U.S. patent application number 10/921613 was filed with the patent office on 2005-04-14 for isolated protein molecule, flh2882, a gpcr showing homology to the 5ht family of receptors.
Invention is credited to Glucksmann, Alexandra, Robison, Keith E..
Application Number | 20050079550 10/921613 |
Document ID | / |
Family ID | 21760935 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050079550 |
Kind Code |
A1 |
Glucksmann, Alexandra ; et
al. |
April 14, 2005 |
Isolated protein molecule, flh2882, a GPCR showing homology to the
5HT family of receptors
Abstract
The present invention provides a G-protein coupled receptor,
flh2882, that shows homology to the 5HT family of receptors. The
present invention further provides flh2882 fusion proteins.
Inventors: |
Glucksmann, Alexandra;
(Lexington, MA) ; Robison, Keith E.; (Wilmington,
MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD, LLP.
28 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
21760935 |
Appl. No.: |
10/921613 |
Filed: |
August 18, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10921613 |
Aug 18, 2004 |
|
|
|
09176075 |
Oct 20, 1998 |
|
|
|
09176075 |
Oct 20, 1998 |
|
|
|
09013634 |
Jan 26, 1998 |
|
|
|
5945307 |
|
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/320.1; 435/325; 435/69.1; 530/350; 530/388.22; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/70578 20130101 |
Class at
Publication: |
435/007.1 ;
435/069.1; 435/320.1; 435/325; 530/350; 530/388.22; 536/023.5 |
International
Class: |
G01N 033/53; C07H
021/04; C07K 014/705; C07K 016/28 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
(a) a polypeptide comprising the amino acid sequence of SEQ ID
NO:2, (b) a polypeptide consisting of the amino acid sequence of
SEQ ID NO:2, and (c) a polypeptide comprising an amino acid
sequence which is 95% identical to SEQ ID NO:2.
2. An isolated polypeptide which is encoded by a nucleic acid
molecule comprising a nucleotide sequence which is at least 95%
identical to the nucleotide sequence of SEQ ID NO:1 or 3.
3. The isolated polypeptide of claim 2 which is encoded by a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:1 or 3.
4. An isolated polypeptide of claim 2 which is encoded by a nucleic
acid molecule comprising the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number 98914.
5. An isolated polypeptide comprising at least 50 contiguous amino
acids of SEQ ID NO:2.
6. The isolated polypeptide of claim 5 comprising at least 100
contiguous amino acids of SEQ ID NO:2.
7. The polypeptide of any one of claims 1 or 2 operatively linked
to a heterologous amino acid sequence.
8. A method for identifying a compound which binds to a polypeptide
comprising the amino acid sequence of SEQ ID NO:2 or a polypeptide
fragment comprising at least 15 contiguous amino acids of SEQ ID
NO:2; the method comprising: i) contacting the polypeptide or the
polypeptide fragment, or a cell expressing the polypeptide or the
polypeptide fragment with a test compound; and ii) detecting
binding of the test compound to the polypeptide or the polypeptide
fragment.
9. The method of claim 8, wherein said fragment comprises at least
40 contiguous amino acids.
10. The method of claim 9, wherein said fragment comprises at least
100 contiguous amino acids.
11. The method of claim 8, wherein said detection is by direct
binding.
12. The method of claim 8, wherein said direct binding is
determined by an immunoprecipitation.
13. The method of claim 8, wherein said direct binding is
determined by a yeast two-hybrid assay.
14. The method of claim 8, wherein said detection is by the use of
a competition binding assay.
15. The method of claim 8, wherein said detection is by the use of
an assay for flh2882 activity.
16. The method of claim 15, wherein said assay for flh2882 activity
is based on phosphatidylinositol turnover.
17. The method of claim 15, wherein said assay for flh2882 activity
is selected from the group consisting of: a GTP binding assay, a G
protein binding assay, a plasma membrane polarization assay, and a
cell proliferation assay.
18. The method of claim 8, wherein said cell expressing said
polypeptide is a neural cell.
19. A kit comprising a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2; b) a polypeptide consisting of the amino acid
sequence of SEQ ID NO:2; and c) a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, wherein the
fragment comprises at least 15 contiguous amino acids of SEQ ID
NO:2; and instructions for use in identifying a compound which
binds to said polypeptide.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/176,075, filed Oct. 20, 1998, now abandoned, which is a
divisional of U.S. Ser. No. 09/013,634, filed Jan. 26, 1998,
granted, the contents of which are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] G-protein coupled receptors (GPCRs) are one of the major
class of proteins that are responsible for transducing a signal
within a cell. GPCRs are proteins that have seven transmembrane
domains. Upon binding of a ligand to the extracellular domain of a
GPCR, a signal is transduced within the cell which results in a
change in a biological or physiological property of the cell.
[0003] GPCRs, along with G-proteins and effectors (intracellular
enzymes and channels which are modulated by G-proteins), are the
components of a modular signaling system that connects the state of
intracellular second messengers to extracellular inputs. These
genes and gene-products are potential causative agents of disease
(Spiegel et al. (1993) J. Clin. Invest. 92:1119-1125; McKusick and
Amberger (1993) J. Med. Genet. 30:1-26). Specific defects in the
rhodopsin gene and the V2 vasopressin receptor gene have been shown
to cause various forms of autosomal dominant and autosomal
recessive retinitis pigmentosa (see Nathans et al. (1992) Annu.
Rev. Genet. 26:403-424), nephrogenic diabetes insipidus (Holtzman
et al. (1993) Hum. Mol. Genet. 2:1201-1204 and references therein).
These receptors are of critical importance to both the central
nervous system and peripheral physiological processes. Evolutionary
analyses suggest that the ancestor of these proteins originally
developed in concert with complex body plans and nervous
systems.
[0004] The GPCR protein superfamily now contains over 250 types of
paralogues, receptors that represent variants generated by gene
duplications (or other processes), as opposed to orthologues, the
same receptor from different species. The superfamily can be broken
down into five families: Family I, receptors typified by rhodopsin
and the beta2-adrenergic receptor and currently represented by over
.sup.20.sup.0 unique members (reviewed by Dohlman et al. (1991)
Annu. Rev. Biochem. 60:653-688 and references therein); Family II,
the recently characterized parathyroid hormone/calcitonin/secretin
receptor family (Juppner et al. (1991) Science 254:1024-1026; Lin
et al. (1991) Science 254:1022-1024); Family III, the metabotropic
glutamate receptor family in mammals (Nakanishi (1992) Science
258:597-603); Family IV, the cAMP receptor family, important in the
chemotaxis and development of D. discoideum (Klein et al. (1988)
Science 241:1467-1472); and Family V, the fungal mating pheromone
receptors such as STE2 (reviewed by Kurjan (1992) Annu. Rev.
Biochem. 61:1097-1129).
[0005] In addition to these groups of GPCRs, there are a small
number of other proteins which present seven putative hydrophobic
segments and appear to be unrelated to GPCRs; however, they have
not been shown to couple to G-proteins. Drosophila express a
photoreceptor-specific protein bride of sevenless (boss), a
seven-transmembrane-segment protein which has been extensively
studied and does not show evidence of being a GPCR (Hart et al.
(1993) Proc. Natl. Acad. Sci. USA 90:5047-5051 (1993)). The gene
frizzled (fz) in Drosophila is also thought to be a protein with
seven transmembrane segments. Like boss, fz has not been shown to
couple to G-proteins (Vinson et al. (1989) Nature 338:263-264).
[0006] G proteins represent a family of heterotrimeric proteins
composed of .alpha., .beta. and .gamma. subunits, which bind
guanine nucleotides. These proteins are usually linked to cell
surface receptors, e.g., receptors containing seven transmembrane
domains, such as the ligand receptors. Following ligand binding to
the receptor, a conformational change is transmitted to the G
protein, which causes the .alpha.-subunit to exchange a bound GDP
molecule for a GTP molecule and to dissociate from the
.beta..gamma.-subunits. The GTP-bound form of the .alpha.-subunit
typically functions as an effector-modulating moiety, leading to
the production of second messengers, such as cyclic AMP (e.g., by
activation of adenylate cyclase), diacylglycerol or inositol
phosphates. Greater than 20 different types of .alpha.-subunits are
known in man, which associate with a smaller pool of .beta. and
.gamma. subunits. Examples of mammalian G proteins include Gi, Go,
Gq, Gs and Gt. G proteins are described extensively in Lodish H. et
al. Molecular Cell Biology, (Scientific American Books Inc., New
York, N.Y., 1995), the contents of which are incorporated herein by
reference.
[0007] GPCRs are a major target for drug action and development.
Accordingly, it is valuable to the field of pharmaceutical
development to identify and characterize previously unknown GPCRs.
The present invention advances the state of the art by providing a
previously unidentified GPCR which is expressed predominantly in
the brain.
SUMMARY OF THE INVENTION
[0008] The present invention is based on the identification of a
novel G-protein coupled receptor (GPCR) that is expressed
predominantly in the brain and nucleic acid molecules that encoded
the GPCR, referred to herein as the flh2882 protein and flh2882
gene respectively. Based on this identification, the present
invention provides: 1) isolated flh2882 protein; 2) isolated
nucleic acid molecules that encode an flh2882 protein; 3)
antibodies that selectively bind to the flh2882 protein; 4) methods
of isolating allelic variants of the flh2882 protein and gene; 5)
methods of identifying cells and tissues that express the flh2882
protein/gene; 6) methods of identifying agents and cellular
compounds that bind to the flh2882 protein; 7) methods of
identifying agents that modulate the expression of the flh2882
gene; and 8) methods of modulating the activity of the flh2882
protein in a cell or organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 depicts the human flh2882 gene sequence (SEQ ID
NO:1).
[0010] FIG. 2 depicts the coding region of the human flh2882 gene
without the 5' and 3' untranslated regions (SEQ ID NO:3).
[0011] FIG. 3 depicts the amino acid sequence of the human flh2882
protein (SEQ ID NO:2).
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention is based on the discovery of a novel
G-protein coupled receptor (GPCR) molecule that is expressed
predominantly in the brain, the flh2882 protein, and nucleic acid
molecules that encode the flh2882 protein, the flh2882 gene or
flh2882 nucleic acid molecule. Specifically, an EST was first
identified in a public database that had low homology to G-protein
coupled receptors. PCR primers were then designed based on this
sequence and a cDNA was identified by screening a human fetal cDNA
library (See Example 1). Positive clones were sequenced and contigs
were assembled. Analysis of the assembled sequence revealed that
the cloned cDNA molecule encoded a GPCR, denoted herein as the
flh2882 protein. The flh2882 protein is a GPCR and plays a role in
and function in signaling pathways within cells that express the
flh2882 protein, particularly brain cells.
[0013] Various aspects of the invention are described in further
detail in the following subsections:
[0014] I. Isolated flh2882 Protein
[0015] The present invention provides isolated flh2882 protein as
well as peptide fragments of an flh2882 protein.
[0016] As used herein, a protein is said to be "isolated" or
"purified" when it is substantially free of cellular when it is
isolated from recombinant and non-recombinant cells, or free of
chemical precursors or other chemicals when it is chemically
synthesized. The language "substantially free of cellular material"
includes preparations of flh2882 protein in which the protein is
separated from cellular components of the cells in which it is
naturally or recombinantly produced. In one embodiment, the
language "substantially free of cellular material" includes
preparations of an flh2882 protein having less than about 30% (by
dry weight) of non-flh2882 protein (also referred to herein as a
"contaminating protein"), more preferably less than about 20% of
non-flh2882 protein, still more preferably less than about 10% of
non-flh2882 protein, and most preferably less than about 5%
non-flh2882 protein. When the flh2882 protein or biologically
active fragment thereof is recombinantly produced, it is also
preferably substantially free of culture medium, i.e., culture
medium represents less than about 20%, more preferably less than
about 10%, and most preferably less than about 5% of the volume of
the protein preparation. The language "substantially free of
chemical precursors or other chemicals" includes preparations of
flh2882 protein in which the protein is separated from chemical
precursors or other chemicals that are involved in the synthesis of
the protein. In one embodiment, the language "substantially free of
chemical precursors or other chemicals" includes preparations of
flh2882 protein having less than about 30% (by dry weight) of
chemical precursors or non-flh2882 chemicals, more preferably less
than about 20% chemical precursors or non-flh2882 chemicals, still
more preferably less than about 10% chemical precursors or
non-flh2882 chemicals, and most preferably less than about 5%
chemical precursors or non-flh2882 chemicals. In preferred
embodiments, isolated proteins or biologically active fragments
thereof lack contaminating proteins from the same animal from which
the flh2882 protein is derived. Typically, such proteins are
produced by recombinant expression of, for example, a human flh2882
protein in a non-human cell.
