detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable
label. For example,
the assay may comprise the steps of combining at least one test compound with
MDDT, either in
solution or affixed to a solid support, and detecting the binding of MDDT to
the compound.
Alternatively, the assay may detect or measure binding of a test compound in
the presence of a
labeled competitor. Additionally, the assay may be carried out using cell-free
preparations, chemical
libraries, or natural product mixtures, and the test compounds) may be free in
solution or affixed to a
solid support.
MDDT of the present invention or fragments thereof may be used to screen for
compounds
that modulate the activity of MDDT. Such compounds may include agonists,
antagonists, or partial
or inverse agonists. In one embodiment, an assay is performed under conditions
permissive for
MDDT activity, wherein MDDT is combined with at least one test compound, and
the activity of
MDDT in the presence of a test compound is compared with the activity of MDDT
in the absence of
the test compound. A change in the activity of MDDT in the presence of the
test compound is
indicative of a compound that modulates the activity of MDDT. Alternatively, a
test compound is
combined with an in vitro or cell-free system comprising MDDT under conditions
suitable for
MDDT activity, and the assay is performed. In either of these assays, a test
compound which
modulates the activity of MDDT may do so indirectly and need not come in
direct contact with the
test compound. At least one and up to a plurality of test compounds may be
screened.
In another embodiment, polynucleotides encoding MDDT or their mammalian
homologs may
be "knocked out" in an animal model system using homologous recombination in
embryonic stem
(ES) cells. Such techniques are well known in the art and are useful for the
generation of animal
models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent
No. 5,767,337.) For
example, mouse ES cells, such as the mouse 129/SvJ cell line, are derived from
the early mouse
embryo and grown in culture. The ES cells are transformed with a vector
containing the gene of
interest disrupted by a marker gene, e.g., the neomycin phosphotransferase
gene (neo; Capecchi,
M.R. (1989) Science 244:1288-1292). The vector integrates into the
corresponding region of the host
genome by homologous recombination. Alternatively, homologous recombination
takes place using
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the Cre-loxP system to knockout a gene of interest in a tissue- or
developmental stage-specific
manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al.
(1997) Nucleic Acids
Res. 25:4323-4330). Transformed ES cells are identified and microinjected into
mouse cell
blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred
to pseudopregnant dams, and the resulting chimeric progeny are genotyped and
bred to produce
heterozygous or homozygous strains. Transgenic animals thus generated may be
tested with potential
therapeutic or toxic agents.
Polynucleotides encoding MDDT may also be manipulated in vitro in ES cells
derived from
human blastocysts. Human ES cells have the potential to differentiate into at
least eight separate cell
lineages including endoderm, mesoderm, and ectodermal cell types. These cell
lineages differentiate
into, for example, neural cells, hematopoietic lineages, and cardiomyocytes
(Thomson, J.A. et al.
(1998) Science 282:1145-1147).
Polynucleotides encoding MDDT can also be used to create "knockin" humanized
animals
(pigs) or transgenic animals (mice or rats) to model human disease. With
knockin technology, a
region of a polynucleotide encoding MDDT is injected into animal ES cells, and
the injected
sequence integrates into the animal cell genome. Transformed cells are
injected into blastulae, and
the blastulae are implanted as described above. Transgenic progeny or inbred
lines are studied and
treated with potential pharmaceutical agents to obtain information on
treatment of a human disease.
Alternatively, a mammal inbred to overexpress MDDT, e.g., by secreting MDDT in
its milk, may also
serve as a convenient source of that protein (Janne, J. et al. (1998)
Biotechnol. Annu. Rev. 4:55-74).
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of MDDT and molecules for disease detection and treatment. In
addition, examples
of tissues expressing MDDT can be found in Table 6 and can also be found in
Example XI.
Therefore, MDDT appears to play a role in cell proliferative,
autoimmune/inflammatory,
developmental, and neurological disorders. In the treatment of disorders
associated with increased
MDDT expression or activity, it is desirable to decrease the expression or
activity of MDDT. In the
treatment of disorders associated with decreased MDDT expression or activity,
it is desirable to
increase the expression or activity of MDDT.
Therefore, in one embodiment, MDDT or a fragment or derivative thereof may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of MDDT. Examples of such disorders include, but are not limited to,
a cell proliferative
disorder such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed
connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers including
adenocarcinoma,
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leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an
autoimmune/inflammatory
S disorder such as acquired immunodeficiency syndrome (AIDS), Addison's
disease, adult respiratory
distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-
candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact
dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia
with lymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic
gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis,
hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia
gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative
colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections, and
trauma; a developmental
disorder such as renal tubular acidosis, anemia, Cushing's syndrome,
achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis, WAGR
syndrome (Wilms'
tumor, aniridia, genitourinary abnormalities, and mental retardation), Smith-
Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia, hereditary
keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,
hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and cerebral
palsy, spina bifida,
anencephaly, craniorachischisis, congenital glaucoma, cataract, and
sensorineural hearing loss; and a
neurological disorder such as epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms,
Alzheimer's disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple
sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain abscess,
subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis,
viral central nervous
system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-
Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and
metabolic diseases of the
nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other developmental
disorders of the central
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nervous system including Down syndrome, cerebral palsy, neuroskeletal
disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord diseases,
muscular dystrophy and other
neuromuscular disorders, peripheral nervous system disorders, dermatomyositis
and polymyositis,
inherited, metabolic, endocrine, and toxic myopathies, myasthenia gravis,
periodic paralysis, mental
disorders including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,
dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive supranuclear palsy,
corticobasal
degeneration, and familial frontotemporal dementia.
In another embodiment, a vector capable of expressing MDDT or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
associated with decreased
expression or activity of MDDT including, but not limited to, those described
above.
In a further embodiment, a composition comprising a substantially purified
MDDT in
conjunction with a suitable pharmaceutical carrier may be administered to a
subject to treat or
prevent a disorder associated with decreased expression or activity of MDDT
including, but not
limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of MDDT
may be
administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of MDDT including, but not limited to, those listed above.
In a further embodiment, an antagonist of MDDT may be administered to a
subject to treat or
prevent a disorder associated with increased expression or activity of MDDT.
Examples of such
disorders include, but are not limited to, those cell proliferative,
autoimmune/inflammatory,
developmental, and neurological disorders described above. In one aspect, an
antibody which
specifically binds MDDT may be used directly as an antagonist or indirectly as
a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissues which
express MDDT.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding MDDT may be administered to a subject to treat or prevent a disorder
associated with
increased expression or activity of MDDT including, but not limited to, those
described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary
sequences, or vectors of the invention may be administered in combination with
other appropriate
therapeutic agents. Selection of the appropriate agents for use in combination
therapy may be made
by one of ordinary skill in the art, according to conventional pharmaceutical
principles. The
combination of therapeutic agents may act synergistically to effect the
treatment or prevention of the
various disorders described above. Using this approach, one may be able to
achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the potential for
adverse side effects.
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An antagonist of MDDT may be produced using methods which are generally known
in the
art. In particular, purified MDDT may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind MDDT.
Antibodies to MDDT may
also be generated using methods that are well known in the art. Such
antibodies may include, but are
not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies,
Fab fragments, and
fragments produced by a Fab expression library. Neutralizing antibodies (i.e.,
those which inhibit
dimer formation) are generally preferred for therapeutic use. Single chain
antibodies (e.g., from
camels or llamas) may be potent enzyme inhibitors and may have advantages in
the design of peptide
mimetics, and in the development of immuno-adsorbents and biosensors
(Muyldermans, S. (2001) J.
Biotechnol.74:277-302).
For the production of antibodies, various hosts including goats, rabbits,
rats, mice, camels,
dromedaries, llamas, humans, and others may be immunized by injection with
MDDT or with any
fragment or oligopeptide thereof which has immunogenic properties. Depending
on the host species,
various adjuvants may be used to increase immunological response. Such
adjuvants include, but are
not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are
especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce
antibodies to
MDDT have an amino acid sequence consisting of at least about 5 amino acids,
and generally will
consist of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides, or
fragments are identical to a portion of the amino acid sequence of the natural
protein. Short stretches
of MDDT amino acids may be fused with those of another protein, such as KLH,
and antibodies to
the chimeric molecule may be produced.
Monoclonal antibodies to MDDT may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma technique, and
the EBV-hybridoma
technique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D.
et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA
80:2026-2030; and
Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity, can be used. (See, e.g.,
Morrison, S.L. et al. ( 1984) Proc.
Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and Takeda,
S. et al. (1985) Nature 314:452-454.) Alternatively, techniques described for
the production of single
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chain antibodies may be adapted, using methods known in the art, to produce
MDDT-specific single
chain antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be
generated by chain shuffling from random combinatorial immunoglobulin
libraries. (See, e.g.,
Burton, D.R. (1991) Proc. Natl. Acad. Sci. USA 88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunoglobulin libraries or panels of highly
specific binding reagents as
disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl.
Acad. Sci. USA
86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for MDDT may also be
generated.
For example, such fragments include, but are not limited to, F(ab~2 fragments
produced by pepsin
digestion of the antibody molecule and Fab fragments generated by reducing the
disulfide bridges of
the F(ab~2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and
easy identification of monoclonal Fab fragments with the desired specificity.
(See, e.g., Huse, W.D.
et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the desired
specificity. Numerous protocols for competitive binding or immunoradiometric
assays using either
polyclonal or monoclonal antibodies with established specificities are well
known in the art. Such
immunoassays typically involve the measurement of complex formation between
MDDT and its
specific antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies
reactive to two non-interfering MDDT epitopes is generally used, but a
competitive binding assay
may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for MDDT. Affinity
is expressed as an
association constant, Ka, which is defined as the molar concentration of MDDT-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium conditions.
The Ka determined for a preparation of polyclonal antibodies, which are
heterogeneous in their
affinities for multiple MDDT epitopes, represents the average affinity, or
avidity, of the antibodies
for MDDT. The Ka determined for a preparation of monoclonal antibodies, which
are monospecific
for a particular MDDT epitope, represents a true measure of affinity. High-
affinity antibody
preparations with Ka ranging from about 109 to 10'2 L/mole are preferred for
use in immunoassays in
which the MDDT-antibody complex must withstand rigorous manipulations. Low-
affinity antibody
preparations with Ka ranging from about 106 to 10' L/mole are preferred for
use in
immunopurification and similar procedures which ultimately require
dissociation of MDDT,
preferably in active form, from the antibody (Catty, D. (1988) Antibodies,
Volume I: A Practical
Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A
Practical Guide to
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Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is generally employed in procedures
requiring precipitation
of MDDT-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity, and
guidelines for antibody quality and usage in various applications, are
generally available. (See, e.g.,
Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention, the polynucleotides encoding MDDT, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, modifications
of gene expression can be achieved by designing complementary sequences or
antisense molecules
(DNA, RNA, PNA, or modified oligonucleotides) to the coding or regulatory
regions of the gene
encoding MDDT. Such technology is well known in the art, and antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding MDDT. (See, e.g.,.Agrawal, S., ed. (1996) Antisense Therapeutics,
Humana Press Inc.,
Totawa NJ.)