[0017] As used herein, an flh2882 protein is defined as a protein
that comprises: 1) the amino acid sequence shown in SEQ ID NO:2 or
an amino acid sequence which is encoded by the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC (American Type
Culture Collection, 10801 University Boulevard, Manassas, Va.
20110-2209); as Accession Number 98914, hereinafter human flh2882
protein; 2) functional and non-functional naturally occurring
allelic variants of human flh2882 protein; 3) recombinantly
produced variants of human flh2882 protein; and 4) flh2882 proteins
isolated from organisms other than humans (orthologues of human
flh2882 protein.)
[0018] As used herein, an allelic variant of human flh2882 protein
is defined as: 1) a protein isolated from human cells or tissues;
2) a protein encoded by the same genetic locus as that encoding the
human flh2882 protein; and 3) a protein that contains substantially
homology to human flh2882.
[0019] As used herein, two proteins are substantially homologous
when the amino acid sequence of the two protein (or a region of the
proteins) are at least about 60-65%, typically at least about
70-75%, more typically at least about 80-85%, and most typically at
least about 90-95% or more homologous to each other. To determine
the percent homology of two amino acid sequences (e.g., SEQ ID NO:2
and an allelic variant thereof) or of two nucleic acids, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in the sequence of one protein or nucleic acid
for optimal alignment with the other protein or nucleic acid). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions are then compared. When a
position in one sequence (e.g., SEQ ID NO:2) is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the other sequence (e.g., an allelic variant of the human
flh2882 protein), then the molecules are homologous at that
position (i.e., as used herein amino acid or nucleic acid
"homology" is equivalent to amino acid or nucleic acid "identity").
The percent homology between the two sequences is a function of the
number of identical positions shared by the sequences (i.e., %
homology=# of identical positions/total # of
positions.times.100).
[0020] Allelic variants of human flh2882 include both functional
and non-functional flh2882 proteins. Functional allelic variants
are naturally occurring amino acid sequence variants of the human
flh2882 protein that maintain the ability to bind an flh2882 ligand
and transduce a signal within a cell. Functional allelic variants
will typically contain only conservative substitution of one or
more amino acids of SEQ ID NO:2 or substitution, deletion or
insertion of non-critical residues in non-critical regions of the
protein.
[0021] Non-functional allelic variants are naturally occurring
amino acid sequence variants of human flh2882 protein that do not
have the ability to either bind ligand and/or transduce a signal
within a cell. Non-functional allelic variants will typically
contain a non-conservative substitution, a deletion, or insertion
or premature truncation of the amino acid sequence of SEQ. ID. NO:2
or a substitution, insertion or deletion in critical residues or
critical regions.
[0022] The present invention further provides non-human orthologues
of human flh2882 protein. Orthologues of human flh2882 protein are
proteins that are isolated from non-human organisms and possess the
same ligand binding and signaling capabilities of the human flh2882
protein. Orthologues of the human flh2882 protein can readily be
identified as comprising an amino acid sequence that is
substantially homologous to SEQ ID NO:2.
[0023] The flh2882 protein is a GPCR that participates in signaling
pathways within cells. As used herein, a signaling pathway refers
to the modulation (e.g., stimulated or inhibited) of a cellular
function/activity upon the binding of a ligand to the GPCR (flh2882
protein). Examples of such functions include mobilization of
intracellular molecules that participate in a signal transduction
pathway, e.g., phosphatidylinositol 4,5-bisphosphate (PIP.sub.2),
inositol 1,4,5-triphosphate (IP.sub.3) or adenylate cyclase;
polarization of the plasma membrane; production or secretion of
molecules; alteration in the structure of a cellular component;
cell proliferation, e.g., synthesis of DNA; cell migration; cell
differentiation; and cell survival. Since the flh2882 protein is
expressed substantially in the brain, examples of cells
participating in an flh2882 signaling pathway include neural cells,
e.g., peripheral nervous system and central nervous system cells
such as brain cells, e.g., limbic system cells, hypothalamus cells,
hippocampus cells, substantia nigra cells, cortex cells, brain stem
cells, neocortex cells, basal ganglion cells, caudate putamen
cells, olfactory tubercle cells, and superior colliculi cells.
[0024] Depending on the type of cell, the response mediated by the
flh2882 protein/ligand binding may be different. For example, in
some cells, binding of a ligand to an flh2882 protein may stimulate
an activity such as adhesion, migration, differentiation, etc.
through phosphatidylinositol or cyclic AMP metabolism and turnover
while in other cells, the binding of the ligand to the flh2882
protein will produce a different result. Regardless of the cellular
activity modulated by flh2882, it is universal that the flh2882
protein is a GPCR and interacts with a "G protein" to produce one
or more secondary signals in a variety of intracellular signal
transduction pathways, e.g., through phosphatidylinositol or cyclic
AMP metabolism and turnover, in a cell. G proteins represent a
family of heterotrimeric proteins composed of .alpha., .beta. and
.gamma. subunits, which bind guanine nucleotides. These proteins
are usually linked to cell surface receptors, e.g., receptors
containing seven transmembrane domains, such as the ligand
receptors. Following ligand binding to the receptor, a
conformational change is transmitted to the G protein, which causes
the .alpha.-subunit to exchange a bound GDP molecule for a GTP
molecule and to dissociate from the .beta..gamma.-subunits. The
GTP-bound form of the .alpha.-subunit typically functions as an
effector-modulating moiety, leading to the production of second
messengers, such as cyclic AMP (e.g., by activation of adenylate
cyclase), diacylglycerol or inositol phosphates. Greater than 20
different types of .alpha.-subunits are known in man, which
associate with a smaller pool of .beta. and .gamma. subunits.
Examples of mammalian G proteins include Gi, Go, Gq, Gs and Gt. G
proteins are described extensively in Lodish H. et al. Molecular
Cell Biology, (Scientific American Books Inc., New York, N.Y.,
1995), the contents of which are incorporated herein by
reference.
[0025] As used herein, "phosphatidylinositol turnover and
metabolism" refers to the molecules involved in the turnover and
metabolism of phosphatidylinositol 4,5-bisphosphate (PIP.sub.2) as
well as to the activities of these molecules. PIP.sub.2 is a
phospholipid found in the cytosolic leaflet of the plasma membrane.
Binding of a ligand to the flh2882 activates, in some cells, the
plasma-membrane enzyme phospholipase C that in turn can hydrolyze
PIP.sub.2 to produce 1,2-diacylglycerol (DAG) and inositol
1,4,5-triphosphate (IP.sub.3). Once formed IP.sub.3 can diffuse to
the endoplasmic reticulum surface where it can bind an IP.sub.3
receptor, e.g., a calcium channel protein containing an IP.sub.3
binding site. IP.sub.3 binding can induce opening of the channel,
allowing calcium ions to be released into the cytoplasm. IP.sub.3
can also be phosphorylated by a specific kinase to form inositol
1,3,4,5-tetraphosphate (IP.sub.4), a molecule which can cause
calcium entry into the cytoplasm from the extracellular medium.
IP.sub.3 and IP.sub.4 can subsequently be hydrolyzed very rapidly
to the inactive products inositol 1,4-biphosphate (IP.sub.2) and
inositol 1,3,4-triphosphate, respectively. These inactive products
can be recycled by the cell to synthesize PIP.sub.2. The other
second messenger produced by the hydrolysis of PIP.sub.2, namely
1,2-diacylglycerol (DAG), remains in the cell membrane where it can
serve to activate the enzyme protein kinase C. Protein kinase C is
usually found soluble in the cytoplasm of the cell, but upon an
increase in the intracellular calcium concentration, this enzyme
can move to the plasma membrane where it can be activated by DAG.
The activation of protein kinase C in different cells results in
various cellular responses such as the phosphorylation of glycogen
synthase, or the phosphorylation of various transcription factors,
e.g., NF-kB. The language "phosphatidylinositol activity", as used
herein, refers to an activity of PIP.sub.2 or one of its
metabolites.
[0026] Another signaling pathway in which the flh2882 protein may
participate is the cAMP turnover pathway. As used herein, "cyclic
AMP turnover and metabolism" refers to the molecules involved in
the turnover and metabolism of cyclic AMP (cAMP) as well as to the
activities of these molecules. Cyclic AMP is a second messenger
produced in response to ligand induced stimulation of certain G
protein coupled receptors. In the ligand signaling pathway, binding
of ligand to a ligand receptor can lead to the activation of the
enzyme adenylate cyclase, which catalyzes the synthesis of cAMP.
The newly synthesized cAMP can in turn activate a cAMP-dependent
protein kinase. This activated kinase can phosphorylate a
voltage-gated potassium channel protein, or an associated protein,
and lead to the inability of the potassium channel to open during
an action potential. The inability of the potassium channel to open
results in a decrease in the outward flow of potassium, which
normally repolarizes the membrane of a neuron, leading to prolonged
membrane depolarization.
[0027] The present invention further provides fragments of flh2882
proteins. As used herein, a fragment comprises at least 8
contiguous amino acids from an flh2882 protein. Preferred fragments
are fragments that possess one or more of the biological activities
of the flh2882 protein, for example the ability to bind to a
G-protein, as well as fragments that can be used as an immunogen to
generate anti-flh2882 antibodies.
[0028] Biologically active fragments of the flh2882 protein include
peptides comprising amino acid sequences derived from the amino
acid sequence of an flh2882 protein, e.g., the amino acid sequence
shown in SEQ ID NO:2 or the amino acid sequence of a protein
homologous to the flh2882 protein, which include less amino acids
than the full length flh2882 protein or the full length protein
which is homologous to the flh2882 protein, and exhibit at least
one activity of the flh2882 protein. Typically, biologically active
fragments (peptides, e.g., peptides which are, for example, 5, 10,
15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more amino acids in
length) comprise a domain or motif, e.g., a transmembrane domain or
G-protein binding domain.
[0029] The isolated flh2882 proteins can be purified from cells
that naturally express the protein, purified from cells that have
been altered to express the flh2882 protein, or synthesized using
known protein synthesis methods. Preferably, as described below,
the isolated flh2882 protein is produced by recombinant DNA
techniques. For example, a nucleic acid molecule encoding the
protein is cloned into an expression vector (as described above),
the expression vector is introduced into a host cell (as described
above) and the flh2882 protein is expressed in the host cell. The
flh2882 protein can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Alternative to recombinant expression, the flh2882
protein or fragment can be synthesized chemically using standard
peptide synthesis techniques. Lastly, native flh2882 protein can be
isolated from cells that naturally express the flh2882 protein
(e.g., hippocampal cells, or substantia nigra cells).
[0030] The present invention further provides flh2882 chimeric or
fusion proteins. As used herein, an flh2882 "chimeric protein" or
"fusion protein" comprises an flh2882 protein operatively linked to
a non-flh2882 protein. An "flh2882 protein" refers to a protein
having an amino acid sequence corresponding to an flh2882 protein,
whereas a "non-flh2882 protein" refers to a heterologous protein
having an amino acid sequence corresponding to a protein which is
not substantially homologous to the flh2882 protein, e.g., a
protein which is different from the flh2882 protein. Within the
context of fusion proteins, the term "operatively linked" is
intended to indicate that the flh2882 protein and the non-flh2882
protein are fused in-frame to each other. The non-flh2882 protein
can be fused to the N-terminus or C-terminus of the flh2882
protein. For example, in one embodiment the fusion protein is a
GST-flh2882 fusion protein in which the flh2882 sequences are fused
to the C-terminus of the GST sequences. Other types of fusion
proteins include, but are not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL fusions, poly-His fusions and Ig fusions. Such fusion proteins,
particularly poly-His fusions, can facilitate the purification of
recombinant flh2882 protein. In another embodiment, the fusion
protein is an flh2882 protein containing a heterologous signal
sequence at its N-terminus. In certain host cells (e.g., mammalian
host cells), expression and/or secretion of an flh2882 protein can
be increased by using a heterologous signal sequence.
[0031] Preferably, an flh2882 chimeric or fusion protein is
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different protein sequences are ligated
together in-frame in accordance with conventional techniques, for
example by employing blunt-ended or stagger-ended termini for
ligation, restriction enzyme digestion to provide for appropriate
termini, filling-in of cohesive ends as appropriate, alkaline
phosphatase treatment to avoid undesirable joining, and enzymatic
ligation. In another embodiment, the fusion gene can be synthesized
by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be
annealed and re-amplified to generate a chimeric gene sequence
(see, for example, Current Protocols in Molecular Biology, eds.
Ausubel et al. John Wiley & Sons: 1992). Moreover, many
expression vectors are commercially available that already encode a
fusion moiety (e.g., a GST protein). An flh2882-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the flh2882 protein.
[0032] The present invention also provides altered forms of flh2882
proteins that have been generated using recombinant DNA or
mutagenic methods/agents. Altered forms of an flh2882 protein can
be generated by mutagenesis, e.g., discrete point mutation or
truncation of the flh2882 protein and recombinant DNA method that
are well known in the art.
[0033] II. Antibodies that Bind to an flh2882 Protein
[0034] The present invention further provides antibodies that
selectively bind to an flh2882 protein. As used herein, an antibody
is said to selectively bind to an flh2882 protein when the antibody
binds to flh2882 proteins and does not substantially bind to
unrelated proteins. A skilled artisan will readily recognize that
an antibody may be considered to substantially bind an flh2882
protein even if it binds to proteins that share homology with a
fragment or domain of the flh2882 protein.
[0035] The term "antibody" as used herein refers to immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
which specifically binds (immunoreacts with) an antigen, such as
flh2882. Examples of immunologically active fragments of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention provides polyclonal and monoclonal
antibodies that bind flh2882. The term "monoclonal antibody" or
"monoclonal antibody composition", as used herein, refers to a
population of antibody molecules that contain only one species of
an antigen binding site capable of immunoreacting with a particular
epitope of flh2882. A monoclonal antibody composition thus
typically displays a single binding affinity for a particular
flh2882 protein with which it immunoreacts.
[0036] To generate anti-flh2882 antibodies, an isolated flh2882
protein, or a fragment thereof, is used as an immunogen to generate
antibodies that bind flh2882 using standard techniques for
polyclonal and monoclonal antibody preparation. The full-length
flh2882 protein can be used or, alternatively, an antigenic peptide
fragment of flh2882 can be used as an immunogen. An antigenic
fragment of the flh2882 protein will typically comprises at least 8
contiguous amino acid residues of an flh2882 protein, e.g. 8
contiguous amino acids from SEQ ID NO:2. Preferably, the antigenic
peptide comprises at least 10 amino acid residues, more preferably
at least 15 amino acid residues, even more preferably at least 20
amino acid residues, and most preferably at least 30 amino acid
residues of an flh2882-protein. Preferred fragments for generating
anti-flh2882 antibodies are regions of flh2882 that are located on
the surface of the protein, e.g., hydrophilic regions.
[0037] An flh2882 immunogen typically is used to prepare antibodies
by immunizing a suitable subject, (e.g., rabbit, goat, mouse or
other mammal) with the immunogen. An appropriate immunogenic
preparation can contain, for example, recombinantly expressed
flh2882 protein or a chemically synthesized flh2882 peptide. The
preparation can further include an adjuvant, such as Freund's
complete or incomplete adjuvant, or similar immunostimulatory
agent. Immunization of a suitable subject with an immunogenic
flh2882 preparation induces a polyclonal anti-flh2882 antibody
response.
[0038] Polyclonal anti-flh2882 antibodies can be prepared as
described above by immunizing a suitable subject with an flh2882
immunogen. The anti-flh2882 antibody titer in the immunized subject
can be monitored over time by standard techniques, such as with an
enzyme linked immunosorbent assay (ELISA) using immobilized
flh2882. If desired, the antibody molecules directed against
flh2882 can be isolated from the mammal (e.g., from the blood) and
further purified by well known techniques, such as protein A
chromatography to obtain the IgG fraction. At an appropriate time
after immunization, e.g., when the anti-flh2882 antibody titers are
highest, antibody-producing cells can be obtained from the subject
and used to prepare monoclonal antibodies by standard techniques,
such as the hybridoma technique originally described by Kohler and
Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981)
J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem
0.255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al.
(1982) Int. J. Cancer 29:269-75), the more recent human B cell
hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the
EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma
techniques. The technology for producing monoclonal antibody
hybridomas is well known (see generally R. H. Kenneth, in
Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer (1981)
Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic
Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a
myeloma) is fused to lymphocytes (typically splenocytes) from a
mammal immunized with an flh2882 immunogen as described above, and
the culture supernatants of the resulting hybridoma cells are
screened to identify a hybridoma producing a monoclonal antibody
that binds flh2882.
[0039] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-flh2882 monoclonal antibody (see,
e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al.
Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited
supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the
ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line. Preferred immortal cell lines are
mouse myeloma cell lines that are sensitive to culture medium
containing hypoxanthine, aminopterin and thymidine ("HAT medium").
Any of a number of myeloma cell lines can be used as a fusion
partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1,
P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells
are fused to mouse splenocytes using polyethylene glycol ("PEG").
Hybridoma cells resulting from the fusion are then selected using
HAT medium, which kills unfused and unproductively fused myeloma
cells (unfused splenocytes die after several days because they are
not transformed). Hybridoma cells producing a monoclonal antibody
of the invention are detected by screening the hybridoma culture
supernatants for antibodies that bind flh2882, e.g., using a
standard ELISA assay.
[0040] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-flh2882 antibody can be identified
and isolated by screening a recombinant combinatorial
immunoglobulin library (e.g., an antibody phage display library)
with flh2882 to thereby isolate immunoglobulin library members that
bind flh2882. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZIP.TM. Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, Ladner et al. U.S. Pat. No.
5,223,409; Kang et al. PCT International Publication No. WO
92/18619; Dower et al. PCT International Publication No. WO
91/17271; Winter et al. PCT International Publication WO 92/20791;
Markland et al. PCT International Publication No. WO 92/15679;
Breitling et al. PCT International Publication WO 93/01288;
McCafferty et al. PCT International Publication No. WO 92/01047;
Garrard et al. PCT International Publication No. WO 92/09690;
Ladner et al. PCT International Publication No. WO 90/02809; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins
et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991)
Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et
al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS
88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
[0041] Additionally, recombinant anti-flh2882 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human fragments, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in Robinson et al. PCT International Application
No. PCT/US86/02269; Akira, et al. European Patent Application
184,187; Taniguchi, M., European Patent Application 171,496;
Morrison et al. European Patent Application 173,494; Neuberger et
al. PCT International Publication No. WO 86/01533; Cabilly et al.
U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) PNAS 84:3439-3443; Liu et al. (1987) J. Immunol.
139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al.
(1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature
314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.
80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et
al. (1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0042] An anti-flh2882 antibody (e.g., monoclonal antibody) can be
used to isolate flh2882 proteins by standard techniques, such as
affinity chromatography or immunoprecipitation. An anti-flh2882
antibody can facilitate the purification of natural flh2882 protein
from cells and recombinantly produced flh2882 protein expressed in
host cells. Moreover, an anti-flh2882 antibody can be used to
detect flh2882 protein (e.g., in a cellular lysate or cell
supernatant) in order to evaluate the abundance and pattern of
expression of the flh2882 protein. The detection of circulating
fragments of an flh2882 protein can be used to identify flh2882
protein turnover in a subject. Anti-flh2882 antibodies can be used
diagnostically to monitor protein levels in tissue as part of a
clinical testing procedure, e.g., to, for example, determine the
efficacy of a given treatment regimen. Detection can be facilitated
by coupling (i.e., physically linking) the antibody to a detectable
substance. Examples of detectable substances include various
enzymes, prosthetic groups, fluorescent materials, luminescent
materials, bioluminescent materials, and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0043] III. Isolated flh2882 Nucleic Acid Molecules
[0044] The present invention further provides isolated nucleic acid
molecules that encode an flh2882 protein, hereinafter the flh2882
gene or flh2882 nucleic acid molecule, as well as fragments of an
flh2882 gene.
[0045] As used herein, the term "nucleic acid molecule" is intended
to include DNA molecules (e.g., cDNA or genomic DNA) and RNA
molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0046] As used herein, an "isolated" nucleic acid molecule is one
that is separated from other nucleic acid molecules that are
present in the natural source of the nucleic acid. Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of
the nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived. For example, in various embodiments, the
isolated flh2882 nucleic acid molecule can contain less than about
5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived
(e.g., a substantia nigra cell). Moreover, an "isolated" nucleic
acid molecule, such as a cDNA molecule, can be substantially free
of other cellular material, or culture medium when produced by
recombinant techniques, or chemical precursors or other chemicals
when chemically synthesized. However, the flh2882 nucleic acid
molecule can be fused to other protein encoding or regulatory
sequences and still be considered isolated.
[0047] The isolated nucleic acid molecules of the present invention
encode an flh2882 protein. As described above, an flh2882 protein
is defined as a protein comprising the amino acid sequence depicted
in SEQ ID NO:2 (human flh2882 protein), allelic variants of human
flh2882 protein, and orthologues of the human flh2882 protein. A
preferred flh2882 nucleic acid molecule comprises the nucleotide
sequence shown in SEQ ID NO:1 or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC (American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209);
on Oct. 2, 1998, as Accession Number 98914. The sequence of SEQ ID
NO:1 corresponds to the human flh2882 cDNA. This cDNA comprises
sequences encoding the human flh2882 protein (i.e., "the coding
region", from nucleotides 184 to 1194 of SEQ ID NO:1), as well as
5' untranslated sequences (nucleotides 1 to 183 of SEQ ID NO:1) and
3' untranslated sequences (nucleotides 1195 to 2581 of SEQ ID
NO:1). Alternatively, the nucleic acid molecule can comprise only
the coding region of SEQ ID NO:1 (e.g., nucleotides 184 to 1194
shown separately as SEQ ID NO:3).
[0048] The human flh2882 gene is approximately 2581 nucleotides in
length and encodes a full length protein having a molecular weight
of approximately 38.7 KDa and which is approximately 337 amino acid
residues in length. The human flh2882 protein is expressed
primarily in the brain, particularly the substantia nigra. Based on
structural analysis, amino acid residues 11-28 (SEQ ID NO:4), 43-62
(SEQ ID NO:5), 80-102 (SEQ ID NO:6), 121-146 (SEQ ID NO:7), 169-190
(SEQ ID NO:8), 247-265 (SEQ ID NO:9), and 280-300 (SEQ ID NO:10)
comprise transmembrane domains. As used herein, the term
"transmembrane domain" refers to a structural amino acid motif
which includes a hydrophobic helix that spans the plasma
membrane.
[0049] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence shown in SEQ ID NO:1 (and
fragments thereof) due to degeneracy of the genetic code and thus
encode the same flh2882 protein as that encoded by the nucleotide
sequence shown in SEQ ID NO:1.
[0050] In another preferred embodiment, an isolated nucleic acid
molecule of the invention comprises a nucleic acid molecule which
is a complement of the nucleotide sequence shown in SEQ ID NO:1,
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number 98914, or a fragment of either of
these nucleotide sequences. A nucleic acid molecule which is
complementary to the nucleotide sequence shown in SEQ ID NO:1 is
one which is sufficiently complementary to the nucleotide sequence
shown in SEQ ID NO:1 such that it can hybridize to the nucleotide
sequence shown in SEQ ID NO:1, thereby forming a stable duplex.
[0051] Orthologues and allelic variants of the human flh2882 gene
can readily be identified using methods well known in the art.
Allelic variants and orthologues of the human flh2882 gene will
comprise a nucleotide sequence that is at least about 60-65%,
typically at least about 70-75%, more typically at least about
80-85%, and most typically at least about 90-95% or more homologous
to the nucleotide sequence shown in SEQ ID NO:1, or to the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number 98914, or a fragment of these nucleotide
sequences. Such nucleic acid molecules can readily be identified as
being able to hybridize, preferably under stringent conditions, to
the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98914, or a fragment of either of these nucleotide
sequences.