In therapeutic use, any gene delivery system suitable for introduction of the
antisense
sequences into appropriate target cells can be used. Antisense sequences can
be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence
complementary to at least a portion of the cellular sequence encoding the
target protein. (See, e.g.,
Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and
Scanlon, K.J. et al. (1995)
9(13):1288-1296.) Antisense sequences can also be introduced intracellularly
through the use of
viral vectors, such as retrovirus and adeno-associated virus vectors. (See,
e.g., Miller, A.D. (1990)
Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol.
Ther. 63(3):323-347.)
Other gene delivery mechanisms include liposome-derived systems, artificial
viral envelopes, and
other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull.
51(1):217-225; Boado,
R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al.
(1997) Nucleic Acids Res.
25( 14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding MDDT may be
used for
somatic or germline gene therapy. Gene therapy may be performed to (i) correct
a genetic deficiency
(e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-
linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672),
severe combined
immunodeficiency syndrome associated with an inherited adenosine deaminase
(ADA) deficiency
(Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995)
Science 270:470=475),
cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et
al. (1995) Hum. Gene
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Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703),
thalassamias,
familial hypercholesterolemia, and hemophilia resulting from Factor VIII or
Factor IX deficiencies
(Crystal, R.G. (1995) Science 270:404-410; Verma, LM. and N. Somia (1997)
Nature 389:239-242)),
(ii) express a conditionally lethal gene product (e.g., in the case of cancers
which result from
unregulated cell proliferation), or (iii) express a protein which affords
protection against intracellular
parasites (e.g., against human retroviruses, such as human immunodeficiency
virus (HIV) (Baltimore,
D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad.
Sci. USA 93:11395-
11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida
albicans and
Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium
falciparum and
Trypanosoma cruzi). In the case where a genetic deficiency in MDDT expression
or regulation
causes disease, the expression of MDDT from an appropriate population of
transduced cells may
alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by
deficiencies in
MDDT are treated by constructing mammalian expression vectors encoding MDDT
and introducing
these vectors by mechanical means into MDDT-deficient cells. Mechanical
transfer technologies for
use with cells in vivo or ex vitro include (i) direct DNA microinjection into
individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated transfection, (iv)
receptor-mediated gene
transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (
1993) Annu. Rev.
Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay, J-L. and H.
Recipon (1998) Curr.
Opin. Biotechnol. 9:445-450).
Expression vectors that may be effective for the expression of MDDT include,
but are not
limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors
(Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La
Jolla CA),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
MDDT
may be expressed using (i) a constitutively active promoter, (e.g., from
cytomegalovirus (CMV),
Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ~3-actin
genes), (ii) an inducible
promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard
(1992) Proc. Natl.
Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769;
Rossi, F.M.V. and
H.M. Blau (1998) Curr. Opin. Biotechnol. 9:451-456), commercially available in
the T-REX plasmid
(Invitrogen)); the ecdysone-inducible promoter (available in the plasmids
PVGRXR and PIND;
Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone
inducible
promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific
promoter or the native
promoter of the endogenous gene encoding MDDT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID
TRANSFECTION KTT, available from Invitrogen) allow one with ordinary skill in
the art to deliver
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polynucleotides to target cells in culture and require minimal effort to
optimize experimental
parameters. In the alternative, transformation is performed using the calcium
phosphate method
(Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation
(Neumann, E. et al.
(1982) EMBO J. 1:841-845). The introduction of DNA to primary cells requires
modification of
these standardized mammalian transfection protocols.
In another embodiment of the invention, diseases or disorders caused by
genetic defects with
respect to MDDT expression are treated by constructing a retrovirus vector
consisting of (i) the
polynucleotide encoding MDDT under the control of an independent promoter or
the retrovirus long
terminal repeat (LTR) promoter, (ii) appropriate RNA packaging signals, and
(iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA sequences and
coding sequences
required for efficient vector propagation. Retrovirus vectors (e.g., PFB and
PFBNEO) are
commercially available (Stratagene) and are based on published data (Riviere,
I. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:6733-6737), incorporated by reference herein. The
vector is propagated in
an appropriate vector producing cell line (VPCL) that expresses an envelope
gene with a tropism for
receptors on the target cells or a promiscuous envelope protein such as VSVg
(Armentano, D. et al.
(1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-
1646; Adam, M.A. and
A.D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998) J. Virol.
72:8463-8471; Zufferey, R.
et al. (1998) J. Virol. 72:9873-9880). U.S. Patent No. 5,910,434 to Rigg
("Method for obtaining
retrovirus packaging cell lines producing high transducing efficiency
retroviral supernatant")
discloses a method for obtaining retrovirus packaging cell lines and is hereby
incorporated by
reference. Propagation of retrovirus vectors, transduction of a population of
cells (e.g., CD4+ T-
cells), and the return of transduced cells to a patient are procedures well
known to persons skilled in
the art of gene therapy and have been well documented (Ranga, U. et al. (1997)
J. Virol. 71:7020-
7029; Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M.L. (1997) J.
Virol. 71:4707-4716;
Ranga, U. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206; Su, L. (1997)
Blood 89:2283-
2290).
In the alternative, an adenovirus-based gene therapy delivery system is used
to deliver
polynucleotides encoding MDDT to cells which have one or more genetic
abnormalities with respect
to the expression of MDDT. The construction and packaging of adenovirus-based
vectors are well
known to those with ordinary skill in the art. Replication defective
adenovirus vectors have proven to
be versatile for importing genes encoding immunoregulatory proteins into
intact islets in the pancreas
(Csete, M.E. et al. (1995) Transplantation 27:263-268). Potentially useful
adenoviral vectors are
described in U.S. Patent No. 5,707,618 to Armentano ("Adenovirus vectors for
gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also Antinozzi,
P.A. et al. (1999)
Annu. Rev. Nutr. 19:511-544 and Verma, LM. and N. Somia (1997) Nature
18:389:239-242, both
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incorporated by reference herein.
In another alternative, a herpes-based, gene therapy delivery system is used
to deliver
polynucleotides encoding MDDT to target cells which have one or more genetic
abnormalities with
respect to the expression of MDDT. The use of herpes simplex virus (HSV)-based
vectors may be
especially valuable for introducing MDDT to cells of the central nervous
system, for which HSV has
a tropism. The construction and packaging of herpes-based vectors are well
known to those with
ordinary skill in the art. A replication-competent herpes simplex virus (HSV)
type 1-based vector has
been used to deliver a reporter gene to the eyes of primates (Liu, X. et al. (
1999) Exp. Eye Res.
169:385-395). The construction of a HSV-1 virus vector has also been disclosed
in detail in U.S.
Patent No. 5,804,413 to DeLuca ("Herpes simplex virus strains for gene
transfer"), which is hereby
incorporated by reference. U.S. Patent No. 5,804,413 teaches the use of
recombinant HSV d92 which
consists of a genome containing at least one exogenous gene to be transferred
to a cell under the
control of the appropriate promoter for purposes including human gene therapy.
Also taught by this
patent are the construction and use of recombinant HSV strains deleted for
ICP4, ICP27 and ICP22.
For HSV vectors, see also Goins, W.F. et al. (1999) J. Virol. 73:519-532 and
Xu, H. et al. (1994)
Dev. Biol. 163:152-161, hereby incorporated by reference. The manipulation of
cloned herpesvirus
sequences, the generation of recombinant virus following the transfection of
multiple plasmids
containing different segments of the large herpesvirus genomes, the growth and
propagation of
herpesvirus, and the infection of cells with herpesvirus are techniques well
known to those of
ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus)
vector is used to
deliver polynucleotides encoding MDDT to target cells. The biology of the
prototypic alphavirus,
Semliki Forest Virus (SFV), has been studied extensively and gene transfer
vectors have been based
on the SFV genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During
alphavirus RNA replication, a subgenomic RNA is generated that normally
encodes the viral capsid
proteins. This subgenomic RNA replicates to higher levels than the full length
genomic RNA,
resulting in the overproduction of capsid proteins relative to the viral
proteins with enzymatic activity
(e.g., protease and polymerase). Similarly, inserting the coding sequence for
MDDT into the
alphavirus genome in place of the capsid-coding region results in the
production of a large number of
MDDT-coding RNAs and the synthesis of high levels of MDDT in vector transduced
cells. While
alphavirus infection is typically associated with cell lysis within a few
days, the ability to establish a
persistent infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN)
indicates that the lytic replication of alphaviruses can be altered to suit
the needs of the gene therapy
application (Dryga, S.A. et al. (1997) Virology 228:74-83). The wide host
range of alphaviruses will
allow the introduction of MDDT into a variety of cell types. The specific
transduction of a subset of
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cells in a population may require the sorting of cells prior to transduction.
The methods of
manipulating infectious cDNA clones of alphaviruses, performing alphavirus
cDNA and RNA
transfections, and performing alphavirus infections, are well known to those
with ordinary skill in the
art.
Oligonucleotides derived from the transcription initiation site, e.g., between
about positions
-10 and +10 from the start site, may also be employed to inhibit gene
expression. Similarly,
inhibition can be achieved using triple helix base-pairing methodology. Triple
helix pairing is useful
because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using
triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et
al. (1994) in Huber, B.E.
and B.I. Can, Molecular and Immunolo i~c-Approaches, Futura Publishing, Mt.
Kisco NY, pp. 163-
177.) A complementary sequence or antisense molecule may also be designed to
block translation of
mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic cleavage.
For example,
engineered hammerhead motif ribozyme molecules may specifically and
efficiently catalyze
endonucleolytic cleavage of sequences encoding MDDT.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides,
corresponding to the region of the target gene containing the cleavage site,
may be evaluated for
secondary structural features which may render the oligonucleotide inoperable.
The suitability of
candidate targets may also be evaluated by testing accessibility to
hybridization with complementary
oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared
by any method known in the art for the synthesis of nucleic acid molecules.
These include
techniques for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite
chemical synthesis. Alternatively, RNA molecules may be generated by in vitro
and in vivo
transcription of DNA sequences encoding MDDT. Such DNA sequences may be
incorporated into a
wide variety of vectors with suitable RNA polymerase promoters such as T7 or
SP6. Alternatively,
these cDNA constructs that synthesize complementary RNA, constitutively or
inducibly, can be
introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half-
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
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ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine,
queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine,
cytidine, guanine, thymine, and uridine which are not as easily recognized by
endogenous
endonucleases.
An additional embodiment of the invention encompasses a method for screening
for a
compound which is effective in altering expression of a polynucleotide
encoding MDDT.
Compounds which may be effective in altering expression of a specific
polynucleotide may include,
but are not limited to, oligonucleotides, antisense oligonucleotides, triple
helix-forming
oligonucleotides, transcription factors and other polypeptide transcriptional
regulators, and non-
macromolecular chemical entities which are capable of interacting with
specific polynucleotide
sequences. Effective compounds may alter polynucleotide expression by acting
as either inhibitors
or promoters of polynucleotide expression. Thus, in the treatment of disorders
associated with
increased MDDT expression or activity, a compound which specifically inhibits
expression of the
polynucleotide encoding MDDT may be therapeutically useful, and in the
treatment of disorders
associated with decreased MDDT expression or activity, a compound which
specifically promotes
expression of the polynucleotide encoding MDDT may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for
effectiveness in
altering expression of a specific polynucleotide. A test compound may be
obtained by any method
commonly known in the art, including chemical modification of a compound known
to be effective
in altering polynucleotide expression; selection from an existing,
commercially-available or
proprietary library of naturally-occurring or non-natural chemical compounds;
rational design of a
compound based on chemical and/or structural properties of the target
polynucleotide; and selection
from a library of chemical compounds created combinatorially or randomly. A
sample comprising a
polynucleotide encoding MDDT is exposed to at least one test compound thus
obtained. The sample
may comprise, for example, an intact or permeabilized cell, or an in vitro
cell-free or reconstituted
biochemical system. Alterations in the expression of a polynucleotide encoding
MDDT are assayed
by any method commonly known in the art. Typically, the expression of a
specific nucleotide is
detected by hybridization with a probe having a nucleotide sequence
complementary to the sequence
of the polynucleotide encoding MDDT. The amount of hybridization may be
quantified, thus
forming the basis for a comparison of the expression of the polynucleotide
both with and without
exposure to one or more test compounds. Detection of a change in the
expression of a polynucleotide
exposed to a test compound indicates that the test compound is effective in
altering the expression of
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the polynucleotide. A screen for a compound effective in altering expression
of a specific
polynucleotide can be carried out, for example, using a Schizosaccharomyces
ombe gene expression
system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al.
(2000) Nucleic Acids
Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al.
(2000) Biochem. Biophys.
Res. Commun. 268:8-13). A particular embodiment of the present invention
involves screening a
combinatorial library of oligonucleotides (such as deoxyribonucleotides,
ribonucleotides, peptide
nucleic acids, and modified oligonucleotides) for antisense activity against a
specific polynucleotide
sequence (Bruice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W.
et al. (2000) U.S.
Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and
equally suitable
for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be
introduced into stem cells
taken from the patient and clonally propagated for autologous transplant back
into that same patient.
Delivery by transfection, by liposome injections, or by polycationic amino
polymers may be achieved
using methods which are well known in the art. (See, e.g., Goldman, C.K. et
al. (1997) Nat.
Biotechno1.15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as humans, dogs, cats,
cows, horses, rabbits, and
monkeys.
An additional embodiment of the invention relates to the administration of a
composition
which generally comprises an active ingredient formulated with a
pharmaceutically acceptable
excipient. Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins.
Various formulations are commonly known and are thoroughly discussed in the
latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing, Easton PA). Such
compositions may
consist of MDDT, antibodies to MDDT, and mimetics, agonists, antagonists, or
inhibitors of MDDT.
The compositions utilized in this invention may be administered by any number
of routes
including, but not limited to, oral, intravenous, intramuscular, intra-
arterial, intramedullary,
intrathecal, intraventricular, pulmonary, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
Compositions for pulmonary administration may be prepared in liquid or dry
powder form.
These compositions are generally aerosolized immediately prior to inhalation
by the patient. In the
case of small molecules (e.g. traditional low molecular weight organic drugs),
aerosol delivery of
fast-acting formulations is well-known in the art. In the case of
macromolecules (e.g. larger peptides
and proteins), recent developments in the field of pulmonary delivery via the
alveolar region of the
lung have enabled the practical delivery of drugs such as insulin to blood
circulation (see, e.g.,
Patton, J.S. et al., U.S. Patent No. 5,997,848). Pulmonary delivery has the
advantage of
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administration without needle injection, and obviates the need for potentially
toxic penetration
enhancers.
Compositions suitable for use in the invention include compositions wherein
the active
ingredients are contained in an effective amount to achieve the intended
purpose. The determination
of an effective dose is well within the capability of those skilled in the
art.
Specialized forms of compositions may be prepared for direct intracellular
delivery of
macromolecules comprising MDDT or fragments thereof. For example, liposome
preparations
containing a cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of
the macromolecule. Alternatively, MDDT or a fragment thereof may be joined to
a short cationic N-
terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to
transduce into the cells of all tissues, including the brain, in a mouse model
system (Schwarze, S.R. et
al. (1999) Science 285:1569-1572).
For any compound, the therapeutically effective dose can be estimated
initially either in cell
culture assays, e.g., of neoplastic cells, or in animal models such as mice,
rats, rabbits, dogs,
monkeys, or pigs. An animal model may also be used to determine the
appropriate concentration
range and route of administration. Such information can then be used to
determine useful doses and
routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
MDDT or fragments thereof, antibodies of MDDT, and agonists, antagonists or
inhibitors of MDDT,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be determined
by standard pharmaceutical procedures in cell cultures or with experimental
animals, such as by
calculating the EDso (the dose therapeutically effective in 50% of the
population) or LDso (the dose
lethal to 50% of the population) statistics. The dose ratio of toxic to
therapeutic effects is the
therapeutic index, which can be expressed as the LDSO/EDSO ratio. Compositions
which exhibit large
therapeutic indices are preferred. The data obtained from cell culture assays
and animal studies are
used to formulate a range of dosage for human use. The dosage contained in
such compositions is
preferably within a range of circulating concentrations that includes the EDSO
with little or no
toxicity. The dosage varies within this range depending upon the dosage form
employed, the
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account include
the severity of the disease state, the general health of the subject, the age,
weight, and gender of the
subject, time and frequency of administration, drug combination(s), reaction
sensitivities, and
response to therapy. Long-acting compositions may be administered every 3 to 4
days, every week,
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or biweekly depending on the half-life and clearance rate of the particular
formulation.
Normal dosage amounts may vary from about 0.1 ,ug to 100,000 fig, up to a
total dose of
about 1 gram, depending upon the route of administration. Guidance as to
particular dosages and
methods of delivery is provided in the literature and generally available to
practitioners in the art.
Those skilled in the art will employ different formulations for nucleotides
than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular cells,
conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind MDDT may be used for
the
diagnosis of disorders characterized by expression of MDDT, or in assays to
monitor patients being
treated with MDDT or agonists, antagonists, or inhibitors of MDDT. Antibodies
useful for
diagnostic purposes may be prepared in the same manner as described above for
therapeutics.
Diagnostic assays for MDDT include methods which utilize the antibody and a
label to detect MDDT
in human body fluids or in extracts of cells or tissues. The antibodies may be
used with or without
modification, and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A
wide variety of reporter molecules, several of which are described above, are
known in the art and
may be used.
A variety of protocols for measuring MDDT, including ELISAs, RIAs, and FACS,
are known
in the art and provide a basis for diagnosing altered or abnormal levels of
MDDT expression.
Normal or standard values for MDDT expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, for example, human subjects,
with antibodies to
MDDT under conditions suitable for complex formation. The amount of standard
complex formation
may be quantitated by various methods, such as photometric means. Quantities
of MDDT expressed
in subject, control, and disease samples from biopsied tissues are compared
with the standard values.
Deviation between standard and subject values establishes the parameters for
diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding MDDT may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide sequences,
complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used
to detect
and quantify gene expression in biopsied tissues in which expression of MDDT
may be correlated
with disease. The diagnostic assay may be used to determine absence, presence,
and excess
expression of MDDT, and to monitor regulation of MDDT levels during
therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide
sequences, including genomic sequences, encoding MDDT or closely related
molecules may be used
to identify nucleic acid sequences which encode MDDT. The specificity of the
probe, whether it is
made from a highly specific region, e.g., the 5'regulatory region, or from a
less specific region, e.g., a
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conserved motif, and the stringency of the hybridization or amplification will
determine whether the
probe identifies only naturally occurring sequences encoding MDDT, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and may have
at least 50%
sequence identity to any of the MDDT encoding sequences. The hybridization
probes of the subject
invention may be DNA or RNA and may be derived from the sequence of SEQ )D
N0:38-74 or from
genomic sequences including promoters, enhancers, and introns of the MDDT
gene.
Means for producing specific hybridization probes for DNAs encoding MDDT
include the
cloning of polynucleotide sequences encoding MDDT or MDDT derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art, are commercially
available, and may
be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as 32P or 35S,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems, and the like.
Polynucleotide sequences encoding MDDT may be used for the diagnosis of
disorders
associated with expression of MDDT. Examples of such disorders include, but
are not limited to, a
cell proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and
cancers including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus,
thyroid, and uterus; an
autoimmune/inflammatory disorder such as acquired immunodeficiency syndrome
(AIDS),
Addison's disease, adult respiratory distress syndrome, allergies, ankylosing
spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia,
autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy
(APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic
dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis,
erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's
syndrome, gout, Graves'
disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,
multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma,
Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of cancer,
hemodialysis, and
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extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal,
and helminthic infections, and
trauma; a developmental disorder such as renal tubular acidosis, anemia,
Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy, epilepsy,
gonadal dysgenesis,
WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental
retardation),
Smith-Magenis syndrome, myelodysplastic syndrome, hereditary mucoepithelial
dysplasia,
hereditary keratodermas, hereditary neuropathies such as Charcot-Marie-Tooth
disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders such as
Syndenharri s chorea
and cerebral palsy, spina bifida, anencephaly, craniorachischisis, congenital
glaucoma, cataract, and
sensorineural hearing loss; and a neurological disorder such as epilepsy,
ischemic cerebrovascular
disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease,
Huntington's disease,
dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic
lateral sclerosis and
other motor neuron disorders, progressive neural muscular atrophy, retinitis
pigmentosa, hereditary
ataxias, multiple sclerosis and other demyelinating diseases, bacterial and
viral meningitis, brain
abscess, subdural empyema, epidural abscess, suppurative intracranial
thrombophlebitis, myelitis and
radiculitis, viral central nervous system disease, prion diseases including
kuru, Creutzfeldt-Jakob
disease, and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
nutritional and
metabolic diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal
hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and
other developmental
disorders of the central nervous system including Down syndrome, cerebral
palsy, neuroskeletal
disorders, autonomic nervous system disorders, cranial nerve disorders, spinal
cord diseases,
muscular dystrophy and other neuromuscular disorders, peripheral nervous
system disorders,
dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic
myopathies,
myasthenia gravis, periodic paralysis, mental disorders including mood,
anxiety, and schizophrenic
disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia,
diabetic neuropathy,
tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia,
Tourette's disorder,
progressive supranuclear palsy, corticobasal degeneration, and familial
frontotemporal dementia .