[0052] Moreover, the nucleic acid molecule of the invention can
comprise only a fragment of the coding region of an flh2882 gene,
such as a fragment of SEQ ID NO:1. The nucleotide sequence
determined from the cloning of the human flh2882 gene allows for
the generation of probes and primers designed for use in
identifying and/or cloning flh2882 gene homologues from other cell
types, e.g., from other tissues, as well as flh2882 gene
orthologues from other mammals. A probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, preferably about
25, more preferably about 40, 50 or 75 consecutive nucleotides of
SEQ ID NO:1 sense, an anti-sense sequence of SEQ ID NO:1, or
naturally occurring mutants thereof. Primers based on the
nucleotide sequence in SEQ ID NO:1 can be used in PCR reactions to
clone flh2882 gene homologues. Probes based on the flh2882
nucleotide sequence can be used to detect transcripts or genomic
sequences encoding the same or homologous proteins. In preferred
embodiments, the probe further comprises a label group attached
thereto, e.g., the label group can be a radioisotope, a fluorescent
compound, an enzyme, or an enzyme co-factor. Such probes can be
used as a part of a diagnostic test kit for identifying cells or
tissue which misexpress an flh2882 protein, such as by measuring a
level of an flh2882-encoding nucleic acid in a sample of cells from
a subject e.g., detecting flh2882 mRNA levels or determining
whether a genomic flh2882 gene has been mutated or deleted.
[0053] In addition to the flh2882 nucleotide sequence shown in SEQ
ID NO:1, it will be appreciated by those skilled in the art that
DNA sequence polymorphisms that lead to changes in the amino acid
sequences of an flh2882 protein may exist within a population
(e.g., the human population). Such genetic polymorphism in the
flh2882 gene may exist among individuals within a population due to
natural allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding an flh2882 protein, preferably a
mammalian flh2882 protein. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of the
flh2882 gene. Any and all such nucleotide variations and resulting
amino acid polymorphisms in an flh2882 gene that are the result of
natural allelic variation are intended to be within the scope of
the invention. Such allelic variation includes both active allelic
variants as well as non-active or reduced activity allelic
variants, the later two types typically giving rise to a
pathological disorder. Moreover, nucleic acid molecules encoding
flh2882 proteins from other species, and thus which have a
nucleotide sequence which differs from the human sequence of SEQ ID
NO:1, are intended to be within the scope of the invention. Nucleic
acid molecules corresponding to natural allelic variants and
non-human orthologues of the human flh2882 cDNA of the invention
can be isolated based on their homology to the human flh2882
nucleic acid disclosed herein using the human cDNA, or a fragment
thereof, as a hybridization probe according to standard
hybridization techniques under stringent hybridization
conditions.
[0054] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 15 nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:1. In other
embodiments, the nucleic acid is at least 30, 50, 100, 250 or 500
nucleotides in length. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% homologous to each other typically remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 65%, more preferably at least about 70%, and even more
preferably at least about 75% or more homologous to each other
typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of
stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. Preferably, an isolated nucleic acid molecule of
the invention that hybridizes under stringent conditions to the
sequence of SEQ ID NO:1 corresponds to a naturally-occurring
nucleic acid molecule. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). In one embodiment, the nucleic acid encodes a natural
human flh2882.
[0055] In addition to naturally-occurring allelic variants of the
flh2882 nucleic acid sequence that may exist in the population, the
skilled artisan will further appreciate that changes can be
introduced by mutation into the nucleotide sequence of SEQ ID NO:1,
thereby leading to changes in the amino acid sequence of the
encoded flh2882 protein, without altering the functional ability of
the flh2882 protein. For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in the sequence of SEQ ID NO:1. A "non-essential" amino
acid residue is a residue that can be altered from the wild-type
sequence of an flh2882 protein (e.g., the sequence of SEQ ID NO:2)
without altering the activity of flh2882, whereas an "essential"
amino acid residue is required for flh2882 protein activity. For
example, conserved amino acid residues, e.g., aspartates, prolines,
threonines, and tyrosines, in the transmembrane domains of the
flh2882 protein are most likely important for binding to ligand and
are thus essential residues of the flh2882 protein. Other amino
acid residues, however, (e.g., those that are not conserved or only
semi-conserved in the transmembrane domain) may not be essential
for activity and thus are likely to be amenable to alteration
without altering flh2882 protein activity.
[0056] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding flh2882 proteins that contain
changes in amino acid residues that are not essential for flh2882
activity. Such flh2882 proteins differ in amino acid sequence from
SEQ ID NO:2 yet retain at least one of the flh2882 activities
described herein. In one embodiment, the isolated nucleic acid
molecule comprises a nucleotide sequence encoding a protein,
wherein the protein comprises an amino acid sequence at least about
30-35%, preferably at least about 40-45%, more preferably at least
about 50-55%, even more preferably at least about 60-65%, yet more
preferably at least about 70-75%, still more preferably at least
about 80-85%, and most preferably at least about 90-95% or more
homologous to the amino acid sequence of SEQ ID NO:2.
[0057] An isolated nucleic acid molecule encoding an flh2882
protein homologous to the protein of SEQ ID NO:2 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into the nucleotide sequence of SEQ ID NO:1, such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into SEQ ID NO:1 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), non-polar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in
flh2882 is preferably replaced with another amino acid residue from
the same side chain family. Alternatively, in another embodiment,
mutations can be introduced randomly along all or part of an
flh2882 coding sequence, such as by saturation mutagenesis, and the
resultant mutants can be screened for an flh2882 activity described
herein to identify mutants that retain flh2882 activity. Following
mutagenesis of SEQ ID NO:1, the encoded protein can be expressed
recombinantly (e.g., as described in Examples 3 and 4) and the
activity of the protein can be determined using, for example,
assays described herein.
[0058] In addition to the nucleic acid molecules encoding flh2882
proteins described above, another aspect of the invention pertains
to isolated nucleic acid molecules which are antisense thereto. An
"antisense" nucleic acid comprises a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an
antisense nucleic acid can hydrogen bond to a sense nucleic acid.
The antisense nucleic acid can be complementary to an entire
flh2882 coding strand, or to only a fragment thereof. In one
embodiment, an antisense nucleic acid molecule is antisense to a
"coding region" of the coding strand of a nucleotide sequence
encoding an flh2882 protein.
[0059] The term "coding region" refers to the region of the
nucleotide sequence comprising codons which are translated into
amino acid residues, e.g., the entire coding region of SEQ ID NO:1
comprises nucleotides 184 to 1194 (shown separately as SEQ ID
NO:3). In another embodiment, the antisense nucleic acid molecule
is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence encoding an flh2882 protein. The term
"noncoding region" refers to 5' and 3' sequences which flank the
coding region that are not translated into amino acids (i.e., also
referred to as 5' and 3' untranslated regions).
[0060] Given the coding strand sequence encoding the flh2882
protein disclosed herein (e.g., SEQ ID NO:1), antisense nucleic
acids of the invention can be designed according to the rules of
Watson and Crick base pairing. The antisense nucleic acid molecule
can be complementary to the entire coding region of flh2882 mRNA,
but more preferably is an oligonucleotide which is antisense to
only a fragment of the coding or noncoding region of flh2882 mRNA.
For example, the antisense oligonucleotide can be complementary to
the region surrounding the translation start site of flh2882 mRNA.
An antisense oligonucleotide can be, for example, about 5, 10, 15,
20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense
nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomet-
hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopente- nyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0061] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding an flh2882 protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule which binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of an antisense nucleic acid molecule of
the invention includes direct injection at a tissue site.
Alternatively, an antisense nucleic acid molecule can be modified
to target selected cells and then administered systemically. For
example, for systemic administration, an antisense molecule can be
modified such that it specifically binds to a receptor or an
antigen expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecule to a peptide or an antibody which
binds to a cell surface receptor or antigen. The antisense nucleic
acid molecule can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of the antisense molecules, vector constructs in
which the antisense nucleic acid molecule is placed under the
control of a strong pol II or pol III promoter are preferred.
[0062] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0063] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave flh2882 mRNA transcripts to thereby
inhibit translation of flh2882 mRNA. A ribozyme having specificity
for an flh2882-encoding nucleic acid can be designed based upon the
nucleotide sequence of an flh2882 cDNA disclosed herein (i.e., SEQ
ID NO:1). For example, a derivative of a Tetrahymena L-19 IVS RNA
can be constructed in which the nucleotide sequence of the active
site is complementary to the nucleotide sequence to be cleaved in
an flh2882-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.
4,987,071 and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,
flh2882 mRNA can be used to select a catalytic RNA having a
specific ribonuclease activity from a pool of RNA molecules. See,
e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[0064] Alternatively, flh2882 gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of the flh2882 gene (e.g., the flh2882 gene promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the flh2882 gene in target cells. See generally,
Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et
al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992)
Bioassays 14(12):807-15.
[0065] IV. Recombinant Expression Vectors and Host Cells
[0066] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
an flh2882 protein (or a fragment thereof). As used herein, the
term "vector" refers to a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression
of genes to which they are operatively linked. Such vectors are
referred to herein as "expression vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. In the present specification, "plasmid" and
"vector" can be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.
[0067] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid
sequence to be expressed. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an
in vitro transcription/translation system or in a host cell when
the vector is introduced into the host cell). The term "regulatory
sequence" is intended to include promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many
types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., flh2882 proteins, altered forms of flh2882 proteins, fusion
proteins, and the like).
[0068] The recombinant expression vectors of the invention can be
designed for expression of an flh2882 protein in prokaryotic or
eukaryotic cells. For example, an flh2882 protein can be expressed
in bacterial cells such as E. coli, insect cells (e.g., using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0069] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein. In one embodiment, the coding
sequence of the flh2882 gene is cloned into a pGEX expression
vector to create a vector encoding a fusion protein comprising,
from the N-terminus to the C-terminus, GST-thrombin cleavage
site-flh2882 protein. The fusion protein can be purified by
affinity chromatography using glutathione-agarose resin.
Recombinant flh2882 protein unfused to GST can be recovered by
cleavage of the fusion protein with thrombin.
[0070] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET Id vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lambda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0071] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid
to be inserted into an expression vector so that the individual
codons for each amino acid are those preferentially utilized in E.
coli (Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0072] In another embodiment, the flh2882 gene expression vector is
a yeast expression vector. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (Baldari, et al., (1987) Embo J
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2
(Invitrogen Corporation, San Diego, Calif.).
[0073] Alternatively, an flh2882 gene can be expressed in insect
cells using, for example, baculovirus expression vectors.
Baculovirus vectors available for expression of proteins in
cultured insect cells (e.g., Sf 9 cells) include the pAc series
(Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL
series (Lucklow and Summers (1989) Virology 170:31-39).
[0074] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987)
EMBO J. 6:187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd,
ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989.
[0075] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, for example the murine hox
promoters (Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0076] The invention further provides a recombinant expression
vector comprising a DNA molecule encoding an flh2882 protein cloned
into the expression vector in an antisense orientation. That is,
the DNA molecule is operatively linked to a regulatory sequence in
a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to flh2882 mRNA.
Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense orientation can be chosen which direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen which direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub, H. et al.,
Antisense RNA as a molecular tool for genetic analysis,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0077] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0078] A host cell can be any prokaryotic or eukaryotic cell. For
example, flh2882 protein can be expressed in bacterial cells such
as E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0079] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0080] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding the flh2882 protein or can be introduced on a
separate vector. Cells stably transfected with the introduced
nucleic acid can be identified by drug selection (e.g., cells that
have incorporated the selectable marker gene will survive, while
the other cells die).
[0081] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) flh2882 protein. Accordingly, the invention further
provides methods for producing flh2882 protein using the host cells
of the invention. In one embodiment, the method comprises culturing
the host cell of invention (into which a recombinant expression
vector encoding an flh2882 protein has been introduced) in a
suitable medium until the flh2882 protein is produced. In another
embodiment, the method further comprises isolating the flh2882
protein from the medium or the host cell.
[0082] The host cells of the invention can also be used to produce
non-human transgenic animals. The non-human transgenic animals can
be used in screening assays designed to identify agents or
compounds, e.g., drugs, pharmaceuticals, etc., which are capable of
ameliorating detrimental symptoms of selected disorders such as
nervous system disorders, e.g., psychiatric disorders or disorders
affecting circadian rhythms and the sleep-wake cycle. For example,
in one embodiment, a host cell of the invention is a fertilized
oocyte or an embryonic stem cell into which flh2882 protein-coding
sequences have been introduced. Such host cells can then be used to
create non-human transgenic animals in which exogenous flh2882 gene
sequences have been introduced into their genome or homologous
recombinant animals in which endogenous flh2882 gene sequences have
been altered. Such animals are useful for studying the function
and/or activity of an flh2882 protein and for identifying and/or
evaluating modulators of flh2882 protein activity. As used herein,
a "transgenic animal" is a non-human animal, preferably a mammal,
more preferably a rodent such as a rat or mouse, in which one or
more of the cells of the animal include a transgene. Other examples
of transgenic animals include non-human primates, sheep, dogs,
cows, goats, chickens, amphibians, and the like. A transgene is
exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous flh2882 gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0083] A transgenic animal of the invention can be created by
introducing flh2882 protein encoding nucleic acid into the male
pronuclei of a fertilized oocyte, e.g., by microinjection,
retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The human flh2882 cDNA
sequence of SEQ ID NO:1 can be introduced as a transgene into the
genome of a non-human animal. Moreover, a non-human homologue of
the human flh2882 gene, such as a mouse flh2882 gene, can be
isolated based on hybridization to the human flh2882 cDNA
(described further above) and used as a transgene. Intronic
sequences and polyadenylation signals can also be included in the
transgene to increase the efficiency of expression of the
transgene. A tissue-specific regulatory sequence(s) can be operably
linked to the flh2882 transgene to direct expression of an flh2882
protein to particular cells. Methods for generating transgenic
animals via embryo manipulation and microinjection, particularly
animals such as mice, have become conventional in the art and are
described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009,
both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and
in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods
are used for production of other transgenic animals. A transgenic
founder animal can be identified based upon the presence of the
flh2882 transgene in its genome and/or expression of flh2882 mRNA
in tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene encoding an
flh2882 protein can further be bred to other transgenic animals
carrying other transgenes.
[0084] To create a homologous recombinant animal, a vector is
prepared which contains at least a fragment of an flh2882 gene into
which a deletion, addition or substitution has been introduced to
thereby alter, e.g., functionally disrupt, the flh2882 gene. The
flh2882 gene can be a human gene (e.g., from a human genomic clone
isolated from a human genomic library screened with the cDNA of SEQ
ID NO:1), but more preferably is a non-human homologue of a human
flh2882 gene. For example, a mouse flh2882 gene can be isolated
from a mouse genomic DNA library using the flh2882 cDNA of SEQ ID
NO:1 as a probe. The mouse flh2882 gene then can be used to
construct a homologous recombination vector suitable for altering
an endogenous flh2882 gene in the mouse genome. In a preferred
embodiment, the vector is designed such that, upon homologous
recombination, the endogenous flh2882 gene is functionally
disrupted (i.e., no longer encodes a functional protein; also
referred to as a "knock out" vector). Alternatively, the vector can
be designed such that, upon homologous recombination, the
endogenous flh2882 gene is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to thereby alter the expression of the endogenous
flh2882 protein). In the homologous recombination vector, the
altered fragment of the flh2882 gene is flanked at its 5' and 3'
ends by additional nucleic acid of the flh2882 gene to allow for
homologous recombination to occur between the exogenous flh2882
gene carried by the vector and an endogenous flh2882 gene in an
embryonic stem cell. The additional flanking flh2882 nucleic acid
is of sufficient length for successful homologous recombination
with the endogenous gene. Typically, several kilobases of flanking
DNA (both at the 5' and 3' ends) are included in the vector (see
for example, Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503
for a description of homologous recombination vectors). The vector
is introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced flh2882 gene has
homologously recombined with the endogenous flh2882 gene are
selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected
cells are then injected into a blastocyst of an animal (e.g., a
mouse) to form aggregation chimeras (see e.g., Bradley, A. in
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.
J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric
embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought to term. Progeny harboring the
homologously recombined DNA in their germ cells can be used to
breed animals in which all cells of the animal contain the
homologously recombined DNA by germline transmission of the
transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and
in PCT International Publication Nos. WO 90/11354; WO 91/01140; WO
92/0968; and WO 93/04169.
[0085] In another embodiment, transgenic non-human animals can be
produced which contain selected systems which allow for regulated
expression of the transgene. One example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.
(1991) Science 251:1351-1355. If a cre/loxP recombinase system is
used to regulate expression of the transgene, animals containing
transgenes encoding both the Cre recombinase and a selected protein
are required. Such animals can be provided through the construction
of "double" transgenic animals, e.g., by mating two transgenic
animals, one containing a transgene encoding a selected protein and
the other containing a transgene encoding a recombinase.
[0086] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al. (1997) Nature 385:810-813 and PCT International
Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell,
e.g., a somatic cell, from the transgenic animal can be isolated
and induced to exit the growth cycle and enter G.sub.o phase. The
quiescent cell can then be fused, e.g., through the use of
electrical pulses, to an enucleated oocyte from an animal of the
same species from which the quiescent cell is isolated. The
reconstructed oocyte is then cultured such that it develops to
morula or blastocyst and then transferred to pseudopregnant female
foster animal. The offspring borne of this female foster animal
will be a clone of the animal from which the cell, e.g., the
somatic cell, is isolated.
[0087] V. Uses and Methods of the Invention
[0088] The nucleic acid molecules, proteins, protein homologues,
modulators, and antibodies described herein can be used in one or
more of the following methods: a) drug screening assays; b)
diagnostic assays particularly in disease identification, allelic
screening and pharmocogenetic testing; c) methods of treatment; d)
pharmacogenomics; and e) monitoring of effects during clinical
trials. An flh2882 protein of the invention can be used as a drug
target for developing agents to modulate the activity of the
flh2882 protein (a GPCR). The isolated nucleic acid molecules of
the invention can be used to express flh2882 protein (e.g., via a
recombinant expression vector in a host cell or in gene therapy
applications), to detect flh2882 mRNA (e.g., in a biological
sample) or a naturally occurring or recombinantly generated genetic
mutation in an flh2882 gene, and to modulate flh2882 protein
activity, as described further below. In addition, the flh2882
proteins can be used to screen drugs or compounds which modulate
flh2882 protein activity. Moreover, the anti-flh2882 antibodies of
the invention can be used to detect and isolate an flh2882 protein,
particularly fragments of an flh2882 protein present in a
biological sample, and to modulate flh2882 protein activity.
[0089] a. Drug Screening Assays:
[0090] The invention provides methods for identifying compounds or
agents that can be used to treat disorders characterized by (or
associated with) aberrant or abnormal flh2882 nucleic acid
expression and/or flh2882 protein activity. These methods are also
referred to herein as drug screening assays and typically include
the step of screening a candidate/test compound or agent to
identify compounds that are an agonist or antagonist of an flh2882
protein, and specifically for the ability to interact with (e.g.,
bind to) an flh2882 protein, to modulate the interaction of an
flh2882 protein and a target molecule, and/or to modulate flh2882
nucleic acid expression and/or flh2882 protein activity.
Candidate/test compounds or agents which have one or more of these
abilities can be used as drugs to treat disorders characterized by
aberrant or abnormal flh2882 nucleic acid expression and/or flh2882
protein activity. Candidate/test compounds include, for example, 1)
peptides such as soluble peptides, including Ig-tailed fusion
peptides and members of random peptide libraries (see, e.g., Lam,
K. S. et al. (1991) Nature 354:82-84; Houghten, R. et al. (1991)
Nature 354:84-86) and combinatorial chemistry-derived molecular
libraries made of D- and/or L-configuration amino acids; 2)
phosphopeptides (e.g., members of random and partially degenerate,
directed phosphopeptide libraries, see, e.g., Songyang, Z. et al.
(1993) Cell 72:767-778); 3) antibodies (e.g., polyclonal,
monoclonal, humanized, anti-idiotypic, chimeric, and single chain
antibodies as well as Fab, F(ab').sub.2, Fab expression library
fragments, and epitope-binding fragments of antibodies); and 4)
small organic and inorganic molecules (e.g., molecules obtained
from combinatorial and natural product libraries).
[0091] In one embodiment, the invention provides assays for
screening candidate/test compounds which interact with (e.g., bind
to) an flh2882 protein. Typically, the assays are recombinant cell
based or cell-free assays which include the steps of combining a
cell expressing an flh2882 protein or a bioactive fragment thereof,
or an isolated flh2882 protein, and a candidate/test compound,
e.g., under conditions which allow for interaction of (e.g.,
binding of) the candidate/test compound to the flh2882 protein or
fragment thereof to form a complex, and detecting the formation of
a complex, in which the ability of the candidate compound to
interact with (e.g., bind to) the flh2882 protein or fragment
thereof is indicated by the presence of the candidate compound in
the complex. Formation of complexes between the flh2882 protein and
the candidate compound can be detected using competition binding
assays, and can be quantitated, for example, using standard
immunoassays.
[0092] In another embodiment, the invention provides screening
assays to identify candidate/test compounds which modulate (e.g.,
stimulate or inhibit) the interaction (and most likely flh2882
protein activity as well) between an flh2882 protein and a molecule
(target molecule) with which the flh2882 protein normally
interacts. Examples of such target molecules include proteins in
the same signaling path as the flh2882 protein, e.g., proteins
which may function upstream (including both stimulators and
inhibitors of activity) or downstream of the flh2882 protein in,
for example, a cognitive function signaling pathway or in a pathway
involving flh2882 protein activity, e.g., a G protein or other
interactor involved in cAMP or phosphatidylinositol turnover,
and/or adenylate cyclase or phospholipase C activation. Typically,
the assays are recombinant cell based assays which include the
steps of combining a cell expressing an flh2882 protein, or a
bioactive fragment thereof, an flh2882 protein target molecule
(e.g., an flh2882 ligand) and a candidate/test compound, e.g.,
under conditions wherein but for the presence of the candidate
compound, the flh2882 protein or biologically active fragment
thereof interacts with (e.g., binds to) the target molecule, and
detecting the formation of a complex which includes the flh2882
protein and the target molecule or detecting the
interaction/reaction of the flh2882 protein and the target
molecule. Detection of complex formation can include direct
quantitation of the complex by, for example, measuring inductive
effects of the flh2882 protein. A statistically significant change,
such as a decrease, in the interaction of the flh2882 protein and
target molecule (e.g., in the formation of a complex between the
flh2882 protein and the target molecule) in the presence of a
candidate compound (relative to what is detected in the absence of
the candidate compound) is indicative of a modulation (e.g.,
stimulation or inhibition) of the interaction between the flh2882
protein and the target molecule. Modulation of the formation of
complexes between the flh2882 protein and the target molecule can
be quantitated using, for example, an immunoassay.
[0093] To perform cell free drug screening assays, it is desirable
to immobilize either the flh2882 protein or its target molecule to
facilitate separation of complexes from uncomplexed forms of one or
both of the proteins, as well as to accommodate automation of the
assay. Interaction (e.g., binding of) of the flh2882 protein to a
target molecule, in the presence and absence of a candidate
compound, can be accomplished in any vessel suitable for containing
the reactants. Examples of such vessels include microtitre plates,
test tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein can be provided which adds a domain that allows the protein
to be bound to a matrix. For example,
glutathione-S-transferase/flh2882 fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the cell lysates (e.g., .sup.35S-labeled) and the
candidate compound, and the mixture incubated under conditions
conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads are washed to
remove any unbound label, and the matrix immobilized and radiolabel
determined directly, or in the supernatant after the complexes are
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of flh2882-binding
protein found in the bead fraction quantitated from the gel using
standard electrophoretic techniques.
[0094] Other techniques for immobilizing proteins on matrices can
also be used in the drug screening assays of the invention. For
example, either the flh2882 protein or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated flh2882 protein molecules can be prepared from
biotin-NHS(N-hydroxy-succinimide) using techniques well known in
the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized in the wells of streptavidin-coated 96 well
plates (Pierce Chemical). Alternatively, antibodies reactive with
an flh2882 protein but which do not interfere with binding of the
protein to its target molecule can be derivatized to the wells of
the plate, and flh2882 protein trapped in the wells by antibody
conjugation. As described above, preparations of an flh2882-binding
protein and a candidate compound are incubated in the flh2882
protein-presenting wells of the plate, and the amount of complex
trapped in the well can be quantitated. Methods for detecting such
complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes
using antibodies reactive with the flh2882 protein target molecule,
or which are reactive with flh2882 protein and compete with the
target molecule; as well as enzyme-linked assays which rely on
detecting an enzymatic activity associated with the target
molecule.