The polynucleotide sequences encoding MDDT may be used in Southern or northern
analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in dipstick,
pin, and multiformat
ELISA-like assays; and in microarrays utilizing fluids or tissues from
patients to detect altered
MDDT expression. Such qualitative or quantitative methods are well known in
the art.
In a particular aspect, the nucleotide sequences encoding MDDT may be useful
in assays that
detect the presence of associated disorders, particularly those mentioned
above. The nucleotide
sequences encoding MDDT may be labeled by standard methods and added to a
fluid or tissue
sample from a patient under conditions suitable for the formation of
hybridization complexes. After
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a suitable incubation period, the sample is washed and the signal is
quantified and compared with a
standard value. If the amount of signal in the patient sample is significantly
altered in comparison to
a control sample then the presence of altered levels of nucleotide sequences
encoding MDDT in the
sample indicates the presence of the associated disorder. Such assays may also
be used to evaluate
the efficacy of a particular therapeutic treatment regimen in animal studies,
in clinical trials, or to
monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
MDDT, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding MDDT, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially purified
polynucleotide is used. Standard values obtained in this manner may be
compared with values
obtained from samples from patients who are symptomatic for a disorder.
Deviation from standard
values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in the
patient begins to approximate that which is observed in the normal subject.
The results obtained
from successive assays may be used to show the efficacy of treatment over a
period ranging from
several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the appearance
of actual clinical symptoms. A more definitive diagnosis of this type may
allow health professionals
to employ preventative measures or aggressive treatment earlier thereby
preventing the development
or further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences
encoding
MDDT may involve the use of PCR. These oligomers may be chemically
synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a
polynucleotide encoding MDDT, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding MDDT, and will be employed under optimized conditions
for identification
of a specific gene or condition. Oligomers may also be employed under less
stringent conditions for
detection or quantification of closely related DNA or RNA sequences.
In a particular aspect, oligonucleotide primers derived from the
polynucleotide sequences
encoding MDDT may be used to detect single nucleotide polymorphisms (SNPs).
SNPs are
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substitutions, insertions and deletions that are a frequent cause of inherited
or acquired genetic
disease in humans. Methods of SNP detection include, but are not limited to,
single-stranded
conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In
SSCP,
oligonucleotide primers derived from the polynucleotide sequences encoding
MDDT are used to
amplify DNA using the polymerase chain reaction (PCR). The DNA may be derived,
for example,
from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
SNPs in the DNA cause
differences in the secondary and tertiary structures of PCR products in single-
stranded form, and
these differences are detectable using gel electrophoresis in non-denaturing
gels. In fSCCP, the
oligonucleotide primers are fluorescently labeled, which allows detection of
the amplimers in high-
throughput equipment such as DNA sequencing machines. Additionally, sequence
database analysis
methods, termed in silico SNP (isSNP), are capable of identifying
polymorphisms by comparing the
sequence of individual overlapping DNA fragments which assemble into a common
consensus
sequence. These computer-based methods filter out sequence variations due to
laboratory
preparation of DNA and sequencing errors using statistical models and
automated analyses of DNA
sequence chromatograms. In the alternative, SNPs may be detected and
characterized by mass
spectrometry using, for example, the high throughput MASSARRAY system
(Sequenom, Inc., San
Diego CA).
SNPs may be used to study the genetic basis of human disease. For example, at
least 16
common SNPs have been associated with non-insulin-dependent diabetes mellitus.
SNPs are also
useful for examining differences in disease outcomes in monogenic disorders,
such as cystic fibrosis,
sickle cell anemia, or chronic granulomatous disease. For example, variants in
the mannose-binding
lectin, MBL2, have been shown to be correlated with deleterious pulmonary
outcomes in cystic
fibrosis. SNPs also have utility in pharmacogenomics, the identification of
genetic variants that
influence a patient's response to a drug, such as life-threatening toxicity.
For example, a variation in
N-acetyl transferase is associated with a high incidence of peripheral
neuropathy in response to the
anti-tuberculosis drug isoniazid, while a variation in the core promoter of
the ALOXS gene results in
diminished clinical response to treatment with an anti-asthma drug that
targets the 5-lipoxygenase
pathway. Analysis of the distribution of SNPs in different populations is
useful for investigating
genetic drift, mutation, recombination, and selection, as well as for tracing
the origins of populations
and their migrations. (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512;
Kwok, P.-Y. and Z. Gu
(1999) Mol. Med. Today 5:538-543; Nowotny, P. et al. (2001) Curr. Opin.
Neurobiol. 11:637-641.)
Methods which may also be used to quantify the expression of MDDT include
radiolabeling
or biotinylating nucleotides, coamplification of a control nucleic acid, and
interpolating results from
standard curves. (See, e.g., Melby, P.C. et al. (1993) J. Immunol. Methods
159:235-244; Duplaa, C.
et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of
multiple samples may be
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accelerated by running the assay in a high-throughput format where the
oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric or
colorimetric response gives
rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as elements on a
microarray. The microarray
can be used in transcript imaging techniques which monitor the relative
expression levels of large
numbers of genes simultaneously as described below. The microarray may also be
used to identify
genetic variants, mutations, and polymorphisms. This information may be used
to determine gene
function, to understand the genetic basis of a disorder, to diagnose a
disorder, to monitor
progression/regression of disease as a function of gene expression, and to
develop and monitor the
activities of therapeutic agents in the treatment of disease. In particular,
this information may be used
to develop a pharmacogenomic profile of a patient in order to select the most
appropriate and
effective treatment regimen for that patient. For example, therapeutic agents
which are highly
effective and display the fewest side effects may be selected for a patient
based on his/her
pharmacogenomic profile.
In another embodiment, MDDT, fragments of MDDT, or antibodies specific for
MDDT may
be used as elements on a microarray. The microarray may be used to monitor or
measure protein-
protein interactions, drug-target interactions, and gene expression profiles,
as described above.
A particular embodiment relates to the use of the polynucleotides of the
present invention to
generate a transcript image of a tissue or cell type. A transcript image
represents the global pattern of
gene expression by a particular tissue or cell type. Global gene expression
patterns are analyzed by
quantifying the number of expressed genes and their relative abundance under
given conditions and
at a given time. (See Seilhamer et al., "Comparative Gene Transcript
Analysis," U.S. Patent No.
5,840,484, expressly incorporated by reference herein.) Thus a transcript
image may be generated by
hybridizing the polynucleotides of the present invention or their complements
to the totality of
transcripts or reverse transcripts of a particular tissue or cell type. In one
embodiment, the
hybridization takes place in high-throughput format, wherein the
polynucleotides of the present
invention or their complements comprise a subset of a plurality of elements on
a microarray. The
resultant transcript image would provide a profile of gene activity.
Transcript images may be generated using transcripts isolated from tissues,
cell lines,
biopsies, or other biological samples. The transcript image may thus reflect
gene expression in vivo,
as in the case of a tissue or biopsy sample, or in vitro, as in the case of a
cell line.
Transcript images which profile the expression of the polynucleotides of the
present
invention may also be used in conjunction with in vitro model systems and
preclinical evaluation of
pharmaceuticals, as well as toxicological testing of industrial and naturally-
occurring environmental
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compounds. All compounds induce characteristic gene expression patterns,
frequently termed
molecular fingerprints or toxicant signatures, which are indicative of
mechanisms of action and
toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S.
and N.L. Anderson
(2000) Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test
compound has a signature similar to that of a compound with known toxicity, it
is likely to share
those toxic properties. These fingerprints or signatures are most useful and
refined when they
contain expression information from a large number of genes and gene families.
Ideally, a genome-
wide measurement of expression provides the highest quality signature. Even
genes whose
expression is not altered by any tested compounds are important as well, as
the levels of expression
of these genes are used to normalize the rest of the expression data. The
normalization procedure is
useful for comparison of expression data after treatment with different
compounds. While the
assignment of gene function to elements of a toxicant signature aids in
interpretation of toxicity
mechanisms, knowledge of gene function is not necessary for the statistical
matching of signatures
which leads to prediction of toxicity. (See, for example, Press Release 00-02
from the National
Institute of Environmental Health Sciences, released February 29, 2000,
available at
http://www.niehs.nih.gov/oc/news/toxchip.htm.) Therefore, it is important and
desirable in
toxicological screening using toxicant signatures to include all expressed
gene sequences.
In one embodiment, the toxicity of a test compound is assessed by treating a
biological
sample containing nucleic acids with the test compound. Nucleic acids that are
expressed in the
treated biological sample are hybridized with one or more probes specific to
the polynucleotides of
the present invention, so that transcript levels corresponding to the
polynucleotides of the present
invention may be quantified. The transcript levels in the treated biological
sample are compared with
levels in an untreated biological sample. Differences in the transcript levels
between the two samples
are indicative of a toxic response caused by the test compound in the treated
sample.
Another particular embodiment relates to the use of the polypeptide sequences
of the present
invention to analyze the proteome of a tissue or cell type. The term proteome
refers to the global
pattern of protein expression in a particular tissue or cell type. Each
protein component of a
proteome can be subjected individually to further analysis. Proteome
expression patterns, or profiles,
are analyzed by quantifying the number of expressed proteins and their
relative abundance under
given conditions and at a given time. A profile of a cell's proteome may thus
be generated by
separating and analyzing the polypeptides of a particular tissue or cell type.
In one embodiment, the
separation is achieved using two-dimensional gel electrophoresis, in which
proteins from a sample
are separated by isoelectric focusing in the first dimension, and then
according to molecular weight
by sodium dodecyl sulfate slab gel electrophoresis in the second dimension
(Steiner and Anderson,
su ra). The proteins are visualized in the gel as discrete and uniquely
positioned spots, typically by
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staining the gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical
density of each protein spot is generally proportional to the level of the
protein in the sample. The
optical densities of equivalently positioned protein spots from different
samples, for example, from
biological samples either treated or untreated with a test compound or
therapeutic agent, are
compared to identify any changes in protein spot density related to the
treatment. The proteins in the
spots are partially sequenced using, for example, standard methods employing
chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein in a spot
may be determined by
comparing its partial sequence, preferably of at least 5 contiguous amino acid
residues, to the
polypeptide sequences of the present invention. In some cases, further
sequence data may be
obtained for definitive protein identification.
A proteomic profile may also be generated using antibodies specific for MDDT
to quantify
the levels of MDDT expression. In one embodiment, the antibodies are used as
elements on a
microarray, and protein expression levels are quantified by exposing the
microarray to the sample and
detecting the levels of protein bound to each array element (Lueking, A. et
al. (1999) Anal. Biochem.
270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788). Detection
may be performed
by a variety of methods known in the art, for example, by reacting the
proteins in the sample with a
thiol- or amino-reactive fluorescent compound and detecting the amount of
fluorescence bound at
each array element.