[0095] In yet another embodiment, the invention provides a method
for identifying a compound (e.g., a screening assay) capable of use
in the treatment of a disorder characterized by (or associated
with) aberrant or abnormal flh2882 nucleic acid expression or
flh2882 protein activity. This method typically includes the step
of assaying the ability of the compound or agent to modulate the
expression of the flh2882 nucleic acid or the activity of the
flh2882 protein thereby identifying a compound for treating a
disorder characterized by aberrant or abnormal flh2882 nucleic acid
expression or flh2882 protein activity. Methods for assaying the
ability of the compound or agent to modulate the expression of the
flh2882 nucleic acid or activity of the flh2882 protein are
typically cell-based assays. For example, cells which are sensitive
to ligands which transduce signals via a pathway involving an
flh2882 protein can be induced to overexpress an flh2882 protein in
the presence and absence of a candidate compound. Candidate
compounds which produce a statistically significant change in
flh2882 protein-dependent responses (either stimulation or
inhibition) can be identified. In one embodiment, expression of the
flh2882 nucleic acid or activity of an flh2882 protein is modulated
in cells and the effects of candidate compounds on the readout of
interest (such as cAMP or phosphatidylinositol turnover) are
measured. For example, the expression of genes which are up- or
down-regulated in response to an flh2882 protein-dependent signal
cascade can be assayed. In preferred embodiments, the regulatory
regions of such genes, e.g., the 5' flanking promoter and enhancer
regions, are operably linked to a detectable marker (such as
luciferase) which encodes a gene product that can be readily
detected. Phosphorylation of an flh2882 protein or flh2882 protein
target molecules can also be measured, for example, by
immunoblotting.
[0096] Alternatively, modulators of flh2882 gene expression (e.g.,
compounds which can be used to treat a disorder characterized by
aberrant or abnormal flh2882 nucleic acid expression or flh2882
protein activity) can be identified in a method wherein a cell is
contacted with a candidate compound and the expression of flh2882
mRNA or protein in the cell is determined. The level of expression
of flh2882 mRNA or protein in the presence of the candidate
compound is compared to the level of expression of flh2882 mRNA or
protein in the absence of the candidate compound. The candidate
compound can then be identified as a modulator of flh2882 nucleic
acid expression based on this comparison and be used to treat a
disorder characterized by aberrant flh2882 nucleic acid expression.
For example, when expression of flh2882 mRNA or protein is greater
(statistically significantly greater) in the presence of the
candidate compound than in its absence, the candidate compound is
identified as a stimulator of flh2882 nucleic acid expression.
Alternatively, when flh2882 nucleic acid expression is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of flh2882 nucleic acid expression. The level of
flh2882 nucleic acid expression in the cells can be determined by
methods described herein for detecting flh2882 mRNA or protein.
[0097] In yet another aspect of the invention, the flh2882
proteins, or fragments thereof, can be used as "bait proteins" in a
two-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.
(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO
94/10300), to identify other proteins, which bind to or interact
with the flh2882 protein ("flh2882-binding proteins" or
"flh2882-bp") and modulate flh2882 protein activity. Such
flh2882-binding proteins are also likely to be involved in the
propagation of signals by the flh2882 proteins as, for example,
upstream or downstream elements of the flh2882 protein pathway.
[0098] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Bartel, P. et al. "Using the Two-Hybrid System
to Detect Protein-Protein Interactions" in Cellular Interactions in
Development: A Practical Approach, Hartley, D. A. ed. (Oxford
University Press, Oxford, 1993) pp. 153-179. Briefly, the assay
utilizes two different DNA constructs. In one construct, the gene
that encodes an flh2882 protein is fused to a gene encoding the DNA
binding domain of a known transcription factor (e.g., GAL4). In the
other construct, a DNA sequence, from a library of DNA sequences,
that encodes an unidentified protein ("prey" or "sample") is fused
to a gene that codes for the activation domain of the known
transcription factor. If the "bait" and the "prey" proteins are
able to interact, in vivo, forming an flh2882-protein dependent
complex, the DNA-binding and activation domains of the
transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ)
which is operably linked to a transcriptional regulatory site
responsive to the transcription factor. Expression of the reporter
gene can be detected and cell colonies containing the functional
transcription factor can be isolated and used to obtain the cloned
gene which encodes the protein which interacts with the flh2882
protein.
[0099] Modulators of flh2882 protein activity and/or flh2882
nucleic acid expression identified according to these drug
screening assays can be used to treat, for example, nervous system
disorders. These methods of treatment include the steps of
administering the modulators of flh2882 protein activity and/or
nucleic acid expression, e.g., in a pharmaceutical composition as
described in subsection IV above, to a subject in need of such
treatment, e.g., a subject with a disorder described herein.
[0100] b. Diagnostic Assays:
[0101] The invention further provides a method for detecting the
presence of an flh2882 protein or flh2882 nucleic acid molecule, or
fragment thereof, in a biological sample. The method involves
contacting the biological sample with a compound or an agent
capable of detecting flh2882 protein or mRNA such that the presence
of flh2882 protein/encoding nucleic acid molecule is detected in
the biological sample. A preferred agent for detecting flh2882 mRNA
is a labeled or labelable nucleic acid probe capable of hybridizing
to flh2882 mRNA. The nucleic acid probe can be, for example, the
full-length flh2882 cDNA of SEQ ID NO:1, or a fragment thereof,
such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to flh2882 mRNA. A preferred agent for
detecting flh2882 protein is a labeled or labelable antibody
capable of binding to flh2882 protein. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof (e.g., Fab or F(ab').sub.2) can be used. The
term "labeled or labelable", with regard to the probe or antibody,
is intended to encompass direct labeling of the probe or antibody
by coupling (i.e., physically linking) a detectable substance to
the probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The term
"biological sample" is intended to include tissues, cells and
biological fluids isolated from a subject, as well as tissues,
cells and fluids present within a subject. That is, the detection
method of the invention can be used to detect flh2882 mRNA or
protein in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of flh2882 mRNA include
Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of flh2882 protein include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. Alternatively, flh2882 protein can be
detected in vivo in a subject by introducing into the subject a
labeled anti-flh2882 antibody. For example, the antibody can be
labeled with a radioactive marker whose presence and location in a
subject can be detected by standard imaging techniques.
Particularly useful are methods which detect the allelic variant of
an flh2882 protein expressed in a subject and methods which detect
fragments of an flh2882 protein in a sample.
[0102] The invention also encompasses kits for detecting the
presence of an flh2882 protein in a biological sample. For example,
the kit can comprise reagents such as a labeled or labelable
compound or agent capable of detecting flh2882 protein or mRNA in a
biological sample; means for determining the amount of flh2882
protein in the sample; and means for comparing the amount of
flh2882 protein in the sample with a standard. The compound or
agent can be packaged in a suitable container. The kit can further
comprise instructions for using the kit to detect flh2882 mRNA or
protein.
[0103] The methods of the invention can also be used to detect
naturally occurring genetic mutations in an flh2882 gene, thereby
determining if a subject with the mutated gene is at risk for a
disorder characterized by aberrant or abnormal flh2882 nucleic acid
expression or flh2882 protein activity as described herein. In
preferred embodiments, the methods include detecting, in a sample
of cells from the subject, the presence or absence of a genetic
mutation characterized by at least one of an alteration affecting
the integrity of a gene encoding an flh2882 protein, or the
misexpression of the flh2882 gene. For example, such genetic
mutations can be detected by ascertaining the existence of at least
one of 1) a deletion of one or more nucleotides from an flh2882
gene; 2) an addition of one or more nucleotides to an flh2882 gene;
3) a substitution of one or more nucleotides of an flh2882 gene, 4)
a chromosomal rearrangement of an flh2882 gene; 5) an alteration in
the level of a messenger RNA transcript of an flh2882 gene, 6)
aberrant modification of an flh2882 gene, such as of the
methylation pattern of the genomic DNA, 7) the presence of a
non-wild type splicing pattern of a messenger RNA transcript of an
flh2882 gene, 8) a non-wild type level of an flh2882-protein, 9)
allelic loss of an flh2882 gene, and 10) inappropriate
post-translational modification of an flh2882-protein. As described
herein, there are a large number of assay techniques known in the
art that can be used for detecting mutations in an flh2882
gene.
[0104] In certain embodiments, detection of the mutation involves
the use of a probe/primer in a polymerase chain reaction (PCR)
(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor
PCR or RACE PCR, or, alternatively, in a ligation chain reaction
(LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which
can be particularly useful for detecting point mutations in the
flh2882-gene (see Abravaya et al. (1995) Nucleic Acids Res
0.23:675-682). This method can include the steps of collecting a
sample of cells from a patient, isolating nucleic acid (e.g.,
genomic, mRNA or both) from the cells of the sample, contacting the
nucleic acid sample with one or more primers which specifically
hybridize to an flh2882 gene under conditions such that
hybridization and amplification of the flh2882-gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample.
[0105] In an alternative embodiment, mutations in an flh2882 gene
from a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
for example, U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0106] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
flh2882 gene and detect mutations by comparing the sequence of the
sample flh2882 gene with the corresponding wild-type (control)
sequence. Examples of sequencing reactions include those based on
techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or
Sanger ((1977) PNAS 74:5463). A variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT International Publication No. WO
94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and
Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).
[0107] Other methods for detecting mutations in the flh2882 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et
al. (1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 217:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton (1993) Mutat. Res. 285:125-144; and
Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79), and movement of
mutant or wild-type fragments in polyacrylamide gels containing a
gradient of denaturant is assayed using denaturing gradient gel
electrophoresis (Myers et al (1985) Nature 313:495). Examples of
other techniques for detecting point mutations include, selective
oligonucleotide hybridization, selective amplification, and
selective primer extension.
[0108] c. Methods of Treatment
[0109] Another aspect of the invention pertains to methods for
treating a subject, e.g., a human, having a disease or disorder
characterized by (or associated with) aberrant or abnormal flh2882
nucleic acid expression and/or flh2882 protein activity. These
methods include the step of administering an flh2882 protein/gene
modulator (agonist or antagonist) to the subject such that
treatment occurs. The language "aberrant or abnormal flh2882
protein expression" refers to expression of a non-wild-type flh2882
protein or a non-wild-type level of expression of an flh2882
protein. Aberrant or abnormal flh2882 protein activity refers to a
non-wild-type flh2882 protein activity or a non-wild-type level of
flh2882 protein activity. As the flh2882 protein is involved in a
pathway involving signaling within cells, aberrant or abnormal
flh2882 protein activity or expression interferes with the normal
regulation of functions mediated by flh2882 protein signaling, and
in particular brain cells.
[0110] The terms "treating" or "treatment", as used herein, refer
to reduction or alleviation of at least one adverse effect or
symptom of a disorder or disease, e.g., a disorder or disease
characterized by or associated with abnormal or aberrant flh2882
protein activity or flh2882 nucleic acid expression.
[0111] As used herein, an flh2882 protein/gene modulator is a
molecule which can modulate flh2882 nucleic acid expression and/or
flh2882 protein activity. For example, an flh2882 gene or protein
modulator can modulate, e.g., upregulate (activate/agonize) or
down-regulate (suppress/antagonize), flh2882 nucleic acid
expression. In another example, an flh2882 protein/gene modulator
can modulate (e.g., stimulate/agonize or inhibit/antagonize)
flh2882 protein activity. If it is desirable to treat a disorder or
disease characterized by (or associated with) aberrant or abnormal
(non-wild-type) flh2882 nucleic acid expression and/or flh2882
protein activity by inhibiting flh2882 nucleic acid expression, an
flh2882 modulator can be an antisense molecule, e.g., a ribozyme,
as described herein. Examples of antisense molecules which can be
used to inhibit flh2882 nucleic acid expression include antisense
molecules which are complementary to a fragment of the 5'
untranslated region of SEQ ID NO:1 which also includes the start
codon and antisense molecules which are complementary to a fragment
of the 3' untranslated region of SEQ ID NO:1. An example of an
antisense molecule which is complementary to a fragment of the 5'
untranslated region of SEQ ID NO:1 and which also includes the
start codon is a nucleic acid molecule which includes nucleotides
which are complementary to nucleotides 171 to 186 of SEQ ID NO:1.
This antisense molecule has the following nucleotide sequence: 5'
CGGGGCGCGCACCATG 3' (SEQ ID NO:11). An additional example of an
antisense molecule which is complementary to a fragment of the 5'
untranslated region of SEQ ID NO:1 and which also includes the
start codon is a nucleic acid molecule which includes nucleotides
which are complementary to nucleotides 180 to 196 of SEQ ID NO:1.