Toxicant signatures at the proteome level are also useful for toxicological
screening, and
should be analyzed in parallel with toxicant signatures at the transcript
level. There is a poor
correlation between transcript and protein abundances for some proteins in
some tissues (Anderson,
N.L. and J. Seilhamer (1997) Electrophoresis 18:533-537), so proteome toxicant
signatures may be
useful in the analysis of compounds which do not significantly affect the
transcript image, but which
alter the proteomic profile. In addition, the analysis of transcripts in body
fluids is difficult, due to
rapid degradation of mRNA, so proteomic profiling may be more reliable and
informative in such
cases.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
sample containing proteins with the test compound. Proteins that are expressed
in the treated
biological sample are separated so that the amount of each protein can be
quantified. The amount of
each protein is compared to the amount of the corresponding protein in an
untreated biological
sample. A difference in the amount of protein between the two samples is
indicative of a toxic
response to the test compound in the treated sample. Individual proteins are
identified by sequencing
the amino acid residues of the individual proteins and comparing these partial
sequences to the
polypeptides of the present invention.
In another embodiment, the toxicity of a test compound is assessed by treating
a biological
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sample containing proteins with the test compound. Proteins from the
biological sample are
incubated with antibodies specific to the polypeptides of the present
invention. The amount of
protein recognized by the antibodies is quantified. The amount of protein in
the treated biological
sample is compared with the amount in an untreated biological sample. A
difference in the amount of
protein between the two samples is indicative of a toxic response to the test
compound in the treated
sample.
Microarrays may be prepared, used, and analyzed using methods known in the
art. (See, e.g.,
Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al.
(1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT application
W095/251116; Shalom D.
et al. (1995) PCT application W095/35505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci. USA
94:2150-2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605,662.)
Various types of
microarrays are well known and thoroughly described in DNA Microarrays: A
Practical Approach,
M. Schena, ed. (1999) Oxford University Press, London, hereby expressly
incorporated by reference.
In another embodiment of the invention, nucleic acid sequences encoding MDDT
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic sequence.
Either coding or noncoding sequences may be used, and in some instances,
noncoding sequences
may be preferable over coding sequences. For example, conservation of a coding
sequence among
members of a mufti-gene family may potentially cause undesired cross
hybridization during
chromosomal mapping. The sequences may be mapped to a particular chromosome,
to a specific
region of a chromosome, or to artificial chromosome constructions, e.g., human
artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial
chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA libraries. (See,
e.g., Harrington,
J.J. et al. (1997) Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-
134; and Trask, B.J.
(1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of
the invention may be
used to develop genetic linkage maps, for example, which correlate the
inheritance of a disease state
with the inheritance of a particular chromosome region or restriction fragment
length polymorphism
(RFLP). (See, for example, Lander, E.S. and D. Botstein (1986) Proc. Natl.
Acad. Sci. USA
83:7353-7357.)
Fluorescent in situ hybridization (FISH) may be correlated with other physical
and genetic
map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-
968.) Examples of genetic
map data can be found in various scientific journals or at the Online
Mendelian Inheritance in Man
(OMIM) World Wide Web site. Correlation between the location of the gene
encoding MDDT on a
physical map and a specific disorder, or a predisposition to a specific
disorder, may help define the
region of DNA associated with that disorder and thus may further positional
cloning efforts.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such as
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linkage analysis using established chromosomal markers, may be used for
extending genetic maps.
Often the placement of a gene on the chromosome of another mammalian species,
such as mouse,
may reveal associated markers even if the exact chromosomal locus is not
known. This information
is valuable to investigators searching for disease genes using positional
cloning or other gene
discovery techniques. Once the gene or genes responsible for a disease or
syndrome have been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
l 1q22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. (1988) Nature 336:577-
580.) The nucleotide
sequence of the instant invention may also be used to detect differences in
the chromosomal location
due to translocation, inversion, etc., among normal, carrier, or affected
individuals.
In another embodiment of the invention, MDDT, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed to a
solid support, borne on a cell surface, or located intracellularly. The
formation of binding complexes
between MDDT and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds
having suitable binding affinity to the protein of interest. (See, e.g.,
Geysen, et al. (1984) PCT
application W084/03564.) In this method, large numbers of different small test
compounds are
synthesized on a solid substrate. The test compounds are reacted with MDDT, or
fragments thereof,
and washed. Bound MDDT is then detected by methods well known in the art.
Purified MDDT can
also be coated directly onto plates for use in the aforementioned drug
screening techniques.
Alternatively, non-neutralizing antibodies can be used to capture the peptide
and immobilize it on a
solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing
antibodies capable of binding MDDT specifically compete with a test compound
for binding MDDT.
In this manner, antibodies can be used to detect the presence of any peptide
which shares one or more
antigenic determinants with MDDT.
In additional embodiments, the nucleotide sequences which encode MDDT may be
used in
any molecular biology techniques that have yet to be developed, provided the
new techniques rely on
properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the preceding
description, utilize the present invention to its fullest extent. The
following embodiments are,
therefore, to be construed as merely illustrative, and not limitative of the
remainder of the disclosure
in any way whatsoever.
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The disclosures of all patents, applications and publications, mentioned above
and below,
including U.S. Ser. No.60/287,152, U.S. Ser. No.60/288,599, U.S. Ser.
No.60/290,364, U.S. Ser.
No.60/293,837, U.S. Ser. No.60/353,284, and U.S. Ser. No.60/351,107 are
expressly incorporated by
reference herein.
EXAMPLES
I. Construction of cDNA Libraries
Incyte cDNAs were derived from cDNA libraries described in the LIFESEQ GOLD
database
(Incyte Genomics, Palo Alto CA). Some tissues were homogenized and lysed in
guanidinium
isothiocyanate, while others were homogenized and lysed in phenol or in a
suitable mixture of
denaturants, such as TR1ZOL (Life Technologies), a monophasic solution of
phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or
extracted with
chloroform. RNA was precipitated from the lysates with either isopropanol or
sodium acetate and
ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to
increase RNA
purity. In some cases, RNA was treated with DNase. For most libraries,
poly(A)+ RNA was isolated
using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN,
Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN). Alternatively,
RNA was
isolated directly from tissue lysates using other RNA isolation kits, e.g.,
the POLY(A)PURE mRNA
purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the
corresponding cDNA
libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed
with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life Technologies),
using the
recommended procedures or similar methods known in the art. (See, e.g.,
Ausubel, 1997, supra, units
5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic
oligonucleotide adapters were ligated to double stranded cDNA, and the cDNA
was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-
1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column
chromatography (Amersham Pharmacia Biotech) or preparative agarose gel
electrophoresis. cDNAs
were ligated into compatible restriction enzyme sites of the polylinker of a
suitable plasmid, e.g.,
PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Life Technologies),
PCDNA2.1 plasmid
(Invitrogen, Carlsbad CA), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid
(Invitrogen),
PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte Genomics, Palo Alto CA), pRARE
(Incyte
Genomics), or plNCY (Incyte Genomics), or derivatives thereof. Recombinant
plasmids were
transformed into competent E. coli cells including XL1-Blue, XL1-BIueMRF, or
SOLR from
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Stratagene or DHSa, DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by
in vivo
excision using the UNIZAP vector system (Stratagene) or by cell lysis.
Plasmids were purified using
at least one of the following: a Magic or WIZARD Minipreps DNA purification
system (Promega);
an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and
QIAWELL 8 Plasmid,
QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP
96 plasmid purification kit from QIAGEN. Following precipitation, plasmids
were resuspended in
0.1 ml of distilled water and stored, with or without lyophilization, at
4°C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct
link PCR in a
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell
lysis and thermal
cycling steps were carried out in a single reaction mixture. Samples were
processed and stored in
384-well plates, and the concentration of amplified plasmid DNA was quantified
fluorometrically
using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II
fluorescence
scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as described in Example II were sequenced as
follows.
Sequencing reactions were processed using standard methods or high-throughput
instrumentation
such as the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the PTC-
200 thermal
cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins
Scientific) or the
MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions
were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied in ABI
sequencing kits such as
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied
Biosystems).
Electrophoretic separation of cDNA sequencing reactions and detection of
labeled polynucleotides
were carried out using the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics); the
ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction
with standard ABI
protocols and base calling software; or other sequence analysis systems known
in the art. Reading
frames within the cDNA sequences were identified using standard methods
(reviewed in Ausubel,
1997, supra, unit 7.7). Some of the cDNA sequences were selected for extension
using the
techniques disclosed in Example VIII.
The polynucleotide sequences derived from Incyte cDNAs were validated by
removing
vector, linker, and poly(A) sequences and by masking ambiguous bases, using
algorithms and
programs based on BLAST, dynamic programming, and dinucleotide nearest
neighbor analysis. The
Incyte cDNA sequences or translations thereof were then queried against a
selection of public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases, and
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BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo
saniens, Rattus norvegicus, Mus musculus, Caenorhabditis elegans,
Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans (Incyte Genomics, Palo Alto
CA); hidden
Markov model (HMM)-based protein family databases such as PFAM, INCY, and
TIGRFAM (Haft,
D.H. et al. (2001) Nucleic Acids Res. 29:41-43); and HMM-based protein domain
databases such as
SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic,
I. et al. (2002)
Nucleic Acids Res. 30:242-244). (HMM is a probabilistic approach which
analyzes consensus
primary structures of gene families. See, for example, Eddy, S.R. (1996) Curr.
Opin. Struct. Biol.
6:361-365.) The queries were performed using programs based on BLAST, FASTA,
BLIMPS, and
HMMER. The Incyte cDNA sequences were assembled to produce full length
polynucleotide
sequences. Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched
sequences, or Genscan-predicted coding sequences (see Examples IV and V) were
used to extend
Incyte cDNA assemblages to full length. Assembly was performed using programs
based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open reading frames
using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences
were translated
to derive the corresponding full length polypeptide sequences. Alternatively,
a polypeptide of the
invention may begin at any of the methionine residues of the full length
translated polypeptide. Full
length polypeptide sequences were subsequently analyzed by querying against
databases such as the
GenBank protein databases (genpept), SwissProt, the PROTEOME databases,
BLOCKS, PRINTS,
DOMO, PRODOM, Prosite, hidden Markov model (HMM)-based protein family
databases such as
PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
Full
length polynucleotide sequences are also analyzed using MACDNASIS PRO software
(Hitachi
Software Engineering, South San Francisco CA) and LASERGENE software
(DNASTAR).
Polynucleotide and polypeptide sequence alignments are generated using default
parameters specified
by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence
alignment program
(DNASTAR), which also calculates the percent identity between aligned
sequences.