This antisense molecule has the following nucleotide sequence: 5'
CACCATGAACTCGTGGG 3' (SEQ ID NO:12). An example of an antisense
molecule which is complementary to a fragment of the 3'
untranslated region of SEQ ID NO:1 is a nucleic acid molecule which
includes nucleotides which are complementary to nucleotides 1195 to
1210 of SEQ ID NO:1. This antisense molecule has the following
sequence: 5' TGAAGGACCGCGCTCC 3' (SEQ ID NO:13). An additional
example of an antisense molecule which is complementary to a
fragment of the 3' untranslated region of SEQ ID NO:1 is a nucleic
acid molecule which includes nucleotides which are complementary to
nucleotides 1189 to 1204 of SEQ ID NO:1. This antisense molecule
has the following sequence:
1 5' TCTGAGTGAAGGACCG 3'. (SEQ ID NO:14)
[0112] An flh2882 modulator that inhibits flh2882 nucleic acid
expression can also be a small molecule or other drug, e.g., a
small molecule or drug identified using the screening assays
described herein, which inhibits flh2882 nucleic acid expression.
If it is desirable to treat a disease or disorder characterized by
(or associated with) aberrant or abnormal (non-wild-type) flh2882
nucleic acid expression and/or flh2882 protein activity by
stimulating flh2882 nucleic acid expression, an flh2882 modulator
can be, for example, a nucleic acid molecule encoding an flh2882
protein (e.g., a nucleic acid molecule comprising a nucleotide
sequence homologous to the nucleotide sequence of SEQ ID NO:1) or a
small molecule or other drug, e.g., a small molecule (peptide) or
drug identified using the screening assays described herein, which
stimulates flh2882 nucleic acid expression.
[0113] Alternatively, if it is desirable to treat a disease or
disorder characterized by (or associated with) aberrant or abnormal
(non-wild-type) flh2882 nucleic acid expression and/or flh2882
protein activity by inhibiting flh2882 protein activity, an flh2882
modulator can be an anti-flh2882 antibody or a small molecule or
other drug, e.g., a small molecule or drug identified using the
screening assays described herein, which inhibits flh2882 protein
activity. If it is desirable to treat a disease or disorder
characterized by (or associated with) aberrant or abnormal
(non-wild-type) flh2882 nucleic acid expression and/or flh2882
protein activity by stimulating flh2882 protein activity, an
flh2882 modulator can be an active flh2882 protein or fragment
thereof (e.g., an flh2882 protein or fragment thereof having an
amino acid sequence which is homologous to the amino acid sequence
of SEQ ID NO:2 or a fragment thereof) or a small molecule or other
drug, e.g., a small molecule or drug identified using the screening
assays described herein, which stimulates flh2882 protein
activity.
[0114] Other aspects of the invention pertain to methods for
modulating an flh2882 protein mediated cell activity. These methods
include contacting the cell with an agent (or a composition which
includes an effective amount of an agent) which modulates flh2882
protein activity or flh2882 nucleic acid expression such that an
flh2882 protein mediated cell activity is altered relative to
normal levels (for example, cAMP or phosphatidylinositol
metabolism). As used herein, "an flh2882 protein mediated cell
activity" refers to a normal or abnormal activity or function of a
cell. Examples of flh2882 protein mediated cell activities include
phosphatidylinositol turnover, production or secretion of
molecules, such as proteins, contraction, proliferation, migration,
differentiation, and cell survival. In a preferred embodiment, the
cell is neural cell of the brain, e.g., a hippocampal cell. The
term "altered" as used herein refers to a change, e.g., an increase
or decrease, of a cell associated activity particularly cAMP or
phosphatidylinositol turnover, and adenylate cyclase or
phospholipase C activation. In one embodiment, the agent stimulates
flh2882 protein activity or flh2882 nucleic acid expression. In
another embodiment, the agent inhibits flh2882 protein activity or
flh2882 nucleic acid expression. These modulatory methods can be
performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo (e.g., by administering the agent to a
subject). In a preferred embodiment, the modulatory methods are
performed in vivo, i.e., the cell is present within a subject,
e.g., a mammal, e.g., a human, and the subject has a disorder or
disease characterized by or associated with abnormal or aberrant
flh2882 protein activity or flh2882 nucleic acid expression.
[0115] A nucleic acid molecule, a protein, an flh2882 modulator, a
compound etc. used in the methods of treatment can be incorporated
into an appropriate pharmaceutical composition described below and
administered to the subject through a route which allows the
molecule, protein, modulator, or compound etc. to perform its
intended function.
[0116] d. Pharmacogenomics
[0117] Test/candidate compounds, or modulators which have a
stimulatory or inhibitory effect on flh2882 protein activity (e.g.,
flh2882 gene expression) as identified by a screening assay
described herein can be administered to individuals to treat
(prophylactically or therapeutically) disorders (e.g., CNS
disorders) associated with aberrant flh2882 protein activity. In
conjunction with such treatment, the pharmacogenomics (i.e., the
study of the relationship between an individual's genotype and that
individual's response to a foreign compound or drug) of the
individual may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by
altering the relation between dose and blood concentration of the
pharmacologically active drug. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds (e.g.,
drugs) for prophylactic or therapeutic treatments based on a
consideration of the individual's genotype. Such pharmacogenomics
can further be used to determine appropriate dosages and
therapeutic regimens. Accordingly, the activity of flh2882 protein,
expression of flh2882 nucleic acid, or mutation content of flh2882
genes in an individual can be determined to thereby select
appropriate compound(s) for therapeutic or prophylactic treatment
of the individual.
[0118] Pharmacogenomics deal with clinically significant hereditary
variations in the response to drugs due to altered drug disposition
and abnormal action in affected persons. See, e.g., Eichelbaum, M.
(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder,
M. W. (1997) Clin. Chem. 43(2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0119] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C
19 quite frequently experience exaggerated drug response and side
effects when they receive standard doses. If a metabolite is the
active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0120] Thus, the activity of flh2882 protein, expression of flh2882
nucleic acid, or mutation content of flh2882 genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of a subject. In addition,
pharmacogenetic studies can be used to apply genotyping of
polymorphic alleles encoding drug-metabolizing enzymes to the
identification of a subject's drug responsiveness phenotype. This
knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
an flh2882 modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0121] e. Monitoring of Effects During Clinical Trials
[0122] Monitoring the influence of compounds (e.g., drugs) on the
expression or activity of flh2882 protein/gene can be applied not
only in basic drug screening, but also in clinical trials. For
example, the effectiveness of an agent determined by a screening
assay, as described herein, to increase flh2882 gene expression,
protein levels, or up-regulate flh2882 activity, can be monitored
in clinical trials of subjects exhibiting decreased flh2882 gene
expression, protein levels, or down-regulated flh2882 protein
activity. Alternatively, the effectiveness of an agent, determined
by a screening assay, to decrease flh2882 gene expression, protein
levels, or down-regulate flh2882 protein activity, can be monitored
in clinical trials of subjects exhibiting increased flh2882 gene
expression, protein levels, or up-regulated flh2882 protein
activity. In such clinical trials, the expression or activity of an
flh2882 protein and, preferably, other genes which have been
implicated in, for example, a nervous system related disorder can
be used as a "read out" or markers of the ligand responsiveness of
a particular cell.
[0123] For example, and not by way of limitation, genes, including
an flh2882 gene, which are modulated in cells by treatment with a
compound (e.g., drug or small molecule) which modulates flh2882
protein/gene activity (e.g., identified in a screening assay as
described herein) can be identified. Thus, to study the effect of
compounds on CNS disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of an flh2882 gene and other genes implicated in the
disorder. The levels of gene expression (i.e., a gene expression
pattern) can be quantified by Northern blot analysis or RT-PCR, as
described herein, or alternatively by measuring the amount of
protein produced, by one of the methods described herein, or by
measuring the levels of activity of an flh2882 protein or other
genes. In this way, the gene expression pattern can serve as an
marker, indicative of the physiological response of the cells to
the compound. Accordingly, this response state may be determined
before, and at various points during, treatment of the individual
with the compound.
[0124] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with a compound (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the compound; (ii)
detecting the level of expression of an flh2882 protein, mRNA, or
genomic DNA in the preadministration sample; (iii) obtaining one or
more post-administration samples from the subject; (iv) detecting
the level of expression or activity of the flh2882 protein, mRNA,
or genomic DNA in the post-administration samples; (v) comparing
the level of expression or activity of the flh2882 protein, mRNA,
or genomic DNA in the pre-administration sample with the flh2882
protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the compound to
the subject accordingly. For example, increased administration of
the compound may be desirable to increase the expression or
activity of an flh2882 protein/gene to higher levels than detected,
i.e., to increase the effectiveness of the agent. Alternatively,
decreased administration of the agent may be desirable to decrease
expression or activity of flh2882 to lower levels than detected,
i.e. to decrease the effectiveness of the compound.
[0125] VI. Pharmaceutical Compositions
[0126] The flh2882 nucleic acid molecules, flh2882 proteins
(particularly fragments of flh2882), modulators of an flh2882
proein, and anti-flh2882 antibodies (also referred to herein as
"active compounds") of the invention can be incorporated into
pharmaceutical compositions suitable for administration to a
subject, e.g., a human. Such compositions typically comprise the
nucleic acid molecule, protein, modulator, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, such media can be
used in the compositions of the invention. Supplementary active
compounds can also be incorporated into the compositions.
[0127] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0128] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0129] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., an flh2882 protein or
anti-flh2882 antibody) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above,
as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0130] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0131] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0132] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0133] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0134] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0135] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0136] The nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) PNAS
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells,
e.g. retroviral vectors, the pharmaceutical preparation can include
one or more cells which produce the gene delivery system.
[0137] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0138] VII. Uses of Partial flh2882 Sequences
[0139] Fragments or fragments of the cDNA sequences identified
herein (and the corresponding complete gene sequences) can be used
in numerous ways as polynucleotide reagents. For example, these
sequences can be used to: (a) map their respective genes on a
chromosome; and, thus, locate gene regions associated with genetic
disease; (b) identify an individual from a minute biological sample
(tissue typing); and (c) aid in forensic identification of a
biological sample. These applications are described in the
subsections below.
[0140] a. Chromosome Mapping
[0141] Once the sequence (or a fragment of the sequence) of a gene
has been isolated, this sequence can be used to map the location of
the gene on a chromosome. This process is called chromosome
mapping. Accordingly, fragments of an flh2882 nucleic acid
sequences can be used to map the location of the flh2882 gene,
respectively, on a chromosome. The mapping of the flh2882 sequence
to chromosomes is an important first step in correlating these
sequence with genes associated with disease.
[0142] Briefly, the flh2882 gene can be mapped to a chromosome by
preparing PCR primers (preferably 15-25 bp in length) from the
flh2882 gene sequence. Computer analysis of the flh2882 gene
sequence can be used to rapidly select primers that do not span
more than one exon in the genomic DNA, thus complicating the
amplification process. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the flh2882 gene sequence will yield an amplified
fragment.
[0143] Somatic cell hybrids are prepared by fusing somatic cells
from different mammals (e.g., human and mouse cells). As hybrids of
human and mouse cells grow and divide, they gradually lose human
chromosomes in random order, but retain'the mouse chromosomes. By
using media in which mouse cells cannot grow, because they lack a
particular enzyme, but human cells can, the one human chromosome
that contains the gene encoding the needed enzyme, will be
retained. By using various media, panels of hybrid cell lines can
be established. Each cell line in a panel contains either a single
human chromosome or a small number of human chromosomes, and a full
set of mouse chromosomes, allowing easy mapping of individual genes
to specific human chromosomes. (D'Eustachio P. et al. (1983)
Science 220:919-924). Somatic cell hybrids containing only
fragments of human chromosomes can also be produced by using human
chromosomes with translocations and deletions.
[0144] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the flh2882 gene sequence to design oligonucleotide
primers, sublocalization can be achieved with panels of fragments
from specific chromosomes. Other mapping strategies which can
similarly be used to map an flh2882 gene sequence to its chromosome
include in situ hybridization (described in Fan, Y. et al. (1990)
PNAS, 87:6223-27), pre-screening with labeled flow-sorted
chromosomes, and pre-selection by hybridization to chromosome
specific cDNA libraries.
[0145] Fluorescence in situ hybridization (FISH) of a DNA sequence
to a metaphase chromosomal spread can further be used to provide a
precise chromosomal location in one step. Chromosome spreads can be
made using cells whose division has been blocked in metaphase by a
chemical like colcemid that disrupts the mitotic spindle. The
chromosomes can be treated briefly with trypsin, and then stained
with Giemsa. A pattern of light and dark bands develops on each
chromosome, so that the chromosomes can be identified individually.