Table 7 summarizes the tools, programs, and algorithms used for the analysis
and assembly
of Incyte cDNA and full length sequences and provides applicable descriptions,
references, and
threshold parameters. The first column of Table 7 shows the tools, programs,
and algorithms used,
the second column provides brief descriptions thereof, the third column
presents appropriate
references, all of which are incorporated by reference herein in their
entirety, and the fourth column
presents, where applicable, the scores, probability values, and other
parameters used to evaluate the
strength of a match between two sequences (the higher the score or the lower
the probability value,
the greater the identity between two sequences).
The programs described above for the assembly and analysis of full length
polynucleotide
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and polypeptide sequences were also used to identify polynucleotide sequence
fragments from SEQ
ID N0:38-74. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization
and amplification technologies are described in Table 4, column 2.
IV. Identification and Editing of Coding Sequences from Genomic DNA
Putative molecules for disease detection and treatment were initially
identified by running the
Genscan gene identification program against public genomic sequence databases
(e.g., gbpri and
gbhtg). Genscan is a general-purpose gene identification program which
analyzes genomic DNA
sequences from a variety of organisms (See Burge, C. and S. Karlin (1997) J.
Mol. Biol. 268:78-94,
and Burge, C. and S. Karlin (1998) Curr. Opin. Struct. Biol. 8:346-354). The
program concatenates
predicted exons to form an assembled cDNA sequence extending from a methionine
to a stop codon.
The output of Genscan is a FASTA database of polynucleotide and polypeptide
sequences. The
maximum range of sequence for Genscan to analyze at once was set to 30 kb. To
determine which of
these Genscan predicted cDNA sequences encode molecules for disease detection
and treatment, the
encoded polypeptides were analyzed by querying against PFAM models for
molecules for disease
detection and treatment. Potential molecules for disease detection and
treatment were also identified
by homology to Incyte cDNA sequences that had been annotated as molecules for
disease detection
and treatment. These selected Genscan-predicted sequences were then compared
by BLAST analysis
to the genpept and gbpri public databases. Where necessary, the Genscan-
predicted sequences were
then edited by comparison to the top BLAST hit from genpept to correct errors
in the sequence
predicted by Genscan, such as extra or omitted exons. BLAST analysis was also
used to find any
Incyte cDNA or public cDNA coverage of the Genscan-predicted sequences, thus
providing evidence
for transcription. When Incyte cDNA coverage was available, this information
was used to correct or
confirm the Genscan predicted sequence. Full length polynucleotide sequences
were obtained by
assembling Genscan-predicted coding sequences with Incyte cDNA sequences
and/or public cDNA
sequences using the assembly process described in Example III. Alternatively,
full length
polynucleotide sequences were derived entirely from edited or unedited Genscan-
predicted coding
sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
Partial cDNA sequences were extended with exons predicted by the Genscan gene
identification program described in Example N. Partial cDNAs assembled as
described in Example
III were mapped to genomic DNA and parsed into clusters containing related
cDNAs and Genscan
exon predictions from one or more genomic sequences. Each cluster was analyzed
using an
algorithm based on graph theory and dynamic programming to integrate cDNA and
genomic
information, generating possible splice variants that were subsequently
confirmed, edited, or
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extended to create a full length sequence. Sequence intervals in which the
entire length of the
interval was present on more than one sequence in the cluster were identified,
and intervals thus
identified were considered to be equivalent by transitivity. For example, if
an interval was present on
a cDNA and two genomic sequences, then all three intervals were considered to
be equivalent. This
process allows unrelated but consecutive genomic sequences to be brought
together, bridged by
cDNA sequence. Intervals thus identified were then "stitched" together by the
stitching algorithm in
the order that they appear along their parent sequences to generate the
longest possible sequence, as
well as sequence variants. Linkages between intervals which proceed along one
type of parent
sequence (cDNA to cDNA or genomic sequence to genomic sequence) were given
preference over
linkages which change parent type (cDNA to genomic sequence). The resultant
stitched sequences
were translated and compared by BLAST analysis to the genpept and gbpri public
databases.
Incorrect exons predicted by Genscan were corrected by comparison to the top
BLAST hit from
genpept. Sequences were further extended with additional cDNA sequences, or by
inspection of
genomic DNA, when necessary.
"Stretched" Sequences
Partial DNA sequences were extended to full length with an algorithm based on
BLAST
analysis. First, partial cDNAs assembled as described in Example III were
queried against public
databases such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases
using the BLAST program. The nearest GenBank protein homolog was then compared
by BLAST
analysis to either Incyte cDNA sequences or GenScan exon predicted sequences
described in
Example IV. A chimeric protein was generated by using the resultant high-
scoring segment pairs
(HSPs) to map the translated sequences onto the GenBank protein homolog.
Insertions or deletions
may occur in the chimeric protein with respect to the original GenBank protein
homolog. The
GenBank protein homolog, the chimeric protein, or both were used as~probes to
search for
homologous genomic sequences from the public human genome databases. Partial
DNA sequences
were therefore "stretched" or extended by the addition of homologous genomic
sequences. The
resultant stretched sequences were examined to determine whether it contained
a complete gene.
VI. Chromosomal Mapping of MDDT Encoding Polynucleotides
The sequences which were used to assemble SEQ ID N0:38-74 were compared with
sequences from the Incyte LIFESEQ database and public domain databases using
BLAST and other
implementations of the Smith-Waterman algorithm. Sequences from these
databases that matched
SEQ )D N0:38-74 were assembled into clusters of contiguous and overlapping
sequences using
assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic
mapping data available
from public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for
Genome Research (WIGR), and Genethon were used to determine if any of the
clustered sequences
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had been previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment
of all sequences of that cluster, including its particular SEQ ID NO:, to that
map location.
Map locations are represented by ranges, or intervals, of human chromosomes.
The map
position of an interval, in centiMorgans, is measured relative to the terminus
of the chromosome's p-
arm. (The centiMorgan (cM) is a unit of measurement based on recombination
frequencies between
chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb)
of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM
distances are based on genetic markers mapped by Genethon which provide
boundaries for radiation
hybrid markers whose sequences were included in each of the clusters. Human
genome maps and
other resources available to the public, such as the NCBI "GeneMap'99" World
Wide Web site
(http://www.ncbi.nlm.nih.gov/genemap/), can be employed to determine if
previously identified
disease genes map within or in proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which RNAs
from a particular cell type or tissue have been bound. (See, e.g., Sambrook,
supra, ch. 7; Ausubel
(1995) su ra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or related
molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genomics). This
analysis is
much faster than multiple membrane-based hybridizations. In addition, the
sensitivity of the
computer search can be modified to determine whether any particular match is
categorized as exact or
similar. The basis of the search is the product score, which is defined as:
BLAST Score x Percent Identity
5 x minimum { length(Seq. 1), length(Seq. 2) }
The product score takes into account both the degree of similarity between two
sequences and the
length of the sequence match. The product score is a normalized value between
0 and 100, and is
calculated as follows: the BLAST score is multiplied by the percent nucleotide
identity and the
product is divided by (5 times the length of the shorter of the two
sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches in a high-
scoring segment pair
(HSP), and -4 for every mismatch. Two sequences may share more than one HSP
(separated by
gaps). If there is more than one HSP, then the pair with the highest BLAST
score is used to calculate
the product score. The product score represents a balance between fractional
overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced only for 100%
identity over the
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entire length of the shorter of the two sequences being compared. A product
score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88% identity and
100% overlap at the
other. A product score of 50 is produced either by 100% identity and 50%
overlap at one end, or
79% identity and 100% overlap.
Alternatively, polynucleotide sequences encoding MDDT are analyzed with
respect to the
tissue sources from which they were derived. For example, some full length
sequences are
assembled, at least in part, with overlapping Incyte cDNA sequences (see
Example III). Each cDNA
sequence is derived from a cDNA library constructed from a human tissue. Each
human tissue is
classified into one of the following organ/tissue categories: cardiovascular
system; connective tissue;
digestive system; embryonic structures; endocrine system; exocrine glands;
genitalia, female;
genitalia, male; germ cells; heroic and immune system; liver; musculoskeletal
system; nervous
system; pancreas; respiratory system; sense organs; skin; stomatognathic
system; unclassified/mixed;
or urinary tract. The number of libraries in each category is counted and
divided by the total number
of libraries across all categories. Similarly, each human tissue is classified
into one of the following
disease/condition categories: cancer, cell line, developmental, inflammation,
neurological, trauma,
cardiovascular, pooled, and other, and the number of libraries in each
category is counted and divided
by the total number of libraries across all categories. The resulting
percentages reflect the tissue- and
disease-specific expression of cDNA encoding MDDT. cDNA sequences and cDNA
library/tissue
information are found in the LIFESEQ GOLD database (Incyte Genomics, Palo Alto
CA).
VIII. Extension of MDDT Encoding Polynucleotides
Full length polynucleotide sequences were also produced by extension of an
appropriate
fragment of the full length molecule using oligonucleotide primers designed
from this fragment. One
primer was synthesized to initiate 5' extension of the known fragment, and the
other primer was
synthesized to initiate 3' extension of the known fragment. The initial
primers were designed using
OLIGO 4.06 software (National Biosciences), or another appropriate program, to
be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the target
sequence at temperatures of about 68°C to about 72°C. Any
stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired, additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art. PCR
was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research,
Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction buffer
containing Mgz+, (NH4)ZS04,
and 2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the following
parameters for primer
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pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C,
2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5
min; Step 7: storage at 4°C. In the
alternative, the parameters for primer pair T7 and SK+ were as follows: Step
l: 94°C, 3 min; Step 2:
94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68°C, S min; Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 ~,1
PICOGREEN
quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes, Eugene OR)
dissolved in 1X TE
and 0.5 ~,1 of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar,
Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a
Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the
concentration of DNA. A 5 ~cl to 10 ~1 aliquot of the reaction mixture was
analyzed by
electrophoresis on a 1 % agarose gel to determine which reactions were
successful in extending the
sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly MA) into pUC 18
vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to fill-in
restriction site
overhangs, and transfected into competent E. coli cells. Transformed cells
were selected on
antibiotic-containing media, and individual colonies were picked and cultured
overnight at 37°C in
384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low DNA
recoveries were reamplified using the same conditions as described above.
Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing
primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM
BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
In like manner, full length polynucleotide sequences are verified using the
above procedure
or are used to obtain 5' regulatory sequences using the above procedure along
with oligonucleotides
designed for such extension, and an appropriate genomic library.
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IX. Identification of Single Nucleotide Polymorphisms in MDDT Encoding
Polynucleotides
Common DNA sequence variants known as single nucleotide polymorphisms (SNPs)
were
identified in SEQ ID N0:38-74 using the LIFESEQ database (Incyte Genomics).
Sequences from
the same gene were clustered together and assembled as described in Example
III, allowing the
identification of all sequence variants in the gene. An algorithm consisting
of a series of filters was
used to distinguish SNPs from other sequence variants. Preliminary filters
removed the majority of
basecall errors by requiring a minimum Phred quality score of 15,,and removed
sequence alignment
errors and errors resulting from improper trimming of vector sequences,
chimeras, and splice
variants. An automated procedure of advanced chromosome analysis analysed the
original
chromatogram files in the vicinity of the putative SNP. Clone error filters
used statistically generated
algorithms to identify errors introduced during laboratory processing, such as
those caused by reverse
transcriptase, polymerise, or somatic mutation. Clustering error filters used
statistically generated
algorithms to identify errors resulting from clustering of close homologs or
pseudogenes, or due to
contamination by non-human sequences. A final set of filters removed
duplicates and SNPs found in
immunoglobulins or T-cell receptors.
Certain SNPs were selected for further characterization by mass spectrometry
using the high
throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at
the SNP sites in
four different human populations. The Caucasian population comprised 92
individuals (46 male, 46
female), including 83 from Utah, four French, three Venezualan, and two Amish
individuals. The
African population comprised 194 individuals (97 male, 97 female), all African
Americans. The
Hispanic population~comprised 324 individuals (162 male, 162 female), all
Mexican Hispanic. The
Asian population comprised 126 individuals (64 male, 62 female) with a
reported parental
breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8%
other Asian.
Allele frequencies were first analyzed in the Caucasian population; in some
cases those SNPs which
showed no allelic variance in this population were not further tested in the
other three populations.
X. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:38-74 are employed to screen
cDNAs,
genomic DNAs, or mIRNAs. Although the labeling of oligonucleotides, consisting
of about 20 base
pairs, is specifically described, essentially the same procedure is used with
larger nucleotide
fragments. Oligonucleotides are designed using state-of-the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
~y 32P~ adenosine triphosphate (Amersham Pharmacia Biotech), and T4
polynucleotide kinase
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextrin bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-based
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hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5%
sodium dodecyl sulfate.
Hybridization patterns are visualized using autoradiography or an alternative
imaging means and
compared.
XI. Microarrays
The linkage or synthesis of array elements upon a microarray can be achieved
utilizing
photolithography, piezoelectric printing (ink jet printing, See, e.g.,
Baldeschweiler, supra.),
mechanical microspotting technologies, and derivatives thereof. The substrate
in each of the
aforementioned technologies should be uniform and solid with a non-porous
surface (Schena (1999),
supra). Suggested substrates include silicon, silica, glass slides, glass
chips, and silicon wafers.
Alternatively, a procedure analogous to a dot or slot blot may also be used to
arrange and link
elements to the surface of a substrate using thermal, UV, chemical, or
mechanical bonding
procedures. A typical array may be produced using available methods and
machines well known to
those of ordinary skill in the art and may contain any appropriate number of
elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalom D. et al. (1996) Genome
Res. 6:639-645;
Marshall, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31.)
Full length cDNAs, Expressed Sequence Tags (ESTs), or fragments or oligomers
thereof may
comprise the elements of the microarray. Fragments or oligomers suitable for
hybridization can be
selected using software well known in the art such as LASERGENE software
(DNASTAR). The
array elements are hybridized with polynucleotides in a biological sample. The
polynucleotides in
the biological sample are conjugated to a fluorescent label or other molecular
tag for ease of
detection. After hybridization, nonhybridized nucleotides from the biological
sample are removed,
and a fluorescence scanner is used to detect hybridization at each array
element. Alternatively, laser
desorbtion and mass spectrometry may be used for detection of hybridization.
The degree of
complementarity and the relative abundance of each polynucleotide which
hybridizes to an element
on the microarray may be assessed. In one embodiment, microarray preparation
and usage is
described in detail below.
Tissue or Cell Sample Preparation
Total RNA is.isolated from tissue samples using the guanidinium thiocyanate
method and
poly(A)+ RNA is purified using the oligo-(dT) cellulose method. Each poly(A)+
RNA sample is
reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/~,1 oligo-(dT)
primer (2lmer), 1X
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first strand buffer, 0.03 units/~.l RNase inhibitor, 500 p,M dATP, 500 p.M
dGTP, 500 ~,M dTTP, 40
p.M dCTP, 40 p,M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The
reverse
transcription reaction is performed in a 25 ml volume containing 200 ng
poly(A)+ RNA with
GEMBRIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in
vitro
transcription from non-coding yeast genomic DNA. After incubation at 37
° C for 2 hr, each reaction
sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of
0.5M sodium
hydroxide and incubated for 20 minutes at 85°C to the stop the reaction
and degrade the RNA.
Samples are purified using two successive CHROMA SPIN 30 gel filtration spin
columns
(CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combining,
both reaction
samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml
sodium acetate, and 300 ml
of 100% ethanol. The sample is then dried to completion using a SpeedVAC
(Savant Instruments
Inc., Holbrook NY) and resuspended in 14 p,1 5X SSC/0.2% SDS.
Microarray Preparation
Sequences of the present invention are used to generate array elements. Each
array element
is amplified from bacterial cells containing vectors with cloned cDNA inserts.
PCR amplification
uses primers complementary to the vector sequences flanking the cDNA insert.
Array elements are
amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a
final quantity greater than 5
p.g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham
Pharmacia
Biotech).
Purified array elements are immobilized on polymer-coated glass slides. Glass
microscope
slides (Corning) are cleaned by ultrasound in 0.1% SDS and acetone, with
extensive distilled water
washes between and after treatments. Glass slides are etched in 4%
hydrofluoric acid (VWR
Scientific Products Corporation (VWR), West Chester PA), washed extensively in
distilled water,
and coated with 0.05% aminopropyl silane (Sigma) in 95% ethanol. Coated slides
are cured in a
110°C oven.
Array elements are applied to the coated glass substrate using a procedure
described in U.S.
Patent No. 5,807,522, incorporated herein by reference. 1 ~,1 of the array
element DNA, at an average
concentration of 100 ng/~,1, is loaded into the open capillary printing
element by a high-speed robotic
apparatus. The apparatus then deposits about 5 n1 of array element sample per
slide.
Microarrays are UV-crosslinked using a STRATALINKER UV-crosslinker
(Stratagene).
Microarrays are washed at room temperature once in 0.2% SDS and three times in
distilled water.
Non-specific binding sites are blocked by incubation of microarrays in 0.2%
casein in phosphate
buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60°C
followed by washes in
0.2% SDS and distilled water as before.
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Hybridization
Hybridization reactions contain 9 ~.I of sample mixture consisting of 0.2 ~g
each of Cy3 and
Cy5 labeled cDNA synthesis products in SX SSC, 0.2% SDS hybridization buffer.
The sample
mixture is heated to 65°C for 5 minutes and is aliquoted onto the
microarray surface and covered
with an 1.8 cmZ coverslip. The arrays are transferred to a waterproof chamber
having a cavity just
slightly larger than a microscope slide. The chamber is kept at 100% humidity
internally by the
addition of 140 ~,1 of SX SSC in a corner of the chamber. The chamber
containing the arrays is
incubated for about 6.5 hours at 60° C. The arrays are washed for 10
min at 45° C in a first wash
buffer (1X SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a
second wash buffer (0.1X
SSC), and dried.
Detection
Reporter-labeled hybridization complexes are detected with a microscope
equipped with an
Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of
generating spectral lines
at 488 nm for excitation of Cy3 and at 632 nm for excitation of CyS. The
excitation laser light is
focused on the array using a 20X microscope objective (Nikon, Inc., Melville
NY). The slide
containing the array is placed on a computer-controlled X-Y stage on the
microscope and raster-
scanned past the objective. The 1.8 cm x 1.8 cm array used in the present
example is scanned with a
resolution of 20 micrometers.
In two separate scans, a mixed gas multiline laser excites the two
fluorophores sequentially.
Emitted light is split, based on wavelength, into two photomultiplier tube
detectors (PMT 81477,
Hamamatsu Photonics Systems, Bridgewater NJ) corresponding to the two
fluorophores.
Appropriate filters positioned between the array and the photomultiplier tubes
are used to filter the
signals. The emission maxima of the fluorophores used are 565 nm for Cy3 and
650 nm for CyS.
Each array is typically scanned twice, one scan per fluorophore using the
appropriate filters at the
laser source, although the apparatus is capable of recording the spectra from
both fluorophores
simultaneously.
The sensitivity of the scans is typically calibrated using the signal
intensity generated by a
cDNA control species added to the sample mixture at a known concentration. A
specific location on
the array contains a complementary DNA sequence, allowing the intensity of the
signal at that
location to be correlated with a weight ratio of hybridizing species of
1:100,000. When two samples
from different sources (e.g., representing test and control cells), each
labeled with a different
fluorophore, are hybridized to a single array for the purpose of identifying
genes that are
differentially expressed, the calibration is done by labeling samples of the
calibrating cDNA with the
two fluorophores and adding identical amounts of each to the hybridization
mixture.
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The output of the photomultiplier tube is digitized using a 12-bit RTI-835H
analog-to-digital
(A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-
compatible PC
computer. The digitized data are displayed as an image where the signal
intensity is mapped using a
linear 20-color transformation to a pseudocolor scale ranging from blue (low
signal) to red (high
signal). The data is also analyzed quantitatively. Where two different
fluorophores are excited and
measured simultaneously, the data are first corrected for optical crosstalk
(due to overlapping
emission spectra) between the fluorophores using each fluorophore's emission
spectrum.
A grid is superimposed over the fluorescence signal image such that the signal
from each
spot is centered in each element of the grid. The fluorescence signal within
each element is then
integrated to obtain a numerical value corresponding to the average intensity
of the signal. The
software used for signal analysis is the GEMTOOLS gene expression analysis
program (Incyte).
Cell Growth and Treatments
The following cell lines were obtained from ATCC (Manassus, VA) and cultured
in media
according to the manufacturer's protocols: PrEC is a primary prostate
epithelial cell line isolated
from a normal donor; PC-3 is a prostate adenocarcinoma cell line isolated from
a 62 year-old male
with grade IV prostate adenocarcinoma metastasized to the bone; DU-145 is a
prostate carcinoma cell
line isolated from a 69 year-old man with widespread metastatic disease. DU-
145 was isolated from
a brain metastasis and has no detectable hormone sensitivity; LNCaP is a
prostate carcinoma cell line
isolated from a lymph node biopsy of a 50 year-old male with metastatic
prostate carcinoma. LNCaP
cells are responsive to 5-alpha-dihydrotestosterone and express androgen
receptors. All cell cultures
were incubated in low serum media 48 hours prior to treatment.
For SEQ ID N0:38, the expression of two components of this polynucleotide
sequence,
having Incyte clone )D 4765137 and Incyte clone ID 1975430, are downregulated
by at least two-
fold in LNCaP cells as compared with PrEC cells. Incyte clone m 4765137 spans
nucleotides 353
through 758 of Incyte polynucleotide 6024998CB 1 (SEQ >D N0:38). Incyte clone
)D 1975430 spans
nucleotides 5789 through 5918 of Incyte polynucleotide 6024998CB 1 (SEQ ID
N0:38).
In an alternative example, microarrays could be used to determine the levels
of expression of
SEQ ID N0:58 in breast, lung, and colon tumor tissues compared to the
respective normal tissues. In
this manner, microarray analysis demonstrated that SEQ )D N0:58 is at least
two-fold
underexpressed in breast cancer and lung tumor tissues (relative to normal
breast and lung tissues);
however, SEQ 1D N0:58 is not underexpressed in colon cancer tissues relative
to normal colon
tissues.
In addition, expression of SEQ 1D N0:58 appears to be downregulated in cells
treated with
steroid hormones, including but not limited to, budesonide, prednisone,
dexamethasone, and catechol,
suggesting that SEQ >D N0:58 expression is closely linked to liver toxicity.
These data suggest that
78
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WO 02/092759 PCT/US02/13392
SEQ ID N0:58 is useful as a genetic marker for the toxicological screening of
pharmaceutical
compositions and xenobiotic compounds that affect liver function.
In an alternative example, For example, SEQ ID N0:66 showed differential
expression in
inflammatory responses as determined by microarray analysis. The expression of
SEQ 1D N0:66
was increased by at least two fold before one hour (early genes) in eight
different cell types treated
with PMA (a broad activator of protein kinase C-dependent (PKC) pathways) and
with Inomycin (a
calcium ionophore that permits the entry of calcium in the cell). The cell
types tested included: K-
562, a myeloid precursor cell line; THP-1, a promonocyte cell line; RPMI 6666,
a B cell lymphoma
cell line; Jurkat, an acute T cell leukemia cell line; and ECV304, an
endothelial cell line. The
response of SEQ ID N0:66 was correlated with that of well studied stress
response genes (AP1 genes
and regulators, early growth response genes, MAP kinase pathway regulators,
and apoptosis
regulators), suggesting that SEQ ID N0:66 has an important role in PKC and
calcium signaling
pathways in many cell types. SEQ ID N0:66 is also useful in diagnostic assays
for inflammatory
responses.
In an alternative example, SEQ ID N0:71 showed differential expression in
specific
dissected brain regions from patients with severe Alzheimer's disease as
compared to normal brains.
The expression of SEQ 1D N0:71 was descreased by at least two fold in
striatum, hippocampus,
cerebellum, and posterior cingulate tissues from patients with severe
Alzheimer's disease as
compared to normal brain tissues. Therefore, SEQ ID N0:71 is useful in
diagnostic assays for
Alzheimer's disease.
In an alternative example, SEQ )17 N0:72 showed differential expression in
breast tumor cell
lines versus normal breast epithelial cells as determined by microarray
analysis. The expression of
SEQ ID N0:72 was decreased by at least two fold in four out of seven breast
tumor cell lines.
Therefore, SEQ 1D N0:72 is useful in diagnostic assays for breast cancer.
XII. Complementary Polynucleotides
Sequences complementary to the MDDT-encoding sequences, or any parts thereof,
are used
to detect, decrease, or inhibit expression of naturally occurring MDDT.
Although use of
oligonucleotides comprising from about 15 to 30 base pairs is described,
essentially the same
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of MDDT. To
inhibit transcription, a complementary oligonucleotide is designed from the
most unique 5' sequence
and used to prevent promoter binding to the coding sequence. To inhibit
translation, a
complementary oligonucleotide is designed to prevent ribosomal binding to the
MDDT-encoding
transcript.
XIII. Expression of MDDT
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Expression and purification of MDDT is achieved using bacterial or virus-based
expression
systems. For expression of MDDT in bacteria, cDNA is subcloned into an
appropriate vector
containing an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA
transcription. Examples of such promoters include, but are not limited to, the
trp-lac (tac) hybrid
S promoter and the TS or T7 bacteriophage promoter in conjunction with the lac
operator regulatory
element. Recombinant vectors are transformed into suitable bacterial hosts,
e.g., BL21(DE3).
Antibiotic resistant bacteria express MDDT upon induction with isopropyl beta-
D-
thiogalactopyranoside (IPTG). Expression of MDDT in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autographica californica
nuclear polyhedrosis virus
(AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of
baculovirus is
replaced with cDNA encoding MDDT by either homologous recombination or
bacterial-mediated
transposition involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription. Recombinant
baculovirus is used to
infect Snodo~tera fru~inerda (Sf9) insect cells in most cases, or human
hepatocytes, in some cases.
Infection of the latter requires additional genetic modifications to
baculovirus. (See Engelhard, E.K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al.
(1996) Hum. Gene Ther.
7:1937-1945.)
In most expression systems, MDDT is synthesized as a fusion protein with,
e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His,
permitting rapid, single-step,
affinity-based purification of recombinant fusion protein from crude cell
lysates. GST, a 26-
kilodalton enzyme from Schistosoma Lnonicum, enables the purification of
fusion proteins on
immobilized glutathione under conditions that maintain protein activity and
antigenicity (Amersham
Pharmacia Biotech). Following purification, the GST moiety can be
proteolytically cleaved from
MDDT at specifically engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffmity
purification using commercially available monoclonal and polyclonal anti-FLAG
antibodies
(Eastman Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on
metal-chelate resins (QIAGEN). Methods for protein expression and purification
are discussed in
Ausubel (1995, supra, ch. 10 and 16). Purified MDDT obtained by these methods
can be used
directly in the assays shown in Examples XVII and XVIII where applicable.
XIV. Functional Assays
MDDT function is assessed by expressing the sequences encoding MDDT at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice
include PCMV SPORT (Life Technologies) and PCR3.1 (Invitrogen, Carlsbad CA),
both of which
contain the cytomegalovirus promoter. 5-10 ~cg of recombinant vector are
transiently transfected into
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
a human cell line, for example, an endothelial or hematopoietic cell line,
using either liposome
formulations or electroporation. 1-2 ~g of an additional plasmid containing
sequences encoding a
marker protein are co-transfected. Expression of a marker protein provides a
means to distinguish
transfected cells from nontransfected cells and is a reliable predictor of
cDNA expression from the
recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP;
Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an
automated, laser optics-
based technique, is used to identify transfected cells expressing GFP or CD64-
GFP and to evaluate
the apoptotic state of the cells and other cellular properties. FCM detects
and quantifies the uptake of
fluorescent molecules that diagnose events preceding or coincident with cell
death. These events
include changes in nuclear DNA content as measured by staining of DNA with
propidium iodide;
changes in cell size and granularity as measured by forward light scatter and
90 degree side light
scatter; down-regulation of DNA synthesis as measured by decrease in
bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular proteins as
measured by reactivity with
specific antibodies; and alterations in plasma membrane composition as
measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. (1994) Flow Cvtometry, Oxford, New York NY.
The influence of MDDT on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding MDDT and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions
of human immunoglobulin G (IgG). Transfected cells are efficiently separated
from nontransfected
cells using magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake
Success NY). mRNA can be purified from the cells using methods well known by
those of skill in
the art. Expression of mRNA encoding MDDT and other genes of interest can be
analyzed by
northern analysis or microarray techniques.
XV. Production of MDDT Specific Antibodies
MDDT substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification
techniques, is used
to immunize animals (e.g., rabbits, mice, etc.) and to produce antibodies
using standard protocols.
Alternatively, the MDDT amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are well
described in the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
Typically, oligopeptides of about 15 residues in length are synthesized using
an ABI 431A
.peptide synthesizer (Applied Biosystems) using FMOC chemistry and coupled to
KLH (Sigma-
81
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, su ra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide and anti-MDDT activity by, for example, binding the peptide or
MDDT to a substrate,
blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat
anti-rabbit IgG.
XVI. Purification of Naturally Occurring MDDT Using Specific Antibodies
Naturally occurring or recombinant MDDT is substantially purified by
immunoaffinity
chromatography using antibodies specific for MDDT. An immunoaffinity column is
constructed by
covalently coupling anti-MDDT antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing MDDT are passed over the immunoaffmity column, and the column
is
washed under conditions that allow the preferential absorbance of MDDT (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/MDDT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope, such
as urea or thiocyanate ion), and MDDT is collected.
XVII. Identification of Molecules Which Interact with MDDT
MDDT, or biologically active fragments thereof, are labeled with'ZSI Bolton-
Hunter reagent.
(See, e.g., Bolton, A.E. and W.M. Hunter (1973) Biochem. J. 133:529-539.)
Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated with the
labeled MDDT, washed,
and any wells with labeled MDDT complex are assayed. Data obtained using
different
concentrations of MDDT are used to calculate values for the number, affinity,
and association of
MDDT with the candidate molecules.
Alternatively, molecules interacting with MDDT are analyzed using the yeast
two-hybrid
system as described in Fields, S. and O. Song (1989) Nature 340:245-246, or
using commercially
available kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
MDDT may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT)
which employs the yeast two-hybrid system in a high-throughput manner to
determine all interactions
between the proteins encoded by two large libraries of genes (Nandabalan, K.
et al. (2000) U.S.
Patent No. 6,057,101).
XVIII. Demonstration of MDDT Activity
5-Oxo-L-prolinase (EC 3.5.2) catalyzes the ATP-dependent cleavage of 5-
oxoproline to
L-glutamate. MDDT 5-oxo-L-prolinase activity is determined in reaction
mixtures containing (final
volume, 0.5 ml) 100 mM Na-Hepes buffer (pH 8.0), 2 mM 5-oxo-L-proline
(containing 100
82
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
cpm/nmol 5-oxo-L-[U-'°C]proline), 5 mM ATP, 8 mM MgCl2, 2 mM P-
enolpyruvate, 150 mM KCI,
2 mM dithiothreitol, pyruvate kinase (5 units), and MDDT. After incubation at
37 °C for 30 min, the
reaction mixtures are treated with 0.1 volume of 1 M HCl and placed at 0
°C for 5 min; an equal
volume of 1 M Tris is then added. Denatured proteins are removed by
centrifugation. Portions of the
neutralized reaction mixtures are then analyzed for ['4C]glutamate by liquid
scintillation counting
after removal of unreacted 5-oxoproline by chromatography on Dowex 50 (H+)
(Williamson, J. M.,
and Meister, A. (1982) J. Biol. Chem. 257:9161-9172). One unit of activity is
defined as the amount
of enzyme needed for the production of 1.0 pmol of glutamate/h under standard
assay conditions.
The amount of ['4C]glutamate produced is proportional to 5-Oxo-L-prolinase
activity of MDDT.
Various modifications and variations of the described methods and systems of
the invention
will be apparent to those skilled in the art without departing from the scope
and spirit of the
invention. Although the invention has been described in connection with
certain embodiments, it
should be understood that the invention as claimed should not be unduly
limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out the invention
which are obvious to those skilled in molecular biology or related fields are
intended to be within the
scope of the following claims.
83
CA 02445366 2003-10-23
WO 02/092759 PCT/US02/13392
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