The FISH technique can be used with a DNA sequence as short as 500
or 600 bases. However, clones larger than 1,000 bases have a higher
likelihood of binding to a unique chromosomal location with
sufficient signal intensity for simple detection. Preferably 1,000
bases, and more preferably 2,000 bases will suffice to get good
results at a reasonable amount of time. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual of Basic
Techniques (Pergamon Press, New York, 1988).
[0146] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on that chromosome, or
panels of reagents can be used for marking multiple sites and/or
multiple chromosomes. Reagents corresponding to noncoding regions
of the genes actually are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0147] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data (such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified through linkage analysis
(co-inheritance of physically adjacent genes), described in, for
example, Egeland, J. et al. (1987) Nature, 325:783-787.
[0148] Moreover, differences in the DNA sequences between
individuals affected and unaffected with a disease associated with
the flh2882 gene, can be determined. If a mutation is observed in
some or all of the affected individuals but not in any unaffected
individuals, then the mutation is likely to be the causative agent
of the particular disease. Comparison of affected and unaffected
individuals generally involves first looking for structural
alterations in the chromosomes, such as deletions or translocations
that are visible from chromosome spreads or detectable using PCR
based on that DNA sequence. Ultimately, complete sequencing of
genes from several individuals can be performed to confirm the
presence of a mutation and to distinguish mutations from
polymorphisms.
[0149] b. Tissue Typing
[0150] The flh2882 gene sequences of the present invention can also
be used to identify individuals from minute biological samples. The
United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed on a
Southern blot to yield unique bands for identification. This method
does not suffer from the current limitations of "Dog Tags" which
can be lost, switched, or stolen, making positive identification
difficult. The sequences of the present invention are useful as
additional DNA markers for RFLP (described in U.S. Pat. No.
5,272,057).
[0151] Furthermore, the sequences of the present invention can be
used to provide an alternative technique which determines the
actual base-by-base DNA sequence of selected fragments of an
individual's genome. Thus, the flh2882 sequences described herein
can be used to prepare two PCR primers from the 5' and 3' ends of
the sequences. These primers can then be used to amplify an
individual's DNA and subsequently sequence it.
[0152] Panels of corresponding DNA sequences from individuals
prepared in this manner can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences due to allelic differences. The sequences of the
present invention can be used to obtain such identification
sequences from individuals and from tissue. The flh2882 gene
sequences of the invention uniquely represent fragments of the
human genome. Allelic variation occurs to some degree in the coding
regions of these sequences, and to a greater degree in the
noncoding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once per each
500 bases. Each of the sequences described herein can, to some
degree, be used as a standard against which DNA from an individual
can be compared for identification purposes. Because greater
numbers of polymorphisms occur in the noncoding regions, fewer
sequences are necessary to differentiate individuals. The noncoding
sequence of SEQ ID NO:1, can comfortably provide positive
individual identification with a panel of perhaps 10 to 1,000
primers which each yield a noncoding amplified sequence of 100
bases. If a predicted coding sequence, such as that in SEQ ID NO:3,
is used, a more appropriate number of primers for positive
individual identification would be 500-2,000.
[0153] If a panel of reagents from the flh2882 gene sequences
described herein is used to generate a unique identification
database for an individual, those same reagents can later be used
to identify tissue from that individual. Using the unique
identification database, positive identification of the individual,
living or dead, can be made from extremely small tissue
samples.
[0154] c. Use of Partial flh2882 Gene Sequences in Forensic
Biology
[0155] DNA-based identification techniques can also be used in
forensic biology. Forensic biology is a scientific field employing
genetic typing of biological evidence found at a crime scene as a
means for positively identifying, for example, a perpetrator of a
crime. To make such an identification, PCR technology can be used
to amplify DNA sequences taken from very small biological samples
such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, or semen found at a crime scene. The amplified sequence can
then be compared to a standard, thereby allowing identification of
the origin of the biological sample.
[0156] The sequences of the present invention can be used to
provide polynucleotide reagents, e.g., PCR primers, targeted to
specific loci in the human genome, which can enhance the
reliability of DNA-based forensic identifications by, for example,
providing another "identification marker" (i.e. another DNA
sequence that is unique to a particular individual). As described
above, actual base sequence information can be used for
identification as an accurate alternative to patterns formed by
restriction enzyme generated fragments. Sequences targeted to the
noncoding region of SEQ ID NO:1 are particularly appropriate for
this use as greater numbers of polymorphisms occur in the noncoding
regions, making it easier to differentiate individuals using this
technique. Examples of polynucleotide reagents include the flh2882
sequences or fragments thereof, e.g., fragments derived from the
noncoding region of SEQ ID NO:1, having a length of at least 20
bases, preferably at least 30 bases.
[0157] The flh2882 sequences described herein can further be used
to provide polynucleotide reagents, e.g., labeled or labelable
probes which can be used in, for example, an in situ hybridization
technique, to identify a specific tissue, e.g., brain tissue. This
can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such flh2882
probes can be used to identify tissue by species and/or by organ
type.
[0158] In a similar fashion, these reagents, e.g., flh2882 primers
or probes can be used to screen tissue culture for contamination
(i.e., screen for the presence of a mixture of different types of
cells in a culture).
[0159] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patent applications, patents, and published patent
applications cited throughout this application are hereby
incorporated by reference.
EXAMPLES
Example 1
Identification of Human flh2882 cDNA
[0160] In this example, the human flh2882 nucleic acid molecule was
identified by screening appropriate cDNA libraries. A non-annotated
EST was first identified and used to screen a human fetal cDNA
library. Several positive clones were identified, sequenced, and
the sequences were assembled. BLAST analysis of nucleic acid
databases in the public domain showed homologies only to the 3'
untranslated region of the flh2882 nucleic acid molecule and the
original EST (GenBank.TM. Accession number T09060).
Example 2
Tissue Expression of the flh2882 Gene
[0161] Northern Analysis Using RNA from Human Tissue
[0162] Human brain multiple tissue northern (MTN) blots, human MTN
I, II, and III blots (Clontech, Palo Alto, Calif.), containing 2
###g of poly A+ RNA per lane were probed with human
flh2882-specific primers (probes). The filters were prehybridized
in 10 ml of Express Hyb hybridization solution (Clontech, Palo
Alto, Calif.) at 68.degree. C. for 1 hour, after which 100 ng of
.sup.32P labeled probe was added. The probe was generated using the
Stratagene Prime-It kit, Catalog Number 300392 (Clontech, Palo
Alto, Calif.). Hybridization was allowed to proceed at 68.degree.
C. for approximately 2 hours. The filters were washed in a 0.05%
SDS/2.times.SSC solution for 15 minutes at room temperature and
then twice with a 0.1% SDS/0.1.times.SSC solution for 20 minutes at
50.degree. C. and then exposed to autoradiography film overnight at
-80.degree. C. with one screen. The human tissues tested included:
heart, brain (regions of the brain tested included cerebellum,
cerebral cortex, medulla, spinal cord, occipital pole, frontal
lobe, temporal lobe, putamen, amygdala, caudate nucleus,
hippocampus, corpus callosum, substantia nigra, subthalamic nucleus
and thalamus), placenta, lung, liver, skeletal muscle, kidney,
pancreas, spleen, thymus, prostate, testis, uterus, small
intestine, colon (mucosal lining), and peripheral blood
leukocyte.
[0163] There was a strong hybridization to human whole brain, and
the substantia nigra indicating that the approximately 2.6 kb
flh2882 gene transcript is expressed in these tissues.
Example 3
Expression of Recombinant flh2882 Protein in Bacterial Cells
[0164] In this example, flh2882 is expressed as a recombinant
glutathione-S-transferase (GST) fusion protein in E. coli and the
fusion protein is isolated and characterized. Specifically, flh2882
is fused to GST and this fusion protein is expressed in E. coli,
e.g., strain PEB 199. As the human protein is predicted to be
approximately 38.7 kDa, and GST is predicted to be 26 kDa, the
fusion protein is predicted to be approximately 64.7 kDa, in
molecular weight. Expression of the GST-flh2882 fusion protein in
PEB199 is induced with IPTG. The recombinant fusion protein is
purified from crude bacterial lysates of the induced PEB 199 strain
by affinity chromatography on glutathione beads. Using
polyacrylamide gel electrophoretic analysis of the protein purified
from the bacterial lysates, the molecular weight of the resultant
fusion protein is determined.
Example 4
Expression of Recombinant flh2882 Protein in COS Cells
[0165] To express the flh2882 gene in COS cells, the pcDNA/Amp
vector by Invitrogen Corporation (San Diego, Calif.) is used. This
vector contains an SV40 origin of replication, an ampicillin
resistance gene, an E. coli replication origin, a CMV promoter
followed by a polylinker region, and an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire flh2882
protein and a HA tag (Wilson et al. (1984) Cell 37:767) fused
in-frame to its 3' end of the fragment is cloned into the
polylinker region of the vector, thereby placing the expression of
the recombinant protein under the control of the CMV promoter.
[0166] To construct the plasmid, the flh2882 DNA sequence is
amplified by PCR using two primers. The 5' primer contains the
restriction site of interest followed by approximately twenty
nucleotides of the flh2882 coding sequence starting from the
initiation codon; the 3' end sequence contains complementary
sequences to the other restriction site of interest, a translation
stop codon, the HA tag and the last 20 nucleotides of the flh2882
coding sequence. The PCR amplified fragment and the pCDNA/Amp
vector are digested with the appropriate restriction enzymes and
the vector is dephosphorylated using the CIAP enzyme (New England
Biolabs, Beverly, Mass.). Preferably the two restriction sites
chosen are different so that the flh2882 gene is inserted in the
correct orientation. The ligation mixture is transformed into E.
coli cells (strains HB101, DH5a, SURE, available from Stratagene
Cloning Systems, La Jolla, Calif., can be used), the transformed
culture is plated on ampicillin media plates, and resistant
colonies are selected. Plasmid DNA is isolated from transformants
and examined by restriction analysis for the presence of the
correct fragment.
[0167] COS cells are subsequently transfected with the
flh2882-pcDNA/Amp plasmid DNA using the calcium phosphate or
calcium chloride co-precipitation methods, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Other suitable
methods for transfecting host cells can be found in Sambrook, J.,
Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of
the flh2882 protein is detected by radiolabelling
(.sup.35S-methionine or .sup.35S-cysteine available from NEN,
Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and
Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA
specific monoclonal antibody. Briefly, the cells are labelled for 8
hours with .sup.35S-methionine (or .sup.35S-cysteine). The culture
media are then collected and the cells are lysed using detergents
(RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM
Tris, pH 7.5). Both the cell lysate and the culture media are
precipitated with an HA specific monoclonal antibody. Precipitated
proteins are then analyzed by SDS-PAGE.
[0168] Alternatively, DNA containing the flh2882 coding sequence is
cloned directly into the polylinker of the pCDNA/Amp vector using
the appropriate restriction sites. The resulting plasmid is
transfected into COS cells in the manner described above, and the
expression of the flh2882 protein is detected by radiolabelling and
immunoprecipitation using an flh2882 specific monoclonal
antibody.
Example 5
Characterization of the Human flh2882 Protein
[0169] In this example, the amino acid sequence of the human
flh2882 protein was compared to amino acid sequences of known
proteins and various motifs were identified.
[0170] The human flh2882 protein, the amino acid sequence of which
is shown in FIG. 3 (SEQ ID NO:2), is a novel protein which includes
337 amino acid residues. Hydrophobicity analysis indicated that the
human flh2882 protein contains seven transmembrane domains between
amino acid residues 11-28 (SEQ ID NO:4), 43-62 (SEQ ID NO:5),
80-102 (SEQ ID NO:6), 121-146 (SEQ ID NO:7), 169-190 (SEQ ID NO:8),
247-265 (SEQ ID NO:9), and 280-300 (SEQ ID NO:10). The nucleotide
sequence of the human flh2882 was used as a database query using
the BLASTN program (BLASTN1.3 MP, Altschul et al. (1990) J. Mol.
Biol. 215:403). The closest hit was to the mouse 5HT5B receptor
(GenBank.TM. Accession Number P31387). The highest similarity is
24/77 amino acid identities.
[0171] Equivalents
[0172] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *